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1. Cancel Help Fixed wave length detector configuration window 46 Fig 62 Fig 63 Fig 64 Fig 65 Setup and Control of Knauer HPLC Systems Detector 5 2500 Configuration Mame 5 2500 Detector Type Flow Cell Mame ls 230025 2400 Detector Type ELS Detector K 2x00 1 Co nfiguration Flow Cell Name k 2501 Detector Type K 2501 bl Serial Port COM1 Serial Number je ta E 23004K 2400 Y Ass Units ma v K 2301 E 2401 E 2500 Flow Cet 10mm Cancel Help Older Knauer single channel detector configuration windows Detector UVD2 1L Configuration r Interface LAN vi El Name Detector UVD2 1L IP Address Obtained using S N Serial Number IP Port 1 0001 Chn Name 1 Channel 1 IW Use S N to identify the instrument Options Y Axis Units mau y MV D2 Lamp I Halogen Lamp Flow Cell Leak Sensor Sensitivity Medium vi Cancel Help UVD2 1L configuration window Detector 10D UVD2 1S Configuration Interface LAN v e Name Type juy 100 vi P Addr Obtained using SN Serial Number IP Port 1 0001 Chn Name 1 Channel 1 V Use S N to identify the instrument Y Axis Units mau y Options IV D2 Lamp Flow Cell Cancel Help 10D UVD2 1S configuration window Detector 2520 Configuration Interface 5 2520 A ddress Obtained using S N
2. Fig 342 FC parameters Relative to previous event single event F Fraction Collection Program 00 Single Event Unconditional 0 00 0 00 Timed next 7 gj 1053 e Event Local Maximum 020 030 Max next r a mala Esrar anal Mavierru un nm nom Ada An r Fig 343 FC parameters Relative to previous event single event ChromGate Preparative Option 243 Use as reference for correction next events Normally any shift in retention times is increasing with the magnitude of the retention Using the reference option ChromGate adapts the shift for the calculation of the expected allowed interval It is possible to set more than one event as reference According to the elapsed run time always the last one will be used for calculation FC Parameters at 4 00 min Signal 3 Relative to previous event min Ascending C Descending Signal Level Unit 25 ignal Level Units ne Expected interval min 0 25 Action Collect To Please notice that the parameters will C nest 0 position 111 be used in the Units selected for the Detection Channel i Use as reference for correction next events Cancel Fig 344 FC parameters Use as reference single event After closing the window the parameter indication will be supplemented by Ref Action selection None Selecting this action option neither the recycling valve nor the fractionation valve will be switched The previous action is continued
3. Knauer Instrument Control Method Options 179 Fig 266 Fig 267 Fig 268 Validation of integration C Reference chromatogram data file View report Gausion seaierce T Baseine Date JT goe 100 Validation of Integration dialog window The test can be carried out using either a Gaussian curve as shown in Fig 266 or a pre selected real chromatogram If you select the latter option the usual chromatogram window is opened enabling the software to integrate this chromatogram Validation of integration Reference chromatogram dea lle D ProectsWELLCH KRCHPL 1 VE ret Valkyibenzoate seterence dat Weve repo C Gaussan sequence FF Chee Validation of Integration dialog window real chromatogram To perform the validation procedure just press the Analyze button All integration parameters are preset and cannot be altered If you had previously changed the integration events the validation will not be performed and the following information box will be displayed x No Validation will be performed The following Integration Events table should be used for the Validation procedure Event Start time Stop Time Value Width 0 000 0 000 0 2 Threshold 0 000 0 000 50 0 No Manual Integration Fixes should be used Please change the Integration Events and try again Validation of Integration attempt with manual integration fixes 180 Fig 269 Fig 270 Knauer Instrument C
4. Check the lamp OFF box to switch off the detector lamp after a measurement is finished This option may be used for the shutdown methods Detectors UVD 2 1L S 2520 and 2550 If the lamp was switched off by the method and you switch off the detector then on the next detector start the lamp is still off it must be switched on manually in the detector s own setup or with the software via Instrument Status Direct Control Action at the end of run UVD 2 1L UVD 2 15 S2520 only The detectors S UVD 2 1L UVD 2 1S and S 2520 allow not only to switch off the lamp at the end of run but also to switch the device into Standby You should switch the lamp on manually before you start the next working session After power up allow 30 minutes for the lamp to stabilize before running samples that may require sensitive measurements 94 Fig 137 Creating an Instrument Control Method Wavelength table The wavelength table is a spreadsheet to enter your time detector program In the first row at time 0 00 min enter the initial wavelength s in nm The editing principle is the same as for the time gradient program described in the chapter Instrument Setup Pumps Pump Program Configuring Events Digital Outputs UVD 2 1L S2520 only This option is given only for the detectors UVD 2 1L and S 2550 Configure the events by pressing the H button which is displayed when you place the cursor on the Events cell The follow
5. Name IP Ai Serial Number IP Port 1 0001 Chn Name 1 Channel 1 V Use S N to identify the instrument m Options MV D2Lamp Halogen Lamp Y Axis Units mau vi Flow Cell OK Cancel Help Coa Hb S 2520 configuration window Setup and Control of Knauer HPLC Systems Fig 66 Fig 67 Fig 68 Detector 2550 Configuration Name 5 2550 Serial Number Channels fi 4 Chn Name 1 Channel 1 Chn Name 2 pocos Chn Name 3 fennel Chn Name 4 ch Annel Options Y D2 Lamp Halogen Lamp Interface R5 232 COM Port COM1 Baud Rate 1115200 m Y Axis Units mau y Flow Cell Detector K 2600 Configuration Name k 2600 Interface Network 1 hi Serial Number Y Ass Units m l Flow Cell Detector 2600 Configuration Name 5 2600 Serial Number 65478 Channels p4 Chn Name 1 Channel 1 Chn Name 2 Channel 2 Chn Name 3 soe gt Chn Name 4 Channel A Cancel Help S 2550 configuration window Channels i Chn Name 1 Poo Chn Name 2 fo Chn Name 3 mm Chr Name H4 Add Info K 2600 configuration window Interface A R5 232 COM Port coma Baud Rate 115200 X Options V D2Lamp I Halogen Lamp IV Shutter Control Y Axis Units m l x Flow Cell Cancel Help S 2600 configu
6. To set the noise and drift select the desired event field then select the start and stop time and value ChromGate includes a large number of additional parameters that can be used to refine the chromatogram These can be applied on a programmed basis or on a manual basis for data that is already collected A detailed discussion on integration is presented in the ChromGate Chromatography Data System Reference Manual 20 Short guide ChromGate KNAUER Instrument Control Generating and Using a Calibration Curve A calibration curve is normally generated via a sequence which is discussed in the chapter Quantitation and Using Sequence Files below Method File Commands Fig 14 A method file can be saved retrieved and edited as desired in the same way as with other Windows applications As an example if the Method dialog box is open and you want to save a new method select File Method Save as and enter the desired name Save Method File As Abbrechen Hite Dateiname v Dateityp Method files met F Description Save Method File as Window Similarly if you want to open a method select File Method Open and enter the file name A printed report describing all aspects of a method can be obtained by selecting File Method Print Quantification and Using Sequence Files Overview Calibration involves the generation of the relationship between the concentration of a compound and the are
7. You can enter the settings either by typing the desired values into the corresponding field or by selecting your choice from the pull down menu which becomes accessible via the xi button appearing in the selected field For details on the spreadsheet operations refer to the section Creating a Time Gradient Program as described in the chapter nstrument Setup Pumps Pump Program The valves program profile will be adapted automatically to the spreadsheet inputs when you save the method If you select any field in the comment column the H button will appear to open a comment window 132 Fig 190 Fig 191 Creating an Instrument Control Method Comments Valves 3 and 4 for column switching only simultanenously to the same positions Setup window Knauer Switching Valves Comments Pretreatment Checking this option the Setup button becomes active Clicking on it the following dialog window will be opened Pretreatment Program le Timecmin Valve 1 Valvex 2 valve 3 Valve xt 4 Commen 2 00 1 3 6 6 y nee ance For pretreatment zero time is considered to be the injection time Please use negative times to program the pretreatment time table Pretreatment setting dialog window The appearance of the valve program corresponds to that of the main valve setup window It must be edited in the same way as described above To add a new line make a right mouse click into the table and sele
8. 11 5 Syringe volume ul 250 x Options Column oven Tray cooling Tray Cancel Help Configuration window for the autosampler 3900 Name Enter a descriptive name for the sampler This name will appear on the autosampler tab when the instrument setup or status windows are displayed Use a name which is unique within the instrument Serial Number Type in the serial number of the detector you are using Device ID Select the device identifier for the autosampler from the drop down list This must match the settings of the autosampler being used To check the device identifier for the autosampler 3900 press the SYSTEM MENU and then SERIAL lt SETTINGS gt buttons on the autosampler keyboard Serial Port Select the number for the communication port on your PC where the autosampler is connected Loop Volume Enter the volume of the installed loop in ul Tubing Volume Type in volume of the installed needle and tubing in ul The valid range is 0 999 ul If the original tube is installed you can find the tubing volume on a label on the tube Syringe Volume Select the volume of installed syringe from the drop down list The choices are 250 ul 1000 ul and 2500 ul Options Column oven Tray cooling Check this box if you want to control the column thermostat and or the tray cooling The appropriate corresponding temperatures are to be entered later as part of the instrument setup Tray Click the Tray but
9. AS Status Searching vial Tray Cooling Current Vial 50 Tray Temperature O Serial Port COM1 Fig 239 Autosampler 3800 status tab Monitor AS Status The status Ready will be displayed when the autosampler is waiting to perform an injection If an injection program is running the actual program step will be indicated After a completed injection the status is described as Analysis timer running during the chromatographic run Current Vial The actual position of the injection needle is shown Tray Temperature C If the cooling option is installed and configured the actual tray temperature will be shown Creating an Instrument Control Method Aux The auxiliary output s status of the AS 3800 is indicated The On state is represented by an active red LED symbol 8 and the Off state is represented by an inactive LED symbol Direct Control Tray Cooling The tray cooling can be switched On or Off as long as the cooling option has been included and configured The AS 3800 does not support to set a temperature Advance Tray Clicking briefly on this button will move the tray to the next position By holding down the Advance Tray button the tray will rotate until the button is released Instrument Status Autosampler Optimas 3900 MM Instrument Status DER System Status gy s106 Y As 3900 BY s 2550 s 2300 5 2400 Monitor Direct Control AS Status Ready Oven Temperature C 20
10. All LAN controlled devices can be added and configured automatically using the Auto Configuration button on the configuration main window Beside this you can let the software search for your already connected and switched on device from the devices configuration window The option Use S N to identify the instrument must be enabled Click on the button to start the search for all connected LAN devices of the currently configured type If more than one will be found all hits will be shown in the window Select the desired device from the shown list Configuration Setup and Control of Knauer HPLC Systems 65 information as syringe loop and tubing volume as well as the tray will be added with default settings Please check if these settings match with the installed option and change it if necessary All configuration data will be downloaded into the autosampler if the instrument will be open As an option you can enter the IP address of your device if the option Use S N to identify the instrument is disabled Please note that in case the device receives the IP address from a DHCP server e g a router the IP address may change if the device is switched off and switched on Loop Volume Enter the volume of the installed loop in ul If the Prep mode option is checked the loop volume is fixed on 10000 ul Tubing Volume Type in volume of the installed needle and tubing in ul The valid range is 0 999 ul If the original tu
11. Displays the library from the method or for Quick enables you to select a library to be used Pre Filters tab When Quick is selected as the Search Mode the items on this tab allow the user to specify search pre filters that will be performed on library spectra prior to the test for similarity All pre filters are optional If Method is selected as the Search Mode the items on this tab are read only and reflect the parameters values on the Library tab of PDA Options Retention Time Range When a Retention Time Range is specified library search will be restricted to those library entries whose retention time is within the specified range Entries outside this range are automatically excluded from the search no similarity calculation is made Entering a value for this is optional Lambda Max When one or more of these values is specified library search will be restricted to those library entries containing a lambda max within 5 nm of one of the specified values Entries without a matching lambda max are automatically excluded from the search no similarity calculation is made Entering values for this is optional Compound Name Filter When a Compound Name Filter is specified library search will be restricted to those library entries whose name contains the specified string as a case insensitive sub string Entries without a matching sub string are automatically excluded from the search no similarity calculation is made Enter
12. Fig 47 39 Pump 5 1000 Configuration Hame 5 1000 Pump Type Gradient Mode lzocratic Interface Network x Serial Number 67890 Head 10 mi Pressure Units MPa pe Material nta Max Head Flow 10 0 rol min Mas Head pressure 40 0 MPa Add Info Cancel Help Pump S 1000 configuration window Pump 10P 20P P2 15 P4 15 Configuration Mame Type Pump 10P y Gradient Mode isocratic Serial Number Head 10 ral hel Interface LAN r El Material nra Mas Head Flow 10 0 mil min Max Head Pressure 40 0 MPa IF Fort 11 0001 W Use S N to identify the instrument a Add Info Cancel Help Pump 10P 20P P2 1S P4 1S configuration window Pump 5 1050 Configuration Mame ls 1050 Gradient Mode Isocratic v Senal Number Head 10 mi its MP r ER E Pressure Units a Material na Max Head Flow 10 0 ml min Max Head Pressure 70 0 MPa IF Port 0001 W Use S M to identify the instrument Di lea AER Cancel Help Pump S 1050 configuration window Pump P2 1L Configuration Mame Pump P2 1L Gradient Mode lsocratic Serial Number Head 100 mi its MP Interface Lan y Bl Pressure Units Mas Head Flow 100 0 ml min Max Head Pressure 40 0 MPa IP Pott 10001 Leak Sensor Sensitivity Medium h Use S N to identify the instrument Config Service Add Info Cancel Help Pump P2 1L configuration win
13. Leak Sensor Sens Medium Add Info Cancel Help Assistant ASM2 1L configuration window tab General If one of the modules should not be controlled it must be removed from the ASM 2 1L configuration There is no option to disable a configured module later on in the Method Setup The configuration window has four tabs one for general settings and one for each of the three device positions left middle and right While the General tab allows for settings for the whole assistant the device tabs give the option to configure the included devices General Assistant Name The name of the assistant ASM2 1L appears automatically You can enter any other descriptive name This name will appear on the assistant tab when the instrument setup or instrument status windows are displayed Setup and Control of Knauer HPLC Systems 61 ae Fig 87 Fig 88 Use a name which is unique within the instrument configuration Serial Number If you have read out the device via LAN the serial number has been added automatically Otherwise type in the serial number of the instrument Interface The Assistant ASM2 1L supports LAN connection only one LAN port for all 3 devices is required Interface SEN egg 2 IP Port 11 0001 MW Use S N to identify the instrument LAN interface dialog As all other LAN controlled devices the assistant ASM2 1L can be added and configured automatically using the Auto Configuration bu
14. MV Air segment Flush volume ul 30 F Enable tray cooling Headspace pressure esa Wash volume ul 300 4 Needle height mm 15 Timed events Alarm buzzer I Run screens Fig 175 Autosampler Triathlon setup window Standard Injection In the Standard Injection section you can enter parameters for the standard actions that can be performed with the autosampler Injection Method Click one of the Flushed Partial or pl Pick up fields to select the injection method 877 The maximum programmable injection volumes are Flushed loop not programmable is equal to the loop volume but needs more sample to fill the loop completely 3 x loop volume for loop volumes lt 100 ul and 2 x loop volume for loop volumes gt 100 ul Partial loop fill 50 of the programmed loop volume ul Pick up Injection volume loop volume 3 x needle volume 2 Syringe Speed and Scale Factor The aspirating speed of the syringe used in injection methods can be adapted depending on the viscosity of the samples the choices are Low Normal and High Alternatively syringe speeds can be reduced by entering a scale factor 1 to 10 The syringe speed will be the scale factor multiplied by the syringe speed The syringe speed during the washing or the rinsing procedures is not affected by this setting Air Segment Check the Air Segment box to enable the use of an air segment between the sample and the wash solvent to reduce th
15. Select the number for the communication port on your PC where the autosampler is connected Port settings In the dialog box for the port settings only the baud rate will be active for selection of 4800 Or 9600 Port settings Baud Rate EST Data Bits Farity Stop Bitz Port settings Vial number For the AS 360 560 and 565 a number in the range from 1 through 65 can be selected In a method only the number of vials entered here are available A wrong number will lead to an error message ES Please enter the vial number between 1 and 65 Vial type Select the volume of the used vials from the drop down list The choices are 1 8 ml 0 5 ml and 0 3 ml Syringe Volume Select the volume of the installed syringe from the drop down list The choices are 250 pl 500 ul 1000 ul and 2500 ul Loop Volume Select the volume of the installed loop from the drop down list The choices are 20 ul 100 pl 500 ul and 1000 ul 78 Setup and Control of Knauer HPLC Systems Configuration Miscellaneous Instruments Configuration Switching Valves Fig 109 The switching valves icon refers to a group of the Knauer switching valves the Valco Vici valves icon to the latest Vici valve drivers Each of them must be configured before using in an instrument method Double click the icon and complete the configuration dialog Valco VICI Switching Valves Configuration Valves number mn Configure valve Valve
16. Time 9 63898 Minutes Amplitude 254 Wavelength nm Wavelength nm Wavelength nm Fig 195 Setup window Auxiliary Traces Any configured HPLC system with at least one pump with pressure transducer a column oven or a flowmeter will be completed by an additional tab for auxiliary traces Only the selected traces will be stored with the run data Setting up a Trigger Any configured HPLC system with at least one detector acquisition channel will be completed by an additional tab for the trigger Click on the Trigger tab to designate the trigger type and to setup the synchronization The trigger type determines how the data sampling and the gradient program s are started nm Instr ument Setup EAS S 1000 E as Triathlon E Switching Valves y Ovens 4050 als 2500 7X Aux Traces pen Trigger Type Y None Sampling starts immediately after clicking on Start Sequence acquisitions do not pause between runs Manual Operator has to press Enter to start the run Sequence acquisitions pause for confirmation between runs External If the data sampling is started from an external trigger select this option The type of trigger is designated in instrument configuration Select when using a contact closure from an autosampler or manual injector Flat FST 008 on Valve at Jemperature FC Fig 196 Setup window trigger tab Trigger Type Select the type of synchronization from the drop down list The choic
17. otherwise the Run Status displays a run time in minutes elapsed from the start of the run Signal uRIU The actual signal is indicated in this field The unit corresponds to the setting in the configuration Direct Control The direct control option must be activated see Fig 249 on page 170 The buttons Flush and Autozero can be pressed to perform the corresponding functions The flush function flushing for 1 min is deactivated during a run The Autozero defines the current signal as a zero level Instrument Status UV Detectors S 2520 S 2500 S 200 K 200 K 2000 1 K 2500 1 The tabs for most of the UV detectors appear identical independent of the features of the detector Unavailable features or functions are only grayed and inaccessible 146 Fig 218 Creating an Instrument Control Method Monitor Direct Control Aun Status 0 97 min Belen O I Lamp fi Signal m l 0 578 Autozero Wavelength nm 254 Wavelength nm 254 Apply Se al Port COM 8 SN 73286 UV Detector status tab example S 2500 The only exception is the Direct Control Area of the fixed wave detectors Direct Control HOLD D2 Lamp i Autozero 630 254 Fig 219 UV Detector status tab Direct Control S 200 Monitor Run Status The status HOLD will be displayed when no program is running otherwise the Run Status displays a run time in minutes elapsed from the start of the run Signal AU The actual
18. 109 Source 53 Actualize flow 237 Add Info 50 Air Segment 113 117 122 Alias see AS 3950 Alltech 650 see Conductivity detector Analog output 154 Analysis Options 37 AS 3800 Configuration 70 111 Instrument Status 161 Setup 111 Wiring Scheme 288 289 AS 3900 Configuration 71 Instrument Status 162 166 Setup 112 Wiring Scheme 284 AS 3950 Configuration 64 Instrument Status 163 Setup 116 Autosampler Configuration 60 63 Instrument status 156 161 Large Injection 128 Setup 107 111 Wiring Scheme 287 288 289 290 Autosampler 3900 see AS 3900 see AS 3900 see AS 3900 see AS 3900 Autosampler 3950 see AS 3950 Autozero 58 96 Auxiliaries 112 126 Axis Offset 94 Axis Units 54 81 82 B Background Correction 227 Bandwidth 102 151 Baseline Check 18 37 136 Basic Marathon see AS 3800 C Calculating Total Purity 227 291 Calibration Curve 20 Chromatogram View 204 Collector reset 236 Column Oven 68 71 135 Configuration 80 Instrument Status 168 Setup 133 Communication port 33 Conductivity detector Configuration 48 Instrument status 154 Setup 104 Configuration System 14 Configuration Acquisition channel 53 Autosampler 60 63 Column oven 80 Detector 45 Flowmeter 81 Fraction collector 230 Interface 31 KNAUER HPLC System 35 Kontron detector 55 56 Manager 5000 79 Pumps 38 Switching Valves 78 User defined Detector 53 Virtual Detector 57 Contour View 199 Control Method Options 170 D DAD Analysis 222 Calculations
19. 144 Vial Volume ml TN Serial Port comi vi Port Settings Add Info Cancel Help Fig 320 Fraction collector configuration window FC Name Enter a descriptive name for the collector This name will appear on the fraction collector tab when the instrument setup or status windows are displayed Use a name which is unique within the instrument Serial Number Type in the serial number of the detector you are using Rack Type Select from the drop down list the required rack type The possible choices depend on the Type of fraction collector The Vials number will be displayed automatically ChromGate Preparative Option 231 Fig 321 Vial Volume ml The rack depended value will be displayed automatically However you are allowed to overwrite it by self defined values lf any fraction is larger than the set volume the next free collection position will be used for continued collection Serial Port Select from the drop down list the serial port number for the communication port on your PC where the collector is connected Interface S 3050 MultiValve fraction collector Foxy R1 R2 only Select the Interface you want to use for communication with the device RS 232 Select the number for the RS 232 communication port COM Port on your PC to which the autosampler is connected LAN S 3050 MultiValve fraction collector To let the software search for your already connected and switched on devic
20. A Tempeian 259 C Pierina 0 0UMPa y Oriri wal nia E Stale Cod la GE ni Chal 87h ANU G Ohnl 240 nm 0000 m l Chn2 323 nn 0000 m l ZA mva SE mva ZC ma 30 va Overs Tengs n a Tey Tempe n a or Help press FL Instrument Status window system status tab method not running 138 Fig 201 Fig 202 Fig 203 Creating an Instrument Control Method E Knawer HM Method Method f met Data Sample 4 dat Project Default Instrument Status E all Epe f r p teid Des epa siete Corro Reports Ey Pr Ps ira cer 1 g O sja Seamus a 5 1000 GH AS 3800 l Jetteam Oven MB K 2600 a 23 7 Flow 1 000 ml if Curert vist 50 a J Fempestue 200 T sasssa Vu Chnt 258 nn 16352 m l ar Presmse 76 MPA recuse AS staur Arken beret nord E Suma ide Chn2 400 ne 0709 mA ZA Bl Se 4 Creed Temgesabuer nia wc 0 0 Instrument Status window system status tab method running For some devices the software reads out the data only during a running method Therefore if no method is running some data are not available n a or might not be the actual data but rather data from a previous run The running method is visualized by the dark dotted lines between the displayed instruments For each pump identified by its name the actual flow and pressure is displayed If a gradient system is configured the solvent composition w
21. A table place holder will appear on your report To view the current results do a right hand mouse click and select the Show Data at Design Time option Adding an SEC Summary Report To add a summary report to your custom report do a right hand mouse click and select Insert Report SEC Summary Report A dialog will appear where you select the trace for the summary GPC SEC Summary Report Options Trace ME Cancel Help Select the trace for the report and click OK A table placeholder will appear on your report To view the current report data click the right mouse button in the table and select Show Data at Design Time SEC Summary information Channel A Processing Start Time min 12 831 Processing Stop Time min 25 859 Number of Slices 39 Weight Average Molecular Mass 552812 Number Average Molecular Mass 76133 Z Average Molecular Mass 1250191 Z 1 Average Molecular Mass 1847926 Polydispersityindex 7 261 Peak Molecular Mass 183506 Z Average Weight Average 2 262 Z 1 Average Weight Average 3 343 Saving an SEC Report Template If you plan to use the same report for all your SEC methods you can save the custom report as a template which can then be opened from SEC Option ES 281 any SEC method To save the template select the File Template Save As command Then enter the name you wish to use for your template To use a stored template for a new method open the template using the File T
22. Advance Tray Triathlon only Clicking briefly on this button will move the tray to the next position By holding down the Rotate Tray button the tray will rotate until the button is released Instrument Status Kontron Autosamplers OP System Status Ly Virtual Detectd d Kontron 360 Monitor Direct Control AS Status Ready Auxiliary 17 On Current ia 5 Position 5 Advance tray Initial fash O Serial Port COM1 Fig 243 Kontron Autosamplers status tab Monitor AS Status The status Ready will be displayed when the autosampler is waiting to perform an injection If an injection program is running the actual program step will be indicated After a completed injection the status is described as Analysis timer running during the chromatographic run Current Vial The actual position of the injection needle is shown Direct Control Auxiliary The auxiliaries 1 or 2 1 4 for 460 465 autosamplers can be selected and activated Position The tray can be moved directly to any entered position Initial Wash Press this button to force the autosampler to perform the needle wash Advance Tray Clicking briefly on this button will move the tray to the next position Instrument Status Miscellaneous Instruments Instrument Status Manager 5000 5050 IF2 I O O System Status 51000 13 AS 3300 EJ JetStream ED Knauer Flowmeter Monitor Direct Control Run Status HOLD AD mv fo ADF 4 fo
23. Air Segment Check the Air Segment box to enable the use of an air segment between the sample and the wash solvent to reduce the amount of flush volume Headspace Pressure Check the Headspace Pressure box to enable the use of headspace pressure in combination with the syringe to transport sample to the loop The compressor will always be used during a wash procedure The accuracy and reproducibility of the autosampler may decrease if headspace pressure is switched off 114 Fig 164 Creating an Instrument Control Method Needle Wash Check the Needle Wash box to enable a wash procedure Alarm Buzzer Check the Alarm Buzzer box to enable the alarm beep which is emitted when an error occurs during a run Run Screens Check the Run Screens box to display the actual run information on the autosamplers display during a run Temperature control If your autosampler is equipped with a tray cooling and or a facility thermo stated column oven AS 3900 only this options can be checked and the desired temperatures can be entered The tray cooling allows to enter a temperature between 4 C and 15 C The AS 3900 oven can only control the temperature at ambient temperature 5 C and up The maximum temperature is 60 C Timed Events These types of events or methods enable you to control other devices via the two auxiliaries contact closures or to activate a new oven set point Each event can be programmed on a time bas
24. L and 5 141 L 5 85 i Single Event Signal Level Slope 0 10 0 00 Land S Waste T EY 6 30 Single Event Signal Level Slope 0 10 0 00 L and 5 142 F EI 6 70 i Single Event Signal Level Slope 0 10 0 00 Land S Waste C E 7 98 Single Event Signal Level Slope 0 10 0 00 L and 5 143 C 8 38 Single Event Local Minimum 0 10 0 00 Min 144 T EI 8 75 Single Event Signal Level Slope 0 10 0 00 Land S Waste F 3 10 00 Single Event Unconditional 0 00 0 00 Timed Waste E Example for a fraction table The colored squares below the graph are to indicate the status in running methods and for fraction tracing However those aspects will be described later in the chapter instrument status of a running control method page 137 Pretreatment The pretreatment option is only shown if a recycling valve has been selected in the fraction collector configuration dialog If you enable the pretreatment check box the button Setup is accessible If you click on the button the pretreatment configuration window will open Here you can select the position of the recycling valve if any other device runs a pretreatment The choices are Waste and Slv Recycling ChromGate Preparative Option 247 Fraction Collector Instrument Status The fraction collector status is shown in two separated tabs one for monitoring the instrument status and direct control options Fig 353 and the other for the rack view Fig 354 MM Instrument Status O System Status
25. S 1000 E AS 3950 EY Ovens 4050 a s 260 S 3050 FC Monitor Direct Control PY Rack View onie Direct Control Single Event vi Status n a 7 Operation Waste vi Set Signal 9 Collect to Next Pos Vial Run Time Settings Vial Volume ml Signal Level C AND OR Slope Fr Time min Expected Interval min 0 10 Fr Volume ml Clear Rack Serial Port COM9 SN n a Fig 353 Fraction Collector status tab Monitor Direct control MM Instrument Status O System Status J 5 1000 sosa Ovens 4050 629 s 2600 E s 3050FC Monitor Direct Contro Rack View Vial 1 4 67 4 98 min 15 47 ml 00 50909090909996 OOOOCOOOOOCO 0000000000 00000090000 0000000000 0000000000 0000000000 SOSGSSGSSOSS o0EMCEGGOP OG Seal Port COM9 SN n a Fig 354 Fraction Collector status tab Rack view In case of the Knauer MultiValve fraction collector both tabs are combined to one Fig 355 Monitor Status The actual operation is displayed It is additionally indicated by the LED color waste gt grey fraction gt green peak recycling gt red or solvent recycling gt blue Signal ChromGate Preparative Option The actual value of the controlling detector channel is displayed Vial The actual fraction vial number is displayed Fr Time min The collection time for the a
26. Select Color or Grayscale for the plot Checking the Wire Mesh box will cause the data to be rendered as a wire frame plot rather than as a solid fill plot Colors Range Select how you want the coloration on the plot to appear When Light amp Dark Range is selected alternating light and dark bands are used When Full Spectrum is selected then a continuous color spectrum is used Background Select the color for the background of the plot Axes Select the color for the plot axes Display This specifies the relative quality of the displayed contour plot Lower Display Detail will result in faster drawing of the plot Print This specifies the relative quality of the printed contour plot Lower Print Quality will result in faster printing Viewing Use these fields to view current rotation settings or to set them manually Light source Selecting this option causes the plot to be shaded as if an external light source were shining upon it Rotation This reports the current level of rotation front to back in the aspect position of the plot When a new value is entered the plot is redrawn to reflect the new value upon exiting the dialog 198 ChromGate PDA Option Elevation This reports the current level of tilt forward or backward in the aspect position of the plot When a new value is entered the plot is redrawn to reflect the new value upon exiting the dialog Roll This reports the current level of roll side
27. Stop Bits Port settings Virtual Fraction Collector Configuration Fig 324 Virtual FC Configuration FC Name Virtual FC Tubing Parameters Serial Number Siv Peak Recycling Rack Type Micra Number of Viale 240 Vial volume mi me serial Part vi Porsetings Add Info Cancel Help Virtual fraction collector configuration window The virtual fraction collector is a tool to optimize the fractionation without need for sample or eluents Therefore only opening and closing the configuration window would be sufficient However you have two areas for individual settings Rack Type Select from the drop down list that rack type best in comparison with your real one The choice is the same as for the Buchi fraction collector Micro with 240 vials standard with 120 and preparative with 48 positions The Vials number will be displayed automatically Vial Volume ml The rack dependent value will be displayed automatically However you are allowed to overwrite it by self defined values If any fraction is larger than the set volume the next free collection position will be used for continued collection Tubing Parameters These settings enable someone to consider the time delay for transporting the sample from the flow cell to the collector valve Here you can adapt it to the real conditions of your system The solvent and peak recycling options you cannot simulate 234 ChromGate
28. The current method custom report template will appear 28 Short guide ChromGate KNAUER Instrument Control Collecting Data General Data Collection Instructions with ChromGate 1857 The user should review the operating manual for each component in the system for warm up requirements preparing the component for operation etc This section includes requirements and suggestions relative to the computer and e line network when ChromGate is used to operate the HPLC system Before starting the chromatographic runs e Make sure that the energy saving feature and the screen saver on the computer are off e Check that all modules are connected to the e line cable e Topower up the system first turn on the PC and finally power up the various modules If a unit loses power or if the communication is broken turn off all components and start over e lfamodule has been operating via the local mode Ul interface make sure that it is not in use when connecting the cable e If you are using a manual injector make sure that the system is in the waiting for injection mode waiting for trigger before injecting e f you are using an autosampler with a single rack the input numbers should be integers If you are using multiple racks input vial numbers using a letter and number e g A1 B3 etc should be used If an incorrect vial number or invalid volume is noted the sequence will stop Collecting Data When you wa
29. Y axis is labeled Consider the formula DU displayed units DU MS Y M Y O MS measured signal in micro Volt Y M Y Axis Multiplier Y O Y Axis Offset Please note that the Offset also will be calculated using the Y Axis Multiplier As an example To change the Offset by 2 mV if the Multiplier is set on 0 001 a value of 2000 must be entered for the Offset Negative Multipliers are allowed Add Info Click the button Add Info to enter more detailed description of the detector Enter additional information or comments to be printed out when you print the instrument configuration When complete click OK to exit the dialog and return to the instrument configuration icon list Setup and Control of Knauer HPLC Systems 55 Configuration Kontron UV Detectors Fig 79 Kontron 332 335 Detectors Configuration Kontron 332 Add Info Type 332 hi Serial Number Kontron 4304 Add Info Type 4304 h Serial Number Kontron 525 Add Info Type 530 Serial Number Serial Port COM1 r Flow Cell Data acquisition Interface Interface Knauer Interface Bos device number 1 Channel 1 Input 1 Anis Units m l bal Channel 2 Input 12 i Cancel Help Kontron detector configuration windows The Kontron detectors are controlled similar to the Knauer detectors via the configured RS232 port For the data acquisition the analog output with an A D car
30. a Instrument Setup y 1050 SY As 3950 5 2550 AX Aux Traces oe Trigger Nn AR nan A770727 07 EL 771420 Wavelength Program Used Channels f a S 2550 W T 254 nm SER 1 Ret Time k Area Area Height 4 000 254 Options 7 at the end of run i 4 iF j Fig 253 Advanced Runtime Settings Trace name changes The option will only change the trace name for new chromatograms ES If enabled this option is valid for all instruments of the Enterprise it is not limited on the currently used instrument or computer That means that the trace name will include the wave length for all instruments and therefore for all methods that will be stored on the mentioned Enterprise machine Download Tab Method The actually set parameters may be even not saved of the current instrument setup tab as well as those of the whole method can be downloaded to the corresponding instruments Select the menu CONTROL DOWNLOAD METHOD or CONTROL DOWNLOAD TAB Knauer Instrument Control Method Options 173 f New Instrument Method My Method 4 met Data None Project Default Inst File Edit View Method Data Sequence Analysis Control Reports window Help si 5 2500 X By GI Preview Run n i gt single Run Strg Umschalt F9 S 1000 EJ as Triathlon E Switching Valves E J E Working Mode Sequence Run 5trg Umschalt F8 lsocratic E Run Queue Control P
31. as Knauer interface box IF2 must be used The serial control only allows the same features as for the detector RF 10Axl All advanced features cannot be supported Please be sure that the detector has a firmware version that allows for serial control E AF A Baseline Check Jo Trager F Acaton channel or Time constart Samp ng e Sutable hor mresa peak wath al bare of Plan tras Acquation delay Lanp OFF al the end of pan Fluorescence Detector RF 10AxI setup window Lamp OFF at the end of run Check the lamp OFF box to switch off the detector lamp after a measurement is finished This option may be used for the shutdown methods This option is supported only for detector firmware versions 3 32 and higher All additional settings are integrated as additional columns into the spreadsheet for the detector time program The creation of the detector 104 Fig 153 Creating an Instrument Control Method time program is similar in principal to that described above in the chapter Instrument Setup Pumps Pump Program The Time Excitation wavelength and Emission wavelength values must be entered manually whereas the options Autozero Event Out and Event Mark can be checked separately for each time line Event Out short circuits the event out terminal for approximately 1 second Event Mark adds an event mark to the analog output The values for the Sensitivity Gain Recorder Range and the Output Mode
32. if the detector UVD2 1S is configured Time Constant Select the time constant for the detector from the drop down list The values are 0 1 0 2 0 5 1 0 2 0 5 0 and 10 0 sec The additional values 0 000 0 005 0 01 0 02 0 05 will only be available if the detector is connected by LAN Smaller time constants are required for high speed analyses good rule is to select a time constant which is the reciprocal of the data rate in Hz e g 2 Hz data rate gt 0 5 sec time constant Sampling rate The sampling rate is the rate at which data will be sampled by the system Click on the down arrow to get a list of the frequencies available for the configuration of your system The choices are 1 2 5 and 10 Hz and if the detector is connected by LAN also 20 50 and 80 Hz For any selected sampling rate the narrowest peak width will be shown for which this sampling rate will be adequate This selection is in Hz data points per second This is the selection for most chromatography applications We recommend having more than 20 data points per peak 30 40 This means that for a peak with a peak width of 20 seconds from peak start to peak end a data rate of 2 Hz is recommended Autozero at start Check the Autozero field if you wish the signal to be zeroed at the moment a measurement is started to define the current signal as the baseline Creating an Instrument Control Method 109 Autozero at WL change No access to
33. j Current Vial Tray Temperature CJ 4 Oven Temperature C Start Needle Wash Tray Temperature C Advance tray SerialPort COMI Fig 240 Autosampler 3900 status tab Monitor AS Status The status Ready will be displayed when the autosampler is waiting to perform an injection If an injection program is running the actual program step will be indicated After a completed injection the status is described as Analysis timer running during the chromatographic run Current Vial The actual position of the injection needle is shown Oven Temperature C If the column oven option has been configured the actual column oven temperature will be shown Tray Temperature C If the tray cooling option has been installed and configured the actual tray temperature will be shown Direct Control The direct control features are not accessible during an injection Oven Temperature C If the column oven option is installed and configured the column oven temperature can be set The entered value will be transferred to the oven Creating an Instrument Control Method 163 by clicking the Apply button The On Off button can be used for enabling disabling the heating control The labeling of the button changes with the actual status Tray Temperature C If the cooling option is installed and configured the tray temperature can be set The entered value will be transferred to the sampler by clicking
34. o 165 Instrument Status Kontron Autosamplers ccooccccocnccccnconcnccnnncnonnnnnnnos 166 Instrument Status Miscellaneous InstruMentS oocccconcccccnocccncnncccncnnnccnnnnnncnnnnnos 166 Instrument Status Manager 5000 5050 1F2 I O ccoccccccconcconnconn 166 Instrument Status Knauer Switching Valves coocccoccccccnccccnnnnonss 167 Instrument Status Column Oven 4050 and Jetstream 168 Instrument Status Flowmeter ocooocccoccncocccccccccccnconcncnncnonnnnnnnnnnnnnnnnnnos 169 Knauer Instrument Control Method OptioOnsS axxrnnennnennnennnennnennnnnnnnnnnnnnnnnnnnnnnnnnnnr 170 General SENGS oi 170 RUMUNE SENOS 42 Se 170 Download Tab Melodia 172 SONEN CONTO ln ia 173 Qualification Procedures das 175 Validation of Imelda ao etc 178 Sele DNS 183 ChromGate System Suitability Setup anrrnnnennnnnnnnnnnnnnnnnnannnnernnnennnnnnnnnnnnnennnnennnnnnnn 185 COPY PIT ee 186 Suitability Calculation SelectiOnN oocccoccccocnconnnconnoconnconcnonnnononnnonnnonnrnnoncnnnnnnnos 186 RUNNING a Suitability TEST user 187 SUNADI REPONS Lapeer 188 ChromGate PDA OPIO EANN 189 PDA Method SUP 189 PASO DIOS LPEN ceca ct ita 189 PIANOS MI iras 191 RDA OPpl ns SPEARS SE 192 PDA Options Multi Chromatogram occcoccncnccncoccccccnnoncncnncnnnnnnnnnnnnnnnconnnnnanononnnnnnnss 194 PDAS Options RAMO ii class 195 PDA NIENS ss esse tere ae seen 196 P
35. wavelength axis to the maximum values To enter a manual range de select the auto scale box and add your own limits or click the Get Limits button to enter the limits displayed on the current 3D graph ChromGate amp PDA Option 199 Absorbance Click Auto scale if you want to have the software automatically scale the absorbance axis to the maximum values To enter a manual range de select the auto scale box and add your own limits or click the Get Limits button to enter the limits displayed on the current 3D graph Labels You can customize the labeling of the 3D plot using the parameters in this area You can select font size color and style using the selections provided 3D Plot Rotation Options Fig 292 A right mouse click on the 3d plot provides a menu containing various rotation options When you select one of these rotation options a checkmark will appear next to the option and that rotation option will become active on the plot Elevate w Rall Rotate Rotate parz Spin DOM Reset Axis Setup Properties The cursor will change to indicate the option is in effect While the option is active you can move the plot in the designated way by holding down the left mouse button and moving the mouse in the desired direction on the plot When you have the plot in the desired position click the right mouse button and turn off the option by selecting it again checkmark removed If you want to return
36. your custom report Click the right hand mouse button on the report and select Insert Graph Data Graph Select the Current Data for the Data Source or another source if desired and then select Mol Wt Dist for the Trace SEC Option Fig 384 Fig 385 279 Data Graph Properties Trace Setup Ais Setup Appearance 4 Reset Scaling Cancel Apply Help Click OK to add the trace to your custom report Note that this trace is slice area vs Molecular Mass You can add annotations to this chromatogram in a similar fashion as you do with other chromatograms It is not possible to overlay this type of trace with a standard chromatogram be cause the scaling is different E Channel Mol WE Dist 1 A ES Slice Area 100000 1e H006 Molecular Weight Adding a Slice Report To add a slice report to your custom report do a right hand mouse click and select Insert Report SEC Slice Report A dialog will appear 280 Fig 386 Fig 387 SEC Option GPC 7 SEC Slice Report Options Trace I Channel A v ET All Slices fe Specified Range Start Time Stop Time 5 35 OK Cancel Help ok Select the trace for which you want to generate the slice report from the drop down list Select the Specified Range button if you want to report slices within a certain time range Enter a Start Time and Stop Time for the range you wish slices to be reported Otherwise select the All Slices button
37. 00 Min after ready Check this option to use a logical trigger signal for starting the system This allows a synchronous system start also without an external trigger source e g autosampler or manual injection valve With 0 00 Min the system will be started if all connected devices have sent a ready to the software For a delayed start enter the desired time Argument X Y X and Y are optional arguments use of these can be switched off A result formula can be calculated without either X nor Y with only X or with both X and Y single Y is forbidden If using X or Y is switched on X and Y signs must be used in the result formula expression After checking this field the pull down menu of the Source becomes accessible The options are Time Data File and External Source Time With this selection no further settings for Argument X are possible The argument value is equal to the current time value Data File Load an already existing chromatogram data file that should be shown starting a run with the virtual detector The value of this argument is calculated using the corresponding point in the selected data file The speed of the data acquisition can be set with the sampling rate If the original chromatogram was acquired for 10 min with 1 Hz the chromatogram will be finished within 5 min if the sampling rate for the virtual detector is set to 2 Hz If no data points left of the original chrom
38. 06 PC PM LAB TEST AS 3950 Stacked injection trigger marker is detected in 6 02 min after start BS System 09 09 201016 19 50 PC PM LAB TEST AS 3950 Stacked injection 5 pl from vial 101 will be done in 5 75 min after start E System 09 09 2010 16 17 06 PC PM L4B TEST AS 3950 Stacked injection trigger marker is detected in 3 02 min after start al System 09 09 2010 16 16 50 PC PM LAB TEST AS 3950 Stacked injection 5 ul from vial 181 vill be done in 2 75 min after start E System 09 09 2010 16 13 41 PC PM L4B TEST Run Queue Start Single Run CEZChrom ElitetEnterprise ProjectsiDefaultiDatal E System 09 09 2010 16 13 40 PC PM LAB TEST Run Queue Add Single Run C EZChrom ElitelEnterprise ProjectsiDefaultiDataiS Autosampler 3950 Stacked injections instrument activity log entry Instrument Setup Triathlon Endurance Autosampler The method parameters required for automatic operation of your autosampler are contained in the Method Instrument Setup Triathlon Endurance tab The control parameters for the autosampler become part of your method and sequence files Creating an Instrument Control Method E instrument Setup E s10 EN AS Triathlon oven 5 4050 5 2500 Aux Traces 3 Trigger Standard inject User defined program Large injection injection method Syringe speed Enable UDP Enable large injections Flushed 5 peed Normal y Partial Local E i Scale factor 1 Cul Pick up Temperature Control
39. 12 Save the sequence Running a Sequence To acquire data using the sequence file you just created click the Sequence Run button on the command ribbon or do a right hand mouse click in the sequence spreadsheet and select Run Sequence The following dialog box will appear 26 Fig 24 eS Short guide ChromGate KNAUER Instrument Control xl Sequence information Sequence name Canes Run 1107 FM tider Helg Ev C All Tower ye 3 lariat Processing mode Hormel gt Bracketing Nene Pito fs Review I Port method reports e F geile reven pales alter 20d huh La Prrt Sequence rapote Er a at ri tees each Gres sei On start Da stop or erar ol E Ta Esgin F Email Pr Run Sequence dialog box Enter the name of your sequence file by typing the name along with path in the Sequence Name field You can also select it from a list of sequence files on your disk by clicking the File button next to the field Leave the other parameters as their defaults Prepare your autosampler to inject your standard sample followed by 3 unknown samples When you are ready to inject your first sample click Start When the sequence is completed you will have acquired and saved the data files for one standard and three unknown runs and generated a simple result report for each unknown sample and a summary report for t
40. 2 MM Dispense 10 ul to Destination a 20 mm Edit 3 Aspirate 100 ul from Reagent Remowe Cancel Autosampler Triathlon edited user defined example program Click the Add button to add one more line to the programmed steps The total number of steps for the user program cannot exceed 240 The Add New Step dialog will appear Creating an Instrument Control Method 125 Add new step Action Add Aspirate Cancel Volume ful 1000 Source irom Sample E Speed y vi Height rar 2 Fig 178 Autosampler Triathlon editing user defined programs new step select the Action from the drop down list The appearance of the dialog will be changed depending on the selected action Action Aspirate 7 pappas Source from Sample Spiinge valve Mel Syringe rea zampe pen dizi from Destination Wait from Wash from Reagent ira fram Aeagent E uiliaries tom Aeagent E ram Aa agent O Fig 179 Autosampler Triathlon UDP action choices Aspirate Aspirates a programmed volume from a sample vial a destination vial a wash vial or one of the reagent vials into the buffer tubing Speed and height of the syringe can be entered The maximum volume that can be aspirated is the total volume of the syringe If more than one consecutive aspirating action from different sources is programmed the total volume of the consecutive aspirating actions cannot exceed the volume of the syringe Dis
41. 222 Configuration 48 50 56 Custom Report 216 Diagnostics 150 Instrument Status 149 Method Setup 189 Option 95 102 185 189 230 Setup 100 Utilities 210 Views 196 Wiring Scheme 290 Dark Current 152 Data Collection 28 Destination Vial 66 75 Detection channel 236 Detector Configuration 45 Connections 57 Instrument Status 145 Program 102 Setup 91 Synchronization 94 Type 48 292 Device ID Autosampler 68 70 71 73 Diagnostics DAD 150 RF 10AxI 153 Digital Output 89 94 140 148 149 150 156 167 168 169 Diode Array see DAD Direct Control AS 3800 162 AS 3900 162 163 166 Column Oven 169 DAD 149 Enabling 170 Fraction collector 248 K 2600 148 Manager 5000 167 Pumps 141 RF 10AxI 153 154 156 RI Detector 145 Save Changes 170 Switching Valves 167 Triathlon Endurance 165 UV Detector 146 Download Tab 172 E Emergency during a run 170 Emission wavelength 153 Endurance Configuration 72 Setup 121 Status 165 Ethernet 35 49 56 Excitation wavelength 153 Export 209 Extend run time 143 144 F Fixed wave length detector 45 Flow Cell 50 55 56 Flowmeter 135 Configuration 81 Instrument Status 169 Setup 134 Fluorescence Detector Configuration 45 Diagnostics 153 Instrument Status 145 152 154 Setup 103 Wiring Scheme 287 Flush Volume 113 117 122 Fraction collector 18 Configuration 230 Direct control 248 Instrument status 247 Program 237 245 Setup 235 Fractionation conditions 238 Index G
42. 3 5601 5 0983 1 1037 Expected 11 544 38644100 2866261 0 01957 12384 2637 3 9601 5 0983 1 1037 Difference 11 0 06 0 00 0 00 0 01 0 00 0 00 0 00 0 00 Calculated 12 6 93 296108 00 26006840 02518 14299 5450 5 1875 6 9554 1 0469 O Expected 12 6 95 29610800 2600684 0 02318 14299 5450 5 1875 6 9554 1 0469 Difference 12 0 00 0 00 0 00 0 00 0 00 0 00 0 00 0 00 Yo Calculated 13 925 21515400 2466452 0 03020 15020 6299 7 2619 5 7046 1 0118 O Expected 13 925 21515400 24684520 O302 15020 6299 T 2619 5 7046 1 0115 D Difference 13 0 04 0 00 0 00 0 00 0 00 0 00 0 00 0 00 Calculated 17 12 74 14950800 22689360 04052 15821 8555 10 3780 1 7915 0 9596 Expected 17 1274 14950800 22659360 04052 15821 8555 10 5780 1 7915 0 9596 O Difference 17 0 03 0 00 0 00 0 01 0 00 0 00 0 00 0 00 Mo Cate Wed Dec OF 16 17 29 2011 Fig 271 Validation report reference chromatogram 182 Calculated Expected Difference o o Calculated Expected Difference o o Calculated Expected Difference o a Calculated Expected Difference o a Calculated Expected Difference o o Calculated Expected Difference o o Calculated Expected Difference Yo Calculated Expected Difference o a Calculated Expected Difference a o Calculated Expected Difference o a Fig 272 Validation report Gaussian trace ChromGate Chromatography Data System 3 3 2 980 Knauer Instrument C
43. 30 7 er e s ropa m iS p ia ES Je ER ere PS EE mu N p a al f g p lt a 3 PC RS232 ports Fig 392 1 serial NULL modem cable A0895 2 serial NULL modem cable A0895 3 serial 25 pin male D cable A1004 4 Trigger AS 3800 Control I O Connector K 2501 Pin 4 N O gt START Pin2 COMM gt gt GROUND Typical Wiring Schemes Fig 393 Pump K 1001 Autosampler 3800 Detector K 2600 Interface Box User Defined Detector RR O N gt Pop pro in ge av y A gt Li ip Analog Out User defined Detector serial Y cable A0755 serial Y cable A0755 serial 25 pin male D cable A1004 Trigger AS 3800 Control I O Connector K 2501 Pin 4 N O gt START Pin 2 COMM gt GROUND Detector signal cable CINCH CINCH A0868 CINCH wire ends G1021 289 290 Pump K 1001 Autosampler Triathlon Detector DAD 2800 Detector K 2301 A DF ut HI ae ul HL EZ TS PCI DAD Interface Fig 394 1 serial Y cable A0755 2 serial one to one cable A0884 3 serial one to one cable A1004 4 DAD communication cable A0887 5 Trigger Triathlon P4 Connector K 1001 Pin 1 N O gt START IN Pin 2 COMM gt GROUND Index Index 3 3D Data 96 98 216 3D Properties 197 3D View 196 A Acquisition 50 100 Hz option 33 Channel 33 91 95 96 98 102 148 Configuration 53 Delay 92 108
44. 400 0000 2 83 074 5573 300 0000 2 83 1554 7704 1600 0000 2 83 2429 3406 2500 0000 2 83 3496 26063 3600 0000 2 83 4761 5570 4900 0000 2 82 6224 9165 6400 0000 2 14 7006 5540 91 00 0000 2 63 9791 8213 10000 0000 2 08 Cap Factor 0 1071 0 1071 0 00 0 7857 0 7857 0 00 1 6786 1 6786 0 00 25714 25714 0 00 3 4643 3 4643 0 00 4 3571 4 3571 0 00 5 2500 5 2500 0 00 6 1429 6 1429 0 00 7 0357 7 0357 0 00 7 9286 7 9286 0 00 Resolutio n 0 0000 0 0000 0 00 2 4644 2 5000 1 42 2 4644 2 5000 1 42 2 4644 2 5000 1 42 2 4644 2 5000 1 42 2 4644 2 5000 1 42 2 4644 2 5000 1 42 2 4650 2 5000 1 40 2 4662 2 5000 1 35 2 4704 2 5000 1 19 Asymmetr y 10 4 0000 4 0000 0 00 4 0000 4 0000 0 00 4 0000 4 0000 0 00 4 0000 4 0000 0 00 4 0000 4 0000 0 00 4 0000 4 0000 0 00 1 0000 4 0000 0 00 4 0009 4 0000 0 09 4 0019 1 0000 0 19 1 0053 4 0000 0 53 Knauer Instrument Control Method Options 183 Generic Drivers Fig 273 Fig 274 Generally spoken any device that supports RS 232 ASCII communication can be controlled with ChromGate software No changes in source code of ChromGate are required but for each device an additional driver is required The configuration of a generic device is to be done by Knauer For each
45. C Leak Sensor Alarm Time 7 14219 Minutes Amplitude 254 Wavelength nm LA S 2500 Whe 5 2600 temperatura PE Fig 192 Setup window column oven 4050 For details on the spreadsheet operations refer to the section Creating a Time Gradient Program as described in the chapter Instrument Setup Pumps Pump Program The oven program profile will be adapted automatically to the spreadsheet inputs Wait before injection If you are working with a temperature program check this option The setting Wait with tolerance C defines the accuracy of the oven temperature allowing a new injection A temperature independent delay between succeeding injections can be set in the field Wait time min The system will wait for that time after having reached the initial temperature and before the next injection is performed Cooler Temperature C Enter the desired temperature Leakage Sensor Alarm Enter the desired sensitivity level Instrument Setup Column Oven Jetstream 1887 Make sure that the column oven Jetstream is switched on before opening the instrument window lts keypad must display choose function Otherwise the oven will not be controlled 134 Fig 193 Creating an Instrument Control Method MM Instrument Setup s 1000 E as Triathlon E Switching vaks El JetStream Oven Temperature Time Temp Bie 30 0 00 Wait Time min 10 00 30 Time 9 97733
46. Detector K 2600 setup window with PDA option 96 Fig 140 Creating an Instrument Control Method Used Channels Check the box es to turn the acquisition for the selected channel s on If the box is not selected no data will be acquired and no time program will be executed for this channel As many channels are activated at least one as many wavelength columns appear in the spreadsheet below You can only enable the number of channels you have activated in the instrument configuration setup of the device The wavelength table is a spreadsheet to enter your time detector program In the first row at time 0 00 min enter the initial wavelength s in nm For up to 50 steps time in minutes you can then define the detector parameters The editing principle including programming the digital outs is the same as for the time gradient program described in the chapter Instrument Setup Pumps Pump Program Autozero at start Check the Autozero field if you wish the signal to be zeroed at measurement start to define the current signal as the baseline You cannot disable this option if you are acquiring 3D data in the advanced mode of the detector Autozero at WL change Check this option if you wish the signal to be zeroed at the moment the wavelength is changed to define the current signal as the baseline Lamp OFF at the end of run Check the Lamp OFF box to switch off the detector lamp after a measurement is finished Th
47. Enterprise Projects Defaults Data file Sample lt 001 gt gt Number of unknown runs in sequence Repetitions per run al Abbrechen lt Zur ck Weiter gt Fertig stellen Sequence Wizard Unknowns 4 Press the blue arrow on the Sample ID line to access a list of identification options for the samples Typically Line Number and Method Name are used so that each sample will be identified with the sequence line number and method name 5 Indicate where the data should be stored using the Data Path field 6 Click on the blue arrow on the Data File line to access how the data file should be named Typically Sample ID is used this will assure that the sample is uniquely identified and easily related to the sample 7 Enter the appropriate number of unknowns the number of repetitions 8 Press next to present the Autosampler screen if you have indicated that the data files are to be acquired initial screen in the wizard this screen is not presented if existing data files are used Unknown vials of sequence First vial Increment by fi r Calibration vials of sequence gt i First vial Increment by fi 3 Sete Autosampler injection volume lo Cancel Finish Sequence Wizard Autosampler 9 Enter the appropriate information and press Next to present Short guide ChromGate KNAUER Instrument Control 25 Sequence Wizard Calib
48. File Instrument Activity Log Display Log E Instrument Activity Log hogget source Activity 04 09 2012 10 56 28 PC PM SWTEST 1 Extending run by 2 00 minutes 04 09 2012 10 54 30 PC PM SWTEST 1 Direct Control S 1050 Gradient has been modified in 6 58 min aft 04 09 2012 10 51 15 PC PM SWTEST 1 Direct Control 5 1050 Gradient has been modified in 3 34 min aft 04 09 2012 10 47 25 PC PM SWTEST 1 Run Queue Start Single Run pc pm laboriic EZChrom Elite Ent 04 09 2012 10 47 24 PC PM SWTEST 1 Run Queue Complete Preview Run Wpc pm laborliciEZChrom El Run Queue Add Single Run Wpc pm laborlicVEZChrom Elite Ente Run Queue Start Preview Run pc pm laborlic EZCh rom Elite E 04 09 2012 10 42 38 PC PM SWTEST 1 04 09 2012 10 42 37 PC PM SWTEST 1 04 09 2012 10 41 36 PC PI Q eer teeter f SJYUVILII VSET IG DIG UVULTU Fig 215 Extend run time window Fig 216 Control Pressure Limits This section allows you to enter the control pressure limits in the units chosen in the pump configuration dialog The pump will automatically shut down its operation when the measured pressure is outside of the limits Min Pressure Enter the minimum pressure allowable for your pump Max Pressure Enter the maximum pressure allowable for your pump Apply Click the button to actualize the selected Min Max pressure limits Events Click the button to set up the event outputs Refer to the chapter Instrument
49. Inj volume 0 Injection Date 26 11 1990 20 49 09 Analysis Method CE ZChrom Elteidethodsinultilevel calibration met Last changed 14 07 1999 21 14 22 Description Data Description Channel A multi callbraton level 2 dat Nane 0 020 Retertba Tine 0 015 000 0 005 0 000 ew labeh Mires KNAUER Area Report Analysis Time 28 02 2012 10 48 35 Multiplier F actor 1 Dilution F actor 1 Sample Amount 1 Channel 4 Results Ret Time K Area Area Height Height ASYmim Width Plates Resolution 571 0 00 170464 26 30 20886 36 67 0 00 0 651 6 55 0 00 139959 21 50 15331 26 92 0 00 0 651 6 84 0 00 306 0 05 59 0 10 0 00 0 168 8 53 0 00 337346 52 05 20673 36 30 0 00 0 927 Totals End of Report Knauer HPLC Offline 22 06 2012 10 50 18 matthias Page 1 of 1 Fig 26 KNAUER Area Report If you wish you can modify the standard report templates or create entirely new reports using the Custom Report capability of ChromGate Client Server Modified standard reports can be stored as templates Custom Reports will be available as part of the method You can create custom method reports and or custom Sequence reports To view the custom report template in the multilevel calibration met file first open the file if it is not already open Use the File Open button followed by Method then select the template from the file list Click the Edit Custom Report button on the command toolbar to access the method custom report editor
50. LAB TEST Run Queue Add Single Run CEZChrom ElitelEnterprise ProjectsiDefaultiDatalStacked Injection with InjModule 3_ 1_001 dat Injection Module Stacked injections instrument activity log entry Injection module instrument status If you open the instrument status you will find an own tab for the injection module ChromGate Preparative Option 255 ME Instrument Status Monitor Direct Control Run Status Injecting 0 19 min Pump Flow ml min 0 000 Pump Flow ml min 3 000 Pump Pressure MPa 0 0 Walve Position 3 Pump LAN 172 16 5 14 10001 SN ABC000094150 O Valve Serial Port COME SN n a Fig 367 Injection Module status window initial injection Monitor Run Status Shows the current run status of the module If no run is performed the status is Hold During the initial injection the status njecting with the time of the current injection duration is shown During the run the run status shows the run time Pump Flow Shows the current flow rate of the injection module s pump Pump Pressure Shows the current pressure of the injection module s pump This option is only shown if the pump is equipped with a pressure transducer Vial Position The vial position can be either L Load or Inject If no sample loop is used that means that the sample will be injected directly on the column the vial position during the injection while the injection module s pump is running is I Inject If
51. Minutes Amplitude 254 Wavelength nm Setup window column oven Jetstream For details on the spreadsheet operations refer to the section Creating a Time Gradient Program as described in the chapter Instrument Setup Pumps Pump Program The oven program profile will be adapted automatically to the spreadsheet inputs compare Fig 192 Wait before injection If you are working with a temperature program check this option The setting Wait with tolerance C defines the accuracy of the oven temperature allowing a new injection A temperature independent delay between succeeding injections can be set in the field Wait time min The system will wait for that time after having reached the initial temperature and before the next injection is performed Instrument Setup Flowmeter Fig 194 The setup of the flowmeters is the same for all supported models is very simple E Instr ument Setup a AE E AS Triathlon E Switching Valves E Oven s 4050 y 5 25 A Knauer Flowmeter 3 Flowmeter Settings Aun time Acquisition delay Setup window Flowmeter Run time Enter the desired run time Acquisition delay Here you can define a delay time between starting the run and the data acquisition Creating an Instrument Control Method 135 Setting up Auxiliary Traces Tr Instr ument Setup E PR E 51000 E As Triathlon E Switching Valves Sy Ovens 4050 6 s 20 Aux Traces trigger EEE
52. P2 1L the gradient mode must correspond with the gradient mode set in the pump The LPG option is not available for S 100 10P 20P P2 1S P4 1S K 120 and K 501 pumps The Isobar mode is only available for the pump P2 1L Interface Serial Port Select from the drop down list the serial port or network number for the communication port on your PC where the instrument is connected To ensure an error free operation each serial controlled device should be connected with an own COM port also for devices that allow to connect more than one device onto the same COM port For pumps K 1000 K 1001 S 1000 and K 1800 preparative pump 1800 the interface Network means the serial port number The pumps K 1000 K 1001 S 1000 and K 1800 preparative pump 1800 only are controlled with ChromGate if the pump s internal Setup and Control of Knauer HPLC Systems 41 Fig 51 Fig 52 E communication in the menu option CONTROL is set to NET baud rate 9600 The pumps S 1050 S 100 allow for communicate via RS 232 or LAN Select the interface you want to use for communication with the device R5 232 Airaga Available interfaces pumps S 1050 S 100 10P 20P P2 1S P4 1S For pump P2 1L only communication via LAN is supported RS 232 Select the number for the RS 232 communication port COM Port on your PC to which the pump is connected If the pump S 1050 should be controlled by RS 232 in the pump s own se
53. Peak Recognition 10 00 Peak Recognition Signal Level Signal Level Signal Slope Spectral Similarity Unconditional Signal Level Signal Slope Signal Level Slope Local Maximum Local Minimum Spectral Similarity Fig 331 Event definition 238 ChromGate Preparative Option Unconditional Signal independent action as selected in the parameter window Fig 332 Signal Level The collection takes place according to set level thresholds Signal Slope The collection takes place according to set slope thresholds Signal Level Slope The collection takes place according to set level and slope thresholds which can be combined in logical and and or combinations Local Maximum The collection will be started by a local maximum Local Minimum The collection will be started by a local minimum Spectral Similarity The collection takes place according to set similarity thresholds Exp interv These fields are only informative according to the settings in the parameter window for the expected possible time shift for the retention Rel time shift These fields are only informative according to the settings in the parameter window if Use as reference for correction next events is activated refer to page 242 Parameters Activating any field in this column an arrow appears Clicking on that a dialog box for the FC parameters at the given time will be opened lts appearance depends as well on the mode as on the
54. Preparative Option Multi Valve Fraction Collector Configuration MultiValve FC Configuration FC name Multivalve FC Tubing Siy Peak Recycling Number of valves 11 Vial volume ml 1100 Number of vials 5 Configure valve 1 kd Seral port COM Valve hype E positions Add Valve Infa B oositions 12 positians ok Cancel Hel 16 positians EH Fig 325 Multi valve fraction collector configuration window Name Enter a descriptive name for the collector This name will appear on the fraction collector tab when the instrument setup or status windows are displayed Use a name which is unique within the instrument Tubing Parameters The same setup procedure as with any other fraction collector must be done However you cannot define the tubing parameters for each included valve One setting will be valid for all cascaded valves Solvent and or Peak Recycling With the Knauer MultiValve fraction collector the optional solvent and or peak recycling an additional recycling valve can be used but it is not necessary Siv Peak Recycling Recycling Seral port COM Configuration Settings Serial number 7 erial number ped Valve type gt Vi w Interface KE titel i T da fler Add Into IP Part 10001 h Use S N to identify the instrument Fig 326 Addi
55. Se for peak recognition Spectral Similarity The settings using the spectral similarity are quite similar to perform The difference is that the similarity values are to be used as thresholds The maximum value is 1 000 for absolute identical spectra FC Parameters at 2 50 min Signal 2 Relative to previous event min Ascending c Descending l Use as reference for correction next events Ref Spectrum ler SpectraWBenzol spd eI Brei Similarity maxs 1 000 10 95 Action Collect To nest 6 position 48 Expected interval min 10 1 ones Fig 338 FC parameters Spectral Similarity single event Additionally you must select the desired reference spectrum Clicking on will open the selection window Open Reference Spectrum Look in E Spectra vi e 1 et Fr Edeenzol E Naphthalin My Recent EThioharnstoff Documents My Computer a File name Naphthalin v Open Files of type Spectrum data spe X Cancel I Open as read only H I Help Fig 339 Spectrum selection window After closing the window Fig 338 the parameter will be designed as Sim Sim or Sim for peak recognition 242 ChromGate Preparative Option FC Parameters at 2 50 min Signal Relative to previous event min Use as reference for conection next events Ref Spectrum let Spectra Benzal sp Ej ere Similarity max 1 000 0 95 Action Collect To v Ascending D
56. Service Pack 1 for EZChrom Elite 3 3 2 gt Install ChremGate Software tad tune ntes gt Install KMAUER FRE Option gt Install the Microsoft HET 3 0 if required gt Online Manual EZChrem Elite Ragu tes Adober bakt Fader gt Online Manual ChromGate Request idabrtil2crabatE Ferda gt Visit the KMALER Website gt Browse the CD gt Exit Fig 1 ChromGate installer start window If EZChrom Elite is installed after a computer restart the Service Pack 2 of EZChrom Elite 3 3 2 must be installed Click on the link Install Service Pack 2 for EZChrom Elite 3 3 2 to start the installer Finally ChromGate as an Add on for the EZChrom Elite software must be installed If the Knauer FRC Option is purchased the fraction collector drivers and the advanced fraction collection functionality must be installed with a separate installer link Install KNAUER FRC Option The Knauer FRC Option can only be installed if ChromGate with the same build number has already been installed 188 Do not insert the USB license dongle into the USB port before the software installation is completed otherwise the license will not be recognized 183 For all installation work login with administrator access Before the installation switch off all running programs especially anti virus software In Windows Vista and Windows 7 you must confirm that the software should be installed Please also login as administrator if the computer ha
57. Setup Pumps section Configuring Events for details Click the Events Apply button to accept the settings This status area is only shown for pumps offering this feature Purge Current pressure MPa 7 3 Purge settings Purge flow ml min od Start Gradient components A Be C OCD 0 ABCD Stop after sec Caution Please open the deaeration screw before starting the pump Purge settings window Click the Purge button to open the purge set up window In Purge flow ml min enter the desired flow rate Click on the Start button to start the pump In the Gradient components section you can select each solvent channel directly or a Mix ABCD You can set a time limit for the purge Enable the Stop after option and enter the desired purging time Creating an Instrument Control Method 145 Instrument Status Detectors Instrument Status RI Detectors S 23 4 00 K 23 4 00 1 The tabs for all RI detectors appear identical independent of the features of the detector Unavailable features or functions are only grayed and inaccessible MN Instrument Status E BK OP System Status E 51000 Ej as 3900 E JetStream Oven md 5 255 5 2300 5 2400 Monitor Direct Control Run Status HOLD Signal uRIU 0 424 Flush Autozero Serial Port COM 9 Fig 217 Rl Detector status tab example S 2300 S 2400 Monitor Run Status The status HOLD will be displayed when no program is running
58. Vial Select the first destination vial position Leave the field blank if no destination vial is used Reagent A B C D Vial Select vial position 1 of the vial positions 1 through 4 for the Reagent A B C D Select No if the reagent vial is not used Configuration Kontron Autosamplers The configuration windows of the Kontron autosamplers 360 460 465 560 and 565 are identical The only difference is the vial number see below Kontron 360 Autosampler Configuration Name Kontron 360 Type 360 Kontron 460 465 Autosamplers Configuration Kontron 460 Type Serial Numb AO AO Mame Kontron FA Type FER ka Serial Number Serial Part COM Port Settings Vial number E Syringe volume ul 250 Vial type 1 8 ml Loop volume ful 20 ad Cancel Help Fig 106 Configuration windows for the Kontron autosamplers Serial Humbe Vial number Vial type Vial number Vial hype Name Enter a descriptive name for the sampler This name will appear on the autosampler tab when the instrument setup or status windows are displayed Use a name which is unique within the instrument Serial Number Type in the serial number of the autosampler you are using Setup and Control of Knauer HPLC Systems 17 Fig 107 Fig 108 Type Select the AS type 460 or 465 respectively 560 or 565 In the 300 series only 360 is possible Serial Port
59. after the operation has already been performed including the operation specified in the Spectrum tab of PDA Options a message box is displayed and the background correction request ignored A background correction is performed as follows e The spectra from the baseline start and baseline stop times for the peak are extracted from the 3D data The Max Plot is used to determine the peak that is used e For each spectrum in the peak a corresponding background spectrum is generated by linear interpolation between the baseline start and baseline stop spectra e These background spectra are subtracted from the original spectra Spectrum Interpolation Spectrum interpolation may be performed on a spectrum as a result of the settings on the Spectrum tab of PDA Options or by selecting Operations gt Interpolate from the Spectrum View context menu Interpolation may only be performed once on a spectrum If a second interpolation is attempted on a spectrum after the operation has already been performed including the operation specified in the Spectrum tab of PDA Options a message box is displayed and the interpolation request ignored Interpolated spectra may not be stored in spectrum libraries The calculation is performed by doing a 10 1 interpolation of the spectrum data points using a cubic spline curve fit This interpolation is performed after the applying any spectral filtering option 1st derivative 2nd derivative or smooth t
60. column e g for maximum delivery of a pump and the percentage values into the AOF Analog Output Factor column The output voltage produced by the Manager 5000 5050 1F2 I O will be the result of Voltage AOF 100 The influence of the Step Mode option is illustrated in the next figure Creating an Instrument Control Method 131 0 00 10 100 1 00 10 50 1 00 10 50 2 00 10 50 3 00 10 100 4 00 10 50 AOF AOF 100 100 50 50 0 min 0 min 0 1 2 3 4 0 1 2 3 4 Fig 188 Analog output profile without and with activated Step Mode Fig 189 Instrument Setup Switching Valves E Instrument Setup Ej Switching Valves Program e rien vave 1 Valve 2 vave 3 vate 4 Coment 6 Setup window Knauer Switching Valves Use this spreadsheet to enter your time program The number of columns corresponds to the number of configured valves in the system In the first row at time 0 00 enter the initial position for each valve Then you can define the states of the valves at the given time The choices for a Knauer 2 position valve are BLANK and L Load and Inject for a Vici 2 position valve BLANK and A Load and B Inject 1 through 6 for a 6 position valve 1 through 8 for a 8 position valve 1 through 10 for a 10 position valve 1 through 12 for a 12 position valve and 1 through 16 for a 16 position valve The BLANK space means that the valve will not change its state at the given time
61. corresponding installed segment Duplicate segment numbers of one type are not allowed However if different segment types are involved each of them shall be counted starting with 1 Transport Vials Enable this box if you want to use ul pick up injections with transport solvent from as many vials as necessary Setup and Control of Knauer HPLC Systems 75 M Transport vials 4 Reagent Standard f W Reagent E Standard 4 y Reagent C E tam E Reagent D Standard lf Destination nm LEVE y Fig 104 Tray configuration transport reagent and destination vials Ja DJ First Transport Vial Enter the first transport vial position Last Transport Vial Enter the last transport vial position All the vials from the first up to the last transport vial must be of the same type Transport Vial Segment Select the type of the segment where the transport vials are installed The choices are Standard A segment LSV B segment Super LSV C segment and Micro vial D segment All the vials with transport solvent must be from one segment type corresponding to the segment definition in the upper part of the dialog If all conditions for the transport vial definition are met the selected vial positions will appear in Fig 102 highlighted in yellow Reagent A B C D Destination Vial Enable the corresponding boxes if you want t
62. depend on the detector type wave length flow rate solvent and other parameters used in the test 186 ChromGate System Suitability Setup 6 If you want a drift test to be performed click on the lower spreadsheet in the column labeled Test Select Drift uV min from the drop down list If you select this box you must enter a Start and Stop time for the test and a Threshold value in uV Min for the acceptable limit 7 When you have completed the System Suitability Setup close the box Copy amp Paste After highlighting sections of the spreadsheet that you wish to copy push Ctrl C to copy the highlighted section of the spreadsheet to the clipboard Shift Insert to paste the clipboard item to where the cell is highlighted Shift Arrows highlights the spreadsheet cells and can move them Suitability Calculation Selection System Suitability calculations require information about your chromatography column These parameters are entered in the Method Advanced Performance tab dialog box 1 Click on Method Advanced Performance tab Enter values for the column parameters shown required for calculation of performance options ul Advanced Method Options Detector 1 Esport Custom Parameters Column Performance Files Advanced Reports Column Information Unretalned peak time 0 25 Minutes Column length p f meters cm Particle diameter p microns Column senal number Column installa
63. down list Setup and Control of Knauer HPLC Systems 49 Fig 72 Fig 73 Fig 74 Detector Type K 2501 Name fam E 2000 a Seal Number ye ne K 2300 K 2400 K 2301 K 2401 SerialPort ree K 2500 Detector type selection For some detectors this field is not accessible because no choices are possible Interface Serial Port Select from the drop down list the serial port or network number for the communication port on your PC where the instrument is connected To ensure an error free operation each serial controlled device should be connected with an own COM port also for devices that allow to connect more than one device onto the same COM port Interface DAD 2700 2800 2850 only For the diode array detectors the choice is PCI and LAN If you control the DAD via the LAN you must either press the button to search for the detector or enter the IP Address of the detector The access to the IP Address field is blocked if PCI was selected Interface UVD 2 1L S 2550 S 2520 10D UVD2 1S only Select the Interface you want to use for communication with the device Available interfaces detector S 2550 S 2520 10D UVD2 1S For detector UVD2 1L only communication via LAN is supported RS 232 Select the number for the RS 232 communication port COM Port on your PC to which the detector is connected If the detectors S 2520 or 2550 should be controlled by RS 232 in the detector s own setup
64. for a peak with a peak width of 20 seconds from peak start to peak end a data rate of 2 Hz is recommended Click on the down arrow to get a list of the frequencies available for the configuration of your system The choices are 1 2 5 and 10 Hz The detectors UVD 2 1L UVD 2 1S S 2550 52520 also support higher data rates 20 50 and 80 Hz for LAN connection only for S 2550 for one channel only In case of the Interface Box Model 96 with activated 100 Hz option or the Manager 5000 5050 or IF2 with activated 50 Hz option see page 33 you can additionally select 20 50 or Model 96 only 100 Hz for the configured User Defined Detector This option is restricted to channel No 1 if the interface while the other channels are not accessible 92 Creating an Instrument Control Method It is recommended that you use the peak width s see Graphical Events Programming or if included peak report to determine the optimal sampling rate for your chromatography Run Time Run time determines the length of time data will be sampled Acquisition Delay Acquisition delay is the interval between the start of the run Trigger and the time when sampling starts for this detector Instrument Setup RI Detectors Fig 135 IS S 23 4 00 K 23 4 00 1 MM Instrument Setup 8 51050 as 3950 I s 2550 63 s 25 8 S 2300 5 2400 ws 2500 Bronkhorst Flowmeter 2 lt Aux Traces Trigger MV Acquisition channel on Time constan
65. have generated the peak table see below then enter the value directly into the Peaks Groups table 22 Peak Short guide ChromGate KNAUER Instrument Control Enter the Reference ID which is the Peak ID s from the peak table This number is obtained from the Peak Table Leave it blank until you have generated the peak table then enter the value directly into the Peaks Groups table Indicate the desired basis for the calculation of the retention time window and the desired value Repeat the above step for each compound in the standard When you have entered information for all standards press Done Press the Peaks Groups Table button E or select the menu sequence Method Peaks Groups to present the calibration peak table Each peak that you have defined will appear as a row in the table along with the retention time and other parameters that you have entered I Group Tables 2600 Channel 1 Named Peaks Groups o Name D Ret Time Window Ret1DH ISTD ID i a Compound 1 5 70958 0 285479 0 Y Compound 2 8 26361 0 413181 Y Fig 16 Peak table 9 10 11 12 Analysis Analysis Sample Method Data path C EZChrom Elite Enterprise Projects Default D f Data file multi calibration level 2 dat Amount values Sample Internal Enter all values needed for the calibration as the concentration for all levels of your compounds If the tabl
66. identical If e g the parity is set to 2 means even parity it must be set in the detector s setup as well in the software If all parameters have been reviewed the display shows RS 232C _ Enter to Enable Press the key lt enter gt The display shows RS 232C Enable for only about 2 seconds then the default screen will be shown Now the detector is in serial control mode and can be controlled by software If the detector is switched Off and On it will start again in RS 232 control mode During the boot procedure the RS 232 control mode will be shown very shortly on the display To switch off the RS 232 control mode the keys lt CE gt und lt del gt must be pressed at the same time Setup and Control of Knauer HPLC Systems 53 eS The driver for the model RF 20A Axs only supports the firmware version 0 9 This firmware is only available through Knauer The control is compatible with model RF 10Axl and will not support the advanced features of the model RF20A Axs 187 The maximum data rate for the RF 10Axl using the serial port is 0 5 Hz for the RF 20A Axs 5 Hz For higher data rates the 1 V Integrator Output with a Knauer interface must be used IS Both detectors may not work on additionally installed serial COM ports It is recommended to connect it with a computer s onboard COM port Configuration User Defined Detectors Fig 77 Fig 78 The user defined detector can be a
67. in ml min Remove calibration curve outliers To temporarily remove an outlier from the calibration curve click on the point with your mouse It will change color and the curve will be re calculated with the point removed Time Volume Select the mode of viewing for your calibration curve Time or Volume If you select Volume you must enter the flow rate so that the curve is displayed correctly Print Click this button to print your current SEC method information Export settings Click this button to select export options and path When you have completed the SEC Setup you are ready to acquire and process your SEC runs You can acquire data either manually single injection or by batch acquisition auto injector See the ChromGate User s Guide for information on creating a batch sequence Zooming on the Calibration curve You can zoom on the SEC Calibration curve by clicking and dragging with the mouse Double click the mouse in the graph area to unzoom the curve view Once you have zoomed in on a chromatogram you can scroll the chromatogram to the right or left without losing the zoom This is done by pressing the lt CTRL gt lt SHIFT gt keys down and moving the mouse until the cursor changes to a hand and dragging it to the left or right You can also scroll the X or Y axis to view features which may be out of the range To do this press the lt CRTL gt lt SHIFT gt keys down while the mouse cursor is outside
68. interval between the start of run Trigger and the time when sampling starts for this channel Trigger Select the Trigger tab to select the type of trigger for the instrument MM Instrument Setup JE fx sj 51050 as 3950 5 2550 Aux Traces 2 Trigger Type None Sampling starts immediately after clicking on Start Sequence acquisitions do hot pause between uns Manual Operator has to press Enter to start the run Sequence acquisitions pause for confirmation between runs External If the data sampling is started from an external trigger select this option The type of trigger it designated in Instrument configuration Select when using a contact closure from an autosampler or manual injector Trigger Type Determines how the data sampling is started None Sampling starts immediately after clicking on Start Sequence acquisitions do not pause between runs Manual Operator has to press Enter to start the run Sequence acquisitions pause for confirmation between runs External If the data sampling is started from an external trigger select this option The type of trigger is designated when the instrument is configured When you have completed the acquisition parameters click the X box in the upper right corner of the dialog box to exit the dialog Single Run Acquisition There are two ways you can acquire data using ChromGate Client Server One way is with a sequence for multiple runs and the other way is to m
69. match Click OK to enter the defined peaks into the SEC Setup table Annotating SEC Chromatograms When you view an SEC chromatogram in the chromatogram window there are a number of annotation options available to you that are specific to SEC samples These are available for you to set when you do a right hand mouse click in the chromatogram window and select Annotations SEC Option Fig 374 Fig 375 265 Add Trace Add Multiple Traces Asis Setup Appearance Full Unzoom Operations j Utilities Graphical Programming Properties A dialog box will appear where you can select both SEC specific and or normal chromatogram annotations To view the SEC specific parameters for annotation select SEC from the drop down list peaks SEC A list of SEC calculated values will appear that you can choose for annotation on your chromatogram To select a parameter double click on it to move it to the right hand box Trace Annotation Properties Annotation rare 11 Current Data Channel Y Avalable 4nnotations Show the following annotations Min Decimals Other Baseline AT Window i SEC Result Range USP Width iM SEC Baseline iM SEC Cumulative Cancel Apply To All Help Note that the standard Baseline is not used in SEC so it should not be selected To annotate the SEC Baseline select the checkbox for this option Once the annotations are selected click OK and the
70. min Set the allowed flow range for this operating mode The allowed absolute minimum is 0 ml min the maximum depend on the selected pump head Pump program Pressure unit Enter the desired system pressure The pump will vary the flow if the pressure in the system will change The pressure unit depends on the unit selected in the pump s instrument configuration The minimum time step between two time lines is 0 25 min 90 Fig 133 Creating an Instrument Control Method Pretreatment not for Kontron pumps Checking this option the Setup button becomes active Clicking on it the following dialog window will be opened Pretreatment Pump Program Time Flow Comp 4 Events ini one 2 00 1 000 100 00000000 Cancel 0 00 1 000 100 00000000 For pretreatment zero time is considered to be the injection time Please use negative times to program the pretreatment time table Pretreatment setting dialog window The appearance of the pump program corresponds to that of the main pump setup window lt must be edited in the same way as described above To add a new line make a right mouse click into the table and select Insert line from the menu The only difference is that negative times must be entered because they are relative to the zero injection time During a pretreatment no data acquisition is possible However the run status during a pretreatment is Equilibrating If a pretreatment is to defi
71. mir Solvent pO Stacked Injections Wil 15 25 Haw Fure H Injection module instrument setup window The injection module will be used for the initial and the stacked injections Injection program The settings in this section will only be used for the initial injection that means for the first injection at the start of the run Injection Volume Enter the desired injection volume in ml Injection Flow 252 Fig 361 Fig 362 ChromGate Preparative Option Enter the flow rate of the injection pump during the injection If no injection is prepared the pump is stopped during a run the pump and valve cannot be controlled beside the programmed stacked injection Injection Duration The duration of the injection is calculated from the injection volume and the injection flow The reduced flow during start and stop of the pump is taken into account with 6 seconds in sum Pump Settings Control Pressure Limits MPa Enter the values for the desired pressure limit 0 will disable the software control of the pressure Keep in mind that the pressure for the sample loading may be different from the system pressure during a run if a different flow rate is used or if you use a sample loop Note that for a pump S 100 without pressure transducer this option is not accessible Solvent Enter the name of the solvent Stacked Injections gt gt The option Stacked injections allow to define additional inje
72. name Valve 1 Valves number fi Add Info Gender gt Configure valve f1 v 6 positions v Valve name Valve 1 Interface RS 232 v Serial number mH COM Port comi vi Valve type 6 positions Baud Rate 19500 vl Add Valve Info Switching Valves Configuration Valve type Add Va OK Cancel Help H Configuration window for the Switching Valves Valves Number Enter the number of valves used in a group In maximum 32 valves can be configured in this configuration window Configure Valve Select the number of the valve you would like to configure You must configure all of the valves Valves Number before leaving the dialog Valve Name Enter a descriptive name for the selected valve This name will appear on the valves tab when the instrument setup or status windows are displayed Use a name which is unique within the instrument Valve Type Select the type of the valve from the drop down list The choices are 2 6 12 and 16 positions for the Valco Vici valves additionally 8 and 10 positions Interface Select the Interface you want to use for communication with the device RS 232 v Fig 110 Available interfaces Knauer Switching Valves Fig 111 RS 232 Select the number for the RS 232 communication port COM Port on your PC to which the valve is connected LAN Knauer Switching Valves only Interface MT y 24 IP Port 11 0001 MW Use S N to ide
73. only the HPG A pump can be selected 3 column The 3 column allows for entering a value for the selected device If no line is selected the header shows If a pump is selected the column header show Flow ml min and the flow for the pump or HPG system can be selected The allowed flow depends on the pump head For a 10 ml pump head the flow rate range is 0 00 9 99 ml min for a 50 ml pump head 0 0 50 0 ml min Please note that for a HPG both pumps must have the same pump head If the detector is selected the column header shows WL nm Following the specifications of the UVD2 1S the allowed wavelength range is 190 500 nm If a valve is selected the column header shows Pos drop down menu will be accessible to select the desired valve position If the ASM2 1L is selected as a device the column header shows Events The field shows now the selected events will be shown for events no option is selected In practice this means that the current status will not be changed The setup will open by clicking the small arrow key The choices for the events are Off On and Pulse If selected 0 represents event Off 1 event On and P Pulse 4 column The 4 column is only to show A if as a device a pump of a HPG is selected This allows for selecting the A part of a gradient while B is calculated by the software 100 A If no pump is selected the header sh
74. option You can define this range by selecting one from the pull down menu Scan A click on this button will start the diagnostic measurement lt will take some time depending on your scan range and speed settings During the scan the info please wait will blink in the head bar The spectrum will be displayed after finishing the scan ae The obtained diagnostic spectrum cannot be saved directly or exported For later comparisons you must take a screenshot and save this in an appropriate way Instrument Status Conductivity Detector Alltech 650 MM Instrument Status E System Status P Alltech 650 Monitor Direct Control Run Status Cell temp setpoint C Cell temperature C Conductance us Range uS Total conductance us Polarity Range us gt Serial Port COM 1 SN 22222 Fig 228 Detector Status tab Conductivity Detector Alltech 650 Monitor Run Status The status HOLD will be displayed when no program is running otherwise the Run Status displays a run time in minutes elapsed from the start of the run The actual cell temperature the conductance and total conductance as well as the range will be displayed Direct Control All settings you have entered in this area will become effective after clicking on the Apply buttons To achieve changes during a run the direct control option must be activated see Fig 249 on page 170 Also an autozero can be initial
75. sequence is used to define the samples indicate how they are to be injected the method used to separate them and how the data is to be calculated A sample sequence can be used to acquire both calibration data as well as data from unknowns and both can be performed in a single sequence In addition a sequence is used to generate a calibration curve and analyze unknowns from stored data To setup a sequence 1 Click File Sequence Sequence Wizard or select Create a Sequence on the Instrument Wizard to access the Method page of the Sequence wizard Sequence Wizard Method Method rojects D efault Method muttilevel calibration met Ga Data File Type amp For acquisition C From existing data files Amount values Sample amount Internal standard amount Multiplication factors f 3 Dilution factors i r R ye T ete sae Sequence Wizard Method 2 Enter the method name 3 Select the mode of data acquisition If the data is to be collected as part of the sequence the Amount Values fields will be activated When you are beginning to develop a sequence ignore them and press Next to present the Sequence Wizard Unknowns If the From existing data files option is selected a dialog box will be presented to select the desired files 24 Fig 19 Fig 20 Short guide ChromGate KNAUER Instrument Control Sequence Wizard Unknowns Sample ID gt Data path C EZChrom Elite
76. signal is indicated in this field Wavelength nm The actual wavelength is indicated in this field Direct Control The direct control option must be activated see Fig 249 on page 170 D2 Lamp The On state of the lamp is represented by an active green LED symbol the Off state is represented by an inactive grey LED symbol af Clicking on the Lamp Off button will after inquiry switch the lamp off and change the button label to Lamp ON Autozero Click this button to define the current signal as the baseline zero level Wavelength nm Specify the wavelength you would like to have at the moment and click the Apply button Creating an Instrument Control Method 147 ME New Instrument Method untitled met Data None Project Cor Ioj x l File Edit View Method Data Sequence Analysis Control Reports Window Help x neper Pr re ere en Ss GS 1 Kontron 430 2 B A HEEL EAT gt o gt EP System Status SP Kontron 430 Monitor E Direct Contral fe New Instrument Method untitled met Data None Project Corin 1 Tes gt li File Edit View Method Data Sequence Analysis Control Reports Window Help la x 4 gt MS 1 kontron 430 2 E ela A 2s e S ol gt s k E EP System Status Y Kontron 430 fl onitor Direct Control Run Status 0 75 min Acquisition Channels Channeli 254 nm 103944 Channel 1 254 Channel 2 350 nm 0350 Channel 2 254 Communi
77. t To solve this equation a user must supply a molecular number average Mn and Molecular Mass average Mw for a single broad Molecular Mass distribution MWD standard Also an F t detector response is needed from a mass concentration detector With these three known values a 282 SEC Option single variable search can be used to find D2 using the following equation M M FO FO op D The usual range for D2 is between 0 and 1 inclusive Therefore golden section search is used to find D2s value For example with a known value for Mw Mn the search routine would substitute 0 5 for D2 If the equation is valid both sides of the equation equal each other the search ends and D2 0 5 If the equation is not valid then values of 0 25 and 0 75 are substituted for D2 to see if either can satisfy the equation If neither can solve the equation then the D2 value that is closest to satisfying the equation is the direction the search should continue Once D2 is solved D1 can be solved directly using one of the following equations b D F t dt MN M F t Exp D t dt D F t Exp D pdt fr t dt With D1 and D2 solved a calibration curve can be created over time Equations Broad Standard Method 2 The second method is to solve for a non linear Molecular Mass calibration curve The curves equation has two unknown Mark Houwink constants K and A It is these constants that must be solved before this curve can be cre
78. the graph area yet near the axis of interest The cursor will change to an up down arrow near the Y axis or a left right arrow near the X axis Moving the mouse in this mode will scroll the graph up down or left right on the axis To restore the original view do a right hand mouse click in the chromatogram window followed by the Full Unzoom command Removing Calibration Outliers You can temporarily remove an outlier from your calibration curve by clicking on the outlier point on the calibration curve When you select an outlier point this way the point will turn red as will its Molecular Mass 278 Fig 383 eS SEC Option information in the spreadsheet and the calibration curve will be re drawn with the point excluded To restore the point to the curve click it again You can also double click on the row number in the spreadsheet to do the same thing SEC Export When you select the Export settings button in the SEC Setup window the following dialog box appears Click the Export Enabled box to turn export functions on Then select the boxes for the data you wish to have exported Enter a path including file name where the export file should be placed SEC Export W Export Enabled Export data W Summary results h Header Slice report SEC Method Export file name Include path Miservervuser export SECOO1 Cancel Help Click OK when you have completed the dialog The data will be exported in
79. the injection is made the valve will switch into L Load position If a sample loop is used the valve will switch to L Load position if the pump is running and fill the sample loop For the injection the valve will switch to Inject position Direct Control Pump Flow During a run the pump cannot be controlled using the pump flow option The buttons Stop and Flow Apply will be inaccessible gt gt If the option Direct control during a run is enabled after the initial injection the gt gt button is accessible lf you click on the button the known stacked injection setup with the default values will open This is allow for create a modified stacked injection table The setup works as described for the instrument setup of the injection module 256 Fig 368 ChromGate Preparative Option Stacked Injections Humber of Injectors Injection Volume ral Injection Flow mlrmin Injection Durator min Time between Injectons min Injection module Stacked injections table setup If you click the Apply button the new table will be executed immediately the program will not check when the last injection was made If the Direct Control during a run option Save changes in time table is enabled all program lines for stacked injections which have not been executed yet during the current run will be replaced by the stacked injections from the direct control whereas lines for stacked injections
80. the sampler by clicking the Apply button The Off button can be used for disabling the cooling control To switch it on again set the required temperature and press the Apply button 164 Creating an Instrument Control Method Reset Transport Vials only with 84 3 vials tray If the tray type 84 3 vials is configured the button Reset Transport Vials allows for reset the transport liquid level of all transport vials vials 85 86 87 to the default value of 8000 ul If you click on that button the software will ask if you wants to reset the filling level of all transport vials to 8000 ul Click lt Yes gt to do so or lt No gt to leave the window without a level reset Please be sure that you also fill the transport vial s because the autosampler adjusts the needle immersion depth dependent on the vials filling level If the vials will not fill the autosampler can aspirate air instead transport liquid from the transport vial The software cannot read out or set the filling level The transport vial filling level will automatically be reset if the autosampler will be switched on Start Needle Wash Press this button to force the autosampler to perform the needle wash Move Tray Clicking briefly on this button will change the tray position FRONT lt gt HOME gt gt The Stacked Injection table can be changed during a run To have access the option Direct Control during a run in Method Runtime settings must be enable
81. the section on contour plot Right click within the chromatogram window to display the pop up menu This menu contains the same options as the basic chromatogram window for all detector types and in addition enables you to overlay chromatograms from different wavelengths and change the Gallery view Overlay Chromatograms When this option is available when you have a chromatogram view and a contour plot displayed simultaneously Mixed View Select Overlay Chromatograms to add chromatograms from different wavelengths to the view whenever you slide the wavelength selection cursor on the contour plot to a new wavelength Max Plot A Max Plot is a chromatogram with each point plotted at its maximum absorbance This plot gives an indication of the appearance of the chromatogram when the wavelengths are optimized for each peak ChromGate amp PDA Option 205 Fig 299 DAD Spectra Max Plot f An O AND AP pe e Peak Purity Plot Fig 300 Fig 301 The Total Peak Purity view displays the purity profile for a chromatogram extracted from the 3D data The view displays purity information for the Max Plot chromatogram that is displayed in the Chromatogram pane of the Mixed View The pane will be blank until the data has been analyzed and a peak has been selected for the purity calculation Time 3 92818 Minutes Amplitude 0 06 Purty Index Purity Threshold Purity Index Purity Index 3 80 3 55 3
82. to side in the aspect position of the plot When a new value is entered the plot is redrawn to reflect the new value upon exiting the dialog Zoom This reports the current level of magnification in the plot When a new value is entered the plot is redrawn to reflect the new value upon exiting the dialog Performance Use Zoom Rotate Bounding Box When this box is checked the plot will be temporarily replaced by a box during zoom and rotation operation When the operation is completed the plot will be redrawn Checking this box will increase performance on computers with slower graphic subsystems 3D Properties Axis Fig 291 This dialog lets you set up axis limits for your 3D plot 3D Data Graph Properties Ei General Axis Setup Limits Time A A Min 0 00 Max 11199 4 Minutes Get Limi Ge mits Wavelength MV Autoscale Min po Max po 4 nm Absorbance MV Autoscale Min 029 Max 5111 mau Labels Font Arial v Size 7 Color C Ste B Z U Cancel Apply Help Limits Enter the limits for the 3D plot Time Click Auto scale if you want to have the software automatically scale the time axis to the maximum values To enter a manual range de select the auto scale box and add your own limits or click the Get Limits button to enter the limits displayed on the current 3D graph Wavelength Click Auto scale if you want to have the software automatically scale the
83. to the original plot view click the right mouse button and select Reset Contour View Fig 293 The Contour Plot also referred to as an Isoabsorbance Plot provides an aerial view of the absorbance of the sample at each wavelength versus time The contour view supplies quick and easy to assimilate information about those wavelengths at which the sample exhibits appreciable absorbance With contour view it is also possible to generate a Chromatogram View for an individual wavelength and a Spectrum View for a given point in time Time 1 67907 Minutes Wavelength 268 nm Right click inside the window to display the pop up menu Select Properties to display the Contour Properties dialog box To generate a chromatogram view from contour view of Mixed View or Mixed View w 3D 200 ChromGate PDA Option 1 Select View PDA Views Mixed View to display the Contour Map Chromatogram and Spectrum 2 Move the cursor to the triangle shaped handle located on the left hand wavelength axis of the Contour Map and press the left mouse button 3 Drag the cursor up or down to the desired wavelength and release the mouse button 4 The chromatogram associated with the specified wavelength is displayed in the Chromatogram View To generate a spectrum view from contour view of Mixed View or Mixed View w 3D 1 Select View PDA Views Mixed View to display the Contour Map Chromatogram and Spectrum 2 Move the cursor to the
84. triangle shaped handle located on the time axis of the Contour Map and press the left mouse button 3 Drag the cursor to the desired peak and release the mouse button 4 The Spectrum associated with the specified retention time value is displayed in the Spectrum View Contour Properties A right mouse click anywhere on the contour plot followed by selecting the Properties button will display a dialog where you can select the way the contour plot is displayed Contour Graph General Axis Setup Style Colors Color Range Full Spectrum z C Grayscale Background py Display Detail Less 4 o More I 1 1 1 1 1 1 I 1 1 I Print Fete Coarse Smooth Lality faster l l A i _ flower Cursors Time 13 71 Minutes Wavelength 430 rim Bandwidth 1 nm Cancel Fig 294 Style Select how you want the plot to appear Grayscale or Color Colors Range Use this to select how the coloration of the plot is to be displayed When Light amp Dark Range is selected alternating light and dark bands are used When Full Spectrum is selected then a continuous color spectrum Is used ChromGate amp PDA Option 201 Background Select the color to be used for the background of the plot Display This specifies the relative quality of the displayed contour plot Less Display Detail will result in faster rendering of the plot Print Quality This specifies the relative quality of the printed contour plo
85. using This serial number should match the serial number of the defined pump Pressure Units Select the pressure units you want to use for the pump from the drop down list The choices are bar MPa and psi Serial Port Select the number for the communication port on your PC where the instrument is connected The selection lt none gt is for simulations No real control of the instrument will be performed Baud rate Select from the drop down list the baud rate you want to use for the pump The choices are 4800 and 9600 The selection must be the same as at the pump itself The default value is 4800 for all Kontron pumps Add Info Click the button Add Info to enter more detailed description of the pump The entered additional information or comments will also be printed out when you print the instrument configuration When complete click OK to exit the dialog and return to the instrument configuration icon list Configuration Knauer Detectors Fig 61 The configuration dialogs of the supported detectors differ only slightly from each other The appearance of the corresponding detector configuration windows is nearly identical Fig 62 through Fig 71 For configuration information regarding the PLATINblue detectors PDA 1 and MW 1 please refer to the PLATINblue system manual UY Detector 200 Configuration Name Uy 200 Serial Port COM 1 y Serial Number 187889 Y Axis Units m u v Flow Cel Add Into
86. which have already been executing during the current run remain untouched If the Direct control during a run option Save changes in time table is not active the new table will be executed but not stored in the instrument setup However in this case the method does not include the correct injection program SEC Option SEG Option Overview 257 Size Exclusion Chromatography SEC is a chromatography technique used to determine the Molecular Mass distribution of large molecules and polymers SEC also referred to as GPC is performed by injecting a sample onto a column comprised usually of rigid polymer gels with known pore sizes As the sample migrates through the column the smaller molecules penetrate the pores of the column and are retained longer than the larger molecules There is no column adsorption involved in SEC as there is in classical partition chromatography Since the size of the molecule is related to its Molecular Mass the elution time or volume can therefore be used to approximate the Molecular Mass of the molecule larger molecules eluting first and smaller molecules eluting last Due to the nature of the types of compounds analyzed using SEC generally high Molecular Mass polymers it is relatively rare to encounter a chromatogram with sharp baseline resolved peaks as is found in partition chromatography The calculations involved are therefore generally designed to produce Molecular Mass distribution MWD or
87. will be based on the slice width you select here The software will force this number to be an integral of the data collection rate during the analysis Baseline Start and Stop Times The baseline start and stop times in minutes can be entered manually or selected graphically using the SEC buttons on the chromatogram toolbar Results Range Start and Stop One or more ranges of the chromatogram must be defined to be used in calculation and reporting of the Molecular Mass distribution Each range is determined by the results range start and stop values The times in minutes can be entered manually or graphically using a stored chromatogram Horizontal Baseline Select this button if you wish the SEC baseline to be drawn as a horizontal line forward from the Baseline Start Time to the Stop Time The horizontal baseline stops when it crosses the data trace A threshold value in microvolt must be specified to ignore any detector baseline noise in the trace that would cause the horizontal projection to stop when it encounters the noise in the trace If the Horizontal Baseline reaches the end of the detected peak before the programmed Stop Time the range will stop where the Horizontal Baseline crosses the data trace Calibration Data Enter values for M and M in the fields provided or k and a then click the Calibrate button The system will calculate k and a or M and M values to be used for the calibration You must have completed the
88. will be explained generally at the end of the section parameters on page 242 Signal Level FC Parameters at 4 00 min Signal Ascending Descending Signal Level Units 25 Expected interval min Please notice that the parameters will be used in the Units selected for the Detection Channel Relative to previous event min Use as reference for correction next events Action Action Collect To C pest 0 position 111 Fig 333 FC parameters Signal Level single event Enter the level threshold value and define it to be on the ascending edge of the peak Than enter the expected possible shift interval for the retention time select the action and the destination vial After closing the window the parameter will be designed as L FC Parameters at 4 50 min Signal Ascending Descending Signal Level Unitz 125 Expected interval min 10 5 Please notice that the parameters will be used in the Units selected for the Detection Channel Relative to previous event min Use as reference for correction next events Acton Action waste o E g laa Waring this event may overlap with one or more events Cancel Fig 334 FC parameters Signal Level single event Enter the level threshold value and define it to be on the descending flange of the peak Than enter the expected possible ChromGate Preparative Option shift interval for the retention time sel
89. window example Jasco FP 920 After completing the generic driver program an instrument tab as for any other instrument will be displayed in the instrument status tab O System Status E 100 Ci reso gt EX WL 240 nm EM WL 400 nm Gain 10 Attenuation 1 Signal 0 00130 LastError 0 Communication active Instrument status tab example Jasco FP 920 Depending on the device a few most important parameters will be monitored and displayed in the status area Direct control is not supported for Generic Drivers Generic drivers only support basic functionality Please ask Knauer for available new Generic drivers or for the requirements for the development of a new Generic driver at software knauer net ChromGate System Suitability Setup 185 ChromGate System Suitability Setup Before ChromGate can make system Suitability calculations you must enter the required acceptance ranges for the peaks of interest This is done in using the Method System Suitability command 1 Click on the Method System Suitability command to view the system Suitability Setup dialog lel System Suitability Setup Detector 1 Compound Plates meter EMG Plates meter DAB Plates meter 40H Plates meter JP End a Fig 278 2 Select the first peak to be used for calculations by highlighting it with the mouse in the Compounds list 3 In the adjacent spreadsheet click on the first field in the Parameter c
90. you are working in offline or online mode Instrument Wizard Knauer HPLC Offline GS Create or modify a method Create or modify a method E Create a sequence Create a sequence EN Process a sequence Run one sample Run a sequence of samples M Show at instrument startup V Show at instrument startup Instrument wizard A offline B online 84 Creating an Instrument Control Method For details on other aspects of method development e g calibration integration and sequence operations please refer to the ChromGate Data System Reference Manual Instrument Setup Fig 120 The instrument setup portion of your method displays how the devices pumps detectors autosamplers switching valves are to be controlled and how data is to be acquired This information is entered in the Instrument Setup dialog Click the Instrument Setup button or select Method Instrument Setup from the menu window will appear which displays the parameters required for the equipment configured for this instrument MN Instrument Setup E E Working Mode Solvent el lzocratic a Control Pressure Limits MPa Mlin 10 0 Max 10 0 No Action at the end of run Pretreatment Solent Type Pump Program 2 ta em eo ane comens min ml nin CH DP 0 00 41 000 100 00000000 10 00 1 000 100 00000000 LD 15 2530 a Flore ES 4050 Wavelength nm Instrument setup window Smartline Pump 105
91. you can enter the IP address of your device if the option Use S N to identify the instrument is disabled Please note that in case the device receives the IP address from a DHCP server e g a router the IP address may change if the device is switched off and switched on We highly recommend using the S N and not the IP address to identify the instrument because devices may automatically receive a new IP address when reset 42 Fig 53 Fig 54 Fig 55 Fig 56 Setup and Control of Knauer HPLC Systems Device Information P Addes 51050 LPG AAA 093600007 Refresh Select LAN interface Device Information window Serial Number If you have read out the device via LAN the serial number has been added automatically Otherwise type in the serial number of the pump you are using The serial number of network controlled pumps is used for the device identification and addressing during communication This serial number must match the serial number of the defined pump If not you will get an appropriate error message if you later try to start in run Head Select the type of pump head you are using from the drop down list The pump head must also be selected in the pump s own menu For the pumps K 1001 K 1000 K 501 and K 120 pumps the choices are 10 ml and 50 ml of different materials steel PEEK ceramics titan Head 10 mi Ceramic IA FS Material Maz Head flow Mas Head pressure A
92. 0 For each of the single configured devices you get a separate tab in the instrument setup window Each of these contains several areas where you can enter parameters related to the individual device In any case you find at the bottom a unique graph part displaying the wavelength flow gradient and possibly other related profiles With a right hand mouse click you get access to multiple setup possibilities of the profile For details see the ChromGate Data System User s Manual The definition and setups of triggers see page 135 and auxiliary traces see page 135 must be performed in a separate tab for the whole instrument not in the tabs of the single devices Creating an Instrument Control Method 85 Instrument Setup Pumps The pump setup window shows several areas Their appearance may depend as well on the selected pump as on the settings while configuring it Working Mode 8 51000 ag s 3200 El Switching Valves mi Bl sor El 120 E kiom Ea k 1800 Working Mode Working Mode isocratic High Pressure Gradient HPG A g som g an g k100 E kaso 1 Working Mode LPG Cycle Time sec 20 Fig 121 Working mode in pump setup window Using the Kontron pump 325 additionally a CAM correction can be set The correction value can be selected from 1 through 5 i Kontron 422 PB Kontron 525 Fig 122 Working mode in Kontron 325 pump setup window In case of isocratic or high pressure gradie
93. 0 nm Retention time range F Min Wavelength step DEG Am Lambda max kl la iz nm Max hits Boo Compound name filter E Similarity threshold Enabled Library F Fig 283 Library Enabled Enter the spectral library to be searched or select from available libraries by clicking the Library field followed by clicking the file button You can select more than one library To enable the library for searching click the Enabled box If this list is empty or no library is enabled no hits will be returned when a search is performed Search parameters Enter the parameters to use for the search Wavelength range Enter the wavelength range to search Wavelength step Enter a step number for the search Larger numbers will make the search faster but if you use too large of a step spectral details may not be picked up Max hits Specify the number of hits that will be reported in the results of a library search Note that this works in conjunction with the Similarity Threshold parameter to limit the number of hits reported Similarity threshold Enter a number for threshold of similarity The library search results will only display matches whose similarity to the unknown exceeds this value Pre filters The options in this group allow you to specify search pre filters that will be performed on library spectra prior to the test for similarity All pre filters are optional Retention time range When values are enter
94. 0 077 m l Scan nm Start End Scan Channel 3 254 nm 0 077 m l Digital Channels nm T Gha 254 ihe WL Apply Lamp Number 001 Lamp Starts 275 Lamp operation time 4354 0 h Ch 3 254 Ch 4 Total lamp time 4354 0 h Digital Outs r e DO Apply Installation date 22 Feb 2002 O Networks jal SN 63945 Detector status tab K 2600 For Help press F1 Running sample 1 Monitor Run Status The status HOLD will be displayed when no program is running otherwise the Run Status displays a run time in minutes elapsed from the start of the run Digital Outs Events The current state for each digital output will be displayed in this control The On state is represented by an active red Light Emission Diode LED symbol 8 the Off state including waiting for pulse is represented by an inactive grey LED symbol 4f Channel 1 2 3 4 The actual signal in the units which have been chosen in the detector configuration dialog at the actual wavelength in nm is displayed for each active channel GLP The GLP data of the detector are displayed in this area The data will be actualized by pressing the Refresh button Direct Control The direct control option must be activated see Fig 249 on page 170 Lamp The On state of the lamp is represented by an active green LED symbol y the Off state is represented by an inactive gray LED symbol af Clicking on the Lamp Off button will after inquiry switch
95. 0 522 525 Pump Configuration Proax 200 0 Communicatio Serial Port Instrument Name Kontron B25 Pump Type 525 Gradient Mode LPG y Head SemiFrep bd Serial Number 55555 Pmax 200 0 Bar Flow O 20 ml min Communication Serial Port COM3 Baud rate 4800 Add Into Cancel Help Serial Port Pressure Units Bar Kontron Pumps configuration window Name The name of the selected pump appears automatically You can enter any other descriptive name for the pump This name will appear on the pump tab when the instrument setup or status windows are displayed Use a name which is unique within the instrument Pump Type Select the type of pump you want to configure from the drop down list Gradient Mode In case of the 3xx pumps it is automatically set to isocratic and for the 5xx pumps to LPG For the 4xx pumps the choices are HPG A HPG B or HPG C Head Select the type of pump head you are using from the drop down list The choices are Analytical Micro and Semi Prep for all Kontron pumps Setup and Control of Knauer HPLC Systems 45 Fig 60 ES EXE Micro Pmax 00 0 Bar H analutical Communication Eks Available pump heads for Kontron pumps Once you have chosen the pump head the related maximum pressure and maximum flow rate are displayed automatically Serial Number Type in the serial number of the pump you are
96. 01 you should set in the detector setup screen EXT CONTROL menu the keyboard mode to be able to control the wavelength from the ChromGate software The detector basic settings as time constant sampling rate etc are described on page 91 MM Instrument Setup J s 1050 S as 3950 3 s 2550 3 s 2520 y s 2300 5 246 Bronkhorst Flowmeter 2 lt Aux Traces R Trigger Y Acquisition channel on Wavelength table Tim Wave Garnes mi length nm e n 0 00 254 Time constant 10 v Sec Options V Aut t start Sampling rate 1 lt Hz ASIDE SES Suitable for minimum peak width at base 0 3330 Min Run time 10 Min I Lamp OFF at the end of run CEN Time constant 100 Sec Sampling rate 1 Hz Suitable for minimum peak width at base of 0 333 Min a MN Instrument Setup s 1050 SY AS 3950 E s 2554 0 00 254 0000 10 00 254 0000 Run time 10 0 Min Acquisition delay 0 0 Min V Autozero at start v atthe end of run Autozero at WL change UV Detector setup window Autozero at start Check the Autozero field if you wish the signal to be zeroed at the moment a measurement is started to define the current signal as the baseline Autozero at WL change No access to this option The grayed area is only a reminder that the autozero will take place at any wavelength change during a run Lamp OFF at the end of run
97. 066868 OOOO Tray configuration 84 3 vials for the autosampler 3950 The 84 3 vial tray for the autosampler 3950 is supported in firmware version 1 14 or higher A firmware upgrade from former firmware versions is not available Left Right tray type Select the type of the installed tray for both sides The choices are 96 low 96 high 384 low 48 vials and 12 vials The selection can be different for each tray side In case of activated prep mode it is fixed on 12 vials If the tray type 84 3 vials is enabled this choice is for both side this tray uses the left and right tray side Only for the tray type 84 3 vials it is possible to select a source vial for the transport liquid in the method setup later The 84 3 vials tray is only available for autosamplers with firmware version 1 14 or higher Autosamplers with a previous firmware version cannot be updated Plate process order The vials of the two trays will be processed either in columns or rows order First Destination Vial Enable this option if you want to use destination vials Enter the first destination vial position For each injection a destination vial will be used for mixing a diluting Reagent A B Vial Enable these options if you want to use reagent vials Enter the corresponding vial position The selected vial will be used for Reagent A and or Reagent B for all injections Use Mix amp Dilute Enable this option to select for each injection one destin
98. 1000 Pump 5 1050 Pump K 120 K 501 fal Pump K 1001 4 E Options Auto Configuration Cancel Help Instrument selection window Several icons will be displayed in the Available Modules box on the left Add modules to be configured by double clicking on each or by clicking once on the icon followed by the green arrow Setup and Control of Knauer HPLC Systems 37 Fig 40 Fig 41 Fig 42 KNAUER HPLC System Available modules Configured modules Pump 10P 20P 7 P2 15 7 P4 15 Detector UVD2 1L ED Kontron 32073227325 Pump Pump P2 1L ED Kontron 4207 4227425 Pump sl Assistant ASMZ1L ED kontron 520 522 525 Pump ed Autosampler 5 3800 Basic Marathon S Autosampler 5 3900 Midas Ed Autozampler Knauer Optimas 229 Autosampler Endurance 229 Autosampler Triathlon Autoszampler 5 3950 eg Kontron 360 Autosampler Kontron 460 465 Autosamplers ere Kontron 560565 Autosamplers AT Options Auto Configuration Cancel Help Instrument selection window with selected items Auto Configuration Using the Auto Configuration button all devices connected via LAN and switched on will be automatically added and configured Auto Configuration En auer LE System Auto Configuratior only LAN capable devices will be detected Interface HAT a IP Port 10001 j Cancel Help Auto Configuration start window All Knauer devices use a single IP port for LAN communication Only devices with
99. 45 Setup 92 status 145 RS232 communications 68 70 71 73 Run Screens 114 123 Run Status Column Oven 168 DAD 149 Flowmeter 169 K 2600 147 148 Manager 5000 167 Pumps 140 RF 10AxI 152 154 155 RI Detector 145 Switching Valves 167 UV Detector 146 Run time Extension 143 144 Run Time 92 108 S S 1000 Setup 85 Status 139 Wiring Scheme 284 S 2x00 Configuration 45 Instrument Status 145 Setup 92 Wiring Scheme 284 Sample Sequence 23 Sample Tray 65 68 70 71 73 Sampling Frequency 91 92 Sampling Rate 91 108 Scale Factor 122 Scan 96 98 101 151 153 Scanning speed 154 Search parameters 190 SEC Acquisition Delay 260 Acquisition Setup 259 Amount Values 262 Applications of 258 Average Time min 268 270 275 Calibration 257 259 271 Channel Status 259 Custom Reports 278 Define SEC Peaks 263 Enable Analysis 268 270 273 275 Equations 281 Index Export 278 Fit Type 269 272 277 Goodness of Fit 269 272 277 Horizontal Line 269 271 274 276 Instrument Setup 259 Intrinsic viscosity 258 271 Mark Houwink equation 258 Molecular Mass 268 270 275 Narrow Standards 257 Number average molecular mass 258 Period 259 Polydispersity 258 Reference Peak Time min 269 272 Reference Window 269 272 Results Range 268 271 273 276 Run Time 259 Sample Data 269 271 Sample Description 262 Sampling Frequency 259 Sampling Rate 259 Setup 267 Single Run Acquisition 260 Start Time min 268 271 273 276 Submit 262 T
100. 5 Fig 366 ChromGate Preparative Option MM Instrument Setup Time 7 49 minutes Amplitude 254 Wavelength nm Hd FSET owr Pup 1800 TT I S Injection Module Stacked injections auxiliary trace MN Stacked Inj Inj Module Time 688262 Minutes Amplitude tau Sacked Inj Jinj Module Injection Module Stacked injections trace For every stacked injection an entry for the sample preparation and the injection will be made ME Instrument ctivity Lo g Synchronize Axes and Zoom Limits System 08 2 PC PM LAB TEST Run Queue Complete Single Run C EZChrom Elite Enterprise Projects Defaut DataiStacked Injection with InjModule 3_ 1_001 dat 08 10 2010 18 23 23 PC PM LAB TEST Inj Module Stacked injection trigger marker is detected in 6 04 min after run start 08 10 2010 18 22 38 PC PM LAB TEST Inj Module Stacked injection with inj volume 2 00 mi flow 3 000 ml min duration 0 72 min will begin in 5 29 min after run start 08 10 2010 18 20 24 PC PM LAB TEST Inj Module Stacked injection trigger marker is detected in 3 06 min after run start 08 10 2010 18 19 38 PC PM LAB TEST Inj Module Stacked injection with inj volume 2 00 mi flow 3 000 ml min duration 0 72 min will begin in 2 29 min after run start 08 10 2010 18 16 02 PC PM LAB TEST Run Queue Start Single Run C EZChrom ElitelEnterprise Projects DefaultiDatalStacked Injection with InjModule 3_ 1_001 dat 08 10 2010 18 16 01 PC PM
101. 58 ren Percha ren ma Tere 0 CONEY brutal Serngitudy Um gt Veaveteregn 270 ren Bandasdh rm Tere 0 DOMENET ruget Arlt dos ett Spectrum View Fig 305 Fig ChromGate PDA Option 1 mA This view displays the spectrum associated with a time on the chromatogram The time of the spectrum to be displayed can be changed from Mixed View by sliding the spectrum selection box on the x axis mu Wavelength 220 333 nm Amplitude 1 512 mall 4 82 Min nknown column test001 Spectrum Properties 306 mA Select this menu item to access the trace properties dialog This enables you to add another trace to the view or change scaling It also lets you selectively remove overlaid traces from the view Up to 3 wavelength maxima and minima can be displayed Use the Annotations command from the right mouse click menu in the Spectrum view and select Lambda Max and or Lambda Min mA Wavelength 261 813 nm Amplitude 3 694 mAU own column test001 Add Trace Annotations m Full Unzoom Clear Overlays Operations Utilities Properties nm Annoin Lac 11 Maren Data 2 82 Min FE Ayadabie Aedo Lambda Mie Lanitrda Mar Zhu la fully annm aons ChromGate amp PDA Option 209 Spectrum Background Correction A background correction can be performed if the current spectrum has been extracted from an integr
102. 9 m l WL 1254 Apply AZ ASM2 1L status tab UV D2 Lamp Status The button shows the current lamp status The On state is represented by a green active Light Emission Diode LED symbol the Off state including initializing heating is represented by an inactive grey LED symbol ss WL Status Creating an Instrument Control Method 159 Fig 235 Fig 236 WL shows two different values the current wave length and the signal The wave length s unit is nm the signal unit depends on the unit that has been chosen in the ASM2 1L configuration for the detector The right handed part of the UV section allows for direct control of the detector independently from the method setup During a run this part is not accessible beside the autozero AZ button WL Direct Control Enter the desired wave length for the detector click on the lt Apply gt button to change the wave length in the detector If the wave length is out of the acceptable wave length range an appropriate error message will be shown Error message Wavelength out of range Lamp OFF Lamp On button Direct Control This button allows for switching off or on the detector lamp The button s labeling indicates what will happen if the button is pressed it does not show the lamp status If the lamp is on the button shows Lamp OFF because the lamp will be switched off if you click on it It shows Lamp ON if the detector lamp is off c
103. 90 3 95 400 4 05 Minutes Reference spectrum Apex at 3 927 Min Total peak purity 0525435 Up slope similarity 0 379592 Threshold 0 950000 Down slope similarity 0 249657 Threshold 0 950000 3 point peak purity 0 314790 Threshold 0 950000 Select Peak for Purity Similarity Display To select the reference spectrum for the purity calculation make sure the data has been analyzed If you are not sure click the analyze button on the toolbar Then click Actions Select Peak from the Mixed View toolbar A dialog will appear that instructs you to select a peak by holding down the Ctrl button and then clicking on a peak from the Max Plot pane The retention time of the reference spectrum is displayed along with a value indicating peak purity Select Peak for Similarity Purity Display Peak apex at 5 61 Min Select Peak Drag the spectrum cursor on the Contour or Chromatogram view to select a peak To select a peak without using this window press Ctrl while clicking In one of those views E 206 ChromGate PDA Option You can continue to select or change the peak When you are finished click the Close button Purity View Properties Fig 302 To set up the properties for the Purity View do a right mouse click and select Properties Purity Yiew Properties General M Override method settings Display information Wavelength range 200 205 rim ET otal purity Wavelength st
104. Apply n Dutput mv 0 Digital Output J Digital Output 1 E Apply Communication interface Interface Manager 5000 Device 1 at COM6 SN 1 Version 1 00 Fig 244 Manager 5000 5050 IF2 status tab Creating an Instrument Control Method 167 Fig 245 Monitor Run Status The status HOLD will be displayed when no program is running otherwise the Run Status displays a run time in minutes elapsed since the start of the run An Output mV The actual output signal is indicated in this field Digital Output Event The current state for the digital output will be displayed in this control The On state is represented by an active red Light Emission Diode LED symbol O The Off state is represented by an inactive LED symbol af Direct Control The output voltage AO mV and the percentage factor values of the AOF can be set The settings become effective by clicking the Apply button resulting in an output voltage AO AOF 100 at the given channel The digital output defines the event possibilities 0 1 and Pulse selectable from the pull down menu The setting becomes effective by clicking the Apply button Instrument Status Knauer Switching Valves Run Status The Run Status displays a run time in minutes elapsed since the start of the run While the method is not running the status HOLD will be displayed Valves Status Direct Control The actual position for each valve is displayed i
105. Direct Control Run Status Waiting for Trigger Events J 2 D2 Lamp D Autozero Chanmell 254 nm 0 000 Channel 2 288 nm 0 000 Digital Channels nm Range 200 400 nm chi 300 cha 300 3 Serial Port COMS SN 75934 Diagnostics Fig 223 DAD Detector status tab example DAD 2600 Monitor Run Status The status HOLD will be displayed when no program is running otherwise the Run Status displays a run time in minutes elapsed from the start of the run Channel 1 2 3 4 The actual signal in the units which have been chosen in the detector configuration dialog at the actual wavelength in nm is displayed for each active channel Direct Control The direct control option must be activated see Fig 249 on page 170 Creating an Instrument Control Method D2 Lamp HAL Lamp The On state of the lamp s is represented by a green active LED symbol 9 the Off state is represented by an inactive gray LED symbol af Clicking on the Lamp Off button will after inquiry switch the lamp off and change the button label to Lamp ON The appearance of the HAL lamp control depends on the individual detector and its configuration Autozero Click this button to define the current signal over the specified spectrum as the baseline zero level Digital Channels nm The number of channels that will be activated depends on how many possibilities are given to set new values for the measuring wavelengths Pre
106. Exclusion Software is an optional package that enables ChromGate Chromatography Data System to perform SEC calculations Once the SEC option is enabled it is possible for the user to configure any one or more of the instruments connected to the system as an SEC instrument It is also possible to perform normal partition chromatography calculations on some instruments while performing SEC on other instruments Note that SEC and partition SEC Option ES 259 chromatography are distinctly different techniques and cannot therefore be performed simultaneously on the same instrument In general performing an SEC analysis involves the following steps 1 Run SEC calibration standard s and use it to set up the calibration information for the SEC samples Method SEC Setup 2 Create a custom report to include the types of SEC reports and plots you want for your analyses 3 Create a sequence if appropriate and run your SEC samples Running an SEC Calibration Standard Before you run your calibration standard s you must first set the acquisition parameters so that all the data are correctly sampled and saved on your disk Do this using Instrument Setup From the instrument window for the chromatograph you intend to use for your SEC run click on the Instrument Setup button or select Method Instrument Setup from the menu Instrument Setup The instrument setup portion of your method tells how you want to acquire the data com
107. Fig 259 Solvent Control dialog box HPG system ES The amount of solvents used will be calculated correctly only under software control Should the pumps have been controlled manually or the bottles have been refilled the actual solvent amount must be corrected Qualification Procedures ChromGate provides a performance qualification tool for checking your HPLC systems This is not a qualification procedure for each individual device pump detector etc but for the whole configured chromatogra phic instrument The procedure is at last a normal method used to determine the measurement reproducibility The PQ procedure is started from the main instrument screen by selecting the menu sequence CONTROL QUALIFICATION PROCEDURES f PQ Method Test PQ met Data None Project Default Performance Qualification TT Eile Edit View Method Data Sequence Analysis Control Reports Window Help A 1 5 2500 y NERA ELLE EAT Ne TOEN G5 Generic PQ Procedure This Performance Qualification Procedure can be used to determine reproducibilty of the measurement results for complete chromatography system Begin new Qualification session Session at 10 October 2006 08 08 34 Session at 10 October 2006 07 49 00 Exit Start Session gt For Help press F1 Fig 260 PQ start screen You have the choice to create a new PQ session method or to select earlier created ones These may be completed or not If you select a not compl
108. File Library Save As and enter a name for the library The lib extension is automatically appended A new easy way to add spectra to an opened library is to select the option Add Spectrum to Spectrum Library from the Actions menu Move the cursor in the chromatogram view to the appropriate position The corresponding time is displayed in the dialog box Simply click on Add to save the spectrum in the library Add Spectrum to Library gt Spectrum at 2 83 Min Tip Drag the spectrum cursor on the Contour or Chromatogran view to select the spectrum Fig 313 Close Library Search How to Perform a Manual Library Search 1 In the Contour map of the Mixed View window drag the vertical cursor to the apex of the peak of interest to display the corresponding spectrum in the spectrum pane Click on the Actions Search library Click either Method to use the library parameters from the method or Quick to enable you to modify the search parameters Before you do the search make sure you have either selected a library in your method or have opened a library to do a quick search Click Search Now to display the Library Search Results window showing the three closest matches in the specified library When appropriate click on the gt gt or lt lt button to display additional matches Spectral Library Search This window appears when you click on the Actions Search Library command from the Mixed Vie
109. For already existing instruments right mouse click on the instrument and select the Configure command The Instrument Configuration dialog will appear 36 Fig 38 Setup and Control of Knauer HPLC Systems Instrument Configuration Instrument name New Instrument Instrument type KMALER HPLE system si OF Cancel Help e Instrument configuration dialog box Instrument name Enter a descriptive name by which the instrument is identified This name will appear under the instrument icon and when the instrument window is displayed Instrument type Select the Instrument type from the drop down list Knauer devices are only available in the Knauer HPLC system type Server name in Client Server only Select the Server name This is the name of the CG server to which the instrument is attached Finally click on Configure to proceed to the instrument configuration where one can define the detectors pumps and further devices for the instrument Instrument Configuration Configuration KNAUER HPLC System Fig 39 When you click the Configure button the following dialog will appear KNAUER HPLC System Available modules Configured modules Assistant ASM2 1L Pump P2 1L Detector UVD2 10 sf PLATIMblue Pump P 1 fh PLATINblue Autosampler AS 1 als PLATINblue Detector Myw 1 J PLATINblue Detector PDA PLATINblue Thermostat T 1 i PLATIMBlue Thermostat T 1 Basic r I Pump 5
110. Fraction Single Event Unconditional Timed Waste F Single Event Unconditional Timed Waste F N Rack configuration Path Create Open E Fig 345 Fraction collector setup window You must either create a new configuration or you can load an already existing one Trying to leave this window without doing so you will get the error message ES Mo Rack configuration File specified Fig 346 Press the Create Open button to open the next window Here you can select an existing file or type in a new file name which should be descriptive for the configured rack Select the Rack configuration file Look ir E Methods vl e re fr B standard rck Select the Rack configuration file My Recent e Documents Look in E Methods e E ex Fa B standardirck 4 My Recent Documents My Computer File name My Network Files of type Places File name B standard X My Network Files of type Rack configuration data rck v Cancel Places ro ate pen as read only Help Fig 347 Selecting creating the rack configuration file In both cases press the open button The window will be closed and the path is displayed in setup window Fig 348 ChromGate Preparative Option 245 Fig 348 Fig 349 Fig 350 Chromatogram window Rack configuration Path C ChromGate Methods B standard rck Select 0 0 5 10 E Fraction E Peak recycling H Solvent recy
111. Generic Drivers 183 GLP 141 148 Gradient modify 142 143 H Halogen Lamp 50 51 56 57 Headspace Pressure 113 118 122 HPG System 174 175 Injection Method 113 117 122 Injection Program 124 Injection Type 111 Injection Valve Wiring Scheme 285 286 Installation ChromGate 8 instrument selection 9 preparative option 11 Instrument Configuration 36 Control 31 Name 36 Setup 84 Status 29 137 Autosampler 156 161 Column Oven 168 DAD 149 Detector 145 Endurance 165 Flowmeter 169 Fraction collectors 247 Manager 5000 166 Pumps 139 RF 10AxI 152 154 Switching Valves 167 Triathlon 165 User defined Detector 155 UV Detectors 145 147 148 Virtual Detector 155 Type 36 Wizard 83 Instrument Configuration 14 Integration Events 19 Time 151 Validation of 178 Integration 19 Interface 40 45 49 55 56 61 231 Configuration 31 Interface Box Configuration 32 Wiring 57 289 Interface Box IF2 33 Interpolate Spectrum 209 IP Address 49 56 Index J Jasco FP 920 183 K K 1001 Configuration 38 Setup 85 Status 139 Wiring Scheme 286 287 289 290 K 120 Setup 85 Status 139 K 1800 Configuration 38 Setup 85 Status 139 K 2600 Direct Control 148 Instrument Status 148 Setup 95 Wiring Scheme 286 289 K 2700 2800 Interface Card Installation 35 Setup 100 K 2x00 1 Configuration 45 Instrument Status 145 Setup 92 Wiring Scheme 287 288 290 K 501 Setup 85 Status 139 Wiring Scheme 288 KNAUER Net 138 Kontron detector Conf
112. HTML format htm to make it easy to view with a browser or for transfer to other programs This export applies only to the current channel If you want to export data from a different channel you must select the channel then set up the export for that channel SEC Custom Reports SEC reports are created using ChromGate Custom Report formatting When you click on the Custom reports button the Custom Report formatter window will appear For details on using the custom report editor see the main ChromGate User s Guide When using the SEC software the following options will be available in the custom report editor that allow you to place SEC specific items in your report Adding an SEC Chromatogram To add an SEC trace to your report do a right hand mouse click on the report form and select Insert Graph Data Graph In the resulting dialog select Current Data if you want the report to display whatever chromatogram is in your current chromatogram window Select the Trace channel for example you wish to display The chromatogram will appear on your report You can adjust the ranges and add annotations by selecting the chromatogram do a right hand mouse click and then select the appropriate command See the main ChromGate User s Guide for details on how to change the appearance of your custom report chromatogram Adding a Molecular Mass Distribution Trace To add a Slice vs Molecular Mass Molecular Mass Distribution trace to
113. I Fig 231 Detector status tab Virtual detector Monitor Run Status The status HOLD will be displayed when no program is running otherwise the Run Status displays a run time in minutes elapsed from the start of the run Creating an Instrument Control Method Signal MAU The actual signal is indicated in this field The units correspond to the setting in the configuration of the user defined or virtual detector Direct Control Signal Apply You can set the detector to a desired signal value for the time being External Trigger ON Pressing this button will give the trigger signal to your system if it is in the position waiting for trigger Instrument Status Assistant ASM2 1L The Assistant status window allows for checking the current status of all configured modules of the Assistant and the direct control The status and direct control options on this window depends on the configured modules Below the possible status options are described ME Instrument Status System Status 3 P21L 3 UVDA ser Monitor Run Status On Events 29299 Pump Flow ml min 1 000 Flow 1 000 Stop Pressure MPa 0 1 PMin 10 0 PMax 10 0 Apply Walve s Valvel ValveM ValveL ValveM System Position 5 3 5 S Events 000 al Apply Pump Purge gt E Lan 192 168 2 145 10001 tal SN FYA121400001 Fig 232 ASM2 1L status tab valve valve pump Monitor Run Status The status On will be displayed whe
114. If the check boxes for Flow and A have been enabled the values for both will be send to the pumps while clicking the lt Apply gt button If you wants to update only the flow or gradient please disable the A check box Click on the lt Stop gt button to set the flow rate to O zero A Direct Control If the corresponding check box is enabled you can change the gradient composition Enter the desired value for HPG pump A and click on the upper lt Apply gt button same line as for the Flow and A values to send the new gradient composition to the pumps The program will complete automatically the value for HPG pump B lf the check boxes for Flow and A have been enabled the values for both will be send to the pumps while clicking the lt Apply gt button If you wants to update only the A please disable the flow check box Pmin Pmax only for P4 1S Direct Control Enter the desired values for the allowed minimum and maximum system pressure Click on the lt Apply gt button to send the pressure limits to the pump lf the pressure limits are out of acceptable pressure range defined by the mounted pump head an appropriate error message will be shown UV The left handed part of the UV section shows the detector status as Deuterium lamp status wave length in nm and signal The signals unit depends on the unit selected in the detector s configuration AU mAU HAU Ll D2 Lamp 3 Lamp OFF WL 254 nm 0 00
115. Ih KNALIER Software ChromGate Chromatographie Software V7057 8 CONTENTS 3 CONTENTS Installation Guide ChromGate 3 3 2 scssssssssssessssssesesessesssessssescsesessesescsessescseseeseseseesees 8 GENE DEMNIONS HE 8 ke Le EET EE osa 8 KNAVER FRC Como OP ON Las STENE 11 Short Guide ChromGate KNAUER Instrument Control araxnuvnnnunannunnvnununnnnunnunuvnunne 13 A NE ud aah NE tue 13 Fi heels cee hate canals ae a oases 14 Instrument CONQUE GRADAR 14 SEN ODINS v NNN 14 Seting UP ia MANI ee od 15 The NESTEN VZ AN 15 TASE MEMO NON een 16 Quantification of the Compound s of Interest ooocccccccnccccnncconnnccnnnnonnnnncnncnonononos 19 NET 19 in A Ne 19 Generating and Using a Calibration Curve oooccoccncnncnccccnnccnnoncncnnnnnnnnnnnnnnnnnnnonenonors 20 Method File GOMMandS agree 20 Quantification and Using Sequence Files ooccccccnccccncoccncoonncncnncncnonaronnnnnnononononos 20 NENNE ee 20 SNE LEVEL C ANDFALION 4 21 Creating a Sample Sequence rrrarrnnanrnnnnennnnnnnnrnnanrnnnnennnnnnnnnnnnnrnnanennnnnnnnnnnnnsennne 23 PRUNING ASEJU E HANN 25 REDOING nara Pee 26 Collecting D 445 28 General Data Collection Instructions with ChromGate rrarrrrarrnnarennnnrnnnrnnnnrnnnne 28 CONG CUING Dala PP 28 Instrument Status of a running Control Metho0d oocccoccccocnccccnconcnconcnnocnnnnnnnnnonos 29 Shutting Down the System a coi 30 Setup and Control o
116. L or S 0 00 Lor S 0 00 Lors 0 00 Lors 0 00 L and 5 0 00 Max 0 00 Max 0 00 Timed Waste Exp interv Rel time p T Fraction Fraction N Fraction min shift min ic Position O NTE Check 1 D Pump not defined y Flow ml min 50 000 Actualize flow E Fraction BB Peak recycling E Solvent recycling Fig 356 Fraction collector instrument setup window Running method elp press F1 Fraction Annotations The fractions as well as the fraction and vial numbers can be shown in the chromatogram Open the chromatogram s annotations right mouse click on the chromatogram Annotation Select Fractions from the drop down menu and add Fraction and or Vial Number using the green arrow key In the menu Other on the bottom of the menu window please enable the option FRC Actual to show the selected annotations Be sure to have selected the correct Trace for your settings E S 2550 Channel 1 Time 31 2057 Minutes Amplitude 979 937 mAU Trace Annotation Properties Annotation Trace 1 Current Data 5 2520 Channel 1 vi Fractions v Available Annotations Other I RT Window Fraction 1 Vial 1 lt 1 Show the following annotations Fraction Number Vial Number Decimals I Group Range IV Show undetected named peaks M FRC Actual 32 Sa gt e 2 o 5 yoo Abbrechen Apply To All Hilfe
117. Molecular Mass table before entering the calibration data Reference Peak Time min Select this box if you are using a retention time reference peak to adjust for changes in flow rate All elution times volumes used in calculations will be offset by the time difference between the value entered here and the time of the reference peak in the sample Enter the expected retention time for the reference peak in minutes Reference Window If you are using a reference peak enter a value for the window for the reference peak A peak falling within this of the expected retention time for the reference peak will be considered the reference peak If two peaks fall within the window the peak closest to the center will be chosen as the retention time reference SEC Option 217 Fit Type Select a calibration curve fit type from the drop down list The choices include Point to point Linear Quadratic Cubic 4th Degree Polynomial and Spline Goodness of Fit The calibration curve for the data in the SEC Setup will be displayed in the calibration curve window The r value which represents goodness of fit of the calibration points to the curve selected will be displayed above the curve value of 1 00 indicates perfect fit Select the Time or Volume button at the bottom of the calibration curve to display the x axis of the curve as either retention time in minutes or retention volume If you select volume enter the flow rate
118. OM1 SN 33333 Instrument status tab example Kontron Pump 320 For Help press F1 The tab consists of two main parts the Monitor and the Direct Control areas Monitor Run Status The status HOLD will be displayed when no program is running otherwise the Run Status displays a run time in minutes elapsed from the start of the run During a Pretreatment the pump status is equilibrating Flow ml min The actual flow is indicated in this field Pressure Control MPa The highlighted actual pressure is shown framed by the settings for the allowed minimum and maximum pressures Components In case of gradient systems HPG or LPG the percentage of components will be shown during the run The components are also indicated by the multi colored bar In the case of isocratic systems the bar is completely red colored showing 100 Events Digital Outputs The current state for each Event Digital Output will be displayed in this control The On state is represented by a red active Light Emission Diode LED symbol 8 the Off state including waiting for pulse is represented by an inactive grey LED symbol af Creating an Instrument Control Method 141 GLP This section only available with Knauer networking pumps displays GLP Good Laboratory Practice related information transferred to or downloaded from the pump Pressing the lt Refresh gt button will actualize this information The extent of the information depend
119. Standard E Up na oF F3Ltel Piltarvigita I sec Attenuation 1 Acquiaition Flucrescence Gatahate 10 Hz 0 0 davelengthEx 240 nm Hove lurgt hee 400 om ACQUisltion Flucrescence TsOn true F STA 10 0 Arquislt Len FLUSESaBESNSS IEON falss gt EGP data acquis H velengt her 240 na VavelenagthEHN 400 fm KG E Pi LT Brno Farger Auta eng comrara Dip gules korp arter FuoieserdrssRar aba Arlt dota tale for Fr nor IL a du spor Farc bin peppery 5 16 Tre 1 Er a atin or Mes Chanel Taka a Ye mysen proper Durai mikin rurekengt 00 ran DO nm apelar h proper Curent ereb raveia Erm 0 ren ber propo Curen g mT T T Fah piofariig Dime itpro re al By L f r Fi Saad lea Fai panna Dif i apenios fie nad By dell hat YE me A ar Aternuston awai Deira sterssten value TT EET NTE NER TT NE davelengthex 240 nm HavelengtheM 400 nm Gain 50 FilterBl Standard i Use FiltarMigital 7 Artenuarion 1 Acquisition Ja Frog Dave la rato re re mf ANAE REAREN ved Evne contralor ine Fo Gan SEY Value k out of range CT EIC LOT 10007 Acquisition a Acquisition HavelangtheX 240 nm Havelaengthen 400 tim I lr LS Ti command On aio fumo 3 Aequiton Fisci bcirca Dalat papers Aerqactin da tala at ASS Se Acountion Fluorsrcesce in copes Set bo ue do sier ete scouiriton foe thin cael Tale ne Instrument setup programming
120. The Diagnostics window appears which allows you to access information and control of important parameters and modules of the device Diagnostics Scan nm max range 200 900 nm NE Start 200 End 600 Spectrum type Emission gt Excitation wavelength 254 Lamp On Scanning speed 600 nm min v Output mode xcitation ene Sensitivity v Recorder range x Lamp Ott Gain x1 v I Analog output Scan Data 2501 300 350 400 450 00 550 00 Emission wavelength nm Diagnostics window of Fluorescence Detector RF 10AxI with an anthracene spectrum Scan nm max range 200 900 nm This area is used to specify spectral parameters used for your test measurements Set the Start and End values within the given limits according to your needs Spectrum type Select the excitation or emission spectrum from the pull down menu Depending on your choice the field description will change with the selection you can enter either the Emission wavelength or the Excitation wavelength Creating an Instrument Control Method Scanning speed Select a value between 24 and 3000 nm min from the pull down menu Sensitivity Gain Output Mode Select the sensitivity level HIGH MED or LOW the gain 1 4 16 and the output mode Emission NOT Corrected Excitation Energy Light Source Corrected from the corresponding pull down menus Analog output An analog output can be activated The Recorder range is combined with this
121. V rense 196 ENN 199 MIXEI VIE ee 203 A A coke t 204 SPM VINN 208 KE OR AE EE EE Ne PEN 210 Spectrum SIMEN TAES 211 Specral Library DENIA sudo 212 How to Collect Spectra for a Library occcocccconnnconcncccnonocnconcnonacononnnonnncnnnncnncnonors 213 How to Add Spectra to a Library occococnccccncoccncccnoccnnnnncncnncnonanonnnnnnnnnnnnnncnncnnnnns 213 HOEN ETNE 214 Special Library SEAN ee 214 CUSTOM REMO NNN 216 PDA INSEN Graph NEM S apene 216 PDA Insert Report Items cooncooncoocnoccnoccnononononononocnononononononononanonanonanenaninon 217 Library Search Report 217 Library Definition Report 218 Spectrum Report 219 Spectral Display 221 6 CONTENTS Pekere eeo eee Rl 221 Analysis he AO lesers ae 222 PDA Analysis and Calculations ennnrnnnennnnennnnvnnnnnnnnnnnnnennnnnnnnnnnnnnnnnnennnnennnnnnnnnnnnnennnne 222 Chromatograms Extracted from the 3D Data rrrannnnnnannnnennnnnnnnrrranennnnennnnennnnennnnnn 222 Multi Chromatogram Channels sarsaran a ar 222 Working CHrOMAlLOGI aN 2 au 2422 223 Spectra Extracted from the 3D Dadta occcconcccoccoccnccccnccncncconnocnnnnnannonannonanonnnnnnanoss 223 PIAS sie ARO AER EP O SEO E 223 Vo 223 Background COMeCOMN ua 223 SPSCUUIM KEN OA I ass 224 Spectrum SMOONING Eee 224 Spectrum Derivatives 00000 A A uden 224 Upslope and Down slope Spectra occcocccccccccccnconcncnocononnnonononncnonanonnnnonononenenonans 225 Library Search Calculations os
122. a max Calculation range 1 30 1950 nm Spectral filtering In this area you designate how spectral filtering if any will be performed Filtering type Choose the type of filtering for the spectral plot The choices are None Smooth 1 Derivative and 2 Derivative Selecting one of these smoothing algorithms can remove noise from the spectrum Background correction If this box is checked then prior to display a correction for spectral background is made as follows 1 The spectra from the baseline start and baseline stop times for the peak are extracted from the data ChromGate amp PDA Option 193 2 For each spectrum in the peak a corresponding background spectrum is generated by linear interpolation between the baseline start and baseline stop spectra 3 This background spectrum is subtracted from the original spectrum IS The baseline start and stop times that are used in calculating background compensation are based on the detected peaks for the channel currently selected Interpolate spectra Select this check box to automatically perform 10 1 interpolation of spectra displayed in the spectrum window using a cubic spline curve fit This interpolation is performed after the applying any spectral filtering option 1st derivative 2nd derivative or smooth to the display spectrum ee Interpolated spectra may not be stored or added to a library The peak spectrum is defined as This specifies if and ho
123. a of the corresponding peak in the chromatogram Once calibration is established it can be used to determine the concentration of the compound of interest in unknowns A single level calibration involves a single sample to calculate the calibration constant the curve is generated from that point and the origin A multi level calibration uses several standards e g 10 ug ml 20 ug ml 50 ug ml 100 ug ml Setting up a calibration involves the following steps 1 Run the chromatograms containing the standards and save them 2 Identify the peaks from the standards 3 Generate a peak table 4 Generate the standard curve The standards used to generate a calibration should be well defined and should be in a matrix that is similar to that containing the samples that you will analyze The standards undergo exactly the same sample preparation process that is used for real samples Short guide ChromGate KNAUER Instrument Control 21 There are two general ways that a standard can be employed e Internal Standard A known amount of the compound used as a standard is added to the sample and the area for the compound of interest is compared to the area of the standard e External Standard The standard is a separate sample and the observed intensity is used to generate the calibration curve directly The application program allows you to generate calibration curves for a number of compounds in a single operation and an analytical repor
124. abel on the tube Syringe Volume Select the volume of installed syringe from the drop down list The choices are 250 ul 1000 wl and 2500 ul If the 2500 ul syringe is selected automatically the Prep mode check box is enabled In the Prep mode the values for the syringe 2500 ul and loop 10000 ul and the 24 vial tray type are fixed Options Tray cooling Prep mode Check the appropriate boxes if you want to control the tray cooling or enable the Prep mode The desired tray temperature can be entered later as part of the instrument setup In the Prep mode the values for syringe 2500 ul and loop 10000 ul and the 24 vial tray type are fixed The tubing volume is set to 45 ul Please check the correct volume of the tube between the needle and the injection valve on the tube s label Tray Click the Tray button to set up the autosampler tray Setup and Control of Knauer HPLC Systems 69 Tray configuration Tray type 04 3 Mialtype Standard Cancel First destination vial mM Fig 97 Tray configuration for the autosampler Knauer Optimas Tray Type Select the type of the installed tray The choices are 84 3 96 and 24 vials In Prep mode the tray type is fixed on 24 vials tray Vial Type Select the type of the vials used The choices are Standard and 2 5 ml The last is used only when lt 84 3 gt or lt 96 gt tray type is selected First Destination Vial Enable this option if you want to use destination
125. able Add Spectrum to Spectral Library ChromGate PDA Option Search Library Opens the Spectral Library Search window Overlay Spectra Allows you to select a spectrum to overlay in the Spectrum view pane Select Peak for Similarity Purity Display Enables you to select a peak for display in the Purity view pane Add to Multi Chromatogram Table Enables you to select a chromatogram to add to the Multi Chromatogram table in the PDA options Add Spectrum to Spectral Library Selecting this menu item allows you to create a new library entry from the spectrum currently displayed in the spectrum pane The spectrum will be added to the currently open library opened by selecting File gt Spectrum Library gt Open A name of the form Spectrum at time xx xx will be used as the Compound Name for the new library entry Mixed View Options Button This button presents options that enable you to toggle the view for the lower right pane of the mixed view is Uptions Show SD Plot Show Peak Similarity and Threshold Plot Show Peak Purity Plot Show Spectrum Similarity Table Fig 298 Show 3D Plot Show Peak Similarity and Threshold Plot Show Peak Purity Plot Show Spectrum Similarity Table Chromatogram View The Chromatogram View may be used alone or as part of the Mixed View displays Generate a chromatogram from the Contour plot as described in
126. acked injections Start sample preparation X XX min before stacked injection Define the time before the injection time the autosampler should start with the sample injection preparation Please make sure that the given time is sufficient to prepare the injection before the injection time arrives otherwise the injection may be delayed If the autosampler is ready to inject it will wait until the programmed time for the stacked injection If the autosampler needs a longer time than programmed it will inject immediately after the injection preparation is finished In this case the next stacked injection will be delayed by this extra time Do not wash the needle between injections from same vial Checking this option prevents the needle wash if the stacked injection is made from the same vial as the previous injection This option may be useful for saving time or increasing reproducibility of injections Stacked injections time table Make a right mouse click into the time table and select Fill table from the corresponding menu Stacked Injections Cancel Number of Injections Sample Vial Injection Volume pl Sample Preparation Time min 0 43 Time between Injections min 1 00 Do not wash needle between injections from same vial Autosampler 3950 Stacked injections table setup Number of Injections Enter the number of stacked injections If you e g enter 2 two stacked injections will be made in
127. actors Autosampler Vial Injection volume Description Activating the baseline check for a single run Instrument Status of a running Control Method If you have the Instrument Control option installed and your instrument configured as a KNAUER HPLC System the instrument control functions appear as part of the Instrument Status area of your instrument window To access the Instrument window where you can set up your devices and methods double click on the instrument icon you wish to start in the Main Menu It may be required that you log in before you can access the instrument window functions For details on other aspects of method development calibration integration and sequence operations see the ChromGate Data System Reference Manual System Status Fig 200 Open the Instrument Status Window using the menu sequence Control Instrument Status The Instrument Status window contains the status tabs for all configured instruments and as shown in the figure below the system status tab providing an overview for the whole system Kraner HPLC Project Default Instrument Status a je Ea E Fle got yew Method paa Sequence Analysis Control Reports Widow Help 5x i F e 1 52550 Channei A AA OG ADD BAO ae O Sytem Stas sim GJ a5 300 delitos Omn M 5 2550 ls 230005 2800 Method Method 1 met Dala Sample 3 dat J n aple e ae f Flower 0 000 riirii P AS awus Rent
128. addition of the initial injection 3 injections at all 120 Creating an Instrument Control Method will be made per run Please make sure that you have enough samples for the entered number of injection Sample Vial Select the vials the stacked injection should be made from Select Same to injection from the same vial as the previous injection Next to inject from the vial number lt n 1 gt from the previous injection or Custom to select free the vial number Injection Volume pl Select the desired injection volume Note that the volume depend from the selected injection mode of the autosampler instrument setup Sample Preparation Time min Define the time before the injection time the autosampler should start with the sample injection preparation Please make sure that the given time is sufficient to prepare the injection before the injection time arrives otherwise the injection may be delayed If the autosampler is ready to inject it will wait until the programmed time for the stacked injection If the autosampler needs a longer time than programmed it will inject immediately after the injection preparation is finished In this case the next stacked injection will be delayed by this extra time Time between Injections min Enter the time between the injections Do not wash needle between injections from same vial Enable this option if you wish to prohibit the needle wash after each injection programmed in the autos
129. ake a single run To make a single data acquisition run you need to specify the method to be used for analysis and a file name for data storage Note In order to use a method for data acquisition its instrument setup should have the acquisition channel turned On and a sampling rate and run time designated To make a single run click the Single Run button or select the Control Single Run command from the menu The following dialog will appear SEC Option Fig 370 Single Run Acquisition Aun information Sample ID Test Run 010 Method EZChrom ElitesMethads multilewel calibration met S Cancel Data path lc enterprise ProjectsiDefaulData Help Data file Test Run 010 dat Print method report g ele Amount values Sample amount NO Internal standard amount TE Multiplication factor ri e Calibrate Calibration level 11 Clear all calibration Clear replicates Clear calibration for level Average replicates Print calibration report Description Run Information This section allows you to specify files for the run Sample ID Enter a Sample ID for the run This can contain text and numbers and is saved with the data file Method Enter the name of the method to be used for data acquisition and processing Include the entire path name if the method is not in your default method directory You can select the method from a list of methods available on your disk by clicking the Fil
130. aks in the sample and will be a Gaussian peak In this case the area of the peak is directly related to the concentration of the compound of interest The analyst could generate a calibration plot using standards of known concentration to determine the concentration area relationship and then use the calibration plot to determine the concentration of unknowns The calibration data is incorporated into the method so that when you select a method to use all of the information that is required for quantization is loaded Integration Fig 13 Integration of the chromatogram requires two basic parameters the width and the threshold which define the starting point and ending point for a peak and to distinguish a peak from noise When a method is established the default values are entered and these can be edited via the Integration Events window which is accessed by selecting Method Integration Events E integ TEK EPT E FG AS Width FS MN Integration Events S 2600 Channel 1 2 Threshold 3 a Integration Events window The Width value is used to calculate a value for bunching or smoothing the data points before the integration algorithm is applied integration works best when 20 points are sampled across a peak The width should be selected for the narrowest peak in the chromatogram The Threshold value is the first derivative of the chromatogram and is used to distinguish the peak from noise and or drift
131. alco Switching Valves from one configuration window even if they are of different type K 6 K 12 or K 16 they are represented together in one tab The detailed description is given on page 131 Baseline Check tab The Baseline Check tab is presented if Baseline Check is selected on the Configuration Options dialog box see section Setting up the Baseline Check on page 136 It is used to ensure that the baseline is satisfactory The Baseline check must be enabled in the Options menu of the instrument configuration Short guide ChromGate KNAUER Instrument Control 19 185 To use this feature select the Baseline Check option in the Single Run dialog box or include Baseline Check in the current Sequence line When the baseline feature is selected the initial conditions for the run will be used to acquire baseline data If the data is satisfactory for all channels the run will be performed if the baseline data is not satisfactory the run will be aborted and a message to that effect will be presented Trigger tab The Trigger tab is used to indicate the action to start a chromatogram The definition of the various options is presented on the tab For details see page 135 Quantification of the Compound s of Interest Overview The change of the signal from a detector is related to the concentration of the eluting compound Under ideal circumstances the peak for each compound of interest will be well resolved from other pe
132. amplers instrument setup for the stacked injections from the same vial If you close the Stacked Injections window with Apply the selected settings will be copied into the Stacked injections table Any existing entries from previous programming will be deleted If you make a right mouse click on the table you can select Fill Down to fill the table with the same settings as in the selected line Insert Line to add a new line Delete Line to delete the selected line Delete Lines to delete all selected lines Fill Table will open the Stacked injection setup again The new table will overwrite the existing one If you select the command Fill table to open the setup dialog for the stacked injection table the window will show default values for all parameters beside the sample preparation time Click on OK to close the Stacked Injections table window the autosampler S 3950 instrument setup window will be shown In the Aux Traces tab a trace Stacked Inj AS 3950 can be enabled The trace will show the start of the sample preparation and the stacked injection Creating an Instrument Control Method 121 Fig 173 Fig 174 MM Instrument Setup tE Autosampler 3950 Stacked injections trace For every stacked injection an entry for the sample preparation and the injection will be made MM Instrument Activity Log user Logged source Activity E System 09 09 2010 16 20
133. and the newer Smartline devices pump S 1050 and detectors S 2550 und S 2520 Older Smartline devices as pump S 1000 detector S 2600 cannot wake up from Standby by a software command also the lamp of the detector cannot be switched on if the detector is in Standby 172 Knauer Instrument Control Method Options Advanced Method Settings Check run time when saving method E W Enabled System Wake Up and all lamps on i Power all devices up at 14 08 12 12 40 49 a August 2012 gt 3 1 2 3 4 5 7 8 9 10 11 12 If this option i 13 15 16 17 18 19 tains the initial WL Oth 20 21 22 23 24 25 26f h comespondin 27 28 29 30 31 1 2 figuration 3 4 5 6 7 89 3 Today 1470872012 Cancel Trace name Fig 252 Advanced Runtime Settings System wake up date setting If enabled this option is valid for the currently used computer Trace name If the option Trace name is enabled the wave length of the initial line of the wave length table becomes part of the trace name in the chromatogram window as well as in the report chromatogram channel name in run report instead of the channel name This belongs to all detectors with a wave length table as UV DAD and fluorescence detectors The channel name in the channel selector will not be changed lit View Method Data Sequence Analysis sa 19 El B 1 52550 Channel 1 Trace name not changed E 52550 254 nm oG Time dn mn mAU Trace name changed
134. are disabled 218 Fig 316 ChromGate PDA Option Limit Peaks to time range This allows the user to restrict the peak selection made above to a time range When checked only peaks within the specified time range will be searched When this box is unchecked the Time Range edit fields are disabled Time Range This allows the user to restrict the peak selection made above to a time range When the Limit to time range box is unchecked these fields are disabled Specific Spectrum button Pressing this button will display a menu allowing the user to specify the spectrum to be searched The menu will allow the user to specify a spectrum based on one of the following choices Current Data Named File or Spectrum Peak Spectral Display The in this group specify the layout of the search results graphs Graph Height This specifies the relative height of a search results graph A value of 100 corresponds to a standard sized graph A larger value may be selected to provide more a detailed plot Graph Properties Pressing this button displays the graph properties dialog as found on the standard Spectrum graph Library Definition Report Selecting this option will insert a Library Definition Report into your custom report To specify parameters for the report do a right mouse click in the report table then select Properties The following dialog will appear Library Definition Report Properties Library E Sente
135. ash Temperature Control Check the Enable tray cooling field if your sampler is equipped with the tray cooling facility and you want to use it Aux iliaries This section allows you to program auxiliary output s for the AS 3800 Enable Aux1 Select this box if time programmable auxiliary output 1 is to be used Aux1 Time ON Enter the time at which auxiliary 1 must switch ON 0 99 min 59 sec Aux1 Time OFF Enter the time at which auxiliary 1 must switch OFF 0 99 min 59 sec Enable Aux2 Select this box if time programmable auxiliary output 2 is to be used Aux2 Time ON Enter the time at which auxiliary 2 must switch ON 0 99 min 59 sec Aux2 Time OFF Enter the time at which auxiliary 2 must switch OFF 0 99 min 59 sec Instrument Setup Autosampler Knauer Optimas 3900 Midas The method parameters required for automatic operation of your autosampler are contained in the Method Instrument Setup Optimas AS3900 tab The control parameters for the autosampler become part of your method and sequence files Creating an Instrument Control Method 113 Fig 163 EEE amp Instrument Setup S 5 100K E as 3900 Oven S 4050 y 2500 2 Aux Traces p Trigger Injection Parameters InSeton method Syringe speed Normal y IV Air segment e Full loop C Partial loopfil Flush volume ul 30 V Headspace pressure pl pick up alain 2 l V Needle wash C None Alarm buzzer
136. at is installed on the left side in the assistant housing from the drop down menu Pump P2 1S If selected you can choose the device name please refer to the device name section below the gradient type none HPG A HPG B and the pump head 10 ml 50 ml Pump P4 1S If selected you can choose the device name please refer to the device name section below the gradient type none HPG A HPG B and the pump head 10 ml 50 ml Due to that the pump P4 1S is equipped with a pressure sensor you can also select the unit for the pressure monitoring MPa bar psi Detector UVD2 1S If selected you can choose the device name please refer to the device name section below and the Y Axis Units AU mAU AU The Flow Cell field allows for entering information regarding the used detector flow cell Knauer Valve If selected you can choose the device name please refer to the device name section below and the number of position Please be sure to select the correct number of position if you select it manually 2 6 12 or 16 position can be selected For one valve in the assistant you can check the Use as a Fraction Collector box lf selected there are additional options You can enter the Vial Volume please be sure to enter the correct volume and setup Tubing and Solvent Peak Recycling Please refer to the chapter Multi Valve Fraction Collector Configuration for detailed information The Number of Vials is fixed an
137. ated To implement method 2 a user must supply a molecular number average Mn and Molecular Mass average Mw for a single broad Molecular Mass distribution MWD standard Also the user must supply a universal calibration curve o t qe E a M 3 p t Given below is an equation with unknown n and BP vai lie M 1 p t not gt l 1 A n Now there are four unknowns K A n and B p A single variable search using the following equation is used to find B and that in turn will allow a direct calculation to find n SEC Option 283 M M F t ont F t pa a a With B solved one of the equations below is used to directly calculate n nf FOO di M F t dt nf F t dt y n fFOSO di With B and n known the equations are solved for A and K to create the curve fe E 1 A 284 Typical Wiring Schemes Typical Wiring Schemes Pump S 1000 Manager 5000 Detector S 2500 Detector S 2300 Autosampler 3900 A A A EE EE j i 4 i M 5000 0 i he 1 agn AN OUT DIG I RELAY S 1000 A mjml A S 2500 M a am TN g Ved oe iE wy Ee 2300 sical E a TELELTIES t ry Fig 388 1 serial NULL modem cable A0895 2 serial Y cable A0755 3 connection cable DIN gt DIN G0649 4 Trigger AS 3800 Control I O Connector M 5000 Pin 4 N O gt DIG Pin5 COMM gt IN Typical Wiring Schemes Manager 5000 User Define
138. ated peak Select the right mouse click Operations Background Correction command from the Spectrum window Then click on the spectrum on which you wish to perform the calculation A background correction for the displayed spectrum will be performed based on the working chromatogram The background corrected spectrum is then displayed along with the original spectrum Please refer to the PDA Analysis and Calculations section for details on how this calculation is performed Background Correction may only be performed once on a spectrum If a second background correction is attempted on an already corrected spectrum a message box is displayed and the operation ignored If the current spectrum has not been extracted from an integrated peak of the working chromatogram selecting this menu item will have no effect on the spectrum Background subtraction must always be the first operation performed on a spectrum If a background correction is attempted on a spectrum after another operation has already been performed including operation specified in the Spectrum tab of PDA Options a message box is displayed and the background correction request ignored Interpolate Spectrum Spectrum Interpolation can be performed if the current spectrum has been extracted from an integrated peak Select the right mouse click Operations Interpolate command from the Spectrum window Then click on the spectrum on which you wish to perform the calculation A 10 1 i
139. ation reagent A and reagent B vial position This allows a higher number of injections if a higher volume of the reagent s is required The Mix Dilute option is only available if you have selected the same tray type for left and right lt is not available for the tray type 84 3 vials Setup and Control of Knauer HPLC Systems 67 Fig 95 Fig 96 Tray configuration Left tray type 45 vials E Right tray type 49 vials E Plate process order Columns m 00000060 00000006 00000060 00000068 00000060 Tray configuration for the autosampler 3950 with option Use Mix amp Dilute For pl pick up no transport vial must be chosen For the tray type 84 3 vials the transport vial can be selected in the method setup for all other tray types the transport liquid will be taken from the bottle with wash solution All configuration settings will be transmitted to the autosampler if the instrument will be opened In difference to the other Spark manufactured autosamplers the configuration data will not be stored permanently into the device and compared with your settings However please make sure that the configuration settings are correct due to that the device will take over your settings without verification When complete click OK to exit the dialog and return to the instrument configuration icon list Configuration Autosampler Knauer Optimas Autosampler Knauer Optimas Configuration Mame AS Serial number Comm
140. ation Type the SEC Setup screen will look similar to the one displayed below 268 Fig 379 SEC Option E SEC Setup Iof x V Enable GPC SEC Analysis 54000 20 7 9200 24 67 34500 221 1250 28 5 152 371 zl Calibration Type Narow y Fit Type Quadratic v R 2 0 994462 Time Slice sec Time 15 2296 Minutes Molecular Weight 150669 Ranges in Minutes 100000 100000 10000 1000 1000 27 803 pill Horizontal Baseline Threshold Sample Data 100 la 10000 Molecular Weight Reference Peak Time min Time Volume Flow Rate ml min Reference Window Print Export Settings Enable Analysis Click this box to enable SEC calculations for the current channel Each channel can have its own analysis conditions Molecular Mass For each calibration peak enter a Molecular Mass that is representative of the calibration standard peak Average Time min Enter the time in minutes of the calibration standard peak If you have used the graphical Define SEC Peaks command the peak times will be entered already for you Note the time is converted to seconds for calibration and Molecular Mass calculations Time Slice Select a time slice width in seconds for the calculations The slice width will determine the frequency of Molecular Mass fractions reported If a Slice Report is generated it will be based on the slice width you select here The software wi
141. atogram the virtual detector will show a value of 0 zero The Settings button will become accessible and clicking on it will bring up the following dialog box 106 Fig 156 Fig 157 Fig 158 Creating an Instrument Control Method Select Data File c ezchrom elitetenterprisetprojectsyd i Shift Time min OK Cancel Virtual detector setup data file selection The very last file selection will be displayed You can either enter a new file or you can use the Browse button To avoid possible signal distortion the Y Axis multiplier in the configuration of the virtual detector must be equal to that of the selected data file The dialog box which appears includes in the upper part the typical Windows searching facilities Additionally you can search using the Sample ID and or the Analyst Open Data File Suchen in Data ta Br m Ed multi calibration level 6 dat E pda estd unknown 002002 dat ef pda estd level 1 E QCOO1 dat E pda estd level 2 E QCOO2 dat E pda estd level 3 E STDOO1 dat sj pda estd level 4 les STDO02 dat E pda estd unknown 001001 dat E SysSuito01 dat I Dateiname multi calibration level 6 dat Dateityp All Files z Find files that match these criteria Sample ID Created any time y Find Now Analyst y Modified any time v New Search Delete Search results Virtual detector setup data file selecti
142. avelength Enter the wavelength of the chromatogram channel Bandwidth Enter the nm range to be averaged in generating the chromatogram channel Configuration of user defined detectors Enter the ratio threshold The threshold is the minimum absorbance value required in the chromatograms of both channels for calculation of the ratio It is a method of eliminating ratio calculation at minor absorbance 196 ChromGate PDA Option values such as those that occur with noise If the threshold value is not met the ratio plot will be zero PDA Views The PDA views provide a variety of ways to view PDA data You can choose to display one view at a time or a combination of views such as 3D and Contour or Mixed with 3D The view is selected using the View PDA View menu or from the PDA toolbar by selecting Views A right mouse click within any of the view windows provides a unique menu of options for that view 3D View Fig 288 Fig 289 The 3D View provides a three dimensional view of absorbance versus time and wavelength Wavelengths of appreciable absorbance and interference which may be invisible in a single wavelength plot are easy to locate with the 3D View The plot can be elevated and rotated around its axis for display from any angle During a run the user must manually refresh this display It does not update automatically Right click inside the 3D view to display the pop up menu that gives you access to p
143. avelength W Autoscale Hlin fon Max poo H nm Absorbance f Autoscale Mir hos H Max s752 m I Labels Fant Arial v Size 7 Color Style B 0 Cancel Help Time Click Auto scale if you want to have the software automatically scale the time axis to the maximum values To enter a manual range de select the auto scale box and add your own limits or click the Get Limits button to enter the limits displayed on the current contour graph Wavelength Click Auto scale if you want to have the software automatically scale the wavelength axis to the maximum values To Contour Axis Setup Absorbance Click Auto scale if you want to have the software automatically scale the absorbance axis to the maximum values To enter a manual range de select the auto scale box and add your own limits or click the Get Limits button to enter the limits displayed on the current contour graph Labels You can customize the labeling of the contour plot using the parameters in this area You can select font size color and style using the selections provided ChromGate amp PDA Option 203 Mixed View Fig 296 This view displays the contour chromatogram and spectrum views along with the fourth pane displaying similarity purity or peak profile DAD Spectra Mixed View P viens Synchronize EH Anange L Actions 3 Options Time 1 67907 Minutes Wavelength 246 nm Wavelength 394 438 nm A
144. average Molecular Mass numbers rather than discreet Molecular Mass values Because the distribution of Molecular Mass is of interest here results often include not only the average Molecular Mass values but also a list of area slices for the sample along with their associated average Molecular Mass s This type of report is called a Slice Report SEC Calibration ChromGate SEC supports three methods for SEC Calibration Narrow Standards calibration Broad Range calibration two versions and Universal calibration The time is converted to seconds for calibration and Molecular Mass calculations Narrow Standards Calibration A calibration curve is created by running a standard sample consisting of compounds of known Molecular Mass s The retention times or volumes of these narrow standards are plotted against the logarithm of the corresponding Molecular Mass s log M Broad Range Calibration Using the Hamielec method there are two methods available for calculation of Broad Range calibrations Broad Range 1 is a linear calibration and requires a SEC column that can provide a linear calibration curve It uses only average MW values of the polymer standard but assumes a linear approximation of GPC calibration curve Mw and Mn values must be known for the polymer standard that can be determined by light scattering and osmotic pressure techniques This method needs one broad MWD standard of the same structure as the u
145. baseline check feature should be used To generate a new instrument configuration e Select File New Instrument in the ChromGate main window create a new instrument file presented by an instrument icon on the right side of the main window e Right click on the instrument icon and select Configure Instrument on the context menu that is presented to access the Instrument Configuration dialog box e Verify that the Instrument type field indicates Knauer HPLC system and press lt Configure gt to open the Knauer HPLC dialog box e Click on the module that you want to include in the configuration and press the green arrow to put that icon onto the right hand side Access the Configuration dialog box for a module by double clicking on it The dialog box contains information about the specific module and lets you edit the configuration information The configuration information must match with the configuration of the device From devices which will be controlled via LAN connection you can read out the configuration e Add and configure all of the modules you want to control until the appropriate configuration has been created e lf additional system options as SEC SYS or PDA are desired access the options dialog box by pressing the lt Options gt button The purchased options must be enabled to make them accessible in the instrument Please refer the next chapter System Options for more information e Once the configuratio
146. be is installed you can find the tubing volume on a label on the tube Syringe Volume The volume of installed syringe can be set on 250 500 or 1000 ul If the Prep mode option is checked the syringe volume is fixed on 2500 ul Options Tray Cooling Check this box if you want to control the tray cooling The appropriate corresponding temperature is to be entered later as part of the instrument setup SSV Solvent Selection Valve Check this box if you have a solvent selection valve installed Prep mode Check this box if you have the preparative instrument installed The loop and syringe volumes then are set to a fixed volume 10 000 ul loop 2 500 ul syringe and the tray type to the 12 vials type Tray Click the Tray button to set up the autosampler tray Tray configuration Left tray type 112 vials Y gt First destination vial 1 0 Right tray type 12 vials W Reagent vial Plate process order Rows bal W Reagent E vial Fig 93 Tray configuration for the autosampler 3950 66 Fig 94 Tray configuration Left tray type Fight tray type First transport vial Last transport vial Setup and Control of Knauer HPLC Systems 84 3 vials First destination vial Reagent A vial m Cancel Transport vials are used for pL PickUp injection only 8 8 O Y oo E OOOO 0 Y 00000090090 VOS 0008 Sue oa O 0O OOOO E 8 0690 6
147. be used for data acquisition You must configure your data acquisition interface before you can acquire analog data using the data system The Knauer interfaces can also be used to control other devices by analog or digital output Interface configuration is accessed through the Tools command on the ChromGate Main Menu Click the Tools Interface Configuration command from the ChromGate Main Menu A window will appear displaying several possible interface devices To configure a device click on the icon to select it then click the lt Properties gt button or double click on the instrument icon 32 Fig 30 Setup and Control of Knauer HPLC Systems Interface Configuration O Help A HPLC Interface Box Kontron Interface Card 35420 A D Selection window for interface configurations Knauer Interface Configuration Fig 31 Fig 32 If you are using either a Knauer Interface Box IF2 or a Manager 5000 5050 click on the corresponding icon and then click Properties A dialog box will appear where you can configure the Interface Box see Fig 32 In regards to Knauer software the Manager 5050 interface module is identical to the Manager 5000 To configure the Manager 5050 please select Manager 5000 If you configure the interface newly the message like Fig 31 will remind you to enter the serial number of the interface device Configuration error E Please specify a serial number Error message
148. bed at the beginning of the chapter Instrument Setup Detectors on page 89 followed by details which are specific to certain detectors RI Detectors S 23 4 00 K 23 4 00 1 ooooccccoccccccococcncococcnnconnonoos page 92 UV Detectors S 200 K 200 ooocccccooccccococcnccccococnonaconconanoncnonanonoos page 92 UV Detectors K 2000 K 2500 rrrnnnnnnnnnnnvnnnnnnennnnnrnenvnnsnennnnsnnen page 92 UV Detectors S 2500 K 2001 K 2501 UVD2 1S rrrrnrrnnnnnnnn page 93 Instrument Setup Kontron Detectors occcoccccoccccccnccncnccacnnnonnnos page 95 K 2600 Dele CON Lur endar page 95 UV Detectors S 2550 S 2520 nannnrannvrnavernnvennnnnnnnnennvnnnnnenn page 80 Diode Array Detectors S 2600 K 2700 DAD 2800 2850 and Kontron DAD 540 rrronnnenrnnnnrvnnnnnnnn page 100 Fluorescence Detector RF 10Axl RF 20A AXS rrnrrrrrnnnrrevrnnnr page 103 Instrument Setup Alltech 650 Conductivity Detector page 104 User defined Detector oococccccocccccccccnnoccnononnnnnnann nn cers page 104 Instrument Setup Virtual Detector oocccoocccconcccconcnccnncncnnnnnnnos page 105 Fig 12 Short guide ChromGate KNAUER Instrument Control MM Instrument Setup Acquisition 11 0 Sec fi Hz 0 333 Min Time constant Sampling rate Suitable for minimum peak width at base of Run time 10 0 Min 0 0 Min Acquisition delay Options An output Scale 10v f I La
149. been successfully loaded on the computer or network and the instrumentation for which it is used has been installed It does not include topics that are relevant to installation interfacing of the unit to other components of the HPLC system major maintenance and other topics that are more properly of interest to the system administrator In addition it is assumed that the chromatographic conditions for the separation and the overall data processing procedures are well understood The automated operation of the system involves the use of e the Method The method describes instrument control data acquisition data processing reporting and exporting of data for a single run It includes all of the parameters that are used to perform ad process a run e the Sequence The sequence is a listing of the samples to be analyzed by the system on an automated basis The user can select the method to be used for each analysis ChromGate includes wizards that lead the user through the generation of the method and the sequence In addition the ChromGate software contains a broad range of features to assist the user and also includes a number of security options This manual is provided to introduce the analyst to the ChromGate program and provides a discussion of some of the most commonly performed operations It is not meant to present a detailed step by step procedure for use of the system or the data processing software There are a numbe
150. can be selected from the pull down menus Fields with selections available will display a combo box button when the field or row is selected Click the button to display the available choices The available choices for the sensitivity are HIGH MED and LOW F or the Output Mode the available choices are Excitation Energy Light Source Correction and Emission NOT corrected Output Mode Emission MOT Corrected Excitation Energy Light Source Correction Emission MOT Corrected pull down menus in the Fluorescence Detector RF 10AxI setup Instrument Setup Alltech 650 Conductivity Detector Fig 154 The Alltech 650 conductivity detector supports the general detector settings described on page 91 E Instrument Setup ED Alltech 650 7X Aux Traces VA Baseline Check 3 Trigger IV Acquisition channel on Options Progr m table 10 Sec Ti 4 0 00 5 00 negative 1000 5 00 negative Time constant Sampling rate 10 Min 0 Min Run time Acquisition delay T emperature Control Temperature C 35 Wait with tolerance C 5 1 Walt time min Time 9 69916 Minutes Amplitude 5 Range u Alltech 650 conductivity detector setup window Temperature Control Enter the desired temperature of the measuring cell the allowed temperature tolerance and the waiting time for conditioning Program table The program table can be edited in the usual way F
151. car da 225 GONG ee A 225 E EOS eoa E E T ai 225 Ratio Chromatogram Calc lati N sissioni a aa aa a aE 226 Simant Ca cua 455 226 Lambda Max Min Calcula Ss N 226 Noise Spectrum CalculatiONS ccoocccconcoconccoonccnnnoconcononccnononnnnonannnnanconancnnanananess 227 Peak Purity Calculations sa 227 6 do pao eed etcieee a a ANE A ERE 22 Calcu ting Total PU save 227 TAES PORN PU stn tscvescetenncchanteceaassacens ne a a a apii 228 SPECTUM EPO are 228 END EDON EET 229 ChromGate Preparative Option ssssssessssssesssesessssssssescsesessenssesescsesstsesneneeeeeenes 230 Fraction Collector Contigurati n usa 230 Virtual Fraction Collector ConfiguratioN ooccccccccoccncoccncccnccncncnacnnnnos 233 Multi Valve Fraction Collector Configuration c ooccccccncoccccccnonocnnnnonos 234 Fraction COMNCCION sep as vrede 235 Fraction Collector Instrument Status ipenn a ana a i 247 Fraction ANNOLALONS ina iba 249 Stacked INE Ne 250 SEG ODPUON suse 257 NM 257 A aa a Mantecsuntotancsaseaadete aimed Meatansustecencsones 257 PAO DIICATIONS Ol DE msi ll lll de a 258 Using ChromGate SEC Software cccccccccecscsesescsceseececescssecerseseesvacereevacereaseneess 258 Running an SEC Calibration Standard oocccoccccccnccccnconcnconnnnncnnnnnncnnnncnanononnnnns 259 SINGIS RUN ACUM e da e ao 260 Denning SEG BasSelNE sarna id idea 263 DENNING SEG Result Ran us dia 263 DETRAS SEC PEAKS save 263 Annotating SEG Ch
152. cation Instrument Acquisition Parameters 2 Simulation Resp time 0 50 v sec Communication 4 0 interface Range 1 000 AUFS Fig 220 E 3 ID Interface Manager 5000 Device 1 at COM3 Cycle time 0 50 v sec Apply SAN 1 Version 1 00 AutoZero EN Ap A ie O E ec For Help press Fl MIN at For Help press F1 MIN at Channel 1 2 The actual signal in the units which have been chosen in the detector configuration dialog at the actual wavelength in nm is displayed for each active channel Communication instrument This field displays the serial port of the detector which is used to control the instrument The active state of the communication with an instrument is represented by the green y symbol If the serial port was selected as lt None gt then no real control of the detector takes place Communication A D interface The active state of the communication with an A D interface is represented by the green y symbol Only in this case you will have access to the Device Information Click the button to display the instrument s connected to that COM port Information about the serial number device name and instrument software versions will appear Device Information Serial No i Software Version Manager 5000 Refresh Fig 221 Information about the connected instrument s Direct Control Acquisition Channels The number of channels that will be activated depends on how many
153. cccccccnconcncnonnnnnnononnconcncnnos 56 Configuration Virtual Detector ccooccccocccconnoconccconcconnocannonanconancnnnnonannnnannonons 57 Detector COMMECION S He 57 Configuration Assistant ASM2 1L rrrnnnornnnonnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnennnnnnnee 60 Configuration Autosampler ccoconcoccnccccnccocncconnonanccnoncnnnnocnnnnannonancnnnnonannnnannonons 63 Configuration Autosampler 3950 rrrarrrrarennnnevnnrevnnrnnnnrnranennnnennnnennnenn 64 4 CONTENTS Configuration Autosampler Knauer OptiMaS occocccccncocnccnncconccnnnnnnos 67 Configuration Autosampler 3800 ooccconcccoccccccnccccnconcncnnnononnnonononanononos 70 Configuration Autosampler 3900 ooccncocccccccccccnconcnconcncnnnononnnonnnconcnonons 71 Configuration Autosampler Triathlon Endurance ccocccccccccnicnncnn 72 Configuration Kontron Autosamplers o ooccccccnccccncoccnconnnonononncnconcnonons 76 Configuration Miscellaneous Instruments cccoocccocccocncccncccnnocncocncnononononanonanonanos 78 Configuration Switching Valves ooocccoccccocnccccnconcnonnnononnnnnnncnncnnnnos 78 Configuration Manager 5000 5050 lF2 occcocccccccccncocncccncocnccnnconnnonons 79 Configuration Column Oven 4050 Column Oven Jetstream 80 Configuration Flowmeter oocccooccccccnccoccnconcncconononnnoncnonncnnnnnnnnnnnnnnncnncnonons 81 Creating an I
154. check conditions General These selections will be valid for all active detectors or detector channels Set the time for which the baseline must meet the set criteria to pass the check The check and the method will be stopped if the system has not passed the check within the set time This time must be at least equal to the set pass time Noise Test Method The noise test will be performed either according to the root of the mean of squares rms method or to ASTM Select your choice For any available detector channel of the configured system a separate line will appear in the spreadsheet Here you can set the thresholds for the noise and drift for each channel separately Any test noise and drift must be enabled separately for any channel Creating an Instrument Control Method 185 Fig 199 137 To use this feature select the Perform Baseline Check option in the Single Run dialog box or include Baseline Check in the current Sequence line Single Run Acquisition Run information Calibrate Cancel Help lt M gt lt OD1 gt gt C EZChrom Elite Enterprise Projects D efault M Ic EZChrom Elite EnterprisesProjects Default D fr Data file lt 1D gt gt Number of reps 1 Amount values Sample ID Method Data path Print method report Sample amount 1 Internal standard amount 1 i FE Y h Ar 4 N Multiplication factors Begin run nme Oi Dilution f
155. chromatogram 274 SEC Option Horizontal Baseline Select this button if you wish the SEC baseline to be drawn as a horizontal line forward from the Baseline Start Time to the Stop Time The horizontal baseline stops when it crosses the data trace A threshold value in microvolt must be specified to ignore any detector baseline noise in the trace that would cause the horizontal projection to stop when it encounters the noise in the trace If the Horizontal Baseline reaches the end of the detected peak before the programmed Stop Time the range will stop where the Horizontal Baseline crosses the data trace Calibration Data Enter values for M and M in the fields provided or D1 and D2 then click the Calibrate button The system will calculate D1 and D2 or M and M values to be used for the calibration Reference Peak Time min Select this box if you are using a retention time reference peak to adjust for changes in flow rate All elution times volumes used in calculations will be offset by the time difference between the value entered here and the time of the reference peak in the sample Enter the expected retention time for the reference peak in minutes Reference Window If you are using a reference peak enter a value for the window for the reference peak A peak falling within this of the expected retention time for the reference peak will be considered the reference peak If two peaks fall within the window th
156. cling Fraction collector setup window This area is important as well for setting up the method as well for the instrument status during the run Here only the setup aspects shall be described If you have performed at least one run with the method the last taken chromatogram will be displayed Independent on this you can load any already stored chromatogram in this area to use it for the fractionation Select Open Data and then the desired chromatogram This will open the usual chromatogram view W Channel A Time 8 75913 Minutes Amplitude 0 00046 volts Opened chromatogram Going back to the setup window the chromatogram will there also appear Moving the cursor inside the graph area it will become a cross hair and additionally a vertical line appears Depending on the position the actual values Time Amplitude level and Slope will be displayed directly above the graph Time 8 48 min Amplitude 0 042 Slope 0 007 Opened chromatogram in FRC setup window Move the cursor to the desired position and double click with pressed Control lt Ctrl gt key In the fraction collection program a new line is 246 Fig 351 Fig 352 ChromGate Preparative Option created and the parameter window for this line is opened The time level and slope values are automatically inserted Complete the parameter settings as necessary close the parameter window Repeat this as often as dif
157. configured to control any user defined instrument necessary for programmed wave length control pump control switching valve control Please note that the Manager 5050 must be configured as a Manager 5000 Manager 5000 IF 2 10 Configuration LAO Name 101 Interface Manager 5000 Device number 1 ha Output 1 Cancel Help Configuration window for the Manager 5000 5050 IF2 I O Name Enter a descriptive name for I O of the manager This name will appear on the manager tab when the instrument setup or status windows are displayed Use a name which is unique within the instrument Interface Select the used interface type Manager 5000 IF2 Manager 5000 if you own a Manager 5050 Device number Select the device number The available choices depend on the global interface configuration as described on page 32 Output Select the channel of the interface in use Setup and Control of Knauer HPLC Systems ES It is not possible to use the cinch connector and the WAGO connector from the same channel for different instruments They are not independent of each other When complete click OK to exit the dialog and return to the instrument configuration icon list Configuration Column Oven 4050 Column Oven Jetstream Select the Smartline column oven 4050 or the Jetstream in the Configured Modules window double click the icon and complete the configuration dialog Column Oven 4050 Configu
158. creen The choices are Inject Vial and Labeled Markers must be programmed because they are not automatically generated as in the regular injection method Refer to the Triathlon Endurance User Manual for the cable connections SSV Port Program the solvent selection valve SSV port position range 1 to 6 Repeat The last x steps will be repeated y times Button functions Click the Add button to complete the operation Creating an Instrument Control Method 127 Fig 180 Fig 181 Click the Edit button to modify the selected step Click the Remove button to delete the selected step Click the Up button to move the selected step up Click the Down button to move the selected step down Click the Save button to save the user defined program or its part in to a file Save As File name tes Save as type User Defined Program Files udp ial Cancel Save steps Program comments Entire program Selected C Inrange 1 h Autosampler Triathlon saving user defined programs You can save Entire Program Selected steps and Steps in the specified Range Enter the name of the file and if wanted your comments to the program Click the Save button in the dialog to complete the operation Click the Open button to read the saved user defined program Look jr 1 Methods Jtestiudn File name test Files of type User Defined Program Files udp bl Cancel Open mode Program commen
159. ct Insert line from the menu The only difference is that negative times must be entered because they are relative to the zero injection time If a pretreatment is to define for more than one instrument pump and or valve drive in a method it is required to set for all of them the same earliest time All pretreatment procedures will start simultaneously If a pump should exactly switch off after 5 minutes of pretreatment include a line with 0 ml min flow into the pretreatment s pump program Otherwise the pump may run for a longer time as it need some seconds to load the new flow rate from the method s normal pump program An example for both A pump should run for exactly 5 minutes before the injection and a valve should switch 2 minutes before injection from position 2 to position 5 means 3 minutes after the pump s pretreatment is started Here are the setup tables create a time table for the valve which starts with 5 minutes Pump 5 04 min 1 ml min 0 04 min 1 ml min 0 02 min 0 ml min 0 00 min 0 ml min Creating an Instrument Control Method 133 Valve 5 04 min pos 2 2 00 min pos 5 0 00 min pos 5 Instrument Setup Column Oven 4050 MM Instrument Setup S s1000 SJ as 3960 E Oven S 4050 BJ 5 2600 Aux Traces 3x Trigger Temperature Wait Before Injection Time Temp Dig Outs Walt with Tolerance C 2 000 300 000 Wait Time min 10 00 300 ooo Cooler Temperature
160. ctions during a run Such injections can be programmed either for the autosampler 3950 or the injection module please refer the Instrument Setup for the autosampler 3950 for details of the stacked Injection setup of the autosampler Click on the gt gt button to open the Stacked Injections table dialog Stacked Injection Table Lancel Initial injection Injection module Stacked injection table Stacked injections time table Make a right mouse click into the time table and select Fill table from the corresponding menu Stacked Injections Humber of Injectors Injection volume ral Injection Flow mlrmin Injection Duratiori mi Time between Injections min Injection module Stacked injections table setup ChromGate Preparative Option 253 Fig 363 Number of Injections Enter the number of stacked injections If you e g enter 5 two stacked injections will be made in addition of the initial injection 6 injections at all will be made per run Please make sure that you have enough samples for the entered number of injection Injection Volume Enter the desired injection volume in ml Injection Flow Enter the flow rate of the injection pump during the injection If no injection is prepared the pump is stopped During a run the injection module s pump can only be controlled using the option Direct control during a run please refer the manual s appropriate chapter Runtime Sett
161. ctor If a Knauer detector is connected via serial or LAN port the autozero and the measurement signal will be transferred via this connection The pictures should only show the principle of connection 60 Setup and Control of Knauer HPLC Systems Configuration Assistant ASM2 1L Fig 86 The Azura assistant ASM2 1L is a modular instrument that allows combining up to 3 devices The following devices can be included pumps P2 1S P4 1S detector UVD2 1S Knauer valve drives with 2 6 12 and 16 positions Valco valve drives with 2 6 8 10 12 and 16 positions Configuration rules Two pumps are only supported as HPG both pumps must have the same pump head three pumps are not supported Only one UV detector is allowed One valve drive can be used as a fraction collector if the Knauer Fraction Collector Control option is installed and the appropriate license option is used Cascading fraction valves is not supported This is also applicable if the valves are installed in different ASM2 1L housings All the devices will be controlled by only one LAN port a serial control via RS 232 is not implemented Pumps cannot run in a HPG if they are installed in different ASM2 1L housings Assistant ASM2 1L Configuration General Left Device Middle Device Right Device Assistant Name las M2 1L Serial Number Interface I IF Port 11 0007 W Use S N to identify the instrument
162. ctual fraction is displayed Fr Volume ml The collected volume of the actual fraction is displayed Direct Control Operation The operation which shall be performed can be selected from the pull down menu The choices are waste peak recycling or solvent recycling It will be executed by clicking on the Set button Collect to The operation fraction collection can be initiated here The destination vial is either defined as next or in case of collect to by its number The collection starts by clicking on the Go button Run Time Settings All operations started in the direct control mode are performed unconditioned as timed events For consecutive conditioned runs the values for level slope and the expected interval will be stored according the settings However in any case it is recommended to check the automatically stored fractionation program before using it Rack View The displayed rack corresponds to the configured one All already used vials will appear green highlighted Moving the cursor across the rack will change the upper descriptive line There the vial number the begin and end of collection and the collected volume is displayed see the arrows in Fig 354 Multi valve fraction collector For this fraction collector the rack view is integrated into the instrument status tab The shape of the rack view again depends on the configured valves If different valves 6 12 or 16 positions are combined the graphical se
163. d Source This column displays the source of the spectrum Current Data refers to working spectra extracted from the current 3D data When spectra come from a stored data file this column will display the source filename Set Reference Pressing this button will designate the spectrum in the currently highlighted row of the table as the new reference spectrum The similarities for all of the rows will be recalculated based on the new reference spectrum Print Pressing this button will output a simple text report representation of the table to the default printer Properties Pressing this button will display a dialog with parameters related to the calculation of similarity 212 ChromGate PDA Option Similarity Table Properties Fig 310 187 Enter the wavelength range and data point spacing in nm for similarity calculations Similarity Table Properti Xx Wavelength range od 400 nm Wavelength step 1 nm Cancel Help During analysis it is possible that a portion of this wavelength range will be outside the range of the acquired data In that case the wavelength range will be truncated to the limits of the acquired data Spectral Library Definition Fig 311 This window appears when you select the File Spectral Library New command and is used to define a spectral library to use with the PDA option In order to define a library you must have the spectra to be added saved on disk as spec
164. d Pressing gt gt button during a run will open the known Stacked Injection setup with the default values For the re programming of the Stacked injection table please refer the instrument setup chapter of the autosampler 3950 If you click on the Apply button the new stacked injection table will be executed immediately the program will not check when the last injection was made If the Direct Control during a run option Save changes in time table is enabled all program lines for stacked injections which have not been executed yet during the current run will be replaced by the stacked injections from the direct control whereas lines for stacked injections which have already been executing during the current run remain untouched If the Direct control during a run option Save changes in time table is not active the new table will be executed but not stored in the instrument setup However in this case the method does not include the correct injection program Creating an Instrument Control Method 165 gt Pressing the gt button opens a window that allows you for perform several service operations Tray position You can move the tray to several positions The choices are Tray Home Needle exchange position and Tray Front Syringe position The syringe can be moved to Home End or Exchange position Valve position The valve can be switch to Inject and Load position Instrument Status Triathlon Endurance A
165. d Pump User Defined Detector manual Injection Valve User defined Detector Analog Out Autozero AN IN AN OUT DIG IN RELAY e DEE M 5000 MADE IN GERMANY ss Analog In User defined Pump Fig 389 1 serial NULL modem cable A0895 2 Detector signal cable CINCH CINCH A0868 CINCH wire ends G1021 3 Trigger Injection Valve K 2600 Contact 1 gt START IN Contact 2 gt GROUND 4 Autozero cable 5 Pump control cable 285 286 Typical Wiring Schemes Pump K 1001 manual Injection Valve Detector K 2600 v gaa PENE HF ge 4 e ip r a e m aii ER Fig 390 1 serial Y cable A0755 2 serial one to one cable A0884 3 Trigger Injection Valve K 2600 Contact 1 gt START IN Contact 2 gt GROUND Typical Wiring Schemes 2 Pumps K 1001 Autosampler Triathlon Detector K 2501 Fluorescence Detector RF 10AxI Fig 391 A OO N gt AAANIINOLI VMS 1 1 Autosampler iii Triathlon 3 T HELE ES LL ULLI TRAV la 13 k or i F TE A serial Y cable serial one to one cable serial NULL modem cable Injection Marker Cable A K 1001A TB 1 287 LA am m mm I PC RS232 ports A0755 A0884 A0895 0830 811 288 Typical Wiring Schemes Pump K 501 Autosampler 3800 Detector K 2301 r re HN HND A Q11 9 im sm mat a
166. d inaccessible depending on the selected number of positions of the used valve and the Solvent Peak Recycling setup If for another valve in the assistant the option Use as a Fraction Collector is already selected for the next valve an error message The fraction collection option is already selected for another valve will be shown Valco Valve Setup and Control of Knauer HPLC Systems 63 2 If selected you can choose the device name please refer to the device name section below and the number of position Please be sure to select the correct number of position if you select it manually 2 6 8 10 12 or 16 position can be selected For one valve in the assistant you can check the Use as a Fraction Collector box If selected there are additional options You can enter the Vial Volume please be sure to enter the correct volume and setup Tubing and Solvent Peak Recycling Please refer to the chapter Multi Valve Fraction Collector Configuration for detailed information The Number of Vials is fixed and inaccessible depending on the selected number of positions of the used valve and the Solvent Peak Recycling setup If for another valve in the assistant the option Use as a Fraction Collector is already selected for the next valve an error message The fraction collection option is already selected for another valve will be shown Device Name The default name corresponds to the selected device type All de
167. d is so low that the solvent spectrum interferes Background compensation Checking this box will cause spectra to be corrected for background using the peak baseline prior to being used in the calculation of purity Similarity View Properties To set up the properties for the Similarity View do a right mouse click and select Properties ChromGate amp PDA Option 207 Fig 303 Similarity Yiew Properties x General e Overide method settings Display information Wavelength range 200 400 nm Total purity 3 point purit Wavelength step fi nm 13 point purity Purity threshold 0 590000 Absorbance threshold 5 QOOOU0 Background compensation Cancel Apply Help By default the Similarity View will use the current method settings To override the method settings click the Override method settings box then enter the values you wish to use Wavelength range Specify the wavelength range over which the purity calculations will be performed for example from 200 nm to 400 nm Wavelength Step Specify the wavelength spacing in nm to be used when purity calculations are performed Purity threshold The comparison of two spectra gives a Similarity Index SI the closer to 1 00 number is the more similar the spectra are The purity threshold is used to eliminate spectra that do not match If the SI is greater than the purity threshold the spectra will be considered
168. d or box must be used Name Enter a descriptive name for the detector This name will appear on the detector tab when the instrument setup or status windows are displayed The default detector name will be suggested automatically by selecting the detector type The name must be unique within the instrument Type Select the type of your detector Serial Port Select the number for the communication port on your PC where the instrument is connected The selection lt none gt is for simulations No real control of the instrument will be performed Serial Number Type in the serial number of the detector you are using Flow Cell Enter a description for the flow cell you are using The description will be printed out when you print the instrument configuration Data acquisition interface The data acquisition is performed as it was described for the user defined detectors via the interface box or the Manager 5000 5050 Select the connected interface from the pull down menu Most of the Kontron detectors do not support data acquisition via serial port therefore the A D converter is required Channel 1 Input Select the connected data channel of your interface 56 Setup and Control of Knauer HPLC Systems Channel 2 Input Select the second connected data channel of your interface It can be deactivated by the selection lt none gt The Kontron x32 detectors are automatically set to lt none gt Y Axis Units Select the typ
169. detector is connected The available choices depend on the global interface configuration as described on page 32 Select the acquisition Input of the interface device It is not possible to use a channel twice in a system A data acquisition channel cannot be used for control commands such as wavelength changes Click the Add Info button to enter additional information Y Axis Units Fill in the units of your signal to be displayed on the y axis e g microvolt AU MPa etc depending on the detector type An interface device acquires your analog signal and stores it in microvolt If you want to store and to display the signal in different units you must use the correct multiplier Y Axis Multiplier ChromGate by default stores a signal in microvolt If you wish to display another unit enter the conversion factor to be applied The following table shows commonly used y axis labels and their corresponding multipliers Y Axis Label Y Axis Multiplier Volts 0 000001 Millivolt 0 001 Microvolt 1 Other Enter a number such that when multiplied by microvolt gives you the desired units For example if you want to label the units and each unit corresponds to 5 microvolt of signal enter 1 5 0 2 Please also note that the Multiplier also will be used for the Y Axis Offset Y Axis Offset Type in the correction offset which will be used for the signal recalculation The Y Axis offset must be given in the same units as the
170. dicated as Intensity without access for changing it This change you can perform in the configuration window only Flow Cell Enter a description for the flow cell you are using The description will be printed out when you print the instrument configuration Channels Up to four channels can be activated According to the selected number the fields Chn Name 1 4 become accessible for editing Setup and Control of Knauer HPLC Systems 57 Halogen Lamp Check this box when the detector is equipped with a halogen lamp Configuration Virtual Detector Fig 81 ae To configure a virtual detector you have only to open and to close the configuration window without any additional entering The Y axis multiplier is only to change if very weak signals are to simulate Virtual Detector Configuration Detector name virtual Detector T Ass unite Ae multiplier 1 Cancel Help Virtual detector configuration window To avoid possible signal distortion the Y Axis multiplier in the configuration of the virtual detector must be equal to that of the selected data file Detector Connections Up to four detectors can be connected to the Interface Box or Manager 5000 5050 1F2 The detector connections are principally identical The following description for the Interface Box can also be used for the Manager 5000 5050 IF2 only the connector positions may differ Always use a shielded twisted pair cable
171. dow The configuration windows for the other Knauer pumps are nearly identical 40 Fig 48 Fig 49 Fig 50 Setup and Control of Knauer HPLC Systems Name The name of the selected pump appears automatically You can enter any other descriptive name for the pump This name will appear on the pump tab when the instrument setup or status windows are displayed Use a name which is unique within the instrument configuration E g for a high pressure gradient system all pumps must have different names Pump Type accessible for Pump K 120 501 only Select the type of pump you want to configure from the drop down list The choices are K 120 and K 501 Pump Type KS01 7 selection option for K 120 K 501 pumps Name Type accessible for Pumps 10P 20P P2 1S P4 1S only Select the type of pump you want to configure from the drop down list The choices are 10P 20P P2 1S and P4 1S The 10P P2 1S is the device without the pressure sensor As already described in the Name section above you must use different names for all of the devices in an instrument configuration Seral Number Interface selection option for pumps 10P 20P P2 1S and P4 1S Gradient Mode Gradient Mode lsocrab lsocratic Interface Head selection options for gradient modes Select the desired gradient mode from the drop down list Please note that for pumps K 1000 1 S 1000 S 1050 preparative pump 1800 K 1800 and
172. e click on the l button to start the search for all connected LAN devices of the currently configured type The option Use S N to identify the instrument must be enabled Select the desired device from the shown list The software will read out the information as serial number and will fill it automatically into the configuration screen To search for an instrument the device must be connected switched on and must have a valid IP address As an option you can enter the IP address of your device if the option Use S N to identify the instrument is disabled Please note that in case the device receives the IP address from a DHCP server e g a router the IP address may change if the device is switched off and switched on LAN Foxy FC R1 R2 There is no option for the Foxy fraction collectors to search via LAN or read out the configuration If selected please enter the IP address of the FC The IP address must match with the IP address in the FC the Foxy FC R1 R2 do not support LAN with DHCP The LAN communication is supported in firmware version 1 01 03 and higher Please note that the touch pad of the Foxy R1 R2 FC will NOT been BLOCKED if the device will be controlled by software The software cannot recognize inputs via the touch pad Therefore it is highly recommended not to use the touch pad under software control Otherwise the vial position of fractions and other information may not been stored correctly in the Dev
173. e that the correct lamp type is selected Leak Sensor Sensitivity UVD2 1L only Select the leak sensor s sensitivity in accordance with your requirements The choices are Off Low Medium and High Shutter Control DAD S 2600 only For a shutter equipped detector check this box if you want dark current correction to be carried out If this option is chosen the detector will close a shutter before every run measure a dark current and then subtract it from all measured spectra Additionally the shutter will prevent the fiber optics for going blind Port Settings Spectrofluorometric detector RF 10Axl RF 20A Axs only Port settings Baud Rate 9600 v Data Bits 8 v Parity Even parity w Stop Bits 1 r Port setting dialog box Select the settings from the pull down menus as shown in Fig 76 Baud Rate 9600 Data Bits 8 Parity Even parity or No parity Stop Bits 1 27 The default value for parity in ChromGate is Even For RF 20A Axs the parity default setting in the device currently is No Please check in the device for the correct setting Refer to the description below how to do this ae A proper connection normally is only possible via onboard COM ports The port settings must coincide with the RS 232 parameters on the detector To control the detector via RS 232 the detector must be set into serial mode as described below also refer the detector s manual 92 Setup and Control of Knaue
174. e which starts at the moment of injection Click the Timed events button to set up the time base methods The time base methods set up dialog will appear Timebase Methods Programmed methods Method type ALIX 2 H Method AL time New 1 AUX ON 0 00 01 lime fo h fi min f30 sec 2 AUX 2 ON 0 00 01 Delete 3 AUM 1 OFF 0 01 00 Switch ON AUM 2 OFF Switch OFF Stop events at time lo h o min 0 sec OF Cancel Autosampler timed events Click the New button to add one more lines to the programmed methods Method type Select one of the available AUX 1 and AUX 2 Every auxiliary AUX 1 through AUX 2 can be set to ON contact closure four times and four times to OFF at a programmed Time It is not possible to program the same auxiliary to ON and OFF at the same time The maximal programmable time for the time base methods is 9 h 59 min 59 sec Click the Commit button to actualize your selection Click the Delete button to remove the selected line from the programmed methods Creating an Instrument Control Method 115 The Stop Events at Time allows setting the end time of the timed events If the Stop Events at Time box is not checked the end time is equal to the analysis time the run time The maximal Stop events time is 9 h 59 min 59 sec If the END time exceeds the programmed run time this END time overrules the analysis time for the autosampler The next injection will be started as soon as the ti
175. e InstallShield Wizard has successfully installed Knauer HPLC System Click Finish to exit the wizard Cancel Fig 4 Click on Finish to finalize the ChromGate installation Installation Guide ChromGate 3 3 2 11 KNAUER FRC control option If you own a fraction collector supported by ChromGate you can install the KNAUER fraction collectors AddOn KNAUER FRC control option This option can only be installed if ChromGate v 3 3 2 is installed To control a fraction collector a KNAUER FRC option license must also be subsequently installed Click on Install KNAUER FRC option to start the installation of the KNAUER FRC control option I KMALIERA ChromGate gt Install ElChrom Elite 3 3 7 Software Read release moles gt Install Service Pack 1 for EZChrom Elite 3 3 2 gt Install ChremGate Software Krad ne note gt Install KNAUER FRE Option gt Install the Microsoft HET 3 0 if required gt Online Manual EZChrem Elite Fog Adobe 2Ar r lboa LE Resd r gt Online Manual ChromGate E squires AdabetAcrabatt Repder gt Visit the KMAUER Website gt Browse the CO gt Exit Fig 5 In the next window the fraction collectors that should be installed can be selected As default all supported fraction collectors will be selected Knauer Fraction Collectors AddOn InstallShield Wizard Select Components Select the components setup will install Select the components
176. e Shutter Start 200 End 400 Bandwidth 1 Mode Absorpti art nd andy ode Absorption EE Signal m l Dark Signal 1 Reset Dark Wavelength nm Diagnostics window of Diode Array Detectors Integration Time Range 12 1000 msec The integration time is the time interval when the detector accumulates light to receive one spectrum This value is a measure of the intensity of light originating from a lamp The more light that passes the optical scheme and the cell the shorter the integration time will be The integration time depends also on the specific spectral interval Scan selected for the measurement since the maximum of intensity may be varied The integration time range is defined by the device parameters see K 2700 2800 device specific data Click the 2 button to determine the optimal integration time You can use this measurement to check quality of the lamp and used optical elements If the software cannot find an optimum integration time the lowest time of the range i e 12 will be shown The integration time will be checked before every run If there not enough light can be detected a single run will be aborted with an appropriate error message In a sequence run the sequence will not be aborted and the data will be acquired but ChromGate will enter a message into the instrument activity log that there was not enough light Lamp Select the lamp you want to check This option
177. e amount of flush volume Headspace Pressure Check the Headspace Pressure box to enable the use of headspace pressure in combination with the syringe to transport sample to the loop The compressor will always be used during a wash procedure IS The accuracy and reproducibility of the autosampler may decrease if headspace pressure is switched off Needle Wash Check the Needle Wash box to enable a wash procedure Flush Volume Enter the amount of sample in ul taken from a vial to preflush the needle before the loop is filled with the sample The default value is 30 ul for an installed tube volume of 15 ul Creating an Instrument Control Method 123 ae Flush volumes smaller than twice the volume of the needle and tubing will result in a decrease of performance of the autosampler Wash Volume Enter the volume of wash solvent in ul to be used The minimum programmable volume is 300 ul Needle Height Enter the distance between the needlepoint and the plate holder in mm default value is 5 mm Temperature control If your Triathlon is equipped with a tray cooling facility this option can be checked and the desired temperature can be entered Alarm Buzzer Check the alarm buzzer box to turn ON the alarm beep given when an error occurs during a run Run Screens Check the run screens box to display the actual run information during a run Timed events These type of events or methods enable you to control optional ISS
178. e button adjacent to the field Data Path Enter a path name where the data acquired for this run will be stored Click the File button to select a path from a list of those on your disk Data File Enter a file name to be used to save the data on disk You can select a path by clicking the File button adjacent to the field It is not possible to use an existing file name unless the file exists in located in a directory whose path contains the term public For example if you data files are saved in a directory entitled C Public Data the ChromGate Client Server files saved in this directory can be overwritten Print Method Report When this box is checked the method report or reports will be printed at the end of the run 262 SEC Option Amount Values In this section you can enter values that affect how the concentrations are calculated If you are making a single data acquisition prior to calibrating your method simply leave these values at the default level Sample Amount The Sample Amount value is used as a divisor during calculation of concentrations lt is intended to compensate for differences between samples due to weighing and when percentages of the total sample are being calculated rather than the amount detected in an injection Internal Standard Amount For calibration runs the Internal Standard Amount is taken from the method Peak Table For unknown runs enter the amount of the Internal S
179. e device must be connected switched on and must have a valid IP address As an option you can enter the IP address of your device if the option Use S N to identify the instrument is disabled Please note that in case the device receives the IP address from a DHCP server e g a router the IP address may change if the device is switched off and switched on When complete click OK to exit the dialog and return to the instrument configuration icon list Configuration Flowmeter Select the flowmeter in the Configured Modules window double click the icon and complete the configuration dialog GJE Flowmeter Configuration Senal Part Mame Knauer Flowmeter Ture Unite Serial Fart COM 1 we Y Ass Units ml min Seral Number E Cancel Help Fig 116 Configuration window for the Knauer and GJC flowmeter Name Enter a descriptive name for the flowmeter This name will appear on the flowmeter tab when the instrument setup or status windows are displayed Use a name which is unique within the instrument Serial Number Type in the serial number of the flowmeter you are using Serial Port Select the number for the communication port on your PC to which the flowmeter is connected Y Axis Units The y axis units are given as ml min When complete click OK to exit the dialog and return to the instrument configuration icon list 82 Setup and Control of Knauer HPLC Syst
180. e displayed above the curve A value of 1 00 indicates perfect fit Select the Time or Volume button at the bottom of the calibration curve to display the x axis of the curve as either retention time in minutes or retention volume If you select volume enter the flow rate in ml min Remove calibration curve outliers To temporarily remove an outlier from the calibration curve click on the point with your mouse It will change color and the curve will be re calculated with the point removed Time Volume Select the mode of viewing for your calibration curve Time or Volume If you select Volume you must enter the flow rate so that the curve is displayed correctly Print Click this button to print your current SEC method information Export settings Click this button to select export options and path When you have completed the SEC Setup you are ready to acquire and process your SEC runs You can acquire data either manually single injection or by batch acquisition auto injector See the ChromGate User s Guide for information on creating a batch sequence SEC Option 273 SEC Setup Broad Range1 Calibration Fig 381 When you select Broad Rangel as the calibration method the SEC Setup will appear as shown below E SEC Setup A E Intrinsic viscosity y b Calibration Type Broad Range 1 s Time Slice Sec fi Ranges in Minutes 500000 500000 Baseine ResukRange a 400000 400000 200000 200000
181. e drop down menu The menu shows a list of possible valve positions For Knauer two position valves the positions are I injection and L load for Valco two position valves A and B and for multi position valves 1 16 depending on the valves configuration That means for a 6 position valves the available positions are 1 6 for a 16 position valve 1 16 GLP The GLP section of the ASM 2 1L status window allows for reading out the GLP data of all modules of the ASM 2 1L GLP Information UY a Lamp operation time 314 5 hours Light intensity I Ref at UV mas 477943 hi Fig 237 ASM2 1L status tab GLP UV Click on the lt Refresh gt button to read out the GLP information of the built in modules You can use the scroll bar to scroll through the GLP data The following GLP information will be shown Instrument data Serial number Firmware version Instrument installation date Last service date Last service code Device working time in hours Pumps Pump head Pump operation time UV Lamp operation time in hours Number of lamp ignitions Optical bandwidth in nm Light intensity Sig at UV max Light intensity Ref at UV max Valve s for Knauer and Valco valves Valve type number of ports and positions Switching cycles System The System section of the ASM 2 1L status window allows for changing the Events of the ASM 2 1L and the access for the UV Diagnostics if an UV detector is configur
182. e established To search for an instrument the device must be connected switched on and must have a valid IP address As an option you can enter the IP address of your device if the option Use S N to identify the instrument is disabled Please note that in case the device receives the IP address from a DHCP server e g a router the IP address may change if the device is switched off and switched on Channels available not for all detectors For multi wavelength detectors more than one channel can be activated According to the selected number the channel fields become accessible for editing The channel name will be shown later in the method window and in the result reports Only channels which have been activated in the detector configuration will be available in the method setup later for data acquisition Serial Number If you have read out the device via LAN the serial number has been added automatically Otherwise type in the serial number of the detector you are using The serial number of network controlled detectors is used for the device identification and addressing during communication This serial number must match the serial number of the defined pump If not you will get an appropriate error message if you later try to start in run Y Axis Units Select the type of units to be displayed on the y axis of your chromatogram from the drop down list For the UV and diode array detectors the choices are AU Absorpti
183. e is complete close Peak Table dialog box by clicking on the x in the upper right corner Save the method Select Analysis Analysis Single Level Calibration from the menu bar to present the following dialog box Single Level Calibration information Calibrate ID PNA STDS p C EZChrom Elite Enterprise Projects D efault M Cancel usle san Help I Print method report Sr td tk ste amount standard amount 1 Multiplication factors 1 1 1 Dilution factors 1 1 1 Fig 17 Analysis Single Level Calibration Window 13 14 15 16 17 Enter the sample identification used for the standard Enter the method that you want to calibrate and the data path name for the data file Enter the name of the calibration data file Click on the Calibration check box and enter a 1 for Calibration level If the method is presently not calibrated it is not necessary to check any of the boxes but if you want to be certain select the Clear all calibration check box Repeat the steps 12 to 16 for all levels you want to calibrate Enter the appropriate calibration level into the Calibration level field Short guide ChromGate KNAUER Instrument Control 23 18 Click Start The peaks will be integrated and calibration curves will be generated The method is now calibrated and can be used to analyze samples Creating a Sample Sequence Fig 18 If you are using an autosampler the
184. e of units to be displayed on the y axis of your chromatogram from the drop down list The choices are AU Absorption Units mAU 0 001 AU or HAU 0 000001 AU and ADC counts Configuration Kontron Diode Array Detectors Fig 80 Kontron DAD 540 545 Configuration Interface LAN y g DAD 540 si Name Serial Number Channels f V Use S N to identify the instrument Options Chn Name 1 Channel 1 Ta M Halogen Lamp Chn Name 2 i E Chn Name 3 Y Ass Units m l y Chn Name 4 Flow Ce Cancel Help Kontron DAD configuration window The Kontron PDA detectors are controlled similar to the Knauer detectors via the configured RS232 port Name Enter a descriptive name for the detector This name will appear on the detector tab when the instrument setup or status windows are displayed The name must be unique within the instrument Serial Number Type in the serial number of the detector you are using Interface For the diode array detectors the choice is PCI and LAN If you control the DAD via the LAN you must enter the IP Address The access to the IP Address field is blocked if PCI was selected Y Axis Units Select the type of units to be displayed on the y axis of your chromatogram from the drop down list The choices are AU Absorption Units mAU 0 001 AU or HAU 0 000001 AU and ADC counts Selecting the ADC counts the signal mode in the Instrument setup page 102 will be in
185. e of viewing for your calibration curve Time or Volume If you select Volume you must enter the flow rate so that the curve is displayed correctly Print Click this button to print your current SEC method information 270 Export settings SEC Option Click this button to select export options and path Zooming on the Calibration curve You can zoom on the SEC Calibration curve by clicking and dragging with the mouse Double click the mouse in the graph area to unzoom the curve view SEC Setup Universal Calibration If you select Universal for the Calibration Type the following screen will appear mi SEC Setup V Enable GPC SEC Analysis Bil ES Average Time Std Mark Std Mark 12 42 Std Mark Houywi 15 35 Std Mark Houywi 16 33 Std Mark Houwi 18 A15 Std Mark Hru 1wwi 500000 220000 160000 ASAAA Calibration Type HEARE Time Slice Sec fi Ranges in Minutes Baseine Result Range 2 start sop sart stop H E Horizontal Baseline Threshold Sample Data Intrinsic Viscosity K 0003 Y Reference Peak Time min 35 Reference Window fi 0 Fig 380 Enable Analysis Click this box to enable SEC calculations for the current channel de Fit Type Cubic v R 2 0 999895 100000 Molecular Weight 20 25 h nutes Flow Rate ml min Export Settings 15 Time Volume Print Each channel can have its own analysis conditions Molecu
186. e peak closest to the center will be chosen as the retention time reference Remove calibration curve outliers To temporarily remove an outlier from the calibration curve click on the point with your mouse It will change color and the curve will be re calculated with the point removed Time Volume Select the mode of viewing for your calibration curve Time or Volume If you select Volume you must enter the flow rate so that the curve is displayed correctly Print Click this button to print your current SEC method information Export settings Click this button to select export options and path SEC Option 275 SEC Setup Broad Range 2 Calibration Fig 382 If you select Broad Range 2 for the Calibration Type the following screen will appear i SEC Setup M E MV Enable GPC SEC Analysis Average Time Std Mark Std Mark 12 42 Std Mark Houwi 15 35 Std Mark Houwi 16 33 Std Mark Houwi 1A R15 Std Mark Hru vi of gt Calibration Type Broad Range 2 y Fit Type Cubic y R 2 0 999895 Time Slice Sec 1 fi Time 10 9336 Minutes Molecular Weight Ranges in Minutes 500000 500000 TJ Baseine Resurange ee 400000 start sop sort stop H uo 200000 T 200000 200000 y gt Horizontal Baseline Threshold Calibration Data Molecular Weight Mn 108563 Muy 127628 K 10 627765 A 10 039318 h nutes IV Reference Peak Time min 35 Time Volume Flow Rate ml
187. e spectra relative to the apex Since spectra taken at different points along a pure peak will look identical they will have high similarity indices The closer the Similarity Index is to 1 0000 the more similar or more pure the peak is deemed to be While it is easy to decide that a peak with a very low Purity value 0 0000 0 8900 is indeed not pure it is more difficult to decide whether peaks with purity values between 0 9000 and 0 9500 are actually contaminated The user s judgment is required to make this determination and may necessitate using other methods to determine purity such as overlaying the spectra or comparing first and second derivatives Spectrum Export The format for spectrum export is as follows Export Type Spectrum Version 2 Sample ID pna_6 1 2 Data File D DatalPNA Level 2 vp dat Method D Methods test met User Name Tontala Spectrum Type Peak Apex Operations Applied Smooth X Axis Title nm Y Axis Title mAU X Axis Multiplier 1 0 Y Axis Multiplier 1 0 Number of Spectra 23 Peak Ret Time 13 440 Number of Points 201 200 193 201 30 202 7 229 PDA Data Export To export the PDA data at first a multichromatogram must be enabled in the PDA options Then elect the new multichromatogram in the channel selector and open Method Advanced In the Data Export tab enable the export and select PDA Data from the drop down menu Add ASCII 3D Data to the export parameters Select the Field sepa
188. e time represented by the second inflection point Library Search Calculations General During analysis if one or more search libraries are defined on the Library tab of PDA Options then an automated library search is performed on every integrated peak of every PDA analysis channel 277 Unless a Library Search Results object is part of the method custom report no automated library searching will done when analysis is performed In this section a Query Spectrum is defined the unknown spectrum that is being searched A Reference Spectrum is defined as a spectrum from a spectrum library file During a search the apex spectrum of the peak the query spectrum is compared to each spectrum contained in the libraries reference spectra to determine the similarity of the query spectrum to the reference spectrum The similarity is quantified through the calculation of a Similarity Index for each query reference pair The Similarity Indices are used to generate a hit list of the best matching entries A perfect match will have a Similarity Index of 1 0000 Similarity indices less than 1 indicate differences in the spectral patterns If the query spectrum and the reference spectrum have different wavelength ranges then the intersection of the two ranges is used in the similarity calculation If the query spectrum and the reference spectrum have different wavelength steps then the higher resolution spectrum is de resolved to match the othe
189. e viscosity of the samples the choices are Low Normal and High The speed of the syringe during washing or rinsing procedures is not affected by this setting Flush Volume Enter the amount of sample in ul taken from a vial to preflush the needle before the loop is filled with the sample The default value is 30 ul for a tube volume of 15ul The preflush will only be done for the injection modes Full loop and Partial loop fill In the flushed loop mode this option is deactivated ee Flush volumes lower than twice the volume of the needle and tubing will result in a decrease of performance of the autosampler Wash times Select the Wash times in number of syringe volumes The valid interval is between 1 and 9 Needle height It defines the penetration depth of the needle or the distance between the vial bottom and the needle tip The valid settings are between 2 and 6 mm with the step 1 mm Air Segment Check the Air Segment box to enable the use of an air segment between the sample and the wash solvent to reduce the amount of flush volume 118 Creating an Instrument Control Method Headspace Pressure Check the Headspace Pressure box to enable the use of headspace pressure in combination with the syringe to transport sample to the loop The compressor will always be used during a wash procedure The accuracy and reproducibility of the autosampler may decrease if headspace pressure is switched off Needle Wash Check the N
190. ecify the nm range to be averaged in generating the analogue signal for each channel The multi chromatogram data is an average of the absorbencies monitored at each wavelength in the wavelength range The wavelength range is equal to the selected wavelength one half of the bandwidth For example given a wavelength of 600 nm and a bandwidth of 4 nm for each sample time the data points from the chromatograms at 598 599 600 601 and 602 nm are added and the sum divided by 5 If in the example above the detector s range is 190 600 nm inclusive the software only averages the points across chromatograms at 598 599 and 600 nm dividing by 3 The range would not be exceeded PDA Options Ratio The X Y Ratio tab is used to set the displays of the Ratio Plot View and the Max Plot The Ratio View displays two PDA wavelength channels and the ratio of those two channels These may be viewed during real time acquisition as well as during post run analysis The flat tops on the ratio peaks are a preliminary indication of peak purity 4 PDA Options E Ek EN Library Purity 4 Spectrum Ta Muli Chtomatogram Ratio Ratio plot Wavelength Bandwidth Channel 1 260 nm E nm Channel 2 280 nen le nm Threshold 11 m l Fig 287 Ratio plot These controls specify the wavelength of the ratio multi chromatogram channels The extracted chromatograms will be centered about the specified wavelength Channel 1 Channel 2 W
191. ect the action waste After closing the window the parameter will be designed as L The red displayed warning message for possible event overlapping must not be a reason to change the event programming but at least to check it It may happen that one event cannot become active In the shown case Fig 333 and Fig 334 the time difference between both events is 0 5 min however both allow a time interval of also 0 5 min leading to this possible overlapping In case of peak recognition mode and event signal level the parameter window only enables you to enter the threshold values for the peak at beginning and end as well as to check the solvent recycling option No further settings are possible The action is set to collect to next After closing the window the parameter will be designed as L without leading sign FC Parameters at 10 00 min Signal g i Solvent recycling Start if signal level i lower than E Action Peak Beg Slope Peak End Slope Action Flease notice that the parameters will be used in the Units selected for the Detection Channel Cancel Fig 335 FC Parameters Signal Level Peak Recognition Signal Slope When using the slope instead of the level the settings are made similarly The only difference is that the slope values defined as signal difference per 1 sec are to be used as thresholds After closing the window the parameter will be designed as S S or S Signal Level Slope The settings usi
192. ed System E vents 000 El Apply UY Diagnostics gt K Fig 238 ASM2 1L status tab System with UV Diagnostics Creating an Instrument Control Method 161 Events Status The three digits show how the events have been set in the Events Digital Output Control menu This may not the current status of the Events if this settings have not been send by clicking the lt Apply gt button The current status of the events will be represented by the LED symbols on the top of the ASM 2 1L status window Click on the l button to setup the events digital outputs Refer to the chapter Instrument Setup Pumps Pump section Configuring Digital Outputs for details Click on the lt Apply gt button to send the settings to the device Pump Purge gt Click on the l button to open the purge setup window Please refer to the chapter Instrument Status Pumps for more information UV Diagnostics gt Click on the l button to open the UV diagnostics window Please refer to the chapter Instrument Status Detectors for more information Instrument Status Autosampler Only one autosampler can be included in any system therefore all supported samplers will be explained separately without cross references Instrument Status Autosampler 3800 Smartline Autosampler 3800 and K 3800 Basic Marathon MM instrument Status oO System Status e 5100 E s 20 JetStream Oven 3 K 2600 Monitor Direct Control
193. ed to the oven by clicking the Set button Cooler Temperature C S 4050 only The oven temperature can be set The entered value will be transferred to the oven by clicking the Set button Leakage Alarm Level S 4050 only The leakage alarm level can be set The entered value will be transferred to the oven by clicking the Set button Digital Outs Events S 4050 only Click on the l button to set up the digital outputs Refer to the chapter described in the chapter Instrument Setup Pumps section Configuring Digital Outputs for details Click the DO Apply button to accept the settings Set Temperature Control S 4050 only Press the Off button to switch the temperature control off It will be switched on again by setting the desired temperature and pushing the Set button GLP S 4050 only The minimum and maximum reached temperatures the total working time and the working time above 150 C will be monitored The read out will be actualized by clicking on the Refresh button Instrument Status Flowmeter A Knauer Flowmeter EP System Status r S 1000 44 AS 3800 El JetStream Oven mf 10 Monitor Run Status HOLD Flow ml min 0 000 Q Serial Port COM 5 Lo SH 58637 Fig 248 Status tab Knauer Flowmeter Monitor Run Status The status HOLD will be displayed when no program is running otherwise the Run Status displays a run time in minutes elapsed from the start of the run F
194. ed in these fields PDA limits its search to library spectra obtained from peaks whose apex is within the specified retention time range This pre filter is optional and may be left blank Lambda max When one or more of these values is specified library search will be restricted to those library entries containing lambda max value s within 5 nm of all of the specified values Entries without matching lambda max ChromGate amp PDA Option 191 value s are automatically excluded from the search no similarity calculation is made Entering values for lambda max is optional Compound name filter If you are searching only for spectra that contain a certain name enter the name here Only spectra containing that name will be searched PDA Options Purity The Purity tab is used to set the parameters necessary to perform on demand peak purity calculations and peak purity calculations that occur as part of analysis PDA Options HER 3j Library EE Purity 4 Spectrum keg Multi Chromatagram Ratio Purity calculations p Perpeak spectrum calculations Wavelength range Ecos 400 am Mi Ip Wavelength step G F nm 13 point puny Purity threshold 0 990000 Absorbance E threshold 500 ES T Background compensation Fig 284 Purity Calculations Wavelength range Specify the wavelength range over which the purity calculations will be performed for example from 200 nm to 400 nm Wavel
195. ed result range Molecular Mass calculations will be made for each range you select Result Range Result Range Start Time BERE Minutes Result Range Stop Time 4 966 Minutes Add Cancel Help ddil Baseline Start Time 7 951 Minutes Baseline Stop Time 0 334 Minutes The specified range will be displayed in the dialog box If the result range falls within a baseline range already entered that baseline range will be displayed Click the Add button to enter the designated result range into your SEC setup Alternatively this information can be entered manually in the SEC Setup screen One or more result ranges are required before an SEC analysis can be performed Define SEC Peaks You can use your stored calibration standard chromatogram s to enter peaks and their retention times into your SEC calibration setup 1 Make sure your calibration standard chromatogram is displayed as the current chromatogram and that it has been analyzed If it has not been analyzed click on the Analyze button 2 Select on the Method Define SEC Peaks command or click the Define SEC Peaks button on the tool bar of the chromatogram window You will be prompted to click your mouse before the first 264 Fig 373 SEC Option SEC peak and after the last SEC peak you wish to enter in your SEC setup peak list Once you have clicked to define the peak region a box will appear where you select options for the defined peaks De
196. eedle Wash box to enable a wash procedure Temperature control If your autosampler 3950 is equipped with a tray cooling facility this option can be checked and the desired temperature 4 22 C can be entered Wait with tolerance C The system becomes ready only if the actual tray temperature is inside the set range Wait time min The waiting time is the equilibrating time in minutes after the tray tempe rature reached the temperature set range before the injection takes place Mix Methods Click the Mix Methods button to create a program of sample preparation The Mix Methods set up dialog will appear It is the same dialog as already described for the AS 3900 starting with Fig 165 on page 115 Please note that for the autosampler 3950 for all tray types except the 83 3 vials tray no transport vial for the ul pick up can be defined The transport liquid will be escaped from the wash solution If the 84 3 vials tray is selected the vials 85 86 or 87 can be selected as a transport vial The transport plug is 2 5 x needle volume needle volume tubing volume please refer to the autosampler configuration chapter The autosampler itself calculates the liquid level in the transport vials It will be reset to 8000 ul every time if the autosampler is switched off and on Additionally the level can be reset from the autosamplers direct control tab The level cannot be read out from the software If the autosampler calc
197. eld information can be cut copied pasted and cleared To open the menu with these commands right click anywhere within the spreadsheet To select a field click on that field to highlight To select a row click on a number in the column to highlight the entire row Certain spreadsheet fields will have choices available for you to select Fields with selections available will display a combo box button when the field or row is selected Click the button to display the available choices Fill Down This enables you to automatically copy spreadsheet information from one field or row down through the rest of the spreadsheet Insert Line This command inserts a copy of the line in the spreadsheet below where the cursor is located Delete Line This command deletes the line in the spreadsheet where the cursor is located Delete Lines This command deletes the selected lines in the spreadsheet The flow and gradient composition profile is displayed in the profile window area below the spreadsheet For the HPG mode the changes made for a gradient program will be automatically transferred to the gradient programs of all the pumps which form this HPG system This does not affect the event configuration which can be setup for all pumps independently Creating an Instrument Control Method 89 Configuring Events Digital Outputs Fig 131 Fig 132 This option is given only for the S 1050 P2 1L S 1000 K 1001 and K 1800 pum
198. emplate Open command ChromGate SEC Equations Weight Average Molecular Mass My D AM XA Number Average Molecular Mass M D A A M Z Average Molecular Mass M gt A M2 AM Z 1 Average Molecular Mass May A M AM Polydispersity Index My Mn Viscosity Average Molecular Mass M 2A M2 A a Where MW Weight Average Molecular Mass Al Area of the slice of interest Mi Average Molecular Mass of the slice of interest MN Number Average Molecular Mass MZ Z Average Molecular Mass Mz 1 Z 1 Average Molecular Mass Mv Viscosity Average Molecular Mass a Mark Houwink Empirical Constant M is only calculated when using universal calibrations with Mark Houwink Constants Equations Universal Calibration Using Universal Calibration Molecular Mass s are determined by first adjusting the calibration points by using the following equations Intrinsic Viscosity n Km Given Sample viscosity Adjusted Molecular Mass M n standara Mlsampie Given Sample Mark Houwink Constants Adjusted Molecular Mass M 10 log n standard K sample a sample 1 Next they are plotted to create a general calibration curve that is used to lookup sample Molecular Mass s Equations Broad Standard Method 1 The goal of method 1 is to solve the following Molecular Mass calibration curve equation To do this the analysis routines solves for the two unknowns D1 and D2 M t D Exp D
199. ems Bronkhorst Flowmeter Configuration Name Bronkhorst Flowmeter Capacity Mode Volume Flow Seral Number Capacity Limit 30 0 Serial Port COM 1 Asis Units ml min A Please adjust the Capacity Limit after changing the Capacity Mode or the 1 G13 Units Cancel Help Fig 117 Configuration window for the Bronkhorst flowmeter Name Enter a descriptive name for the flowmeter This name will appear on the flowmeter tab when the instrument setup or status windows are displayed Use a name which is unique within the instrument Serial Number Type in the serial number of the flowmeter you are using Serial Port Select the number for the communication port on your PC to which the flowmeter is connected Capacity Mode Select the desired capacity mode from the drop down menu The choices are Volume Flow and Mass Flow The Y Axis unit will change dependently from the selected capacity mode Capacity Limit Set the capacity limit which depends from the flowmeter model and the selected capacity mode y axis units Y Axis Units The available y axis units depend from the selected capacity mode When complete click OK to exit the dialog and return to the instrument configuration icon list Creating an Instrument Control Method 83 Creating an Instrument Control Method Fig 118 Fig 119 If you have the Instrument Control option installed and your instrument is configured as a KNAUER HPLC Sy
200. eneral detector settings described on page 91 Instrument EE GEER PEK S 1000 Ey Fe Trigger Acquisition Scan nm Time constant x 3 Sec Start 200 End 400 Bandwidth 1 Sampling rate 17 y Hz Signal mode Suitable for minimum peak width at base of 0 333 Min 7 Acqua 3 data Run time 10 0 Min ener dell br a Min Wavelength Program Used channels V 1 2 Options Bandwidth nm E BRAD Scale 10V ins o Time Wave Events min length 1 conpra Comments i NE Lamp OFF at the end of run 0 00 254 00 254 00 F Autozero at WL change Time 9 93671 Minutes Amplitude 254 Wavelength nm Detector S 2600 setup window with PDA option Options An output no access with Kontron DAD 540 You can activate the option to select the scaling of the analogue output signal This option is not compatible with a set sampling rate of 10 Hz The following error message will be displayed when the window will be closed Error message Scale The selected value represents the full scale Values are selectable from 0 001 AU up to 10 AU For the Kontron DAD 540 it is fixed on 10 AU Autozero at WL change Check this option if you wish the signal to be zeroed at the moment the wavelength is changed to define the current signal as the baseline At the run start always an Autozero is performed Creating an Instrument Control Method 101 Fig 149 Lamp OFF at the end of run The op
201. ength step Specify the wavelength spacing in nm to be used when purity calculations are performed Purity threshold The Threshold setting is used to modify the impact of the noise spectrum on total peak purity calculations It has no effect on the calculation of three point purity Higher values for the Threshold setting increase the weight of spectral noise on the purity calculation This has the effect of raising the computed similarity values for spectra and thus increasing the total peak purity value Absorbance threshold This value represents the percentage of peak height that spectra will be included in purity calculation Spectra in sections of the peak that do not exceed this threshold will not be included in the purity calculation This provides a method of eliminating spectra where the concentration of the compound is so low that the solvent spectrum interferes Background compensation Checking this box will cause spectra to be corrected for background using the peak baseline prior to being used in the calculation of purity Per peak spectrum calculations Checking any of these boxes indicates that the indicated value will be calculated on a per peak basis during analysis The result of this 192 ChromGate PDA Option calculation will then be available in reports and as chromatogram annotations Disabling values that are not of interest will speed up analysis If a box is unchecked and the field appears in a run re
202. entation and contains information specific to the use of the optional instrument control software Overview of Instrument Control The instrument control software for the Knauer HPLC System enables you to enter instrument control parameters that become part of the method The parameters are then executed in real time when samples are acquired using the data system The software allows you to set and control parameters as pressures flows and gradients for pumps wavelengths and scans for detectors injection parameters for autosamplers on the Knauer HPLC System and monitor these in real time Configuration device communication port some of the devices have two communication ports on the rear panel for controlling the device by the computer One is an RS 232 port also called serial or COM port the other is an Ethernet port also called LAN Local Area Network Beside the Knauer valve drives for all the devices the desired communication port must be defined in the device Please pay attention to the corresponding notes in this manual and refer to the device s manual for more information how the desired communication port must be configured Please note that for some devices the functionality depends on the selected communication port Configuring the Interface Beside all detectors directly supported in ChromGate any detector or sensor producing an analog signal which can be digitized with an A D converter or Interface Box can
203. ents not controlled via Knauer net the serial port number is shown Creating an Instrument Control Method 139 Fig 204 Fig 205 Fig 206 9 Se al Pot COM E SH 73266 KNAUER serial port instrument status For instruments that will be controlled via LAN the IP address is shown 2 LAN 172 16 5 14 10001 EA SN CNAOS0600026 KNAUER LAN instrument status SN XXXXX This field displays the serial number of the instrument which was entered in the configuration window It should be the same as in the list in Fig 206 where the serial numbers are read out from the instrument The serial numbers serve as instrument identifiers an error message will be generated in the case that the method is started with incorrectly entered serial numbers In this case the serial number must be corrected in the instrument configuration The active state of the communication with an instrument is represented by the green y symbol Only in this case you will have access to the Device Information Click the button to display instrument s connected to that COM port Information about the serial number device name and instrument software versions will appear Device Information Device Software Version Reftesh Information about the connected instrument s Instrument Status Pumps The tabs for all types of pumps appear identical independent of the features of the pump The embedding of the pump into an HPG or LPG system will al
204. ep me nm 13 point purity Purity threshold 0 350000 pego SOOO Background compensation Cancel Help By default the Similarity View will use the current method settings To override the method settings click the Override method settings box then enter the values you wish to use Wavelength range Specify the wavelength range over which the purity calculations will be performed for example from 200 nm to 400 nm Wavelength Step Specify the wavelength spacing in nm to be used when purity calculations are performed Purity threshold The comparison of two spectra gives a Similarity Index SI the closer to 1 00 number is the more similar the spectra are The purity threshold is used to eliminate spectra that do not match If the Sl is greater than the purity threshold the spectra will be considered pure In general a spectra with an Sl greater than 0 9900 would have a high probability of being the same as the apex spectra A SI greater than 0 9000 but less than 0 9900 shows some similarity but would need to be evaluated with care and caution Sl less than 0 9000 should be considered non similar Absorbance threshold This value represents the percentage of peak height that spectra will include in purity calculation Spectra in sections of the peak that do not exceed this threshold will not be included in the purity calculation This provides a method of eliminating spectra where the concentration of the compoun
205. er must be installed on all computers The TCP IP protocol can be installed using the standard procedure with the Windows installation disks the Net version 3 installer can be found on the ChromGate Installation Disc For the installation of the ChromGate software you must be logged onto the network domain in which you will be working only for Client Server installation and the person performing the installation must have administrator rights All other software especially virus detection software must be shut down ChromGate 3 3 2 is shipped on one DVD lt includes the original EZChrom Elite 3 3 2 from Agilent the Service Pack 2 of EZChrom Elite 3 3 2 and the Knauer Add ons as drivers and additional program options The XXXX for the ChromGate version is the internal build number e g 1787 and may change if a new build will be released A new build may include patches and or the support of additional devices The license is stored on a USB license dongle labeled with ChromGate and the serial number of the license Installation First EZChrom Elite 3 3 2 must be installed from the ChromGate installation Disc Click on the link Install EZChrom Elite 3 3 2 software to start the installer Please refer to the EZChrom Elite installation guide to install EZChrom Elite Installation Guide ChromGate 3 3 2 9 J KMALDEA ChromGate gt Install E2Chrom Elite 3 3 7 Software Read release moles gt Install
206. ered to be Two spectra are considered to be identical when the SI value is close to 1 Lambda Max Min Calculations A lambda max min is defined as a local maximum minimum of the absorbance values of a spectrum The user must define the wavelength range over which the calculation should be performed To compute n lambda max min values for a spectrum the software will find the n local maxima minima with the largest smallest absorbance values The calculation range of Lambda Min Max is based on the X axis settings for the graph in the Axis Setup context menu item of the graph PDA Analysis and Calculations 227 Noise Spectrum Calculations A noise spectrum is used in Purity calculations to compensate for solvent and detector effects The spectrum is computed using a 3D data file and a user supplied retention time range The noise spectrum is an array of data points over the spectrum range Each data point in the noise spectrum is 3 standard deviations of the mean of the absorbance values for that wavelength over the user supplied time range of the 3D data Peak Purity Calculations Peak purity calculations use the spectra from 3D PDA data to assess the purity of a chromatographic peak Spectra from the peak are compared to the apex spectrum to compute a measure of uniformity of the spectra Background Correction 27 Background Correction may only be performed on a spectrum as a result of the settings on the Spectru
207. erface card ISA AD Setup and Control of Knauer HPLC Systems 35 K 2700 2800 Interface Card and Driver Installation ChromGate communicates with the Diode Array Detectors K 2700 and 2800 via either a PCI interface card or LAN Installation of the necessary PCI card is described in the detector manual Please note that the driver is not released for Windows Vista or Windows 7 For other PDA detectors S 2800 S 2850 only the LAN interface will work Configuring the KNAUER HPLC System Fig 36 Fig 37 In order to access commands for control of Knauer instruments and autosamplers you must configure them appropriately To access the configuration screens locate the icon on the main menu for the instrument you want to configure You must have system administration privileges in order to configure an instrument ChromGate Client Server File Edit View Tools Help Bi e J DEE Current locationfgroup is Lab 1 The Enterprise Current User He Lab 1 Available 2 New Instrument Available ChromGate main screen Select File New to create new instruments or groups a ChromGate Client Server wie Edit View Tools Help Location troup Oper Instrument onfgroup is The Enterpr Configure j Rename Delete Properties System Activity Log I Exit Creating new Instrument or Location Group If you have selected Instrument the Instrument Configuration dialog see Fig 38 appears
208. es are None Manual and External None Time program s and Sampling start immediately after clicking on Start Sequence acquisitions do not pause between runs Manual Operator must press Enter to start the run Sequence acquisitions pause for confirmation between runs External Program execution and data sampling are started from an external trigger if this option is selected Further settings are not necessary Sequence acquisitions pause for external trigger signal between runs 136 Creating an Instrument Control Method Setting up the Baseline Check Fig 197 Fig 198 This check is only available if you have activated it according to the next figure For configuration of the system refer to page 38 Configuration Options General C Baseline Check Cancel Help Activation of the baseline check option Any configured HPLC system with activated baseline check option will be completed by an additional tab for this check Click on the baseline check tab to define the check conditions E Instrument Setup E 5100 E As Triathlon E Switching Valves Ej Oven s 4050 y 5 2500 2 Aux Trads VI Baseline Check 2 Trigger General To pass the baseline must meet the test criteria for 15 0 minutes Stop checking baseline if conditions are not met after 30 0 minutes Noise test method ims X Enable Threshold Enable Threshold lil Noise Test Noise Drift Test Drift hr Defining the baseline
209. es active for changing This defines the alternating measuring time for the two channels The data acquisition is not performed truly simultaneously The cycle time you can set within the range of 0 40 through 1 00 seconds with 0 05 s increments For the x32 detectors the option Used Channel 2 is not available and the cycle time is not displayed at all The wavelength table is a spreadsheet to enter your time detector program In the first row at time 0 00 min enter the initial wavelength s in nm The editing principle is the same as for the time gradient program described in the chapter Instrument Setup Pumps Pump Program Instrument Setup K 2600 Detector The detector K 2600 supports the general detector settings described on page 91 The appearance of some of the details of the K 2600 detector tab depends on the availability of a PDA option MM Instrument Setup 3 K 1001 SJ as 390 8 ram Aux Traces 3 Trigger Time constant 1 0 v Sec Used Channels v 1 lv 2 Sampling rate ios d iis ATTEN anaes min length 1 length 2 5000 fr Pi Comments Suitable for minimum peak width at base of 0 333 Min 0 00 254 254 Fon 00 Run time 10 Min 10 00 254 254 7 On 00 Acquisition delay 0 Min Scan nm Range 190 740 nm Start 200 End 500 Advanced V Acquire 3D data IV I Lamp OFF at the end of run F Autozero at WL change Time 9 89494 Minutes Amplitude 254 Wavelength nm Fig 139
210. escending C pest 0 position 48 Expected interval min 0 1 mes Fig 340 FC parameters Spectral Similarity Peak Recognition Local Maximum Minimum If the mode single event is selected you can also use local maxima or minima to define action conditions FC Parameters at 2 00 min Signal Relative to previous event min Use as reference for conection next events Noise threshold Units 11 0 Action Expected interval min E Sein Collect To gt f next 0 position Local Maximum Please notice that the parameters will be used in the Units selected for the Detection Channel Cancel Fig 341 FC parameters Local Maximum single event Special settings in parameter window These settings are not available in the peak recognition mode Relative to previous event If you check this option you have to enter the relative time shift from the previous event In the Exp time column this time difference is automatically considered The time in this row is displayed blue colored indicating that there is no further access for free editing FC Parameters at 1 30 min Signal z W Relative to previous event min 0 3 Use as reference for correction next events Noise threshold Unita 11 0 Action Expected interval min 10 2 Action Collect Ta El f nest 0 position Local Maximum Pleaze notice that the parameters will be used in the Units selected for the Detection Channel
211. ese tabs you get the possibility of direct control interaction with the individual instruments This is even possible during a method is running however this option must be enabled while configuring the system refer to Knauer instrument control method options on page 170 Shutting Down the System The system can be automatically shut down at the end of a sequence by including a shutdown method at the end of a sequence discussion of sequence types is presented in Chapter 4 of the reference manual The shutdown method should include the following e Column Oven temperature value should be below ambient e Pump conditions the flow for all pumps should be 0 0 ml min and 100 A and no events For new pumps as Smartline pump 1050 also a Standby option is available e Deselect any timed events and Mix methods e Setthe Trigger to None Now an autosampler will not be controlled and will not perform an injection e Detector activate the Lamp Off option or if supported the Standby option Setup and Control of Knauer HPLC Systems 31 Setup and Control of Knauer HPLC Systems This manual contains detailed information on the use of the ChromGate instrument control software option for the Knauer Instruments It contains information on how to configure set up and run samples automatically from the ChromGate Chromatography Data System This manual is intended to be a supplement to the general ChromGate Data System docum
212. esired time and enable the scan by clicking the check box Please note that during a scan the measurement signal s cannot be acquired In the chromatogram you will find a plateau for the scanning time It is not allowed to enable a scan in the first and last line of the time table The time between two scan must be 0 33 min in minimum Time Yave Wave Dig Outs z A min length 1 length2 gt comer Ore nl j 00 254 294 7 On 300254 ila DO 10 00 24 254 7 On 00 Fig 142 Detector K 2600 time based scan 98 Fig 143 Creating an Instrument Control Method Instrument Setup S 2550 Detector The detector S 2550 supports the general detector settings described on page 91 MM instrument Setup E 5100 E AS 3950 Sy Ovens 4050 6 s 2600 lt Aux Traces py Trigger Time constant 1 000 Sec Used Channels H V 2 Sampling rate 1 Hz Suitable for minimum peak width at base of 0 333 Min 0 00 254 254 On 0000 Runtime for 1000 254 254 7 On 0000 Acquisition delay loo Min Scan nm Range 190 900 nm Start 200 End 500 IV Acquire 3D data Lamp OFF at the end of run Use quick scans F Autozero at WL change Detector S 2550 setup window Used Channel Check the box es to turn the acquisition for the selected channel s on Only channels that have been selected in the instrument configuration of the detector are available If the box is not selected no data will be acqu
213. eted one you can continue with it Selecting a completed one the final Report will be displayed 176 Fig 261 Knauer Instrument Control Method Options The delete button becomes active if one of the earlier sessions is selected f PQ Method Test PQ met Data None Project Default Performance Qualification File Edit View Method Data Sequence Analysis Control Reports Window Help a SME 1 s 2500 OBR E kg E ATA Conditions Settings Measurements Active channel S 2500 ba Active pump 51000 v Test type Custom Test y Mobile phase Methanol Water 80 20 Gradient A 1100 B fo C lo D lo Temperature ambient Flow 0 8 mL min Detector 254 nm Acquisition Time 3 min omoa C For Help press F1 PQ configure screen Simultaneously the instrument setup screen and the PQ configure screen will be opened If more than one detector channel and or pump are configured you have to select the active ones via the drop down menus Next you can select either any customized method or the Knauer Test Kit Test type Custom Test E Custom Test Knauer Test Kit If you have selected the Knauer Kit no further entries are to do in this screen You have no access for editing For custom tests you have to enter the parameters according to your method Clicking on Continue or selecting Settings on the left hand side will open the next dialog window Knauer Instrument Control Met
214. ew Fig 308 The Ratio view displays two PDA wavelength channels and the ratio of those two channels These may be viewed during real time acquisition as well as during post run analysis The flat tops on the ratio peaks are a preliminary indication of peak purity The ratio wavelengths and parameters are set in the Method PDA Options Ratio tab The Y axis of the ratio chromatogram is auto scaled to 1 3 DAD Spectra Ratio Plot F views w S Synchronize EX Arange Channel 1 Wavelength 260 nm Bandwidth 8 nm Time 0 0260893 h Amplitude 0 175 mAU Right click within the window to display the pop up context menu The context menu is the same as for a standard chromatogram graph window PDA Utilities The Utilities menu allows displayed spectra to be printed copied saved or exported The Utilities menu is available from the right mouse click menus of various PDA views Print select Print to automatically print the currently displayed spectra Copy to Clipboard Select Copy to Clipboard to copy the displayed spectra to the Clipboard The contents of the Clipboard may then be pasted into other software Save trace Select Save trace to save the spectrum to a file with an spc extension for later inclusion in a library or report ChromGate amp PDA Option 211 Spectrum Similarity Table Fig 309 The Spectrum Similarity table is displayed in the PDA Mixed View when the Options Mode is set to Show Spectrum Similarit
215. f Knauer HPLC Systems r nnennnnnnnnnvnnnnnnnnnnnnnennnnennnnnnnnnnnnnnnnnnennnne 31 Overview of Instrument Control rnnnrnnnnnnnnnnnanrnnnnvnnnnnnnnrnnanennnnennnnnnnnnnnanrnnnnennnnnnnnee 31 Configuration device communication port occoocncoccncccnncccnccncncnncncnnnnnnnnnnnnnnoncnnnnos 31 Configuring the IMIS ACE eee 31 Knauer Interface Configurati0N occccoccoconcoconccocnccnnnononnonanccnancnnnnonnnnonannonannnnanonos 32 Kontron Interface ConfiguratiON oooccccccnccccnconcncoonnnncnnoncncnncnonanonnnnnonnnnnnnnonaninnnninos 34 K 2700 2800 Interface Card and Driver Installation o cococcccccnconnnccnnnnos 35 Configuring the KNAUER HPLC SysteM ccooccccccccccnccccnccnonccocnonanccnanncnnnonannonannnnono 35 Instrument COMIGUATION si Se 36 Configuration KNAUER HPLC SysStemM oocccoccccccncoccncccnncncnconcncnncncnnnnnnnnonnnononcnnnnos 36 Configuration Knauer PUMPS occccocccccnncccnnoncnoconnnnannononnnnnnnnnnnonannnnnnnonnnnnannnnaninnans 38 Configuration Kontron PUMPS sisan a a 44 Configuration Knauer Detectors cooocccoccccccnconcnconcncnnnnnnnnnonnnooncncnnnnnnnnonnnnnonenonass 45 Configuration User Defined Detectors oocccconncoccccocnccccnconcnonanoconnnnonnnnnnnnoncnonons 53 Configuration Kontron UV Detectors occccoccccccnccccncocnnconnconcnonanncnnnonnnnnnnnnnonenonoos 99 Configuration Kontron Diode Array Detectors oocccocccc
216. f Run screens Mix methods Temperature Control Enable column oven control Enable tray cooling Wavelength nm Wavelength nm Autosampler 3900 setup window Injection Method Select the injection method by clicking on one of the available options Full loop Partial loop fill ul Pick up or None The maximum programmable injection volumes are Flushed loop not programmable is equal to the loop volume but needs more sample to fill the loop completely 3 x loop volume for loop volumes lt 100 ul and 2 x loop volume for loop volumes from 100 ul to 500 ul 1 5 loop volume for loop volumes gt 500 ul Partial loop fill 50 of the programmed loop volume ul Pick up Injection volume loop volume 3 x needle volume 2 Syringe Speed The aspirating speed of the syringe used in injection methods can be adapted depending on the viscosity of the samples the choices are Low Normal and High The speed of the syringe during washing or rinsing procedures is not affected by this setting Flush Volume Enter the amount of sample in ul taken from a vial to preflush the needle before the loop is filled with the sample The default value is 30 ul for a tube volume of 15ul In the flushed loop mode this option is deactivated Flush volumes lower than twice the volume of the needle and tubing will result in a decrease of performance of the autosampler Wash times Enter the wash volume in number of syringe volumes
217. ferent events has to be programmed For all lines added in this way as a comment DC operation will be displayed ME instrument Setup J 51050 Y Inj Module Y as 3950 3 s 2550 E S 3050FC 9 Aux Traces UL Baseline Check I Trigger Detection Channel 5 2550 Channel 1 M Collector Reset gt Pump Is 1050 vi Flow ml min 1 000 Update Flow Fraction Collection Program Exp interv Rel time Fraction Fraction T Single Event Unconditional Timed Waste Single Event Signal Level Slope p L and S next F DC Operation m mina tona Single Event Waste Relative to previous event min Use as reference for correction of next events Action Action Collect To x next C position Rack configuratidn Path iLaborspiEZChrom Elite Ente Time 5 51 min Amplitude 0 002 Slope 0 001 Please notice that the parameters will be used in the Units selected for the Detection Channel 0 Pretreatment B Fraction BB Peak recycling BB Solvent recycling Wavelength nm Wavelength nm Programming the fraction table In Fig 352 an example for the fraction program of the above chromatogram is shown Fraction Collection Program Exp time Exp interv Rel time p ai Fraction Fraction c a min min shift min arameters Position Check sk i D EE 0 00 i Single Event Unconditional 0 00 0 00 Timed Waste 1 5 51 Single Event Signal Level Slope 0 10 0 00
218. fferent you should view your current chromatogram trace in a different window than the Mol Wt Dist trace To do this select the View Tiled command from the menu bar The Mol Wt SEC Option Fig 378 267 Dist Trace will appear in a separate window Notice the axes are Molecular Wt Vs Slice Area for this trace E Channel A Mol WE Dist 1 A ES 100000 1e H006 te 007 Molecular Weight SEC Setup SEC Setup involves entering Molecular Mass information about your standard which will subsequently be used to calculate Molecular Mass numbers for your unknown samples SEC baselines result ranges and peaks can be entered automatically with graphic programming using a stored calibration standard These values can also be entered manually You must then complete the SEC Setup by entering the information required for the selected SEC calibration method and calculations The information displayed in the SEC Setup screen will vary depending on the Calibration Type selected Narrow Broad1 Broad2 or Universal For details see the section describing the calibration method of choice When you have completed the SEC Setup you are ready to acquire and process your SEC runs You can acquire data either manually single injection or by batch acquisition auto injector See the ChromGate User s Guide for information on creating a batch sequence SEC Setup Narrow Standard Method When you select the Narrow Standard method for Calibr
219. fine Peaks Options fe Add Tolerance E C Replace E C Average Add Choose this option to add the selected peaks to the current SEC calibration peak table Existing peaks in the table will be maintained Replace Choose this option to selectively replace peaks in the current SEC calibration table The tolerance is used to determine if an existing time should be replaced with a new time If the tolerance is 0 the numbers must be an exact match The larger the tolerance percentage is the larger the variance can be between the new value and the existing value and still be considered a match Average Choose this option to selectively average new times in the current SEC calibration table with existing ones The tolerance is used to determine if an existing time should be average with the new time If the tolerance is 0 the numbers must be an exact match The larger the tolerance percentage is the larger the variance can be between the new value and the existing value and still be considered a match Only five trial values will be used in the average If you try to average a sixth time value the oldest trial time will be thrown out Tolerance Tolerance is used to determine matching values when using the replace or average options If the tolerance is 0 the numbers must be an exact match The larger the tolerance percentage is the larger the variance can be between the new value and the existing value and still be considered a
220. for missing serial number Click OK and perform the required settings The same message will appear if you try to close the setup window without an entered serial number Interface setup Number of boxes 1 Configure box 1 Serial number bbbbb Manufacturer Knauer Serial Port COME gt Model 5 5000 Box type Channel Range m 256W 50Hz Cancel Help Dialog box for configuring the Manager 5000 5050 The Knauer Interface Box and the Manager 5000 5050 require the same parameters to be set up Therefore the configuration dialogs for both devices are nearly identical Setup and Control of Knauer HPLC Systems 33 Fig 33 Number of boxes Select the number of boxes used in the system ChromGate supports up to 4 Knauer Interface Boxes Configure box Select the box you wish to configure Each box has to be configured separately Serial Port Manager 5000 5050 Interface Box IF2 Select the number for the communication port on your PC to which the Manager 5000 5050 or the Interface Box IF2 is connected Interface Box Model 96 The Knauer Interface Box supports the binary Knauer Net The networks 1 2 correspond to the number of the utilized communication port COM1 COM2 Select the number for the communication port on your PC to which the Interface Box is connected You can use the Interface Box or the Manager 5000 5050 to acquire signals from different instruments at the same time It i
221. ft for the next fraction The Empty vial check tests before the injection if enough empty vials for the fractions are left Enter the number of vials needed for one run The software will check then before the injection if this number of vials is left If not the system will not start the next run Stacked Injections Restart from first vial of current run If stacked injections are used check this box if you wants the collector to start for every stacked injection with the same vial as for the previous runs for the fraction collection This is an option if you inject the same sample several times and want to collect the fractions of the same substances from every run in the same fraction vials ChromGate Preparative Option 237 Adjust FRC table after stacked injection This option allows you to copy the already executed part of the fraction table for the next injection if a stacked injection is performed In this case you will have the same collection conditions for every injection without any manual intervention This makes sense if you use Single Event parameters which otherwise should have to be programmed separately for every injection If you use the peak recognition mode just program the collection condition for the whole run time the Adjust FRC table option leave unchecked Pump If more than one pump is configured in the system select the used one from the drop down list for volume and delay calculations Update Fl
222. g Check this box if you want to control the tray cooling The setting of the appropriate temperature will be done later as part of the instrument setup Tray Click the Tray button to set up the autosampler tray The tray configuration dialog will depend on the autosampler type see below Setup and Control of Knauer HPLC Systems The Triathlon Trays Since the Triathlon can be equipped with a mix of four available types of tray segments it is necessary to define the tray configuration Tray configuration segment Type Humber Standard Md Transport vials PT and Reagent 1 Standard y Reagent E LS Standard y Reagent E et Standard v Reagent D lz Standard y Destination fo Standard y Fig 102 Tray configuration for the autosampler Triathlon 1 2 3 5 E 8 Segment The Triathlon tray consists of eight segments of different types Segment Type Select the type of the corresponding installed segment The choices are Standard A segment LSV B segment Super LSV C segment and Micro vial D segment The selected type will be shown in the tray configuration window Segment Type Mumber 1 frends sl 0900 2 Standard idl 2 y 3 Standard 3 y 4 Standard 4 7 5 Micro vial D 11 E f 8 Standard LS E Fig 103 Tray configuration displaying different tray types Segment Number Select the number from 1 to 8 of the
223. h 59 min 59 sec Click the Commit button to actualize your selection Click the Delete button to remove the selected line from the programmed methods The Stop Events at Time allows setting the end time of the timed events lf the Stop Events at Time box is not checked the end time is equal to the analysis time the run time The maximal Stop events time is 9 h 59 min 59 sec If the END time exceeds the programmed run time this END time overrules the analysis time for the autosampler The next injection will be started as soon as the timed events program has finished User Defined Program UDP The User Defined Method is a user defined combination of all possible programming actions of the Triathlon or the Endurance like aspirating from or dispensing to a certain position moving the syringe switching the syringe valve switching the compressor ON or OFF switching the injection valve and the optional ISS valves wait and programming all inputs and outputs You can build your own sample handling sequence within the possibilities of the Triathlon or the Endurance Enable UDP Check the Enable UDP box to use the user defined method This selection sets the standard injection parameters inactive UDP Setup Click the Setup UDP button to set up the user defined method The User Defined Program dialog will appear User Defined Program Program steps Mo Action Values Speed Height Add q Aspirate 10 ul from Sample
224. he Direct control during a run section of the Runtime Settings is enabled Please refer the section Runtime Settings in the Knauer Instrument Control Options for more information lt Flow amp Gradient Apply gt Pressing this button will send the current flow and gradient settings for execution to the pump Gradient Components Specify the composition A B you would like to have at the moment and click the lt Gradient Apply gt button The components field is applicable for the HPG High Pressure Gradient or LPG Low Pressure Gradient mode Depending on how many components are configured the corresponding number of fields A B will be accessible The lt Gradient Modify gt button is active only during a run Clicking on it opens the following window 142 Fig 209 Fig 210 Fig 211 Creating an Instrument Control Method Set New Gradient Modify the running time program Current run time 0 26 min Time 0 00 Flow 0 000 1100 B 0 a o 2D o Commit Line Delete Line Enter the new gradient line in form Time min Flow ml min B Z C D Send Gradient Freeze the actual gradient for 10 00 min will be performed immediately Freeze Gradient If the run time is to be prolongated use the menu Control Extend run min mimin 36 EN 00 95 5 EA 26 95 5 3 150 5 La 300 so 1 2 Gradient Time min Gradient modify window In t
225. he appropriate chapters Injection module configuration The injection module will be installed with the Knauer FRC AddOn In the instrument configuration dialog select the injection module and add it to the Configured modules Note that it is not allowed to add an autosampler and the injection module Injection Module Configuration Mame ni Module L Serial Number Use Sample Loop Type of Valve Pump Configuration Interface Type of Pump 5 100 v A n COM Port z Baud Rate S600 hi Serial Number Pump Head Cancel Help Injection module configuration window The injection module consists of a pump and a Knauer 2 position switching valve Optional an injection loop can be used As a pump a pump P 2 1L P 2 15 P 4 1S S 100 S 1050 or 1800 can be selected If ChromGate Preparative Option 251 Fig 360 selected click on the Lbutton The pump configuration window will open Please configure the pump accordingly the pump configuration described in the appropriate chapter Note that you cannot configure the same pump as for the solvents However it is not possible to use e g one channel of an LPG solvent pump for the injection module Also configure the valve as described in the appropriate chapter If you want to use a sample loop enable the check box Use Sample Loop The injection module also allows for using the AZURA ASM 2 1L as an injection module if a pump and a valve have been installed The in
226. he box is automatically enabled because these models always have a pressure sensor while models 10P P2 1S do not have this component here the box is disabled Add Info Click the button Add Info to enter more detailed description of the pump Additional Information Flow in the system is limited to Smil due to securnkty reasons pump setup Cancel Additional information box 44 Setup and Control of Knauer HPLC Systems Enter additional information or comments to be printed out when you print the instrument configuration Without the correct serial number the communication with the pumps cannot be established When complete click OK to exit the dialog and return to the instrument configuration icon list Configuration Kontron Pumps Fig 59 For each pump 320 322 325 420 422 425 or 520 522 525 in the Configured Modules window double click the icon and complete the configuration dialog You have to make sure that all settings correspond to the settings at the pumps their selves The configuration windows of all Kontron pumps Fig 59 look very similar that they can be described together Kontron 320 322 325 Pump Configuration Instrument Mame Kontron 320 Pump Type 2320 Gradient Mode Kontron 4207 4227425 Pump Configuration Head Fmax 400 0 E Communicatio Instrument Hame Kontron 422 Pump Type 472 Gradient Mode HFG kl Head Kontron 52
227. he gradient spread sheet the elapsed lines are grayed The red line shows the actual gradient status The yellow ones are that which are open for modifying The Gradient Modify allows for changing the gradient table by adding new lines of freezing the gradient for a defined time To modify the gradient program enter the time for the new line and the desired values in the fields on the top of the window To change an already existing time line enter this time in the Time field and the new values Clicks on lt Commit Line gt insert the new or modified line into the gradient program The changes by one or more committed lines becomes only active send for execution to the pump by a mouse click on the button lt Send Gradient gt You will see then the new line s in the gradient spread sheet Due to that the complete gradient in the pump will be replaced with a new one the run time on the pump s display will start with time 0 00 min This will not touch the method run time Set New Gradient Modify the running time program Current run time 0 26 min CI ose Time 0 00 Flow 0 000 24 1100 XB 0 C 0 D 0 Commit Line Delete Line Enter the new gradient line in form Time min Flow ml min 44 B Z C D Send Gradient Gradient table modify window To delete any line of the gradient program enter the time of the line to be deleted and press the lt Delete Line gt and then the lt Send Gradient gt button You ca
228. he sequence Since you have not yet defined a sequence summary report do not check the box to print sequence reports Reporting Fig 25 ChromGate Client Server comes with a complete suite of report templates that can be used without modification to generate reports To see an example of one of these reports use the Reports View Area command from the menu bar Make sure your current chromatogram has been analyzed first The standard report will appear in a window on your screen Area Report Data File C Programme CGiCromG atelDatalmulti calibration level 6 dat El Method C Programme CGi CromG ate HBYMethoduntitled met Acquired 05 06 1992 Printed 08 07 2003 08 26 20 Retention Time Channel Results Retention Time rea Area Height Height 0 237 3824 0 11 88 0 03 0 897 13416 0 54 1647 0 49 1 095 7713 0 23 1097 0 33 ni Area Report If your method contains no defined custom report the system will use the standard report formats to print reports when required Short guide ChromGate KNAUER Instrument Control 27 Knauer has added three reports in a fresh design Knauer Area Report Knauer ESTD Report Knauer ISTD Report which also can be found as standard reports in the Report menu HPLC SMB Osmometry Ih KNALIER Date File CAE ZChrom ElitelEnterprise P rojedsiD atamutt calibration level 2 dat Sample Name PNA STDS Acq Operator demo Location N A Acq Instrument Knauer HPLC Offline
229. height of a search results graph A value of 100 corresponds to a standard sized graph A larger value may be selected to provide more a detailed plot Graph Properties Pressing this button displays the graph properties dialog as found on the standard graph Spectrum Report This will display a table showing spectra extracted from peaks of the chromatogram Based on the selection on the Spectrum tab of PDA Options the report will contain either the apex spectrum an average of the upslope apex and down slope spectra or several averaged spectra To set parameters for this report do a right mouse click on the table then select Properties The following dialog will appear 220 Fig 318 ChromGate PDA Option Spectrum Report Properties Report peak spectra Report Information Detected on PEER l Lambda max f Limit to named peaks only Lambda min Limit to time range Purity Fe all Spectral display W Show lambda maxima paa foo s Show lambda minima Background correction Filtering type Mone v Graph Properties Cancel Help Report peak spectra Detected on This allows the user to select the channel or channels from which peaks will be detected Limit to named peaks only This allows the user to restrict the peak selection made above to a time range When checked only peaks within the specified time range will be searched When this box is unchecked the Time Range edi
230. hod Options 177 Fig 262 Fig 263 Fig 264 f PQ Method Test PQ met Data None Project Default Performance Qualification File Edit View Method Data Sequence Analysis Control Reports Window Help SME 1 5 2500 E EIA EAS AER E Conditions Settings Measurements Injection Configuration Injection Volume 20 ul Number of Vials 2 Mi Injections per Vial 3 y Vial Positions 1 1141 Z faz Peak detection Configuration IV Use Peak Time Windows Peaks count 1 gt Peak Begin min End min 1 11 75 2 25 Results Processing Integration Width 0 2 min Integration Threshold 50 Allowable RSD 5 fo coed tes NA gt L For Help press F1 PQ setting screen Here the injection configuration the peak detection configuration and the result processing are to define The peak detection configuration is predefined and not access is possible as far you have selected the Knauer Kit The result processing parameters define the thresholds for passing the test Clicking on the continue button first a consistence test is performed If this test failed you will get the hints what changes are necessary FE PO Method Test PQ met Data None Project Default Performance Qualification EEK TT File Edit View Method Data Sequence Analysis Control Reports Window Help x ass 1 5 2500 BEe REBEE LEAN NSSTO HK E Conditions Settings Measurements O System is no
231. ht g 100000 Horizontal Baseline Threshold Calibration Data Molecular Wei Mn fio8010 Mw 127656 Calibrate D1 16117483 p2 0 281250 V Reference Peak Time min 35 Reference Window 10 Print Export Settings 12 5 15 0 17 5 20 0 22 5 h nutes Time Volume Flow Rate ml min Enable Analysis Click this box to enable SEC calculations for the current channel Each channel can have its own analysis conditions Molecular Mass Table The Broad Range 2 method is a linear calibration method that does not utilize the Molecular Mass Table Enter values in the Calibration Data area Time Slice Select a time slice width in seconds for the calculations The slice width will determine the frequency of Molecular Mass fractions reported If a Slice Report is generated it will be based on the slice width you select here The software will force this number to be an integral of the data collection rate during the analysis Baseline Start and Stop Times The baseline start and stop times in minutes can be entered manually or selected graphically using the SEC buttons on the chromatogram toolbar Results Range Start and Stop One or more ranges of the chromatogram must be defined to be used in calculation and reporting of the Molecular Mass distribution Each range is determined by the results range start and stop values The times in minutes can be entered manually or graphically using a stored
232. ibrary search e Working Spectrum spectrum extracted from the Contour plot on the Mixed View display This chromatogram is displayed in the Spectrum View e Specific Analysis related capabilities are for these spectra are detailed below Analysis Spectra The following applies to the analysis spectra e Prior to being used elsewhere all analysis spectra extracted from the 3D data are filtered according to the settings on the Spectrum tab of PDA Options Working Spectrum The following apply to the working spectrum e Working spectra extracted from the 3D data are never filtered according to the settings on the Spectrum tab of PDA Options e The working Spectrum is not available as a trace in Custom Reports Background Correction Background Correction may only be performed on a spectrum as a result of the settings on the Spectrum tab of PDA Options the settings on the Purity tab of PDA Options or by setting the properties of the Peak Purity Pane Background subtraction is always the first operation performed on a spectrum lf a background correction is attempted on a spectrum after another operation has already been performed including an operation PDA Analysis and Calculations specified in the Spectrum tab of PDA Options a message box is displayed and the background correction request ignored Background subtraction may only be performed once on a spectrum lf a second background correction is attempted on a spectrum
233. ic control of devices When using a DAD K 2700 or K 2800 with a PCI interface card the driver for the interface card must be installed This driver can be found on the ChromGate CD in ChromGatelDrivers If the interface card has been installed after a re start Windows will ask for the driver Start ChromGate once in Demo Mode by clicking Start All programs Chromatography EZChrom Elite default settings refer the EZChrom Elite installation guide Close ChromGate Insert the USB license dongle into a free USB port of the enterprise machine Start ChromGate The license will be recognized and can be used Short guide ChromGate KNAUER Instrument Control 13 Short Guide ChromGate KNAUER Instrument Control Overview This section is provided to assist the operator in the day to day operation of Knauer HPLC systems using ChromGate software It focuses mainly on routine data acquisition and processing and is to be used in conjunction with the documentation provided with the ChromGate software and the HPLC system The full software description can be found in the online help The online help will be installed along with the software You can open it using the desktop link for the online help or with the help menu in a ChromGate window Additionally li will open in the context of the currently opened ChromGate window if you press the key lt F1 gt on the computer keyboard This chapter assumes that the software has
234. ice Monitor Show Rack The communication port of the fraction collector S 3050 must be select using a switch on the collector s rear panel Tubing Parameters Tubing Parameters r Volumetnc delay fe Fixed time min 0 00 C Capillary Length fom fai Inner Diameter mri f LLI C Capillary Volume cm3 fi EN Tubing parameters setup ChromGate Preparative Option These settings enable someone to consider the time delay for transporting the sample from the flow cell to the collector valve If the option Use recycling valve is checked the capillary from the flow cell via the recycling valve to the fractionation valve must be taken into account If you activate the option Fixed time you can enter this time delay directly This value will be valid even if the flow rate is changed Entering the Capillary Volume directly or calculating it by entering the Capillary Length and Inner Diameter the delay time will be adapted to the programmed flow rate of the method Solvent and or Peak Recycling For an optional solvent and or peak recycling for most fraction collectors an additional recycling valve is necessary As a recycling valve only a Knauer 6 port valve can be used The Knauer fraction collector 3050 includes a valve for recycling For a multi valve fraction collector some ports of the fractionation valve can be defined for the recycling To define a separate recycling valve check the option Use recycling val
235. iguration 55 Setup 95 Interface 34 Pumps see also Pumps Kontron Autosamplers Configuration 76 Setup 128 Kontron DAD 540 100 Kontron detector Configuration 56 Kontron Pumps Configuration 44 L Lambda Max Min Calculations 226 Lamp OFF 93 96 98 101 103 109 Library Definition Report 218 Library Search 214 225 license dongle 12 Local Maximum 242 Login 83 Loop Volume 65 68 70 71 73 77 293 M Manager 5000 Configuration 79 Instrument Status 166 Interface Configuration 32 Setup 130 Wiring 57 284 Marker 126 153 Method 13 Window 16 Wizard 15 Midas see AS 3900 see AS 3900 Mix Methods 115 118 119 Mixed View 203 Multi valve fraction collector 62 63 234 248 Multi Chromatogram View 207 N Needle Height 123 Needle Wash 112 114 118 122 125 Noise Test 136 O Output profile 131 P P1x0 Setup 85 Status 139 Partial loopfill 70 PDA see DAD Peak Purity calculations 22 Recognition 237 241 Recycling 232 234 Table 22 221 Performance qualification 175 Report 178 Plate Type 76 Preflush 112 Preparative option Installation 11 Pressure Limits 85 144 Units 43 45 51 61 62 63 Pump Configuration 38 Head 42 44 Setup 85 Status 139 Type 40 44 Purity Calculations 191 Q Quantification 19 294 R Rack configuration 243 Ratio Chromatogram 226 Ratio View 210 Recycling valve 232 Reference Signal 152 Report 26 Performance qualification 178 RF 10AxI see Fluorescence detector RI Detector Configuration
236. ill also be shown For each detector channel the output values and as far as available the wavelength is shown Similarly the relevant data for all included instruments will be displayed e g the actual switching positions in the case of the valves The single instrument tabs of this window provide more detailed information They also enable direct system control On these tabs you have the possibility of directly controlling the individual instruments This is even possible while a method is running however this option must be enabled when configuring the system refer to the Knauer instrument control method options on page 170 MM instrument Status O System Status MD S 1000 As Triathlon M s 2600 Monitor Direct Control Run Status HOLD Flow ml min ooo Stop Flow Apply Flow ral min 1 000 Flow amp Gradient Apply Pressure Control MPa i 55 40 0 Gradient Components Components Aa Foo B Gradient Apply Events IDA y ASS cr fo Diz 0 diet Mi GLP Refresh Control Pressure Limits MPa Min 0 0 Max 0 0 Apply Events 00000000 Events Apply Network 2 SN 44444 Purge Instrument status tab example Smartline Pump 1000 All of the instrument tabs provide communication status information at the bottom The KNAUER Net status and the communication port assigned for communication is displayed ID Networks af SN 78322 KNAUER Net instrument status For instrum
237. ill be set to default values The Instrument Wizard The Instrument Wizard is used to lead you through the steps required to create a new method or modify an existing one create a new sequence or run a method or sequence The Instrument Wizard will always start if you open an instrument You can also start it from the opened instrument window with a click on the Wizard icon Fig 9 Fig 10 Short guide ChromGate KNAUER Instrument Control Instrument Wizard New Instrument Create or modify a method Create a sequence Run one sample Run sequence of samples V Show at instrument startup Instrument Wizard select Create or modify a method to bring up the method window Method Wizard New Instrument Oo Create a new method a Modify the current method Modify a method on disk Method Wizard Click the appropriate button to present the Method window which can be edited as described in the section The Method Window If you are more familiar with the menus you can ignore the wizard If you don t want to start the Wizard always if you open the instrument disable the checkbox Show at instrument startup The Method Window Overview The Method window includes a tab for each instrument in the system on which you can select the particular run parameters The information on a given tab is essentially the same as if you were to program the component on a standalone basis The reader should review inf
238. ime Slice 268 271 273 276 Trigger 260 Trigger Type 260 Universal calibration 258 Use Constants 270 275 Viscosity average molecular mass 258 Weight average molecular mass 258 Z average molecular mass 258 Sequence 13 Files 20 Run 25 Wizard 23 Serial Number 42 61 Server name 36 Setup AS 3900 112 AS 3950 116 Autosampler 107 111 Baseline Check 136 Column Oven 133 Conductivity detector 104 DAD 100 Flowmeter 134 Fraction collector 235 Instrument 84 Kontron detectors 95 Manager 5000 130 RF 10AxI 103 Switching Valves 131 Trigger 135 Short Guide 13 Shutter 152 Shutter Control 51 Signal Mode 102 152 Similarity 222 Calculations 226 Threshold 190 View properties 206 Index Single event 237 Single Level Calibration 21 Solvent Control 173 Recycling 232 234 Selection Valve SSV 65 73 Spectral Analysis 185 189 230 Spectral Library 212 Spectral similarity 238 241 Spectrum 152 Spectrum Background Correction 209 Spectrum Export 228 229 Spectum View 208 SSV 65 Step Mode 130 Switching Valves Configuration 78 Instrument Status 167 Setup 131 Syringe Speed 113 117 122 Syringe Volume 65 68 71 73 77 System Options 14 Shutting Down 30 Status 137 T Temperature control 104 Threshold 19 Time Constant 91 108 Time Program 102 Timed Events 114 123 Traces 135 Transport Vial 14 Tray Configuration Endurance 15 Triathlon 14 Tray Cooling 65 68 70 71 73 Triathlon Configuration 72 Setup 121 S
239. ing a value for this is optional Custom Report A variety of PDA information can be placed in a custom report These items are inserted in the report by placing the cursor at the location where you want to insert the item then do a right mouse click to access the custom report menus PDA Insert Graph Items The Insert Graph menu has the following additional items available when the PDA Option is enabled 3D Data Graph and Contour Graph 3D Data Graph The 3D View provides a three dimensional view of absorbance versus time and wavelength Wavelengths of appreciable absorbance and interferences which may be invisible in a single wavelength plot are easy to locate with the 3D View Select this function to automatically enter the 3D map into the report Click the right mouse button within the 3D map and select Properties to display the 3D Properties dialog box and enter appropriate changes The 3D map can be elevated and rotated around its axis for display from any angle These functions work the same as in the 3D view window ChromGate amp PDA Option 217 Insert Contour Graph A data file containing PDA data may include a Contour Plot in a custom report The contour view also referred to as Isoabsorbance plot provides an aerial view of the absorbance of the sample at each wavelength vs time The contour view supplies quick and easy to assimilate information about those wavelengths at which the sample exhibits appreciable absorba
240. ing dialog will appear Events Digital Outputs Control Events Mio i 1 Relay Of r gt TE All Off 2TTL jor Sein 2TTL jot gt 24 Off bl Cancel Configuring Digital Outputs The Events Digital Outputs correspond to the outputs available on the rear panel of the pump Select the state for each Event you want to set from the drop down list The choices are Off 0 On 1 and Pulse P Refer to the pump s user manual for details about digital outputs parameters Use the All On or All Off buttons to switch all of the outputs either on or off The standard version of the K 2001 detector is delivered with a deuterium lamp as well as with 4 filters for the wavelengths 200 220 254 and 280 nm For the K 2001 you can only use these wavelengths in the time program An optional tungsten halogen lamp is also available Two additional wavelength filters of your choice can also be built in In this case you can use those corresponding wavelengths in the time program The Start Input of the detectors K 2501 and S 2500 will also be used to start an internal wave length program If the detector is used for synchronization input when working under software control the internal wave length program also will be started after receiving the synchronization signal trigger As a result of this fact the wave length for the measurement may be different from the wave length programmed in the detector s instrument set
241. ing from your chromatograph The default acquisition data rate of 1 Hz is adequate for SEC runs You can adjust the slice width for slice reports independently in the SEC setup part of the method Detector 1 x For each detector channel x configured on the instrument define the following acquisition information Acquisition Channel On Select this box to turn the acquisition for this channel ON If this box is not selected no data will be acquired for this channel Sampling This is the rate at which data will be sampled by the system You can choose how you want to specify the sampling rate When you select a sampling rate a prompt will appear indicating the narrowest peak width for which this sampling rate will be adequate It is recommended that you use the Graphical Events Programming to determine the optimum sampling rate for your chromatography Frequency This selection is in Hz samples per second This is the selection for most chromatography applications Click on the down arrow to get a list of the frequencies available for the configuration of your system Period When you select this type of sampling you must select the number of seconds or milliseconds between data points Enter the value then select whether the period is in milliseconds msec or Seconds Run Time Run Time determines the length of time data will be sampled 260 Fig 369 SEC Option Acquisition Delay Acquisition Delay is the
242. ingle run to be executed immediately after the current sequence run record in the Run Queue After this sample is finished the sequence will resume If the chromatogram is not integrated at the end of the run or if you were expecting a report and none was printed check the Method Properties section of your method to make sure data analysis and reporting are turned on for this method SEC Option 263 Defining SEC Baseline Fig 371 You can use your stored SEC calibration standard chromatogram to establish the SEC baseline for the analysis With the chromatogram displayed in the chromatogram window click on the SEC baseline button from the toolbar Then click the mouse on the chromatogram at the beginning and then the end of the desired SEC baseline SEC Baseline Start Time EE Minutes Add Stop Time 17 023 Minutes Lancel Help The range you selected will be displayed in the dialog Click Add to add this baseline range to your SEC method setup Alternatively this information can be entered manually in the SEC Setup screen An SEC baseline is required before SEC analysis can be performed Defining SEC Result Ranges Fig 372 You can use your stored SEC calibration standard chromatogram to establish the SEC result range s for the analysis With the chromatogram displayed in the chromatogram window click on the SEC Range button on the toolbar Then click the mouse at the beginning and end of each desir
243. ings page 141 Injection Duration The duration of the injection is calculated from the injection volume and the injection flow The reduced flow during start and stop of the pump is taken into account with 6 seconds in sum Time between Injections min Enter the time between the injections The value must be higher than the time calculated for the injection duration Click the Apply button to apply the settings into the Stacked injections table Stacked Injection Table if Uze stacked injection Time Inj Yolume Inj Flows Initial injection Injection module Stacked injection table If you make a right mouse click on the table you can select Fill Down to fill the table with the same settings as in the selected line Insert Line to add a new line Delete Line to delete the selected line Delete Lines to delete all selected lines Fill Table will open the Stacked injection setup again The new table will overwrite the existing one If you select the command Fill table to open the setup dialog for the stacked injection table the window will show default values for all parameters beside the sample preparation time Click on OK to close the Stacked Injections table window the injection module instrument setup window will be shown In the Aux Traces tab a trace Stacked Inj Inj Module can be enabled The trace will show the start of the sample preparation and the stacked injection 254 Fig 364 Fig 36
244. interrupted This leads to a plateau shape for that time and thus to a false quantitative result When this option is activated the software interpolates a reasonable course of the chromatogram The option is limited for runs with a time constant lt 1s Warning This option leads to a better shaped chromatogram however the quantitative results are still not fully reliable For accurate and reliable qualitative and quantitative results the run should be performed twice with and without scanning Scan if a peak is detected At any detected peak the software will perform a scan automatically if this option is activated All further settings in this area are valid for the whole method You may decide whether the scan shall be performed At the peak apex or At the beginning of the peak with the given Delay min With the Slope HAU sec and at Height AU settings you can define the thresholds necessary for evaluating the signal as a peak The unit MAU depends on the configuration setting Use channel defines the data channel where the analysis is performed You cannot change this selection during a run However you can change the measuring wavelength of this channel as often as necessary Scan column in time table If the option Acquire 3D data is enabled a new column Scan will be available in the time table for the detector K 2600 If you want to perform a time defined scan enter a new line into the table with the d
245. ion Injection volume The injection volume has to be entered for the AS 360 560 and 565 in fact as a set of three parameters The possible values depend on the configured syringe They are summarized in the following table Syringe Air Gap Sample Into Loop Sum 250 5 to 120 Oto 230 240 500 5 to 240 Oto 470 480 1000 5 to 480 Oto 950 960 2500 5 to 800 Oto 1590 1600 Includes the air gap twice Air Gap A defined Air segment is used to separate the sample plug from the wash liquid in the sampler lines Sample Defines the actual sample volume of sample taken from the sample vial Into Loop Defines the volume required to transport the sample into the sample loop Aux Two auxiliary outputs four for AS 460 and 465 can be programmed At the given times a pulse will be sent to the selected output number Mix Methods This button appears for the AS 460 and 465 only Click the Mix Methods button to create a program of sample preparation The Mix Methods set up dialog will appear Creating an Instrument Control Method Fig 183 Fig 184 Fig 185 129 Mix methods Mis program Hi Method Parameters OK Cancel Kontron AS 460 465 Mix Methods Clicking the New button first the method type drop down list becomes active Click the New button to add one more line to the programmed methods select the Method Type from the drop down list The choices are Add Mix and Wait Method type Add bi Am
246. ired and no time program will be executed for this channel As many channels are activated at least one as many wavelength columns appear in the spreadsheet below The wavelength table is a spreadsheet to enter your time detector program In the first row at time 0 00 min enter the initial wavelength s in nm The editing principle including programming the digital outs is the same as for the time gradient program described in the chapter Instrument Setup Pumps Pump Program Autozero at WL change Check this option if you wish the signal to be zeroed at the moment the wavelength is changed to define the current signal as the baseline Action at the end of run The detector S 2550 allows not only to switch off the lamp at the end of run but also to switch the device into Standby You should switch on the lamp either manually or from the direct control window before you start the next working session After power up allow 20 minutes for the lamp to stabilize before running samples that may require sensitive measurements Scan nm Range xxx yyy nm This section allows you to specify the scan spectral interval in nm Start Enter the start wavelength for the scan in nm End Enter the end wavelength for the scan in nm Acquire 3D data You have access to this selection field only with the PDA option The displayed range depending on the detector type and the installed lamps will only be updated if you open that wind
247. is not enabled for the K 2700 detector The choices are Deuterium and Halogen lamps Scan nm Range 189 1024 nm This area is used to specify spectral parameters used for your test measurements The range displays the minimal and maximal wavelengths available for the detector The minimal and maximal wavelengths are the limits which are defined by the configuration of the detector The Start of Scan is the minimal wavelength with a default value of 200 nm The End of Scan is the upper limit of the scans to be measured with a default value of 500 nm The Bandwidth is a measure of the range over which the calculations and measurements are used to calculate the intensity for a particular wavelength For example with an 11 nm bandwidth the intensity for 250 nm would be calculated from 245 nm to 255 nm The wider bandwidth Creating an Instrument Control Method results in an increased signal to noise ratio because the multiple measurements cause the random noise to be cancelled out That is if the noise is truly random more measurements will decrease the noise because for each positive excursion in the noise will have a good likelihood of being cancelled by a negative excursion But the signal since it is not random will remain the same over multiple measurements Bandwidth is a tradeoff with a high value decreasing the noise but also decreasing the possible resolution which can be measured 5 nm is the default value but if your pea
248. is option may be used for the shutdown methods You should switch the lamp on either manually or from the direct control window before you start the next working session After power up allow 30 minutes for the lamp to stabilize before running samples that may require sensitive measurements Scan nm Range 190 740 nm This section allows you to specify the scan spectral interval in nm Start Enter the start wavelength for the scan in nm End Enter the end wavelength for the scan in nm The scan section limits Start and End are important even if no PDA option is available All measuring wavelengths in the used channels 1 to 4 must be within these limits Acquire 3D data You have access to this selection field only with the PDA option The Advanced button becomes accessible if the Acquire 3D data check mark is set Scan nm Range 190 740 nm Start 200 End 500 Advanced i Acquire 3D data Typicaldatasme 1 42 MB Detector K 2600 setup window with PDA option A click on the Advanced button will open the following dialog box Creating an Instrument Control Method 97 Fig 141 K 2600 Advanced Settings W Scan iba peak is detected Atthe peak apes using Slope m ll zec 0 010 f At the beginning lite Sele Sa a Height ml 0 100 Use channel H3 Detector K 2600 Advanced setup window Interpolate peak profile after scan While scanning the data acquisition of the detector is
249. is value requires that a reference spectrum be included in the peak table See the section on Analysis and Calculations for details Spectral Display The controls in this group specify the content and labeling of the spectrum graphs Show Lambda Maxima Checking this box will annotate each spectrum graph with the 3 largest lambda max absorbance Show Lambda Minima Checking this box will annotate each spectrum graph with the 3 largest lambda minima absorbance Background Correction Checking this will cause each apex spectrum to be corrected for background using the chromatographic baseline prior to being used elsewhere in analysis Refer to the PDA Analysis and Calculations section for details on the formula used Filtering Type This allows the user to specify a mathematical filtering function to be performed on all spectra extracted from the 3D data during analysis Refer to the PDA Analysis and Calculations section for details on the formula used for each filter Graph Height This specifies the relative height of the spectrum graph A value of 100 corresponds to a standard sized graph larger value may be selected to provide more a detailed plot Graph Properties Pressing this button displays the graph properties dialog as found on the standard Spectrum graph Peak Table When using the PDA option certain columns will be added to the peak table that enable you to analyze peaks from the PDA detector Detectio
250. ized Instrument Status User Defined Detector The detector status tab of a user defined detector displays only the monitor area No direct control is possible Creating an Instrument Control Method 155 Monitor O System Status T User Defined Detector Monitor Run Status HOLD Signal mv 0 598 Communication 44D interface 3 Interface Manager 5000 Device 3 at COM15 S N 83014 Version 1 05 Fig 229 Detector status tab user defined detector Run Status The status HOLD will be displayed when no program is running otherwise the Run Status displays a run time in minutes elapsed from the start of the run Communication A D interface The active state of the communication with an instrument again is represented by the green y symbol Only in this case you will have access to the KNAUER Net Device Information Click the l button to display a list of Knauer networking instruments which are connected to that COM port A list containing serial numbers device names and instrument software versions for Knauer networking instruments will appear Device Information Serial No Device Software Version Manager 5000 1 05 Fig 230 Device information for connected Manager 5000 interface module Instrument Status Virtual Detector o System sin w DM Virtual FC Monitor Direct Control Run Status HOLD Signal i 0 0 Apply Signal 0000 mv External Trigger ON i virtual Bos 3 0 For Help press F1 M
251. ks have substantially wider UV spectra you can increase the bandwidth value in ChromGate to obtain a lower noise level Mode Select the signal mode for your measurements from the drop down list The choices are Absorption and Intensity Open Close Shutter A click on this button will open close the shutter The label will be changed accordingly Filter On Off S 2600 only You can click on this button depending on whether or not you are using an edge filter Dark Signal Click the button to measure the dark current This operation is enabled only for devices equipped with a shutter Set Reference Click the button to measure a spectrum to be used as a reference spectrum Spectrum Click the button to measure an absorption or intensity spectrum depending on the signal mode selected for the measurement The measured spectrum will be shown in the spectrum view Instrument Status Fluorescence Detector RF 10Axl RF 20A MM Instrument Status O System Status EJ RF 1084d Status HOLD DirectControl Emission nm 550 Excitation wavelength 254 Emission wavelength 550 Excitation nm 254 E Sensitivity MED h Autozero Signal Gain x1 v Event out Sensitivity Recorder range x1 v Event mark Output mode Emission not corrected Y Output Mode Emission not corrected Autozero Serial Port COM3 SN 47235 Diagnostics Gain Fig 226 Detector Status tab Fluorescence Detector RF 10AxI S
252. lar Mass For each calibration peak enter a Molecular Mass that is representative of the calibration standard peak Average Time min Enter the time in minutes of the calibration standard peak If you have used the graphical Define SEC Peaks command the peak times will be entered already for you Note the time is converted to seconds for calibration and Molecular Mass calculations Use Constants Select the constants to be used in the calibration either Mark Houwink or Viscosity from the drop down selection Based on your selection the appropriate columns in your spreadsheet will become active SEC Option 271 Std Mark Houwink k If you have selected Mark Houwink for the Use Constants field enter the k value for the standard in this field Std Mark Houwink a If you have selected Mark Houwink for the Use Constants field enter the a value for the standard in this field Intrinsic Viscosity If you have selected Intrinsic Viscosity for the Use Constants field enter the value here Otherwise this field is not active Time Slice Select a time slice width in seconds for the calculations The slice width will determine the frequency of Molecular Mass fractions reported If a Slice Report is generated it will be based on the slice width you select here The software will force this number to be an integral of the data collection rate during the analysis Baseline Start and Stop Times The baseline start and st
253. le are used to adjust the calibration curve to better represent the sample being analyzed 212 SEC Option All samples analyzed with a Universal Calibration must have the Mark Houwink constants or intrinsic viscosity specified here If you analyze samples with different values you must create a new method with the values for those samples Reference Peak Time min Select this box if you are using a retention time reference peak to adjust for changes in flow rate All elution times volumes used in calculations will be offset by the time difference between the value entered here and the time of the reference peak in the sample Enter the expected retention time for the reference peak in minutes Reference Window If you are using a reference peak enter a value for the window for the reference peak A peak falling within this of the expected retention time for the reference peak will be considered the reference peak If two peaks fall within the window the peak closest to the center will be chosen as the retention time reference Fit Type Select a calibration curve fit type from the drop down list The choices include Point to point Linear Quadratic Cubic 4th Degree Polynomial and Spline Goodness of Fit The calibration curve for the data in the SEC Setup will be displayed in the calibration curve window The r value which represents goodness of fit of the calibration points to the curve selected will b
254. licking the button will switch on the lamp While the deuterium lamp is heating the inaccessible button shows Heating AZ button Direct Control If you click on this button the detector will perform an autozero which means the current signal will be set to 0 zero This button will also be active during a run please be careful then Clicking this button if a peak elutes will make the peak information area maybe also retention time unusable Valve s The left handed part of the Valve s section shows the valves current position Valve s Valvel Valve Valvel Valve Position 3 3 ASM2 1L status tab Valve s Position Status The current position of all configured valves will be shown For each valve the name given in the configuration is shown If the AMS2 1L is configured using the Auto Configuration option the default name for a valve is Valve followed by an extension for the valve s position in the ASM2 1L case L left M middle R right For Knauer two position valves the position can be either I injection or L load for Valco two position valves A or B For multi position valves the position can be 1 16 depending on the valves configuration The right handed part of the Valve s section allows for direct control of the valve s independently from the method setup During a run this part is not accessible Creating an Instrument Control Method Click on the small arrow key Tlofth
255. ll be offset by the time difference between the value entered here and the time of the reference peak in the sample Enter the expected retention time for the reference peak in minutes Reference Window If you are using a reference peak enter a value for the window for the reference peak A peak falling within this of the expected retention time for the reference peak will be considered the reference peak If two peaks fall within the window the peak closest to the center will be chosen as the retention time reference Fit Type Select a calibration curve fit type from the drop down list The list includes Point to point Linear Quadratic Cubic 4th Degree Polynomial and Spline Goodness of Fit The calibration curve for the data in the SEC Setup will be displayed in the calibration curve window The r value which represents goodness of fit of the calibration points to the curve selected will be displayed above the curve A value of 1 00 indicates perfect fit Select the Time or Volume button at the bottom of the calibration curve to display the x axis of the curve as either retention time in minutes or retention volume If you select volume enter the flow rate in ml min Remove calibration curve outliers To temporarily remove an outlier from the calibration curve click on the point with your mouse It will change color and the curve will be re calculated with the point removed Time Volume Select the mod
256. ll force this number to be an integral of the data collection rate during the analysis Baseline Start and Stop Times The baseline start and stop times in minutes can be entered manually or selected graphically using the SEC buttons on the chromatogram toolbar Results Range Start and Stop One or more ranges of the chromatogram must be defined to be used in calculation and reporting of the Molecular Mass distribution Each range is determined by the results range start and stop values The times in minutes can be entered manually or graphically using a stored chromatogram SEC Option 269 Horizontal Baseline Select this button if you wish the SEC baseline to be drawn as a horizontal line forward from the Baseline Start Time to the Stop Time The horizontal baseline stops when it crosses the data trace A threshold value in microvolt must be specified to ignore any detector baseline noise in the trace that would cause the horizontal projection to stop when it encounters the noise in the trace If the Horizontal Baseline reaches the end of the detected peak before the programmed Stop Time the range will stop where the Horizontal Baseline crosses the data trace Sample Data If you have selected Narrow standards calibration this field will appear empty Reference Peak Time min Select this box if you are using a retention time reference peak to adjust for changes in flow rate All elution times volumes used in calculations wi
257. lot rotation and axis setup features Elevate w Holl Rotate Rotate rd Spin Zoom Reset Avis Setup Properties 3D Plot movement selections Click on the type of plot movement you wish to use elevate roll rotate rotate XYZ or spin A checkmark will appear next to the selected movement option Once you have selected one of the options you will be returned to the plot and the cursor will indicate the type of movement selected Hold down the left mouse button and move the cursor in the direction you wish to move the plot The plot will move as you move the cursor The movement option will remain in effect until you turn it off When finished click the right mouse button and de select the movement option If you wish to return to the original view select the Reset command These values can be viewed or changed using the 3DPlot Properties dialog ChromGate amp PDA Option 197 Fig 290 3D Properties This dialog allows you to set the properties of the 3D plot 3D Data Graph Properties General Ass Setup Style Colors amp Color Range Full Spectrum h C Grayscale Background EE gt na Wire Mesh Axes C Display Detail Less mc More Print Quality Corre r rm Smooth ualiky faster slower Viewing Performance f Light Source Use z00m rotate bounding box Rotation 45 Elevation 45 Fall lo oom 100 as Cancel Style
258. low ml min The actual flow will be shown in units as defined in the instrument setup 170 Knauer Instrument Control Method Options Knauer Instrument Control Method Options General Settings Some general method settings belong exclusively to the Knauer instrument control They are accessible by the METHOD menu Runtime Settings The runtime setting options are general settings The selection is valid for all ChromGate methods until it is changed Runtime Settings A If emergency during run i detected then Cancel W Switch all lamps off W Switch all ovens off Direct Control during rur Advanced Enabled F Fig 249 Knauer Instrument Control Method Option Runtime Settings Emergency during a run options In the case that an emergency situation is detected the software can be forced to stop all pumps and or to switch off all lamps and or switch off all ovens If enabled this option is valid for the currently used computer Direct Control during a run If this option is enabled all devices can be controlled manually using the Direct control section of the device s instrument status tab The direct control during a run option is a valuable tool for method development but can also lead to the possibility for unintended changes to the method For this reason it is recommended that this option not be enabled especially for routine methods All changes during a run will be added to the instrument acti
259. ls W i W 2 H Ha Bandwidth nm 8 fg ae Br a Time e Dig Out Comments EN he cas ena Commerts O00 254 254 00 la 1000 254 254 00 Detector S 2600 wavelength program table Used Channel Check the box es to turn the acquisition for the selected channel s on If the box is not selected no data will be acquired and no time program will be executed for this channel As many channels are activated at least one as many wavelength columns appear in the spreadsheet below Bandwidth nm The bandwidth is a measure of the range over which the calculations and measurements are used to calculate the intensity for a particular wavelength For example with a 10 nm bandwidth the intensity for 250 nm would be calculated from 245 nm to 255 nm A wider bandwidth results in an increased signal to noise ratio because the multiple measurements cause the random noise to be cancelled out That is if the noise is truly random more measurements will decrease Creating an Instrument Control Method 103 Fig 152 the noise because for each positive noise excursion there is a good likelihood that it will be cancelled by a negative excursion But since the signal is not random it will remain the same over multiple measurements A high bandwidth value will decrease the noise level but also decrease the possible resolution which can be measured The default value is 8 nm but if your peaks have substantially wider UV spectra
260. lves have the two positions 1 6 and 1 2 Wait The WAIT action can be used to let the autosampler wait for a programmed time with maximum of 9 h 59 min 59 sec During WAIT the needle turns back to home position if the next action is not at the same position Should the needle be required to remain in the current position it is necessary to program a dummy step after the WAIT in which an aspirate or dispense action of 0 ul is programmed at the actual position Compressor With the compressor action the compressor is activated to transfer air pressure to a vial The compressor will stay active until it is switched OFF The compressor will be automatically switched off at the end of the needle wash routine if a needle wash is used Auxiliaries With the Auxiliaries action the four standard auxiliaries contact closures can be controlled see time base events Wait for Input The autosampler will wait for one of the four inputs to become HIGH or LOW before continuing with the next step Prog Output The Prog Output action is used to define two programmable outputs contact closures These are similar to the auxiliaries Code Output The Code Output action is used to program the output to the connector P3 TIMED OUTPUTS on the rear panel of the autosampler This is a HEX output in the range 0 to 15 F Markers The markers normally generated by the autosampler are not active in the user defined method but can be programmed in this s
261. m tab of PDA Options the settings on the Purity tab of PDA Options or by setting the properties of the Peak Purity Pane Background subtraction is always the first operation performed on a spectrum Ifa background correction is attempted on a spectrum after another operation has already been performed including an operation specified in the Spectrum tab of PDA Options a message box Is displayed and the background correction request ignored Background subtraction may only be performed once on a spectrum Ifa second background correction is attempted on a spectrum after the operation has already been performed including the operation specified in the Spectrum tab of PDA Options a message box is displayed and the background correction request ignored A background correction is performed as follows 1 The spectra from the baseline start and baseline stop times for the peak are extracted from the 3D data The Max Plot is used to determine the peak that is used 2 For each spectrum in the peak a corresponding background spectrum is generated by linear interpolation between the baseline start and baseline stop spectra 3 These background spectra are subtracted from the original spectra While compensation for background will provide more precise purity calculations it can slow down re analysis of large data files Calculating Total Purity Total purity for an integrated peak is calculated as follows 1 The purity calculati
262. me Settings Fig 251 Check run time when saving method ChromGate allows different run times for all devices If you wants to have the same run time for all devices and let the software check if e g after a change all devices have the same run time enable this option If there are different run times the software displays an error message with the run time of all devices The run times must be equalized before you can save the message Run time check Failed One or more instruments have different run time S2550 15 00 min 51050 10 00 min Error message Run time check failed If enabled this option is valid for all instruments of the Enterprise it is not limited on the currently used instrument or computer That means that the run time will checked for all instruments and therefore in all methods that will be stored on the mentioned Enterprise machine System wake up and all lamps on If the option System wake up and all lamps on is enabled and a date and time is selected all device switched to Standby and the lamps of all connected detectors will be switched on at the selected time The method must be opened and the detectors switched on or in Standby at this time The wake up date time you can set by direct editing the time in the date and time line If you click on the arrow key H you can select a date from a monthly overview refer to the picture below Please note that Standby only works for all Azura
263. med events program has finished Mix Methods Click the Mix Methods button to create a program of sample preparation The Mix Methods set up dialog will appear Mix methods flis program Method type Add Mis Hew Beer wa N Method Parameters Cancel Fig 165 Autosampler 3900 3950 Mix Methods Fig 166 Clicking the New button first the method type drop down list becomes active The mix method can contain a maximum of 15 programmable lines including the END OF MIX command Click the New button to add one more line to the programmed methods Select the Method Type from the drop down list The choices are Add Mix and Wait Method type Add Method type Add mount Amount ES Fram From Sampe To To Destinatior Sample Destination Commit Commit Autosampler 3900 3950 Mix Step Add Specify the Amount of a liquid in ul max the syringe volume to be aspirated from a source choices are Sample Reagent A Reagent B and Wash and dispensed to a target choices are Sample and Destination The aspirate and dispense speed depends on the selected syringe and syringe speed The reagents and transport solvent are located at the following positions on the tray Transport solvent vial 85 Reagent A vial 86 Reagent B vial 87 116 Creating an Instrument Control Method Method type Mis Amon Mix times EFI Fig 167 Autosampler 3900 3950 Mix S
264. min Reference Window 10 Print Export Settings a 15 20 25 Enable Analysis Click this box to enable SEC calculations for the current channel Each channel can have its own analysis conditions Molecular Mass Enter a series of Molecular Mass s that will be used in determining the broad range calibration curve Average Time min Enter the time in minutes for the Molecular Mass specified If you have used the graphical Define SEC Peaks command the peak times will be entered already for you Note the time is converted to seconds for calibration and Molecular Mass calculations Use Constants Select the constants to be used in the calibration either Mark Houwink or Viscosity from the drop down selection Based on your selection the appropriate columns in your spreadsheet will become active Std Mark Houwink k If you have selected Mark Houwink for the Use Constants field enter the k value for the standard in this field Std Mark Houwink a If you have selected Mark Houwink for the Use Constants field enter the a value for the standard in this field 216 SEC Option Intrinsic Viscosity If you have selected Intrinsic Viscosity for the Use Constants field enter the value here Otherwise this field is not active Time Slice Select a time slice width in seconds for the calculations The slice width will determine the frequency of Molecular Mass fractions reported If a Slice Report is generated it
265. mmunication port on your PC where the autosampler is connected Serial Number Type in the serial number of the detector you are using Loop Volume Enter the volume of the installed loop in ul Sample Tray Select the type of the installed sample tray The choices are 96 vials of 1 5 ml and 48 vials of 5 0 ml Tray cooling Select this box if you have a built in Peltier cooling option installed and want to cool down your samples The temperature setting will be done within the instrument setup Partial loop fill Check this box to select the partial loop fill injection mode for the Basic Marathon Plus only Add Info Click the button Add Info to enter a more detailed description of the autosampler Enter additional information or comments to be printed out when you print the instrument configuration When complete click OK to exit the dialog and return to the instrument configuration icon list The autosampler 3800 must be set in to serial mode to be controlled by software Press the buttons lt F gt and lt 4 gt on the autosamplers keypad the display will show then Serial Mode Press FO to exit Setup and Control of Knauer HPLC Systems 71 Fig 99 Configuration Autosampler 3900 Smartline Autosampler 3900 and K 3900 Midas Autosampler 5 3900 Midas Configuration Mame 3900 Serial number 44444 Communication Device ID 60 Loop volume ul 11 OG Serial Part COM1 Tubing volume ul
266. mp OFF at the end of run F Autozero at WL change Scan nm Start 200 End 400 Bandwidth 1 Signal mode M Acquire 3D data Wavelength Program Used channels IV 1 T 2 Bandwidth nm E Events c nt min length 1 0AP SES 000 254 00 10 00 254 00 Advanced Time 0 0103671 Minutes Amplitude 254 Wavelength nm Wavelength nm Wavelength nm UV Detector S 2600 tab Autosampler Tabs Different autosamplers can be controlled by the ChromGate software Any method window will include the tab for the configured one to setup the needed injection parameters You will find detailed setup information for the autosamplers starting on page 111 Fraction Collector Tabs Several fraction collectors including the Knauer multi valve FC and a virtual one can be controlled by the ChromGate software Any method window will include the tab for the configured one to setup the needed control parameters You will find detailed setup information for the fraction collectors on page 235 Miscellaneous Instrument Tabs Further instruments to be controlled by this software are Manager 5000 5050 IF2 analog digital outputs of the Knauer A D converters Knauer Switching Valves Valco Switching Valves Column oven 4050 Jetstream oven Flowmeter For each of them a tab will be shown in the method window as far as the instrument has been configured If you have included more the one of the Knauer or V
267. mplitude 0 324 mAU 250 To zoom in on a portion of either the contour plot chromatogram or spectrum hold the left mouse button down move the mouse over the plot until the area of interest is highlighted and release the mouse button To quickly move to the previous level of zoom double click on the plot To zoom out to the full plot after multiple Zooming operations use Ctrl Z or use Shift double click in the window or click the right mouse button anywhere in the window and select Full Unzoom from the pop up menu Synchronize 27 You can synchronize the axis and zoom limits with the other two plots using the Synchronize button on the PDA toolbar When the contour plot is zoomed the time range of the chromatogram and the wavelength range of the spectrum plot are automatically adjusted to the same range as the contour When synchronized zooming has been performed the x axis plot limits for the chromatogram and spectrum plot are changed To unzoom these plots to the limits of the data you must select Full Unzoom from the contour plot context menu Mixed View Arrange Button If you have changed the size of the 4 views clicking on the Arrange button will arrange all the views in the same size again Mixed View Actions Button Fig 297 This button presents the following actions Actions El Options Search Library Overlay Spectra Select Peak for Similarity Purity Display Add to Multi Chromatogram T
268. n Select the basis for the identification of the peak If you choose Ret Time only the retention time will be used for identification of the peak If you choose Ret Time with Spectral Confirm the Similarity of the peak s spectrum to that of a designated reference spectrum will be used in addition to the retention time as the basis of peak identification Spectrum If you want Similarity to be used as a basis for peak identification then click on the arrow to the right of this field to specify the stored reference spectrum to be used for comparison During identification this reference spectrum is compared to the peak apex spectrum and a similarity index is 222 PDA Analysis and Calculations computed A peak is considered identified if this calculated similarity index is at least the value specified in the Similarity column of the peak table If Similarity is not specified as a basis for peak identification then this field is ignored Similarity If Similarity is specified as a basis for peak identification then this field specifies required minimum similarity index for a peak to be considered identified During identification the reference spectrum see previous section is compared to the peak apex spectrum and a similarity index is computed A peak is considered identified if the calculated similarity index is at least the value in this field If Similarity is not specified as a basis for peak identification then this field i
269. n also freeze the current gradient status for a time you have entered by pressing the lt Freeze Gradient gt button Freeze the actual gradient for 10 00 min will be performed immediately Freeze Gradient If the run time is to be prolongated use the menu Control Extend run Gradient modify window freeze gradient This will extend the pump table and therefore also the pump s run time In the following example the gradient 100 A 0 B 0 A 100 B in 10 minutes will be freeze for 2 minutes from 3 34 5 34 minutes at 3 34 minutes the freeze command was sent The gradient freeze will be sent and executed if you click on the lt Freeze Gradient gt button Creating an Instrument Control Method Set New Gradient 143 Modify the running time program current run time 2 82 min Time 0 00 Flow 0 000 xa 100 ze fo 2c fo xp fo Freeze the current gradient for 1300 min will be performed immediately If the run time is to be extended use Control Extend run from the menu Commit Line Delete Line Enter the new gradient line in the form Time min Flow ml min B 3C D Send Gradient Freeze Gradient 10 Comp B Comp C fi 0 D 72 28 0 0 100 0 Ee KAT ARRET eeen Th Torir AE ereget i beaccsaencsesteteaaabeaee Lar AE Fk 5 4 er p 0 5 10 Gradient Time min Close Fig 212 Gradient modify window f
270. n and desired options have been selected press OK to return to the Instrument Configuration dialog box indicate the desired name of the instrument and press OK to return to the main screen An existing configuration can be edited by right clicking on the icon for that configuration and selecting Configure Instrument If you alter a configuration make sure that you first make a copy of the existing instrument if you want to maintain both configurations System Options The System Options dialog box is used to access the following e System Suitability used to calculate a number of parameters pertaining to the overall efficiency of the system such as the resolution repeatability peak asymmetry number of theoretical Short guide ChromGate KNAUER Instrument Control 15 plates noise and drift A detailed description of the setup of this feature you can find in the ChromGate System Suitability Setup chapter below e SEC used to calculate a number of parameters for SEC GPC related chromatography lt adds special calibration methods and report options Please find more in the SEC Option chapter in this manual and in the original manual Elite SEC found on the installation DVD in EZChrom Elite 3 3 2 English Manual Optional Software Manuals e PDA used to calculate a number of parameters that are related to the use of photodiode array detection detailed discussion of this feature is presented in the chapters ChromGate PDA O
271. n ml min the gradient components in and pressure depending on the selected unit in MPa bar or psi Flow Status The flow shown here is the current flow of both pumps set either by direct control or a method Components Status The Components show the current pump gradient set either by direct control or a method Pressure only one pump has a pressure sensor Status The shown pressure is the current system pressure measured by the pump s pressure unit Please note that also a pressure may be measured if the pump is stopped e g if another pump is running or if the flow path is blocked 158 Fig 234 Creating an Instrument Control Method The right handed part of the HPG section allows for direct control of the pumps independently from the method setup Please note that only A can be entered the B component of the gradient will be completed automatically by the software Only a parameter the check box is enabled for can be edited and the changed value will be send by clicking the lt Apply gt button Flow Direct Control To enter a flow enable the Flow check box If enabled you can enter a flow which will be the resulting flow of both pumps Click on the upper lt Apply gt button same line as for the Flow and A values to start the pumps or change the current flow If the flow rate is out of the acceptable flow range defined by the mounted pump head an appropriate error message will be shown
272. n of your autosampler are contained on the Method Instrument Setup AS 3800 tab The control parameters for the autosampler become part of your method and sequence files Instrument setup 10 as 30 3 Oven 5 4050 5 2500 2 Aux Traces PA Trigger Injection type Aux Dispenser controlled Preflush volume pil 30 Time ON E nia B l i I Enable Aux C Pressure controlled Flush time sec 1 Pa Time OFF 0 min 0 F Needle wash Time ON fo min o Temperature Control Enable Aux2 E enod Time OFF 0 min fo Time 9 39201 Minutes Amplitude 254 Wavelength nm WL 2500 Temperature PC Flow E e lt En S E Wavelength nm Autosampler 3800 setup window Injection Type The AS 3800 can perform injections based on the flushed loop or partial loop filling for the Plus only principles Loop filling can be done on a volume basis controlled by the digital dispenser system or on a time 112 Creating an Instrument Control Method basis based on the headspace pressure of the sample You can choose between the dispenser controlled or pressure controlled injection type Dispenser Controlled Injection For the dispenser controlled injection specify the Preflush Volume 30 999 pl Pressure Controlled Injection For the pressure controlled injection specify the Flush Time 1 999 seconds Check the Needle Wash button to select needle w
273. n the ASM 2 1L is ready to run pump is found valves have been initialized detector lamp is on The status On is also shown if a Pretreatment is running please refer to Pretreatment section on page 89 During a run the status Run and the current run time in minutes will be shown The Off status will be shown if no connection with the ASM 2 1L can be established or if the detector lamp is switched off Events Digital Outputs The current state for each Event Digital Output will be displayed in this control The On state is represented by a red active Light Emission Diode LED symbol 8 the Off state including waiting for pulse is represented by an inactive grey LED symbol af Pump The left handed part of the Pump section shows the pump status as flow in ml min and pressure depending on the selected unit in MPa bar or psi Creating an Instrument Control Method 157 Fig 233 Flow ml min Status The flow shown here is the current pump flow set either by direct control or a method Pressure unit only for P4 1S Status The shown pressure is the current system pressure measured by the pump s pressure sensor The unit depends on the pressure unit selected in the pump s configuration MPa bar or psi Please note that also a pressure may be measured if the pump is stopped e g if another pump is running or if the flow path is blocked If only a pump without a pressure sensor is configured the pressure sec
274. n the Position row To switch the valves specify the required positions and click the Set Position button The choices are BLANK and I Inject and L Load for a 2 position valve 1 through 6 for a 6 position valve 1 through 12 for a 12 position valve and 1 through 16 for a 16 position valve The BLANK space means that the valve will not change its state after pressing the Set Position button MM Instrument Status P System Status El Valves Run Status HOLD Valves Status Direct Control eneren teen 2 pos 2 pos 12 pos Position set Postion o Valve Valve 2 Valve 3 2 pos 2 pos1 12 p03 3 Communication Status tab switching valves If there is for any reason the communication to a valve interrupted the corresponding field for the valve position is highlighted and the position is indicated as zero 168 Creating an Instrument Control Method Instrument Status Column Oven 4050 and Jetstream Fig 246 Fig 247 ME Instrument Status O System Status 5 1000 lt 7 as 3950 amp Oven S 4050 Y AF 1041 Monitor Direct Control Run Status Temperature C 30 Set Min reached temperature 5 0 C Max reached temperature 143 6 C State Ott Cooler Temperature C 15 Set Working time 519h Temperature za E Leak Alarm Level 30 Set Cooler Temperature 109 C Digital Outs Events 000 z DO Set Digital Outs Event
275. nce Changing parameters for the contour graph in a custom report works exactly as in the Contour view PDA Insert Report Items Fig 315 When the PDA option is enabled the following report items will be available to insert into a custom report Library Search Report Purity Report and Spectrum Report Library Search Report This command will insert a table of library search results in your custom report To modify the parameters of the report do a right mouse click in the report table then select Properties The following dialog will appear Library Search Report Properties Search libraries for f Apex spectra of peaks Detected on Al PDA Traces hv h Limit to named peaks only Limit to time range Min C specific spectrum p Spectral display Graph height 100 Z Graph Properties OF Cancel Il Search Libraries for This specifies what peaks should be searched for the report When Apex Spectra of Peaks is selected the Specific Spectrum button is disabled When A Specific Spectrum is selected the other controls of this group are disabled Detected on This allows the user to select the channel or channels from which peaks will be detected Limit to named peaks only This allows the user to restrict the peak selection made above to a time range When checked only peaks within the specified time range will be searched When this box is unchecked the Time Range edit fields
276. nd the format and tabs is dependent on the configuration of the system Open the Instrument Status Window using the menu sequence Control Instrument Status The Instrument Status window contains the status tabs for all configured instruments and as shown in the figure below the system status tab providing an overview for the whole system 30 Fig 29 Short guide ChromGate KNAUER Instrument Control ME instriment Status System Status y 5 1000 EJ AS Triathlon MS s 2600 Flow 1 000 ml rmirn G AS Status Flushing J iS Chn1 254 nm 0 000 m l a FA mY Pressure 5 7 MPa Junuama E ESF Current Vial 1 Chn2 288 nm 0 000 m l Lins ZA 60 B 40 Tray Temperature n a ZE 0 2D 0 mur m Chn4 n a AAA waiting for Trigger Instrument Status window system status tab of a running method The running method is visualized by the dark dotted lines between the displayed instruments For each pump identified by name and serial number the actual flow pressure is displayed If a gradient system is configured the solvent composition also will be shown For each detector channel the wavelength and output values are shown Similarly for all included instruments the relevant data will be displayed e g the actual switching positions in case of the valves All these information you also find on the single instrument tabs of this window for details see pages 139 169 On th
277. ne for more than one instrument pump and or valve drive in a method it is required to set for all of them the same earliest time All pretreatment procedures will start simultaneously If a pump should exactly switch off after 5 minutes of pretreatment include a line with 0 ml min flow into the pretreatment s pump program Otherwise the pump may run for a longer time as it need some seconds to load the new flow rate from the method s normal pump program An example for both A pump should run for exactly 5 minutes before the injection and a valve should switch 2 minutes before injection from position 2 to position 5 means 3 minutes after the pump s pretreatment is started Here are the setup tables create a time table for the valve which starts with 5 minutes Pump 5 04 min 1 ml min 0 04 min 1 ml min 0 02 min 0 ml min smallest time step is 0 06 minutes for pump 1800 P2 1L or 0 02 min for all other pumps 0 00 min 0 ml min Valve 5 04 min pos 2 2 00 min pos 5 0 00 min pos 5 Creating an Instrument Control Method 91 Instrument Setup Detectors All Detectors Fig 134 In most cases you find at the bottom a unique graph part displaying the wavelength flow gradient or other related profiles With a right hand mouse click you get access to multiple setup possibilities of the profile For details see the ChromGate Data System User s Manual If the detector lamp is disabled the event outpu
278. ng the slope and the level are again quite similar to perform The only difference is that both the level and slope values are to be used as thresholds FC Parameters at 6 00 min ES Signal 2 Relative to previous event min f Ascending 0 Descending Signal Level Units 25 Use as reference for correction next events Action Actor Collect To C nest 0 position 12 Signal Slope Unitz min i 0 Expected interval min 10 2 Please notice that the parameters will be used in the Units selected for the Detection Channel Cancel Fig 336 FC parameters Signal Level Slope single event Additionally you have to select either the and option meaning that both conditions must been exceeded or the or option meaning that only one of these conditions must be exceeded This can be defined differently for the ascending and descending flange of the peak ChromGate Preparative Option 241 FC Parameters at 10 00 min Signal Peak Beg Level Units 50 f AND OR Peak Beg Slope Units min 10 Action Actor Peak End Level Units 20 C AND OF Peak End Slope Units mink f Please notice that the parameters will be used in the Units selected for the Detection Channel Cancel Fig 337 FC parameters Signal Level Slope Peak Recognition After closing the window the parameter will be designed for example as L and S L or S for single events and Lb or Sb Le or
279. nknown_sample It should also be possible to use two different Molecular Mass standards with two known MW values in any combination of Mn and Mw The Broad Range 2 method is a non linear calibration utilizing a broad range standard 258 SEC Option Universal Calibration Universal calibration allows a narrow range polystyrene standard calibration curve to be used to calibrate for a broader range of polymers Using the relationship between the hydrodynamic volume the product of the Molecular Mass M and the intrinsic viscosity n of the polymer and its elution time the narrow range calibration curve can be adjusted to more closely fit the sample being analyzed The intrinsic viscosity n of a polymer is experimentally determined from directly measuring the viscosity of the polymer solution It is related to the polymer Molecular Mass through the Mark Houwink equation nl Km where K and a are constants that vary with polymer type solvent and temperature Tables have been published that contain K and a constants for a wide variety of polymers Thus using published values for K and a or the measured viscosity of the polymer one can determine Molecular Mass information for the sample Applications of SEC SEC is used commonly for quality control of high Molecular Mass polymers Molecular Mass distribution is indicative of the physical properties of the polymer Such properties include strength flexibility and tackiness am
280. nnennnnnnannnnnnennanennnnnnnnrnnanennnnennn 107 Instrument Setup AUTOSQAMplers oocccoocccocccocncoccconococnnonononononnnonnnnononanonanonanenanes 111 Instrument Setup Autosampler 3800 ocoocccccccccncocncocncocnconnncnnnnanonanos 111 Instrument Setup Autosampler Knauer Optimas 3900 Midas 112 Instrument Setup Autosampler 3950 AliasS ooocccocnccccnccccncnno 116 Instrument Setup Triathlon Endurance Autosampler ooccoo 121 Instrument Setup Kontron Autosamplers coocccocncocncccncccnccnncnonononos 128 Instrument Setup Miscellaneous InstruMentS coocccocccocncocnconnconnconnccononanonanos 130 Instrument Setup Manager 5000 5050 1F2 VWOQ rrrunnnunnnannnanenanennnen 130 Instrument Setup Switching Valves ooccccccccoccccconocccnncncncnncnnnnos 131 Instrument Setup Column Oven 4050 rrrurnrarnnnrnnarnnnrrnnrrnarennrennnennner 133 Instrument Setup Column Oven Jetstream ooccocccccncccccccncconiconinnnos 133 Instrument Setup Flowmeter rrranrnnnnrnnnnrnnnnnnnnnnnanennnnennnnnnnnnnnnnennnne 134 Setting up Auxiliary Traces oocooncoccncccncccncccnoconoconoconononocanonnnonanonnnnnnnncnnnnnnnnnnnnnns 135 SOUND STIGE aia o 135 Setting up the Baseline Check r rrrrnnnnnnnnnannnanenanenanennnenanenanenanenanennnennnennnennnennee 136 Instrument Status of a running Control Metho0d cccoccccocnccccnccccnc
281. nstrument Control Method anarennevnnnevnnnvnnnnnnnnnnnnnennnnennnnnnnnnnnnnennnnennnnennn 83 IVS UIA IN SEP o ins 84 HSTUIMEN SE UP PUM DS uke 85 Instrument Setup Detectors oocccocnccocncconnoconnononconanoconnonnnnonannonannnnnnonannnnaninnons 91 All Detectors 91 Instrument Setup RI Detectors cooocccocnccocnccocnccccnonanconanccnanonanonnnnnos 92 Instrument Setup UV Detectors S 200 K 200 rrrrrnrrvrnnrrnvnnrrnvnnnen 92 Instrument Setup UV Detectors K 2000 K 2500 rrrrrrnnrrnvnnrrnvnnnnn 92 Instrument Setup UV Detectors UVD 2 1L UVD 2 185 S 2520 S 2500 K 2001 K 2501 venninne eiseege 93 Instrument Setup Kontron Detectors oocccoccconccocococncociconoconoconicononos 95 Instrument Setup K 2600 Detector ooccoocccocococicocncococonoconoconoconocononos 95 Instrument Setup S 2550 Detector oocccccccocccocccocncocncocnconnnocncononononos 98 Instrument Setup Diode Array Detectors S 2600 DAD 2850 DAD 2800 K 2700 and Kontron DAD 540 545 100 Instrument Setup Fluorescence Detector RF 10AxI RF 20A 103 Instrument Setup Alltech 650 Conductivity Detector 104 Instrument Setup User defined Detector ooccocccocnconncccncconiconononos 104 Instrument Setup Virtual Detector ooccocccocncocncocnconncconccononanonanos 105 Instrument Setup Assistant ASM2 1L rrrrannnnanenn
282. nt no setting can be made In LPG mode except the Kontron x25 pumps you can define the cycle time of the valves via the drop down list Control Pressure Limits Control Pressure limits MPa Mir 0 0 Max 400 Fig 123 Pressure limits in pump setup window This section allows you to enter the control pressure limits in the units which have been chosen in the pump configuration dialog The pump will automatically shut down its operation when the measured pressure IS out of the limits This option is not accessible in the case of the pump K 120 10P P2 1S or S 100 without pressure transducer Min Pressure Enter the minimum pressure allowable for your pump The zero setting disables the shut down due to minimum pressure Max Pressure Enter the maximum pressure allowable for your pump The default value always corresponds to the configured pump head setting above this value is not allowed If for the min and max limit 0 is set the software will not check for the pressure and the limits will only be monitored by the pump internally 86 Fig 124 Fig 125 Fig 126 Creating an Instrument Control Method Error message when pressure limits are out or range Solvent s EE 1800 5 Pump 420 amp 51000 E K 1800 E K Solwvent s methanol 15 E water C acetonitrile Solvent description in pump setup window The number of displayed rows depends on the configuration Isocratic sy
283. nt to collect data click on the desired instrument to present the Instrument Wizard dialog box Fig 9 on page 16 and select the desired operation If you select Run one sample the Single Run Acquisition dialog box will be presented Enter the desired Sample ID method and name e g sample dat and then press Start The chromatogram will be collected as described in the method During the data acquisition the chromatogram will appear in the window as shown below Short guide ChromGate KNAUER Instrument Control 29 E Channel A E BK Time 6 32264 Minutes Amplitude 0 001752 volts Fig 27 Single run data acquisition window lf you have selected Run a sequence of Samples the following dialog box will be presented Cs xl Sequence Harman App gt gt gt LAA Sequence name i Cancel Ain ange PS Help All Tower 7 Printing eon Al I Prim method reporis Print sequence reports Begin ran Email rac pient Ol T On start Da stop or eror i 4 AGRA Fig 28 Sequence Run Acquisition dialog box Enter the Sequence name and press Start The chromatogram for the first run will be collected when the conditions have been met Instrument Status of a running Control Method During Data acquisition you can access the status of the system by selecting Control Instrument Status on the main menu The window that will be presented is shown below a
284. nterpolation will be performed on the spectrum The new interpolated spectrum is then displayed along with the original spectrum Please refer to the PDA Analysis and Calculations section for details on how this calculation is performed Refer to the Data Analysis and Calculations section for details on how the values are computed Export Selecting this menu item will display a File Save As dialog When a valid filename is entered and Ok is pressed the currently displayed spectrum will be exported as an ASCII data file Refer to the Spectrum Export section for details on the data file format Overlay Spectra You can overlay spectra using the Actions Overlay Spectra command from the Mixed View toolbar When you select this option a dialog appears where you can enter the retention time from which you want to extract a spectrum You can also overlay spectra quickly by moving the wavelength cursor in the Contour or Chromatogram view to the desired wavelength To remove the spectra you have added to the view use the Clear overlays command from the right mouse click menu in the Spectrum view You can also clear selected spectra using the Trace Setup dialog accessed by doing a right mouse click in the view and then selecting Properties 210 Fig 307 ChromGate PDA Option Owerlay Spectra px Spectrum at 5 50 Miri Tip Drag the spectrum cursor on the Contour or Chromatogran view to select the spectrum Close Ratio Vi
285. ntify the instrument LAN interface dialog Setup and Control of Knauer HPLC Systems 79 To let the software search for your already connected and switched on device click on the button to start the search for all connected LAN devices of the currently configured type The option Use S N to identify the instrument must be enabled Select the desired device from the shown list The software will read out the information as serial number and will fill it automatically into the configuration screen To search for an instrument the device must be connected switched on and must have a valid IP address As an option you can enter the IP address of your device if the option Use S N to identify the instrument is disabled Please note that in case the device receives the IP address from a DHCP server e g a router the IP address may change if the device is switched off and switched on Add Valve Info Click the button Add Valve Info to add the valve specific description or comments for the selected valve Add Info Click the button Add Info to enter more detailed description of the valve group Enter additional information or comments to be printed out when you print the instrument configuration When complete click OK to exit the dialog and return to the instrument configuration icon list Configuration Manager 5000 5050 IF2 Fig 112 As with user defined detectors the Manager 5000 IF2 device must be
286. ny channels may be selected as the analysis channel for quantitative information e When an analysis is performed all multi chromatogram channels are automatically analyzed PDA Analysis and Calculations 223 e Each multi chromatogram channel is an average of the absorbencies monitored at each wavelength in the wavelength range The wavelength range is equal to the selected wavelength one half of the bandwidth Working Chromatogram The following apply to the working chromatogram e In the Channel Selection drop down list a single entry is added that identifies the channel as applying to the working chromatogram e This channel has a separate Integration Events Table Manual Integration Table Export Table and Performance Table associated with it These are separate from the tables of the Multi Chromatogram Channels above e When an analysis is performed the working chromatogram will automatically be analyzed e The working chromatogram is an average of the absorbencies monitored at each wavelength in the wavelength range The wavelength range is equal to the selected wavelength one half of the bandwidth e The working chromatogram is not available as a trace in Custom Reports Spectra Extracted from the 3D Data Two types of spectra may be extracted from the 3D data e Analysis Spectra One or more spectra automatically extracted from the 3D data during analysis and used for peak identification similarity and or l
287. o the spectrum Spectrum Smoothing Spectrum smoothing may be performed on a spectrum as a result of the settings on the Spectrum tab of PDA Options or by selecting Operations gt Smooth from the Spectrum View context menu Interpolation may be performed repeatedly on a spectrum The calculation is performed by doing a 9 point Savitsky Golay smooth on the spectrum data points Spectrum Derivatives Calculation of the 1 and 2 derivatives of a spectrum may be performed on a spectrum as a result of the settings on the Spectrum tab of PDA Options or by selecting Operations gt Smooth from the Spectrum View context menu Derivatives may be computed repeatedly on a spectrum The absorbance values of the 1 derivative of a spectrum are computed by calculating the differences between adjacent absorbance values to create a new spectrum The 2 derivative of a spectrum is defined as the 1 derivative of the 1 derivative of the spectrum PDA Analysis and Calculations 225 Upslope and Down slope Spectra The upslope and down slope spectra of a peak are identified by calculating the second derivative of the portion of the chromatogram containing the peak The two times at which the second derivative plot crosses zero are known as the inflection points For normal peaks i e non Negative peaks the Upslope Spectrum is the spectrum at the time represented by the first inflection point while the Down slope Spectrum is the spectrum at th
288. o use the destination vial or vials with Reagent A B C D Reagent A B C D Destination Vial Position Select the Reagent A B C D destination vial position Reagent A B C D Destination Vial Segment Select the type of the segment where the corresponding vials are installed The choices are Standard A segment LSV B segment Super LSV C segment and Micro vial D segment The Endurance Trays Tray configuration Plate type foals 2 3 L 5 7 8 Plate process order Rows O E O O Cy First transport vial Ho 11 12 13 14 15 Last transport vial py First destination vial fF rene 90000000 aan 00000000 Reagent C vial r 00000000 Y cm Fig 105 Tray configuration for the autosampler Endurance Setup and Control of Knauer HPLC Systems Plate Type Select the type of the installed plate The choices are 96 low wells 96 high wells 384 low wells and 48 vials Plate Process Order Select the order of vial processing The choices are in Rows and in Columns First Transport Vial Select the first transport vial position The vial can be placed in any of the positions 1 through 4 Select No if the transport vials are not used Last Transport Vial Select the last transport vial position lt 4 and gt first transport vial position The transport vials must be placed in a continuous row Select No if the transport vials are not used First Destination
289. od 87 No Acton at the end of run No Acton Flow Cf Standb rie Flow Con eee roy Fig 127 Possible action at the end of run for pump S 1050 P2 1L Pretreatment This option enables several pretreatment operations of pumps and valves before injection and starting the data acquisition For setting up see below on page 89 Solvent Type S 1050 P2 1L only Select in the Solvent Type menu the used solvent for a proper compression and flow correction Note that for an LPG pump only one solvent type can be selected Solvent Type Salvent s Default fl LE a E fi FETT fl it ra Cancel Fig 128 Solvent type settings for pump S 1050 P2 1L Fig 129 Pump Program Accordingly the spreadsheet for the pump program is displayed Up to four columns for the composition will be displayed Additionally for pumps with digital outputs the option for digital outs is given in a separate column The spreadsheets of the S 100 10P 20P P2 1S P4 1S K 120 K 501 and the Kontron pumps do not include this column Pump Program E EEG Comp A Comp a pomp C ans Outs EU 0 00 al 100 Oo 00000000 A ET 10 po 1 000 100 D O 00000000 Pump Program Time Flow Comp E E min mlnin 96 HEG O po 1 000 100 B 1000 1 000 100 Pump program in pump setup window Pump 1800 LPG B K 501 isocratic For all time programs the minimum time step between two line
290. of Knauer HPLC Systems analog input voltage to be digitized by an A D converter Use the Signal Range to optimize the signal to noise ratio of the measured signal Manufacturer and Model This area is for information only It contains manufacturing information about the box which may be required in service situations Kontron Interface Configuration Fig 34 Fig 35 If you are using a Kontron interface card click on the corresponding icon and then click Properties A dialog box will appear where you can configure the interface see Fig 34 If you configure the interface newly the message like Fig 31 on page 32 will remind you to enter the serial number of the interface device Interface setup Number of PC cards 12 Configure card 1 Card type Manufacturer Kontron Serial number 1 2345 Cancel Help Dialog box for configuring the Kontron interface card PCI AD Number of PC cards Select the number of PC cards used in the system ChromGate supports up to 4 interfaces Configure card Select the card you wish to configure Each card has to be configured separately Card type Select either ISA AD or PCI AD In case of selected PCI AD the setup window is changed to the following appearance Interface setup Number of PC cards Configure card Card type Base lO Address Memory Address Ox0D0000 Serial number 1 2345 Cancel Help Dialog box for configuring the Kontron int
291. of those devices the new generated driver has to be installed If the driver is installed its icon will be present in the list of instruments for configuration KNAUER HPLC System Available modules Configured modules get m Knauer Flowmeter g Switching Valves E Jasco FP 920 Options Cancel Help Instrument selection window example Jasco FP 920 For each item on the left hand side the corresponding parameters are shown and described right Jasco FP 920 Configuration Property Id comm Rs232Port Baudrate Instrilame Value com y Traces Signal traceName Type outputsUnits YAsisMult Description Communication port Cancel Help Instrument configuration window example Jasco FP 920 These configuration windows are strongly depended on the given type of instrument Some settings are fixed They will appear grayed with no access The setup programming is done using a script language as demon strated below The upper part of the window is for the programming inputs whereas the lower one gives the instrument specific programming information e g the allowed ranges for different parameters Before any program is saved it is checked for its correctness If this check fails you will get a corresponding error message 184 Fig 275 Fig 276 Fig 277 Knauer Instrument Control Method Options Wave langt hEX 40 rm davolanathEN 400 nm Gain 1 FilterBC
292. ollectors Control option FRC must not been enabled here Whenever a configuration with a fraction collector is started an installed FRC license will be activated Configuration Knauer Pumps For each pump in the Configured Modules window double click the icon and complete the configuration dialog For configuration information regarding the PLATINblue pump P 1 please refer to the PLATINblue system manual If the pump S 1050 should be controlled by RS 232 in the pump s own setup the Interface must be set to RS232 115200 Please refer to the pump s manual chapter Setup menu for more information The pumps K 1000 K 1001 S 1000 and K 1800 preparative pump 1800 only can be controlled with ChromGate if the pump s internal communication in the menu option CONTROL is set to NET baud rate 9600 To control a pump K 501 in the pump s internal setup the menu option CONTROL must be set to Ext RS232 The firmware version must be v 1 23 or higher If you are operating a K 1001 pump fitted with a firmware version lt 5 x you must select the K 1000 icon for configuration This is due to the status information which the older firmware does not support Otherwise you will get error messages when the pump starts running For those pumps no advanced status information and no pressure trace recording are available Setup and Control of Knauer HPLC Systems Fig 44 Fig 45 Fig 46
293. olume but needs more sample to fill the loop completely 3 x loop volume for loop volumes lt 100 ul and 2 x loop volume for loop volumes from 100 ul to 500 ul 1 5 loop volume for loop volumes gt 500 ul Partial loop fill 50 of the programmed loop volume ul pick up Injection volume loop volume 3 x needle volume 2 Transport pos only available for ul pick up with tray type 84 3 vials Select the position of the vial with the transport liquid Available are vials 85 86 and 87 The transport plug is 2 5 x needle volume needle volume tubing volume please refer to the autosampler configuration chapter The autosampler itself calculates the liquid level in the transport vials It will be reset to 8000 ul every time if the autosampler is switched off and on Additionally the level can be reset from the autosamplers direct control tab The level cannot be read out from the software If the autosampler calculates that the vial is empty the method sequence will stopped Dependent from the transport liquid level the autosampler will adapt the needle immersion depth Therefore it is important to fill the transport vials whenever the autosampler was switch off or the transport liquid level was reset manually If a vial is selected as the transport vial the configuration of the vial as a Reagent A or B vial will be ignored Syringe Speed The aspirating speed of the syringe used in injection methods can be adapted depending on th
294. olumn A drop down list of available parameters is displayed Select a parameter from the list If you do not wish to perform a suitability test on any given peak simply leave the Parameter fields blank Similarly if you do not wish to have one of the test criteria used for example RSD leave it blank Some parameters have a choice of calculation methods These parameters have the calculation method displayed after the parameters in parenthesis e g Plates Meter JP indicates Plates Meter calculated using the Japanese Pharmacopeia calculation For details on these calculations see the equations section 4 For each parameter selected enter a minimum value Min maximum value Max and maximum allowed percent relative standard deviation RSD 5 If you want a noise test to be performed click on the lower spreadsheet in the column labeled Test Select one of the noise calculations Noise rms ASTM Noise rms or 6 sigma Noise rms from the drop down list If you select this box you must enter a Start and Stop time for the test and the Threshold value to determine acceptable limit The RMS noise value for the portion of chromatogram between the Start and Stop times will be calculated and compared to the Threshold value to determine whether the test Passed or Failed Note that the times you enter for the noise test should be representative of a baseline area of your chromatogram where not peaks elute Keep in mind that the noise may
295. on Use the Trace column fields to select the required trace if more than one is available in the data file After checking the Shift Time min option you can enter the desired time difference e g for correcting time delays of serial arranged detectors External Source The Settings button also becomes accessible Clicking on it the following dialog box appears Select Channel Virtual detector setup channel selection You can select from the pull down menu one of the detectors or detector channels accessible in the configured system Creating an Instrument Control Method 107 IS Fig 159 To avoid possible signal distortion the Y Axis multiplier in the configuration of the virtual detector must be equal to that of the selected User Detector Argument Y This option becomes accessible if the Argument X field is activated The setting dialogs for Y are than the same as for X Formula X Y You can either fill in the desired formula or you can select it from the pull down menu providing all of the formulas used before Formula possibilities Following functions constants and operations will be supported Functions SIN COS TG ASIN ATG EXP LG log10 LN In SH hyperbolic sine CH hyperbolic cosine TH hyperbolic tangent SQRT ABS RAND randomizer Constants Pl 1 3 141592653 E e 2 71828183 Operations Addition Subtraction Multiplica
296. on Units mAU 0 001 AU or HAU 0 000001 AU For the RI detectors the only choice is uRIU and for the spectrofluorometric detector the units are given as a u arbitrary units For DAD s you have additionally also the choice ADC counts Selecting this the signal mode in the Instrument setup page 102 will be indicated as Intensity without access for changing it This change you can perform in the configuration window only Flow Cell Enter a description for the flow cell you are using The description will be printed out when you print the instrument configuration Add Info Click the button Add Info to enter more detailed description of the detector Setup and Control of Knauer HPLC Systems 51 Fig 75 Fig 76 Additional Information The detector 12 equipped with halogen lanp for visible wavelength range For UV range choose deuterium lamp Cancel Additional information box Enter additional information or comments to be printed out when you print the instrument configuration When complete click OK to exit the dialog and return to the instrument configuration icon list Halogen Lamp available not for all detectors Check this box when the detector is equipped with a halogen lamp Also if you have read out a detector equipped with halogen lamp the option will not be enabled automatically The detectors S 2600 and S 2520 can be equipped either with a deuterium or a halogen lamp Please be sur
297. on range of the peak is determined by starting from the peak apex and working toward peak start and peak end Each chromatographic point in the peak is compared to the absorbance threshold The start and stop values of the range are defined as the point at which the absorbance drops below the absorbance threshold 2 For each point in the calculation range a similarity index is calculated by comparing the spectrum for that point to the peak apex spectrum PDA Analysis and Calculations 3 All spectra whose similarity index meets or exceeds the Purity Threshold will be considered similar to the apex spectra 4 A sum is calculated of the chromatographic area represented spectrally similar points 5 The Purity Index is calculated by dividing this sum by the total area over the calculation range of the peak The purity index will have a range of between 0 000000 and 1 000000 Three Point Purity Three Point Purity is calculated by comparing the spectrum at the apex of a peak Point 1 with the spectra at the up slope Point 2 and down slope Point 3 of the peak The up slope spectrum is defined as the spectrum located 20 of the peak width from the peak start The down slope spectrum is defined as the spectrum located 20 of the peak width from the peak end Time up Time start TIME apex Time start 1 5 Time gown Time apex Time eng TIME apex 4 5 Similarity indices are generated for the up slope and down slop
298. on the pump s display can be changed for the P2 1L in the pump s own menu all others will always show MPa Leak Sensor Sensitivity P2 1L only Select the leak sensor s sensitivity in accordance with your requirements The choices are Off Low Medium and High Config Service P2 1L 51050 only Click the button lt Config Service gt to enter the Pump Configuration Setup window of the pump This will only work if the pump is connected via LAN Pump Configuration Service Head 110 ml Gradient Mode LFG Bd Components WwAlWw Biv Cl D Cancel Pump Configuration Setup window of pump S 1050 Using the lt Read gt button you can read out the pump head and gradient configuration If this is done also the lt Write gt button will be active You can change the settings for pump head and gradient and send these settings to the pump using the lt Write gt button Note that if the auto detection for the pump head RFID based pump head recognition is activated in the pump the pump head cannot be changed Leaving the configuration window with the lt OK gt or lt Cancel gt button will leave the window with the last sent setting Pressing the lt OK gt button will not send any changed settings Pressing the lt Cancel gt button will not recover the original settings Pressure Sensor S 100 only If your pump S 100 is equipped with a pressure sensor please enable the corresponding check box For pumps 20P P4 1S t
299. oncnncnnncncncnncnnonos 137 SIGE NESJE os 137 HStumen Status PINPS AAN 139 instrument Status Detectors LEGGE 145 Instrument Status RI Detectors S 23 4 00 K 23 4 00 1 145 Instrument Status UV Detectors S 2520 S 2500 S 200 K 200 K 20001 K250 ss ee 145 Instrument Status Kontron Detectors 3xx 4xx and 5xXX 008 147 Instrument Status fast scanning UV Detector K 2600 148 Instrument Status Diode Array DetectorS oooccconcccoccccccnccccnconcnnnnos 149 Instrument Status Fluorescence Detector RF 10Axl RF 20A 152 Instrument Status Conductivity Detector Alltech 650 154 Instrument Status User Defined Detector arurrnrarnrnvananrnnnenennnvenenen 154 CONTENTS 5 Instrument Status Virtual Detector oocccoccccccnccccnconcnccncnnonnnnnnnnnnnnos 155 Instrument Status Assistant ASM2 1L rornrrnnnrnanennnnennnnnnnnnnranennnnennnnennnnnnnnennnne 156 Instrument Status Autosampler occcoccccocccconccccnnoncnccncnononnonanncnannonnnonnnnnannnnono 161 Instrument Status Autosampler 3800 rrrnrrnnnrrrnnrrranennarennnnennnnnnnnnnn 161 Instrument Status Autosampler Optimas 3900 rrranrrnarennnnennnnennnnnn 162 Instrument Status Autosampler 3950 rrrrnrennnrennnrrnnnrnnnnennnnennnnennnnnn 163 Instrument Status Triathlon Endurance Autosampler
300. ong others Because the shape of the broad SEC peaks can vary In many cases simply examining the average Molecular Mass number is not adequate to characterize the polymer as two polymers can have the same average Molecular Mass but very different weight distribution It is therefore important to evaluate a range of different Molecular Mass calculations as well as examine the actual peak shape overlay comparisons are helpful here to get a good indication of polymer quality The various different Molecular Mass numbers are used to characterize different properties of polymers as follows Mn Number average Molecular Mass is used in determination of flexibility and tackiness of samples which is a function of the proportion of low Molecular Mass material Mw Weight average Molecular Mass is indicative of the strength of the polymer as it gives an indication of the proportion of high Molecular Mass material in the sample Mz Z average Molecular Mass is used to determine brittleness and indicates the proportion of very high Molecular Mass material in the sample My Viscosity average Molecular Mass is used to relate the average viscosity of the sample to its Molecular Mass Mw Mn Polydispersity is an indication of how homogeneous the sample is A low number indicates more homogeneity or narrow Molecular Mass distributions Higher numbers indicate more complex polymers Using ChromGate SEC Software The ChromGate Size
301. only a marker is set This is useful especially in combination with the option relative to the previous event Thus for instance a collection can be started at a defined time after the set conditions are met Fraction Position This field is only informative according to the settings in the parameter window Either the defined vial position is displayed or next for the first free vial position Further possibilities are Waste Peak recycling Solvent Recycling and in case of action None 0 00 is displayed In case of Collect Slices always next is shown Fraction Check If this option is checked any run will be interrupted if the defined fractionation conditions will not met It is not possible directly to continue such an interrupted run In case of peak recognition mode this option cannot be used Rack configuration path In any method the fraction collector program must connected to a rack configuration The rack configuration file contains information about collected fractions in one or several runs Opening the fraction collector tab of the setup window first time no rack configuration path will be displayed 244 ChromGate Preparative Option MM Instrument Setup S 5 1000 E as 3950 Ej Ovens 4050 s 2600 S 3050 FC XX Aux Traces 2 Trigger Detection Channel ls 260011 vi MV Collector Reset gt Pump s 1000 vi Flow ml min 1 000 Update Flow Fraction Collection Program Exp interv Rel time Fraction
302. ontrol Method Options The only integration parameter which can be modified is the baseline drift when using the Gaussian sequence Check this option to consider a baseline drift of 10 Press the Analyze button to repeat the validation procedure Validation of integration C Reference chromatogram data file View report Gaussian sequence W Baseline Dritt 2 Close Validation of Integration with baseline drift If you press the View report button first you will get the following message Error ES ES Please analyze the data Error message After a few moments calculation time the Gaussian sequence will also appear in the background window In both cases the performed integration will now be visualized by the baseline Press the View report button to open the automatically created validation report text file 181 Knauer Instrument Control Method Options ChromGate Chromatography Data System 3 3 2 980 Validation report of the Integration Algorithms Reference file alkylbenzoate reference dat Calculation Mode USF Pk Ret Time Height Area USP Width Theor Cap Resolutio Asymmetr min pAU pAU serc min Plates Factor n Y 10 Calculated 10 452 44707800 28238260 01665 11787 0400 30357 2 4088 1 1216 Expected 10 452 44707800 28238260 01665 11787 0400 3 0357 2 4088 1 1216 Difference 10 0 00 0 00 0 00 0 02 0 00 0 00 0 00 0 00 o Calculated 11 54 38844100 2866261 0 01957 12581 2637
303. ontrol Method Options Validation report of the Integration Algorithms Calculation Mode USP Pk 1 10 10 10 Inspector matthias Ret Time min 1 00 2 00 2 00 0 00 3 00 3 00 0 00 4 00 4 00 0 00 5 00 5 00 0 00 6 00 6 00 0 00 7 00 7 00 0 00 8 00 8 00 0 00 9 00 3 00 0 00 10 00 10 00 0 00 Date Thu Dec 08 09 22 11 2011 Height pAU 999996 00 1000000 0 0 0 00 499997 00 S00000 00 0 00 249996 00 250000 00 0 00 125000 00 125000 00 0 00 62500 00 62500 00 0 00 31 250 00 31 250 00 0 00 15625 00 15625 00 0 00 7808 00 7812 00 0 05 3902 00 3906 00 0 10 1948 00 1953 00 0 26 Reference file Gaussian trace baseline drift 0 Area USP Width pAU ser 15039481 OG 15039769 BS 0 00 1519692 0 75196604 5 2 0 00 37596825 0 37599424 1 0 00 1879914 0 1879971 2 1 0 00 939956 00 939985 60 0 00 469964 00 469992 50 0 01 234969 00 234996 40 0 01 117266 D0 117490 66 0 17 29550 00 sof 45 4 0 33 29140 00 29372 BF 0 79 TELE A IT min 0 4058 0 4000 1 44 0 4058 0 4000 1 44 0 4058 0 4000 1 44 0 4058 0 4000 1 44 0 4058 0 4000 1 44 0 4058 0 4000 1 44 0 4058 0 4000 1 44 0 4056 0 4000 1 40 0 4054 0 4000 1 34 0 4042 0 4000 1 06 Theor Plates 97 1724 100 0000 2 83 300 5902
304. op times in minutes can be entered manually or selected graphically using the SEC buttons on the chromatogram toolbar Results Range Start and Stop One or more ranges of the chromatogram must be defined to be used in calculation and reporting of the Molecular Mass distribution Each range is determined by the results range start and stop values The times in minutes can be entered manually or graphically using a stored chromatogram Horizontal Baseline Select this button if you wish the SEC baseline to be drawn as a horizontal line forward from the Baseline Start Time to the Stop Time The horizontal baseline stops when it crosses the data trace A threshold value in microvolt must be specified to ignore any detector baseline noise in the trace that would cause the horizontal projection to stop when it encounters the noise in the trace If the Horizontal Baseline reaches the end of the detected peak before the programmed Stop Time the range will stop where the Horizontal Baseline crosses the data trace Calibration Select Universal for the calibration type ES If Universal Calibration is selected the Mark Houwink constants or intrinsic viscosity of the sample s must be entered to display an adjusted calibration curve Sample Data Use this area to input K and a or measured intrinsic viscosity values for your samples These values in conjunction with the Mark Houwink constants intrinsic viscosity specified in the calibration tab
305. or Reset gt Pump S 1000 y Flow ml min 11 000 Update Flow Fraction Collection Program Rel time p E Fraction Fraction shift min ASS Position Check Single Event Unconditional J Timed Waste CO Single Event Unconditional l Timed Waste Comments Fraction collector setup window upper part Detection Channel Select from the drop down list of active detector channels that one to be responsible for fractionations Collector Reset This option must be checked for cumulative collection from repeating runs Advanced collector settings With a click on the 2 button the Advanced collector settings window will open Advanced Collector Settings Rack Settings Stacked Injections Exchange rack if full Restart from first vial of current run i Adjust FAC table after stacked injection Min allowed empty vials 0 Adjust FAC table in 0 00 mir after The run will start only if the number of empty stacked injection vials exceeds the minimum number of allowed empty vials Cancel Fig 329 Advanced collector settings Rack Settings If the option Exchange rack if full is enabled automatically a new empty rack will be created if the current one is full A copy of the full rack will be stored the data from the fractions will not be lost This allows changing the rack manually during the sequence without starting the sequence again Is this option disabled a sequence will stop if no empty vial is le
306. or any time window you can select the range and the polarity of the signal via drop down lists The choice for the range is 0 01 0 05 0 10 0 50 1 00 5 00 10 50 100 500 1000 and 5000 US Instrument Setup User defined Detector For any user defined detector only the general detector settings described on page 91 can be set Creating an Instrument Control Method 105 Instrument Setup Virtual Detector Fig 155 A virtual detector is not a real instrument but a software tool having special functions For instance it is possible to carry out simulations or corrections of chromatograms with this detector The result will be stored as normally created chromatogram The virtual detector requires the same general settings as any other detector as described on page 91 left hand side of the setup window They have to been set also for the virtual detector MX Instrument Setup 23 Virtual Detector Trigger Detector Settings Argument Settings MW Acquisition channel on V Argument Time constant 1 0 w Sec ONE Data File p Sampling rate E v Hz Data File c ezchrom eliteventerprisetprojectsyd Status 1014 points loaded Trace Name Charm Run time 10 Min Y Multiplier 1e 006 Acquisition delay 0 Min gt Argument Y Source mme DEG Resulting Formula lt Y Ext Trigger ON in fi Min after ready E y Advanced Virtual detector setup window Ext Trigger On in 0
307. or is interrupted This leads to a plateau shape for that time and thus to a false quantitative result For accurate and reliable qualitative and quantitative results the run should be performed twice with and without scanning Scan column in time table If the option Acquire 3D data is enabled a new column Scan will be available in the time table for the detector S 2550 If you want to perform a time defined scan enter a new line into the table with the desired time and enable the scan by clicking the check box Please note that during a scan the measurement signal s cannot be acquired In the chromatogram you will find a plateau for the scanning time It is not allowed to enable a scan in the first and last line of the time table The time between two scan must be 0 1 min in minimum 100 Fig 146 Creating an Instrument Control Method Time Wave WAYE c A min length 1 length2 gt Pa ente 0 00 254 254 On 0000 3 00 254 254 0000 10 oo 254 254 On 0000 Detector S 2550 time based scan Instrument Setup Diode Array Detectors Fig 147 Fig 148 S 2600 DAD 2850 DAD 2800 K 2700 and Kontron DAD 540 545 The S 2600 S 2850 S 2800 K 2700 and K 2800 detectors are fully functional DAD diode array detector or PDA photo diode array detectors with the capability to simultaneously collect data over the entire UV and visible spectra These detectors support the g
308. ormation about parameter selection that is provided in the instruction manual for each module Pump Tabs The Pump tab contains several areas where you can enter parameters related to operating mode control pressure limits and flow programs The tab for the Smartline Pump 1000 is presented below the tabs for other pumps are very similar Short guide ChromGate KNAUER Instrument Control 17 Fig 11 amp instrument Setup 6 5 1000 6 as 3950 EJ Ovens 4050 E 5 2600 2 Aux Traces Fe Trigger Working Mode Solvent s LPG Cycle Time sec 20 Control Pressure Limits Pa Min 10 0 Max 40 0 F Flow OFF at the end of run Pretreatment tit Pump Program cary eam od oo co comer Commer min emani 3 OM PE 0 00 1 000 100 0 0 0 00000000 10 00 1 000 100 0 0 0 00000000 Time 0 0103671 Minutes Amplitude 254 Wavelength nm WL1 5 2600 WL2 5 2600 Temperature PC Flow 5 1000 o gt o Wavelength nm amp o Wavelength nm Instrument setup tab Smartline Pump 1000 Details for the Control Settings Control Pressure Limits as well as the creation of a Time Table are described in the section Instrument Setup Pumps on page 84 Detector Tabs The format of the detector tab is dependent on the nature of the specific detector A typical tab is shown below Some of the parameters are common to essentially all detectors and are descri
309. ount ul 1 From 1 h To 1 Ar gap ul Commit Kontron AS 460 465 Mix Step Add Specify the Amount of a liquid in ul max the syringe volume to be aspirated from a source vial 1 to 110 and dispensed to a target vial 1 to 110 Additionally the air gap can be defined To continue click the Commit button and then the New button to program the next step Method type blis Amount ul 100 Mix times Aur gap ful qo Commit Kontron AS 460 465 Mix Step Mix The programmed Amount ul max the syringe volume will be aspirated and dispensed three times Again the air gap can be defined The mixing step is performed in that vial which is specified in the last adding step by add to 130 Creating an Instrument Control Method Wait lo h lo mir i sec Eonnn t Fig 186 Kontron AS 460 465 Mix Step Wait Wait a programmed period of time x xx xx 0 00 00 9 59 59 before continuing with the next step reaction time Click the Commit button to complete the operation Click the Delete button to delete the selected step Instrument Setup Miscellaneous Instruments Instrument Setup Manager 5000 5050 IF2 I O Instrument Setup EEK Step Mode Fig 187 Setup window Manager 5000 5050 IF2 Edit the spreadsheet as already described for other time programs e g as described in the chapter Instrument Setup Pumps Pump Program Enter the maximum output voltage in the voltage
310. ow The set flow of the selected pump after the last actualization will be displayed If the flow is not constant for the whole method the value of 0 000 will be shown Please enter then the flow rate for the time the fractions will be collected The flow is important to calculate the time delay between detecting a component in the detector and the approach to the fraction valve as well as the filling level of the vials Fraction Collection Program The editing principles are again the same as it was described for the pump gradient programming Some fields are free for entering data whereas others are either only informative without access or you can select the possible options via a drop down list Exp Time min Enter the desired time values Mode Fraction Collection Program Exp time Ga woe et EN Single Event Unconditional a 1 Peak Recognition Ewent Unconditional Fig 330 Fractionation mode selection The single event option is to select for programming a detailed exact fractionation program whereas the peak recognition is preferably for use in method development Every new detected peak according to the defined thresholds see Parameters on page 238 will be collected in the next empty vial The selected mode will influence the further setting possibilities Event The possible options depend on the selected mode Fraction Collection Program Fraction Collection Program Exp int m r 0 00
311. ow online The Advanced button becomes accessible if the Acquire 3D data check mark is set Creating an Instrument Control Method 99 Fig 144 Fig 145 Scan nm Range 190 900 nm Start 200 End 500 W Acquire 3D data F Use quick scans Detector S 2550 Scan setup window with PDA option A click on the Advanced button will open the following dialog box 2550 Advanced Settings Cancel At the peak apex using Slope m4U sec At the beginning ithe peak sal ki eit Height m l Delay min Channel Detector S 2550 Advanced setup window Scan if a peak is detected At any detected peak the software will perform a scan automatically if this option is activated All further settings in this area are valid for the whole method You may decide whether the scan shall be performed At the peak apex or At the beginning of the peak with the given Delay min With the Slope uAU sec and at Height HAU settings you can define the thresholds necessary for evaluating the signal as a peak The unit MAU depends on the configuration setting Use channel defines the data channel where the analysis is performed You cannot change this selection during a run However you can change the measuring wavelength of this channel as often as necessary The option Continuous quick scan cannot be enabled This option is not available at this time Warning While scanning the data acquisition of the detect
312. ows Pretreatment This option enables several pretreatment operations of pumps and valves before injection and starting the data acquisition For setting up refer the Pretreatment section on page 89 Assistant Setup FRC Setup lf a valve is configured as a fractionation valve the Assistant setup includes two tabs one for the Assistant and one for the fractionation valve Please refer to the section Fraction Collector Setup for detailed information for the fraction valve setup MultiValve FC Creating an Instrument Control Method 111 Fig 161 E instrument Setup E ASM2 1L AN Baseline Check Assistant Setup FRC Setup Detection Channel na channels IV Colector Reset gt Pump not defined y Flow ml min 1 000 noder in Fraction Collection Program ve me AREAS min shift min Position Check Single Event Unconditional 0 00 0 00 Timed Waste 8 Single Event Unconditional 0 00 0 00 Timed Waste E Rack configuration Path Create Open 5 6 Fraction Bi Peak recycling Solvent recycling ASM 2 1L setup window for fractionation valve Instrument Setup Autosamplers Fig 162 In any system you can include only one autosampler Therefore all supported samplers will be explained separately without cross references Instrument Setup Autosampler 3800 Smartline Autosampler 3800 and K 3800 Basic Marathon The method parameters required for automatic operatio
313. parameters you may not wish to display them when you search Search Now When you click this button a library search will be performed on the selected spectra using the parameters and the number of hits requested will be displayed Search Spectrum Click the arrow to select a new spectrum to search on You can choose from the current data or from a stored spectrum file The current spectra source will be displayed Search Parameters This tab contains search parameters If you have elected to use the Method search parameters the parameters displayed will be those in your current method in the PDA Options Library tab If you have elected to do a Quick search the parameters will be default parameters Wavelength Range These specify the wavelength range over which the library search will be performed Wavelength Step This specifies the data point spacing to be used when a library search is performed Max Hits This specifies the number of hits that will be reported in the results of a library search Note that this works in conjunction with the Similarity Threshold parameter to limit the number of hits reported Similarity Threshold Specifying a value for this control will cause the library search results to only display matches whose similarity to the unknown exceeds this value ChromGate PDA Option Note that this control works in conjunction with the Max Hits parameter to limit the number of hits reported Library
314. paration may not be shown The number of displayed positions also may succeed the actual existing number of collection positions due to the rectangular arrangement I MM Instrument Status 46 System Status Multi alve FC Monitor Direct Control Monitor Status WASTE Signal Vial Fr Time min Fr Volume ml Communication Recycling Valve COMA Fig 355 Multi Valve fraction collector status tab with addtl recycling valve ChromGate Preparative Option 249 Differing from other instrument setup windows that of the fraction collector is also valuable during the run In the lower part the measured chromatogram will be displayed as it is in progress Additionally the areas of fractions peak recycling and solvent recycling are shown by highlighting in the corresponding colors Refer to the chapter nstrument Setup Fraction Collector beginning page 235 and Fig 348 on page 245 E Instrument Setup La VD Virtual FC EG Trigger Detection Channel VD y VW Collector Reset Fraction Collection Program Exp time min Single Event Single Event Single Event Single Event Single Event Single Event Single Event Single Event Signal Level Slope Signal Level Slope Signal Level Slope Signal Level Slope Signal Level Slope Local Maximum Local Maximum Unconditional Iii 0 10 0 10 0 10 0 10 0 10 0 10 0 10 0 00 0 00
315. pectral Background Correction would be checked in the Method PDA Options Spectrum tab 3 Using the contour plot in the Mixed view window drag the vertical cursor to the apex of a peak to display the corresponding spectrum in the spectrum view See also Extracting Spectra and Chromatograms from the Contour 4 Right click within the spectrum view to display the Spectrum pop up menu select Utilities Save Trace and enter a file name in the dialog box Repeat this procedure for each spectrum to be added to the library Files are saved automatically with the spc extension To add the 1st or 2nd Derivative of a spectrum to the library select the appropriate filter in the Method PDA Options Spectrum tab and repeat steps 3 and 4 How to Add Spectra to a Library 1 When all of the spectra are saved as spc files select File Library New to display the Library Definition dialog box and create a new library Alternatively select File Library Open to add spectra to an existing library 2 Click in the Spectrum File cell of row 1 to display the Open dialog box Double click on the appropriate spc file in the list box The spc 214 ChromGate PDA Option file name is entered into that cell and the associated spectrum is simultaneously displayed A component name and comment may be entered in the appropriate columns Repeat this procedure with Subsequent rows in the table until all spectrum files are entered into the library 3 Select
316. pense Dispenses a programmed volume from the buffer tubing into the sample vial a destination vial the waste a wash vial or one of the reagent vials Speed and height of the syringe can be entered Syringe Valve This action controls the inter connections of the syringe to one of its three ports NEEDLE connection to the sample needle WASH connection to the wash solvent bottle WASTE connection to the syringe waste tubing Syringe This action controls the movement of the syringe LOAD loads the syringe with the programmed volume UNLOAD unloads the syringe with the programmed volume HOME initializes the syringe to its HOME position The volume previously aspirated will be dispensed to the last programmed position Needle Wash With this action a needle wash can be performed The content of the buffer tubing will not be rinsed to waste before the washing procedure Creating an Instrument Control Method starts The programmed volume of wash solvent is used to wash the needle at the wash position The wash position may be contaminated with the contents of the buffer tubing which may generate cross contamination To prevent contamination of the wash position program a dispense to waste action before programming a wash action Valve The positions of the installed high pressure valves are programmed in this screen The injector valve has two positions INJECT and LOAD If the ISS option is installed the two va
317. port it will be reported as zero Total Purity Checking this box will cause total peak purity to be calculated on a per peak basis during analysis The result of this calculation will then be available in reports and as chromatogram annotations If this box is unchecked and a report contains the field a zero will be reported 3 Point Purity Checking this box will cause 3 point peak purity to be calculated on a per peak basis during analysis The result of this calculation will then be available in reports and as chromatogram annotations If this box is unchecked and a report contains the field a zero will be reported PDA Options Spectrum Fig 285 The Spectrum tab is used to specify the types of filtering and processing to be performed on spectra that are extracted from the 3D data during analysis as well as to spectra displayed in the PDA Spectrum View The processing specified on this page is performed prior to any use of the spectra in the software including display searching and reporting 4 PDA Options de Library Purity 4 Spectrum ke Multi Chramatagram Ratio Spectral filtenng Per peak spectrum calculations Filtering type PE 2 Up slope similarity Background correction Down slope similarity Interpolate spectra e Lambada max The peak spectrum is defined as 4 The peak apex spectrum C The avg of the upslope apex and downslope spectra C The avg of every spectra across the peak Lambd
318. possibilities are given to set new values for the measuring wavelengths Pressing the Apply button forces the wavelength change on the detector channels Acquisition Parameters Select the Resp time from the drop down list Smaller Resp time or time constants are required for high speed analyses good rule is to 148 Fig 222 Creating an Instrument Control Method select a time constant which is at least one tenth of the peak width at the base of the narrowest peak of interest The range can be set to define the range of the analog detector outputs The choice is 0 100 0 200 0 500 1 000 and 2 000 Absorption Units Full Scale AUFS The Kontron x30 and x35 detectors can be used in a dual channel mode As far as the option Used Channel 2 is checked the otherwise not accessible cycle time becomes active for changing The cycle time you can set within the range of 0 40 through 1 00 seconds with 0 05 s increments For the x32 detectors the option Used Channel 2 is not available and the cycle time is not displayed at all f New Instrument Method tower met Data 07 03 2005 16 18 06 dat Project Corinna Test 1 Instr SEE TT Eile Edit View Method Data Sequence Analysis Control Reports Window Help x B eHh ssm 7 SRNA ae gt gt OF A O System Status 23 K 1800 23 K 1800 EY AS Triathlon ae Vario 2000 FC ES ontrol Monitor Run Status 1 97 min Digital Outs Lamp Q AutoZero Channel1 254 nm
319. ps The spreadsheets of the S 100 10P 20P P2 1S P4 1S K 120 K 501 and the Kontron pumps do not include this column Configure the events by pressing the button which is displayed when you place the cursor on the Events cell The following dialog will appear Events Digital Outputs Control Events ano 1 TTL Of y 5 TTL Off y 56 3 OC Off y 7 Relay Off 4 TTL fof 8 Relay or gt Cancel Configuring Digital Outputs The Events Digital Outputs correspond to the outputs available on the rear panel of the pump Select the state for each Event you want to set from the drop down list The choices are Off 0 On 1 and Pulse P Refer to the pump s user manual for details about digital outputs parameters Use the All On or All Off buttons to switch all of the outputs either on or off Constant pressure mode Isobar P2 1L only If in the instrument configuration the gradient mode for the pump P2 1L is set to Isobar the setup windows looks different ME instrument Setup E P21L 63 UVD21L M ASM21L 9 Aux Traces I Trigger Constant Pressure Mode Flow Limits ml min Pump Prograrn iat i Time Pressure Events mT mer of fn oe 0 8 00000000 E 0 8 00000000 Control Pressure Limits MPa Min 0 0 Max 0 0 No Action y at the end of run Pretreatment Solvent Type Instrument setup window P2 1L constant pressure mode isobar Constant Pressure Mode Flow Limits ml
320. ption starting on page 185 and PDA Analysis and Calculations on page 222 The original software manual PDA Analysis can be found on the installation DVD in EZChrom Elite 3 3 2 English Manual Optional Software Manuals e Baseline Check used to verify that the baseline is stable enough to provide useful chromatograms detailed discussion of this feature is presented in the chapter Setting up the Baseline Check on page 136 Setting up a Method A method is a set of parameters that describes the instrumental conditions for a chromatographic separation and includes information about data processing and reporting It includes the parameters for the operation of each of the various modules in the HPLC system such as the pumps detectors oven and the autosampler which are downloaded to the various modules for use when the separation run is initiated The Method window is used to set up a method and is accessed via the Method wizard The Method window includes a tab for each component of the system and the tab presents various parameters that are available via the control panel of that module The various tabs that are presented in the Method window are described in the chapter The Method Window Method files can be saved met and retrieved as well as imported from or exported to another instrument in the same manner as other Windows files Please note that for devices which are not configured in the original instrument the parameters w
321. pure In general a spectra with an Sl greater than 0 9900 would have a high probability of being the same as the apex spectra A Sl greater than 0 9000 but less than 0 9900 shows some similarity but would need to be evaluated with care and caution Sl less than 0 9000 should be considered non similar Absorbance threshold This value represents the percentage of peak height that spectra will include in purity calculation Spectra in sections of the peak that do not exceed this threshold will not be included in the purity calculation This provides a method of eliminating spectra where the concentration of the compound is so low that the solvent spectrum interferes Background compensation Checking this box will cause spectra to be corrected for background using the peak baseline prior to being used in the calculation of purity Multi Chromatogram View This displays multiple chromatographic plots of absorbance versus time each at a different wavelength Maximize the PDA display and select View PDA Views Multi Chromatogram or use the PDA toolbar Views to display all of the chromatograms in their respective channels which were specified in the Method PDA Options Multi Chromatogram tab Right click within the window to display the pop up context menu 208 Fig 304 IDAD Spectra Mulli Chromatogram eo Diprion GIO E Arange 1 Varner ft 240 rm Baca ren Tira RAED kraner Merida m li 2 V reim gh 2
322. r method Connect one pole of the trigger cable to one of the connectors labeled D IN1 and the other pole to the connector labeled D GND digital ground Autozero Signal Connections If your detector has an autozero option and you want to use the feature to zero the baseline signal at the start of data acquisition you must connect an autozero signal cable to the Interface Box The autozero signal is connected to one of the digital output connectors on the rear panel of the Interface Box Connect the ground connector of the instrument to the D GND digital ground labeled connector of the Interface Box and the AUTOZERO connector of the instrument to the connector labeled D OUT3 The connectors D GND and D OUT3 must be situated on the same connector strip Setup and Control of Knauer HPLC Systems 59 Fig Fig 85 Data acquisition cable INTEGRATOR S tv On 7 3 i i en Tr i 15 VA CAUTION A DISCONNECT POWER SA BEFORE OPENING Alas MADE IN GERMANY ATTENTION UTS de 4 JE OE N ms 8 LI esp AAN AAA Data acquisition cable ett o INTEGRATOR ne N ot Any T O00 Sh p 15VA CAUTION A DISCONNECT POWER A BEFORE OPENING Ala MADE IN GERMANY ATTENTION m 10 i Wiring example Manager 5000 5050 IF2 and detector These examples show the connection lines between the Interface Box or Manager 5000 5050 IF2 and a detector as an example the Knauer K 2501 dete
323. r HPLC Systems Before you open the ChromGate instrument you must switch the detector in to serial mode RF 10 Axl Press the function key lt func gt on the detector s keypad as often as RS232C will be displayed Confirm with E key The display will show CONNECT also confirm with E key If you press the function while CONNECT will be displayed you will enter to the COM connection table Changed values must be confirmed by pressing the key E to move to the next parameter press the function key Parameter Value 2 means even parity The COM settings here and in the software must be identical If e g the parity is set to 0 means no parity it must be set in the detector s setup as well in the software If all parameters have been entered the display shows the message CLOSE KEY Press key E to leave the setup To switch the device to serial control mode please follow the steps above To leave the serial mode press the shift and CE del key at the same time RF 20A Axs Press the function key lt func gt until the display shows SYSTEM _ Enter to Select Press the lt enter gt key Press function key until the display shows RS 232 PARAM _ Enter to select Press the lt enter gt key Now the settings for the serial interface can be reviewed and modified if necessary To move to the next parameter press the function key The COM settings here and in the software must be
324. r of sources for further information including e The Operator Manuals and Instruction Manuals that are provided with the modules in the system e The documentation provided with ChromGate Data System particularly the User s Guide available on the ChromGate DVD in EZChrom Elite 3 3 2 English Manual e Release notes provided with the software available on the ChromGate DVD in ChromGate ChromGate e Documentation provided with Microsoft Windows Configuring the System 14 Short guide ChromGate KNAUER Instrument Control Configuring a System Instrument Configuration For more detailed information please refer to the chapter Instrument Configuration on page 36 Configuring the system involves the selection of the instruments and features that are to be used during installation each component of the HPLC system is registered on the network Once an instrument configuration has been made it can be easily retrieved There are two aspects to system configuration e Indicating the modules to be used in a given configuration As an example if the laboratory contains both a Model K 1001 pump and a K 1800 pump and only the Model K 1001 pump is to be used for a given separation setting the instrument configuration includes selecting this pump and choosing various pump parameters the type of pump head that is used and the desired pressure units e Indicating the options that should be used with the method e g if the
325. r spectrum before being used in the similarity calculation Pre Filters A pre filter is a criterion on a reference spectrum that must be met before that spectrum is used in similarity calculations One or more pre filters may be specified in the search parameters When multiple pre filters are specified a reference spectrum must meet all of the individual pre filters in order to be considered for similarity calculations Reference spectra that do not meet all of the pre filter criteria are automatically excluded from calculations and from being a candidate for a hit list No similarity calculation is performed on these spectra The following pre filters are supported Retention Time Range When a Retention Time Range is specified a reference spectrum is excluded unless the retention time for the spectrum in the library is within the specified range Lambda Max Up to three Lambda Max values may be specified When one or more Lambda Max values are specified a reference spectrum is excluded PDA Analysis and Calculations unless all of the specified maxima are within 5 nm of a maxima for the spectrum in the library Compound Name When a Compound Name is specified a reference spectrum is excluded unless the specified string is a case insensitive sub string of the compound name for the spectrum in the library Ratio Chromatogram Calculation The ratio chromatogram plot consists of data points calculated as follows Ratio Pt ab
326. ram annotations If this box is unchecked and a report contains the field a zero will be reported Peak Apex Similarity applies only to named peaks and its use requires that a reference spectrum be included in the peak table Upslope Similarity Checking this box causes upslope similarity to be calculated on a per peak basis during analysis Upslope similarity compares the peak apex spectrum to the spectrum at the peak inflection point on the upslope side ChromGate PDA Option to the left of the apex The result of this calculation will then be available in reports and as chromatogram annotations If this box is unchecked and a report contains the field a zero will be reported Down slope Similarity Checking this box causes down slope similarity to be calculated on a per peak basis during analysis Down slope similarity compares the peak apex spectrum to the spectrum at the peak inflection point on the down slope side to the right of the apex The result of this calculation will then be available in reports and as chromatogram annotations If this box is unchecked and a report contains the field a zero will be reported Lambda max Checking this box indicates causes the lambda max wavelength at which the highest absorbance occurs to be computed for each peak of each multi chromatogram extracted from the 3D data during analysis The result of this calculation will then be available in reports and as chromatogram annotation
327. ration Name Oven 5 4050 Interface R5 232 Serial Number 99873 COM Port COM 4 v Temp Units e Baud Rate 3600 v Cancel Help Fig 113 Configuration window for the column oven 4050 Name Enter a descriptive name for the oven This name will appear on the oven tab when the instrument setup or status windows are displayed Use a name which is unique within the instrument Serial Number Type in the serial number of the device you are using Temperature units column oven 4050 only Select from the drop down list either C or F Interface Select the Interface you want to use for communication with the device RS 232 v L N Fig 114 Available interfaces column oven 4050 RS 232 Select the number for the RS 232 communication port COM Port on your PC to which the column oven is connected LAN Interface SEN Mg y 24 IP Part 11 0001 MW Use S N to identify the instrument Fig 115 LAN interface dialog To let the software search for your already connected and switched on device click on the button to start the search for all connected LAN devices of the currently configured type The option Use S N to identify the instrument must be enabled Select the desired device from the shown list The software will read out the information as serial number and will fill it automatically into the configuration Setup and Control of Knauer HPLC Systems 81 screen To search for an instrument th
328. ration i x Calibration ID me ry Dl Calibration path C NEZChrom Elite Enterprise Projects Default Calibration file A A a gt 2202 Number of calibration levels er 2 E Repetitions per level hz x a Clear all calibration at start of sequence i Create a separate row in the sequence for each repetition I Multiple calibration sets lt Number of Unknown runs between sets ee rterspersescalibratiog Vials With Unknown vials C Reuse calibration vials from first calibration set Cancel Finish Fig 21 Sequence Wizard Calibration 10 Enter the desired information and press Next to access the Reports page System Suitability Run calibration as system suitability M Firstcalibration setonl Ail calibration sets ES QC Check Standard E After every fro unknowns set OC check standard I Include method contents report Cancel lt Back nen Finish Fig 22 Sequence Wizard Reports 11 Enter the desired information and press Finish to present a spreadsheet that describes the overall sequence ml Sequence testi seq l Summary Begin elf Oma gt ef 1 gt testmetfel 2 fSummayRun Oa Her testmet 3 Summary Run Oa Her testmet 4 Summary Run Oma gt VP ter testmet SummaryRun Oma lt A OE SummayEnd Ova AilxD gt testmet AE AAA A FE OE ER best met Fig 23 Sequence Wizard Description of a finished sequence
329. ration window 47 Setup and Control of Knauer HPLC Systems Detector K 2700 Configuration Interface LAN v e Name 5 2800 Serial Number Detector Type ls 2800 vi S 2800 V Use S N to identify the instrument 5 2850 Channels 1 Options lv Halogen Lamp Chn Name 1 Channel 1 Chn Name 2 Y Shutter Control Chn Name 3 Channel 3 Y Axis Units m l vi Chn Name H4 Flow Cell OK Cancel Help Camel Help Fig 69 DAD configuration windows Detector RF10Axl Configuration Mame RRA Serial Number NA mr Serial Port COM y Port Settings Anis Unite Pen Flow Cell Kr sm mm gt Cancel Help Fig 70 RE 10Axl RF 20A AXS detector configuration window Alltech 650 Conductivity Detector Configuration Name lltech 650 ar CA Serial Port COMI Flow Cell Te gt YaAsisUnite fi Cancel Help Fig 71 Alltech 650 Conductivity detector configuration window Name Enter a descriptive name for the detector This name will appear on the detector tab when the instrument setup or status windows are displayed The default detector name will be suggested automatically by selecting the detector type E Use a name which is unique within the instrument Detector Type For some detectors the correct model type can be selected in the configuration window Select the type of detector you want to configure from the drop
330. rator and the path for the export files Store the method If you analyze PDA data with the modified method the export file will be created If you open the file in Microsoft Excel note that for each wavelength a separate column is required Excel supports up to Excel version 2003 256 columns If the scan range includes more than 256 wavelength steps not all will shown 230 ChromGate Preparative Option ChromGate Preparative Option To use the ChromGate Preparative Option it has to be installed separately as described in KNAUER FRC control option on page 8 Then the implemented fraction collectors will appear in the device list for creating instruments KMAUER HPLC System ALLE Available modules Configured modules Multi ake FE Virtual FC FL Fost Options Cancel Help Fig 319 Fraction collector selection window Fraction Collector Configuration The configuration windows are the same for different fraction collectors Therefore the procedure is the same FC Smartline 3050 Configuration Racks FC Name 5 3050 FC Tubing Parameters Rack Type 1 12 mm vials flow Serial Number 12 mm vias owl Slv Peak Recycling Interface RS 232 E FC Foxy Jr Configuration COM Port COM1 y Baud Rate 19200 FC Name Foxy Jr FC Tubing Parameters Serial Number PO Sly Peak Recycling Rack Type 12 13 mm tubes v Number of Vials
331. rbance max value is displayed in these columns ChromGate amp PDA Option 213 Fig 312 Retention time When the spectrum was extracted from a chromatographic run the software automatically identifies the retention time of the spectrum and enters the value in this field When desired the value in this field may be edited This field can also be used as a search pre filter Comment Enter any descriptive information desired related to the spectrum This field is used only for display and documentation purposes Library Notes This field is used to document relevant information about the library as a whole e g documentation about run conditions or general sample information Fields in existing library entries may be edited by selecting the fields with the cursor Right click on a row in the library table to display a pop up menu used to cut paste copy and insert and delete lines from the library as necessary Spectrum Information Select the retention time at which you wish to use the spectrum or the name of the peak whose spectrum is to be used Spectrum Information Spectrum Information x Spectrum at Retention Time 13 7 Min Named peak Acenaphthylene coed t coca tp How to Collect Spectra for a Library 27 1 Perform an acquisition using a sample with known components or standards to be placed in the library 2 Perform an analysis using the appropriate integration parameters Ideally S
332. reeze gradient for 2 minutes entered Set New Gradient Modify the running time program current run time 5 38 min Time 00 00 Flow 0 000 za 100 ze fo 2c fo p 0 Freeze the current gradient for 10 00 min will be performed immediately If the run time is to be extended use Control Extend run from the menu 10 Flow min m min 1 000 1000 100 o 0 EAN OS A BY 000 33 0 we Commit Line Delete Line Enter the new gradient line in the form Time min Flow ml min 24 2B 2 C 2 D Send Gradient Freeze Gradient 5 10 Gradient Time min Close Fig 213 Gradient modify window freeze gradient for 2 minutes executed The pump table now has two new lines 3 34 and 5 34 minutes with the frozen gradient settings The gradient graphics on the right handed side also shows the new gradient The pump table now ends after 12 minutes instead of 10 In this case you should prolong the set run time via the menu sequence Control extend run IN our example it is required to extend the run time by 2 minutes You must close the Set New Gradient window to modify the method s run time Extend Run Extend run by 2 00 Minutes Cancel Help Fig 214 Extend run time window 144 Creating an Instrument Control Method All direct control settings will also be stored in the Instrument Activity Log To see the log click on
333. ressure Limits MPa 10 0 ar 140 0 Show Autosampler Trav Instrument Status Baseline Check Flow OFF at the end of run Download Method F F gape os Download Tab Time Flow Comp Events mini Cmimin 36 DMP Qualification Procedures a OG I 1 000 100 00000000 Solvent Control A nn 1 Ann 100 ANNAN Fig 254 Knauer Instrument Control Control Download Method If the device s is are not properly connected the Instrument Activity Log will open and show the appropriate error message s Error Switching Valves Set position of the valve failed Error JetStream Oven Error Error setting temperature F ig 2 5 5 5 1000 Cannot download device settings The instrument with the given serial number is not found on the communication port chosen The action starts operation of devices but not the data acquisition ES In case of Download Tab only a single pump will be controlled even for an HPG system Solvent Control The Solvent Control option is accessible only with the pumps S 1000 K 1001 and K 1800 It is accessible via the menu sequence CONTROL SOLVENT CONTROL MM Solvent Control Dar gt Amount of solventis mi I ritene a ral Wie 500 0 E 450 0 O am Waring The amount of solvents used wil be correctly calculated only under software control Should the pumps have been controled manually the actual solvent amount must be corrected Fig 256 Solvent Control windo
334. romalogr AMS iii 264 A aati a seh asa Neters 267 SEC Setup Narrow Standard Method occcoccccoccccccnccocnconcncnncnnonnnnnononncncnncnnnnos 267 SEC Setup Universal Calibration oocccocnccocncconnoconnonanncnonocnanonannncannnnanonnono 270 SEC Setup Broad Range1 CalibratiON oocccoccccocnccccncccnnnoncnnnnnononnnonononanonnnos 273 SEC Setup Broad Range 2 CalibratiON ooccccocnccocnconcnccocononnnononononnconcnonans 275 CONTENTS T SEC CUSTOM ROON S saraap En p Aa a a EESAN 278 ChromGate SEC EQU Stripes did 281 TYDICAlWWIFINO OD CHOMES isso 284 291 8 Installation Guide ChromGate 3 3 2 Installation Guide ChromGate 3 3 2 General Definitions ChromGate is the Knauer OEM version of the original EZChrom Elite software from Agilent Technologies The computers must be running under Windows 7 Professional or Ultimate 32 bit Windows Vista Business or Ultimate 32bit or Windows XP Prof Service Pack 2 for EZChrom Elite must be installed Principally 64 bit or Home versions of Windows are not supported The licenses are to be installed on the enterprise machine The enterprise machine is also the host of all system configurations instruments and the user and project management data A server is the computer that is connected to the HPLC system A computer that controls the server in a Client Server system is called client The TCP IP protocol and Microsoft s framework Net version 3 or high
335. rprise Projects4PD DE Cancel Help Enter or select the library you wish to include in the report then click OK Purity Report Selecting this option will insert a Purity Report into your custom report To specify parameters for the report do a right mouse click in the report table then select Properties The following dialog will appear ChromGate amp PDA Option 219 Fig 317 Purity Report Properties Report peak purity lt All PDA Traces gt Detected on W Limit to named peaks only Limit to time range Po Min Graph Properties Graph height 1100 z Cancel Help Detected on This allows the user to select the channel or channels from which peaks will be detected Limit to named peaks only This allows the user to restrict the peak selection made above to a time range When checked only peaks within the specified time range will be searched When this box is unchecked the Time Range edit fields are disabled Limit Peaks to time range This allows the user to restrict the peak selection made above to a time range When checked only peaks within the specified time range will be searched When this box is unchecked the Time Range edit fields are disabled Time Range This allows the user to restrict the peak selection made above to a time range When the Limit to time range box is unchecked these fields are disabled Graph Height This specifies the relative
336. s JJ Set Temperature Control se al Por COM21 ce SN 99625 Status tab column oven 4050 MM Instrument Status EEK O System Status E 5 1000 SI as 39000 E JetStream Oven ad s 2550 3 s 2300 5 2400 Monitor Direct Control Run Status HOLD Temperature C 25 State Standby Mode Temperature 26 0 C SerialPort COM10 SN n a sl Status tab column oven Jetstream To achieve the communication with the column oven Jetstream its control unit must display choose function as it does after powering up Otherwise communication can be established but the oven will not execute commands sent by software Monitor Run Status The status HOLD will be displayed when no program is running otherwise the Run Status displays a run time in minutes elapsed since the start of the run State The state On Idle or Off will be indicated if the oven will be controlled or not Temperature C The actual oven temperature will be shown Cooler Temperature C S 4050 only The actual cooler temperature will be shown Digital Outs Events S 4050 only The current state for the digital outs will be displayed in this control The On state is represented by an active red Light Emission Diode LED symbol 9 The Off state is represented by an inactive LED symbol sf Creating an Instrument Control Method 169 Direct Control Temperature C The oven temperature can be set The entered value will be transferr
337. s sqrt abs abs abs abs where abs the absorbance in chromatogram 1 at this wavelength abs the absorbance in chromatogram 2 at this wavelength If the absorbance of any point in chromatogram Tor chromatogram 2 is less than the threshold the Ratio Pt for that wavelength is set to 0 Similarity Calculations The Similarity Index Sl compares two spectra across the wavelength range defined in Method gt PDA Setup gt Library A Library Search is performed using the Similarity Index determined as follows A spectrum is considered as an array of absorbencies at each wavelength a 01 a 02 a On Where a i is the absorbance at The spectrum can also be represented as an n dimensional vector with each absorbance corresponding to one dimension of the vector as follows S a 14 a La a En When two spectra are obtained from the same compound the ratio between corresponding elements in S1 and S2 is constant consequently the vectors have the same direction In such a case the angle between the two vectors would be 0 As a general rule the greater the angle between the two vectors the less similarity they have This relationship is calculated as follows SI cos O where The angel between the two vectors The closer the SI value is to 1 the more similar two spectra are consid
338. s If this box is unchecked and a report contains the field a zero will be reported Lambda max Calculation range This allows the user to restrict the wavelength range over which the Spectrum Max Plot will be calculated Default is the detector acquisition range PDA Options Multi Chromatogram The Multi Chromatogram tab is used to specify data channels within the 3D data for integration and quantization It allows you to select wavelengths to display on the Multi Chromatogram plot When you select an Enabled box enter a wavelength and bandwidth to be used for one plot After you have set up several wavelengths you can disable them temporarily by clicking the Enabled box such that the check mark is not displayed The Multi Chromatogram display is selected from the View Gallery The Multi Chromatogram view is not functional unless one or more valid wavelengths are enabled in the Multi Chromatogram Definition tab PDA Options 22 Library Purity 4 Spectrum f Muli Chromatogram ia Ratio Enabled Wavelength Bandwidth 1 Subtract baseline chromatogram Wavelength p rmi Bandwidth nm Fig 286 Enabled To enable a wavelength click the checkbox To disable a wavelength click the checkbox again to remove the checkmark Only enabled wavelengths will appear in the multi chromatogram view ChromGate amp PDA Option 195 Wavelength Enter the wavelength you want to view Bandwidth Sp
339. s detector Bw Enter the bandwidth of the reference wavelength Limits are 1 to 32 nm in 1 nm steps Scan nm Range xxx yyy nm The displayed range depending on the detector type and the installed lamps will only be updated if you open that window online 102 Creating an Instrument Control Method Scan nm Start 200 End 400 Bandwidth 1 Signal mode han MW Acquire 3D data Fig 150 DAD setup scan range Fig 151 This section allows you to specify the scan spectral interval in nm The given range limits are read out from the used instrument Therefore they may differ from the shown values Start Enter the start wavelength for the scan in nm End Enter the end wavelength for the scan in nm Bandwidth The Bandwidth is the wave length range that will be used to measure a data point For the spectral acquisition it is highly recommended to use a bandwidth of 1 nm The scan section limits Start and End are important even if no PDA option is available All of the measuring wavelengths in the Wavelength Program for the used channels 1 to 4 must be within these limits Signal Mode The active signal mode is already selected in the instrument configuration see page 50 The choices are Absorption and Intensity For the Kontron DAD 540 it is fixed on Absorption Acquire 3D data You have access to this selection field only with the PDA option Wavelength Program wavelength Program Used channe
340. s ignored Analysis Channel Specify which Diode Array wavelength channel is to be used for analysis of the peak The choices will be those specified in Instrument Setup DAD PDA Analysis and Calculations This section describes the various types of calculations that are related to PDA data and how the data is analyzed ae In order to maintain accuracy during the application of multiple operations all calculations are performed using double precision floating point numbers Chromatograms Extracted from the 3D Data Two types of chromatograms may be extracted from the 3D data e Multi Chromatogram Channels One or more chromatograms defined on the Multi Chromatogram tab of PDA Options e Working Chromatogram A single chromatogram extracted from the Contour plot on the Mixed View display This chromatogram is displayed in the Chromatogram View Specific Analysis related capabilities are for these chromatograms are detailed below Multi Chromatogram Channels The following apply to multi chromatogram channels e In the Channel Selection drop down list each multi chromatogram channel has a unique identifier that includes the wavelength and bandwidth of the channel e Each Multi Chromatogram Channel has a separate Integration Events Table Manual Integration Table Export Table and Performance Table associated with it e All Multi Chromatogram Channels share a common peak and group table Within the peak and group tables a
341. s is 0 02 min except for the preparative pumps P2 1L 1800 here the minimum time step is 0 06 min For the pump P2 1L in isobar mode the smallest time step is 0 25 minutes Use the spreadsheet in the pump setup view to enter your time gradient program The number of columns shown depends on the defined system isocratic LPG or HPG and the number of included components 88 Fig 130 Creating an Instrument Control Method In the first row at time 0 00 min enter the initial flow in ml min solvent composition in and digital events Then for time in minutes you can define the pump parameters For KNET pumps up to 50 steps are allowed with an accepted minimum time difference of 0 02 minutes between two lines The complete program is transferred or uploaded to the pump when you start a run All time programs are displayed in a spreadsheet similar to the one shown below Although the information in the fields will vary the spreadsheets always support certain basic features Comp B Comp Comp D Events 18 Pump Program Time Flow Comp amp min mimin 0 00 1 000 75 7 0 00000000 4 000 75 18 7 0 00000000 Fill Down 1 000 65 28 7 0 00000000 aye 1 000 100 0 0 0 00000000 Delete Line Delete Lines Editing tools for the time gradient program Each row is assigned a program line number and time in minutes followed by columns for information for each time in the program Rows and fi
342. s is indicated by monitoring the performance qualification TE vp Method PO Measurements 7 110 10 06 OG 22 59 met Dita PO Measurements amp 2 10 10 06 08 22 591 dal Peon ject Det A F T Elo p y Method Dato Jeguenor Ay Corfrol Roperts Window tinh w OMG 1 vinual Detea r QA TOD EN kg ar tow se DAT B Cauttios i Seter D Nesarrmeres D Samote miecton 1 D Saree miecken 2 A PERFORMANCE QUALIFICATION REPORT System Configuration Dutecior Vetus Detecta S N not defred a durei Test condition Mote Prise MOON 8030 Graden AL 100 E 0 O 0 O 0 Temperarure ambient Ep w Qa nmila Ing voksne MD js Intergatior wite 02 threshokd 50 BEGAN Tb 175 main Results Performed Injections 2 Detected Peas i Test Reni Succeeded Notes Caculated mama RSD is 0 000 Mama Midi RSD amp Po P j Pret Repot Fig 265 Performance qualification report The chromatograms of the single session runs can be inspected by selecting them on the left hand side They are now permanently stored in Installation folder Enterprise OEM Performance Qualification Generic PQ Procedures Validation of Integration This tool is a hardware independent test of the installed software package that indicates how reliably the integration of the chromatograms will be performed To carry out this test select any method setup window to gain access to the menu sequence METHOD OPTIONS VALIDATION OF INTEGRATION
343. s necessary to connect the interfaces to separate communication ports on a PC See the Typical Cabling Wiring chapter Fig 389 on page 285 Serial Number Type in the serial number of the Interface Box Manager 5000 5050 you are using The serial number is used for the device identification and addressing during serial communications This serial number should match the serial number of the Interface Box For Knauer HPLC Boxes with an S N higher than 65000 only the last 4 digits of the S N must be entered e g 5567 instead of 65567 Box type The only choice is Model 96 No others are selectable Channel Four data acquisition channels are normally available However only channel one will be available if you have checked the 100 Hz sampling rate option in model 96 or 50 Hz in case of Manager 5000 5050 IF2 see Sampling rate on page 91 Interface setup Number of boxes 1 Configure box 1 Serial number 7777 Manufacturer Knauer Serial Port Network Model Box type Channel Range e 104 v Cancel Help Dialog box for configuring the Model 96 Interface Box with activated 100 Hz option Range Select the Signal Range for the channel chosen from the channel drop down list The choices are 10 V 1 V 0 1 V 0 01 V for the Interface Box and 2 56 V 1 28 V 0 64 V 0 16 V for the Manager 5000 5050 IF2 The Signal Range specifies the maximum 34 Setup and Control
344. s on the given pump Direct Control The direct control area will appear disabled as long as the direct control option is not activated Except during a run it can be activated via the menu sequence METHOD OPTIONS RUNTIME SETTINGS see Fig 249 on page 170 The direct control enables someone to control the instrument directly even during a running method Changes made to the settings and applied during a run will be automatically stored in the method The corresponding new lines in the spreadsheets are marked with the comment DC Op Direct Control Operation Applied changes in a not running method will not be saved However it will be recorded in the Activity Log Flow ml min Specify the flow in ml min you would like to have at the moment and click the lt Flow Apply gt button to send the flow to the pump If an LPG pump is used also the gradient setting will be send lt Flow Apply gt The Flow Apply button will send the flow entered into the Flow field to the pump lt Stop gt The lt Stop gt button will interrupt delivery while the run time continues The pump remains in slave mode This button is also active during a run to stop the pump e g In an emergency situation This will not stop the whole method only the pump s flow Due to that this is a method change the current method will be changed The Stop will be added to the currently running method if the option Save changes in time table of t
345. s rebooted after installation otherwise the installation will not be completed To install ChromGate click on the link Install ChromGate software The components which should be installed can be selected in the next window As default all components which ChromGate supports are selected Installation Guide ChromGate 3 3 2 Knauer HPLC System InstallShield Wizard Select Components Select the components setup wall install Select the components you want to install and deselect the components you do not want to install Description Pump P2 15 UY Detector UVDZ TL Assistant ASM2 1L PLATIN blue fe Autosampler A5 1 Pump P 1 Detector kv Detector PDA 1 B n l Manager M 38 40 ME of space required on the C drive 2033 09 MB of space available on the C drive Ntkall amp tneld Fig 2 Click on Next gt to confirm this window In the next window click on Install to start the installation To cancel the installation procedure click Cancel Knauer HPLC System InstallShield Wizard Ready to Install the Program The wizard is ready to begin installation Click Install to begin the installation If you want to review or change any of your installation settings click Back Click Cancel to exit the wizard Inmdalsheld Fig 3 The following window will be displayed when the installation has completed Knauer HPLC System InstallShield Wizard InstallShield Wizard Complete Th
346. se the window If you close the window without having accepted any changes those will not become valid The actual volume of solvent in each bottle is displayed The eluent volume of the symbolized bottles displayed depends on the actual volume and bottle size E Solvent Control Amount of solvent s ml Ela TB eo 750 0 g 500 0 5 1000 4 S N 33333 S 1000 8 S N 44444 Warning The amount of solvents used will be corectly calculated only under software control Should the pumps have been controlled manually the actual solvent amount must be corrected Solvent Control window binary HPG system If you are working with a high pressure gradient system again you can enter the values for all components in the same window However only that component will be accessible to which the pump was selected Knauer Instrument Control Method Options 175 Solvent Control L If the option On is checked then the amount of solvent s will be continuously monitored The HPLC system 8 will be shutdown as soon as the amount for even one solvent is smaller than the given shutdown level S 1000 6 S N 44444 X Accept 5 1000 4 5 N 33333 5 1000 B 5 N 44444 bustic warning Shutdown Bottle Close volume ml level ml level ml size ml Warning The amount of solvents used will be correctly calculated only under software control Should the pumps have been controlled manually the actual solvent amount must be corrected
347. selected event Unconditional FC Parameters at 4 00 min Signal 2 Relative to previous event min Unconditional Use as reference for correction next events Action Expected interval min Seen Collect To gt f next 0 position Waste Collect To Peak Recycling Solvent Recycling None Collect Slices E Cancel Fig 332 FC parameters unconditional only single event Action This selection as far as accessible is independent on the set mode unconditional signal level The choices are Waste Collect To Peak Recycling Solvent Recycling None Collect Slices ChromGate Preparative Option 239 Waste The eluate will be wasted Collect To The eluate will be led to the set position Peak Recycling The eluate will be led back to the column peak shaving Solvent Recycling The eluate will be collected in a recycling container because its purity is better than the set limits None Neither the recycling valve nor the fractionation valve will be switched The previous action is continued only a marker is set Collect Slices The eluate will be collected in set volume fractions into the next free positions You have only to select the type of action and in case of collect to the destination vial After closing the window the parameter will be designed as Timed The options Relative to previous event and Use as reference for correction next events
348. so not change the appearance of this window tab Unavailable features and functions will only be disabled and inaccessible In case of the Kontron pumps the GLP data and the Events are not displayed 140 Fig 207 Fig 208 Creating an Instrument Control Method ME Instrument Status System Stat Ces 51009 J AS Triathlon md s ssi Monitor Direct Control aa Run Status HOLD Flow ml min fo 000 Stop Flow Apply Pressure Control MPa i Bs 40 0 Gradient Components Component 2 EE E J Events Peele O O O O ee C 3 lo D 4 Control Pressure Limits MPa Total working time 4350h 21m 11s Total pump cycles 7360193 Min 0 0 Max 0 0 Apply Power index 1565402 40 MPah Installation date 27 Jul 2004 Events 00000000 Events Apply Network o SN 44444 f kontron Method kon met Data None Project Customer_hb Instrument Status E BIK File Edit View Method Data Sequence Analysis Control Reports Window Help Era EJ BRAM ER zg ET on O System Sta E komen 320 39 Kontron 422 ED Kontron 525 ED Kontron 322 ED Kontron 325 Monitor Direct Control Run Status HOLD Flow ml min 1 000 Stop Flow Apply Flow ml min 1 000 Gradient Components Pressure Control Bar 0 0 O 00 0 A ho B jo Gradient Apply Components not defined C fo Control Pressure limits MPa Min 0 0 Max 0 0 Apply 3 Serial Port C
349. ssigned to any detector or sensor producing an analog signal which can be digitized with A D converters or Interface Boxes Such a detector could be any UV RI or fluorescence detector or some type of pressure flow or temperature sensor etc For each user defined detector in the Configured Modules window double click the icon and complete the configuration dialog User Defined Detector Configuration Detector Signal Name User Detector Ags unita mv Model Analog El T Axlz multiplier 10 001 Serial Number Anis offset lo Interface IF2 r sl Add Info Cancel Help User defined detector configuration window Detector Name Enter a descriptive name for the detector This name will appear on the detector tab when the instrument setup or status windows are displayed Use a name which is unique within the instrument Detector Model Select the model of the detector from the drop down list planned future option Interface Select the A D interface for the detector you have already configured please see chapter Knauer Interface Configuration on page 32 and click the button to set up the acquisition channel Acquisition channel configuration Eg Device number Input Mote that you are not allowed to select channel which is already used Cancel Acquisition channel configuration window 54 Setup and Control of Knauer HPLC Systems Select the Device number to which the
350. ssing the WL Apply button forces the wavelength change on the detector channels Events Digital Outs Click the button to set up the digital outputs Refer to the chapter Instrument Setup Pumps Pump section Configuring Digital Outputs for details Click the Events Apply button to accept the settings For detectors without the feature of digital outputs this area is grayed and inaccessible ae The D lamp should be switched on manually from the Direct Control window before starting the next working session After power up allow 30 minutes for the lamp to stabilize before running samples that may require sensitive measurements Info Click this button to display the device specific data in the detector information tab Spectrometer Information Spectrometer Identification 5 2600 DAD Manufacturer Knauer eae lee a UV no halogen lamp installed 56 Integration time range 13 200 me Coefficient A IA eee B 1 451168 0 000894 190 501 nm Instrument power cycles Instrument working hours Fig 224 Detector information window Diagnostics Click this button to use the diagnostic features of the ChromGate software The Diagnostics window appears which allows you to access information and to control important parameters and modules of the device Creating an Instrument Control Method 151 Fig 225 Diagnostics Integration Time Range 13 200 146 7 mec Scan nm Range 189 505 rim Clos
351. stalled modules in the ASM 2 1L have own IP ports which allows for separate control The configuration does to allow to select an ASM 2 L or search for the modules by using the button the pump and valve must be configured in the Injection Module configuration manually To use an ASM pump or valve in the injection module select the appropriate pump or valve model in the Injection Module configuration disable the option Use S N to identify the instrument and enter manually the serial number and IP address of the ASM 2 1L device Now change the IP port for the modules pump and valve lf the ASM 2 1L has IP port 10001 the module installed on the left side has IP port 10002 the middle module 10003 and the module installed on the right handed side the IP port 10004 If the IP port of the ASM 2 1L has been changed in the ASM s own setup the IP ports of the build in modules will be changed automatically they will always have the next possible IP ports However before you enter the IP ports of the pump and valve please check for the IP port of the used ASM 2 1L device Injection module instrument setup If you open the configured instrument and the instrument setup you will find an own tab for the injection module MM Instrument Setup E E El ES Pump 18000 Inj Module I Injection Frogram Pump Settings Injection Volume i DD mi Control Pressure Limits MPa Injection Flow 1 000 ER Min 0 0 Max 0 0 Injection Duration
352. stem the instrument control functions appear as part of the Instrument Setup area of your instrument window There are two ways to access the Instrument window in which you can set up your devices and methods 1 Double click on the instrument icon you wish to start from the main screen The window will be opened in the online mode 2 Right click on that icon and select the online or offline opening mode It may be required to log in before you can access the instrument window functions User name hans Password Project Customer hb Cancel Help Login dialog box Enter your name and your password and select the project from the pull down menu and then press the Login button Make sure that all devices of the selected instrument are switched on and connected to the described communication ports of the computer before opening the instrument window The keypad of the column oven Jetstream must display choose function If a device is not switched on or connected to the computer if opening the instrument window please close the instrument window switch or connect the device and reopen the instrument window Otherwise the correct communication of the devices with the software can be affected All of the functions and procedures described in this chapter can be performed in the off line operation mode The instrument wizard will appear the first time you open the instrument window Its shape depends on whether
353. stems only show the component A High pressure gradients show up to four components corresponding to the system configuration In case of low pressure gradient pumps three P2 1L preparative pump 1800 and Kontron 42x or four S 1050 S 1000 K 1000 K 1001 components can be defined ChromGate does not distinguish the two different LPG valve blocks for the preparative pumps P2 1L and 1800 refer the pumps manual The working limits of these blocks are three solvents with a maximum summarized flow of 220 ml min or two solvents with a maximum summarized flow of 1000 ml min Be sure to select the correct gradient mode for the connected valves otherwise the pump or valves may damaged In the K 501 pump s internal setup screen in the CONTROL menu you should set the Ext RS232 mode to be able to control the pump from the ChromGate software Using the Kontron pump 322 additionally the Compression stage can be selected The choices are Low Medium and High ED Kontron 322 de sl Compression stage EEMS gt Medium High Kontron 325 2 Aue Traces Solvent description in Kontron 322 pump setup window Flow OFF at the end of run Checking this option the flow will be set to zero after elapsing the runtime Action at the end of run S 1050 P2 1L only The pumps S 1050 and P2 1L allow not only setting the flow to zero but also to switch the pump into Standby after the run Creating an Instrument Control Meth
354. switch off the detector lamp after a measurement is finished This option may be used for the shutdown methods Time program The time program show the program lines for all configured modules Please refer to the setup information of the separate instruments pump detector valve MultiValve FC for the specific setup options If a HPG is configured there will only be shown a line for Pump A The header of the 3 column will show the unit for the module selected in the table For pumps the header shows Flow ml min for the detector WL nm and for valves Pos The 4 column only shows YA if a HPG is configured The smallest allowed time step in the time program is 0 02 minutes Show All Lines The drop down menu allows for showing either the lines for all configured modules or for just one of the modules The available modules depend on the configured modules for the Assistant The option Show ASM2 1L lines allows for programming the 3 events of the Assistant 110 Creating an Instrument Control Method Time min Add a new time line with the time you want to change a module s setting If you have added a line select the module you want to program in the device column If you want to change settings for several devices at the same time you must add separate lines for each module Device The device column allows for selecting one of the configured modules or the Assistant If a HPG is configured
355. t 10 Sec Options V Autozero at start Sampling rate 1 Hz Suitable for minimum peak width at base 0 3330 Min Run time 10 Min Acquisition delay 0 Min RI Detector setup window The only additional setting is the optional Autozero at start Check the Autozero field if you wish the signal to be zeroed at the moment a measurement is started to define the current signal as the baseline Instrument Setup UV Detectors S 200 K 200 The detectors K 200 and S 200 support settings that for all of the detec tors described on page 91 The only additional setting is the optional Autozero at start Check the Autozero field if you wish the signal to be zeroed at the moment a measurement is started to define the current signal as the baseline Because these detectors have a fixed wave length of 254 nm this setting is shown without access for changing Instrument Setup UV Detectors K 2000 K 2500 In the detector s setup screen EXT CONTROL menu you should set the keyboard mode to be able to control the wavelength from the ChromGate software The detectors K 2000 and K 2500 do not support additional commands Therefore the detector tab gives no access to further settings than that for all of the detectors described on page 91 Creating an Instrument Control Method 93 Instrument Setup UV Detectors UVD 2 1L UVD 2 18 S 2520 S 2500 K 2001 K 2501 277 Fig 136 For the detectors K 2001 25
356. t Coarse Print Quality will result in faster printing Cursors The values in these boxes reflect the current contour cursor positions When you change the values the cursors on the plot will be changed when you exit the dialog or when you click the Apply button Time This specifies the time position X Value of the cursor When a value is entered the X cursor is updated to the new position upon exiting the dialog Wavelength This specifies the wavelength position Y Value of the cursor When a value is entered the Y cursor is updated to the new position upon exiting the dialog Bandwidth This control specifies the wavelength band that will be averaged when a chromatogram is extracted from the contour plot The extracted chromatogram is an average of the absorbencies at each wavelength in the wavelength band The wavelength band is equal to the selected wavelength see above one half of the bandwidth Contour Axis Setup You can change the setup of the axes for the contour plot using this dialog If the Auto scale box is selected the software will automatically set the axis parameter lf you want to enter the parameter manually de select the Auto scale box then enter the range values or click the Get Limits button to bring in the limits from the current view 202 Fig 295 ChromGate PDA Option Contour Graph General Axis Setup Limits Time M Autoscale kin pon Mar Fado H Minutes aoto OOo Oooo W
357. t can be generated printed and exported as desired Single Level Calibration To set up a single level calibration 1 Collect a chromatogram containing the standard and save it The concentration of the compound in the standard should be similar to that in the samples that you want to analyze 2 Open the stored data file 3 Press the Analyze button Lal to integrate the chromatogram and show the baseline 4 Press the Define Single Peak button A to present the Define Single Peak dialog box The retention time in the upper right corner will refer to the first peak in the chromatogram Define Single Peak Retention time 0 394444 Minutes Peak name Conc level gt 0 Units ISTD ID Ref ID Current peak 1 Retention time window Total peaks 15 Relative Absolute Next gt gt Fig 15 Single peak definition 5 Press the Next gt gt button until the peak corresponding to the standard is selected As an alternative you can simply click on the peak in the chromatogram with the cursor 6 Complete the dialog box for the first standard Enter the peak name Indicate the concentration level For a single level concentration enter 1 for the level and indicate the concentration of the standard Indicate the desired unit e g ug ml Enter the internal standard D if you are using the internal standard method This number is obtained from the peak table Leave it blank until you
358. t fields are disabled Limit Peaks to time range This allows the user to restrict the peak selection made above to a time range When checked only peaks within the specified time range will be searched When this box is unchecked the Time Range edit fields are disabled Time Range This allows the user to restrict the peak selection made above to a time range When the Limit to time range box is unchecked these fields are disabled Report information Checking any of these controls indicates that the indicated value will be calculated and printed to the right of the graph Unchecking values that are not of interest will speed up analysis Peak Area Checking this indicates that the peak area should be printed to the right of the spectrum graph Lambda Max Checking this indicates that the three largest lambda max values should be printed to the right of the spectrum graph Lambda Min Checking this indicates that the three largest lambda min values should be printed to the right of the spectrum graph Total Purity Checking this indicates that total peak purity should be printed to the right of the spectrum graph ChromGate amp PDA Option 221 3 Point Purity Checking this indicates that 3 point peak purity should be printed to the right of the spectrum graph Similarity Checking this indicates that peak apex similarity to the reference spectrum should be printed to the right of the spectrum graph The use of th
359. t have a valid IP address As an option you can enter the IP address of your device if the option Use S N to identify the instrument is disabled Please note that in case the device receives the IP address from a DHCP server e g a router the IP address may change if the device is switched off and switched on Valve type The drop down list offers 2 6 12 and 16 position valves Select for each valve the desired type If you read out the valve via LAN the appropriate valve type has been selected automatically Vial Volume ml Enter any desired vial volume If any fraction is larger than the set volume the next free collection position will be used for continued collection Vial number The Vial number will be displayed automatically Fraction Collector Setup In any system you can include only one fraction collector However the setup window is independent on the configured fraction collector Therefore they will be explained together This window differs in one respect from other setup windows It is not only important for setting up the fraction collector You need it also during the runs However those aspects will be described later in the chapter instrument status of a running control method page 137 236 Fig 328 ChromGate Preparative Option ME Instrument Setup s 1000 Ej as 3950 Ej Ovens 4050 a s 2649 S 3050 FC Jk Aux Traces Je Trigger Detection Channel S 2600 1 vi V Collect
360. t ready to begin measurements Some configuration steps are not performed or test parameters are not correct Check the list below for details e Injection volume 20 00 is invalid For specified injection method volume should equal 100 pul oe Je et IL m A Ax i For Help press F1 PQ consistence check failed f PQ Method Test PQ met Data None Project Default Performance Qualification File Edit View Method Data Sequence Analysis Control Reports Window Help ass 1 5 2500 BEe REBEE LEAN Gove HK E Conditions Settings Measurements i System is ready to begin measurements under following conditions e Injecting 100 uL with Alias amp utosampler e Active channel S 2500 configured for 254 nm e Flow is 0 8 mL min e Acquisition Time is 3 min co gt ed J Maw M VX pe i For Help press F1 PQ consistence check passed Knauer Instrument Control Method Options Clicking on the Start gt button starts the automatic performance of the whole PQ procedure It will run like a usual sequence The direct control should not being used while running the PQ sequence While the data acquisition is running you can add traces of other runs as usual but with one exception You cannot add traces of earlier runs of the same PQ session because they are at this time only temporarily stored and not accessible The final storage takes place at the end of the session Thi
361. tandard in your unknown sample Multiplication Factor Enter a multiplication factor to be used for this run All analyzed peaks will be multiplied by this factor Calibrate This box is only used for calibration of partition chromatography samples It is not used in SEC runs When you have completed the Single Acquisition Run dialog box click Start to begin the acquisition The current data will appear in the chromatogram window as it is acquired and stored on disk At the end of the run the chromatogram will be analyzed according to the method parameters and a report generated if specified If the sample is not analyzed at the end of the acquisition click the Analyze button if you wish to view the results Sample Description When you click the Description button you can enter a text description for the sample that is saved in the data file The description can be viewed from the Open File dialog or when the file is open as the current data file using the Data Properties command Submit This button appears when data is currently being acquired using a sequence or single run The Submit button allows you to submit a single acquisition to be run at the completion of the current run The run is entered at the end of the Run Queue if you are currently running a sequence of runs Submit Priority This button will appear when data is currently being acquired using a sequence When you click Submit Priority you can submit the s
362. tatus The status HOLD will be displayed when no program is running otherwise the Run Status displays a run time in minutes elapsed from the start of the run The actual excitation wavelength emission wavelength signal value sensitivity gain and the selected output mode will be displayed Creating an Instrument Control Method 153 Fig 227 Direct Control All settings you have entered in this area will become effective after clicking on the Set button To achieve changes during a run the direct control option must be activated see Fig 249 on page 170 Excitation wavelength Emission wavelength You can enter the values of the excitation and emission wavelengths Sensitivity Gain Recorder Range Output Mode The time constant sensitivity level HIGH MED or LOW the gain 1 4 16 recorder range and the output mode Emission NOT Corrected Excitation Energy Light Source Corrected can be selected from the corresponding pull down menus Press the Set button to send the parameters to the detector Autozero Activate this option to perform an autozero at the start of the diagnostic measurement Once the Autozero button has been pressed the autozero is performed immediately Event Out Event Mark Event Out short circuits the event out terminal for approximately 1 second Event Mark adds an event mark to the analog output Diagnostics Click this button to use the diagnostic features of the ChromGate software
363. tatus 165 Wiring Scheme 287 290 Trigger 58 135 295 Tubing parameters 231 Tubing Volume 65 68 71 73 U USB license dongle 12 User defined Detector Configuration 53 Setup 104 Status 155 Wiring Scheme 285 289 User defined Injection Program 124 UV Detector Configuration 45 Instrument Status 145 147 Setup 92 V Validation of Integration 178 Vial Position 66 69 72 75 Virtual Detector Configuration 57 Formula 107 Setup 105 Status 155 Virtual fraction collector 233 W Wake Up option 149 Wash Times 113 117 Wash Volume 123 Wavelength table 94 Width 19 Wiring Interface box 57 289 Manager 5000 57 Wiring Schemes 284 Y Y Axis Units 50 56 O Wissenschaftliche Ger tebau Dr Ing Herbert Knauer GmbH All rights reserved The information in this document is subject to change without prior notice Translation of the original German edition of this manual 2012 10 02 Printed in Germany on environmentally friendly paper from sustainable forests ChromGate are registered trademark of Dr Ing Herbert Knauer GmbH See up to date manuals online www knauer net downloads HPLC SMB Osmometry Wissenschaftliche Geratebau Phone 49 30 809727 0 Dr Ing Herbert Knauer GmbH Telefax 49 30 8015010 Hegauer Weg 38 E Mail info knauer net 14163 Berlin Germany Internet www knauer net Ih KNALIER KNAUER 2012 V7057 8 0 05 10 12 Koe
364. tegrated stream switching 155 6 Solvent selection valve 55 Trav cooling Trap i Cancel Help Configuration windows for the autosamplers Triathlon and Endurance Name Enter a descriptive name for the sampler This name will appear on the autosampler tab when the instrument setup or status windows are displayed Use a name which is unique within the instrument Serial Number Type in the serial number of the autosampler you are using Device ID Select the device identifier for the autosampler from the drop down list This must match the settings of the autosampler being used To check the device identifier for the Triathlon or Endurance autosampler press the SYSTEM MENU and then lt COMMUNICATION SETTINGS gt buttons on the autosampler keyboard Serial Port Select the number for the communication port on your PC where the autosampler is connected Loop Volume Enter the volume of the installed loop in ul Tubing Volume Enter the volume of the installed needle and tubing in ul If the original tube is installed you can find the tubing volume on a label on the tube Syringe Volume Select the volume of the installed syringe from the drop down list The choices are 100 pl 250 ul 500 ul 1000 pl and 10000 ul The 10000 ul syringe can only be selected when a user defined injection method is used Solvent Selection Valve SSV Option Check this box if you have a solvent selection valve installed Tray Coolin
365. tep Mix According the number of Mix Times 1 9 times the programmed Amount ul max the syringe volume will be aspirated and dispensed If no destination vial is used the mixing step is performed in the sample vial Wait lo h mi min O sec Commi Fig 168 Autosampler 3900 3950 Mix Step Wait Wait a programmed period of time x xx xx 0 00 00 9 59 59 before continuing with the next step reaction time Click the Commit button to complete the operation Click the Delete button to delete the selected step Instrument Setup Autosampler 3950 Alias MM Instrument Setup E m x Injection Parameters asi da Syringe speed Normal i Ar segment fe Full loop Partial loopfill Flush volume pl 130 iW Headspace pressure pl pick up Wash times 2 I Needle wash Needle height mm 20 Temperature Control Enable tray cooling ra Mis methods Stacked injections WAL 523509 gt WES JB 2550 Flor 5 050 044 5 1650 Fig 169 Autosampler 3950 setup window Creating an Instrument Control Method 117 Injection Method Select the injection method by clicking on one of the available options Full loop Partial loop fill or ul pick up If ul pick up is selected a drop down menu for the Transport vial position Transport pos is available IS The maximum programmable injection volumes are Flushed loop not programmable is equal to the loop v
366. the Interface must be set to RS232 115200 Please refer to the detector s manual chapter Setup menu for more information The detectors 2550 2520 and 10D UDV2 1S have a limited data rate max 10 Hz with a serial RS 232 connection The maximum data rate for a detector is only available via LAN connection for data acquisition for the detector S 2550 only on one channel The detector S 2600 must be set in its own setup in CONTROL to RS232 binary baud rate 115200 LAN Interface LR Mg y 2 IP Port 1 0001 MW Use S N to identify the instrument LAN interface dialog 50 Setup and Control of Knauer HPLC Systems All LAN controlled devices can be added and configured automatically using the Auto Configuration button on the configuration main window Beside this you can let the software search for your already connected and switched on device from the devices configuration window The option Use S N to identify the instrument must be enabled Click on the l button to start the search for all connected LAN devices of the currently configured type If more than one will be found all hits will be shown in the window Select the desired device from the shown list The software will read out the information as serial number and will fill it automatically into the configuration screen For all Knauer LAN devices port 10001 is selected by default Don t change this setting otherwise no communication can b
367. the Apply button The On Off button can be used for enabling disabling the cooling control The labeling of the button changes with the actual status Start Needle Wash Press this button to force the autosampler to perform the needle wash Advance Tray Clicking briefly on this button will move the tray to the next position Instrument Status Autosampler 3950 MM Instrument Status O System Status jy 5 1050 J User Defined Detector MultiWalve FC Monitor Direct Control AS Status Ready Tray Temperature C 4 Current Vial Tray Temperature C Reset Transport Vials Start Needle Wash Move Tray 3 Serial Port COM9 al SN 80079 gt Fig 241 Autosampler 3950 status tab Monitor AS Status The status Ready will be displayed when the autosampler is waiting to perform an injection If an injection program is running the actual program step will be indicated After a completed injection the status is described as Analysis timer running during the chromatographic run Current Vial The actual position of the injection needle is shown Tray Temperature C If the tray cooling option has been installed and configured the actual tray temperature will be shown Direct Control The direct control features are not accessible during an injection Tray Temperature C If the cooling option is installed and configured the tray temperature can be set The entered value will be transferred to
368. the lamp off and change the button label to Lamp ON Creating an Instrument Control Method 149 Autozero Click this button to define the current signal as a zero level Scan nm This section is disabled for changes It only provides information on how the scan spectral interval is specified Changes must be made from the instrument setup window Fig 150 on page 102 It is not possible to modify this function during a run Digital Channels nm The number of channels that will be activated depends on how many possibilities are given to set new values for the measuring wavelengths Pressing the WL Apply button forces the wavelength change on the detector channels Digital Outs Events Click the button to set up the digital outputs Refer to the chapter Instrument Setup Pumps Pump section Configuring Digital Outputs for details Click the DO Apply button to accept the settings For detectors without the feature of digital outputs this area is grayed and inaccessible ae The Dz lamp should be switched on manually from the direct control window or using the Wake Up option from the K 2600 before starting the next working session After power up allow 30 minutes for the lamp to stabilize before running samples that may require sensitive measurements Instrument Status Diode Array Detectors S 2600 DAD 2850 DAD 2800 K 2700 and Kontron DAD 540 545 DE Instrument Status O System Status E 5 1000 E as nE Monitor
369. the same IP port as selected in the Auto Configuration window will be found The default IP address is 10001 It can be changed manually in the device s setup We recommend reviewing the configuration of all automatically added devices Devices connected via RS 232 serial connection will not be found and must be added manually Now configure or review each module detector pump autosampler or event configuration separately Not configured instruments may be indicated by a question mark on the corresponding icons If you try to exit the window with OK and one or more instruments are still not configured a message like Fig 42 will appear ChromGate Client Server A Autosampler 5 3950 has not been configured and must be configured or deleted Example of an operation message to complete configuration Options The Options button is used to initiate any optional analysis software for this instrument 38 Fig 43 Setup and Control of Knauer HPLC Systems A list of available analysis options will be presented Configuration Options General Ts patem Suitability SEC PDA Baseline Check Abbrechen Hilfe analysis options window Click the box es to select the optional software you want to enable for this instrument The additional licenses are required beside the Baseline Check For more information regarding the option please check the System Options part above on page 14 The Knauer Fraction C
370. this option The grayed area is only a reminder that the autozero will take place at any wavelength change during a run Pump HPG Pressure Limits unit only for pump P4 1S This section is only accessible if a pump P4 1S Pump or two pumps as a HPG with including a pump P4 15 HPG have been configured The shown pressure unit depends on the unit selected in the pump configuration MPa bar psi Min Enter the minimum pressure allowable for your pump The zero setting disables the shut down due to minimum pressure Max Enter the maximum pressure allowable for your pump The default value always corresponds to the configured pump head setting above this value is not allowed If for the min and max limit 0 is set the software will not check for the pressure and the limits will only be monitored by the pump internally Options The settings accessible in this section depend on the configured modules Standby at the End of Run If the standby option is checked the Assistant will switch into Standby if a run is finished The pump motor and the detector lamp will be switched off as well as the Assistant s display will show the Standby status of the Assistant If this option is selected all other options of the Options section become inaccessible Flow OFF at the end of run Checking this option the flow will be set to zero after elapsing the runtime Lamp s OFF at the End of Run Check the lamp OFF box to
371. tion Division Power unary plus and minus Advanced Pressing this button a dialog box appears to create a time table for changing the formula during a run Advanced Settings Dialog Cancel Virtual detector setup time table Instrument Setup Assistant ASM2 1L Due to the modular system there are a lot of possible configurations for the ASM2 1L The setup window will look different depending on the configured modules For all possible modules the setup is described below 108 Creating an Instrument Control Method ME Instrument Setup Run time 10 0 Min Time Program Show All Linez Acquisition delay 0 0 Min Time Device Comments min LIN 254 Uy 0 00 0 00 Waler Time constant 100 sec 0 00 Pump 0 000 Sampling rate 1 Hz W Autozero at start Autozero at WL change Pump Pressure Limits MPa Mlin 10 0 Mas 10 0 Options Standby at the End of Run T Flow Off at the End of Bun Pretreatment Lamplz Off at the End of Run oh Fig 160 ASM 2 1L setup window for pump valve detector Run time Run time determines the run time of all devices of the Assistant Acquisition delay only for UVD2 1S Acquisition delay is the interval between the start of the run Trigger and the time when sampling starts for this detector This option will only be shown if a detector is configured in the ASM 2 1L UV only for UVD2 1S This section only appears
372. tion date Column description M Calculate performance parameters for this channel Calculation methods iw LISP JEMG IDAE BP EP ASTM ChromGate System Suitability Setup 187 2 Select a method for calculation of the performance values by clicking on the Calculation Method down arrow 3 Choices include USP EMG Exponential Modified Gaussian DAB JP and AOH Area Height method The calculations used are given in the equations section For BP EP and ASTM methods choose DAB 4 Click on the Calculate Performance Parameters box to enable the suitability calculations for the method Running a Suitability Test Once you have completed the System Suitability Setup and Performance dialogs create a Sequence or modify an existing Sequence to include one or more system suitability runs according to the needs and SOP s of your laboratory If you want the System Suitability Report to print automatically at the end of the sequence you must also select the Print Sequence Reports option from the Sequence Properties dialog box For example following USP standards five replicate standards are run at the beginning of the sequence These are designated as system suitability standards in the sequence At the end of the sequence suitability calculations are made and EZChrom Elite Client Server generates a system suitability report Ss Suit Begin y 0 Suitability iltilevel Calibration met calibratio Sys Suit Std O Suitabili
373. tion information regarding the PLATINblue autosampler AS 1 please refer to the PLATINblue system manual 64 Fig 90 Fig 91 Fig 92 Setup and Control of Knauer HPLC Systems Configuration Autosampler 3950 Smartline Autosampler 3950 Alias Autosampler 3950 Configuration Name AS 3950 Serial number Interface RS 232 v COM Port COM1 v Baud Rate 9600 Tubing volume pl Loop volume pil Syringe volume yl Options I Tray cooling Prep mode 120 Tray SSV Cancel Help Configuration window for the autosampler 3950 Name Enter a descriptive name for the sampler This name will appear on the autosampler tab when the instrument setup or status windows are displayed Use a name which is unique within the instrument Serial Number You must type in the serial number from the manufacturer s label on the rear panel of the autosampler not the Knauer S N Interface Select the Interface you want to use for communication with the device Please note that the autosampler 3950 can only have RS 232 or LAN interface The communication port on the autosamplers rear panel is labeled as Communication Available interfaces Smartline autosampler 3950 RS 232 Select the number for the RS 232 communication port COM Port on your PC to which the autosampler is connected LAN Interface ILAN S l Use S N to identify the instrument LAN interface dialog
374. tion is disabled for the K 2700 detector Check the Lamp OFF box to switch the detector lamp off after a measurement is finished This option may be used for the shutdown methods You should switch the lamp on from the direct control window before you start the next working session After power up allow 30 minutes for the lamp to stabilize before running samples that may require sensitive measurements Advanced Pressing the Advanced button opens the window for reference correction Advanced Reference Correction Sample By O Rei He Ha Ha reference correction window Referring to a wavelength with practically zero absorption the drift due for instance to a too short heating time of the lamp can be eliminated Reference Correction According to the configuration up to 4 wavelengths can be monitored from the 3D data stream Select the checkbox to activate the associated channel Sample The sample wavelength is defined in the Acquisition tab Bw The bandwidth for the sample channels are also defined in the Acquisition tab Ref Reference is the wavelength at which a reference absorbance is measured as a function of time The reference wavelength compensates for fluctuations caused by changes in baseline absorbance for example due to changes in solvent composition during gradient elution The selected wavelength must be higher than the sample wavelength and be inside of the scan range for thi
375. tion is shown grayed out The right handed part of the Pump section allows for direct control of the pump independently from the method setup During a run this part is not accessible Flow ml min Direct Control Enter the desired flow for the pump and click on the lt Apply gt button to start the pump or change the current flow lf the flow rate is out of the acceptable flow range defined by the mounted pump head an appropriate error message will be shown Click on the lt Stop gt button to set the flow rate to O zero Pmin Pmax only for P4 1S Direct Control Enter the desired values for the allowed minimum and maximum system pressure The pump will immediately stop if the maximum pressure value is exceeded or if the pressure is below the minimum value for 30 seconds The unit depends on the pressure unit selected in the pump s configuration MPa bar or psi Please check for the pressure unit shown in the Status section of the pump Click on the lt Apply gt button to send the pressure limits to the pump If the pressure limits are out of acceptable pressure range defined by the mounted pump head an appropriate error message will be shown HPG only if an HPG is configured HPG Flow ml mini 1 000 Flow ml mint I A z lv Sep Components 100 0 60 Apply Pressure MP al MES Fin 0 0 FMax 0 0 Apply ASM2 1L status tab HPG The left handed part of the HPG section shows the pump status as flow i
376. tional recycling valve setup For an additional valve its communication port serial or LAN must be configured Sly Peak Recycling Recycling Volumetric delay a fa me W Use own ports for recycling Peak recycling port 10 S lv recycling port 11 Add Info Fig 327 Own port recycling valve setup for 12 position valve ChromGate Preparative Option 235 For use of own ports no further settings are required Valves number Enter the number of valves which build up the multi valve fraction collector The number is in practical patterns unlimited Configure Valve The drop down list offers as many positions as the valve number was entered above The following settings have to be made for each valve separately Interface Select the Interface you want to use for communication with the device RS 232 Select the number for the RS 232 communication port COM Port on your PC to which the autosampler is connected LAN To let the software search for your already connected and switched on device click on the l button to start the search for all connected LAN devices of the currently configured type The option Use S N to identify the instrument must be enabled Select the desired device from the shown list The software will read out the information as serial number and will fill it automatically into the configuration screen To search for an instrument the device must be connected switched on and mus
377. to connect each detector to the Autozero channel 1 To an integrator TE channel 2 output of a detector channel 3 e a channel 4 om FUSE 2A SLOW 58 IS Fig 83 Setup and Control of Knauer HPLC Systems The integrator inputs 5 and 6 as well as the I O channels WAGO connectors 5 and 6 of the Interface Box are not accessible by ChromGate To an integrator output of a detector Trigger y O AN IN AN EEE DIG IN RELAY 2 O sm HA channel 1 ua RS 232 o ms sk channel 2 mm channel 3 Pz EUT MADE IN GERMANY gt A S channel 4 GROUND 0 3 Cable connections to the Manager 5000 5050 or IF2 If you want your instrument to trigger the start of data acquisition or to perform an autozero you must take care for making the trigger signal and autozero connections The digital inputs and outputs used for the connections are situated on WAGO connectors adjacent to the Integrator Inputs Each of the WAGO connectors corresponds to one of the data acquisition channels Trigger Signal Connections If you want your instrument to trigger the start of data acquisition on your ChromGate system using the Interface Box you must connect a trigger signal cable to the Interface Box The trigger signal should be connected to one of the digital input connectors on the rear panel of the Interface Box The digital input must correspond to that one selected in you
378. ton to set up the autosampler tray 12 Setup and Control of Knauer HPLC Systems Tray configuration Tray type Vial type Standard First destination vial Fig 100 Tray configuration for the autosampler 3900 Tray Type Select the type of the installed tray The choices are 84 3 96 and 24 vials Vial Type Select the type of the vials used The choices are Standard and 2 5 ml The last is used only when lt 84 3 gt or lt 96 gt tray type is selected First Destination Vial Enable this option if you want to use destination vials First Destination Vial Position Enter the first destination vial position When complete click OK to exit the dialog and return to the instrument configuration window When complete click OK to exit the dialog and return to the instrument configuration icon list Configuration Autosampler Triathlon Endurance The configuration windows of the autosamplers Triathlon and Endurance are identical The only difference is in the tray setup window accessed by pressing the Tray button Setup and Control of Knauer HPLC Systems 73 Fig 101 Autosampler Endurance Configuration Mame AS Endurance Serial number god Autosampler Iriathlon Configuration Mame AS Triathlon Serial number Communication Device ID 20 Loop volume ul 1100 Serial Port COM1 Tubing volume ful 115 Syringe volume ul 250 Options Integrated stream switching 55 4 I In
379. tra files spc or you can create a library from the spectra obtained during the current run Spectral Library Definition PNA503 lib Wave From Wave To Time E Spectrum Data Source Component Hame Comment Acenapthene spc Acenapthene 400 5 23 Background Subtracted from 229 Trace Acenaphthylene spe Acenaphthylene 400 4415 Background Subtracted from 334 Trace Anthracenespc Anthracene 400 5 76 Background Subtracted from 334 Trace Library notes This library was created from the PN amp _Level_ dat demo files All spectra were background corrected from their peak start and stop points determined from their respective multi chromatogram trace Spectrum Data Source Click on the arrow in this field to select either Current Data or Spectrum File to be used to select the spectrum to be added to the library To add a spectrum from a spectrum file the spectrum must have been previously saved as a file with the spc extension typically using the Utilities Save Trace function of the pop up menu in the Spectrum pane If you chose to enter a spectrum from the current data file a dialog will appear where you must select the retention time at which you wish to select the spectrum Component Name Enter the chemical name or description of the spectrum This field is used for display and search pre filter purposes Lambda Max From To The wavelength range of the spectrum over which the software will calculate the lambda abso
380. ts To print the Suitability Report click on Reports Print Sequence Custom Report and select the System Suitability Report choice Note that in order to have your report printed automatically at the end of the sequence you must have selected the Print Sequence Reports option in the Sequence Properties dialog Suitability Reports EZChrom Elite Client Server provides a default suitability report template This report template can be used as is or it can be customized in the Sequence Custom Report editor ChromGate amp PDA Option 189 System Suitability Report Page 1 of 1 Sequence CESC rom Elite SEQUENCE Siutbality seg User System Printed VENT 9 43 24 AM SYSTEM 15 NOT SUITABLE Channel 4 Conmpound Parameter Nim Max w RSD Acetmingp hen estd 2 000 11 000 tpperm 48000 00 S0000 00 5 000 k ID pund Parameter n Law I w RSD Stane Acetomingp hen estd 13 009 10 000 50 046 OF 444 Suna iht 10 000 Passed Suma nt 10 000 Passed Suntab ity 10 000 Passed Suab ihn 10 000 Passed Summa hint S0 046 FAILED FAILED tpperm 49016 15 4J6025 98 J9 63 66 3410 Suab inh 4J97E5 66 Passed Suntah ity 49 76 5 66 Passed Suab iht 49763 66 Passed Suab iht 4J97E5 66 Passed Suab iht JH02592 Passed Channel 4 Sample ID Test tartihi in Stop rim Threshold Result Stat Suab iy Pratt 0 000 0 012 1000 0010 600 000 Passed Suntab iby Drift 0 000 0 012 1000 000 600 000 Passed Suntah ity Dritt 0 000 0 012 10001 0010 600 000 Passed Suab wh
381. ts f Ovenatite existing program Insert to begin f Append to end E Insert to focused positior Autosampler Triathlon opening a user defined program Select the file with the user defined program and the Open Mode The choices are Overwrite existing program Insert to begin Append to end and Insert to focused position Click the Open button in the dialog to complete the operation and to return to the main autosampler method set up dialog Click the Store UDP button to store the user defined method in the battery backup memory of the autosampler with the entered number of the local program 128 Creating an Instrument Control Method Large Injection Check the Enable large injections box to use this option for injections larger than 1 ml This selection sets the standard injection parameters inactive It cannot be combined with any user defined injection program Instrument Setup Kontron Autosamplers MM Instrument Setup q s r Kontron 560 B Oven 5 4000 a K 200 4 s 2800 gt Aux Trac a Aux V Needle wash Air gap ul B Sample volume ul feo Into loop ul jo E Instrument Setup 3 E Kontron 460 jYoven s 4000 aa k 200 J s 2800 2 lt Aux Traces 3e Trigger Fig 182 Kontron Autosamplers setup window Injection type Needle wash The standard procedure involves two needle wash cycles after each injection Checking this option additionally a needle wash is performed before each inject
382. ts cannot be controlled anymore they will stay in the last set status The detector tab contains several areas where you can define the acquisition Some of them are independent on type and model of the detector Time constant 10 1 v Sec Sampling rate 1 v Hz Suitable for minimum peak width at base 0 3330 Min Run time 11 1 Min rcquaition delay lo Min Detector type independent settings Acquisition channel on Check the acquisition channel on box to enable data acquisition for the detector This function is unavailable when using diode array detectors the S 2550 or the K 2600 Time Constant Select the time constant for the detector from the drop down list The choices are 0 1 0 2 0 5 1 0 2 0 5 0 and 10 0 sec The detectors UVD 2 1L UVD 2 1S S 2550 52520 allow additional values 0 000 0 005 0 01 0 02 0 05 for LAN connection only Smaller time constants are required for high speed analyses A good rule is to select a time constant which is the reciprocal of the data rate in Hz e g 2 Hz data rate gt 0 5 sec time constant Sampling rate This is the rate at which data will be sampled by the system For any selected sampling rate the narrowest peak width will be shown for which this sampling rate will be adequate This selection is in Hz data points per second This is the selection for most chromatography applications We recommend having more than 20 data points per peak 30 40 This means that
383. tton on the configuration main window All included device will be recognized and configured Beside this you can let the software search for your already connected and switched on ASM2 1L from the devices configuration window The option Use S N to identify the instrument must be enabled Click on the l button to start the search for all ASM2 1L instruments Leak Sensor Sensitivity Select the leak sensor s sensitivity in accordance with your requirements The choices are Off Low Medium and High Add Info Click the button Add Info to enter more detailed description of the pump Additional Information Flow in the system is limited to Sml due to security reasons pump setup Cancel Additional information box If you have already read out the assistant instrument using lt Auto Configuration gt or the l button you may not configure your assistant instrument manually This is the recommended approach The automatic configuration eliminates the risk of a faulty configuration Nevertheless the manual configuration will be described below Left Device Due to that for all three device positions the same devices and settings can be selected the options will only be described for the Left Device tab 62 Fig 89 Setup and Control of Knauer HPLC Systems Device Name Valco Valve Cancel Help Configuration windows left device tab device type selection Device Type Select the device type th
384. tup the Interface must be set to RS232 115200 Please refer to the pump s manual chapter Setup menu for more information LAN Interface EAN ea a IP Port 1 0001 MW Use S N to identify the instrument LAN interface dialog All LAN controlled devices can be added and configured automatically using the Auto Configuration button on the configuration main window Beside this you can let the software search for your already connected and switched on device from the devices configuration window The option Use S N to identify the instrument must be enabled Click on the l button to start the search for all connected LAN devices of the currently configured type Please refer the picture below for the information which will be displayed for the found devices If more than one will be found all hits will be shown in the window Select the desired device from the shown list The software will read out the information as serial number pump head and gradient mode from the pump and will fill it automatically into the configuration screen For pumps P2 1L and S 1050 the gradient mode can be changed using ChromGate if necessary Please see the corresponding section below For all Knauer LAN devices port 10001 is selected by default Don t change this setting otherwise no communication can be established To search for an instrument the device must be connected switched on and must have a valid IP address As an option
385. ty Multilevel Calibration met iuli calibre Sus Suit Std O Suitabilitys Multilevel Calibration met iuli calibre Sie Sult Std O Suitability Multilevel Calibration met ialti calibra Sie Suit End O Suitability Multilevel Calibration met rulti calibre Fig 280 Run Type is selected by clicking on the Run Type field then selecting the appropriate sample type from the choices provided 188 ChromGate System Suitability Setup Sample Run Typels r Run Type Parameters Clear Response Factors for Level Print Response Factors Average Replicates Clear Replicates No parameters necessary Begin System Suitability System Suitability Standard JEnd System Suitability Begin Summary Summary Run JEnd Summary JQC Check Standard Begin Calibration JEnd Calibration Fig 281 The first sample in your suitability set should be designated as Begin System Suitability Additional suitability standards should be designated as System Suitability Standards and the final sample in your suitability set should be designated as End System Suitability Multiple sample run types can be selected for a given sample A System Suitability Report will be generated at the end of the set of suitability standards when the sequence is run To view the Suitability Report on screen click Reports View Sequence Custom Report choice The System Suitability Report will appear in a list of available Sequence Custom Repor
386. ulates that the vial is empty the method sequence will stopped Dependent from the transport liquid level the autosampler will adapt the needle immersion depth Therefore it is important to fill the transport vials whenever the autosampler was switch off or the transport liquid level was reset manually If a vial is selected as the transport vial the configuration of the vial as a Reagent A or B vial will be ignored Stacked injections The option Stacked injections allow to define additional injections during a run Such injections can only be programmed either for the autosampler 3950 or the injection module please refer the FRC section chapter Stacked Injections of this manual Click on the Stacked injections button to open the Stacked Injections table dialog Enable the check box Use stacked injection Creating an Instrument Control Method 119 Stacked In Table Injection method max inj volume 100 pl Camel Start sample preparation 0 25 min before stacked injection Do not wash the needle between injections from same vial Time Inj olume TE 13 0 00 lt IlNJ_POS gt slh YOL Initial injection A The actual time s of the stacked injection s may deviate from that programmed Fig 170 Autosampler 3950 Stacked injection table Fig 171 Injection method The injection method is the same defined in the instrument setup of the autosampler It cannot be selected separately for the st
387. unication Device ID 60 Loop volume pil 1 OG Serial Port COM1 Tubing volume pl 5 Syringe volume pl 250 Options Tray cooling Prep mode Trap Cancel Help Configuration window for the autosampler Knauer Optimas Name Enter a descriptive name for the sampler This name will appear on the autosampler tab when the instrument setup or status windows are displayed Use a name which is unique within the instrument Serial Number Type in the serial number of the detector you are using Setup and Control of Knauer HPLC Systems Device ID Select the device identifier for the autosampler from the drop down list This must match the settings of the autosampler being used To check the device identifier for the autosampler Knauer Optimas press the SYSTEM MENU and then SERIAL buttons on the autosampler keyboard The default identifier is 60 Serial Port Select the number for the communication port on your PC where the autosampler is connected On the autosamplers rear panel use the port S2 IN to connect the autosampler with the computer It is recommended to switch all DIP switches for the port to OFF position for a proper communication Loop Volume Enter the volume of the installed loop in ul If the Prep mode is enabled the loop volume is fixed on 10000 ul Tubing Volume Type in volume of the installed needle and tubing in ul The valid range is 0 999 ul The volume is noted on a l
388. up menu Therefore it is highly recommend to delete all program lines of the detector s internal wave length program please refer to the detector s manual and for the first line time 0 which cannot be deleted to set the wave length to the same value as in the software Refer to the section setting up a trigger on page 135 for details Creating an Instrument Control Method 95 Instrument Setup Kontron Detectors 3xx 4xx and 5xx Piti MM instrument Setup OB Kontron 4 ul OB Kontron 4304 YB Kontron 432 OB Kontron 435 I p Trigger Resp time 0 05 w Sec Used Channels 7 Sampling rate 1 y Hz Time Wave Wave min lenoth 1 length2 Comments Suitable for minimum peak width at base 0 333 Min D DO 254 350 Range 1 000 AUFS 10 00 254 350 Cycle time 1 00 Sec Run Time 10 Min Acquisition Delay lo Min IV Autozero at start Fig 138 Kontron Detector setup window Except the not available option Acquisition Channel On Off the general detector settings as described on page 91 can be made Additionally the range can be set to define the range of the analog detector outputs The choice is 0 100 0 200 0 500 1 000 and 2 000 Absorption Units Full Scale AUFS The Kontron x30 and x35 detectors can be used in a dual channel mode As far as the option Used Channel 2 is checked a second wavelength column will appear in the spreadsheet below Also the otherwise not accessible cycle time becom
389. utosampler MN Instrument Status O System Status E 5 1000 amp AS Triathlon BH s 2600 Monitor Direct Control AS Status Filling sample loop Tray Temperature C Spi Current Vial 1 Tray Temperature CJ n a Serial Port COM1 Fig 242 Autosampler Triathlon status tab Monitor AS Status The status Ready will be displayed when the autosampler is waiting to perform an injection If an injection program is running the actual program step will be indicated After a completed injection the status is described as Analysis timer running during the chromatographic run Current Vial The actual position of the injection needle is shown Tray Temperature C If the tray cooling option has been installed and configured the actual tray temperature will be shown Direct Control The direct control features are not accessible during an injection Tray Temperature C If the tray cooling option has been installed and configured the tray temperature can be set The entered value will be transferred to the sampler by clicking the Apply button The On Off button can be used for enabling disabling the cooling control The labeling of the button changes with the actual status Start Initial Wash Press this button to force the autosampler to perform the needle wash Creating an Instrument Control Method Move Plate Endurance only Clicking briefly on this button will move the tray to the next position
390. vailable pump heads for K 1001 K 1000 K 501 and K 120 pumps For the pumps S 1000 1050 S 100 and 10P 20P P2 1S P4 1S only 10 ml or 50 ml is to select not considering the material The pump P2 1L and the preparative pump 1800 can be equipped with four different pump heads with 100 250 500 or 1000 ml respectively In case of titanium inlays also select the only available steel pump head for the preparative pump 1800 Head Material Fil Max Head flow TU mmn Available pump heads S 1000 1050 100 Head 100 ral Head 100 ml steel Loe OG ml stee Pressure Units 100 ml steel 250 mi Material 250 ml steel 300 ml Max Head Flow 1000 mi EREE C jai Max Head Flow 500 ml steel Max Head Pressure B Max Head Pressure 1000 mi steel Available pump heads P2 1L A preparative pump 1800 B Once you have chosen the pump head the related information about material maximal flow and maximal pressure is displayed automatically If you have read out the device via LAN the pump head type has been selected automatically Setup and Control of Knauer HPLC Systems 43 Fig 57 Fig 58 Pressure Units Select the pressure units you want to use for the pump from the drop down list The choices are MPa bar and psi All control pressure fields in the pump tab of the instrument method window as well as the instrument status window will be displayed in the selected pressure units The pressure unit
391. valves and other devices via auxiliary contact closures or a binary output BCD or HEX Click the Timed events button to set up the time base methods The time base methods setup dialog will appear Timebase Methods Programmed methods HN Method AL time Stop events at time E h jo min sec cancel Fig 176 Autosampler Triathlon Time base Methods Click the New button to add one more line to the programmed methods Select the Method Type from the drop down list The choices are AUX 1 AUX 2 AUX 3 AUX 4 ISS A ISS B CODE Out and SSV Port Every auxiliary AUX 1 through AUX 4 can be set to ON contact closure four times and four times to OFF at a programmed Time It is not possible to program the same auxiliary to ON and OFF at the same time The ISS valve can only be programmed if the optional ISS valves are installed The valves ISS A and ISS B can be set to positions 6 1 four times and four times to 1 2 at a programmed Time The SSV port can only be programmed if the SSV option is installed Set the time and the SSV port number a value between 1 and 6 124 CAUT ION Fig 177 Creating an Instrument Control Method Eight SSV port switch commands are possible in the time base program The Code event needs a time and a value between 0 and 15 F hexadecimal output Eight code output commands are possible in the time base program The maximal programmable time for the time base methods is 9
392. ve and click on the l button For the separate recycling valve a COM port or the IP address is to define The settings for the volumetric delay will be displayed without access for changing The settings are valid as well for the recycling valve as for the fractionation valve Therefore the capillary between the recycling valve and the fraction collector should be as short as possible Only for the Knauer fraction collector 3050 and the multi valve fraction collectors the option Use own ports for recycling will be displayed If you want to use this option click into the check box for this option For the multi valve fraction collector you will find that the number of available ports for fractionation will be reduced by 2 Additionally the ports numbers for solvent and peak recycling will be shown It is not possible only to enable one of the recycling options Siv Peak Recycling Volumetric delay Recycling EE Es este fa Seral port COM Recycling Valve Configuration Settings Serial number li al TI Valve type Enma e Interface ES ddi merma IP Part 1 0001 h Use S N to identify the instrument Fig 322 Recycling valve setup ChromGate Preparative Option 233 Fig 323 The dialog box for the port settings will be active only for fraction collectors with different options Port Settings Baud Rate Cancel Data Bits Parity
393. vials First Destination Vial Position Enter the first destination vial position When complete click OK to exit the dialog and return to the instrument configuration window When complete click OK to exit the dialog and return to the instrument configuration icon list IES The autosampler Knauer Optimas must be set in to serial mode to be controlled by software Press the lt Serial gt button to switch the autosampler into serial mode 70 Fig 98 Setup and Control of Knauer HPLC Systems Configuration Autosampler 3800 Smartline Autosampler 3800 and K 3800 Basic Marathon Autosampler 5 3800 Basic Marathon Configuration Mame 3800 Serial number 33333 Communication Device ID 10 h Loop volume ul 20 Serial Port COM1 sample tray 36 vials of 1 5 mi Options Tray cooling Partial loopfil Configuration window for the autosampler 3800 Name Enter a descriptive name for the sampler This name will appear on the autosampler tab when the instrument setup or status windows are displayed Use a name which is unique within the instrument Device ID Select the device identifier for the autosampler from the drop down list This must match the settings of the autosampler being used To check the device identifier for the Smartline Autosampler 3800 or the K 3800 Basic Marathon press the PROG END button on the autosampler keyboard once in serial mode Serial Port Select the number for the co
394. vice names must be unique in an instrument configuration Therefore enter different names for the same device types e g if a high pressure gradient is used If the configuration has been read out by Auto Configuration or using the 1 button the software will name devices from the same type automatically with different names For valves a letter in regard of the valve s position in the assistant will be added e g a valve in left position will be named as Valvel Pumps will get an A or B increment for HPG A or B pump However you can edit the automatically given name as well as the HPG position for the pumps Middle Device Due to that for all three device positions the same devices and settings can be selected the options will only be described for the Left Device tab Right Device Due to that for all three device positions the same devices and settings can be selected the options will only be described for the Left Device tab If one of the modules should not be controlled it must be removed from the ASM 2 1L configuration There is no option to disable a configured module later on in the Method Setup Configuration Autosampler For each system only one autosampler will be accepted Select the autosampler in the Configured Modules window double click the icon and complete the configuration dialog For configuration of the Injection Module please refer the FRC section chapter Stacked Injections For configura
395. vity log but cannot be stored in the original acquisition method attached to the data file This may not meet your GLP GMP requirements Save Changes in time table This option can only be enabled disabled if the Enabled checkbox for Direct Control during a run is activated If the Save changes Option is checked any method changes by direct control will be saved in the method Otherwise the changes only can be monitored in the instrument activity log and in the audit trail refer to the EZChrom reference manual Due to that the changes cannot be stored in the method which is attached as the original acquisition method to the data file we recommend reanalyzing the chromatogram with the changed method All methods used to analyze a chromatogram also will be added to the chromatogram data file and can be extracted as From Results in the Open Data dialog Knauer Instrument Control Method Options 171 lt Advanced gt If you click the lt Advanced gt button three additional options are available Advanced Method Settings Check run time when saving method E W Enabled System wake up and all lamps on W Power all devices up at 14 08 12 12 48 49 Trace name Cancel If this option te checked the trace name contains the Initial WL Otherwise it contains the name of the corresponding channel as defined in the configuration Fig 250 Knauer Instrument Control Method Option Advanced Runti
396. w quaternary LPG To reach the dialog box for editing the settings either click on the Change button or select the menu sequence 174 Fig 257 Fig 258 Knauer Instrument Control Method Options METHOD CONFIGURE SOLVENT Solvent Control E Ifthe option On is checked then the amount of solvent s will be continuously monitored The HPLC system will be shutdown as soon as the amount for even one solvent is smaller than the given shutdown level 5 100074 S N 33333 v Components Acoustic warning Shutdown Bottle level ml level ml size mi 1000 0 1000 0 1000 0 1000 0 Warning The amount of solvents used will be correctly calculated only under software control Should the pumps have been controlled manually the actual solvent amount must be corrected Solvent Control dialog box quaternary LPG If you are working with a low pressure gradient system you can enter the values for all components in the same window You can activate the option for each of the components separately As many solvents are configured as many components will be accessible Consequently an isocratic system will give access only to component Check the On option to get a continuously monitored solvent amount for each pump separately With this option activated you have access to entering the values for the bottle size actual volume the acoustic warning level and the shutdown level Accept the entered values and clo
397. w spectra extracted from the 3D data are averaged before being exported or used in the Spectrum Report Spectrum Averaging is the calculation of a spectrum whose absorbance values are the average of the absorbencies The peak apex spectrum When this is selected the spectrum at the apex of the peak will be used without averaging The avg of upslope apex and down slope spectra When this is selected three spectra the spectra at the upslope inflection point the apex and the down slope inflection point are averaged The avg of every n spectra across the peak When this is selected enter a value for n Every nth spectrum from the peak is extracted beginning at the start of the peak and continuing to the end of the peak These extracted spectra are averaged and become the spectrum that is reported or exported Per peak spectrum calculations Checking any of these boxes indicates that the indicated value will be calculated on a per peak basis during analysis The result of this calculation will then be available in reports and as chromatogram annotations Unchecking values that are not of interest will soeed up analysis If a box is unchecked and the field appears in a run report it will be reported as zero Similarity Checking this box causes the Peak Apex Similarity to the reference spectrum to be calculated on a per peak basis during analysis The result of this calculation will then be available in reports and as chromatog
398. w window It allows you to perform a library search on a spectrum you have selected in the contour map and also lets you select a spectrum from the current data or stored spectrum file to search ChromGate amp PDA Option 215 Fig 314 Spectral Library Search Of Hits Displaved 3 Search Quick y 31 Search Now 3 Display Params 3 Hits Found Hit 1 Library Eten PNA4503 lib Source Acenaphthylene s Component Acenaphthylene Params Pre Filters 22 250 275 300 Retention time 4 15 Min y Wavelength Wavelength range 200 400 nm Source Current Data Spectrum 5 18 Min Wavelength step fi nm Unknown Hit 2 5 Library E ten PNA503 lib Max hits 3 E Source 4cenapthene spc E Component 4cenapthene Similarity threshold Retention time 5 23 Min 225 250 27 3 Similarity 0 24 Wavelength Library E tenterpriselProjects PDAPNASD3 li 5 Hit 3 Library E ten PNA503 lib Source Anthracene spe Unknown Component Anthracene Retention time 5 76 Min 225 250 275 300 Similarity 0 09 Wavelength Hits Displayed Select the number of hits you wish to display Hits will be displayed in order of similarity Search Select Method if you want to use the parameters from the current method for your search Select Quick if you want to enter or change the parameters yourself before the search Display Parameters Click this button if you want to display the parameters If you always use the method
399. y Drift 0 000 0 012 1000 000 600 000 Passed Suma iby Drift 0 000 0 012 10001 0010 428 571 Passed Summa iht Moise 0 000 0 012 5 000 0 000 Passed Suab iht Moie 0 000 0 012 5 000 0 000 Passed Suab ihi Nore 0 000 0 012 5 000 0 000 Passed Sunna iby Moise 0 000 0 012 5 000 0 000 Passed Suab iht Moie 0 000 0 012 5 000 0 000 Passed Fig 282 ChromGate PDA Option The PDA Option enables you to view and analyze spectra generated with a diode array detector or a K 2600 on your LC In order to use the PDA Option you must have control software installed for your LC that contains PDA detector capabilities PDA Method Setup In addition to instrument control parameters that control the operation of your PDA detector there are setup options for the PDA available in the method These include setting up libraries spectral filtering definition of multiple wavelengths for multi wavelength display and setup of two wavelengths to be used for the Ratio view These options are available from the main menu by selecting Method PDA Options PDA Options Library Using this tab you can enter library parameters for spectral library searches that will be saved as part of your method When you do a spectral library search using method parameters these parameters will be used ChromGate PDA Option PDA Options EEK Library lt Purity 4 Spectrum en Multi Chromatagram Ratio Search parameter Pre filterz Wavelength range oo 40
400. y Table Spectra are automatically added to and deleted from this table whenever a spectrum is added to and deleted from the Spectrum pane of the Mixed View Spectrum Name Similarity Spectrum Source v 5 80Min 1 0000 Current Data pna spe 0 0510 C jenterpriselPr 5 90 Min 0 4373 Current Data ne Set Reference Print Properties Spectrum Name This column displays identifications for each of the spectra added to the table For spectra extracted from the 3D data the identification includes the time For spectra extracted from a chromatogram based on a peak name the identification includes the peak name For spectra loaded from a file the identification includes the retention time A check mark next to the colored line for a spectrum indicates that the spectrum will serve as the Reference Spectrum for the similarity calculation Spectra may be added to the table in the Mixed View by selecting the Add to Similarity Table from the Actions button menu Similarity This column displays the similarity of the spectrum on that row relative to the Reference Spectrum The Reference Spectrum is determined by double clicking on a row of the table or by highlighting a row and pressing the Set Reference button Until a reference spectrum has been selected the similarity for all spectra in the table will be zero Refer to the PDA Analysis and Calculations section for details on how this calculation is performe
401. y will appear on your chromatogram as shown below Note If you have not entered an SEC Baseline or Result Range into your SEC method yet it will not appear on the chromatogram 266 Fig 376 Fig 377 SEC Option fee Channel A Pi Time 30 0263 M nutes Amplitude volts 0 6 OO 0 5 I 0 5 0 4 0 4 Y 0 35 20 3 q 0 2 T 0 2 Ca o i F un Hl 0 1 E 20 1 0 0 0 0 O 0 10 0 12 5 15 0 17 5 20 0 27 5 25 0 27 5 50 0 h nutes You can change the colors and fonts of these annotations from the Appearance dialog Right hand mouse click and select Appearance Slice vs Mol Wt Trace Molecular Mass Distribution To create a Slice vs Molecular Mass Molecular Mass Distribution trace open a data file and analyze it Select the View Overlay command from the menu bar Click the right hand mouse button on the chromatogram and select Add Trace Select the Current Data for the Data Source or another source if desired and then select a Mol Wt Dist for the Trace Then Scale to the largest peak and click OK Note This can also be added using the Rt Mouse click Properties command New Trace Properties New Trace Annotation Appearance Data Source Current Data Trace Channel z Channel 4 scale To Chanel Mol we Diti v Min Y Max Units 2 Offset Scale Y Offset Y Scale OF Cancel Apply Help Because the scales are totally di
402. you can increase the bandwidth value in ChromGate to obtain a lower noise level Each discrete channel can use its own wavelength program and can be measured with its own bandwidth so you can always obtain the lowest noise conditions while still having a low enough bandwidth to not degrade the resolution measured This means that the diode array detectors can always be optimized for maximum performance The scan parameters can be set to the values which are optimal for doing the scans and yet the discrete channels can be measured independently at their optimal values The wavelength table is a spreadsheet to enter your time detector program In the first row at time 0 00 min enter the initial wavelength s in nm The editing principle including programming the digital outs S 2600 only is the same as for the time gradient program described in the chapter Instrument Setup Pumps Pump Program Instrument Setup Fluorescence Detector RF 10AxI RF 20A The Fluorescence Detectors RF 10Axl and RF 20A Axs support the general detector settings described on page 91 The detector RF 10Axl only supports a fixed sampling rate of 0 5 Hz For higher sampling rates the data acquisition via the 1V integrator output and an A D converter as Knauer interface box IF2 must be used The detector RF 20A Axs supports sampling rates up to 0 5 Hz For higher sampling rates the data acquisition via the 1V integrator output and an A D converter
403. you want to install and deselect the components you do not want to install Description FES FC Buechi FC Vario 2000 FC Vario 4000 FC Foxy dr FC Foxy Al FC Foxy Az FC Buechi C 660 hel virtual FC 2 60 MB of space required on the C drive 1950 82 MB of space available on the E drive Fig 6 Click on Next gt to confirm the window In the next window click on Install to start the installation To cancel the installation procedure click Cancel Fig 7 Fig 8 Installation Guide ChromGate 3 3 2 Knauer Fraction Collectors AddOn InstallShield Wizard Ready to Install the Program The wizard is ready to begin installation Click Install to begin the installation IF you want to review or change any of your installation settings click Back Click Cancel to exit the wizard Cas JE The following window will be displayed when the installation has completed Knauer Fraction Collectors AddOn InstallShield Wizard InstallShield Wizard Complete The InstallShield Wizard has successfully installed Knauer Fraction Collectors 4ddOn Click Finish to exit the wizard Click on Finish to finalize the KNAUER fraction collectors AddOn installation The installer for the Generic Drivers can be found in ChromGate GenericDrivers Disk1 The installation is only required if you wants to control one of the devices supported via the Generic Driver Generic drivers only allow for bas
404. ze 28 75 29 00 29 25 29 50 29 75 30 00 30 25 30 50 30 75 31 00 31 25 Fig 357 Chromatogram FRC Annotation Minutes Fraction 16 Vial 16 Fraction 17 Vial 17 Fraction 19 Vial 19 Fraction 20 Vial 20 32 00 Fraction 21 Vial 21 250 ChromGate Preparative Option To change the settings for colors font type etc open the chromatogram s appearance right mouse click on the chromatogram Appearance Select the correct detector channel in Trace and select the annotation you want to change the settings for Appearance Properties Appearance Scheme l Save s Item Subitem f1 Current Data 5 2520 Channel 1 y Line Style Size Color Solid y Thin EJ Peak Stop Tick USP Width RT Window RT Window undet FAC Start Tick FAC Stop Tick FRC Annotation FRC Actual 1 FAC Actual 2 Abbrechen Hilfe Fig 358 Chromatogram FRC Appearance Please note that for report previews as well as for pdf prints the fraction color will not be shown correctly always pink yellow If the report is been printed with a physical printer the colors are as selected in the Appearances menu Stacked Injection Fig 359 The option Stacked Injection allows to perform injection during a run As an injection device the autosampler 3950 or the Injection Module can be used For the configuration and instrument setup of the autosampler 3950 please refer t

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