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Agilent 1200 Series Purification System

1. Displays the Select Study to Open dialog box which allows you to select an existing study to open Closes the current study Displays the Save Study As dialog box which allows you to save a study under a new name or in a different folder Displays the Report dialog box which allows you to select a report to produce and a destination for the output Toggles the display of the Agilent 1200 Series autosampler graphical display disabled in Select Mode Agilent 1200 Series Purification System User Manual 131 4 132 Menus and Views E E E l E E amp E eee Toggles the display of the Agilent 1200 Series fraction collector graphical display disabled in Select Mode Toggles the display of a read only version of the Study Parameters dialog box disabled in Select Mode Allows you to toggle the display of the Study List Samples or the Study List Injections disabled in Select Mode Toggles the display of the Results Panel containing the injection results In Select mode this tool displays a snapshot of the current study Displays the Start Study dialog box which allows you to run or reprocess a complete study or a partial study Pauses a running study at the end of the current run this might be useful for adding or removing samples or fractions to resume the study press Start Stops a running study at the end of the current run any remaining injections are not completed Aborts a study immedia
2. Pooling you specify the same method multiple times Methods The Methods panel shows the method s that will be applied to the selected samples The methods will be applied in the listed sequence you can change the sequence using the up and down arrows or remove a method from the list using the X tool Injection Parameters In the injection parameters group you specify the injection volume for each method if different from the method parameters The Injection Parameters group also displays the number of pools specified Run Method If The Run Method If group for LC MSD only allows you to reinject and rerun a sample using a second Agilent ChemStation method if certain conditions related to the purity of the sample are met The conditions are e All the target masses are found e Some at least one but not all target masses are found e None of the target masses are found A target mass is found if the purity calculation for that target mass exceeds the qualifier level as specified in the Edit Method panel Expected masses The masses to expect can be filled in using their absolute mass or using their molecular formula The masses are used for mass based fraction Agilent 1200 Series Purification System User Manual 151 5 Study Setup Modifying Samples You can modify the samples in the study for example to associate additional methods change injection parameters or change the sample name or descri
3. 100 00 350 00 100 1 0 100 010 lon Mode API ES Peakwidth 0 08 min Cycle Time 0 78 sec cycle A d l aste m A E E X Time Filter Scan Data Storage Signal 2 Condensed M Mode Scan 2 Polarity Positive gt Active Signals tti 0n Mass Range Frag Gain Thres Step Ik 1 Scan lt Off Low High mentor hold size n 1 o 00 M 100 00 1000 00 70 1 0 150 0 10 n Acquisition Parameters Figure 126 MSD Signal Parameters Table 27 MSD Spray Chamber parameters see also Figure 127 on page 267 and Figure 128 on page 268 Parameter Value Drying Gas Temp 350 Drying Gas Flow 10 L min for Electrospray source 5 L min for API source 266 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 Table 27 MSD Spray Chamber parameters see also Figure 127 on page 267 and Figure 128 on page 268 continued Parameter Value Neb Pressure 35 psig Vaporizer Temp if APCI source 400 C VCap Positive 3000 V VCap Negative 3000 V not used in acquisition Corona Current Positive if APCI source 4uA Corona Current Negative if APCI source 15 uA not used in acquisition MSD Spray Chamber Figure 127 MSD Electrospray Spray Chamber parameters Agilent 1200 Series Purification System User Manual 267 11 Checkout Procedures MSD Spray Chamber Figure 128 MSD APCI Spray Chamber parameters Table 28 MSD Frac
4. Close Study Closes the current study Specify Report Displays the Specify Report dialog box which allows you to set up the reporting parameters for the study see Specifying the Report on page 144 Report Displays the Report dialog box which allows you to select a report to produce and a destination for the output Export This dialog is used to manually export CSV files see Chapter 5 Exporting to a CSV file starting on page 160 Agilent 1200 Series Purification System User Manual 125 4 Menus and Views Exit Close the Purification Hi Throughput software Run Control Edit 126 The following items are available in the Run Control menu Start Study Displays the Start Study dialog box which allows you to run or reprocess a complete study or a partial study Pause Study Allows you to pause a study e g for adding samples The study can be resumed by pressing Start Study again Stop Study Stops a running study at the end of the current run any remaining injections are not completed Abort Aborts a study immediately any remaining injections are not completed Status Info Displays the Purify Status panel which shows the current status of the system The following items are available in the Edit menu which is available in Select Mode only Agilent 1200 Series Purification System User Manual Menus and Views 4 Add Sample s When sample locations are selected i
5. Selection ChemStore None gt C EEA Operator Agilent C Method Description Uv based system checkout _ Bun Method IF gij Method None Al lf tf Allmasses found Shut Down i Poa lt None gt nd C Some masses found Wo masses found m Default Sample Name Format Fraction Collection and Recovery Format Check Fraction Start Loc NextLoc ain Example CheckP1 4 01 CheckVIAL 103 Number of pools kNone gt H Recovery Start Loc None 7 Cancel lt Back Finish Help Figure 106 Study parameters for UV triggered PS checkout Agilent 1200 Series Purification System User Manual 11 239 11 Checkout Procedures 5 Specify the report as shown in Figure 107 Use Next and Previous to navigate through the Study Setup Wizard i Specify Report 15 EDIT ea E Hegi Abundance EDA BEI Meo Abed J fa Msp Figure 107 Report parameters for UV triggered PS checkout 240 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 Step 4 Adding Sample s to the study 1 Fill vial with the isocratic checkout sample 2 Select the sample location on the Sampler panel and add the sample to the study see Figure 108 Sample s Renove Samples Modit samperi Figure 108 Adding the sample to the study 3 Select the uvpsfr m method to apply to the selected sample see Figure 109 Ada Sample s
6. Vp Vp2 Vp1 to y Vp2 Figure 48 Step 2 Delay Volume Calculation Maximum flow rates to avoid missing peaks Because of the effect of the detector peakwidth settings on the delay time of a peak the use of higher flow rates than those recommended in Table 8 on page 115 to Table 11 on page 116 will result in peaks being missed unless you take into account the analog signal delay The maximum flow is calculated as Maximum flow Delay volume signal delay time Agilent 1200 Series Purification System User Manual Instrument Parameters 3 If you don t have enough volume available between your detector and the fraction collector to operate at the desired flow rate extra tubing volume must be added The minimum required delay volume is calculated as Minimum required delay volume analog signal delay x flow Table8 Analytical Fraction Collector with DAD or MWD Peakwidth min Response Time Rate Hz Analog signal Max Flow Rate sec delay sec in ml min 50 pl capillary lt 0 01 0 1 20 0 05 60 0 gt 0 01 0 2 20 0 15 20 0 gt 0 03 0 5 10 0 5 6 0 gt 0 05 1 0 5 1 25 2 4 gt 0 10 2 0 2 5 2 75 11 gt 0 20 4 0 1 25 5 9 0 5 gt 0 40 8 0 0 62 11 9 0 25 gt 0 85 16 0 0 31 23 9 0 13 Table9 Analytical Fraction Collector with VWD Peakwidth min Response Time Rate Hz Analog signal Max Flow Rate sec delay sec in ml min 50 pl capillary lt 0 005 lt 0 12 20 0 07 42 9 gt 0 005 0 12 20 0 1
7. 145 hardware 42 height tube 68 Help 130 help menu 130 hotspot 190 191 HUB 29 humidity 18 Importing 159 information sample 150 study 141 injection order 142 overlap 64 66 parameters 151 status 155 type 62 64 65 injection volume 62 64 155 maximum 58 60 61 injections deleting 163 injector configuration 57 program 62 64 65 setup 61 installation 44 installed trays 58 60 61 68 Installing 27 installing a thermostatted module interface cables 31 power cable 31 safety 31 Agilent 1200 Series Purification System User Manual installing the Purification System interface cables 27 power cable 27 safety 27 installing the purification system flow connections 34 installing the thermostatted fraction collector preparation 31 installing the thermostatted Purification System safety 27 inteface board LAN 27 interactive report 201 interconnection cable thermostat 32 into location 69 L LAN interface board 27 LC CE ChemStation 42 LC MSD ChemStation 42 LC MSD 28 license 43 46 49 144 line frequency 18 voltage 18 locations reserved 69 sample 148 M ainpass 64 ake up pump 29 mass window 92 mass based fraction collection kit 28 ass based fraction collection 77 asses target selected 87 33 3 3 maximum flow rate 114 injection volume 58 60 61 peak width 89 volume 74 maximum flow rates 114 maximum peak duration
8. Jo 20 Threshold 7 fi 00 0 Full Scale volts 0 1 comet __ Hob Figure 45 Fraction Collection Parameters dialog box with delay volume entry 32 Save the method NOTE If you anticipate running samples at a variety of flow rates it is best to calibrate at the highest likely flow rate first and install the appropriate length tubing This way there will be sufficient delay for the lower flow rates and new lengths will not need to be cut Agilent 1200 Series Purification System User Manual 111 3 Instrument Parameters Making Your Own Calibration Method You make your own calibration method by editing one of the default calibration methods 1 Load the default calibration method D CALAS M in the case of an analytical system and DGCALPS M in the case of a preparative system 2 Change the method parameters flow runtime solvent composition injection volume or detector parameters to suit your adapted calibration procedure CAUTION The detector peakwidth has an influence on the delay time of the peak therefore we recommend that you use the maximum flow rates for the peakwidths given in Table 8 on page 115 or Table 9 on page 115 Use of higher flow rates will result in missed peaks unless the analog signal delay is taken into account Minimum required delay volume signal delay x flow 3 Save the method with a new name in the method folder for your instrument 4 Follow the appropriate procedure as descri
9. The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend If software is for use in the performance of a U S Government prime contract or subcon tract Software is delivered and licensed as Commercial computer software as defined in DFAR 252 227 7014 June 1995 or as a commercial item as defined in FAR 2 101 a or as Restricted computer soft ware as defined in FAR 52 227 19 June 1987 or any equivalent agency regulation or contract clause Use duplication or disclo sure of Software is subject to Agilent Tech nologies standard commercial license terms and non DOD Departments and Agencies of the U S Government will receive no greater than Restricted Rights as defined in FAR 52 227 19 c 1 2 June 1987 U S Government users will receive no greater than Limited Rights as defined in FAR 52 227 14 June 1987 or DFAR 252 227 7015 b 2 November 1995 as applicable in any technical data Safety Notices CAUTION A CAUTION notice denotes a haz ard It calls attention to an operat ing procedure practice or the like that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met A WARNING notice denotes a hazard
10. and those of the fraction collector are shown on the right You use this view to add your samples to the study list which you can display in a separate window see Figure 57 Figure 57 Autosampler and Fraction Collector Views Agilent 1200 Series Purification System User Manual 147 Study Setup Sampler Panel Single Location Complete Row or 148 Column Rectangular Section You use the sampler panel to add samples to the study list The following actions are possible in the sampler view Selecting Sample Locations You can use the following mouse actions separately or in combination to select sample locations Click on the location with the left mouse button Click on the row or column label with the left mouse button Draw a rectangle around the required locations Deselecting Sample Locations Clicking on a selected sample location with the left mouse button toggles the location selection Location Selection Indicators Table 13 explains the color indicators used to show the state of each location in the Sampler panel Table 13 Sample Location Indicators Line Color Fill Color Meaning Blue Gray The location has not been selected or has been deselected White Gray The location has been selected but not yet added to the study list Blue Dark blue The location has been added to the study list White Dark blue The location has been added to the study list and has been selected for example fo
11. gt 5 seconds see Figure 44 Print Close Calibrate Detector Cum Delay Vol ull Calc Delay Vol ul DAD1 G13154 US64400562 937 14 ice oe ae ae Cur Delay Time sec Calc Delay Time sec MSD 11 991 Figure 44 Delay time greater than 5 seconds 27 Press Calibrate if you want to store the new calculated delay volume for the MSD in the Fraction Collector Configuration see also Figure Configuration on page 67 28 Press Print to get a hard copy of the report 29 Stop the splitter 30 Return to the Method and Run Control View and load the method you will be using to run your samples Select Fraction Collection from the More MSD sub menu under the Instrument menu 110 Agilent 1200 Series Purification System User Manual Instrument Parameters 3 31 Press Parameters and enter the final calculated delay time that appears in the report in the Collector Delay field first converting it to minutes In this case you would enter 11 991 60 0 20 minutes see Figure 45 Fraction Collection Parameters Fi xj Model Type FE Hardware Configuration Peak Timing Min Peak width min fo oo Time Limit min fi 5 00 Max Peak Width min fo 50 MSD m z Detection Peak Slope counts sec 500 Collector Delay min fo 20 Threshold counts 20000 Mass Window fi 0 Analog Output Range kilo counts volt feooo Other Detector Peak Slope my sec fi 0 Collector Delay min
12. study 136 pause 126 pause study 126 pausing a study 173 peak detection parameters 72 detectors 72 slope 90 timing 89 peak duration 71 peak width 93 98 112 114 maximum 89 minimum 89 peak based fraction collection 71 peakwidth 115 physical specifications 18 plug channel 32 plumbing 34 pooling 180 parameters 181 properties 182 pools number 143 position needle 69 post time 74 power cable 28 consumption 18 cords 15 requirements 14 switch 28 power cords 15 278 preconfigured well plate types 53 prefetch sample vial 63 preparative scale system configuration 24 prerequisites 52 preview 73 report 201 properties pooling 182 pump make up 29 purification hi throughput reports 205 software 43 urify menu 47 urity 43 purity calculation 43 Q qualifier level 151 rate 115 recovery 143 recovery fraction collector 75 recovery location 155 156 regional settings 42 report ChemStation 144 202 interactive 201 preview 201 specification 144 specify 125 reprocess a study 172 176 reserved locations 69 response time 115 Result Panel 129 result panel 123 191 results study 175 review study data 184 rinse fraction collector needle 74 rinse needle 74 Run Control 126 run control 172 menu 126 S safety standards 18 sample description 155 flush out factor 64 information 150 list 208 location 148 155 name 155 name format 142
13. syringe 58 tube 68 vessel tray 177 W wash needle 62 64 65 vial 62 64 66 waste drainage 28 tube 31 weight 15 weight specifications 16 well plate 60 61 69 dimensions 54 well plate types 53 configuring 54 preconfigured 53 user configured 54 Window 129 window menu 129 wizard 139 working mode 71 72 workspace 121 280 Agilent 1200 Series Purification System User Manual www agilent com In This Book This user s guide contains information about the Agilent 1200 Series Purification System The user s guide describes the following e Installing Your Agilent 1200 Series Purification System Installing the Software e Instrument Parameters e Menus and Views e Study Setup e Running Studies e Pooling e Data Review e Reports e Directory Structure and File Naming e Checkout Procedures Agilent Technologies 2006 Printed in Germany 02 06 G2262 90010 Agilent Technologies
14. to Study uvpstr m uyvpstr m Figure 109 Applying the uvpsfr m method Agilent 1200 Series Purification System User Manual 241 11 Checkout Procedures 4 Add as description Isocratic Sample as in Figure 110 and click OK lsocratic Sample Figure 110 Adding a sample description The selected sample is marked as a filled blue circle 5 In the fraction collection panel assign a fraction start position to an empty tube The Study List is updated as shown in Figure 111 P viaLi CheckviAL psf lt Method gt Figure 111 Study List after assigning the fraction start position Step 5 Starting the study 1 Start the study by clicking the tool 2 Select Run Whole Study During the run you can observe the chromatogram by clicking the tool 242 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 Step 6 Reviewing the results 1 When the study run is finished verify that you have four fractions collected in the tubes 2 Verify that you have four green fraction locations on the Fraction Collector panel as in Figure 112 The sampler and fraction collector panel views depend on the configuration the ones shown here are examples only 000E w D a opti 0000 Y i e000 000G Vials Study List Injections Location Name Description IniectionNo Pool Method EE ae es oe Collected Se w
15. 1200 Series Diode Array Detector Detector with analytical flow cell Software e Agilent ChemStation Software G2170AA A 09 01 or Higher e Agilent Purification Hi Throughput Software G2262AA A 01 01 Column e Zorbax Eclipse XDB C8 4 6 mm x 15 cm 5 um Part number 993967 906 220 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 Solvents e HPLC grade Bi distilled water HPLC grade Acetonitrile Sample e Isocratic Checkout Sample Part number 01080 68704 e Delay calibrant part number G1946 85020 Step 1 Calibrating the Delay Volume Before running the fraction collection the delay volume must be determined This procedure determines the delay volume relative to the configured detector See Delay Volume Calibration on page 93 for the correct procedures Agilent 1200 Series Purification System User Manual 221 11 Checkout Procedures Step 2 Preparing the LC system 1 Fill the solvent bottles with HPLC grade bi distilled water Channel A and HPLC grade acetonitrile Channel B In the case of an Isocratic pump fill the bottle with a mixture of 10 water and 90 acetonitrile 2 Turn on all the units 3 Purge the channels 4 Enter the LC method parameters as shown in Table 17 on page 222 5 Save the method with the name uvasfr m 6 Pump the water acetonitrile 10 90 mobile phase through the column for 10 minutes at a flow rate of 1 5 ml min 7 Allow the detector at least 15 mi
16. 184 save 125 saving 164 start 126 starting 172 status 174 177 stop 126 stopping 173 Study Base Path 129 Study List Fraction Wells 128 study list fraction wells 190 Study List Injections 128 study list injections 189 Study List Samples 128 study run summary 207 Study Wizard 146 study wizard 139 supported configurations 42 supported operating systems 42 switch power 28 syringe volume 58 Agilent 1200 Series Purification System User Manual Index system configuration analytical scale 21 preparative scale 24 T target mass 151 target masses 87 temperature 18 non operating 18 operating 18 test tube tray 68 thermostat electrical connections 33 thermostat interconnection cable 32 threshhold 71 72 73 91 thumbnail 145 204 tickmarks 175 time limit 90 time based fraction collection 72 timetable 71 72 toolbar 131 tree view 121 trigger mode 71 73 fraction 71 trigger parameters 73 trigger type 88 triggering 72 tube height 68 volume 68 waste 31 U UIB 28 uninstall 49 universal interface box 28 unpacking modules 19 upper threshhold 71 73 upslope 71 73 user configured well plate types 54 279 Index V vessel filling volume 177 view menu 128 mode 120 View Mode 128 viewing data in ChemStation 196 voltage 18 volume delay 69 injection 62 64 155 maximum 74 maximum injection 58 60 61 seat capillary 58 60 61
17. 2 gt 0 20 4 gt 0 4 8 Rate Hz 2 5 1 25 0 62 0 31 Analog signal delay sec 0 58 1 31 2 84 5 97 12 3 Instrument Parameters 3 Max Flow Rate in ml min 500 pl capillary 52 22 9 10 6 5 0 2 4 In Table 10 and Table 11 the figures shown in bold text are the default values The 500 ul capillary is 0 8 mm i d x 1000 mm Agilent part no G1364 68711 Agilent 1200 Series Purification System User Manual 117 3 Instrument Parameters 118 Agilent 1200 Series Purification System User Manual This chapter describes the menu structure and toolbar buttons Agilent 1200 Series Purification System User Manual 4 Menus and Views Mode 120 Main Panel 121 Menu Bar 125 Tool Bar 131 ee Agilent Technologies 119 4 Menus and Views Mode The Purification Hi Throughput software can be used in two modes each of which has its own menu structure and toolbar buttons View Mode You use View mode to view the details of a study once it has been created and run You cannot create edit or run a study in View mode Select Mode Edit You use Select mode to create a new study or edit an existing study and to run a study You cannot view the results of a study in Select mode 120 Agilent 1200 Series Purification System User Manual Main Panel Explorer Menus and Views 4 The main panel of the Purification Hi Throughput application is divided into two main areas the explorer at the left and
18. 24 Time Rinse Fraction Collection Needle Auxiliary Max fill volume per location Stoptime hie p ej min I At the start of collection as configured H ml Posttime Off min Between fraction collection I Store temperature signal I Store delay sensor signal l Store UIB signal Figure 24 More fraction collector setup parameters 8 In the Time group specify a stop time the stop time should always be later than the end of the last peak which should be collected plus the delay time between detector and fraction collector and a post time for the fraction collector 9 In the Rinse Fraction Collector Needle group select when to rinse the needle You can select to rinse the needle before the start of fraction collection and or between the collection of fractions or not at all 10 In the Auxiliary group specify the maximum volume per location and select the signals to store You can select the maximum volume to be as configured in the Fraction Collection Configuration dialog box or override this value by specifying a lower maximum volume in the field 74 Agilent 1200 Series Purification System User Manual Instrument Parameters 3 Multiple Fraction Collectors The Agilent ChemStation supports the installation of up to three fraction collectors which can be used in series for fraction collection plus a fourth fraction collector which will then be used as a recovery fraction collector automatically Multiple fra
19. 3 Running the Checkout Method 1 From the Agilent ChemStation RunControl menu select Sample Info then select manual data file name and enter the data file name msaschk1 d then specify the appropriate vial location for the checkout sample then click Run Method Step 4 Review Results 1 When the checkout method run is finished verify that you have three fractions collected in the vials wells or tubes The checkout sample contains four components but you will probably get separation into only three peaks two of the compounds will co elute 2 Verify that you have three fractions indicated on the MSD TIC in the Agilent ChemStation report or in Agilent ChemStation Data Analysis see Figure 123 on page 259 3 If you have not successfully collected the three fractions corresponding to the three peaks evaluate the data file in Agilent ChemStation Data Analysis for the proper threshold triggering level Update the threshold value in the MSD Fraction Collection Parameters dialog box and rerun the checkout 258 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 Figure 123 MSD triggered AS checkout results Agilent 1200 Series Purification System User Manual 259 11 Checkout Procedures Step 5 Reanalyze Fraction 2 1 Reanalyze the fraction that was collected in the second vial well or tube using the same conditions used for the checkout method msdasfr m but with the Use MSD for Mass
20. 71 menu bar 125 purify 47 method 155 176 parameters 176 source path 137 Method Source Path 129 method target masses 87 methods assigning 150 available 151 inimum peak width 89 issing parts 19 issing peaks 114 ode fraction collection 81 fraction trigger 71 modifying study 167 molecular formula 161 weight 161 more 74 MS signals 85 MSD m z detection 90 multi instrument configuration 43 multiple draw 58 multiple fraction collectors 35 75 configuration 76 setup 76 multiple graphics 145 Agilent 1200 Series Purification System User Manual Index name data file 155 180 operator 141 sample 155 study 139 name format sample 142 needle movement 69 position 69 rinse 74 wash 62 64 65 new study 125 139 new study run 168 non operating altitude 18 non operating temperature 18 number of fractions 72 of pools 143 0 offline software 44 online software 44 online tickmarks plot 175 open study 125 opening a study 165 operating altitude 18 operating systems 42 operating temperature 18 operator name 141 Options 129 options menu 129 order fraction collector 75 injection 142 overlap injection 64 66 cycle 63 P packaging damaged 19 277 Index parameters fraction collection 88 injection 151 method 176 peak detection 72 pooling 181 study 123 141 182 185 partial study 156 168 172 parts and materials 19 path method 137
21. 73 You can set up to 99 repetitions 3 2 1 3 FE 1 Figure 73 Pooling with three repetitions The technique for pooling that is described in this chapter generates pools only from identical injection positions same sample in the sampler If you want to pool from different samples you may do so by injecting from different positions samples in the sampler and then using the identical fixed fraction start location for each injection for more details please refer to Chapter 5 Assigning the Fraction Start Position starting on page 153 Since pooling is a study parameter that is the number of repetitions is set up in the study parameters the pooling conditions apply to all samples and methods in the study run The pooling run creates multiple data files form each sample location Since the data file names must be unique the last two digits of the data file name are used as a Pooling Counter which is incremented with each repetition of the Agilent 1200 Series Purification System User Manual CAUTION Pooling 7 sample injection see Chapter 10 Directory Structure and File Naming Figure 74 shows a Study List Injections for a run with three samples each pooled three times Note the data file naming in which the fourth character denotes the injection number and the final character denotes the pool number rr 2 t 3 i Wai Pred viai 2 F agterat m ODODO r Wal 2 Povia 12 3
22. A 01 01 Column e 9 46 mm x 50 mm SB C18 Part number 846975 202 Solvents e HPLC grade Bi distilled water e HPLC grade Acetonitrile Agilent 1200 Series Purification System User Manual 235 11 Checkout Procedures Sample e Isocratic Checkout Sample Part number 01080 68704 e Delay calibrant part number G1946 85020 Step 1 Calibrating the Delay Volume Before running the fraction collection the delay volume must be determined This procedure determines the delay volume relative to the configured detector See Delay Volume Calibration in Delay Volume Calibration on page 93 for the correct procedures Step 2 Preparing the LC system 1 Fill the solvent bottles with HPLC grade bi distilled water Channel A and HPLC grade acetonitrile Channel B Turn on all the units Purge the channels Enter the LC method parameters as shown in Table 19 Save the method with the name uvpsfr m ao oo FF WwW N Pump the water acetonitrile 835 65 mobile phase through the column for 10 minutes at a flow rate of 10 ml min Allow the detector at least 15 minutes to provide a stable baseline Insert an appropriate tray in the Fraction Collector Table 19 LC Method Parameters for UV triggered PS Fraction Collection checkout Device Parameter Value Injector Injection Volume 50 ul Pump Flow Rate 10 0 ml min Channel A Water 35 236 Agilent 1200 Series Purification System User Manual Checkout Proced
23. Checkout Procedures Table 23 MSD Spray Chamber parameters see also Figure 119 and Figure 120 on page 255 continued Parameter Value Vaporizer Temp if APCI source 400 C VCap Positive 3000 V VCap Negative 3000 V not used in acquisition Corona Current Positive if APCI source 4uA Corona Current Negative if APCI source 15 uA not used in acquisition IMSL Spray Chamber Figure 119 MSD Electrospray Spray Chamber parameters 254 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 MSD Spray Chamber Figure 120 MSD APCI Spray Chamber parameters Table 24 MSD Fraction Collection parameters see also Figure 121 on page 256 and Figure 122 on page 257 Parameter Value FC Mode Use method target masses MS Signals Signal 1 Detectors MS Only Adducts M H 1 Selected Base Masses 278 0 284 0 310 0 Min Peak Width 0 00 min Max Peak Width 1 00 min Agilent 1200 Series Purification System User Manual 255 11 256 Checkout Procedures Table 24 MSD Fraction Collection parameters see also Figure 121 on page 256 and Figure 122 on page 257 continued Parameter Value Time Limit 1 00 min Peak Slope 0 Threshold 100000 counts Collector Delay Mass Window Analog Output Value taken from MSD Delay Calibration procedure 1 0 2000 not used for the checkout This value may need to be adjusted based on the actual da
24. Criteria The study stops when the study has no injections left that have no data associated and you have stopped the study The study is aborted when you abort the study or the Agilent ChemStation is aborted either automatically for example because of a leak or through user intervention Agilent 1200 Series Purification System User Manual 173 6 Running Studies Study Status 174 The Purify Status panel see Figure 70 allows you to view the status of the Purification Hi Throughput software and the current study You toggle the display status of the Purify Status panel by using the RunControl menu RunControl gt Status Info Alternatively you can choose the i tool from the toolbar or press Ctrl P Purify Status Sequence Running Data Acquisition Running Acquisition StudpAgilent Checkout Sample CheckVIAL 1 Location VIAL 1 Datafile00010200 d Method uvasfr m Figure 70 Purify Status panel The upper two panels of the Purify Status panel show the status of the Purification Hi Throughput software the lower part of the display shows the status of the current study The upper two panels of the status panel are color coded in the same way as the Agilent ChemStation status panel When the Purify Status panel is displayed it is always displayed on top of all other applications so that it is always in view The Purify Status panel is also displayed when you switch to the Agilent ChemStation Agilent 1200
25. Manual Data Review 8 Result Panel The Result panel see Figure 76 contains tabular and graphical views if the fractions from the selected injection The table of fractions is identical with the table of fractions in the Study List Injections see Study List Fraction Wells on page 190 The graphical view of the collected fractions shows the acquired chromatogram from the trigger detector with superimposed vertical lines marking the start green and end red of fraction collection Each fraction is annotated with the location A list of injections selected as a Hotspot can be individually reported or exported to CSV Figure 76 Result Panel Agilent 1200 Series Purification System User Manual 191 8 Data Review Configuring the Result Panel The information displayed in the Result Panel can be configured by selecting Setup Result Panel from the View menu see Figure 77 this menu item is available only if the Result panel menu item is already checked Setup Result Panel MSD Sanas m Tieg Jo Show wher evarable Byelsy sianale Average Spi Figure 77 The View menu 192 Agilent 1200 Series Purification System User Manual Data Review 8 Tabular If Tabular is checked then the results table if available is displayed in the upper part of the Result Panel UV Detector signals The following UV detectors and signals are supported e HP35900E Analog Digital converter
26. Open the Windows Start menu and choose Settings 2 Select the Control Panel and choose Add Remove Programs 3 Select the Purification Software from the list and click Add Remove When you remove the software the data is not affected when you re install the software you can access the data as before Agilent 1200 Series Purification System User Manual 49 2 Installing the Software 50 Agilent 1200 Series Purification System User Manual Agilent 1200 Series Purification System User Manual 3 Instrument Parameters Well Plate Types 53 Autosamplers 57 Fraction Collector 67 Multiple Fraction Collectors 75 Setting Up for Mass based Fraction Collection 77 Delay Volume Calibration 93 This chapter describes how to set up the instrument parameters that are necessary to run a study ee Agilent Technologies 51 3 Instrument Parameters Prerequisites Pump s Detector s UV and Column Compartment if present are Setup This chapter deals only with the setup and configuration of the modules that require special handling related to purification topics The setup of pumps detectors and the thermostatted column compartment is not described If you need help about how to setup any of these modules please refer to your Agilents ChemStation s Online Help 52 Agilent 1200 Series Purification System User Manual Instrument Parameters 3 Well Plate Types The high performance autosampler the dual loop autosampler and frac
27. Series Purification System User Manual Running Studies 6 Online Tickmarks You can view the results of the current study run Online while the fractions are being collected Open the Online Plot in the Agilent ChemStations Method amp Run Control View and select to display tickmarks for the Online chromatogram as shown in Figure 71 Figure 71 An example of an Online plot with tickmarks Agilent 1200 Series Purification System User Manual 175 6 Running Studies Reprocessing Studies You apply reprocessing to study runs that have been completed The injections in the completed study run already have data associated with them you use the reprocessing option to reprocess the existing data using modified method parameters You must use the same Agilent ChemStation method to reprocess the data you cannot use a different method You can however change the parameters of the original method If you want to retain the original method unchanged you must save it with a different name in the Agilent ChemStation You cannot reprocess a study that is marked as read only to remove the read only flag use Windows Explorer CAUTION Reprocessing overwrites the original data so if you want to retain the original results you must make a copy of the results file before you reprocess the data You control the reprocessing of a complete study or a partial study from the Start Study dialog box see Figure 72 Select Start Mode C Run wh
28. Study Parameters 185 Sampler 186 Fraction Collector 187 Show Fraction Scheme 187 Study Parameters 188 Study List Samples 188 Study List Injections 189 Study List Fraction Wells 190 Result Panel 191 Configuring the Result Panel 192 View Mode Tools 194 Viewing data in Agilent ChemStation 196 9 Reports 199 Introduction 200 Producing Reports Interactively 201 Agilent ChemStation Reporting 202 Fraction Annotation 202 Fraction Table 203 Graphical Reporting 204 Agilent 1200 Series Purification System User Manual 9 Contents Purification Hi Throughput Reports 205 Study Run Summary 207 Sample List 208 Fraction List 209 10 Directory Structure and File Naming 211 Directory Structure 212 The Data Folder 213 Data File Naming 214 11 Checkout Procedures 217 Introduction 218 UV triggered AS Fraction Collection Checkout 220 What you will Need 220 Step 1 Calibrating the Delay Volume 221 Step 2 Preparing the LC system 222 Step 3 Creating anew study 224 Step 4 Adding Sample s to the study 228 Step 5 Starting the study 229 Step 6 Reviewing the results 231 Step 7 Reanalyzing fraction 3 233 UV triggered PS Fraction Collection Checkout 235 What you will Need 235 Step 1 Calibrating the Delay Volume 236 Step 2 Preparing the LC system 236 Step 3 Creating anew study 238 Step 4 Adding Sample s to the study 241 Step 5 Starting the study 242 Step 6 Reviewing the results 243 Step 7 Reanalyzing fraction 3 245 MSD trigg
29. at the left of the display You cannot open a study that has been write protected Agilent 1200 Series Purification System User Manual 165 5 166 Study Setup On line Agilent ChemStation To run a study its configuration must match the current Agilent ChemStation configuration absolutely If the configuration of the study you open is compatible with the current Agilent ChemStation configuration you can acquire view and reprocess data However if there is any mismatch between the study configuration and the current Agilent ChemStation configuration the study will not run In the case of a mismatch of configurations you can view and reprocess data but you cannot acquire data Off line Agilent ChemStation Compatibility of study and Agilent ChemStation configurations is unnecessary in the case of off line operation In off line mode you can view and reprocess data but you cannot acquire data Agilent 1200 Series Purification System User Manual Study Setup 5 Closing a Study Ca You choosing the ai tool from the toolbar or use the File menu to close a study that you have selected in the explorer at the left of the display File gt Close Study You cannot close a study that is currently running acquiring data Modifying a Study You can modify a study in either on line or off line mode If the study has no data you can modify the study parameters the study list and the reporting parameters If the study alread
30. from that injection are also selected and the sample from which they were collected is highlighted on the Sampler panel The Study Lists and Result panel show the results of the selected injection of the sample Show Fraction Scheme Displays the fraction collection pattern Collection Order as defined in the Agilent ChemStation configuration of the fraction collector only for studies that were setup with Agilent ChemStation A 10 01 and higher and Purify A 02 01 and higher Agilent 1200 Series Purification System User Manual 187 8 Data Review Study Parameters Displays the study poarameters as defined during the study setup Study List Samples The Study List Samples in View mode is identical with the Study List Samples displayed in Edit mode see The Study List on page 155 The selected sample is denoted by an arrow in the left column of the table Location The location of the sample in the sampler Name The sample name according to the specified naming convention Description The sample description entered in the Description field of the Add Samples to Study dialog box The Injection group columns describe the parameters for each injection Method The method used for this injection Volume The injection volume by default the injection volume specified in the method denoted by lt Method gt Data File The name of the data file that will be produced only the first injection if pooling is specified R
31. it is reset to its default display width Agilent 1200 Series Purification System User Manual 123 4 Menus and Views Changing the sequence of the columns You can change the sequence of the columns either by a drag and drop method or by using a drop down menu To change the sequence by the drag and drop method 1 Click and hold the left mouse button in the column heading 2 Drag the column outline to its desired position and release the mouse button The column is inserted into the table at this position To change the sequence using the drop down menu 1 Click the left mouse button in the column heading A down arrow is displayed at the right of the heading 2 Click the down arrow to reveal the drop down menu containing a list of all columns in the table or sub table 3 Select the name of the column that you want to move to this position The positions of the current column and the selected column are switched 124 Agilent 1200 Series Purification System User Manual Menu Bar File Menus and Views 4 The following items are available in the File menu New Study Starts the Study Wizard which allows you to set up a new study see Creating a New Study on page 139 Open Study Displays the Select Study to open dialog box which allows you to select an existing study to open Save Study As Displays the Save Study As dialog box which allows you to save a study under a new name or in a different folder
32. least one peak detector detects a peak Recovery Location Recovery Volume 0 Frac Well Location Volume BecinTime EndTime Reason Mass 3 p1 xin main l l 1 1 l Vial 1 170 1 5246 1 6354 Peakdet 2 i 1 Vial 2 180 2 0919 2 2115 Peakdet 3 1 1 Vial 3 222 3 6152 3 7652 Peakdet 4 1 l Vial 4 290 5 2150 5 4083 Peakdet Figure 83 Fraction Table To include a fraction table with the Agilent ChemStation report you mark the Fraction Table check box which is available when you have selected to produce Agilent ChemStation Data Analysis reports Graphical Reporting In some cases it is convenient to plot multiple graphics for example chromatograms on a single page The Purification Hi Throughput software allows you to print up to 81 graphics nine columns x nine rows on one page These thumbnails can be useful for a first visual review of the study data or for reporting pooling runs To produce such thumbnail graphics you mark the Plot Multiple Graphics on a Single Page check box and mark the check boxes of the graphics you want to print in this way You use the Plot fields to specify the number of rows horizontal and columns vertical on each page You can either enter the number of rows and or columns in the field or use the up and down arrows to increase or decrease the number 204 Agilent 1200 Series Purification System User Manual Reports 9 Purification Hi Throughput Reports To print the reports s
33. of the delay volume calibration and gives instructions and warnings 8 Click Continue For the G1364A the Info field gives the instruction to connect the needle capillary with the rinse port see Figure 38 To do so remove the needle from the arm and disconnect the needle tubing AFC Delay Volume Calibration Status Window x Calibration method DGCALPS1 M Please connect needle capillary with rinse port Figure 37 An instruction in the status window for the G1364A Agilent 1200 Series Purification System User Manual 99 3 100 Instrument Parameters For the G1364B the Info field gives the instruction to install the needle adapter to the needle arm see Figure 38 AFC Delay Yolume Calibration Status Window Please install FDS adapter and close the 8 8 _ Continue __Cancet He Figure 38 An instruction in the status window for the G1364B 9 When you have closed the door click Continue again to start the delay volume calibration The selected method is loaded and the delay volume calibration sample is injected Agilent 1200 Series Purification System User Manual Instrument Parameters 3 10 When the calibration run has finished disconnect the needle capillary from the rinse port G1364A or the needle adapter from the arm G1364B and click Continue The Calibration Results panel see Figure 39 is displayed Figure 39 Calibration Results panel The Calibratio
34. page 266 and Figure 28 on page 268 Save the method with the name msdpsfr m 8 Pump water acetonitrile 10 90 mobile phase through the column for 10 minutes at a flow rate of 10 ml min Insert an appropriate tray in the fraction collector 10 Enter a split ratio of 3 into the MRA active splitter and start the splitter At 10 ml min this corresponds to a 250 1 split ratio Table 25 LC Method Parameters for MSD triggered PS Fraction Collection checkout Device Parameter Value Injector Injection Volume 50 ul Quaternary or Binary Pump Flow Rate 10 0 ml min Channel A Water 90 Channel B Acetonitrile 10 Stop Time 5 min Post Time 2 min Pump Timetable 3 min 60 B Isocratic Pump for make up Channel A 50 50 Water 100 flow to LC MSD Acetonitrile with 0 1 Acetic or Formic Acid Flow Rate 0 5 ml min Agilent 1200 Series Purification System User Manual 263 11 264 Checkout Procedures Table 25 LC Method Parameters for MSD triggered PS Fraction Collection checkout Device Parameter Value Column Thermostat Column Temperature 40 C Detector Wavelength VWD 254 nm Wavelength DAD MWD 254 16 nm 360 100 nm Fraction Collector see Setup Fraction Collector Peak based Use MSD for Figure 125 Mass Based Fraction Collection Report Destination Printer Report Style Short Set up Fraction Collector Instrument 1 x O Fraction Trigger Mode Off C Use Timetable Peak based Max Peak Duratio
35. pump flow of 0 5 ml min Agilent 1200 Series Purification System User Manual 249 11 250 Checkout Procedures Step 2 Preparing the LC system 1 ao ol A Ww Fill the solvent bottles for the quaternary or binary pump with HPLC grade bi distilled water Channel A and HPLC grade acetonitrile Channel B Fill the solvent bottle for the isocratic make up pump with 50 50 water acetonitrile with 0 1 acetic or formic acid 1 ml of acid 1 liter of solvent Turn on all the units Purge the channels Enter the LC method parameters as shown in Table 21 Enter the MSD parameters as shown in Table 22 on page 252 Table 23 on page 253 and Table 24 on page 255 Save the method with the name msdasfr m 8 Pump water acetonitrile 10 90 mobile phase through the column for 10 minutes at a flow rate of 3 ml min Insert an appropriate tray in the fraction collector 10 Enter a split factor of 3 into the MRA active splitter and start the splitter At 3 ml min this corresponds to a 75 1 split ratio Table 21 LC Method Parameters for MSD triggered AS Fraction Collection checkout Device Parameter Value Injector Injection Volume 50 ul Quaternary or Binary Pump Flow Rate 3 0 ml min Channel A Water 90 Channel B Acetonitrile 10 Stop Time 5 min Post Time 2 min Pump Timetable 3 min 60 B Isocratic Pump for make up Channel A 50 50 Water 100 flow to LC MSD Acetonitrile with 0 1 Acetic or Formic Acid Flow Rat
36. selected the parameters in the MSD Fraction Collection panel for the Agilent ChemStation method control the fraction collection Other UV or analog UIB signals can be configured in the Set up Fraction Collector panel and these signals can be used along with the MSD signal for triggering fraction collection See Fraction Collector on page 67 for a discussion of the other parameters and functions in the Setup Fraction Collector panel If your system is configured for mass based fraction collection two dialog boxes are used to set up the Agilent ChemStation to monitor and even trigger the process based on mass or time e MSD Fraction Collection Dialog Box e MSD Fraction Collection Parameters Dialog Box Agilent 1200 Series Purification System User Manual 79 3 Instrument Parameters MSD Fraction Collection Dialog Box The primary dialog box MSD Fraction Collection lets you set up a single or multiple signal Agilent ChemStation method using one or two detectors and a selected fraction collector The parameters in the MSD Fraction Collection dialog box are grouped as follows see Figure 27 FC Mode MS Signals Detectors Adducts Method Target Masses Fraction Collection xj MFE Mode MS Signals Detectors None Monitor Polarity Mode MS only Tic Sigal Positive Scan ther only 2 Use method target masses Signal2 JV Negative Scan EIME a Use sample
37. sgial m 0000003 lt Matted I WAL Poe Viet 5 ti agiert m OOIT lt Medsed 7 ai gt Posi viti PeT MaLa Povia sphere m OCOD charted I 1 TPT AO 1 STELLI Pied on ie Tree iPtcor 22 ia Tew EESE it 200 Tae i MEESI m 203 240 Tew Figure 74 Study List Injections with pooled samples Since pooling always collects the same fraction into the same container you must set up the pooling parameters for example the number of pools to ensure that the containers you have configured are not over filled Agilent 1200 Series Purification System User Manual 181 7 Pooling Pooling Properties The following properties apply to the pooling facility of the Purification Hi Throughput software e One sample can be pooled up to 99 times This is a restriction imposed by the data file naming conventions of the sequence engine e An injection marked for pooling is repeated consecutively as specified in the pooling parameters e Pooling applies to all injections samples in the study or partial study that have a fraction collection method e Ifthe study list contains a mixture of fraction collection methods and non fraction collection methods the fraction collection methods are pooled and the non fraction collection methods are run as single injections e You can specify pooling for each study run individually including the number of repetitions To track pooled injections the repetition number is stored as an ext
38. target masses rala E ENS onother Import mass info from 3 e Signal4 F Time based r Positive Adducts r Negative Adducts Method Target Masses OM OCM Selected I M H 1 P M H A Edit Mass naa P M NH4 18 I M Cl 35 37 M M Na 23 J M formate 45 M M K 39 J M TFA 113 a M scalar a M oai Insert M M Remove J M M Charge State Charge State ARA m S D iS Cancel Help Figure 27 The MSD Fraction Collection dialog box Agilent 1200 Series Purification System User Manual Instrument Parameters 3 FC Fraction Collection Mode None No fraction collection If you select this mode most of the fields in the dialog box are disabled grayed You use this option when you don t want fraction collection to be part of the method TIC You use this option to trigger fraction collection based on TIC peaks If you select TIC under a Purify system the Adducts and the Selected Base Masses sections and the entire Detectors section are inactive as in Figure 28 Fraction Collection E xj r FC Mode MS Signals r Detectors C None Monitor Polarity Mode MS only TIC Signall W Fasitive Scan Other only C Use method target masses Gianal2 f Negative Span CMS andother Use sample target masses peang Sigal3 7 HS or other Import mass info from i Sigal4 7 Time based m Positive Adducts FH T M H 1 J M NH4 18 F M Nal23 F M K 29 Negative Adducts Ey Se
39. the corrugated waste tubing into a waste container 7 Place the module sampler or fraction collector on top of the thermostat Make sure that the module sampler or fraction collector is correctly engaged in the thermostat locks Agilent 1200 Series Purification System User Manual 31 1 Installing Your Agilent 1200 Series Purification System 8 Remove the plastic cover from the tray base place the air channel adapter 1 into the module sampler or fraction collector tray base Make sure the adapter is fully pressed down This ensures that the cold airstream from the thermostat is correctly guided to the tray area of the module sampler or fraction collector Place the plug channel 2 on top of the air channel adapter Both devices must be installed correctly to assure proper operation of the instrument a M ta ee Figure 8 Installation of G1330A B Thermostat in the Purification System 9 Install the tray you have ordered for your module sampler or fraction collector 10 Ensure the power switch ON the front of the purification system is OFF and the power cables are disconnected 11 Connect the cable G1330 81600 between the module sampler or fraction collector and the thermostat see Connections at the Rear of the Thermostatted Module Sampler or Fraction Collector on page 33 Do not disconnect or reconnect the sampler fraction collector to thermostat interconnection cable when the power cords are connecte
40. the module identifier changes from 1 to 2 or 3 6 The Agilent ChemStation must be shutdown and restarted to activate the changes Configuring Multiple Fraction Collectors Each fraction collector is configured individually and has its own entry in the fraction collector context menu and the More Fraction Collector sub menu of the Instrument menu For details of configuring the individual fraction collectors see Configuration on page 67 Setting Up Multiple Fraction Collectors Multiple fraction collectors are set up as a single fraction collector the selected setup parameters apply to all configured fraction collectors For details of setting up the fraction collectors see Setup on page 70 Agilent 1200 Series Purification System User Manual Instrument Parameters 3 Setting Up for Mass based Fraction Collection Beginning with LC MSD Agilent ChemStation rev A 10 01 mass based fraction collection can be setup and run through either just the Agilent ChemStation software or it can be run through a study with the Purification Hi Throughput software if the G2263AA Mass Based Fraction Collection Add on Software is installed First ensure that the LC MSD Agilent ChemStation is configured for fraction collection From the Agilent ChemStation Method and Run Control view select Instrument gt Configure 1200 MSD Ensure that the Fraction Collection Enabled check box is marked as in Figure 25 MSD Configur
41. the study tree and make the necessary modifications Setting Up a Partial Study 168 You may want to run only a selection of the samples in the study list if for example you have imported the study list but do not have a complete set of samples or if you want to reprocess a group of samples with modified method parameters You set the partial study up in Edit mode Agilent 1200 Series Purification System User Manual Study Setup 5 Adding Samples to a Partial Study To assign samples to a partial study 1 Inthe sampler view select the sample locations that contain the samples that you want to add to the partial study 2 Click with the right mouse button in the sampler view to display the context menu and select Add Sample s to Partial Study from the context menu options Samples that are part of a partial study are marked in the Partial Study column of the Study List see Figure 68 It is also possible to add samples to a partial study directly in this table click to set a tickmark ijl Study Lest Samples fc ose Toate TA Frachor Slant vaL Mu Vial Lagiertt m Q0010000 d Done m NextLocation VOLT Mul VIAL agher 2 n oo QOUIUTOUd Done F m NedLocaton vaL Mul VAAL Laglert im cMethod gt LOnrennnn d Dane F m NeatLocation Lei Vial 2 Mus Vial egiert2m lt Method gt 0000da Done CE m NextLocation VIAL 3 Muh VIAL agiertl in lt Method gt NINN d Done Ei NedLocator VAAL 3 Mudi VIA
42. wl head or ALS G2260A Column delivered with the ALS optional Multi Draw kit available for Injection volumes up to 1800 ul Dual loop Injection valve to 4b 5065 9930 Autosampler Column delivered with the G2258A dual loop autosampler thermostatted Version of the Prep ALS G2261A Prep Manual From Manual 4c G2262 87300 1 Injection loops of Injector 0101 1232 Injector to Column delivered w the valve variable sizes available V Column Holder Column Organizer Column to detector 5 G2260 87301 needed for SST cell only G1383A flow cell delivered with the ALS VI UV Detectors DAD G1315B C Detector flow cellto 6 see fraction fraction collector collector or or MRA if installed 7 5063 6527 delivered with the detector MWD G1365B C 38 Agilent 1200 Series Purification System User Manual Installing Your Agilent 1200 Series Purification System 1 Table 5 Flow Connections in an Preparative Scale Purification System see Figure 4 on page 24 and Figure 5 on page 25 continued No Type of Module Available Choices Flow Connection Capillary Tubing Comments VII Fraction Collector G1364B PS Inlet and Outlet of 6 G1364 68711 delivered with the the fraction Collector fraction collector VIII Make up Pump Isocratic Pump Pump purge valve to 3 0890 1762 In Combination with an for LC MSD G1310A MRA delivered with the LC MSD only mass based fraction collector kit IX Flow Splitter MRA Mass Rate MRA to fraction 6 see fractio
43. you select continuous flow collection available only with well plates not for capped vessels the flow is diverted to waste only for long needle movements for example in the case of the 96 well plate A12 to B1 so no sample is lost However there is a small risk of cross contamination through splashing 8 In the Needle Movement group select where you want the needle to be positioned for fraction collection If you select to move the needle into location the needle moves the specified depth into the container This selection is required for capped vials or if surface tension causes the solution to remain at the top of the well in 384 well plates Additional time is needed for the needle to move into location If you select to move the needle above location the needle does not move down into the container the needle moves across the tray with the specified distance to the specified vessel height This selection is not suitable for capped vials The drop setup mode is not available for analytical scale AS or preparative scale PS fraction collectors 9 If well plates are installed in the fraction collector specify the type of well plates and their positions in the Well Plates group If 384 well plates are installed you can also select to treat these in sections as four 96 well plates You can also specify reserved locations which will not be used for fraction collection Agilent 1200 Series Purification System User Manua
44. you should see only one peak as in Figure 114 on page 246 to Figure 116 on page 247 Please note the difference between the flow cells Agilent 1200 Series Purification System User Manual 245 11 Checkout Procedures The retention times of the peaks will differ depending on multiple parameters like system configuration capillary volumes type and state of column and method parameters such as flow rate composition detector signal settings The absorbance scale will depend on the flow cell you have installed DAD 4S Sig 254 10 Ref S00 100 CHECKDOG D Norm 3 mm Path Length 70 in Ph a S o os 1 15 2 25 3 Figure 114 Fraction 3 after reanalysis 3 mm path length 246 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 0 3 mm Path Length Figure 115 Fraction 3 after reanalysis 0 3 mm path length 0 06 mm Path Length Figure 116 Fraction 3 after reanalysis 0 06 mm path length Agilent 1200 Series Purification System User Manual 247 11 Checkout Procedures MSD triggered AS Fraction Collection Checkout The checkout procedure consists of the following activities e Preparing the Agilent 1200 Series LC and LC MSD e Setting the checkout method parameters e Running the checkout and collecting fractions e Reviewing the results and reanalyzing a fraction What you will need Instruments e Agilent 1200 Series high performance autosampler or Agilent 1200 Series stan
45. 1200 Series Purification System User Manual 107 3 108 Instrument Parameters 6 Set the flow of the make up pump to the flow rate you will be using for your analysis 7 Set the active splitter to a setting of 3 8 Save the method to a new name If the Delay Sensor is not already installed remove the tray installed in the fraction collector 10 Install the Delay Sensor 11 Go to Diagnostics menu select Delay volume Calibration from the Fraction Collector sub menu within the Maintenance menu 12 Press Change method to select the method you created The new method name appears on the info line 13 Start the active splitter 14 Press Continue to execute the Delay Volume Calibration 15 When prompted on the Info line disconnect the needle from the transport arm and disconnect the tubing for the PS version of G1364A or install the FDS adapter to the fraction collector arm G1364B 16 Connect the tubing of the transport arm to the union in the rinse port G1364A only 17 Close the door and press Continue The selected method is loaded and the Delay Calibration Sample Injected 18 When prompted disconnect the tubing from the rinse port union G1364A or remove the FDS adapter from the arm G1364B 19 If the Calibration Run is finished press OK 20 Examine the value for the MSD Calculated Delay time The delay time should be at least 5 seconds but may in fact be negative see Figure 43 Agilent 1200 S
46. 20010925 000 2001091 4 0005 20010831 0035 20010830 0035 Study Base Path F purify data demo o cs C Figure 54 Study Selection dialog box 140 Agilent 1200 Series Purification System User Manual Study Setup 5 Setting Up the Study Parameters The study parameters dialog box see Figure 55 contains five groups of parameters Not all groups of parameters are relevant to all studies for example the Run Method If parameters are relevant only to studies that use an MSD and are not available grayed out when no MSD is configured Setup Study Parameters Study Information Injection Order Ooo Order injection by 2 Tni Operator fagientusel Method Beet AI Run Method Ir B Method None H IS masses found Eiran kNone gt fad Some masses found C Wo masses found Default Sample Name Format Fraction Wollection and Recover Format Sample i Fraction Stet oe y 2 11 ocation Example Sample 1401 ee TEA F Sample 0103 Number of pools None Recovery Start Loc None nd Help Cancel lt Previous L se Finish Figure 55 Study Parameters Study Information The Software automatically detects if Agilent ChemStore is installed with your Agilent ChemStation If this is the case you have the choice to generate a new Study during study setup as a Agilent ChemStore study as well In this case you can choose from a list of available Agilent ChemStore studies The Oper
47. 4 21 4 gt 0 01 0 25 10 0 29 10 3 gt 0 025 0 5 5 0 58 5 2 gt 0 05 1 2 5 1 31 2 3 gt 0 10 2 1 25 2 84 11 Agilent 1200 Series Purification System User Manual 115 3 116 Instrument Parameters Table9 Analytical Fraction Collector with VWD continued Peakwidth min Response Time Rate Hz Analog signal Max Flow Rate sec delay sec in ml min 50 pl capillary gt 0 20 4 0 62 5 97 0 50 gt 0 4 8 0 31 12 3 0 24 In Table 8 and Table 9 the figures shown in bold text are the default values The 50 ul capillary is 0 25 mm i d x 1000 mm Agilent part no G1364 68712 Table 10 Preparative Fraction Collector with DAD or MWD Peakwidth min Response Time Rate Hz Analog signal Max Flow Rate sec delay sec in ml min 500 pl capillary lt 0 01 0 1 20 0 05 600 gt 0 01 0 2 20 0 15 200 gt 0 03 0 5 10 0 5 60 gt 0 05 1 0 5 1 25 24 gt 0 10 2 0 2 5 2 75 10 9 gt 0 20 4 0 1 25 5 9 5 1 gt 0 40 8 0 0 62 11 9 2 5 gt 0 85 16 0 0 31 23 9 1 3 Table 11 Preparative Fraction Collector with DAD or MWD Peakwidth min Response Time Rate Hz Analog signal Max Flow Rate sec delay sec in ml min 500 pl capillary lt 0 005 lt 0 12 20 0 07 429 gt 0 005 0 12 20 0 14 214 gt 0 01 0 25 10 0 29 103 Agilent 1200 Series Purification System User Manual Table 11 Preparative Fraction Collector with DAD or MWD continued Peakwidth min Response Time sec gt 0 025 0 5 gt 0 05 1 gt 0 10
48. 4 Examine the value for the MSD Calculated Delay time Agilent 1200 Series Purification System User Manual 103 3 Instrument Parameters CAUTION The delay time should be at least 5 seconds but may in fact be negative see Figure 40 Ei Fraction Collector 1 Calibration Results Print Close Calibrate Curr Delay Vol ul Calc Delay Vol ul DAD1 G13154 US64400562 Oooo Curr Delay Time sec Calc Delay Time sec 0 294 Detector MSD Figure 40 Delay Volume Calibration results 15 If the measured delay time is greater than 5 seconds you can skip to step 20 otherwise continue with step 16 If the measured delay time is significantly greater than 5 seconds you should continue with the calibration at step 16 to avoid losing fractions at the end of the run 104 16 Determine how much additional time is needed to create a 5 second delay For example if the time displayed is 1 sec a six second delay is needed 17 Using the main pump flow rate determine the desired additional delay volume For example at 3 ml min and a six second delay an additional 3 0 x 6 60 or 0 3 ml is required 18 Cut off a sufficient piece of the supplied ETFE tubing Part No G1968 60500 to provide the needed volume The tubing s volume is 8 1 ul cm In this case 37 cm would be required 19 Add the tubing between the splitter and the fraction collector or if multiple fraction collectors are insta
49. 84 316 P1 D 62 FI_D62 puri_scn m puri_sim m C16H9C1N402S P1 G 61 FI_G61 puri_pos m puri_neg m 276 278 284 316 Figure 67 CSV file viewed in Notepad Import the CSV file as described in Figure Exporting to a CSV file on page 160 Agilent 1200 Series Purification System User Manual Study Setup 5 Deleting Samples Injections from the Study List You can delete samples injections from the study list using either the sampler view or the study list table You cannot delete sample injections that have associated Agilent ChemStation data To delete samples injections from the study list using the sampler view 1 In the sampler view select the sample locations that contain the samples that you want to delete from the study list 2 Click with the right mouse button in the sampler view to display the context menu and select Remove Sample s from the context menu options Alternatively press Del To delete samples injections from the study list using the study list table 1 Select the line s in the study list table that contains the sample s injection s that you want to delete 2 Select Remove Sample s from the Edit menu Edit gt Set Method Source Path Agilent 1200 Series Purification System User Manual 163 5 Study Setup Saving a Study Studies are saved automatically when any changes are made The changes you make cannot be canceled or undone You can save a copy of a study with a different name so t
50. ADC for connection of non 1200 detectors signal ADC1A e Agilent Fraction Collector UIB signal AFC10 e Agilent G1315B C DAD signal DADIA e Agilent G1321A FLD signal FLD1A e Agilent G1365B C MWD signal MWD1A e Agilent G1314B C VWD signal VWD1A If the Show when Available item is unchecked the signals of the checked detectors are displayed in the Result Panel if they have been acquired in the run If the signals of the checked detectors have not been acquired in the run an empty graphic is shown indicating that this signal was not found If the Show when Available item is checked all available UV detector signals in the current data file are displayed MS Detector signals The Agilent G1946D and G1956B MSD have the ability to acquire a TIC for up to four channels TIC 1 4 The Agilent ChemStation method determines which TIC will be acquired and stored in the data file Menu items MSD 1 4 correspond to the four TIC 1 4 If the Show when Available item is unchecked the TICs corresponding to the checked MSD 1 4 signals will be displayed in the Result Panel if the MSD signals have been acquired in the run If the checked MSD signal has not been acquired in the run an empty graphic is shown indicating that this signal was not found If the Show when Available item is checked the TICs corresponding to all the acquired MSD signals in the current data file are displayed To view the TICs in one graphic using the same time a
51. After connecting all inter module tubings and capillaries re install the front covers of all of the modules Agilent 1200 Series Purification System User Manual 35 1 Installing Your Agilent 1200 Series Purification System Flow Connections in an Analytical Scale System Table 4 Flow Connections in an Analytical Scale Purification System see Figure 1 on page 21 and Figure 2 on page 22 No Type of Module Available Choices Flow Connection Capillary Tubing Comments l Solvent Container Solvent Cabinet Bottle head to 1 G1311 60003 Up to 4 different 5062 8581 comes with every pump degasser or pump inlet delivered with the pump solvents amber or transparent bottles Il Degasser Standard or micro Degasser outlet to 2 G1322 67300 Only if the optional Degasser pump inlet delivered with the degasser is installed G1322A G1379B degasser lll Pump Isocratic pump Pump purge valve to 3 G1315 87311 Solvent inlet at Active G1310A Quarternary pump injection valve delivered with the detector Inlet valve needs Adapter 0100 1847 Solvent inlet at the G1311A MCGV Binary pump Solvent inlet at the AlVs G1312A with Adapt 0100 1847 or at optional Solvent Selection Valve IV Sampling Device Autosampler ALS Injection valve to 4a G1313 87305 Optional 900 ul head or G1329A TCC peltier delivered with the Multi Draw kit available autosampler for Injection volumes up to 1800 ul high perfor
52. Agilent 1200 Series Purification System User Manual cii Agilent Technologies Notices Agilent Technologies Inc 2006 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number G2262 90010 Edition 02 06 Printed in Germany Agilent Technologies Hewlett Packard Strasse 8 76377 Waldbronn Warranty The material contained in this docu ment is provided as is and is sub ject to being changed without notice in future editions Further to the max imum extent permitted by applicable law Agilent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchantability and fitness for a par ticular purpose Agilent shall not be liable for errors or for incidental or consequential damages in connec tion with the furnishing use or per formance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms the warranty terms in the sep arate agreement shall control Technology Licenses
53. Based Fraction Collection deselected in the Setup Fraction Collector dialog box Save the method as msdrean1 m 2 You should see only one peak in the chromatogram of the reanalyzed fraction If you do not see the peak in the TIC generate EICs for masses 279 285 and 311 and verify that there is a single peak in one of the EICs see Figure 124 Figure 124 EICs after reanalysis of fraction 2 260 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 MSD triggered PS Fraction Collection Checkout The checkout procedure consists of the following activities e Preparing the Agilent 1200 Series LC and LC MSD e Setting the checkout method parameters e Running the checkout and collecting fractions e Reviewing the results and reanalyzing a fraction What you will need Instruments e Agilent 1200 Series Preparative Autosampler optional thermostatted version e Two Agilent 1200 Series Preparative Pumps used in a binary pump configuration e Agilent 1200 Series Isocratic Pump with solvent bottle to be used as the make up pump for the LC MSD e Agilent 1200 Series Fraction Collector Preparative Scale optional thermostatted version e Agilent 1200 Series LC MSD G1946B C or G1956A B e G1968C Mass Based Fraction Collection Kit Software e Agilent LC MSD ChemStation Software G2710AA rev B 01 01 or higher Column e Zorbax SB C18 9 4 mm x 5 0 cm 5um part number 846975 202 included in G1968C mass
54. Help Figure 95 Report parameters for UV triggered AS checkout Agilent 1200 Series Purification System User Manual 227 11 Checkout Procedures Step 4 Adding Sample s to the study 1 Fill a well or vial with the isocratic checkout sample 2 Select the sample location on the Sampler panel and add the sample to the study see Figure 96 wy amp Add Sample s Remove Samples Modi Sample Figure 96 Adding the sample to the study 3 Select the uvasfr m method to apply to the selected sample see Figure 97 Add Sample s to Study m Sample Information m Available Methods Methods p Injection Parameters Location tmeohh2o m T Injection Volume Method VIAL 1 merck560 m merckend m Nema merckm m Pooling None gt merckme m CheckVIAL 1 midegtst m on multisig m Description mydegley m purity m feanaly m sysperf m syssuit m Figure 97 Applying the uvasfr m method 4 Add as description Isocratic Sample as in Figure 98 and click OK Description isocratic Sample Figure 98 Adding a sample description 228 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 The selected sample is marked as a filled blue circle 5 In the fraction collector panel assign a fraction start position to an empty vial well or tube The Study List is updated as shown in Figure 99 NOTE The sampler and fraction collector panel views depend o
55. It calls attention to an operating procedure practice or the like that if not correctly per formed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated condi tions are fully understood and met Agilent 1200 Series Purification System User In This Guide This user s guide describes the installation and operation of the Purification System 1 Installing Your Agilent 1200 Series Purification System This chapter contains site requirements and installation of the purification system 2 Installing the Software This chapter contains instructions and other information to help you install and start the Purification Hi Throughput software on your ChemStation PC 3 instrument Parameters This chapter describes how to set up the CS instrument parameters that are necessary to run a study 4 Menus and Views This chapter describes the menu structure and toolbar buttons 5 Study Setup This chapter explains what a study is and describes the process of setting up a study including an explanation of all the necessary parameters 6 Running Studies This chapter describes the options for running and reprocessing studies 7 Pooling This chapter explains the concept of pooling and describes how to set up a pooling run 8 Data Review This chapter describes the options available in the software for reviewing the study data Agilent 1200 Series Purifica
56. L ageri m lt Method gt COPE d Dane m Next ocation Figure 68 Study List with samples for a partial study Removing Samples from a Partial Study To remove samples from a partial study 1 In the sampler view select the sample locations that contain the samples that you want to remove from the partial study 2 Click with the right mouse button in the sampler view to display the context menu and select Remove Sample s from Partial Study from the context menu options It is also possible to remove samples from a partial study directly in the table click to remove a tickmark Agilent 1200 Series Purification System User Manual 169 5 Study Setup 170 Agilent 1200 Series Purification System User Manual This chapter describes the options for running and reprocessing studies Agilent 1200 Series Purification System User Manual 6 Running Studies Run Control 172 Study Status 174 Online Tickmarks 175 Reprocessing Studies 176 ee Agilent Technologies 171 6 Run Control 172 Running Studies You control the running of a study either from the RunControl menu or from the toolbar Starting a Study You can start a study in either Select mode or View mode You start the study from the Start Study dialog box see Figure 69 which you display using the RunControl menu RunControl gt Start Study Alternatively you can choose the tool from the toolbar or press the F5 function key r Select Start Mo
57. MWD Fraction Trigger Mode Destination Report Style 254 16 nm 360 100 nm Off Printer Short On the printed chromatogram you should see only one peak as in Figure 103 Agilent 1200 Series Purification System User Manual 233 11 Checkout Procedures The retention times of the peaks will differ depending on multiple parameters like system configuration capillary volumes type and state of column and method parameters such as flow rate composition detector signal settings 234 S a ge a us DAD A Sig 254 16 Ret 360 100 DEFAULT D mn Figure 103 Fraction 3 after reanalysis Agilent 1200 Series Purification System User Manual Checkout Procedures 11 UV triggered PS Fraction Collection Checkout The Checkout Procedure consists of the following activities e Preparing the Agilent 1200 Series LC e Setting the study parameters e Starting the analysis and collecting fractions e Reanalyzing the fractions What you will Need Instruments e Agilent 1200 Series Preparative Autosampler optional thermostatted version e Agilent 1200 Preparative Pump e Agilent 1200 Series Fraction Collector PS optional thermostatted version e Agilent 1200 Series Multi wavelength Detector with preparative flow cells or 1200 Series Diode Array Detector with preparative flow cells Software e Agilent ChemStation Software G2170AA A 09 01 or Higher e Agilent Purification Hi Throughput Software G2262AA
58. Method and Run Control View if necessary View gt Method and Run Control 3 Open the Configure Well Plate Types dialog box from the Instrument menu Instrument gt Configure Well Plate Types 4 Click New to add a new line to the list of user configured well plates Enter the dimensions of the new well plate in the fields use the horizontal scroll bar to access the fields to the right Straight well plate Staggered well plate Figure 14 Well plate dimensions 54 Agilent 1200 Series Purification System User Manual Instrument Parameters 3 Table 7 Well plate dimensions Label Description Definition Limits Rows Number of rows on the plate up to 16 Columns Number of columns on the plate up to 24 Volume Volume in ul of a sample vessel well A Row distance Distance in mm between the centers of two adjacent rows B Column Distance in mm between the centers of two distance adjacent columns C Plate length X size in mm at the bottom of the plate 127 75 0 25 mm SBS Standard D Plate width Y size in mm at the bottom of the plate 82 50 0 25 mm SBS Standard E Plate height Height in mm from the bottom to the top of up to 47 mm the plate F Row offset Distance in mm from the left edge bottom to the center of the first hole A1 G Column offset Distance in mm from the back edge bottom to the center of the first hole A1 J Well depth Distance in mm from the top of the plateto up to 45 mm the bottom of the well I
59. More Injector gt Configuration Configuring the Standard Autosampler Supported standard autosamplers are e Autosampler G1329A Agilent 1200 Series Purification System User Manual 57 3 58 Instrument Parameters e Preparative autosampler G2260A Injector Configuration Instrument 1 Figure 15 Standard autosampler configuration dialog box To configure the standard autosampler refer to Figure 15 1 Inthe Volumes group specify the syringe volume and seat capillary volume of your autosampler configuration The maximum injection volume is calculated automatically from these entries 2 If the seat capillary volume is greater than the syringe volume then multiple draw is enabled and you can specify a time between draws in the Waittime field The default wait time is 2 seconds 3 Ifthe Agilent ChemStation is Online the installed trays are automatically detected and displayed If the Agilent ChemStation is Offline you can specify the trays that are installed Agilent 1200 Series Purification System User Manual Instrument Parameters 3 Configuring the high performance autosampler Injector Configuration Instrument 1 x Trays ret TA Well Plates Plate 1 front none x Plate 2 back none M olumes ruse BCD port for Syringe 100 0 M ul Location Seat Capillary 2 3 yi ul annie H Binary Output Max Inj Yolume 100 pl Figure 16 High performance
60. Sampler Panel 128 sampler panel 122 148 186 samples adding 149 deleting 163 save study 125 saving a study 164 seat capillary volume 58 60 61 Select Mode 128 select mode 120 selected masses 87 setup fraction collector 70 injector 61 more 74 multiple fraction collectors 76 Setup Result Panel 129 Show Fraction Scheme 128 show fraction scheme 187 Show Messages 129 site requirements 14 slope 72 snapshot 175 software ChemStation 42 purification hi throughput 43 space bench 15 Agilent 1200 Series Purification System User Manual specification report 144 specifications dimensions 17 physical 18 weight 16 specify report 125 splitter flow 29 spreadsheet 158 162 stack configuration 20 21 22 23 24 25 26 front view 21 22 24 25 rear view 23 26 standard autosampler 57 standards safety 18 start location 143 recovery 143 start study 126 starting a study 172 status injection 155 study 174 177 status info 126 stop study 126 stop time 74 stopping a study 173 storage 18 study 136 aborting 173 base path 136 close 125 closing 167 deleting 164 description 142 editor 138 information 141 list 122 123 136 140 155 158 188 modifying 167 name 139 new 125 139 open 125 opening 165 parameters 123 136 141 182 185 partial 168 172 pause 126 pausing 173 reprocess 172 reprocessing 176 results 175 review
61. al No Stopl a j1 49 1 VIAL 3 1033 33 1 VIAL 4 1502 08 Figure 112 Panels after fraction collection Agilent 1200 Series Purification System User Manual 243 11 Checkout Procedures 3 Review the results by clicking the tool see Figure 113 on page 244 NOTE The retention times of the peaks will differ depending on multiple parameters like system configuration capillary volumes type and state of column and method parameters such as flow rate composition detector signal settings The absorbance scale will depend on the flow cell you have installed Result Panel C Purify data 20030723_000 D ata 00010500 d Figure 113 UvV triggered PS checkout results 244 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 Step 7 Reanalyzing fraction 3 1 Reanalyze the fraction that is collected in the third tube under the conditions specified in Table 20 and save the method as verify m Table 20 LC method parameters for UV triggered PS fraction collection checkout verification Device Parameter Value Injector Injection Volume 100 wl Pump Flow Rate 10 0 ml Channel A Water 35 Channel B Acetonitrile 65 Stop Time 2 5 min Pump Timetable 2 5 min 75 B Detector Wavelength VWD 254 nm Wavelength DAD MWD 254 16 nm 360 100 nm Fraction Collector Fraction Trigger Mode Off Report Destination Printer Report Style Short On the printed chromatogram
62. alculates masses from the molecular formulas Agilent 1200 Series Purification System User Manual 161 5 162 Study Setup If you are using Microsoft Excel set up your spreadsheet with all the expected masses for each sample in the same cell separated by commas as in column D of Figure 66 You must enter molecular formulas with the correct capitalization of the elements E1 Microsoft Excel purify1_csv G9 File Edit View Insert Format Tools Data Window Help OSH GRY BRS 4 SrA MA H baf ale A B ale C D well position sample name method exp masses P1 A 01 FI_AD1 puri_pos m puri_neg m 270 278 284 310 P1 A 02 FI_AD2 puri_pos m puri_neg m C1IQHSCIN4025 P1 D 01 Fl_DO1 puri_scn m puri_sim m 270 278 284 310 P1 D 02 FI_D02 puri_scn m puri_sim m C1QHSCIN4025 P1 G 01 FI_GO1 puri_pos m puri_neg m 270 278 284 310 7 Figure 66 Microsoft Excel file with masses Export your Excel spreadsheet in csv file format Export to the path specified in File gt Import Study List The CSV file from Figure 66 on page 162 as viewed in Notepad is shown in Figure 67 Note that multiple values per cell and per field are grouped within quotation marks 8 purify1_cs Notepad File Edit Search Help well position sample name method exp masses P1 A 61 FI_A61 puri_pos m puri_neg m 276 278 284 316 P1 A 62 FI_A62 puri_pos m puri_neg m C1GH9C1N402S P1 D 61 FI_D61 puri_scn m puri_sim m 276 278 2
63. anges all the open windows in the display in a cascade Agilent 1200 Series Purification System User Manual 129 4 Menus and Views Help 130 Tile Horizontal Arranges all the open windows in the display as horizontal tiles Tile Vertical Arranges all the open windows in the display as vertical tiles Arrange Icons Arranges all minimized windows as icons at the bottom of the display Arrange Windows Arranges all minimized windows to their standard positions in the display Minimize All Minimizes all open windows and arranges them as icons at the bottom of the display The following items are available in the Help menu Contents Displays the Purification Hi Throughput Software Help contents Search for Help on Displays the Purification Hi Throughput Software Help index About Purify Displays the information about the Purification Hi Throughput Software Agilent 1200 Series Purification System User Manual Tool Bar E E t oo o wi fe Menus and Views 4 The toolbar contains tools that allow you quickly and easily to navigate through the Purification Hi Throughput software Some of the tools are common to both View Mode and Select Mode others are Mode specific The following tools are common to both View Mode and Select Mode Switches to View Mode Switches to Select Mode Starts the Study Wizard which allows you to set up a new study see Creating a New Study on page 139
64. anted evaporation of fractions in the fraction collector e g Agilent 1200 Series thermostatted column compartment G1316A B or Agilent 1200 Series diode array detectors G1315B C Agilent 1200 Series Purification System User Manual 21 1 Installing Your Agilent 1200 Series Purification System IX Flow Splitter VIII Make up waste Pump X LC MSD waste waste waste Figure 2 Recommended Stack Configuration Analytical Scale Purification System Including LC MSD Front View To identify the inter module capillaries and tubing as labelled in Figure 2 please refer to NOTE Table 4 on page 36 NOTE he fraction collector should never be positioned on top of a module that generates heat This could lead to an unwanted evaporation of fractions in the fraction collector e g Agilent 1200 Series thermostatted column compartment G1316A B or Agilent 1200 Series diode array detectors G1315B C 22 Agilent 1200 Series Purification System User Manual Installing Your Agilent 1200 Series Purification System 1 For more details about the installation of any of the modules that are used only if the G1946C D or G1956A B LC MSD is part of the system G1310A Make up pump G1390A UIB G1968 64102 MRA please refer to the G1968C Mass Based Fraction Collection Kit Installation Instructions manual available in the G1968C Mass Based Fraction Collection kit E E GPIB or LAN A ee to LC ChemStation mA g Analog signal to record
65. are Figure 10 Select Components Figure 10 Choosing the software to install 4 Select any additional components that you want to install Figure 11 NOTE You must have the necessary licenses for all the options you select You can only install either the Offline OR the Online version of the SW on one instrument 44 Agilent 1200 Series Purification System User Manual Installing the Software 2 Select Components Figure 11 Selecting additional components You can add a G2263AA license product for mass based fraction collection in combination with either G2262AA or G2265AA NOTE The G2264AA Purity Calculation for MS data add on software product is not supported with the CS B 01 01 and Purify B 01 01 However the corresponding functionality is built into the CS product There is no additional G2264AA license required for the B 01 01 Purification SW and CS B 01 01 Agilent 1200 Series Purification System User Manual 45 2 Installing the Software 5 Enter the license numbers for each component in the appropriate fields Figure 12 Check License for G2262AA Purify Online Software Figure 12 Entering license numbers 6 You can select to install the Purification Hi Throughput software in any folder of your choice but we recommend that you accept the default folder 7 Restart your computer to put all the changes made during installation into effect 46 Agilent 1200 Series Purification System U
66. are displayed in View mode are selectable either using the toolbar or using the items of the View menu Six panels are available The panels are interactive so that when you select a sample in the Sampler Study Parameters Sampler Fraction Collector Study List Samples Study List Injections Result panel it is also selected in both study lists and when you select a sample in one of the study lists it is also highlighted on the Sampler panel The fractions for the selected sample or injection are displayed simultaneously in the Study List Injections and the Result panel Study Parameters The study parameters panel is a read only version of the Setup Study Parameters panel see Figure 55 on page 141 It shows the parameters that were set up when the study was created Agilent 1200 Series Purification System User Manual 185 8 186 Data Review Sampler The Sampler panel shows a graphical representation of the autosampler trays and samples for the study Table 15 explains the indicators used to show the state of each location in the Sampler panel Table 15 Sampler Location Indicators Line Color Fill Color Meaning Blue Gray There is no sample at this location location not selected added White Gray Location selected but not yet added to a study Blue Dark blue There is a sample assigned at this location White Dark blue The location has a sample assigned and has been selected Blue Light blue T
67. are used WARNING To avoid personal injury keep fingers away from the needle area in the sampling device and fraction collector during purification system operation Do not attempt to insert or remove a vial tray or a plate during the operation of the sampling device and fraction collector 1 Install the LAN interface board in one of the purification system modules G1315B C DAD or G1365B C MWD if present See Exchanging or Installing the Optional Interface BCD Board in any of the reference manuals of the modules in your purification system 2 Remove the adhesive tape which covers the front doors 3 Open the front doors 4 Remove the transport protection foam To do so please remove the side door also Be careful not to damage any of the mechanics and especially tubing inside the instrument 5 Position the modules on top of each other in the recommended stacks and order of the modules see Figure 1 on page 21 to Figure 6 on page 26 Make sure that all instruments are nicely aligned and centered on top of each Agilent 1200 Series Purification System User Manual 27 1 28 Installing Your Agilent 1200 Series Purification System other to guarantee proper functioning of the leak drainage system in case of a leakage Every pump must have a separate waste tubing installed at its purge valve Every stack must have a waste drainage installed at the bottom A fraction collector must have an additional waste drainage i
68. ation Available Modules Configured Modules G1946C L G1956B SL Mass Mass MSD Spectrometer J MSD Spectrometer 4 4 G1946D SL MSD Spectrometer IX Fraction Collection Enabled G1956A VL Mass MSD Spectrometer SS Figure 25 Configuring the MSD Agilent 1200 Series Purification System User Manual 71 3 Instrument Parameters Next still in the Agilent ChemStation Method and Run Control View select Instrument gt Set up Fraction Collector Select Peak based from the Fraction Trigger Mode group and mark the Use MSD for Mass based Fraction Collection check box in the Peak Detectors group as in Figure 26 Set up Fraction Collector Instrument 1 xi Fraction Trigger Mode Off Use Timetable Peak based Max Peak Duration 0 5 min Peak Detectors Detector Working Mode Up Slope Unit s Down Slope Unit s Threshold Unit Upper Threshold Unit Unit 1 _pan1 of ST TO 3000 000 sA T of a a 3000 000 Fraction is collected when a peak is detected by C all selected peak detectors atleast one selected peak detector rTimetable Insert Time Trigger Mode Max Peak Duration min of Fractions Timeslices min Append u Copy Paste l Figure 26 Setting up the fraction collector for mass based fraction collection 78 Agilent 1200 Series Purification System User Manual Instrument Parameters 3 When the Use MSD for Mass based Fraction Collection option is
69. ator field contains by default the current Windows user name however you can change the operator name in this field if necessary The operator name can be up to 29 characters long The Operator name is also transferred to the Agilent ChemStation and is printed on the Agilent ChemStation reports Agilent 1200 Series Purification System User Manual 141 5 142 Study Setup Selection Location Method If you are creating a study using an existing study as a template the Description field contains the text from the template which you can edit If you are creating a new study manually this field is empty so that you can add a description of the new study In the shutdown field you can select the shutdown options for the instrument at the end of the study Standby PumpAllOff LampAllOff or a user editable shutdown macro Default Sample Name Format The default sample name consists of a prefix which you supply and a location the sample vial well position which you denote by either a or a character where gives the abbreviated location Purify location format and gives the full location Agilent ChemStation location format as shown in Table 12 Table 12 Sample Name Formats Vials Well Plates SAMP 0 vial location Plate row column SAMP 0001 SAMP 1A01 SAMP VIAL number Plate row column SAMP VIAL 1 SAMP P1 A 01 Since the complete sample name including the location identifier can be a ma
70. autosampler configuration dialog box Agilent 1200 Series Purification System User Manual 59 3 Instrument Parameters To configure the high performance autosampler refer to Figure 16 on page 59 1 Ifthe Agilent ChemStation is on line the installed trays are automatically detected and displayed If the Agilent ChemStation is off line you can specify the trays that are installed 2 In the Well plates group specify the type of well plates that are installed In the Volumes group specify seat capillary volume of your high performance autosampler configuration The maximum injection volume is calculated automatically from these entries Configuring the dual loop autosampler Injector Configuration Instrument 1 lx Trays left Wellplate 50mm r Well Plates Plate 1 front 1 5 ialPlate Plate 2 back 1 5 ialPlate bd Volumes use BCD port for Lower Loop 5000 00 w Upper Loop 50 od ul Location Binary Output Seat Capillary lower loop 20 0 yl Seat Capillary upper loop 20 0 ul Cancel Help Figure 17 Dual loop autosampler configuration dialog box Agilent 1200 Series Purification System User Manual Instrument Parameters 3 To configure the dual loop autosampler refer to Figure 17 on page 60 1 Ifthe Agilent ChemStation is on line the installed trays are automatically detected and displayed If the Agilent ChemStation is off lin
71. based fraction collection kit Agilent 1200 Series Purification System User Manual 261 11 Checkout Procedures Solvents e HPLC grade Bi distilled water e HPLC grade Acetonitrile e Acetic or Formic acid Sample e Delay calibrant part number G1946 85020 included in G1968C mass based fraction collection kit e Electrospray LC Demo Sample part number 59987 20033 included in G1968C mass based fraction collection kit Step 1 Calibrating the Delay Time Before running the fraction collection the delay time between the MSD detector and the fraction collector must be determined See Delay Volume Calibration on page 93 for the procedure to determine the delay time This value is entered into the MSD Fraction Collection Parameters panel For the delay calibration method specify an LC flow of 10 0 ml min and a make up pump flow of 0 5 ml min 262 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 Step 2 Preparing the LC system 1 O oO A Q Fill the solvent bottles for the Preparative pumps with HPLC grade bi distilled water Channel A and HPLC grade acetonitrile Channel B Fill the solvent bottle for the isocratic make up pump with 50 50 water acetonitrile with 0 1 acetic or formic acid 1 ml of acid 1 liter of solvent Turn on all the units Purge the channels Enter the LC methods as shown in Table 25 Enter the MSD parameters as shown in Figure 26 on page 265 Figure 27 on
72. bed in the previous sections for running the method Delay Volume Calculations UV Detector Figure 46 shows how the delay volume calibration results are generated 112 Agilent 1200 Series Purification System User Manual Instrument Parameters 3 Diverter valve Waste Fraction delay sensor Figure 46 Overview of delay volume calibration The first step in the delay volume calculation is the calculation of the delay time This calculation is shown in Figure 47 tp tFC t0 mAL to l 70 Detector tp delay time w o RT of peak at fraction coll 12504 fy i trc RT of peak at fraction collector 1000 750 l I to RT of peak in Detector l l l l 0 01 0 03 o4 05 0g a E LE mn l l nau I tec 350 l WO l I Fraction delay sensor m i oo l 0 l 100 I 50 0 A H Do o Y 402 03 n4 05 06 07 08 03 min tp Figure 47 Step 1 Delay Time calculation Agilent 1200 Series Purification System User Manual 113 3 114 Instrument Parameters The calculation of the delay volume is shown in Figure 48 In Figure 48 the delay times tp and tps and the volumes Vp and Vpg include the volume of the valve which is corrected for in the delay volume calculation Diverter valve Waste Vp1 Delay volume to be calculated Vp2 Internal volume of the diverter valve plus the valve to needle tubing plus the rinse port to delay sensor tubing v flow rate tp tpi tp2 and Vp Vo1 Vp2 Vp1
73. cations You assign recovery locations in the study setup wizard during the generation of a study The recovery location is then denoted as a filled dark green circle in the fraction collector panel Recovery locations can also be setup interactively in the fraction collector panel left mouse click to select a location and then right mouse click on this selected location to bring up a menu that allows to toggles between a fixed position as a start location and the next available free position Agilent 1200 Series Purification System User Manual 157 5 Study Setup Using CSV Files 158 A CSV file is a table that has been saved as simple text The contents of each cell of the table is converted to text and separated from its neighboring cells by commas Each row of the table ends with a carriage return All common spreadsheet programs for example Microsoft Excel allow you to save the data as a CSV file You can set up your study list either the complete study list or selected columns and rows of the study list in your spreadsheet program save it as a CSV file then import it directly into the study Setting up the CSV file To set up the CSV file 1 Decide which columns of the study list you want to import into your study and set them up in your spreadsheet program You can use column headings in your spreadsheet you specify the number of header lines to ignore when you import the CSV file 2 Add the sample data to the
74. ction collectors must be installed with a G1160A 12 position 13 port Multiple Purpose Switching Valve No matter how many fraction collectors are installed only one fraction collector icon is displayed in the instrument diagram Configuring the Multiple Purpose Switching Valve The configuation of the valve is done automatically by the Agilent ChemStation Specifying the Fraction Collector Order Fraction collectors are identified in the order that they appear in the list of configured modules The one that appears first is given the identifier 1 and is used to control the switching valve and will be used first for fraction collection To specify the fraction collector order 1 Switch the Agilent ChemStation to Full Menus if necessary View gt Full Menu 2 Switch to Method and Run Control View if necessary View gt Method and Run Control 3 Open the Configuration dialog box Figure 21 on page 68 from the Instrument menu Instrument gt Configure Agilent 1100 1200 Series access 4 Select a fraction collector from the Configured Modules list and click Identify The module identifies itself by displaying a color cycle on the module status indicator Agilent 1200 Series Purification System User Manual 75 3 76 Instrument Parameters 5 Use Up and Down to position the selected instrument in the required order in the list As you switch the position of the selected module from first fraction collector to second or third
75. ction volume by default the injection volume specified in the method denoted by lt Method gt The recovery location for this injection The volume of sample recovered The selected injection is denoted by an arrow in the left column of the table Agilent 1200 Series Purification System User Manual 189 8 Data Review Study List Fraction Wells The Study List Fraction Wells is available only in View Mode after the study has been run Table of Fraction Wells Sapte Location Hot Spot Sample Name Method Pool Well No Volume Datafile Start Stop Reason Masses The location of the fraction in the fraction collector A location of interest can be selected as a HotSpot for Reporting Preview Export to CSV The sample name according to the specified naming convention The method used for this injection Number of Pools Number of Wells for overfilling The injection volume by default the injection volume specified in the method denoted by lt Method gt Datafile for current injection Start time of fraction collection as shown in the chromatogram corrected by used delays Stop time of fraction collection as shown in the chromatogram corrected by used delays Reason for fraction collection trigger Masses found and used for fraction collection The selected injection is denoted by an arrow in the left column of the table 190 Agilent 1200 Series Purification System User
76. ction with needle wash specify the wash vial in the adjacent field If you choose to use an injector program press Edit to display the current injector program for editing 62 Agilent 1200 Series Purification System User Manual Instrument Parameters 3 2 In the Injection group you can also choose an optimization program for fast throughput If you choose Overlap Injection Cycle the next sample is drawn into the autosampler while the current sample is being run You specify the time delay between sample injection and sample drawing If you choose Prefetch Sample Vial the next sample vial is picked up while the current sample is being run You specify the time delay between sample injection and vial collection 3 In the Auxiliary group specify the Draw and Eject speeds and the Draw Position offset to default position is higher is deeper of the autosampler For thermostatically controlled autosamplers you can also select to store the temperature Setting Up the high performance autosampler Figure 19 High performance autosampler setup dialog box Agilent 1200 Series Purification System User Manual 63 3 64 Instrument Parameters To set up the high performance autosampler refer to Figure 19 1 In the Injection group specify the injection type and injection volume You can choose to make a standard injection an injection with a needle wash or to use an injector program If you choose an in
77. ctions to the Purification System Preparation Purification System is installed in the LC system Parts required Parts from the accessory kits of each module see accessory kit contents in the chapters 1 of the modules reference manuals plus parts from the G1968C Mass Based Fraction collection Kit if the G1946C D or G1956A B LC MSD is part of the system WARNING When opening capillary or tube fittings solvents may leak out Please observe appropriate safety procedures for example goggles safety gloves and protective clothing as described in the material handling and safety data sheet supplied by the solvent vendor especially when toxic or hazardous solvents are used WARNING This system should only be used with solvents that have an ignition temperature higher than 200 C The following tables and Figure 1 on page 21 to Figure 6 on page 26 give a rough overview of the plumbing and cabling of your purification system only For a more detailed layout of flow and electronic connections to the modules that are part of your purification system please refer to the installation sections in each of the reference manuals delivered with every module If your stack configuration differs from the recommended ones the capillaries delivered with the modules may not be long enough In this case it is possible to order additional capillaries of various lengths separately If multiple capillaries are connected to each other
78. d ee wal No StopTim 1 VIAL 1 57 67 2 01 2 06 H 1 VIAL 2 47 2 20 2 24 1 1 VIAL 3 52 33 2 63 2 68 fiew Study Agilent Checkout Well VIAL 1 CheckYlAL1 uvasfr m 00010200 d Figure 101 Panels after fraction collection Agilent 1200 Series Purification System User Manual 231 11 Checkout Procedures 3 Review the results in the CS Data Analysis View see Figure 102 on page 232 NOTE The retention times of the peaks will differ depending on multiple parameters like system configuration capillary volumes type and state of column and method parameters such as flow rate composition detector signal settings ETTU COOU NARMA ENO Emgang Rate 5 ORT men GEE ml SEE a eo Figure 102 UV triggered AS checkout results as shown in the CS data analysis view 232 Agilent 1200 Series Purification System User Manual Step 7 Reanalyzing fraction 3 Checkout Procedures 11 1 Reanalyze the fraction that was collected in the third vial well or tube under the conditions specified in Table 18 and save the method as verify m Table 18 LC method parameters for UV triggered AS faction collection checkout verification Device Parameter Value Injector Injection Volume 20 ul Pump Flow Rate 0 8 ml Channel A Water 10 Channel B Acetonitrile 90 Stop Time 4 min Column Thermostat Column Temperature 40 C Detector Wavelength VWD 254 nm Fraction Collector Report Wavelength DAD
79. d to either of the two modules This will damage the electronics of the modules 32 12 Connect the power cables to the power connectors 13 Connect the CAN cable to other Agilent 1200 Series modules Agilent 1200 Series Purification System User Manual Installing Your Agilent 1200 Series Purification System 1 14 Turn ON power by pushing the button at the lower left hand side of the purification system 15 The module sampler or fraction collector is turned ON when the line power switch is pressed and the green indicator lamp is illuminated The module sampler or fraction collector is turned OFF when the line power switch is protruding and the green light is OFF To disconnect the module sampler or fraction collector from the line unplug the power cord The power supply still uses some power even if the power switch at the front panel is turned OFF To avoid personal injury keep fingers away from the needle area during purification system operation Do not attempt to insert or remove a vial tray or a plate during the operation of the sampling device and fraction collector Sampling device or Fraction Collector to Thermostat cable G1330 81600 r A EMNI N AU CAN bus AC Power Figure 9 Connections at the Rear of the Thermostatted Module Sampler or Fraction Collector Agilent 1200 Series Purification System User Manual 33 1 Installing Your Agilent 1200 Series Purification System Flow Conne
80. dard autosampler optional thermostatted version e Agilent 1200 Series Quaternary or Binary Pump with Agilent 1200 Series Vacuum Degasser e Agilent 1200 Series Isocratic Pump with solvent bottle to be used as the make up pump for the LC MSD e Agilent 1200 Series Fraction Collector Analytical Scale optional thermostatted version e Agilent 1200 Series LC MSD G1946B C or G1956A B e G1968C Mass Based Fraction Collection Kit Software e Agilent LC MSD ChemStation Software G2710AA rev B 01 01 or higher Column e Zorbax SB C18 9 4 mm x 5 0 cm 5um part number 846975 202 included in G1968C mass based fraction collection kit 248 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 Solvents e HPLC grade Bi distilled water e HPLC grade Acetonitrile e Acetic or Formic acid Sample e Delay calibrant part number G1946 85020 included in G1968C mass based fraction collection kit e Electrospray LC Demo Sample part number 59987 20033 included in G1968C mass based fraction collection kit Step 1 Calibrating the Delay Time Before running the fraction collection the delay time between the MSD detector and the fraction collector must be determined See Delay Volume Calibration on page 93 for the procedure to determine the delay time This value is entered into the MSD Fraction Collection Parameters panel For the delay calibration method specify an LC flow of 3 0 ml min and a make up
81. de Run Whole Study Run Partial Study Cancel C Reprocess Whole Study mrde methods Reor Figure 69 Start Study dialog box In on line mode the upper part of the Select Start Mode group is active This enables you to run a complete study or if you have identified samples for a partial study to run the partial study In off line mode the lower part of the Select Start Mode group is active This enables you to reprocess a complete study or if you have identified samples for a partial study to reprocess the partial study Agilent 1200 Series Purification System User Manual Running Studies 6 Stopping a Study You can stop a study that is running either by choosing the a tool from the toolbar or by using the RunControl menu RunControl gt Stop Study The study stops at the end of the current run any remaining injections are not completed Pausing a Study You can pause a study that is running either by choosing the n tool from the toolbar or by using the RunControl menu RunControl gt Pause The study is paused and you can add samples or modify the current study to resume the study press the start study button Aborting a Study You can abort a study that is running either by choosing the 1 tool from the toolbar or by using the RunControl menu RunControl gt Abort The study stops immediately any remaining injections are not completed Study Stop Abort
82. delay volume reduction automatic 64 deleting injections 163 samples 163 study 164 delivery checklist 19 description study 142 detection parameters 72 detector peakwidth 93 98 112 114 detectors 86 peak 72 dimensions 17 well plate 54 directory structure 212 discrete fraction collection 69 downslope 71 73 drainage waste 28 draw position 63 64 66 speed 63 64 66 Dual loop autosampler 65 E edit menu 126 eject speed 63 64 66 electrical connections thermostat 33 environment 14 18 equilibration time 64 66 Existing Study 140 Exit 126 explorer 121 Export 125 export to CSV 205 Exporting 160 276 F file menu 125 filling information 177 flow connections 34 analytical scale system 36 preparative scale system 38 flow rate maximum 114 flow splitter 29 flush out factor 64 66 sample 64 flushport 64 66 format sample name 142 fraction annotation 202 list 209 start position 153 table 203 trigger mode 71 fraction collection 143 enabled 77 mass based 77 mode 81 parameters 88 peak based 71 time 72 time based 72 fraction collector configuration 67 order 75 panel 122 153 187 setup 70 fraction collectors multiple 35 75 Fraction Controller Panel 128 fraction delay sensor 93 Fraction Preview 73 frequency 18 fuses 14 G G1160A 28 35 G1310A 29 G1390A 28 G1968C 28 graphical reporting 204 graphics multiple
83. e you can specify the trays that are installed 2 In the Well plates group specify the type of well plates that are installed 3 In the Volumes group specify the volume of the upper and the lower loop The maximum injection volume is calculated automatically from these entries 4 Inthe Volumes group specify the seat capillary volume for the upper and the lower loop of your dual loop autosampler configuration The maximum injection volume is calculated automatically from these entries Setup You set up the injector using the Setup Injector dialog box 1 Display the Injector context menu right click on the Injector icon of the system diagram 2 Select Set up Injector to open the Setup Injector dialog box Alternatively you can use the Agilent ChemStation menus 1 Switch to Method and Run Control View if necessary View gt Method and Run Control 2 Open the Set up Injector dialog box from the Instrument menu Instrument gt Set up Injector Agilent 1200 Series Purification System User Manual 61 3 Instrument Parameters Setting Up the Standard Autosampler Set up Injector Instrument 1 Ea Figure 18 Standard autosampler setup dialog box To set up the standard autosampler refer to Figure 18 1 In the Injection group specify the injection type and injection volume You can choose to make a standard injection an injection with a needle wash or to use an injector program If you choose an inje
84. e 0 5 ml min Agilent 1200 Series Purification System User Manual Checkout Procedures 11 Table 21 LC Method Parameters for MSD triggered AS Fraction Collection checkout Device Parameter Value Column Thermostat Column Temperature 40 C Detector Wavelength VWD 254 nm Wavelength DAD MWD Fraction Collector see Setup Fraction Collector Figure 117 on page 251 Report Destination Report Style 254 16 nm 360 100 nm Peak based Use MSD for Mass Based Fraction Collection Printer Short Set up Fraction Collector Instrument 1 x Fraction Trigger Mode Off C Use Timetable Peak based Max Peak Duration 0 5 min Peak Detectors Detector DADI Working Mode Fraction is collected when a peak is detected by xk i C all selected peak detectors at least one selected peak detector Up Slope Unit s Down Slope Unit s Threshold Unit Upper Threshold Unit Unit 1 ow 50 so 5o 2 up of s 5 s 3000 000 3000 000 rTimetable Time Trigger Mode Max Peak Duration min of Fractions Timeslices min Insert Append Copy oO Paste Figure 117 Fraction Collector parameters Agilent 1200 Series Purification System User Manual 251 11 252 Checkout Procedures Table 22 MSD Signal Parameters see also Figure 118 Parameter Value Use MSD Enabled Tune file Atunes tun Stop Time AsPump Time Filt
85. e different expected masses for each sample will be used as the base masses for MSD Fraction Collection The license number for the G2263AA Mass Based Fraction Collection add on software must be installed in order to be able to enter expected masses for samples in the study Agilent 1200 Series Purification System User Manual Instrument Parameters 3 When preparing to start a run the Agilent ChemStation adds an entry to the logbook if this option has been selected and the CSV file is missing or corrupted Fraction Collection s Exi r FC Mode MS Signals r Detectors C None Monitor Polarity Mode MS only Bye Sigal JV Positive Scan C Other only 2 Use method target masses Sigal2 JV Negative Scan EME aAa Use sample target masses aada ta EMS orohar Import mass info from i Signal4 F Time based C Chem32 CORE basemass _ r Positive Adducts r Negative Adducts Method Target Masses FM B Selected V M H 1 M M H 1 Edit Mass M M NH4 18 J M Cl 35 37 M M Na 23 M formate 45 M M K 39 JV M TFA 113 I Me r rie Jnsert M M Remove M M D M Charge State Charge State Parameters m r3 m iS OK Cancel Help Figure 31 MS Signals The Fraction Collection dialog box with Import mass info selected Select 1 4 MSD signals to be monitored If a signal has not been activated in the Set Up MSD Signals dialog box its check box is disabl
86. e fraction collection it is possible to specify individual start locations for each sample for more details please refer to Assigning the Fraction Start Position on page 153 Number of Pools You can specify the number of injections that are to be pooled see Chapter 7 Pooling You can pool up to 99 injections Recovery Start You can choose the recovery start location see Chapter 5 Recovery Location Locations This option is only available if you have a valid configuration for Recovery Agilent 1200 Series Purification System User Manual 143 5 144 Study Setup Specifying the Report The final stage of the creation of a new study is the specification of the reporting options The parameters available in the Specify Report dialog box see Figure 56 depend on the configuration of your system For example the Extended Sampler Report and Sample Information groups are NOT available because this revision of the SW does not support the Purity Calculations license Specify Report m ChemStation Reporting V ChemStation Data Analysis report Include JV Fraction annotation Label each well IV Fraction table Fraction labels IV Plot multiple graphics on a single page Graphics Chromatogram Plot 2 Horizontal I Mass spectrum 2 Vertical m m Automated Purify Reporting Report Printer PDF file Landscape Studyrun summary Vv E E Sample list Vv f E Fraction list Vv E E Purity calculati
87. e group select how fraction collection is to be triggered If you select OFF no fractions will be collected with this method If you select Use Timetable the Timetable group is activated If you select Peak based fraction collection fractions will be collected based on the peak detection parameters Working mode Upslope downslope lower and upper threshold Specify a maximum peak duration in minutes to prevent continuous collection if the baseline drifts and the signal does not drop below the specified threshold value Agilent 1200 Series Purification System User Manual 71 3 Instrument Parameters Peak Detector Parameters With Working Mode you determine whether a selected peak detector is not used for fraction triggering OFF or whether fractions are collected based on the Slope parameters only or Threshold parameters only It is also possible to collect on Threshold and Slope at the same time A peak start is triggered then if both the Upslope value AND the Threshold are exceeded The peak end will be triggered when the signal falls below the Threshold OR the Downslope value is exceeded OR if you exceed the max peak Duration as entered in the Fraction Trigger Mode Above the Upper Threshold a peak will always be collected independent of the current slope for use with saturated peaks 4 In the Peak Detectors group specify which of the configured detectors will be used for peak detection and specify the peak detection pa
88. eadsheet with column headings specify the number of header lines to skip You can either edit the field directly or use the up and down arrows to increase or decrease the number of rows Agilent 1200 Series Purification System User Manual 159 5 160 Study Setup 5 Click OK to close the dialog box and import the CSV file If there is no mismatch between the fields in the CSV file and the study list the CSV file is imported and the Sampler panel and Study List are updated to show the imported samples A log file is generated and saved in the same directory as the CSV file If there is a mismatch between the fields in the CSV file and the study list the import is aborted and an error log is generated and displayed on the screen z Study List Samples J Sample riection eo a i as at ea a Checkout injection 1 time m 10 Checkout injection 1 time m 20 x P1A01 Sample blank time m 10 lde m L P1401 Sampe blank timem 10 ide m Figure 64 Study List after importing from a CSV file Exporting to a CSV file To export a Sample or Fraction List to a CSV file 1 Open the Export dialog box using the File menu File gt Export Study The Export CSV files dialog box is shown in Figure 65 You can also specify a CSV file with the help of this dialog as report destination as part of your report setup Agilent 1200 Series Purification System User Manual Study Setup 5 Exp
89. ecovery The recovery location for this injection Recovery Vol The volume of sample recovered Status Sample status Partial Selected if this sample is part of a partial study Fraction Start Fraction Start position for this sample The selected injection is denoted by an arrow in the left column of the table 188 Agilent 1200 Series Purification System User Manual Data Review 8 Study List Injections The Study List Injections which is available only in View Mode after the study has been run contains two tables The upper table lists all the samples that have been run in the sequence that they were run the lower table lists all fractions that were collected for a selected injection Table of Injections The table of samples contains two groups of columns The Sample group columns describe the sample parameters Location Name Description The location of the sample in the sampler The sample name according to the specified naming convention The sample description entered in the Description field of the Add Samples to Study dialog box The Injection group columns describe the parameters for each injection Injection No Pool Method Data File Volume Recovery Loc Recovery Vol The injection number for this sample The pool number for this injection The method used for this injection The name of the data file that will be produced only the first injection if pooling is specified The inje
90. ed Agilent 1200 Series Purification System User Manual 85 3 86 Instrument Parameters MS only Detectors Specifies use of the MSD as the only detector for fraction collection If you have selected Use Method Target Masses you need to specify base masses in the Selected Base Masses group see Method Target Masses on page 87 if you have selected Import Mass Info the base masses are imported from the specified CSV file Note that when the optional Purify system is running the entire Detectors section is disabled and MS only is automatically selected You configure time based fraction collection through the Setup Fraction Collection dialog box for the Fraction Collector module if a fraction collector is present in the system see Figure 26 on page 78 Adducts The Adducts group has separate sections for Positive and Negative ions Adducts in the positive ion section will be monitored in all positive ion signals and adducts in the negative ion section will be monitored in all negative ion signals Predefined and user defined adducts can be added or deleted by marking the corresponding check boxes The predefined adducts for positive ion include Base mass M Hydrogen H M 1 Ammonium NH4 M 18 Sodium Na M 23 Potassium K M 39 For negative ion a hydrogen abstraction M 1 is also predefined You can define other adducts by activating a positive or negative ion check box and entering the nu
91. emo OK EM ak data Cancel C 20010830 003 C 20010831 003 C 20010914 000 C 20010925 000 20010926 000 Help Drives SJ f ChemStation m Network Figure 51 The Set Study Base Path dialog box 136 Agilent 1200 Series Purification System User Manual Study Setup 5 You must enter a complete path name including sub folders and the folder name must conform to MS DOS file and folder naming conventions that is maximum 11 alphanumeric characters for folders and 8 3 alphanumeric characters for files If the location you specify does not exist you can create the path and folder directly at this time You display the Set Study Base Path dialog box by using the Options menu Options gt Set Study Base Path Setting the Method Source Path By default the source of the methods that are available for your study is the Agilent ChemStation methods folder for the configured instrument You can however specify a different folder as the source of the available methods using the Choose Folder dialog box You display the Choose Folder dialog box see Figure 52 from which you select the methods folder by using the Options menu Options gt Set Method Source Path w Choose Folder x Select Method Source Path Path F HPCHEM 1 METHODS Folders an Cancel EJHPCHEM F 9 56SLSCCI M C SeSLSCES M C 56SLSCPI M C 56SLSMCI M C 56SLSMES M zi Help Drives 3 f ChemStation
92. ension to the file name with the injection information in the study database Setting Up a Study for Pooling You set up the number of repetitions of each sample in the Fraction Collection group of the Setup Study Parameters dialog box see Setting Up the Study Parameters on page 141 You can pool between 2 and 99 injections Fraction Collection and Recovery Fraction Start Loc NextLocation v Number of pools 3 times Recovery Stat boe yone Figure 75 Pooling set up in the Study Parameters dialog box 182 Agilent 1200 Series Purification System User Manual Agilent 1200 Series Purification System User Manual 8 Data Review Overview 184 Data View Panels 185 This chapter describes the options available in the software for reviewing the study data ee Agilent Technologies 183 184 8 Data Review Overview You can review any open study see Opening a Study on page 165 either as a stand alone task or as a background task while another study is running You can select the results to review in several ways e selecting a location from the Sampler panel e selecting a fraction from the Fraction Collector panel e selecting a sample from one of the Study Lists No matter which way you select the sample all panels are updated to mark the selected sample and its fractions Agilent 1200 Series Purification System User Manual Data View Panels Data Review The data view panels that
93. ent 1200 Series Purification System User Manual 36 38 Contents 3 Instrument Parameters 51 Prerequisites Pump s Detector s UV and Column Compartment if present are Setup 52 Well Plate Types 53 Preconfigured Well Plate Types 53 Configuration of Well Plate Types 54 Autosamplers 57 Configuration 57 Setup 61 Fraction Collector 67 Configuration 67 Setup 70 Multiple Fraction Collectors 75 Configuring the Multiple Purpose Switching Valve 75 Configuring Multiple Fraction Collectors 76 Setting Up Multiple Fraction Collectors 76 Setting Up for Mass based Fraction Collection 77 MSD Fraction Collection Dialog Box 80 Fraction Collection Parameters Dialog Box 88 Delay Volume Calibration 93 Procedure for a Fraction Collector AS G1364A 050 or G1364C with an UV detector 94 Procedure for a Fraction Collector PS G1364A or G1364B with an UV detector 98 Procedure for a Fraction Collector AS with an MSD 102 Procedure for a Fraction Collector PS with an MSD 107 Making Your Own Calibration Method 112 Delay Volume Calculations UV Detector 112 Maximum flow rates to avoid missing peaks 114 Agilent 1200 Series Purification System User Manual 4 Menus and Views 119 Mode 120 Main Panel 121 Configuring the Tables 123 Menu Bar 125 File 125 Run Control 126 Edit 126 View 128 Options 129 Window 129 Help 130 ToolBar 131 View Mode 133 Select or Edit Mode 134 5 Study Setup 135 What is a Study 136 Setting t
94. ents The Checkout Procedure consists of the following activities e Preparing the Agilent 1200 Series LC e Setting the study parameters e Starting the analysis and collecting fractions e Reanalyzing the fractions UV triggered PS fraction collection checkout procedure see page 235 This checkout procedure verifies the operation of the Agilent Fraction Collector PS G1364B used with the Agilent Purification Hi Throughput software and in combination with the associated hardware components The Checkout Procedure consists of the following activities e Preparing the Agilent 1200 Series LC e Setting the study parameters e Starting the analysis and collecting fractions e Reanalyzing the fractions MSD triggered AS fraction collection checkout procedure see page 248 This checkout procedure verifies the operation of the Agilent Fraction Collector AS G1364C and the Agilent Mass Based Fraction Collection Kit G1968C used with the Agilent Purification Hi Throughput software and in combination with the associated hardware components Agilent 1200 Series Purification System User Manual Checkout Procedures 11 The checkout procedure consists of the following activities e Preparing the Agilent 1200 Series LC and LC MSD e Setting the checkout method parameters e Running the checkout and collecting fractions e Reviewing the results and reanalyzing a fraction MSD triggered PS fraction collection checkout procedure see page 261 T
95. equently there are no voltage selectors in the rear of the purification system modules There are also no externally accessible fuses because automatic electronic fuses are implemented in the power supplies WARNING To disconnect the purification system modules from line power unplug the power cord of each module The power supplies still use some power even if the power switches ON the front panel of the modules are turned OFF WARNING Shock hazard or damage of your instrumentation can result if the devices are connected to a line voltage higher than specified 14 Agilent 1200 Series Purification System User Manual Installing Your Agilent 1200 Series Purification System 1 Power Cords Your purification system modules are delivered with power cords which match the wall sockets of your particular country or region The plug on the power cord which connects to the rear of the instrument is identical for all types of power cords Never operate your instrumentation from a power outlet that has no ground connection Never use power cords other than the power cords designed for your region Never use cables other than the ones supplied by Agilent Technologies to ensure proper functionality and compliance with safety or EMC regulations Bench Space The purification system dimensions and weight see Table 1 on page 16 and Table 2 on page 17 allow the instruments to be placed on almost any laboratory bench The instru
96. equiring a minimum slope protects against a sloping baseline that might go above threshold If the slope is set to zero then only the threshold is used Signal must exceed threshold abundance and slope to start Fraction collection starts when the signal level at a given point exceeds the threshold AND the slope is greater than the specified peak slope in counts second Signal must fall below threshold abundance AND have negative slope less than threshold to stop Agilent 1200 Series Purification System User Manual Instrument Parameters 3 The signal slope decreases to zero and becomes negative on the down side of the peak After the negative slope is over the specified peak slope again then the peak ends when a given point is below the threshold AND the negative slope is lower than the specified peak slope Slope calculation For a given point P3 the slope is calculated using two points midway between P1 and P2 Peak detection with unresolved peaks A peak is considered terminated when a different ion becomes the largest even if the peak stop criteria have not been met This feature allows peaks that are not well resolved to be separated This allows you to have lower thresholds than would otherwise be practical catching more of the peak If two ions are being monitored that are very close to each other in abundance there is a danger that a peak could be split into two or more vials While this is not desirable it is better t
97. er CAN Bus cable a i power Figure 3 Recommended Stack Configuration Analytical Scale Purification System UV Vis Detector Based Rear View Agilent 1200 Series Purification System User Manual 23 1 Installing Your Agilent 1200 Series Purification System Preparative Scale System Configurations Solvent bottles Prep Pump A Column organizer Prep Pump B Fraction collector Injection device UV Vis Detector Figure 4 Recommended Stack Configuration Preparative Scale Purification System UV Vis Detector Based Front View To identify the inter module capillaries and tubing as labelled in Figure 4 please refer to NOTE Table 5 on page 38 24 Agilent 1200 Series Purification System User Manual Installing Your Agilent 1200 Series Purification System 1 IX Flow Splitter X LC MSD wast waste waste Figure 5 Recommended Stack Configuration Preparative Scale Purification System Including LC MSD Front View To identify the inter module capillaries and tubing as labelled in Figure 5 please refer to Table 5 on page 38 The fraction collector should never be positioned on top of a module that generates heat This could lead to an unwanted evaporation of fractions in the fraction collector e g Agilent 1200 Series thermostatted column compartment G1316A B or Agilent 1200 Series diode array detectors G1315B C For more detail
98. er Enabled Data Storage Condensed Peak Width 0 08 min Scan Speed Override Disabled Active Signals Signal 1 Polarity Positive Scan SIM Scan Scan Parameters Start Time 0 00 min Mass Range 100 350 Fragmentor 100 Gain 1 0 Threshold 100 Step Size 0 10 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 Set Up MSD Signals x MSD Control MSD X Use MSD FIA Disabled General Tune File atunes tun M lon Mode API ES Peakwidth 0 08 min Cycle Time 0 78 sec cycle Scan Speed Override IX Time Filter Scan Data Storage Condensed x Active Signals x 1 Scan r2 B O 4 Acquisition Parameters C Display EIC Parameters Signal Settings Sioat 1 Mode Scan x Polarity Positive x cycle time 00 0 On Mass Range Frag Gain Thres Step Off Low High mentor hold size _ 0 00 M 100 00 350 00 100 1 0 100 0 10 Time min Signal 2 z Mode Scan x Polarity Positive 7 z On Mass Range Frag z Thres Step Uira Off Low High mentor hold size 1 __ 0 00 M 100 00 1000 00 70 1 0 150 0 10 Figure 118 MSD Signal Parameters Table 23 MSD Spray Chamber parameters see also Figure 119 and Figure 120 on page 255 Parameter Value Drying Gas Temp 350 Drying Gas Flow 10 L min for Electrospray source 5 L min for API source Neb Pressure 35 psig Agilent 1200 Series Purification System User Manual 253 11
99. ered AS Fraction Collection Checkout 248 10 Agilent 1200 Series Purification System User Manual What you willneed 248 Step 1 Calibrating the Delay Time Step 2 Preparing the LC system Step 3 Running the Checkout Method Step 4 Review Results Step 5 Reanalyze Fraction 2 MSD triggered PS Fraction Collection Checkout What you willneed 261 Step 1 Calibrating the Delay Time Step 2 Preparing the LC system Step 3 Running the Checkout Method Step 4 Review Results Step 5 Reanalyze Fraction 2 Index 275 Agilent 1200 Series Purification System User Manual Contents 261 11 Contents 12 Agilent 1200 Series Purification System User Manual Agilent 1200 Series Purification System User Manual 1 Installing Your Agilent 1200 Series Purification System Site Requirements 14 Optimizing the Stack Configuration and Plumbing of Your Purification System 20 Installing the Purification System 27 Installing a Thermostatted Module in Your Purification System 31 Flow Connections to the Purification System 34 This chapter contains site requirements and installation of the purification system ee Agilent Technologies 1 Installing Your Agilent 1200 Series Purification System Site Requirements A suitable site environment is important to ensure optimum performance of your purification system Power Considerations The purification system power supplies have wide ranging capabilities see Table 3 on page 18 Cons
100. eries Purification System User Manual Instrument Parameters 3 Ei Fraction Collector 1 Calibration Results Print Close Calibrate Curr Delay Vol ul Calc Delay Vol ul DAD G13154 US64400562 eee Detector Curr Delay Time sec Calc Delay Time sec MSD 0 294 Figure 43 Delay Volume Calibration results 21 If the measured delay time is greater than 5 seconds you can skip to step 27 otherwise continue with step 22 CAUTION If the measured delay time is significantly greater than 5 seconds you should continue with the calibration at step 22 to avoid losing fractions at the end of the run 22 Determine how much additional time is needed to create a 5 second delay For example if the time displayed is 1 sec a six second delay is needed 23 Using the main pump flow rate determine the desired additional delay volume For example at 20 ml min and a six second delay an additional 20 0 x 6 60 or 2 0 ml is required 24 Cut off a sufficient piece of the supplied ETFE tubing Part No G1968 60500 to provide the needed volume The tubing s volume is 8 1 ul cm In this case 247 cm would be required Agilent 1200 Series Purification System User Manual 109 3 Instrument Parameters 25 Add the tubing between the splitter and the fraction collector or if multiple fraction collectors are installed between the splitter and the G1160A valve 26 Perform the calibration again Verify the time is sufficient
101. erlap the injection either after the sample is flushed out or after a specified time In the Rinse group you can specify how much solvent is used to rinse the syringe the buffer loop and the seat capillary In the Auxiliary group specify the Draw and Eject speeds and the Draw Position offset to default position is higher is deeper of the dual loop autosampler Specify an equilibration time which is the time that the needle stays in the vial after drawing the sample This allows the vacuum caused by drawing the sample to equilibrate Specify a Sample Flush Out factor to ensure that the sample is thoroughly flushed out of the syringe and past the capillary seat and valve to prevent sample carry over when using the overlapped injection mode For thermostatically controlled dual loop autosamplers you can also select to store the temperature Select Vial Well bottom sensing to ensure that the needle detects non uniform well bottoms and adjusts the depth of the needle to 1 mm above the detected bottom of the well Agilent 1200 Series Purification System User Manual Instrument Parameters 3 For more details see the User Manual of the Agilent 1200 Series dual loop autosampler Fraction Collector There are two aspects to the fraction collector parameters Configuration is the process of characterizing the physical configuration of the fraction collector for example by specifying the trays that are installed
102. f it has a flow delay sensor installed part of the internal tray All modules of the purification system must be installed in a horizontal position Ensure that the power switch at the front of each module in the purification system is OFF Connect the power cable to the power connector at the rear of each of the purification system modules Connect the CAN cables between each of the Agilent 1200 Series modules in your purification system CAN cables long PN 5181 1519 short PN 5181 1516 10 An optional universal interface box UIB G1390A for communication to the G1946C D or G1956A B LC MSD or a non 1200 with 35900E A D Converter or Agilent 1200 Series non UV Vis detector must be connected via CAN If the UIB is used to process an analog signal from an LC detector then connect the analog signal cable from the LC detector to the Analog In BNC connection on the front of the UIB 11 If one of the Agilent 1200 Series switch valves e g the G1160A Agilent 1200 Series 12 position 13 port valve for the operation of multiple fraction collectors is part of the system it must be connected to the system via CAN cable and powered via CAN DC IN cable connected to the CAN DC IN port at the valve and CAN DC OUT port at a G1361A preparative pump or a G1367B high performance autosampler or G1364B C fraction collector 12 The optional G1946C D or G1956A B LC MSD needs a remote start cable connection 05989 60080 to one of the A
103. f you use well plates with inlets caps and septa you have to specify the distance from the top of the septa to the bottom of the inlets Well X size Size of the well in x direction Plate length If min 3 7 mm you use well plates with inlets caps and min 3 0 mm with septa you have to specify the x size of the position accuracy septa alignment micro scale Well Y size Size of the well in y direction Plate width If down to 3 7 mm you use well plates with inlets caps and septa you have to specify the y size of the septa min 3 0 mm with position accuracy alignment micro scale Agilent 1200 Series Purification System User Manual 55 3 Instrument Parameters Table 7 Well plate dimensions continued Label Description Definition Limits Bottom size For round wells the relative of the top and 1 0 cylindrical well bottom of the well 0 0 conical well Square Click in the field to specify whether the wellis Yes rectangular rectangular or round No round oval Is well plate Click in this field to specify if this is a well Yes well plate plate or not Relevant for continuous flow No Vial Tray or operation Eppendorf tray 6 Export the new configuration You can export the new configuration either to the preconfigured well plate file config wpt or to a separate configuration file We recommend that you set up a separate configuration file for your non standard well plates this file can then also be used
104. fad Network Figure 52 The Method Source Path dialog box Agilent 1200 Series Purification System User Manual 137 5 Study Setup Setting Up a New Study Studies are set up in the study editor the study setup consists of three steps 1 Creating the study 2 Setting up the study parameters 3 Specifying the report 4 Completing the Setup You are guided through this process by the Study Wizard CAUTION Before you create a study ensure that the sampler and fraction collector configurations in the Agilent ChemStation match the Agilent 1200 Series configurations see Instrument Parameters on page 51 otherwise you cannot use the study to acquire data 138 Agilent 1200 Series Purification System User Manual Study Setup 5 Creating a New Study You start the study wizard in Select Mode by choosing the ol tool from the toolbar or by using the File menu File gt New Study The new study is created by giving it a name and entering the sample locations The new study name can be a maximum of 30 characters long but must be restricted to alphanumeric characters with no special characters The name you choose does not have to be unique because the creation date of the study confers uniqueness to the name You can choose to enter sample locations manually or base the new study on an existing study see Figure 53 Create New Study Enter a name for the study ee r How to create the study Ma
105. files are organized and how data files are named ot Agilent Technologies 211 10 Directory Structure and File Naming Directory Structure The Purification Hi Throughput directory structure is very simple as shown in Figure 88 e All files associated with the Purification Hi Throughput software are in a Purify folder directly under the root e The Purify folder contains two sub folders the Bin folder contains all the files for the operation of the Purification Hi Throughput software the Data folder see The Data Folder on page 213 contains the data for all study runs acquired by the Purification Hi Throughput software All folders in the Purify subdirectory should be accessed for viewing only Any manual modifications in these folders might result in corruptive data or an unusable Software 2 Desktop My Computer 9 3 Floppy 4 epee System C Program Files 3 Purify E Bin H data ey Recycled Temp g Winnt Figure 88 Directory tree for a ChemStation installation including Purification Hi Throughput 212 Agilent 1200 Series Purification System User Manual Directory Structure and File Naming 10 The Data Folder The data folder see Figure 89 contains sub folders for each of the study runs acquired by the Purification Hi Throughput software Study run folders are named by date in the format YYYYMMDD run where YYYY is the year MM is the month DD is the day and run is the stud
106. fraction that was collected in the second vial well or tube using the same conditions used for the checkout method msdpsfr m but with the Use MSD for Mass Based Fraction Collection deselected in the Setup Fraction Collector dialog box Save the method as msdrean2 m 2 You should see only one peak in the chromatogram of the reanalyzed fraction If you do not see the peak in the TIC generate EICs for masses 279 285 and 311 and verify that there is a single peak in one of the EICs see Figure 132 MSO1 TIC MS File REANO001 D APLES Pos Scan Frag 100 M501 285 EIC 234 7 236 7 REAND001 0 APLES Pos Scan Frag 100 0000 40000 20000 o 1 2 3 4 min M301 31t EIC 960 7 911 7 REANDOO1 D AP ES Pes Scan Frag 100 aa 3000 ae M 1000 v o x e 1 2 3 a min s Figure 132 ElCs after reanalysis of fraction 2 274 Agilent 1200 Series Purification System User Manual Index Numerics 35900E 28 A A D Converter 28 abort 126 aborting a study 173 active flow splitter 29 actual fill volume 177 adapter air channel 32 adding samples 149 adducts 86 air channel adapter 32 air circulation 15 altitude non operating 18 operating 18 ambient non operating temperature 18 ambient operating temperature 18 analog output range 92 analog signal delay 115 analytical scale system configuration 21 annotation fractions 202 assigning methods 150 automatic delay volu
107. g box together with some additional header information about the study The report is produced on a single page StudyNeme Combinason Defeuh Semple Neme Combi Study Pat F puritycintaiem 01200109725 0008 Injecten orderby Selection StudyRunw 1 Shutdown STANDBY Deser iptum Run Method Nona a her tore Dissblod gt FrectionStertLoe 1P1 A01 Operate Aglert Number ofPasts None E StartOate 26 Sep 01 9 01 25 AM Recovery Stet Loc None z StopMode Completed Agilert 1100 Well Piate Sampler z Yay Vray dorip on Visiedexcription 0 Wella 8 Sanpla Vy Iwana act amp oc s Worgeent o our 3 s w y Agilent 1100 Afraco AFC EPRO0000071 y Yraydemeripfon r deserigtion Wals FF ilied 4 Raya AW ep lite Tray Naty tent 3A R E Vong ten o Woeoeer QO Acme a Figure 85 Study Run Summary Report Agilent 1200 Series Purification System User Manual 207 9 Reports Sample List The Sample List report see Figure 86 contains the header information about the study together with a table containing information about the samples in the study The table contains six columns Sample Location the location of the sample in the autosampler Sample Name the name you gave to the sample when you set up the study Sample the description of the sample that you entered when you set up the study Description Inj the number of the injection in the study run Method the name of the method used for this injecti
108. g box which allows you to modify the run parameters of the selected samples 134 Agilent 1200 Series Purification System User Manual Agilent 1200 Series Purification System User Manual Pa 6 ee 5 e Study Setup J What is a Study 136 Setting Up a New Study 138 Adding Samples to the Study List 147 Using CSV Files 158 Deleting Samples Injections from the Study List 163 Saving a Study 164 Deleting a Study 164 Opening a Study 165 Closing a Study 167 Modifying a Study 167 Setting Up a New Study Run 168 Setting Up a Partial Study 168 This chapter explains what a study is and describes the process of setting up a study including an explanation of all the necessary parameters gg Agilent Technologies 135 5 Study Setup What is a Study A study is a collection of samples from each of which one or more injections is made For each injection any valid Agilent ChemStation method can be used A study consists of the list of injections the study list together with parameters that apply globally to all injections in the study list Studies are configuration specific Once a study has been created it can be run only ona Agilent ChemStation with the identical configuration Setting the Study Base Path By default a study is created in the default Purify data folder but you can specify a different folder in the Set Study Base Path dialog box see Figure 51 Select Study Base Path Path IF purify data d
109. gilent 1200 Series system modules and the additional fraction collector control cable G1968 60002 delivered with the mass based fraction collection kit G1968C to the Universal Interface Box UIB G1390A to be operated and controlled with the Agilent Purification Software 13 The optional G1946C D or G1956A B LC MSD needs LAN connection to the Agilent ChemStation PC for control and operation to the detector Agilent 1200 Series Purification System User Manual Installing Your Agilent 1200 Series Purification System 1 14 If both the optional G1946C D or G1956A B LC MSD and the rest of Agilent 1200 Series purification system are operated via LAN communication an additional HUB has to be installed between the system LC MS detector and PC 15 An optional Active Flow Splitter MRA PN G1968 64102 to use the LC MSD in combination with a make up pump typically G1310A must be powered via 24V DC universal power supply shipped with the MRA The power output of the universal power supply must be connected to the MRA The power cord for the operation of the universal power supply must be connected to the socket in the wall The active splitter can be turned OFF and ON through either software control manually or via external contacts meeds the G1351A External contacts board installed in the BCD board slot in one of the Agilent 1200 Series modules and the G1103 61611 general purpose cable For more details about the installation of a
110. gure 30 The Fraction Collection dialog box with Use sample target masses selected Agilent 1200 Series Purification System User Manual 83 3 Instrument Parameters Import mass info 84 When you select this option the ability to enter base masses manually is disabled Figure 19 The Browse button is enabled so that you can use a CSV comma separated values file to import base masses You need to select the CSV file using Browse you cannot type a CSV file path and name directly into the field The CSV file is a one line file with the following format BaseMass1 BaseMass2 BaseMass3 A special CSV file C HPCHEM CORE BASEMASS CSV where C is the disk volume which contains the Agilent ChemStation software exists when the LC MSD Agilent ChemStation is used with the Purification Hi Throughput software At the beginning of each run the Purification software will update this BASEMASS CSV file with base masses corresponding to the expected masses specified in the study list for the sample that is to be run Since each sample will likely contain different compounds with different molecular ions different base masses would need to be specified for each sample Instead of selecting Use Method Info and entering just one set of base masses in the MSD Fraction Collection dialog box select Import Mass Info and specify the CSV file C HPCHEM CORE BASEMASS CSV Then in the study enter the expected masses for each sample In this way th
111. hat you can make changes without modifying the original study parameters You save a study by choosing the g tool from the toolbar or by using the File menu File gt Save Study As You type the name and if necessary a new path for the copy of the study in the Save Study As dialog box Deleting a Study Once a study has been created it cannot be deleted from within the Purification Hi Throughput software 164 Agilent 1200 Series Purification System User Manual Study Setup 5 Opening a Study Ca You open a study by choosing the tool from the toolbar or by using the File menu File gt Open Study The Select Study to Open dialog box that is displayed see Figure 54 on page 140 contains a tabular list of all available studies in the current study base path The list contains all studies in the base path irrespective of their compatibility with the current Agilent ChemStation configuration The table contains four columns Study The name of the study Created The date and time of creation of the study Modified The date and time of the last modification to the study Study directory The folder in which the study data and methods are stored You can select one study to open from the list any study that is currently open is closed before the selected study is opened Previous studies that have been opened in the current session since the Purification Hi Throughput software was started are listed in the explorer
112. he Study Base Path 136 Setting the Method Source Path 137 Setting Up a New Study 138 Creating a New Study 139 Setting Up the Study Parameters 141 Specifying the Report 144 Completing the Study Setup 146 Adding Samples to the Study List 147 Sampler Panel 148 Adding Samples 149 Assigning Methods 150 Modifying Samples 152 Agilent 1200 Series Purification System User Manual Contents Contents Fraction Collector Panel 153 The Study List 155 Recovery Locations 156 Assigning Recovery Locations 15 7 Using CSV Files 158 Setting up the CSV file 158 Importing the CSV file 159 Exporting toa CSV file 160 Importing Expected Masses from a CSV File Deleting Samples Injections from the Study List Saving a Study 164 Deleting a Study 164 Opening a Study 165 Closing a Study 167 Modifying a Study 167 Setting Up a New Study Run 168 Setting Up a Partial Study 168 Adding Samples to a Partial Study 169 Removing Samples from a Partial Study 169 Running Studies 171 Run Control 172 Starting a Study 172 Stopping a Study 173 Pausing a Study 173 Aborting a Study 173 Study Stop Abort Criteria 173 Study Status 174 Online Tickmarks 175 161 163 Agilent 1200 Series Purification System User Manual Contents Reprocessing Studies 176 Resetting the Vessel Fill Volumes 177 7 Pooling 179 What is Pooling 180 Pooling Properties 182 Setting Up a Study for Pooling 182 8 DataReview 183 Overview 184 Data View Panels 185
113. he location has been added to a partial study for example for reprocessing White Light blue The location has been added to a partial study and has been selected for example for modifying When you select a sample location in the Sampler panel the fraction locations from all injections of that sample are highlighted on the fraction collector panel the Study Lists and Result panel show the results of the last injection of the sample Agilent 1200 Series Purification System User Manual Data Review 8 Fraction Collector The Fraction Collector panel shows a graphical representation of the fraction collector trays with the locations that have been used to collect fractions Table 16 explains the indicators used to show the state of each location in the Fraction Collector panel Table 16 Fraction Collector Location Indicators Line Color Fill Color Meaning Green Gray The location has not been used to collect fractions empty White Gray The location has been selected Green Green The location has been used for fraction collection Black Green The location has been used to collect a fraction from the selected sample Gray Gray The location has been specified as a reserved location in the fraction collector configuration Dark Green Grey Empty recovery location Black Dark Green The location has been used as a recovery location When you select a fraction location in the Fraction Collector panel all fractions
114. his checkout procedure verifies the operation of the Agilent Fraction Collector PS G1364B and the Agilent Mass Based Fraction Collection Kit G1968C used with the Agilent Purification Hi Throughput software and in combination with the associated hardware components The checkout procedure consists of the following activities e Preparing the Agilent 1200 Series LC and LC MSD e Setting the checkout method parameters e Running the checkout and collecting fractions e Reviewing the results and reanalyzing a fraction Agilent 1200 Series Purification System User Manual 219 11 Checkout Procedures UV triggered AS Fraction Collection Checkout The Checkout Procedure consists of the following activities e Preparing the Agilent 1200 Series LC e Setting the study parameters e Starting the analysis and collecting fractions e Reanalyzing the fractions What you will Need Instruments e Agilent 1200 Series high performance autosampler or Agilent 1200 Series standard autosampler Agilent 1200 Series preparative autosampler or Agilent 1200 Series dual loop autosampler optional thermostatted version e Agilent 1200 Series Isocratic or Quaternary or Binary Pump with Agilent 1200 Series Vacuum Degasser e Agilent 1200 Series Fraction Collector AS optional thermostatted version e Agilent 1200 Series Variable Wavelength Detector with analytical flow cell or Agilent 1200 Series Multi wavelength Detector with analytical flow cell or Agilent
115. ication System 1 Installing a Thermostatted Module in Your Purification System Preparation Locate bench space Provide power connections Unpack the module sampler or fraction collector and the thermostat Parts required Purification System modules and thermostat Power cords for the other cables see below and ChemStation and or Control Module G1323B 1 Place the thermostat on the bench 2 Remove the front cover and route the condensation drain tube to a waste container WARNING Make sure the condensation drain tube runs down into a waste container without any upwards bends or curves Free and unrestricted flow of the condensation into a waste container must be guaranteed Make sure that the condensation drain tube is always above the liquid level in the container If the tube is located in liquid the condensed water cannot flow out of the tube and the outlet is blocked Any further condensation will then remain in the instrument This may damage the instruments electronics 3 Remove the adhesive tape which covers the side and front doors if present 4 Open the front door of each of the modules 5 Remove the transport protection foam 6 Install the corrugated waste tube in the plastic port at the front bottom center of the module sampler or fraction collector and route down into a waste container Slide the waste tubing coming from the internal tray if present through the plastic port and the corrugated waste tube Route
116. ion annotations tickmarks and a fraction collection table consisting of the fraction number location start and stop time of the fraction collection the collected volume the reason for trigger and additional file information Figure 79 Data viewed in the ChemStation 196 Agilent 1200 Series Purification System User Manual Data Review 8 The functions in Agilent ChemStation Data Analysis can be used to further evaluate the data Agilent 1200 Series Purification System User Manual 197 8 Data Review 198 Agilent 1200 Series Purification System User Manual Agilent 1200 Series Purification System K User Manual 6 ee 9 e Reports e Introduction 200 Agilent ChemStation Reporting 202 Purification Hi Throughput Reports 205 This chapter describes the reporting options that are provided by the Purification Hi Throughput software ee Agilent Technologies 199 9 Reports Introduction You specify the reports that you want to produce in the Specify Report dialog box see Figure 80 You can output reports from both the Agilent ChemStation and the Purification Hi Throughput software i Specify Report a4 E M Backoround masses M a a openee lors E Area I Height M Abundance Tallestpeak I 2nd Tallest peak To Tallestpeak I 2nd Tallest peak CE D E G H Figure 80 The Specify Report dialog box 200 Agilent 1200 Series Purification System User Manual Repor
117. it is recommended to use the zero dead volume union PN 0100 0900 AS or 5022 2133 PS for capillary to capillary connections 34 Agilent 1200 Series Purification System User Manual Installing Your Agilent 1200 Series Purification System 1 Every pump must have a separate waste tubing installed at its purge valve Every stack must have a waste drainage installed at its bottom A fraction collector must have an additional waste drainage if it has a flow delay sensor installed part of the internal tray If a G1330B ALS thermostat is part of the system it must always be installed underneath the module that it is supposed to be thermostatted any Agilent 1200 Series injector or fraction collector If a G1328A B Manual Injector and or a G1383A Column Organizer is installed it should be installed between the two Agilent 1200 Series modules stacks If you want to operate multiple fraction collectors in parallel up to three this will be possible by installing the G1160A Agilent 1200 Series 12 position 13 port valve between the installed UV detector and the fraction collectors The length of all connecting tubings from the G1160A Agilent 1200 Series 12 position 13 port valve to the fraction collectors should be identical same delay volume required to use the same delay volume in the configuration of each fraction collector Otherwise it will be necessary to measure and configure the delay volume of each fraction collector individually
118. jection with needle wash the Needle Wash group is activated and you can specify either Flushport in which case the needle is flushed with mobile phase for a specified time or the location of a wash vial and a number of washes If you choose to use an injector program press Edit to display the current injector program for editing In the High Throughput group you can specify to activate automatic delay volume reduction and or overlapped injection If you activate automatic delay volume reduction the flow from the injector is switched from mainpass to bypass after the injection This reduces the delay volume for low volume techniques If you activate automatic delay volume reduction specify the point at which the switch takes place by using a fixed time or setting the Sample Flush Out factor in the Auxiliary group If you select Enable Overlapped Injection the next sample is drawn into the high performance autosampler while the current sample is being run You can specify to overlap the injection either after the sample is flushed out or after a specified time In the Auxiliary group specify the Draw and Eject speeds and the Draw Position offset to default position is higher is deeper of the high performance autosampler Specify an equilibration time which is the time that the needle stays in the vial after drawing the sample This allows the vacuum caused by drawing the sample to equilibrate Specify a Samp
119. l 69 3 Instrument Parameters The specified reserved locations apply to all installed plates Setup You set up the fraction collector using the Setup Fraction Collector dialog box 1 Display the Fraction Collector context menu right click on the Fraction Collector icon of the system diagram 2 Select Set up Fraction Collector to open the Setup Fraction Collector dialog box Alternatively you can use the Agilent ChemStation menus 1 Switch to Method and Run Control View if necessary View gt Method and Run Control 2 Open the Set up Fraction Collector dialog box from the Instrument menu Instrument gt Set up Fraction Collector 70 Agilent 1200 Series Purification System User Manual Instrument Parameters 3 Set up Fraction Collector Instrument 1 xi Fraction Trigger Mode Off Peak based Max Peak Duration 0 5 min Peak Detectors Detector Working Mode Up Slope Unit s Down Slope Unit s Threshold Unit Upper Threshold Unit Unit 1 SSCS SSO 5 3000 000 eL us o aw 3000 000 Fraction is collected when a peak is detected by Use MSD for Mass based Fraction Collection all selected peak detectors at least one selected peak detector Timetable Time Trigger Mode Max Peak Duration min of Fractions Timeslices min 1 04o Timebsed Tt pend a A E E tbl Figure 22 Fraction collector setup dialog box 3 In the Fraction Trigger Mod
120. le Flush Out factor to ensure that the sample is thoroughly flushed out of the syringe and past the capillary seat and valve to prevent sample carry over when using the automatic delay volume reduction mode or the overlapped injection mode For thermostatically controlled high performance autosamplers you can also select to store the temperature Agilent 1200 Series Purification System User Manual Instrument Parameters 3 Select Vial Well bottom sensing to ensure that the needle detects non uniform well bottoms and adjusts the depth of the needle to 1 mm above the detected bottom of the well Setting up the dual loop autosampler Set up Injector Instrument 1 Injection Plug Settings Use Loop upper C Faloppataly wth 500 u F Draw Plug betore and after the sample C Overt loop by toctor T Pag Volme 7000 wl Standard Inection Oraw Plug hom Injection with Neede Wash ow C Une Iniector Program C Location Fiat High Throughout Neede Wash Arse n Finrpon Rinse Vome 40 tenes T Enable Oveelagped Ingecton g J Tene 50 I Rice Draw Spend 20000 plies Rinse Erect Speed 20000 nil min Aupchary Time Draw Speet 500 pimin Equites Time 20 tet ei ti ki ErctSpoeed S000 l min M Stove Temperate Draw Postion 00 7 VialWel bottom serang oK Cancel Help Figure 20 Dual loop autosampler setup dialog box To set up the dual loop autosampler refer to Figure 20 1 In the Injection group specify the injection
121. lected E idic EdtMass J M Cl 35 37 F M formate 45 To M TFA 173 Method Target Masses M M M H l M M l T M l M H M M l Insert Remove Parameters Cancel Help The MSD Fraction Collection dialog box with TIC selected N Charge State Charge State lt 4 L 3 m2 mis Figure 28 Agilent 1200 Series Purification System User Manual 81 3 Instrument Parameters Use method target masses When you select this option Figure 30 all of the Agilent ChemStation parameters for fraction collection are taken from this dialog box including the selection of base masses and adducts When preparing to start a run the Agilent ChemStation adds an entry to the logbook if this option has been selected and the base mass values are missing Fraction Collection M M M M M D M M M M M z Charge State ra Charge State m a Insert Remove r FC Mode m MS Signals r Detectors C None Monitor Polarity Mode MS only C TIC Signal1 M Positive Scan C Other only 3 Use method target masses Sigal2 JV Negative Scan Ee andother Use sample target masses T a EMS onother C Import mass info from i T Signal4 F Time based r Positive Adducts r Negative Adducts m Method Target Masses VM OM Selected M M H 1 M M H 1 i 5 210 0 270 0 Edit Mass 5780 M M NH4 18 I M Cl 35 37 284 0 M M Na 23 I M fo
122. lent Technologies sales and service office Agilent 1200 Series Purification System User Manual 19 1 Installing Your Agilent 1200 Series Purification System Optimizing the Stack Configuration and Plumbing of Your Purification System 20 You can ensure optimum performance and minimum delay volume for your purification system by installing the modules in one of the following configurations Figure 1 on page 21 Figure 2 on page 22 and Figure 3 on page 23 show the configuration recommended for the analytical scale purification system Figure 4 on page 24 Figure 5 on page 25 and Figure 6 on page 26 show the configuration recommended for the preparative scale purification system Agilent 1200 Series Purification System User Manual Installing Your Agilent 1200 Series Purification System 1 Analytical Scale System Configurations I Solvent bottles Il Degasser optional for all pumps except the Quat Pump G1311 Ill Pump IV Sampling device V Column compartment 1 UV Vis 7 VII Fraction collector w e 5 g 4 on a w waste waste Figure 1 Recommended Stack Configuration Analytical Scale Purification System UV Vis Detector Based Front View To identify the inter module capillaries and tubing as labelled in Figure 1 please refer to Table 4 on page 36 The fraction collector should never be positioned on top of a module that generates heat This could lead to an unw
123. les the display of the Agilent 1200 Series fraction collector graphical display Show Fraction Scheme Displays the order in which the fractions are collected in the current setup Study List Samples Toggles the display of the Study List containing sample details Study List Injections Toggles the display of the Study List containing injection details Study List Fraction Wells Toggles the display of the Study List containing fraction well details of your fraction collector Agilent 1200 Series Purification System User Manual Menus and Views 4 Result Panel Toggles the display of the Result Panel containing the injection results of the selected sample Setup Result Panel Displays a menu that allows you to set up the results display This item is available only when the Result Panel is displayed Options The following items are available in the Options menu Set Study Base Path Displays the Choose Folder dialog box which allows you to select a folder in which to store your study see Setting the Study Base Path on page 136 Set Method Source Path Displays the Choose Folder dialog box which allows you to select the folder that contains the methods for your study see Setting the Method Source Path on page 137 Show Messages Toggles the state of the message line at the bottom of the display ON or OFF Window The following items are available in the Window menu Cascade Arr
124. lled between the splitter and the G1160A valve Agilent 1200 Series Purification System User Manual Instrument Parameters 3 20 Perform the calibration again Verify the time is sufficient gt 5 seconds see Figure 41 Ei Fraction Collector 1 Calibration Results Print Close Calibrate Detector Cum Dele Vol ll Cale Delay Vol ul DAD1 G13154 US64400562 937 14 lt n os eo ey a ea aa Cur Delay Time sec Calc Delay Time sec MSD 2 11 991 Figure 41 Delay time greater than 5 seconds 21 Press Calibrate if you want to store the new calculated delay volume for the MSD in the Fraction Collector Configuration see also Configuration on page 67 22 Press Print to get a hard copy of the report 23 Stop the splitter 24 Return to the Method and Run Control View and load the method you will be using to run your samples Select Fraction Collection from the More MSD sub menu under the Instrument menu 25 Press Parameters and enter the final calculated delay time that appears in the report in the Collector Delay field first converting it to minutes In this case you would enter 11 991 60 0 20 minutes see Figure 42 Agilent 1200 Series Purification System User Manual 105 3 Instrument Parameters Fraction Collection Parameters Ea Model Type Trigger Type Positive Step _ Peak Timing Min Peak Width min OXI Time Limit min 15 00 Max Peak Width min 0 50 MSD m
125. lts is now done using the G2727AA ChemStation Data Browser software See the Online Help in Agilent ChemStation for more information on the estimated purity calculations Agilent 1200 Series Purification System User Manual 145 5 Study Setup Completing the Study Setup The final panel of the Study Wizard contains a check box that allows you to automatically import a study list from a CSV file When you mark the Import Study List check box then click Finish to complete the study setup the Import Study List dialog box is displayed which allows you to specify the name and location of the CSV file and its layout For details of how to set up a study list as a CSV file and import it see Using CSV Files on page 158 During the study setup you can at any time click Previous to review or modify the study parameters You can also Cancel the study setup without saving the study End of Wizard The study test has been created Select wells in the Sampler Panel Then use the Right Mouse Button to add methods to it Help Cancel lt Previous Enn 146 Agilent 1200 Series Purification System User Manual Study Setup 5 Adding Samples to the Study List When the study setup has been completed and the Study Wizard closed the software displays a view containing graphical representations Views of the current autosampler and fraction collector configurations The wells or trays of the autosampler are shown on the left
126. mance autosampler G1367B Prep ALS G2260A Injection volumes up to 5ml with Multi draw kit thermostatted Versions of the ALS or WPS Manual Injector From Manual 4b G1328 87600 Injection loops of G1328A B Injector to Column delivered with the manual injector variable sizes available 36 Agilent 1200 Series Purification System User Manual Table 4 page 22 continued Installing Your Agilent 1200 Series Purification System 1 Flow Connections in an Analytical Scale Purification System see Figure 1 on page 21 and Figure 2 on No Type of Module Available Choices Flow Connection Capillary Tubing Comments V Column Compartment Thermostatted Col Comp G1316A or Column Organizer G1383A TCC Peltier to column Column to detector flow cell 5 G1316 87300 delivered with the TCC 6 G1312 67305 to detector delivered with the pump Column Switching Valve available as an option VI UV Detectors VWD G1314B C DAD G1315B C MWD G1365B C Detector flow cell to fraction collector or MRA if installed 7a b see fraction collector or 8 5063 6527 delivered with the detector VII Fraction Collector a G1364C AS or b G1364B PS Inlet and Outlet of the fraction Collector 7a G1364 68712 7b G1364 68711 delivered with the fraction collector VIIL Make up Pump for LC MSD lsocratic Pump G1310A Pump purge valve
127. mber or to add to the base mass For example to monitor a TFA trifluoroacetic acid adduct in negative ion mode you select one of the boxes under the Negative heading and enter 113 The result would monitor the base mass in the negative ion signals which would be described as M 113 Agilent 1200 Series Purification System User Manual Instrument Parameters 3 The adduct limits are 1 0 999 9 for either Positive or Negative signals Note that the number of adducts you select multiplied by the number of base masses you select should not exceed 16 Method Target Masses When the MSD is used as a detector and FC Mode is Use Method Info you must enter one or more base masses in the Method Target Masses group of the dialog box using the edit buttons Insert and Remove The selected mass is displayed in the Selected list Enter a mass and click Insert to add it to the Selected list To edit a mass in the list select it and the mass is displayed in the edit field To remove a mass select it and click Remove Agilent 1200 Series Purification System User Manual 87 3 Instrument Parameters Fraction Collection Parameters Dialog Box This dialog box lets you set parameters that control how the data is processed for fraction collection control The settings include which detectors are used peak detection criteria and delay times Fraction Collection Parameters Model ppe FL Hardware Other Eontiquration Wager ly
128. me reduction 64 autosampler 57 auxiliary 74 available methods 151 base path study 136 baseline drift 71 bench space 15 bottom sensing 65 66 bypass 64 c cable CAN 28 connecting APG remote 23 26 connecting CAN 23 26 connecting GPIB 23 26 connecting LAN 23 26 connecting the ChemStation 23 26 connecting the power 23 26 connections 30 33 power 28 thermostat interconnection 32 cabling 34 calculation delay volume 112 calibration delay volume 93 CAN cable 28 CAN DC IN 28 CAN DC OUT 28 capillaries 34 carry over 64 66 channel plug 32 ChemStation report 144 202 software 42 ChemStore 42 141 chromatogram 145 close study 125 closing a study 167 Agilent 1200 Series Purification System User Manual collection mode 69 order 69 collector delay 91 column sequence 124 width 123 condensation 18 configuration analytical scale system 21 fraction collector 67 injector 57 multi instrument 43 multiple fraction collectors 76 preparative scale system 24 stack 20 well plate types 54 configurations 42 connections cable 30 33 flow 34 considerations 14 continuous flow collection 69 corrugated waste tube 31 cross contamination 69 CSV 205 CSV file 146 158 161 current study 121 D damaged packaging 19 data file name 155 180 214 data folder 213 data view panels 185 275 Index delay volume 64 69 calculations 112 calibration 93
129. ments require an additional 2 5 cm 1 0 inch of space on either side approximately 8 cm 3 1 inches at the rear for the circulation of air and room for electrical connections If the thermostatted version of a sampler or fraction collector is used an additional 25 cm 10 inches of space on either side of the stack including the thermostat for the circulation of air and approximately 8 cm 8 1 inches at the rear for electrical connections are required Ensure that each module of the purification system is installed in a horizontal position Agilent 1200 Series Purification System User Manual 15 1 Installing Your Agilent 1200 Series Purification System If a complete Agilent 1200 Series system is to be installed on the bench make sure that the bench is designed to carry the weight of all the modules For a complete purification system it is recommended to position the modules in two stacks See Recommended Stack Configuration Analytical Scale Purification System UV Vis Detector Based Front View on page 21 Be aware of the additional bench space that is needed and the large weight that has to be carried if a G1946C D or G1956A B LC MSD is part of your purification system Be aware of the additional space and power connection that are needed to operate the LC MSD s rough pump The G1946C D or G1956A B LC MSD needs a pure Nitrogen gas supply for its operation Weight Specifications Table 1 Weight Specifications for Agile
130. n 0 5 min 1 2 Peak Detectors papi of 50 so 5o 3000 000 E OT SOT Detector Working Mode Up Slope Unit s Down Slope Unit s Threshold Unit Upper Threshold Unit Unit uB 3000 000 Fraction is collected when a peak is detected by xk i C all selected peak detectors at least one selected peak detector r Timetable Time Trigger Mode Max Peak Duration min of Fractions Timestlices min Cut Figure 125 Fraction Collector parameters Agilent 1200 Series Purification System User Manual Checkout Procedures 11 Table 26 MSD Signal Parameters see also Figure 126 Parameter Use MSD Tune file Stop Time Time Filter Data Storage Peak Width Scan Speed Override Active Signals Polarity Scan SIM Scan Parameters Start Time Mass Range Fragmentor Gain Threshold Step Size Value Enabled Atunes tun AsPump Enabled Condensed 0 08 min Disabled Signal 1 Positive Scan 0 00 min 100 350 100 1 0 100 adjust as appropriate to eliminate spectral noise 0 10 Agilent 1200 Series Purification System User Manual 265 11 Checkout Procedures Set Up MSD Signals x MSD Control MSD Signal Settings x l cluza HD Signal 1 E Frag np StopTine 2 lt Punp z ae Mode Scan x Polarity Positive hal cycle time 100 0 FIA Disabled Gancral aan ae kes ret Frag Gain T Step fronel ee ow ig mentor ol size atunes tun z 1___0 00 M
131. n In Combination with an Attenuator collector collector plus tubing LC MSD only G1968 64102 or for additional volume G1968 64104 G1968 60500 standalone delivered with the mass based fraction collector kit X LC MS Detector 1946 C D MRA to LC MSD 8 0890 1762 Optional detector G1956 A B delivered with the LC MSD Agilent 1200 Series Purification System User Manual 39 1 40 Installing Your Agilent 1200 Series Purification System Agilent 1200 Series Purification System User Manual Agilent 1200 Series Purification System User Manual 2 Installing the Software Before you start 42 Product Structure 43 Software Installation 44 Starting the Purification Hi Throughput Software 47 Adding New Licenses or Uninstalling the SW 49 This chapter contains instructions and other information to help you install and start the Purification Hi Throughput software on your Agilent ChemStation ee Agilent Technologies 41 2 Installing the Software Before you start 42 Before you start the installation of the Purification Hi Throughput software Rev B 01 01 or higher you must complete the following tasks 1 Install the hardware Agilent 1200 Series Liquid Chromatograph Agilent 1200 Series LC MSD optional Agilent 1200 Series Fraction Collector Universal Interface box optional 2 Make all necessary electrical connections 3 Install the Agilent ChemStation Software revision B 01 01 or higher as described in
132. n Results panel contains a results table and the acquired chromatograms from the detector s and the fraction collector delay sensor The results table contains e the name and serial number of the detector e the current delay volume stored in the fraction collector configuration e the calculated delay volume from the calibration You can click Print to print the Calibration Results panel Agilent 1200 Series Purification System User Manual 101 3 Instrument Parameters 11 If you want to store the calculated delay volume in the fraction collector configuration click Calibrate A confirmation dialog box is displayed when the delay volume has been stored click OK to close the confirmation If you do not want to store the calculated delay volume in the fraction collector configuration click Close A confirmation dialog box is displayed click OK to close the confirmation or Cancel button return to the Calibration Results panel If two or three fraction collectors are configured you can calibrate the delay volume for the first fraction collector only the delay volume calibration menu is accessible only for Fraction Collector 1 which is the fraction collector that appears first in the list of configured Agilent 1200 Series modules The calculated delay volume is used for all fraction collectors therefore the volume tubing between the selection valve and each installed fraction collector must be identical You still have the p
133. n the configuration the ones shown here are examples only pT viaL1 CheckviAL Figure 99 Study List after assigning the fraction start position Step 5 Starting the study 1 Start the study by clicking the D tool 2 Select Run Whole Study During the run you can observe the chromatogram by clicking the tool see Figure 100 NOTE The retention times of the peaks can differ Agilent 1200 Series Purification System User Manual 229 11 230 Checkout Procedures Data File C Purify data checkout 20010723 000 Data 00010200 D Injection Date 7 23 2003 5 46 15 PN Semple Nase CheckViaL 1 Jon Operator Aqilest Sequence Pile MPCREM I SEQUENCE 2 Method t 1 Perafy devavebeckot 20030723 000 nethods urasrr m Last changed 7 23 2003 44 531 PM by Agilent UV based AS fraction Collection System Checkout oe Volume Letter 31 2 011 evPosteat Agilent 1200 Series Purification System User Manual GR oye a Poe lo IN a ne Figure 100 The chromatogram as displayed during a run Checkout Procedures 11 Step 6 Reviewing the results 1 When the study run is finished verify that you have four fractions collected in the vials or wells or tubes 2 Verify that you have four green fraction locations on the fraction collector panel as in Figure 101 G 0 Vials 0 vials Study List Injections Se P EVIA T L CheckVIAL uvasfr m pee Collecte
134. n the sampler panel displays the Add Sample s dialog box which allows you to specify one or more methods for the selected samples and add the samples to the study Remove Sample s Removes the selected samples from the study Modify Sample Displays the Modify Sample s dialog box which allows you to modify the run parameters of the selected samples Add Sample s to Partial Study Adds the selected samples to a partial study Remove Sample s from Partial Study Removes the selected samples from the partial study Fraction Start Location for Selected Sample Marks the next available location for the start of fraction collection of the selected sample This can be set to lt Next Location gt lt Next Plate gt or a lt fixed position gt Fraction Start Location for the Current Study Marks the next available location for the start of the current study This can be set to lt Next Location gt lt Next Plate gt or a lt fixed position gt Agilent 1200 Series Purification System User Manual 127 4 Menus and Views View 128 The following items are available in the View menu View Mode Switches to View Mode see View Mode on page 120 Select Mode Switches to Select Mode see Select Mode Edit on page 120 The following items are available in View Mode only Sampler Panel Toggles the display of the Agilent 1200 Series autosampler graphical display Fraction Controller Panel Togg
135. nce on the delay time of the peak therefore we recommend that you use the maximum flow rates for the peakwidths given in Table 10 on page 116 or Table 11 on page 116 Use of higher flow rates will result in missed peaks unless the analog signal delay is taken into account Minimum required delay volume signal delay x flow Agilent 1200 Series Purification System User Manual 93 3 94 Instrument Parameters Procedure for a Fraction Collector AS G1364A 050 or G1364C with an UV detector 1 Place a vial containing the Delay Sensor Calibrant Part No G1946 85020 in position 1 of the Autosampler Remove the installed column and connect the capillaries with a zero dead volume connector Connect a bottle of water to Channel A 4 Switch to Diagnosis View if necessary 5 Open the AFC Delay Volume Calibration status window see Figure 33 on page 94 from the Fraction Collector sub menu of the Maintenance menu Maintenance gt Fraction Collector gt Delay Volume Calibration AFC Delay Volume Calibration Status Window Calibration method DGCALAS M OO Figure 33 Analytical AFC Delay Volume Calibration status window The method to be used for the delay volume calibration is displayed DGCALAS M is the default calibration method if you want to change to a different method click Change Method and select a new method See also Making Your Own Calibration Method on page 112 The Info field show
136. ng a StudyList e Study Run Control starting stopping run status displaying e Study Explorer step through data acquired by a study e Data Viewer view the data of an injection made by the ChemStation Chromatograms fractions e Reporting The product license structure is as shown in Table 6 Table 6 Purification Hi Throughput Software License Structure License Product Description Required License G2262AA Purification Hi Throughput Software Module Base software for G2170AA or sampler fraction collector and time peak based collection G2710AA G2263AA Mass Based Fraction Collection add on software G2262AA G2264AA Purity Calculation for MS data add on software Not This product is not supported with the CS B 01 01 and Purify B 01 01 supported However the corresponding functionality is built into the CS product G2265AA Stand alone Purification Hi Throughput data analysis software the Offline CS stand alone copy for creating studies data review and reprocessing Agilent 1200 Series Purification System User Manual 43 2 Installing the Software Software Installation Installation Process To install the Purification Hi Throughput software 1 Place the Purification Hi Throughput CD ROM in the CD ROM drive 2 From the Windows Start menu run the file D Purify Setup exe where D is the drive letter of your CD ROM drive 3 Follow the on screen instructions and choose to install either the on line or off line softw
137. ng of the splitter becomes partially restricted Mass Window Mass Window is a mass range in amu to monitor For example for mass 195 4 and a Mass Window of 1 0 the ChemStation monitors from 194 4 to 196 4 daltons Default 1 0 Analog Output Range kilo counts volt Analog Output Range is a scaling factor for the analog output signal This value should be set so that the largest expected peak yields a voltage that matches the fraction collector s maximum input voltage For example if the fraction collector expects a maximum voltage of 10 mV and the largest expected peak is 200 000 counts the scaling factor can be calculated as follows 00 counts x 1000 0 01 volts 20000 counts x 1000 7 vo Default 2000 kilo counts volts Other Detector These are parameters that determine whether a sample is detected in the analog input signal This entire section is disabled for a Purify system Agilent 1200 Series Purification System User Manual Instrument Parameters 3 Delay Volume Calibration The delay volume calibration measures the delay volume or time if an LC MSD is used for the delay calibration between a detector a source of a peak trigger and a fraction collector The procedure determines the delay volume relative to all configured detectors It is available for a Fraction Collector AS or for a Fraction Collector PS with an additional Fraction Delay Sensor installed CAUTION The detector peakwidth has an influe
138. nt 1200 Series purification system modules Module Specification Online Vacuum Degasser G1322A G1379B 7 0 kg 15 4 Ibs Pumps G1310A G1311A G1312A G1361A 15 5 kg 34 0 Ibs Maximum Samplers G1329A G1367B G2258A G2260A 15 5 kg 29 8 Ibs Thermostatted Column Compartment G1316A B 10 2 kg 22 5 Ibs UV Vis Detectors G1314B C G1315B C G1365B C 11 5 kg 26 0 Ibs LC MSD including foreline pump approx 96 8 kg 212 7 Ibs Fraction Collector G1364B C 13 5 kg 29 8 lbs Thermostat for samplers or fraction collector G1330B 18 5 kg 40 7 Ibs 16 Agilent 1200 Series Purification System User Manual Installing Your Agilent 1200 Series Purification System 1 Dimensions Table 2 Dimension Specifications for Agilent 1200 Series purification system modules Module Specification height x width x depth Online Vacuum Degasser G1322A G1379B 80 x 345 x 435 mm 3 1 x 13 5 x 17 inches Pumps G1310A G1311A G1312A G1361A 200 x 345 x 435 mm 8 0 x 13 5 x 17 inches Maximum Samplers G1329A G1367B G2258A G2260A 200 x 345 x 435 mm 8 0 x 13 5 x 17 inches Thermostatted Column Compartment G1316A 140 x 410 x 435 mm 5 5 x 16 1 x 17 inches UV Vis Detectors G1314B C G1315B C G1365B C 140 x 345 x 435 mm 5 5 x 13 5 x 17 inches LC MSD requires extra space below the bench for foreline pump 575 x 730 x 690 mm 23 x 29 x 27 inches Fraction Collector G1364B C 200 x 345 x 435 mm 8 0 x 13 5 x 17 inche
139. nually Use existing study Keep Tiray Configurator Keen Studbiist Study Help Cancel Ernst Figure 53 Creating a New Study Creating the Study Manually When you create the new study manually you specify sample names and methods for each of the samples in the sample tray individually Agilent 1200 Series Purification System User Manual 139 5 Study Setup Using an Existing Study If you choose to base your new study on an existing study you can choose to keep the tray configuration from the existing study If you choose to keep the tray configuration you can also choose to keep the study list from the existing study You select the study that you want to use as a template browse displays the Select Study to Open dialog box see Figure 54 which contains a list of all available studies with their creation and modification dates and directory names Select Study to Open Recovery Locations Purity Check Peak_Time_01 Combination Overill Reserved Locations Multiple Injections Pooling 20 10 55 19 2003 07 08 10 55 16 2003 07 08 10 55 11 2003 07 08 10 54 48 2003 07 08 10 54 43 2003 07 08 10 54 39 2003 07 08 10 54 33 2003 07 08 10 54 28 2003 07 08 10 54 22 21 30 11 14 40 2003 04 02 15 46 38 2003 06 30 09 51 14 2003 05 06 08 58 47 2002 08 26 15 37 03 2003 01 17 17 56 58 2002 08 26 15 29 56 2002 08 30 08 42 32 2003 07 08 15 15 58 20011114 000 20011106 008 20011018 000 20010926 000
140. nutes to provide a stable baseline 8 Insert an appropriate tray in the Fraction Collector Table 17 LC Method Parameters for UV triggered AS Fraction Collection checkout Device Parameter Value Injector Injection Volume 1 pl Pump Flow Rate 0 8 ml min Channel A Water 10 Channel B Acetonitrile 90 Stop Time 4 min Column Thermostat Column Temperature 40 C Detector Wavelength VWD 254 nm Wavelength DAD MWD 254 16 nm 360 100 nm Fraction Collector Fraction Trigger Mode Peak based Max Peak Duration 0 5 min Upslope 5 mAU Downslope 10 mAU Threshold 100 mAU 222 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 Table 17 LC Method Parameters for UV triggered AS Fraction Collection checkout Device Parameter Value Report Destination Printer Report Style Short Agilent 1200 Series Purification System User Manual 223 11 224 Checkout Procedures Step 3 Creating a new study 1 Create a new study base path with the name Checkout see Figure 92 Select Study Base Path Path Ic Purify data checkout Purity data Cancel Help Drives E c ChemStation 7 Network PP Ee Figure 92 Creating a new study base path 2 Click new study Agilent 1200 Series Purification System User Manual Checkout Procedures 11 3 Give the study the name Agilent Checkout see Figure 93 Create New Study fe Jo Keep Tray Configuration Figure 93 Creating the ne
141. ny of the modules that are used only if the G1946C D or G1956A B LC MSD is part of the system G1310A Make up pump G1390A UIB G1968 64102 MRA please refer to the G1968C Mass Based Fraction Collection Kit Installation Instructions manual available in the G1968C Mass Based Fraction Collection kit 16 Install the trays you have ordered for your sampler and fraction collector s 17 Turn ON power by pushing the button at the lower left hand side each of the purification system modules Agilent 1200 Series Purification System User Manual 29 1 Installing Your Agilent 1200 Series Purification System CAN cable to previous module o eE ee e Ld a Control of Thermostat THERMO fr CAN c OUT CONFIG anes CAN DC Out RS 232 CAN bus A BMN AC Power TAU CAN cable to next module Figure 7 Cable Connections Rear View of a G1367B or G1364B C as an Example A module of the purification system is turned ON when the line power switch is pressed and the green indicator lamp is illuminated A module is turned OFF when the line power switch is protruding and the green light is OFF WARNING To disconnect the purification system modules from the line unplug the power cord Otherwise the power supplies still uses some power even if switches at the front panels are turned OFF 30 Agilent 1200 Series Purification System User Manual Installing Your Agilent 1200 Series Purif
142. o have a single peak in two vials than two peaks in one vial Threshold counts The signal must be above this value for a sample to be collected If Peak Slope is above zero then both the threshold and peak slope criteria must be met to collect a peak Default 2000 counts Collector Delay min Collector Delay is the time interval between when the signal peak enters the MSD and when it enters the fraction collection hardware This value is always at least 0 01 min later than the value for Min Peak Width Default 0 01 min When a destructive detector such as a mass spectrometer or evaporative light scattering detector ELSD is used a splitter must be included so that the sample can be analyzed while still passing most of the sample along to the fraction collector The splitter alters the flow rate through both so a simple volume flow rate calculation cannot be used The split ratio for a given splitter can vary based on the solvent composition viscosity and any changes in tubing restriction that may occur over time Agilent 1200 Series Purification System User Manual 91 3 92 Instrument Parameters In fact the split ratio can change over time during a gradient run As a result the actual delay times need to be measured for any changes in flow rate solvent composition or tubing and connections Refer to Delay Volume Calibration on page 93 for details The split ratio may also change over time if any of the tubi
143. oles tudy Gl Bun Partial Study Reprocess Whole Study C Reprocess Partial Study Figure 72 Start Study dialog box for Reprocessing 176 Agilent 1200 Series Purification System User Manual Running Studies 6 The commands for reprocessing studies are identical to those for running a study However you can reprocess a study in both on line and off line versions of the Agilent ChemStation and the Purification Hi Throughput software Resetting the Vessel Fill Volumes You can reset the actual fill level of the vessels in your tray by removing the tray from the fraction collector and re inserting it By doing so the instrument will assume that all positions in the re inserted tray are empty Agilent 1200 Series Purification System User Manual 177 6 Running Studies 178 Agilent 1200 Series Purification System User Manual Agilent 1200 Series Purification System User Manual 6 ee 7 e Pooling e What is Pooling 180 Pooling Properties 182 Setting Up a Study for Pooling 182 This chapter explains the concept of pooling and describes how to set up a pooling run ot Agilent Technologies 179 7 Pooling What is Pooling 180 Pooling is a technique that is used to collect larger amounts of one or more fractions from each sample In the pooling study each sample is run multiple times using the same ChemStation method the fractions are always collected into the same vessels see Figure
144. olume did not overfill the well the location of the fraction in the fraction collector the collection start time of the fraction the collection end time of the fraction the trigger reason for the start of fraction collection Masses used for fraction trigger MSD only Fraction Volumes are only visible if the display type is Portrait Agilent 1200 Series Purification System User Manual 209 9 Reports ARo silica nee Sie i 3 3 SEENI REE EES CO OS I Fraction List Siudy Name Combination Defa Sampie Name Comer Study Path F SUNtVcatWEMOOCIONIE CO jection order by Selecion x Study Ren t Sharm STANDGY Dsonpiion Fan Method Mine z Li 5i ChemStore Cisatiee Fraction Start Loc 1 P 1 401 zi Oper stor mior Number of Poots lt wne R Start Date 26 Sep 01 90125 AM Recovery Start Loc Aone 1 8 PT AoT s 108 Tne 21m 10 195 Te a 311 403 16 2 Tme 2 1 1P1 A0Os 202 2 Te 2191 005 261 318 Time 311 08 32 35 Tee Figure 87 Fraction List Report Depending on the number of samples in the study and the number of fractions in each sample the Fraction List report may be many pages long 210 Agilent 1200 Series Purification System User Manual Agilent 1200 Series Purification System User Manual 6 ee 10 e Directory Structure and File Naming e Directory Structure 212 The Data Folder 213 Data FileNaming 214 This chapter explains how the Purification Hi Throughput
145. on Volume the injection volume of this sample Datafile the name of the datafile produced StartFC Start position for the fraction collection Ol amp ewe O aooaa sii S Sample List Study Name Combnston Dotat Sample Name Cori Study Path F Spurtydataisemo2001 0376 000 injection order by Selecton Siudy kuns 1 Stetdown STANDOY Description Fam Method None ChemSore lt Oisatied gt Fraction Stat Loc 11 401 Opan abot Agie Mumnber of Poots None Start Dete 26 Sep 01 9 01 25AM Recovery Start Loc None Stop Mode Competed Figure 86 Sample List Report Depending on the number of samples in the study the Sample List report may be several pages long 208 Agilent 1200 Series Purification System User Manual Reports 9 Fraction List Sample Location Sample Name Method Pool Fract No Well No Location Start Stop Reason Masses Fraction Volumes The Fraction List report see Figure 87 on page 210 contains the header information about the study together with a table containing information about the fractions collected in the study The table contains eleven columns the location of the sample in the autosampler the name you gave to the sample when you set up the study the name of the method used for this injection the number of the pool always 1 if no pooling was set up the number of the fraction the number of the well for this fraction always 1 if the fraction v
146. on Depth 5 0 mm CilContioaaus ion above location Distance 2 0 mm C drop setup Distance 0 5 mm Well Plates Plate Type 1 pean Reserved Locations Installed Plates Row s X Plate 1 front left X Plate 3 front right Column s O O X Plate 2 back left X Plate 4 back right Single o Location s Figure 21 Fraction collector configuration dialog box 4 The Trays group displays the trays that are installed in the fraction collector If the Agilent ChemStation is Online the installed trays are automatically detected and displayed If the Agilent ChemStation is Offline you can specify the trays that are installed If a test tube tray is installed specify the tube volume and height Specify a volume 10 less than the maximum volume to prevent over filling Agilent 1200 Series Purification System User Manual Instrument Parameters 3 5 In the Fraction Delay Volume group specify a delay volume in ul for each of the detectors in use see Delay Volume Calibration on page 93 6 Inthe Collection Order group select the collection pattern that you want to use 7 In the Collection Mode group select to collect discrete fractions or continuously available for all supported vessels and trays If you select to collect discrete fractions the diverter valve switches the flow to waste before the needle moves to the next collection position so a small amount of sample is lost If
147. ons L E E CSY Settings m Fraction list report T Include chromatograms m Purity calculations report m Sampler Score T Totals m Method Information I Adducts and Dimers Backaround masses T Purity Calculation parameters P fultiply Charged lors I lonization polarity r Sample Information Signals m Purity MSD 1 M Area To Hegi e Abundance M MSD 2 ae M Msp 3 Retentiontime M wsp4 M Tellestpeak Ia Zadifallest peat T Uv Highest m z M ADC o Tellestpeak MA Zadifellest peat Figure 56 Report Parameters Agilent ChemStation Reporting cwot tee In the Agilent ChemStation Reporting group you specify the parameters to add Fraction Collection items to the standard data analysis reports that are generated by the Agilent ChemStation See the Agilent ChemStation on line help for more details Agilent 1200 Series Purification System User Manual Study Setup 5 Agilent ChemStation Data Analysis Report You mark this check box to specify that you want to produce the standard Agilent ChemStation reports at the end of the study run When you mark this check box you can also choose to include either annotation of the fractions on your chromatograms or a printed fraction table or both If the fraction annotation option is selected you also have the option to label each well or to add fraction labels For details of the ChemStation reporting options
148. ort CS File s r Select Exports Export to M Sample list F Path JF purify data demo 20010831 0034C Ext csv x I Hotspots on J Purity calc erii Header M Seperator J 7 Delimiter 7 m Preview Export Fraction list 7 StudyName SampleLocation SampleName MethodName Cc Multiple Injections VIAL 1 Multi VIAL 1 agilent1 m i i Multiple Injections VIAL 1 Multi VIAL 1 agilent m Ei Multiple Injections VIAL 1 Multi VIAL 1 agilent1 m Ei Multiple Injections VIAL 1 Multi VIAL 1 agilent1 m m Multiple Injections VIAL 1 Multi VIAL 1 agilent2 m p Multiple Injections VIAL 1 Multi VIAL 1 agilent2 m E Multiple Injections VIAL 2 Multi VIAL 2 agilent1 m m Multiple Injections VIAL 2 Multi VIAL 2 agilent1 m fa Multiple Injections VIAL 2 Multi VIAL 2 agilent1 m E Multiple Injections VIAL 2 Multi VIAL 2 agilent m 4 Eoo Loe e gt Figure 65 Export CSV files dialog box 2 First select whether you want to export a sample or a fraction list to a CSV file purity calculations is not supported with this Rev of the SW 3 Specify a destination directory file extension header separator and delimiter 4 Click OK to close the dialog box and export the CSV file Importing Expected Masses from a CSV File Expected masses can be imported from a CSV file either as monoisotopic molecular weights or as molecular formulas The software automatically c
149. ossibility to manually change the volumes for each fraction collector separately Procedure for a Fraction Collector AS with an MSD CAUTION 1 Place a vial with the Delay Sensor calibrant Part No G1946 85020 in position 1 of the Autosampler 2 Remove the installed column and connect the capillaries with a zero dead volume connector or a mixer Part No 79835 87330 Connect a bottle of water to Channel A of the main pump Connect a bottle of water with 0 1 acetic acid or ammonium formate to the make up pump 5 Load the method DGCALAS M and adjust the flow in the main pump to the flow you will be using for the analysis Unlike for UV detectors the delay calibration for the MSD needs to be performed whenever the flow rate is changed 102 Agilent 1200 Series Purification System User Manual Instrument Parameters 3 6 Set the flow of the make up pump to the flow rate you will be using for your analysis 7 Set the active splitter to a setting of 3 8 Save the method to a new name Go to Diagnostics menu select Delay volume Calibration from the Fraction Collector Sub menu within the Maintenance menu 10 Press Change method to select the method you created The new method name appears on the info line 11 Start the active splitter 12 Press Continue to execute the Delay Volume Calibration The selected method is loaded and the Delay Calibration Sample Injected 13 When the Calibration Run is finished press OK 1
150. pe Positive Step Wither Detector Peak Slope my sec Eollector Delay miri Threshold 2 Full Scale volts Figure 32 The Fraction Collection Parameters dialog box Model Type The Model Type shows the configuration of your FC Hardware and the Trigger Type In a Purify system this entire section is disabled 88 Agilent 1200 Series Purification System User Manual Instrument Parameters 3 Peak Timing These parameters are used to set limits on the expected peak width or to advance to the next fraction collection vial if the peak must span multiple vials If the fraction collector is being controlled via an analog signal these parameters have no effect Min Peak Width min This parameter is used to eliminate spikes in the chromatogram If a mass peak is narrower than the minimum value the fraction collection relay is not triggered Note that the minimum peak width must be less than the MSD Collector Delay If the delay is less than the minimum peak width there is no way to cancel the fraction collection signal Default 0 0 min Max Peak Width min This parameter protects against peaks that never reach the peak termination conditions This might happen if a rising baseline prevents a signal from going below threshold Default 0 50 min When the Max Peak Width time is reached fraction collection for the current fraction stops This is different than the Time Limit parameter where
151. pecific to the Purification Hi Throughput software you mark the relevant check boxes in the Automated Purify Reporting group of the Specify Report dialog box see Figure 56 on page 144 You can select to report the following items e Study Run Summary e Sample List e Fraction List For each report you can choose to print the report on the current printer in either portrait default or landscape format and or save the report as a file in PDF and or Export to CSV format Figure 84 Exporting a report to a CSV file Agilent 1200 Series Purification System User Manual 205 9 Reports Press the CSV Settings Export button in the report setup to specify a CSV file to export to e Choose whether you want to export the sample list and or fraction list Purity Calculations is not supported with CS B 01 01 and Purify B 01 01 e Choose a file destination and type header separator and delimiter e Specify the report Details for the sample list and or fraction list You can select fields by moving them from the list of available fields to selected fields You can apply multiple sort patterns e In the Options tab you can apply several different filter options e In the Preview tab you can preview the CSV files as currently defined 206 Agilent 1200 Series Purification System User Manual Reports 9 Study Run Summary The Study Run Summary report see Figure 85 contains the information from the Study Parameters dialo
152. ple Information B selected Sample isocratic sample m Available Methods r Methods Injection Parameters T Injection Volume Method Pooling fNone gt Mass Foma m on x x ERA Help Cancel OK Figure 59 Add Samples to Study Dialog Box Sample Information The Sample Information group shows the location s name format and description of the selected sample location s If a single sample location has been selected the Location field displays the exact location and the Name Format is displayed as the sample name in the default format see Default Sample Name Format on page 142 You can edit the sample name and add a description of the selected sample If a group of sample locations has been selected the Location field displays the number of selected locations and the Name Format is displayed as the default prefix and location symbol You can add a description which is applied to all selected samples Agilent 1200 Series Purification System User Manual Study Setup 5 Available Methods The Available Methods panel displays a list of all the methods in the Method Source by default the Agilent ChemStation Methods folder You select one or more methods to apply to the selected samples from this list and use the tool to transfer it to the Methods panel If you want to make multiple injections of the same sample location without pooling see Chapter 7
153. ption For LC MS systems you can also change the expected masses However if you are modifying a sample for reprocessing you can change only the sample name and description To modify a sample 1 If necessary click the z tool to switch to Select mode 2 In the sampler view select the sample location that contains the sample that you want to modify 3 Click with the right mouse button in the sampler view to display the context menu and select Modify Sample from the context menu options Samples can also be modified directly in the sample list Alternatively use the Edit menu Edit gt Modify Sample The Modify Sample dialog box that is displayed has the same configuration at the Add Samples to Study dialog box see Figure 59 on page 150 Parameters that are available for modification are active parameters that are fixed are inactive grayed 152 Agilent 1200 Series Purification System User Manual Study Setup 5 Fraction Collector Panel You use the fraction collector panel to assign fraction start position and recovery locations see Recovery Locations on page 156 The following actions are possible in the fraction collector view Assigning the Fraction Start Position You assign the fraction start position from the context menu of the sampler and the fraction collector panel You can select any valid position on the fraction collector tray that is any position that is not a recovery location or a reserved loca
154. r modifying Blue Light blue The location has been added to a partial study White Light blue The location has been added to a partial study and has been selected for example for modifying Agilent 1200 Series Purification System User Manual Study Setup 5 ted ts i 2 2 ee A E OO OOOO 00000000 HaQaaoe ow eS Q oO Q Q 20000 Q QO Q QO Q O O O SOOOHHGOOHOO Figure 58 Sample Location Indicators Samples that have been added to the study list can be removed as long as there is no data associated with them Removing samples from the study list removes all injections of the sample Adding Samples To add samples to the study list 1 In the sampler view select the sample locations that contain the samples that you want to add to the study 2 Click with the right mouse button in the sampler view to display the context menu and select Add Sample s from the context menu options Alternatively use the Edit menu Edit gt Add Sample s Agilent 1200 Series Purification System User Manual 149 5 150 Study Setup Assigning Methods When you add samples to a study each sample must be assigned one or more methods The Add Sample s to Study dialog box Figure 59 which is displayed when you add one or more samples to the study list allows you to select the method that is assigned to the selected sample s Add Sample s to Study Location Name Format Description r Sam
155. rameters for each selected detector You also select whether fraction collection is triggered when any detector detects a peak or only when all detectors detect a peak 5 The Timetable allows you to switch fraction collection ON and OFF during the run and to change the trigger type and parameters In addition to the peak based trigger the Timetable also allows you to set up time based fraction collection based on either a number of fractions or a specified fraction collection time 72 Agilent 1200 Series Purification System User Manual Instrument Parameters 3 6 Click on Fraction Preview see Figure 24 to determine how a given sample would have been collected by the fraction collector with the current settings The fraction preview is only active if you have selected a trigger mode and set trigger parameters Here you can interactively change values drag Upslope Downslope Upper Threshold Threshold for the collection of fractions and see what impact these changes have on a chromatogram which you have previously loaded from any Agilent ChemStation data file Changes in the chromatogram are transferred to this table Zoom In Zoom Out Pointer Upslope Downslope Up Threshold Threshold Load CS data file Transfer changes for preview to Settings Figure 23 Fraction Preview Agilent 1200 Series Purification System User Manual 73 3 Instrument Parameters 7 Click More to access additional set up parameters see Figure
156. reen The location has been assigned White Green The location has been assigned and selected Dark green Gray The location can be used as a recovery location see Recovery Locations on page 156 Dark green Dark green The location has been assigned as a recovery location White Dark green The location has been assigned as a recovery location and selected Gray Gray The location cannot be used for fraction collection either because of the tray type or because the location has been specified as a reserved location in the fraction collector configuration see Configuration on page 67 Agilent 1200 Series Purification System User Manual Study Setup 5 The Study List Location Name Description Recovery Method Volume Data File Status The Study List samples which is displayed by default in the lower part of the display in Edit mode lists all the injections that have been added to the study in the sequence in which they will be run The Study List Samples contains a table with two groups of columns see Figure 60 a a ae a ae Location Name Description Method Volume Expected Data Fie Status_ Partia Run Fraction _ Multi VIAL agilentl m lt Method gt 0001000 Done NextLocatic Multi VIAL agilent2 m lt Method gt 00010101 Done NextLocatic Multi VIAL agilent1 m lt Method gt 0002000 Done NextLocatic Multi VIAL agilent2 m lt Method gt 00020101 Done NextLocatic Mul
157. required delay volume signal delay x flow 98 There are some differences in this procedure depending wether you use the G1364A or the G1364B version Please follow the instructions for the correct version of the instrument Place a vial containing the Delay Sensor Calibrant Part No G1946 85020 in position 1 of the Autosampler Remove the installed column and connect the capillaries with a zero dead volume connector 3 Connect a bottle of water to Channel A 4 Remove the installed tray 5 Install the delay sensor G1364A only The delay sensor is already pre installed in the G1364B Switch to Diagnosis View if necessary Open the AFC Delay Volume Calibration status window see Figure 36 from the Fraction Collector sub menu of the Maintenance menu Maintenance gt Fraction Collector gt Delay Volume Calibration Agilent 1200 Series Purification System User Manual Instrument Parameters 3 AFC Delay Yolume Calibration Status Window Calibration method DGCALPS M ee Chae cd Figure 36 Preparative AFC Delay Volume Calibration status window The method to be used for the delay volume calibration is displayed DGCALPS M is the default calibration method if you want to change to a different method click Change Method and select a new method See also Procedure for a Fraction Collector PS G1364A or G1364B with an UV detector on page 98 The Info field shows the current status
158. rmate 45 P M K 39 M M TFA 113 Figure 29 82 The Fraction Collection dialog box with Use method target masses selected Agilent 1200 Series Purification System User Manual Parameters Cancel Help Use sample target masses Instrument Parameters When you select this option the Method Target Masses section is disabled Figure 30 The target masses will instead be read from the Target Masses field 3 in the Sample Info dialog box if a single method is run for a sample or from the Target Masses column in the sequence table if a sequence of samples is run In either the Sample Info dialog box or the sequence table multiple target masses should be separated by semicolons Fraction Collection r FC Mode MS Signals m Detectors C None Monitor Polarity Mode MS only SE Sigal JV Positive Scan f Other only C Use method target masses Signal2 JV Negative Scan EAE andother C Use sample target masses Signal T C MS orother Import mass info from j _ Signal4 F Time based E m Positive Adducts M M IV M H 1 P M NH4 18 M M Na 23 M M K 39 M M D M r Negative Adducts JM I M H 1 P M Cl 35 37 I M formate 45 D M TFA 113 M M M z Charge State Charge State z z Method Target Masses Edt Mass Insert Remove Parameters Cancel Help Fi
159. ry II Pollution Degree 2 For indoor use only Wide ranging capability For the LC MSD For the LC MSD Maximum for each module For the LC MSD For the LC MSD Non condensing For storing the purification system 18 Agilent 1200 Series Purification System User Manual Installing Your Agilent 1200 Series Purification System 1 Unpacking the Purification System If you need to ship any module of the purification system at a later date always use the correct shipping boxes and protection foam parts Damaged Packaging Upon receipt of your purification system inspect the shipping containers for any signs of damage If the containers or cushioning material are damaged save them until the contents have been checked for completeness and the purification system has been mechanically and electrically checked If the shipping container or cushioning material is damaged notify the carrier and save the shipping material for the carrier s inspection CAUTION If there are signs of damage to the purification system please do not attempt to install the purification system Delivery Checklists Ensure all parts and materials have been delivered with each of the purification system modules For this compare the shipment contents lists in the reference manual of each of the modules that are part of your purification system with the checklist included in each instrument box Please report missing or damaged parts to your local Agi
160. s Thermostat for samplers or fraction collector G1330B 140 x 345 x 435 mm 5 5 x 13 5 x 17 inches Agilent 1200 Series Purification System User Manual 17 1 Installing Your Agilent 1200 Series Purification System Environment and Physical Specifications Your purification system will work within specifications at ambient temperatures and relative humidity as described in Table 3 CAUTION Do not store ship or use your purification system under conditions where temperature fluctuations may cause condensation within the purification system modules Condensation will damage the system electronics If any of your purification system modules was shipped in cold weather leave it in its box and allow it to warm up slowly to room temperature to avoid condensation Table3 Physical Specifications for Agilent 1200 Series purification system modules Type Specification Comments Line voltage Line frequency Power consumption apparent power Ambient operating temperature Ambient non operating temperature Rel Humidity Operating altitude Non operating altitude Safety standards IEC CSA UL 100 240 VAC 10 180 220 VAC 5 220 264 VAC 5 50 or 60 Hz 5 200 300 VA 1500 VA max 4 55 C 41 131 F 15 35 C 59 95 F 40 70 C 4 158 F lt 95 at 25 40 C 77 104 F Up to 2000 m 6500 ft Up to 4600 m 14950 ft Installation Catego
161. s about the installation of any of the modules that are used only if the G1946C D or G1956A B LC MSD is part of the system G1310A Make up pump G1390A UIB G1968 64102 MRA please refer to the G1968C Mass Based Fraction Collection Kit Installation Instructions manual available in the G1968C Mass Based Fraction Collection kit Agilent 1200 Series Purification System User Manual 25 1 Installing Your Agilent 1200 Series Purification System CAN Bus cable GPIB or LAN to LC ChemStation a Analog signal to recorder Figure 6 Recommended Stack Configuration Preparative Scale Purification System UV Vis Detector Based Rear View 26 Agilent 1200 Series Purification System User Manual Installing Your Agilent 1200 Series Purification System 1 Installing the Purification System Preparation Locate bench space Provide power connections Unpack the purification system modules Parts required Purification System modules Power cord for the other cables see below and Agilent ChemStation and or Control Module G1323B plus the Agilent Purification SW WARNING When opening capillary or tube fittings solvents may leak out Please observe appropriate safety procedures for example goggles safety gloves and protective clothing as described in the material handling and safety data sheet supplied by the solvent vendor especially when toxic or hazardous solvents
162. s displayed when the delay volume has been stored click OK to close the confirmation If you do not want to store the calculated delay volume in the fraction collector configuration click Close A confirmation dialog box is displayed click OK to close the confirmation or Cancel to return to the Calibration Results panel If two or three fraction collectors are configured you can calibrate the delay volume for the first fraction collector only the delay volume calibration menu is accessible only for Fraction Collector 1 which is the fraction collector that appears first in the list of configured Agilent 1200 modules The calculated delay volume is used for all fraction collectors therefore the volume tubing between the selection valve and each installed fraction collector must be identical You still have the possibility to manually change the volumes for each fraction collector separately in the configure screen of the ChemStation for each module Agilent 1200 Series Purification System User Manual 97 3 Instrument Parameters Procedure for a Fraction Collector PS G1364A or G1364B with an UV detector CAUTION The detector peakwidth has an influence on the delay time of the peak therefore we recommend that you use the maximum flow rates for the peakwidths given in Table 10 on page 116 or Table 11 on page 116 Use of higher flow rates will result in missed peaks unless the analog signal delay is taken into account Minimum
163. s the current status of the delay volume calibration and gives instructions and warnings Agilent 1200 Series Purification System User Manual Instrument Parameters 3 6 Click Continue The Info field gives the instruction to turn on the pumps see Figure 34 AFC Delay Volume Calibration Status Window _ Continue __Cancet Hei Figure 34 An instruction in the status window 7 When you have turned ON the pumps click Continue again to start the delay volume calibration The selected method is loaded and the delay volume calibration sample is injected Agilent 1200 Series Purification System User Manual 95 3 Instrument Parameters 8 When the calibration run has finished click OK The Calibration Results panel see Figure 35 is displayed Figure 35 Calibration Results panel The Calibration Results panel contains a results table and the acquired chromatograms from the detector s and the fraction collector delay sensor The results table contains e the name and serial number of the detector e the current delay volume stored in the fraction collector configuration e the calculated delay volume from the calibration You can click Print to print the Calibration Results panel 96 Agilent 1200 Series Purification System User Manual Instrument Parameters 3 9 If you want to store the calculated delay volume in the fraction collector configuration click Calibrate A confirmation dialog box i
164. see Agilent ChemStation Reporting on page 202 Plot Multiple Graphics on a Single Page You can print up to 81 graphics nine columns x nine rows of chromatograms mass spectra and or TICs on a single page Such thumbnail graphics can be useful for a first visual review of the study data You mark this check box to specify that you want to print multiple graphics and mark the check boxes of the graphics you want to print in this way You use the Plot fields to specify the number of rows and columns on each page Automated Purify Reporting For the specific Purify reports you can select the items to include select either portrait default or landscape format and select to produce the study results additionally as a PDF file and or as a CSV comma separated values file that you can import into a spreadsheet You also specify the destination of the PDF and CSV files default is the study folder and select to overwrite existing files For details of the Purify reporting options see Purification Hi Throughput Reports on page 205 The Export button allows you to export reports to CSV files The Sampler Summary and Sampler Extended report functions are no longer available with revision A 02 01 or B 01 01 of the Purification Hi Throughput software The estimated purity calculation function has been incorporated into the LC MSD ChemStation rev A 10 01 or B 01 01 software and the reporting of the estimated purity calculation resu
165. ser Manual Installing the Software 2 Starting the Purification Hi Throughput Software To start the Purification Hi Throughput software 1 If this is the first time you have started the Agilent ChemStation after installing the Purification Hi Throughput software switch the Agilent ChemStation to Full Menus View gt Full Menu This action inserts the Purify menu into the Agilent ChemStation menu bar Once the Purify menu is present you can access it from both full and short menus 2 Switch to Method and Run Control View or Data Analysis View View gt Method and Run Control View gt Data Analysis 3 Start the software from the Purify menu Purify gt Start Software Agilent 1200 Series Purification System User Manual 47 2 Installing the Software The Purification Hi Throughput software starts in a new window see Figure 13 eS SU ap bebe fof sabe of eb foe tate fopetot Figure 13 New Purification Hi Throughput Window 48 Agilent 1200 Series Purification System User Manual Installing the Software 2 Adding New Licenses or Uninstalling the SW If you want to add new licenses to your Purification Hi Throughput software for example Mass Based Fraction Collection you need to uninstall the existing Purification Hi Throughput software and re install the software including the new licenses To see which licenses are installed display the About Purify panel from the Help menu To uninstall the software 1
166. shald 1 Figure 130 Fraction Collection Parameters for MSD Agilent 1200 Series Purification System User Manual 271 11 Checkout Procedures Step 3 Running the Checkout Method 1 From the Agilent ChemStation RunControl menu select Sample Info then select manual data file name and enter the data file name mspschk1 d then specify the appropriate vial location for the checkout sample then click Run Method Step 4 Review Results 1 When the checkout method run is finished verify that you have three fraction collected in the vials wells or tubes The checkout sample contains four components but you will probably get separation into only three peaks two of the compounds will co elute 2 Verify that you have three fractions indicated on the MSD TIC in the ChemStation report or in Agilent ChemStation Data Analysis see Figure 131 on page 273 3 If you have not successfully collected the three fractions corresponding to the three peaks evaluate the data file in Agilent ChemStation Data Analysis for the proper threshold triggering level Update the threshold value in the MSD Fraction Collection Parameters dialog box and rerun the checkout 272 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 Figure 131 MSD triggered AS checkout results Agilent 1200 Series Purification System User Manual 273 11 Checkout Procedures Step 5 Reanalyze Fraction 2 1 Reanalyze the
167. spreadsheet You can use all the editing facilities of the spreadsheet program to facilitate the process see Figure 62 A 1 Location Method s Sample Name Sample Description Volume 2 vial 1 time m Checkout injection 1 10 3 vial 1 time m Checkout injection 1 20 4 P1 A 01 time m time m blank 10 5 R i D Je Figure62 A CSV file created in Microsoft Excel 3 Save the spreadsheet as a CSV file with the extension csv Agilent 1200 Series Purification System User Manual Study Setup 5 Importing the CSV file To import a Study List from a CSV file 1 Open the Import Study List dialog box using the File menu File gt Import Study List The Import Study List dialog box is shown in Figure 65 CSV File E S purify esy m CS Layout Available Fields Comment field gt Injection Volume Sample Name Kj Sample Description Expected Masses Number of header lines to skip 0 Help Cancel OK Figure 63 Import Study List dialog box 2 Click the button at the right of the CSV File field to display the file selection dialog box and select the CSV file you want to import 3 Set up the Fields in CSV File list to be identical with the columns in your CSV file in the correct sequence Click lt to transfer the selected item from the right panel to the left panel and gt to transfer the selected item from the left panel to the right panel 4 If you have set up your spr
168. ta acquired To obtain the proper thresh old after the first run has completed go into Agilent ChemStation Data Analysis and generate EICs for masses 279 285 and 311 Note the abundance level at 10 of the EIC peaks Take the lowest value from the three EICs and enter this into the Threshold field Fraction Collection x gt FC Mode m MS Signals m Detectors fC None Monitor Polarity Mode MS only ae Sigal Positive Scan Other only k Use method target masses Sigal2 J Negative Scan C MS and other Use sample target masses Sigala Gives Import mass info from pecs Signal4 F Time based r Positive Adducts m Negative Adducts _ pr Method Target Masses cM cM Selected Vv M H 1 T HEA P 3 faio 0 278 0 Edit Mass 5940 P M NH4 18 IV M Cl 35 37 M M Na 23 I M formate 45 P M K 39 I M TFA 113 B a gis EE Insert T M iMi Remove T M T M Charge State Charge State R E a a B Cancel Help Figure 121 Fraction Collection Setup for MSD Agilent 1200 Series Purification System User Manual Checkout Procedures 11 Fraction Collection Parameters Model Type FL harawere Other Configuration I igger lype Positive Step Uther Detector slope my sec hreshald 72 Figure 122 Fraction Collection Parameters for MSD Agilent 1200 Series Purification System User Manual 257 11 Checkout Procedures Step
169. tation report you mark the Agilent ChemStation Data Analysis Report check box Fraction Annotation When you select to include fraction annotation the beginning and end of each fraction is marked on your chromatograms as in Figure 82 The beginning of the collection of each fraction is marked with a green vertical line the end of collection is marked by a red vertical line The dotted horizontal line is the threshold that you specified in the peak detection parameters In addition you can choose whether you want to label each well and each fraction also Agilent 1200 Series Purification System User Manual Reports 9 DAD1 A Sig 254 4 Ref 360 100 DATAW0030100 D Figure 82 Fraction Annotation To include fraction annotation you mark the Fraction Annotation check box which is available when you have selected to produce Agilent ChemStation Data Analysis reports Fraction Table When you select to include a fraction table with your report a list of all the fractions taken during the run is added to the Agilent ChemStation data analysis report The header of the fraction table also gives detailed information of the fraction collection parameters see Figure 83 Agilent 1200 Series Purification System User Manual 203 9 Reports Fraction Information Study UV based AS DataPath C Purify Data UVAS 20010724 001 Data OQ010000 D Fraction collection peak controlled Maximm peak duration 0 5 min Practions are collected when at
170. tely any remaining injections are not completed Displays the Purify Status panel which shows the current status of the system Displays the Purification Hi Throughput Software Help contents Agilent 1200 Series Purification System User Manual Menus and Views 4 View Mode The following tools are specific to View Mode Shows the details for the first injection on the previous sample plate Shows the details for the first injection on the next sample plate Kl Shows the details for the previous sample E Shows the details for the next sample E Shows the details for the previous injection E Shows the details for the next injection ls Shows the details for the previous pool Shows the details for the next pool E Shows the details for the previous target disabled if no target masses are available A Shows the details for the next target disabled if no target masses are available E Loads the current data file into the Agilent ChemStation and switches to the Agilent ChemStation Agilent 1200 Series Purification System User Manual 133 4 Menus and Views Select or Edit Mode The following tools are specific to Select Mode et Ki Displays the Add Sample s dialog box which allows you to specify one or more methods for the selected samples and add the samples to the study Da ol Removes the selected samples from the study e Displays the Modify Sample s dialo
171. the fraction collector advances to the next position and continues collecting the fraction when the Time Limit is reached Time Limit is not used with the Purify system Agilent 1200 Series Purification System User Manual 89 3 90 Instrument Parameters Time Limit min This parameter is used to prevent a fraction collection vial overflow The correct value can be calculated from the fraction collection vial volume and the LC flow rate as follows Vial volume ml Time limit min ime limit min Flow rate ml min Note that many fraction collectors can be programmed to advance the vials based on time If this function is to be controlled from the fraction collector then set the time limit to a number much larger than the Max Peak Width Default 15 00 min This parameter is disabled on Purify systems On Purify systems the fraction collector module and the Agilent ChemStation software know the volume of the installed fraction collection vessel and overflow of the vessel is automatically prevented MSD m z Detection These parameters determine whether a sample is collected Peak Slope counts sec The signal must be above this value for a sample to be collected Default 500 counts sec For mass based fraction collection the specified ions base mass plus adduct mass are extracted from the scan If multiple ions are being monitored the largest ion at any point in time is evaluated to see if criteria are met R
172. the injection order might not correspond to the sample numbering then the next injection might be different than the next sample Shows the results for the previous pool This tool is available only if multiple pools were acquired in the study e Shows the results for the next pool This tool is available only if multiple pools were acquired in the study A Shows the results for the previous target disabled if none or only one target mass is specified for a sample Clicking this button is equivalent to placing the pointer one row up in the Target Masses table in the Results Panel EA Shows the results for the next target disabled if none or only one target mass is specified for a sample Clicking this button is equivalent to placing the pointer one row down in the Target Masses table in the Results Panel Agilent 1200 Series Purification System User Manual 195 8 Data Review Viewing data in Agilent ChemStation To view data for a particular sample in ChemStation Data Analysis 1 Click on the sample in the Sample Panel to select it 2 Click the Show data in ChemStation button on the right side of the toolbar An offline Agilent ChemStation session is started if the Agilent ChemStation is not already running and the Agilent ChemStation switches to the Data Analysis view The UV and MSD signals corresponding to the data file for the selected sample are loaded see Figure 79 and displayed including fract
173. the reference manual Installing your Agilent ChemStation Verify that the software is operational Supported Operating Systems are Windows 2000 with Service Pack 3 OR Windows XP with Service Pack 1 LC CE Agilent ChemStation G2170AA G1601A Optional LC MSD Agilent ChemStation G2710AA Optional Agilent ChemStore G1410A G2181BA G2186BA B 03 02 or greater The system must meet all requirements of the above and any other installed used software on the system Regional settings English United States Number format default for English United States Decimal symbol Number format default for English United States Digit grouping symbol In addition to the requirements of the Agilent ChemStation there must be one sampler configured e g 1200 Series autosampler G1329A or high performance sampler G1367B Any other module like a fraction collector or a detector is optional but can be required for the availability of functionality Agilent 1200 Series Purification System User Manual Installing the Software 2 4 Make sure that you are familiar with the Agilent 1200 Series LC LC MSD if installed and the Agilent ChemStation software Multi instrument Agilent ChemStation configuration is not allowed in combination with the Purification Hi Throughput Software Product Structure The Purification Hi Throughput Software includes the following functions e Study Editor creating and modifyi
174. the workspace at the right Above the two main areas are the menu bar and toolbar below are the status bar and message line if displayed see Figure 49 Toolbar Workspace Message line Se inla IOA E Status bar Figure 49 Purification Hi Throughput Main Panel Explorer The Explorer contains a tree view of all the studies that have been opened in the current session that is since the application was started The current study is highlighted in blue the study node expands to show the methods and samples that are associated with the study You can re open any one of the studies in the explorer by selecting it The explorer is cleared when the software is re started Agilent 1200 Series Purification System User Manual 121 4 Menus and Views Workspace The content of the workspace depends on the current mode of the software In Select mode the workspace contains e the Sampler panel a graphical representation of the configured autosampler the Fraction Collector panel s a graphical representation of the configured fraction collector s e the Study List Samples a table containing the information of each of the samples in the study Figure 50 shows an example of the workspace in Select mode Figure 50 The workspace in Select mode 122 Agilent 1200 Series Purification System User Manual Menus and Views 4 The items that are displayed in the workspace in Select mode are fixed and you cannot change
175. them You can however minimize or maximize each of the items resize the panels and adjust their positions in the workspace In View mode you can select the worksheet items to display In addition to the items displayed in Select mode see above you can display e the Study Parameters dialog box which is a read only version of the Setup Study Parameters dialog box This contains all the set up parameters for the current study see Setting Up the Study Parameters on page 141 for an explanation of the parameters e The Study List Injections which contains the details of all the injections in the study and the fractions collected for each of them The Result Panel which contains details of all the results in the study You can also configure the Results Panel from the View menu Configuring the Tables The width and sequence of the columns in all tables can be customized Changing the width of a column To change the width of a column 1 Place the mouse pointer over the right side of the column heading so that it changes to a horizontal double headed arrow 2 Hold the left mouse button down and adjust the width of the column To minimize the column width 1 Click the right mouse button in the column heading The width of the column and any columns beneath it is minimized To reset a minimized column 1 Click the right mouse button in the minimized column heading The width of the column and any columns beneath
176. ti VIAL agilentl m lt Method gt 00030001 Done NextLocatic Multi VIAL agilent2 m lt Method gt 00030101 Done NextLocatic Figure 60 Study List Samples Sample group The sample group columns describe the sample parameters The location of the sample in the sampler The sample name according to the specified naming convention The sample description entered in the Description field of the Add Samples to Study dialog box The recovery location if any for the sample Injection group The injection group columns describe the parameters for each injection or set of injections if pooling is specified The method to be used for this injection The injection volume by default the injection volume specified in the method denoted by lt Method gt The name of the data file that will be produced only the first injection if pooling is specified The status of the injection Agilent 1200 Series Purification System User Manual 155 5 156 Checked if the sample is specified for a partial study Recovery Locations You use recovery locations to collect the baseline eluent between fractions into a separate funnel see Figure 61 ls Figure 61 Recovering baseline eluent in a recovery location You can collect the baseline between fractions into funnels if a funnel tray is installed in the fraction collector into vials if in your fraction collector a vial tra
177. tion collector can accept well plates that are either in a list of standard preconfigured plates or new plate configurations that were set up by you Preconfigured Well Plate Types The list of preconfigured well plate types contains those well plates that are supported as standard by the Agilent 1200 Series high performance autosampler and Agilent 1200 Series fraction collector The table of well plate types shows the dimensions of the wells and well plate in accordance with Figure 14 on page 54 To view the list of the preconfigured well plate types display the System Well Plate Types dialog box 1 Switch the Agilent ChemStation to Full Menus if necessary View gt Full Menu 2 Switch to Method and Run Control View if necessary View gt Method and Run Control 3 Open the System Well Plate Types dialog box from the Instrument menu Instrument gt Preconfigured Well Plate Types Agilent 1200 Series Purification System User Manual 53 3 Instrument Parameters Configuration of Well Plate Types If the well plate that you want to use is not in the list of preconfigured well plates you can add it to a list of user configured well plates by specifying accurately the dimensions of the plate in accordance with Figure 14 on page 54 and Table 7 on page 55 You configure a new well plate type in the Configure Well Plate Types dialog box 1 Switch the Agilent ChemStation to Full Menus if necessary View gt Full Menu 2 Switch to
178. tion or that has not already been used for fraction collection on this tray Select the sample s that should be modified with a mouse click on the sampler panel the selected sample s will be displayed with a white circle in the sampler panel Start Fraction at To display the context menu click the right mouse button in the sampler panel Next Location If you choose Next Location this will start the fraction collection at the next available location for the currently selected sample s Start Fraction at To display the context menu click the right mouse button in the fraction lt Selected collector panel If you choose Selected Location this will start the fraction Location gt collection at the selected location for the currently selected sample s for example 1 P2 A03 starts fraction collection at location A03 on the second plate of the first fraction collector CAUTION If you remove and replace the tray the information about locations that have already been used for fraction collection are reset lost Agilent 1200 Series Purification System User Manual 153 5 154 Study Setup Location Indicators Table 14 explains the color indicators used to show the state of each location in the Fraction Collector panel Table 14 Fraction Collector Location Indicators Line Color Fill Color Meaning Green Gray The location has not been assigned or has been unassigned White Gray The location has been selected Green G
179. tion Collection parameters see also Figure 129 on page 270 and Figure 130 on page 271 Parameter Value FC Mode Use method target masses MS Signals Signal 1 Detectors MS Only Adducts M H 1 Selected Base Masses 278 0 284 0 310 0 Min Peak Width 0 00 min Max Peak Width 1 00 min 268 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 Table 28 MSD Fraction Collection parameters see also Figure 129 on page 270 and Figure 130 on page 271 continued Parameter Value Time Limit 1 00 min Peak Slope 0 Threshold 100000 counts Collector Delay Value taken from NMSD Delay Calibration procedure Mass Window 1 0 Analog Output 2000 not used for the checkout This value may need to be adjusted based on the actual data acquired To obtain the proper thresh old after the first run has completed go into ChemStation Data Analysis and generate EICs for masses 279 285 and 311 Note the abundance level at 10 of the EIC peaks Take the lowest value from the three EICs and enter this into the Threshold field Agilent 1200 Series Purification System User Manual 269 11 Checkout Procedures i Fraction Collection M Ej m Ej CU U U U E lt H Figure 129 Fraction Collection Setup for MSD 270 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 Fraction Collection Parameters Uther Detector Peak Slope my sec hre
180. tion System User Manual 3 10 11 Reports This chapter describes the reporting options that are provided by the Purification Hi Throughput software Directory Structure and File Naming This chapter explains how the Purification Hi Throughput files are organized and how data files are named Checkout Procedures This chapter describes the checkout procedures that are used to verify correct operation of hardware and software Agilent 1200 Series Purification System User Contents Contents 1 Installing Your Agilent 1200 Series Purification System 13 Site Requirements 14 Power Considerations 14 Power Cords 15 Bench Space 15 Weight Specifications 16 Dimensions 17 Environment and Physical Specifications 18 Unpacking the Purification System 19 Damaged Packaging 19 Delivery Checklists 19 Optimizing the Stack Configuration and Plumbing of Your Purification System 20 Analytical Scale System Configurations 21 Preparative Scale System Configurations 24 Installing the Purification System 27 Installing a Thermostatted Module in Your Purification System 31 Flow Connections to the Purification System 34 Flow Connections in an Analytical Scale System Flow Connections in a Preparative Scale System 2 Installing the Software 41 Before you start 42 Product Structure 43 Software Installation 44 Installation Process 44 Starting the Purification Hi Throughput Software 47 Adding New Licenses or Uninstalling the SW 49 Agil
181. to MRA 3 0890 1762 delivered with the mass based fraction collector kit In Combination with an LC MSD only IX Flow Splitter MRA Mass Rate Attenuator G1968 64102 or G1968 64104 standalone MRA to fraction collector 7a b see fraction collector plus tubing for additional delay volume G1968 60500 delivered with the mass based fraction In Combination with an LC MSD only collector kit X LC MS Detector 1946 C D MRA to LC MSD 9 0890 1762 Optional detector G1956 A B delivered with the LC MSD Agilent 1200 Series Purification System User Manual 37 1 Installing Your Agilent 1200 Series Purification System Flow Connections in a Preparative Scale System Table5 Flow Connections in an Preparative Scale Purification System see Figure 4 on page 24 and Figure 5 on page 25 No Type of Module Available Choices Flow Connection Capillary Tubing Comments l Solvent Container Solvent Cabinet Bottle head to pump 1 G1361 60002 Up to 4 different 5062 8581 comes inlet delivered with the solvents amber or with every pump pump transparent bottles Il Pump Prep Pump G1361A PumpAtoPumpB 2 Interconnections Only for binary setup isocratic see prep Pump Manual lll Pump 2nd Prep Pump Pump purge valve to 3 G1361 67302 G1391A for binary injection valve delivered with the gradients pump IV Sampling Device Prep Autosampler Injection valve to 4a G2260 87300 With std 900
182. to export configuration data to another Agilent ChemStation You can import your configuration file or a file from a different ChemStation to review or modify well plate configurations However you do not need to import the file to add a new well plate configuration The import function allows you to import only files of the wpt file format 56 Agilent 1200 Series Purification System User Manual Instrument Parameters 3 Autosamplers There are two aspects to the autosampler parameters Configuration is the process of characterizing the physical configuration of the autosampler for example by specifying the hardware trays syringe and seat capillary that are installed Setup is the process of specifying the method parameters for injection conditions for example injection volume injector program and syringe parameters Configuration You configure the injector using the Injector Configuration dialog box 1 Display the Injector context menu right click on the Injector icon of the system diagram 2 Select Configuration to open the Injector Configuration dialog box Alternatively you can use the Agilent ChemStation menus 1 Switch the Agilent ChemStation to Full Menus if necessary View gt Full Menu 2 Switch to Method and Run Control View if necessary View gt Method and Run Control 3 Open the Injector Configuration dialog box from the More Injector sub menu of the Instrument menu Instrument gt
183. together with any reserved locations the delay volume and the collection conditions for example collection order collection mode Setup is the process of specifying which detectors and which parameters will be used for triggering fraction collection Configuration You configure the fraction collector using the Fraction Collector Configuration dialog box 1 Display the Fraction Collector context menu right click on the Fraction Collector icon of the system diagram 2 Select Configuration to open the Fraction Collector Configuration dialog box Alternatively you can use the Agilent ChemStation menus 1 Switch the Agilent ChemStation to Full Menus if necessary View gt Full Menu 2 Switch to Method and Run Control View if necessary View gt Method and Run Control 3 Open the Fraction Collector Configuration dialog box Figure 21 on page 68 from the More Fraction Collector sub menu of the Instrument menu Instrument gt More Fraction Collector gt Configuration Agilent 1200 Series Purification System User Manual 67 3 68 Instrument Parameters Fraction Collector Configuration Instrument 1 Trays Tray A 4 WellPlates x Tube Volume ml 0 00 Tube Height mm jo Fraction Delay Volumes Collection Order Shortest path j _ j Detector Volume pl Row by row eoan O Column by O Shortest path column by row Collection Mode Needle Movement OW ae ee into locati
184. ts 9 Producing Reports Interactively You can also produce individual Purification Hi Throughput software reports interactively either as hardcopy or as PDF or CSV files To produce a report interactively you use the Report dialog box see Figure 81 which you display using the File menu File gt Report Alternatively you can press Ctrl P to display the dialog box r Select report s M Studyrun summary I Fi st IV Sample list J Purity calculations Cancel r Output to Screen Portrait e Printer C Landscape PDF File Acrobat Jv Prat ih color Help Figure 81 Report dialog box Using the Report dialog box you can select a report to produce and select where to send the output to Unlike automated reporting you can preview the interactive report on the screen before printing it The preview window also allows you to magnify or contract the report so that you can see a complete page in the preview window Agilent 1200 Series Purification System User Manual 201 9 Reports Agilent ChemStation Reporting 202 The standard Agilent ChemStation reports can be produced as specified in the Agilent ChemStation method or enhanced with the inclusion of fraction annotation on the chromatogram and or a print out of the fraction table In addition you can choose for the fraction annotations whether you also want to label each well and the fractions To print a standard Agilent ChemS
185. type You can choose to make a standard injection an injection with a needle wash or to use an injector program You have to specify if you want to use the lower loop or the upper loop for your injection The dual loop autosampler offers two loop filling modes Agilent 1200 Series Purification System User Manual 65 3 66 Instrument Parameters fill loop partially with a specified injection volume This mode is intended for preparative work and will provide a high sample recovery Overfill loop by a specified factor This mode is intended for analytical work and will result in a high injector precision and reproducibility If you choose an injection with needle wash the Needle Wash group is activated and you can specify either Flushport in which case the needle is flushed with mobile phase for a specified time or the location of a wash vial and a number of washes If you choose to use an injector program press Edit to display the current injector program for editing In the Plug Settings group you can choose to draw an air or solvent plug before and after your sample Specify the volume of your plugs and whether it should be an air plug or a solvent plug form a specified vial location In the High Throughput group you can specify to use overlapped injections If you select Enable Overlapped Injection the next sample is drawn into the dual loop autosampler while the current sample is being run You can specify to ov
186. ures 11 Table 19 LC Method Parameters for UV triggered PS Fraction Collection checkout Device Parameter Value Channel B Acetonitrile 65 Stop Time 2 5 min Pump Timetable 2 5 min 75 B Detector Wavelength VWD 254 nm Fraction Collector Report Wavelength DAD MWD Fraction Trigger Mode Max Peak Duration Upslope Downslope Threshold Destination Report Style 254 16 nm 360 100 nm Peak based 0 5 min 5 mAU 10 mAU 10 mAU Printer Short Agilent 1200 Series Purification System User Manual 237 11 238 Checkout Procedures Step 3 Creating a new study 1 Create a new study base path with the name Checkout see Figure 104 Select Study Base Path Path Ic Purify data checkout Purity J data Cancel Help Drives E c ChemStation 7 Network Ir EE Figure 104 Creating a new study base path 2 Click new study 3 Give the study the name Agilent Checkout see Figure 105 Agilent 1200 Series Purification System User Manual Checkout Procedures Create New Study Enter a name for the study Agilent Checkout m How to create the study Manually Use existing study F Keep Tray Gontiquration talk eep Studylist Study Cancel Finish Help Figure 105 Creating the new study 4 Complete the study parameters as shown in Figure 106 Setup Study Parameters Study Information Injection Order ee Order injection by
187. w study Agilent 1200 Series Purification System User Manual 225 11 Checkout Procedures 4 Complete the study parameters as shown in Figure 94 Setup Study Parameters fe s 2 UV based system checkout FL a e e Non JID guridi fourd io esses ound NextLocation E Figure 94 Study parameters for UV triggered AS checkout 226 Agilent 1200 Series Purification System User Manual Checkout Procedures 11 5 Specify the report as shown in Figure 95 Use Next and Previous to navigate through the Study Setup Wizard Specify Report ChemStation Reporting r Fractionlist Report M Include chromatograms M ChemStation Data Analysis report Include JV Fraction annotation JV Label each well M Fraction table I Fraction labels au f sa sass i V Plot multiple graphics on a single page MA Tcal M Graphi Graphics Chomatogam Plot f 7 Horizontal ee SE ee T Mass spectrum E Vertical Tee aaua E Bi A Eurip Calculation parameters TA tltislyiiherged or Automated Purify Reporting fis Iohteetion plenty Report Printer PDF file Landscape Sample Information Study Run Information Iv F a Signal Purity S ist Vv E BD SIN palettes ae EEn Fraction List v ms a F wsp2 E ia a Poco Retentiontime F Hs04 E Tallestpeak M 2nd Tallest pee ai Highest m z F ADe Po allestoesk Po Zadifallestnesk Export Cancel lt Back Finish
188. ximum of 15 characters long the prefix can be a maximum of 12 characters with the short location or eight characters with the full location Injection Order In the Injection Order group you specify the sequence of the injections in the study You can choose from Samples are injected in the order that they were added to the Study List Samples are injected according to their position in the autosampler tray for example 1 1 to 10 96 Samples injections are arranged by method sorted by modification date so that all samples using the same method are run together Agilent 1200 Series Purification System User Manual Study Setup 5 Fraction Collection and Recovery In the Fraction Collection group you specify the default start position in the fraction collector and also specify the number of pools the number of repetitions for fraction collection injections If this dialog is greyed out you don t have a valid active fraction collector configuration available from your Agilent ChemStation Frac Start You can choose one start location per study for the fraction collection in the Location study setup to start at the next available location on the current plate at the first available location on the next plate if more than one plate is configured or at a specific location When you add samples to a study you can also specify individual fraction start locations for each sample In addition to the study start location for th
189. xis the Overlay Mode menu item can be checked Agilent 1200 Series Purification System User Manual 193 8 194 Average Spectra If this menu item is checked an average spectrum is displayed below the TIC View Mode Tools To view data in other samples in this study you can either click on the desired sample in the Sampler diagram or use the View mode navigation tools available along the toolbar These tools are available only if the View mode button has been selected on the far left side of the toolbar Figure 78 File Rui ul R Figure 78 The View mode tool The following tools are specific to View Mode Shows the results for the first injection on the previous sample plate This tool will be available only if the study was run using a high performance autosampler or a dual loop autosampler Shows the results for the first injection on the next sample plate This tool will be available only if the study was run using a high performance autosampler or a dual loop autosampler el Shows the details for the previous sample gt lel Shows the details for the next sample Agilent 1200 Series Purification System User Manual Data Review 8 Ey J Shows the results for the previous injection Since the injection order might not correspond to the sample numbering then the previous injection might be different than the previous sample gt El Shows the results for the next injection Since
190. y run index for that day starting with 000 Thus 20010925 002 represents the third study run on 25 September 2001 Purify E Bin data 9 20010830 000 f 4 20010830 001 C 20010830 002 C 20010830 003 4 20010831 002 C 20011002 000 C 20011011 000 EE E E Figure 89 Study runs in the data folder Agilent 1200 Series Purification System User Manual 213 10 Directory Structure and File Naming Data File Naming Within each study run folder are sub folders containing the data and methods associated with the study run Data files for a study run are contained in folders with the extension d They have the general format SSSSNNPP d where SSSS is the sample number starting with 0001 NN is the injection number starting with 00 and PP is the pool number starting with 01 If no pooling has been specified then PP is always 00 Thus 00030200 d represents the third injection of the third sample no pooling and 00030102 d represents the second pool of the second injection of the third sample Figure 90 shows a list of the data files from a study run where no pooling was specified but each sample was injected twice a 20010831 002 a Data E 00010000 d C 00010100 d C 00020000 d E 00020100 d C 00030000 d C 00030100 d Methods E agilent1 m E agilent2 m E def_lc m E time m Figure 90 Data files from a study run without pooling 214 Agilent 1200 Series Purification System User Manual Director
191. y Structure and File Naming 10 Figure 91 shows the list of data files from a study run with three pools a 20010830 003 Data E 00010001 d 00010002 d 00010003 d C 00020001 d 00020002 d 00020003 d 00030001 d C 00030002 d C 00030003 d Methods agilentt m E p p Figure 91 Data files from a study run with pooling Agilent 1200 Series Purification System User Manual 215 10 Directory Structure and File Naming 216 Agilent 1200 Series Purification System User Manual Agilent 1200 Series Purification System User Manual 11 Checkout Procedures Introduction 218 UV triggered AS Fraction Collection Checkout 220 UV triggered PS Fraction Collection Checkout 235 MSD triggered AS Fraction Collection Checkout 248 MSD triggered PS Fraction Collection Checkout 261 This chapter describes the checkout procedures that are used to verify correct operation of hardware and software aa Agilent Technologies 217 11 Checkout Procedures Introduction 218 This chapter describes four different checkout procedures These procedures are used to verify the operation of the Agilent Purification Hi Throughput software and its associated hardware components UV triggered AS fraction collection checkout procedure see page 220 This checkout procedure verifies the operation of the Agilent Fraction Collector AS G1364C used with the Agilent Purification Hi Throughput software and in combination with the associated hardware compon
192. y contains data you can modify the study list to add or remove samples injections and the reporting parameters only You cannot make modifications to a study that is running unless you Pause the study before doing so Agilent 1200 Series Purification System User Manual 167 5 Setting Up a New Study Run You can set up a new study run within an existing study the new study run has the name Study Run lt n gt where lt n gt is incremented for each new study run The new study run opens with an empty study list You can configure the new study run to contain some or all of the samples in the existing study and you can add new samples in exactly the same way as when you set up a new study You can also add or remove methods from the study including the methods associated with the existing samples You can also modify the study parameters and the reporting parameters for the new study run To set up a new study run 1 If necessary open the study from you want to set up a new study run see Opening a Study on page 165 2 In the study tree select the study run from you want to set up the new study run 3 Switch to Edit mode and select the samples that you want to include in the new study run 4 Open the context menu for the Sampler panel right mouse click and select Add Sample s to New StudyRun 5 If you want to add more samples to the new study run or modify the samples you have added select New Study Run from
193. y is installed in addition to wellplate trays or if your system has a dedicated recovery fraction collector Using the recovery option should be applied in case the separation and the fraction collection regimes become un coordinated Baseline recovery also helps to avoid the consequences of operator error for example leading to a peak of interest in a rare sample being missed Recovery locations are set up as part of the study setup with the study setup wizard This option is only active if your system has a configuration valid for the use with the recovery option see below The following properties apply to recovery locations Agilent 1200 Series Purification System User Manual Study Setup 5 e Recovery locations are available only when either one fraction collector with a funnel tray is configured or you can recover into vials if in your fraction collector a vial tray is installed in addition to a wellplate tray or if your system has a dedicated recovery fraction collector e Only one recovery start location can be allocated per study run and one individual recovery location will then be used for every sample in thus study run e The non fraction collected portion of all injections of the sample flows into the specified recovery location If no recovery location is specified the non fraction collected portion is directed to waste e You cannot use a recovery location for fraction collection Assigning Recovery Lo
194. z Detection Peak Slope counts sec 500 Collector Delay min fo 20 Threshold counts 2000 Mass Window 1 0 Analog Output Range kilo counts volt 2000 Other Detector Peak Slope mv sec fio Collector Delay min 10 01 Threshold 2 15 0 Full Scale volts o 1 H Figure 42 Fraction Collection Parameters dialog box with delay volume entry 26 Save the method If you anticipate running samples at a variety of flow rates it is best to calibrate at the highest likely flow rate first and install the appropriate length tubing This way there will be sufficient delay for the lower flow rates and new lengths will not need to be cut 106 Agilent 1200 Series Purification System User Manual Instrument Parameters 3 Procedure for a Fraction Collector PS with an MSD 1 Place a vial with the Delay Sensor Calibrant Part No G1946 85020 in position 1 of the Autosampler 2 Remove the installed column and connect the capillaries with a zero dead volume connector or a mixer Part No 79835 87330 3 Connect a bottle of water to Channel A of the main pump Connect a bottle of water with 0 1 acetic acid or ammonium formate to the make up pump 5 Load the method DGCALPS M and adjust the flow in the main pump to the flow you will be using for the analysis CAUTION Unlike for UV detectors the delay calibration for the MSD needs to be performed whenever the flow rate is changed Agilent

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