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Agilent 1260 Infinity Quaternary LC VL System Manual

1. 80 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Optimization of the Agilent 1260 Infinity Quaternary LC VL 3 However you have to be aware that there are additional tolerance of gasket thickness and its compression ratio which is supposed to be very small in comparison with the machining tolerance Set the Detector Parameters VWD 1 Set peakwidth as close as possible to the width at half height of a narrow peak of interest 2 Choose the sample wavelength at a longer wavelength than the cut off wavelength of the mobile phase at a wavelength where the analytes have strong absorptivity if you want to get the lowest possible detection limit at a wavelength with moderate absorptivity if you work with high concentrations and preferably where the spectrum is flat for better linearity 3 Consider to use time programming to further optimization Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 81 3 Optimization of the Agilent 1260 Infinity Quaternary LC VL Optimizing Detection with VWD 82 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference ee 4 System Setup and Installation e E e j Installing Software 84 Installing the Modules 85 Priming the System 86 Integration Into the Network 90 This chapter includes information on software installation stack configurations and prepar
2. gt Brukt batteri returneres appararleverandoren 104 Bij dit apparaat zijn batterijen geleverd Wanneer deze leeg zijn moet u ze niet weggooien maar inleveren als KCA Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Appendix 6 Radio Interference Cables supplied by Agilent Technologies are screened to provide optimized protection against radio interference All cables are in compliance with safety or EMC regulations Test and Measurement If test and measurement equipment is operated with unscreened cables or used for measurements on open set ups the user has to assure that under operating conditions the radio interference limits are still met within the premises Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 105 6 Appendix Sound Emission Manufacturer s Declaration This statement is provided to comply with the requirements of the German Sound Emission Directive of 18 January 1991 This product has a sound pressure emission at the operator position lt 70 dB e Sound Pressure Lp lt 70 dB A e At Operator Position e Normal Operation e According to ISO 7779 1988 EN 27779 1991 Type Test 106 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Appendix 6 Solvent Information Flow Cell To protect optimal functionality of your flow cell e Avoid the use of alkaline solutions pH gt 9 5 which can attack quartz and thus impair the optical p
3. e Overlapped injection mode e Increase of draw and eject speed for large injection volumes e Add at last run a blank if overlapped injection is used To reduce the injection time the detector balance has to be set to OFF Precise Injection Volume Injection Volumes Less Than 2 pL When the injection valve switches to the BYPASS position the mobile phase in the sample loop is depressurized When the syringe begins drawing sample the pressureof the mobile phase is decreased further If the mobile phase is not degassed adequately small gas bubbles may form in the sample loop during the injection sequence When using injection volumes lt 2 uL these gas bubbles may affect the injection volume precision For best injection volume precision with injection volumes lt 2 uL use of an Agilent 1260 Infinity degasser is recommended to ensure the mobile phase is adequately degassed Also using the automated needle wash see Optimization for Lowest Carry over on page 54 between injections reduces carry over to a minimum further improving the injection volume precision Draw and Eject Speed Draw Speed The speed at which the metering unit draws sample out of the vial may have an influence on the injection volume precision when using viscous samples If the draw speed is too high air bubbles may form in the sample plug affecting precision The default draw speed is 200 uL min This speed is suitable for the majority of applications
4. For storing the module For indoor use only 1 This temperature range represents the technical specifications for this instrument The mentioned temperatures may not be suitable for all applications and all types of solvents Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Specifications 2 Table3 Module specific physical specifications Module Weight Dimension width x depth x height Power consumption G1311B C 11 kg 24 Ibs 140 x 345 x 435 mm 5 5 x 13 5 x 17 inches 180 VA 55 W 188 BTU Quaternary pump G1329B Autosampler 14 2 kg 32 Ibs 200 x 345 x 435 mm 8 x 13 5 x 17 inches 300 VA 200 W 683 BTU G1330B Thermostat 20 7 kg 46 lbs 140 x 345 x 435 mm 5 5 x 13 5 x 17 inches 260 VA 210 W 717 BTU G1316B 11 2 kg 25 Ibs 140 x 345 x 435 mm 5 5 x 13 5 x 17 inches 320 VA 150 W 512 BTU Thermostatted Column Compartment G4212B DAD 11 5 kg 26 Ibs 140 x 345 x 435 mm 5 5 x 13 5 x 17 inches 160 VA 130 W 444 BTU G1314F VWD 11 kg 24 Ibs 140 x 345 x 435 mm 5 5 x 13 5 x 17 inches 220 VA 85 W 290 BTU Using the autosampler at high environmental temperatures may cause the rear panel to become hot gt Do not use the autosampler at environmental temperatures higher than 50 C 122 F Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 39 2 Specifications Performance Specifications Performance Specifications G1311C Table 4 Performance Specifica
5. Note that this method can never be overwritten with new parameters Hence clicking on L ds Save will re direct you into the Save As function so that you must enter a different method name Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 93 5 Quick Start Guide Preparing the System Configuring the Online Plot 1 If the Online Plot window is not visible Click View gt Online Signals gt Signal Window 1 to display the window Online Plot DAD1A DAD Signal A 2 To configure the desired signal s in the Online Plot window click Change The Edit Signal Plot setup page opens Edit Signal Plot Available Signals Selected Signals PMP1B BinPump Pressure PMP1C BinPump Flow PMP1D BinPump Solvent Ratio 4 sd PMP1E BinPump Solvent Ratio B HM14 TCC Left Temperature HMI TEC Right Temperature PER 3 DAD Signal B DADIC DAD Signal C Window DAD14 DAD Signal x axis 3 x min Type acquired y axis range 100 B mau draw zero line auto y adjust Offset 10 gt Praction Collector p Method Settings I Show fraction collection ticks Use method settings Apply to Method OK Cancel Help 3 In the Available Signals box highlight the required signal s and click Add to move them to the Selected Signals box 94 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Quick Start Guide 5 4 To
6. tf Agilent Technologies Notices Agilent Technologies Inc 2010 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 61311 90310 Edition 06 2010 Printed in Germany Agilent Technologies Hewlett Packard Strasse 8 76337 Waldbronn This product may be used as a com ponent of an in vitro diagnostic sys tem if the system is registered with the appropriate authorities and com plies with the relevant regula tions Otherwise it is intended only for gen eral laboratory use 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 connection with the furnishing use or perfor mance of this document or of any information contained herein Should Agilent and the user have a separate written agreem
7. The detector measures absorbance simultaneously at wavelengths from 190 to 640 nm A UV lamp provides good sensitivity over the whole wavelength range If you know little about the analytes in your sample store all spectra over the full wavelength range This provides full information but fills up your disk space rather quickly Spectra can be used to check a peak s purity and identity Spectral information is also useful to optimize wavelength settings for your chromatographic signal 68 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Optimization of the Agilent 1260 Infinity Quaternary LC VL 3 The detector can compute and store at run time up to 8 signals with these properties e sample wavelength the center of a wavelength band with the width of sample bandwidth BW and optionally e reference wavelength the center of a wavelength band with the width of reference bandwidth The signals comprises a series of data points over time with the average absorbance in the sample wavelength band minus the average absorbance of the reference wavelength band Signal A in the detector default method is set to sample 254 0 4 reference 360 0 100 that is the average absorbance from 252 256 nm minus the average absorbance from 310 410 nm As all analytes show higher absorbance at 252 256 nm than at 310 410 nm this signal will show you virtually every compound which can be detected by UV absorbance Many compoun
8. however when using viscous samples set the draw speed to lower speed for optimum results A DRAW statement in an injector Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Optimization of the Agilent 1260 Infinity Quaternary LC VL 3 program also uses the draw speed setting which is configured for the autosampler Eject Speed The default eject speed setting is 200 uL min When using large injection volumes setting the eject speed to a higher value speeds up the injection cycle by shortening the time the metering unit requires to eject solvent at the beginning of the injection cycle when the plunger returns to the home position An EJECT statement in an injector program also uses the eject speed setting which is configured for the autosampler A faster eject speed shortens the time required to run the injector program When using viscous samples a high eject speed should be avoided Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 59 3 Optimization of the Agilent 1260 Infinity Quaternary LC VL Choice of Rotor Seal Vespel Seal for standard valves only The standard seal has sealing material made of Vespel Vespel is suitable for applications using mobile phases within the pH range of 2 3 to 9 5 which is suitable for the majority of applications However for applications using mobile phases with pH below 2 3 or above 9 5 the Vespel seal may degrade faster leading to reduced seal lifetim
9. 1260 Infinity Quaternary LC VL Manual and Quick Reference 1 Introduction Introduction to the Agilent 1260 Infinity Quaternary LC VL 8 Concept of the Quaternary LC VL System 8 System Properties 8 Features of the Agilent 1260 Infinity Quaternary LC VL 10 System Components 11 Optimizing the Stack Configuration 12 One Stack Configuration 12 Two Stack Configuration 15 Quaternary pump 17 Hydraulic Path 18 Autosampler 19 Sequences 21 Thermostatted column compartment 24 Detector 25 Diode Array Detector DAD 25 Variable Wavelength Detector VWD 30 This chapter gives an introduction to the Agilent 1260 Infinity Quaternary LC VL the underlying concepts and the features of the Agilent 1260 Infinity Quaternary LC VL gig Agilent Technologies 7 1 introduction Introduction to the Agilent 1260 Infinity Quaternary LC VL Concept of the Quaternary LC VL System The Agilent 1260 Infinity Quaternary LC VL offers the most flexibility for solvent selection and automation in HPLC method development research and all HPLC applications requiring continuous access to a wide range of solvent choices The availability to rapidly switch between methods using different solvents and the capability of using binary ternary or quaternary solvent gradients make the Agilent 1260 Infinity Quaternary LC VL the most flexible system on the market System Properties The Agilent 1260 Infinity Quaternary LC VL is ideally suited for multi method high throughput
10. Max Light Cartridge Cell 10 mm V o 1 0 ul with flow of 0 5 ml min LC grade water or Max Light Cartridge Test Cell Linearity Linearity is measured with caffeine at 265 nm 4 nm with slit width 4 nm and TC 1 s or with RT 2 s with Max Light Cartridge Cell 10 mm V o 1 ul gt 2 0 AU 5 typical 2 5 AU 5 The specifications are based on the standard RFID tag lamp 5190 0917 and may be not achieved when other lamp types or aged lamps are used ASTM drift tests require a temperature change below 2 C hour 3 6 F hour over one hour period Our published drift specification is based on these conditions Larger ambient temperature changes will result in larger drift Better drift performance depends on better control of the temperature fluctuations To realize the highest performance minimize the frequency and the amplitude of the temperature changes to below 1 C hour 1 8 F hour Turbulences around one minute or less can be ignored Performance tests should be done with a completely warmed up optical unit gt two hours ASTM measurements require that the detector should be turned on at least 24 h before start of testing Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 47 2 48 Specifications Specification Conditions VWD ASTM Standard Practice for Variable Wavelength Photometric Detectors Used in Liquid Chromatography Reference conditions Standard flow cell path length 10 mm
11. Quick Reference Quick Start Guide 5 Preparing the system Loading the Default Method The ChemStation has a default method named DEF_LC M which is loaded at first execution or whenever a new blank method template is required It contains default settings for all modules With this procedure you load the method DEF_LC M You can use it to set all parameters to default settings or to get a blank method template before setting up a new method 1 Go to Method and Run Control view of the ChemStation 2 On the menu bar select Method gt New Method and select DEF_LC M from the context menu Alternatively you can use the Load Method icon A under the menu bar or double click the method name DEF_LC M in the Methods tab of the Navigation Pane Fie RunControl Instrument Method Sequence View Abort Help v Sequences fed veF_ic s ras Se Method and Run Control Method and Run Control re PHENONESZ M a Main Automation Controls Single Sample HiP ALS ale BinPump al TEC 3 Offline W Q Offline Offiine Q DGALSTST M a QW DGcaLas m ua F QW DGcaLOQI M A 4 W vecaLoge m g 55 aw a QW DGCALOQ3 M Miia QI DGCALOQ4 M QJ DGCALOQS M QW DGcaLoQ6 m M1 necai ps M The default method DEF_LC M has a set of default parameters which can then be modified to create a new method For instance the flow rate is set to zero and the Method Information and Method History are blank
12. Specifications G1314F Type Specification Comments Drift lt 1 104 AU h at 230 nm Under specified condtions See Specification Conditions VWD on page 48 below the table Linearity gt 2 5 AU 5 at 265 nm Under specified condtions See Specification Wavelength accuracy Maximum sampling rate Band width Flow cells Electronic Temperature Control ETC Control and data evaluation Time programmable Spectral tools Analog outputs 1nm 80 Hz G1314F 6 5 nm typical Standard 14 pL volume 10 mm cell path length and 40 bar 588 psi pressure maximum High pressure 14 uL volume cell path length and 400 bar 5880 psi pressure maximum Micro 2 uL volume 3 mm cell path length and 120 bar 1760 psi pressure maximum Semi micro 5 pL volume 6 mm cell path length and 40 bar 588 psi pressure maximum For improved baseline stability in instable environment Agilent ChemStation B 04 02 SP2 or above G1314F Instant Pilot G4208A with firmware B 02 11 or above G1314F Wavelength Reference and Sample scan balance steps lamp on off Stop flow wavelength scan Recorder integrator 100 mV or 1 V output range 0 001 2 AU one output Conditions VWD on page 48 below the table Self calibration with deuterium lines verification with holmium oxide filter All flow cells have RFID tags for unambitious identification Can be repaired on component level Control and data evaluation Co
13. almost completely eliminated resulting in significantly less baseline drift T Agilent G4212 60008 Figure 13 Max Light Cartridge Flow Cell Slit The fixed slit combines the required optical functions slit and shutter in a simple and compact component The slit width is directly controlled by the micro processor of the instrument and is fixed to 4 nm Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Introduction 1 Detector Grating and Diode Array The combination of dispersion and spectral imaging is accomplished by using a concave holographic grating The grating separates the light beam into all its component wavelengths and reflects the light onto the photodiode array The diode array is a series of 1024 individual photodiodes and control circuits located on a ceramic carrier It has a wavelength range from 190 640 nm and the sampling interval is 0 5 nm Figure 14 Grating and diode array Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 29 1 Introduction Variable Wavelength Detector VWD Features G1314F The Agilent variable wavelength detectors described in this manual is designed for highest optical performance GLP compliance and easy maintenance with data rate up to 80 Hz for standard HPLC deuterium lamp for highest intensity and lowest detection limit over a wavelength range of 190 to 600 nm optional flow cell cartridges standard 10 mm 14 u
14. channel is to be replaced by another solvent that is not compatible solvents are immiscible or one solvent contains a buffer it is necessary to follow the procedure below to prevent clogging of the pump by salt precipitation or residual liquid droplets in parts of the system Description 1 Purging solvent s see Table 13 on page 90 Remove the column and replace it by a ZDV fitting Prepare bottles with appropriate intermediate solvents see Table 13 on page 90 1 Ifthe channel is not filled with a buffer proceed to step 4 Place the solvent intake filter into a bottle of water Flush the channel at a flow rate suitable for the installed tubing typically 3 5 mL min for 10 min 4 Modify the flow path of your system as required for your application For delay volume optimization see the Rapid Resolution System manual Buffer salt of aqueous buffers may precipitate in residual isopropanol Capillaries and filter may be cloged by precipitating salt gt Don t perform steps 5 to 7 for channels run with aqueous buffer as solvent Replace the solvent bottle by a bottle of isopropanol Flush the channel at a flow rate suitable for the installed tubing typically 3 5 mL min for 5 min Swap the bottle of isopropanol with a bottle of solvent for your application Repeat steps 1 to 7 for the other channel s of the pump Install the desired column set the required composition and flow rate for your application and equilibrat
15. concentration the linearity limit of the detector is then seen at about 2 AU path length so for the 6 cm Max Light Cartridge Cell the linearity limit is 333 mAU cm 2 In AU that is equal to x C x d like normal done in the past now for recalculation to your concentration C the path length must be considered Therefore flow cells with longer path lengths yield higher signals Although noise usually increases little with increasing path length there is a gain in signal to noise ratio When increasing the path length the cell volume could increase Depending on the peak volume this could cause more peak dispersion As a rule of thumb the flow cell volume should be about 1 3 of the peak volume at half height To determine the volume of your peaks take the peak width as reported in the integration results multiply it by the flow rate and divide it by 3 This may result in problems when the used peak width is set to large and all peaks are filtered accordingly Traditionally LC analysis with UV detectors is based on comparing measurements with internal or external standards To check photometric accuracy of the Agilent detector it is necessary to have more precise information on path lengths of the detector flow cells Part Number 64213 60008 G4213 60007 Path Length Cell Volume 0 1 0 cm 1 0 pL 6 0 cm 4 0 uL 66 Peak width response time Response time describes how fast the detector signal follows a sudden chan
16. during optimization of a method or when analyzing unique samples However when running many samples of the same type the large size of data files with all spectra may become a burden The detector provides functions to reduce the amount of data yet retaining the relevant spectral information Range Only the wavelength range where the compounds in your sample absorb contains information that is useful for purity checks and library searches Reducing the spectrum storage range saves disk space Step Most substances have broad absorbance bands Display of spectra peak purity and library search works best if a spectrum contains 5 to 10 data points per width of the absorbance bands For anisic acid the example used before a step of 4 nm would be sufficient However a step of 2 nm gives a more optimal display of the spectrum Threshold Sets the peak detector Only spectra from peaks higher than threshold will be stored when a peak controlled storage mode is selected Margin for Negative Absorbance The detector adjusts its gain during balance such that the baseline may drift slightly negative about 100 mAU In some special case for example when gradient with absorbing solvents are used the baseline may drift to more negative values Only for such cases increase the margin for negative absorbance to avoid overflow of the analog to digital converter Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Optimizatio
17. salt will fall back into the salt solution and will be dissolved When using the pump in a different configuration e g D salt solution A organic solvent the salt can fall into the port of the organic solvent and may lead to performance problems When using salt solutions and organic solvents it is recommended to connect the salt solution to one of the bottom ports of the MCGV and the organic solvent to one of the upper gradient valve ports It is best to have the organic channel directly above the salt solution channel Regular flushing with water of all MCGV channels is recommended to remove all possible salt deposits in the valve ports When to Use the Seal Wash Option Highly concentrated buffer solutions will reduce the lifetime of the seals and pistons in your pump The seal wash option allows to maintain the seal lifetime by flushing the back side of the seal with a wash solvent The seal wash option is strongly recommended when buffer concentrations of 0 1 M or higher will be used for long time periods in the pump The active seal wash upgrade can be ordered as G1398A The seal wash option comprises a support ring secondary seal gasket and seal holder for both piston sides A wash bottle filled with water isopropanol 90 10 is placed above the pump in the solvent cabinet and the peristaltic pump moves a flow through the pump head removing all possible buffer crystals from the back of the pump seal Running dry is the wors
18. visible detector based on a diode array and the correct choice of a reference wavelength setting quantitative detection is possible To suppress Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 73 3 74 Optimization of the Agilent 1260 Infinity Quaternary LC VL caffeine the reference wavelength must be set to 282 nm At this wavelength caffeine shows exactly the same absorbance as at 222 nm When the absorbance values are subtracted from each another any indication of the presence of caffeine is eliminated In the same way hydrochlorothiazide can be suppressed if caffeine is to be quantified In this case the wavelength is set to 204 nm and the reference wavelength to 260 nm Figure 23 on page 74 shows the chromatographic results of the peak suppression technique The trade off for this procedure is a loss in sensitivity The sample signal decreases by the absorbance at the reference wavelength relative to the signal wavelength Sensitivity may be decreased by as much as 10 30 Hydrochlorothiazide Hydrochlorothiazide and caffeine and caffeine Wavelength 204 nm Wavelength 222 nm No reference No reference Hydrochlorothiazides Caffeine suppressed uppressed Wavelength 204 nm Wavelength 222 nm Reference 260 nm Reference 282 nm 6 2 8 1 0 1 2 6 2 8 1 8 1 2 Time min Time min Figure 23 Peak Suppression Using Reference Wavelength Ratio Qualifiers for Selective Detection of Compound Classes Ratio q
19. workflows It offers Convenient access to four solvents for isocratic or gradient analysis for rapid method development and speed up preparation of mobile phases and flushing the HPLC system Pressure range up to 400 bar A wide flow range up to 10 mL min and a delay volume of 800 1100 uL supports narrow bore standard and semi preparative applications Easy programming and control through the Instant Pilot G4208A requires firmware B 02 08 or above p n G4208 67001 or through an Agilent Data System Included micro vacuum degasser offers high degassing efficiency for trouble free operation and highest performance and completely eliminates the need for helium sparging Direct front access for quick exchange of maintenance parts Fast problem identification by self diagnostics built in log books and preprogrammed test methods Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Introduction 1 e Early maintenance feedback EMF that continuously tracks long term instrument usage and user defined limits with feedback message when limit is exceeded e Upgradeability and expandability with the complete range of Agilent 1200 Infinity Series HPLC modules e Agilent Data System helps you manage your lab for best chromatographic quality with intuitive diagnostic and monitor capabilities and alert functions to notify you of problems Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 9 1 introdu
20. 260 Infinity Quaternary LC VL Manual and Quick Reference Optimization of the Agilent 1260 Infinity Quaternary LC VL 3 Flow Cell Path Length Lambert Beer s law shows a linear relationship between the flow cell path length and absorbance Absorbance log T log exCxd where T is the transmission defined as the quotient of the intensity of the transmitted light divided by the intensity of the incident light Ip e is the extinction coefficient which is a characteristic of a given substance under a precisely defined set of conditions of wavelength solvent temperature and other parameters C mol L is the concentration of the absorbing species d cm is the path length of the cell used for the measurement Therefore flow cells with longer path lengths yield higher signals Although noise usually increases little with increasing path length there is a gain in signal to noise ratio For example in Figure 29 on page 80 the noise increased by less than 10 but a 70 increase in signal intensity was achieved by increasing the path length from 6 mmFigure 29 on page 80 to 10 mm When increasing the path length the cell volume usually increases in the example from 5 13 uL Typically this causes more peak dispersion As demonstrated this did not affect the resolution in the gradient separation in the example shown below As a rule of thumb the flow cell volume should be about 1 3 of the peak volume at ha
21. 60 Infinity a Quaternary LC VL Optimizing the Pump 50 Operational Hints for the Vacuum Degasser 50 Operational Hints for the Multi Channel Gradient Valve MCGV 50 When to Use the Seal Wash Option 51 Choosing the Right Pump Seals 52 Optimize the Compressibility Compensation Setting 52 Optimizing the Autosampler 54 Optimization for Lowest Carry over 54 Fast Injection Cycle and Low Delay Volume 57 Precise Injection Volume 58 Choice of Rotor Seal 60 Optimizing the Thermostatted Column Compartment 61 Optimizing the Detector Regarding to the System 62 Delay Volume and Extra Column Volume 62 Optimizing Detection with DAD 63 Introduction 63 Optimization Overview 64 Optimizing for Sensitivity Selectivity Linearity and Dispersion 65 Optimizing Selectivity 73 Warm up of the Detector 76 Optimizing Detection with WWD 78 Optimizing the Detector Performance 78 Match the Flow Cell to the Column 78 Set the Detector Parameters VWD 81 This chapter considers how to apply the theory and use the features of the LC system to develop optimized separations ae Agilent Technologies 49 3 Optimization of the Agilent 1260 Infinity Quaternary LC VL Optimizing the Pump 50 Operational Hints for the Vacuum Degasser Operational Hints for the Vacuum Degasser If you are using the vacuum degasser for the first time if the vacuum degasser was switched off for any length of time for example overnight or if the vacuum degasser lines are emp
22. A G1315A G1365A G1315B C G1365B C G1600A G7100A G1314A B C G1314D EF G4286 90A B C 1120 1220 Wavelength Optical Accuracy Bandwidth The variation in Measured Wavelength depends on the different Optical Bandwidth May 19 2010 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 109 6 Appendix Agilent Technologies on Internet 110 For the latest information on products and services visit our worldwide web site on the Internet at http www agilent com Select Products Chemical Analysis It will provide also the latest firmware of the modules for download Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Index Index configuration one stack 12 A absorbance Beer Lambert 79 active seal wash 51 Agilent 1260 Infinity Quaternary LC VL features 10 Agilent oninternet 110 algae 107 alternative seal material 52 analog out put 45 analog signal output 44 analog output 40 array diode 29 B band width 6 5nm 45 battery safety information 104 beam splitter 35 Beer Lambert law 65 Beer Lambert absorbance 79 boards photodiode boards ADC 35 bypass 22 C choice of vials and caps 58 communications 46 composition precision 40 composition range 40 compressibility compensation 40 52 Configuration two stack 15 configuring online plot 94 control and data evaluation 43 45 cool down time 41 correction factors
23. L high pressure 10 mm 14 uL micro 3 mm 2 uL semi micro 6 mm 5 uL are available and can be used depending on the application needs other types may be introduced later easy front access to lamp and flow cell for fast replacement electronic identification of flow cell and lamp with RFID Radio Frequency Identification tag for unambiguous identification lamp information part number serial number production date ignitions burn time cell information part number serial number production date nominal path length volume maximum pressure built in electronic temperature control ETC for improved baseline stability and built in holmium oxide filter for fast wavelength accuracy verification This detectors cannot be operated with a G1323B Control Module Use the Instant Pilot G4208A as local controller For specifications refer to Performance Specifications G1314F on page 44 30 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Introduction 1 Optical System Overview The optical system of the detector is shown in the figure below Its radiation source is a deuterium arc discharge lamp for the ultraviolet UV wavelength range from 190 to 600 nm The light beam from the deuterium lamp passes through a lens a filter assembly an entrance slit a spherical mirror M1 a grating a second spherical mirror M2 a beam splitter and finally through a flow cell to the sample diode The beam
24. Optimized cell geometry for less peak dispersion for narrow bore applications More reliable and robust peak integration process automated due to less baseline noise drift refractive index and thermal effects especially under ultra fast gradient conditions RFID tracking technology is used for the UV lamp and the Max Light cartridge flow cells Multiple wavelength and full spectral detection at 80 Hz sampling rate keeping up with the analysis speed of ultra fast LC Fixed 4 nm slit for rapid optimization of sensitivity linearity and spectral resolution provides optimum incident light conditions Improved Electronic temperature control ETC provides maximum baseline stability and practical sensitivity under fluctuating ambient temperature and humidity conditions Additional diagnostic signals for temperature and lamp voltage monitoring Easy exchange of flow cell by cartridge design For specifications see Performance Specifications G4212B on page 43 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 25 1 Introduction Detector Optical System The optical system of the detector is shown in Figure 11 on page 26 2 Figure 11 Optical System of the Detector UV lamp Lamp mirror Flow cell Fold mirror Micro slit aoa oO FF O N Grating 7 Array The illumination source is a deuterium arc discharge lamp 1 for the ultraviolet UV wavelength range Its light is focused by a
25. a rate 43 wavelength accuracy 43 wavelength bunching 43 wavelength range 43 spectra acquisition 72 tools 45 standards 38 stepper motor 35 suppression quantifying 73 system setup and installation network Integration 90 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference optimizing stack configuration 12 85 system 26 turningon 92 T temperature control 45 temperature range 41 time constant versus response time 48 transport mechanism 19 U UV radiation 108 V vial racks 19 vials 19 viscous samples 58 58 Ww warm up of detector 76 warm up time 41 waste electrical and electronic equipment 103 wavelength accuracy 43 45 bunching 43 range 190 600nm 44 range 43 WEEE directive 103 weight 39 113 www agilent com In This Book This manual contains technical reference information about the Agilent 1260 Infinity Quaternary LC VL The manual describes the following e introduction product description system optimization e setup and installation e quick start guide Agilent Technologies 2010 Printed in Germany 06 2010 G1311 90310 EE Agilent Technologies
26. aced well between the heat exchanger fins Keep the left and right heat exchanger temperature the same unless you do specific applications e Assure that the front cover is always closed Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 61 3 Optimization of the Agilent 1260 Infinity Quaternary LC VL Optimizing the Detector Regarding to the System 62 Delay Volume and Extra Column Volume The delay volume is defined as the system volume between the point of mixing in the pump and the top of the column The extra column volume is defined as the volume between the injection point and the detection point excluding the volume in the column Extra Column Volume Extra column volume is a source of peak dispersion that will reduce the resolution of the separation and so should be minimized Smaller diameter columns require proportionally smaller extra column volumes to keep peak dispersion at a minimum In a liquid chromatograph the extra column volume will depend on the connection tubing between the autosampler column and detector and on the volume of the flow cell in the detector The extra column volume is minimized with the Agilent 1290 Infinity Agilent 1260 Infinity LC System due to the narrow bore 0 12 mm i d tubing the low volume heat exchangers in the column compartment and the Max Light cartridge cell in the detector Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Optimization o
27. and electronic appliances starting with 13 August 2005 This product complies with the WEEE Directive 2002 96 EC marking requirements The affixed label indicates that you must not discard this electrical electronic product in domestic household waste Product Category With reference to the equipment types in the WEEE Directive Annex I this product is classed as a Monitoring and Control Instrumentation product Do not dispose off in domestic household waste To return unwanted products contact your local Agilent office or see www agilent com for more information Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 103 6 Appendix Lithium Batteries Information Lithium batteries may not be disposed off into the domestic waste Transportation of discharged Lithium batteries through carriers regulated by IATA ICAO ADR RID IMDG is not allowed Danger of explosion if battery is incorrectly replaced gt Discharged Lithium batteries shall be disposed off locally according to national waste disposal regulations for batteries gt Replace only with the same or equivalent type recommended by the equipment manufacturer Lithiumbatteri Eksplosionsfare ved fejlagtig handtering Udskiftning ma kun ske med batteri af samme fabrikat og type gt Lever det brugte batteri tilbage til leverand ren Lithiumbatteri Eksplosionsfare Ved udskiftning benyttes kun batteri som anbefalt av apparatfabrikanten
28. anner as specified in this manual Safety Standards This is a Safety Class I instrument provided with terminal for protective earthing and has been manufactured and tested according to international safety standards Operation Before applying power comply with the installation section Additionally the following must be observed Do not remove instrument covers when operating Before the instrument is switched on all protective earth terminals extension cords auto transformers and devices connected to it must be connected to a protective earth via a ground socket Any interruption of the protective earth grounding will cause a potential shock hazard that could result in serious personal injury Whenever it is likely that the protection has been impaired the instrument must be made inoperative and be secured against any intended operation Make sure that only fuses with the required rated current and of the specified type normal blow time delay and so on are used for replacement The use of repaired fuses and the short circuiting of fuse holders must be avoided Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 101 102 Appendix Some adjustments described in the manual are made with power supplied to the instrument and protective covers removed Energy available at many points may if contacted result in personal injury Any adjustment maintenance and repair of the opened instrument under volta
29. anual and Quick Reference 43 2 Specifications Table 7 Performance Specifications G4212B Type Specification Comments Analog outputs Recorder integrator 100 mV or 1 V output range 0 001 2 AU one output Communications Controller area network CAN RS 232C APG Remote ready start stop and shut down signals LAN Safety and maintenance Extensive diagnostics error detection and display through control module and ChemStation leak detection safe leak handling leak output signal for shutdown of pumping system Low voltages in major maintenance areas GLP features Early maintenance feedback EMF for continuous tracking of instrument usage in terms of lamp burn time with user setable limits and feedback messages Electronic records of maintenance and errors Verification of wavelength accuracy with the emission lines of the deuterium lamp Housing All materials recyclable Performance Specifications G1314F Table8 Performance Specifications G1314F Type Specification Comments Detection type Double beam photometer Light source Deuterium lamp Wavelength range 190 600 nm The UV lamp is equipped with RFID tag that holds lamp typical information Short term noise 0 25 10 AU at 230 nm G1314F Under specified condtions See Specification Conditions VWD on page 48 below the table 44 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Specifications 2 Table8 Performance
30. ath Cator Filter plus Holmin Oxide Kilter Shutter Cutoff Filter Figure 17 Filter Assemby The filter assembly has two filters installed and is processor controlled OPEN nothing in light path at A lt 370 nm CUTOFF cut off filter in light path at A gt 370 nm HOLMIUM holmium oxide filter for wavelength check SHUTTER for measurement of dark current of photo diodes A photo sensor determines the correct position Mirror Assemblies M1 and M2 The instrument contains two spherical mirrors M1 and M2 The beam adjustable is vertically and horizontally Both mirrors are identical 34 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Introduction 1 Grating Assembly The grating separates the light beam into all its component wavelengths and reflects the light onto mirror 2 The stepper motor reference position is determined by a plate fitted onto the motor shaft interrupting the beam of a photo sensor The wavelength calibration of the grating is done at the zero order light position and at 656 nm which is the emission line of the deuterium lamp Beam Splitter Assembly The beam splitter splits the light beam One part goes directly to the sample diode The other part of the light beam goes to the reference diode Photo Diodes Assemblies Two photo diode assemblies are installed in the optical unit The sample diode assembly is located on the left side of the optical u
31. ations 43 44 pHrange 40 photodiode assemblies 35 boards 35 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Index photometric accuracy 66 80 physical specifications humidity 38 line voltage and frequency 38 operating temperature 38 safety information 38 piston chamber 17 power consumption 39 pressure pulsation 40 52 pressure range 52 pressure operating range 40 priming with a syringe 50 witha pump 88 with the pump 50 programmable slit width 43 proportioning valve high speed 17 Radio Frequency Identification flow cell and lamp 30 radio interference 105 recommended pH range 40 recyclable material 44 response time peak width 66 response time versus time constant 48 RFID Radio Frequency Identification 30 S safety class 101 safety information lithium batteries 104 safety general information 101 symbols 100 sample and reference wavelength 68 sampling rate datarate 45 sampling sequence 21 sealwash 17 51 when touse 51 seal alternative material 52 selectivity optimization 73 setable flow range 40 slit width 43 slit fix 28 solvents 107 sound emission 106 source lens assembly 33 specifications analog signal output 44 communications 44 control and data evaluation 43 diode width 43 flow cell 43 GLP features 44 linear range 43 noise and drift ASTM 43 noise and linearity 47 48 performance 44 programmable slit width 43 safety and maintenance 44 signal datarate 43 spectra dat
32. atted column compartment 24 The Agilent 1260 Infinity Thermostatted Column Compartment is a stackable temperature controlled column compartment for LC It is used for heating and cooling to meet extreme requirements of retention time reproducibility The main features are e Peltier heating and cooling from 10 degrees below ambient up to 80 C with high heating and cooling speeds for maximum application flexibility and stability e Holds up to three 30 cm columns and optimized design gives minimum dead volumes and maximum efficiency Two independently programmable heat exchangers contribute volumes of only 3 uL and 6 uL e Electronic column identification module as standard for GLP documentation of column type and major column parameters e Optional high quality Rheodyne column switching valves with ceramic stator face assemblies for prolonged lifetime For specifications see Performance Specifications G1316A on page 41 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Detector Introduction 1 Diode Array Detector DAD Features G4212B The detector is designed for highest optical performance GLP compliance and easy maintenance It includes the following features Maximum of 80 Hz data acquisition rate Higher sensitivity for conventional LC as well as ultra fast applications by using next generation optical design Increased sensitivity with 60 mm Max Light cartridge flow cell
33. bility compensation default setting is 100 x 106 bar for the pump This setting represents an average value Under normal conditions the default setting reduces the pressure pulsation to values below 1 of system pressure that will be sufficient for most applications and for all gradient analyses For applications using sensitive detectors the compressibility settings can be optimized by using the values for the various solvents described in Table 9 on page 53 If the solvent in use is not listed in the compressibility tables when using isocratic mixtures of solvents and if the default settings are not sufficient for your application the following procedure can be used to optimize the compressibility settings When using mixtures of solvents it is not possible to calculate the compressibility of the mixture by interpolating the compressibility values of the pure solvents used in that mixture or by applying any other calculation In these cases the following empirical procedure has to be applied to optimize your compressibility setting 1 Start the pump with the required flow rate 2 Before starting the optimization procedure the flow must be stable Check the tightness of the system with the pressure test Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Optimization of the Agilent 1260 Infinity Quaternary LC VL 3 3 Your pump must be connected to a data system or Instant Pilot with which the pressure and ripple can be m
34. can be connected directly to the column 3 Setting the peak width response time Use peak width according Choosing a Flow Cell on page 65 as starting point Set the peak width close to the width of a narrow peak of interest in your chromatogram 4 Setting wavelength and bandwidth Sample wavelength Never miss a peak by the use of a browser wavelength like 250 nm with 100 nm bandwidth Select specific wavelength with reduced bandwidth if you need selectivity e g 250 10 nm and 360 100 nm as reference wavelength Set the sample wavelength to a peak or valley in the spectrum to get best linearity for high concentrations Reference wavelength Select the reference wavelength with broad bandwidth 30 100 nm wavelength range where your analytes have little or no absorbance e g sample at 254 nm reference at 320 nm peak resolution versus sensitivity chromatographic resolution peak resolution versus sensitivity versus disk space sensitivity versus selectivity sensitivity versus linearity baseline drift due to RI effects 64 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Optimization of the Agilent 1260 Infinity Quaternary LC VL 3 Choosing a Flow Cell The Max Light Cartridge Cell with path length 10 mm and volume o 1 0 ul covers a wide range of applications e all column diameter down to at least 2 1 mm ID or even less e applications with peak dispersion Peakwidth x flow d
35. configure the individual settings for each signal highlight the signal in the Selected Signal box and set the required values in the lower half of the page In addition to the detector signals parameter traces such as temperature and pressure can also be plotted With Apply to Method the settings in this page can be stored into the method The Online Plot window behaves like electronic chart paper continuously recording the output from the detector s and other output parameters The signals are drawn at the right of the window and move away to the left Up to 90 min of past data is accessible This is useful for checking the baseline and looking at previous injections The X and Y axis scales can be adjusted directly with the up down buttons on each axis The Adjust button in the Online Plot window moves the current point on the selected signal to the zero line The selected signal is indicated by the color of the Y axis labels A particular signal may be selected by clicking on the signal or by clicking on the relevant signal description at the top of the plot The Balance button zeroes all detectors when pressed Changes made in the Online Plot page do not in any way affect the data stored into the individual data files Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 95 5 96 Quick Start Guide Purging the Pump Purge the pump if e The pump has been primed for the first time The pump is to be p
36. ction Features of the Agilent 1260 Infinity Quaternary LC VL The Agilent 1260 Infinity Quaternary LC VL is designed to offer the greatest flexibility for performing analytical liquid chromatography using all types of current and emergent column technologies The quaternary system as described in this manual offers 10 Gradients of up to 4 different solvents Pressure range up to 400 bar Sophisticated pump control to deliver very low chromatographic noise and very low acoustic noise for better results and better working environment Degasser and automatic purge valve integrated into pump module Variable volume autosampler with reduced delay volume reduced carryover and the option to operate as a fixed loop autosampler Thermostated column compartment with a pressure range up to 600 bar Choice of detectors a set of different flow cells is available for different detectors to fit application needs regarding flow ranges nano scale micro scale standard and preparative applications and pressures Diode array detector with greatly enhanced sensitivity and baseline stability using cartridge cell system with optofluidic waveguides data collection rate up to 80 Hz with full spectral information or Variable wavelength detector Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Introduction 1 System Components Solvent cabinet The solvent cabinet is a case to keep four bottles with solvent standing frame
37. ction type 1024 element photodiode array Light source Wavelength range Short term noise ASTM Single and Multi Wavelength Drift Linear absorbance range Wavelength accuracy Wavelength bunching Slit width Diode width Signal data rate Spectra Data rate Flow cells Control and data evaluation Local Control Test and diagnostic software Deuterium lamp 190 640 nm lt 3x 10 AU at 230 nm 4 nm lt 0 5 x 10 AU hr at 230 nm gt 2 0 AU 5 at 265 nm 1nm 2 400 nm G4212B 4 nm 0 5 nm 80 Hz G4212B 80 Hz G4212B Max Light Cartridge Cell 10 mm V o 1 0 pl 60 bar 870 psi pressure maximum Max Light Cartridge Cell 60 mm V o 4 0 uL 60 bar 870 psi pressure maximum Max Light Cartridge Test Cell Data System 1 Agilent ChemStation for LC 2 EZChrom Elite 3 MassHunter Agilent Instant Pilot G4208A Agilent LabAdvisor Equipped with RFID tag that holds lamp typical information see Specification Conditions below see Specification Conditions below see Specification Conditions below After recalibration with deuterium lines Programmable in steps of 1 nm Fixed slit pH range 1 0 12 5 solvent dependent Cartridge type equipped with RFID tags that holds cell typical information For G4212B 1 B 04 02 DSP3 or above 2 3 3 2 SP2 or above 3 B 04 00 and B 03 01 SP2 or above B 02 11 or above B 01 03 SP4 or above Agilent 1260 Infinity Quaternary LC VL M
38. d and stable so that they can be connected by solvent tubings to the LC System Quaternary pump The quaternary pump generates gradients by low pressure mixing from four individual solvent channels Autosampler with without thermostat The autosampler is specifically designed for the Agilent 1200 Infinity Series system for increased analysis speed with sensitivity resolution and precision Thermostatted column compartment This stackable temperature controlled column compartment is used for heating and cooling in order to meet extreme requirements of retention time reproducibility Diode array or variable wavelength detector Signals triggered by UV absorption are sampled to be converted to electrical signals in order for display and software handling The Agilent 1260 Infinity Quaternary LC VL are described in more detail in the following sections All modules are stackable see One Stack Configuration on page 12 and Two Stack Configuration on page 15 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 11 1 Introduction Optimizing the Stack Configuration You can ensure optimum performance by installing the system in following configurations These configurations optimize the system flow path ensuring minimum delay volume One Stack Configuration Ensure optimum performance by installing the modules of the Agilent 1260 Infinity LC System in the following configuration see Figure 1 on page 13 and Figu
39. d after injection to ensure minimum carry over The module uses an analytical head providing injection volumes from 0 1 to 100 uL for pressures up to 600 bar The six port injection valve unit only 5 ports are used is driven by a high speed hybrid stepper motor During the sampling sequence the valve unit bypasses the autosampler and directly connects the flow from the pump to the column During injection and analysis the valve unit directs the flow through the autosampler which ensures that the sample is injected completely into the column and that any sample residue is removed from the metering unit and needle from before the next sampling sequence begins Different valves are available Control of the vial temperature in the thermostatted autosampler is achieved using the additional Agilent 1260 Infinity ALS thermostat Details of this module are given in the Agilent 1260 Infinity Autosampler Thermostat manual Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Introduction 1 Sequences Sampling sequence The movements of the autosampler components during the sampling sequence are monitored continuously by the autosampler processor The processor defines specific time windows and mechanical ranges for each movement If a specific step of the sampling sequence can t be completed successfully an error message is generated Solvent is bypassed from the autosamplers by the injection valve during the sampling sequ
40. dit Entire Method Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 3 Contents Contents Introduction 7 Introduction to the Agilent 1260 Infinity Quaternary LC VL 8 Features of the Agilent 1260 Infinity Quaternary LC VL 10 System Components 11 Optimizing the Stack Configuration 12 Quaternary pump 17 Autosampler 19 Thermostatted column compartment 24 Detector 25 Specifications 37 Physical Specifications 38 Performance Specifications 40 Optimization of the Agilent 1260 Infinity Quaternary LC VL 49 Optimizing the Pump 50 Optimizing the Autosampler 54 Optimizing the Thermostatted Column Compartment 61 Optimizing the Detector Regarding to the System 62 Optimizing Detection with DAD 63 Optimizing Detection with WWD 78 System Setup and Installation 83 Installing Software 84 Installing the Modules 85 Quick Start Guide 91 Preparing the System 92 Setting Up the Method 97 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Contents 6 Appendix 99 Safety 100 The Waste Electrical and Electronic Equipment Directive 103 Lithium Batteries Information 104 Radio Interference 105 Sound Emission 106 Solvent Information 107 UV Radiation 108 Declaration of Conformity for HOX2 Filter 109 Agilent Technologies on Internet 110 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Contents 6 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Agilent
41. ds show absorbance bands in the spectrum Figure 19 on page 70 shows the spectrum of anisic acid as an example To optimize for lowest possible detectable concentrations of anisic acid set the sample wavelength to the peak of the absorbance band that is 252 nm and the sample bandwidth to the width of the absorbance band that is 30 nm A reference of 360 100 is adequate Anisic acid does not absorb in this range If you work with high concentrations you may get better linearity above 1 5 AU by setting the sample wavelength to a valley in the spectrum like 225 nm for anisic acid Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 69 3 Optimization of the Agilent 1260 Infinity Quaternary LC VL 52 sample wavelength 252 nm Anisic acid j 421 Reference bandwidth 100 nm 5 lt 324 o o n gt 284 E 30 nm 2 bandwidth z Reference wavelength 360 i j 7 r T T T T 220 248 262 280 388 320 340 360 380 422 Wavelength nm Figure 19 Optimization of Wavelength Setting A wide bandwidth has the advantage of reducing noise by averaging over a wavelength range compared to a 4 nm bandwidth the baseline noise is reduced by a factor of approximately 2 5 whereas the signal is about 75 of a 4 nm wide band The signal to noise ratio for a 30 nm bandwidth is twice that for a 4 nm bandwidth in our example Bandwidth 30 nm 12 nm 4nm Figure 20 Influence of Bandwidth on Signal and No
42. e Tefzel Seal for standard valves only For mobile phases with pH below 2 3 or above 9 5 or for conditions where the lifetime of the Vespel seal is drastically reduced a seal made of Tefzel is available Tefzel is more resistant than Vespel to extremes of pH however is a slightly softer material Under normal conditions the expected lifetime of the Tefzel seal is shorter than the Vespel seal however Tefzel may have the longer lifetime under more extreme mobile phase conditions PEEK Seal for preparative injection valve only The preparative injection valve has a sealing material made of PEEK This material has high chemical resistance and versatility It is suitable for application using mobile phases within a pH between 1 and 14 Strong oxidizing acids such as concentrated nitric and sulfuric acids are not compatible with PEEK 60 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Optimization of the Agilent 1260 Infinity Quaternary LC VL 3 Optimizing the Thermostatted Column Compartment For best performance results of the column compartment e Use short connection capillaries and place them close to the heat exchanger This will reduce heat dissipation and external band broadening e Use the left heat exchanger for small volume columns for example 2 3 mm i d columns at flow rates of less than 200 uL min e For even lower band broadening the heat exchanger can be by passed and the column is pl
43. e the system for approx 10 minutes prior to starting a run Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 89 4 90 System Setup and Installation Table 13 Choice of Priming Solvents for Different Purposes Activity Comments After an installation When switching between reverse phase and normal phase both times After an installation To clean the system when using buffers After changing aqueous solvents After the installation of normal phase seals P N 0905 1420 Ethanol or methanol HPLC grade water HPLC grade water Hexane 5 isopropanol Best solvent to flush air out of the system Miscible with almost all solvents Alternative to isopropanol second choice if no isopropanol is available Best solvent to re dissolve buffer crystals Best solvent to re dissolve buffer crystals Good wetting properties Integration Into the Network For network integration of your system refer to user manuals of your modules chapter LAN Configuration Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference ik Start Guide a 7 Preparing the System 92 Turning the System ON 92 Loading the Default Method 93 Configuring the Online Plot 94 Purging the Pump 96 Setting Up the Method 97 This chapter provides information on data acquisition and data analysis with the Agilent 1260 Infinity Quaterna
44. ect the check box to make Solvent B active B Initial value 65 Stop Time 6 min Max Pressure Limit 400 bar b Click the sign to open the Timetable c Add a line select Change Solvent Composition and set B to 80 d Other parameters can remain at default settings Click OK to exit the window The changes are sent to the pump module 3 Right click the autosampler area and select Method in the context menu a In the Method page for the 1260 Infinity Autosampler enter the following parameters Injection volume 1 0 ul Injection with Needle Wash Mode Flush Port Time 6 s b Other parameters can remain at default settings Click OK to exit the window The changes are sent to the autosampler module Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 97 5 98 Quick Start Guide 4 Right click the Thermostatted Column Compartment TCC area and select Method in the context menu a In the Method page for the 1260 Infinity TCC enter the following parameters Left Temperature 40 C Right Temperature Combined b Other parameters can remain at default settings Click OK to exit the window The changes are sent to the TCC module Right click the Diode Array Detector area and select Method in the context menu a In the Method page for the 1260 Infinity DAD enter the following parameters Use Signal Turn all signals except Signal A off by clearing the check boxes Signal A 250 n
45. eedle and needle seat capillary This can help to have faster cycle times especially if low flow rates have to be used like it is mandatory in narrow bore and micro bore HPLC Having the valve in bypass position can increase the carry over in the system The injection cycle times also depend on the injection volume In identically standard condition injecting 100 ul instead of 1 ul increase the injection time by approximately 8 sec In this case and if the viscosity of the sample allows it the draw and eject speed of the injection system has to be increased For the last injection of the sequence with overlapped injections it has to be considered that for this run the injection valve is not switched as for the previous runs and consequently the injector delay volume is not bypassed This means the retention times are prolonged for the last run Especially at low flow rates this can lead to retention time changes which are too big for the actual calibration table To overcome this it is recommended to add an additional blank injection as last injection to the sequence Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 57 3 58 Optimization of the Agilent 1260 Infinity Quaternary LC VL General Recommendations for Fast Injection Cycle Times As described in this section the first step to provide short cycle times are optimizing the chromatographic conditions If this is done the autosampler parameter should be set to
46. efore the output was set to zero The characteristic wavelengths 249 nm A and 224 nm Az were found from the spectra shown in Figure 24 on page 75 The ratio range was set at 2 2 4 2 2 10 Only when the ratio between 249 and 224 nm was within this range is the signal plotted Of all four peaks only the third fulfilled the criterion Figure 25 on page 75 The others were not plotted Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 75 3 Optimization of the Agilent 1260 Infinity Quaternary LC VL Warm up of the Detector Give the optical unit enough time to warm up and stabilize gt 60 minutes The detector is temperature controlled After turn on of the detector it goes through a cycle of different states 0 to 0 5 minutes the heater control is OFF and the heater element runs at 0 duty cycle 0 5 to 1 minutes the heater control is OFF and the heater element runs at 66 duty cycle This first minute is used as self test of the heater functionality 1 to 30 minutes the heater control is OFF and the heater element runs at 40 duty cycle e After 30 minutes the heater control is ON and is working with optimized parameters to get the optical unit into the optimal temperature window stabilized This cycle starts e when the detector is turned off on e when the lamp is turned off on to ensure that the temperature control operates in a defined control range The times to stabilize the baseline ma
47. ence The sample vial is selected by a gripper arm from a static sample rack or from external vial positions The gripper arm places the sample vial below the injection needle The required volume of sample is drawn into the sample loop by the metering device Sample is applied to the column when the injection valve returns to the mainpass position at the end of the sampling sequence The sampling sequence occurs in the following order 1 The injection valve switches to the bypass position 2 The plunger of the metering device moves to the initialization position 3 The gripper arm moves from the home position and selects the vial At the same time the needle lifts out of the seat The gripper arm places the vial below the needle The needle lowers into the vial The metering device draws the defined sample volume The needle lifts out of the vial on ow A If the automated needle wash is selected see Using the Automated Needle Wash on page 54 the gripper arm replaces the sample vial positions the wash vial below the needle lowers the needle into the vial then lifts the needle out of the wash vial 9 The gripper arm checks if the safety flap is in position 10 The gripper arm replaces the vial and returns to the home position Simultaneously the needle lowers into the seat 11 The injection valve switches to the mainpass position Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 21 1 22 Int
48. ent 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 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 dis closure of Software is subject to Agilent Technologies 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 hazard It calls attention to an operating procedure practice or the like that if not correctly per formed or adhered to could result in damage to the
49. er in series design which comprises all essential functions that a solvent delivery system has to fulfill Metering of solvent and delivery to the high pressure side are performed by one pump assembly which can generate pressure up to 400 bar Degassing of the solvents is done in a built in vacuum degasser Solvent compositions are generated on the low pressure side by a high speed proportioning valve MCGV The pump assembly includes a pump head with a passive inlet valve and an outlet valve A damping unit is connected between the two plunger chambers A purge valve including a PTFE frit is fitted at the pump outlet for convenient priming of the pump head An active seal wash optional is available for applications using concentrated buffers as solvents Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 17 1 Introduction Quaternary pump Hydraulic Path From solvent bottles Figure 5 Hydraulic Path of the Quaternary Pump 18 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Introduction 1 Autosampler Autosampler Analytical head Sampling Unit Transport assembly ASM board Power supply Vial Tray Gripper Arm Needle seat Switching Valve Figure 6 Overview of the Autosampler The Agilent 1260 Infinity Autosampler is designed to offer the well established Agilent flow through design with variable volume injection and to achieve extremely low carryover The small h
50. ests e Attempts to identify supported LAN based instruments that are powered on and connected to your PC or lab s network e Automatically suggests replacements parts and troubleshooting tasks for some common instrument problems Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference System Setup and Installation 4 Installing the Modules Installing the System Modules For details of installation procedures for the modules refer to the individual module manuals These manuals also contain information on specifications maintenance and parts Stack Configuration You can ensure optimum performance by installing the system in one stack and two stack configurations One Stack Configuration on page 12 and Two Stack Configuration on page 15 These configurations optimize the system flow path ensuring minimum delay volume Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 85 4 System Setup and Installation Priming the System When Parts required Preparations Initial Priming Before a new degasser or new solvent tubing can be used it is necessary to prime the system Isopropanol IPA is recommended as priming solvent due to its miscibility with nearly all HLPC solvents and its excellent wetting properties Description 1 lsopropanol Connect all modules hydraulically as described in the respective module manuals Fill each solvent bottle with 100 mL isopropanol Switch
51. f the Agilent 1260 Infinity Quaternary LC VL 3 Optimizing Detection with DAD Introduction The detector has a variety of parameters that can be used to optimize performance Depending on whether signal or spectral data need to be optimized different settings are recommended The following sections describe optimization for e signal sensitivity selectivity and linearity e spectral sensitivity and resolution DAD only and e disk space required for storing data The information in this chapter should be seen as a basic introduction to diode array detector techniques Some of these techniques may not be available in the instrument software controlling the detector How to Get the Best Detector Performance The information below will guide you on how to get the best detector performance Follow these rules as a start for new applications It gives rules of thumb for optimizing detector parameters Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 63 3 Optimization of the Agilent 1260 Infinity Quaternary LC VL Optimization Overview Table 10 Optimization Overview Parameter Impact 1 Selection of flow cell Choose flow cell according to used column see Choosing a Flow Cell on page 65 2 Connection of flow cell For flow rates from 0 5 ml min connect column using the zero dead volume fittings of the detector For small column i d e g 1 mm the inlet capillary of the micro flow cell
52. ficantly higher than expected the following procedure can be used to clean the needle seat Go to MORE INJECTOR and set needle to home position e Pipette an appropriate solvent on to the needle seat The solvent should be able to dissolve the contamination If this is not known use 2 or 3 solvents of different polarity Use several milliliters to clean the seat e Clean the needle seat with a tissue and remove all liquid from it RESET the injector Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Optimization of the Agilent 1260 Infinity Quaternary LC VL 3 Fast Injection Cycle and Low Delay Volume Short injection cycle times for high sample througput is one of the most important requirements in analytical laboratories In order to shorten cycle times you can e shorten the column length e use high flow rates e apply a steep gradient Having optimized these parameters further reduction of cycle times can be obtained using the overlapped injection mode Overlapped Injection Mode In this process as soon as the sample has reached the column the injection valve is switched back to bypass and the next injection cycle starts but waits with switching to mainpass until the actual run is finished You gain the sample preparation time when using this process Switching the valve into the bypass position reduces the system delay volume the mobile phase is directed to the column without passing sample loop n
53. flow 1 mL min LC grade methanol Noise 0 15 10 AU G1314E D 0 25 10 AU G1314F at 230 nm TC 2 s RT 2 2 TC Linearity Linearity is measured with caffeine at 265 nm The specification are based on the the standard RFID tag lamp G1314 60101 and may be not achieved when other lamp types or aged lamps are used ASTM drift tests require a temperature change below 2 C hour 3 6 F hour over one hour period Our published drift specification is based on these conditions Larger ambient temperature changes will result in larger drift Better drift performance depends on better control of the temperature fluctuations To realize the highest performance minimize the frequency and the amplitude of the temperature changes to below 1 C hour 1 8 F hour Turbulences around one minute or less can be ignored Performance tests should be done with a completely warmed up optical unit gt one hour ASTM measurements require that the detector should be turned on at least 24 hours before start of testing Time Constant versus Response Time According to ASTM E1657 98 Standard Practice of Testing Variable Wavelength Photometric Detectors Used in Liquid Chromatography the time constant is converted to response time by multiplying by the factor 2 2 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference ece 3 Optimization of the Agilent 12
54. for flow cells 66 80 cutoff filter 34 D data evaluation and control 43 data rate sampling rate 45 dead volume 41 declaration of conformity 109 delay volume 40 62 detectiontype 44 detection compound classes 74 detector warmup 76 dimension 39 diode width 43 draw speed 58 58 DRAW 58 drift ASTM and noise 43 drift 45 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference dual piston in series design 17 E ejectspeed 58 58 EJECT 58 electronic waste 103 entrance slit assembly 33 extra column volume description 62 F features GLP 41 46 safety and maintenace 41 46 safety and maintenance 44 flow cell max light cartridge 28 solvent information 107 flow accuracy 40 flow cell correction factors 66 80 specifications 43 types and data 45 with RFID tag 32 flow precision 40 flow range 40 G GLP features 44 gradient formation 40 grating assembly 35 111 Index holmium oxide declaration of conformity 109 filter 34 hydraulic system 40 information on UV radiation 108 injection volume precision 58 injection volumes less than 2 ul 58 injection sequence 22 injection valve 19 injection volumes 58 Instant Pilot G4208A 30 internet 110 introduction to the autosampler 19 introduction 30 to optical system 31 L lamp type 44 uv 27 with RFID tag 33 linear range 43 linearity 45 specifications 47 line voltage and frequency 38 lithium batteries 104 loading default 93
55. ge of absorbance in the flow cell The detector uses digital filters to adapt response time to the width of the peaks in your chromatogram These filters do not affect peak area nor peak symmetry When set correctly such filters reduce baseline noise significantly Figure 18 on page 67 but reduce peak height only slightly In addition these filters reduce the data rate to allow Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Optimization of the Agilent 1260 Infinity Quaternary LC VL 3 optimum integration and display of your peaks and to minimize disk space required to store chromatograms and spectra DAD A Sig 250 100 Rem300 100 042 12AINOI_PW_160H 0 DADI A Sige20 100 Ret200 100 042 12ANOI_Pw_25 0 mau Response time 2 s PW gt 0 10 min 2 0 s response time 2 5 Hz PW lt 0 0016 min 0 016 s response time 160 Hz Response time 0 016 s Slit width 4 nm o os 1 15 2 25 3 35 o a5 mis Figure 18 Influence of Response Time on Signal and Noise Table 11 on page 68 lists the filter choices of the detector To get optimum results set peak width as close as possible to a narrow peak of interest in your chromatogram Response time will the be approximately 1 3 of the peak width resulting in less than 5 peak height reduction and less than 5 additional peak dispersion Decreasing the peak width setting in the detector will result in less than 5 gain in peak height but baseline noise will i
56. ge should be avoided whenever possible When inevitable this has to be carried out by a skilled person who is aware of the hazard involved Do not attempt internal service or adjustment unless another person capable of rendering first aid and resuscitation is present Do not replace components with power cable connected Do not operate the instrument in the presence of flammable gases or fumes Operation of any electrical instrument in such an environment constitutes a definite safety hazard Do not install substitute parts or make any unauthorized modification to the instrument Capacitors inside the instrument may still be charged even though the instrument has been disconnected from its source of supply Dangerous voltages capable of causing serious personal injury are present in this instrument Use extreme caution when handling testing and adjusting When working with solvents please observe appropriate safety procedures e g goggles safety gloves and protective clothing as described in the material handling and safety data sheet by the solvent vendor especially when toxic or hazardous solvents are used Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Appendix 6 The Waste Electrical and Electronic Equipment Directive Abstract The Waste Electrical and Electronic Equipment WEEE Directive 2002 96 EC adopted by EU Commission on 13 February 2003 is introducing producer responsibility on all electric
57. ing the system for operation Agg Agilent Technologies 83 4 System Setup and Installation Installing Software 84 Installing the Software Controller and Data System For details of installation procedures for the software refer to the detector manual and the software manuals Installing the Agilent Lab Advisor Software For details of installation procedures for the Agilent Lab Advisor software refer to the software documentation on the Lab Advisor DVD Agilent Lab Advisor replaces and extends upon the diagnostic functions that were formerly only in the ChemStation software Agilent Lab Advisor is a Windows based application that continuously monitors instruments in the lab in real time and increases productivity through automatic notification of maintenance and service needs with the use of advanced counters This allows a problem to be fixed before it impacts results The software includes an extensive suite of user information and documentation a set of calculators and tools to help set up calibrate and maintain your instrument and tests and diagnostic routines to verify proper performance Agilent Lab Advisor also provides feedback and solutions for any instrument errors that may arise The software will work with or without Agilent data systems The software monitors LC module status e Early Maintenance Feedback to determine the need for upgrade or replacement In addition the software Automates useful t
58. into the specified wash vial before injection For example 1 DRAW 5 ul 2 NEEDLE WASH vial 7 3 INJECT Line 1 draws 5 ul from the current sample vial Line 2 moves the needle to vial 7 Line 3 injects the sample valve switches to main pass Using an Injector Program The process is based on a program that switches the bypass grove of the injection valve into the flow line for cleaning This switching event is performed at the end of the equilibration time to ensure that the bypass grove is filled with the start concentration of the mobile phase Otherwise the separation could be influenced especially if microbore columns are used Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 55 3 56 Optimization of the Agilent 1260 Infinity Quaternary LC VL For example Outside wash of needle in vial 7 before injection Injector program Draw x x y ul from sample NEEDLE WASH vial 7 Inject Wait equilibration time see text above Valve bypass Wait 0 2 min Valve mainpass Valve bypass Valve mainpass Overlapped injection together with additional injection valve switching is not possible General Recommendation to Lowest Carry over e For samples where needle outside cannot be cleaned sufficiently with water or alcohol use wash vials with an appropriate solvent Using an injector program and several wash vials can be used for cleaning In case the needle seat has got contaminated and carry over is signi
59. ise 70 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Optimization of the Agilent 1260 Infinity Quaternary LC VL 3 Because the detector averages absorbance values that are calculated for each wavelength using a wide bandwidth does not negatively impact linearity The use of a reference wavelength is highly recommended to further reduce baseline drift and wander induced by room temperature fluctuations or refractive index changes during a gradient An example of the reduction of baseline drifts is shown in Figure 21 on page 71 for PTH amino acids Without a reference wavelength the chromatogram drifts downwards due to refractive index changes induced by the gradient This is almost completely eliminated by using a reference wavelength With this technique PTH amino acids can be quantified in the low picomole range even in a gradient analysis Z y lt q Ta c 1pmol each i 2 TE i a a Wavelength 267 nm Reference 380nm Wavelength 267nm No reference Time min Grad 0 02 m KH P0 ACN from 12 ACN to 45 ACN in 12 min Figure 21 Gradient Analysis of PTH Amino Acids 1 pmol each with and without Reference Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 71 3 72 Optimization of the Agilent 1260 Infinity Quaternary LC VL Optimizing Spectral Acquisition DAD only Storage of all spectra consumes a lot of disk space It is very useful to have all spectra available
60. ited to the following TLVs Threshold Limit Values according to the American Conference of Governmental Industrial Hygienists Table 15 UV Radiation Limits Exposure day Effective Irradiance 8 hours 0 1 pW cm 10 minutes 5 0 W cm Typically the radiation values are much smaller than these limits Table 16 UV Radiation Typical Values Position Effective Irradiance Lamp installed 50 cm distance average 0 016 pW cm2 Lamp installed 50 cm distance maximum 0 14 pW cm2 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Appendix 6 Declaration of Conformity for HOX2 Filter Declaration of Conformity for HOX2 Filter We herewith inform you that the Declaration of Conformity Holmium Oxide Glass Filter used in Agilents absorbance detectors listed in the table below meets the requirements of National Institute of Standards and Technology NIST to be applied as certified wavelength standard According to the publication of NIST in J Res Natl Inst Stand Technol 112 303 306 2007 the holmium oxide glass filters are inherently stable with respect to the wavelength scale and need no recertification The expanded uncertainty of the certified wavelength values is 0 2 om Agilent Technologies guarantees as required by NIST that the material of the filters is holmium oxide glass representing the inherently existent holmium oxide absorption bands Test wavelengths Product Number 79883A 79854A G1306
61. l the liquid through the degasser Reattach the tubing to the solvent selection valve Pump 30 mL isopropanol to remove residual air bubbles Switch to the next solvent channel and repeat steps 5 and 6 until all channels have been purged Turn the flow off and close the purge valve Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 87 4 System Setup and Installation Regular Priming When When the pumping system has been turned off for a certain time for example overnight air will rediffuse into the solvent channel between the vacuum degasser and the pump Solvents containing volatile ingredients will slightly lose these if left in the degasser without flow for a prolonged period of time Preparations Switch the system on The purge tool of the LabAdvisor or Instrument Utilities can be used for automatically purging the pump 1 Open the purge valve of your pump by turning it counterclockwise and set the flow rate to 5 mL min Flush the vacuum degasser and all tubes with at least 10 mL of solvent Repeat step 1 and 2 for the other channel s of the pump Set the required composition and flow rate for your application and close the purge valve 5 Pump for approximately 10 minutes before starting your application 88 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference When Parts required Preparations CAUTION System Setup and Installation 4 Changing Solvents When the solvent of a
62. lamp mirror 2 onto the entrance of the Max light cartridge flow cell 3 with optofluidic waveguides The light leaves the Max light cartridge flow cell at the other side and is focused by the fold mirror 4 through the slit assembly 5 onto a holographic grating 6 light being dispersed onto the diode array 7 This allows simultaneous access to all wavelength information 26 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Introductio 1 Detector Lamp The light source for the UV wavelength range is a long life UV lamp with RFID tag As a result of plasma discharge in low pressure deuterium gas the lamp emits light over the 190 nm to approximately 800 nm wavelength range Figure 12 UV Lamp Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 27 1 28 Introduction Max Light Cartridge Flow Cell The detector allows easy access to flow cells via a cartridge A variety of optional flow cells can be inserted using the same quick simple mounting system A Max Light Cartridge Cell 10 mm V o 1 0 uL and a Max Light Cartridge Cell 60 mm V o 4 uL are available For testing of the detector a Max Light Cartridge Test Cell is available The optical principle of the Max Light Cartridge cell is based on opto fluidic waveguides Nearly 100 light transmission is achieved by utilizing total internal reflection in a non coated silica fiber Compromising refractive index and thermal effects are
63. lf height To determine the volume of your peaks take the peak width as reported in the integration results multiply it by the flow rate and divide it by 3 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 79 3 Optimization of the Agilent 1260 Infinity Quaternary LC VL Analysis of pesticide standard as 6 mm optical path length oO E 10 mm optical path length io 2 j r Nl ho UAL Uh ak a I KW 1 poe y V J HE d y v J rr oe EAA A A aA n 18 20 30 Time min Figure 29 Influence of Cell Path Length on Signal Height Traditionally LC analysis with UV detectors is based on comparing measurements with internal or external standards To check photometric accuracy of the Agilent 1200 Series Infinity Variable Wavelength Detector it is necessary to have more precise information on path lengths of the VWD flow cells The correct response is expected response correction factor Please find below the details of the Agilent 1200 Infinity Series Variable Wavelength Detector flow cells Table 12 Correction factors for Agilent VWD flow cells Part number Path length actual Correction factor Standard flow cell 10 mm 14 pL p n G1314 60186 10 15 0 19 mm 10 10 15 Semi micro flow cell 6 mm 5 pL p n G1314 60183 6 10 0 19 mm 6 6 10 Micro flow cell 3 mm 2 pL p n G1314 60187 2 80 0 19 mm 3 2 8 High pressure flow cell 10 mm 14 uL p n G1314 60182 10 00 0 19 mm 6 5 75
64. low volume injections 58 M mainpass 22 max light cartridge 112 flow cell 28 metering device 58 method settingup 97 mirror assemblies 34 multi draw option 19 negative absorbance 72 network integration 90 noise and drift ASTM 43 Noise and Linearity specifications 47 48 noise short term 44 0 online plot configuring 94 operating temperature 38 operational hints vacuum degasser 50 operational pressure range 40 optical unit flow cell 32 optical unit beam splitter assembly 35 entrance slit assembly 33 filter assembly 34 filter 34 grating assembly 35 lamp 33 mirrors 34 photodiode assemblies 35 photodiode boards 35 source lens assembly 33 stepper motor 35 optical 26 optimization detector performance 78 detector performance 63 for sensitivity selectivity linearity dispersion 65 how to get the best performance 63 margins for negative absorbance 72 of selectivity 73 overview 64 peak width 66 sample and reference wavelength 68 spectra acquisition 72 stack configuration 12 85 optimizing the performance 61 optimizing performance automated needle wash 58 delay volume 58 delay volume adjustment 58 inject valve seal 58 low volume capillary kit 58 maintenance 58 optimizing flow cell 65 overview pump 17 overview optical path 31 optical system 31 system overview 31 P peak width response time 66 performance specifications autosampler 42 performance optimization 63 78 optimizing 61 specific
65. m bw 100 nm ref 360 nm bw 100 nm Peak width 0 012 min 0 25 s Response 20 Hz b In the Advanced section set Spectrum Store to All c Other parameters can remain at default settings Click OK to exit the window The changes are sent to the DAD module All the required module parameters have now been entered Select Method gt Save Method As SO 1 M to save the method with a new name The ChemStation will not allow the method to be saved as DEF_LC M so that the default method template is not altered Allow the system to equilibrate for at least 10 min and check that the baseline in the Online Plot is stable before starting the analysis Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 6 Appendix Safety 100 The Waste Electrical and Electronic Equipment Directive 103 Lithium Batteries Information 104 Radio Interference 105 Sound Emission 106 Solvent Information 107 UV Radiation 108 Declaration of Conformity for HOX2 Filter 109 Agilent Technologies on Internet 110 This chapter provides addition information on safety legal web and the Edit Entire Method ee Agilent Technologies 99 6 Appendix Safety Safety Symbols Table 14 Safety Symbols Symbol Description The apparatus is marked with this symbol when the user should refer to the instruction manual in order to protect risk of harm to the operator and to
66. n of the Agilent 1260 Infinity Quaternary LC VL 3 Optimizing Selectivity Quantifying Coeluting Peaks by Peak Suppression In chromatography two compounds may often elute together A conventional dual signal detector can only detect and quantify both compounds independently from each other if their spectra do not overlap However in most cases this is highly unlikely With a dual channel detector based on diode array technology quantifying two compounds is possible even when both compounds absorb over the whole wavelength range The procedure is called peak suppression or signal subtraction As an example the analysis of hydrochlorothiazide in the presence of caffeine is described If hydrochlorothiazide is analyzed in biological samples there is always a risk that caffeine is present which might interfere chromatographically with hydrochlorothiazide As the spectra in Figure 22 on page 73 shows hydrochlorothiazide is best detected at 222 nm where caffeine also shows significant absorbance It would therefore be impossible with a conventional variable wavelength detector to detect hydrochlorothiazide quantitatively when caffeine is present WL1 204 nm caffeine WL2 222 nm hydrochlorothiazide WL3 260 nm reference to suppress hydrochlorothiazide WL4 282 nm reference to suppress caffeine WL3 W4 b 1k va 582 38k WLI WL Wavelength nm Figure 22 Wavelength Selection for Peak Suppression With a UV
67. ncrease by a factor of 1 4 for a factor of 2 response time reduction Increasing peak width response time by factor of two from the recommended setting over filtering will reduce peak height by about 20 and reduce baseline noise by a factor of 1 4 This gives you the best possible signal to noise ratio but may affect peak resolution Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 67 3 Optimization of the Agilent 1260 Infinity Quaternary LC VL Table 11 Peak Width Response Time Data Rate Peak width at half Response Signaldata Scan data rate Scan data rate Scan data rate Scan data rate height min s rate Hz HZ HZ HZ HZ 126 pts scan 251 pts scan 501 pts scan gt 501 pts scan lt 0 0016 0 016 1602 1602 80 40 20 gt 0 0016 0 03 1602 1602 80 40 20 gt 0 003 0 062 80 80 80 80 40 gt 0 006 0 12 40 40 40 40 40 gt 0 012 0 25 20 20 20 20 20 gt 0 025 0 5 10 10 10 10 10 gt 0 05 1 0 5 5 5 5 5 gt 0 10 2 0 2 5 2 5 2 5 2 5 2 5 gt 0 20 4 0 1 25 1 25 1 25 1 25 1 25 gt 0 40 8 0 0 625 0 62 0 625 0 625 0 625 gt 0 85 16 0 0 3125 0 31 0 3125 0 3125 0 3125 Values in the User Interface may be rounded 2 64212A only The maximum spectra scan rate depends on the data points per scan see Table 11 on page 68 Running at 160 Hz the spectra scan data rate is reduced automatically if the spectra scan data rate is more than 251 points scan Sample and Reference Wavelength and Bandwidth
68. nit The reference diode assembly is located in the front of the optical unit Photo Diode ADC analog to digital converter The photo diode current is directly converted to digital data direct photo current digitalization The data is transferred to the detector main board The photo diode ADC boards are located close to the photo diodes Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 35 1 36 Introduction Detector Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 7 ee 2 7 e Specifications e a e a Physical Specifications 38 Performance Specifications 40 Specification Conditions 47 This chapter provides information about specifications for the LC system ee Agilent Technologies 37 2 Specifications Physical Specifications 38 Table2 General p hysical specifications Type Specification Comments Line voltage Line frequency Ambient operating temperature Ambient non operating temperature Humidity Operating altitude Non operating altitude Safety standards IEC CSA UL EN 100 240 VAC 10 50 or 60 Hz 5 4 55 C 32 131 F 40 70 C 4 158 F lt 95 at 25 40 C 77 104 F Up to 2000 m 6562 ft Up to 4600 m 15092 ft Wide ranging capability See warning Hot rear panel on page 39 Non condensing
69. nt Pilot Detector Column compartment Autosampler Fu ENX c S 7 E oo T 2 I Sais Thermostat for the ALS optional Solvent cabinet Degasser optional Pump Figure 3 Two stack configuration front view Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 15 1 Introduction Optimizing the Stack Configuration LAN to control software CAN Bus cable to Instant Pilot Thermo cable optional ae Remote cable AC Power CAN Bus cable AC Power Figure4 Two stack configuration rear view 16 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Introduction 1 Quaternary pump The quaternary pump is based on a two channel dual plung
70. ntrol only Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 45 2 Specifications Table8 Performance Specifications G1314F Type Specification Comments Communications Safety and maintenance GLP features Housing LAN card integrated on main board Controller area network CAN RS 232C APG Remote ready start stop and shut down signals Extensive diagnostics error detection and display through Instant Pilot and Data System leak detection safe leak handling leak output signal for shutdown of pumping system Low voltages in major maintenance areas Early maintenance feedback EMF for continuous tracking of instrument usage in terms of lamp burn time with user settable limits and feedback messages Electronic records of maintenance and errors Verification of wavelength accuracy with built in holmium oxide filter RFID for electronics records of flow cell and UV lamp conditions path length volume product number serial number test passed usage All materials recyclable 46 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Specifications 2 Specification Conditions Specification Conditions DAD ASTM Standard Practice for Variable Wavelength Photometric Detectors Used in Liquid Chromatography Reference conditions e Wavelength 230 nm 4 nm with Reference Wavelength 360 nm 100 nm Slitwidth 4 nm TC 2 s or with RT 2 2 TC ASTM e
71. ocratic and also during a gradient analysis The residual amount of sample remaining on the outside of the needle after injection may contribute to carry over in some instances When using small injection volumes or when injecting samples of low concentration immediately after samples of high concentration carry over may become noticeable Using the automated needle wash enables the carry over to be minimized and prevents also contamination of the needle seat Using the Automated Needle Wash The automated needle wash can be programmed either as injection with needle wash or the needle wash can be included into the injector program When the automated needle wash is used the needle is moved into a wash vial after the sample is drawn By washing the needle after drawing a sample the sample is removed from the surface of the needle immediately 54 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Optimization of the Agilent 1260 Infinity Quaternary LC VL 3 Uncapped Wash Vial For best results the wash vial should contain solvent in which the sample components are soluble and the vial should not be capped If the wash vial is capped small amounts of sample remain on the surface of the septum which may be carried on the needle to the next sample Injector Program with Needle Wash The injector program includes the command NEEDLE WASH When this command is included in the injector program the needle is lowered once
72. onitored otherwhise connect a signal cable between the pressure output of the pump and a recording device for example 339X integrator and set parameters Zero 50 Att 2 3 Chart Speed 10 cm min 4 Start the recording device with the plot mode 5 Starting with a compressibility setting of 10 x 10 bar increase the value in steps of 10 Re zero the integrator as required The compressibility compensation setting that generates the smallest pressure ripple is the optimum value for your solvent composition Table9 Solvent Compressibility Solvent pure Compressibility 10 6 bar Acetone Acetonitrile Benzene Carbon tetrachloride Chloroform Cyclohexane Ethanol Ethyl acetate Heptane Hexane Isobutanol lsopropanol Methanol 1 Propanol Toluene Water 126 115 95 110 100 118 114 104 120 150 100 100 120 100 87 46 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 53 3 Optimization of the Agilent 1260 Infinity Quaternary LC VL Optimizing the Autosampler Optimization for Lowest Carry over Several parts of an injection system can contribute to carry over e needle outside e needle inside e needle seat e sample loop e seat capillary e injection valve The autosampler continuous flow through design ensures that sample loop needle inside seat capillary and the mainpass of the injection valve is always in the flow line These parts are continuously flushed during an is
73. osampler G1329B Type Specification Pressure Operating range 0 60 MPa 0 600 bar 0 8850 psi GLP features Early maintenance feedback EMF electronic records of maintenance and errors Communications Safety features Injection range Replicate injections Precision Minimum sample volume Carryover Sample viscosity range Sample capacity Injection cycle time Controller area network CAN GPIB IEEE 448 RS232C APG remote standard optional four external contact closures and BCD vial number output Leak detection and safe leak handling low voltages in maintenance areas error detection and display 0 1 100 uL in 0 1 pL increments recommended 1 L increments Up to 1500 pL with multiple draw hardware modification required 1 99 from one vial Typically lt 0 25 RSD of peak areas from 5 100 pL Typically lt 1 RSD of peak areas from 1 5uyL 1 pL from 5 pL sample in 100 pL microvial or 1 uL from 10 pL sample in 300 uL microvial Typically lt 0 1 lt 0 05 with external needle cleaning 0 2 50 cp 100 x 2 mL vials in 1 tray 40 x2 mL vials in tray 15 x 6 mL vials in tray Agilent vials only 50 s for draw speed 200 pL min ejection speed 200 pL min injection volume 5 pL 42 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Performance Specifications G4212B Specifications 2 Table 7 Performance Specifications G4212B Type Specification Comments Dete
74. own to 2 ul example pw 0 04 min at flow 0 1 ml min gives peak dispersion of 0 04 min x 0 1 ml min 0 004 ml 4 ul If higher sensitivity is necessary the Max Light Cartridge Cell with path length 60 mm and volume 6 4 uL can be used This cell enhances the detector by lowering the limit of detection LOD by a factor of about 3 depending on the application Optimizing for Sensitivity Selectivity Linearity and Dispersion Flow Cell Path Length Lambert Beer s law shows a linear relationship between the flow cell path length and absorbance Absorbance log T log exCxd where T is the transmission defined as the quotient of the intensity of the transmitted light I divided by the intensity of the incident light Ip is the extinction coefficient which is a characteristic of a given substance under a precisely defined set of conditions of wavelength solvent temperature and other parameters C mol L is the concentration of the absorbing species and d cm is the path length of the cell used for the measurement The detector can now output the signal in two forms 1 In Absorbance divide by the path length AU cm that is then similar to x C Advantage samples with same concentration have same peak height also at cells with different path lengths Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 65 3 Optimization of the Agilent 1260 Infinity Quaternary LC VL The upper limit of
75. product or loss of important data Do not proceed beyond a CAUTION notice until the indicated condi tions are fully understood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly performed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indi cated conditions are fully under stood and met Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference In This Book In This Book This book describes the Agilent 1260 Infinity Quaternary LC VL 1 Introduction This chapter gives an introduction to the Agilent 1260 Infinity Quaternary LC VL the underlying concepts and the features of the Agilent 1260 Infinity Quaternary LC VL 2 Specifications This chapter provides information about specifications for the LC system 3 Optimization of the Agilent 1260 Infinity Quaternary LC VL This chapter considers how to apply the theory and use the features of the LC system to develop optimized separations 4 System Setup and Installation This chapter includes information on software installation stack configurations and preparing the system for operation 5 Quick Start Guide This chapter provides information on data acquisition and data analysis with the Agilent 1260 Infinity Quaternary LC VL 6 Appendix This chapter provides addition information on safety legal web and the E
76. protect the apparatus against damage Indicates dangerous voltages Indicates a protected ground terminal Indicates eye damage may result from directly viewing the light produced by the deuterium lamp used in this product The apparatus is marked with this symbol when hot surfaces are available A and the user should not touch it when heated up im alerts you to situations that could cause physical injury or death gt Do not proceed beyond a warning until you have fully understood and met the indicated conditions CAUTION A CAUTION alerts you to situations that could cause loss of data or damage of equipment gt Do not proceed beyond a caution until you have fully understood and met the indicated conditions 100 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Appendix 6 General Safety Information The following general safety precautions must be observed during all phases of operation service and repair of this instrument Failure to comply with these precautions or with specific warnings elsewhere in this manual violates safety standards of design manufacture and intended use of the instrument Agilent Technologies assumes no liability for the customer s failure to comply with these requirements WARNING Ensure the proper usage of the equipment The protection provided by the equipment may be impaired gt The operator of this instrument is advised to use the equipment in a m
77. re 2 on page 14 This configuration optimizes the flow path for minimum delay volume and minimizes the bench space required 12 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Introduction 1 Optimizing the Stack Configuration Solvent cabinet oe e Pump S 900 000 828 Fy oo Local user interface oe p Autosampler Thermostatted column compartment Detector Figure 1 Recommended Stack Configuration Front View Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 13 1 Introduction Optimizing the Stack Configuration AC power CAN bus cable to local user interface Remote cable CAN bus cable LAN to control software location depends on detector EEEEELLJ Analog detector signal 1 or 2 outputs per detector Figure 2 Recommended Stack Configuration Rear View 14 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Introduction 1 Two Stack Configuration To avoid excessive height of the stack when the autosampler thermostat is added to the system it is recommended to form two stacks Some users prefer the lower height of this arrangement even without the autosampler thermostat A slightly longer capillary is required between the pump and autosampler See Figure 3 on page 15 and Figure 4 on page 16 Insta
78. ro Micro High Pressure Maximum pressure 40 4 40 4 120 12 400 40 bar Path length 10 conical 6 conical 3 conical 10 conical mm Volume 14 5 2 14 uL Inlet i d 0 17 0 17 0 12 0 17 mm Inlet length 750 750 310 310 mm Outlet i d 0 25 0 25 0 17 0 25 mm Outlet length 120 120 120 120 mm Materials in contact with solvent SST quartz PTFE PEEK SST quartz PTFE SST quartz PTFE SST quartz Kapton 32 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Introduction 1 Lamp The light source for the UV wavelength range is a deuterium lamp As a result of plasma discharge in a low pressure deuterium gas the lamp emits light over the 190 600 nm wavelength range The lamp has an integrated RFID tag that contains the lamp specific information e g part number burn time A RFID tag reader reads out this information and transfers it to the user interface Source Lens Assembly The source lens receives the light from the deuterium lamp and focuses it onto the entrance slit Entrance Slit Assembly The entrance slit assembly has an exchangeable slit The standard one has a 1 mm slit For replacement and calibration purposes to optimize the alignment a slit with a hole is needed Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 33 1 introduction Filter Assembly The filter assembly is electromechanically actuated During wavelength calibrations it moves into the light p
79. roduction Autosampler Injection sequence Before the start of the injection sequence and during an analysis the injection valve is in the mainpass position Figure 7 on page 22 In this position the mobile phase flows through the autosamplers metering device sample loop and needle ensuring all parts in contact with sample are flushed during the run thus minimizing carry over Figure 7 Mainpass Position When the sample sequence begins the valve unit switches to the bypass position Figure 8 on page 22 Solvent from the pump enters the valve unit at port 1 and flows directly to the column through port 6 Figure 8 Bypass Position Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Introduction 1 Autosampler Next the needle is raised and the vial is positioned below the needle The needle moves down into the vial and the metering unit draws the sample into the sample loop Figure 9 on page 23 Figure 9 Drawing the Sample When the metering unit has drawn the required volume of sample into the sample loop the needle is raised and the vial is replaced in the sample tray The needle is lowered into the needle seat and the injection valve switches back to the mainpass position flushing the sample onto the column Figure 10 on page 23 1 Yt Figure 10 Mainpass Position Sample Injection Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 23 1 Introduction Thermost
80. roperties of the flow cell Use of Solvents Observe the following recommendations on the use of solvents e Brown glass ware can avoid growth of algae e Avoid the use of the following steel corrosive solvents Solutions of alkali halides and their respective acids for example lithium iodide potassium chloride and so on High concentrations of inorganic acids like sulfuric acid and nitric acid especially at higher temperatures Gif your chromatography method allows replace by phosphoric acid or phosphate buffer which are less corrosive against stainless steel Halogenated solvents or mixtures which form radicals and or acids for example 2CHCl O gt gt 2COCI 2HC1 This reaction in which stainless steel probably acts as a catalyst occurs quickly with dried chloroform if the drying process removes the stabilizing alcohol Chromatographic grade ethers which can contain peroxides for example THF dioxane di isopropylether such ethers should be filtered through dry aluminium oxide which adsorbs the peroxides Solvents containing strong complexing agents e g EDTA Mixtures of carbon tetrachloride with 2 propanol or THF Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 107 6 Appendix UV Radiation 108 Emissions of ultraviolet radiation 200 315 nm from this product is limited such that radiant exposure incident upon the unprotected skin or eye of operator or service personnel is lim
81. ry LC VL Agilent Technologies 91 5 Quick Start Guide Preparing the System Turning the System ON If the system is not already fully on with the software showing Ready status follow these steps 1 Turn on the computer system and wait for the Windows desktop to appear 2 Turn on the electrical power to the LC modules using the button at the lower left of each module A green power on light will be visible in the center of the button 3 Start the control software on the computer by clicking the icon if configured Alternatively you can select Start gt All Programs gt Agilent ChemStation gt Agilent ChemStation Instrument 1 Online As more than one instrument system may be connected to the computer the number 1 2 indicates the system number The ChemStation software opens in the Method and Run Control view The modules are initially in Standby mode and Not Ready status except for the autosampler which immediately initializes and becomes Ready 4 To switch on each module individually right click the relevant icon and select Switch module name on from the context menu Alternatively you can turn on all modules simultaneously in the system by clicking the System On Off button in the bottom right of the system diagram The system status changes from Not Ready yellow indication to Ready green indication after a short delay as the setpoints are attained 92 Agilent 1260 Infinity Quaternary LC VL Manual and
82. t case for a wash seal and drastically reduces its lifetime The seal will build up sticky layers on the surface of the piston These sticky layers will also reduce the lifetime of the pump seal Therefore the tubes of the wash option should always be filled with solvent to prolong the lifetime of the wash seal Always use a mixture of LC grade water 90 and isopropanol 10 as wash solvent This mixture prevents growth of algae or bacteria in the wash bottle and reduces the surface tension of the water Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 51 3 52 Optimization of the Agilent 1260 Infinity Quaternary LC VL Choosing the Right Pump Seals The standard seal for the pump can be used for most applications However applications that use normal phase solvents for example hexane are not suited for the standard seal and require a different seal when used for a longer time in the pump For applications that use normal phase solvents for example hexane we recommend using polyethylene pump seals PE seals pack of 2 p n 0905 1420 and Wash Seal PE p n 0905 1718 These seals have less abrasion compared to the standard seals Polyethylene seals have a limited pressure range of 0 200 bar When used above 200 bar their lifetime is reduced significantly DO NOT apply the seal wear in procedure performed with new standard seals at 400 bar Optimize the Compressibility Compensation Setting The compressi
83. the system on When opening capillary or tube fittings solvents may leak out The handling of toxic and hazardous solvents and reagents can bear health risks gt 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 86 The purge tool of the LabAdvisor or Instrument Utilities can be used for automatically purging the pump If the pump is not able to aspirate the solvent from the bottles a syringe can be used to draw the solvent manually through tubing and degasser When priming the vacuum degasser with a syringe the solvent is drawn through the degasser tubes very quickly The solvent at the degasser outlet will therefore not be fully degassed Pump for approximately 10 minutes at your desired flow rate before starting an analysis This will allow the vacuum degasser to properly degas the solvent in the degasser tubes Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference oF O N System Setup and Installation 4 Open the purge valve of the pump Set the flow rate to 5 mL min Select channel Al Turn the flow on Observe if the solvent in the tubing of channel A1 is advancing towards the pump If it isn t disconnect the solvent tubing from the solvent selection valve attach a syringe with a syringe adapter and pul
84. through the flow cell is absorbed depending on the solutions in the cell in which UV absorption takes place and the intensity is converted to an electrical signal by means of the sample photodiode Part of the light is directed to the reference photodiode by the beam splitter to obtain a reference signal for compensation of intensity fluctuation of the light source A slit in front of the reference photodiode cuts out light of the sample bandwidth Wavelength selection is made by rotating the grating which is driven directly by a stepper motor This configuration allows fast change of the wavelength The cutoff filter is moved into the lightpath above 370 nm to reduce higher order light Deuterium lamp am a Filter assembly Entrance slit Mirror M1 Lens Sample diode Flow cell Grating Mirror M2 Beam splitter eference diode Figure 15 Optical Path of the Variable Wavelength Detector Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 31 1 introduction Flow Cell A variety of flow cell cartridges can be inserted using the same quick and simple mounting system The flow cells have an integrated RFID tag that contains the flow cell specific information e g part number cell volume path length A RFID tag reader reads out this information and transfers it to the user interface LUA N RFID tag Figure 16 Flow Cell with RFID tag Table 1 Flow Cell Data STD Semi mic
85. tion Agilent 1260 Infinity Quaternary Pump VL G1311C Type Hydraulic system Setable flow range Flow range Flow precision Flow accuracy Pressure Pressure pulsation Compressibility compensation Recommended pH range Gradient formation Delay volume Composition range Composition precision Control and data evaluation Analog output Communications Specification Dual piston in series pump with proprietary servo controlled variable stroke drive floating pistons 0 001 10 mL min in 0 001 mL min increments 0 2 10 0 mL min lt 0 07 RSD or lt 0 02 min SD whatever is greater based on retention time at constant room temperature 1 or 10 pL min whatever is greater pumping degassed H30 at 10 MPa Operating range 0 40 MPa 0 400 bar 0 5880 psi up to 5 mL min Operating range 0 20 MPa 0 200 bar 0 2950 psi up to 10 mL min lt 2 amplitude typically lt 1 0 or lt 3 bar at 1 mL min isopropanol at all pressures gt 10 bar 147 psi User selectable based on mobile phase compressibility 1 0 12 5 solvents with pH lt 2 3 should not contain acids which attack stainless steel Low pressure quaternary mixing gradient capability using proprietary high speed proportioning valve 600 900 uL dependent on back pressure 0 95 or 5 100 user selectable lt 0 2 RSD at 0 2 and 1 mL min Agilent control software For pressure monitoring 2 mV bar one ou
86. tput Controller area network CAN RS 232C APG Remote ready start stop and shut down signals LAN optional 40 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Specifications 2 Performance Specifications G1316A Table5 Performance Specifications Thermostatted Column Compartment Type Specification Comments Temperature range Temperature stability Temperature accuracy Column capacity Warm up cool down time Dead volume Communications Safety and maintenance GLP features Housing 10 degrees below ambient to 80 C up to 80 C flow rates up to 5 mL min 0 15 C 0 8 C 0 5 C With calibration Three 30 cm 5 minutes from ambient to 40 C 10 minutes from 40 20 C 3 uL left heat exchanger 6 uL right heat exchanger Controller area network CAN RS 232C APG Remote ready start stop and shut down signals LAN via other 1260 Infinity module Extensive diagnostics error detection and display through Instant Pilot and Agilent data system leak detection safe leak handling leak output signal for shutdown of pumping system Low voltages in major maintenance areas Column identification module for GLP documentation of column type All materials recyclable Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 41 2 Specifications Performance Specifications G1329B Table 6 Performance Specifications Agilent 1260 Infinity Standard Aut
87. ty you should prime the vacuum degasser before running an analysis The vacuum degasser can be primed either by drawing solvent through the degasser with a syringe or by pumping with the quaternary pump Priming the degasser with a syringe is recommended when e vacuum degasser is used for the first time or vacuum tubes are empty or e changing to solvents that are immiscible with the solvent currently in the vacuum tubes Priming the vacuum degasser by using the quaternary pump at high flow rate is recommended when e quaternary pump was turned off for a length of time for example during night and volatile solvent mixtures are used or e solvents have been changed For more information see the Service Manual for the Agilent 1200 Series vacuum degasser Operational Hints for the Multi Channel Gradient Valve MCGV In a mixture of salt solutions and organic solvent the salt solution might be well dissolved in the organic solvent without showing precipitations However in the mixing point of the gradient valve at the boundary between the two solvents micro precipitation is possible Gravity forces the salt particles to fall down Normally the A channel of the valve is used for the aqueous salt solution and the B channel of the pump is used for the organic solvent If used Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Optimization of the Agilent 1260 Infinity Quaternary LC VL 3 in this configuration the
88. ualifiers can be used where in a complex sample only one particular class needs to be analyzed a parent drug and its metabolites in a biological sample for example Another example is the selective analysis of derivatives after pre or post column derivatization Specifying a signal ratio that is typical for the sample class is one way of selectively plotting only those peaks that are of interest The signal output remains at zero so long as the ratio is out of the user specified ratio range When the ratio falls within the range the signal output corresponds to the normal absorbance giving single clear peaks on a flat baseline An example is shown in Figure 24 on page 75 and Figure 25 on page 75 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Optimization of the Agilent 1260 Infinity Quaternary LC VL 3 Select 2 characteristic 1005 wavelengths aa Signal WL1 WL2 20 8a 78 p 7 o Terphenyl 684 s Biphenyl 4e4 B a S 28 Nn n 1a f Wl2 WLI 222 248 260 28a 38a 320 Wavelength nm Figure 24 Wavelength Selection for Ratio Qualifiers Signals at 250 nm Biphenyl o Terphenyl No selectivity With ratio qualifier 250 222 nm 3 520 m ao Time min Figure 25 Selectivity by Ratio Qualifiers In a four component mixture only biphenyl was recorded The other three peaks were suppressed because they did not meet the ratio qualifier criterion and ther
89. urged with fresh solvent before using the system or when the solvent is to be exchanged for another The pump has been idle for a few hours or more air may have diffused into the solvent lines and purging is recommended e The solvent reservoirs are refilled and the pump requires purging to fill the system with fresh solvent If different solvents are to be used ensure that the new solvent is miscible with the previous solvent and if necessary use an intermediate step with a co miscible solvent isopropanol is often a good choice check with a solvent miscibility table For details on the purging procedure refer to Priming the System on page 86 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Quick Start Guide 5 Setting Up the Method This section shows how to quickly set the method conditions for an analysis The default method DEF_LC M has been loaded ready to prepare the new method Now the key parameters can be edited to create the new method 1 To quickly access the Method page for each module right click in the system diagram for the module and select Method from the context menu Each of the modules will be set up in this way 2 Right click the pump area and select Method in the context menu a Inthe Method page for the 1260 Infinity Quaternary Pump enter the following parameters Flow rate 1 5 ml min Solvent A Select Water from the compressibility drop down list Solvent B Sel
90. y vary from instrument to instrument and depends on the environment The example below was done under stable environmental conditions The figures below show the first two hours of a detector warm up phase The lamp was turned on immediately after turn on of the detector 76 Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference Optimization of the Agilent 1260 Infinity Quaternary LC VL 3 Optimizing Detection with DAD Figure 26 Detector Warm up 15 hour Baseline 230 4 360 100 SLW 4 RT 2s Board Temperature ja Figure 27 Detector Warm up 2 hour Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 77 3 Optimization of the Agilent 1260 Infinity Quaternary LC VL Optimizing Detection with VWD Optimizing Detection with VWD Optimizing the Detector Performance The detector has a variety of parameters that can be used to optimize performance The information below will guide you on how to get the best detector performance Follow these rules as a start for new applications It gives a rule of thumb for optimizing the detector parameters Match the Flow Cell to the Column Standard HPLC Applications 0 025 min Micro flow cell 0 05 min Semimicro flow cell a gt ieee tse d flow rate 0 05 0 2 ml min 0 2 0 4 ml min 0 4 0 8 ml min 1 2 ml min ote 01 5 ml min Internal column diameter Figure 28 Choosing a Flow Cell Standard HPLC Applications 78 Agilent 1
91. ydraulic volume of the flow path is suited to fast gradients and the ability to use overlapped injections and automatic delay volume reduction ADVR contribute to faster cycle times and even faster gradient delivery to the column The system draws exactly the set volume of sample solution without waste and achieves high reproducibility across the whole range of possible injection volume The autosampler is controlled from G4208 A Instant Pilot or from the Agilent Data System Three sample rack sizes are available The standard full size rack holds 100 x 1 8 mL vials while the two half size racks provide space for 40 x 1 8 mL Agilent 1260 Infinity Quaternary LC VL Manual and Quick Reference 19 1 20 Introduction vials and 15 x 6 mL vials respectively Any two half size rack trays can be installed in the autosamplers simultaneously A specially designed sample rack holding 100 x 1 8 mL vials is available for use with thermostatted autosamplers The half size racks trays are not designed for an optimal heat transfer when they are used with a thermostatted autosampler The autosamplers transport mechanism uses an X Z Theta movement to optimize vial pick up and return Vials are picked up by the gripper arm and positioned below the sampling unit The gripper transport mechanism and sampling unit are driven by motors Movement is monitored by optical sensors and optical encoders to ensure correct operation The metering device is always flushe

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