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Agilent 1260 Infinity Binary LC

1. Spee ULLA j T Spectrum task toe T 2 se w rT ni J Navigation panel no pamena d aa A 4 j ee tes pen ont mants iatan mte an T J 96 1260 Infinity Binary LC System User Guide Quick Start Guide 5 Data Analysis View To open a chromatogram in the Data Analysis view 1 Launch an offline ChemStation 2 Click Data Analysis in the bottom left of the screen 3 In the Navigation Panel find the data directory containing the data files All the single injection data are represented as a subset called Single Runs Double click Single Runs to load these data files into the Navigation Table 4 Select a file in the Navigation Table and double click it to load the chromatogram into the viewer 1260 Infinity Binary LC System User Guide 97 5 Quick Start Guide Integrating a Signal 1 Select the Integration Task Tool see figure below The Integrate icon and the Set Integration Events Table icon are highlighted in the figure shown below op Calibration ul Signal ay Purify N Spectrum EJ IN Report Short DAD1A Sig 254 MO 005 0105 D J7 El Ey D I Ad AA if HN LALIN fel M DADI A Sig 254 4 Ref 550 100 DEMO 005 0105 D mU 5 Method Manual Events I 100 Initial Events For All Signals J 80 Integration Events Value ii Tangent Skim Mode Sta
2. Pressure Sensor Low delay configuration for 2 1 mm inner diameter columns Figure 13 It is important to remember to set the correct parameter in the pump auxiliary screen This ensures that the correct compressibility values are always applied for the mobile phases used Calibration curves are available for most common solvents 1260 Infinity Binary LC System User Guide 43 3 Optimization of the Agilent 1260 Infinity Binary LC Medium delay volume configuration to achieve highest UV sensitivity For high sensitivity UV applications an additional 200 uL mixer Low volume mixer 200 uL 5067 1565 can be installed to reduce any residual mixing noise This small mixer gives the lowest UV baseline noise even under extreme gradient conditions See Figure 14 on page 44 Medium delay volume 320 ul for ultra fast and superior UV sensitivity with 2 1 mm ID columns 600 bar Damper Pressure Sensor Figure 14 Medium delay volume configuration for 2 1 mm ID columns with highest UV sensitivity 44 1260 Infinity Binary LC System User Guide Optimization of the Agilent 1260 Infinity Binary LC 3 Optimum Instrument Configuration for 3 and 4 6 mm i d Columns Standard delay volume configuration for highest UV sensitivity and direct method transferability The relative column volumes for 3 mm and 4 6 mm inner diameter columns are about two and five times larger respectively than for the
3. Item p n 1 G1312 87303 1 G1312 87304 2 G1313 87304 2 01090 87610 3 G1316 80002 3 G1316 80003 4 01090 87611 5 G1315 87312 6 5062 2462 1260 Infinity Binary LC System User Guide System Setup and Installation Description Capillary ST 0 17 mm x 400 mm S S Pump to autosampler Capillary ST 0 17 mm x 700 mm S S Pump to cooled Autosampler Capillary ST 0 12 mm x 180 mm S S Autosampler to TCC Capillary ST 0 12 mmx 280 mm S S cooled Autosampler to TCC or to item 3 if installed Heater long up 0 12 mm i d 1 6 uL internal volume Heater long down 0 12 mm i d 1 6 uL internal volume Capillary ST 0 12 mmx 105 mm S S TCC to Column not needed if item 3 is installed Capillary ST 0 12 mm x 150 mm S S Column to DAD Tube PTFE 0 8 mm x 2 m re order 5 m DAD to waste 4 75 4 System Setup and Installation Priming the System When Parts required Preparations Initial Priming Before a degasser or solvent tubing can be used it is necessary to prime the system lsopropanol is recommended as priming solvent due to its miscibility with nearly all HPLC 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 the system on When opening capillary or tube fittings solvents may leak out The handling of toxic and hazardous solvents an
4. ite Agilent Technologies Notices Agilent Technologies Inc 2006 2008 2011 2013 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 61312 90303 Edition 02 2013 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 regulations Otherwise it is intended only for gen eral laboratory use Software Revision This guide is valid for revision A 01 04 of Agilent OpenLAB CDS Windows is U S registered trademarks of Microsoft Corporation 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 dam
5. line frequency 23 1260 Infinity Binary LC System User Guide line voltage 23 loading default 84 low delay volume configuration 43 M medium delay volume configuration 44 Method Information 112 113 method default 111 edit entire method 111 settingup 92 single injection 94 mixer 11 network integration 70 non operating altitude 23 non operating temperature 23 0 online plot configuring 85 operating Altitude 23 operating temperature 23 optimization achieving higher resolution 53 achieving higher sensitivity 60 chromatographic separation 31 column use 62 conditions for HPLC 31 detector sensitivity 62 injection volumes 47 optimized configuration 2 1 mmi d columns 43 3 and 4 mm i d columns 45 optimizing resolution chromatographic conditions 59 column compartment 56 data rate 58 extra column volume 57 optimizing flow cell 63 overlapped injections 49 P performance specifications 24 pH range 24 physical specifications 23 power consumption 23 pressure sensor 14 pressure operating range 24 pulsation 24 priming witha pump 78 principle of operation 14 purging 87 Q quick start guide introduction 82 recommended pH range 24 report specification 99 resolution increase 53 Optimization 53 retention factor 32 Run Time Checklist 112 S safety class 102 safety general information 102 standards 23 1260 Infinity Binary LC System User Guide Index symbols 104 sensitivity inst
6. DATA NODAMPNOMIX1 00X21 30000009 D DAD1 A Sig 245 10 Ref 360 80 E CHEM32 1 DATA NODAMPNOMIX1 00X21 30000005 D DAD1 A Sig 245 10 Ref 360 80 E CHEM32 1 DATA WMIXANDDAM PADVRX21 ADVR30000005 D DADI A Sig 245 10 Ref 360 80 E CHEM32 1 DATA REPROVIAL30 30000023 D mAU 80 Low delay volume setup ADVR Low delay volume set up ua setup ADVR es setup 0 1 2 3 4 5 6 7 Min 30 20 10 Figure 17 Reduction of the delay volume The lower the flow rate the greater the negative impact that can be expected from delay volume In Figure 17 on page 49 a 2 1 mm inner diameter column was used at a flow rate of 0 6 mL min The delay volume is reduced step by step from the lowest trace to the top trace The influence on total run time and the impact especially on peak width and the heights of the first peaks is obvious The drawback of overlapped injection and automatic delay volume reduction is that the autosampler is not in the flow path for the complete run time For very sticky compounds this could lead to higher carry over and or compound discrimination 1260 Infinity Binary LC System User Guide 49 3 50 Optimization of the Agilent 1260 Infinity Binary LC Carry over is the percentage of compound that remains in the parts of the instrument that come into contact with the sample and is not flushed onto the column for analysis It also means that this percentage is lost for
7. applications some of which are described in the Chapter Optimization 10 1260 Infinity Binary LC System User Guide The Agilent 1260 Infinity Binary LC Product Description 1 1260 Infinity Binary Pump G1312B The binary pump comprises two identical pumps integrated into one housing Binary gradients are created by high pressure mixing Pulse damper and mixer can be bypassed for low flowrate applications or whenever a minimal transient volume is desirable Typical applications are high throughput methods with fast gradients on high resolution 2 1 mm columns The pump is capable of delivering flow in the range of 0 1 5 mL min against up to 600 bar A solvent selection valve optional allows to form binary mixtures isocratic or gradient from one of two solvents per channel Active seal wash optional is available for use with concentrated buffer solutions Principle of operation The binary pump is based on a two channel dual piston 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 two pump assemblies which can generate pressure up to 600 bar Each channel comprises a pump assembly including pump drive pump head active inlet valve with replaceable cartridge and outlet valve The two channels are fed into a low volume mixing chamber which is connected via a restriction capillary coil to a damping u
8. e Avoid or minimize the use of solvents that may corrode parts in the flow path Consider specifications for the pH range given for different materials like flow cells valve materials etc and recommendations in subsequent sections Material Information Materials in the flow path are carefully selected based on Agilent s experiences in developing highest quality instruments for HPLC analysis over several decades These materials exhibit excellent robustness under typical HPLC conditions For any special conditions please consult the material information section or contact Agilent Disclaimer Subsequent data were collected from external resources and are meant as a reference Agilent cannot guarantee the correctness and completeness of such information Data is based on compatibility libraries which are not specific for estimating the long term life time under specific but highly variable conditions of UHPLC systems solvents solvent mixtures and samples Information can also not be generalized due to catalytic effects of impurities like metal ions complexing agents oxygen etc Apart from pure chemical corrosion other effects like electro corrosion electrostatic charging especially for non conductive organic solvents swelling of polymer parts etc need to be considered Most data available refers to room temperature typically 20 25 C 68 77 F If corrosion is possible it usually accelerates at higher temperatures If in do
9. 2D LC For detailed descriptions of these applications please refer to the Valve Solution User Guide 1260 Infinity Binary LC System User Guide 21 1 The Agilent 1260 Infinity Binary LC Product Description Specifications 22 The modular design of the 1260 Infinity Binary LC allows you to configure a system that exactly meets your individual application requirements This individual configuration can be different from the standard configuration which is described in this System User Guide The physical and performance specifications of the 1260 Infinity Binary Pump are shown below Information on the specifications of other modules in your system can be found in the respective module user manuals 1260 Infinity Binary LC System User Guide Table 1 Physical Specifications The Agilent 1260 Infinity Binary LC Product Description 1 Physical Specifications 1260 Infinity Binary Pump G1312B Type Specification Comments Weight 15 5 kg 34 Ibs Dimensions 180 x 345 x 435 mm height x width x depth 7 x 13 5 x 17 inches Line voltage 100 240 VAC 10 Wide ranging capability Line frequency 50 or 60 Hz 5 Power consumption 220 VA 74 W 253 BTU Maximum Ambient operating temperature Ambient non operating temperature Humidity Operating altitude Non operating altitude Safety standards IEC CSA UL 4 55 C 39 131 F 40 70 C 40 158 F lt 95 rh at 40 C 104 F
10. 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 ia PMP1D BinPump Solvent Ratio 4 f PMP1E BinPump Solvent Ratio B HM1A TCC Left Temperature HM1B TCC Right Temperature a lt Remove Signal B DADIC DAD Signal C m Window x axis 3 x min F draw zero line DAD1A DAD Signal A Type acquired y axis range 100 mau I auto y adjust Offset 10 Fraction Collector m Method Settings F Use method settings Apply to Method OK Cancel Help _ Show fraction collection ticks 1260 Infinity Binary LC System User Guide 85 5 86 Quick Start Guide 3 In the Available Signals box highlight the required signal s and click Add to move them to the Selected Signals box 4 To 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 c
11. F of pages Quantitative result sorted by Signal lw Portrait w g Sample info on each page Add fraction table and ticks O Landscape Add chromatogram output Add summed peaks table Add sample custom fields to sample info Add compound custom fields Response Multi page Landscape Report layout for uncalibrated peaks l Pages O Separately With calibrated peaks O Do not report Signal options Destination File setting Printer File prefix Report Screen Eile Unique PDF file name OK Cancel Help Figure 33 Specify Report Screen To setup a simple area report with Classic Reporting which prints to the printer and to a PDF file enter the following settings in these sections of the Specify Report screen On the Reporting settings tab Report mode Use Classic Reporting Style Report Style Short Quantitative results sorted by Signal Add Chromatogram Output Checked 1260 Infinity Binary LC System User Guide 133 6 Appendix Chromatogram Output Portrait Size Time axis 100 of page Response axis 40 of page Destination Printer Checked Screen Unchecked File Checked File Setting PDF Checked Unique PDF file name Checked On the Quantitation settings tab e Calculation mode Calculate Percent Based on Area Cli
12. Method Lx amp Sampler amp Sampler Injector Program oa Column Comp v DAD X Instrument Curves lis Flow Advanced Timetable empty 0 000 mL min function centric v Solvents j 5 Time min A BR lan a ae nS i z oo 1000 00 0 000 400 00 B Pressure Limits Min 0 00 ba Max 400 00 bar Stoptime Posttime As Iniector No Limit Off 6 min min Add Remove Clear All ClearEmpty J Show timetable graph oK Apply Cancel Help Figure 27 Setup Method screen Binary Pump tab Flow sets the flow rate up to 5 mL min For the example separation 4 mL minis used If the back pressure briefly reaches the maximum pressure setting the flow will be reduced for a few seconds to lower the pressure If the pressure continues to be limited in this way an error condition will be created and the flow will be stopped 1260 Infinity Binary LC System User Guide 119 120 Appendix Solvents defines the mobile phases that are available and the percentage proportions that are pumped on the two channels A and B On each channel a drop down box allows the selection of a solvent from a list so that the pump control uses the optimum compressibility settings This will optimize the flow characteristics as described in Optimum Instrument Configuration for 2 1 mm i d Columns on page 43 A second text box allows a description of the mobile phase to be entered If the sol
13. Setup and Installation Installing Software Installing the Software Controller and Data System For details of installation procedures for the software refer to the detector manual and the software manuals 68 1260 Infinity Binary LC System User Guide System Setup and Installation 4 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 the diagnostic functions that were formerly available 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 Early Maintenance Feedback to determine the need for upgrade or replacement In addition the software e Automates
14. The Agilent 1200 Infinity Series Multi Method Development Solution facilitates complete automation of this time consuming selection process making method development and method transfer an easy and reliable task ZORBAX 1 8 um RRHT columns use the same chemistry as ZORBAX columns with 3 5 and 5 um particles As a result for any particular ZORBAX phase the 5 0 3 5 and 1 8 um particles provide identical selectivity which allows easy fast and secure bidirectional method transfer between conventional LC and UHPLC 1260 Infinity Binary LC System User Guide 33 2 Benefits of small particle size columns 34 Faster Chromatography There are several advantages of having shorter run times High Throughput labs now have higher capacity and can analyze more samples in less time More samples in less time also means lower costs For example by reducing the analysis time from 20 min per sample to 5 min the cost for 700 samples is reduced by 79 Table 3 on page 34 Table 3 Time and cost savings over 700 runs Cycle time 20 min cycle time 5 min cycle time Runs 700 700 Approx costs analysis 10 58 2 24 Approx cost 700runs 7400 1570 Cost savings 5830 Time 10 days 2 5 days 1 solvents 27 1 disposal 2 1 labor 30 h 2 24 hours day The Agilent cost savings calculator provides an easy way to calculate the cost savings by switching from conventional HPLC to UHPLC using 1 8 um particle size colum
15. The corrosion of flow cell windows can negatively affect measurement results For a pH greater than 12 the use of flow cells with sapphire windows is recommended Gold Gold is inert to all common HPLC solvents acids and bases within the specified pH range It can be corroded by complexing cyanides and concentrated acids like aqua regia Zirconium Oxide Zr04 Zirconium Oxide is inert to almost all common acids bases and solvents There are no documented incompatibilities for HPLC applications Platinum Iridium Platinum Iridium is inert to almost all common acids bases and solvents There are no documented incompatibilities for HPLC applications Fluorinated polymers PTFE PFA FEP FFKM Fluorinated polymers like PTFE polytetrafluorethylene PFA perfluoroalkoxy and FEP fluorinated ethylene propylene are inert to almost all common acids bases and solvents FFKM is perfluorinated rubber which is also resistant to most chemicals As an elastomer it may swell in some organic solvents like halogenated hydrocarbons TFE PDD copolymer tubings which are used in all Agilent degassers except G1322A are not compatible with fluorinated solvents like Freon Fluorinert or Vertrel They have limited life time in the presence of Hexafluoroisopropanol HFIP To ensure the longest possible life with HFIP it is best to dedicate a particular chamber to this solvent not to switch solvents and not to let dry out the chamber For opti
16. Up to 2000 m 6562 ft Up to 4600 m 15091 ft Installation category II Pollution degree 2 Non condensing For storing the module For indoor use only 1260 Infinity Binary LC System User Guide 23 1 24 The Agilent 1260 Infinity Binary LC Product Description Performance Specifications Table 2 Performance Specifications of the Agilent 1260 Infinity Binary Pump G1312B Type Specification Comments Hydraulic system Setable flow range Flow range Flow precision Flow accuracy Pressure operating range Pressure pulsation Compressibility compensation Recommended pH range Gradient formation Delay volume Composition range Two dual piston in series pumps with servo controlled variable stroke drive power transmission by gears and ball screws floating pistons Set points 0 001 5 mL min in 0 001 mL min increments 0 05 5 0 mL min lt 0 07 RSD or 0 02 min SD whateveris based on retention time at greater constant room temperature 1 or 10 pL min what ever is greater pumping degassed H 0 at 10 MPa 100 bar Operating range 0 60 MPa 0 600 bar 0 8700 psi up to 5 mL min lt 2 amplitude typically lt 1 3 or lt 0 3 MPa 3 bar whatever is greater at 1 mL min isopropanol at all pressures gt 1 MPa 10 bar 147 psi Low delay volume configuration lt 5 amplitude typically lt 2 Pre defined based on mobile phase compress
17. User Guide Appendix 6 Setting Up a Method using Edit Entire Method Method Information The Method Information screen can also be directly accessed through the menu Method gt Method Information or by right clicking on the graphical user interface This box allows information about the method to be entered This information will be displayed above the system diagram on the Method and Run Control screen whenever this method is loaded and resident in memory Method Information Instrument 2 Method Comments This method runs the test sample to demonstrate how to run an analysis Figure 24 Method Information 1260 Infinity Binary LC System User Guide 113 114 Appendix Instrument Acquisition Setup Instrument Method The Setup Method screen can be directly accessed through the menu Instrument gt Setup Instrument Method or by right clicking on the graphical user interface on any module icon and then selecting Method in the context menu This next stage in Edit Entire Method is the Setup Method screen with six tab dividers for different modules or functions The tabs are High Performance Autosampler HiP ALS HiP ALS Injector Program Binary Pump BinPump Thermostatted Column Compartment TCC e Diode array Detector DAD Instrument Curves To move between the tabs click on the tab name at the top of the screen When parameter changes have been entered they can be immediately sent to the instrum
18. done in two ways manually by disconnecting and reconnecting capillaries automatically using a 600 bar 2PS 6PT valve optional 1260 Infinity Binary LC System User Guide Optimization of the Agilent 1260 Infinity Binary LC 3 Optimum Instrument Configuration for 2 1 mm i d Columns Disconnect only here Low delay volume 120 ul delay Low delay volume configuration to achieve shortest gradient delay for ultra fast gradient separations In the low delay volume configuration of the Agilent 1260 Infinity Binary Pump the damper and mixer are bypassed to reduce the pump delay volume to about 120 uL Figure 13 on page 43shows the flow path connections for this configuration This provides the shortest gradient delay for ultra fast gradient separations In order to take full advantage of the electronic damping control which replaces the physical volume damping it is important to select the respective Enhanced Solvent Compressibility function in the auxiliary screen of the pump menu To minimize peak dispersion the low dispersion kit Low dispersion kit G1316 68744 must be installed This kit includes short 0 12 mm i d capillaries and low dispersion heat exchangers 1 6 uL and 1 5 uL for the thermostatted column compartment To maintain resolution in the UV detector a low volume flow cell should be used See Choosing a Flow Cell on page 63 for flow cell recommendations Flow Path Mixing T Pressure ll Sensor
19. final step of the sampling sequence is the inject and run step The six port valve is switched to the main pass position and directs the flow back through the sample loop which now contains a certain amount of sample The solvent flow transports the sample onto the column and separation begins This is the beginning of a run within an analysis In this stage all major performance influencing hardware is flushed internally by the solvent flow For standard applications no additional flushing procedure is required Flush the Needle Before injection and to reduce the carry over for very sensitive analysis the outside of the needle can be washed in a flush port that is located behind the injector port on the sampling unit As soon as the needle is on the flush port a peristaltic pump delivers some solvent during a defined time to clean the outside of the needle At the end of this process the needle returns to the injection port 1260 Infinity Binary LC System User Guide The Agilent 1260 Infinity Binary LC Product Description 1 1290 Infinity Thermostatted Column Compartment G1316C The Agilent 1290 Infinity Thermostatted Column Compartment TCC controls the temperature between 10 C below ambient and up to 100 C at 2 5 ml min and 80 C at up to 5 ml min respectively The temperature stability specification is 0 05 C and the accuracy specification 0 5 C with calibration This is achieved by a combination of conduction f
20. 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 1260 Infinity Binary LC 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 1260 Infinity Binary LC System User Guide 41 3 42 Optimization of the Agilent 1260 Infinity Binary LC Delay Volumes in the Agilent 1260 Infinity Binary LC Table 4 on page 42 and Table 5 on page 42show the component volumes which contribute to system delay volume in the Agilent 1260 Infinity Binary LC System Table4 Delay volumes of the 1260 Infinity Binary LC modules Components Delay Volume pL Binary Pump 120 Binary Pump 600 800 Low volume mixer 200 Mixer 400 Autosampler 270 Low dispersion heat exchanger 1 6 Built in heat exchanger 3 and6 1 in low delay volume configuration with bypassed damper and mixer 2 in standard delay volume configuration Table5 Delay volumes of 1260 Infinity Binary LC configurations System Configuration Delay Volume pL Low delay volume configuration Pump 120 Autosampler Medium delay volume configuration Pump 320 Standard delay volume configuration Pump 600 800 Switching between configurations can be
21. maximize peak height and minimize baseline noise Any reduction in peak dispersion will help to maintain peak height and so extra column volume should be minimized by use of short narrow internal diameter connection capillaries and correctly installed fittings Using smaller inner diameter columns should result in higher peak height and is therefore ideal for applications with limited sample amounts If the same sample amount can be injected on a smaller i d column then the dilution due to column diameter will be less and the sensitivity will increase For example decreasing the column i d from 4 6 mm to 2 1 mm results in a theoretical gain in peak height of 4 7 times due to the decreased dilution in the column For a mass spectrometer detector the lower flow rates of narrow columns can result in higher ionization efficiencies and therefore higher sensitivity Detector Settings The detector has a number of parameters that are used to optimize performance The following sections describe how the detector parameters affect performance characteristics e Flow cell affects sensitivity e Wavelength and bandwidth affect sensitivity selectivity and linearity Slit Width affects sensitivity spectral resolution and linearity e Peak Width affects sensitivity and resolution 1260 Infinity Binary LC System User Guide Optimization of the Agilent 1260 Infinity Binary LC 3 Choosing a Flow Cell Several flavors of the Max Light Cartridge
22. o e e ee 1 7 e The Agilent 1260 Infinity Binary LC Product Description Features of the Agilent 1260 Infinity Binary LC 8 System Components 10 1260 Infinity Binary Pump G1312B 11 1260 Infinity High Performance Degasser G4225A 14 1260 Infinity High Performance Autosampler G1367E 15 1290 Infinity Thermostatted Column Compartment G1316C 17 1260 Infinity Diode Array Detector G4212B 19 1200 Infinity Series Quick Change Valves 21 Specifications 22 This chapter discusses the features of the 1260 Infinity Binary LC Ee Agilent Technologies 1 The Agilent 1260 Infinity Binary LC Product Description Features of the Agilent 1260 Infinity Binary LC The design concept of the 1260 Infinity Binary LC is to provide a liquid chromatograph offering ultra fast and high resolution separation capability and yet which retains full functionality for standard HPLC applications Therefore it provides full backwards compatibility for your established HPLC and UHPLC methods The use of sub two micron STM particles means that for high flowrates or long columns additional pressure is required to drive the mobile phase through the column The flowpath of the 1260 Infinity Binary LC is optimized to produce minimal backpressure and ZORBAX RRHT columns have an engineered particle size distribution that produces significantly less backpressure than other STM columns The design features and benefits of the Agilent 1260 Infinit
23. of organic solvent ion strength and pH can be manipulated to achieve the shortest possible retention and highest selectivity Slow Ad or Desorption Non linear isotherms Chemical Equilibrum pH Pressure CHEMISTRY Stationary and Mobile Phase Properties Solute Properties Temperature Particle Size Porosity Column Dimensions Flow Velocity Temperature Figure 8 Selecting optimal conditions for HPLC 1260 Infinity Binary LC System User Guide 31 2 32 Introduction Resolution can be described as a function of three parameters column efficiency or theoretical plates N e selectivity a e retention factor k According to the resolution equation Figure 9 on page 32 the selectivity has the biggest impact on resolution Figure 10 on page 32 This means that the selection of appropriate mobile and stationary phase properties and temperature is critical in achieving a successful separation Figure 9 Resolution equation R f a 4 3 f N 2 f k 1 20000 40000 60000 80000 N 1 05 1 10 1 15 1 20 a 5 10 15 20 k Figure 10 Effect of plate number separation factor and retention factor on R 1260 Infinity Binary LC System User Guide Introduction 2 No matter whether the UHPLC separation method is being newly developed or simply transferred from an existing conventional method it is clearly beneficial to have a wide choice of stationary phase chemistries available in a range of column
24. parameters Injection volume 1 0 ul Injection with Needle Wash Mode Flush Port Time 6 s b Other parameters can remain at their default settings Click OK to exit the window The changes are sent to the autosampler module 92 1260 Infinity Binary LC System User Guide Quick Start Guide 5 4 Right click the Thermostatted Column Compartment TCC area and select Method in the context menu a In the Method page for the 1290 Infinity TCC enter the following parameters Left Temperature 40 C Right Temperature Combined b Other parameters can remain at their default settings Click OK to exit the window The changes are sent to the TCC module 5 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 254 nm bw 4 nm ref 550 nm bw 100 nm Peak width 0 02 b In the Advanced section set Spectrum Store to All c Other parameters can remain at their default settings Click OK to exit the window The changes are sent to the DAD module 6 All the required module parameters have now been entered Select Method gt Save Method As and enter ISO 1 M to save the method with a new name The ChemStation does not allow the method to be saved as DEF_LC M so that the default method template is not altered 7 Allow the sy
25. quantitative measurement it is discriminated The carry over can be measured by injecting pure solvent after the sample run is finished Discrimination and carry over can become even more important if the analyte compounds are non polar and the start of the gradient contains a high percentage of water In the worst case the non polar compound precipitates at the surface of contact Small plugs of for example dimethylsulfoxide before and after the sample plug can help to minimize this problem For overlapped injection or automated delay volume reduction the time before the injection valve is switched to the bypass mode should be increased using the flush out factor to 20 This extends the time during which the autosampler delay volume is flushed with mobile phase How to Achieve High Throughput The injection can be optimized for speed remembering that drawing the sample too fast can reduce the reproducibility Marginal gains are to be made here as the sample volumes used tend towards the smaller end of the range in any case A significant portion of the injection time is the time taken with the needle movements to and from the vial and into the flush port These manipulations can be performed while the previous separation is running This is known as overlapped injection and it can be easily turned on from the autosampler setup screen in the control software The autosampler can be told to switch the flow through the autosampler to bypass aft
26. resolution is always to test different stationary phases and to select the column with the best separation This is the parameter that is of most importance for resolution e The second step is to use long columns or even coupled columns to increase the plate number e A third step is to shift peaks to higher retention factors For k values of 5 to 10 the impact is significant With higher k values the effect is very low In practice this means that longer columns with appropriate selectivity give better resolution 1260 Infinity Binary LC System User Guide 55 3 Optimization of the Agilent 1260 Infinity Binary LC Optimum Instrument Configuration for High Resolution Column Compartment The column compartment can be used in its standard version for 4 6 mm inner diameter columns At flow rates above 2 mL min and temperatures above 60 C the column effluent should be cooled down to the temperature of the detector by using the 1 5 uL cooler heater in the column compartment This ensures lowest noise level with UV detectors even at 5 mL min and 80 C see Figure 18 on page 56 With post column coolin Noise PtoP 0 8289 mA No post column cooling S N for peak 4 482 Noise PtoP 1 5905 mAU S N for peak 4 392 Cooling needed gt 2 ml min 02 04 0 6 08 1 min Figure 18 Influence of post column cooling PCC on baseline noise If 2 1 mm inner diameter columns are used at low flow rates the small heating dev
27. separations to have small delay volumes especially with narrow bore columns that is 2 1 mm i d as often used with mass spectrometric detection As an example in HPLC methods using 5 um packing material flow rates of 1 ml min are typically used in a 4 6 mm i d column and about 0 2 ml min ina 2 1 mm i d column same linear velocity in the column On a system with a typical delay volume of 1000 ul and using a 2 1 mm column there would be an initial hidden isocratic segment of 5 min whereas on a system with 600 ul delay volume the delay would be 3 min These delay volumes would be too high for run times of one or two minutes With sub two um packings the optimum flow rate from the Van Deemter curve is a little higher and so fast chromatography can use three to five times these flow rates yielding delay times of about one minute However the delay volume must be reduced further to achieve delay times which are a fraction of the intended run time This is achieved with the Agilent 1260 Infinity Binary LC due to the low delay volume of the pump flow path and low volume of the flow path through the autosampler 1260 Infinity Binary LC System User Guide Optimization of the Agilent 1260 Infinity Binary LC 3 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
28. 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 manner 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 102 1260 Infinity Binary LC System User Guide Appendix 6 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
29. usability and paves the way for ultra high throughput multi method and automated method development solutions A low dispersion tubing kit and low volume flow cells minimize peak dispersion for narrow bore columns Baseline robustness and fast spectral acquisition at data rates up to 80 Hz through the new optical design of the 1260 Infinity Diode Array Detector Different UV detector flow cells for use with 2 1 3 0 and 4 6 mm inner diameter columns are available including the revolutionary Agilent Max Light cartridge flow cell with 60 mm optical path length typical noise lt 0 6 uAU cm for ultra sensitivity in detection A stepwise upgrade from 1100 Series or 1200 Series to Agilent 1260 Infinity Binary LC is possible for example a 1100 Series Detector or 1200 Series Column Compartment can be further used in combination with a 1260 Infinity Binary Pump 1260 Infinity Binary LC System User Guide 9 1 The Agilent 1260 Infinity Binary LC Product Description System Components Numerous system configurations of the 1260 Infinity Binary LC are possible tailored to the needs of your individual application requirements A few configurations are described in more detail in this manual see System Setup and Installation on page 67 The modules that are described in the following sections are typical components of a 1260 Infinity Binary LC In addition to these core components individual solutions are available for specific
30. useful tests 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 1260 Infinity Binary LC System User Guide 69 4 System Setup and Installation 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 Integration Into the Network For network integration of your system refer to user manuals of your modules chapter LAN Configuration 70 1260 Infinity Binary LC System User Guide System Setup and Installation 4 Capillary and Tubing Connections in Flow Path Depending on the system configuration capillaries of different lengths and diameters are used These are described below See module manuals for module internal capillary and tubing connections 1260 Infinity Binary LC System User Guide 71 4 System Setup and Installation Installing the Modules Connections for Standard Delay Volume Configuration Figure 21 on page 72 shows capillary and tubing connections in the flow path for the standard delay volume configuration of the 1260 Infinity Binary LC It Solvent cabinet i i q q Degasser Sp e Pump Aa SS Saar 1 e 1 Autosampl
31. 1260 Infinity Binary LC 6 Appendix This chapter provides additional information on safety legal and web and about setting up a method 1260 Infinity Binary LC System User Guide 3 Contents Contents 1 The Agilent 1260 Infinity Binary LC Product Description 7 Features of the Agilent 1260 Infinity Binary LC 8 System Components 10 Specifications 22 Introduction 27 Theory of Using Smaller Particles in Liquid Chromatography 28 Benefits of small particle size columns 34 Frictional Heating 37 Optimization of the Agilent 1260 Infinity Binary LC 39 How to Configure the Optimum Delay Volume 40 How to Achieve Higher Injection Volumes 47 How to Achieve Shorter Cycle Times 48 How to Achieve Lowest Carry over 51 How to Achieve Higher Resolution 53 How to Achieve Higher Sensitivity 60 How to Prevent Column Blockages 65 System Setup and Installation 67 Installing Software 68 Installing the Modules 70 Quick Start Guide 81 About the Quick Start Guide 82 Preparing the System 83 Data Acquisition in Method and Run Control View 88 Data Analysis 96 1260 Infinity Binary LC System User Guide Contents 6 Appendix 101 Safety Information 102 Solvent Information 105 Agilent Technologies on Internet 110 Setting Up a Method using Edit Entire Method 111 1260 Infinity Binary LC System User Guide Contents 6 1260 Infinity Binary LC System User Guide 1260 Infinity Binary LC System User Guide KP amp e
32. 35 min Figure 16 Comparison Agilent 1200 Series and Agilent 1260 Infinity LC system on one Configuration 46 1260 Infinity Binary LC System User Guide Optimization of the Agilent 1260 Infinity Binary LC 3 How to Achieve Higher Injection Volumes The standard configuration of the Agilent 1260 Infinity Autosampler can inject a maximum volume of 100 uL with the standard loop capillary To increase the injection volume the Multidraw upgrade kit G1313 68711 can be installed With the kit you can add a maximum of 400 uL or 1400 uL to the injection volume of your injector The total volume is then 500 uL or 1500 uL for the 1260 Infinity Autosampler with 100 uL analytical head Note that the delay volume of your autosampler is extended when using the extended seat capillaries from the multi draw kit When calculating the delay volume of the autosampler you have to double the volume of the extended capillaries The system delay volume due to the autosampler will increase accordingly Whenever a method is scaled down from a larger column to a smaller column it is important that the method translation makes an allowance for reducing the injection volume in proportion to the volume of the column to maintain the performance of the method This is to keep the volume of the injection at the same percentage volume with respect to the column This is particular important if the injection solvent is stronger more eluotropic than the starting mobile phase
33. Flow Cell are available see Table 7 on page 63 Table 7 Specifications for Max Light Cartridge Flow Cells Cartridge Cells Max Light Cartridge Cell 10 mm V o 1 0 uL G4212 60008 Max Light Cartridge Cell Bio inert 10 mm V o 1 0 pL G5615 60018 Max Light Cartridge Cell 60 mm V o 4 0 pL G4212 60007 Max Light Cartridge Cell Bio inert 60 mm V o 4 0 pL G5615 60017 HDR Max Light Cartridge Cell 3 7 mm V o 0 4 uL G4212 60032 ULD Max Light Cartridge Cell 10 mm V o 0 6 pL G4212 60038 Max Light Cartridge Test Cell G4212 60011 Maximum 70 bar 1015 psi Maximum Operating Pressure MOP Pressure 150 bar 2175 psi Maximum Incidential Pressure MIP 2 pH range 1 0 12 5 solvent dependent 1 Maximum Operating Pressure MOP The maximum pressure at which the system can operate continuously under normal conditions 2 Maximum Incidential Pressure MIP The maximum pressure which the system can experience during a short time Normal Applications The Max Light Cartridge Cell 10 mm V o 1 0 uL G4212 60008 covers a wide range of applications all column diameter down to at least 2 1 mm ID or even less applications with peak dispersion Peakwidth x flow down 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 High Sensitiviy If higher sensitivity is necessary the Max Light Cartridge Cell 60 mm V o 4 0 uL G4212 60007
34. ION 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 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 1260 Infinity Binary LC System User Guide In This Guide In This Guide This manual covers the Agilent 1260 Infinity Binary LC 1 The Agilent 1260 Infinity Binary LC Product Description This chapter discusses the features of the 1260 Infinity Binary LC 2 Introduction This chapter gives an introduction to the Agilent 1260 Infinity Binary LC and the underlying concepts 3 Optimization of the Agilent 1260 Infinity Binary LC 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 installation of the modules and preparing the system for operation 5 Quick Start Guide This chapter provides information on data acquisition and data analysis with the
35. When temperature is within ss UE eg T When temperature is within moe C Posttime Of Figure 28 122 Setup Method screen Thermostatted Column Compartment tab Temperature defines the temperature of the left and right hand side column holders which can be independently controlled or linked together by clicking the Combined radio button on When combined the settings for the left side control both sections and this is certainly necessary when the column is longer than 15 cm and needs to be supported by both sections The two sides can be operated separately when it is required to have two columns operating at different temperatures this can be implemented when a column switching valve is also installed to switch between them Another use of separate temperature zones is when the column is operated at a high temperature e g above 60 C on one side and the heat exchanger on the other side is used to cool the eluent before it enters the detector thereby reducing noise due to thermal effects in the flow cell Selecting the As Detector Cell option will automatically read the temperature from the cell in the detector 1260 Infinity Binary LC System User Guide Appendix 6 The temperature of each zone can be set from 5 C to 100 C and the user should check that the column is suitable for operation at that temperature Agilent ZORBAX RRHT StableBond phases can be used at the higher end of the range Th
36. ages 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 agreement with warranty terms covering the material in this document that conflict with these terms the warranty terms in the sep arate agreement shall control Technology Licenses 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 CAUT
37. and any increase will affect the separation particularly for early running peaks low retention factor In some cases it is the cause of peak distortion and the general rule is to keep the injection solvent the same or weaker than the starting gradient composition This has a bearing on whether or by how much the injection volume can be increased and the user should check for signs of increased dispersion wider or more skewed peaks and reduced peak resolution in trying to increase the injection size If an injection is made in a weak solvent then the volume can probably be increased further because the effect will be to concentrate the analyte on the head of the column at the start of the gradient Conversely if the injection is in a stronger solvent than the starting mobile phase then increased injection volume will spread the band of analyte down the column ahead of the gradient resulting in peak dispersion and loss of resolution Perhaps the main consideration in determining injection volume is the diameter of the column as this will have a large impact on peak dispersion Peak heights can be higher on a narrow column than with a larger injection on a wider column because there is less peak dispersion With 2 1 mm i d columns typical injection volumes might range up to 5 to10 ul but it is very dependent on the chemistry of the analyte and mobile phase as discussed above In a gradient separation injection volumes of about 5 of the column v
38. and so on are used for replacement The use of repaired fuses and the short circuiting of fuse holders must be avoided 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 voltage 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 observe appropriate safety procedures for example goggles safety gloves and protective clothing as described in the material handling and safety data sheet by the
39. bile phase starting slowly at 0 1 mL min for a 2 1 mm inner diameter column 0 2 mL min for a 3 0 mm inner diameter column and 0 4 mL min for 4 6 mm inner diameter Increase the flow rate to the desired flow over 5 minutes 9 Once the pressure has stabilized attach the column to the detector 10 Equilibrate the column and detector with 10 column volumes of the mobile phase prior to use 1 5 mL depending on column size 11 Avoid over pressure Check the pressure range of your gradient which may be 100 130 bar or more before starting any sequence 1260 Infinity Binary LC System User Guide 65 3 Optimization of the Agilent 1260 Infinity Binary LC How to Prevent Column Blockages For 2 1 mm Frits use inserts with small cone For 4 6 mm Frits use inserts with big cone Figure 20 Protection for 4 6 and 2 1 mm id columns packed with 1 8 um particles inlet frit with 0 2 um pore size 66 1260 Infinity Binary LC System User Guide 1260 Infinity Binary LC System User Guide a e e e00 ee 4 7 e System Setup and Installation e e Installing Software 68 Installing the Modules 70 Installing the System Modules 70 Integration Into the Network 70 Capillary and Tubing Connections in Flow Path 71 Priming the System 76 This chapter includes information on software installation installation of the modules and preparing the system for operation ea Agilent Technologies 67 4 System
40. 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 1260 Infinity Binary LC System User Guide 63 3 Optimization of the Agilent 1260 Infinity Binary LC Ultra Low Dispersion The Max Light Cartridge ULD cell can be used with the G4212A DAD and G4212B DAD The cell is a requirement for the Ultra Low Dispersion Kit solution which currently exists as 1290 Infinity Ultra Low Dispersion Kit 5067 5189 The cell should be part of the ultra low dispersion solution High Dynamic Range The Max Light Cartridge HDR cell can be used with the G4212A DAD and G4212B DAD The cell is required as a part of the High Dynamic Range HDR solution which will be introduced March April 2013 To protect the flow cell against overpressure e g in systems with LC MS install Inline Pressure Relief Valve Kit G4212 68001 see Agilent 1200 Infinity Series Diode Array Detectors manual 64 1260 Infinity Binary LC System User Guide Optimization of the Agilent 1260 Infinity Binary LC 3 How to Prevent Column Blockages Columns packed with sub 2 micron particles also need frits with small pore size to prevent packing material being swept out This immediately brings increased risk of blocking these frits with particulates from the sample mobile phase or and from the instrument itself To protect the column additional small filters Figure 20 on page 66 can be used in
41. ck OK to exit and the next screen in the Edit Entire Method process will open 134 1260 Infinity Binary LC System User Guide Appendix Setting Up a Method using Edit Entire Method Instrument Curves Instrument Curves Instrument 2 Select curves to overlay Instrument Data Curves Figure 34 Instrument Curves screen The Instrument Curves checkboxes allow these recorded parameters to be overlaid as a graph on the chromatogram 1260 Infinity Binary LC System User Guide 135 6 Appendix Run Time Checklist The Run Time Checklist can also be directly accessed through the menu Method gt Run Time Checklist or by clicking on the Run Time Checklist icon at the top right of the screen Run time Checklist Instrument 2 Check Method Sections to Run Data Acquisition V Standard Data Analysis C Customized Data Analysis Macro C Save GLP Data C Post Run Command Macro C Save Method with Data Figure 35 Run Time Checklist Screen The Run Time Checklist selects whether the method should run both data acquisition and data analysis and also offers an opportunity to link macro commands or programs into the work flow at various points In most cases the Data Acquisition and Standard Data Analysis check boxes are checked If no data analysis is required for example in a series of method development runs Standard Data Analysis can be unchecked so that no report is produced and the data can be visually asses
42. d reagents can carry health risks gt Observe appropriate safety procedures for example wear 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 76 The purge tool of the Lab Advisor can be used to purge the pump automatically If the pump is not able to draw in the solvent from the bottles use a syringe to move 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 1260 Infinity Binary LC System User Guide oF O N 1260 Infinity Binary LC 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 pull the liquid through the degasser Reattach the tubing to the solvent selection valve Pump 30 mL isopropanol to remove residual air bubbles Switch to t
43. e Agilent Max Light cartridge cell dramatically increases the light transmission by utilizing the principle of total internal reflection along a non coated fused silica capillary achieving a new level of sensitivity without sacrificing resolution through cell volume dispersion effects This design minimizes baseline perturbations that are caused by refractive index or thermal effects and results in more reliable integration of peak areas Mirror Grating Optofluidic waveguide Deuterium lamp Max light cartridge cell Slit 1024 element diode array Figure 5 The light path through the DAD The module also incorporates electronic temperature control to further enhance the resistance to ambient temperature effects Although the hydraulic volume of the Max Light cartridge cell is very small the path length is a standard 10 mm However for even higher sensitivity the alternative Agilent Max Light high sensitivity cell is available with a path length of 60 mm Cells are easily exchanged by sliding them in or out of the cell holder and they are auto aligned in the optical bench The DAD light source is a deuterium lamp and the operating wavelength range that is covered is 190 to 640 nm This is detected by a diode array comprising 1024 diodes The entrance to the spectrograph is through a fixed optical slit of 4 nm 1260 Infinity Binary LC System User Guide 19 1 20 The Agilent 1260 Infinity Binary LC Product Descri
44. e pump effluent is led directly to the column Prior to injection the outside surfaces of the needle are washed with fresh solvent This is achieved using the flush port of the autosampler and prevents contamination of the needle seat The flush port of the autosampler is refilled with fresh solvent by a peristaltic pump that is installed in the autosampler housing The flush port has a volume of about 680 uL and the pump delivers 6 mL min Setting the wash time to 10 s means that the flush port volume is refilled more than once with fresh solvent which is sufficient in most cases to clean the outside of the needle 1260 Infinity Binary LC System User Guide Optimization of the Agilent 1260 Infinity Binary LC 3 How to Achieve Higher Resolution Increased resolution in a separation will improve the qualitative and quantitative data analysis allow more peaks to be separated or offer further scope for speeding up the separation This section explains how resolution can be increased by examining the following points e Optimize selectivity e Smaller particle size packing e Longer columns e Shallower gradients faster flow e Minimal extra column volume Optimize injection solvent and volume e Fast enough data collection Resolution between two peaks is described by the resolution equation l a 1 k 1 Rs y N rA a k where e R resolution e N plate count measure of column efficiency e o selectiv
45. e setting for the UV detector must be selected appropriately Selecting a data rate that is too low results in increased peak width and lower resolution see Figure 19 on page 58 PW 0 30 sec Yon og ee 80 Hz ff ARNA De a Kea Ne easy Ne SINGH Aa i AU NC a aaa PW 0 33 sec MILA Ue PW 0 42 sec AA KIMI UL 10 Hz AAN PW 1 24 sec INS 5 Hz o 0 1 0 2 03 0 4 0 5 min Figure 19 Peak width vs UV data Rate Table 6 on page 59 shows the dependency of peak width resolution and peak capacity on the data rate For fast and ultra fast LC high data rates are needed otherwise the separation that is achieved on the column is destroyed in the detector 58 1260 Infinity Binary LC System User Guide Optimization of the Agilent 1260 Infinity Binary LC 3 Table6 Relation between data rate and chromatographic performance Data Rate Peak Width Resolution Peak Capacity 80 Hz 0 300 2 25 60 40 Hz 0 329 2 05 55 20 Hz 0 416 1 71 45 10 Hz 0 666 1 17 29 5 Hz 1 236 0 67 16 80 Hz data rate compared with 20 Hz data rate results in improvements in Peak Width 30 e Resolution 30 e Peak capacity 40 Apparent column efficiency 70 Chromatographic Conditions As already stated previously chromatographic conditions depend on the compounds to be analyzed But here also some rules of thumb exist Moderate flow rates should be used but recent experiments have sho
46. e temperature is controlled to 0 15 C down to 10 C below ambient although it should be noted that there are very few applications operating below 12 15 C Avoid using the TCC at such low temperature that condensation of water from humid air occurs as this will trigger the leak sensor Column Switching Valve is an active option only when a valve is fitted between the column holders Three types of valve are available 2 position 6 port used to switch between 2 columns 2 position 10 port used for alternating column regeneration 8 position 9 port used for multiple column selection in MDS 1260 Infinity Binary LC System User Guide 123 6 Appendix Setting Up a Method using Edit Entire Method Diode Array Detector Tab H Binary Pump Sampler Sampler Injector Program a Column Comp Y DAD x Instrument Curves Signals Advanced Use Wave Band Reference Reference Spectrum Signal length width Wavelength Bandwidth Store None z Signal v 254 0 40 gt Y 3600 gt 1000 gt nm Signal B v 210 0 40 v 3600 gt 1000 nm Range from 190 0 to 400 0 nm Signal C a 2402 40 36002 1000 gt nm Sher FORE em Signal D z 2300 40 m 3600 gt 1000 gt nm P E a Signal E vi 260 0 gt 40 V 360 0 gt 100 0 gt nm ies Signal F 273 0 4 0 360 0 lt 100 0 lt nm Analog Output Signal G 80 0 40 360 0 gt 100 0 nm Output 1 Signal H 250 0 40 360 0 100 0 nm Z
47. eals from solvent bottle A bottle B Pistons Pistons Pump head A Pump head B Figure2 The Hydraulic Path of the Binary Pump with Bypassed Damper and Mixer 1260 Infinity Binary LC System User Guide 13 1 The Agilent 1260 Infinity Binary LC Product Description 1260 Infinity High Performance Degasser G4225A The Agilent 1260 Infinity High Performance Degasser model G4225A comprises four separate vacuum chambers with semipermeable tubings a vacuum pump and control assembly When the vacuum degasser is switched on the control assembly turns on the vacuum pump which generates a low pressure in the vacuum chambers The pressure is measured by a pressure sensor The vacuum degasser maintains the low pressure by a controlled leak in the air inlet filter and a regulation of the vacuum pump using the pressure sensor The LC pump draws the solvents from their bottles through the semipermeable tubes of the vacuum chambers When solvents pass through the vacuum chambers any dissolved gas in the solvents permeates through the tubings into the vacuum chambers The solvents will be degassed when leaving the outlets of the vacuum degasser Control Sensor oo circuit 4 separate vacuum chambers Solvent Vacuum container Figure Overview only one of the four solvent channels is shown 14 1260 Infinity Binary LC System User Guide The Agilent 1260 Infinity Binary LC Product Description 1 1260 Infinity High Perfo
48. em Setup and Installation Table 8 Choice of Priming Solvents for Different Purposes Activity Solvent Comments After an installation lsopropanol Best solvent to flush air out of When switching between lsopropanol the system reverse phase and normal Miscible with almost all phase both times solvents After an installation To clean the system when using buffers After changing aqueous solvents After the installation of normal phase seals PE seals pack of 2 0905 1420 Ethanol or methanol HPLC grade water HPLC grade water Hexane 5 isopropanol 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 1260 Infinity Binary LC System User Guide 1260 Infinity Binary LC System User Guide 5 Quick Start Guide About the Quick Start Guide 82 Preparing the System 83 Turning the System ON 83 Loading the Default Method 84 Configuring the Online Plot 85 Purging the Pump 87 Data Acquisition in Method and Run Control View 88 Method Parameters for System Checkout with Isocratic Checkout Sample 88 Setting Up the Method 92 Running the Method for a Single Injection 94 Data Analysis 96 Data Analysis View 97 Integrating a Signal 98 Specify the Report 99 This chapter provides information on data acquisition and data analysis with the 1260 Infinity Binary LC Ee A
49. ent by clicking Apply or when all tabs have been completed clicking 0K will send all parameters to the modules close the screen and move on to the next stage The parameter entry tabs look similar in all the control programs ChemStation EZChrom MassHunter et al due to the Agilent concept of common RC Net drivers for the instrument modules To run the example separation as with most methods it is not necessary to change every available parameter but for completeness they are described in the next sections 1260 Infinity Binary LC System User Guide Appendix 6 Method of G1367E PPAANOO005 x Injection Mode Advanced Injection volume i uL Ausiliary Standard injection Draw speed 100 0 gt p min Injection with needle wash Eject speed 100 0 pl min Draw position 00 mm NESTS Equilibration time ea sec a Sample flush out factor 5 0 times injection volume Tin V Vial Well bottom sensing Time Locati High throughput Repeat Automatic delay volume reduction Stoptime Posttime Enable overlapped injection fhen le Is Flushed Out As Pump No Limit of After Period Of Time min min Tut X 4 E Injection Cleaning X Figure 25 e Injection Mode Setup Method screen High Performance Autosampler tab Injection volume sets the volume to be injected example 1 ul Standard injection indicates that no external needle wash is done Injection with needle wash is used to reduce potent
50. er Thermostat 2 Thermostatted column compartment 3 4 Detector DAD 5 Figure 21 Capillary and tubing connections in flow path standard delay volume configuration 72 1260 Infinity Binary LC System User Guide Item p n 1 G1312 87303 1 G1312 87304 2 G1367 87304 2 01090 87306 3 G1316 87300 4 G1315 87311 5 5062 2462 5 5062 8535 1260 Infinity Binary LC System User Guide System Setup and Installation Description Capillary ST 0 17 mm x 400 mm S S Pump to autosampler Capillary ST 0 17 mm x 700 mm S S Pump to cooled Autosampler Capillary ST 0 17 mm x 250 mm S S Autosampler to TCC Capillary ST 0 17 mm x 380 nm S S Cooled Autosampler to TCC Capillary ST 0 17 x 90 mm S S TCC to Column Capillary ST 0 17 mm x 380 mm S S Column to DAD Tube PTFE 0 8 mm x 2 m re order 5 m DAD to waste Waste accessory kit VWD to waste 4 73 4 System Setup and Installation Installing the Modules Connections for Medium and Low Delay Volume Configuration Figure 22 on page 74 shows capillary and tubing connections in the flow path for medium and low delay volume configurations of the 1260 Infinity Binary LC Solvent cabinet Degasser Pump Autosampler Thermostat Thermostatted column compartment Detector DAD 6 Figure 22 Capillary and tubing connections in flow path medium and low delay volume configurations 74 1260 Infinity Binary LC System User Guide
51. er the injection has been made and then after for example 3 minutes into a 4 minutes run to start the process of aspirating the next sample and preparing for injection This can typically save 0 5 to 1 minute per injection 1260 Infinity Binary LC System User Guide Optimization of the Agilent 1260 Infinity Binary LC 3 How to Achieve Lowest Carry over Carryover is measured when residual peaks from a previous active containing injection appear in a subsequent blank solvent injection There will be carryover between active injections which may lead to erroneous results The level of carryover is reported as the area of the peak in the blank solution that is expressed as a percentage of the area in the previous active injection The Agilent 1260 Infinity Autosampler is optimized for lowest carryover by careful design of the flow path and use of materials in which sample adsorption is minimized A carryover figure of 0 002 should be achievable even when a triple quadrupole mass spectrometer is the detector Operating settings of the autosampler allow the user to set appropriate parameters to minimize carryover in any application involving compounds liable to stick in the system The following functions of the autosampler can be used to minimize carryover Internal needle wash External needle wash e Needle seat backflush Injection valve cleaning The flow path including the inside of the needle is continuously flushed in normal operati
52. erature 23 analog signal output 25 analysis data 96 automatic delay volume reduction 49 binary pump 11 buffer solutions 11 C calculator costs 34 carry over 50 51 changing solvents 79 column blockages prevent 65 column temperature 37 thermostatting 37 columns Sub 2 micron particles 34 compensation compressibility 24 composition accuracy 25 composition precision 25 composition range 24 138 compressibility compensation 24 configuration low delay volume 43 medium delay volume 44 standard delay volume 45 configuring online plot 85 control and data evaluation 25 control assembly 14 cycle time instrument configuration 48 D data analysis 112 data analysis 96 data evaluation and control 25 DEF_LCM 111 delay volume automatic switching 42 description 40 example 40 design 11 detector achieving higher sensitivity 62 dimensions 23 diode array detector description 19 dual piston in series design 11 E extra column volume description 41 F flow accuracy 24 flow cell 63 flow precision 24 flow range 11 operational 24 setable 24 frequency range 23 frictional heating 37 G gradient formation 24 high pressure mixing 11 humidity 23 hydraulic system 24 injection volume achieving higher volumes 47 installing data system 68 Lab Advisor 69 software controller 68 Instrument Acquisition 112 integration events table 98 integration 98 internet 110 introduction to the degasser 14 L
53. erefore reduces the efficiency losses but a higher column outlet temperature has to be accepted The raised temperature may have an effect on selectivity At lower back pressure performance losses due to frictional heat are minimized so that 4 6 or 3 mm inner diameter sub 2 micron columns still deliver superior efficiencies compared with the respective 2 1 mm inner diameter columns In summary the use of sub two micron packing material offers benefits of increased efficiency higher resolution and faster separations The features of the Agilent 1260 Infinity Binary LC are discussed in the chapter Product Description The chapter Optimization considers how to apply the theory and use these features to develop optimized separations 1260 Infinity Binary LC System User Guide 37 2 introduction Frictional Heating 38 1260 Infinity Binary LC System User Guide 1260 Infinity Binary LC System User Guide 3 Optimization of the Agilent 1260 Infinity Binary LC How to Configure the Optimum Delay Volume 40 Delay Volume and Extra Column Volume 40 Delay Volumes in the Agilent 1260 Infinity Binary LC 42 Optimum Instrument Configuration for 2 1 mm i d Columns 43 Optimum Instrument Configuration for 3 and 4 6 mm i d Columns 45 How to Achieve Higher Injection Volumes 47 How to Achieve Shorter Cycle Times 48 How to Achieve High Throughput 50 How to Achieve Lowest Carry over 51 How to Achieve Higher Resolution 53 Optimum Instrument Configurat
54. ero Offset EH Peakwidth Attenuation 1000 v mAU gt 0 10 min 2 0 time 2 5 H S ind 2 Os eel a Margin for negative Absorbance Stoptime Posttime 100 mau As Pump Injector Of Autobalance Lamps on required for acquisition 1 00 gt min 1 00 min Prerun vV UV Lamp Postrun Timetable empty I Show timetable graph Figure 29 Setup Method screen Diode Array Detector tab Signals Up to eight separate signals chromatograms can be recorded To mark a signal for collection check the Use Signal box for that signal define the wavelength and bandwidth and if a reference signal is required also check and define that box Wavelength sets the central wavelength nm of the signal Bandwidth sets the width nm of the signal 124 1260 Infinity Binary LC System User Guide Appendix 6 Reference Wavelength sets the central wavelength nm of the reference band which is subtracted from the analytical signal Reference Bandwidth sets the width nm of the reference band e Peakwidth sets the data collection rate and signal filtering Stoptime Posttime are set to As Pump Injector and Off and these values are usually taken care of in the Pump tab However a detector stop time that is different from the pump stop time can be set if it is required to stop data analysis before the end of the run which is defined in the pump This can be the case when a gradient equilibration ramp has been set at
55. eters 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 B under the menu bar or double click the method name DEF_LC M in the Methods tab of the Navigation Pane File RunControl Instrument Method Sequence View Abort Help v Sequences 3 fed veF_ic s ras Se Method and Run Control Method and Run Control re PHENONES2 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 QJ DGCALOQ3 M e QJ DGCALOQ4 M QJ DGCALOQS M QW DGcaLoQ6 m M1 necaips 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 NA I Note that this method can never be overwritten with new parameters Hence clicking on de Save will re direct you into the Save As function so that you must enter a different method name 84 1260 Infinity Binary LC System User Guide Quick Start Guide 5 Preparing the System Configuring the Online Plot 1 If the Online Plot window is not visible
56. formats Agilent offers more than 140 ZORBAX 1 8 um Rapid Resolution High Throughput RRHT columns 14 selectivity choices 15 to 150 mm long 2 1 3 0 and 4 6 mm internal diameters Additionally to the ZORBAX columns PoroShell columns with nine selectivity choices are available for use with the Agilent 1260 Infinity Binary LC This enables the optimum stationary phase to be selected so that the selectivity is maximized The resolution flow rate and analysis time can be optimized by selecting the appropriate column length and diameter and operation with longer STM columns has become more accessible than ever before PoroShell columns are so called superficially porous particle SPP columns In contrast to totally porous silica columns these SPP columns have a solid core 1 7 um in diameter and a porous silica layer 0 5 um thickness surrounding it Speed and resolution of PoroShell columns are comparable to sub two micron columns with up to 50 less backpressure PoroShell columns have enjoyed a recent resurgence in smaller particle sizes than the older pellicular particle columns The current interest in this technology is driven by its re introduction in smaller particle sizes such as the sub 3 micron sizes for use in typical small molecule reversed phase separations Many laboratories perform an extensive screening process to select the best combination of stationary phase mobile phase and temperature for their separations
57. front of the column It is also recommended that the sample is thoroughly filtered or and centrifuged and that any kind of particulate matter in the mobile phases is avoided To ensure best results follow these simple usage guidance 1 Install and run the column only in the flow direction marked on the column 2 Use only solvents that are high quality chromatography grade Filter all aqueous buffers and all samples through an appropriate 0 2 um filter before use 4 Replace bottles of mobile phase buffer every 24 48 h do not add mobile phase to the bottle always use a new bottle 5 Do not use a high buffer salt mobile phase gt 50 mM in combination with high acetonitrile concentrations due to possible precipitation 6 An in line filter is recommended to catch particulates and extend column life Use the suitable 1260 Infinity In line filter for your column 1260 Infinity LC in line filter 2 1 mm 0 2 um pore size filter max 600 bar connecting capillary 70 x 0 12 mm SS 5067 1551 for 2 1 i d columns 1260 Infinity LC in line filter 4 6 mm 0 2 um pore size filter max 600 bar connecting capillary 90 x 0 17 mm SS 5067 1553 for 4 6 mm or 3 0 mm i d columns Change the filter when the pressure increases by 10 7 Purge the pumps the connections up to the column of any buffer containing mobile phases and flush through 5 mL of solvent before attaching the column to the instrument 8 Flush the column with compatible mo
58. gilent Technologies 81 5 Quick Start Guide About the Quick Start Guide 82 This chapter provides information on running the Agilent 1260 Infinity Binary LC It can be used as a guide to quickly running a first analysis after installation serving both as a tutorial example and a check on the overall functioning of the system It also includes more detailed information about method parameters This example demonstrates setting up and running an analysis using the columns and checkout sample that are provided with the Agilent 1260 Infinity Binary LC The example refers to menus and commands in OpenLAB CDS ChemStation Edition but identical functions are also available on the alternative control options such as OpenLAB CDS EZChrom Edition and MassHunter software In the following the terms ChemStation and EZChrom always refer to Agilent OpenLAB CDS ChemStation Edition and Agilent OpenLAB CDS EZChrom Edition respectively The starting point assumes that the system has been installed turned on and initially primed see Initial Priming on page 76 The UV lamp should be turned on at least 30 minutes before starting any quantitative work 1260 Infinity Binary LC System User Guide Quick Start Guide 5 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 T
59. h column temperatures are recommended to enable high flow rates to be used and to shorten run time even further Zorbax SB columns can be used up to 90 C at low pH Alternating column regeneration Even shorter cycle times can be achieved by using a column regeneration valve in combination with a regeneration pump Using this setup the regeneration of the column that is used previously takes place during the analysis on the second column This shortens cycle time significantly Using two columns two pumps and one 2 position 10 port valve allows switching between these columns for shortest cycle times from injection to injection Typically columns of the same chemistry and the same batch provide a retention time precision that allows data processing using the same calibration table Please refer to the Valve Solution User Guide for a more detailed description of the alternating column regeneration 1260 Infinity Binary LC System User Guide Optimization of the Agilent 1260 Infinity Binary LC 3 Automatic delay volume reduction ADVR The Agilent 1260 Infinity High Performance Autosampler offers the possibility of performing overlapped injections OI and or automatic delay volume reduction ADVR This means that the injection valve is switched out of the flow path after the sample has reached the top of the column This reduces the delay volume significantly see Figure 17 on page 49 DAD1 A Sig 245 10 Ref 360 80 E CHEM32 1
60. hart 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 1260 Infinity Binary LC System User Guide Quick Start Guide 5 Purging the Pump Purge the pump if The pump has been primed for the first time The pump is to be purged 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 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 sol
61. he next solvent channel and repeat steps 5 and 6 until all channels have been purged Turn the flow off and close the purge valve System User Guide 77 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 If solvents containing volatile components are left in the degasser without flow for a prolonged period there will be a slight loss of the volatile components Preparations Switch the system on The purge tool of the Lab Advisor 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 4 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 78 1260 Infinity Binary LC System User Guide When Parts required Preparations CAUTION System Setup and Installation 4 Changing Solvents When the solvent of a 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 preci
62. hecking the relevant box These are primarily used for diagnostic purposes They are Pump Pressure Flow A B Composition can be useful for overlaying the gradient profile ona chromatogram Thermostatted Column Compartment Left Right temperature e Detector Board temperature Optical unit temperature UV lamp anode voltage Appendix 6 Data Analysis Signal Details The Signal Details screen can also be directly accessed in Method and Run Control view right click on the graphical user interface on the Calibration icon and then select Signal Details in the context menu In the Data Analysis view it can be accessed through the menu Calibration gt Signal Details The Signal Details screen is the next step in Edit Entire Method and it tells Data Analysis which of the acquired signals to process The drop down box lists the available signals including the analytical signals defined in the detector settings and recorded parameters such as temperature flow composition pressure and diagnostic traces Select a signal and click Add to Method to transfer it to the Signal Details table shown in the lower part of the screen You can select any or all of the acquired detector signals for processing If no signals are selected the table is empty in this situation ChemStation will default to processing all of the acquired detector signals Sometimes the user might edit an existing method to create a new method and is faced
63. ial carryover This is the recommended option and is configured in the next entry Needle Wash if selected above Mode determines how the outside of the needle will be rinsed either actively in the Flush Port or by dipping into a specified Wash Vial Time in seconds the peristaltic pump connected to the flush port will pump the rinse solvent It then pumps for a further 15 s to clear the flush port Location determines which vial or well plate will be used if Wash Vial was selected Vials should not have a septum i e they should be open to avoid transfer of carryover material on the septum 1260 Infinity Binary LC System User Guide 115 6 Appendix Repeat determines if Wash Vial was selected how many times the needle dips into the vial default 3 maximum 5 Stop Time Post Time are set to No Limit Off and these values are taken care of in the pump tab Advanced Auxiliary Draw speed is the speed that the sample is pulled into the needle Default value is 100 ul min It should be slowed down for viscous samples or for greater precision with small sample volumes lt 2 uL Eject speed is the speed of delivery from the needle Draw position is the vertical offset from the nominal injection position of 10 mm above the bottom of a vial This is about half way up a 2 ml vial so should be given a negative offset to take the sample from near the bottom of the vial for example a value of 7 mm would
64. ibility 1 0 12 5 solvents with pH lt 2 3 should not contain acids which attack stainless steel High pressure binary mixing Standard delay volume configuration measured with water at 600 800 uL includes 400 uL mixer 1 mL min water caffeine dependent on back pressure tracer Low delay volume configuration 120 uL settable range 0 100 recommended range 1 99 or 5 pL min per channel whatever is greater 1260 Infinity Binary LC System User Guide Table 2 The Agilent 1260 Infinity Binary LC Product Description 1 Performance Specifications of the Agilent 1260 Infinity Binary Pump G1312B Type Specification Comments Composition precision Composition accuracy Control Local control Analog output Communications Safety and maintenance GLP features Housing lt 0 15 RSD or lt 0 04 min SD whatever is greater 0 35 absolute at 2 mL min at 10 MPa 100 bar Agilent control software e g ChemStation EZChrom OL MassHunter Agilent Instant Pilot For pressure monitoring 1 33 mV bar one output Controller area network CAN RS 232C APG Remote ready start stop and shut down signals LAN optional Extensive support for troubleshooting and maintenance is provided by the Instant Pilot Agilent Lab Advisor and the Chromatography Data System Safety related features are leak detection safe leak handling leak output signal for shutdown of
65. ice should be used for lowest extra column volume 56 1260 Infinity Binary LC System User Guide Optimization of the Agilent 1260 Infinity Binary LC 3 Extra column Volume To maintain the high resolution achieved on the column the extra column volume especially after the column should be as low as possible For 4 6 mm inner diameter columns the standard delay configuration should be used for the pump see Figure 15 on page 45 e For 2 1 mm inner diameter columns the low delay configuration should be used for the pump and the low dispersion kit should be installed in the column compartment For highest UV sensitivity the short mixer is also recommended See Figure 13 on page 43 and Figure 14 on page 44 The injection volume is also of importance especially if the sample is dissolved in an organic solvent In this case the gradient should start with a low percentage of organic phase to focus the compounds at the top of the column This avoids peaks dispersion due to the injection The 10 mm Max Light cartridge cell has a low dispersion volume o volume 1 0 uL and no further volume optimization is required In situations where the alternative 60 mm Max Light high sensitivity cell is used to get higher sensitivity the cell volume is optimized for the use with 3 mm and 4 6 mm inner diameter columns 1260 Infinity Binary LC System User Guide 57 3 Optimization of the Agilent 1260 Infinity Binary LC Data Rate The data rat
66. ile phase and A B and C are constants that are related to the different dispersive forces The A term relates to eddy diffusion or multiple flow paths through the column B relates to molecular diffusion along the column axis longitudinal C relates to mass transfer of the analyte between the mobile and stationary phases The separation is at its most efficient when H is at a minimum The effect of each individual term and the combined equation are shown in Figure 6 on page 29where the plate height is plotted against the linear flow rate through the column This type of plot is known as a Van Deemter Curve and is used to determine the optimum flow rate Gninimum point of the curve for best efficiency of separation for a column I J lt renee x 2 large particle small particle a T E A 2 2 Resulting Van Deemter curve 5 2 2 E Resistance to Mass Transfer H min Multipath Term Eddy Diffusion rs eve ese U op Longitudinal diffusion Linear flow u Figure 6 A hypothetical Van Deemter curve 1260 Infinity Binary LC System User Guide 29 2 30 Introduction The van Deemter plots in Figure 7 on page 30 show that reducing particle size increases efficiency Switching from commonly used 3 5 um and 5 0 um particle sizes to 1 8 um particles offers significant performance improvements The 1 8 um particles give two to three times lower plate height values and proportionately higher efficiencies This allows a sh
67. imetable graph displays the timetable changes graphically when the box is ticked Stop Timedefines the overall time for the separation or run and is sometimes referred to as the Run Time by some users This is the time counted in minutes since the injection was made when the run ends which means that data acquisition will stop the flow composition and other system settings will revert to the initial values for the method and the system will become available to make the next injection It should always be at least as long as the last entry in the timetable or else the run will stop and revert to starting conditions before the timetable events are complete Stop Time can be set to No Limit in which case the user must manually stop the run While all the system modules have Stop Time parameters the Stop Time in the pump is considered to be the master and the other modules will usually be set to follow this value Post Time defines a countdown period after the end of a run during which the next injection is prevented It allows time for the system to re equilibrate after a gradient run For an isocratic method it can be set to Off For a gradient method the value can be determined experimentally by observing the baseline behavior but typically will need time for the delay volume of the system plus at least three to five column volumes to be flushed through the system Pressure Limits control the behavior of the pump regarding pressu
68. injection which would cause a reduced peak height In order to achieve this the sample should be dissolved in a solvent composition of lower elution strength than the mobile phase e The column temperature should not be too low to avoid long retention of the peaks on the column This also creates peak dispersion and a lower peak height e The data rate of the UV detector should be selected according to the actual peak width Higher than necessary data rates should be avoided because of higher noise levels e Available UV detectors are the Agilent 1260 Infinity Diode Array Detector G4212B and the Agilent 1260 Infinity Variable Wavelength Detector G1314F with data rates of 80 Hz and significantly lower noise and drift levels Choosing a Flow Cell on page 63 gives an overview of the flow cells available with the G4212B DAD For information on flow cells used with the G1314F VWD please refer to the User Manual of the Agilent 1260 Infinity Variable Wavelength Detector Chromatographic Conditions For an optimum signal to noise ratio it is of advantage if the peaks elute quickly Elevated flow rates should be used to elute the peaks at low k values e Faster gradients can also be applied to elute the peaks at low k values 1260 Infinity Binary LC System User Guide 61 3 62 Optimization of the Agilent 1260 Infinity Binary LC Columns Sensitivity is specified as a signal to noise ratio S N and hence the need to
69. ion Long columns packed with smaller particles result in higher efficiency and higher resolution This is important for analysis of complex samples from metabolomics or proteomics studies Also applications such as impurity profiling can benefit from higher separation power Even the LC MS analysis of drugs in biological fluids can benefit from the higher peak capacity because of the reduced interference from ion suppression In general higher separation power provides more confidence in the analytical results Peak capacity 694 LC with GC resolution 20 Absorbance mAU 1 A Nh IA Pr UW kaha kj M l W huuh A IWAL 5 10 15 20 25 30 35 40 Time min Figure 12 Peak capacities of more than 700 can be achieved using a ZORBAX RRHT SB C18 column 2 1 x 150 mm 1 8 um to analyze a tryptic digest of BSA 36 1260 Infinity Binary LC System User Guide Introduction 2 Frictional Heating Forcing mobile phase through the column at higher pressure and higher flow rates generates heat The resulting temperature gradients radial and longitudinal can have an impact on the column efficiency Power F p where F is the flow rate and p is the pressure Powerful column thermostatting for example using a water bath generates a strong radial temperature gradient which leads to significant loss in column efficiency Still air column thermostatting reduces the radial temperature gradient and th
70. ion for High Resolution 56 How to Achieve Higher Sensitivity 60 Optimum Instrument Configuration for High Sensitivity 61 Choosing a Flow Cell 63 How to Prevent Column Blockages 65 This chapter considers how to apply the theory and use the features of the LC system to develop optimized separations Apg Agilent Technologies 39 3 Optimization of the Agilent 1260 Infinity Binary LC How to Configure the Optimum Delay Volume 40 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 Delay Volume In gradient separations this volume causes a delay between the mixture changing in the pump and that change reaching the column The delay depends on the flow rate and the delay volume of the system In effect this means that in every HPLC system there is an additional isocratic segment in the gradient profile at the start of every run Usually the gradient profile is reported in terms of the mixture settings at the pump and the delay volume is not quoted even though this will have an effect on the chromatography This effect becomes more significant at low flow rates and small column volumes and can have a large impact on the transferability of gradient methods It is important therefore for fast gradient
71. ity between two peaks k retention factor of second peak formerly called capacity factor The term with the most significant effect on resolution is the selectivity a and practically varying this term involves changing the type of stationary phase C18 C8 phenyl nitrile etc the mobile phase and temperature to maximize the selectivity differences between the solutes to be separated This is a substantial piece of work which is best done with an automated method development system which allows a wide range of conditions on different columns and mobile phases to be assessed in an ordered scouting protocol This section considers how to get higher resolution with any chosen stationary and mobile phases If an automated method development system was used in the decision on phases it is likely that short columns were used for fast analysis in each step of the scouting 1260 Infinity Binary LC System User Guide 53 3 54 Optimization of the Agilent 1260 Infinity Binary LC The resolution equation shows that the next most significant term is the plate count or efficiency N which can be optimized in a number of ways N is inversely proportional to the particle size and directly proportional to the length of a column and so smaller particle size and a longer column will give a higher plate number The pressure rises with the inverse square of the particle size and proportionally with the length of the column Resolution increases wi
72. lumn Poroshell 120 EC C18 3 0 x 50 mm 2 7 um 699975 302 Column Poroshell 120 EC C18 4 6 x 50 mm 2 7 um 699975 902 Temperature 40 C Detector Signal A Wavelength 250 nm Bandwidth 4 nm Reference Wavelength 360 nm Reference Bandwidth 100 nm 1260 Infinity Binary LC System User Guide 89 5 Quick Start Guide Table 10 Method parameters for first separation run low delay volume configuration Module Parameter Setting Pump Solvent A Water Solvent B Acetonitrile Flow rate 0 75 mL min Solvent 40 A 60 B Composition Stop Time 1 2 min 1 min post time Autosampler Injection Tul Column Column Column SB C18 4 6x 50 mm 1 8 um 600 bar Compartment 827975 902 Temperature 40 C Detector Signal Wavelength 250 nm Bandwidth 4 nm Reference Wavelength 360 nm Reference Bandwidth 100 nm Checkout specifications four peaks are visible in the chromatogram Intensity Test pass Wavelength Calibration 1 nm 90 1260 Infinity Binary LC System User Guide Quick Start Guide 5 Checkout Preparation 1 uy Oo Oo A 1260 Infinity Binary LC Fill solvent bottle A with HPLC grade Water Fill solvent bottle B with HPLC grade Acetonitrile If installed purge each channel of the on line degasser using the supplied syringe Open the purge valve of the pump and purge each channel at 5 mL min This should be sufficient to remove air in the system Install the supplied column into the column c
73. m particle size columns can be used in pursuit of two main objectives 1 Faster Chromatography Short columns with sub two micron particles offer the opportunity to dramatically reduce analysis time by increasing the flow rate without losing separation performance 2 Higher Resolution Long columns with sub two micron particles provide higher efficiency and therefore higher resolution which is required for the separation of complex samples The pressure that is needed to drive solvent through a column containing STM sub two micron particles rises rapidly as flow rate is increased for faster separations and very rapidly as the length of the column increases for more resolution Thus the acceptance of STM columns has been synonymous with the development of UHPLC systems that is HPLC systems offering higher pressures than the 400 bar norm that was extant since the early days of HPLC Today Agilent offers the 1290 Infinity LC for highest UHPLC requirements with pressures up to 1200 bar 28 1260 Infinity Binary LC System User Guide Introduction 2 The Theory Separation efficiency in HPLC can be described by the van Deemter equation Figure 6 on page 29 This results from the plate height model that is used to measure the dispersion of analytes as they move down the column H is the Height Equivalent to a Theoretical Plate sometimes HETP dp is the particle size of the column packing material uo is the linear velocity of the mob
74. mizing the life of the pressure sensor do not leave HFIP in the chamber when the unit is off 1260 Infinity Binary LC System User Guide Appendix Sapphire Ruby and Al 03 based ceramics Sapphire ruby and ceramics based on aluminum oxide AlgOz are inert to almost all common acids bases and solvents There are no documented incompatibilities for HPLC applications 1260 Infinity Binary LC System User Guide 6 109 6 Appendix Agilent Technologies on Internet For the latest information on products and services visit our worldwide web site on the Internet at http www agilent com 110 1260 Infinity Binary LC System User Guide Appendix 6 Setting Up a Method using Edit Entire Method A method in the ChemStation contains all the parameters for Data Acquisition controlling the system and Data Analysis processing the data to give quantitative and qualitative results The parameters are accessed through a series of screens which each focus on one module or function These screens are accessed by clicking an icon on the graphical user interface GUI or through the menu bar with its drop down menus A new method can be created by loading and editing an existing method or it can be created by loading and editing the blank template method DEF_LC M To change only a few parameters you can go directly to the relevant setup pages for the parameters to be altered Less experienced users may find it easier to use the Edit E
75. mps and for normal phase applications in 1260 Infinity pumps Polyethylene has a good stability for most common inorganic solvents including acids and bases in a pH range of 1 to 12 5 It is compatible to many organic solvents used in chromatographic systems like methanol acetonitrile and isopropanol It has limited stability with aliphatic aromatic and halogenated hydrocarbons THF phenol and derivatives concentrated acids and bases For normal phase applications the maximum pressure should be limited to 200 bar 1260 Infinity Binary LC System User Guide Appendix 6 Tantalum Ta Tantalum is inert to most common HPLC solvents and almost all acids except fluoric acid and acids with free sulfur trioxide It can be corroded by strong bases e g hydroxide solutions gt 10 diethylamine It is not recommended for the use with fluoric acid and fluorides Stainless Steel SST Stainless steel is inert against many common solvents It is stable in the presence of acids and bases in a pH range of 1 to 12 5 It can be corroded by acids below pH 2 3 It can also corrode in following solvents Solutions of alkali halides their respective acids for example lithium iodide potassium chloride and so on and aqueous solutions of halogens High concentrations of inorganic acids like nitric acid sulfuric acid and organic solvents especially at higher temperatures replace if your chromatography method allows by phosphoric acid o
76. ndard i Tail Peak Skim Height Ratio 0 00 eo Front Peak Skim Height Ratio 0 00 Skim Valley Ratio 20 00 i Baseline Correction Advanced 40 a Peak to Valley Ratio 500 00 J 5 a D g 20 8 i w Specific Events For Signat 4 A ANo Fal DAD Default X J a r T T r r rr 1 2 2 4 5 rin 4 r Time Integration Events Value Time Area Height Width Area Symmetry Initial Slope Sensitivity 5 1 0 747 301 105 9 0 0452 29 738 _0 718 Initial Peak Width 0 02 2 1 023 275 9 76 8 0 0539 27 257 0 648 Initial Area Reject 5 3 2 571 178 2 26 9 0 1013 17 604 0 634 Initial Height Reject 1 4 5 856 257 1 16 9 0 2255 25 401 0 676 Initial Shoulders OFF 98 2 Click the Set Integration Events Table icon wf to open the table as shown Set Baseline Correction to Advanced for gradient runs 4 Set Slope Sensitivity to 5 Higher numbers will integrate steeper peaks and ignore less steep peaks 5 Set the Peak Width value to the narrowest peak of interest approximately 0 02 in this case 6 Area Reject and Height Reject can be set to reject the smallest peaks 7 Click the Integrate icon aia to update the results using these new settings 8 Exit from the Integration Events table using the green tick icon B 1260 Infinity Binary LC System User Guide Specify the Report Quick Start Guide 5 Data Analysis 1 On the menu bar click Report gt Specify Report to display the window shown in the figure below Specify Report Instrume
77. nit and a mixer A pressure sensor monitors the pump pressure A purge valve with integrated PTFE frit is fitted to the pump outlet for convenient priming of the pumping system 1260 Infinity Binary LC System User Guide 11 1 The Agilent 1260 Infinity Binary LC Product Description System Components Pump outlet g Inlet valve from solvent bottle A 12 Damper Purge valve Mixer 4 gt Pressure sensor to waste Outlet Mixing Inlet valve Chamber from solvent Seals bottle B Seals Pistons Pistons Pump head A Pump head B Figure 1 The Hydraulic Path of the Binary Pump with Damper and Mixer 1260 Infinity Binary LC System User Guide The Agilent 1260 Infinity Binary LC Product Description 1 System Components Damper and mixer can be bypassed for lowest delay volume of the binary pump This configuration is recommended for low flow rate applications with steep gradients Figure 2 on page 13 illustrates the flow path in low delay volume mode For instructions on how to change between the two configurations see the G1312B Binary Pump User Manual NOTE Bypassing the mixer while the damper remains in line is not a supported configuration and may lead to undesired behavior of the binary pump Purge valve Pump outlet Py Pressure sensor to waste Outlet valve Inlet valve Mixing chamber _ i Be from solvent Seals S
78. ns This calculator is available on the Agilent Technologies web site along with a method translator calculator www chem agilent com The results are presented graphically and in tabular form Shorter run times also deliver faster answers This is important in process control and rapid release testing Instead of waiting hours to release a single batch of a drug all the system suitability calibration and sample analysis can now be done in less than an hour Rapid answers are also important for synthetic chemists using open access LC MS systems for compound confirmation and reaction control Shorter run times can also accelerate the method development process significantly 1260 Infinity Binary LC System User Guide Column Length mm 150 100 75 50 30 15 Column Efficiency N 5 pm 12 500 8 500 6000 4 200 N A N A Figure 11 Column Column Efficiency Efficiency N 3 5 um N 1 8 um 21 000 35 000 14 000 23 250 10 500 17 500 7 000 12 000 4 200 6 500 2 100 2 500 Efficiency N Pressure Analysis Time Peak Volume Solvent Usage Introduction Analysis Time Reduction 33 50 67 80 90 Relation between particle size efficiency and analysis time Analysis time can be shortened without sacrificing column efficiency by optimizing particle size and pressure 1260 Infinity Binary LC System User Guide 2 35 2 Introduction Higher Resolut
79. nt 1 Reporting settings Quantitation settings Report mode Use Intelligent Reporting Use Classic Reporting Style Report style Short Quantitative result sorted by C Sample info on each page V Add chromatogram output C Add sample custom fields to sample info Report layout for uncalibrated peaks O Separately With calibrated peaks Destination File setting Printer Screen File Unique PDF file name File prefix Report V Add fraction table and ticks C Add summed peaks table Add compound custom fields O Do not report Chromatogram output of pages Portrait w g Landscape Response Multi page Landscape Pages Signal options 1260 Infinity Binary LC System User Guide m 99 5 100 Quick Start Guide Data Analysis Specify Report Instrument 1 Reporting settings Quantitation settings Calculation mode Calculate Based on Area ISTD correction E Use multiplier and dilution factor with ISTDs Calculation factors Use sample data From Data File Compound ISTD Amount Amount 0 Multiplier Dilution 2 With the example settings shown in the figures above you can produce an Area Percent report on the screen 3 In the Destination section selec
80. ntire Method function as this automatically steps through the pages This is accessed through the menu Method gt Edit Entire Method and opens the Check Method Sections to Edit dialog Edit Method instrument 2 Check Method Sections to Edt M Method Informatie E Insteumant Acqusitios Data Analpees M Bun Time Checklist Cancel a Figure 23 Check Method Sections to Edit This dialog summarizes the sections that will be viewed and offers an opportunity to bypass certain parts by deselecting them 1260 Infinity Binary LC System User Guide 111 112 Appendix Depending on the selected parts the function sequentially shows several screens Method Information comprises a text description about the method Instrument Acquisition comprises injector parameters pump parameters oven parameters detector parameters and instrument curves Data Analysis comprises signal details integration parameters and reporting parameters Run Time Checklist comprises the parts of the method that will be executed During Edit Entire Method clicking OK will close the current entry screen and move on to the next screen It is a one way process If you inadvertently pressed OK before all entries have been made then use Cancel and restart Edit Entire Method Alternatively carry on and return to the unfinished screen at the end Clicking Cancel offers a button to Skip the remaining screens 1260 Infinity Binary LC System
81. nuous or peak controlled basis this applies to the ChemStation software Some software packages e g EZChrom only support continuous collection of all spectra and the peak controlled options do not appear Spectral collection and signal collection are independent operations that are performed by the detector firmware and are not dependent on the computer software extracting data from the 3D data matrix The rate at which spectra are taken is determined by the Peakwidth setting and eight spectra are taken during the time that is specified in Peakwidth The firmware does peak detection on signal A only to determine when peak controlled spectra should be saved For multiple signals it may be necessary to have signal A set as a broadband detector to ensure that peak spectra are available for all the different wavelength signals Store controls the spectral collection mode with the following options None no spectra are stored Apex Baselines three spectra are taken at the start apex and end of peak Apex Slopes Baselines five spectra are taken at the start upslope apex downslope and end of peak Allin Peak all the available spectra within a peak are stored All all spectra throughout the run are stored Every 2nd Spectrum stores only alternate spectra that are acquired throughout the run Range Spectra can be saved across the full range of the detector 190 nmto 640 nm or any reduced range that is chosen as a
82. olume might be achieved while maintaining good resolution and peak dispersion 1260 Infinity Binary LC System User Guide 47 3 Optimization of the Agilent 1260 Infinity Binary LC How to Achieve Shorter Cycle Times 48 Shorter cycle times can be achieved by selecting a short column with good selectivity The column dimensions are also determined by the detection system that is used For UV detection 3 0 mm inner diameter columns are ideal because here the highest linear velocities can be obtained With 4 6 mm inner diameter columns high linear velocities can also be reached but the maximum flow rate is limited to 5 mL min The pump should be used in its standard delay volume configuration see Figure 13 on page 43 for 4 6 mm inner diameter and 3 0 mm inner diameter columns For 2 1 mm inner diameter columns the low delay volume configuration should be used In addition when using 2 1 mm inner diameter columns the low dispersion kit should be installed to provide lowest extra column volume For highest UV sensitivity it is recommended in addition to use the short mixer Low volume mixer 200 uL 5067 1565 Chromatographic conditions strongly depend on the compounds that are to be analyzed but some rules of thumb can be used to achieve short run times The flow rates should be as high as possible depending on the required resolution back pressure and the detection system used Steep gradients should be used Hig
83. ompartment Flush the flow cell with HPLC grade water for 5 min Run a Wavelength Calibration and Intensity Test where applicable Allow the system to equilibrate at the checkout conditions listed in Table 9 on page 89 and Table 10 on page 90 for 10 min Prepare a vial of the isocratic checkout sample System User Guide 91 5 Quick Start Guide Setting Up the Method This section shows how to quickly set the method conditions for an analysis Prerequisites 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 In the Method page for the 1260 Infinity Binary Pump enter the following parameters Flow rate 4 0 ml min Solvent A Select Water from the compressibility drop down list Solvent B Select the check box to make Solvent B active Set the value for B to 60 Stop Time 1 min Post Time 1 min b Other parameters can remain at their 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
84. on providing good elimination of carryover for most situations Automated delay volume reduction ADVR will reduce the delay volume but will also reduce the flushing of the autosampler and should not be used with analytes where carryover might be a problem To ensure lowest carry over consider the following recommendations e Always use the autosampler with the injection valve in mainpass position e Flush the exterior of the needle with an appropriate solvent using the flush port The flush time should be a minimum of 10 s If possible reduce the draw speed to 10 uL min e Use Agilent capped 2 mL vials Screw cap vial 2 mL 5182 0556 e Ifthe seat is contaminated use an appropriate seat flush procedure Use flushing solvents that are capable of dissolving the sample compounds e Use acidic mobile phases for basic compounds 1260 Infinity Binary LC System User Guide 51 3 52 Optimization of the Agilent 1260 Infinity Binary LC Flushing and cleaning of the autosampler to achieve near zero carry over During the injection routine the sample loop the inside of the needle the seat capillary and the main channel of the injection valve are in the flow path and remain there throughout the duration of the run This means that these parts are flushed continuously with mobile phase during the complete analysis It is only during aspiration of the sample that the injection valve is switched out of the flow path In this position th
85. orter column to be used without sacrificing resolution and hence the analysis time is also reduced by a factor of two to three The increased efficiency is derived to a large extent from the reduction in multiple flow paths as a result of the smaller particles this leads to a smaller A term eddy diffusion In addition smaller particles mean shorter mass transfer times reducing the C term and it can be seen that the overall effect is a much reduced loss of efficiency as the flow rate increases the slope of the line is reduced This means that the separation on smaller particles can be further accelerated by increasing the flow rates without significantly reducing efficiency 0 0045 0 0040 0 0035 0 0030 0 0025 5 0 pm 0 0020 HETP cm plate 0 0015 3 5 ym 0 0010 0 0005 18pm 0 0000 2 ml min 5 0 ml min 0 0005 f f f T 0 0 0 2 0 4 0 6 0 8 1 0 1 2 1 4 1 6 Interstitial linear velocity Hg cm sec Figure 7 Van Deemter curve for different particle sizes 1260 Infinity Binary LC System User Guide Introduction 2 A chromatographic separation can be optimized based on physical parameters of the HPLC column such as particle size pore size morphology of the particles the length and diameter of the column the solvent velocity and the temperature In addition the thermodynamics of a separation can be considered and the properties of the solute and the stationary and mobile phases percentage
86. pitation or residual liquid droplets in parts of the system p n Description 1 Purging solvent s see Table 8 on page 80 1 5022 2184 Union ZDV Remove the column and replace it by a ZDV fitting Prepare bottles with appropriate intermediate solvents see Table 8 on page 80 1 Ifthe channel is not filled with 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 Buffer salt of aqueous buffers may precipitate in residual isopropanol Capillaries and filter may be clogged by precipitating salt gt Flush solvent lines containing high concentration of salts first with water before introducing organic solvent gt Do not perform steps 5 to 7 for channels running with aqueous buffer as solvent 5 Replace the solvent bottle by a bottle of isopropanol 6 Flush the channel at a flow rate suitable for the installed tubing typically 3 5 mL min for 5 min 7 Swap the bottle of isopropanol with a bottle of solvent for your application 8 Repeat steps 1 to 7 for the other channel s of the pump 9 Install the desired column set the required composition and flow rate for your application and equilibrate the system for approx 10 minutes prior to starting a run 1260 Infinity Binary LC System User Guide 79 4 80 Syst
87. place the tip of the needle 3 mm above the bottom of the vial Equilibration time is a delay time between drawing the sample and moving the needle Sample flush out factor determines how long the autosampler would wait after injection before allowing the valve to turn to bypass This ensures that the sample zone has cleared from the needle seat and injection valve Default value is 5 Vial Well bottom sensing is an alternative to using draw position offset The needle slowly moves down until it touches the bottom of the vial or well and then lifts up 1 mm This is a versatile way of ensuring the needle is close to the bottom of the vial but it takes slightly longer to complete the injection and should not be used if there are particulates at the bottom of the vial which might block the needle 116 1260 Infinity Binary LC System User Guide Appendix 6 Advanced High Throughput Automatic delay volume reduction ADVR switches the injection valve from mainpass to bypass after the injection has taken place and a volume defined by the sample flush out factor has swept through the injector This reduces the delay volume of the system by about 70 ul and allows the gradient changes to reach the column sooner Enable overlapped injection also switches the injection valve from mainpass to bypass after the injection has taken place either after the sample has flushed out of the injector or at some specified later time in the run The injecto
88. ppropriate by the user This also reduces the number of data to be stored Step controls the interval nm of the data that are stored in a spectrum and hence affects the spectral resolution seen The default setting of 2 nm is a good choice for most applications Threshold determines that spectra are not stored for peak heights below this value MAU 1260 Infinity Binary LC System User Guide Appendix 6 Advanced Analog Output The 1260 Infinity DAD has one analog signal output connector for use with data systems that do not accept a digital input The following may be set Zero Offset places the zero level at a set percentage of the output signal hence allowing some scope for negative drift Attenuation scales the set absorbance to the full output Advanced Margin for Negative Absorbance The default setting is 100 mAU which means the detector has sufficient dynamic range considering where the zero level was set to measure down to this value To measure larger negative peaks or follow a baseline with a strong negative drift the value needs to be adjusted downwards to prevent the signal flat lining at the bottom of the range However it should not be changed without good reason as making it more negative will increase baseline noise and reduce the range available for measuring positive peaks Advanced Autobalancesets the absorbance level to zero at all wavelengths that is balances all points on the spect
89. ption The chromatographic signals are extracted from the diode array data within the firmware of the module Up to eight individual signals can be defined Each of them comprises a signal wavelength a diode bunching bandwidth and if required a reference wavelength and bandwidth Signals can be output at 80 Hz 80 data points second for accurate recording of the fastest narrowest chromatographic peaks At the same time the module can also output full range spectra to the data system at the same rate of 80 Hz For regulated laboratories it is important that all the method parameters are recorded The 1260 Infinity DAD not only records the instrument setpoints but also has RFID tags radio frequency identification tags incorporated into the lamp and flow cell cartridge so that the identity and variables of these important components are also recorded by the system 1260 Infinity Binary LC System User Guide The Agilent 1260 Infinity Binary LC Product Description 1 1200 Infinity Series Quick Change Valves Agilent 1200 Infinity Quick Change Valves support a variety of challenging valve applications Each valve head comes as a complete kit containing all required capillaries additional low dispersion heat exchangers and other parts Some typical applications for Quick Change Valves are Dual column selection Sample enrichment e Sample clean up Alternating column regeneration Special applications such as method development or
90. pumping system and low voltages in major maintenance areas Early maintenance feedback EMF for continuous tracking of instrument usage in terms of seal wear and volume of pumped mobile phase with pre defined and user settable limits and feedback messages Electronic records of maintenance and errors All materials are recyclable at 0 2 and 1 mL min based on retention time at constant room temperature water caffeine tracer Revision B 02 00 or above For use with flow rates below 500 ul min or for use without damper and mixer a vacuum degasser is required All specification measurements are done with degassed solvents 1260 Infinity Binary LC System User Guide 25 1 26 The Agilent 1260 Infinity Binary LC Product Description Specifications 1260 Infinity Binary LC System User Guide 1260 Infinity Binary LC System User Guide e00 eer 2 r Introduction Theory of Using Smaller Particles in Liquid Chromatography 28 Benefits of small particle size columns 34 Frictional Heating 37 This chapter gives an introduction to the Agilent 1260 Infinity Binary LC and the underlying concepts gg Agilent Technologies 27 2 Introduction Theory of Using Smaller Particles in Liquid Chromatography Introduction In 2003 Agilent introduced the first commercially available totally porous silica columns with 1 8 um particles In combination with the Agilent 1260 Infinity Binary LC the sub two micron 1 8 u
91. r phosphate buffer which are less corrosive against stainless steel e Halogenated solvents or mixtures which form radicals and or acids for example 2 CHCl 02 gt 2 COCly 2 HCI This reaction in which stainless steel probably acts as a catalyst occurs quickly with dried chloroform if the drying process removes the stabilizing alcohol e 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 e Solutions of organic acids acetic acid formic acid and so on in organic solvents For example a 1 solution of acetic acid in methanol will attack steel e Solutions containing strong complexing agents for example EDTA ethylene diamine tetra acetic acid Mixtures of carbon tetrachloride with 2 propanol or THF Diamond Like Carbon DLC Diamond Like Carbon is inert to almost all common acids bases and solvents There are no documented incompatibilities for HPLC applications 1260 Infinity Binary LC System User Guide 107 108 Appendix Fused silica and Quartz Si04 Fused silica is used in 1290 Infinity Flow Cells and capillaries Quartz is used for classical flow cell windows It is inert against all common solvents and acids except hydrofluoric acid and acidic solvents containing fluorides It is corroded by strong bases and should not be used above pH 12 at room temperature
92. r specific to different signals from the same detector Key parameters in this table are Slope Sensitivity represents the slope and curvature of the baseline needed to mark the start and end of a peak Peak Width the width at half height of the narrowest peak of interest should be entered This helps the integrator to distinguish between noise and very small peaks Area Reject Height Reject values that control rejection from the results of peaks whose area or height falls below these values Integration OFF ON suppresses integration between set limits Almost always used to inhibit integration in the region from injection to solvent front or unretained peak marker Lines such as Integration OFF ON are added to the table using the icons at the top of the window Click OK to exit and the next screen in the Edit Entire Method process will open 132 1260 Infinity Binary LC System User Guide Appendix 6 Specify Report The Specify Report screen can also be directly accessed in Method and Run Control view by right clicking on the graphical user interface on the Report icon and then selecting Specify Report in the context menu In the Data Analysis view it can be accessed through the menu Report gt Specify Report or the Specify Report task icon Specify Report Instrument 1 Reporting settings Quantitation settings Report mode O Use Intelligent Reporting Use Classic Reporting Style Chromatogram output Report style E
93. r will then aspirate the next sample in readiness for the next injection thus reducing the overall cycle time and increasing the sample throughput e Injection Valve Cleaning Injector Cleaning allows the injection system to be flushed with solvent Injection Valve Cleaning allows the valve to be switched at setpoints during the run to minimize carryover when problem compounds are injected 1260 Infinity Binary LC System User Guide 117 6 Appendix Setting Up a Method using Edit Entire Method High Performance Autosampler HiP ALS Injector Program Tab Setup Method Qe HIP ALS Injector Program EM BinPump sf TCC 7 DAD Instrument Curves Md Draw default volume from sample with default speed using default offset f Delete Clear all Move up Move down Figure 26 Setup Method screen HiP Autosampler Injector Program tab This allows specialized injection procedures to be constructed which involve the manipulation of aliquots from multiple vials as for instance in pre column derivatization Reagent chemicals are automatically mixed with the sample to enhance detectability or sensitivity A commonly used example is the derivatization of amino acids with OPA and FMOC reagents For details please refer to the Agilent 1260 Infinity High Performance Autosampler manual 118 1260 Infinity Binary LC System User Guide Appendix 6 e M et ting Up a 10d using Edit En Binary Pump Tab Setup
94. re The maximum pressure of the 1260 Infinity Binary Pump is 600 bar but some columns can only withstand lower pressure and by setting this value here the column will be protected The pump will generate an error condition if this pressure is exceeded Any run in progress will be stopped and the pump switched to standby mode with no flow Information about the maximum pressure for a particular column is supplied with the column Agilent ZORBAX RRHT columns are suitable for operation at 600 bar The Low Pressure limit is Off when the value is zero but at any other value the pump will generate an error if the pressure goes below this value during operation This is used as an alternative safeguard when the column is not inside a module with a leak sensor or in case the system is pumped dry A value of 10 to 20 bar for minimum pressure limit is typical 1260 Infinity Binary LC System User Guide 121 6 Appendix Setting Up a Method using Edit Entire Method Thermostatted Column Compartment TCC tab Setup Method HiP ALS S24 HiP ALS Injector Program Temperature Left Not Controlled 400 I As Detector Cell Column Switching Valve Poit1 gt 2 Stoptime As Pump Injector A 1 00 sj BinPump TCC 37 DAD Instrument Curves Timetable Advanced Enable Analysis Right Not Controlled F 20 0 t V when front door open As Detector Cell Left Right 5 With any temperature D With any temperature
95. rmance Autosampler G1367E Features The 1260 Infinity High Performance Autosampler features an increased pressure range up to 600 bar enabling the use of today s column technology sub two micron narrow bore columns with the Agilent 1260 Infinity Binary LC Increased robustness is achieved by optimized new parts high speed with lowest carry over by flow through design increased sample injection speed for high sample throughput increased productivity by using overlapped injection mode and flexible and convenient sample handling with different types of sample containers such as vials and well plates Using 384 well plates allows you to process up to 768 samples unattended Autosampler Principle 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 is not completed successfully an error message is generated Solvent is bypassed from the autosampler by the injection valve during the sampling sequence The needle moves to the desired sample position and is lowered into the sample liquid in the sample to allow the metering device to draw up the desired volume by moving its plunger back a certain distance The needle is then raised again and moved onto the seat to close the sample loop Sample is applied to the column when the injection val
96. rom contact with the thermostat vanes still air temperature in the column environment and most importantly by pre heating or cooling the mobile phase by passing it through a heat exchanger before entering the column There are two independent temperature zones in each TCC which can work together for long columns up to 300 mm length or work at different temperatures for short columns of 100 mm length or less The module comes with a 1 6 ul low dispersion heat exchanger and each valve kit contains additional low dispersion heat exchangers for each column The low dispersion heat exchangers up to 4 can be mounted flexibly inside the TCC For conventional HPLC operation 3 ul and 6 ul built in heat exchangers are also available Each TCC can accommodate one internal valve drive to facilitate valve switching applications from simple switching between two columns to alternating column regeneration sample preparation or column back flushing Each valve head comes as a complete kit containing all required capillaries additional low dispersion heat exchangers and other parts The switching valves have exceptional ease of use and flexibility when making connections to the valve When pressed the drive unit of the Quick Change Valve slides forward for easy access see Figure 4 on page 18 left Alternative valve heads can be interchanged by the user on the drive mechanism for different applications see Figure 4 on page 18 right Note the RFID tag on
97. rum to zero and hence also zeroes the baseline signal Prerun is selected to balance just before the start of the run which is the normal situation Sometimes Postrun is alternatively selected to balance at the end of the run after the postrun time has expired For example if the signal always shows a negative drift and the user prefers the run to end at zero absorbance then this will set the correct zero level for the following run It will not retrospectively change the run at the end of which it performed the balance Advanced Lamps on required for acquisition the 1260 Infinity DAD has one UV lamp and it needs to be on for analysis so this box should be checked 1260 Infinity Binary LC System User Guide 127 Instrument Curves Tab E setup Method HIP ALS Q HIP ALS Injector Program EM BinPump eg TCC DAD X Instrument Curves BinPump v Pressure bar lt Flow ml min K Solvent Ratio lt Solvent Ratio B TCC Left Temperature C _ Right Temperature C DAD Board Temperature C Optical Unit Temperature C _ UV Lamp Anode Voltage V C Show timetable graph OK Apply Cancel Help Figure 30 Setup Method screen Instrument Curves tab The instrument curves tab allows monitored data streams other than detector signals to be stored with the data by c
98. rument configuration 61 optimization 60 setable flowrange 24 setup instrument method 114 solvent selection valve 11 solvents change 79 specification physical 23 specifications analog signal output 25 communications 25 control and data evaluation 25 performance 24 standard delay volume configuration 45 sub 2 micron particles 34 system components diode array detector 19 thermostatted column compartment 17 system setup and installation network Integration 70 system turningon 83 T theoretical plates 32 Thermostatted Column Compartment description 17 V vacuum degasser 25 vacuum pump 14 van Deemter Equation 29 voltage range 23 WwW weight 23 139 www agilent com In This Book This manual contains information about the Agilent 1260 Infinity Binary LC The manual describes the following Product description e introduction specifications e system optimization e system setup and installation quick start guide Agilent Technologies 2006 2008 2011 2013 Printed in Germany 02 2013 G1312 90303 fe Agilent Technologies
99. same length 2 1 mm i d columns and the flow rates that are used are also proportionally higher Therefore the standard delay volume of the binary pump will not result in a significantly higher gradient delay Flow Path Mixing T Disconnect only here 400 pl Mixer Pressure Sensor Std delay volume y 600 800 pl T 1 Purge Valve Figure 15 Standard delay volume configuration for 3 and 4 6 mm ID columns with highest UV sensitivity 1260 Infinity Binary LC System User Guide 45 3 Optimization of the Agilent 1260 Infinity Binary LC The standard delay volume configuration is also the configuration which provides direct method transferability from the Agilent 1100 and 1200 Series LC system to the 1260 Infinity Binary LC or vice versa Figure Figure 16 on page 46 shows two overlaid chromatograms of a method for analysis of paracetamol and related impurities The method was transferred from an Agilent 1200 Series LC system to an Agilent 1260 Infinity Binary LC with the chromatographic conditions column mobile phase pump settings injection volume column temperature detector settings left unchanged As can be seen a seamless method transfer is possible mAU Paracetamol 1004 RT shift lt 1 4 80 J Imp B 604 Imp K Imp A Imp H 404 f Imp J 4 Agilent 1200 Series Imp 20 5 4 Agilent 1260 Infinity E A A Foto ae ae a ao nt ae ee eo ro 7 8 hk Ce en 8k 0 5 10 15 20 25 30
100. sed later in the Data Analysis view To link in a macro program at one of the access points the relevant box is checked and the name of the macro is typed in the text box to the right The software looks for the macro in the C Chem32 Core directory include the path if it is located elsewhere 136 1260 Infinity Binary LC System User Guide Appendix 6 The access points in the work flow of the method are Pre Run Command Macro e Customized Data Analysis Macro Post Run Command Macro Save Method with Data saves a copy of the method in the data file and names it RUN M This is not needed if the ChemStation is operated in the usual configuration as the software always saves the method in the data file all versions since B 02 01 Only if the ChemStation has been configured such that Unique Sequence Folder Creation is turned off and hence methods are not routinely copied into the data file this option will be relevant As this is the final screen in the process clicking OK will exit from Run Time Checklist and from the Edit Entire Method process The method should now be saved into the master methods directory by default C Chem32 1 Methods using File gt Save As gt Method or Method Menu gt Save Method As 1260 Infinity Binary LC System User Guide 137 Index Index A active seal wash 11 ADVR 49 Agilent oninternet 110 alternating column regeneration 48 ambient non operating temperature 23 ambient operating temp
101. solvent vendor especially when toxic or hazardous solvents are used 1260 Infinity Binary LC System User Guide 103 6 Appendix Safety Symbols Table 11 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 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 WARNING aia 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 104 1260 Infinity Binary LC System User Guide Appendix 6 Solvent Information Observe the following recommendations on the use of solvents e Follow recommendations for avoiding the growth of algae see pump manuals e Small particles can permanently block capillaries and valves Therefore always filter solvents through 0 4 um filters
102. stem to equilibrate for at least 10 min and check that the baseline in the Online Plot is stable before starting the analysis 1260 Infinity Binary LC System User Guide 93 5 94 Quick Start Guide Running the Method for a Single Injection This section shows how to run a single injection of the isocratic checkout sample using the conditions entered in the previous section Analyses on the ChemStation can be run in two modes Run Method single injections for example in interactive method development using the parameter settings current at the time Run Sequence automated series of injections from multiple vials possibly with multiple methods For further details refer to the ChemStation manuals 1 Click the Select Run Method Task icon a 2 Ifrequired method conditions are not currently loaded select Method gt Load Method or the icon si under the menu bar to load them If changes have been made to a loaded method and not yet saved this is indicated by a yellow asterisk on the method status icon The injection can be made without first saving those parameters changes The ChemStation always stores a copy of the acquisition parameters in the data file as ACO TXT to ensure the preservation of the original method parameters 3 Place the sample vial in position 1 this is the front position of the 10 x 2 ml vial positions on the right hand side of the sample tray 4 Select Run Control gt Sample Info and en
103. t Printer for a paper copy and select File and PDF to obtain a useful PDF report file stored into the datafile the data file with D suffix is actually a directory The report file can be viewed directly in ChemStation or it can be found in the directory using the normal Windows File Explorer 4 Save the method once again to ensure the report settings are stored for future use by the method When the method is used again these integration events and report settings will be used to produce the report This completes a brief look at using the Data Analysis part of the ChemStation software Consult the ChemStation manuals and the online help system for further details about the powerful features of the ChemStation 1260 Infinity Binary LC System User Guide 1260 Infinity Binary LC System User Guide 6 Appendix Safety Information 102 Solvent Information 105 Material Information 105 Agilent Technologies on Internet 110 Setting Up a Method using Edit Entire Method 111 Method Information 113 Instrument Acquisition 114 Data Analysis 129 Run Time Checklist 136 This chapter provides additional information on safety legal and web and about setting up a method ae Agilent Technologies 101 6 Appendix Safety Information 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
104. tegration Events or the Edit Integration Events task icon Single Runs PHENONES H Integration amp Calibration ul Signal fil Pu iu as ik Report Short Fy Lo DAD1 A Sig 242 ONE MIX 008 D v EN RS SiE CSES Eu M Method Manual Events Initial Events For All Signals Integration Events Yalue Tangent Skim Mode Standard Tail Peak Skim Height Ratio 0 00 Front Peak Skim Height Ratio 0 00 Skim Valley Ratio 20 00 Baseline Correction Classical Specific Events For Signal DAD Default ind Time Integration Events Value Initial Slope Sensitivity 5 Initial Peak Width 0 05 Initial Area Reject 5 Initial Height Reiect 1 Initial Shoulders OFF Figure 32 Edit Integration Events screen Integration calibration and reporting are the data analysis part of the method Integration parameters and the calibration table are easier to setup once data have been acquired and the data are being reviewed in Data Analysis view The integration events can be optimized at that time and default settings are often used for the initial acquisition runs 1260 Infinity Binary LC System User Guide 131 6 Appendix The Edit Integration Events screen has two tables Initial Events For All Signals contains events integration parameters that apply to all signals acquired with the method Specific Events For Signal contains events which are specific for one type of detector o
105. ter the required sample parameters For example Subdirectory optional Name Pattern Vial Location Sample name Comment 5 Ifthe system is already equilibrated and the baseline is stable click Run Method in the Sample Info page to start the injection Alternatively click OK and when ready select Run Control gt Run Method The item will be added to the Run Queue and then automatically started from there 1260 Infinity Binary LC System User Guide Quick Start Guide 5 Data Acquisition in Method and Run Control View 6 The injection is made and the chromatogram appears in the Online Plot The data acquisition will stop when the Stop Time is reached The chromatogram should look similar to the one below DAD1 A Sig 254 4 Ref 550 100 DEMO 005 0105 D mU 1260 Infinity Binary LC System User Guide 95 5 Quick Start Guide Data Analysis A method in the ChemStation contains all the parameters for data acquisition controlling the system and data analysis processing the data to give quantitative and qualitative results This section looks briefly at integration and reports in data analysis so that the separations generated earlier in this chapter can be integrated and printed For more detailed information on data analysis including the use of calibration for quantification refer to the ChemStation manual Method source Navigation table Integration task Waa
106. th the square root of N so doubling the length of the column will increase resolution by a factor of 1 4 What is achievable depends on the viscosity of the mobile phase as this relates directly to the pressure Methanol mixtures will generate more back pressure than acetonitrile mixtures Acetonitrile is often preferred because peak shapes are better and narrower in addition to the lower viscosity but methanol generally yields better selectivity certainly for small molecules less than about 500 Da The viscosity can be reduced by increasing the temperature but it should be remembered that this can change the selectivity of the separation Experiment will show if this leads to increase or decrease in selectivity As flow and pressure are increased it should be remembered that frictional heating inside the column will increase That can lead to slightly increased dispersion and possibly a small selectivity change both of which could be seen as a reduction in resolution The latter case might be offset by reducing the temperature of the thermostat by a few degrees and again experiment will reveal the answer The van Deemter curve shows that the optimum flow rate through an STM column is higher than for larger particles and is fairly flat as the flow rate increases Typical close to optimum flow rates for STM columns are 2 ml min for 4 6 mm i d and 0 4 ml min for 2 1 mm i d columns 1260 Infinity Binary LC System User Guide Optimization of
107. the Agilent 1260 Infinity Binary LC 3 In isocratic separations increasing the retention factor k results in better resolution because the solute is retained longer In gradient separations the retention is described by k in the following equation pefo F 100 A B V S m where k meank value tg time length of gradient or segment of gradient min F flow ml min Vn column delay volume e A B change in fraction of solvent B during the gradient e S constant ca 4 5 for small molecules This shows that k and hence resolution can be increased by having a shallower gradient 2 to 5 min change is a guideline higher flow rate and a smaller volume column This equation also shows how to speed up an existing gradient if the flow is doubled but the gradient time is halved k remains constant and the separation looks the same but happens in half the time Any reduction in extra column volume will reduce dispersion and give better resolution This is already optimized in the 1260 Infinity Binary LC with narrow bore 0 12 mm i d capillaries check that the shortest length is used between column and detector and the Max light cartridge flow cell Finally any gains in resolution must be preserved by having data collection which is fast enough to accurately profile the narrow peaks In summary the following steps should be considered to increase resolution The first step in improving
108. the end of the gradient For instance the B may rise to 95 at 10 min and it is expected that all peaks have eluted from the column The run is essentially at an end but an extra gradient segment is added which takes B back down to the initial value over two minutes to gently start the re equilibration of the column No useful data is expected during this downwards ramp so the detector stop time is set to 10 min and data collection stops while the pump stop time is 12 min to allow the downwards ramp to complete This is a matter of user choice and some accept that the final minutes of the chromatogram cannot contain useful data but record it anyway to avoid the inconvenience of having a different stop time for the detector The detector stop time does not override the pump and cause the run to end as would an earlier stop time in any other module hence the convenience of setting stop time to As Pump Injector Timetable operates in the same way as other modules add a line select the function to be changed and enter the new values for that function The changes for the detector will take place immediately at the specified time The following functions can be changed during the run Balance Change Signal Change Threshold Change Peak detector Peakwidth Change Spectra Acquisition Mode Change Contacts 1260 Infinity Binary LC System User Guide 125 126 Appendix Advanced Spectrum Spectra can be saved during the run on a conti
109. top of the valve head 1 all specifications are valid for distilled water at ambient temperature of 25 C setpoint at 40 C and a flow range from 0 2 to 5 ml min 1260 Infinity Binary LC System User Guide 17 1 The Agilent 1260 Infinity Binary LC Product Description System Components Figure 4 Quick change valve in TCC Up to three TCC can be clustered to allow advanced applications such as switching between eight columns for automated method development or to make additional columns available for different applications Thus the column to be used becomes a simple method parameter This requires two 8 position 9 port valve heads one each in two of the TCCs Clustered TCC are represented by the software as one unit with one interface for ease of operation Further improvements compared to earlier designs include better thermal insulation better capillary guides and a door open sensor so that methods can define that the door must be closed especially useful for low or high temperature methods 18 1260 Infinity Binary LC System User Guide The Agilent 1260 Infinity Binary LC Product Description 1 1260 Infinity Diode Array Detector G4212B The 1260 Infinity Diode Array Detector DAD is a new optical design using a cartridge cell with optofluidic waveguide technology offering high sensitivity with low dispersion a wide linear range and a very stable baseline for standard or ultra fast LC applications Th
110. ubt please consult technical literature on chemical compatibility of materials 1260 Infinity Binary LC System User Guide 105 106 Appendix PEEK PEEK Polyether Ether Ketones combines excellent properties with regard to chemical resistance mechanical and thermal stability It is stable in a pH range of 1 to 12 5 and inert to many common solvents There are several known incompatibilities with chemicals such as chloroform methylene chloride THF DMSO gt 1 strong acids nitric acid gt 10 sulphuric acid gt 10 trichloroacetic acid sulfonic acids halogenes or aequous halogene solutions phenol and derivatives cresols salicylic acid etc When used above room temperature PEEK is sensitive to bases and various organic solvents which can cause it to swell Polyimide Agilent uses semi crystalline polyimide for rotor seals in valves and needle seats in autosamplers One supplier of polyimide is DuPont which brands polyimide as Vespel which is also used by Agilent Polyimide is stable in a pH range between 1 and 10 and in most organic solvents It is incompatible with concentrated mineral acids e g sulphuric acid glacial acetic acid DMSO and THF It is also degraded by nucleophilic substances like ammonia e g ammonium salts in basic conditions or acetates Polyethylene PE Agilent uses UHMW ultra high molecular weight PE PTFE blends for yellow piston and wash seals which are used in 1290 Infinity pu
111. ure and click the Parameter box to reveal the entry box for the value If timetable entries are made out of logical sequence the entries are automatically re ordered into time order Timetable lines can be edited directly and the Cut Paste and Remove buttons can be used to add and remove lines Multiple lines can be added to give a series of linear gradient segments to create any gradient profile To set up a simple gradient first clear the timetable with the Clear all button if it is not already empty then add a line and enter the time and solvent composition required If a step gradient is required it can be formed by making two entries with the before step and after step settings separated by 0 01 min This is often used to rapidly elute strongly retained peaks from a column near the end of a run by increasing the stronger solvent and or the flow rate in a step gradient for example the B might be stepped up from 75 to 95 It is not necessary to enter the settings for 1260 Infinity Binary LC System User Guide Appendix 6 time 0 00 min into the timetable these values are picked up from other setpoints on this screen However some users like to see a complete list in the timetable and make an entry for 0 00 min There is no problem with this but if the initial conditions are ever changed then the new settings must be entered in both the timetable and the setpoints in the Solvents section of the screen Show t
112. urn 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 Technologies gt OpenLAB gt OpenLAB Control Panel Select the relevant instrument in the navigation pane under Instruments and click Launch online 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 1260 Infinity Binary LC System User Guide 83 5 Quick Start Guide 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 param
113. ve returns to the mainpass position at the end of the sampling sequence The standard sampling sequence occurs in the following order The injection valve switches to the bypass position The plunger of the metering device moves to the initialization position The needle lock moves up The needle moves to the desired sample vial or well plate position The needle lowers into the sample vial or well plate The metering device draws the preset sample volume The needle lifts out of the sample vial or well plate The needle is then moved onto the seat to close the sample loop oo nr oa FF amp wWN The needle lock moves down 10 The injection cycle is completed when the injection valve switches to the mainpass position If needle wash is required it will be done between step 7 and 8 1260 Infinity Binary LC System User Guide 15 1 16 The Agilent 1260 Infinity Binary LC Product Description Injection Sequence Before the start of the injection sequence and during an analysis the injection valve is in the mainpass position In this position the mobile phase flows through the autosampler metering device sample loop and needle ensuring all parts in contact with sample are flushed during the run thus minimizing carry over When the sampling sequence begins the valve unit switches to the bypass position Solvent from the pump enters the valve unit at port 1 and flows directly to the column through port 6 The
114. vent selection valve is installed in the pump each channel has two solvent options and the correct option for the method is selected with the radio button to the left of the solvent description The pump will form binary mixtures of the selected A and B channels for example A2 and B1 it is not possible to mix Al with A2 or B1 with B2 The value that is entered for the proportions of A and B defines the composition of an isocratic method or they define the starting conditions of a gradient method and the equilibration conditions between gradient runs Only the value of B is entered A will then update to show 100 minus B when the cursor is moved For the example separation set A to water and B to acetonitrile at 60 A will be 40 Timetable details the changes that should occur during the run to the percentage composition of A and B in the mobile phase or if required to the flow rate and maximum pressure allowed The timetable makes linear changes to the parameters between the given setpoints The settings that are made elsewhere on this screen act as the initial conditions and will only change if an entry is made in the timetable For example if the flow is constant during the run then there is no need to make an entry about the flow in the timetable To make an entry in the timetable click the Add button to add a line to the timetable enter the time of the setpoint select the type of entry from the drop down list composition flow press
115. vent 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 Regular Priming on page 78 1260 Infinity Binary LC System User Guide 87 5 Quick Start Guide Data Acquisition in Method and Run Control View 88 Method Parameters for System Checkout with Isocratic Checkout Sample For checkout of the 1260 Infinity Binary LC a test run with an isocratic test mixture Agilent isocratic checkout sample 01080 68704 will be performed on the column ordered and delivered with your system Agilent isocratic checkout sample 01080 68704 contains the following components dissolved in methanol dimethyl phthalate e diethyl phthalate biphenyl o terphenyl The method parameters for separation of this test mixture are summarized in Table 9 on page 89 for the standard delay volume configuration and in Table 10 on page 90 for the low delay volume configuration 1260 Infinity Binary LC System User Guide Quick Start Guide 5 Table9 Method parameters for first separation run standard delay volume configuration Module Parameter Setting Pump Solvent A Water Solvent B Acetonitrile Flow rate 4 0 mL min Solvent 40 A 60 B Composition Stop Time 1 min 1 min post time Autosampler Injection Tul Column Column Column Eclipse Plus C18 4 6 x 100 mm 3 5 pm Compartment 959961 902 Co
116. with a Parameter Mismatch error when attempting to run the method What has happened is that in the old method the Signal Details contained a specified signal for example 250 nm with 8 nm bandwidth and this is changed in the new method to 254 nm 12 nm for example The Signal Details table still contains the original details and is being told to process a signal which is no longer acquired Highlighting the old signal in the table and using the Delete Row button will correct the problem If a system uses multiple detectors such as the diode array detector and a mass spectrometer the Signal Description lines allow delay times for the downstream detector to be entered so that the software can align the peaks from the different detectors 1260 Infinity Binary LC System User Guide 129 6 Appendix Setting Up a Method using Edit Entire Method Signal Details Instrument Z Available Signals DAD1 A Sig 250 100 Ref off Insert Row Append Row Delete Row Signal Description Align Peak1 Peak 2 Align Window Figure 31 Signal details 130 1260 Infinity Binary LC System User Guide Appendix 6 Edit Integration Events The Edit Integration Events screen can also be directly accessed in Method and Run Control view by right clicking on the graphical user interface on the Integration Events icon and then clicking Edit Integration Events in the context menu In the Data Analysis view it can be accessed through the menu Integration gt In
117. wn that elevated flow rates can also be advantageous for improved separation For 4 6 mm inner diameter columns packed with sub 2 micron particles a flow rate of 2 mL min is recommended as a starting value For 2 1 mm inner diameter sub 2 micron columns 0 4 mL min is a good starting point e Moderate gradients should be used for example 2 5 gradient change per minute The column temperature is an additional parameter for optimization Temperature can influence a separation and should not be overlooked 1260 Infinity Binary LC System User Guide 59 3 Optimization of the Agilent 1260 Infinity Binary LC How to Achieve Higher Sensitivity The sensitivity of a separation method is linked to the choice of stationary and mobile phases as good separation with narrow peaks and a stable baseline with minimal noise are desirable The choice of instrument configuration will have an effect and a major impact is the setup of the detector 60 1260 Infinity Binary LC System User Guide Optimization of the Agilent 1260 Infinity Binary LC 3 Optimum Instrument Configuration for High Sensitivity General Considerations e To achieve lowest baseline noise the standard delay volume configuration is recommended for the 1260 Infinity Binary Pump e Here also the injection volume and the sample dissolution solvent are important Care must be taken that the compounds are focused at the top of the column to avoid peak dispersion due to the
118. y Binary LC are e The configurable delay volume down to 120 uL in the 1260 Infinity Binary Pump combined with a flow range from 0 05 up to 5 mL min at pressures up to 600 bar provides universal applicability from narrow bore 2 1 mm ID to standard bore 4 6 mm ID columns matching the needs for both LC and LC MS The standard delay volume configuration of the 1260 Infinity Binary Pump allows you to run not only UHPLC but also conventional HPLC methods without compromising performance or changing chromatographic patterns The next generation flow through design of the 1260 Infinity High Performance Autosampler achieves highest precision for a wide range of injection volumes from 0 1 up to 100 uL without changing sample loops It is designed for high sample throughput low carryover and fast injection cycles High temperature up to 100 C on certain columns allows more selectivity flexibility and reduces solvent viscosity to allow even faster separation In the 1290 Infinity Thermostatted Column Compartment different heater 1 6 uL and cooling elements 1 5 uL for low extra column volume can be installed The temperature is adjustable from 10 C below ambient up to 100 C 8 1260 Infinity Binary LC System User Guide The Agilent 1260 Infinity Binary LC Product Description 1 The new pull out valve drive design and user exchangeable Quick Change valves in the 1290 Infinity Thermostatted Column Compartment boosts

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