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Agilent 1260 Infinity Bio-inert Quaternary LC

1. Aggregates Time min Figure 18 Aggregation analysis of a monoclonal antibody Bio inert Quaternary LC Typical Bio inert LC Applications 4 Peptide Mapping Rapid Resolution LC at 600 bar The Agilent 1260 Infinity Bio inert Quaternary LC offers RRLC capability combined with low surface activity especially for critical samples In combination with the Agilent Eclipse Plus 1 8 um particle columns or with the Poroshell 120 stationary phases high resolution and superior peak capacities are achieved in order to confirm the identity of the analyzed NBE drug ina QA QC environment mAU Agilent ZORBAX Eclipse Plus C18 2 4 6 8 10 12 14 16 18min Figure 19 Peptide map with Agilent Zorbax Eclipse Plus C18 Column Bio inert Quaternary LC 61 4 Typical Bio inert LC Applications Application Examples 62 Bio inert Quaternary LC Bio inert Quaternary LC Specifications e Performance Specifications 64 Solvent Information 71 This chapter provides information about specifications of the 1260 Infinity Bio inert Quaternary LC system a Agilent Technologies 63 5 Specifications Performance Specifications For detector specifications refer to the respective User Manual Diode Array Detector G1315C e Multiple Wavelength Detector G1365C Fluorescence Detector G1321B 64 Bio inert Quaternary LC Specifications 5 Specifications Agilent 1260 Infinity Bio inert Quaternary Pump G5611A Specif
2. Remote cable CAN bus cable LAN to control software location depends on detector Analog detector signal 1 or 2 outputs per detector Figure 2 Recommended Stack Configuration Rear View 16 Bio inert Quaternary LC Two Stack Configuration Introduction 1 Optimizing the Stack Configuration To avoid excessive height of the stack for example when the autosampler thermostat or fraction collector is added to the system we recommend that you form two stacks A slightly longer capillary is required between the pump and autosampler see Figure 3 on page 17 and Figure 4 on page 18 Instant Pilot control module Detector Column compartment Autosampler E a E mF g a E 2 PO Thermostat for the ALS optional e e Solvent cabinet Pump Figure 3 Bio inert Quaternary LC Two stack configuration front view 17 1 Introduction Optimizing the Stack Configuration LAN to control software CAN Bus cable to Instant Pilot Thermo cable optional CAN Bus cable AC Power Figure4 Two stack configuration rear view 18 Bio inert Quaternary LC Introduction 1 Bio inert Quaternary pump G5611A Seal wash not shown Pump head Outlet valve Purge valve Active inlet valve MCGV Degasser Figure 5 Overview of the bio inert quaternary pump The Agilent 1260 Infinity Bio inert Quaternary LC pump is
3. gt Discharged Lithium batteries shall be disposed off locally according to national waste disposal regulations for batteries gt Replace only with the same or equivalent type recommended by the equipment manufacturer Lithiumbatteri Eksplosionsfare ved fejlagtig handtering Udskiftning ma kun ske med batteri af samme fabrikat og type gt Lever det brugte batteri tilbage til leverand ren Lithiumbatteri Eksplosionsfare Ved udskiftning benyttes kun batteri som anbefalt av apparatfabrikanten gt Brukt batteri returneres appararleverandoren 80 Bij dit apparaat zijn batterijen geleverd Wanneer deze leeg zijn moet u ze niet weggooien maar inleveren als KCA Bio inert Quaternary LC Appendix 6 Radio Interference Cables supplied by Agilent Technologies are screened to provide optimized protection against radio interference All cables are in compliance with safety or EMC regulations Test and Measurement If test and measurement equipment is operated with unscreened cables or used for measurements on open set ups the user has to assure that under operating conditions the radio interference limits are still met within the premises Bio inert Quaternary LC 81 6 Appendix Sound Emission Manufacturer s Declaration This statement is provided to comply with the requirements of the German Sound Emission Directive of 18 January 1991 This product has a sound pressure emission at the operato
4. For details on the purging procedure refer to Priming the System on page 41 52 Bio inert Quaternary LC Quick Start Guide 3 Preparing the System Active Seal VVash The active seal vvash helps to keep the pump heads clean and prevents salt crystals precipitating and thus ensures longer piston seal lifetime Therefore the use of the active seal vvash is alvvays recommended vyhen operating vvith bio molecules and buffer solutions You must select this function in the control menu of the pump Control DER Sequence Setup WCXFEB15 M Quat Pump O off Single Wash Duration 1 0 min Periodic Period 505 min on for Ka min Tuesday February 15 2011 12 0000P bar Pressure Limit Composition C D 0 0 0 0 NA 2E AN ma in Figure 15 Specify Seal Wash in Pump Control menu Bio inert Quaternary LC 53 3 Quick Start Guide Setting Up the Method Setting Up the Method This section shovys hovy 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 nevv 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 module is set up in this way a CEM ENCES QU t MATIN so PEST s N E P Om deM 0 Sta
5. 74 Specifications Flow Cell To protect optimal functionality of your flow cell Standard flow cell bio inert 10 mm 13 uL 120 bar 12 MPa for MWD DAD includes Capillary Kit Flow Cells BIO p n G5615 68755 G5615 60022 PEEK ceramic sapphire PTFE for 1260 Infinity Diode Array Detectors G1315C D The recommended pH range of the cell is 1 13 short term 14 Max Light Cartridge Cell Bio inert 60 mm V s 4 0 uL G5615 60017 and Max Light Cartridge Cell Bio inert 10 mm V s 1 0 uL G5615 60018 PEEK fused silica for 1260 and 1290 Infinity Diode Array Detectors G4212A B The recommended pH range of the cell is 1 12 solvent dependent Bio inert flow cell 8 uL 20 bar pH 1 12 includes Capillary Kit Flow Cells BIO p n G5615 68755 G5615 60005 PEEK fused silica PTFE for 1260 Infinity Fluorescence Detector G1321B The recommended pH range of the cell is 1 12 solvent dependent If the flow cell is transported while temperatures are below 5 C it must be ensured that the cell is filled with alcohol to avoid damage by freezing water Aqueous solvents in the flow cell can build up algae Therefore do not leave aqueous solvents sitting in the flow cell Add a small percentage of organic solvents for example about 5 of acetonitrile or methanol Bio inert Quaternary LC Bio inert Quaternary LC 6 Appendix Safety 76 The Waste Electrical and Electronic Equipment Directive 79 L
6. For solvent compatibility refer to section Solvent information for parts of the 1260 Infinity Bio inert LC system in the manual 70 Bio inert Quaternary LC Specifications 5 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 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 Solvent information for parts of the 1260 Infinity Bio inert LC system For the Agilent 1260 Infinity Bio inert LC system Agilent Technologies uses highest quality materials see Bio inert Materials on page 12 in the flow path also referred to as wetted parts which are widely accepted by life scientists as they are known for optimum inertness to biological samples and ensure best compatibility to common samples and solvents over a wide pH range Explicitly the complete flow path is free from stainless steel and free from other alloys containing metals such as iron nickel cobalt chromium molybdenum or copper which can interfere with biological samples The flow downstream of the sample introduction contains no metals whatsoever However t
7. Specifications 5 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 a mixture of concentrated hydrochloric and nitric acid Zirconium Oxide Zirconium Oxide ZrO9 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 PTFE PTFE polytetrafluorethen is inert to almost all common acids bases and solvents There are no documented incompatibilities for HPLC applications Sapphire Ruby and Al 03 based ceramics Sapphire ruby and ceramics based on Al503 are inert to almost all common acids bases and solvents There are no documented incompatibilities for HPLC applications Data above were collected from external resources and are meant as a reference Agilent cannot guarantee the completeness and correctness of such information Information can also not be generalized due to catalytic effects of impurities like metal ions complexing agents oxygen etc Most data available refers to room temperature typically 20 25 C 68 77 F If corrosion is possible it usually increases at higher temperatures If in doubt consult additional resources Bio inert Quaternary LC 73 5
8. flow path being biologically inert All solvent lines in the pump are made of titanium making them completely resistant to corrosion All capillaries and fittings throughout the autosampler column compartment and detectors are completely metal free so that bio molecules in the sample come in contact only with ceramics or PEEK This minimizes secondary interactions of proteins and peptides with metallic surfaces which could cause peak tailing low recovery and decreased column lifetime 8 Bio inert Quaternary LC Introduction 1 System Properties Bio inert Quaternary LC Titanium gold pump solvent lines with quaternary solvent capability for online buffer mixing from up to four solvents at 600 bar Flow rates up to 10 mL min in gradient operation and bio inert fraction collection for automated bio purification and semi prep work with larger column dimensions 3 mm 4 6 mm 7 2 mm 10 mm ID columns Integrated micro vacuum degasser offers high degassing efficiency for trouble free operation and highest performance and completely eliminates the need for helium sparging Bio compatible valves for solvent selection or column switching for easy method development 2 pos 6 port 4 column selector 12 pos 13 port valve Bio inert flow cells for MWD DAD and FLD Mix and match compatibility with existing Agilent systems for full flexibility to meet future requirements Direct front access for quick exchange of maintenance parts Fast ide
9. 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 The purge tool of the LabAdvisor or Instrument Utilities 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 Bio inert Quaternary LC 41 2 42 System Setup and Installation oF O N 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 Al 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 the next solvent channel and repeat steps 5 and 6 until a
10. Infinity Bio inert Analytical scale Fraction Collector G5664A manual Bio inert Quaternary LC 11 1 Introduction Bio inert Materials For the Agilent 1260 Infinity Bio inert LC system Agilent Technologies uses highest quality materials in the flow path also referred to as wetted parts which are widely accepted by life scientists as they are known for optimum inertness to biological samples and ensure best compatibility with common samples and solvents over a wide pH range Explicitly the complete flow path is free of stainless steel and free of other alloys containing metals such as iron nickel cobalt chromium molybdenum or copper which can interfere with biological samples The flow downstream of the sample introduction contains no metals whatsoever Max Light Cartridge Cell Bio inert 60 mm V s 4 0 uL G5615 60017 and Max Light Cartridge Cell Bio inert 10 mm V s 1 0 uL G5615 60018 offer highest sensitivity for bio inert reverse phase applications Please note that at low salt SEC or ion exchange chromatography potentially peak tailing might occur and therefore for these applications the universal Bio inert DAD G1315C or D or MWD G1365C or D is recommended Table1 Bio inert materials used in Agilent 1260 Infinity Systems Module Materials Agilent 1260 Infinity Bio inert Quaternary Pump Titanium gold platinum iridium ceramic G5611A ruby PTFE PEEK Agilent 1260 Infinity Bio inert High Performanc
11. System Components 10 Bio inert Materials 12 Optimizing the Stack Configuration 14 One Stack Configuration 15 Two Stack Configuration 17 Bio inert Quaternary pump G5611A 19 Hydraulic Path 21 Autosampler G5667A 22 Bio inert Manual Injector G5628A 24 Thermostatted column compartment G1316C with Bio inert heat exchangers 25 Bio inert Flow Cells for Detectors 27 Fraction Collector G5664A 29 This chapter gives an introduction to the Agilent 1260 Infinity Bio inert Quaternary LC and explains underlying concepts and features of the system Ee Agilent Technologies 1 Introduction Concepts of the 1260 Infinity Bio inert Quaternary LC System The Agilent 1260 Infinity Bio inert Quaternary LC System can withstand harsh conditions for bio analytic and biopurification applications while maintaining the performance of a modern UHPLC instrument This is possible through the careful design of all components that are in contact with mobile phase and sample Problems often associated with large bio molecules are non specific surface interactions analyte discrimination and metal ion release which can cause decreased column lifetime peak tailing or lack of resolution In addition high salt concentration and extreme pH values cause corrosion and decrease robustness and instrument uptime The Agilent 1260 Infinity Bio inert Quaternary LC System is based on the industry proven Agilent 1200 Infinity Quaternary LC system with the entire
12. and Installation 2 Table2 Choice of Priming Solvents for Different Purposes Activity Solvent Comments After an installation Isopropanol Best solvent to flush air out of When switching between Isopropanol the system reverse phase and normal Miscible vvith almost all phase both times solvents After an installation Ethanol or methanol Alternative to isopropanol second choice if no isopropanol is available To clean the system when HPLC grade vvater Best solvent to re dissolve using buffers buffer crystals After changing aqueous HPLC grade vvater Best solvent to re dissolve solvents buffer crystals After the installation of normal Hexane 5 isopropanol Good wetting properties phase seals PE seals pack of 2 0905 1420 Storage of the system Never leave the system with buffers containing high salt concentrations or with cleaning solutions such as HCl or NaOH in place Always flush and purge the system sufficiently with water before storage For longer periods of storage use water with 5 10 organic solvent for example isopropanol to prevent algae growth Bio inert Quaternary LC 45 2 46 System Setup and Installation Installing the Modules Bio inert Quaternary LC Bio inert Quaternary LC Preparing the System 48 Turning the System ON 48 Loading the Default Method 49 Configuring the Online Plot 50 Purging the Pump 52 Active Seal Wash 53 Setting Up the Method 54 e ee Quick Start Guide
13. capillaries to keep the flow path free of steel and to ensure pressure stability to at least 600 bar If in doubt consult the available literature about the chemical compatibility of PEEK Titanium Titanium is highly resistant to oxidizing acids for example nitric perchloric and hypochlorous acid over a wide range of concentrations and temperatures This is due to a thin oxide layer on the surface which is stabilized by oxidizing compounds Reducing acids for example hydrochloric sulfuric and phosphoric acid can cause slight corrosion which increases with acid concentration and temperature For example the corrosion rate with 3 HCl about pH 0 1 at room temperature is about 13 um year At room temperature titanium is resistant to concentrations of about 5 sulfuric acid about pH 0 3 The addition of nitric acid to hydrochloric or sulfuric acids significantly reduces corrosion rates Titanium is subject to corrosion in anhydrous methanol which can be avoided by adding a small amount of water about 3 Slight corrosion is possible with ammonia gt 10 Fused silica Fused silica is inert against all common solvents and acids except hydrofluoric acid It is corroded by strong bases and should not be used above pH 12 at room temperature 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 Bio inert Quaternary LC
14. e This chapter provides information on data acquisition and data analysis vvith the Agilent 1260 Infinity Bio inert Quaternary LC Bea Agilent Technologies 47 3 Quick Start Guide Preparing the System Turning the System ON If the system is not already fully on with the software showing Ready status follow these steps 1 Turn on the computer system and wait for the Windows desktop to appear 2 Turn on the electrical power to the LC modules using the button at the lower left of each module A green power on light will be visible in the center of the button 3 Start the control software on the computer by clicking the icon if configured Alternatively you can select Start gt All Programs gt Agilent 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
15. leaky 210 recommended180 range Second and subsequent installations of a capillary to a soft connector 1 When tightening the fitting for the second and subsequent times again start from the finger tight position which is not necessarily a vertical wrench position and rotate the wrench by 135 180 Staying below 135 grey arrow could be insufficiently tight enough more than 180 red arrow could damage the capillary 0 ossibly eaky ror 135 180 recommended range Bio inert Quaternary LC Summary for Second Step Table 11 Summary for second step Appendix 2ndstep First installation Subsequent installations Hard connectors 0 ossibly eaky D 135 180 recommended range Soft connectors ossibly eaky 210 recommended 180 range possibly leaky 90 ecommended range e possibly leaky 135 180 recommended range Bio inert Quaternary LC 89 6 Appendix Removing Capillaries CAUTION Potential damage of capillaries 3 Do not remove fittings from used capillaries To keep the flow path free of stainless steel the front end of the capillary is made of PEEK Under high pressure or when in contact with some solvents PEEK can expand to the shape of the connector where the capillary is installed If the capillary is removed this may become visible as a small step In such cases do not try to pull the fit
16. 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 Bio inert Quaternary LC In This Book In This Book This manual describes the Agilent 1260 Infinity Bio inert System For detailed information on bio inert modules see the separate manuals 1 Introduction This chapter gives an introduction to the Agilent 1260 Infinity Bio inert Quaternary LC and explains underlying concepts and features of the system 2 System Setup and Installation This chapter includes information on software installation stack configurations and how to prepare the system for operation 3 Quick Start Guide This chapter provides information on data acquisition and data analysis with the Agilent 1260 Infinity Bio inert Quaternary LC 4 Typical Bio inert LC Applications This chapter gives an overview on typical Bio inert LC application examples in protein characterization 5 Specifications This chapter provides information about specifications of the 1260 Infinity Bio inert Quaternary LC system 6 Appendix This
17. short delay as the setpoints are attained 48 Bio inert Quaternary LC Quick Start Guide 3 Loading the Default Method The ChemStation has a default method named DEF_LC M which is loaded at first execution or whenever a new blank method template is required It contains default settings for all modules With this procedure you load the method DEF_LC M You can use it to set all parameters to default settings or to get a blank method template before setting up a new method 1 Go to Method and Run Control view of the ChemStation 2 On the menu bar select Method gt New Method and select DEF_LC M from the context menu Alternatively you can use the Load Method icon Kul under the menu bar or double click the method name DEF_LC M in the Methods tab of the Navigation Pane East Standby ZZ ide E de BB i A gw s aai B 3 00 00 ae Gea F Flow 0000 ml min Pressure 127 bar 000 Pressure Limit 600 00 bar 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 Note that this method can never be overwritten with new parameters Hence clicking on Save will re direct you into the Save As function so that you must enter a different method name Bio inert Quaternary LC 49 3 Quick Start Guide Preparing the System Co
18. 210 bar max PEEK pH range 1 13 short term 141 For solvent compatibility refer to section Solvent information for parts of the 1260 Infinity Bio inert LC system in the manual 68 Bio inert Quaternary LC Specifications 5 Specifications Agilent 1260 Infinity Analytical Bio inert Fraction Collector G5664A Table 6 Specifications Agilent 1260 Infinity Analytical Bio inert Fraction Collector G5664A Type Specifications Delay volume Maximum system flow Fraction containers Fraction trays Cooling Trigger modes Trigger source Diverter valve Max pressure Approximately 50 uL 10 mL min Shallow or deep well plates up to 48 mm height 96 or 384 format Test tubes up to 48 mm height Autosampler Vials 2 mL and 6 mL Eppendorf safe lock tubes 0 5 mL 1 5 mL 2 5 mL Full trays cover complete fraction collector 4 well plates 40 x 20 mL test tubes 30 mm od 48 mm height 60 x 15 mL test tubes 25 mm od 48 mm height 126 x 8 mL test tubes 16 mm od 48 mm height 215 x 5 mL test tubes 12 mm od 48 mm height Trays with possibiity to add 1 aditional half tray 2 well plates 10 x 2 mL vials one possible additional half tray 100 x 2 mL vials one possible additional half tray Half trays up to 3 per fraction collector 15 x 6 mL vial 40 x2 mL vial Plates for well plate trays 2 or 4 per fraction collector depending on well plate tray Eppendorf safe lock tubes 27 x 0
19. 5 mL 1 5 mL or 2 5 mL 24 test tubes 18 mm OD 54 x 2 mL vial 15 x 6 mL vial Optional Time slices and peak threshold up downslope upper threshold and timetable Boolean logic for different detector signals combination of different modes manual trigger Supported with Agilent 1200 Series Instant Pilot Agilent 1200 Infinity Series VWD MWD and DAD detectors Agilent 6100 Series Quadrupole LC MS ELSD FLD RID third party detectors require UIB 3 2 valve switching time lt 100 ms 6 bar diverter valve Bio inert Quaternary LC 69 5 Specifications Table6 Specifications Agilent 1260 Infinity Analytical Bio inert Fraction Collector G5664A Type Specifications Environment 4 55 C constant temperature lt 95 humidity non condensing Materials in flow path PEEK Ceramic PTFE pH range 1 13 short term 14 For solvent compatibility refer to section Solvent information for parts of the 1260 Infinity Bio inert LC system in the manual Only one type of well plates can be used at a time in one tray Specifications Agilent 1260 Infinity Bio inert Manual Injector G5628A Table 7 Specifications Agilent 1260 Infinity Bio inert Manual Injector G5628A Type Specifications Injection valve 2 position 6 port Manual Injection Valve Loops PEEK 20 uL standard 5 uL 5 mL optional Materials in flow path PEEK PTFE ceramic pH range 1 13 short term 141
20. 5 mm hex wrench for fixing the fitting maximum torque 0 8 Nm Bio inert Quaternary LC 39 2 System Setup and Installation Installing the Modules CAUTION Installation of the Bio inert Zero Dead Volume ZDV Union The Bio inert ZDV p n 5067 4741 union has two different connectors where capillaries need to be installed in the correct sequence Otherwise an inset of the union may be damaged and the connection may not be tight Potential leak or damage of the Bio inert ZDV Union gt To avoid leaks or a damage to the Bio inert ZDV union follow the procedure below in the prescribed sequence 1 Install the capillary at the end marked with a 2 Install the second capillary at the other end ring indentation 40 Bio inert Quaternary LC System Setup and Installation 2 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 Isopropanol 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 and reagents can carry health risks
21. Agilent 1260 Infinity Bio inert Quaternary LC System manual Notices Agilent Technologies Inc 2011 2012 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 65611 90301 Edition 11 2012 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 Warranty The material contained in this docu ment is provided as is and is sub ject to being changed without notice in future editions Further to the max imum extent permitted by applicable law Agilent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchantability and fitness for a par ticular purpose Agilent shall not be liable for errors or for incidental or consequential damages in connection with the furnishing use or perfor mance of this document or of any information contained herein Should Agile
22. C 23 1 Introduction Bio inert Manual Injector G5628A 24 The Agilent 1260 Bio inert Manual Injector can be used for manual operation or use of large injection volumes It offers a standard injection volume of 20 uL optional 5 uL to 5 mL and ensures highest injection accuracy Agilent 1260 Bio inert Manual Injector uses a Bio inert 6 port sample injection valve 5067 4158 Sample is loaded into the external 20 uL sample loop through the injection port at the front of the valve The valve has a PEEK injection seal A make before break passage in the stator ensures that the flow is not interrupted when the valve is switched between the INJECT and LOAD positions and back again Agilent 1260 Bio inert Manual Injector is based on the Manual Injector G1328C For further information refer to the Agilent 1260 Infinity Bio inert Manual Injector Manual G5628A Bio inert Quaternary LC Introduction Thermostatted column compartment G1316C with Bio inert heat exchangers Thermostatted column compartment G1316C with Bio inert heat exchangers Tag readers Valve head mm olumn clip Figure 8 Thermostatted column compartment with column identification system Bio inert Quaternary LC 25 1 1 26 Introduction The Agilent 1290 Infinity Thermostatted Column Compartment is a stackable temperature controlled column compartment for LC It is used for heating and cooling to meet extreme requirements of retentio
23. D G4212B This detector is highly sensitive for applications such as anion exchange and SEC analysis at pH lt 8 Two Bio inert flow cells are available a 10 mm and a 60 mm cell Both have the same cartridge design as the non Bio inert flow cells for this detector Higher sensitivity for conventional LC as well as ultra fast applications by using next generation optical design Increased sensitivity with 60 mm Max Light cartridge flow cell Optimized cell geometry for less peak dispersion in narrow bore applications RFID tracking technology is used for the UV lamp and the Max Light cartridge flow cells Easy exchange of flow cell by cartridge design For specifications of flow cells see Specifications Bio inert LC capillaries connectors detector flow cells solvent heating elements and valves on page 68 Other Detectors Bio inert flow cells for Multiple Wavelength Detector G1365C and Fluorescence Detector G1321B are available Materials Used are described in detail in Specifications Bio inert LC capillaries connectors detector flow cells solvent heating elements and valves on page 68 28 Bio inert Quaternary LC Introduction 1 Fraction Collector G5664A Fraction Collector G5664A U JI ait Y o N 9 END Needle ceramic Diverter valve not shown Internal tray holder Overview of fraction collector G5664A Figure 10 Bio inert Qu
24. LC Appendix 6 General Safety Information The following general safety precautions must be observed during all phases of operation service and repair of this instrument Failure to comply with these precautions or with specific warnings elsewhere in this manual violates safety standards of design manufacture and intended use of the instrument Agilent Technologies assumes no liability for the customer s failure to comply with these requirements WARNING Ensure the proper usage of the equipment The protection provided by the equipment may be impaired gt The operator of this instrument is advised to use the equipment in a 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 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 inoperati
25. MRM 1 505 5 2 170 CE 36 V MRM 2 505 5 2 201 2 CE 20 V fragmentor 150 V delta EMV 200 V For solvent compatibility refer to section Solvent information for parts of the 1260 Infinity Bio inert LC system in the manual Bio inert Quaternary LC 5 67 5 Specifications Specifications Bio inert LC capillaries connectors detector flow cells solvent heating elements and valves Table5 Specifications Bio inert LC capillaries connectors detector flow cells solvent heating Capillaries and connectors complete system Materials in flow path Metal cladded outside PEEK PTFE pH range 1 13 short term 14 600 bar DAD MWD flow cells G5615 60022 for G1315 C D and G1365 C Materials in flow path PEEK sapphire window pH range 1 13 short term 14 Diode array detector G4212A B with bio inert flow cells 10 mm G4212 60008 or 60 mm G4212 60007 high sensitivity flow cells Materials in flow path PEEK Fused Silica pH range 1 12 Fluorescence detector G1321B with bio inert flow cell G5621 60005 Materials in flow path PEEK Fused Silica pH range 1 12 Bio inert heating elements G5616 60050 9 pL for G1316C Materials in flow path PEEK pH range 1 13 short term 141 Stand alone bio inert valves in G1316C and G1170A Universal Actuator 2 position 6 port valve head G5631A 600 bar max PEEK ceramic 4 column selection valve head G5639A 600 bar max PEEK ceramic 12 position 13 port valve head G4235A
26. als 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 Automates useful tests 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 Bio inert Quaternary LC System Setup an
27. aternary LC 29 1 30 Introduction Fraction Collector G5664A Injection of delay calibration sample UV detector Diverter valve no column VVaste mAU 1400 1000 600 200 0 0 1 2 3 4 5 i 6 7 8 9 Time min Fraction delay sensor FDS FDS Fraction collector 0 1 2 3 4 5 6 7 8 9 Time min Figure 11 Injection of delay calibration sample The Agilent 1260 Infinity Bio inert Fraction Collector provides patented fraction delay calibration and time or peak triggered fraction collection for best recovery and purity of proteins A wide range of collection vessels such as vials microtiter plates or custom vessels give highest flexibility Flow rates up to 10 mL min enable separations with large diameter columns Bio inert Quaternary LC a Bio inert Quaternary LC e a ete 6OO do 2 5 209 System Setup and Installation e 8 e Installing Softvvare 32 Installing the Modules 33 Capillary and Tubing Connections in Flow Path 33 Installing Capillaries 36 Priming the System 41 Storage of the system 45 This chapter includes information on software installation stack configurations and how to prepare the system for operation ee Agilent Technologies 31 2 System Setup and Installation Installing Software 32 Installing the Software Controller and Data System For details of installation procedures for the software refer to the detector manual and the software manu
28. chapter provides addition information on safety legal and web Bio inert Quaternary LC 3 Contents Contents 1 Introduction 7 Concepts of the 1260 Infinity Bio inert Quaternary LC System 8 System Properties 9 System Components 10 Bio inert Materials 12 Optimizing the Stack Configuration 14 Bio inert Quaternary pump G5611A 19 Autosampler G5667A 22 Bio inert Manual Injector G5628A 24 Thermostatted column compartment G1316C with Bio inert heat exchangers 25 Bio inert Flow Cells for Detectors 27 Fraction Collector G5664A 29 System Setup and Installation 31 Installing Software 32 Installing the Modules 33 Quick Start Guide 47 Preparing the System 48 Setting Up the Method 54 4 Typical Bio inert LC Applications 57 Characterization of bio therapeutics 58 Application Examples 59 Specifications 63 Performance Specifications 64 Solvent Information 71 Bio inert Quaternary LC Contents 6 Appendix 75 Safety 76 The Waste Electrical and Electronic Equipment Directive 79 Lithium Batteries Information 80 Radio Interference 81 Sound Emission 82 UV Radiation 83 Declaration of Conformity for HOX2 Filter 84 Installation of Stainless Steel Cladded PEEK Capillaries 85 Agilent Technologies on Internet 91 Bio inert Quaternary LC Contents 6 Bio inert Quaternary LC Bio inert Quaternary LC 1 Introduction Concepts of the 1260 Infinity Bio inert Quaternary LC System 8 System Properties 9
29. d Installation 2 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 Capillary and Tubing Connections in Flow Path Figure 12 on page 34 shows capillary and tubing connections in the flow path For details and necessary parts refer to the individual module manuals Bio inert Quaternary LC 33 2 System Setup and Installation Installing the Modules Connections to be installed Preinstalled connections nai NAI PTFE Titanium PTFE Titanium PEEK Titanium i PTFE PEEK al Hi T a PEEK PEEK PEEK or PTFE Stainless steel cladded peak capillaries Figure 12 Capillary and tubing connections in flow path Normal PEEK capillaries are very sensitive to high pressures For the bio inert LC system Agilent uses stainless steel cladded PEEK capillaries which keep the flow path free of metal as required for bio inert applications and ensure pressure stability to at least 600 bar These capillaries are equipped with UHP FF fittings see Installing UHP FF Fittings on page 37 for handling instructions 34 Bio inert Quaternary LC System Setup and Installation 2 Optionally a bio in
30. d 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 solvent vendor especially when toxic or hazardous solvents are used Bio inert Quaternary LC Appendix 6 The Waste Electrical and Electronic Equipment Directive Abstract The Waste Electrical and Electronic Equipment WEEE Directive 2002 96 EC adopted by EU Commission on 13 February 2003 is introducing producer responsibility on all electric and electronic appliances starting with 13 August 2005 This product complies with the WEEE Directive 2002 96 EC marking requirements The affixed label indicates that you must not discard this electrical electronic product in domestic household waste Product Category With reference to the equipment types in the WEEE Directive Annex I this product is classed as a Monitoring and Control Instrumentation product Do not dispose off in domestic household waste To return unwanted products contact your local Agilent office or see www agilent com for more information Bio inert Quaternary LC 79 6 Appendix Lithium Batteries Information Lithium batteries may not be disposed off into the domestic waste Transportation of discharged Lithium batteries through carriers regulated by IATA ICAO ADR RID IMDG is not allowed Danger of explosion if battery is incorrectly replaced
31. dule manuals for module internal capillary and tubing connections Bio inert Quaternary LC 35 2 System Setup and Installation Installing Capillaries Agilent Technologies will introduce new UHP FF Fittings planned for 2013 The new fittings are designed for improved robustness and ease of use Previous fittings require careful handling Therefore it is important to know which fittings are used in the system The figure below illustrates the differences between new and previous capillaries 5 mm fitting Figure 13 New bio inert capillary and UHP FF fitting Figure 14 Previous bio inert capillary and fitting with nose planned for 2013 For handling instructions of capillaries and fittings used in modules before delivery of the new UHP FF fittings planned for 2013 refer to Installation of Stainless Steel Cladded PEEK Capillaries on page 85 36 Bio inert Quaternary LC System Setup and Installation 2 Installing the Modules Installing UHP FF Fittings 1 Slide the fitting on the capillary Let the capillary jut out 5 mm Capillary Fitting Bio inert Quaternary LC 37 2 System Setup and Installation Installing the Modules 2 Insert the fitting to the receiving port and push the capillary to the bottom of the port 3 Finger tighten the nut into the port until snug 38 Bio inert Quaternary LC System Setup and Installation 2 Installing the Modules 4 Use Fitting mounting tool 5043 0915 or a
32. e Autosampler Upstream of sample introduction G5667A Titanium gold PTFE PEEK ceramic Downstream of sample introduction PEEK ceramic Agilent 1260 Infinity Bio inert Manual Injector PEEK ceramic G5628A Agilent 1260 Infinity Bio inert Analytical Fraction Collector PEEK ceramic PTFE G5664A 12 Bio inert Quaternary LC Table1 Bio inert materials used in Agilent 1260 Infinity Systems Introduction 1 Module Materials Bio inert Flow Cells Standard flow cell bio inert 10 mm 13 pL 120 bar 12 MPa for MWD DAD includes Capillary Kit Flow Cells BIO p n G5615 68755 G5615 60022 for Agilent 1260 Infinity Diode Array Detectors DAD G1315C D Max Light Cartridge Cell Bio inert 10 mm V s 1 0 pL G5615 60018 and Max Light Cartridge Cell Bio inert 60 mm V s 4 0 uL G5615 60017 for Agilent 1200 Infinity Series Diode Array Detectors DAD G4212A B Bio inert flow cell 8 uL 20 bar pH 1 12 includes Capillary Kit Flow Cells BIO p n G5615 68755 G5615 60005 for Agilent 1260 Infinity Fluorescence Detector FLD G1321B Bio inert heat exchanger G5616 60050 for Agilent 1290 Infinity Thermostatted Column Compartment G1316C Bio inert Valve heads Bio inert Connection capillaries PEEK ceramic sapphire PTFE PEEK fused silica PEEK fused silica PTFE PEEK steel cladded G4235A G5631A G5639A PEEK ceramic Al 03 based Upstream of sample introduction Titanium Downstream of
33. ert fraction collector may be installed For details refer to the Agilent 1260 Infinity Bio inert Analytical scale Fraction Collector Manual Depending on the system configuration one may need capillaries of different lengths To achieve optimal LC results the following different bio inert capillaries are available Bio inert capillaries p n Description G5611 60500 Capillary 400 x 0 17 mm titanium Bio inert pump to autosampler G5611 60502 Capillary 900 x 0 17 mm titanium Bio inert pump to thermostatted autosampler G5667 60500 PEEK SST capillary bio inert 0 17 mm ID 400 mm long autosampler to thermostatted column compartment TCC G5615 68755 Capillary Kit Flow Cells BIO includes Peek Capillary i d 0 18 mm 1 5 m Ig and PEEK Fittings 10 PK p n 5063 6591 TCC to DAD G5664 68712 Analytical tubing kit 0 25 mm i d PTFE ESD DAD to fraction collector For other connections following stainless steel cladded PEEK capillaries are available for bio inert applications PK ST capillaries p n Description G5667 60502 Capillary PK ST 0 17 mm x 100 mm male to male pre swaged G5667 60503 Capillary PK ST 0 17 mm x 150 mm male to male pre swaged G5667 60504 Capillary PK ST 0 17 mm x 200 mm male to male pre swaged G5667 60505 Capillary PK ST 0 17 mm x 300 mm male to male pre swaged G5667 60500 Capillary PK ST 0 17 mm x 400 mm male to male pre swaged G5667 60501 Capillary PK ST 0 17 mm x 500 mm male to male pre swaged See mo
34. ervievv on typical Bio inert LC application examples in protein characterization te Agilent Technologies 57 4 Typical Bio inert LC Applications Characterization of bio therapeutics Characterization of bio therapeutics Physico chemical characterization and confirmation plays a crucial role in the New Biological Entity NBE and biotherapeutics workflow to ensure drug safety and efficacy Agilent offers a broad spectrum of tools for all assays in order to fulfill the regulatory requirements For the LC based tests the Agilent 1260 Infinity Bio inert Quaternary solution offers a high degree of flexibility to address the needs of a quality control environment as well as flexibility for method development in SEC ion exchange peptide mapping confirmation and glycan analysis Therapeutic monoclonal antibodies mABs Figure 16 on page 58 are characterized by a variety of assays to ensure drug safety and efficacy The Agilent 1260 Infinity Bio inert Quaternary solution is a flexible tool addressing the major requirements A Antigen binding site Pyroglutamate Heavy chain Hinge region disulfide bonds Fab region Ed Light chain 4 Deamidation or oxidation 3a a ee Fc region Truncation lysine n v Glycosylation Figure 16 General structure of Therapeutic Antibodies 58 Bio inert Quaternary LC Application Examples Typical Bio inert LC Applications 4 lon chromatography of charge variants
35. f the window and move away to the left Up to 60 min of past data is accessible This is useful for checking the baseline and Bio inert Quaternary LC 51 3 Quick Start Guide 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 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 solvent and if necessary use an intermediate step with a co miscible solvent isopropanol is often a good choice check with a solvent miscibility table
36. he system p n Description 1 Purging solvent s see Table 2 on page 45 1 5022 2184 Union ZDV Remove the column and replace it by a ZDV fitting Prepare bottles with appropriate intermediate solvents see Table 2 on page 45 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 8 5 mL min for 10 min 4 Modify the flow path of your system as required for your application For delay volume optimization see the Rapid Resolution System manual Buffer salt of aqueous buffers may precipitate in residual isopropanol Capillaries and filter may be 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 44 5 Replace the solvent bottle by a bottle of isopropanol 6 Flush the channel at a flow rate suitable for the installed tubing typically 8 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 Bio inert Quaternary LC System Setup
37. here are no materials that combine suitability for versatile HPLC instrumentation valves capillaries springs pump heads flow cells etc with complete compatibility with all possible chemicals and application conditions This section recommends the preferred solvents Chemicals that are known to cause issues should be avoided or exposure should be minimized for example for short term cleaning procedures After potentially aggressive chemicals have been used the system should be flushed with compatible standard HPLC solvents Bio inert Quaternary LC 71 5 72 Specifications PEEK PEEK Polyether Ether Ketones combines excellent properties with regard to biocompatibility chemical resistance mechanical and thermal stability and is therefore the material of choice for biochemical instrumentation It is stable in the specified pH range and inert to many common solvents There is still a number of known incompatibilities with chemicals such as chloroform methylene chloride THF DMSO strong acids nitric acid gt 10 sulphuric acid gt 10 sulfonic acids trichloroacetic acid 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 As normal PEEK capillaries are very sensitive to high pressure especially under such conditions Agilent uses stainless steel cladded PEEK
38. highly resistant to corrosion and provides a titanium gold solvent flow path It generates gradients by low pressure mixing from four individual solvent channels It is based on a two channel dual plunger in series design which comprises all essential functions that a solvent delivery system has to fulfill Metering of solvent and delivery to the high pressure side are performed by one pump assembly which can generate pressure up to 600 bar and deliver flow rates up to 10 mL min The pump can also be run in Emulation Mode 400 bar for compatibility with classic modules Bio inert Quaternary LC 19 1 20 Introduction The solvents are degassed in a built in vacuum degasser Solvent compositions are generated on the low pressure side by a high speed proportioning valve MCGV The pump assembly includes a pump head with an active inlet valve and an outlet valve A damping unit is connected between the two plunger chambers For convenient priming of the pump head a purge valve with PTFE frit is fitted at the outlet of the pump head The Agilent 1260 Infinity Bio inert Quaternary pump has an active seal wash Rinsing seals and pistons prevents crystallization of non volatile highly concentrated buffers The seal wash option comprises a support ring secondary seal gasket and seal holder for both piston sides A wash bottle filled with water isopropanol 90 10 is placed above the pump in the solvent cabinet The peristaltic pump moves a flo
39. ications Agilent 1260 Infinity Bio inert Quaternary Pump G5611A Table 3 Type Specifications Hydraulic system Setable flow range Recommended flow range Flow precision Flow accuracy Pressure Pressure pulsation Compressibility compensation Recommended pH range Gradient formation Composition range Composition precision Communications Materials in flow path Active seal wash Dual piston in series pump with proprietary servo controlled variable stroke drive floating pistons and active inlet valve integrated 4 channel degassing unit 0 001 10 mL min in 0 001 mL min increments 0 2 10 mL min lt 0 07 RSD or lt 0 02 min SD whichever is the greater based on retention time at constant room temperature 1 or 10 pL min whichever is the greater Operating range up to 60 MPa 600 bar 8700 psi up to 5 mL min Operating range up to 20 MPa 200 bar 2950 psi up to 10 mL min lt 2 amplitude typically lt 1 3 at 1 mL min isopropanol at all pressures gt 1 MPa 10 bar User selectable based on mobile phase compressibility 1 13 short term 14 Low pressure quaternary mixing gradient capability using proprietary high speed proportioning valve Delay volume 600 900 pL dependent on back pressure 0 95 or 5 100 user selectable lt 0 2 RSD at 0 2 and 1 mL min Controller area network CAN RS 232C APG Remote ready start stop and shut down signal
40. ithium Batteries Information 80 Radio Interference 81 Sound Emission 82 UV Radiation 83 Declaration of Conformity for HOX2 Filter 84 Installation of Stainless Steel Cladded PEEK Capillaries 85 First Step Finger tight Fitting 86 Second Step Installation to Connector 86 Removing Capillaries 90 Agilent Technologies on Internet 91 This chapter provides addition information on safety legal and web Apg Agilent Technologies 75 6 Appendix Safety Safety Symbols Table8 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 ahaa alerts you to situations that could cause physical injury or death 3 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 3 Do not proceed beyond a caution until you have fully understood and met the indicated conditions 76 Bio inert Quaternary
41. ll channels have been purged Turn the flow off and close the purge valve Bio inert Quaternary LC System Setup and Installation 2 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 LabAdvisor or Instrument Utilities can be used for automatically purging the pump 1 Open the purge valve of your pump by turning it counterclockwise and set the flow rate to 5 mL min Flush the vacuum degasser and all tubes with at least 10 mL of solvent Repeat step 1 and 2 for the other channel s of the pump 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 Bio inert Quaternary LC 43 2 System Setup and Installation When Parts required Preparations CAUTION 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 precipitation or residual liquid droplets in parts of t
42. n time reproducibility The main features are e Bio inert heating elements peltier heating and cooling from 10 C below ambient up to 80 C with high heating and cooling speeds for maximum application flexibility and stability Holds up to three 30 cm columns without heating elements and optimized design gives minimum dead volumes and maximum efficiency Up to 6 additional bio inert heat exchangers contribute only 9 uL Electronic column identification module as standard for GLP documentation of column type and major column parameters Optional high quality Rheodyne column switching valves with ceramic stator face assemblies for prolonged lifetime For specifications see Specifications Bio inert LC capillaries connectors detector flow cells solvent heating elements and valves on page 68 Bio inert Quaternary LC Introduction 1 Bio inert Flow Cells for Detectors Diode Array Detector DAD 1 Agilent 1260 Infinity DAD VL G1315C This detector with robust Bio inert flow cell pH 1 14 is the standard detector for universal Bio molecule applications It features 80 Hz detection rates RFID tags and a wide pH range for highest robustness Ceramic cell inside Saphire window not shown A fa PEEK Ferrules Inert unions PEEK capillaries Figure 9 Overview of bio inert flow cell G1315C Bio inert Quaternary LC 27 1 Introduction 2 Agilent 1260 Infinity DA
43. ndby E J g die M e Idie ME amp kozi A 4 J PR 10 01 IN 50 0 50 0 KS 8 8 0 000 ml min Vial 2 Ciba oana 5g a Gj Ga noapenchlledied Temperature 4 C Qoxrresnn 23 51 C 24 75 C Q orrors Flow 0000 mi min Pressure 1 27 bar Ripple 000 QO EL morenoi _Pressure Limit 600 00 bar 54 Bio inert Quaternary LC Quick Start Guide 3 2 Right click the pump area and select Method in the context menu a In the Method page for the 1260 Infinity Quaternary Pump enter the following parameters Flow rate 1 5 ml min Solvent A Select Water from the compressibility drop down list Solvent B Select the check box to make Solvent B active B Initial value 65 Stop Time 6 min Max Pressure Limit b Click the sign to open the Timetable c Add a line select Change Solvent Composition and set B to 80 d Other parameters can remain at 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 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 4 Right click the Thermostatted Column Compartment TCC area and select Method in the co
44. nfiguring the Online Plot 1 Ifthe Online Plot window is not visible click View gt Online Signals gt Signal Window 1 to display the window 50 Bio inert Quaternary LC Quick Start Guide 3 Preparing the System 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 HM14 TCC Left Temperature HM1B TCC Right Temperature F F DAD1B DAD Signal B DADIC DAD Signal C lt Remove m Window DADIA DAD Signal A x axis 3 Hmn Type acquired y axis range 100 Hm U draw zero line TD auto y adjust Offset 10 H Method Settings w Fraction collection ticks E Use method settings Apply to Method OK Cancel Help 3 In the Available Signals box highlight the required signal s and click Add to move them to the Selected Signals box 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 NOTE In addition to the detector signals parameter traces such as temperature and pressure can du pen 4 also be plotted With Apply to Method the settings in this page can be stored into the method The Online Plot window behaves like electronic chart paper continuously recording the output from the detector s and other output parameters The signals are drawn at the right o
45. ng for the second and subsequent times again start from the finger tight position which is not necessarily a vertical wrench position and rotate the wrench by 90 135 Staying below 90 grey arrow could be insufficiently tight more than 135 red arrow could damage the capillary o ossibly eaky 90 recommended range o Second Step Installation to Soft Connectors Use this procedure for soft connectors which are typically made from PEEK These are the following connections to and from all bio inert valves injection valve in the autosampler and valves in the thermostatted column compartment and 1290 Infinity Valve Drive bio inert ZDV unions detector flow cells multi draw upgrade kit capillary to capillary connections for example for heat exchangers Bio inert Quaternary LC 87 Appendix to the autosampler needle and to PEEK columns like many bio inert columns For the installation of bio inert ZDV unions see Installation of the Bio inert Zero Dead Volume ZDV Union on page 40 First installation of a capillary to a soft connector 1 When tightening a fitting for the first time start from the finger tight position which does not necessarily need to be a vertical wrench position and rotate the wrench by 180 210 Staying below 180 grey arrow will not be sufficiently tight more than 210 red arrow could damage the capillary possibly
46. nt 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 CAUTION notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly per formed
47. ntext menu a In the Method page for the 1260 Infinity TCC enter the following parameters Left Temperature 40 C Right Temperature Combined b Other parameters can remain at their default settings Click OK to exit the window The changes are sent to the TCC module Bio inert Quaternary LC 55 3 56 Quick Start Guide 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 VL enter the following parameters Use Signal Turn all signals except Signal A off by clearing the check boxes Signal A 250 nm bw 100 nm ref 360 nm bw 100 nm Peak width 0 012 min 0 25 s Response 20 Hz b In the Advanced section set Spectrum Store to All c Other parameters can remain at their default settings Click OK to exit the window The changes are sent to the DAD module All the required module parameters have now been entered Select Method gt Save Method As SO 1 M to save the method with a new name The ChemStation does not allow the method to be saved as DEF_LC M so that the default method template is not altered Allow the system to equilibrate for at least 10 min and check that the baseline in the Online Plot is stable before starting the analysis Bio inert Quaternary LC Bio inert Quaternary LC Typical Bio inert LC Applications e Characterization of bio therapeutics 58 Application Examples 59 This chapter gives an ov
48. ntification of problems by self diagnostics built in log books and preprogrammed test methods Widest pH compatibility pH 1 13 High corrosion resistance 1 Introduction System Components 10 Solvent cabinet The solvent cabinet is a tray for the storage of four solvent bottles for stable and secure containment of solvent bottles during LC operation Bio inert Quaternary pump G5611A The Agilent 1260 Infinity Bio inert Quaternary LC pump is highly resistant to corrosion and consists of titanium gold solvent lines It generates gradients by low pressure mixing from four individual solvent channels For further information refer to the Agilent 1260 Infinity Bio inert Quaternary LC Pump G5611A manual Autosampler G5667A The Agilent 1260 Infinity High performance Bio inert Autosampler handles vials and microtiter plates and enables injection volumes from 0 1 100 uL The ceramic needle PEEK needle seat and stainless steel clad PEEK capillaries ensure highest injection accuracy and precision In addition the needle wash capability ensures minimum carryover To protect labile compounds from degradation during analysis or storage the autosampler is cold room compatible alternatively a cooling module can be added For manual injections the Agilent 1260 Infinity Bio inert Manual Injector is available for single injections or larger injection volumes For further information refer to the Agilent 1260 Infinity Bio ine
49. of monoclonal antibodies With the Agilent 1260 Infinity Bio inert Quaternary LC and the polymeric BioMab WCX columns Agilent offers a unique single vendor solution for highest performance requirements The combination of column technology from 10 um particles to 1 7 um particles and biocompatibility results in increased robustness superior resolution and prolonged column lifetime In ion exchange applications the LC system is often operated at high salt concentrations or in extreme pH ranges Agilent recommends that solutions be replaced after usage to prevent crystallization of salts or to avoid additional stress on system components Absorbance AU 0 60 0 50 0 30 0 20 0 00 aJ Acidic Isoforms Figure 17 Agilent Bio MabNP10 column Bio inert Quaternary LC Basic Isoforms 24 26 28 30 32 34 Time min High resolution separation of acidic and basic charge variants using the 59 4 Typical Bio inert LC Applications Size exclusion chromatography for purity and aggregation analysis With the Agilent 1260 Infinity Bio inert Quaternary LC and Bio SEC 5 and Bio SEC 3 columns robust performance and high reproducibility are obtained under different buffer conditions with or without detergents In combination with a variety of detectors such as UV and fluorescence impurities can be easily resolved and detected with superior sensitivity Absorbance AU Monomer Possible Dimer 0 010
50. ors on page 87 a wrench is used to rotate the fitting relative to the finger tight position by a defined angle For each of the cases mentioned above there is a recommended range in which the fitting is tight Staying below this range could create a leak either a visible one or a micro leak potentially biasing measurement results Exceeding the recommended range could damage the capillary Alternatively a torque wrench may be used The target torque for all connections is about 0 7 Nm When using a torque wrench read instructions for that tool carefully as wrong handling may easily miss the correct torque Second Step Installation to Hard Connectors Use this procedure for hard connectors made from metal titanium or ceramics In the system these are connections to and from the analytical head of the autosampler connections to injection valve and needle and to a metal column 86 Bio inert Quaternary LC Appendix 6 First installation of a capillary to a hard connector 1 When tightening a fitting for the first time start from the finger tight position which is not necessarily a vertical wrench position and rotate the wrench by 135 180 Staying below 135 grey arrow will be insufficiently tight more than 180 red arrow could damage the capillary possibly leaky D 180 recommended range Second and subsequent installations of a capillary to a hard connector 1 When tightening the fitti
51. r position lt 70 dB Sound Pressure Lp lt 70 dB A At Operator Position Normal Operation According to ISO 7779 1988 EN 27779 1991 Type Test 82 Bio inert Quaternary LC Appendix 6 UV Radiation Emissions of ultraviolet radiation 200 315 nm from this product is limited such that radiant exposure incident upon the unprotected skin or eye of operator or service personnel is limited to the following TLVs Threshold Limit Values according to the American Conference of Governmental Industrial Hygienists Table9 UV radiation limits Exposure day Effective irradiance 8h 0 1 uW cm 2 10 min 5 0 W cm Typically the radiation values are much smaller than these limits Table 10 UV radiation typical values Position Effective irradiance Lamp installed 50 cm distance average 0 016 W cm Lamp installed 50 cm distance maximum 0 14 pW cm2 Bio inert Quaternary LC 83 Appendix Declaration of Conformity for HOX2 Filter Declaration of Conformity for HOX2 Filter 84 We herewith inform you that the Declaration of Conformi Holmium Oxide Glass Filter used in Agilents absorbance detectors listed in the table below meets the requirements of National Institute of Standards and Technology NIST to be applied as certified wavelength standard According to the publication of NIST in J Res Natl Inst Stand Technol 112 303 306 2007 the holmium oxide glass filters are inherently stable wi
52. ration 33 0 online plot configuring 50 optimization stack configuration 14 overview pump 19 P peptide mapping 61 priming withapump 43 properties system 9 radio interference 81 S safety classl 77 safety information Bio inert Quaternary LC lithium batteries 80 safety general information 77 symbols 76 solvent information 71 solvents change 44 sound emission 82 system setup and installation network Integration 33 optimizing stack configuration 14 system turningon 48 T thermostatted column compartment overview 25 U UV radiation 83 VV vvaste electrical and electronic equipment 79 WEEE directive 79 Bio inert Quaternary LC Index 93 www agilent com In This Book This book describes the Agilent 1260 Infinity Bio inert Quaternary LC System The manual describes the following Introduction e System Setup and Installation Quick Start Guide Typical Bio inert LC Applications Optimiziation of the Agilent 1260 Infinity Bio inert Quaternary LC Specifications e Appendix Agilent Technologies 2011 2012 Printed in Germany 11 2012 G5611 90301 EE Agilent Technologies
53. rt High Performance Autosampler G5667A manual Bio inert Quaternary LC Introduction 1 Thermostatted column compartment G1316C The Agilent 1290 Infinity Column Compartment includes bio inert heating elements to pre heat the solvent before it reaches the column The column compartment houses either four short columns or two columns up to 30 cm in length Agilent Quick Change valves are available for more complex workflows such as column switching column screening or method development For further information refer to the Agilent 1290 Infinity Thermostatted Column Compartment G1316C manual Diode array multiple wavelength detector or other detector choices G1315C D G4212A B G1321B Agilent offers a variety of detection options to address the requirements of protein research applications or of assays to assess the integrity of bio therapeutic compounds Bio inert flow cells are available for MWD DAD and FLD For further information refer to the Agilent 1260 Infinity Diode Array and Multiple Wavelength Detector Agilent 1200 Infinity Series Diode Array Detectors G4212A B and Agilent 1260 Infinity Fluorescence Detector G1321B manual Fraction Collector with Cooling Option G5664A The Agilent 1260 Infinity Bio inert Fraction Collector provides patented fraction delay calibration and time or peak triggered fraction collection for best recovery and purity of proteins For further information refer to the Agilent 1260
54. s LAN optional Titanium Gold Platin lridium Sapphire PEEK PTFE Included 1 manual Bio inert Quaternary LC For solvent compatibility refer to section Solvent information for parts of the 1260 Infinity Bio inert LC system in the 65 5 Specifications Specifications Agilent 1260 Infinity Bio inert High Performance Autosampler G5667A Table 4 Specifications Agilent 1260 Infinity Bio inert High Performance Autosampler G5667A Type Specification Injection range Precision Injection Accuracy Pressure range Sample viscosity range Sample capacity Injection cycle time Carry over pH range Sample cooling Materials in flow path GLP features Communications Safety features Metering device 0 1 100 pL in 0 1 pL increments Up to 40 pL with reduced injection volume kit hardware modification required Up to 1500 UL with multiple draw hardware modification required Typically lt 0 25 RSD from 5 100 ul Typically lt 0 5 RSD from 2 5 pL Typically lt 0 7 RSD from 1 2 pL volume Measured with injections of caffeine in water 1 10 uL n 10 Up to 600 bar 8700 psi 0 2 5 cp 2 x well plates MTP 10 x 2 0 mL vials 108 x 2 mL vials in 2 x 54 vial plate plus 10 additional 2 mL vials 30 x 6 mL vials in 2 x 15 vial plate plus 10 additional 2 mL vials 54 Eppendorf tubes 0 5 1 5 2 mL in 2 x 27 Eppendorf tube plates Typically lt 17 s using the follo
55. sample introduction Agilent uses stainless steel cladded PEEK capillaries which keep the flow path free of steel and provide pressure stability to more than 600 bar To ensure optimum bio compatibility of your Agilent 1260 Infinity Bio inert LC system do not include non inert standard modules or parts to the flow path Do not use any parts that are not labeled as Agilent Bio inert For solvent compatibility of these materials see Solvent information for parts of the 1260 Infinity Bio inert LC system on page 71 Bio inert Quaternary LC 13 1 Introduction Optimizing the Stack Gonfiguration You can ensure optimum performance by installing the system in one of the follovving configurations These configurations optimize the system flovy path ensuring minimum delay volume 14 Bio inert Quaternary LC Introduction 1 One Stack Configuration Install the modules of the Agilent 1260 Infinity Bio inert LC System in the configuration shown in Figure 1 on page 15 and Figure 2 on page 16 This configuration optimizes the flow path for minimum delay volume and minimizes the bench space required 3 fa Solvent cabinet Instant Pilot control module Autosampler Detector Figure 1 Recommended Stack Configuration Front View Bio inert Quaternary LC 15 1 Introduction Optimizing the Stack Configuration I mM CAN bus cable to local user interface
56. talled carefully in order to keep them tight without damaging them by over tightening Handling of stainless steel cladded PEEK capillaries Be careful when installing stainless steel cladded PEEK capillaries The correct torque must be applied to avoid leaks potentially causing measurement problems or damage to the capillary gt Follow the procedure below for a correct installation Installation procedure The amount of force torque needing to be applied to install the capillary depends on the female connector to which the capillary is installed and whether the material of that connector is soft or hard Compared with hard connectors a greater tightening angle is required for soft connectors to achieve same torque e whether the capillary is installed for the first time or subsequent times For the first time a greater tightening angle needs to be applied The installation consists of two steps In the first step the fitting is installed finger tight without using tools Finger tight means that the fitting will grip and hold the capillary This brings the fitting to the appropriate start position marked as 0 below for the second step Bio inert Quaternary LC 85 6 Appendix First Step Finger tight Fitting 1 Tighten the fitting using your fingers Second Step Installation to Connector In the second step Second Step Installation to Hard Connectors on page 86 or Second Step Installation to Soft Connect
57. th respect to the wavelength scale and need no recertification The expanded uncertainty of the certified wavelength values is 0 2 om Agilent Technologies guarantees as required by NIST that the material of the filters is holmium oxide glass representing the inherently existent holmium oxide absorption bands Test wavelengths Product Number 79883A 79854A G1306A Wavelength Optical Accuracy Bandwidth G1315A G1365A G1315BIC G1365B C G1600A G7100A G1314A B C G1314D EF G4286 90A B C 1120 1220 The variation in Measured Wavelength depends on the different Optical Bandwidth P N 89550 90501 May 19 2010 Revision H 417 Agilent Technologies Il Effective by May 19 2010 Bio inert Quaternary LC Appendix 6 Installation of Stainless Steel Cladded PEEK Capillaries CAUTION This installation procedure applies for capillaries and corresponding fittings used in modules delivered before January 2013 For current capillaries and fittings see Installing UHP FF Fittings on page 37 The 1260 Infinity Bio inert LC system uses PEEK capillaries that are cladded with stainless steel These capillaries combine the high pressure stability of steel with the inertness of PEEK They are used in the high pressure flow path after sample introduction loop needle seat capillary through the thermostatted column compartment heat exchangers to the column Such capillaries need to be ins
58. ting from the capillary as this can destroy the front part of the capillary Instead carefully pull it to the rear During installation of the capillary the fitting will end up in the correct position ON ee Rear Front Figure 20 Capillary fitting 90 Bio inert Quaternary LC Appendix 6 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 Bio inert Quaternary LC 91 Index Index A active seal wash 53 Agilent oninternet 91 algae 74 battery safety information 80 bio inert materials 12 bio therapeutics characterization 58 C changing solvents 44 chromatography size exclusion 60 component autosampler 10 detector 11 fraction collector 11 pump 10 solvent cabinet 10 ce 11 components system 10 concepts bio inert 8 configuration one stack 15 two stack 17 configuring online plot 50 92 D declaration of conformity 84 default method loading 49 E electronic vvaste 79 F flovv cell 74 solvent information 74 fraction collector overview 29 H holmium oxide declaration of conformity 84 I information on UV radiation 83 installing Lab Advisor 32 internet 91 ion exchange truncation 59 L lithium batteries 80 loading default 49 M manual injector 24 mapping peptide 61 materials bio inert 12 method settingup 54 N network integ
59. ve and be secured against any intended operation Make sure that only fuses with the required rated current and of the specified type normal blow time delay and so on are used for replacement The use of repaired fuses and the short circuiting of fuse holders must be avoided Bio inert Quaternary LC TI 78 Appendix Some adjustments described in the manual are made with power supplied to the instrument and protective covers removed Energy available at many points may if contacted result in personal injury Any adjustment maintenance and repair of the opened instrument under 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 an
60. w through the pump head removing all possible buffer crystals from the back of the pump seal For further information refer to the Agilent 1260 Infinity Bio inert Quaternary LC Pump G5611A manual Bio inert Quaternary LC Hydraulic Path Figure 6 Bio inert Quaternary LC Introduction Bio inert Quaternary pump G5611A From solvent bottles Hydraulic Path of the Quaternary Pump 1 21 1 Introduction Autosampler G5667A Autosampler G5667A Metering device Loop capillary Transport assembly Needle carrier Needle Injection valve Needle wash peristaltic pump Figure 7 Autosampler G5667A 22 Bio inert Quaternary LC Introduction 1 The Agilent 1260 Infinity High performance Bio inert Autosampler handles vials and microtiter plates It injects volumes from 0 1 100 uL and is extendable to 900 uL The ceramic needle PEEK needle seat and wash and stainless steel clad PEEK capillaries ensure the highest injection accuracy and precision with minimum carryover To protect labile compounds from degradation during analysis or storage the autosampler is cold room compatible alternatively you can add a cooling module The Agilent 1260 Infinity High performance Bio inert Autosampler is based on the High Performance Autosampler G1367E For further information refer to the Agilent 1260 Infinity Bio inert High Performance Autosampler G5667A manual Bio inert Quaternary L
61. wing standard conditions Default draw speed 100 pL min Default eject speed 100 L min Injection volume 5 pL Typically lt 0 004 For measurement conditions see Li 2 3 1 13 short term 144 Optional with G1330B 4 40 C Upstream of sample introduction titanium gold PTFE PEEK ceramic Downstream of sample introduction PEEK ceramic Early maintenance feedback EMF electronic records of maintenance and errors Controller area network CAN RS232C APG remote standard optional four external contact closures and BCD vial number output Extensive diagnostics can be done with the help of the Control Module and Agilent LabAdvisor Diagnostic Software leak detection and safe leak handling low voltages in maintenance areas error detection and display Metering pump in high pressure flow path 66 Bio inert Quaternary LC Specifications 1 Chromatographic conditions Column Agilent ZORBAX SB C18 2 1 x 50 mm1 8 um p n 827700 902 mobile phase A 0 1 TFA in water B 0 1 TFA in acetonitrile isocratic B 35 flow rate 0 5 mL min temperature 30 C UV detection Sample 1200 ng l chlorhexidine dissolved in mobile phase A 1 uL injected and measured on G4212A DAD 10 mm cell Wavelength 257 nm 4 nm ref 360 nm 16 nm slit 4 nm 10 Hz MS detection Sample 50 ng pl chlorhexidine dissolved in mobile phase A 1 pL injected and measured on Agilent 6460 QQQ in specified conditions

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