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Agilent Technologies 1290 Infinity II 2D-LC Solution, User Manual

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Agilent Technologies 1290 Infinity II 2D-LC Solution User Manual Download Page 186

7

Method Development of Active Solvent Modulation (ASM)

Method Development of Active Solvent Modulation (ASM)

2D-LC User Guide

186

Method Development of Active Solvent 
Modulation (ASM)

ASM method development helps finding the optimal dilution of 1D solvents in the 
sample loop for best 2D resolution at lowest cycle time.
After switching on the ASM functionality (see 

“Method Parameters”

on page 187), 

execute the steps in the following order:

•

“Optimize the Dilution by Using ASM Capillaries”

on page 188

•

“Optimize the Sample Loop Flush”

on page 189

•

“Include the ASM Phase to the 2D Gradient”

on page 190

•

“Optimize Dilution Through Method Settings”

on page 191

Summary of Contents for 1290 Infinity II 2D-LC Solution

Page 1: ...Agilent 1290 Infinity II 2D LC Solution MassHunter Acquisition for TOF and Q TOF User Guide 2D LC User Guide...

Page 2: ...ement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used orcopied only in accordance with the terms of such license...

Page 3: ...s chapter describes the hardware and software installation of the Agilent 1290 Infinity II 2D LC Solution The 2D LC instrument can be used with the software described in this document The installation...

Page 4: ...Diagnostics This chapter gives an overview about the troubleshooting and diagnostic features and the different user interfaces 11 Error Information This chapter describes the meaning of error messages...

Page 5: ...Cutting and High Resolution Sampling 2D LC 19 Comprehensive 2D LC LCxLC 30 Triggering of 2D LC 32 Active Solvent Modulation ASM 35 3 Compatibility Matrix 45 Supported Chromatographic Data Systems 46 S...

Page 6: ...sition 11 123 6 Method Parameters 125 Method Editor 2D LC 126 Set the 2D LC Method parameters 127 Preview 2D LC 154 Set up a Peak Based Experiment Graphically 168 Setup 2D Gradient Graphically 173 Set...

Page 7: ...ion for MassHunter 230 GC Image Basic Information 256 10 Troubleshooting and Diagnostics 275 Overview of the Module s Indicators and Test Functions 276 User Interfaces 277 Agilent Lab Advisor Software...

Page 8: ...arts for Maintenance 334 2D LC Loops 335 2D LC Capillaries 336 ASM Capillaries 337 Pressure Release Kit 338 2D LC Easy Starter Kit 339 Valve Drive Parts 340 Valve Driver Parts Infinity II 341 Valve He...

Page 9: ...ul Mode Combinations 373 Solvent Elution Modes 374 Practical Issues 380 15 Appendix 382 General Safety Information 383 Waste Electrical and Electronic Equipment WEEE Directive 389 Radio Interference 3...

Page 10: ...2D LC User Guide 10 1 Introduction Product Description 11 Features 12 Terms related to 2D LC 14 This chapter describes the product of Agilent 1290 Infinity II 2D LC Solution...

Page 11: ...solved in one dimension at all or good enough within a short time Comprehensive 2D LC offers unmatched peak capacity for complex samples or sample matrices 2D LC can be used for desalting samples for...

Page 12: ...dimension are completely independent by using Agilent multiple heart cutting valves for highest storage capacity for up to 12 cuts at one time fast and parallel analysis High resolution sampling is a...

Page 13: ...stem can be prepared for a run by interactively and automatically flushing both dimensions flow paths and all sample loops The progress of a 2D run including parking of cuts in sample loops of deck va...

Page 14: ...nal chromatography which provides two dimensional data Two dimensional data is data that has a first and a second dimension For example 2D results can have chromatograms and peaks in each dimension A...

Page 15: ...22 Comprehensive 2D LC LCxLC 30 Triggering of 2D LC 32 Concept of Peak Triggering 32 Concept of Time Triggering 34 Active Solvent Modulation ASM 35 Introduction to Active Solvent Modulation ASM 35 Op...

Page 16: ...and separates it by 1D column A 2D LC Valve Injector connects the first dimension to the second dimension and stores sample peaks intermediately These sample peaks are re injected to the second dimen...

Page 17: ...C LC LC In LC LC the total eluent from the first dimension is injected on to the column in the second dimension Heart cutting 2D LC LC LC In LC LC only parts of the eluent from the first dimension are...

Page 18: ...iency Typically longer columns with higher separation efficiency are used in 2 D column NOTE Heart Cutting 2D LC LC LC is the method of choice if the samples to analyze are known or to improve confide...

Page 19: ...tandard heart cutting techniques is that peaks cannot be sampled while a second dimension gradient is still running In the examples shown here the gradient from the second dimension is analyzing the f...

Page 20: ...multiple heart cutting 2D LC Here the sampling loops on the 2D LC valve are exchanged with 6 position 14 port selection valves which are equipped with six loops each In this configuration a peak can b...

Page 21: ...n Sampling 2D LC Peaks that are cut out and stored during a run are analyzed consecutively in the second dimension even when the first dimension is still running To avoid carry over the peaks are anal...

Page 22: ...As long as the second dimension is free any next cut from the first dimension will be always directly transferred to the second dimension and analysed This means The first 1 D cut will be always dire...

Page 23: ...ns either if the peak end is detected signal falls below threshold or slope or if the settable cut size has been exceeded whatever comes first The purpose of the cut size is delaying the parking such...

Page 24: ...on the right side of the bar If you want to move the cut you can do it graphically by moving the bar with help of the mouse or you can change the start time in the sampling table You can find more in...

Page 25: ...p for consecutive snips stores the same sample volume First and last loop cannot be used for parking Solvent transfer from 1D to 2D can be reduced Cut number 5 cannot be parked entirely in the sample...

Page 26: ...ng example for HiRes sampling In High Resolution sampling the first loop is a bypass position When switching to the second loop for the first cut unknown content may be parked in the first loop which...

Page 27: ...High Resolution Sampling 2D LC Cut number 5 cannot be parked entirely in the sample loop otherwise cut 6 would go partially to the transfer capillary and would therefore be lost or spoil cut 5 Cut 5 s...

Page 28: ...Guide 28 For parking cut 6 into the sample loop the cut first needs to be moved from the 2D LC Valve to the deck valve Cut 7 will be parked in loop B2 Last loop is required for flow through while oth...

Page 29: ...le loop corresponding to a loop filling below 80 Because of the parabolic flow profile a filling greater than 80 will cause samples going to waste The minimum cut size is given by the transfer volume...

Page 30: ...While the first loop is being filled in the first dimension the contents of the second loop is analyzed in the second dimension the switching valve then switches the second loop into the first dimens...

Page 31: ...ent of the first dimension is injected onto the column in the second dimension using two sampling loops alternatingly by switching a modulation valve This will be repeated from the start to the end of...

Page 32: ...olumn Further peaks eluted from the 1 D column during the second dimension gradient time are ignored Figure 9 Principles of peak triggered LC LC Relevant parameters for peak triggering Concept of Peak...

Page 33: ...switches under the following conditions whichever comes first If the Sampling time has elapsed or Figure 11 Peak triggering concept elapsed sampling time If the signal falls below threshold or slope F...

Page 34: ...Guide 34 Concept of Time Triggering Time triggered LC LC One or more parts of the first dimension in given time frames are directly injected onto the 2 D column Figure 13 Principles of time triggered...

Page 35: ...tes the content of the sampling loop sample and 1 D solvent with weak 2 D solvent before it reaches the 2 D column and therefore improves the separation in the second dimension see Figure 17 on page 3...

Page 36: ...D Bonus RP 50 x 2 1 mm 1 8 m H2O acetonitrile gradient 0 2 formic acid weak solvent 3 acetonitrile 400 L min EICs from conventional 2D LC undiluted Figure 15 Analysis of pesticides using a HILIC sepa...

Page 37: ...capillary in parallel flow path schematic view 1D Solvent in the sample loop is partially diluted by 2D solvent from the 2 D pump Introducing a parallel flow through an ASM capillary strongly dilutes...

Page 38: ...flow path looks inside the 2D LC valve ASM The flow coming from the 2 D pump splits up at valve port 10 One part goes through the sample loop in deck A and carries parked sample cuts and 1 D solvent...

Page 39: ...e with sample loop flow path Once the ASM phase has finished which is a settable method parameter the analytical gradient starts As opposed to a dilution with a permanent by pass the ASM capillary is...

Page 40: ...2 Concepts of 2D LC Active Solvent Modulation ASM 2D LC User Guide 40 Figure 22 Switching cycle of the ASM valve countercurrent mode...

Page 41: ...two additional positions in step 2 and 4 In both steps the ASM capillary is in the second dimension and dilutes solvent in deck A and B respectively Table 1 Switching cycle position names in the softw...

Page 42: ...tion schematic view The flow rates F through these parallel capillaries depend on the different backpressures p of the capillaries in use The backpressure of a capillary depends on the capillary lengt...

Page 43: ...kit see note above one can simplify the formulae for the calculation of split ratio and ASM factor as follows NOTE Usage of the ASM capillary kit results in the following situation The capillaries in...

Page 44: ...e Otherwise increasing the modulation time may require reducing the 1 D flow rate to fill the same sample loop volume This would change 1 D chromatography The ASM solution requires backpressure from c...

Page 45: ...e Languages 50 PC Requirements 51 Licensing 52 This chapter provides information about installation and execution prerequisites regarding hardware firmware and the operating system Agilent 1290 Infini...

Page 46: ...nt folder which LC modules are supported This software has been tested successfully with 12 LC modules Please note that complex systems can increase memory consumption in MassHunter which may decrease...

Page 47: ...2D LC User Guide 47 3 Compatibility Matrix Supported Drivers Supported Drivers Table 2 Supported drivers Firmware MassHunter Version LC and CE Driver Version A B C D 7 34 11 or higher 3 4 or higher...

Page 48: ...re the same as for the corresponding Agilent MassHunter CDS revision Windows 10 Professional 64 bit 1909 Windows 10 Enterprise 64 bit 1809 Not shipped by Agilent Windows 10 LTSC 64 bit 1809 Not shippe...

Page 49: ...dentifier and XXX FW re vision NOTE Agilent releases LC firmware updates for so called firmware sets All Agilent LC instrument firmware sets have been designed and tested to be truly and strictly back...

Page 50: ...Available Languages The embedded Agilent 2D LC Software is available in English and has been tested with English versions of operating systems and CDSs NOTE Not all CDSs support all available languag...

Page 51: ...recommended For further details see the respective recommendations and instructions in the software documentation Table 4 PC Requirements Type Requirement PC RAM 32 GB Min except 64 GB for 6546 Hard...

Page 52: ...tation of the corresponding software There it is described how a license is generated and installed in the control panel of the software Usually the corresponding license authorization codes and or li...

Page 53: ...Workstation 93 This chapter describes the hardware and software installation of the Agilent 1290 Infinity II 2D LC Solution The 2D LC instrument can be used with the software described in this documen...

Page 54: ...asy Starter Kit Internal part not orderable G1680 63721 Network LAN Switch 5500 1300 Capillary ST 0 12x85 M M ASM 5500 1301 Capillary ST 0 12x170 M M ASM 5500 1302 Capillary ST 0 12x340 M M ASM 5500 1...

Page 55: ...x 105 mm SL SL 2 5500 1245 Capillary ST 0 17 mm x 400 mm SI SI 2 5500 1251 Capillary ST 0 12 mmX 400 mm SL SL NOTE Depending on the set up of you instrument extra parts and capillaries might be requir...

Page 56: ...I Bio Multisampler Column Compartment G7116B 1290 Infinity II Multicolumn Thermostat G1316C 1290 Infinity Thermostatted Column Compartment Detector G7117A B C 1260 1290 Infinity II Diode Array Detecto...

Page 57: ...SM functionality MHC Valves G4236A 007 G4243A 007 Multiple Heart Cutting Kit Contains two MHC valve heads G4242A 2D LC Multiple Heart Cutting Upgrade Kit Kit to upgrade MHC valves to an existing 2D LC...

Page 58: ...ector G7117A B C 1260 1290 Infinity II Diode Array Detector G7114A B 1260 1290 Infinity II Variable Wavelength Detector G7115A 1260 Infinity II Diode Array Detector WR G7165A 1260 Infinity II Multiple...

Page 59: ...supported G6160A InfinityLab LC MSD iQ supported 61xxB LC MS family requires smart card for update 61x0B to 61x0C via upgrade kit G2735N 61x5B to 61x5C via upgrade kit G4934C G6150B MS Module not supp...

Page 60: ...r both dimensions then Vial or Multisampler The sampler must be placed on top of the pumps The right stack consists of one or two column compartments and one or two standard UV detectors Depending on...

Page 61: ...alve and if selected the MHC decks are mounted on external valve drives G1170A For a SHC configuration the 2D LC valve G4236A is attached to the upper pump of the stack In case of a MHC configuration...

Page 62: ...3 self cutting tapping screws 2 Mount the valve heads on the G1170A external valve drives 3 Clamp the first external valve drive with the MHC valve on top 4 Attach the adapter profile on each of the...

Page 63: ...C In addition the Active Solvent Modulation valve G4243A is only available for the MHC configuration An overview of all available 2D LC modes can be found in Optional hardware configurations Table 5 o...

Page 64: ...require the standard 2D LC valve G4236A The valve can be conveniently attached to any Infinity II pump that is installed For a SHC configuration transfer capillaries 6a 6b are not necessary since MHC...

Page 65: ...igurations both the standard 2D LC valve G4236A and the ASM valve head G4243A are supported The valves can be conveniently attached to any Infinity II pump in the stack Depending on the valve head tha...

Page 66: ...y II High Speed Pump G7120A Agilent 1290 Infinity II Bio High Speed Pump G7132A 1290 Infinity Binary Pump G4220A See Figure 31 on page 73 2 1290 Infinity 1290 Infinity Quaternary Pump G4204A 1290 Infi...

Page 67: ...ion achieved For the standard 2D LC Valve both concurrent and countercurrent operation is possible Schematics in this chapter will reflect a concurrent direction If you want to connect the 2D LC Valve...

Page 68: ...4 Installation Hardware Installation 2D LC User Guide 68 Figure 29 Standard 2D LC valve G4236A with MHC 1300 bar counter current...

Page 69: ...it from 1D column 1 D detector 105 x 0 17 500 x 0 12 5500 1240 5500 1157 Capillary ST 0 17x105 SL SL Capillary ST 0 12x500 SL S 4 6a transfer capillary to MHC Valve IN deck B 170 x 0 12 5500 1270 Capi...

Page 70: ...current configuration of the ASM Valve For the concurrent setup please refer to concurrent configuration of the ASM 2D LC Valve in the 2D LC Software You find the Valve topology configuration screen...

Page 71: ...dware Installation Figure 30 Schematic representation of the ASM 2D LC Valve G4243A in countercurrent flow NOTE Against the example shown in the figure above for 1200 bar MHC Valves that have a differ...

Page 72: ...ease kit from 1 D column 1 D detector 105 x 0 17 500 x 0 12 5500 1240 5500 1157 Capillary ST 0 17x105 SL SL Capillary ST 0 12x500 SL S 5 9 from 2 D pump 280 x 0 17 5067 4608 Capillary ST 0 17x280 SX S...

Page 73: ...4 105 x 0 17 5500 1240 Capillary ST 0 17x105 SL SL 6a 4 2D LC Valve 1 Deck IN Deck Out 2D LC Valve 8 2D LC Valve 5 Deck IN Deck Out 2D LC Valve 4 170 x 0 12 5500 1270 Capillary ST 0 12x170 S M 6b 4 AS...

Page 74: ...105 x 0 17 5500 1240 Capillary ST 0 17x105 SL SL 6a 4 2D LC Valve 1 Deck IN Deck Out 2D LC Valve 8 2D LC Valve 5 Deck IN Deck Out 2D LC Valve 4 170 x 0 12 5500 1270 Capillary ST 0 12x170 S M 6b 4 ASM...

Page 75: ...17 5500 1240 Capillary ST 0 17x105 SL SL 6a 4 2D LC Valve 1 Deck IN Deck Out 2D LC Valve 8 2D LC Valve 5 Deck IN Deck Out 2D LC Valve 4 170 x 0 12 5500 1270 Capillary ST 0 12x170 S M 6b 4 ASM Valve 7...

Page 76: ...t contain separate 1 D and 2 D MCTs TCCs See Figure 35 on page 78 C Setups in which the 1 D column is hosted in an Integrated Column Compartment ICC Longer capillary 5500 1170 for Quick Connect Fittin...

Page 77: ...tion A1 2 Adapter capillary 2 to column switching valve Port IN Adapter column switching valve Port OUT to capillary 3a 75x0 12mm G1316 87326 SST Capillary 75x0 12mm f m ns 0 8 must be purchased separ...

Page 78: ...mber of Capillary Connection L x ID mm P N Description 3b 1 1 D column to 1 D DAD 280 x 0 12 5067 4651 Capillary ST 0 12x280 SL SX 8 1 2 D column in 2 D MCT to 2 D DAD 280 x 0 12 5067 4651 Capillary S...

Page 79: ...0 12x105mm 5500 1238 Capillary ST0 12x105 SL SL provided with ICC C2 1 Heat exchanger out to column InfinityLab Quick Connect Fitting 0 12x280mm 5500 1170 Capillary ST 0 12x280 must be purchased sepa...

Page 80: ...list of supported valves can be found in Table 8 on page 66 More information is available in the following sections Method Parameters on page 125 Run the System on page 192 Figure 37 Setup D Recommen...

Page 81: ...tector to T piece 0 12 x 400 5067 4606 Capillary ST 400x0 12 S SH D2 1 T piece 0100 0969 1 16in Tee SST Low Dead Volume D3 1 Capillary from MS to T piece self cut 0 12 x 400 0890 1915 Capillary PEEK 0...

Page 82: ...400 SL SL 8 1 1 D MCT TCC to 1 D DAD 280 x 0 12 5067 4651 Capillary ST 0 12x280 SL SX E1 2 1 D column to 1 D 2 D Switching Valve 1 2 D column to 1 D 2 D Switching Valve 3 120 x 0 12 5067 4652 Capillar...

Page 83: ...illary ST 0 12x280 SL SX F1 2 1 D column to 1 D 2 D Switching Valve 1 2 D column to 1 D 2 D switching valve 3 120 x 0 12 5067 4652 Capillary ST 0 12x 120 SX SX F2 1 Connection 1 D 2 D switching valve...

Page 84: ...7x105 SL SL Capillary ST 0 12x500 SL S 4 Sample Loop IN 5004 0036 180 L Loop 2D LC as an example 5 Sample Loop OUT 5004 0036 180 L Loop 2D LC as an example 6 1 To 2 D column 0 12 x 400 5500 1251 Capil...

Page 85: ...Capillary ST 0 17x105 SL SL Capillary ST 0 17x150 SL SL Capillary ST 0 17x400 SI SI To 2D LC valve Valve port 3 From DAD cell out To damper capillary T piece Parts required p n Description 1 G4236 60...

Page 86: ...to install the valve head and connecting capillaries see Replace Valve Heads G1170A on page 327 3 Insert the pressure release assembly to the leak tray orientation as shown 4 Push the pressure release...

Page 87: ...are using For further details please refer to the CDS documentation In general however the following applies Default License MassHunter License for details please refer to the MH documentation Additio...

Page 88: ...ense is activated and you can remove the USB Dongle and store in a safe place Deactivate the License Deactivation Steps in LabAdvisor For the deactivation of the license on the 2D LC pump e g you want...

Page 89: ...st critical updates and security fixes run Windows Update Make sure that Windows Update is completed before you continue 1 Install the Data Acquisition program 2 Install the Qualitative Analysis progr...

Page 90: ...d User Guides The small selection of TOF and Q TOF documents listed helps to get further acquainted with the MassHunter software MassHunter Workstation Software Installation Quick Start Guide This gui...

Page 91: ...intenance Guide animated Use this animated guide to help maintain and troubleshoot your 6200 Series TOF and 6500 Series Q TOF LC MS MassHunter Workstation Administration Guide This guide includes admi...

Page 92: ...lent YouTube channel at https www youtube com user agilent To access Agilent University visit http www agilent com crosslab university to learn about training options which include online classroom an...

Page 93: ...the most instrument settings like the IP Addresses are already defined 1 Open the Control Panel 2 Double click the Configure Instrument tool OR Right click and select Configure Instrument 3 Select Mod...

Page 94: ...the instrument use the Instrument Configuration dialog OPTIONAL a To change the name of the instrument type a new Instrument name b To configure the LC instrument click Device Config Figure 43 MassHun...

Page 95: ...44 Auto Configuration window of a full 2D LC solution with two MHC valves and a diverter valve 1 Check Select 2D LC System in Cluster Options 2 Uncheck the 1 D pump in Available Modules 3 To create a...

Page 96: ...D pump Add and select 1 D and 2 D detectors and define the transfer volume Configure the different valves like 2D LC Valve Head MHC decks if multiple valve heads are available and diverter valve Capil...

Page 97: ...t is set for delay calculation in 2D LC OpenLab DA Plugin 2 D offset between Sample Loop and detector ensures that start and end of a cut chromatogram are aligned displayed correctly NOTE This action...

Page 98: ...ndard valve or Capillary 0 12x170 1 9 L 5500 1376 for ASM valve To define the ASM capillary e g default Capillary 0 12x170 1 9 L 5500 1301 for ASM valve ASM factor 3 6 To finish leave the 2D LC Cluste...

Page 99: ...llowing Sampler Iso Pump 1 D Bin Pump 1D MCT 2D MCT 1D DAD 2 D DAD For an example see Figure 46 on page 100 2 It is recommended to change order of column compartments and detectors Use the arrow A mea...

Page 100: ...4 Installation Software Installation 2D LC User Guide 100 Figure 46 Naming in the Device Configuration for the 1 D detector Figure 47 Arrangement of the module UI in the dashboard...

Page 101: ...ommunication you have to select the detector in the UI and click Additional connection Then type in the second LAN address and check Use auxiliary connection Figure 48 Set up another LAN Connection fo...

Page 102: ...ide 102 5 If instrument is configured successfully click OK Figure 49 Successful Instrument Configuration of a 2D LC Q TOF instrument NOTE If you want to change the 2D LC cluster configuration later r...

Page 103: ...D LC User Interface 108 2D LC Valves Online Monitor in the 2D LC User Interface 114 Method Editor Window 116 Sample Run Window 117 Worklist Window 118 Tune Window 119 Instrument Details 120 Use Module...

Page 104: ...online icon OR To start the data acquisition double click the Control Panel icon When you first start the Data Acquisition software the main window appears Preparations To start your instrument you ne...

Page 105: ...ing Set up acquisition methods Run samples interactively or automatically Monitor instrument status and monitor runs Figure 50 Overview of windows that are available in MassHunter Workstation Data Acq...

Page 106: ...board is the common UI element for instrument control The driver is responsible for hardware related features plugged in to the CDS software These are for example the following Instrument configuratio...

Page 107: ...he instrument The possible values for Status are shown in the following figure You also set nonmethod control and configuration parameters for the LC devices and the MS instrument Figure 52 Instrument...

Page 108: ...the drive of the binary pump is off Click the green On button in the UI will activate the pump Figure 54 2D LC help information Additional Information in the 2D LC User Interface The instrument dashb...

Page 109: ...status 2D LC Valve position The info represents the current 2D LC Valve status In the current setup an ASM Valve Position 1 5 is installed see Connecting the 2D LC Valve ASM G4243A on page 70 Deck A...

Page 110: ...ays the pump s Control dialog box Method Displays the pump s Method Setup dialog box only visible in OpenLab and ChemStation Set Error Method Sets the method that is loaded if an error occurs to the m...

Page 111: ...uction in the technical note Best Practices for Using an Agilent LC System Prime If conditioning for 15 min cannot remove air from the pump heads the Prime function can help The module draws 20 times...

Page 112: ...s the configuration of the sample loop transfer capillaries and ASM capillaries Figure 58 Modify Transfer Volumes windows allows the configuration of the transfer volume Modify Transfer Volumes Displa...

Page 113: ...r volume possibility for the 2 D detector defines the volume between the 2D LC valve and the second dimension detector flow cell This is the result set for delay calculation in 2D LC OpenLab DA Plugin...

Page 114: ...displays the status of the 2D LC valve The following illustrations show some examples so that you can see what is happening at any time during operation Figure 59 No sampled parked cuts mobile phase t...

Page 115: ...D LC Valves Online Monitor in the 2D LC User Interface Figure 62 Flush indicated by red beam moving along Figure 63 Hovering over analysis loop indicates time passed and time remaining in seconds Figu...

Page 116: ...de 116 Method Editor Window In the Method Editor window you enter acquisition parameters for the method see Method Parameters on page 125 NOTE To decouple the Method Editor from UI double click the Me...

Page 117: ...5 2D LC Data Acquisition in MassHunter Workstation 11 Sample Run Window Sample Run Window In the sample run window you enter sample information to run individual samples interactively and you can star...

Page 118: ...18 Worklist Window With the worklist window you enter sample information for multiple samples When you run the worklist the samples are automatically run in the order listed in the worklist You can ad...

Page 119: ...of the automated tuning algorithms or you can manually tune the instrument Manual tuning can result in a less than optimal tune however if you perform a manual tune Agilent recommends that you only m...

Page 120: ...to obtain this information exist Use Module List to Obtain Instrument Details on page 120 Use Instrument Configuration Report to Obtain Instrument Details on page 121 Use Module List to Obtain Instrum...

Page 121: ...a Acquisition in MassHunter Workstation 11 Instrument Details Use Instrument Configuration Report to Obtain Instrument Details 1 Start the Data Acquisition program 2 Select the Instrument Configuratio...

Page 122: ...5 2D LC Data Acquisition in MassHunter Workstation 11 Instrument Details 2D LC User Guide 122 3 Click Screen 4 Click OK...

Page 123: ...at take place in a InfinityLab LC MSD instrument Therefore there is a log file in which the processes are logged This log file provides important data for the analysis of the system View the logbook 1...

Page 124: ...on 11 2D LC User Guide 124 Configure logbook notification If you get more logbook notifications than is useful to you you can change the type of notifications that are displayed 1 Click on Filters in...

Page 125: ...8 Use the Advanced 2D Pump Settings 152 Preview 2D LC 154 Further Graphical Explanation 162 Set up a Peak Based Experiment Graphically 168 Setup 2D Gradient Graphically 173 Setup Second Dimension Grad...

Page 126: ...Stoptime on page 130 Posttime see Define the Posttime on page 132 Sampling table see Edit the Sampling Table on page 133 2D Gradient see Define the 2D Gradient on page 139 Flush Gradient see Use the...

Page 127: ...ng this mode see Heart Cutting 2D LC LC LC on page 18 In contrast to Heart Cutting which uses the continuous flowthrough principle in high resolution sampling HiRes the Multiple Heart Cutting MHC valv...

Page 128: ...ient will be repeated The parameter Modulation time is only used in the Comprehensive mode The 2D Gradient stop time reflects the maximal duration of the gradient in 2 D the smallest value is 0 01 min...

Page 129: ...nge 0 5 0 mL min This setting defines the flow in the 2nd dimension being used while 2D LC is active within 2 D time segments where mode is not equal to Off 2 To set and use idle flow select check box...

Page 130: ...lds 2 For each solvent click the down arrow and select a calibrated solvent from the drop down list You can also enter additional information for example about buffers in the adjacent field Define the...

Page 131: ...of all other modules in the system must be set to As Pump As Injector except the 1D pump module that should set the Stop Time Modus As Injector No Limit NOTE If an alert triangle pops up in the chroma...

Page 132: ...lvent composition for example after gradient elution use the post time The instrument remains in a post run state during the post time to delay the start of the next analysis 1 Check the Posttime radi...

Page 133: ...of the buttons Add Remove Clear all Cut Copy Pause For example when you are using the Add button a single cut parking event is generated In this event line you can define the different parameters lik...

Page 134: ...me Based Comprehensive Define a time based Comprehensive run LCxLC in the sampling table Start Peak based Define the Start time of a Peak based Heart cutting run MHC or HiRes in the sampling table A b...

Page 135: ...ps the cut size value in HiRes can be changed t V 80 F 40 L Sample Loop 0 8 0 6 mL min 1D flow 3 2 s Cut s In MHC mode only one cut is allowed Therefore the number of cuts is unavailable In HiRes mode...

Page 136: ...ling HiRes click the down arrow Figure 74 Parameter window in the sampling table for High Resolution Sampling HiRes settings and Multiple Heart Cutting Settings MHC NOTE if you use the optimization fu...

Page 137: ...s soon as possible Selecting Delayed Analysis is delayed until there is an available time slot Selecting Ignored The cut is not analyzed 3 To specify that analysis of one or more cuts should be given...

Page 138: ...lute time range where the system creates equidistant cuts Comprehensive run starts at the given time The modulation time determines the cut size 2 Enter the stop time of the comprehensive measurement...

Page 139: ...lysis time and equilibration time is the 2D LC cycle time which is shown in the Modulation 2D LC 2D Gradient section The values in the 2 D Gradient Phase are synchronized with the Analytical Gradient...

Page 140: ...to flush the sample loop during the ASM phase 1 To use ASM mark the ASM check box This action activates the Active Solvent Modulation The ASM Factor is a read only value and cannot be changed NOTE Fo...

Page 141: ...loop three times is typically enough and the recommended default Less time may be sufficient and can be verified during optimization The user interface displays how long the flushing will take NOTE Wh...

Page 142: ...n cycle in the second dimension After that time the solvent composition gradient for the next cut will be repeated Loop volume The Loop volume represents the configured sample loop volume NOTE The inf...

Page 143: ...d time after the start of the 2 D analysis and switched back to waste when the 2 D analysis has finished Set up the Gradient Time Table for the Analytical Gradient Use this section to set up the eluen...

Page 144: ...ear All Cut Copy Paste Clicking e g the Add button generates a single analytical gradient event where you can define the time for the change and the solvent composition NOTE To visually verify the Ana...

Page 145: ...olvent composition in the Time min field The shifted gradient composition changes linearly from one setpoint to the next Change the solvent composition at a specified time The time axis relates to the...

Page 146: ...shifted 2D gradient click one of the following buttons Add Remove Clear All Cut Copy Paste Clicking e g the Add button generates a single gradient shift event where you can define the time for the cha...

Page 147: ...o button Customize flush gradient and use the table to set up the custom gradient a Specify the duration time in the Analysis duration field The equilibration value is read only and is defined in the...

Page 148: ...box is selected the peak trigger settings are used to trigger the sampling and parking of cuts from the first dimension This means that the areas of interest must be predefined by the method See sampl...

Page 149: ...e 2D LC Valve is triggered and switches to cut the fraction The 2D LC Valve will switch to the next position when the UV signal falls below this value or the cut size elapse OPTIONAL b Or set the Slop...

Page 150: ...LC Valve stops cutting the fraction by switching the valve to the next position For more complex problems like two overlapping peaks it is possible to combine slope and threshold collection The two pe...

Page 151: ...d This method detects peaks based on upper threshold values only The height of the peak ensures that collection is not switched off even for a saturated signal that might be expected to do so When the...

Page 152: ...olvent flow to protect your analytical column For the G4220A B Binary Pumps and G7120A High Speed Pump you can set individual values for Flow ramp up and Flow ramp down OPTIONAL 2 Select the Required...

Page 153: ...rotects the system against overpressure Min is the minimum pressure limit at which the pump will switch itself off This situation can occur when a solvent reservoir is empty The minimum pressure limit...

Page 154: ...lbar The right window which can be toggled on and off shows either the 2 D gradient profile or the flush gradient profile Both gradient profiles can be edited interactively The Preview is synchronized...

Page 155: ...Guide 155 6 Method Parameters Preview 2D LC Figure 78 Selection drop down menu to select shift volume or shift time Displays a data file selection box that allows you in the next steps to select a 1 D...

Page 156: ...s recorded must be maintained NOTE If further 1D detectors are used as reference chromatogram the transfer volume must be corrected for these detectors To do so enter the shift volume or the shift tim...

Page 157: ...e MHC This tool can only be used if heart cutting is selected in the 2D LC Operation mode The function is used with the Add Delete or Sample all function This tool switches to High Resolution Sampling...

Page 158: ...n of this function Cancel this activation by repeated double clicking the tool To remove a single cut from the reference chromatogram and the sampling table mark the single cut and then click the tool...

Page 159: ...he real run time The same task can be achieved by double clicking the vertical stop line in the preview This tool resets all zoomed graphics to their normal magnification Zoom out For zooming in press...

Page 160: ...ers Preview 2D LC 2D LC User Guide 160 Figure 82 Preview of the display of the analytical 2 D gradient in purple and the flush in orange NOTE If the 2D gradient view is activated in the main window th...

Page 161: ...ient preview in the main panel is deactivated For manually changing the threshold setting in the preview see Using this tool will toggle the display of the 2 D analytical gradient panel at the right o...

Page 162: ...me In the following example the Stoptime is too short to analyze all cuts Therefore you must change the stop time to 57 min as indicated by 2 D gradients For example use the optimization tool or doubl...

Page 163: ...valve and the end of parking the peak Cuts can be marked and moved to another position in the preview window HiRes cuts time based HiRes cuts time based are visualized and marked as light green bars...

Page 164: ...MHC cuts HiRes cuts peak based are displayed graphically in blue bars Hovering over the bars gives you the option to move the HiRes Sampling Move cut You can Increase or decrease the cut size Grab th...

Page 165: ...s gives you the option to increase or decrease the cut series indicated by green highlights You can resize by Clicking the highlighted series and dragging the edge along Dragging one of the inner edge...

Page 166: ...D gradient i e time of analysis is indicated Setup 2 D gradient graphically The initial 2D gradient in the Analytical Gradient preview by double click purple line adds a purple ball which can be move...

Page 167: ...y the name of the Y axis in B Threshold If the threshold is activated you can grab this line and shift up and down to adjust the threshold This measure will also update Peak Trigger settings If you ho...

Page 168: ...eached peak start the 2D LC modulator starts sampling A sampling time can be defined which determines the max sampling time for peak based cuts If peak end is detected before the sampling time has fin...

Page 169: ...this example G7117A and Signal A are selected 4 Select MHC or HiRes The icon corresponds to the peak based operation In this example HiRes is selected 5 Double click one of the grey triangles located...

Page 170: ...based event in the preview you can also use the add icon or do a right click somewhere into the preview and press add cut In the sampling table you can add the events start and end peak based see Use...

Page 171: ...must be adjusted To adjust the stop time the stoptime button must be clicked pressed Then the Stop time has been prolonged to ensure that all predicted cuts can be 2D analyzed Figure 86 Cut 3 4 and 5...

Page 172: ...ide 172 Figure 88 Example shows result of displayed in the MassHunter Qual 1D Chromatogram Cut markers NOTE This example is based on a prediction For experiments with unknown outcome you have to add a...

Page 173: ...purple line Figure 89 Analytical gradient This adds a gradient point to the line purple point This gradient point can be moved To add annother gradient point double click on the purple line again To...

Page 174: ...activates the threshold line To adjust the threshold grab this line and shift up or down This will update the peak trigger settings If you hover over the threshold line the slope is also displayed at...

Page 175: ...on the line in the Analytical Gradient window you can set more anchor points to adjust the analytical gradient even better Gradient Shift 1D Time Shifted 2 D gradient The setup of the shifted gradien...

Page 176: ...overing over cycle time and or time for ASM flush out displays actual time in second Three digits for comprehensive mode More Info 2D LC Valve Online monitoring Hovering over analysis loop indicates t...

Page 177: ...t this leads to an increased number of 2 D cycles Multi Inject allows to define a HiRes group to being injected at once which means the content of the loops is transferred to the 2 D column before a s...

Page 178: ...ution sampling 5 cuts with only one analytical gradient for all cuts NOTE Multi Inject works similar to an injection from a large sample loop Large injection volumes can negatively affect 2 D separati...

Page 179: ...RTS will be adjusted accordingly Setup an IRTS experiment for heart cutting mode 1 Upload the chromatogram into the preview 2 Define the UV threshold peak trigger such that the expected IRTS is predic...

Page 180: ...own in sampling table For the first IRTS like in this example the value is 1 Figure 96 Parameters of the IRTS defined as peak based MHC NOTE The IRTS will be 2D analyzed unless you mark the field Refe...

Page 181: ...time based cuts automatically get the Reference Index value and are so linked to IRTS with this index Figure 97 Time based cut with Reference Index 1 and Reference Factor 2 The Reference Factor can be...

Page 182: ...ing a run can be compensated with the help of the IRTS and dynamic cut shifting The chromatograms below shows the results obtained from MassHunter Qual The top 1D chromatogram shows the original refer...

Page 183: ...ethod Parameters Additional Information Figure 99 Shift of IRTS 2 686 min to earlier RT 2 234 min which is compensated by the system Figure 100 Shift of IRTS 2 235 min to later RT 2 686 min which is c...

Page 184: ...2 2D LC Online Monitor in the user interface Modulation Information Hovering over Cycle time and or time for ASM flush out displays actual time in second 3 digits for comprehensive mode 2D LC Valve On...

Page 185: ...ulation ASM 186 Method Parameters 187 Optimize the Dilution by Using ASM Capillaries 188 Optimize the Sample Loop Flush 189 Include the ASM Phase to the 2D Gradient 190 Optimize Dilution Through Metho...

Page 186: ...optimal dilution of 1D solvents in the sample loop for best 2D resolution at lowest cycle time After switching on the ASM functionality see Method Parameters on page 187 execute the steps in the follo...

Page 187: ...hod Parameters Advanced settings of 2D LC method parameters allow switching on and off the use of the ASM functionality If this option is off it works as a standard 2D LC valve without dilution If thi...

Page 188: ...ing best results Longer capillaries reduce shorter capillaries increase the dilution of 1D solvent in the sample loop Install and configure different ASM capillaries see Configure the ASM Valve on pag...

Page 189: ...Optimize the Sample Loop Flush Activate ASM in the software and set Flush sample loop to 3 0 times NOTE Flushing the sample loop 3 times is typically enough and the recommended default Less time may...

Page 190: ...t s why the ASM phase shifts the analytical gradient start An ASM phase of for example 0 68 min based on selected ASM capillary flush factor and 2 D flow rate shifts all times by 0 68 min compared to...

Page 191: ...before the 2D column If for example the original analytical gradient started at 20 B you may use an ASM phase of for example 2 B for diluting 1D solvent more strongly during the ASM phase by changing...

Page 192: ...e for Multiple Heart Cutting 2D LC 206 Run the Checkout Familiarization Procedure for High Resolution LC LC 210 Run the Checkout Familiarization Procedure for Comprehensive LCxLC 214 Run the Checkout...

Page 193: ...e the starter sample are delivered together with the full package to ensure a smooth familiarization and checkout procedure With the given method peaks will overlap in the first dimension and will be...

Page 194: ...w rate of the mobile phase being used Therefore it is usually worth retuning the mass spectrometer after the LC conditions have been determined 1 Perform an Auto Tune 2 To clean the source and flush t...

Page 195: ...igure the 2D LC System Prerequisites The familiarization refers to the driver based 2D LC solution The 2D LC software requires at least the following CDS versions MassHunter 11 OpenLab 2 6 For further...

Page 196: ...six Sample Loops 2D LC ASM Valve with two MHC valves each with six Sample Loops MHC or HiRes 2D LC Valve with two MHC valves each with six Sample Loops 2D LC ASM Valve with two MHC valves each with si...

Page 197: ...cessary 3 Load the given reference method 4 Check modes Heart cutting or Comprehensive and all other important parameters in the method before starting the run NOTE If you want to load and use a 1D me...

Page 198: ...912 24 9 Atrazine desethyl 006190 65 4 Chlorotoluron 015545 48 9 Diuron 000330 54 1 Hexazinone 051235 04 2 Linuron 000330 55 2 Metazachlor 067129 08 2 Methabenzthiazuron 018691 97 9 Metobromuron 00306...

Page 199: ...To prepare sample 1 1000 add 4 L 2D LC starter sample to 3996 L dilution solvent 4 mL sample 1 1000 is prepared Parts required p n Description 5190 6895 2D LC starter sample 1 x 2 mL G2453 85060 Formi...

Page 200: ...n solvent add 720 L methanol to 2880 L Mobile Phase A 3600 L dilution solvent 20 methanol in mobile phase A is prepared 2 To prepare sample 1 10 add 400 L 2D LC starter sample to 3600 L dilution solve...

Page 201: ...ommended conditions in 1D HPLC for SHC MHC and HiRes 2D LC Parameter Value 1D Column Compartment MCT Column RRHD SB C18 2 1x 100 mm 1 8 m 1200 bar 858700 902 Column temperature 40 C Stop time As pump...

Page 202: ...temperature 40 C Stop time As pump No limit 2 D Pump Heart Cutting time or peak based Mobile Phase A Water 0 2 formic acid Mobile Phase B Methanol Flow Rate 1 0 mL min Idle flow not used Stop time 40...

Page 203: ...nt Shifted Gradient Shift 1D 10 B 0 00 min 60 B 1 25 min 30 B 20 min Flush gradient not used 2D Detector DAD Diode array 254 nm Bandwidth 4 nm Reference Wavelength 360 nm Reference Bandwidth 100 nm Pe...

Page 204: ...isition Mode MS1 Min Range m z 50 Max Range m z 1000 Scan Rate spectra sec 3 00 Instrument Parameters Gas Temp 250 C Gas Flow 11 L min Nebulizer 40 psig SheathGasTemp 350 C SheathGasFlow 12 L min Scan...

Page 205: ...5190 6895 1 10 for only UV Checkout 1 100 for LCMS Checkout or 1 1000 for LCMS Checkout diluted with Methanol Water 20 80 v v with 0 1 formic acid 3 If necessary subsequently edit or optimize the met...

Page 206: ...itions in 1D HPLC for SHC MHC and HiRes 2D LC Parameter Value 1 D Column Compartment MCT Column RRHD SB C18 2 1x 100 mm 1 8 m 1200 bar 858700 902 Column temperature 40 C Stop time As pump No limit 1 D...

Page 207: ...emperature 40 C Stop time As pump No limit 2 D Pump Heart Cutting time or peak based Mobile Phase A Water 0 2 formic acid Mobile Phase B Acetonitrile Flow Rate 1 mL min Idle flow not used Stop time 40...

Page 208: ...ump No limit Table 19 Recommended conditions in 2D HPLC for multiple heart cutting Parameter Value Table 20 Recommended conditions in 2D LC MS Parameter Value Ion Source Atmospheric pressure electrosp...

Page 209: ...Water 20 80 v v with 0 1 formic acid 3 If necessary subsequently edit or optimize the method Nozzle Voltage 300 V Fragmentor 120 Skimmer1 45 OctopoleRFPeak 750 ReferenceMasses Ref Mass Enabled Enabled...

Page 210: ...ns in 1D HPLC for SHC MHC and HiRes 2D LC Parameter Value 1 D Column Compartment MCT Column RRHD SB C18 2 1x 100 mm 1 8 m 1200 bar 858700 902 Column temperature 40 C Stop time As pump No limit 1 D Pum...

Page 211: ...L min Idle flow not used Stop time 40 min will not automatically prolonged if peaks in 2D are not work off Post time 6 min Sampling Table Start 11 15 min 10 cuts Cut Size 3 2 Figure 109 HiRes The Cut...

Page 212: ...isition Mode MS1 Min Range m z 50 Max Range m z 1000 Scan Rate spectra sec 3 00 Instrument Parameters Gas Temp 250 C Gas Flow 11 L min Nebulizer 40 psig SheathGasTemp 350 C SheathGasFlow 12 L min Scan...

Page 213: ...90 6895 1 10 for only UV Checkout 1 100 for LCMS Checkout or 1 1000 for LCMS Checkout diluted with Methanol Water 20 80 v v with 0 1 formic acid 3 If necessary subsequently edit or optimize the method...

Page 214: ...parameters have been optimized for this standard configuration Parameters can deviate slightly for your system Run the experiment and subsequently edit or optimize the method for your setup Table 24 E...

Page 215: ...d Mobile Phase B Methanol Flow Rate 0 1 mL min Stop time 40 min Post time 10 min Mobile Phase Gradient 40 B 0 00 min 60 B 34 min 90 B 34 5 min Autosampler Injection Volume 2 Lfor Standard LC 0 5 L Pos...

Page 216: ...bile Phase A Water 0 2 formic acid Mobile Phase B Acetonitrile Flow Rate 2 5 mL min Idle flow not used Stop time ca 43 min will not automatically prolonged if peaks in 2D are not work off Post time 6...

Page 217: ...00 Instrument Parameters Gas Temp 250 C Gas Flow 11 L min Nebulizer 40 psig SheathGasTemp 350 C SheathGasFlow 12 L min Scan Segment 1 Scan Source Parameters Vcap 3500 V Nozzle Voltage 300 V Fragmentor...

Page 218: ...ut diluted with Methanol Water 20 80 v v with 0 1 formic acid 3 If necessary subsequently edit or optimize the method Min Height 1000 counts Reference Masses Positive 121 05087300 922 00979800 Chromat...

Page 219: ...s in 1D HPLC ASM MHC Parameter Value 1 D Column Compartment MCT Column RRHD SB C18 2 1x 100 mm 1 8 m 1200 bar 858700 902 Column temperature 40 C Stop time As pump No limit 1 D Pump Mobile Phase A Wate...

Page 220: ...1x 50 mm 1 8 m 1200 bar 857768 901 Column temperature 40 C Stop time As pump No limit 2 D Pump Heart Cutting time or peak based Figure 110 Time based Figure 111 Peak based The Cut Time MHC can vary s...

Page 221: ...Cycle time 2 12 with ASM ON and ASM Factor 3 Analytical gradient Shifted Gradient Shift 1D 3 B 0 00 min 3 B 0 37 min 10 B 0 38 min 60 B 1 62 30 B 6 min 2 D Detector DAD Diode array 254 nm Bandwidth 4...

Page 222: ...isition Mode MS1 Min Range m z 50 Max Range m z 1000 Scan Rate spectra sec 3 00 Instrument Parameters Gas Temp 250 C Gas Flow 11 L min Nebulizer 40 psig SheathGasTemp 350 C SheathGasFlow 12 L min Scan...

Page 223: ...rmic acid 3 If necessary subsequently edit or optimize the method Min Height 1000 counts Reference Masses Positive 121 05087300 922 00979800 Chromatograms Chrom Type Label Offset Y Range TIC TIC 15100...

Page 224: ...have been optimized for this standard configuration Parameters can deviate slightly for your system Run the experiment with ASM OFF and subsequently edit or optimize the method for your setup Table 3...

Page 225: ...obile Phase B Methanol Flow Rate 0 1 mL min Stop time 40 min Post time 6 min Mobile Phase Gradient 20 B 0 00 min 100 B 50 min 80 B 7 00 min Autosampler Injection Volume 2 Lfor Standard LC 0 5 L Positi...

Page 226: ...Pump Comprehensive Figure 112 Comprehensive Mobile Phase A Water 0 2 formic acid Mobile Phase B Methanol Flow Rate 2 5 mL min Stop time 40 min will not automatically prolonged if peaks in 2D are not w...

Page 227: ...z 1000 Scan Rate spectra sec 3 00 Instrument Parameters Gas Temp 250 C Gas Flow 11 L min Nebulizer 40 psig SheathGasTemp 350 C SheathGasFlow 12 L min Scan Segment 1 Scan Source Parameters Vcap 3500 V...

Page 228: ...method AutoRecalibration Average Scans 1 Detection Window ppm 100 ppm Min Height 1000 counts Reference Masses Positive 121 05087300 922 00979800 Chromatograms Chrom Type Label Offset Y Range TIC TIC...

Page 229: ...in MassHunter Acquisition 230 MassHunter Qualitative Analysis Software 234 GC Image Basic Information 256 Overview 259 Installation 260 Use GCImage Software 261 Investigate the effects of using differ...

Page 230: ...High Resolution Sampling an extra selection step is required in the data acquisition This measure will order the generated 2D cuts correctly which will facilitate the display and data analysis later o...

Page 231: ...the Method Editor start the 2D LC File Splitter Automation function Figure 113 Method Editor in MassHunter Acquisition 1 Select the check box 2D LC File Splitter Automation in Method Editor DA This se...

Page 232: ...retention time from the 1 D run In case it was forgotten to activate DA Re run sample worklist with DA Reprocessing Tool This function is automatically installed with the Acquisition Software and can...

Page 233: ...valuated with the MassHunter Qualitative Analysis Software 10 0 Figure 114 File structure in MassHunter with parent data files and corresponding folder w cuts 2D LC File d This file stores the complet...

Page 234: ...ualitative analysis methods can also be performed on 2D LC data However to make it easier to get started with 2 D data we have listed some different workflows as examples The 2D LC instrument in this...

Page 235: ...2D LC User Guide 235 9 Data Analysis 2D LC Data Analysis Data Evaluation for MassHunter 2 Right click Chromatogram Results and select Extract Other Chromatograms DAD 1...

Page 236: ...Extract Other Chromatograms 2DLC Cut marker in 1D for DAD May be automated using Method Automation Workflow NOTE By default the Agile2 integrator is chosen to integrate UV chromatograms To Integrate...

Page 237: ...D LC User Guide 237 9 Data Analysis 2D LC Data Analysis Data Evaluation for MassHunter 4 Mark DAD 1 and cut marker and press show highlighted signals button Figure 115 1 D signal overlaid w cut marker...

Page 238: ...LC Data Analysis Data Evaluation for MassHunter 2D LC User Guide 238 Workflow 1D UV data extraction Alternative 2 1 Open Data File 2D LC file d in this case Test_Comp r001 d The string of 2D TIC chro...

Page 239: ...2D LC User Guide 239 9 Data Analysis 2D LC Data Analysis Data Evaluation for MassHunter 2 Go to Actions and select Extract All non MS Chromatograms...

Page 240: ...Data Analysis 2D LC Data Analysis Data Evaluation for MassHunter 2D LC User Guide 240 3 Mark DAD 1 and cut marker and press show highlighted signals button Figure 116 1 D signal overlaid w cut marker...

Page 241: ...2D LC User Guide 241 9 Data Analysis 2D LC Data Analysis Data Evaluation for MassHunter Workflow 2D MS Data 1 Open extracted 2D cuts from cut folder 2 D TIC chromatograms appear in thew Sample Table...

Page 242: ...Evaluation for MassHunter 2D LC User Guide 242 2 Work with 2 D MS data as with 1 D data e g ESI extraction NOTE Only one cut can be highlighted in the sample table for extraction purposes highlightin...

Page 243: ...Analysis 2D LC Data Analysis Data Evaluation for MassHunter Workflow Compare 2D UV and MS data Alternative 1 1 Load the 2D LC experiment 2 Mark a single cut in Sample Table Figure 117 Sample Table Fig...

Page 244: ...or MassHunter 2D LC User Guide 244 3 Right click Chromatogram Results and Extract Other Chromatograms 2D DAD signals those with cut in their name Figure 119 Chromatogram Results Cut02 at 9 52 Figure 1...

Page 245: ...tract Chromatograms 2DLC Cut marker in 2D NOTE This cannot be automated because the name of the DAD trace has the cut in it thus cut 3 does not contain any data with a name of cut 2 NOTE Cut markers i...

Page 246: ...er Guide 246 5 DAD data can now be compared with MS traces OPTIONAL 6 In case you want to shift chromatograms for alignment of UV and MS traces use Adjust Delay Time Figure 124 Adjust Delay Time Figur...

Page 247: ...for MassHunter 7 Then the retention time for MS1 and DAD2 was entered Figure 125 Retention Time Value for Peak1 MS RT 3 047min and DAD2 2 972 min 8 By pressing the Calculate delay from RT button and...

Page 248: ...ta Analysis 2D LC Data Analysis Data Evaluation for MassHunter 2D LC User Guide 248 9 Press play button Adjust Delay Time in Data to align chromatograms Figure 127 Overlay of the aligned two chromatog...

Page 249: ...a Analysis Data Evaluation for MassHunter Workflow Compare 2D UV and MS Alternative 2 1 Load eight HiRes cuts from a 2D LC High Resolution experiment Figure 128 Files with results of the eight HiRes c...

Page 250: ...assHunter 2D LC User Guide 250 Figure 130 HiRes experiment 2 To extract the same EIC s across all cuts highlight the EIC s and use the Use Highlighted Chromatograms Extract from Data Files function Fi...

Page 251: ...2D LC User Guide 251 9 Data Analysis 2D LC Data Analysis Data Evaluation for MassHunter Figure 132 Highlighted chromatograms Extract From Data Files function...

Page 252: ...n for MassHunter 2D LC User Guide 252 Figure 133 Extracted EIC chromatograms from all HiRes cuts NOTE The Use Highlighted ChromatogramsExtract from Data Files function is also accessible by right clic...

Page 253: ...ser Guide 253 9 Data Analysis 2D LC Data Analysis Data Evaluation for MassHunter 3 Mark ALL cuts in Sample Table As with 1D data under Actions select Extract All Non MS Chromatograms Figure 134 Select...

Page 254: ...2D LC Data Analysis Data Evaluation for MassHunter 2D LC User Guide 254 4 In the Data Navigator highlight the data to compare and click show highlighted signals Figure 135 Comparison of Extracted EIC...

Page 255: ...yed is relative to 1D time scale i e displayed when they were analyzed This means that the original retention time from the DA of the acquisition method is retained see Presets in MassHunter Acquisiti...

Page 256: ...omprehensive 2D LC data special software is recommended Agilent recommends GC Image LCxLC edition Software from GC Image LLC Nebraska USA A trial download can be found on www GCImage com as well as an...

Page 257: ...257 9 Data Analysis GC Image Basic Information Figure 138 Display of two dimensional LC data with a one dimensional data analysis software Figure 139 2D LC plot of the optimized separation of 26 polyp...

Page 258: ...display of the separation of the 26 compound standard mixture The first dimension separation takes 40 minutes and each second dimension separation takes 39 seconds The back side shows a generated fir...

Page 259: ...C Edition short GC Image is a software for visualization and data analysis of full comprehensive two dimensional liquid chromatograms M8700AA GC Image LCxLC Edition for UV and Single Quad measurements...

Page 260: ...V only detection 2 Double click the chosen executable and follow the instructions on the screen 3 Activate the software with the USB key Insert the USB dongle and wait The driver will install automati...

Page 261: ...ware are the following Import 2D ChemStation data files Setting the modulation period Choosing a color mapping Navigate in the display Navigate in the display Detect peaks Blobs Basic knowledges 1 Sta...

Page 262: ...9 Data Analysis GC Image Basic Information 2D LC User Guide 262 2 Import the UV signal from the second dimension detector...

Page 263: ...2D LC User Guide 263 9 Data Analysis GC Image Basic Information 3 Import parameters...

Page 264: ...9 Data Analysis GC Image Basic Information 2D LC User Guide 264 4 Fit view...

Page 265: ...2D LC User Guide 265 9 Data Analysis GC Image Basic Information 5 Correct Baseline 6 Shift phase 7 Zoom into an interesting region by using the right mouse button and dragging over the display...

Page 266: ...colors LC Image offers refined possibilities for optimizing the color scales Play around with settings for improving the contrast 9 Select a data range 10 By clicking the Show 3D perspective button or...

Page 267: ...2D LC User Guide 267 9 Data Analysis GC Image Basic Information 11 View single 2D chromatograms 12 Select blobs...

Page 268: ...a is very similar to those of UV measurements Additionally you can for example filter to a certain mass range range limit that you are interested in 2 By clicking on Show 1D view you can display the T...

Page 269: ...part of the available two dimensional separation space will be occupied In such a case shifted gradients in the second dimension can be used to enlarge the accessible two dimensional separation space...

Page 270: ...ulation method with repeating gradients from 5 95 B as shown below 3 Run the comprehensive 2D LC analysis The resulting separation should look similar to the one shown below NOTE Notice how the peaks...

Page 271: ...tion in 2D a shallower 2D gradient e g from 25 75 B could be used The setup of this 2 D method is shown below this is just shown for explanation you do not need to run this method The separation resul...

Page 272: ...not eluted in one modulation cycle from the second dimension column wrap around see marked area To be able to use even shallower gradients in the second dimension to further improve the separation and...

Page 273: ...se shift the second dimension gradients For this purpose keep the valve loop configuration as well as the 1D pump method the same In Instrument Setup 2D LC set up a 2D pump and modulation method with...

Page 274: ...e stepwise shifting of the second dimension gradients is performed at times when no peaks are eluting from the first dimension column In case your resulting separation looks different from the one sho...

Page 275: ...nostic 286 Decluster the 2D LC Cluster 286 Pump Head Leak Test for the 2D Pump 287 Pump Leak Rate Test for the 2D Pump 288 System Pressure Test for the 2D Pump 289 2D LC Capillary Leak Test 291 Replac...

Page 276: ...of the Module s Indicators and Test Functions 2D LC User Guide 276 Overview of the Module s Indicators and Test Functions For an overview of the module s indicators and test functions refer to the man...

Page 277: ...vary The preferred tool for troubleshooting and diagnostics should be Agilent Lab Advisor Software see Agilent Lab Advisor Software on page 278 The current Agilent OpenLab ChemStation Agilent OpenLab...

Page 278: ...included with every Agilent 1200 Infinity Series and Agilent InfinityLab LC Series instrument The Lab Advisor Advanced features can be unlocked by purchasing a license key and include real time monito...

Page 279: ...tures which can be used to get more details and information out of your 2D LC System For further details like the diagnostic buffer the module info purge pump etc please check the manuals of each modu...

Page 280: ...e De activate the license on the current module e g to transfer the license to a different pump module Parts required p n Description 5017 0006 USB Dongle Software required Agilent Lab Advisor Softwar...

Page 281: ...2D LC License is active Figure 142 2D LC Mode is active The hardware dongle is installed The 2 D pump is configured as a 2D LC cluster The 2D LC solution if ready for use The 2D LC License is inactive...

Page 282: ...le to the 2 D pump USB socket 2 In the Lab Advisor Software select Special command from the menu 3 Click the Remove 2D LC License button Figure 144 2D LC License Dongle Status information 2D LC licens...

Page 283: ...ample Loop Generic Transfer Capillary and Generic ASM Capillary to the fields Figure 145 2D LC generic capillaries configuration The Volume L of the specified capillaries is automatically calculated 3...

Page 284: ...ree valves One 2D LC valve Two MHC valves Figure 146 Example of a 2D LC instrument with a 2D LC ASM valve and two MHC valves When Control the behavior of the 2 D pump and the 2D LC valves Software req...

Page 285: ...Advisor Instrument Control 4 To switch the position of the valve select the required Position from the drop down list Figure 147 Example of a 2D LC instrument with a selected MHC valve Deck A Figure...

Page 286: ...ear clustering configuration data press the Run button Figure 149 Firmware Declustering of the 2D LC 1290 binary pump When Replacement of one of the cluster partners Software required Agilent Lab Advi...

Page 287: ...t the 2 D pump 3 Select the Pump Head Leak Test Figure 150 Pump Head Leak Test for the 2D LC 1290 binary pump 4 Press the Run button and follow the instructions in the software When Diagnostic of the...

Page 288: ...stic from the menu 2 Select the 2 D pump 3 Select the Pump Leak Rate Test Figure 151 Pump Leak Rate Test for the 2D LC 1290 binary pump 4 Press the Run button and follow the instructions in the softwa...

Page 289: ...Select Service Diagnostic from the menu 2 Select the 2 D pump When Leaks in the system flow path Tools required Description Wrench 1 4 1 5 inch Parts required Description Blank nut Software required A...

Page 290: ...agnostics Lab Advisor Service Diagnostic 2D LC User Guide 290 3 Select the System Pressure Test Figure 152 System Pressure Test for the 2D LC 1290 binary pump 4 Press the Run button and follow the ins...

Page 291: ...ect the 2 D pump 3 Select the 2D LC Capillary Leak Test 4 Press the Run button and follow the instructions in the software When Leak in the 2D LC valve Tools required Description Wrench 1 4 1 5 inch P...

Page 292: ...alidated revision or if a new module with newer firmware is added to a system or if third party control software requires a special version Tools required Description Agilent Lab Advisor software Part...

Page 293: ...d whid 69761 2 For loading the firmware into the module a Select the folder on the hard drive where the Firmware package is stored b Connect the Lab Advisor Software to your 2D LC instrument Figure 15...

Page 294: ...whether the cause of the error is either The application method or The 2D LC instrument For a recommended approach to isolate the cause of the issue see the graphic below All examples use symbols as d...

Page 295: ...ing Figure 154 Example for a strategy to identify the application method or instrument as root cause for issues in 2D LC chromatography After ruling out the application method as the cause of the issu...

Page 296: ...long with the location of each problem s respective troubleshooting procedure are listed below Pressure too high on page 297 Pressure too low on page 298 Peak area and peak height related on page 299...

Page 297: ...er Guide 297 10 Troubleshooting and Diagnostics The Basic Principle of Troubleshooting Pressure too high Figure 155 Example for a strategy to eliminate issues related to too high pressure in 2D LC ins...

Page 298: ...bleshooting and Diagnostics The Basic Principle of Troubleshooting 2D LC User Guide 298 Pressure too low Figure 156 Example for a strategy to eliminate issues related to too low pressure in 2D LC inst...

Page 299: ...de 299 10 Troubleshooting and Diagnostics The Basic Principle of Troubleshooting Peak area and peak height related Figure 157 Example for a strategy to eliminate issues related to peak problems in 2D...

Page 300: ...eshooting and Diagnostics The Basic Principle of Troubleshooting 2D LC User Guide 300 Retention time related Figure 158 Example for a strategy to eliminate issues related to retention time in 2D LC in...

Page 301: ...e 301 10 Troubleshooting and Diagnostics The Basic Principle of Troubleshooting Missing signal linearity Figure 159 Example for a strategy to eliminate issues related to missing signal linearity in 2D...

Page 302: ...ubleshooting and Diagnostics The Basic Principle of Troubleshooting 2D LC User Guide 302 Drifting signal Figure 160 Example for a strategy to eliminate issues related to drifting signal in 2D LC instr...

Page 303: ...LC User Guide 303 10 Troubleshooting and Diagnostics The Basic Principle of Troubleshooting Signal noisy Figure 161 Example for a strategy to eliminate issues related to signal noise in 2D LC instrume...

Page 304: ...tact your local Agilent Technologies service representative Table 36 Recommended Tests for 2D LC System Troubleshooting Pump Column Compartment Autosampler Valve Detector 2D LC Instrument Pressure Tes...

Page 305: ...ation Sensor Short 311 Fan Failed 312 Leak 312 Module Specific Error Messages 313 Initialization of Valve Failed 313 Valve Switching Failed 314 Valve Tag Violation 314 Pressure Cluster Partner Missing...

Page 306: ...EDs get red and the run will be stopped Depending on the module type this stop is implemented differently For example for a pump the flow will be stopped for safety reasons For a detector the lamp wil...

Page 307: ...e Suggested actions 1 The analysis was completed successfully and the timeout function switched off the module as requested Check the logbook for the occurrence and source of a not ready condition Res...

Page 308: ...a CAN connection to the system Fix the leak in the external instrument before restarting the module 2 Leak detected in an external instrument with a remote connection to the system Fix the leak in th...

Page 309: ...one or more of the modules is no longer recognized as being connected to the system the error message is generated Probable cause Suggested actions 1 Not ready condition in one of the instruments con...

Page 310: ...perature A leak is detected when solvent cools the leak sensor causing the leak sensor current to change within defined limits If the current falls outside the lower limit the error message is generat...

Page 311: ...error message is generated Compensation Sensor Short Error ID 0080 The ambient compensation sensor NTC on the mainboard in the module has failed open circuit The resistance across the temperature com...

Page 312: ...from the two temperature sensors leak sensor and board mounted temperature compensation sensor are used by the leak algorithm to determine whether a leak is present When a leak occurs the leak sensor...

Page 313: ...the movement couldn t be performed properly or it was not noticed correctly by the sensor Probable cause Suggested actions 1 Mechanical problems Friction too high or blockages on the valve drive s mot...

Page 314: ...ry to identify the source of trouble by installing a different valve head if possible Contact your Agilent Service representative 2 Defect Sensor on the Valve Drive Motor Check valve head for correct...

Page 315: ...ted actions 1 Communication issues Check the CAN cable connections of the modules 2 Configuration mismatch Check and correct if necessary the valve configuration and presence of defined pressure clust...

Page 316: ...uggested actions 1 Communication issues Check the CAN cable connections of the modules Check if the hosted module is present 2 Configuration mismatch Check if the firmware on the entire stack is out o...

Page 317: ...the Module 322 Correcting Leaks 323 Correcting Leaks G7116B 323 Correcting Leaks G1170A 323 Replace Valve Heads 324 Replace Valve Heads G7116B 324 Replace Valve Heads G1170A 327 Replacing Parts of th...

Page 318: ...to Maintenance The 2D LC solution is designed for easy maintenance The most frequent maintenance can be done from the front with the modules in place in the system stack Examples are maintenance of t...

Page 319: ...r use of the products in violation of applicable laws rules or regulations Use your Agilent products only in the manner described in the Agilent product user guides WARNING Electrical shock Repair wor...

Page 320: ...sheet supplied by the vendor and follow good laboratory practice The volume of substances should be reduced to the minimum required for the analysis Do not operate the instrument in an explosive atmos...

Page 321: ...s Procedure Typical Frequency Notes Cleaning the Module If required Correcting Leaks If a leak has occured Check for leaks Maintain the Column Switching Valve If valve leaks Replace Valve Heads If the...

Page 322: ...nts for cleaning purposes They can cause damage to plastic parts WARNING Liquid dripping into the electronic compartment of your module can cause shock hazard and damage the module Do not use an exces...

Page 323: ...0A 1 Use a pipette and tissue to dry the leak sensor area 2 Observe the capillary connections and the valve for leaks and correct if required When If a leakage has occurred at the heat exchanger or at...

Page 324: ...nd storage For protecting the actuator a dummy valve head part of Transportation Lock Kit G1316 67001 can be used instead of a functional valve Do not touch parts inside the actuator CAUTION Column Da...

Page 325: ...emoving them from your valve head When working with these substances observe appropriate safety procedures for example by wearing goggles safety gloves and protective clothing as described in the mate...

Page 326: ...ad onto the valve drive such that the lobe fits to the groove 6 Screw the valve head onto the valve drive using the union nut 7 Install all required capillary connections to the valve 8 Push the valve...

Page 327: ...e head can be used instead of a functional valve Do not touch parts inside the actuator NOTE For a correct installation of the valve head the outside pin red must completely fit into the outside groov...

Page 328: ...e to turn the valve head until you feel that the two pins snap into the grooves Now you should feel additional resistance from the valve drive while continuously turning the valve head until the pin f...

Page 329: ...ser Guide 329 12 Maintenance Replace Valve Heads 3 Install all required capillary connections to the valve 4 Power on or power cycle your module so the valve head gets recognized during module initial...

Page 330: ...f required stator face in place on the stator head Reinstall the stator head 10 Insert the stator screws in the stator head Tighten the screws alternately two turns at a time until the stator head is...

Page 331: ...ator ring available for service only 5 Rotor seal 6 Bearing ring 7 Spanner nut available for service only NOTE Figure 162 on page 331 illustrates replacement parts for the valve heads with the 6 colum...

Page 332: ...r Parts required p n Description 2 2110 1486 Fuse 2 AT250 V WARNING Electrical shock Disconnect the module from line power before changing a fuse or trying to open the hatch of the power input socket...

Page 333: ...gently lift the outer plastic housing of the power inlet socket using a flat screwdriver 4 Pull out the fuse drawer as shown 5 Replace the defect fuse s 6 Slide in the fuse drawer and push till it fi...

Page 334: ...erter Valve 348 Additional Parts for the MS Diverter Valve Setup 349 Valve Options Overview for G7116B 350 Additional Heater Devices 352 Additional Heater Devices for G7116B 353 Accessories and Consum...

Page 335: ...5437 60 L Loop 2D LC 5067 5426 80 L Loop 2D LC 5004 0036 180 L Loop 2D LC 5500 1238 Capillary ST 0 12 mm x 105 mm SL SL Bypass Capillary p n Description 5067 6643 Capillary ST 0 35x104 mm M M 10 5067...

Page 336: ...illary ST 0 12 mm x 600 mm S SL 01078 87305 Capillary 0 17 mm x 80 cm male fit 5065 4454 Fitting screw long front and back ferrules 10 pk G1316 60005 Low Dispersion Heat Exchanger Double Assy G7116 60...

Page 337: ...M Capillaries ASM Valve Capillary Replacement Kit p n Description 5500 1300 Capillary ST 0 12x85M M ASM 5500 1301 Capillary ST 0 12x170M M ASM 5500 1302 Capillary ST 0 12x340M M ASM 5500 1303 Capillar...

Page 338: ...2 2 3 4 5 Item p n Description G4236 60010 2D LC Pressure Release Kit 0100 0969 TEE ST 1 16 inch Low Dead Volume Not shown 1 5021 1816 Capillary i d 0 17 mm 105 mm lg 2 5022 2184 Union stand LC flow n...

Page 339: ...rter Kit 2D LC Easy Starter Kit p n Description 5190 6895 2D LC starter sample 1 x 2 mL G2453 85060 Formic Acid Reagent Grade 5 mL 5 cc 858700 902 RRHD SB C18 2 1x100 mm 1 8 m 1200 bar 857768 901 RRHD...

Page 340: ...scription 1 5043 0275 Clamp guide For attaching the valve to a rail assembly 2 5067 4792 Leak sensor assembly External leak sensor 3 5043 0271 Holder leak plane 4 5043 0270 Leak plane 5 5068 0106 Span...

Page 341: ...Left IF II G For G7116A B Not shown 2 5067 5685 Clamp Guide Kit IF II 3 5067 4792 Leak sensor assembly External leak plane 4 5043 0271 Holder leak plane 5 5043 0270 Leak plane 6 2110 1486 Fuse 2 AT25...

Page 342: ...ion 13 port Selector valve as an example The valves can vary in their appearance and do not necessarily include all of the illustrated parts Neither every spare part is available for each flavor of th...

Page 343: ...38 Technical specifications Max Pressure 1300 bar Liquid Contacts Stainless Steel PEEK Connections Accepts 10 32 male threaded and M4 fittings p n Description 8710 2462 Hex Key Driver 3 32 inch 5023...

Page 344: ...ortant If scratches or heavy wear is visible during the inspection the stator must be replaced p n Description 1 5067 4244 2D LC Valve Head 1300 bar 1 5067 5440 Calibrated loop kit for 2D LC 1 5067 61...

Page 345: ...h 1 5500 1300 Capillary ST 0 12x85M M ASM 1 5500 1301 Capillary ST 0 12x170M M ASM 1 5500 1302 Capillary ST 0 12x340M M ASM 1 5500 1303 Capillary ST 0 12x680M M ASM 4 5500 1376 Capillary ST 0 12x170M...

Page 346: ...ce Vale Options Overview for 2D LC 2D LC User Guide 346 5067 4273 6 column selector valve head 1300 bar p n Description 5 5068 0089 Stator screws 1 1535 4045 Bearing ring 1 5068 0242 Rotor Seal PEEK 1...

Page 347: ...legacy p n Description 5068 0186 Rotor Seal Vespel 5068 0115 Stator 1535 4857 Stator screws 10 pk 1535 4045 Bearing ring p n Description 1 5067 4142 Valve head 6 column selector 1200 bar 6 5067 5926 C...

Page 348: ...4282 2 position 6 port valve head 800 bar 5067 4730 2 10 Cap kit 0 17 mm 5067 4249 2 6 Cap Kit 0 12 mm incl QC HEx 5067 4250 2 6 Cap Kit 0 12 mm incl LD HEx 5067 6597 2 6 Cap Kit 0 17 mm incl QC HEx p...

Page 349: ...high pressure valve head 1200 bar 5068 0008 Rotor Seal Vespel p n Description 5067 4118 2 position 10 port ultra high pressure valve head 1200 bar 5068 0012 Rotor Seal Vespel p n Description G4212 60...

Page 350: ...18 Port Valve 1300 bar 5068 0200 PEEK 5068 0199 5068 0089 n a 5067 4241 2 Position 6 Port Valve 1300 bar 5068 0207 PEEK 5068 0006 1535 4857 5068 0120 5067 4240 2 Position 10 Port Valve 1300 bar 5068 0...

Page 351: ...116B Valve Head Rotor Seal Stator Head Stator Screws Stator Ring 5067 4121 8 Position 9 Port Valve 1200 bar 5068 0002 Vespel 5068 0001 1535 4857 5068 0127 5067 4117 2 Position 6 Port Valve 1200 bar 50...

Page 352: ...ces For details see Table 42 on page 353 Table 41 Heat Exchanger Overview Flow rate 0 075 mm i d capillary 0 12 mm i d capillary 2 mL min Ultra low Dispersion G7116 60021 Internal volume 1 0 L Standar...

Page 353: ...and fittings Table 42 InfinityLab Quick Connect Heat Exchanger Item Description Quick Connect Heat Exchanger Standard G7116 60015 Quick Connect Heat Exchanger Ultra Low Dispersion G7116 60021 NOTE Us...

Page 354: ...cable Agilent module to module 0 5 m 5063 6527 Tubing Silicon Rubber 1 2 m ID OD 6 9 mm 5500 1191 InfinityLab Quick Turn Capillary ST 0 12 mm x 280 mm long socket 5067 5966 InfinityLab Quick Turn Fitt...

Page 355: ...ST 0 12 mm x 280 mm long socket Capillary from column outlet to DAD no fittings G7116 60006 Divider Assembly MCT For separating different temperature zones between left and right heater elements 5067...

Page 356: ...ce InfinityLab Quick Connect and Quick Turn Fittings 2D LC User Guide 356 InfinityLab Quick Connect and Quick Turn Fittings For further info check either the consumables catalog or Important Customer...

Page 357: ...y ST 0 075 mm x 150 mm 5067 6164 InfinityLab Quick Connect Assy ST 0 075 mm x 220 mm 5067 6165 InfinityLab Quick Connect Assy ST 0 075 mm x 280 mm 5067 5957 InfinityLab Quick Connect Assy ST 0 12 mm x...

Page 358: ...b Capillary ST 0 12 mm x 105 mm 5500 1172 InfinityLab Capillary ST 0 12 mm x 150 mm 5500 1171 InfinityLab Capillary ST 0 12 mm x 220 mm 5500 1170 InfinityLab Capillary ST 0 12 mm x 280 mm 5500 1179 In...

Page 359: ...nance InfinityLab Quick Connect and Quick Turn Fittings InfinityLab Quick Turn Fitting Figure 168 InfinityLab Quick Turn Fitting p n Description 5067 5966 InfinityLab Quick Turn Fitting 5043 0924 Fron...

Page 360: ...mm x 700 mm S SX 5067 4648 2 D pump to valve 1 Capillary ST 0 12 mm x 90 mm S SX 5067 4649 Valve to heat exchanger 2 Capillary ST 0 12 mm x 150 mm SL SX 5067 4650 Short column to valve 2 Capillary ST...

Page 361: ...bout training options which include online classroom and onsite delivery A training specialist can work directly with you to help determine your best options A useful Agilent Resource Center web page...

Page 362: ...364 Resolution 365 Peak Capacity 368 2D as detector 371 Successful Mode Combinations 373 Solvent Elution Modes 374 Practical Issues 380 This chapter gives the theoratical background of 2D LC and descr...

Page 363: ...ve resolution and sensitivity or to decrease analysis time Figure 169 Peak capacity relationship between peak capacities of orthogonal first and second dimension The most importand benefit of 2D LC ov...

Page 364: ...d to the second dimension Orthogonality The 2D LC separation power depends the fact that the two selectivity mechanisms in the different separation stages must be as different as possible If the mecha...

Page 365: ...1D LC Resolution RS can be described as a function of three parameters Column efficiency or theoretical plates N Selectivity Retention factor k Figure 170 Resolution equation This means that the sele...

Page 366: ...of 2 D resolution measurement Slice for resolution top and 2 dimensional contour plot bottom The distance between two spots in the contour plot may be calculated by the Pythagorean expression For the...

Page 367: ...that peaks are Gaussian Figure 173 2D resolution peak to valley ratio relation Table 44 Definitions Symbol Denotation R Resolution t Difference in retention time maxima of two components Average stan...

Page 368: ...peaks are usually not distributed randomly over the chromatogram and often overlap Or in other words In practice peaks don t fill the available separation space evenly This is the reason why the numb...

Page 369: ...aks In 2D LC the single component zone is A0 2 r2 and an area of component free space of 2r 2 As a result For every two component free widths in one dimension four component free areas are required in...

Page 370: ...the second column and detector are just a unique type of chemically selective detector of what comes out of the first column see 2D as detector on page 371 The peak width observed on the second colum...

Page 371: ...ally the 2 D of 2D LC operates like a chemically sensitive detector for the peaks that elute from the 1D column Thus 2D LC may be understood as a three step process 1D separation 1 Sampling of the 1D...

Page 372: ...cted to second column 3 Peaks of second column separation are detected and reconstructed 1st peak from 1st dimension 3rd peak from 1st dimension First dimension Second dimension 1 2 3 1 3 Peak reconst...

Page 373: ...rizes the advantages and disadvantages of combinations of normal phase NP reverse phase RP ionexchange IEC and size exclusion chromatography SEC for 2D LC operation Table 45 Mode combinations in 2D LC...

Page 374: ...focuses on the effects of elution modes for 2 D separation The following elution modes for 2 D separation are commonly used Gradient A standard gradient of solvent A vs solvent B for the second dimens...

Page 375: ...dient From each 2 D separation to the next the start B and end B values of the individual 2 D gradients will be increased in a defined way Additionally the gradient span can be increased from each 2 D...

Page 376: ...14 Theoretical Background Solvent Elution Modes 2D LC User Guide 376 Isocratic All second dimension separations will be carried out in an isocratic mode Figure 178 Isocratic mode...

Page 377: ...ocratic Nearly isocratic conditions are used in each 2 D separation with slightly increasing solvent strength in each successive run The 2D pumping system is fed with a shallow gradient in eluent comp...

Page 378: ...lution modes in the 2 D pros and conts Criterion Gradient Shifted gradient Isocratic Advancing isocratic Peak capacity Superior Inferior Diversity of samples complex samples Superior Inferior Baseline...

Page 379: ...ved in shifted gradient mode is larger than g achieved in standard gradient elution mode This can lead to an improved peak detection and improved separation See D Li and O J Schmitz Use of Shift Gradi...

Page 380: ...sm for achieving peak focusing Ratio of column diameter in the two dimensions Causes significant analyte dilution effects Goals of the analytical method Chosen parameter depend on what is important in...

Page 381: ...sufficiently phase selective Detection methods Compared to mass spectrometry DAD based UV detection is faster cheaper and offers higher reproducibility thus mass spectrometry offers additional increas...

Page 382: ...plosive Atmosphere 385 Do Not Remove the Instrument Cover 385 Do Not Modify the Instrument 385 In Case of Damage 385 Solvents 386 Safety Symbols 387 Waste Electrical and Electronic Equipment WEEE Dire...

Page 383: ...for the customer s failure to comply with these requirements Safety Standards This is a Safety Class I instrument provided with terminal for protective earthing and has been manufactured and tested ac...

Page 384: ...or EMC regulations Make all connections to the unit before applying power NOTE Note the instrument s external markings described under Safety Symbols on page 387 WARNING Missing electrical ground Ele...

Page 385: ...ble gases or fumes Explosion hazard Do not operate the instrument in the presence of flammable gases or fumes WARNING Instrument covers removed Electrical shock Do Not Remove the Instrument Cover Only...

Page 386: ...nts methanol and ethanol keep the solvent temperature at least 25 C 45 F below the boiling point Do not operate the instrument in an explosive atmosphere Do not use solvents of ignition Class IIC acco...

Page 387: ...see instrument label Flammable Material For Sample Thermostat which uses flammable refrigerant consult Agilent Information Center User Manual before attempting to install or service this equipment Al...

Page 388: ...cts that could be damaged by strong magnetic fields Indicates a pinching or crushing hazard Indicates a piercing or cutting hazard Table 48 Symbols WARNING A WARNING alerts you to situations that coul...

Page 389: ...product complies with the European WEEE Directive marking requirements The affixed label indicates that you must not discard this electrical electronic product in domestic household waste NOTE Do not...

Page 390: ...to ensure proper functionality and compliance with safety or EMC regulations Test and Measurement If test and measurement equipment is operated with equipment unscreened cables and or used for measure...

Page 391: ...2D LC User Guide 391 15 Appendix Sound Emission Sound Emission Sound pressure Sound pressure Lp 70 db A accroding to DIN EN 27779 Schalldruckpegel Schalldruckpegel Lp 70 db A nach DIN EN 27779...

Page 392: ...ate buffer which are less corrosive against stainless steel halogenated solvents or mixtures which form radicals and or acids for example 2CHCl3 O2 2COCl2 2HCl This reaction in which stainless steel p...

Page 393: ...our Website for the latest version at http www agilent com cs library Support Patches SSBs 2D LC html Software Release Bulletin SRB The SRB is an excerpt from the SSB which lists issues which have be...

Page 394: ...x Agilent Technologies on Internet 2D LC User Guide 394 Agilent Technologies on Internet For the latest information on products and services visit our worldwide web site on the Internet at http www ag...

Page 395: ...com Agilent Technologies Inc 2021 Edition 05 2021 Document No D0004828 Rev A In This Book The manual describes the following introduction installing configuring using data analysis safety and related...

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