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ekspert™ nanoLC 400 Systems Operator Guide

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1. Current Tray Autosampler Method Definitions Run Sequence Trayi ss Autosampler Methods Sequential OAs Available v Synchronized Multi Channel Device Idle Driver Waiting for method LC Methods Flush Equilibrate when Idle Valve Position Load ISSA Position Load 10 ISSB Position Load 1 10 ss29890 Tey 73 Elapsed Time 00 queued Time 00 41 12 900908 Pause Click File gt Save As and type Trap Loading in the File name field Click Save 4 Inthe first line in the run table double click the Autosampler Method field and select the autosampler method created above 5 Inthe Tray column type 1 and in the Vial column type C01 Alternatively specify the vial location by clicking the vial position on the picture of the sample tray in the Run Manager window bottom left corner In the Channel column specify Loading Pump Double click the LC Method field and select the loading pump method created above Operator Guide ekspert nanoLC 400 Systems 53 of 174 System Overview 8 Inthe next line ofthe Run Manager table leave the autosampler method tray and vial location empty 9 Inthe Channel column specify Gradient 1 10 Double click the LC Method field and select the gradient method created above 11 Foreach sample to be run create the two lines in the table as shown in Figure 3 35 Figure 3 35 Run Manager Window Two
2. 100 Flow Mode Conserved flow O Independent flow 1 Profile Editor 70 OA B 60 Runtime 35 min 90 50 Total flowrate 40 300 NLATIN 30 time min View Audit Trail ekspert nanoLC 400 Systems Operator Guide 140 of 174 Basic Methods Figure F 13 LC Method Settings Dialog Gradient Table for Trap and elute Method Gradient 1 B LC Method Settings Selected Method Name Gradient1 Pump_15minGradient Summary Run Conditions Gradient Profile Gradient Table Time min View Audit Trail Operator Guide Flow Mode Conserved flow O Independent flow Profile Editor Total flowrate 300 nLmin ekspert nanoLC 400 Systems 141 of 174 Basic Methods Figure F 14 Run Manager Trap and elute Method Eile Edit View Devices System Suitability Help Run Table default ini Run Autosampler LE i Other Aa Method Tray Vial Method Channel Data Filename Status AS3 1 ul pickup trap elute 1 co1 LoadingPump_trap_loading Loading Pump Gradient 1 15min gradient Gradient 1 R Current Tray Autosampler Method Definitions Run Sequence Trayi Aa Autosampler Methods
3. Side A v Side B Total Volume 9 pL purge cycles Flush Flowrate 0 nL min Flush Now g Apply to all channels Apply to all channels C Automatically pur ge amplifiers when mobile phases change Automatically flush system when mobile phases change Create New Fluid 3 Purge the mobile phases a Inthe Purge Settings group select the Side A or Side B check box as appropriate Operator Guide ekspert nanoLC 400 Systems 65 of 174 System Maintenance b Inthe purge cycles field type 20 c Click Purge Now Flush the System In the Flush Settings group in the Total Volume field type 50 uL 2 Setthe Flush Flowrate based on the configuration e For a low flow module configuration type 1000 nL min e Fora high flow module configuration type 10 uL min 3 Connect one end of a length of tubing to the appropriate mobile phase outlet on the side of the pump Fora high flow configuration use 360 um outside diameter 0 d 50 um inside diameter i d tubing Fora low flow configuration use 360 um o d 25 um i d tubing e Or use the supplied PEEKsil tubing 4 Insert the other end of the tubing in a waste vial Caution Potential System Damage Make sure that no LC columns are connected before proceeding with this operation Flushing the system with a column connected could over pressure the system create external leaks and damage the column
4. Caution Potential System Damage There are no user serviceable components or assemblies inside the ekspert nanoLC 415 and 425 systems Service of any internal parts or assemblies requires an AB SCIEX trained Field Service Employee review product Safety Data Sheets for specific instructions Stop the spill or leak only if it is safe to do so Use appropriate personal protective equipment and absorbent to contain the spill and dispose of following local regulations When replacing tubing or fittings on the ekspert nanoLC 415 and 425 systems or the ekspert nanoLC 400 autosampler exposure to solvents may occur It is therefore recommended that appropriate safety procedures be followed and personal protective equipment be used according to the applicable Material Safety Data Sheets supplied by the solvent vendor A WARNING Biohazard Toxic Chemical Hazard In the event of a chemical spill Basic Troubleshooting Steps 1 Step back and look at the overall system Is something obvious causing the problem For example is the instrument unplugged or improperly connected 2 Compare the current system operation with the way the system operated before the problem started Identify conditions such as pressures power settings or flowrates that are different from when the system was operating normally For example if the output pressure is usually 2500 psi for a certain method is the system pressure currently in the same range or drastical
5. Click Flush Now Click OK to close the Mobile Phases dialog Replace the Seal Wash The ekspert nanoLC 400 systems have an integrated automatic seal wash system We recommend periodic inspection of the seal wash reservoir to make sure it is clean and has not evaporated or spilled Make sure that both lines are in the reservoir liquid Periodically replace the liquid reservoir to prolong long life for the pump seals and improve uptime Required Materials 95 5 HPLC grade water isopropanol Fill the seal wash bottle with a mixture of HPLC grade water and 5 isopropanol 2 Insert the seal wash tubing 2 in the bottle making sure that the tubing is submerged in the liquid 3 Tighten the cap and put the bottle in the solvent tray Re initialize the Pressure Transducers 1 Remove fittings from the outlets on all channels 2 Click System gt Hardware Diagnostics ekspert nanoLC 400 Systems Operator Guide 66 of 174 System Maintenance 3 Inthe top right corner of the dialog select the channel 4 Click the Flow Calibration tab 5 Select the Re lnitialize Transducers check box and then click Start Figure 5 2 Hardware Diagnostics Dialog Flow Calibration Tab Recurring Events Channel Remind me to run diagnostic tests once a month Gradient Flow Calibration Calibration Values External AD r Transducers Re Initialize Transducers OK 08 01 12 Gradient 1 Calibrated r Calibrate C
6. 3 Cycle the syringe several times by pushing and pulling on the syringe plunger while holding the syringe tip below the liquid meniscus Fill the syringe one final time leaving the plunger pulled out Examine the syringe body for air bubbles e If airis present repeat the steps to cycle the syringe ekspert nanoLC 400 Systems Operator Guide 74 of 174 System Maintenance e If air is removed install the syringe as described in Replace the Syringe on page 72 e If air reappears consider changing the prime solvent and repeat the procedure Refer to Best Practices and Troubleshooting on page 83 if air continues to be an issue gt Tip Although air may be present in the syringe it may not impact injection precision or CVs significantly Careful evaluation of injection performance has shown that small gt amounts of air in the syringe do not significantly impact analytical performance Recently mixed solvents can heat and de gas due to enthalpy changes De gas solvent mixtures or allow them to sit for at least 24 hours after mixing to minimize air bubbles Premixed solvents are more likely to be at equilibrium gt Tip Solvent choice may be application dependent When running common reversed phase methods put the syringe tip in the sample loading solvent reservoir often A or aqueous solvent It is also possible to use the solvent in the A channel aqueous Remove stubborn bubbles with degassed 1 1 isopr
7. B LC Method Settings Selected Method Name Gradient pump_15minGradient v Save Print Summary Run Conditions Gradient Profile Gradient Table Flow Mode Conserved flow O Independent flow Profile Editor OA B Runtime 35 min Total flowrate 300 nLimin point 40 15 time min View Audit Trail ekspert nanoLC 400 Systems Operator Guide 52 of 174 System Overview Note The mobile phase composition can be set by clicking and dragging points on the graph or by setting the of A or B in the Profile Editor group 10 When the method is complete click Save 11 Click OK to close the editor Create the Run Table The Run Table ties together an autosampler and one or more LC methods with a sample vial and tray position Use the Run Table to specify descriptive information related to the sample or analysis This section creates a Run Table to run two samples with a trap and elute method 1 Inthe Run Manager window click Edit gt Erase Table Figure 3 34 New Run Table Run Manager jek File Edit View Devices System Suitability Help Run Table TrapLoading ini Run Autosampler LE Run ie v Method Tray Vial Method Channel Replicates v sigent41 5 425 1 ulPickup Gradi1 Gradient 4 CO1 LoadingPump trap loading Loading Pump Gradient1 pump_1 5minGradient Gradient 1
8. Stop Change the run duration Select the number of minutes to extend or shorten the run Stop the run The run stops and the pump resets View a dialog containing information about the sample run and other acquisition information saved with the data file Not available during a run Open the Direct Control dialog Use this dialog to change the mobile phase composition flowrate valve position and column oven temperature when the system is not performing a run ekspert nanoLC 400 Systems Operator Guide 56 of 174 System Overview Add or Remove Traces in the Acquisition Window 1 Inthe Acquisition window click System gt Appearance Settings to open the Appearance Settings dialog Figure 3 37 Figure 3 37 Appearance Settings Dialog Flow Data V NE Qa nmin v HE oe nmin FE Ototal nmin E EE Profile A nL min 7 EE Profile B nL min V i 5 A D channel Aux A D mV Plot axes Pressure V ME Column Pc psi E NE Amp A Pa psi BEER Amp B Pb psi psi bar Absorbance FE Chroma 1 mau T O Chroma 2 mAu ER Chroma 3 mau E NEE Chroma MAX mau scale 1 offset 0 Contours and Shading Restore Defaults 2 Select the items to view in the plot and optionally set the colors for the traces by clicking on the color box 3 Click Apply and then OK to save the changes and close the dialog Operator Guide ekspert nanoLC 4
9. Symptom Injection valve does not switch positions Possible Cause Method is faulty Corrective Action Review or recreate the method looking for errors in the injection steps Actuator is faulty e Verify that the valve can be toggled with the Direct Control dialog in the Eksigent control software Contact AB SCIEX Technical Support No flow coming out of the port Valve is plumbed incorrectly Verify the plumbing configuration and reconfigure if needed Ports are plugged Use a syringe to manually flush each port with cleaning solvent Remove the stator and sonicate for 15 minutes in 100 methanol Contact AB SCIEX Technical Support When no column is connected system pressure Pc is unusually high Ports are plugged Use a syringe to manually flush each port with cleaning solvent Remove the stator and rotor seal and sonicate for 15 minutes in 100 methanol Contact AB SCIEX Technical Support Ends of tubing PEEKsil are crushed Replace tubing and do not over tighten fittings Mixing tee on pump is plugged Contact AB SCIEX Technical Support Fluid leaking from the valve Operator Guide Ferrule not properly seated in the port Check the tubing connection and fittings and verify the ferrule is properly seated Rotor seal is scratched Contact AB SCIEX Technical Support ekspert nanoLC 400 Systems 1
10. The files inside are named with the channel and date stamp for example ek1_122807 In this example ek1 indicates the channel from which the data was collected that is channel 1 The second part of the name indicates the time the data was created that is 12 28 07 pm 5 Click the appropriate data file and click Open The Acquisition window shows the various pressure and flow data 6 Select the data to be displayed a Click System gt Appearance Settings b Select the data to be shown The color of each parameter can be changed by clicking the color and selecting a new one ekspert nanoLC 400 Systems 88 of 174 Operator Guide Best Practices and Troubleshooting 7 Send the TXT file in an email to the FSE Autosampler Diagnostics The Diagnostics tab of the Autosampler Configuration dialog allows the user to track the pressure of the autosampler flow path Use the Diagnostics tab to observe the pressures that occur during method development The current pressure mBar shows below the Set Zero button A graphical display of the pressure is on the right side of the tab The current pressure may be positive or negative due to minor fluctuations Figure 6 5 A peak to peak drift of 50 mBar to 50 mBar is not unusual when the autosampler is at rest Figure 6 5 Autosampler Configuration Window Diagnostics Tab Autosampler Configuration Configuration Direct Control Maintenance Diagnostics FlowPath Pre
11. 25 um i d 360 um o d Gradient 1 syringe dispenser column MS sample needle Load 1 10 1 6 Inject 1 2 1 2 10 port valve not used Operator Guide ekspert nanoLC 400 Systems 117 of 174 Standard Plumbing Diagrams Figure D 4 Trap and elute 25 um i d 360 um o d koadig pump 50 um i d 360 um o d syringe Gradient 1 dispenser sample column MS needle Load 1 10 1 6 EE Inject 1 2 1 2 waste ekspert nanoLC 400 Systems Operator Guide 118 of 174 Autosampler Method Editor Use the autosampler Method Editor dialog to create ekspert 400 autosampler methods of varying complexity Basic and Advanced editors are available In the basic editor methods are created quickly and they meet most of the requirements for LC MS experiments For more complex methods refer to Create an Advanced Method on page 122 Create a Basic Method 1 Inthe Run Manager window click Autosampler Methods 2 Optional To open an existing file click File gt Open Figure E 1 Method Editor Dialog Ij Editing AS3 trap elute 1 uLAS3 staf File r Injection Type Direct Injection Trap Elute C Multiplex Gradient Pump Channel Gradient Pump 1 Gradient Pump 2 r Optional Valves None SS 4 Valve 155 B Valve cHiPLC m Sample Pickup uLPickUp FullLoop Sample pick up volume a yL Needle height 2
12. Basic Methods ekspert nanoLC 400 Systems 135 of 174 Basic Methods Figure F 8 LC Method Settings Dialog Run Conditions for Trap and elute Method B LC Method Settings Selected Method Name LoadingPump_trap_loading Summary Run Conditions Gradient Profile Gradient Table Pre Run Flush column for 0 1 g First establish a column pressure of Sample Injection O Nore minutes using 100 initial flowrate conditions 3000 psi Standard Sample valve opens prior to beginning Flow Profile and remains open Metered Inject QO Rapid Inject Post Run C Flush column for 12000 View Audit Trail ekspert nanoLC 400 Systems 136 of 174 nL of sample at 100 nL of sample at maximum flowrate maintaining initial mixture conditions minutes using initial flowrate conditions ending flowrate conditions Operator Guide Basic Methods Figure F 9 LC Method Settings Dialog Gradient Profile for Trap and elute Method B LC Method Settings Selected Method Name LoadingPump_trap_loading Summary Run Conditions Gradient Profile Gradient Table 30 Flow Mode 27 lsocratic Profile Editor 24 21 18 15 time min View Audit Trail Operator Guide ekspert nanoLC 400 Systems 137 of 174 Basic Method
13. If necessary fill the wash bottles with the appropriate solvent Reagent 1 Mobile Phase A Wash 50 50 methanol water In the Autosampler Configuration dialog click the Direct Control tab In the Wash group click Inside sample needle wash A system wash washes the inside needle Click Start Observe the progress of the liquid and bubbles in the tubing Repeat step 4 and step 5 until there are no bubbles in the syringe The syringe may require more flushes than other components of the flow path The liquid path may be flushed in one or two system washes The syringe may require multiple inside needle washes Click OK If air continues to be present in the syringe remove the syringe to manually remove the air Refer to Prime the Syringe ekspert nanoLC 400 Systems 71 of 174 System Maintenance Replace the Syringe The ekspert nanoLC 400 systems include a standard 50 uL syringe If a different size syringe is installed specify the volume in the Autosampler Configuration dialog The installed syringe must fit the volume of the injection needle and loop After replacing the syringe refer to Prime the Syringe on page 74 1 In the Run Manager window click Devices gt Autosampler Device Settings 2 On the Maintenance tab in the Exchange group click Syringe Figure 5 6 Autosampler Configuration Window Maintenance Tab Autosampler Configuration Configuration Direct Control Tray Tray Position Home Curr
14. Item Description 1 Control processor Electronically controlled pressure source Flowmeter PD OO N Replaceable flow module Operator Guide ekspert nanoLC 400 Systems 107 of 174 Theory of Operation How the MFC Plus System Works For each mobile phase 1 The flowmeter continuously calculates the flowrate and sends a signal to the PID control processor The control processor is a tunable proportional integral derivative PID controller A PID controller is a control loop feedback device that automatically adjusts one variable in a system in an attempt to hold another variable at a specified setpoint For the ekspert nanoLC 400 systems the process controller adjusts the pressure to maintain the specified flowrate 2 The PID control processor sends out a voltage signal to the controller at the pressure source The signal is proportional to the pressure required to achieve the desired flowrate during the gradient 3 The controller at the pressure source changes the pressure as needed to generate the required flowrate or gradient Pressure in the system is generated by connecting laboratory air or nitrogen to a pneumatic amplifier that can produce an amplification in pressure For example 100 psi incoming air pressure from the house air system can be used to produce hydraulic pressure extending from 0 psi to more than 10 000 psi Calculating the Flowrate The flowrate of each mobile phase is d
15. PUMCING PUMP 20866 bok eed Seed ie HYDE REDE sk earth 26 Flush the arie EE OE han an aan E EE OE EK 27 Replace the Flow Module Cartridges EE EG ee EE Ee ee ee 28 Set the Column Oven Temperature EE EE SE Ee ee eee ee 29 Configure the A D Converter 2 2 0 0 RE EER RE eee 30 Set the Scale and A D Input Voltage Range Selection 31 Set the Data Acquisition Rate for Including the A D Converter Detector Stream in an LC Method ER RE RE ER RR RE EE eee 32 Configure the Appearance Settings in the Eksigent Control Software 33 Equilibrate the System ie ee ees se nenn ER RE ER ees 35 Verify the Flowrate tits RE RE ergeben 36 Prepare to Run a Sequence EE SS ee ee ee ee 38 Confirm the Autosampler Configuration ee EE EE cee eee 38 Create Autosampler and LC Methods Direct Injection 39 Create the Autosampler Method 0 0000 SS EE EE ees 40 Create the LC Method Gradient 00 0 cee eee eee 42 Standard and Metered Injection Parameters 00000 eee 44 Create Autosampler and LC Methods Trap and Elute ee Ge Ge ee ee ee ee ee 45 Create the Autosampler Method SEE ES SS EE SS SS ee ee 45 Create the LC Method Loading Pump ee EE 00 eee eee eee 47 Operator Guide ekspert nanoLC 400 Systems 5 of 174 Contents Create the LC Method Gradient EE EE EE EE EE GE EE ee ee ee 50 Creat
16. e Binary gradient pumping system with loading pump The flowrates depend on the installed flow modules e Eksigent control software e ekspert M nanoLC 400 autosampler Autosampler solvent tray Figure 2 1 ekspert nanoLC 425 system Item Description 1 Solvent tray 2 Autosampler 3 Pump Operator Guide ekspert nanoLC 400 Systems 15 of 174 Introduction Accessory Options Depending on the system purchased it may include the following accessory options cHiPLC system PN 950 00070 e Column oven and mounting kit PN 5019593 e External A D converter PN 5019951 ekspert nanoLC 400 Systems Operator Guide 16 of 174 System Overview This section provides an overview of the basic operations of the Eksigent ekspert nanoLC 400 system It is written for standard nanoLC 415 and 425 systems Some sections describe features which may not be included in the nanoLC 415 system For information on the cHiPLC system refer to the cHiPLC System Operator Guide Note Prior to completing any procedures in this section review the Regulatory and Safety Information Turn On the System Caution Potential System Damage If any component of the system is unplugged from the AC mains make sure that the power switch on the back of the component is in the 0 position prior to connecting the component to AC mains Failure to do so could result in component damage 1 If required co
17. ekspert nanoLC 400 Systems Operator Guide 4 0f 174 Contents Chapter 1 Regulatory and Safety Information ee ee ee Ek ek ek ee 9 About this Guide AE eens 9 Symbols and Conventions EE EE EE EE nenne ER RR ees 9 Safety Instructions a de aas EER wie N ER hata ne N RE OR ER ERGE aed 10 Qualitied Personnel u MERE RE RR RE DS DR Doe DES bee RE Da Sen 12 Equipment Use and Modification iss Ee Ee ke eee 12 Regulatory Compliance 2 02 sess EERS BERE kh one KERR ED RE GE EE EE EE 12 Symbols and Labels EE RE EE SR Cee SEE RES SEERDE RE a ae 13 System Disposal Waste Electrical and Electronic Equipment 14 Related Documentation 00 EE EE cee 14 Technical Support 2 2222 nennen een een 14 Chapter 2 Introduction H nen ann nn nn ee 15 ses RAS AE RE EE RE EO N RO RE 16 Chapter 3 System Overview ss see ek kk se nennen AR RR AR nenn 17 Turn On the System nee Ba MEE Ee EERS EE Re Ee de Br ee KEN RE 17 Channel Assignments in the Eksigent Control Software 19 Autosampler Configuration 22222 EE ee EE EE en 19 Replace the Mobile Phases is EE EE EE EE EE eee eee ke ke ke nenn 21 Specify the Maximum Flowrate EE ES cece Ge ee ee ee 23 Prime the Pump iss EE EE EE ES EG EG Es EG ER nennen nennen een 23 Prim a Dy PUMP 12228 EE ER TE EE IK 24 Exchange the Solvent isi k EER ER 22 000 ER RR RR ER KAR ERLA OER EER ER 25
18. nanoLC 400 Port Address 23 Firmware FPGA PCB Serial Number Hardware Settings USB COM Auto Detect w v 950 110 1 04 00 950 100 1 04 00 950 601 000 014 110002 Hardware Settings Loop Volume 10 Needle Tubing Volume 36 Syringe Yolume 50 Wash Speed 3 Front Tray 1 108 Yials Back Tray 2 Log Counters Injector valve ISS A 3672 0 Syringe valve 13697 Syringe 19878 Power On Time hours 0 Refresh The standard settings in the Hardware Settings group should be as shown in the table Table 3 2 Hardware Settings Field Value Loop Volume 10 uL Needle Tubing Volume 3 6 uL Syringe Volume 50 uL Wash Speed 2 uL s Front Tray 108 vials The wash speed used by the method is dependent on the wash speed settings Loop volume must match the currently installed loop If the sample loop size is changed the Loop Volume configuration must be modified to reflect this change New autosampler methods must also be created for this new sample loop size ekspert nanoLC 400 Systems 20 of 174 Operator Guide System Overview Tray Cooling The sample temperature compartment provides a constant environment for all samples The temperature range is 4 C to 40 C To minimize temperature fluctuations set the required temperature one hour before loading the samples in the sample compartment For biological samples 6 C is advisable If only a constant tempera
19. then complete the following steps a Close the Eksigent control software b Disconnect and connect the USB cable c Open the Eksigent control software d Confirm that communication is established between the LC and the computer using autodetect If communication is still not established then disconnect the USB cable from the LC f Close the Eksigent control software g Turn off the LC and computer h i j D Turn on the LC and computer When the computer has fully started connect the USB cable to the LC Wait 2 minutes and then close and open the Eksigent control software k Confirm that communication is established between the LC and the computer If communication has not been established restart the system again or contact an AB SCIEX FSE for assistance 9 Ifa message similar to an error opening COM x appears complete the following steps a Click Cancel The software opens in Demo Mode b Click System gt Instrument Configuration c Select the lowest COM port and click OK The system uses autodetect by default Manually select a port Repeat this step using sequentially higher COM ports until the correct COM port is found 10 When the system is communicating with the software in the Acquisition window click Run Manager a Ifa message similar to WARNING no autosampler detected appears then click Cancel b Inthe Run Manager window click Devices gt Autosampler Type and verify tha
20. 162 Analysis Method for the Chip Column 00000 0c eee 166 Create the Acquisition Methods and Batch SES EE EE SE Se Ee 167 Create the Acquisition Methods 0 0c cc ees 167 Create the Acquisition Batch ES EES SS es 167 Load the Mobile Phases 0 0000 ee ee ee ee ee ee 168 Run the Batch EE os saci cued anne men nee 168 Tips for Optimizing Your Own Experiments iss ss se ss ke se se nen 169 Set Up with Two CHiPLC Systems is Es SE EE ee 169 Index eae a hat reset OR EE eae RE N EE EN 171 ekspert nanoLC 400 Systems Operator Guide 8 of 174 Regulatory and Safety Information About this Guide This guide is intended for laboratory technicians who are responsible for day to day operation and maintenance of an Eksigent ekspert nanoLC 400 system It is assumed that the user of this guide is familiar with standard laboratory terminology Note Read these safety instructions and the rest of this guide before using the A system Symbols and Conventions The following conventions are used throughout the guide Table 1 1 Symbols and Conventions Pictorial Description The danger sign warns about a hazard It calls attention to a procedure or practice which if not adhered to could results in injury or loss of life Do not proceed beyond a danger sign until the indicated conditions are fully understood and met The warning sign denotes a hazard It cal
21. 55 control buttons 56 run status viewing 55 Run table creating 53 runs starting 54 stopping 56 viewing run status 55 Operator Guide Index S safety instructions 10 standards 12 sample loop replacing 79 sample pickup 120 sample preparation techniques 84 scheduled maintenance 63 seal wash replacing 66 Setpoint temperature column oven 29 settings configuring autosampler 19 shorten a run 56 solvents exchanging 25 replacing the wash bottle 77 standard injection parameters 44 starting runs 54 stopping a run 56 stopping runs 56 storing the system 81 symbols and labels 13 syringe flushing 71 replacing 72 system alerts log 87 cleaning and inspecting 64 disconnecting 59 equilibrating 35 148 flushing 66 installing at a new location 59 moving 59 preparing for storage 81 symbols and labels 13 turn on 17 waster disposal 64 system diagnostics 84 system maintenance 63 system storage 81 T technical support obtaining 14 temperature setting column oven 29 theory of operation 107 ekspert nanoLC 400 Systems 173 of 174 Index timer 36 torque wrench 28 traces adding 57 removing 57 transducers re initializing pressure transducers 66 trap and elute creating methods 45 trap and elute methods 120 tray cooling 21 troubleshooting 83 autosampler 100 autosampler valve 101 column oven 100 flow control system 96 injection valve 101 system initialization 96 troubleshooting guidelines 95 turn on
22. A 1 Purge and flush all pumps 2 Purge Reagent 1 on the nanoLC 400 autosampler and change to 97 ammonium formate and 3 mobile phase B Run the Batch Place the sample vial in the autosampler 2 Click the Submit tab of the Batch Acquisition dialog in the Analyst software then click Submit to add the samples to the queue Click View Queue in the toolbar to open the Queue Manager Local dialog Click Equilibrate to equilibrate the nanoLC system and the mass spectrometer Monitor the progress of the run e View the LC chromatogram and spectral data in Explore mode in the Analyst software e View flow rate and pressure information in the Acquisition window of the Eksigent control software Caution Potential System Damage To prevent damage to the cHiPLC system and the columns do not allow system pressure Pc to exceed 4000 psi In the Eksigent control software set the upper limit for pressure in the Advanced tab of the Instrument Configuration dialog ekspert nanoLC 400 Systems Operator Guide 168 of 174 2 D Reverse phase Reverse phase Liquid Chromatography Workflow Tips for Optimizing Your Own Experiments e For the high pH column Gradient 1 e Adjust B and time for the Gradient 1 LC methods but make sure that the run time for Gradient 1 and the Loading pump is the same e Try different columns to increase throughput separation or loading capacity adjusting methods as needed If
23. AB SCIEX Refer to the mass spectrometer Safety Guide or System User Guide for instructions for safely connecting to the protective earth on the LC MS system N WARNING Electrical Shock Hazard Do not change the external or internal ekspert nanoLC 400 Systems Operator Guide 10 of 174 Regulatory and Safety Information WARNING Electrical Shock Hazard Make sure that a protective earth grounding cable is connected between the injection valve s sample loop and an appropriate grounding point at the LC MS source This supplementary grounding will reinforce the safety configuration specified by AB SCIEX WARNING Environmental Hazard Follow a regulated approved waste disposal program Never dispose of flammable or toxic solvents into a municipal sewage z system Do not allow flammable or toxic solvents to accumulate WARNING Puncture Hazard Do not operate the autosampler without the front cover properly installed To avoid injury during operation keep hands and loose objects away from the autosampler needle and syringe assembly WARNING Personal Injury Hazard Use caution when working with any PEEKsil fused silica or polymeric tubing under pressure Always wear proper eye protection when near the tubing Do not use tubing that has been severely stressed or kinked Do not use tubing in particular PEEK or DuPont Tefzel tubing with tetrahydrofuran THF dimethylsulfoxide DMSO chlorinated organic so
24. Analyst software For more information refer to the Analyst Software Getting Started Guide or System User Guide available from the Start menu Complete the following steps in the order in which they appear 1 Verify the Hardware Profile Enable the Column Oven Equilibrate the System Create the Autosampler and LC Methods Create the Acquisition Method and the Batch Submit the Batch Monitor the Run NOONAN Operator Guide ekspert nanoLC 400 Systems 145 of 174 Working with Analyst Software Verify the Hardware Profile The active Hardware Profile must include the autosampler and the Eksigent control software to perform a run using Analyst software The FSE should configure the system during installation However if a different computer is being used or the Analyst software has been uninstalled then the Hardware Profile may not be correct 1 Open the Analyst software 2 On the Navigation bar under Configure double click Hardware Configuration 3 In the Hardware Configuration Editor window click each hardware profile to open it and locate a profile that contains a mass spectrometer the autosampler and the Eksigent control software If an appropriate profile does not exist create a profile as described in Create a Hardware Profile on page 147 4 Ifthe profile exists but does not have a green check to the left click Activate Profile The active profile shows with a check the Eksigent control
25. Dialog Column Oven Heater Setpoint 35 C Start Stop 3 Click Start The oven will come to temperature quickly However the column can take up to 30 minutes to stabilize to the oven temperature es Note Do not operate the oven for an extended period of time without a closing the oven compartment i Equilibrate the System Make sure that the LC column is connected In the Eksigent control software click System gt Direct Control In the Pump Direct Control group click the Conserved Flow option and set A to 95 and B to 5 This is the mobile phase composition used for equilibration 4 Set the Total flowrate based on the configuration e Type 200 nL min for the low flow configuration e Type 2 uL min for the high flow configuration In the Pump Direct Control group click Start to begin equilibration In the Valve Direct Control group flush the injection valve by alternately clicking Load Position and Inject Position Wait approximately 10 minutes for the system to equilibrate and then click Stop Click Close ekspert nanoLC 400 Systems Operator Guide 148 of 174 Working with Analyst Software Create the Autosampler and LC Methods Create the autosampler method and a trap and elute method for the LC The LC methods contains the conditions used for separating the sample including flowrate flow mode and mobile phase gradient Refer to Create Autosampler and LC Methods Tra
26. Injector Inject for Time h mm ss 0 20 00 Start Gradient 2 Switch Injection valve to Load Clean Up 5x 2 D Reverse phase Reverse phase Liquid Chromatography Workflow ekspert nanoLC 400 Systems 157 of 174 2 D Reverse phase Reverse phase Liquid Chromatography Workflow The Elution Method This method waits for the pumps to be ready then starts Gradient 1 and waits 25 minutes as the sample elutes from the high pH column and is diluted by the Loading Pump The diluted sample is then loaded onto the trap column After the fraction is loaded the method starts Gradient 2 The cHiPLC system valve switches to connect the trap to the chip column to elute the sample from the chip column In the example experiment this method runs three times once for each fraction from the high pH column Figure H 5 Autosampler Elution Method Command 1 Initialize 2 Wait 3 Wait 4 Wait 5 Start 6 Start 7 Wait 8 Start 10 Description Autosampler Device for Gradient 2 ready to start for Gradient 1 ready to start for Loading Pump ready to start Start Gradient 1 Start Loading Pump for Time h mm ss 0 25 00 Start Gradient 2 ekspert nanoLC 400 Systems 158 of 174 Operator Guide 2 D Reverse phase Reverse phase Liquid Chromatography Workflow Create the LC Methods These methods are for the example experiment and should be modified as needed for your experiment Create the following methods in t
27. Loading pump d Inthe Purge Settings group select the solvent A or B to be changed e Inthe mobile phase bottle pull the inlet frit out of the liquid so that the filter can be cleared of old solvent f Specify 5 purge cycles and click Purge Now g As the pump draws liquid up the line an air gap forming in the line at one end of the reservoir h Remove the old bottle when the end of the line is clear of solvent i Replace the old bottle with a new bottle and place the mobile phase filters into the new liquid 3 Proceed to step 2 in the section Prime a Dry Pump Purge the Pump Purging rapidly replaces the solvent in the pump Repeat this procedure for all channels if necessary Prerequisite Procedure e Replace the Mobile Phases 1 Inthe Acquisition window click System gt Mobile Phases 2 Verify that the software settings for solvent composition match the actual solvents in the bottles Binary mixture A sets the composition for Solvent A mobile phase Binary mixture B sets the composition for Solvent B mobile phase 3 If necessary repeat step 2 for Gradient 2 and Loading pump 4 Click More to open the Purge Settings and Flush Settings ekspert nanoLC 400 Systems Operator Guide 26 of 174 System Overview Figure 3 9 Mobile Phases Dialog Purge and Flush Settings Mobile Phases Channel 4 Gradient vl 1 Binary mixture A Binary mixture B Aqueous Solution Aqueous Solut
28. Loop or uL Pick Up HL Pick Up The more common method is the uL Pick Up method In this method a sample volume less than the loop volume is loaded between two bands of transport liquid and centered in the loop ekspert nanoLC 400 Systems Operator Guide 120 of 174 Autosampler Method Editor The uL Pick Up cycle is as follows 1 Wash the needle 2 Pick up the first transport liquid volume typically 30 uL 3 Pick up the sample volume often less than 1 2 the loop volume 4 Pick up the second transport volume typically the needle volume plus the required volume to center the sample in the loop The uL Pick Up method can reliably pick up and inject sub uL volumes of sample In addition almost all of the sample aspirated can be injected onto the trap or column This method is a better choice than Full Loop if sample is limited Significant amounts of transport solvent are present in the loop at injection The impact of the transport solvent composition can be an important consideration in method development We recommend for reversed phase LC methods that aqueous solvents be used for example Solvent A Matching the transport solvent to initial column equilibration and sample solvent composition is considered a good starting point Note The use of 100 water even with acid can allow microbial growth in the solvent reservoir We recommend that the user wash the bottle and change the solvent regularly when u
29. O S DODDO Pin Description Input3 ground Input3 Input2 ground Input2 Input1 ground ND OO PR Oo Oo INput1 ekspert nanoLC 400 Systems Operator Guide 114 of 174 D5033460 C Standard Plumbing Diagrams This appendix includes sample plumbing diagrams for two ekspert nanoLC systems e ekspert nanoLC 415 Systems on page 115 e ekspert nanoLC 425 Systems on page 117 ekspert nanoLC 415 Systems Refer to the appropriate figures to plumb the following configurations e Figure D 1 Direct On column Injection e Figure D 2 Trap and elute Figure D 1 Direct On column Injection 25 um i d 360 um o d Gradient 1 syringe dispenser column MS sample needle Load 1 10 1 6 Inject 1 2 1 2 10 port valve not used Operator Guide ekspert nanoLC 400 Systems D5033460 C 115 of 174 Standard Plumbing Diagrams Figure D 2 Trap and elute 25 um i d 360 um o d Loading pump 60 um i d 360 um o d syringe Gradient 1 dispenser sample column MS needle Load 1 10 1 6 Inject 1 2 1 2 waste ekspert nanoLC 400 Systems Operator Guide 116 of 174 Standard Plumbing Diagrams ekspert nanoLC 425 Systems Refer to the appropriate figures to plumb the following configurations e Figure D 3 Direct On column Injection e Figure D 4 Trap and elute Figure D 3 Direct On column Injection
30. Samples Run Manager File Edit View Devices System Suitability Help Run Table TrapLoading ini Autosampler LE Sample Run A Method Tray Vial Method Channel Name Replicates sigent41 5 425 1 ulPickup Grad1 Gradient 1 001 LoadingPump_trap_loading Loading Pump Gradient1 pump_15minGradient Gradient 1 sigent415 425 1 ulPickup Grad1 Gradient 1 c01 LoadingPump_trap_loading Loading Pump Gradient1pump 15minGradient Gradient 1 Current Tray Autosampler Method Definitions Run Sequence i Trayi a Autosampler Methods Sequential O As Available O Synchronized Multi Channel Device Idle Driver VVaiting for method LC Methods Valve Position Load ISSA Position Load 10 ISSB Position Load 10 Terre rarae ment S star FlushEquilibrate when Idle E sed Time 00 00 00 Pause yeued Time 01 22 24 12 Select the Run check box to specify the lines to run 13 In the Run Sequence group select Sequential mode 14 Optional Select the Flush Equilibrate when Idle check box to start flow of the pump at the conditions specified in the Pre Run flush section of each LC method To edit the first two lines clear the Flush Equilibrate when Idle check box 15 Click File gt Save to save the Run Table Start the Run Select the samples to be analyzed by placing a check mark in the box to
31. Selection is not taking advantage of the full dynamic range of the device s A D conversion Use the live view of the inputs to the A D device to adjust and optimize these values for the ideal signal to noise 5 Click Close Set the Data Acquisition Rate for Including the A D Converter Detector Stream in an LC Method When the A D converter is enabled the Detector tab shows in the LC Method Settings dialog ekspert nanoLC 400 Systems Operator Guide 32 of 174 System Overview On the Detector tab specify an Acquisition Rate Figure 3 14 LC Method Settings Dialog Detector Tab B LC Method Settings Selected Method Summary Run Conditions Gradient Profile Gradient Table Detector Data Acquisition Acquisition rate v Hz is possible to go higher for fast chromatography with very narrow peak shapes A 10 Hz rate means that a 3 second wide peak is sampled 30 times Gi Tip 10 Hz is a good starting place for many chromatographic systems It Configure the Appearance Settings in the Eksigent Control Software Open the Eksigent control software Click System gt Appearance Settings Click the number of boxes in the External A D converter group that are wired with inputs from the detector Operator Guide ekspert nanoLC 400 Systems 33 of 174 System Overview Figure 3 15 Appearance Settings Dialog External A D Channel Check Box Fksigent Control Software DEI Aw a0 my Z Appty
32. Sequential O As Available E O synchronized Multi Channel Device Idle Driver Waiting for method teMenoss Flush Equilbrate when ide Valve Position Load ISSA Posttion Load 10 ISSB Position Load t 10 Tray 28 4 C Analysis Methods amp Elapsed Time 00 00 00 ekspert nanoLC 400 Systems Operator Guide 142 of 174 Figure F 15 Run Manager Trap and elute Method Multiple Runs Run Manager Eile Edit View Devices System Suitability Help Basic Methods Run Table default ini segr un Autosampler LE Other a 7 Method Tray Vil Method Channel Data Filename Status 1 Y 4831 ul pickup trap elute 1 co1 LoadingPump_trap_loading Loading Pump Queued Z rg Gradient 1 15min gradient Gradient 1 Queued 3 7 4831 ul pickup trap elute 1 c01 LoadingPump_trap_oading Loading Pump Queued 4 7 Gradient 1 15min gradient Gradient 1 Queued 5 6 7 8 3 10 12 13 14 15 16 17 18 F 1 19 20 2 2 23 24 25 26 27 28 29 30 3 32 33 34 3 x Current Tray Autosampler Method Definitions Run Sequence Trayi A Sequential v O As Available O Synchronized Multi Channel Device Idle Driver Waiting for method LC Me
33. The A side is typically filled with an aqueous solution To prevent microbial growth prior to removing and storing the module flush the module with an organic solution such as isopropanol Refer to Prime the Pump on page 23 for information on how to quickly exchange a solvent in the pump Required Materials Small break over torque wrench PN 5019560 Caution Potential System Damage Only use the break over wrench supplied with the system Over torquing is easily possible and will damage parts and void the warranty One of the following flow modules Nano gradient flow module 0 1 uL min to 1 uL min PN 5018236 Low micro gradient flow module 1 uL min to 10 uL min PN 5018237 High micro gradient flow module 5 uL min to 50 uL min PN 5018238 Loading pump isocratic flow module 1 uL min to 50 uL min PN 5018239 1 Make sure that the current pumping solvent is miscible with the contents of the flow module Replace solvents if necessary Turn off the system Remove the flow module cartridge ekspert nanoLC 400 Systems Operator Guide 28 of 174 System Overview 5 Pull down the slide on the flow module cartridge to protect the fluidic connectors during storage Push up the slide on the new flow module cartridge Insert the new flow module cartridge in the system It can only go in one way Using the calibrated torque wrench secure the cartridge Hold the wrench only by the break over
34. WARNING Biohazard or Toxic Chemical Hazard In the event of a chemical spill review product Safety Data Sheets for specific instructions Stop the spill or leak only if it is safe to do so Use appropriate personal protective equipment and absorbent to contain the spill and dispose of following local regulations WARNING Electrical Shock Hazard Only use fuses of the type and current rating specified Do not use repaired fuses or by pass the fuse holder WARNING Electrical Shock Hazard Plug the instrument into a receptacle that is connected to protective earth and that has adequate current capacity grounding connections Tampering with or disabling these connections could create a safety hazard and damage the system The instrument as shipped is properly grounded in accordance with normal safety regulations WARNING Electrical Shock Hazard Do not turn the system on if any kind of electrical damage is suspected Instead disconnect the power cord and evaluate the system WARNING Electrical Shock Hazard Continue to exercise caution as capacitors inside the system may still be charged even after the system has been turned off WARNING Electrical Shock Hazard Disconnect power cords from the power supply before attempting any type of maintenance WARNING Electrical Shock Hazard The combination of the pump and autosampler with a LC MS system may require additional safety measures as described by
35. Wb Eg Appendix G Working with Analyst Software iss ss ss ks sk ann Example Experiment SS SS ee ee ee ee ee ee ee ee Verity the Hardware Profile 22 ras ME Ens Re EE ein EE Create a Hardware Profile 0 0 SS ce es Enable the Column Oven 0 0 ccc ee nena Equilibrate the System se EE EE Re Re EE nee Create the Autosampler and LC Methods SS EE eee Create the Acquisition Method and the Batch SS EE SS ss eee Create the Acquisition Method EE cece Se ee ee nn Create the Acquisition Batch SEE SS ees Subimitthe Batch a tea es 2 cee a RS GE dees EG tan Rte na wed Monitor the RUN soes Be EE ar De Were ER SEEN ed Ge a hale Gee oR eS Appendix H 2 D Reverse phase Reverse phase Liquid Chromatography Workflow 0 00 c cece eee RR RR RR ER RR eee The Example Experiment 000 cece cee eee Workflow Steps 2 000 cece eee eee eens PIUMB ANG System 05 20 REGS BAe BED HE ate be hee ead cis bee TORS Create the Autosampler Methods 2000 EE EE e eee eee The Injection Method ar Bee eee ee ea eee eS Operator Guide Contents ekspert nanoLC 400 Systems 7 of 174 Contents The Elution Method 0 0 0 0 ccc ee ee ee ee ee 158 Create the LC Methods 0 0 0 cece eens 159 Methods for Loading the Sample on the High pH Column 159 Methods for Eluting the Sample from the High pH Column
36. axes settings to all channels More Options 00 f i 0 017 0 018 0 02 0 022 0 023 0 025 0 027 0 028 0 03 0 032 Minutes 4 Make sure that the Internal A D channel check box is clear The ekspert nanoLC 400 system does not have an internal A D channel Once configured the A D data stream should be visible on the screen Refer to the Eksigent Control Software User Guide for more information ekspert nanoLC 400 Systems Operator Guide 34 of 174 D5033460 C Equilibrate the System System Overview Prerequisite Procedure e Replace the Mobile Phases Use the Direct Control dialog to equilibrate the system following system power up a change of solvent or a change of column Open the Autosampler Configuration window to toggle the injection valve between load and inject positions Toggling flushes the sample loop and interconnecting ports 1 Inthe Acquisition window select the appropriate channel in the top right corner of the window 2 Click System gt Direct Control Figure 3 16 Direct Control Dialog Direct Control Pump Direct Control Waiting for LC Method A Total flowrate Conserved Flow 30 10 400 Independent Flow Q 400 Montor Baseine Column Oven Heater Setpoint 40 C nL min nL min Close Channel 5 Gradient 1 3 Make sure that Conserved Flow is selected and set A and B to 90 and 10
37. control computer ekspert nanoLC 400 Systems Operator Guide 30 of 174 System Overview Figure 3 12 A D Converter Item Description 1 Screwterminalpins 21 40 2 LED 3 Screw terminal pins 1 20 4 USB connector If the LED is on with a steady green then the device is connected to a computer or external USB hub If the LED blinks once then a USB command has been received by the device Continuously blinking indicates an analog input scan is taking place Set the Scale and A D Input Voltage Range Selection Open the Eksigent control software 2 Select System gt Hardware Diagnostics 3 On the External A D tab select the A D Input Voltage Range Selection from the list Operator Guide ekspert nanoLC 400 Systems D5033460 C 31 of 174 System Overview Figure 3 13 Hardware Diagnostics Window External A D Tab Hardware Diagnostics Recurring Events Channel _ Remind me to run diagnostic tests once a month E Ta Flow Caikration Calibration Values Detector Diagnostics AJD Readings live AJD Input Voltage Range Selection Channel 10000 0 10 to 10 Yolts v Channel 135 0 Channels 94809 Scale N 4 Optional In the Scale field specify a scaling factor Many devices output either 1 V to 1 V or 10 V to 10 V Select a scaling factor based on the output range settings in the detector If the data appears noisy once running then the Input Voltage Range
38. ekspert nanoLC 400 Systems 13 of 174 Regulatory and Safety Information System Disposal Waste Electrical and Electronic Equipment Do not dispose of system components or subassemblies including computer parts as unsorted municipal waste Follow local municipal waste ordinances for proper disposal provisions to reduce the environmental impact of WEEE waste electrical and electronic equipment To safely dispose of this equipment contact an FSE for instructions European Union customers Contact a local AB SCIEX Customer Service office for complimentary equipment pick up and recycling Related Documentation e Site Planning Guide available from www eksigent com e Eksigent Control Software User Guide installed with the Eksigent control software Analyst Software Getting Started Guide or System User Guide installed with the Analyst software Technical Support AB SCIEX and its representatives maintain a staff of fully trained service and technical specialists located throughout the world They can answer questions about the instrument or any technical issues that may arise For more information visit the web site at www absciex com ekspert nanoLC 400 Systems Operator Guide 14 of 174 Introduction The Eksigent ekspert nanoLC 400 systems are designed for HPLC applications that employ direct pumping at flowrates of several hundred nL min up to uL min The ekspert nanoLC 415 and 425 systems include
39. flowrate for pump B 12 Disconnect the calibration assembly 13 Repeat this procedure as necessary for other channels Prepare to Run a Sequence Use the Run Manager to queue samples to be run in sequence The sequence typically contains one or more LC methods and autosampler method Autosampler methods and LC methods are created and saved so that they are available to be selected and run in the Run Manager Sequence Tip Make sure that the ekspert nanoLC 400 autosampler is selected in the Devices gt AutoSampler Type menu within the Run Manager before continuing to build an gt autosampler method Confirm the Autosampler Configuration During installation the FSE configures the autosampler in the Eksigent control software Confirm that the ekspert nanoLC 400 autosampler configuration has not changed Refer to Figure 3 19 for typical values for the Hardware Settings ekspert nanoLC 400 Systems Operator Guide 38 of 174 Figure 3 19 Autosampler Configuration Dialog Configuration Tab Autosampler Configuration Configuration System Definition ekspert nanoLC 400 Port USB COM Auto Detect v Address 23 v Firmware 950 110 1 04 00 FPGA 950 100 1 04 00 PCB 950 601 000 014 Serial Number 110002 Direct Control Maintenance Diagnostics Hardware Settings Loop Volume 40 Needle Tubing Volume 36 Syringe Yolume 50 Wash Speed 2 Front Tray 1 108 Yials Back Tray 2 Log Counters Injec
40. in the LC method 1 Inthe Run Manager window in the LC channel column select an appropriate channel Click LC Methods 3 Inthe Name field type a method name and click Save 4 Optional Specify HPLC column information appropriate for the experiment This information is stored with the LC method file 5 Onthe Run Conditions tab in the Pre Run group select the Flush column check box 6 Specify a time appropriate to equilibrate the pump connected tubing and column using 100 initial flowrate conditions This is the pre run equilibration time and the duration is often experimentally determined Five minutes is a typical setup starting point Figure 3 22 LC Method Settings Dialog Run Conditions Tab B LC Method Settings Selected Method Name Gradient1 15min Gradient Summary Run Conditions Gradient Profile Gradient Table Pre Run Flush column for 5 minutes using 100 initial flowrate conditions First establish a column pressure of 3000 psi Sample Injection O None Standard Sample valve opens prior to beginning Flow Profile and remains open Metered Inject nL of sample at 100 initial flowrate conditions mee i 0000 QO Rapid Inject nL of sample at maximum flowrate maintaining initial mixture conditions Post Run Fi Flush column for 0 5 minutes using 100 ending flowrate conditions View Audit T
41. mm 10 pL wait 3 m Leading Volume Wait Time 2 min pL Trailing Volume 1 pL gt Wash Syringe wash cycles 5 x Advanced Editor Test on B1 Ok 3 Select the required options O Tip To save a method without closing the dialog click File gt Save N i Operator Guide ekspert nanoLC 400 Systems 119 of 174 Autosampler Method Editor 4 Click OK to save the method Method Editor Options The Method Editor dialog has several options for configuring a method Injection Type Direct Inject Perform direct injections by placing the sample into the sample loop in load position and then toggling the valve to inject the sample directly onto the column Trap and Elute Trap and elute methods are common in proteomics and involve two valves Initially the sample is placed into the sample valve of the autosampler as with the Direct Injection method Downstream from the sample valve an ISS A valve in the autosampler is plumbed with a sample trapping column The Loading pump channel of the pump is used for pre equilibration transport and loading and washing of the sample The trapping column is pre equilibrated in aqueous solvent to ensure binding of the sample to the trap The gradient pump and column are plumbed into the ISS A valve The Gradient pump is used to elute the sample from the trap to the analytical column when the ISS A valve is toggled Refer to Standard Plumbing Dia
42. more than three fractions are used to elute the high pH column for each additional fraction e Create a Gradient 1 method to elute the fraction from the column Create an Analyst software acquisition method for the fraction Add another run to the acquisition batch e For the chip column Gradient 2 develop additional analysis methods with the same duration to improve separation of individual fractions e For later fractions consider increasing the percentage of the organic mobile phase for the initial conditions of the gradient Set Up with Two cHiPLC Systems If you have two cHiPLC systems you can use a chip column for the first separation refer to Figure H 14 Operator Guide ekspert nanoLC 400 Systems 169 of 174 2 D Reverse phase Reverse phase Liquid Chromatography Workflow Figure H 14 System Configuration for a 2 D Reverse phase Reverse phase Workflow with Two cHiPLC Systems Sample injection Gradient 2 pH 2 5 300 nL min 0 2 x 150 mm chip C18 column 0 075x 150 mm chip C18 column 1 pL min Gradient 1 PH 9 8 7 ul min Loading pump pH 2 5 Substitute a cHiPLC column PN 5015480 for the InertSustain 0 2 mm x 150 mm capillary column for the high pH phase Use the methods described above ekspert nanoLC 400 Systems Operator Guide 170 of 174 Numerics 2 D reverse phase reverse phase workflow 153 A accessory options 16 acquisition batch creating 150
43. respectively This is the mobile phase composition used for equilibration Use Conserved Flow to choose a total flowrate and percent composition Use Independent Flow to select independent flowrates for A and B Conserved Flow is most commonly used with the ekspert nanoLC 400 systems Set the Total flowrate to 400 nL min or other appropriate flowrate In the Acquisition window click Devices gt Autosampler Device Settings b In the Injection Valve group flush the switching valve by alternately clicking Load Position and Inject Position to release trapped air 4 5 Click Start to start the pump flowing and begin equilibration 6 7 Flush the autosampler switching valve a Click the Direct Control tab Operator Guide ekspert nanoLC 400 Systems 35 of 174 System Overview The sample loop is inline when the valve is in the Inject Position and out ofthe LC flow path in Load Position The solvent that is in the loop when the valve is switched out of line remains there Figure 3 17 Autosampler Configuration Dialog Direct Control Tab Autosampler Configuration Configuration Dir Maintenance Diagnostics Temperature Settings Tray Cooling setpoint 10 PC Injection Valve ISS A Valve ISS B Valve Relnitialize Load Load 1 10 Load 71 10 O inject O Inject 1 2 O Inject 41 2 Wash System wash Inside sample needle wash volume uL 2000 O Inside sample needle wash O Outside sample needle wash OFil
44. submitting 151 acquisition information viewing 56 acquisition method creating 149 Acquisition window adding traces 57 removing traces 57 add time to a run 56 Advanced Autosampler Editor 124 alerts log 87 Analyst software hardware profile creating 147 verifying 146 autodetect 60 autosampler configuration settings 19 creating advanced methods 122 creating methods 119 external interface connections 111 maintenance 70 71 maintenance schedule 63 pin assignments 112 preparing for storage 81 replacing the syringe 72 tray cooling 21 troubleshooting 100 autosampler diagnostics 89 Autosampler Method Editor 120 autosampler methods 2 D reverse phase reverse phase work flow 156 creating 45 autosave 58 autosave files 88 AUX 113 Operator Guide B break over torque wrench 28 C calibrating flowmeters 67 calibration pipettes 68 cartridges replacing flow module 28 channel assignments 19 cleaning the system 64 column heater See column oven column oven enabling 147 setting the temperature 29 specifying Setpoint temperature 29 troubleshooting 100 configuring the autosampler 19 conserved flow 35 contact information 14 creating autosampler methods 45 Run table 53 creating advanced autosampler methods 122 D data files viewing 58 diagnostics autosampler 89 dimensions 103 Direct Control dialog opening 56 direct injection 120 disconnecting the system 59 disposal of electrical and electronic equipment 14 disp
45. that channel e 5 L min for a high flow channel e 500 nL min for a low flow channel For the loading pump set channel A to 100 Click Start When the liquid front reaches the black mark on the pipette begin timing e With the high flow calibration assembly the time it takes for the liquid meniscus to travel from the black stripe to the end of the capillary should be 240 seconds e With the low flow calibration assembly the time it takes for the liquid meniscus to transit across two segments of the capillary 2 uL should be 240 seconds 8 Click Stop in the Direct Control dialog when the liquid front reaches the end of the pipette or the appropriate black mark on the pipette 9 Inspect the flowrate to confirm that the system is working within an acceptable range Operator Guide ekspert nanoLC 400 Systems 37 of 174 System Overview Table 3 4 Flowrate Difference from Time sec Action Expected Time lt 5 228 to 252 Do nothing 5 to 20 192 to 228 or Recalibrate the flowmeters Refer to 252 to 288 Calibrate the Flowmeters on page 67 gt 20 lt 192 or gt 288 Refer to Table 6 3 on page 96 in Best Practices and Troubleshooting Contact an AB SCIEX FSE ifthe discrepancy persists 10 Disconnect the calibration assembly and blow out the liquid inside the pipette using a pipette bulb or can of compressed air 11 Set channel B to 95 and repeat steps 5 through 10 to verify the
46. the command on that line Delete Use the Delete button to delete an existing line in the autosampler method ekspert nanoLC 400 Systems Operator Guide 124 of 174 Autosampler Method Editor Edit Use the Edit button to show a list of available options for the command function 1 Click a line in the Command column or field 2 Click Edit A list of available options for that command function opens Command Functions Table E 2 Sample Autosampler Advanced Editor Command and Functions Command Description Available Options Initialize Initializes Autosampler No user configurable values Wait Waits for an event e Wait for Input high or low 1 2 3 or 4 on Autosampler rear VO inputs e Wait for Loading pump to be ready to start Wait for Loading pump to complete injection Wait for Gradient 1 ready to start e Wait for Gradient 1 injection complete e Wait for Gradient 2 ready to start e Wait for Gradient 2 injection complete e Wait for Gradient 1 valve to Load Position e Wait for Gradient 1 valve to Inject Position e Wait for Gradient 2 valve to Load Position e Wait for Gradient 2 valve to Inject Position Get Sample Loads injection Loop Two modes uL Pick Up or Full Loop wit sample Sample Pick Up volume e Needle Height from bottom in mm 2mm is recommended Speed 0 2 uL sec to 15 uL sec Speed depends on sample viscosity temperature loop i d s etc The speed to Get Samples from 1 uL
47. the pump e Push in on the red plastic ring while pulling out on the tubing The system is still pressurized so it may be difficult to remove the tubing 7 Disconnect the communication cables for the LC and autosampler from the computer 8 Disconnect the tubing from the pump to the liquid junction of the source 9 Carefully move the entire system to the new location Install the System at the New Location Install the Eksigent control software on the new computer 2 Connect the pump and autosampler communication cables to any available communication ports on the computer 3 Connect the gas tubing to the pump by pushing the tubing straight into the fitting on the back of the pump 4 Connect the gas tubing to a source of gas clean dry compressed air or nitrogen regulated to 100 psi Connect the column or cHiPLC system outlet tubing to the electrospray source 6 Connect all components that is pump autosampler and cHiPLC system as applicable to the AC mains supply 7 Turn on each component In the bottom right corner of the computer monitor a message similar to Your new device is installed and ready to use will appear Wait for this message before continuing 8 Open the Eksigent control software Operator Guide ekspert nanoLC 400 Systems 59 of 174 Moving the System Communication with the USB COM port on the LC system is established using autodetect Ifthe LC does not autodetect the COM port
48. timer Note Make sure that the computer clock is accurate A secondary timer may be required to verify timing Figure 5 5 Flow Direction High flow End Timer 11 When the fluid front reaches the end of the pipette click Stop 12 Click Next 13 Disconnect the calibration assembly and dispose of the liquid in the pipette 14 Click Finish 15 Repeat this procedure for the low flow channel side A and B Tip For the low flow channel bring the liquid front to the start line and then time the flow for 2 uL 2 divisions on the calibration pipette on channel side A Wait for the flow to travel to the third division 1 uL and then time the flow for 2 divisions 2 uL on channel side B Autosampler Maintenance Basic cleaning and routine maintenance is the responsibility of the customer Maintenance services are available from AB SCIEX Perform the following maintenance procedures to make sure that the autosampler is functioning optimally e Flush the Syringe and Liquid Path on page 71 ekspert nanoLC 400 Systems Operator Guide 70 of 174 System Maintenance Replace the Syringe on page 72 Replace the Sample Needle on page 76 Replace the Wash Bottle Solvent on page 77 Replace the Puncturing Air Needle on page 78 Replace the Sample Loop on page 79 Flush the Syringe and Liquid Path Operator Guide In the Run Manager window click Devices gt Autosampler Device Settings
49. to 5 uL for most purposes is 1 uL sec to 2 uL sec Aspirate Draws liquid into Volume from Sample Table or Fixed 0 uL needle tubing just under syringe volume Speed 0 2 uL sec to 10 uL sec From Position Sample Table Position Fixed or Reagent 1 to 4 e Needle Height in mm from bottom or top Operator Guide ekspert nanoLC 400 Systems 125 of 174 Autosampler Method Editor Table E 2 Sample Autosampler Advanced Editor Command and Functions Continued Command Description Available Options Dispense Pushes sample out of Volume from Sample Table or Fixed 0 needle tubing uL just under syringe volume Speed 0 2 to 10 uL sec To Position Sample Table Position Fixed or Reagent 1 to 4 e Needle Height in mm from bottom Valve Sets ekspert 400 Sample Valve INJECT autosampler Valve i State Sample Valve LOAD e ISS A LOAD 10 1 e ISS A INJECT 1 2 e ISS B LOAD 10 1 e ISS B INJECT 1 2 Output Sets ekspert 400 Output 1 ON wen Hardware Output 1 OFF Output 2 ON Output 2 OFF Output 3 ON Output 3 OFF Output 4 ON Output 4 OFF Output 5 ON Output 5 OFF e Output 6 TTL ON e Output 6 TTL OFF Start Starts 415 425 LC e Start Loading Pump eng Start Gradient 1 Start Gradient 2 Needle Wash Needle Wash Number of cycles 1 to 5 are typical Note Sample valve state not specified and must set to Load Inj first Compressor
50. 0 initial flowrate conditions First establish a column pressure of 3000 psi Sample Injection None O Standard Sample valve opens prior to beginning Flow Profile and remains open O Metered Inject nL of sample at 100 initial flowrate conditions ORapid Inject nL of sample at maximum flowrate maintaining Initial mixture conditions Post Run C Flush column for 1 minutes using 100 ending flowrate conditions View Audit Trail 6 Inthe Sample Injection group click None Refer to Create the LC Method Gradient on page 50 7 Inthe Post Run group make sure that the check box is cleared On the Gradient Table tab specify the loading pump parameters e Type the appropriate Qa flowrate and Time In this method Time is the amount of time that the Loading pump is flowing through the sample loop pushing liquid over to the trap column Make sure that this time is long enough to properly load the sample onto the trap and wash away any salts or detergents ekspert nanoLC 400 Systems Operator Guide 48 of 174 System Overview Figure 3 29 LC Method Settings Dialog Gradient Table Tab B LC Method Settings Selected Method Name LoadingPump_trap_loading lt j Summary Run Conditions Gradient Profile Gradient Table Flow Mode lsocratic View Audit Trail 9 On the Gradient Profile tab review the gradient to make sure it is correct Operator Guide ekspert nanoLC 4
51. 00 Systems 49 of 174 System Overview Figure 3 30 LC Method Settings Dialog Gradient Profile Tab LC Method Settings Selected Method Name LoadingPump_trap_loading v Summary Run Conditions Gradient Profile Gradient Table 50 Flow Mode 27 lsocratic Profile Editor OP Runtime 5 min 25 3 35 4 time min Delete View Audit Trail OK Cancel 10 When the method is complete click Save Create the LC Method Gradient The conditions used for separating the sample are specified in the LC method 1 Inthe Run Manager window select an appropriate channel in the LC channel column Click LC Methods 3 In the Name field type a method name and click Save Optional Specify column information appropriate for the experiment This information is stored with the LC method file ekspert nanoLC 400 Systems Operator Guide 50 of 174 System Overview 5 Onthe Run Conditions tab in the Pre Run group select the Flush column check box and specify a time appropriate to eduilibrate the pump connected tubing and column using 100 initial flowrate conditions Figure 3 31 LC Method Settings Dialog Run Conditions Tab LC Method Settings Selected Method Name CH1_45min_300nLmin_column_trap Summary Run Conditions Gradient Profile Gradient Table Pre Run Flush column for 0 1 minutes using 100 initial flowrat
52. 00 Systems 57 of 174 System Overview View the Data Files Previously collected data files can be opened reviewed and processed flow graphs from previous runs Such information as pressure traces flowrates and percent compositions for A and B may be displayed This allows for diagnostic information to be rapidly displayed and interrogated Refer to Figure 3 36 to see the Acduisition window displaying total pressure at the outlet and flowrates for Gradient 1 channels A andB 7 Note Unused channels not pumping solvent may be used to open run pressure and 1 Click File gt Open 2 Click the data file and then click Open The default location for data files is C Program Files Eksigent NanoLC autosave ekspert nanoLC 400 Systems Operator Guide 58 of 174 Moving the System This section describes how to move the Eksigent ekspert 400 nanoLC system from one location to another if required It assumes that the new location includes a mass spectrometer with a new computer and that the system is on a wheeled cart Complete disassembly of the system is not required Disconnect the System at the Original Location Close the Eksigent control software and mass spectrometer software if open Turn off the pump and autosampler Turn off the cHiPLC system if applicable Disconnect each of the above components from the AC mains supply Turn off the gas supply Oot DM Remove the gas tubing from the back of
53. 01 of 174 Best Practices and Troubleshooting Table 6 6 Autosampler Valve Continued Symptom Inconsistent flowrate Possible Cause Internal leakage in valve Corrective Action Contact AB SCIEX Technical Support Ports are plugged Use a syringe to manually flush each port with cleaning solvent Remove the stator and sonicate for 15 minutes in 100 methanol Contact AB SCIEX Technical Support System does not initiate an injection System flow is unstable Purge the pump and equilibrate the system Flow stabilization set too low Set the flow stabilization limit to greater than 100 nL min on the Advanced tab of the Instrument Configuration dialog ekspert nanoLC 400 Systems 102 of 174 Table 6 7 AID Converter Symptom Possible Cause Corrective Action No signal Cables not connected Check all cables at both ends and make sure they are connected Power not connected Check power LED and reconnect the cable LED not on Communication is lost TO restore communication disconnect the USB cable and then reconnect it Operator Guide System Specifications ekspert nanoLC Pump Specifications Dimensions WxDxH 22 inches x 26 inches x 9 inches 56 cm x 66 cm x 23 cm Weight 26 kg 57 Ibs Working temperature 15 C to 30 C Power e Input line voltage 100 VAC to 240 VAC 250 VAC Input line frequency 50 60 Hz In
54. 05 00015 see note below cHiPLC 200 um ID x 6 mm trap column ChromXP C18 3 um 120 A PN 5015841 e cHiPLC 75 um ID x 150 mm chip column ChromXP C18 3 um 120 A PN 804 0001 e 1 32 inch PEEK nut with glass filled PEEK ferrule PN 5019621 10 pack e Either of the following e Capillary tubing 50 um ID 360 um OD PN 910 00002 2 m and a 1 32 inch sleeve PN 910 00088 10 pack PEEKsil tubing 50 um ID 1 32 inch OD PN 205 00041 50 cm or PN 205 00038 15 cm capacity but may require changes to the LC and autosampler methods Select a column that is stable at high pH such as an InertSustain 200 um ID x 150 mm capillary column from GL Science PN 5020 15537 7 Note Other columns may be used to increase throughput separation or loading 1 Plumb the system as shown in Figure H 2 and Figure H 3 ekspert nanoLC 400 Systems Operator Guide 154 of 174 2 D Reverse phase Reverse phase Liquid Chromatography Workflow 2 Place a mixing tee in the flow path after the high pH column and before port 6 using e 1 32 inch mixing tee e 1 32 inch PEEK nut with ferrule e Either of the following e PEEKSil tubing 50 um ID 1 32 inch OD e Capillary tubing 50 um ID 360 um OD and a 1 32 inch sleeve 3 Connect the loading pump into the mixing tee Figure H 2 Plumbing Diagram Autosampler Valve Sample injection Autosampler Gradient 1 syringe High pH coum a gt Valve port 6 Loading pump O
55. 1 Montor Baseine Valve Direct Control Load Position Load Position Inject Position Column Oven Heater Setpoint 39 C Close Make sure the selected Channel is connected to the autosampler Click Conserved Flow In the A field type 95 and in the Total flowrate field type 1000 nL min Click Start to start the flow and flush the valve ports and capillary On the Direct Control tab for the autosampler rotate the autosampler valve to Inject This flushes air out of the new sample loop Continue flushing until liquid is observed from the open end of the new sample loop Connect the other end of the loop to Port 5 of the autosampler valve Click Stop to stop the flow ekspert nanoLC 400 Systems 79 of 174 System Maintenance Replace the Rotor Seal Required Materials e 3 mm hex key The autosampler is equipped with an injection valve and optionally ISS valves Figure 5 12 Valve with Rotor Seal Exploded Item Description 1 Metal plate 2 Black plastic connector plate 3 18 32 x 3 8 inch bolts 2 4 M4 2 5 6 7 Valve body Seal Stator Disconnect all tubing from the valve Only the sample loop can stay in place Remove the three screws that fasten the valve stator Gently open the valve and take out the rotor seal Clean or replace the seal on the two pins Put the stator on the valve following the orientation on th
56. 74 AlDGONYVEIER ran De sr Me GET es RE OE ERG Ba Appendix B Theory of Operation ss ss ss sk RE RE RE ke ke en ke ke ee Microfluidic Flow Control PluS si see a ee ea RR How the MFC Plus System Works ie EE EE 0c eee eee nen Calculating the Flowrate sac oso ek eg ane EE ee Ga eb Eer UR ae ean rd ie Re er Bee Appendix C External Interface Connections iss EE 0 00020 EE Ee ee ekspert nanoLC 400 Pump saaana anaana ane Pin Assignment As RE EL ses erkenne ekspert nanoLC 400 Autosampler ss EE EE EE EE EE EE ek ee ee Pin Assignment ai nee este PRE DR EE BERE Be ee AID Converter EE GER er Pod E Ge RE GE BA ee Pin Aseigriment ss AR erraten Appendix D Standard Plumbing Diagrams ss sae eek ks se ER RR RR RR ee ekspert nanoLC 415 Systems ii EER EE sea nn nn ekspert nanoLC 425 Systems 2 22 00 unseren RR Ee ke Appendix E Autosampler Method Editor iss ss ss ss EE Ee ee se Create a Basic Method EE EE SE SE SE SE GE GE SE se ernennen Method Editor Options EG Se Ge eee Create an Advanced Method is EE EE EE SE SE EE Ee es eee Advanced Editor Options ii cece nennen Direct Inject Method Commands ss EE EE eee ees Appendix F Basic Methods ER ER ER RR ER RR ER ee Direct Inject Method arie BES EE EN de ES ss BE eh or MADE SDA E Trap and elute Method EE SS SE SS eee ee se ee ee aa Gradient 1 Method su SE RR Ke areas aa ee
57. Application Click Software Application lt not configured gt Click OK Click Setup Device In the Name field click the autosampler Click OK 4 Click Add Device to add the Eksigent control software a 9205 f In the Device Type list click Software Application and then click Software Application lt not configured gt Click OK Click Setup Device to open the Software Application Settings window Click Eksigent 1 and then click OK Repeat step i to step iv for the Loading pump For a nanoLC 425 system repeat step i to step iv for Gradient 2 5 Click Add Device to add the mass spectrometer a b In the Device Type list click Mass Spectrometer Click the appropriate mass spectrometer in the list and then click OK Gi Tip The correct instrument is usually highlighted in the list j gt 6 Click OK to save the profile and close the Create New Hardware Profile window Enable the Column Oven The column oven is an optional accessory If the system does not include a column oven skip this section The temperature of the column compartment can be regulated to maximum temperature of 60 C 1 In the Eksigent control software click System gt Direct Control 2 Inthe Column Oven Heater group in the Setpoint field Figure G 2 type 35 Operator Guide ekspert nanoLC 400 Systems 147 of 174 Working with Analyst Software Figure G 2 Column Oven Heater Group of the Direct Control
58. C 400 Systems Operator Guide 128 of 174 Basic Methods This appendix includes screen captures to illustrate the steps to create a basic method e Direct Inject Method e Trap and elute Method Direct Inject Method Figure F 1 Method Editor Dialog Direct Inject Method Ij Editing AS3 trap elute 1 uLASZ File j Injection Type Direct Injection Trap Elute 5 C Multiplex r Gradient Pump Channel Gradient Pump 1 Gradient Pump 2 r Optional Valves None SS 4 Valve SS B Valve cHIPLE M Sample Pickup wl Pick Up Full Loop fe Sample pick up volume 1 pl 2 ee Needle height J 2 mm 9 Loop volume Wait MA Wait Time 2 min pee ME Trailing Volume 8 1 pl p Wash Syringe wash cycles 5 N Test on B1 Ok Advanced Editor Operator Guide ekspert nanoLC 400 Systems D5033460 C 129 of 174 Basic Methods Figure F 2 Advanced Method Editor Dialog Direct Inject Method ekspert nanoLC 400 Method Editor Description ekspert nanoLC 400 Systems 130 of 174 Autosampler Device for Gradient 1 ready to start Pre wash 1x pL Pickup IpL 0 5 ples 2 mm fror Gradient 1 Injector Inject for Gradient 1 injection complete Injector Load Clean Up 5x wi Test on B1 Ok Operator Guide Basic Methods Figure F 3 LC Method Settings Dialog Run Conditions for Direct Inject
59. Eksigent control software System gt Mobile Phases Table 6 1 Mobile Phase Mixtures Binary Mixture A Binary Mixture B Modifier Gradient 1 100 water 100 acetonitrile 0 1 formic acid Gradient 2 100 water 100 acetonitrile 0 1 formic acid Loading Pump 100 water N A 0 1 formic acid Remove air from the mobile phase bottles weekly by purging the system a minimum of 20 times per channel Keep the pump seal wash bottle filled with 5 isopropanol and change the solutions quarterly Empty the waste bottle once a week or more often if needed Check the flowrate daily Re initialize the pressure transducers weekly Pre swage all fittings ekspert nanoLC 400 Systems 83 of 174 Best Practices and Troubleshooting When cutting silica wash the end with methanol and flow solution through the cut end before connecting the silica Sample Preparation Techniques In general the standard practices and procedures for reversed phase LC MS experiments using electrospray mass spectrometry also apply to the use of the ekspert nanoLC 400 system including the optional cHiPLC system Use HPLC or MS grade solvents at all times Avoid the use of non volatile salts and buffers such as CHAPS phosphate TRIS HEPES and perchlorates These additives will foul the electrospray source and mass spectrometer orifice Centrifuge spin all samples at 10 000 RPM for 5 minutes to remove particulates fro
60. Manager dialog verify the number assigned to the COM port is less than 16 e If not assign the COM port with a lower number Change the COM port setting in the software Contact AB SCIEX Technical Support Constant loud hissing sound from the system Table 6 3 Gas leaks from the gas inlet fitting Flow Control System e Verify the gas tubing is properly connected to the gas inlet fitting e Tighten the gas inlet fitting Contact AB SCIEX Technical Support Symptom System pressure Pc and pump pressures Pa amp Pb show pressure though the flow is off ekspert nanoLC 400 Systems 96 of 174 Possible Cause Incorrect zero setting for pressure sensors Corrective Action Re initialize the pressure transducers refer to Re initialize the Pressure Transducers on page 66 Operator Guide Table 6 3 Flow Control System Continued Best Practices and Troubleshooting Symptom No liquid out of waste line when purging Possible Cause Air trapped in the pump Pump is not primed properly e Tubing is not submerged in mobile phase bottle BoB filters plugged Corrective Action e Purge and flush the system refer to Purge the Mobile Phases on page 65 e Replace the BoB filters Internal filters are plugged Contact AB SCIEX Technical Support Leak in the system prior to the purge valve Contact AB SCIEX Technical Sup
61. Method B LC Method Settings Selected Method Name Gradient 1 1 5min gradient Summary Run Conditions Gradient Profile Gradient Table Pre Run Flush column for 0 1 o First establish a column pressure of Sample Injection O Nore minutes using 100 initial flowrate conditions 3000 psi O Standard Sample valve opens prior to beginning Flow Profile and remains open Metered Inject nL of sample at 20000 O Rapid Inject nL of sample at maximum flowrate maintaining initial mixture conditions Post Run C Flush column for View Audit Trail Operator Guide 125 initial flowrate conditions minutes using ending flowrate conditions ekspert nanoLC 400 Systems 131 of 174 Basic Methods Figure F 4 LC Method Settings Dialog Gradient Profile for Direct Inject Method B LC Method Settings Selected Method Name Gradient 1 1 5min gradient Summary Run Conditions Gradient Profile Gradient Table da Flow Mode A Conserved flow O Independent flow Profile Editor Oa B Runtime 30 min Total flowrate nLANIN 18 21 time min View Audit Trail ekspert nanoLC 400 Systems 132 of 174 Operator Guide Basic Methods Figure F 5 LC Method Settings Dialog Gradient Table for Direct Inject Method LC Met
62. Needle Air Path e Off Compressor 50 power 100 power Unload Syringe Pushes contents of No user configurable settings syringe out to waste ekspert nanoLC 400 Systems Operator Guide 126 of 174 Autosampler Method Editor Direct Inject Method Commands The commands for a typical direct inject method are explained below Figure E 3 Advanced Method Editor Dialog Direct Inject Method ioi se File codes Insert Delete Edit Command Description Initialize Autosampler Device Wait for Gradient 1 ready to start 3 Needle Wash Pre wash 1x Get Sample pL Pickup IpL 0 5 uL s 2 mm fror 5 Start Gradient 1 Valve Injector Inject Wait for Gradient 1 injection complete Valve Injector Load Needle Wash Clean Up 5x Lael Test on B1 Ok Initialize Autosampler Device Zeros the pressure sensor in the ekspert 400 autosampler flushes the Reagent Position with solvent puts the sample injection valve into load and then sets other autosampler functions to initialization states Wait for Gradient 1 ready to start Tells the system to wait until the pump reaches the method flowrate and stabilized before proceeding The user can set a number of system parameters related to being READY These are flowrate stability column temperature distance from set point and pressure limits These values are set in the LC method or other parts of the 415 or 425 hardware configura
63. PUT 4 GND OUTPUT GND OO CO N OO A O N gt oO ekspert nanoLC 400 Systems Operator Guide 112 of 174 External Interface Connections AUX I O Connector The Relay output connector provides 5 relay contacts and a 24 V output The 24 V output is protected against overcurrent and short circuit The relay output connections are freely programmable in the control software The following table shows the Relay Output connector pin out Relay 1a and 1b contacts act simultaneously They can be used to control 2 separate instruments Figure C 5 AUX VO Pin Assignment Pin Description Relay 1a Normally open Relay 1a Closed Relay 1a Normally closed Relay 1b Normally open Relay 1b Closed Relay 1b Normally closed Relay 2 Normally open Relay 2 Closed 9 Relay 3 Normally open 10 Relay 3 Closed 11 Relay 4 Normally open 12 Relay 4 Closed 13 Relay 5 Normally open 14 Relay 5 Closed 15 24 V OUT 500 mA max 16 GND CO N OO A O NI gt Operator Guide ekspert nanoLC 400 Systems 113 of 174 External Interface Connections AID Converter Connect the cables input leads between the converter and the device Connect the USB cable from the converter to the ekspert nanoLC 400 system Pin Assignment Figure C 6 A D Converter Pin Assignment S S S Q 8 8 8 8 IS S
64. Pump Send a signal to start running the Loading pump method 8 Valve Injector Inject Toggle the autosampler 6 port valve to inject and place the sample loop in line with the Loading pump flow 9 Wait for Loading This step waits for the Loading pump method to Pump Complete complete In this method this moves the sample from the injection loop on to the trap column and rinses the trap 10 Valve ISS A Inject The ISS A valve switches to Inject putting the Gradient 1 flow through the trap and then the column 11 Start Gradient Pump1 Signals the Gradient 1 method to start running 12 Valve Injector Load Toggles the injection valve back to Load so that the autosampler wash routine can run through the loop 13 Needle Wash Clean Up 5x The autosampler repeats the wash cycle 5 times 6 To modify method steps click a step and then click Edit Operator Guide ekspert nanoLC 400 Systems 123 of 174 Autosampler Method Editor e Click Insert to add lines commands e Click Delete to delete lines commands e Click Reset to return to the Method Editor dialog Caution Potential System Damage When possible use the basic autosampler Method Editor to create a method Incorrect use of Advanced Editor settings can damage the autosampler Methods created in the Advanced Editor must be hardware safe and analytically reliable Review advanced methods carefully before use 7 Click File gt Save next to the name to save t
65. X makes no other warranty of any kind whatsoever expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose whether arising from a statute or otherwise in law or from a course of dealing or usage of trade all of which are expressly disclaimed and assumes no responsibility or contingent liability including indirect or consequential damages for any use by the purchaser or for any adverse circumstances arising therefrom For research use only Not for use in diagnostic procedures The trademarks mentioned herein are the property of AB Sciex Pte Ltd or their respective owners Eksigent is a division of AB Sciex LLC AB SCIEX is being used under license 2013 AB Sciex Pte Ltd Printed in USA AB Sciex Pte Ltd Blk 33 04 06 Marsiling Ind Estate Road 3 Woodlands Central Indus Estate SINGAPORE 739256 ekspert nanoLC 400 Systems Operator Guide 2 of 174 Revision Log Revision Reason for Change Date A First release of document September 2012 B Corrected plumbing diagrams Revised reference to torgue March 2013 wrench C Updated document template Added appendix with 2 D November 2013 reverse phase reverse phase workflow Added tip about degassing mobile phases Updated default volume for sample loop Updated index Operator Guide ekspert nanoLC 400 Systems D5033460 C 3 of 174 Revision Log eksigent part of AB SCIEX
66. ace the Syringe 35 8 ENE re ea Re VD iR ed RR Re 72 Primethe Syringe ss ER ER re EO EE EE RE Ee oe 74 Replace the Sample Needle Ee ee ee 76 Replace the Wash Bottle Solvent EE Se ee ee 77 Replace the Puncturing Air Needle EE seen nennen 78 Replace the Sample Loop 22 EE EE EE EE cece eee eee 79 Replace the Rotor Seal EE EE ee ee ee 80 DIOLS Vie System sis ea ee haha OE ate a SE RED dee bP MSS AG PD man 81 Prepare the nanoLC 400 Pump for Storage i e Ee ee dee 81 Prepare the Autosampler for Storage ss EE EE EE EE EE EE ee eee 81 Chapter 6 Best Practices and Troubleshooting sis Ek Ra EER RR Re 83 Best Practices st a ea ie OE DORS Ke ern 83 Sample Preparation Techniques EES SE SEE SS eee eee 84 system Diagnostics is RR usa ar nn ek ed 84 Generate System Diagnostics for Service EE EE ee ee 85 Autosampler Diagnostics ii seen rennen nenn 89 Example Flow Path Pressure SE SS SS cece eee ee eens 90 Generate Autosampler Diagnostics for Service EE Ee nn 93 Troubleshooting Guidelines 2 000 cece eee ee 95 Troubleshooting Tables 22 0000 cece eee 96 Appendix A System Specifications 2020 nennen eee 103 ekspert nanoLC Pump Specifications 2 000000 20 103 ekspert nanoLC 400 Autosampler Specifications 104 ekspert nanoLC 400 Systems Operator Guide 6 of 1
67. adient Summary Run Conditions Gradient Profile Gradient Table 100 Flow Mode Conserved flow O Independent flow SO 50 Profile Editor 70 A B 60 Runtime 25 min 50 Total flowrate 40 4 300 nLimin 30 point 4 2 v 40 d t 415 min time min Note Set the mobile phase composition by clicking and dragging points on the graph or by setting the of A or B in the Profile Editor group Standard and Metered Injection Parameters Standard Signals the autosampler to place the valve in the Inject position at the start of the run and to switch back to Load at the end of the run Metered Switches the valve to Inject for a defined volume prior to the start of a run then to Load during the run Example Metered injection of 15 000 nL with a flowrate of 3 uL imin specified in the Gradient Table tab At the start of the Metered injection the 6 port valve on the autosampler switches from Load to Inject The remains in Inject until 15 uL 15 000 nL has flowed through the loop and then back to Load At the specified flowrate of 3 uL min this Metered injection should take approximately 5 minutes to complete ekspert nanoLC 400 Systems Operator Guide 44 of 174 System Overview Create Autosampler and LC Methods Trap and Elute Complete the following procedures 1 Create the Autosampler Method 2 Create the LC Method Loadi
68. ading_Iniection_7uL_20min 2 Summary Run Conditions Gradient Profile Gradient Table View Audi Tia Operator Guide ekspert nanoLC 400 Systems D5033460 C 161 of 174 2 D Reverse phase Reverse phase Liquid Chromatography Workflow Methods for Eluting the Sample from the High pH Column The three methods for Gradient 1 are step gradients with each method more hydrophobic than the previous method The methods run for 25 minutes at a flowrate of 1000 nL min Figure H 9 Fraction 1 Gradient 1 Method 14 Selected Method Name Gradient1_Elution_1uLperMin_14percent v Save Print Summary Run Conditions Gradient Profile Gradient Table Flow Mode Conserved flow 97 independent flow 34 86 14 Profile Editor 23 86 14 Total flowrate 23 1 97 3 1000 nL min Delete View Audit Trail ok Cancel ekspert nanoLC 400 Systems Operator Guide 162 of 174 2 D Reverse phase Reverse phase Liquid Chromatography Workflow Figure H 10 Fraction 2 Gradient 1 Method 25 Selected Method Name Gradient1_Elution_1uLperMin_25percent x Summary Run Conditions Gradient Profile Gradient Table View Audi Tia Operator Guide ekspert nanoLC 400 Systems 163 of 174 2 D Reverse phase Reverse phase Liquid Chromatography Workflow Figure H 11 Fraction 3 Gradient 1 Method 50 Selected Method Name Gradient1_Elution_1uLperMin_50percent x Summary Run Conditions Gradient Pro
69. agnifier is often required to observe the numbers Identify the numbers prior to creating or modifying connectors to the pump When removing the connector cover to solder new connections the location of the pins may appear reversed Double check the orientation prior to soldering The common grounds are all tied together and can be used interchangeably ekspert nanoLC 400 Autosampler Figure C 3 Autosampler Back Panel Item Description 1 Inputs Outputs TTL 2 Auxiliaries 3 RS232 4 MultiLink serial interface 5 Ethernet 6 USB Operator Guide ekspert nanoLC 400 Systems 111 of 174 External Interface Connections Pin Assignment The Eksigent ekspert 400 autosampler has two I O connectors e TTL connector e AUX connector Caution Potential System Damage AB SCIEX will not accept any liability for damages directly or indirectly caused by connecting this instrument to equipment that does not meet relevant safety standards Refer to Figure C 3 on page 111 for location of the connectors TTL VO Connector Inputs 1 and 2 are reserved for synchronization with switching of the injection valve during load and inject steps Indicate in the Eksigent control software which input must be used Synchronization with the load step is the default Figure C 4 TTLVO Pin Assignment Pin Description INPUT 1 sync ready to load GND INPUT 2 sync ready to inject GND INPUT 3 GND IN
70. alibrate Flowmeter Ch 1 Canceled 06 30 12 canceled Set Response Normal DR n dra DR Leak Check Start Leak Test missing missing missing r Usage Information arj Total Sample Injections 0 ar Total Flowmeter Usage 0 aR Fiter Usage mL 0 00 A warning dialog opens to confirm that there is no residual pressure on the pump 6 Confirm that the outlet fittings from the mixing tees on all channels are removed and then click OK A status dialog shows the progress of the re initialization When the system indicates that it is at ambient pressure click OK Repeat this procedure for all channels that is complete the procedure for Gradient 1 Gradient 2 and the Loading pump if available 9 When the re initialization process is complete close the Hardware Diagnostics dialog and return to the Acquisition window Calibrate the Flowmeters Calibrating flowmeters consists of measuring the velocity of a liquid front in a tube of known volume Calibrate the flowmeters as needed for custom solvents First calibrate flowmeter A then calibrate flowmeter B Caution Calibrate the flowmeter if the flowrate is more than 5 and less than 20 outside of the acceptable range Refer to Verify the Flowrate on page 36 Operator Guide ekspert nanoLC 400 Systems 67 of 174 System Maintenance Required Materials Flowrate calibration kit for 1 uL min to 30 uL min flowrate PN 801 00063 Fl
71. ally prime the pump Refer to Prime the Pump 6 Fora wet pump proceed to purge and flush the system Refer to Purge the Pump and Flush the Pump Specify the Maximum Flowrate The Maximum Flowrate is automatically set when a flow module is inserted For most nanoflow applications 1000 nL min is appropriate as shown in Figure 3 7 Figure 3 7 Hardware Options Tab Maximum Flowrate Instrument Configuration System Device 170 Advanced Hardware Options Device Customization channel Maximum Flowrate 1000 nLimin a 1 g Monitor A 20000 B 20000 uL Mobile Phase Storage Loop X C Spectrometer Fi Single Wavelength Detector Column Oven Heater C Solvent Selection Allow Customization MS Control C Turn pumps off when in system Standby Mode Export Settings 1 2 3 4 5 Prime the Pump Click System gt Instrument Configuration On the Hardware Options tab click the correct channel Select the Allow Customization check box Type a Maximum Flowrate in the field Click OK Priming is required for dry pumps It should not be required under normal operating conditions This priming procedure may also be used to rapidly exchange solvents Priming the pump requires manual intervention This procedure involves manually drawing solvent through the fluidic path with a 5 mL syringe While the priming syringe minimizes leachables and extractables with commonly used HPLC solvent systems the solvent drawn i
72. amples to the queue 3 Click View Queue in the toolbar to open the Queue Manager Local dialog Click 7 Equilibrate to equilibrate the LC system and the mass spectrometer 5 When the equilibration is finished click a Start Sample to begin the run Monitor the Run e View the LC chromatogram and spectral data in Explore mode in the Analyst software View flowrate and pressure information in the Acquisition window of the Eksigent control software Operator Guide ekspert nanoLC 400 Systems 151 of 174 Working with Analyst Software eksigent part of AB SCIEX ekspert nanoLC 400 Systems Operator Guide 152 of 174 2 D Reverse phase Reverse phase Liquid Chromatography Workflow This appendix describes an example experiment geparating complex digested protein mixtures with a 2 D separation using the Eksigent cHiPLC system and the Eksigent ekspert nanoLC 425 system configured with a loading pump and two nanoflow gradient pumps Use the example experiment as a starting point for your own experiments The first dimension is a high pH reverse phase separation and the second dimension is a low pH reverse phase separation This workflow takes advantage of the fact that peptides contain both basic and acidic groups which change their charge state and thus their relative hydrophobicity at different pH values Figure H 1 shows the setup for the workflow Figure H 1 System Configuration for a 2 D Reverse ph
73. ase Reverse phase Workflow Sample injection Gradient 2 pH 2 5 300 nL min AS Loop Load 0 2x 150mm C18 column 0 075x 150 mm chip C18 column 1 uL min Gradient 1 pH 9 8 Loading pump pH 2 5 Note This workflow can also be performed using two cHiPLC systems refer to Set Up with Two cHiPLC Systems on page 169 Operator Guide ekspert nanoLC 400 Systems 153 of 174 2 D Reverse phase Reverse phase Liquid Chromatography Workflow The Example Experiment The example experiment consists of a 1 uL sample injected onto the high pH pH 9 6 column The sample is eluted in three fractions using a step gradient The percentage of organic in the mobile phase increases for each fraction After the sample is eluted from the high pH column the pH is adjusted to 2 5 before it enters the trap and chip columns The sample is eluted from the chip column with a water acetonitrile gradient and is then analyzed on a mass spectrometer Workflow Steps Follow these steps 1 Plumb the System Create the Autosampler Methods Create the LC Methods Create the Acquisition Methods and Batch Load the Mobile Phases Run the Batch ou RO ND Plumb the System The system is plumbed as for a standard trap and elute experiment with the addition of the mixing tee after the high pH column Required Materials e 1 32 inch mixing tee PN 200 00316 300 um ID x 150 mm column ChromXP C18 3 um 120 A PN 8
74. ces and Troubleshooting Figure 6 4 Alerts Log Window Log Information B Alerts_2012_03_28 Log Log Information archive E Alets 201203 3 Tristan Williams 2012 03 28 21 55 50 CHO a preg el Tristan Williams 2012 03 28 21 55 41 CHO E Ale ts_2012_04_0 Tristan Williams 2012 03 28 21 29 32 CHO Alerts 2012 04 O H Channel01 I Channel02 E Channel03 E Channel04 E Channel05 3 Channel06 am ag F Channel07 Channel08 Sample Queue System Alerts log Channel01 log SA Channl09 lan dil HE Alerts Creation Date 3 28 2012 9 29 32 PM El Alerts 2012 03 2 Last Modified 3 30 2012 1 56 57 PM E Alerts_ File Location C Program Files Eksigent nn we pas wo jeans slolx Epon C Excel Expat por Message ERROR Registry value an ERROR Registry value an ERROR Registry value an 2 Don t show pop up Alerts Select Log OK Autosave Files When a sample is run the pressure and flowrate data is automatically saved to a text file These Autosave files can be helpful when diagnosing issues with the system 1 Open the Eksigent control software 2 Click File gt Open 3 Navigate to C Program Files Eksigent NanoLC autosave Inside the autosave folder are folders named in YYYYMMDD format This is the date that the data inside the folders was created 4 Open the corresponding folder
75. ct mobile phase setting Check the settings in the Mobile Phases Settings dialog and modify them as needed Pump controller out of tune Contact AB SCIEX Technical Support Flow temperature is not stable Contact AB SCIEX Technical Support System responds sluggishly when changing flowrates ekspert nanoLC 400 Systems 98 of 174 Incorrect mobile phase setting Check the settings in the Mobile Phases Settings dialog and modify them as needed Controllers not tuned properly Contact AB SCIEX Technical Support Operator Guide Table 6 3 Flow Control System Continued Best Practices and Troubleshooting Symptom Inaccurate flowrate with no signs of leakage Possible Cause Incorrect mobile phase setting Corrective Action Check the settings in the Mobile Phases Settings dialog and modify them as needed Incorrect K values Calibrate the flowmeters Refer to Calibrate the Flowmeters on page 67 System pressure Pc is unusually low but flowrate is OK Loose connection after mixing tee Check all connections for leaks System pressure Pc is low and flowrate is OK but pump pressures Pa and Pb are high Incorrect k values Calibrate the flowmeters Refer to Calibrate the Flowmeters on page 67 Flow module is plugged Replace flow module Refer to Re initialize the Pressure Transducers on page 66 Internal filters are plugged Contac
76. d provides symmetrical sealing without major creases grooves or areas that are thinner than 1 mm Caution Potential Component Damage Do not overtighten the syringe The Teflon seal is soft and will flow under pressure and repeated overtightening of the syringe will cause the seal to crush and deform Overtightening the syringe will lead to early seal failure The syringe should be tightened until it meets the seal then very gently turned until it fully seals 7 Note To return the syringe to the normal position click OK in the Exchange syringe af dialog Tips for Installing the Syringe When installing a new syringe we recommend that the syringe seal provided with the new syringe be replaced e Prior to installing the syringe fill it with solvent to remove air After installing the syringe perform several needle washes to fill the syringe e After installing a new syringe reset the log counts for the syringe In the Autosampler Configuration window on the Configuration tab in the Log Counters group click Reset Prime the Syringe When installing a syringe flush the air from the syringe prior to installation Use this procedure to prime the syringe and dislodge any air bubbles that remain after flushing with the Direct Control automatic syringe flushing feature 1 Remove the syringe from the autosampler Refer to Replace the Syringe on page 72 2 Put the tip of the syringe in an appropriate solvent bottle
77. ditor dialog specify the method parameters as shown in Figure E 1 The volumes in Table E 1 can be modified but the general format should remain the same for a given method type Table E 1 Autosampler Trap and Elute Method Commands Step Command Description Comments 1 Initialize Autosampler Places the autosampler valve in load position Device zeros the aspiration path pressure sensor moves the tray to allow needle to flow to waste and starts logging aspiration path pressure 2 Valve ISS A Load Valve allows control of a valve on the autosampler The ISS A valve is placed in Load position to place the trap in line with the Loading pump flow 3 Wait for Gradient 1 Wait for Gradient 1 ready to start after Ready equilibrating The ready tolerance is set in System Instrument Configuration Set the flowrate variations that are tolerated prior to start 4 Needle Wash Pre wash 1x This quick prewash makes sure the needle and flow path are filled with liquid and free of air prior to sample pick up 5 Get Sample Volume Needle In uL Pick Up method the sample is between two Height style uL plugs of transfer liquid In Full Loop method the Pick Up or Full autosampler picks up 30 uL of transport volume Loop and then follows that with 3 times the loop and needle volume from the sample vial 6 Wait for Loading Once Gradient 1 is ready step Previous wait for Pump ready to the Loading pump to become ready start 7 Start Loading
78. e conditions Fi First establish a column pressure of 3000 psi Sample Injection O None Standard Sample valve opens prior to beginning Flow Profile and remains open O Metered Inject NL of sample at 100 initial flowrate conditions Q Rapid Inject laL of sample at maximum flowrate maintaining initial mixture conditions Post Run Fi Flush column for 0 5 minutes using 100 ending flowrate conditions Delete View Audit Trail 6 Inthe Sample Injection group click Standard Refer to Standard and Metered Injection Parameters on page 44 Make sure that the check box in Post Run group is cleared On the Gradient Table tab specify the gradient parameters as shown in Figure 3 da e Add new steps by clicking the gt gt to the left of the table e Delete steps by clicking the X e Set the Total flowrate on the right side of the dialog Operator Guide ekspert nanoLC 400 Systems 51 of 174 System Overview Figure 3 32 LC Method Settings Dialog Gradient Table Tab B LC Method Settings Selected Method Name Gradient pump_15minGradient v Save Print Summary Run Conditions Gradient Profile Gradient Table Flow Mode o Conserved flow O Independent flow Profile Editor Total flowrate 300 nLimin View Audit Trail 9 On the Gradient Profile tab review the gradient to make sure it is correct Figure 3 33 LC Method Settings Dialog Gradient Profile Tab
79. e in the wash speed Operator Guide ekspert nanoLC 400 Systems 91 of 174 Best Practices and Troubleshooting Figure 6 7 Flow Path Pressure Trace During an Inside Sample Needle Wash Series 1 5 10 and 15 uL sec Autosampler Configuration Configuration Direct Control Maintenance FlowPath Pressure Pressure mBar 12 Zero value mBar 1232 Upper Limit mBar 7000 Lower Limit maar 1000 Reset Chart Status Device Idle Driver Waiting for method Valve Position Inject ISSA Position Inject 2 ISSB Position Inject 2 Trav 8 C ekspert nanoLC 400 Systems 92 of 174 Pressure mBar 0 Di Aa External WO output 1 C Output 2 C Output 3 3 5 8 Time min C Output 4 C Output 5 C Output 6 7 8 9 10 Operator Guide Best Practices and Troubleshooting Generate Autosampler Diagnostics for Service 1 Open the Eksigent control software 2 Click Run Manager 3 Click Devices gt Autosampler Device Settings 4 On the Diagnostics tab click Reset Chart Figure 6 8 Autosampler Configuration Dialog Diagnostics Tab x Configuration Direct Control Maintenance Diagnostics r FlowPath Pressure 1 0 Pressure mBar 0 _ 08 Set Zero a E 06 Zero value mBar 1013 g Upper Limit mBar 7000 Lower Limit mBar 500 gt oa 0 0 00 1 0 Reset Chart Time min m Status r External VO Device pi
80. e instrument labels Install and fasten the three screws NO a PWN gt Connect all tubing to the valve ekspert nanoLC 400 Systems Operator Guide 80 of 174 System Maintenance 8 Initialize the autosampler to make sure that the valve is in the Inject position 9 Perform a standard wash The autosampler is now ready for use Store the System If the system needs to be stored for a period of time or shipped to a different location prepare the pump and autosampler for storage Retain the original packaging and use it to store and ship the system Contact AB SCIEX customer support for shipping supplies Refer to Moving the System on page 59 for information on moving the system in the same location Prepare the nanoLC 400 Pump for Storage 1 Change the mobile phases to an alcohol or acetonitrile for both A and B channels on all pumps Note Removing the aqueous phases is required to reduce the possibility al of bacterial growth Flush the flow modules A and B 3 Purge and flush the system to remove all water and mobile phases Refer to Flush the Syringe and Liquid Path on page 71 4 Label the system for storage to make sure that the next user is aware that the system contains alcohol or acetonitrile Prepare the Autosampler for Storage Thoroughly flush the system Disconnect and remove all tubing except for the sample loop Turn off the autosampler EA If the samples contained biohazardous
81. e the Run Table is EE EE EG EE Ee Ee Ee ke ke ke ke ke een 53 SIAM TS RUN 222 2 Es MERE RD ss ON tied ae Sodas eae gata 54 View the Run Status in the Run Manager Window 2005 55 Stop a Run in the Run Manager Window 00 eee eee ee eee 56 Control Buttons su Gas Nak ala Gn ae Re kie aad 56 Add or Remove Traces in the Acquisition Window 57 View the Data Files 20 0 0 c ccc tees 58 Chapter 4 Moving the System 0 c eee eee ER RR RR RR AR RR RR ee 59 Disconnect the System at the Original Location 00000 59 Install the System at the New Location 0 0 cee eee eee 59 Chapter 5 System Maintenance 0 0 nennen nn 63 Recommended Maintenance Schedule 0 000 eee eee 63 Dispose of System Waste ies se EE EE ER ee eee 64 Pump Maintenance a ccc ee ee ee ee ee ee ee 64 Clean and Inspect the System 0 EE EE eee EE EE EE se ee ds nn 64 Purge the Mobile Phases iss EE EE EE EE EE se Ee ke ee eee eee 65 Flastrilie System ss gate EER EE BA KA EA EN au Be BRA EDE oe ROME Bl ES 66 Replace the Seal Wash EE EE EE 0c eee 66 Re initialize the Pressure Transducers ss EE EE Ee ke ke ee 66 Calibrate the Flowmeters SE ES EE GE GE Ge ee Ge ee ee eee 67 Autosampler Maintenance EE cece ete eee 70 Flush the Syringe and Liquid Path ee SS cece eee eee 71 Repl
82. ekspert nanoLC 400 Systems Operator Guide D5033460 C November 2013 This document is provided to customers who have purchased AB SCIEX equipment to use in the operation of such AB SCIEX equipment This document is copyright protected and any reproduction of this document or any part of this document is strictly prohibited except as AB SCIEX may authorize in writing Software that may be described in this document is furnished under a license agreement lt is against the law to copy modify or distribute the software on any medium except as specifically allowed in the license agreement Furthermore the license agreement may prohibit the software from being disassembled reverse engineered or decompiled for any purpose Warranties are as stated therein Portions of this document may make reference to other manufacturers and or their products which may contain parts whose names are registered as trademarks and or function as trademarks of their respective owners Any such use is intended only to designate those manufacturers products as supplied by AB SCIEX for incorporation into its equipment and does not imply any right and or license to use or permit others to use such manufacturers and or their product names as trademarks AB SCIEX warranties are limited to those express warranties provided at the time of sale or license of its products and are AB SCIEX s sole and exclusive representations warranties and obligations AB SCIE
83. el ekspert nanoLC 400 Systems Operator Guide 166 of 174 2 D Reverse phase Reverse phase Liquid Chromatography Workflow Create the Acquisition Methods and Batch Create the Acquisition Methods In the Analyst software create four acquisition methods one for injecting the sample and three for the separation and analysis of the three fractions Create the acquisition methods as shown in Table H 1 using the autosampler and LC methods created previously For the MS Methods create two methods appropriate for the mass spectrometer One method should have a 1 min acquisition and the other should have 85 min Table H 1 Acduisition Methods for a Three Fraction 2 D Reverse Phase Reverse Phase Separation Acquisition Autosampler Gradient Gradient Loading MS Method Method Method Pump 1 Pump 2 Pump Name Method Method Method Sample Autosampler Gradient 1 Gradient 2 Loading Pump Method with Injection Injection Injection Injection Injection 1 min Method Method Method Method acquisition duration Fraction 1 Fraction 1 Gradient 1 Method 14 Method with Fraction 2 Autosampler Fraction 2 Gradient 2 Loading Pump 85 min Elution Method Gradient 1 P Analysis Elution acquisition Method 25 Method Method duration Fraction 3 Fraction 3 Gradient 1 Method 50 For experiments with more than three fractions in the first dimension an additional acquisition method is needed for each fraction C
84. elow channel procedures and entering the requested information accurately Lo ading Failure to do so may result in malfunction of the instrument DO NOT P re use values from previous calibrations Proceed deliberately ump Step 1 The following mobile phases are currently selected as active on this system Please verify If incorrect Cancel and make the appropriate selections in the Method Menu Mobile Phase A 100 Aqueous Solution 20 uLidivision wil calibrate at 5 Lamin If the Mobile Phases are not correct click Cancel and then make the necessary changes in the Mobile Phases dialog Repeat these steps Assemble the high flow calibration assembly with the correct pipette as shown in Figure 5 4 Figure 5 4 20 uL Calibration Assembly High flow mozi Attach the assembly to the pump outlet In Step 2 of the Flowmeter Calibration wizard specify the pipette size Refer to Table 5 4 to select the appropriate pipette size and flowrate for calibrating the channel e High flow channel Set the pipette size to 20 uL division ekspert nanoLC 400 Systems 69 of 174 System Maintenance e Low flow channel Set the pipette size to 1 uL division 8 Click Next to start the flow in channel A 9 In Step 3 of the Flowmeter Calibration wizard specify the appropriate Volume 10 Wait for the liquid front to cross the black line on the pipette and then click Start to begin the
85. ent mm 0 Reagent Solvents Align syringe plunger 2 mm from upper position Exchange Tra The Exchange syringe dialog opens and the syringe moves to the middle position ekspert nanoLC 400 Systems Operator Guide 72 of 174 Figure 5 7 Syringe 3 Follow the on screen instructions Operator Guide Item Description 1 Syringe valve 2 Nut 3 Syringe clips System Maintenance a Turn the nut at the top of the syringe counter clockwise to remove the syringe ekspert nanoLC 400 Systems 73 of 174 System Maintenance b Apply tension to the underside of the syringe grasp the barrel and then pull it forward out of the clips Be careful not to bend the plunger c Verify that the syringe seal is uniform and that it properly seals the tip and the valve body The syringe seal a small white Teflon disk sits between the end of the glass syringe tip and the syringe valve body This seal is at the top of the cavity from which the syringe was removed The seal is difficult to observe in situ A small angled mirror is useful for examining its state Alternatively remove the seal for examination by using the tip of a plastic Eppendorf style pipette tip or a similar tool Insert the narrow plastic tip into the valve orifice Aim the tip at the center of the syringe seal and move or rock the tool to dislodge the seal After removing the seal examine it to make sure that it is uniform an
86. ent 1 DB 25 connection Gradient 2 DB 25 connection 425 only Loading pump DB 25 connection Fan Column oven connection USB port Solvent inlets Power supply connection ol OO N OO BO ND gt Power switch Gas input a O Purge waste and pump seal lines Operator Guide ekspert nanoLC 400 Systems 109 of 174 External Interface Connections Pin Assignment Table C 1 Pin Assignments for DB 25 Connector ekspert nanoLC 400 Systems 110 of 174 Pin Number Function Pin Number Mate Top Row large side 1 Aux4 14 2 Aux3 15 3 Valve Load Aux2 16 4 Valve Inject Aux1 17 5 Valve out 18 6 Run out 19 7 Prk out 20 8 Rdy out 21 9 Common ground 10 Not used 11 Not used 12 Prk in 9 22 25 13 Run in 9 22 25 Bottom Row small side 14 Aux4 1 15 Aux3 2 16 Valve Load Aux2 3 17 Valve Inject Aux1 4 18 Valve Out 5 19 Run out 6 20 Prk out 7 21 Rdy out 8 22 Common ground 23 Common ground 24 Common ground 25 Common ground Operator Guide External Interface Connections Figure C 2 25 pin DB Connector Male Fitting The 25 pin DB connector in Figure C 2 is a male connector viewed end on that is from the exposed male pin side and not from the hidden solder post side D connectors have very small numbers inscribed on them indicating the pin numbers a high power m
87. ered from VWR PN BJLC452 2 5 0 1 Formic Acid Water HPLC grade water with 0 1 formic acid PN BJLC441 2 5 0 1 Formic Acid Acetonitrile acetonitrile with 0 1 formic acid Table 3 3 Typical Mobile Phase Mixtures Binary Mixture A Binary Mixture B Modifier Gradient 1 100 water 100 acetonitrile 0 1 formic acid Gradient 2 100 water 100 acetonitrile 0 1 formic acid Loading Pump 100 water N A 0 1 formic acid As shown in Figure 3 6 the mobile phase outlets are located on the right side of the back of the pump The gradient pump outlet or outlets is located at the top Figure 3 6 Back Panel Pump Mobile Phase Outlets 5 Required Materials e HPLC grade mobile phase A e HPLC grade mobile phase B e Waste container that can contain at least 100 mL of solvent Discard old solvents that are in the mobile phase bottles Clean the bottles with appropriate solvents 3 Fill bottle A with mobile phase A and bottle B with mobile phase B Refer to Table 3 3 Place the tubing and filters in the bottles and secure the lids on the bottles Place the refilled bottles on top of the autosampler in the solvent tray ekspert nanoLC 400 Systems Operator Guide 22 of 174 System Overview Caution Potential System Damage If the pump is dry manu
88. ert nanoLC 400 Systems 11 of 174 Regulatory and Safety Information WARNING Potential System Damage Do not disconnect an electrical assembly while power is applied to the system Once the power is turned off wait approximately 30 seconds before disconnecting an assembly WARNING Potential System Damage Do not expose the system to excessive voltage fluctuations or power surges The system contains a number of sensitive electronic components that may be damaged Qualified Personnel After installing the system the Field Service Employee FSE uses the Customer Familiarization Checklist to familiarize the customer on system operation cleaning and basic maintenance Only trained personnel shall install operate and maintain the equipment Equipment service shall only be conducted by FSEs or AB SCIEX authorized dealers Contact AB SCIEX Technical Support for more information or visit www absciex com Equipment Use and Modification Use the system indoors in a laboratory that complies with the environmental conditions recommended in the Site Planning Guide If the system is used in an environment or in a manner not prescribed by AB SCIEX the protection provided by the equipment can be impaired Unauthorized modification or operation of the system may cause personal injury and equipment damage Caution Potential System Damage Contact the manufacturer prior to making changes or modifications to the system Changes or
89. es x 11 5 inches x 3 inches 26 cm x 29 cm x 7 5 cm Weight A D Converter 4 5 pounds 2 kg Power via USB Dimensions WxDxH 3 2 inches x 3 1 inches x 1 1 inches 82 mm x 79 mm x 27 mm Number of channels 3 Analog in 10 V to 10 V or 1 Vto1V Communication USB 2 0 full speed USB 1 1 compatible Maximum sampling rate Operator Guide 100 Hz ekspert nanoLC 400 Systems 105 of 174 System Specifications eksigent part of AB SCIEX ekspert nanoLC 400 Systems Operator Guide 106 of 174 Theory of Operation The ekspert M nanoLC 415 and 425 systems are ultra high pressure liquid chromatography systems optimized for nanoflow and microflow LC MS MS analysis The systems incorporate microfluidic flow control MFC Plus to generate precise LC gradients at nano and micro flowrates Microfluidic Flow Control Plus MFC Plus has the following benefits Use of fast flow measurement and feedback to a pressure source pump to ensure excellent flow rate accuracy and precision e Excellent flow measurement precision ensures long term retention time stability e Interchangeable flow modules allow for a wide flow rate range Built in diagnostics for improved up time and serviceability The components of a binary gradient MFC Plus system are shown schematically in Figure B 1 Figure B 1 Schematic Showing Microfluidic Flow Control MFC System Components
90. etermined by measuring differential pressure across a calibration module of known geometry giving it a fixed flow conductance The flowrate is given as Q k AP u where Q flowrate k flow conductance AP differential pressure u viscosity of mobile phase The flowrate measurement is made only on the individual mobile phases While these mobile phases may themselves be mixtures for example water and acetonitrile the mixture fractions and therefore mobile phase viscosity remain constant The system software contains mixture viscosity parameters for a wide variety of frequently used mobile phases The internal temperature of the system is controlled and measured to allow determination of temperature corrected viscosity To verify system operation the flow conductance k is measured and corrected using the flowmeter calibration routine in the software ekspert nanoLC 400 Systems Operator Guide 108 of 174 External Interface Connections This appendix shows the external interface to other components The external interface connections synchronize sample injection with data collection The connector pin assignments are also described ekspert nanoLC 400 Pump Figure C 1 Pump Back Panel o Q soe d 0 i x L o s J amp is fe JU EG E A Jp IAN Item Description Gradi
91. file Gradient Table View Auch Trl ekspert nanoLC 400 Systems 164 of 174 Operator Guide 2 D Reverse phase Reverse phase Liquid Chromatography Workflow This method for the Loading pump runs for 25 minutes as it dilutes the sample and lowers the pH before the sample enters the trap and the chip column Figure H 12 Loading Pump Elution Method Selected Method Name Loading Elution 7uL 25min 2 Summary Run Conditions Gradient Profile Gradient Table View Audit Trail Operator Guide ekspert nanoLC 400 Systems D5033460 C 165 of 174 2 D Reverse phase Reverse phase Liquid Chromatography Workflow Analysis Method for the Chip Column The example experiment uses a single method to elute the sample from the chip column using the Gradient 2 pump After the sample is eluted it is analyzed on the mass spectrometer This method is a gradient from 5 B to 40 B at a flow rate of 300 nL min for 60 minutes followed by a short wash at high organic and then a return to the original column conditions Figure H 13 Gradient 2 Analysis Method Selected Method Name Gradient2_Analysis_300nLperMin v Save Print Summary Run Conditions Gradient Profile Gradient Table Flow Mode Conserved flow Independent flow Valve Inject 60 60 40 62 10 90 69 10 90 Total flowrate 72 95 5 300 nl min Valve Load Profile Editor Delete View Audit Trail OK Canc
92. ge the pressure e Changing the tubing i d or length can also change the pressure e Clogging of the needle will change the pressure readings that are observed e Exceeding the pressure limit settings of the flow path halts the autosampler with an error The graphical display may be helpful for autosampler operational diagnosis Monitor the aspiration path pressure in order to isolate and identify the source of a problem The autosampler pressure can be used in a variety of approaches for example to determine if an aspiration needle is clogged or if the aspiration speed is too high for a given loop and sample viscosity Tip Note and document the standard flow path pressure variation during an injection cycle The values will depend on many variables for example syringe speed loop i d loop length transport solvent viscosity sample viscosity wash solvent viscosity and temperature It is possible to build a method with different wash sample aspiration and dispense speeds One way to minimize run time is to speed up syringe motion Note The gradual partial clogging of a needle can be detected prior to a total failure a Two needles or loops might be compared to see if one is clogged Example Flow Path Pressure Figure 6 6 shows an example of how the Diagnostics tab may be used It illustrates the impact of varying the wash speeds on flow path pressure In this example 100 water with a 3 uL loop is used N
93. gee Note Refer to Create an Advanced Method on page 122 for a detailed description of how to create an advanced method Create the LC Method Loading Pump In the LC Channel column select Loading pump 2 Inthe Run Manager window click LC Methods Figure 3 27 LC Method Settings Dialog Summary Tab LC Method Settings Selected Method Name LoadingPump_trap_loading Summary Run Conditions Gradient Profile Gradient Table Method Identification Method ID 99 Column Information Manufacturer eksigent particle size 5 am Type N A diameter 75 am Serial Number n A length 15 cm Sample Injection Flow Profile Standard Duration 5 min Detection External Detector Auxillary AD channel available Delete View Audit Trail 3 In the Name field type a method name and click Save Optional Specify column information appropriate to the experiment This information is stored with the LC method file 5 On the Run Conditions tab in the Pre Run group select the Flush column check box and specify a time of 0 1 minutes using 100 initial flowrate conditions Operator Guide ekspert nanoLC 400 Systems 47 of 174 System Overview Figure 3 28 LC Method Settings Dialog Run Conditions Tab B LC Method Settings Selected Method Name CH1 45min 300nLmin column trap Summary Run Conditions Gradient Profile Gradient Table Pre Run Flush column for 0 1 minutes using 10
94. grams on page 115 Figure D 2 to Figure D 4 The injection cycle in a trap and elute method is as follows 1 Load The sample is loaded into the ekspert 400 autosampler sample loop 2 Inject The loading pump moves sample out of the sample valve The loading pump continues pumping to move the sample onto the trapping column installed on the ISS A valve 3 The loading pump continues pumping to wash the bound sample The ISS valve toggles to the Inject position placing the trap column in the gradient flow path 5 Trap Elute The gradient starts and then elutes the compounds from the trap onto the analytical column and then to the mass spectrometer ion source Gradient Pump Channel The nanoLC 415 systems that have only a Gradient 1 pump cannot run methods that use the Gradient 2 pump Users can create a method that uses a Gradient 2 pump but this method will not run on a nanoLC 415 system The nanoLC 425 system offers the choice of using either of the two gradient pumps in a method Make sure to match the method with the plumbing and requirements Optional Valves The Optional Valves options are only available for trap and elute methods and cHiPLC system methods that require multiple valve control The standard ekspert 400 autosampler configuration has one ISS valve ISS A The valve of the optional cCHiPLC system can also be controlled in the Optional Valves section Sample Pickup Samples can be picked up using either Full
95. handle to obtain the calibrated results 9 Flush the module with a minimum of 50 uL of the current solvent When you change the modules the pump may seem to have zero pressure on Pc Because Pc is used to calculate flow the channel may initially not seem to pump It is actually pumping out the air introduced during the module change Once the air is gone Pc will come up the liquid begins flowing through the channel and the software shows normal pressures Set the Column Oven Temperature The column oven is an optional component It is associated with a single Channel On the back panel the column oven connects to Channel 1 1 Click System gt Direct Control 2 Inthe Direct Control dialog select Gradient 1 3 Type the column Setpoint temperature for example 40 C Figure 3 10 Direct Control Dialog Setpoint Temperature Direct Control Pump Direct Control waiting for LC Method Channel Total flowrate Conserved Flow 90 10 400 nLimin 4 Gradient Independent Flow 400 nLimin z 1 Monitor Baseine Column Oven Heater Setpoint 40 C Close 4 In the Column Oven Heater group click Start Operator Guide ekspert nanoLC 400 Systems 29 of 174 System Overview The column begins to heat The current temperature is visible in the upper right corner of the Acquisition window for Gradient 1 when the oven is installed and run on Figure 3 11 Acquis
96. he LC Method Settings dialog e Methods for Loading the Sample on the High pH Column e Methods for Eluting the Sample from the High pH Column e Analysis Method for the Chip Column Methods for Loading the Sample on the High pH Column This method for Gradient 1 runs for 20 minutes at 1 uL min Figure H 6 Gradient 1 Injection Method Selected Method Name Gradienti_Inject_1uLperMin v Print Summary Run Conditions Gradient Profile Gradient Table Flow Mode Conserved flow Independent flow Profile Editor Total flowrate 1000 nL min Delete View Audit Trail OK Cancel Operator Guide ekspert nanoLC 400 Systems 159 of 174 2 D Reverse phase Reverse phase Liquid Chromatography Workflow This method for Gradient 2 acts as a placeholder in the Analyst software acquisition method Figure H 7 Gradient 2 Injection Method Selected Method Name Gradient2_Injection_1uLpermin hd Summary Run Conditions Gradient Profile Gradient Table Flow Mode Conserved flow Independent flow Profile Editor Total flowrate 300 nL min Delete View Audit Trail OK Cancel ekspert nanoLC 400 Systems Operator Guide 160 of 174 2 D Reverse phase Reverse phase Liquid Chromatography Workflow This method for the Loading pump also acts a placeholder in the Analyst software acquisition method Figure H 8 Loading Pump Injection Method Selected Method Name Lo
97. he method Advanced Editor Options The Advanced Editor extends the functionality of the editor Use the Advanced Editor to add or edit steps to methods created in the editor We strongly recommend first creating a method using the Method Editor and then modifying it with the Advanced Editor Although the Advanced Editor offers the most functionality methods generated by the Method Editor are suitable for most applications To learn more about autosampler programming create wizard based methods save them and then open them using the Advanced Editor In the Advanced Editor review the existing functions parameter settings and sequences Caution Potential System Damage Before using the Advanced Editor it is important to know how each function works Hardware damage may occur by improper use of the features in the Advanced Editor The following options are available in the Advanced Editor File Use the File option to save open and name methods Insert Use the Insert option to add a line to the autosampler method After inserting a line associate it with a command function Refer to Command Functions on page 125 1 Click in any part of a line and then click Insert A new line is inserted above the existing line Click in the Command field for any of the lines to open a menu command selector 3 Scroll to one of the available commands and then release the mouse to change the line to that command 4 Configure or edit
98. he oven is enabled in software Temperatures Faulty Table 6 5 Autosampler Cable loose Tighten loose cables Hardware failure Contact AB SCIEX Technical Support Symptom Eksigent control software does not recognize the autosampler when Run Manager starts Possible Cause Communication error between the autosampler and computer Corrective Action Verify the USB cable is securely connected to the autosampler communication port Software may be configured for a different COM port than the autosampler is using If not using USB determine which COM port is configured for the software on the System tab of the Instrument Configuration dialog Ifneeded select a different port Autosampler aspirating too little volume Partial clog in the aspiration path Replace the sample needle e Replace the buffer tubing e Monitor the pressure during an injection cycle with old and new components Contact AB SCIEX Technical Support Autosampler not recognized message when activating the hardware profile in the Analyst software ekspert nanoLC 400 Systems 100 of 174 Communication problem between autosampler and Analyst software Turn the autosampler on and off to reactivate the hardware profile and the COM port setting e Open the Analyst software Operator Guide Table 6 6 Autosampler Valve Best Practices and Troubleshooting
99. hod Settings LoadingPump_Trap_Loading Operator Guide ekspert nanoLC 400 Systems D5033460 C 133 of 174 Basic Methods Trap and elute Method Figure F 6 Method Editor Dialog Trap and elute Method Editing AS3 trap elute 1 uLASZ File lalx Injection Type Direct Injection Trap Elute C Multiplex r Gradient Pump Channel Gradient Pump 1 Gradient Pump 2 Optional Valves None cHiPLC ISS A Valve 155 B Valve M Sample Pickup pl PickUp FullLoop DE Sample pick up volume 1E Needle height 2 mm Loop volume 10 pl Wait Wait Time 2 min ia am Trailing Volume 8 1 pl Wash Syringe wash cycles 5 Advanced Editor Test on B1 Ok ekspert nanoLC 400 Systems 134 of 174 Operator Guide Figure F 7 Advanced Method Editor Dialog Trap and elute Method Command ekspert nanoLC 400 Method Editor Description Initialize Valve Wait Needle Wash Get Sample Wait Start Valve Wait Valve Start Valve Needle Wash Operator Guide Autosampler Device ISS A Load for Gradient 1 ready to start Pre wash 1x uL Pickup 1pL 0 5 pL s 2 mm from bottom for Loading Pump ready to start Loading Pump Injector Inject for Loading Pump complete ISS A Inject Gradient Pump 1 Injector Load Clean Up 5x Test on B1 Ok
100. i v 2500 2500 2500 Zero offset psi 555 574 606 Control Parametres Field Service At A B B gain 0 424 042 0245 0 241 int 1 0 1 0 1 0 1 0 slope Bauen 022 022 offset 750 98 225 96 683 88 643 4 Take a screen capture of this dialog by pressing Shift PrintScreen or Function PrintScreen depending on the keyboard setup Operator Guide ekspert nanoLC 400 Systems 85 of 174 Best Practices and Troubleshooting Open WordPad and create a document Paste the screen capture in the document Save the document o no a Send the document in an email to the FSE Leak Test Diagnostic 1 Open the Eksigent control software 2 Click System gt Hardware Diagnostics 3 Inthe Leak Check group click Start Leak Test 4 Follow the on screen instructions Figure 6 2 Leak Test Prerequisites Window Leak Test Prerequisites gq x Please verify the following 1 flowmeter calibration is recent and correct 2 the pumps have been purged fiushed and have no air bubbles 3 the pressure transducers have been zeroed recently 4 The pump outlet has been plugged If all of these conditions have been met please proceed If not press Cancel and return to this diagnostic when ready 5 Click OK to start the test ekspert nanoLC 400 Systems Operator Guide 86 of 174 Best Practices and Troubleshooting Alerts Log The Alerts Log window contains alerts and errors generated by the system The FSE may use this
101. iew Acquire Tools Explore Window Script Help BEHSR amp BB DO acauire Mode va n Tn id T ax E Bi quatro testing vise KE OR GH Configure A Security Configuration GH Hardware Configuration amp Report Template Editor UP Tune and Calibrate A Compound Optimization AY Instrument Optimization oxi Acquisition method Acquisition Method Ef Mass Spec 29 999 min E Period 29 999 min SB m BS Eksigent 453 Pay Eksigent Gradient 1 3 Eksigent Gradient 2 BS Eksigent Loading Pump Software Application Properties Path C Program Files Eksigent Nanol C settings method Filename CH1 45min 300nL min column trap ini i Manual Tuning FA Acquire 1 X IDA Method Wizard JE Build Acquisition Method Fe Build Acquisition Batch 27 Express View AR Explore Open Data File amp Open Compound Database LZ Quantitate EF Build Quantitation Method X Quantitation Wizard 7 Review Results Table Companion Software Eksigent as3 E Eksigent Gradient 1 E Eksigent Gradient 2 E Eksigent Loading Pump E Reporter 32 For Help press F1 User Name AMFRMW GSTEK1 abservice Di Analyst Data Idle E3 Idle 4 Select the LC method s a Inthe Acquisition Method window click Loading Pump b Click Browse to view the available methods c Select the loading pump method created above and then click Open d Repeat these steps for the gradient 1 me
102. in on a particular area of the chromatogram click the display and drag a box around the area of interest to enlarge that area To zoom back out right click and select Zoom Out or Back Status information such as A B and Time Remaining is shown at the bottom of the screen during the run Status bars at the top of the window show the actual flowrate for pump A Qa and pump B Qb and several pressures View the Run Status in the Run Manager Window During a run the color of the row in the run table indicates the status e White the run can be started e Light green equilibrating e Dark green running e Red stopped e Yellow an error occurred e Gray completed Operator Guide ekspert nanoLC 400 Systems D5033460 C 55 of 174 System Overview Stop a Run in the Run Manager Window 1 Click Stop to stop the run 2 To start again either e Click Start to start the run again beginning at the next row in the run table or e Click 3 Reset to clear the status of every row in the run table and then click Start to start the run again at the first row in the run table Control Buttons Table 3 5 shows the buttons available in the Acquisition window Table 3 5 Control Buttons in the Acquisition Window Click To Pause the run The system is maintained with its current conditions indefinitely until the run is stopped or resumed To resume the run click Resume To stop the run click 8
103. indow after changing the tray type Figure 3 20 Run Manager Window Run Manager File Edit View Devices Help Run Table default ini Run Autosampler Le Other A Method Tray Vial Method Channel Data Filename Comments Status Direct_Injection_1ul_Gradient1 1 408 Gradient1 15min Gradient Gradient 1 Queued Direct_Injection_1ul_Gradient1 1 A06 Gradient1 15min Gradiert Gradient 1 Queued Direct Injection 1ul Gradient1 11807 Gradient1 15min Gradient Gradient 1 Queued Direct Injection 1ul Gradient1 1405 Gradient1 15min Gradient Gradient 1 Queued Direct_Injection_1ul_Gradient1 1403 Gradient1 15min Gradient Gradient 1 Queued Direct_Injection_1ul_Gradient1 11H09 Gradient1 15min Gradient Gradient 1 Queued Current Tray Autosampler Method Definitions Run Sequence Sequential Trayi A Autosampler Methods v Device Idle Driver Waiting for method Valve Position Inject Tray 25 7 C Pause LC Methods Analysis Methods Flush Equilibrate when Idle OAs Available O Synchronized Multi Channel 5 stat Elapsed Time 00 00 00 Queued Time 03 30 00 3 Make sure that the Run Manager window shows the columns in Figure 3 20 e If they do not show then click Edit gt Choose Columns and select the appropriate column headings Click OK 4 Inthe Run Manager window click Autosampler Me
104. information to determine the root cause of an issue 1 Open the Eksigent control software 2 Click View gt System Logs gt Alerts Figure 6 3 Alerts Log Window oix Log Information LI archive Bf Aletts log Creation Date 4 12 2012 9 17 31 AM C Excel E Channeld log Last Modified 4 12 2012 9 18 02 AM Export ane ChannelD2 log File Location C Program Files Eksigent NanoLC logs 4lerts log Channel03 log Channel04 log User Date Channel Message Channel05 log Tristan Williams 2012 04 12 09 18 02 CHO ERROR Registry value and Default value for LCEditorType not found A de a e Tristan Williams 2012 04 12 09 17 31 CHO ERROR Registry value and Default value for LCEditorType not found A de Channel06 log 5 Channel07 lag a Channel08 log Sample Queue log IE System log 4 gt Don t show pop up Alerts Select Log OK 3 Click Export to export the alerts to PDF or Microsoft Excel format 4 Send the exported file in an email to the FSE of the Alerts Log window Figure 6 4 and then expanding the Alerts Log folder The Alerts Log folder contains files for each day when there was an alert or error To see the information for a particular day highlight the appropriate date and follow the on screen instructions 7 Note Alternatively view historical data by expanding the archive folder on the left side Operator Guide ekspert nanoLC 400 Systems 87 of 174 Best Practi
105. ing air needle 5 Install a new needle assembly Make sure the needle is secured by the needle retainer Connect the needle to the injection valve using the correct ferrule and nut On the Autosampler Configuration dialog Direct Control tab click Reinitialize 8 Perform awash routine to clean the new needle Replace the Wash Bottle Solvent 1 Remove the cap of the wash solvent bottle 2 Empty the contents into an approved and appropriate waste container 3 Fill the wash solvent bottle with 50 50 methanol water 4 Replace the cap 5 Perform a wash routine Operator Guide ekspert nanoLC 400 Systems 77 of 174 System Maintenance Replace the Puncturing Air Needle The puncturing air needle is the outer of two needles through which the sample needle passes The puncturing air needle is used to puncture caps and septa and to apply air pressure to the sample vial Tubing for Pressure Assisted Sample Aspiration PASA and wash liquid is connected to the puncturing air needle assembly Caution Puncture Hazard Handle needles with care as they are sharp and may injure the operator Remove the sample needle Refer to Replace the Sample Needle on page 76 2 Disconnect the needle dryer tubing and wash liguid tubing from the autosampler wash pump assembly 3 Rotate the puncturing air needle assembly 1 4 turn clockwise until the connections face forward Figure 5 10 Puncturing Air Needle Removal 4 Pull d
106. ing the System eksigent part of AB SCIEX ekspert nanoLC 400 Systems Operator Guide 62 of 174 System Maintenance This section describes procedures to perform routine maintenance on the Eksigent ekspert M nanoLC 400 systems including e Pump Maintenance on page 64 e Autosampler Maintenance on page 70 For routine maintenance information for the cHiPLC system refer to the cHiPLC System Operator Guide Caution Potential System Damage There are no user serviceable components or assemblies inside the ekspert nanoLC 400 pump Service of any internal parts or assemblies should be completed by a trained Field Service Employee FSE Recommended Maintenance Schedule Basic cleaning and routine maintenance is the responsibility of the customer Maintenance services are available from AB SCIEX To promote reliable performance perform the following procedures at the specified interval Table 5 1 ekspert nanoLC 415 and 425 Pumps Maintenance Procedure Frequency Re initialize the Pressure Transducers Weekly Purge the Mobile Phases Monthly Replace the Seal Wash Monthly Clean and Inspect the System Quarterly Replace the Bottom of Bottle Filters Yearly Calibrate the Flowmeters As needed Table 5 2 ekspert nanoLC 400 Autosampler Maintenance Procedure Frequency Flush the Syringe and Liquid Path Weekly Replace the Syringe As needed Replace the Sample Needle As needed Replace
107. ion Comments Modifiers for mixture A Comments Modifiers for mixture B Mobile Phase Change Purge Settings Flush Settings Side A v Side B Total Volume uL purge cycles Flush Flowrate 0 nLimin Flush Now g Apply to all channels Apply to all channels C Automatically purge amplifiers when mobile phases change Automatically flush system when mobile phases change Create New Fluid OK Apply Cancel a Inthe Purge Settings group select the Side A or Side B check box for the pump to purge Set the number of purge cycles to 20 c Click Purge Now The pump begins to execute purge cycles While the pump is purging verify that the mobile phases are pulled through the mobile phase tubing to the pump d Locate the waste tubing of the pump being purged After about 8 purges the mobile phase should be purged through the waste tubing Flush the Pump Flushing replaces the solvent in the capillaries connecting the pump to the pump outlet through the flow modules Repeat this procedure for all channels if necessary Prerequisite Procedure e Purge the Pump Disconnect the tubing from the pump outlet Connect a waste line to the pump outlet 3 In the Acquisition window select the appropriate channel in the top right corner of the window Click System gt Mobile Phases Click More 6 In the Flush Settings group type 50 uL for the Total Volume O
108. ition Window Column Oven Temperature Eksigent Control Software File View System Analysis Help channel Gradient Sa il 5 Running 0 7 Qa Ob Oc Pa Pb Po A B Total Flowrate 404 nLAmin mm E 4 Runtime 00 00 24 01 00 00 A 891 B 109 LC Method Direct Control Sample Seguence 360 44 0 404 702 51 0 40 98 7 Filename ek1 152005 nL min psi Powe 500 Refer to the Eksigent Control Software User Guide for information on using the column oven to explore the impact of varying column temperature in the Run Manager window The Run Manager window allows a queue to be set up and different column temperatures to be entered for each run Caution Potential System Damage Do not leave the column oven cover open for long periods of time while the column oven is operating The heater unit is designed to operate as a closed system Leaving the cover open with the heater on at high temperatures may damage the heater system Configure the A D Converter An optional A D converter is required for connection to external detectors such as UV or fluorescence detectors The FSE installs the optional A D converter during installation of the ekspert nanoLC 400 system The section assumes the ekspert nanoLC 400 system is connected to a computer and that the Eksigent control software has been installed By default all the software is installed for the A D device when the Eksigent software package is installed on the
109. l Transport Start 8 Allow the system to equilibrate for approximately 10 minutes or until it is well flushed and pressure on Pc has stabilized Verify the Flowrate Before operating the system verify that the flowrate is properly calibrated This is done by measuring the time it takes to move a liquid front through a graduated capillary of known volume Required Materials Flow calibration assembly PN 801 00063 for high flow rate channel includes 20 uL pipettes Flow calibration assembly PN 801 00064 for low flow rate channel includes 5 uL graduated pipettes e Timer Re initialize the Pressure Transducers 2 Attach the appropriate flow calibration assembly to the appropriate mobile phase outlet 3 In the Eksigent control software click System gt Direct Control ekspert nanoLC 400 Systems Operator Guide 36 of 174 System Overview Figure 3 18 Direct Control Dialog Flowrate Check Direct Control Pump Direct Control Waiting for LC Method Channel Total flowrate Conserved Flow 95 5 500 nLimin an ee ME Gradient O Independent Flow 500 nLimin 1 C Monitor Baseline Start se Column Oven Heater Setpoint 40 TE Close In the top right corner of the dialog select the appropriate channel 5 Inthe Direct Control dialog for a Gradient pump set channel A to 95 and an appropriate Total flowrate for
110. ls attention to a procedure or practice which if not adhered to could result in severe injury or damage or destruction of parts or all of the equipment Do not proceed beyond a warning sign until the indicated conditions are fully understood and met The caution signal word denotes a hazard It calls attention to a procedure or practice which if not adhered to could result in damage or destruction of parts Caution or all of the equipment Do not proceed beyond a caution sign until the indicated conditions are fully understood and met The tip sign signals relevant information Read this information as it might be helpful The note sign signals additional information It provides advice or a suggestion to support equipment use This symbol indicates that the waste of electrical and electronic equipment must not be disposed as unsorted municipal waste and must be collected separately Please contact an AB SCIEX field service employee for information concerning the decommissioning of equipment Pf a Operator Guide ekspert nanoLC 400 Systems 9 of 174 Regulatory and Safety Information Safety Instructions The following safety instructions apply to the ekspert nanoLC 400 systems document Using this instrument in a manner not specified by AB SCIEX may A WARNING Personal Injury Hazard Use this product only as specified in this result in personal injury or damage to the instrument
111. lvents concentrated mineral acids such as nitric phosphoric or sulfuric acids or any related compounds lamp Possible eye injury due to hazardous optical radiation A WARNING Ocular Hazard Do not look directly into the autosampler cabinet Caution Potential System Damage Contact the manufacturer prior to making changes or modifications to the system Changes or modifications to this unit not expressly approved by the manufacturer could void the instrument warranty and render the system inoperable Caution Potential System Damage Handle the system with care Electrical damage may have occurred if any part of the system shows visible signs of damage exposure to liquids or of having been transported under severe stress Caution Potential System Damage Do not replace the on board lithium battery The battery maintains the autosampler firmware when the system is turned off It should only be replaced by a factory authorized service engineer Caution Potential System Damage Do not store the system for prolonged periods under extreme conditions for example subjected to heat water etc as damage may occur Caution Potential Data Loss Do not rely on analytical results from the system when a change is determined When a change is observed in the retention of a particular compound the resolution between two compounds or peak shapes immediately determine the reason for the changes Operator Guide eksp
112. ly higher or lower 3 Identify any symptoms which vary from normal system operation e System power on and initialization initialization fails System diagnostics re initialize pressure transducers calibration Flowrate in each channel high low erratic Output pressure Pc high low erratic 4 For each symptom perform the corrective actions in the troubleshooting tables which follow If this process does not correct the problem contact AB SCIEX Technical Support Operator Guide ekspert nanoLC 400 Systems 95 of 174 Best Practices and Troubleshooting Troubleshooting Tables Table 6 2 System Initialization Symptom Power LED on front panel is not on Possible Cause Power supply is not turned on or connected Corrective Action Connect and turn on the power supply No power at outlet Repair the electrical outlet Power LED has failed but system response OK Contact AB SCIEX Technical Support No connection dialog or Not connected in Acquisition window Not Connected Total Flowrate 0 000 yL min Runtime 00 00 00 00 00 00 A 0 B 0 Inj Viv Load LC Method default Sample Sequence Filename Communication error between computer and LC system e Verify the instrument USB cable is securely connected to the computer USB port Reboot the computer and cycle the power on the instrument In the list of COM ports in the Windows Device
113. m the sample solution Dilute all samples to a maximum of 5 organic modifier to prevent sample precipitation in the chromatographic system and at the electrospray source This also ensures binding to the stationary phase Make sure that detergents used during sample preparation are reduced to a concentration less than 0 01 prior to analysis System Diagnostics The diagnostic capabilities of the ekspert nanoLC 400 systems are designed to maintain peak system performance Status Bars The status bars and the text displayed in the Eksigent control software Acduisition window provide general diagnostic information for routine operations We recommend that the Pc pressure readings for the chromatographic method be monitored Become familiar with standard operation conditions Knowledge of pressures under various conditions is useful when troubleshooting For example you can evaluate whether system properties have changed by connecting a standard 750 mm x 20 um i d piece of PEEKsil to a pump outlet and pumping at a flowrate of 500 nL min with different A B ratios and documenting the pressures Hardware Diagnostics Running hardware diagnostics is part of routine system maintenance Refer to Recommended Maintenance Schedule on page 63 Calibration Values The Calibration Values tab of the Hardware Diagnostics window summarizes the current k values of the flow meter the gain and zero offsets of pressure transducers and the PID parameters fo
114. materials then remove and throw away the needle and other flow path materials in accordance with an approved waste disposal program 5 Make sure that the foam block is inserted in the tray location to prevent the tray from moving 6 Make sure that the accessory kit box is inserted tightly in the shipping box to prevent the autosampler from moving Operator Guide ekspert nanoLC 400 Systems 81 of 174 System Maintenance eksigent part of AB SCIEX ekspert nanoLC 400 Systems Operator Guide 82 of 174 Best Practices and Troubleshooting This section describes best practices and the built in diagnostic capabilities of the ekspert TM nanoLC 400 systems It also includes troubleshooting guidelines and tables for the most common symptoms Best Practices Operator Guide Keep the system turned on and fluid flowing at all times Use LC MS grade pre made solvents such as those from Burdick Jackson These solvents can be ordered from VWR e PN BJLC452 2 5 0 1 Formic Acid Water HPLC grade water with 0 1 formic acid e PN BJLC441 2 5 0 1 Formic Acid Acetonitrile Acetonitrile with 0 1 formic acid Verify that the gas flowrate and pressure are consistent Do not disconnect the gas supply Avoid biological growth e Change solutions frequently e Include 0 1 formic acid in all mobile phase bottles Verify that the mobile phase solutions in the bottles match the mobile phase and composition values in the
115. modifications to this unit not expressly approved by the manufacturer could void the instrument warranty and render the system inoperable Contact an AB SCIEX representative for more information on servicing the instrument Regulatory Compliance This system complies with the standards and regulations listed in this section Applicable labels have been affixed to the system For more information refer to the Declaration of Conformance included with the system Canada e Safety CAN CSA C22 2 No 61010 1 e Electromagnetic Compatibility ICES 001 e Safety EN 61010 1 e Electromagnetic Compatibility EN 55011 Class A and EN 61326 1 e WEEE 2002 96 EEC ekspert nanoLC 400 Systems Operator Guide 12 of 174 Regulatory and Safety Information USA e Safety UL 61010 1 e Electromagnetic Compatibility FCC PG 15 Class A International Safety lEC 61010 1 Electromagnetic Compatibility CISPR 11 Class A IEC 61326 1 Symbols and Labels Table 1 2 Symbols and Labels External Labels Definition Caution Risk of needle stick puncture Take care that air circulation is not impeded WARNING Possible eye injury due to hazardous optical radiation Do not look directly into the cabinet lamp Do not dispose of equipment as unsorted municipal waste WEEE 1 gt ee Direct Current Sa Eq A Amperes current V Volts voltage V A Volts Amperes power Operator Guide
116. ng Pump 3 Create the LC Method Gradient Create the Autosampler Method The parameters used for loading the sample into the injection valve and for rinsing the autosampler syringe and sample needle are stored in the autosampler method This section creates an autosampler method appropriate for loading a trap column using the loading pump and running a gradient with Gradient 1 1 Place the sample vial containing the standard test mixture in vial position C01 of autosampler 108 vial tray 2 Inthe Acquisition window click Run Manager Figure 3 25 Run Manager Window Run Manager File Edit View Devices System Suitability Help Run Table default ini Autosampler LC Other A Method Tray Vial Channel Status Eksigent415 425 1 ulPickup Grad Gradient 1 B01 LoadingPump_trap_loading Loading Pump Standard std Queued Gradient pump_15minGradient Gradient 1 Standard std Queued Current Tray Autosampler Method Definitions Run Sequence OO0O008000 ee Trayi A Autosampler Methods Sequential O As Available v O Synchronized Multi Channel 000006 LC Methods J Flush Equilibrate when Idle EES S Run Manager window click Devices gt Autosampler Device Settings and gt select the tray type Open the Run Manager window after changing the tray type Q Tip If the current tray does not display a picture of the 108 vial tray in the 3 Make su
117. nge body when disconnecting it from the system h Once the internal pressure of the syringe has been relieved remove the syringe from the pump by unscrewing the adapter tubing piece from the bulkhead on the front of the pump 5 Repeat these steps for each pump line to be primed Exchange the Solvent If the pump is wet miscible solvents may be changed To introduce a solvent identify and introduce mutually miscible intermediate solvents prior to introducing the final solvents Caution Potential System Damage Do not follow one solvent with an immiscible solvent Doing so may lead to a variety of undesirable results and unpredictable behavior and potential instrument damage If the pump is wet introduce an air gap in the lines holding the existing liquid This gap can stop the new solvent from being contaminated with existing solvent in the line 1 In the Eksigent control software stop all flows gt Tip Attaching the syringe at the beginning of this procedure helps prevent leaks from the priming port when the prime valve is switched 2 Introduce an air gap a ldentify the bottle and line to be changed Operator Guide ekspert nanoLC 400 Systems 25 of 174 System Overview b Place a new labelled bottle filled with solvent near the solvent bottle to be changed out c Inthe Eksigent control software click System gt Mobile Phases gt MORE and specify the channel that is Gradient 1 Gradient 2 or
118. nnect the system to the AC mains using the provided power supply Figure 3 1 Back Panel Pump I O Switch in Off Position a ofa a J a 2 Turn on the nanoLC 400 pump using the I O switch on the back panel The green LED on the front of the system illuminates 3 Turn on the autosampler using the I O switch on the back panel Operator Guide ekspert nanoLC 400 Systems 17 of 174 System Overview Figure 3 2 Back Panel Autosampler I O Switch 4 Turn on the computer and log in 5 Open the Eksigent control software After the system completes initialization the Acquisition window opens as shown in Figure 3 3 Refer to the Eksigent Control Software User Guide for detailed information on the features of the Acquisition window ekspert nanoLC 400 Systems Operator Guide 18 of 174 System Overview Figure 3 3 Acquisition Window rksigent Control Software Ele View System Analysis Help Waiting tor LC Method amp Total Flowrate 0 nL min m oe D a Runtime 00 00 00 00 00 00 A 0 Inj Viv Load LC Method tw_elute Sample Sequence Filename a Ob Pa Pb Pe B channel E Gradient s1 LEI 09 00 0 0 00 1 0 00 00 Column nLimin psi Power 255 C Gradient 1 Pc psi Qa nL min Gb nLimin G total nLJmin Profile B nLAmin 0 67 0 83 al Minutes Channel Assignments in the Eksige
119. nt Control Software The ekspert nanoLC 415 and 425 systems have two or three pump channels respectively The channels are identified in this guide and in the software as Gradient 1 Gradient 2 and Loading pump Refer to Table 3 1 for channel assignments In the software select the channel in an active dialog or window by clicking the arrow beside the channel The channel typically shows in the top right corner of a dialog or window Table 3 1 Channel Assignments with Nanoflow Modules in the Gradient Channels System Gradient 1 Gradient 2 Loading Pump 415 Nanoflow 100 nL min to N A Microflow 1 uL min to 1000 nL min 50 uL min 425 Nanoflow 100 nL min to Nanoflow 100 nL min to Microflow 1 uL min to 1000 nL min 1000 nL min 50 uL min Note The flow rates in Table 3 1 are for the default flow modules If different flow modules are installed on the system the flow rates will differ Autosampler Configuration The AB SCIEX FSE configures the autosampler during installation Verify the Configuration Settings 1 Open the Run Manager Operator Guide ekspert nanoLC 400 Systems 19 of 174 System Overview 2 Click Devices gt Autosampler Device Settings The Autosampler Configuration dialog opens Figure 3 4 Autosampler Configuration Dialog Configuration Tab Autosampler Configuration Configuration Direct Control Maintenance Diagnostics System Definition ekspert
120. nto Operator Guide ekspert nanoLC 400 Systems 23 of 174 System Overview and forced out of the syringe during priming will enter the pump flow path Contamination may occur during these steps Caution Potential System Contamination Failure to keep the priming syringe and connecting tubing free of contamination will lead to potential plugging and contamination of the ekspert nanoLC system Follow good LC MS laboratory practices priming syringe will result in compressed gas and liquids Be prepared for unexpected spray for example the syringe or fittings may slip and liquid may spray A WARNING Personal Injury Hazard Use safety glasses at all times Use of the Required Materials supplied e 5 mL GasTight priming syringe e Prime valve connector tubing and fittings e Waste container minimum 100 mL not supplied Prime a Dry Pump Identify the bottles solvent lines and solvents to be changed 2 Attach the priming syringe to the front of the pump for the solvent being changed The syringe should be empty prior to connection to the system Connect the syringe with the attached connector tubing and adapters The threaded end of the connection tubing plumbs into the pump and the luer lock fitting connects to the syringe Refer to Figure 3 8 Figure 3 8 Front Panel Pump Manual Prime Ports i D j i D 3 Open
121. on page 65 ekspert nanoLC 400 Systems 97 of 174 Best Practices and Troubleshooting Table 6 3 Flow Control System Continued Symptom Inability to reach desired flowrate Possible Cause Internal filters are plugged Corrective Action Contact AB SCIEX Technical Support Flowrate setpoint too high for system back pressure In the Direct Control dialog decrease the flowrate Air pressure is too low Verify that the air supply has pressure of 100 psi Flowrate will not stabilize at start of run Flowrate setpoint too high for system back pressure In the Direct Control dialog decrease the flowrate Unstable back pressures due to column connections or spray tip Disconnect downstream plumbing components to see if flow stabilizes and to identify source of back pressure fluctuations Unable to meet required flowrate within specified tolerance Increase the flow stabilization limit in the Advanced tab of the Instrument Configuration dialog Verify the flow stability refer to Autosampler Maintenance on page 70 Internal filters are plugged Purge the system refer to Purge the Mobile Phases on page 65 and observe the flow of waste through the waste tubing after approximately 8 purges If the waste flow is very low or intermittent the filter should be changed Contact AB SCIEX Technical Support Flowrate will not stabilize during a run Incorre
122. opyl alcohol water The solvent visible in the syringe body is not the sample that will be injected and will eventually flush out during syringe washes Use of 100 organic solvent for priming can lead to bubble formation immediately after reinstallation of the syringe This is because of the initial mixing between the 100 organic solvent and the aqueous solvent within the autosampler flow path Use of non standard reversed phase solvent systems may require a different solvent for priming the autosampler Operator Guide ekspert nanoLC 400 Systems 75 of 174 System Maintenance Replace the Sample Needle Replace the sample needle in the snap in needle mount 1 Inthe Run Manager window click Devices gt Autosampler Device Settings 2 On the Maintenance tab in the Exchange group click Needle Figure 5 8 Autosampler Configuration Window Maintenance Tab Autosampler Configuration Configuration Direct Control Tray Syringe Adjust Tray Position Home Current mm 0 Reagent Solvents Align syringe plunger 2 mm from upper position Exchange d Needle gt ee ee 3 Disconnect the needle from the injection valve ekspert nanoLC 400 Systems Operator Guide 76 of 174 System Maintenance 4 While lifting the needle retainer at the top pull the sample needle out of the puncturing air needle Figure 5 9 Sample Needle Item Description 1 Needle retainer 2 Sample needle 3 Punctur
123. osing of system waste 64 documentation additional 14 E Eksigent control software channel assignments 19 equilibrating the system 35 exchanging solvents 25 external interface connections 109 ekspert nanoLC 400 Systems 171 of 174 Index F flow calibration assembly 36 flow control system troubleshooting 96 flow module cartridges 28 replacing 28 flow rate calculating 108 verifying 36 flowmeters calibrating 67 flushing pumps 27 syringe and liquid path 71 flushing the syringe 71 flushing the system 66 frequency maintenance schedule 63 full loop injections 120 G generating diagnostics for service 85 gradient channels channel assignments 19 creating methods 50 H hardware diagnostics 85 Hardware Diagnostics dialog 66 hardware profile creating 147 verifying 146 j injection parameters metered 44 standard 44 injection types 120 injection valve 35 troubleshooting 101 inspecting the system 64 installing system 59 L LC methods 2 D reverse phase reverse phase work flow 159 ekspert nanoLC 400 Systems 172 of 174 loading pumps creating methods 47 M maintaining the system 63 maintenance autosampler 70 calibrating flowmeters 67 cleaning the pumps 64 flushing the system 66 pumps 64 purging mobile phases 65 re initializing the pressure transducers 66 replacing the flow module cartridge 28 replacing the needle 76 replacing the pierce needle 78 replacing the r
124. ote the extended flat top and relatively low maximum pressure 250 mBar Because the speed is slow the pressure is low In addition the slow speed causes the upper plateaus to take longer as the syringe takes longer to empty before restroking The first stroke of every wash is dumped to waste through the syringe valve and not passed through the pressure gauge or the needle flow path ekspert nanoLC 400 Systems Operator Guide 90 of 174 Best Practices and Troubleshooting Figure 6 6 Flow Path Pressure Trace During an Inside Sample Needle Wash Series at 1 5 and 10 uL sec Autosampler Configuration Configuration Direct Control Maintenance FlowPath Pressure Pressure mBar 6 Zero value mBar 1232 Upper Limit mBar 7000 Pressure mBar Lower Limit mBar 1000 Reset Chart Status Device Idle Driver VWaiting for method Valve Position Inject ISSA Position Inject 2 ISSB Position Inject 2 Trav 8 1 C External I O C Output 1 C Output 2 C Output 3 3 4 Time min C Output 4 C Output 5 C Output 6 High High High High In Figure 6 6 the maximum pressure is much higher and the length of time to stroke the syringe shorter Note that there are slightly more than three strokes for this and the next example as the wash was not stopped by the user after the three strokes Figure 6 7 shows higher pressure and shorter duration than the 10 uL sec example due to an increas
125. otor seal 80 replacing the sample loop 79 replacing the seal wash 66 replacing the syringe 72 replacing the wash bottle solvent 77 schedule 63 Maximum Flowrate 23 metered injection parameters 44 methods creating advanced autosampler 122 creating an autosampler 119 creating autosampler 45 creating for gradient channels 50 creating for loading pumps 47 trap and elute 45 mobile phases replacing 21 moving the system 59 MS acquisition method creating 149 MS acquisition starting 151 N nanoflow modules channel assignments 19 needle replacing 76 replacing the pierce 78 O oven See column oven P pierce needle Operator Guide replacing 78 plumbing diagram direct on column experiments 115 trap and elute experiments 116 pressure transducers re initializing 66 priming wet pumps 25 pumps external interface connections 109 flushing 27 maintenance 64 maintenance schedule 63 pin assignments 110 preparing for storage 81 purging 26 purging pumps 26 purging mobile phases 21 maintenance 65 R regulatory compliance 12 re initializing pressure transducers 66 replacing flow module cartridges 28 replacing mobile phases 21 replacing seal wash 66 replacing the needle 76 replacing the pierce needle 78 replacing the rotor seal 80 replacing the sample loop 79 replacing the syringe 72 replacing the wash bottle solvent 77 rotor seal replacing 80 run changing duration 56 stopping 56 Run Manager window
126. own the puncturing air needle assembly from the needle holder Orient the new puncturing air needle so that the connections face forwards Install the new puncturing air needle assembly by pressing it upwards into the needle holder with the connections facing forward 7 Turn the assembly counter clockwise 1 4 turn until the connections point to the right side of the holder and the assembly clicks in place 8 Connect the air supply and wash solvent tubing to the wash pump assembly Install the sample needle Refer to Replace the Sample Needle on page 76 ekspert nanoLC 400 Systems Operator Guide 78 of 174 System Maintenance Replace the Sample Loop The stainless steel sample loop 10 uL is pre installed and is located at ports 2 and 5 on the ekspert 400 nanoLC autosampler valve If a clog is suspected change the sample loop Required Materials e Pre cut 10 uL stainless steel sample loop PN 5016578 oo Non 10 11 Operator Guide Remove the existing loop Connect one end of the new loop to Port 2 of the autosampler valve This is the upstream side of the loop Leave the other end disconnected during the initial flush In the Eksigent control software click System gt Direct Control Figure 5 11 Direct Control Dialog Channel 1 Example Direct Control Ed Pump Direct Control Not Connected Channel A B Total flowrate Conserved Flow 100 0 20 nLimin radiant Independent Flow Q 500 nLimin B
127. owrate calibration kit for 50 nL min to 1000 nL min flowrate PN 801 00064 e Timer Different flowrates require different pipettes to accurately calibrate the flowmeters Choose the appropriate calibration pipettes when calibrating the high flow and low flow channels Table 5 4 Calibration Pipette Requirements High flow Channel Low flow Channel Calibrated Pipette Size 20 uL 1 uL division 5 uL total Calibration Flowrate 5 uL min 500 nL min Calibration Volume 20 uL side 2 uL side N Tip The following procedure is written for the high flow channel When calibrating the low flow channel using a 5 uL pipette save time by connecting the pipette to the pump and quick priming the pipette to approximately 1 cm before the first black line Do this by holding down the Quick Prime button lower left corner until the liquid reaches about 1 cm from the start mark of the calibrated pipette gt 1 Click System gt Hardware Diagnostics Select the high flow channel in the top right corner of the dialog 3 Click Calibrate Flowmeter Ch The Flowmeter Calibration wizard opens In Step 1 ofthe Flowmeter Calibration wizard verify that the Mobile Phases are correct and then click Next ekspert nanoLC 400 Systems Operator Guide 68 of 174 Operator Guide System Maintenance Figure 5 3 Flowmeter Calibration Wizard Step 1 Flowmeter Calibration CAUTION Flowmeter calibration requires following the b
128. p and Elute on page 45 Make sure the system is plumbed appropriately Refer to Standard Plumbing Diagrams on page 115 for plumbing diagrams Create the Acquisition Method and the Batch This procedure assumes familiarity with the Analyst software For more information press F1 to view the help or refer to the Analyst Software Getting Started Guide or System User Guide available from the Start menu Create the Acquisition Method 1 On the Navigation bar under Acquire double click Build Acquisition Method to create the acquisition method 2 Select the mass spectrometer method a Inthe Acquisition Method window click Mass Spec b Specify the parameters for the mass spectrometer acquisition c Click Edit Parameters to set the Source Gas parameters Table G 2 Source Gas Parameters for 200 nL min to 500 nL min Flowrates Parameter Suggested Ranges Curtain Gas CUR 720 to 25 not less than 20 lon Source Gas 1 GS1 2 to 15 lon Source Gas 2 GS2 0 Temperature TEM 150 lon Spray Voltage ISV 2000 to 2400 d Click OK to save the source and gas parameters 3 Select the autosampler method and then click Open Operator Guide ekspert nanoLC 400 Systems 149 of 174 Working with Analyst Software Figure G 3 Acduisition Method Selecting the Autosampler Method E Analyst Acquisition Method D Analyst Data Projects Quatro Testing Acquisition Methods Operators Guide method N Ele Edit V
129. perator Guide ekspert nanoLC 400 Systems 155 of 174 2 D Reverse phase Reverse phase Liquid Chromatography Workflow Figure H 3 Plumbing Diagram cHiPLC Connectors and Valve 3 2 1 Column Trap Trap Elute 1D A 7 A Waste OA BO 4 og MS Union 10 B DO 5 OE High pH column Filter or chip Gradient 2 1C Create the Autosampler Methods Two methods are reguired one for sample injection and one for elution For each method in the Method Editor dialog select Trap Elute in the Injection Type section and then use the Advanced Method Editor dialog to create the following methods refer to Create an Advanced Method on page 122 The Injection Method This method is used to load 1 uL of sample onto the high pH column The method washes the autosampler needle waits for the pumps to be ready then aspirates and injects the sample After injection the method waits 20 minutes until the sample is loaded onto the column ekspert nanoLC 400 Systems Operator Guide 156 of 174 Figure H 4 Autosampler Injection Method File Command Description 1 Initialize 9 Start 10 Valve 11 Wait 12 Start Operator Guide Autosampler Device Switch Injection valve to Load Pre wash 1x for Gradient 2 ready to start for Gradient 1 ready to start for Loading Pump ready to start uL Pick Up 1 pb 2 mm from bottom Start Gradient 1 Start Loading Pump
130. perator Guide ekspert nanoLC 400 Systems 27 of 174 System Overview 7 Specify an appropriate Flush Flowrate for the maximum flow of the channel For high flow channels type 10 uL min for the Total Flowrate e For low flow channels type 1000 nL min for the Total Flowrate Caution Potential System Damage Be sure to disconnect all traps columns and filters before proceeding Flushing the system with a column connected could damage the column or create leaks in external connections Click Flush Now After the flush sequence ends click OK to close the Mobile Phases dialog Replace the Flow Module Cartridges The flow module cartridges are interchangeable and may be replaced as needed to change the pump flowrate range Caution Potential System Damage Handle the flow modules with care Flow modules require careful use and storage Use of a tool other than the supplied break over torque wrench on the flow modules may result in damage to the unit and system If the tool is misplaced order a new one from Eksigent Damage to the flow module from misuse including failure to use only the supplied torque wrench may void the instrument warranty Store flow modules in a dust free environment to avoid plugging Caution Potential System Damage Never leave solvents with non volatile salts in the flow module when removing the cartridge Doing so will damage the flow module N Tip Each module has an A and B side
131. port Faulty check valve Contact AB SCIEX Technical Support Pump restrokes frequently Pump has reached end of stroke alert message opens Air is trapped in the pump Purge the pump refer to Purge the Mobile Phases on page 65 Pump remains on long enough to prompt a restroke For the current flowrate calculate the time to pump 600 uL Verify that the pump restrokes at approximately that time interval Internal leak or check valve is leaking Contact AB SCIEX Technical Support Pump does not restroke at the end of a run Pump restroke delay is too short Pump stroke sensor not working correctly Contact AB SCIEX Technical Support Pump does not purge 600 uL per stroke Leak in instrument Inspect all connections for leaks Air in the pump Purge the pump to clear any air If the air does not clear verify that the inlet frits are submerged in the reservoir and not drawing air Faulty check valve Contact AB SCIEX Technical Support Purge output drips slowly Internal filters are plugged Contact AB SCIEX Technical Support No flowrate with 100 power indicated System pressure Pc and pump pressures Pa and Pb are all low Operator Guide No gas to system Connect 100 psi clean dry air to the instrument air inlet System not properly purged and flushed Purge and flush the system Refer to Purge the Mobile Phases
132. put current 4 A Maximum pressure 10 000 psi Flowrate range Loading pump 1 to 50 uL min e Gradient with nano flow module 100 to 1000 nL min up to 2 uL min at reduced maximum pressure e Gradient with low micro flow module 1 to 10 uL min up to 20 uL min at reduced maximum pressure Gradient with high micro flow module 5 to 50 uL min Retention time precision lt 0 35 RSD 500 nL min with nanoflow module installed and column at constant temperature 0 2 C Flowrate accuracy lt 1 500 nL min with nano flow module installed Gradient delay volume lt 25nL Wetted parts 316 stainless steel Titanium PEEK fused silica and FEP VO 1x USB for instrument control Each pump channel e 2x TTLin e 8x contact closure out Software Operator Guide Eksigent control software for standalone use Eksigent control software with drivers for LC MS use AB SCIEX Analyst software Thermo Scientific Xcalibur software ekspert nanoLC 400 Systems 103 of 174 System Specifications ekspert nanoLC 400 Autosampler Specifications Dimensions WxDxH 13 inches x 26 inches x 19 inches 33 cm x 66 cm x 49 cm Weight 33 pounds 15 kg Working temperature 15 C to 30 C Power 100 VAC to 240 VAC 450 VA Maximum pressure 10 000 psi Sample capacity 108 vials or two microtiter plates 96 high low or 384 low Injection valve 6 po
133. r the pump Document these values before and after calibrations as part of maintaining a good instrument log Note any changes These values should be similar from diagnostic to diagnostic Large changes may indicate an issue ekspert nanoLC 400 Systems Operator Guide 84 of 174 Best Practices and Troubleshooting Generate System Diagnostics for Service Occasionally it may be useful for the AB SCIEX FSE to review system diagnostics information prior to scheduling an on site visit Reviewing this information may help the FSE diagnose a particular issue and help make sure that the correct replacement parts if any can be ordered The following diagnostics may be sent to the FSE e Hardware Diagnostics Alerts Log e Autosave Files Hardware Diagnostics Regularly perform hardware diagnostics to monitor the system Refer to Recommended Maintenance Schedule on page 63 1 Open the Eksigent control software 2 Click System gt Hardware Diagnostics 3 On the Calibration Values tab select the channel by clicking the arrows in the top right corner of the dialog Figure 6 1 Calibration Values Tab Hardware Diagnostics Recurring Events Channel C Remind me to run diagnostic tests once a month 4 Gradient v Flow Calibration 2 Flowmeter 1203171 Pump A Pump B Geometry factor 0 00434 0 00291 k value 0 4902 0 8537 temperature and viscosity corrected Pressure Sensors Pump A Pump B Column Scale Parameter ps
134. rail ekspert nanoLC 400 Systems Operator Guide 42 of 174 System Overview 7 Inthe Sample Injection group click Metered This option signals the injection valve to be placed in the inject position for the duration of the Metered Injection that is 10000 nL Refer to Standard and Metered Injection Parameters on page 44 8 Make sure that the check box in the Post Run group is cleared 9 On the Gradient Table tab specify the gradient parameters as shown in Figure 3 23 e Add new steps to the table by clicking the arrows gt gt to the left of the table e Delete steps by clicking the X e Set the Total flowrate on the right side of the dialog e If events such as Lamp on off Valve state toggles or Output switching are required specify them in the Events column Figure 3 23 LC Method Settings Dialog Gradient Table Tab B LC Method Settings Selected Method Name Gradient1 15min aradient Summary Run Conditions Gradient Profile Gradient Table Flow Mode Conserved flow O Independent flow Profile Editor Total flowrate 300 nLimin 10 On the Gradient Profile tab review the gradient to make sure it is correct 11 When the method is complete click Save 12 Click OK to close the editor Operator Guide ekspert nanoLC 400 Systems 43 of 174 System Overview Figure 3 24 LC Method Settings Dialog Gradient Profile Tab B LC Method Settings Selected Method Name Gradient1 1 5min gr
135. re the columns in Figure 3 25 appear in the Run Manager window Operator Guide ekspert nanoLC 400 Systems 45 of 174 System Overview If necessary click Edit gt Choose Column and select the appropriate columns for display 4 Inthe Run Manager window click Autosampler Methods Figure 3 26 Method Editor Dialog Ij Editing AS3 trap elute 1 uLAS3 j lol x File M Injection Type C Direct Injection Trap Elute Multiplex M Gradient Pump Channel Gradient Pump 1 Gradient Pump 2 r Optional Valves C None 155 A Valve 1SS B Valve cHiPLC M Sample Pickup Sample pick up volume pl CO uLPickUp FullLoop EI Needle height 2 mm LI Loop volume 10 pl Leading Volume Wait Time min Trailing Volume 8 1 pL Wash Syringe wash cycles 5 Advanced Editor Test on B1 Ok 5 Click File gt Save as and save the method with a new name 6 Edit the method as required Injection Type Trap Elute Gradient Pump Channel Gradient Pump 1 Optional Valves ISS A Valve Sample Pickup uL Pick Up Sample pick up volume 1 uL Needle height 2 mm description of the features in the autosampler Method Editor dialog j Note Refer to Method Editor Options on page 120 for a detailed ekspert nanoLC 400 Systems Operator Guide 46 of 174 System Overview 7 Save the method 8 Click OK to close the Method Editor dialog Wee
136. reate the Acquisition Batch In the Analyst software create an acquisition batch with the following runs Table H 2 Acquisition Batch Runs for One Sample Run Acquisition Purpose Method 1 Load Sample Inject the sample and load it on the high pH column 2 Fraction 1 Elute and analyze fraction 1 3 Fraction 2 Elute and analyze fraction 2 4 Fraction 3 Elute and analyze fraction 3 The first run Run 1 will yield no data because no sample is loaded onto the chip column Operator Guide ekspert nanoLC 400 Systems 167 of 174 2 D Reverse phase Reverse phase Liquid Chromatography Workflow During runs 2 through 4 the sample aspirated in Run 1 is eluted from the high pH column loaded on and then eluted from the chip column and analyzed by the mass spectrometer For experiments with more than one sample all four runs are required for each in the sample table Note Depending on the mass spectrometer additional runs for calibration may be required in the batch Follow the recommendations in the Operator Guide for the system Load the Mobile Phases Table H 3 Mobile Phases for the 2 D Reverse phase Reverse phase Separation Channel Mobile Phase A Mobile Phase B Gradient1 150 mM ammonium formate adjust pH 80 acetonitrile 20 mobile phase A to 9 6 with ammonium hydroxide Gradient 2 Water 0 1 formic acid Acetonitrile 0 1 formic acid Loading Pump Water 0 1 formic acid N
137. rt e 1 32 inch fitting 0 15 mm bore e Port to port volume lt 65 nL e Max pressure 10 000 psi e Stator Stainless steel with diamond like coating Rotor Vespel Column switching valves e One standard built in option for mounting a second valve 10 port e 1 32 inch fitting 0 15 mm bore e Port to port volume lt 65 nL Maximum pressure 10 000 psi e Stator Stainless steel with diamond like coating Rotor Vespel Injection volume Programmable from 100 nL to 10 uL with 10 uL loop e Larger injection volumes possible with optional sample loops Injection needle 0 15 mm PEEKsil 3 6 uL Dispenser syringe 50 uL Reproducibility Full loop pickup 0 5 RSD e uL pickup lt 1 RSD for injections gt 1uL e Carry over lt 0 01 with standard wash Sample tray cooling 4 C to 40 C 2 C Wetted parts ekspert nanoLC 400 Systems 104 of 174 Stainless steel PEEK glass Teflon Kalrez fused silica diamond like coating Operator Guide System Specifications VO RS232 serial or USB 5 programmable relay outputs 4 programmable TTL inputs Software Eksigent software for standalone use Eksigent software with drivers for Analyst AB SCIEX and Xcalibur Thermo for LC MS Autosampler Solvent Tray Outside dimensions WxDxH 13 inches x 14 inches x 3 5 inches 33 cm x 36 cm x 9 cm Inside dimensions WxDxH 10 5 inch
138. s Figure F 10 LC Method Settings Dialog Gradient Table for Trap and elute Method B LC Method Settings LoadingPump_Trap_Loading ekspert nanoLC 400 Systems Operator Guide 138 of 174 D5033460 C Gradient 1 Method Figure F 11 LC Method Settings Dialog Run Conditions for Trap and elute Method Gradient 1 B LC Method Settings Selected Method Name Gradient Pump_15minGradient Summary Run Conditions Gradient Profile Gradient Table Pre Run Flush column for 0 1 _ First establish a column pressure of Sample Injection O Nore minutes using 100 initial flowrate conditions 3000 psi Standard Sample valve opens prior to beginning Flow Profile and remains open O Metered Inject nL of sample at 12000 Q Rapid Inject nL of sample at maximum flowrate maintaining initial mixture conditions Post Run C Flush column for View Audit Trail Operator Guide 100 initial flowrate conditions minutes using ending flowrate conditions Basic Methods ekspert nanoLC 400 Systems 139 of 174 Basic Methods Figure F 12 LC Method Settings Dialog Gradient Profile for Trap and elute Method Gradient 1 B LC Method Settings Selected Method Name Gradient1 Pump_15minGradient Summary Run Conditions Gradient Profile Gradient Table
139. sing solvents favorable to growth of organisms Full Loop Injections Full loop injections use loop overfilling to encourage maximum reproducibility This method can consume considerable sample If the sample loop volume is 10 uL then approximately 35 uL is aspirated while only 10 uL is available for injection The remaining sample is washed out and wasted during the later part of the injection cycle This method is known for high reproducibility but consumes a lot of the sample The Full Loop injection cycle is as follows 1 Wash the flow path through the needle to make sure of a consistent air free injection system Draw up a volume of transport solvent 3 Draw up a volume of sample ideally equal to three times the loop volume plus the needle volume In the standard autosampler configuration the volume of the sample loop is 10 uL and the volume of the sample needle is 3 6 uL This recommended aspiration volume for a full loop injection is therefore 3 x 10 uL 3 6 uL 33 6 uL Wait Wait times are enabled for MultiPlex dual column methods only Wash The number of syringe wash cycles The ekspert 400 autosampler has a defined wash routine that cleans the inner and outer needles using the syringe wash solvent reservoir contents This is typically 50 50 methanol water Operator Guide ekspert nanoLC 400 Systems 121 of 174 Autosampler Method Editor Create an Advanced Method The Advanced Editor dialog Figure E 2 pro
140. software opens the autosampler status window opens and the Eksigent control software Acquisition window opens Figure G 1 Figure G 1 Eksigent Control Software Acquisition Window When started from Analyst Software Showing LC Methods Button fksigent part of AB SCIEX File View System Analysis Help Waiting for LC Method 2 Total Flowrate 0 000 L min s O D 4 Runtime 00 00 00 00 00 00 df amp 0 BO Inj Viv Load LC Method default Sample LC Methods Sequence oo oo oo 00 oo Column Filename Lmin psi Power 32 77 C If the Acquisition window does not open indicating that the Eksigent control software did not open do the following a Close the Analyst software b Select Start gt Programs gt Eksigent gt Driver Configuration c Uninstall and then reinstall the Analyst software drivers d Open the Analyst software ekspert nanoLC 400 Systems Operator Guide 146 of 174 Working with Analyst Software Note Do not open the Eksigent control software manually Instead allow the Analyst software to open the Eksigent control software When the Eksigent control software is opened independently the LC Methods button shows as the Run Manager button Create a Hardware Profile Click New Profile to open the Create New Hardware Profile window Type a name for the profile in the Profile Name field Add the autosampler a 9295 In the Device Type list click Software
141. specting all fluid connections e Look for dried deposits that may indicate a slow leak As needed tighten any loose connections 3 Identify and correct the source of any leaks e Ifa fluidic connection is broken replace the fitting and flush the system Inspect the new connection to make sure that no leaks are present ekspert nanoLC 400 Systems Operator Guide 64 of 174 System Maintenance Purge the Mobile Phases WARNING Environmental Hazard Always follow appropriate safety procedures and local requirements when handling or disposing of waste chemicals Refer to the Material Safety Data Sheets for the mobile phases We recommend replacing solvents each month After replacing the solvent purge and flush the system Table 5 3 Mobile Phase Mixtures Binary Mixture A Binary Mixture B Modifier Gradient 1 100 water 100 acetonitrile 0 1 formic acid Gradient 2 100 water 100 acetonitrile 0 1 formic acid Loading Pump 100 water N A 0 1 formic acid 1 Inthe Eksigent control software click System gt Mobile Phases 2 Click More to open additional options in the dialog Figure 5 1 Mobile Phases Window Purge and Flush Settings Mobile Phases Channel Binary mixture A Binary mixture B Gradient 1 Aqueous Solution Aqueous Solution Comments Modifiers for mixture A Comments Modifiers for mixture B Mobile Phase Change Purge Settings Flush Settings
142. ssure Pressure mBar 4 Zero value mBar 1232 Pressure mBar Upper Limit mBar 7000 Lower Limit mBar 1000 Time min Status External WO Device Idle Driver Waiting for method _ Output1 oupa 4 Valve Position Load C Output2 oupas ISSA Position Load 10 ISSB Position Load 10 ClOutput3 _ Output 6 Trav 8 C Input 1 High Input 2 High Input 3 High Input 4 High The Set Zero button takes the current ambient pressure reading for the flow path and uses that for zero Click Set Zero when the autosampler is not performing operations that might pressurize or depressurize the flow path After clicking Set Zero the zero value or offset displays Minor variations in pressure readings due to slight zero offset errors should not impact normal operations Atmospheric pressure also plays a role in the zero and it is expected to change with the weather Set the Upper Limit and Lower Limit for the autosampler flow path The autosampler flow path is separate from the high pressure LC flow path and the pressure values are much lower Syringe motion greatly affects pressure changes in the flow path Operator Guide ekspert nanoLC 400 Systems 89 of 174 Best Practices and Troubleshooting e Changing the wash aspiration or dispensing speeds can raise or lower the pressure during parts of the injection cycle e Changing the composition of the solvent moved by the syringe can chan
143. t AB SCIEX Technical Support System pressure Pc is very high Tubing or fitting is plugged e Starting at the end of the liquid flow path remove each tube or fitting one at a time until the pressure drops dramatically e Replace the plugged item Excess flow noise Air is trapped in the pump Purge the pump Refer to Purge the Mobile Phases on page 65 Pump controller out of tune Contact AB SCIEX Technical Support Measured flow does not follow the flow profile Pump controller out of tune Contact AB SCIEX Technical Support Pump time response is set incorrectly Adjust the pump time response in the Calibrate group of the Flow Calibration tab Hardware Diagnostics dialog Pump pressures Pa and Pb maximized to approximately 12 000 psi at 100 pump power Operator Guide Incorrect gain setting for pressure Verify the pump parameters on the Calibration Values tab Hardware Diagnostics dialog Incorrect zero setting for pressure sensors Re initialize the pressure transducers Refer to Re initialize the Pressure Transducers on page 66 ekspert nanoLC 400 Systems 99 of 174 Best Practices and Troubleshooting Table 6 4 Column Oven Symptom Column oven not heating Possible Cause Not plugged in Corrective Action Verify that the cable from the oven is connected to the back of the pump Not enabled in the software Verify that t
144. t the correct autosampler is selected for example the Ekspert 400 11 Click Devices gt Autosampler Device Settings Autodetect should be selected If autodetect is selected and there is an issue then in the Device Settings dialog in the Port list select the lowest COM port and click OK This will typically be COM1 if using the RS 232 connection If using USB it will be a higher number If this is the correct COM port configuration for communication with the autosampler then the autosampler initializes If it is not the correct port then try the next COM port until the correct setting is found 12 When the correct COM port settings have been configured for both the autosampler and pump close the Run Manager window 13 Close the Eksigent control software 14 Using the Eksigent driver utility install and configure the mass spectrometer device drivers ekspert nanoLC 400 Systems Operator Guide 60 of 174 Moving the System a Click Start gt All Programs gt Eksigent gt Driver Configuration b Click the appropriate check boxes c Click Apply The status of each driver should change from Not Registered to Registered d Click OK to close the window Some mass spectrometers require additional steps to configure the drivers Refer to the software user manual accompanying the mass spectrometer 15 Open the mass spectrometer software Operator Guide ekspert nanoLC 400 Systems 61 of 174 Mov
145. the Wash Bottle Solvent As needed Replace the Puncturing Air Needle As needed Replace the Sample Loop As needed Replace the Rotor Seal As needed Operator Guide ekspert nanoLC 400 Systems 63 of 174 System Maintenance Dispose of System Waste Properly dispose of any effluent waste in an appropriate chemical waste container Waste generated by the 10 port column switching valve as a result of high flow sample loading is collected in a waste vial Periodically empty the pump purge waste container located on the top ofthe ekspert nanoLC 400 autosampler WARNING Environmental Hazard Always follow appropriate safety procedures and local requirements when handling or disposing of waste chemicals Refer to the Material Safety Data Sheets for the mobile phases Pump Maintenance Basic cleaning and routine maintenance is the responsibility of the customer Maintenance services are available from AB SCIEX Complete maintenance tasks for the pump as prescribed in the Recommended Maintenance Schedule This section includes the following procedures e Clean and Inspect the System e Purge the Mobile Phases e Replace the Seal Wash e Re initialize the Pressure Transducers e Calibrate the Flowmeters Clean and Inspect the System 1 Inspect the waste and wash reservoirs for evidence of biological growth or precipitation 2 Inspect the system fluidics and electronic connectors e Look for evidence of fluid leaks by in
146. the purge valves a In the Acquisition window click System gt Mobile Phases b Click More c Click Prime ekspert nanoLC 400 Systems Operator Guide 24 of 174 System Overview 4 Follow the instructions in the window to prime the pump a Stop all flow b On the front panel turn the manual prime valve to Fill for the appropriate channel c Fill the syringe with solvent by pulling the syringe until the syringe is full This step pulls solvent from the bottle through the line into the back of the instrument through the internal tubing and finally into the syringe body It takes about 5 mL with a dry line for liquid to reach the syringe from the solvent reservoir d Turn the manual prime valve to Prime gt Tip Hold the syringe vertically with the plunger above the syringe tip to make sure that any air that is in the syringe is not forced back into the pump when the plunger is depressed in subsequent steps e Slowly but firmly push the liquid into the pump When approximately 2 5 mL of liquid has been pushed into the pump liquid should begin entering the waste container through the waste purge external lines This is an indication of proper priming f When liquid is entering the waste container turn the manual prime valve to Run g The syringe may have some residual pressure in it Unload or depressurize the syringe by pulling back on the plunger This prevents spilling or spraying solvent from the syri
147. the right of the appropriate row numbers in the Run Table Selecting the Flush Equilibrate when Idle check box initiates the pre run flush for the first method With this option selected the system continues to flush after the sequence as defined by the pre run flow conditions in the LC method for each channel Initiate the run by clicking Start The Start button then shows as a red Stop that can be used to abort the run at any point during the analysis ekspert nanoLC 400 Systems Operator Guide 54 of 174 System Overview After the flowrate stabilizes the sample injection process begins For the Trap loading run table created earlier in this section the Loading pump method runs first The Gradient pump will flow if the Flush Equilibrate when idle check box is selected but the method will not run yet While the run is in progress the Acquisition window shows the specified flow profiles for solvents A and B as well as their actual flowrates Q To add or delete traces from the display click System gt Appearance Settings in the Acquisition window and select the desired items Refer to Figure 3 37 on page 57 Figure 3 36 Acquisition Window Real time View of Pressure and Flow Data e 2008 Eksigent Technologies Fle View System Analysis Help tor LC Method Ge Go Pe Pe Pa A 5 u Total Fiowrate 0 174 planin a G pen n 4 Recta 00 90 08 1 0000 00 Sencie 08 82 00 029000 oa Cum Ee Pore 26 07 C To zoom
148. the system 17 U uL pick up 120 user guides additional 14 V verifying hardware profile 146 verifying flow rate 36 viewing data files 58 W wash replacing seal 66 wash bottle replacing the solvent 77 waste disposal 64 waste disposal electrical and electronic equipment 14 weight 103 wet pumps priming 25 Z zeroing pressure transducers 66 ekspert nanoLC 400 Systems 174 of 174 Operator Guide
149. thod 5 Click File gt Save and then type a name for the acquisition method Create the Acquisition Batch 1 On the Navigation bar under Acquire double click Build Acquisition Batch to create the acquisition batch 2 Specify the required information in the Samples tab of the Batch Editor window a Inthe Set field type Test Table and then click Add Set b Click Add Samples and add samples to the batch as shown in Figure G 4 c Inthe Acquisition group select the acquisition method created above ekspert nanoLC 400 Systems Operator Guide 150 of 174 Working with Analyst Software d For each sample in the table click Plate Code and select 108 vial by row from the list Figure G 4 Sample Tab Plate Code Column ES Analyst Batch Editor Quatro Testing New Batch Alle ter pew Acquire Jods Expire Winde Zenit tim Qsusea WE 2 amp Acquire Mode v OE ouatrovte a a Te id Te ar song mx Semele Locations usehitalion Submt Select Method for Sample Set 15 min adent rore Dis Quart Bemove Set s giste ethe Method Eater Add Samples ods Botch Sospt Sampie mama mac Coe User Name AMPRMV GSTEX1 abservice Submit the Batch Place the sample vials in positions 1 and 2 of tray 1 of the autosampler 2 Inthe Batch Acquisition dialog click the Submit tab and then click Submit to add the s
150. thods ekspert nanoLC 400 Systems 40 of 174 Operator Guide Operator Guide System Overview Figure 3 21 Method Editor Dialog u Editing Direct_Injection_1ul_Gradient1 aS3 DER File Injection Type Direct Injection O Trap Elute Multiplex Gradient Pump Channel Gradient Pump 1 Gradient Pump 2 Optional Yalves None Sample Pickup pl Pick Up Full Loop Sample pick up volume 1 pL Needle height 2 mm Wait Wash Syringe wash cycles Leading Volume 45 pl Trailing Volume 95 pl Advanced Editor Edit the method as required Sample Pickup uL Pick Up Needle height 2 mm Save the method Click File gt Save as and save the method with a new name Injection Type Direct Injection Gradient Pump Channel Gradient Pump 1 Sample pick up volume 1 uL typically Gradient Channel specific Use a name that clearly states the channel and other information related to the method The example method file name Figure 3 20 includes the information required to identify the method Gi Tip Autosampler methods created with the Method Editor dialog are Click OK to close the Method Editor dialog ekspert nanoLC 400 Systems 41 of 174 System Overview Note Refer to Create an Advanced Method on page 122 for a detailed description of how to create an advanced method Create the LC Method Gradient The conditions used to separate the sample are specified
151. thods FlushiEquilibrate when Idle Valve Position Load ISSA Position Load1 10 ISSB Position Load 1 10 mer d Time 00 00 00 Queued Time 01 10 12 Operator Guide ekspert nanoLC 400 Systems 143 of 174 Basic Methods eksigent part of AB SCIEX ekspert nanoLC 400 Systems Operator Guide 144 of 174 Working with Analyst Software This appendix provides a brief tutorial for the ekspert nanoLC 415 and 425 systems using Analyst software The procedures described in this appendix assume the system and Analyst software have already been properly installed and initialized Example Experiment The experiment consists of two samples each injected three times on a 5 cm C18 column heated to 35 C The mobile phases are water and acetonitrile The aqueous channel for the pump side A is filled with Mobile Phase A The organic channel side B is filled with Mobile Phase B shown in Table G 1 Table G 1 Mobile Phase Mixtures Channel Binary Mixture A Binary Mixture B Gradient 1 Water 0 1 formic acid Acetonitrile 0 1 formic acid Gradient 2 Water 0 1 formic acid Acetonitrile 0 1 formic acid Loading pump Water 0 1 formic acid N A The following procedures walk through an example experiment using the Analyst software Use these instructions when using the mass spectrometer and the ekspert nanoLC 415 or 425 system as an integrated system 7 Note These instructions assume familiarity with the
152. tion external to the autosampler method The ready state may also depend on other method and installed options If Gradient 1 is not ready possibly due to an error then the autosampler will not continue to the next step in the method Operator Guide ekspert nanoLC 400 Systems 127 of 174 Autosampler Method Editor Needle Wash Pre wash 1x A quick wash prior to sample pickup promotes an air free flow path This step is not strictly mandatory as the flow path typically does not have air while in operation Get Sample uL Pick up In this step the autosampler aspirates the sample into the sample loop Start Gradient 1 After the Sample is in the loop and Gradient 1 is ready the two previous steps the Gradient 1 method is allowed to start Valve Injector Inject Switches the sample valve Wait for Gradient1 injection complete For a Standard Injection the autosampler waits until the LC method for Gradient 1 finishes before continuing with the injection wash steps For a Metered Injection the autosampler waits for the pump to push the volume defined in the LC method After the injection is finished the wash will proceed even during the LC run Valve Injector Load Sets the sample injector loop into the Load position prior to washing Needle Wash Clean Up 5x Washes the inner and outer needles five times each using the standard needle wash routine and the syringe wash solvent reservoir contents ekspert nanoL
153. tor Valve 3672 ISS A 0 Syringe valve 13697 Syringe 19878 Power On Time hours 0 Refresh System Overview Parameter Loop Volume Typical Value 10 uL dependent on the installed hardware Needle and Tubing Volume 3 6 uL Syringe Volume 50 uL standard installed syringe volume Wash Speed 2 uL min 7 Note With the exception of Wash Speed the values in Figure 3 19 are dependent on the hardware installed d Create Autosampler and LC Methods Direct Injection Complete the following procedures 1 Create the Autosampler Method 2 Create the LC Method Gradient Operator Guide ekspert nanoLC 400 Systems 39 of 174 System Overview Create the Autosampler Method The parameters used for loading the sample into the injection valve loop and for rinsing the syringe and sample needle are stored in the autosampler method This procedure creates an autosampler method appropriate for loading the sample loop using direct on column injection and running a gradient with Gradient 1 1 Place the sample vial containing the sample or standard test mixture in vial position C01 of the autosampler 108 vial tray 2 Inthe Acquisition window click Run Manager Tip If the current tray does not display a picture of the 108 vial tray in the ee Run Manager window then click Devices gt Autosampler Device Settings and select the tray type Open the Run Manager w
154. ture is required set a temperature close to ambient This saves on power consumption and reduces condensation and the associated waste disposal issues Figure 3 5 Autosampler Configuration Dialog Direct Control Tab Autosampler Configuration Configuration fi Maintenance Diagnostics Temperature Settings M Tray Cooling setpoint 10 PC Injection Valve ISS A Valve ISS B Valve Relnitialize Load Load 1 10 Load 1 10 O inject O inject 11 2 O Inject 1 2 Wash System wash Inside sample needle wash volume uL 2000 O Inside sample needle wash O Outside sample needle wash OO Fil Transport Replace the Mobile Phases WARNING Environmental Hazard Always follow appropriate safety procedures and local reduirements when handling or disposing of waste chemicals Refer to the Material Safety Data Sheets for the mobile phases AB SCIEX recommends replacing solvents each month After replacing the solvent purge and flush the system Tip Sonicate or sparge the mobiles phases with helium to degas before adding them to the bottles gt Table 3 3 describes typical mobile phase mixtures for ekspert nanoLC 415 and 425 systems The nanoLC 415 system does not have a Gradient 2 channel Operator Guide ekspert nanoLC 400 Systems 21 of 174 System Overview Caution Potential System Damage Use LC MS grade pre made solvents such as those from Burdick Jackson These solvents can be ord
155. vec samel EHEN Output 1 P Output 4 Valve Position M ouput JT Outputs outputs T Output 6 Operator Guide ekspert nanoLC 400 Systems 93 of 174 Best Practices and Troubleshooting 5 On the Direct Control tab click System wash 6 In the Inside sample needle wash volume field type 1000 Figure 6 9 Autosampler Configure Dialog Direct Control Tab x m Temperature Settings F Tray Cooling setpoint fe 7 Hat Cap setpoint feo EG Reset Errors m injection Vave _ I 88 4 Vale S5 BWValye Relnitialize Load Load 1 10 Load 1 10 Inject Inject 1 2 Inject 71 2 m Wash System wash Inside sample needle wash volume Inside sample needle wash Syringe Solvent Outside sample needle wash Wash Solvent Fill Transport sn se Click Start Wait for the wash procedure to stop 9 Click the Diagnostics tab and take a screen capture of the tab ekspert nanoLC 400 Systems 94 of 174 Send the screen capture in an email to the FSE Operator Guide Best Practices and Troubleshooting Troubleshooting Guidelines When troubleshooting the ekspert nanoLC 400 systems follow these safety practices Caution Potential System Damage Once the power to the system is turned off wait approximately 30 seconds before disconnecting an assembly To avoid damaging electrical parts do not disconnect an electrical assembly while the system is on
156. vides additional flexibility e It includes an in depth view of the steps the autosampler is going to execute e It allows for modification and configuration of the method to optimize all aspects of the injection process Caution Potential System Damage When possible use the basic autosampler Method Editor to create a method Incorrect use of Advanced Editor settings can damage the autosampler Methods created in the Advanced Editor must be hardware safe and analytically reliable Review advanced methods carefully before use 1 Inthe Run Manager window click Autosampler Methods 2 Optional To open an existing file click File gt Open 3 Select the basic options in the Method Editor dialog 4 Click Advanced Editor in the bottom left corner Figure E 2 Advanced Method Editor Dialog Showing Trap and elute Method Details ali File codes Insert Delete Edit Command Description 1 Initialize Autosampler Device ISS A Load for Gradient 1 ready to start 4 Needle Wash Pre wash 1x 5 Get Sample uL Pickup Sp 0 5 ples 2 mm from bottom for Loading Pump ready to start 7 Start Loading Pump Injector Inject for Loading Pump complete 10 Valve ISS A Inject _ 11 Start Gradient Pump 1 _ 12 Valve Injector Load _ 13 Needle Wash Clean Up 5x 14 Test on B1 Ok ekspert nanoLC 400 Systems Operator Guide 122 of 174 Autosampler Method Editor 5 Inthe Advanced E

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