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User Manual - California State University, Fresno

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1. Tray 0 C Slow m oo a gt Pause Figure 5 16 Run Manager Window Two Samples step 10 Check all the lines you wish to run under the Run column step 17 Run the sequence in Sequential mode Click Flush Equilibrate when Idle to start all the pumps if you wish Note f you need to edit the first two lines un check flush equilibrate to do so step 12 Now that the run table has been defined Figure 5 16 save it by selecting File gt Save 5 8 Starting a Run Samples to be analyzed are selected by placing a check mark in the box to the right of the appropriate row numbers in the Run Table For this series of runs make sure there are check marks in the boxes for the first four rows of the Test Table created in the previous section Checking the Flush Equilibrate when Idle will initiate the pre run flush for the first method With this options selected the system will continue to flush at the end of the sequence Initiate the sequence of analyses by clicking on Start After the start button has been pressed it will change to a red Stop button that can be used to abort the run at any point during the analysis Once the flow rate has stabilized the sample injection process will begin Channel 1 will run first then Channel 2 will start at the conclusion of the Channel 1 run While the run is in progress the Acquisition Window can display the specified flow profiles for solvents A and B as well as their a
2. 82 Operator s Manual Default methods Dual Trap Column Configuration Pump Channel 2 Trap 1 Same as previous method except injection NONE instead of injection Standard LC Method Settings LC Method Settings Selected Method Selected Method Name Ch2_trap 2 Name Ch2_trap 2 Summary Run Conditions Flow Profile Flow Table Summary Run Conditions Flow Profile Flow Table hoo Flow Mode Conserved flow O Independent flow Pre Run Flush column for 0 minutes using 100 initial flowrate conditions go 80 Profile Editor 79 Oa OB Sample Injection 60 Runtime None 45 min O Standard Sample valve opens prior to beginning Flow Profile and remains open Total flowrate O Metered Inject nL of sample st 100 initial flowrate conditions 0 25 pLimin 15000 Q Rapid Inject nL of sample at maximum flowrate maintaining initial mixture conditions point 2 v Post Run Flush column for 0 5 minutes using 100 ending flowrate conditions 18 25 27 HS time min View Audit Trail ak Cancer Bette view Audit Trai LC Method Settings Selected Method Name Ch2_trap 2 v Save Print Summary Run Conditions Flow Profile Flow Table Flow Mode Time min A 0 98 25 60 10 32 10 Total flowrate 98 0 25 HLAmin Conserved
3. Figure 6 4 System Alerts Window nanoLC Operator s Manual 611 00012 Revision A1 Appendix A Spare Parts and Consumables A 1 Consumables nanoLC 1D and nanoLC 2D 800 00006 Accessories kit for 1D Includes calibration kit syringe fittings capillary tubing and cables 801 00025 Accessories kit for 1Dplus and 2D Includes mobile phase reservoirs calibration kits priming tool and syringe fittings capillary tubing and cables 801 00002 Flow rate calibration kit with 20 uL calibrated pipettes 10 for 1 30 L min flow rate 801 00020 Replacement calibration pipettes 20 uL for 1 30 L min 10 pkg 801 00006 Flow rate calibration kit with 5 uL calibration pipettes 5 for 50 1000 nL min flow rate 910 00007 Replacement calibration pipettes 5 uL for 50 1000 nL min 10 pkg 801 00003 Pump priming tool kit includes 10 mL syringe 1D 910 00020 Fitting FingerTight 1 16 PEEK 10 pkg 910 00021 Fitting hex 1 16 SS 0 43 long 10 pkg 920 00006 PEEK hex nut 10 pkg 910 00023 Ferrule 1 16 PEEK 10 pkg 910 00026 Ferrule 1 16 SS 10 pkg 920 00004 Fitting hex 1 16 SS 3 4 long 10 pkg 920 00005 Fitting hex 1 16 PEEK 34 long 10 pkg 920 00008 Sleeve FEP green 395 um ID 1 16 OD 10 pkg 200 00165 Fitting NanoTight 1 16 PEEK 0 90 long with Tefzel ferrule 910 00022 Fitting MicroTight 0 025 PEEK 10 pkg 920 00002 Ferrule MicroTight for 0 025 10 pkg 200 00002 MicroTig
4. nanoLC Operator s Manual 611 00012 Revision A1 28 Operator s Manual Waiting for pressure sensors to settle _ Figure 4 4 Auto zero Status Window step 4 When the auto zeroing process is complete exit the diagnostics window and return to the Acquisition Window step 5 Repeat for both channels if not conducted simultaneously 4 6 Checking Flow Stability The flow stability of the A and B channels can be determined in a similar fashion to the procedure used for zeroing pressure transducers as described in Section 4 5 by selecting the Check Flow Stability diagnostic test Two screens will be observed during the running of the test Figure 4 5 Initially the process requires the controllers to stabilize for 60 seconds This is then followed by a 30 second examination of the control Step 1 Step 2 Stabilizing Controllers Step 1 2 Please wait about 60 seconds Examining Control Step 2 2 Please wait about 30 seconds Figure 4 5 Checking Flow Stability Progression Repeat this for both channels 4 7 Autotuning Flow Controllers The flow controller autotune should be run on a monthly basis to optimize the performance of the system To avoid the possibility of overpressure disconnect the capillary at the mixing tee fitting prior to performing the autotune procedure CAUTION step 1 When the Autotune button is selected in the diagnostic window the system s response to changes in flow rate is monitored
5. ready click Start Quick Start Guides 79 Default methods Dual Trap column configuration Autosampler E Autosampler Settings Autosampler Procedure System Configuration Name micropickup_1ul_simultaneous start Eksigent AS 1 Output 1 OFF Initialize LC channel 1 Output 2 OF F Initialize LC channel 2 Valve Injector Load Switch AS injector valve to Load po Aspirate Reagent 1 Speed 1 Height Pick up Reagent with specified volu Aspirate Sample Speed 1 Height Pick up Sample with specified volur Aspirate Reagent 1 Speed 1 Height Pick up Reagent with specified volu Output 1 ON Start LC run on channel 1 Output 2 ON Start LC run on channel 2 Wait for Input 1 LOVW Wait for valve trigger signal LOWY fre 1 2 3 4 5 6 y 8 Valve Injector Inject Switch AS injector valve to Inject pc Wait for Input 1 HIGH Wait for valve trigger signal HIGH frt Valve Injector Load Switch AS injector valve to Load po Dispense Waste Speed 5 Height Dispense specified volume from syr Needle Wash Perform needle wash END 80 Operator s Manual Default methods Dual Trap Column Configuration Pump Channel 1 Method Settings Chisimutaneous smins m swe _ Pie j Quick Start Guides 81 Default methods Dual Trap Column Configuration Pump Channel 2 Trap 1 amp LC Method Settings LC Method Settings ener SC ee ie J Flow Profile al
6. WARNING gt WARNING gt WARNING The following safety practices apply to the optional nanoLC AS1 Autosampler system Changes or modifications to this unit not expressly approved by Exsigent Technologies could void the instrument warranty and render the system inoperable Use of this equipment in a manner not approved by Exsigent Technologies may inhibit its safety protection When you use the nanoLC Autosampler system follow generally accepted procedures for quality control and methods development When you use the nanoLC Autosampler system for chromatographic analyses and observe a change in the retention of a particular compound the resolution between two compounds or peak shapes immediately determine the reason for the changes Do not rely on the analytical results until the cause of the change is determined Only use fuses of the type and current rating specified Do not use repaired fuses or by pass the fuse holder The supplied power cord must be used with a power outlet containing a protective ground contact Do not change the external or internal grounding connections Tampering with or disabling these connections could create a safety hazard and or damage the nanoLC Autosampler system The instrument as shipped is properly grounded in accordance with normal safety regulations The combination of a nanoLC Autosampler system with a LC MS system may require additional safety measures as described by the LC MS syst
7. The conditions used for separating the sample are stored in the LC method This section will create an analysis method called LC Test LC methods are accessed for editing by clicking on LC Method Editor located in the Run Manager window Configuration menu then the LC Method editor appears as described in this section If NanoLC 2D is chosen then the 2D Editor will appear This editor allows for the easy and rapid con figuration of multi step on line 2D methods Each multi step experiment is saved as a single method file and can be programmed on a single line of the Run Table Please see both the 2D section of the software manual as well as Appendix C for further description of the 2D valve configurations Q Note For the NanoLC 2D If NanoLC 2D Parallel is chosen in the System gt Instrument step 1 Click LC Methods on the Run Manager window to display the Method Settings dialog box Figure 5 8 amp LC Method Settings Selected Method Name Chi_trap loading Summary Run Conditions Flow Profile Flow Table Method Identification Method ID 99 Column Information Manufacturer New Objectives trap column particle size 5 am Type c 18 diameter 400 um Serial Number NA length 05 Jom Sample Injection Flow Profile Standard Duration 5 min Detection External Detector Auxillary 4 D channel available View Audit Trail Figure 5 8 LC Method Editor Summary Tab nanoLC Operator s Manua
8. The instrument site should be within about 6 m 20 ft of the air nitrogen regulator When using compressed air Exsigent Technologies strongly recommends an air supply having a dew point of less than 4 5 C 40 F When using dry nitrogen or compressed air Exsigent Technologies strongly recommends the use of air filtration to 5 um e g for compressed gas supplied at less than 150 psi a Wilkerson F18 filter and regulation to a working pressure of 100 psi e g for compressed gas supplied at less than 150 psi a Wilkerson R18 regulator or a Wilkerson B18 combination regulator filter If hydrocarbons are suspected in the air supply i e air supplied from an oiled compressor Exsigent Technologies strongly recommends the regulator be followed with a coalescing filter suitable for particle removal to 0 01 ym e g a Wilkerson M18 coalescing filter Note Always follow manufacturer s specifications in selecting and operating gas filters and regulators Note Always follow manufacturer s specifications for connecting mounting and orienting gas filters and regulators Note Always perform proper maintenance of traps filters and coalescing filters per manufacturer s specifications Liquids collected in filters and coalescing filters must be drained before the liquid level exceeds the manufacturer s specifications nanoLC Operator s Manual 611 00012 Revision A1 Safety and Site Requirements 5 1 4 4 Bench Space Requirement The nanoLC system
9. c ssscsecsssssstesssetssseetetsseensetenenees 3 2 Loading Mobile PASOS ssiri terrissa 3 3 Preparing the nanoLC Autosampler csessescecseseeseesesteneetesnenseteatensetentenseteates 3 4 Flushing the Autosampler Syringe and Liquid Path 3 5 Connecting the nanoLC to the nanoLC Autosampler 3 6 Verifying the Flow Ratte ccscsssssssssssmsssessssessssscssnsscsensscsensnssneensacsenaceensaees Chapter 4 Routine Maintenance ssssssssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 28 4 1 Recommended Maintenance uu scenes cesses cessesseseessnsansneansensneenesnees 4 2 Disposing of Waste 4 3 Changing the Sample LOOP cece ect nennensneenescee nese neeees te 4 4 Replacing Capillary Connections cece cesses nananensnseneseeees 4 5 Zeroing the Pressure Transducers 4 6 Checking Flow Stability ressis 4 7 Autotuning Flow Controllers cscs ieee ineessssesnas ne 4 8 Calibrating the Flow Meters 4 9 Cleaning and Inspecting the InStrUMenNt c cece nents nesses 31 nanoLC Operator s Manual 611 00012 Revision A1 V Chapter 5 Quick start Guide ssssssssssonnsunnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn OO 5 1 5 2 5 3 5 4 5 5 5 6 5 7 5 8 5 9 Powering up the System sssssssssssssrssnnnsnsrnrrnnsnsnrnrnnnunnnnnrinnunsnnnrrenonnnnnrenonnrnreene Purging and Flushing with New Solvents Equilibrating the SysSteMisnpssssronnsiueni aana Creating an Autosampler Method sssssssssssssrrsssrrenssrrensnrninsnrnensnninannnnnnnnnina
10. the meniscus or an air bubble to transit from the black stripe to the end of the capillary should be 4 minutes With the low flow calibration assembly the time it takes for the meniscus or an air bubble to transit across two segments of the capillary 2 uL should be 4 minutes Press Stop when the fluid front reaches the end of the pipette step 6 f the flow rate falls outside of the acceptable range gt 5 re calibrate the flow meters per the procedure found in Section 4 8 step 7 Disconnect the calibration assembly and blow out the liquid inside the pipette using a pipette bulb or can of compressed air Direct Control Pump Direct Control Not Connected Channel A Total flowrate Conserved Flow 100 Jo 5 uLimin O Independent Flow Q 5 pLimin Valve Direct Control Load Position Load Position Inject Position Figure 3 6 Direct Control Window Flow Rate Check step 8 Set channel B to 100 and repeat steps 5 and 6 to verify the flow rate for pump B step 9 Disconnect the calibration assembly nanoLC Operator s Manual 611 00012 Revision A1 Chapter 4 Routine Maintenance Chapter 4 describes the general procedures used to properly maintain the nanoLC system Topics included in this chapter include e recommended maintenance Section 4 1 e disposing of waste Section 4 2 e changing the sample loop Section 4 3 e replacing capillary connections Section 4 4 e zeroing the pressure trans
11. 16 wrenches Re install the filter assemblies and tighten the stainless steel tubing connections into the filter assemblies Turn on the power to the nanoLC and establish communication Purge out or remove the old solvent from the mobile phase reservoirs Refill with new solvent and purge at least 20 times on each pump Disconnect any tubing at the outlet of Channel 1 and Channel 2 Flush 100uL through Channel 1 and Channel 2 Test the nanoLC for leaks a Plug the outlet of CH1 and CH2 b Inthe service menu ramp the power in steps 25 50 100 Check system for leaks in fittings inside the box Repair any leaks then ramp the power again to test c Hold the system at 100 power for 10 minutes Repair any leaks and then stop the flow Index A Acquisition Window 47 Air Supply Requirements 4 Autosampler Configuration Window 36 Autosampler Method Creating 37 Autosampler Method Editor Window 38 Autosampler Safety Practices 2 Cc Calibration Pipette Guide 31 Calibration Values 50 Calibration Values Tab 50 Capillary Connections Replacing 25 Cleaning 31 Configuring nanoLC System 11 Configuring the nanoLC Autosampler 13 Connecting Power 9 Connecting the PC 9 Consumables 59 Creating a New Run Table 45 DEMO MODE 11 Diagnostics 49 Direct Control window 35 Direct Control Window 26 Equilibrating the System 35 Error Messages 58 External Interface 63 F Flow Calibration Window
12. Injection 39 Troubleshooting 49 Troubleshooting Checklist 52 Troubleshooting Guidelines 51 U Unpacking 7 65
13. Out 0 When using the nanoLC Autosampler one can also handshake with other components such as a MS detector using the Ready Out from that device This will allow the LC queue to stop if the peripheral is not ready To do this connect the Ready Out from the peripheral to the In 3 and Gnd of the AS1 Input P6 cable Add the following line to the beginning of the AS1 method Wait for Input 3 LOW Note If the Ready Out from the peripheral is a TTL high trigger instead of TTL low or contact closure change the line to 3 HIGH The AS method will not proceed beyond the first line until it has received a ready signal If you disconnect the peripheral be sure to remove this line from the AS method or to short In 3 to ground nanoLC Operator s Manual 611 00012 Revision A1 Appendix C Valve Configuration Diagrams This Appendix will show several possible configurations for the nanoLC These sketches can be used as guides when configuring the plumbing for your own instrument Figure C 1 shows an automated 2D configuration with 2 parallel traps The flow on the traps is unidirectional le loo i High Flow SCX colu Channel Waste AS syringe Low Flow Channel Sample AS needle 50 um ID capillary 25 um ID capillary RP trap valve in inject position valve in load position Figure C 1 2D Configuration with 2 Parallel Traps nanoLC Operator s Manual 611 00012 Revision A1 70 Operator
14. a sample run click Run lt 8 Manager 1800 1620 1440 78 Operator s Manual Run Manager File Edit Yiew Devices System Suitability Help micropickup_1ul_simulttaneous start Ch1_wash_equilibrate trap loading Queued Ch2_trap 1 Queued micropickup_1ul_simultaneous start Ch1_wash_equilibrate trap loading Queued Ch2_trap 2 Queued micropickup_1ul_simuttaneous start Ch1_wash_equilibrate trap loading Queued Ch2_trap 1 Queued micropickup_1ul_simuttaneous start Ch1_wash_edquilibrate trap loading Queued Ont on fe Wn Ch2_trap 2 NAN n A A LA i Queued JOP eee Current Tra Autosampler Tray 1 Method Definitions Autosampler Methods LC Methods Analysis Methods Run in Synchronized Multi Channel mode Sequence line 1 Load trap 2 Line 2 Gradient Trap 1 Line 3 Load trap 1 Line 4 Gradient trap 2 vv Run Sequence O Sequential OAs Available Synchronized Multi Channel O FlushEquilibrate when Idle g start Elapsed Time 00 00 00 Queued Time 03 00 00 Click on Flush Equilibrate when Idle to start equilibrating pumps at initial gradient conditions Click Flush Equilibrate to start equilibrating at_ gt Flush quilibrate when Idle initial gradient conditions 3 Once system is Elapsed Time 00 00 00 Queued Time 01 05 00
15. and adjustments are made to the controllers PID proportional integral derivative loop step 2 A prompt window alerts you that the autotune is about to begin Figure 4 6 nanoLC Operator s Manual 611 00012 Revision A1 Routine Maintenance 29 AutoTune Alert Any devices or columns connected to the pump outlet may be subjected to high pressures Please disconnect any devices that could be damaged Figure 4 6 Autotune Prompt step 3 This is followed by status windows Figure 4 7 which alert you that the system is preparing to Auto tune and then performing the autotune These adjustments improve the system s response time and flow rate accuracy Step 1 Step 2 The 4 flow controllers are being tuned Please wait Figure 4 7 Autotune Status Bars Channel A step 4 Upon completion close the diagnostics window and return to the main screen step 5 Repeat for both channels if not conducted simultaneously Moving the slider to the right will change the pump to respond faster under damped Moving the Q Note The slider below the Auto Tune Controllers button is used to change the pump time response slider to the left will change the pump to respond slower over damped 4 8 Calibrating the Flow Meters The flow meters should be calibrated quarterly or when the gradient separation performance seems to be drifting Calibration of the flow meters consists of measuring the velocity of a liquid front in a tube of
16. be made to the channel 2 low flow gradient side of the instrument One can also choose to synchronize MS data with the beginning of the injection To do this connect the Valve Out pin 6 and the Ground terminals to the MS contact closure nanoLC Operator s Manual 611 00012 Revision A1 External Interface 67 B 3 3 Triggering Peak Parking Peak parking on the nanoLC system can also be triggered externally using the back panel I O connectors Connect the ground of the triggering contact closure to a Ground terminal on the back panel and connect the second wire of the contact closure to the Park In terminal pin 9 The Park Out terminal pin 4 can also be used to monitor the state of Peak Parking The Peak Parking Toolbox in the nanoLC software is used to configure the Peak Parking flow rate flow rate reference typically column and provide for hold and lockout times on triggering Peak Parking can be configured on ThermoFinnigan MS systems that provide a configurable con tact closure output This variable contact closure can be configured in the Xcalibur software to be activated based on selected criteria including ion intensities and ion inclusion lists A number of systems may also allow a fixed time or duration contact closure that can be configured in the MS software to trigger peak parking These may be useful for well defined experiments that will benefit from peak parking at a known time not MS dependent signals B 3 4 Ready
17. flow O Independent flow x a nj Profile Editor ajajs o 45 98 BS lSlola a View Audit Trail Hints for Dual Trap columns 1 Channel 1 and 2 methods should run the same length of time 2 If pre washing traps is desired change Channel 1 sample injection type to NONE and use timed events a VLV TTL low starts the injection b VLV TTL high ends the injection 3 Contact Eksigent Technologies Support for guidance with complex HPLC methods Appendix E Replacing Internal Instrument Filters Replace solvent filters and check for leaks step 1 step 2 step 3 step 4 step 5 step 6 step 7 step 8 step 9 step 10 step 11 Power off AS 1 autosampler and remove output input serial and power cables Disconnect tubing connections from the autosampler valve and remove the waste tubing from the waste container Remove the autosampler from the top of the nanoLC and set aside Power off the nanoLC Open the cover of the nanoLC and visually check for any signs of leaks corrosion etc If any of the pumps appear to have leaked follow the procedure to replace the pump seals found in Appendix A of this document Remove the internal instrument filter assemblies with a 9 16 and 1 4 wrench Open the filter assemblies and remove the filter frits Rinse the filter assemblies with methanol and replace with new filter frits Tighten the filter frits in place finger tight 1 4 turn with two 7
18. for initial operation or for operation following an extended period of non use Topics covered in this chapter include e hardware components and functions Section 3 1 e loading mobile phases Section 3 2 e preparing the nanoLC Autosampler Section 3 3 e flushing the sample loop and sample needle Section 3 4 e connecting the nanoLC to the nanoLC Autosampler Section 3 5 e verifying the flow rate Section 3 6 3 1 Hardware Components and Functions This section provides a general description of the key components of the nanoLC systems and their various functions Mobile phase outlets are located on the right side of the nanoLC system Each channel has one mobile phase outlet which can be connected to the autosampler and a 10 port column switching valve The 10 port Column Switching Valve is used to switch between reverse phase traps that are con nected to the valve for rapid sample loading Refer to Appendix D for different valve configurations An Optional 6 port injection valve is available instead of the nano LC Autosampler for manual sample loading Refer to Appendix D for valve configurations 3 2 Loading Mobile Phases The procedure for loading mobile phases will be described for a single binary gradient system for reverse phase chromatography If you have a NanoLC 2D system complete this procedure for both channels 1 and 2 It is suggested that you use mobile phase A described below in both reservoirs of channel 1 and
19. the flow stream System does not initiate an injection nanoLC Operator s Manual Quick start Guide 57 Table 6 1 cont Troubleshooting Checklist Possible Cause The ports are plugged The ends of fused silica connected to the port might be crushed or not cut properly Ferrule was not seated properly in the port Rotor seal is scratched Internal leakage in the valve Valve tubing connections are not made correctly gap dead volume between sleeves and port The ports are plugged Bubbles trapped in the valve Valve tubing connections are not made correctly gap dead volume between sleeves and port System flow is unstable Flow stabilization is set too low Autosampler is configured with wait for injection but the LC method is used with no injection 611 00012 Corrective Action Manually flush each port with cleaning solvent using a syringe If flushing does not clean the port contact Eksigent Technologies Service Representative to replace the valve Check the ends under a microscope Cut the ends if needed Check the tubing connection and make sure the ferrule is seated properly Contact Eksigent Technologies Technical Support for assistance Contact Eksigent Technologies Technical Support for assistance Check tubing connections Make sure the cuts at the end of the capillaries are square Manually flush each port with cleaning solvent using a syringe or contact your Eksigent Technologie
20. use the suggested A and B mobile phases for the low flow gradient in channel 2 If you have a nanoLC 1D system complete this procedure for channel 1 using the mobile phases described below It is suggested that you use mobile phase A in the single reservoir for channel 2 for high flow sample loading nanoLC Operator s Manual 611 00012 Revision A1 16 Operator s Manual Required tools and materials e Mobile phase reservoirs p n 800 00105 e Clean degassed HPLC grade mobile phase A water with 2 acetonitrile and 0 1 formic acid suggested e Clean degassed HPLC grade mobile phase B acetonitrile with 2 water and 0 1 formic acid suggested e nanoLC priming tool p n 801 00003 e 15 mL centrifuge tube step 1 Clean all 50 mL mobile phase reservoirs with appropriate solvents step 2 Close the orange valve on the reservoir by turning it to the perpendicular position step 3 Fill reservoir A with mobile phase A and reservoir B with mobile phase B step 4 Install both reservoirs and open each valve by rotating to a vertical position Purging and flushing the pumps are critical operations to get maximum performance from a new instrument Purging rapidly replaces the solvent in the pumps while flushing replaces the solvent in the capillaries connecting the pumps to the sample injector Mobile Phase Mobile Phase Reservoir 2A Reservoir 1B Mobile Phase Mobile Phase Reservoir 2B Reservoir 1A Figure 3 1 Top Panel Reservoirs
21. vial tray in the Run Manager Window select the Devices gt Autosampler Device Settings menu to set the tray type You will need to restart the Run Manager after changing the tray type step 4 If you do not see the columns shown in Figure 5 6 choose Edit gt Choose Column from the menu and select the appropriate columns for display step 5 Click Autosampler Methods to bring up the Autosampler Method Editor Window Figure 5 7 nanoLC Operator s Manual 611 00012 Revision A1 38 Operator s Manual E Autosampler Settings Autosampler Procedure System Configuration Name micropickup_1ul_Trap loading ea Ea pi 3 4 5 6 ie 8 9 Apa peop pe fs aA BON FG hs Save Output Output Valve Aspirate Aspirate Aspirate Output Wait for Input Valve Wait for Input Valve Output Dispense Needle Wash END 1 OFF 2 OFF Injector Load Reagent 1 Sample Reagent 1 1 ON 1 LOWW Injector Inject 1 HIGH Injector Load 2 ON Waste Eksigent AS 1 1 Height 1 Height 1 Height Initialize LC channel 1 Initialize LC channel 2 Switch AS injector valve to Load position 1 6 Pick up Reagent with specified volume Total aspirate volume needs to be less tr Pick up Sample with specified volume Total aspirate volume needs to be less the Pick up Reagent with specified volume Total aspirate volume needs to be less tr Start LC run on channel 1 Wait for valve trigger signal LOVY from LC channel 1 Switch AS injector v
22. 1 to start the method 8 Wait for input 1 Low Wait for an input signal injection signal from Channel 1 before continuing autosampler method 9 Valve Injector Inject Place autosampler valve in the inject position 10 Wait for input 1 High Wait for injection signal from LC Channel 1 to turn off indicating the loop injection is complete 11 Valve Injector Load Place autosampler valve in the load position 12 Output 2 On Send Output 2 relay signal to LC Channel 2 signaling LC Channel 2 to start the gradient method 13 Dispense 25uL Waste 5 0 Dispense aspirated volume to waste Total aspirate and dispense volumes must be equal 14 Needle wash 50uL Perform 50uL needle wash 15 End Method end step 8 To modify method steps click on the step and change the volume by typing over Modify the operation or parameter by clicking on the step and choosing a new value in the drop down menu step 9 Add new steps by clicking on the gt gt arrows to the left of the line Delete a step by clicking on the xX nanoLC Operator s Manual 611 00012 Q Note for an autosampler configured with the standard 25uL syringe Revision A1 40 Operator s Manual step 10 Change the Needle Wash volume to 100 uL step 11 Needle Wash 100 pL step 12 Type Autosampler Test in the Name field and click on Save next to the name to store the method step 13 Click OK button to close the Autosampler Method Editor Window 5 5 Creating an LC Method Channel 1
23. 30 Flow Controllers Autotuning 28 Flow Meters Calibrating 29 Flow Profile Tab 42 44 nanoLC Operator s Manual Flow Rate Verification 21 Flow Stability Checking 28 Flow Table 44 Flushing 34 Flushing the Autosampler Liquid Path 20 Flushing the Autosampler Syringe 20 Hardware Diagnostics 49 Hardware Diagnostics Window 27 Initialization 15 Inspecting 31 Installation 7 Installing Software 10 Instrument Configuration Window 11 Instrument Settings 10 Interface Connections 63 L LC Method Creating 40 43 LC Method Editor 40 Loading Mobile Phases 15 Maintenance 23 MS Data Collection Triggering 66 nanoLC Autosampler Connecting 20 Preparing 19 O Other Autosamplers 66 Other Instruments Connecting 65 611 00012 Revision A1 86 Operator s Manual P vV PC 9 Valve Configuration Peak Parking Diagrams 69 Triggering 67 Valve Connectors 65 Placement 8 Verifying Flow Rate 21 Power Requirements 4 Viewing Data Files 47 Powering up 33 Pressure Transducers W Zeroing 26 Purging 34 Waste Disposal 24 Q Quick start Guide 33 Ready Out 67 Remote Interface 64 Replacement Parts 60 Run Conditions 43 Run Conditions Tab 41 Run Manager Window 13 Two Samples 46 Run Table Creating 45 S Safety 1 Safety Conventions 1 Sample Loop Changing 24 Site Requirements 1 Spare Parts 59 Starting a Run 46 Support 3 System Alerts 58 T Template for Micropickup
24. 70266 0 0 62330 00 Figure 6 1 Hardware Diagnostics Calibration Values Tab The values should be documented before and after as part of maintaining a good instrument log and noting changes These values should be very similar from diagnostic to diagnostic large changes can indicate a problem nanoLC Operator s Manual 611 00012 Revision A1 Quick start Guide 51 6 3 General Troubleshooting Guidelines To avoid the possibility of electric shock never disconnect an electrical assembly while power is applied to the system After turning power off wait about ten seconds before disconnecting an CAUTION assembly To prevent injury always observe good laboratory practices when you handle solvents change tubing or operate the system Know the physical and chemical properties of the solvents Refer to CAUTION the Material Safety Data Sheets for the solvents in use There are no user serviceable components or assemblies inside the nanoLC Service or any internal parts or assemblies requires a Factory Qualified Service Technician CAUTION When troubleshooting the nanoLC system follow these safety practices The basic steps for nanoLC system troubleshooting are step 1 Step back and look at the overall system Is something obvious causing the problem For example is an instrument unplugged or improperly connected step 2 Compare current system operation with the way the system operated before the problem started
25. Identify conditions pressures power settings flow rates that are different than they were when the system was operating normally For example if the output pressure is usually 500 psi with a certain method is the system pressure currently in the same range or drastically higher possibly caused by a plug or lower possibly caused by a leak step 3 Identify in the order listed below the symptom that varies from normal system operation e System power on and initialization initialization fails e System diagnostics flow stability controller tuning e Flow rate in each channel high low erratic e Output pressure high low erratic step 4 For each isolated symptom identify a list of possible causes using the troubleshooting checklist be low The troubleshooting in section 6 5 allows you to narrow down the possible causes of a symp tom and find suggested corrective actions step 5 If this process does not correct the problem contact Eksigent Technologies Technical Support nanoLC Operator s Manual 611 00012 Revision A1 52 Operator s Manual 6 4 Troubleshooting Checklist Table 6 1 Troubleshooting Checklist Symptom System Initialization Power LED on front panel is not on Front panel power LED is on but software fails to recognize the instrument s presence Liquid leaking at the bottom of the mobile phase reservoirs Loud hissing sound from the instrument nanoLC Operator s Manual Possible Cause Pow
26. Manual Quick start Guide 53 Table 6 1 cont Troubleshooting Checklist Possible Cause Pressure transducers zero offsets are incorrect Air trap in the pump Internal filters are plugged Leak in the system prior to the purge valve Air trapped in the pump Pump remains on long enough to prompt a re stroke Check valve is leaking Pump restroke delay is too short Leak in instrument Internal filters are plugged No air to system System not properly primed and flushed Internal filters are plugged Flow rate set point too high for system back pressure Air pressure too low 611 00012 Corrective Action Zero pressure transducers Section 4 5 Prime the pump Section 3 2 Contact Eksigent Technologies Technical Support for assistance Check all connections Prime the pump Section 3 2 Check the duration of time between re strokes to see if the pump re stroke was appropriate Contact Eksigent Technologies Technical support for assistance Contact Eksigent Technologies Technical Support for assistance Contact Eksigent Technologies Technical Support for assistance Contact Eksigent Technologies Technical Support for assistance Connect 100 psi clean dry air to the instrument s air inlet Prime and flush system Section 3 2 Contact local Eksigent Technologies Technical Support to replace internal filters Reset flow rate to a lower level Establish correct air pressur
27. Microliter Pickup Direct Loading Aspirate Aspirate Aspirate Output Wait for Input 1i 2 3 4 5 6 7 8 9 Valve Wait for Input Valve Dispense Needle Wash END 1 OFF 2 OFF Injector Load Reagent 1 Sample Reagent 1 2 0N 2 LOW Injector Inject 2 HIGH Injector Load Waste System Configuration Eksigent NanoLC AS 1 1 Height 1 Height 1 Height 5 Height Initialize LC channel 1 Initialize LC channel 2 Switch AS injector valve to Load position 1 6 0 Pick up Reagent with specified volume Total aspirate volume nee 5 Pick up Sample with specified volume Total aspirate volume need 0 Pick up Reagent with specified volume Total aspirate volume nee Start LC run on channel 2 Wait for valve trigger signal LOW from LC channel 2 Switch AS injector valve to Inject position 1 6 Wait for valve trigger signal HIGH from LC channel 2 Switch AS injector valve to Load position 1 6 0 Dispense specified volume from syringe to Waste Perform needle wash OK Cancel 76 Operator s Manual Default methods Direct injection to column with Channel 2 Pump Channel 2 FS LC Method Settings X LC Method Settings Selected Method Selected Method Nam oiz ngaten I Ce ee e Summary Run Conditions Flow Profile Flow Table Summary Run Conditions Flow Profile Flow Table Pre Run joo M Flush column for 15 minutes using 100 initial flowrate conditions Fl
28. ailable Synchronized Multi Channel LC Methods Flush Equilibrate when Idle 5 T T T mi 5 F CE e Gh at an ime 00 00 00 RRR bee d ME 5 ime 01 31 00 x SF ow m oo a gt Figure 2 9 Run Manager Window Select Devices gt Autosampler Type gt NanoLC AS1 and the following message will appear Eksigent Run Manager Figure 2 10 Run Manager shutdown prompt Click Yes to close the program Click on Run Manager again and Select Devices gt Autosampler Device Settings Set the COM port for the Autosampler nanoLC Operator s Manual 611 00012 Revision A1 14 Operator s Manual Autosampler Configuration Configuration Direct Control System Definition Optional Settings Eksigent AS 1 Tray Cooling setpoint Serial Port v C Air Segment C Headspace Pressure Rinse with Valve in Inject Position Baudrate 9600 v Address 4 Tray Type 96 High w Syringe Speed Scale Factor 0 Tray Cooling Soy Needle Height mm Feeder Loop Volume 40 pL Tubing Volume 2 4 HL Fraction Collection Mode Syringe Yolume 25 pL Figure 2 11 Autosampler Device Settings The system is not yet configured for operation You must first complete the instructions in System Initialization prior to system operation nanoLC Operator s Manual 611 00012 Revision A1 Chapter 3 System Initialization Chapter 3 describes procedures used to prepare a system
29. alve to Inject position 1 2 Wait for valve trigger signal HIGH from LC channel 1 Switch AS injector valve to Load position 1 6 Start LC run on channel 2 0 Dispense specified volume from syringe to Waste Perform needle wash Figure 5 7 Autosampler Method Editor Window step 6 Select a method from the drop down method similar to the one above Highlight the name of the method rename it and click Save to create a new method step 7 All Eksigent autosampler methods should contain the following steps in the same order The volumes can be modified but the general format should remain the same for a given method type In this example a trap loading autosampler method is indicated nanoLC Operator s Manual 611 00012 Revision A1 Quick start Guide 39 Table 5 1 Template for Micropickup Injection Trap Loading Autosampler Method Step Operation Volume Parameter Speed Height Description 1 Output 1 Off Ensure output 1 relay is in off state 2 Output 2 Off Ensure output 2 relay is in off state 3 Valve Injector Load Place autosampler valve in load position 4 Aspirate 19uL Reagent 1 1 0 Aspirate from Reagent 1 vial position 5 Aspirate TuL Sample 1 1 5 Aspirate from Sample vial use appropriate height for sample vial 1 closest to bottom 5 5 mm from bottom of vial 6 Aspirate 5uL Reagent 1 1 0 Aspirate from Reagent 1 vial position 7 Output 1 On Send Output 1 relay signal to LC Channel 1 signaling LC Channel
30. arity is configurable from within the Control software The VLV out contact goes to OV closes when the injection valve moves from the Load position to the Inject position Its polarity is NOT software configurable The ABS out contact outputs an analog signal corresponding to the nanoLC s detector absorbance The wavelength monitored and the voltage per absorbance unit is configurable from within the Control software The RUN in contacts allows remote starting of a method Its polarity is configurable from within the Control software The external trigger should be a pulse type and does not need to be held in the trigger state The PRK in contact allows remote triggering of the peak parking function The instrument will re main in the peak parking state until the contact s state is changed Its input polarity is configurable from within the Control software ONDA WN Figure B 2 External Interface Connector The first AUX in contact is included for future expansion but is not currently used AUX in The second AUX in contact is also included for future expansion The GND contact is a system ground connection used in combination with other contacts such as ABS out 611 00012 Revision A1 External Interface 65 B 2 Valve Connectors B 2 1 Remote Interface The rear panel also features a Valve connector used as an electrical interface between the nanoLC and various valves Figure B 3 24 V The 24V contact supplies the 24V p
31. ctual flow rates Qa and Qb Traces can be added or deleted from the display by clicking on System gt Appearance in the Acquisition Window Figure 5 1 and selecting the desired items To zoom in on a particular area of the chromatogram click on the display and drag a box around the area of interest This will enlarge that area of the chromatogram To zoom back out right click and select Zoom Out or Back nanoLC Operator s Manual 611 00012 Revision A1 Quick start Guide 47 2007 Eksigent Technologies DEMO MODE DER File Yiew System Analysis Help Not Connected Qa Qb Pa Pb Po A B channel VOO imeroner duno z LC Method ch2_inj_gradient Sample Run Manager Sequence 1000 1000 1000 1000 1000 1000 1000 Filename ek2_021227 pLimin psi Power Channel 2 UV Sig MAU Po psi Qa nLimin Qb nLimin Q total n Limin Profile A nLimin Figure 5 17 Acquisition Window Flow Profiles Status information such as A B and Time Remaining are also displayed at the bottom of the screen during the run Status bars at the top of the screen display the actual flow rate for pump A Qa and pump B Qb in nL min and pressure in psi for pump A Pa pump B Pb and column Pc 5 9 Viewing the Collected Data Files Previously collected data files can be re opened reviewed and re processed step 7 To view the data file collected from the first chromatogram click on File gt Open then select the data fi
32. ducers Section 4 5 e checking flow stability Section 4 6 e autotuning flow controllers Section 4 7 e calibrating flow meters Section 4 8 e cleaning and inspecting the instrument Section 4 9 e replacing internal instrument filters Appendix E 4 1 Recommended Maintenance The nanoLC system is designed and built for long term robust use in an active laboratory environment To ensure reliable performance the following procedures should be performed at the specified interval Table 4 1 Recommended Maintenance Procedure Frequency Instructions Changing Mobile Phase As needed Section 3 2 Zero pressure transducers Monthly Section 4 5 Checking flow stability Quarterly Section 4 6 Autotune flow controllers As needed Section 4 7 Waste disposal As needed Section 4 2 Replace interconnecting capillaries As needed Section 4 4 Change sample loop As needed Section 4 3 Calibrate flow meters Quarterly Section 4 8 Clean and inspect system Quarterly Section 4 9 Change internal instrument filters Annually Appendix E nanoLC Operator s Manual 611 00012 Revision A1 24 Operator s Manual 4 2 Disposing of Waste The user will need to properly dispose of the contents of any effluent waste in an appropriate chemical waste container For typical nanospray experiments waste from the 10 port column switching valve due to high flow sample loading will be collected in a waste vial The pump purge waste container located on the left side of the in
33. e 100 psi Revision A1 54 Operator s Manual Symptom Flow rate will not initialize at start of run Flow rate will not stabilize during a run System responds sluggishly when changing flow rates Inaccurate flow rate with no signs of leakage System pressure Pc is unusually low but flow rate is OK System pressure Pc is low and the flow rate is OK but pump pressures Pa amp Pb are high nanoLC Operator s Manual Table 6 1 cont Troubleshooting Checklist Possible Cause Flow rate set point too high for system back pressure Erratic flow rate due to bubbles in system Unable to meet required flow rate within specified tolerance One or both of the Internal solvent filters may be plugged Erratic flow rate due to bubbles in system Incorrect mobile phase setting Pump controller is out of tune Flow temperature is not stable Incorrect mobile phase setting Pump controller is out of tune Flow temperature is not stable Incorrect mobile phase setting Incorrect k values Loose connection after mixing tee Incorrect k values Flow module is plugged 611 00012 Corrective Action Reset flow rate to a lower level Prime and flush system Section 3 2 Relax flow tolerance setting section 3 2 Run the flow stability diagnostic to verify flow control Section 4 6 Autotune flow controllers System gt Hardware Diagnostics gt Auto Tune Controllers Section 4 7 Flush syste
34. e green 400 00106 Serial cable for instrument control and data collection 800 00056 External control synchronization cable 910 00001 Line air supply 25 1 4 300 00007 Internal air line black polyethylene 18 100 00041 Fitting air supply 1 4 compression 920 00088 Luer lock fitting for mobile phase valve set of 2 1D A 3 Replacement Parts AS 1 Autosampler 801 00044 PM kit for AS 1 with Titanium valve Includes pre puncturing needle fused silica needle 25 uL syringe and rotor 801 00045 PM kit for AS 1 with PAEK valve Includes pre puncturing needle fused silica needle 25 uL syringe and rotor 620 00005 Wash bottle bracket 620 00021 Air pre puncturing needle 800 00044 Fused silica needle 2 4 uL Bio 620 00139 PEEKsil needle 2 5 uL 800 00045 Fused silica needle 5 4 uL Bio 620 00027 Wash solvent vial with cap amp screws 200 00208 PEEKSil sample loop 1 uL 620 00039 PEEKSil sample loop 10 pL 620 00052 Belt 25 0 x 1 2 620 00053 Syringe valve 5 4 amp 5 620 00059 Power cable Europe 620 00060 Power cable USA 620 00064 Syringe 25 uL 620 00140 Teflon seal between syringe and syringe valve 910 00045 Replacement tip for 25 uL syringe 5 pkg 620 00065 Syringe 100 pL 620 00072 48 vial adapter 800 00059 NanoLC AS1 P6 input cable 800 00060 NanoLC AS1 P1 output cable 800 00063 Buffer tubing 500 uL 800 00064 Buffer tubing 50 uL nanoLC Operator s Manual 611 00012 Revision A1 Appendix B External Interface B 1 Interface Conn
35. e capillary sleeve and fitting into the port until both bottom out While lightly pressing the capillary and sleeve into the fitting with one hand use the other hand or the 1 4 wrench to tighten the fitting Open the Direct Control window by clicking System gt Direct Control from the Control software s Acquisition Window Figure 4 1 nanoLC Operator s Manual 611 00012 Revision A1 26 Operator s Manual Direct Control Pump Direct Control Not Connected A Total flowrate Conserved Flow 50 6 Limin O Independent Flow 1a 10 uL min C Monitor Baseline Valve Direct Control Load Position Load Position Inject Position Figure 4 1 Direct Control Window step 9 Set solvent A to 50 solvent B to 50 and Total flow rate to an appropriate value for the channel you are connecting step 10 Click the Start button and flush the capillary for 5 minutes 4 5 Zeroing the Pressure Transducers Before zeroing the pressure transducers it is advisable to open the outlet fittings from the mixing tees on all channels This will ensure there is no residual pressure on the outlet of the system Zeroing the pressure transducers should be performed on a monthly basis To initiate the pressure transducer zeroing procedure start the Control software and access the diagnostics screen by selecting System gt Hardware Diagnostics It is very important that the instructions below are followed precisely Attempting to
36. ections Appendix B describes the external interface to other instrumentation in order to synchronize sample injection with data collection The connector pin assignments are described below The connector is located on the rear panel of the nanoLC Figure B 1 Rear Panel External Interface and Valve Connectors nanoLC Operator s Manual 611 00012 Revision A1 64 Operator s Manual B 1 1 Remote Interface An enlarged drawing of the External Interface Connector shows that 12 lines are available Figure B 2 These will be described in turn A D in GND Run out PRK out RDY out VLV out ABS out RUN in PRK in AUX in GND nanoLC Operator s Manual The A D in is a 24 bit A D input for collecting the signal from an external detector using the nanoLC s data system GND is a system ground connection used in combination with other contacts such as RUN out The RUN out contact is used to trigger an external device at the time the gradient is started Its polarity is configurable from within the Control software The logic state changes at the be ginning of the gradient end of injection when using metered injection mode and stays in on state during gradient The PRK out contact changes state when the peak parking function is actuated Its polarity is configurable from within the Control software The RDY out contact changes state when the pump is ready to start run e g at end of prerun flush Its pol
37. eksigent operator s manual nanoLcC 1D plus system nano e 2D system nanoLC Operator s Manual 611 00012 Revision A1 Notice Support At Eksigent Technologies we are committed to providing the highest level of support to our users To obtain answers to questions about any of our products report problems or suggest improvements please visit www Eksigent com For on site service support and training please contact your local Eksigent Technologies sales representative or customer service representative or send an e mail to support eksigent com Notice The information in this document is subject to change without notice and should not be construed as a commitment by Eksigent Technologies Eksigent Technologies assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Eksigent Technologies be liable for incidental or consequential damages in connection with or arising from the use of this document Eksigent Technologies makes no warranties or representations as to the fitness of this equipment for any particular purpose and assumes no responsibility or contingent liability including indirect or consequential damages for any use to which the purchaser may put the equipment described herein or for any adverse circumstances arising therefrom This document is provided to customers who have purchased Eksigent Techno
38. em vendor Detailed instructions for the safe grounding on the LC MS system are outlined in the corresponding vendors operating installation manual Exsigent Technologies recommends using a grounding cable connected between the injection valves sample loop and an appropriate grounding point at the LC MS source This supplementary grounding will reinforce the safety configuration specified by the LC MS system vendor nanoLC Operator s Manual 611 00012 Revision A1 gt WARNING gt WARNING gt CAUTION gt CAUTION gt CAUTION gt WARNING gt WARNING gt WARNING gt WARNING gt CAUTION Safety and Site Requirements 3 Do not turn the instrument on if you suspect that it has incurred any kind of electrical damage Instead disconnect the power cord and contact an Exsigent Technologies representative for a product evaluation Do not attempt to use the instrument until it has been inspected and approved for use Electrical damage may have occurred if the system shows visible signs of damage exposure to liquids or of having been transported under severe stress Damage can also result if the instrument is stored for prolonged periods under extreme conditions e g subjected to heat water etc Disconnect the power cord from its power supply before attempting any type of maintenance Continue to exercise caution as capacitors inside the instrument may still be charged even after the
39. ent Internal C System shut down if idle more than 120 min Display Options C Display flow profile setpoint values instead of measured flow values in the status area Export Settings Figure 2 8 Instrument Configuration Window step 3 Select the appropriate COM port for the nanoLC from the dropdown list step 4 Exit from the Control software step 5 Launch the control software The Instrument Configuration Window Figure 2 8 is used to indicate which components are installed It also sets the communications protocol and configures the system to work with other connected devices Several instrument performance parameters are also set in this window Select System gt Instrument Config from the Control Software s Acquisition Window to access the Instrument Configuration Window Make sure that one of the following systems is selected in the Device field e nanoLC 1D plus e nanoLC 2D The computer is configured at the factory to use COM1 to communicate with the nano LC system If the serial cable is connected to a different serial port change the setting in the on this page to indicate the correct COM port In the Injection Valve field select Eksigent Internal If an auxiliary A D input such as a UV detector is connected to the system specify the voltage range the external device will provide under Aux A D In the Advanced Options box type an acceptable idle time after which the system will be shut down and the
40. ep 12 Once the method is complete click Save to save the analysis method 5 6 Creating an LC Method Channel 2 step 1 Open the LC Method editor and type over the name of the method to create a new method Click Save step 2 If you wish enter any column information appropriate for your experiment This information is information al and stored with the LC method file step 3 Click on the Run Conditions tab of the LC Method Editor window to obtain the Run Conditions tab Figure 5 12 LC Method Settings Selected Method Name CH2 gradient Summary Run Conditions Flow Profile Flow Table Pre Run Flush column for 0 minutes using 100 initial flowrate conditions Sample Injection O None Standard Sample valve opens prior to beginning Flow Profile and remains open O Metered Inject EET nL of sample at 250 initial flowrate conditions O 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 Delete View Audit Trail Figure 5 12 LC Method Editor Run Conditions Tab step 4 Put a check mark in the Pre Run Flush column check box and specify a time of O minutes to flush the column using 100 of the initial flow rate conditions step 5 Select Standard in the Sample Injection region This will cause the 10 port valve to be placed in the inject position for the durat
41. er 2006 Eksigent Technologies DEMO MODE File View System Analysis Help Not Connected n D Runtime 00 00 00 00 00 00 amp 0 BO InjYiv Load LC Method avp_ch2_30min grad Run Manager petal To start a sample run click Run A_n Manager 1800 1620 1440 nanoLC Operator s Manual 611 00012 Revision A1 74 Operator s Manual Run Manager File Edit View Devices Help Run Table ddddefault ini Autosampler Sample HA Tray Vial Aspirate uL Channel Name 7 Microliter Pickup Direct Loading CH2_inj_gradient Current Tray Autosampler Method Definitions Run Sequence Autosampler Methods Sequential O As Available O Synchronized Multi Channel LC Methods O Flush quilibrate when Idle Analysis Methods amp El Time 00 00 00 e ed Time 01 05 00 Tray 1 Select Autosampler method Tray Vial LC Method and Channel 2 Click on Flush Equilibrate when Idle to start equilibrating pumps at initial gradient conditions Run Sequence Sequential O As Available bt oO Synchronized Multi Channel Click Flush Equilibrate to start equilibrating at gt 7 FlushiEquilibrate when Idle initial gradient conditions 3 _Start_Je Once system is ready click Start Elapsed Time 00 00 00 Queued Time 01 05 00 Quick Start Guides 75 Default methods Direct injection to column with Channel 2 Autosampler E Autosampler Settings Autosampler Procedure Name
42. er cord not connected No power at outlet Power LED has failed but system response OK Communication error between computer and an LC system The reservoirs were not placed in the pump adapter properly Air leaks from the air inlet fitting 611 00012 Corrective Action Connect power cord Repair electrical outlet Contact Eksigent Technologies Technical Support for assistance Verify that the instrument s serial cable is securely connected to the com puter s COM1 serial port see Section 2 5 Reboot computer and cycle power on instrument Contact Eksigent Technologies Technical Support for assistance Verify the stopcock is placed properly and tightly screwed in Verify the air tubing is connected properly to the gas fitting Tighten the air inlet gas fitting Contact Eksigent Technologies Technical Support for assistance Revision A1 Symptom Flow Control System System pressure Pc and or pump pressures Pa amp Pb show pressure even though the flow is off No liquid out of waste line when purging Pump restrokes frequently Pump has reached end of stroke error message occurs Pump does not restroke at the end of a run Pump flushes out quickly but does not deliver 600 uL per stroke Purge output drips out slowly No flow rate with 100 power indicated System pressure Pc and pump pressures Pa amp Pb are all low Inability to reach desired flow rate nanoLC Operator s
43. eseeeseseeeseseeseseeneessseeneeeaenes B 3 2 Triggering MS Data Collection B 3 3 Triggering Peak Parking ssessssssssssssennsssurnnnnnsnsnrnrnenunnnnnrrnnunsnnnnrensnnnnnrene B 3 4 Ready Out scsiinissstiasireaiisansisecrmmniateiesine an a Appendix C Valve Configuration Diagrams sssssssssssessesseees 69 Appendix D Quick Start Guides sssssssssssssssssssssssesssssssssssssssessssssees A O D 1 D 2 Quick Start guide Direct to Column Injection Quick Start guide Dual Trap Column Method Appendix E Replacing Internal Instrument Filters 100 83 DRO EREE RE E EE E E E E E A E E E E OO nanoLC Operator s Manual 611 00012 Revision A1 Chapter 1 Safety and Site Requirements Chapter 1 describes safety conventions safety procedures and site requirements necessary for proper operation of the nanoLC 1D plus and nanoLC 2D systems Topics covered in this chapter include e safety conventions Section 1 1 e nanoLC safety practices Section 1 2 e nanoLC Autosampler safety practices Section 1 3 e site requirements Section 1 4 1 1 Safety Conventions The following symbols are used in the manual This label calls attention to a procedure which if not correctly executed could result in injury or loss of life Do not proceed beyond a DANGER sign until the indicated conditions are fully understood and met This label calls attention to a procedure which if not correctly executed could
44. for the NanoLC 2D System step 5 Select System gt Mobile Phases from the main window of the control software to present the Mobile Phases dialog box Figure 3 2 nanoLC Operator s Manual 611 00012 Revision A1 step 6 step 7 System Initialization 17 Set the composition for mobile phase A Enter the correct solvent composition for Mobile Phase A the A mobile phase will generally be mostly water Set the composition for mobile phase B Enter the correct solvent composition for Mobile Phase B the B mobile phase will generally be mostly acetonitrile Mobile Phases Solvent 1A Binary mixture A Aqueous Solutio Aqueous Solution Comments Modifiers for mixture A 0 1 Formic Acid Solvent 1B Binary mixture B us Solution Acetonitrile v Comments Modifiers for mixture B 0 1 Formic Acid Figure 3 2 Mobile Phases Window step 8 Click the More button to present the purge and flush settings Figure 3 3 Mobile Phases Solvent 1A Binary mixture A Aqueous Solutior Aqueous Solution 3 Comments Modifiers for mixture A 0 1 Formic Acia Solvent 1B Binary mixture B Aqueous Solution Acetonitrile Comments Modifiers for mixture B 0 1 Formic Acid Mobile Phase Change Purge Settings v SideA v Side B 20 purge cycles Apply to all channels Automatically purge amplifiers when mobile phases change Automatically flush sys
45. he pump Section 3 2 Autotune flow controllers System gt Hardware Diagnostics gt Auto Tune Controllers Section 4 7 Autotune flow controllers System gt Hardware Diagnostics gt Auto Tune Controllers Section 4 7 Adjust the pump time response in Hardware Diagnostics Check the setting in Calibration Values Window in Hardware Diagnostic menu 1200 for 6000 psi transducers see Section 6 3 Check the setting in Service menu should be 500 200 see Section 6 3 Check the air pressure 100 psi Revision A1 56 Operator s Manual Symptom Autosampler Software does not recognize nano LC Autosampler when Run Manager is started nano LC Autosampler does not trigger instrument to start a run Software does not trigger nano LC Autosampler to switch valve position Injection Valve Injection valve does not switch positions No flow coming out from the port nanoLC Operator s Manual Table 6 1 cont Troubleshooting Checklist Possible Cause Communication error between computer and nano LC Autosampler Software may be configured to use a different COM port nano LC Autosampler is not in serial mode Output cable is not connected properly Loose wire connection at the instrument I O plug Input cable is not connected properly Loose wire connection at the instrument I O plug Valve is not connected to the actuator Valve is connected to the wrong channel Valve is not co
46. ht union PEEK 200 00044 Plug MicroTight PEEK black 920 00003 Nut female MicroTight 10 pkg 910 00043 Sleeve PEEK yellow 180 um ID 0 025 OD 10 pkg 910 00024 Sleeve PEEK orange 405 um ID 1 16 OD 10 pkg 910 00025 Sleeve PEEK green 395 um ID 0 025 OD 10 pkg 200 00138 Mixing tee ferrule for 365 um OD capillary 1 pkg nanoLC Operator s Manual 611 00012 Revision A1 60 Operator s Manual A 2 Replacement Parts nanoLC 1D nanoLC 1D and nanoLC 2D 200 00020 Internal pump filter element 200 00032 Internal pump filter holder and element 200 00047 Replacement rotor for 6 port PEAK injection valve 0 25 mm 200 00048 Replacement stator 6 port PEAK injection valve 0 25 mm 200 00058 Syringe adapter fill port PEEK NanoLC 1D injection valve 200 00061 PEEK sample loop 2 uL 200 00092 PEEK sample loop 5 pL 200 00164 PEEK sample loop 10 uL 200 00093 PEEK sample loop 20 uL 200 00063 PEEK sample loop 50 uL 200 00074 Syringe adapter fill port SS NanoLC 2D injection valves 200 00083 10 port valve titanium stator 200 00130 Replacement stator for Titanium 10 port valve 200 00212 Replacement stator for PAEK 10 port valve 200 00131 Replacement stator for Titanium 6 port valve 200 00159 Replacement rotor for Titanium 10 port valve 200 00211 Replacement rotor for PAEK 10 port valve 200 00160 Replacement rotor for Titanium 6 port valve 200 00167 Front panel mixing tee with fittings and ferrules 300 00000 Solvent
47. instrument has been turned off To avoid damaging electrical parts do not disconnect an electrical assembly while power is applied to the nanoLC Autosampler system Once the power is turned off wait approximately 30seconds before disconnecting an assembly This instrument contains a number of sensitive electronic components that may be damaged if exposed to excessive line voltage fluctuations and or power surges There are no operator serviceable or replaceable parts inside the nanoLC Autosampler system or its power supply If either unit is not functioning contact an Eksigent Technologies representative Never try to repair or replace any of the instrument s components not described in the manual without the assistance of an Exsigent Technologies representative To avoid injury during nanoLC Autosampler system operation keep hands and loose objects away from the autosampler arm and syringe assembly Do not operate the nanoLC Autosampler system without the safety shield properly installed At all times observe safe laboratory practices when handling solvents changing tubing or operating the nanoLC Autosampler system in order to avoid injury Know the physical and chemical properties of the solvents you use See the solvent manufacturers Material Safety Data Sheets for any solvent being used Use caution when working with any polymer tubing under pressure e Always wear proper eye protection when near pressurized polymer tubing e Do n
48. inutes Direct Control Pump Direct Control Not Connected Channel A Total flowrate Conserved Flow 50 20 pLimin O Independent Flow Q 20 Limin Montor Baseine 00 00 00 Yalve Direct Control Load Position Load Position Inject Position Close Figure 3 5 Direct Control Window Flushing CH1 Autosampler Connection Stop the flow by clicking Stop 3 6 Verifying the Flow Rate step 1 step 2 step 3 step 4 Before operating the system it is suggested that you verify that the flow rate 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 tools e Flow calibration assembly p n 801 00002 for high flow rate channel includes 20 uL pipettes e Flow calibration assembly p n 910 00006 for low flow rate channel includes 5 uL graduated pipettes Re initialize the pressure transducers see section 4 5 Attach the appropriate flow calibration assembly to the outlet of channel 1 or 2 Select System gt Direct Control from the Eksigent Control Software Acquisition Window Set channel A to 100 and an appropriate flow rate for that channel Figure 3 6 e 5 uL min for high flow channel e 500 nL min for low flow channel nanoLC Operator s Manual 611 00012 Revision A1 22 Operator s Manual step 5 Click the Start button and begin timing With the high flow calibration assembly the time it takes for
49. ion of the Channel 2 run step 6 Leave the check mark box for Post Run Flush column empty step 7 Click on the Flow Table tab to set the gradient parameters step 8 Enter the gradient parameters you wish to run Add new steps by clicking on the gt gt to the left of the table Delete steps by clicking on the X Set the overall flow rate on the right side nanoLC Operator s Manual 611 00012 Revision A1 44 Operator s Manual amp LC Method Settings Selected Method Name CH2 gradient Summary Run Conditions Flow Profile Flow Table Flow Mode Time min Conserved flow O Independent flow Profile Editor Total flowrate o3 pLitmin View Audit Trail Figure 5 13 LC Method Editor Flow Table Tab step 9 Select the Flow Profile tab to present the Flow Profile dialog box Figure 5 8 amp LC Method Settings Selected Method Name CH2 gradient Summary Run Conditions Flow Profile Flow Table Flow Mode a TT conserved tow O Independent flow Profile Editor Oa B Runtime 30 min Total flowrate 10 3 pLAmin 18 21 24 27 30 time min View Audit Trail Figure 5 14 LC Method Editor Flow Profile Tab nanoLC Operator s Manual 611 00012 Revision A1 5 7 Quick start Guide 45 step 10 Use the flow profile tab to survey the gradient you created to make sure it is correct The mobile phase composition can also be set by clicking and draggi
50. known diameter Selecting the Calibrate Flow meters test will bring up a dialog box with step by step instructions for performing the test Required tools e flow calibration assembly p n 801 00002 or 801 00006 necting the pipette to the system and prefilling the pipette to approximately 5 um before the first black Q Note If calibrating the low flow channel using the 801 00002 assembly you can save time by con line using either Direct Control or the Mobile Phases gt Flush Now feature step 1 Select System gt Hardware Diagnostics from the Control software Acquisition Window Selecting the Calibrate Flow meter test will bring up a dialog box with step by step instructions for performing the test nanoLC Operator s Manual 611 00012 Revision A1 30 Operator s Manual step 2 step 3 step 4 step 5 step 6 Flowmeter Calibration CAUTION Flowmeter calibration requires following the below channel procedures and entering the requested information accurately Failure to do so may result in malfunction of the instrument DO NOT re use values from previous calibrations Proceed deliberately 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 Mobile Phase B 100 Aqueous Solution 100 Acetonitrile 20 uLidivision ie will calibrate at 5 L min Cancel Figu
51. l 611 00012 Revision A1 Quick start Guide 41 step 2 To create a new method type over the name of the method and click Save step 3 If you wish enter any column information appropriate for your experiment This information is information al and stored with the LC method file step 4 Click on the Run Conditions tab of the LC Method Editor window to obtain the Run Conditions tab Figure 5 9 amp LC Method Settings Selected Method Name Ch _trap loading Summary Run Conditions Flow Profile Flow Table Pre Run Flush column for 0 minutes using 100 initial flowrate conditions Sample Injection O Nore Standard Sample valve opens prior to beginning Flow Profile and remains open Metered Inject nL of sample at 100 initial flowrate conditions Q Rapid Inject nL of sample at maximum flowrate maintaining initial mixture conditions Post Run C Flush column for 0 minutes using 100 ending flowrate conditions Delete View Audit Trail Figure 5 9 LC Method Editor Run Conditions Tab step 5 Put a check mark in the Pre Run Flush column check box and specify a time of 0 1 minutes to flush the column using 100 of the initial flow rate conditions step 6 Select Standard in the Sample Injection region This will cause the injection valve to be placed in the inject position for the duration of the Channel 1 run step 7 Leave the check mark box for Post Run Fl
52. lacement of the System Place the nanoLC system on a lab bench in a location with convenient access to power and a source of 100 psi 6 9 bar regulated clean dry air or nitrogen The front of the instrument should be accessible at all times The sides and back of the instrument should be clear to accommodate mobile phase reservoir placement and attachment and computer cable attachments 2 4 Connecting to the Air Supply Operation of the instrument requires connection to a source of 100 psi 6 9 bar regulated clean dry air or nitrogen as described in the section on Site Requirements Air Connection Figure 2 1 Rear Panel Air Connection Required tools and materials e Clean dry source of air or nitrogen at 100 psi 6 9 bar e In line moisture trap with 1 4 end fittings e 6mm or 14 air supply line included e Quick connect adaptor to 6 mm or 1 4 supply line included step 1 Connect the supplied air line to a source of 100 psi 6 9 bar regulated clean dry air or nitrogen using the supplied quick connect adaptor Connect the other end of the air line to the air connection on the back of the nanoLC system see Figure 2 1 step 2 Turn on the air source to the nanoLC system step 3 Test for air leaks Turn off air supply and repair any air leaks that are found nanoLC Operator s Manual 611 00012 Revision A1 System Installation 9 2 5 Connecting the PC Required materials e Computer keyboard and mouse e na
53. ld Rd Dublin CA 94568 tel 925 560 2600 fax 925 560 2700 web www eksigent com Copyright 2007 Eksigent Technologies LLC All rights reserved Printed in the United States of America nanoLC Operator s Manual 611 00012 Revision A1 Table of Contents Chapter 1 Safety and Site Requirements csssse seen 1 1 1 Safety CONVENLIONS ssis isiin 1 2 nanoLC Safety Practices 1 3 nanoLC Autosampler Safety Practices 1 4 Site Requir ments icici lie aa a in eS 1 4 1 nanoLC Power Requirement sesen 1 4 2 nanoLC Autosampler Power Requirements 1 4 3 Air Supply Requirements cesses 1 4 4 Bench Space Requirement 1 4 5 Environment Requirements Chapter 2 System Installation sssnssnssnnnnnnnnnnnnnnnnnunnnnnnnnnnnnnnnnnnnnnnn 7 2 1 nanoLC System Overview uses nseiesee cesar onsen neeneeneee 7 2 2 Unpacking the nanoLC System 2 3 Placement of the System ss ssssesssssesesuusuneesesuununeesosuununeesouuueeeeuunnnuereeuunnuureeessasunererassss 2 4 Connecting to the Air SUPPLY eens nese nese nneseeceneseas nesses oneeneneeneeneee 2 5 Connecting the PC 2 6 Connecting the nanoLC to Power 2 7 Installing Software and Instrument SettingS c sss eeseesneseesnesee ese 10 2 8 Configuring the nanoLC System 2 9 Configuring the nanoLC Autosampler uu cesses sesnsestscesnesesnsees cease 13 Chapter 3 System Initialization s sssussuseunnennnunnnnnnunnnnnnnnnnnnnnnnnnnnn 3 1 Hardware Components and FUNCtions
54. le The nanoLC has an option to collect an external signal e g a UV detector through the A D input on the 12 pin I O connector This data will be stored along with the flow profile data in the data file The Control software also includes a data analysis package and this analysis can be applied to the collected A D signal See the software manual for further instructions on analyzing data files with the software nanoLC Operator s Manual 611 00012 Revision A1 Chapter 6 Diagnostics and Troubleshooting Chapter 6 describes the built in diagnostic capabilities of the nanoLC system along with the most common troubleshooting procedures Topics covered in this chapter include e overview of hardware diagnostics Section 6 1 e calibration values Section 6 3 e general troubleshooting guidelines Section 6 4 e troubleshooting checklist Section 6 5 e error messages and system alerts Section 6 6 6 1 Overview of Hardware Diagnostics The nanoLC system includes a number of diagnostic capabilities designed to maintain peak system performance The status bars and the text displayed in the Control software acquisition window provide general diagnostic capability for routine operation It is a good practice to keep track of Pe pressure readings for the desired chromatographic method Running the Hardware diagnostics is part of routine instrument maintenance and should be performed in the following schedule e Every Month Re initialize press
55. lect the autosampler method micropickup_1uL_trap loading from the drop down menu step 6 Next enter 1 for the tray and A1 for the sample Vial location Alternatively vial location can be entered by clicking at the vial on the picture of the vial tray in the Run Manager window step 7 Double click on the LC Method field and select CH1 Trap Loading Indicate Channel 1 under the Channel column step 8 Inline 2 leave the autosampler method tray and vial location blank Double click on the LC Method field and choose CH2 gradient Indicate Channel 2 under the channel column step 9 For each sample you wish to run duplicate these two lines in succession In Figure 5 16 two sample injections are indicated nanoLC Operator s Manual 611 00012 Revision A1 46 Operator s Manual Run Manager File Edit View Devices System Suitability Help Run Table trap loading ini Autosampler LC Other Method Tray Vial Method Comments Status micropickup_4ul_Trap loac 1 401 Cht_trap loading Queued CH2 gradient Queued micropickup_1ul_Trap loac 1 402 Cht trap loading Queued CH2 gradient Queued 8 Current Tray Autosampler Method Definitions Run Sequence 1 2 3 46678 99 HOMIE wayi i Autosampler Methods Sequential OAs Available O Synchronized Mutti Channel LC Methods 7 FlushEquilibrate when Idie Analysis Methods amp Start Elapsed Time 00 00 00
56. logies equipment to use in the operation of such equipment This document is copyright protected and any reproduction of this document or any part of this document is strictly prohibited except as Eksigent Technologies may authorize in writing Equipment described in this document is protected under one or more patents filed in the United States and other countries Additional patents are pending Software described in this document is furnished under a license agreement It 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 Portions of this document may make reference to other manufacturers products which may contain parts that are patented and may contain parts whose names are registered as trademarks and or function as trademarks Any such usage is intended only to designate and describe those manufacturers products and does not imply any right and or license use or permit others to use such product names as trademarks All products and company names mentioned herein may be the trademarks of their respective owners Eksigent is a registered trademark owned by Eksigent Technologies in the United States and certain other countries For Research Use Only Not for use in diagnostic procedures Eksigent Technologies 5875 Arno
57. losures A variety of configurations are possible using the inputs and outputs of the nanoLC described above One suggested configuration is described here Setup up the injection method and sample sequence using the autosampler interface front panel or other software Setup a matching sequence of chromatographic methods in the Run Manager of the nanoLC Also make sure that LC Wait for Contact Closure is checked under the System menu in the Run Manager Window Make sure that the analysis time per sample time between autosampler injections in the autosampler method is long enough to allow for the nanoLC method run time plus any pre run flush equilibration for the next run Connect the inject marker out from the autosampler to the Run Input pin 8 and Ground pin 7 of the nanoLC Start the sequence by starting the Run Manager sequence and then the autosampler After the autosampler initiates its own sequence for dispensing sample it will trigger the nanoLC to start its chromatographic run The nanoLC also provides a Ready Out pin 5 which can be used as an input to other autosamplers to synchronize runs and error check operation Note The optimized injection routines that can be utilized with the nanoLC Autosampler will in general not be available with other autosamplers While the rapid inject or metered injection routines of the nanoLC method can be used to increase the flow rate during sample loading one will not be able to switch the sa
58. m if flow is unstable Section 3 2 If flow is still unstable calibrate flow meters Section 4 8 Contact Eksigent Technologies Technical Support for assistance Prime and flush system Section 3 2 Check the Mobile Phases setting Section 3 2 Autotune flow controllers Section 4 7 Contact Eksigent Technologies Technical Support for assistance Check the Mobile Phases setting section 3 2 Autotune flow controllers System gt Hardware Diagnostics gt Auto Tune Controllers see section 4 7 Check the Mobile Phases setting section 3 2 Perform flow meter calibration System gt Hardware Diagnostics gt Calibrate Flow meter see Section 4 8 Check all connections for leaks Perform flow meter calibration System gt Hardware Diagnostics gt Calibrate Flow meter Section 4 8 Contact Eksigent Technologies Technical Support for assistance Revision A1 Symptom Excess flow noises Measured flow does not follow the flow profile Pa and Pb maximized to lt 5300 psi with 100 pump power nanoLC Operator s Manual Quick start Guide 55 Table 6 1 cont Troubleshooting Checklist Possible Cause Trapped air in the pump Pump controller is out of tune Pump controller is out of tune Pump time response is set incorrectly Incorrect gain setting for pressure Incorrect zero setting for pressure sensors Inline Air pressure too low 611 00012 Corrective Action Prime t
59. mple loop out of the flow path during the gradient The may lead to significantly increased gradient delay times and decreased response times of the system B 3 2 Triggering MS Data Collection While integrated control of the nanoLC system through the Xcalibur and Analyst software packages will be available most MS systems will allow synchronization of data collection through the use of contact closures The following describes the general configuration for this synchronization In the MS software package choose the option to start data collection with an external contact closure This can be applied for a single MS run or to a sequence or batch of data collection methods When conducting multiple runs in a sequence table please configure the Run Manager of the nanoLC software with the corresponding sequence of methods Set the Run Output configuration to contact closure box checked in the nanoLC soft ware menu One must then identify the hardware interface for the MS contact closure input This is typically either a terminal block mounted on the side of the MS instrument or a pair of wires in an interface cable supplied with the MS To synchronize MS data with the beginning of the gradient after the injection connect the Run Out pin 3 and the Ground terminals to the MS contact closure If one lead of the MS trigger is ground make sure to connect it to the Ground connection of the nanoLC For most applications this connection will
60. n check the box Select the appropriate signal input and output settings based on the requirements of the other instruments connected to the Eksigent nano LC system nanoLC Operator s Manual 611 00012 Revision A1 System Installation 13 Flow Stabilization Limits specifies the degree of flow rate stability required before a gradient will begin For most applications a setting of 100 nL min should be sufficient However for high sensitivity applications this parameter should be set closer to 20 nL min The Pressure Limits box allows for the specification of a maximum system pressure If the column pressure exceeds this limit the run will be automatically stopped and no further injections will take place There is no loss of sample if the system shuts down For most applications a value of 3500 psi is recommended Click OK to go back to the Acquisition Window 2 9 Configuring the nanoLC Autosampler Click on the Run Manager button to bring up the Run Manager Window for configuring the autosampler Figure 2 9 Run Manager DER File Edit View Devices System Suitability Help Run Table ddddefaultini Autosampler Other x Method Tray Vial Channel Status method tray vial method channel No Sample asmethod 1 JA01 gradient 1 Queued Current Tray Autosampler Method Definitions Run Sequence 1 29 46 SOTO TOT Trayi a Autosampler Methods Sequential v OAs Av
61. nfigured in nanoLC software Faulty actuator LED is not on The valve is plumbed incorrectly The ports are plugged 611 00012 Corrective Action Verify that the RS 232 cable is securely connected to the S2 In port on nano LC Autosampler see Section 2 6 Verify that the software is configured for nano LC Autosampler to connect to the correct COM port see Section 2 6 Using the front panel of nano LC Autosampler set it to serial mode Menu gt Serial see Section 2 6 Verify the output cable is connected to P1 connector at the back of nano LC Autosampler Make sure the wire ends are securely screwed in the I O plug Verify the input cable is connected to P6 connector at the back of nano LC Autosampler Make sure the wire ends are securely screwed in the I O plug Connect the cable from valve to actuator at the back of the instrument Check and reconnect cable from actuator to the correct channel In Instrument Configuration set injection valve as internal Section 2 9 Replace actuator Verify the plumbing configuration and reconnect if needed see Appendix C Manually flush each port with cleaning solvent using a syringe If flushing does not clean the port contact Eksigent Technologies Service Representative to replace the valve Revision A1 Symptom System pressure Pc with no column connected is unusually high Fluid leaking from the valve Inconsistent flow rate Bubbles in
62. ng points on the graph or by setting the of A or B in the Profile Editor region step 11 Once the method is complete click Save to save the analysis method Click OK to close the editor Creating the Run Table The Run Table ties together an autosampler and one or more LC method with a sample vial and tray position You can also enter descriptive information related to the sample or analysis This section will create a Run Table to run two samples with a trap and elute method step 1 If not already in the Run Manager click the Run Manager button located on the Eksigent Control Software s Acquisition Window Figure 5 1 step 2 Create a new blank Run Table by selecting Edit gt Erase Table Figure 5 15 Run Manager File Edit View Devices System Suitability Help Run Table trap loading ini Autosampler Method Tray Channel Status Current Tray Autosampler Method Definitions Run Sequence 1 2 9 40107m 0m OOT Trey a Autosampler Methods Sequential O As Available O Synchronized Multi Channel LC Methods Flush quilibrate when Idle Analysis Methods amp Start Tray 0 C SS Sow oo all gt Figure 5 15 Creating a New Run Table step 3 Select File gt Save As and type trap loading in the File name field step 4 Click Save step 5 In the first line in the Run Table double click on the Autosampler Method field and se
63. nge of solvent or change of column The injection valve can also be toggled between load and inject positions to flush the injection valve loop and interconnecting ports Select System gt Direct Control from the main Control software Acquisition Window nanoLC Operator s Manual 611 00012 Revision A1 36 Operator s Manual step 1 step 2 step 3 step 4 Direct Control Pump Direct Control Not Connected Channel A B Total flowrate Conserved Flow 9 so 50 300 nLimin O Independent Flow Q 300 nLimin v 2 Montor Baseine Valve Direct Control Load Position Load Position Inject Position Figure 5 3 Direct Control Window Ensure that the Conserved Flow radio button is selected set A to 50 and B to 50 This will be the mobile phase composition used for equilibration Set the Total flow rate to 300 nL min Click on Start to start the pumps and begin equilibration Flush the switching valve or an injection valve connected directly to the Eksigent nano LC instrument by alternately clicking on the Load Position and Inject Position buttons in the Valve Manual Control area To switch the valve in the nano LC Autosampler use the Autosampler direct control in the run manager under Devices gt Autosampler Device Settings gt Direct Control Tab Figure 5 5 Autosampler Configuration Configuration Plate Position Initial Wash Syringe Position Needle Wash ren 5 u Syringe Valve Position Er
64. nnnnnannne Creating an LC Method Channel 1 Creating an LC Method Channel 2 Creating the Run Table sisiiaiiiieaiiia Starting a RUN ssssssssssssssssrnrrensssrnrrernsne Viewing the Collected Data Files essss ssssessrnsssnssssrrnsnensusnnnnrenunnnnnnnenunnnnnnnensnnnne Chapter 6 Diagnostics and Troubleshooting 01s000000000049 6 1 6 2 6 3 6 4 6 5 Overview of Hardware Diagnostics sesssssssssessrrsrersnrsrnnnensnrnrnnrunsnnnrnnnensnnnrrnrenns Calibration Valu s ssrcinnisanitanianaicannncaanianinniadhanniinnnannnats General Troubleshooting Guidelines Troubleshooting Checklist uo cscs cence ise cnssnnensneenscsenesesieseascseas Error Messages and System Alerts Appendix A Spare Parts and Consumables ss ssesssssseeee D9 A 1 A 2 A 3 Consumables nanoLC 1D and nanoLC 2D ssssssssssssssssssssssssrrssrrssrrennreennsenn 59 Replacement Parts nanoLC 1D nanoLC 1D and nanoLC 2D 4 60 Replacement Parts AS 1 AutoSampler c csesssesessesessesteseeseseeseeseetsseeneetesenes 61 Appendix B External Interface cssscssssssssssssssssnccesssnsnssssseeeeeeseenns OG B 1 B 2 B 3 Interface COMME CtiONS ssecessessessseesessssesseseseeesesnsseesesneeeeseaeesesnsneeseseaeetaeneeseaneaes B 1 1 Remote Interface Valve ConneCtOrsiisiaideisen etna die enema aii aie B 2 1 Remote Interface Connecting to Other Instruments B 3 1 Other AUtoSamplers cs eseesssseseeseseessseees
65. noLC system serial cable included PC I O Connector Figure 2 2 Rear Panel PC I O Connector Connect one end of the RS 232 cable to an available COM port connector on the computer COM1 is recommended as it is selected as the default in the software configuration The other end of the cable should be connected to the 9 pin connector labeled PCI O on the rear panel of the nanoLC system see Figure 2 2 Tighten the retaining screws at both ends to secure the cable in place 2 6 Connecting the nanoLC to Power Required materials e 24 VDC power supply included Insert the supplied 24 VDC power supply s plug into the connector located on the back of the instrument Figure 2 3 Plug the line voltage cord into an appropriately grounded line voltage outlet Turn the nanoLC on using power switch on the back of the unit 24 VAC Power Figure 2 3 Rear Panel Power Connection nanoLC Operator s Manual 611 00012 Revision A1 10 Operator s Manual 2 7 Installing Software and Instrument Settings Required materials e CD with control software e disk with system settings step 1 Insert the CD into the CD drive and install the Control Software see software manual for additional information on installation step 2 After installing software but before starting the software insert the disk with the system settings shipped with new instrument step 3 Locate the file titled EKsetting reg in the Settings subfolde
66. nsert the Teflon tubing nanoLC Operator s Manual 611 00012 Revision A1 20 Operator s Manual 3 4 Flushing the Autosampler Syringe and Liquid Path step 1 step 2 step 3 step 4 step 5 On the front panel of the autosampler select SYRINGE Select SYRINGE END and SYRINGE HOME repeatedly until the syringe is full of liquid with no bubbles Once no further bubbles are observed click ESC and return to the main menu Press WASH on the keypad to execute an initial wash Repeat twice for a dry liquid flow path Press menu SERIAL to return the Autosampler to Serial Mode Open the Run Manager and verify the AS 1 state is Idle This verifies the serial communication is active and the autosampler is connected 3 5 Connecting the nanoLC to the nanoLC Autosampler step 1 step 2 step 3 step 4 Required tools and materials e Two orange 1 16 PEEK sleeves p n 910 00024 e Two 1 16 PEEK fittings and ferrules p n 920 00006 and 910 00023 e Green PEEK sleeve p n 910 00025 e Nut and ferrule for 0 025 fitting p n 920 00002 and 920 00003 e Two meters of 360mm OD 50mm ID silica capillary p n 910 00002 e Capillary cutter p n 200 00096 Note This section assumes connection of the nanoLC Autosampler using the high flow channel of the nanoLC instrument If you are connecting with the low flow channel through the nanoLC Autosampler e g for direct loading experiments use 25 um ID capillary for the connections t
67. o reduce delay volume Two meters of 360mm OD 25mm ID silica capillary p n 910 00008 are provided Use the capillary cutter to ensure a clean cut on one end of the 50 um ID capillary and connect it to the CH1 outlet of the nanoLC using the 0 025 green PEEK sleeve ferrule and nut Cut the capillary at an appropriate length approximately 40 cm to connect from the CH1 outlet to the Pump connection on the injection valve of the nano LC Autosampler Using an 1 16 orange PEEK sleeve and two piece connector attach the capillary to the nanoLC Autosampler Please make a clean cut at the end of the capillary and to fully seat the capillary and sleeve in the fitting Connect the remaining piece of 50 um ID capillary to the Column port of the nanoLC Autosampler injection valve using a second 1 16 orange PEEK sleeve and two piece fitting Cut the 50 um capillary to a length appropriate to your experiment This will most likely be a con nection to port 1 or 2 of the 10 port switching valve using one of the plumbing diagrams shown in Appendix D nanoLC Operator s Manual 611 00012 Revision A1 step 5 step 6 step 7 step 8 System Initialization 21 Open the Direct Control Window Fig 3 5 by clicking System gt Direct Control from the Control software s Acquisition Window Set solvent A and B to 50 50 and Total flow rate to 20 L min for NanoLC 1D use 100 A Click Start to flush the valve ports and capillary Flush for 10 m
68. o the device Cancel Figure 2 6 Serial Port Communications Error Note If the nanoLC system is connected to the PC s COM1 port and the error message still appears re boot the PC and re start the software If the nanoLC is connected to a communications port other than COM 1 the instrument configuration will need to be changed step 1 Launch the control software The COM error in Figure 2 8 will appear Click on Cancel A second window will appear see Figure 2 7 Click on OK to enter the software in DEMO MODE Eksigent The program will proceed in DEMO MODE All communications to the device have been disabled Check the Instrument Configuration if you believe there is an error Figure 2 7 Running the Software in Demo Mode step 2 Select System gt Instrument Config from the Control Software s Acquisition Window to access the Instrument Configuration Window see Figure 2 8 The Instrument Configuration Window Figure 2 8 is used to indicate which components are installed It also sets the communications protocol and configures the system to work with other connected devices Several instrument performance parameters are also set in this window nanoLC Operator s Manual 611 00012 Revision A1 12 Operator s Manual Instrument Configuration System Device 1 0 Advanced System Configuration Eksigent Device NanoLC 2D Parallel COM port com3 Injection Valve Eksig
69. orial which should be useful in understanding the normal operation of the Eksigent nano LC The procedures described in this chapter presume that the system has already been properly installed and initialized as described in chapters 2 and 3 Topics in this chapter include e powering up the system Section 5 1 e purging and flushing with new solvents Section 5 2 e equilibrating the system Section 5 2 e creating an autosampler method Section 5 3 e creating an LC method Section 5 4 e creating a run table Section 5 5 e starting a run Section 5 6 e viewing the collected data file Section 5 7 5 1 Powering up the System If the system is not already on turn on the nanoLC power switch mounted on the rear panel The green LED on the front of the instrument should illuminate and the injection valve should initialize If the autosampler is not already on turn on the power switch on the nanoLC Autosampler Turn on the computer log in to Windows and launch the Control Software by double clicking the software icon After initialization the Control software s Acquisition Window of the Control software will be displayed Figure 5 1 channels of fluid control They are denoted in this manual and in the software as channel numbers 1 and 2 Throughout the software common windows are used to display or control these channels To select the channel appropriate to that window when available simply click on the up or down arrow
70. ot use polymer tubing that has been severely stressed or kinked e Do not use polymer tubing in particular PEEK or DuPont Tefzel tubing with tetrahydrofuran THF dimethylsulfoxide DMSO chlorinated organic solvents concentrated mineral acids such as nitric phosphoric or sulfuric acids or any related compounds An on board lithium battery maintains the autosampler firmware when the instrument is turned off Because it is hard wired in place it should only be replaced a factory authorized service engineer nanoLC Operator s Manual 611 00012 Revision A1 4 Operator s Manual 1 4 Site Requirements This section describes the requirements for power air space and environment for operation of your instrument 1 4 1 nanoLC Power Requirements The nanoLC is powered by a 24 VDC external power supply Only the universal AC DC adapter and power cord supplied with the instrument should be used The external adapter permits operation from any line voltage between 100 240 VAC 47 63 Hz and 4A 1 4 2 nanoLC Autosampler Power Requirements Line voltage e 115 VAC 15 20 50 Hz 60 Hz 250 VA e 230 VAC 15 20 50 Hz 60 Hz 250 VA Fuses e For 115 VAC two 5 0 AT fuses 4 x 11 4 UL CSA For 230 VAC two 2 5 AT fuses 5 x 20 mm IEC 127 All fuses UL listed and CSA certified 1 4 3 Air Supply Requirements Operation of the instrument requires connection to a source of 100 psi 6 9 bar regulated clean dry air or nitrogen
71. ow Mode Conserved flow O Independent flow Profile Editor Oa OB Runtime 130 min 90 80 70 Sample Injection ONone Standard Sample valve opens prior to beginning Flow Profile and remains open Metered Inject 10000 nL of sample at 100 initial flowrate conditions 40 Rapid Inject nL of sample at maximum flowrate maintaining initial mixture conditions 60 50 Total flowrate 03 pLAmin 30 20 Post Run Flush column for 0 5 minutes using 100 ending flowrate conditions 15 time min View Audit Trail LC Method Settings Selected Method Name CH2_ini_gradient Summary Run Conditions Flow Profile Flow Table Time min Flow Mode ime min Conserved flow O Independent flow Profile Editor Total flowrate 03 uL min View Audit Trail Quick Start Guides 77 D 2 Quick Start guide Dual Trap Column Method From CH1 Pump MS Interface Column Autosampler 6 port Valve Waste From CH2 Pump Outlet To start a run in Eksigent software open the Run Manager 2006 Eksigent Technologies DEMO MODE File View System Analysis Help Not Connected n Runtime 00 00 00 00 00 00 AO BO Inj iv Load LC Method avp_ch2_30min grad Run Manager pole To start
72. ower for the valve actuator Figure B 3 External Valve Connector GND The GND contact is a system ground for the valve connection It is shared between the power sup ply for the valve actuator and the trigger lines VLV1 The VLV1 contact is the trigger line to move the valve to position 1 the LOAD position by default It is held low 0 V momentarily to trigger valve actuation to this position VLV2 The VLV2 contact is the trigger line to move the valve to position 2 the INJECT position by de fault It is held low O V momentarily to trigger valve actuation to this position B 3 Connecting to Other Instruments Other Autosamplers The active state TTL low 0 V or TTL high 5 V can be set for most I O connections in the Instrument Configuration menu It is important that the ground lead of the peripheral be connected to the ground connection on the nano LC In addition use of the nanoLC Autosampler provides a number of additional I O signals with additional programming flexibility Note For all of the methods provided with the nanoLC and nanoLC Autosampler and for all of the configurations discussed in this document it is assumed that all of the inputs and outputs are configured as contact closures that are active in the closed state nanoLC Operator s Manual 611 00012 Revision A1 66 Operator s Manual B 3 1 Other Autosamplers The nanoLC can be integrated with autosamplers from other vendors through the use of contact c
73. r step 4 Double click on the file EKsettings reg to install the instrument settings into the registry of the computer A window will pop up asking you if you really want to write to the registry Figure 2 4 Registry Editor 2 Are you sure you want to add the information in C Program Files Eksigent NanoLC settings EkSettings reg to the registry Figure 2 4 First Registry Prompt Window step 5 Click Yes A second window will popup indicating that you have successfully written to the registry Figure 2 5 step 6 Click OK step 7 The factory settings for your instrument should now be loaded into the registry You should still configure the system section 2 9 in case the COM port or other settings need to be adjusted from the factory defaults Registry Editor 5 Information in C Program Files Eksigent NanoLC settings EkSettings reg has been successfully entered into the Figure 2 5 Second Registry Prompt Window registry nanoLC Operator s Manual 611 00012 Revision A1 System Installation 11 2 8 Configuring the nanoLC System Launch the Control Software from your computer s Programs menu or from the Control Software icon on the desktop If you did not connect the serial cable to COM1 or the instrument power is not turned on a COM Error dialog box Figure 2 6 will appear Eksigent COM Error x No Eksigent device was Found on this computer Please check your serial connections and RESET POWER t
74. rates of several hundred nL min The fully integrated system includes binary gradient pumps an additional pump or second binary gradient pump system temperature controlled flow modules and a column switching valve The system is compatible with the nano LC Autosampler This chapter introduces the hardware and software features of the nanoLC systems 2 2 Unpacking the nanoLC System step 1 Inspect the shipping cartons for damage or evidence of mishandling If external damage is evident notify the carrier before opening the cartons step 2 Cut the tape and open the flaps on the top of the nanoLC system shipping box Remove the outer shipping box step 3 Open the inner shipping box and remove the foam packing material from the top of the nanoLC system step 4 Carefully lift the nanoLC system out of the box Grasp the sides of the instrument do not use the protective bag to lift the instrument A second person may be needed to help slide the carton and foam inserts off the instrument nanoLC Operator s Manual 611 00012 Revision A1 8 Operator s Manual step 5 Place the instrument on a lab bench step 6 Check the contents of the accessory kit against the contents checklist in Appendix A to confirm that all items are included step 7 The checklist for parts included with the nanoLC Autosampler is included in the nanoLC Autosampler manual step 8 Unpack the computer and monitor and verify that no parts are missing or damaged 2 3 P
75. rator s Manual 611 00012 Revision A1 Routine Maintenance 31 step 7 Use Table 4 2 to choose the proper calibration pipettes when calibrating the high flow and low flow channels of the nanoLC Table 4 2 Calibration Pipette Guide High Flow Channel Low Flow Channel Calibrated Pipette Size 20 uL 1 pL division 5uL total Calibration Flow Rate 5 L min 500 nL min Calibration Volume 20 uL side 2 uL side 201L from black line to end of pipette cr ee ees 2pL along two lines on SuL pipette Figure 4 9 Flow Calibration Pipettes 4 9 Cleaning and Inspecting the Instrument Unplug the external power supply from the power source before cleaning and internal inspection CAUTION step 1 Clean the outside of the instrument by wiping down with a cloth slightly dampened with water and a small amount of liquid dish soap step 2 Visually inspect the system fluidics and electronic connectors on a quarterly basis Look for evidence of fluid leaks by checking all fluid connections Also look for dried deposits that may indicate a slow leak step 3 Identify and correct the source of any leaks if found If a fluidic connection is broken replace the fitting and re flush the system Inspect the new connection to ensure that no leaks are present Dabbing a laboratory wipe around fluid connections is a good method to identify slow leaks nanoLC Operator s Manual 611 00012 Revision A1 Chapter 5 Quick start Guide Chapter 5 offers a brief tut
76. re 4 8 Flow Calibration Window Verify that mobile phases specified are correct If incorrect click Cancel to close the Flow meter Calibration Window Make the necessary changes in the Mobile Phases Window perform step 1 again and then click Next Attach the flow calibration assembly with appropriate pipette to either the mixing tee or after the valve or column Select the appropriate pipette size and flow rate for calibrating that channel see table 4 2 20 pL division in the pipette size to set the calibration flow rate to 5 L min for the high flow channel or 1 pL division in the pipette size to set the calibration flow rate to 500 nL min for the low flow channel Click Next to start the flow in channel A Enter the appropriate volume requested in step 3 of the Flow Calibration Window dialog Fig 4 8 Wait until the liquid front travels to the black line mark on the pipette and press Start to begin timing For Channel 1 time how long it takes for the liquid front to travel 20 uL For Channel 2 time how long it takes for the liquid front to travel 2 uL Press Stop when the fluid front reaches the end of the pipette or the appropriate mark Then click Next If necessary disconnect the calibration assembly and dry out the liquid inside the capillary Repeat the measurement for calibration of the B channel flow meter Verify that the instructions were followed exactly and that all values entered are correct and then click finish nanoLC Ope
77. requires clear bench space of at least the following dimensions e nanoLC 1D plus 21 53 cm wide x 20 51 cm deep x 19 48 cm high allowing excess space for cables e nanoLC 2D 21 53 cm wide x 24 61 cm deep x 19 48 cm high allowing excess space for cables With the nanoLC Autosampler the height requirement for both systems is 27 69 cm This bench space requirement does not accommodate the computer keyboard mouse and monitor 1 4 5 Environment Requirements The instrument is designed to operate in an environment with ambient temperatures between 20 and 30 C 68 86 F and non condensing humidity nanoLC Operator s Manual 611 00012 Revision A1 Chapter 2 System Installation Chapter 2 describes the recommended procedure for unpacking and installing the nanoLC system Topics covered in this chapter include e nanoLC system overview Section 2 1 e unpacking the nanoLC system Section 2 2 e placement of the system Section 2 3 e connecting to the air supply Section 2 4 e connecting the PC Section 2 5 e installing the nanoLC Autosampler Section 2 6 e connecting the nanoLC to power Section 2 7 e installing software and instrument settings Section 2 8 e configuring the nanoLC system Section 2 9 e configuring the nanoLC Autosampler Section 2 10 2 1 nanoLC System Overview The nanoLC systems are designed for HPLC applications that employ direct pumping at flow
78. result in personal injury Do not proceed beyond a WARNING sign until the indicated conditions are fully understood and met gt gt WARNING gt This label calls attention to a procedure which if not correctly executed could result in damage to the CAUTION equipment Do not proceed beyond a CAUTION sign until the indicated conditions are fully understood and met This label calls attention to important information Read this information before continuing 1 2 nanoLC Safety Practices The following safety practices apply to the nanoLC system Use of this equipment in a manner not approved by Exsigent Technologies may inhibit its safety protection gt CAUTION Perform periodic leak checks on all lines and fittings nanoLC Operator s Manual 611 00012 Revision A1 2 Operator s Manual gt WARNING o When replacing capillaries or fittings on the nanoLC system 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 Do not allow flammable and or toxic solvents to accumulate Follow a regulated approved waste disposal pro gram Never dispose of flammable and or toxic solvents into a municipal sewage system 1 3 nanoLC Autosampler Safety Practices gt CAUTION gt CAUTION POe CAUTION gt
79. ror Conditions Wash Needle Waste Injection Valve Figure 5 5 Autosampler Configuration Window step 5 Allow the system to equilibrate for approximately 10 minutes nanoLC Operator s Manual 611 00012 Revision A1 Quick start Guide 37 5 4 Creating an 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 will review an autosampler method appropriate for loading a trap with Channel 1 and running a gradient with Channel 2 step 1 Place the sample vial containing the standard test mixture in vial position A1 of autosampler 48 vial tray step 2 Click Run Manager in the Acquisition Window to open the Run Manager display Figure 5 6 File Edt View Devices Help Run Table Test Table ini a Run Autosempler Le Other A ah Method Tray Vial Method Status 1 w micropickup_tul 1141 _ Rapki Lose 2lloscis sample fom autosampler onto trap column Queued 2 w Ektion Gradient 1 ehtes sample from trap and column into MS GQueued 3 4 0 5 6 o 7 go 8 3 O 10 mi v Currend Tray Stetus Method Definitions Run Sequence Tray 1 ES Autosampler Methods i ential C As Available Synchronized Mti Channel LC Methods FiusshEguilorste when ide Analysis Methods amp emn e Figure 5 6 Run Manager Window step 3 If you do not see a picture of the 48
80. s next to the channel number display in the upper right of the window Q Note On multichannel systems Both the nanoLC 1D plus and the NanoLC 2D instrument have two nanoLC Operator s Manual 611 00012 Revision A1 34 Operator s Manual 2006 Eksi Figure 5 1 Acquisition Window 5 2 Purging and Flushing with New Solvents If solvent has been sitting for more than 2 weeks the solvent should be replaced with fresh solvent then purged and flushed If the solvent is less than two weeks old proceed to step 5 3 step 1 To discard old solvent in the reservoir bottles one of two methods can be used remove the bottle and pour out the old solvent or purge the old solvent through to waste Figure 5 2 nanoLC Operator s Manual 611 00012 Revision A1 Quick start Guide 35 Grip stopcock Empty pull up Replace Loosen Figure 5 2 Emptying Solvent Reservoirs step 2 Pour new mobile phase into the bottle Purge the HPLC at least 10 times Depress the check valve while purging to dislodge bubbles in the pump head as shown in figure 3 3 1 step 3 After the pump is thoroughly purged flush Select System gt Mobile Phases from the main control window Click More and flush the system 100uL on Channel 1 and Channel 2 See section 3 2 for more information on flushing 5 3 Equilibrating the System The Control software s Direct Control window Figure 5 3 can be used to equilibrate the system following system power up a cha
81. s Manual Figure C 2 shows a rapid sample loading configuration with 2 parallel traps The flow on the traps is unidirectional sample loop High Flow Channel Waste AS syringe Low Flow Channel Sample AS needle 50 um ID capillary 25 um ID capillary RP trap valve in inject position valve in load position Figure C 2 Loading Configuration with 2 Parallel Traps Valve Configuration Diagrams 71 Figure C 3 shows a rapid sample loading configuration with 1 trap The flow on the traps is unidirectional sample loop High Flow Channel Waste AS syringe Low Flow Channel Sample AS needle 50 um ID capillary 25 um ID capillary valve in inject position valve in load position Figure C 3 Loading configuration with 1 trap unidirectional 72 Operator s Manual Figure C 4 shows a rapid sample loading configuration with 1 parallel trap The flow on the traps is bidirectional sample loop High Flow Channel Waste AS syringe 50 pm ID capillary 25 um ID capillary valve in inject position valve in load position Figure C 4 Loading configuration with 1 trap bidirectional Appendix D Quick Start Guides D 1 Quick Start guide Direct to Column Injection Direct to column sample injection Plumbing schematic From CH2 Pump Outlet MS Interface Column Autosampler 6 port Valve To start a run in Eksigent software open the Run Manag
82. s Service Representative to replace the valve Attach a high restrictor at the port to flush out the trapped air bubbles Check tubing connection Purge both pumps and re equilibrate system Section 3 2 Set the Flow Stabilization Limit in instrument configuration to gt 100 nL min System gt Instrument Configuration Section 2 9 Change LC method to with injection or change the AS method without wait for injection command see Sections 5 3 and 5 4 Revision A1 58 Operator s Manual 6 5 Error Messages and System Alerts System alerts can be displayed by selecting View gt System Logs gt Alerts These alerts Figure 6 4 are reminders of an action that needs to be taken such as to run the diagnostic tests or refill the reagent storage loops Pressing Clear Alerts will erase all alerts Note that this does not mean that the recommended alert action has been completed 021523 PM 08 0203 Channel 1 Hemender It has been over one month since the system has had diagnostic checks performed ther tum off this reminder in the Diggnostics Menu or calibrate the Howmeters 67 01 1014 PM 09003 Channel 1 Reminder It has been over one month since the system has had diagnostic checks performed Either tun off thes reminder in the Diagnostics Menu or calibrate the flowmeters am 63 01 13 04 PM 09 03 03 Channel 1 Reminder It has been over one month since the system has had b Reset Mobile Phase Loops Chess Hats
83. strument will also need to be emptied periodically Unscrew the thumbscrew holding the clamp around the waste bottle Remove the bottle from the clamp and unscrew the cap from the bottle Pour the contents into an appropriate chemical waste container Always follow appropriate safety procedures when handling or disposing waste chemicals See the solvent Material Safety Data Sheets for more information CAUTION 4 3 Changing the Sample Loop Required tools and materials e Laboratory wipes e Clean HPLC grade methanol e 1 4 open end wrench e Sample loop e Two 1 16 HPLC fittings p n 920 00006 and 910 00023 Note This sample loop may be on the optional 6 port sample injection valve or on the nano LC Autosampler step 1 Use the 1 4 wrench or fingertight fittings to remove the installed sample loop step 2 Insert one end of the new sample loop through a 1 16 fitting and ferrule and into the injection valve port Make sure the end of sample loop is flat and fully inserted into the valve port Do not reuse fittings from other valves as the port depth can vary from valve to valve step 3 Attach the other end of the sample loop to the opposite valve port step 4 Open the Direct Control Window by clicking System gt Direct Control from the Control software s Acquisition Window step 5 Set solvent A to 50 solvent B to 50 and Total flow rate to 6 L min Figure 4 1 step 6 Click Start step 7 Toggle the injection val
84. tem when mobile phases change Flush Settings Volume 50 pL Total Flowrate 10 pL nin Apply to all channels Create New Fluid OK Apply Cancel Figure 3 3 Mobile Phases Window Advanced step 9 Purge the bubbles from each pump using the priming tool using the following steps a Under Purge Settings select only one pump to purge i e channel 1 A Set the number of purge cycles to 20 b Click Purge Now The pump will begin to execute purge cycles While the pump is purging insert the priming tool all the way into the pump and depress the check valve nanoLC Operator s Manual 611 00012 Revision A1 18 Operator s Manual step 10 step 11 step 12 step 13 step 14 step 15 step 16 step 17 step 18 step 19 Hold down the priming tool and look for slight rise in the liquid in the J bottle When no more bubbles come out remove the tubing and let the pump purge a few more times Figure 3 3 Priming Tool Usage c After bubbles cease to come up into the bottle allow the pump to continue purging for the rest of the 20 cycles d Repeat steps a c for all the pumps separately to ensure each pump is adequately primed Enter 10 for purge cycles select Side A and de select Side B Click Purge Now to purge the A pump Collect the purge output in a 15 mL graduated centrifuge tube The pump should deliver a total of approximately 6 mL of solvent 600 pL per purge cycle
85. ure transducers e Every three months Autotune the controllers e Every three months Calibrate the flow meters Refer to section 4 5 4 7 and 4 8 for details on running these diagnostics nanoLC Operator s Manual 611 00012 Revision A1 50 Operator s Manual 6 2 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 for the pumps The following settings are typical for a nanoLC and are shown for example only The window on the left is for channel 1 and the window on the right is for channel 2 Hardware Diagnostics xl Recurring Events Channel Remind me to run diagnostic tests once a month 3 1 Flow Calbration _Callration vadues Flow Normalized Adud Ka Volue 0 09247 10 3800 Kb Value 0 09066 101776 Fressure Sensors a Oe Peal psi 1200 1200 1200 Zero pal S78 So 281 PD A a B B gan 0306 0233 0372 035 w 10 10 10 10 pac O14 Dit O14 045 ofiset 67563 00 6151 00 Recurring Everts gt Remind me to run diagnostic tests ence a month Caibration Values Hardware Diagnostics x Channel 32 Flow Ca bration Flow Normalized Acus Ko Vaus 000856 1 0085 Ko Velux 0 00369 10817 Pressure Sensors Pa Pb Pe Poal ipsi 1200 1200 1200 Zero pst 573 578 254 PD A A gt e gan OF 03n 022 O29 it 140 10 10 10 peDac O14 O14 O14 Ois offset
86. ush column empty step 8 Click on the Flow Table tab to set the gradient parameters step 9 Enter the flow rate time and percentages of A and B that are appropriate For a 2D system that has the same mobile phase present for Channel 1 A and B use 50 A and 50 B For a 1D system use 100 A nanoLC Operator s Manual 611 00012 Revision A1 42 Operator s Manual LC Method Settings Selected Method Name Ch1_trap loading x Summary Run Conditions Flow Profile Flow Table Time min Flow Mode ime min Conserved flow O Independent flow Profile Editor Total flowrate 5 Limin View Audit Trail Figure 5 10 LC Method Editor Flow Table Tab step 10 Select the Flow Profile tab to present the Flow Profile dialog box Figure 5 8 LC Method Settings Selected Method Name cht _trap loading w Summary Run Conditions Flow Profile Flow Table 100 Flow Mode Conserved flow 30 O Independent flow ey Profile Editor 70 O A B 60 Runtime 5 min 50 Sar Total flowrate 40 5 pLimin 30 20 25 3 3 5 time min View Audit Trail Figure 5 11 LC Method Editor Flow Profile Tab nanoLC Operator s Manual 611 00012 Revision A1 Quick start Guide 43 step 11 The mobile phase composition can also be set by clicking and dragging points on the graph or by setting the of A or B in the Profile Editor region st
87. ve between the load and the inject position several times to flush out any loose particles step 8 Look for any signs of leakage indicating a loose connection Remake any connections that indicate a leak step 9 Click Stop nanoLC Operator s Manual 611 00012 Revision A1 Routine Maintenance 25 4 4 Replacing Capillary Connections step 1 step 2 step 3 step 4 step 5 step 6 step 7 step 8 Required tools and materials e Laboratory wipes e Clean HPLC grade methanol e 1 4 Allen wrench e Transfer capillary For the high flow channel use 50 um ID p n 910 00002 For the low flow channel use 25 um ID p n 910 00008 e Microtight fitting e Green PEEK sleeve p n 910 00025 e Black microtight ferrule p n 920 00002 e Tan microtight nut p n 920 00003 e 1 16 sleeved fitting e 1 16 OD Orange PEEK sleeve p n 910 00024 e 1 16 PEEK fitting nut p n 920 00006 e 1 16 PEEK fitting ferrule p n 910 00023 Use the 1 4 Allen wrench or your fingers to remove the current fitting ferrule and sleeve Remove the fitting and ferrule from the capillary and discard the old capillary Cut the capillary to the appropriate length Ensure a clean end cut and clean with methanol Insert one end of a new capillary through the appropriate PEEK sleeve Slide the end of the capillary and the PEEK sleeve through the compression fitting and ferrule Make sure the sleeve extends through the ferrule Slide th
88. waste seal wash bottle 250 mL 400 00104 Power supply input 100 240VAC 3A output 24VDC 4A NanoLC 1D plus amp NanoLC 2D 620 00060 Power cable North America 400 00078 Power cable European 920 00020 1 25 A fuses 5 pkg 800 00020 Flow meter module standard for NanoLC 1D 800 00223 Flow meter module 1Dplus channel 2 1 20 L min 800 00054 Flow meter module standard for NanoLC 2D channel 1A amp 1B 800 00055 Flow meter module standard for NanoLC 2D channel 2A amp 2B 800 00076 Flow meter module standard for NanoLC 1D channel 2A amp 2B 800 00077 Flow meter module standard for NanoLC 1D channel 1A amp 1B 910 00066 Replacement capillaries kit 1D Plus Includes all SS fused silica capillaries used in a 1D Plus with fittings 910 00067 Replacement capillaries kit 2D Includes all SS and fused silica capillaries used in a 2D with necessary fittings 910 00063 Replacement capillaries kit 1D Plus Includes all SS capillaries used in a 1D Plus with necessary fittings 910 00064 Replacement capillaries kit 2D Includes all SS capillaries used in a 2D with necessary fittings 800 00058 Waste reservoir clamp and thumbscrew 800 00105 Solvent reservoir 50mL clear nanoLC Operator s Manual 611 00012 Revision A1 Spare Parts and Consumables 61 800 00141 Solvent reservoir 50 mL amber 800 00194 Solvent reservoir 100 mL clear 800 00200 Connector 12 pin electrical I O green 400 00196 Connector 4 pin electrical switching valv
89. x 10 cycles Next de select Side A and select Side B Click the Purge Now button to purge the B pump Collect the purge output for pump B and verify that the total volume is approximately 6 mL Enter 100 for the uL Flush volume and select a flow rate appropriate for the maximum flow of the channel For high flow channels select 10 for the uL min Total Flow rate For low flow channels select 6500 for the nL min Total Flow rate Ensure that the outlets of the pump are disconnected before proceeding Flushing the system with a column connected could over pressure the system and create leaks Click the Flush Now button Repeat the above steps for the other channel After the flush sequence ends click OK to close the Mobile Phases window nanoLC Operator s Manual 611 00012 Revision A1 System Initialization 19 3 3 Preparing the nanoLC Autosampler The nanoLC Autosampler is the standard autosampler installed with nanoLC system The autosampler should be pre assembled and aligned by a qualified Eksigent Technologies service representative Refer to the nanoLC Autosampler User Manual for a detailed description on its operation Required tools and materials e Clean HPLC grade water e Clean HPLC grade isopropanol Figure 3 4 Eksigent nanoLC 1D Shown with Nano LC Autosampler step 7 Fill the wash bottle with a 20 80 mixture of isopropanol water that has been degassed step2 Place the wash bottles on the wash station bracket and i
90. zero the pressure transducers while there is still residual pressure on the system will lead to inaccurate flow rates nanoLC Operator s Manual 611 00012 Revision A1 Routine Maintenance 27 Hardware Diagnostics Recurring Events C Remind me to run diagnostic tests once a month Calibration Values Auto Diagnose Re Initialize Transducers OK 06 12 06 Ch 1 Calibrated C Check Flow Stability OK 0411 05 Qa 7 01 Qb 7 99 nLimin Start Diagnostics Flow Metering and Control Calibrate Flowmeter Ch 1 Canceled 03 23 07 canceled Auto Tune Controllers OK 0642 06 Po toh vate ep Pump time response 40 Under Damped faster Usage Information Total Sample Injections 37 Total Flowmeter Usage 101 4 Flowmeter Serial EK00500 001 101 75 Figure 4 2 Hardware Diagnostics Window Reinitialize Transducers step 1 In diagnostics check the Re Initialize Transducers box step2 Click Start Diagnostics A message will appear Fig 4 3 warning that the procedure should only be performed if there is no residual pressure on the system Eksigent Severe Warning Any residual pressure in the channels will be treated as zero which will have severe consequences for flow rates and your data Are you sure the channels are ready Cancel Figure 4 3 Residual Pressure Warning step 3 Once the system indicates that it is at ambient pressure click OK and a status window will indicate that auto zero is in progress Figure 4 4

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