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EASY-nLC Series Xcalibur Data System 2.1 or Later User Guide

1. 0 00e000 47 iv EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific Preface This guide describes how to configure the Thermo EASY nLC virtual instrument VI control software from the Thermo Foundation platform set up the liquid chromatography LC parameters for LC MS instrument methods in the Instrument Setup window of the Thermo Xcalibur data system and set up the sample position settings and injection volumes for an injection sequence from the Sequence Setup view in the Xcalibur data system IMPORTANT To establish communication with the EASY nLC instrument and to avoid the premature termination of data acquisition during sequence runs set up the data system computer as described in Data System Computer Settings on page viii Contents e Related Documentation e System Requirements e Data System Computer Settings e Cautions and Special Notices Contacting Us To suggest changes to the documentation or to the Help Complete a brief survey about this document by clicking the button below Thank you in advance for your help SURVEY Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System v Preface Related Documentation Thermo Fisher Scientific provides these manuals as PDF files on the data system computer and on the USB flash drive that ships with the EASY nLC instrument EASY nLC Series Preinstallation Requirements Guide e EASY nLC Series G
2. Clicking a position turns it green The first position that you click becomes the initial position in the sequence table The sequence editor ignores the option selected in the Sample Selection Layout area and fills in the remaining rows in the order of your selections Click OK to return to the New Sequence Template dialog box To select contiguous vials or wells in sequential order by using the Select button In the Sample Positions Click on Plate or Select Button area click Clear All This clears the Initial Position box and any selected positions on the Plate Rack and the Vial Rack In the Sample Selection Layout area select either the Row First or the Column First option In the Number of Samples to Select box type the number of sample vials or wells Click Select The Plate Rack area displays the selected positions in green Click OK to return to the New Sequence Template dialog box for the Xcalibur Data System Thermo Scientific 4 Creating and Running Sequences Running Sequences Running Sequences After you create a sequence with the appropriate data file names instrument methods and injection positions you can run one or more rows of the sequence from the Sequence Setup view Torun a single sample a range of samples or the entire sequence set 1 In the Sequence Setup view do one of the following e To inject a single sample select the specific row in the sequence table Then choose Actions
3. 3 Creating Xcalibur Instrument Methods Setting Up the EASY nLC Instrument Method Parameters Gradient Page Parameters Table 7 describes the parameters on the Gradient page of the EASY nLC view in the Instrument Setup window Table 7 Gradient page parameters Parameter Description Table Time Specifies the run time when the instrument reaches the conditions specified in the flow and B columns Duration Specifies the length of time between the previous and current time points Range 0 01 to 9999 59 minutes Flow nL min Specifies the mobile phase flow rate for the current time point Range 1 to 2000 nL min B Specifies the percent composition of solvent B in the mobile phase for the current time point Buttons Add Adds a row to the gradient program Del Deletes the selected row from the gradient program Up Moves the selected row up Down Moves the selected row down Graph Flow nL min The y axis on the left side of the graph displays the flow scale A black line represents the change in flow rate with time AB mixture B The y axis on the right side of the graph displays the percentage of solvent B A red line represents change in B with time Time min Thermo Scientific The x axis displays the run time of the gradient program EASY nLC Series User Guide for the Xcalibur Data System 27 3 Creating Xcalibur Instrument Methods Setting Up the EASY nLC Instrumen
4. User Guide for the Xcalibur Data System Xcalibur 2 1 or later 60053 97230 Revision C November 2013 DOCUMENTATION 2013 Thermo Fisher Scientific Inc All rights reserved EASY nLC EASY Column AFC Foundation IFC and LTQ XL are trademarks and Xcalibur and LTQ are registered trademarks of Thermo Fisher Scientific Inc The following are registered trademarks in the United States and other countries Microsoft and Windows are registered trademarks of the Microsoft Corporation McAfee and VirusScan are registered trademarks of McAfee Inc All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries Thermo Fisher Scientific Inc provides this document to its customers with a product purchase to use in the product operation This document is copyright protected and any reproduction of the whole or any part of this document is strictly prohibited except with the written authorization of Thermo Fisher Scientific Inc The contents of this document are subject to change without notice All technical information in this document is for reference purposes only System configurations and specifications in this document supersede all previous information received by the purchaser Thermo Fisher Scientific Inc makes no representations that this document is complete accurate or error free and assumes no responsibility and will not be liable for any errors omissions damage or loss that might result from any use
5. 0 to 137 pL IMPORTANT The maximum allowable entry is 137 pL however for Xcalibur sequence runs the sample loading volume plus the precolumn equilibration volume plus the analytical column equilibration volume must be less than 120 pL See Taking Corrective Action for Run Time Errors on page 45 Flow uL min Specifies the flow rate at which pump A pumps solvent A through the instrument to push the sample out of the loop and onto the precolumn or analytical column If the Flow box is empty the instrument runs at the set pressure Range 0 to 100 pL min Max Pressure bar Specifies the maximum pressure for the sample loading step If the Max Pressure box is empty the instrument runs at the set flow rate EASY nLC II range 0 to 280 bar EASY nLC 1000 range 0 to 980 bar Tip If both the Max Pressure and Flow boxes are empty the instrument runs at the maximum instrument pressure Leave these boxes empty if you want the instrument to run at the highest possible flow rate that is allowed under the maximum instrument pressure limit 24 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific 3 Creating Xcalibur Instrument Methods Setting Up the EASY nLC Instrument Method Parameters Gradient Page Use the Gradient page to set up the gradient program see Figure 10 To open the Gradient page 1 Open the EASY nLC view of the Instrument Setup window 2 Click the Gradient tab If you do n
6. this dialog box from the EASY nLC Configuration dialog box only the Sample Position Layout and Initial Position parameters are available Table 5 Sample Position Selector dialog box parameters Sheet 1 of 2 Parameter Description Plate Rack Use this area to select or clear the sample positions Vial Rack For an EASY nLC instrument with an ASC model autosampler standard in the EASY nLC 1000 instrument you can select or clear a sample position from the additional 6 vials Note The additional vials are located on rack 1 and are called 1 V1 to 1 V6 Sample Selection Layout Row First When you select the Row First option the data system fills the sequence table with the sample positions row by row Column First When you select the Column First option the data system fills the sequence table with the sample positions column by column Note The sequence editor ignores the Sample Selection Layout option when you select the vial or well positions by clicking a set of samples in the Plate and Vial Rack areas 18 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific 2 Setting Up the Foundation Instrument Configuration Setting Up the Sample Layout and Start Position Table 5 Sample Position Selector dialog box parameters Sheet 2 of 2 Parameter Description Sample Positions click on plate Initial Position Specifies the first vial or microplate well position in the sequence table
7. 11 12 13 14 15 16 2 gt D wn a N e m amp amp 19 20 21 22 23 24 3 D 25 26 27 28 29 30 31 32 4 E 33 34 35 36 37 38 39 40 5 F 41 42 43 44 45 46 47 48 6 Sample selection layout Sample positions dick on plate or Select button Row first Initial position Al Number of samples to select 48 ince Ca EASY nLC Series User Guide for the Xcalibur Data System 41 4 Creating and Running Sequences Setting Up the Injection Positions with the Sample Position Selector 42 EASY nLC Series User Guide Follow one of these procedures e To select vials or wells by using the Plate Rack and Vial Rack areas e To select contiguous vials or wells in sequential order by using the Select button To set up the remaining parameters in the New Sequence Template dialog box refer to the Sequence Setup view Help Click OK to create the sample sequence The sequence information appears in the Sequence Setup view on the Xcalibur Home Page see Figure 14 on page 40 To select vials or wells by using the Plate Rack and Vial Rack areas In the Sample Positions Click on Plate or Select Button area click Clear All This clears the Initial Position box and any selected positions on the Plate Rack and the Vial Rack Select contiguous or noncontiguous vials or wells in a sequential or nonsequential order by clicking the positions on the Plate Rack and Vial Rack areas in the order that you want the samples to be injected
8. GoTo Help Aa sm e baw e Info view icon G Expand collapse icon Run time steps for the EASY nLC instrument During a run the MS detector remains in the Waiting for Contact Closure state until the LC instrument sends the start data acquisition signal Pump A i 40000 00 Position i 132 69 Pressure bar z 56 70 Real flow nl min 15000 00 Pump B Desired flow nl min 0 00 Status of the Py le E instrument Real flow nl min 181 17 components Desired flow nl min 0 00 Position ul IBA Pressure bar 0 30 A Position z 16 CENTER 48 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific Index A About box touch screen software 11 Acquisition Server error messages 45 autosampler model 12 15 Auto sampler page 31 blank injections 40 C Change gt Tray Name command 35 Change Instruments In Use dialog box 43 Column first option Sample Position Selector dialog box 42 communication issues caused by Ethernet cable or incorrect IP addresses 7 caused by virus scanner software 5 testing the Ethernet connection 11 computer See data system computer configuration parameters EASY nLC 14 contact closure cable 44 47 current user touch screen software 11 Custom option autosampler wash step 33 Cycles box autosampler wash step 33 data acquisition premature termination of v data system computer connecting to EASY nLC instrument 8 po
9. Rules Details dialog box 7 Click OK to close the Access Protection Properties dialog box and accept the new settings 8 Close the VirusScan Console application 6 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific p Setting Up the Foundation Instrument Configuration This chapter describes how to set up the instrument configuration for the EASY nLC device driver Before you set up the instrument configuration connect the EASY nLC instrument to the data system computer turn on the EASY nLC instrument and log in to the instrument as an administrator Then use the Thermo EASY nLC Configuration dialog box of the Thermo Foundation Instrument Configuration window to set up and test the network connection for the EASY nLC instrument to specify the tray type that you plan to use and to identify the mobile phase solvents in solvent reservoir bottles A and B Contents e Connecting the EASY nLC Instrument to the Data System Computer e Adding the System Devices to the Instrument Configuration e Setting Up the EASY nLC Configuration e Setting Up the Sample Layout and Start Position Connecting the EASY nLC Instrument to the Data System Computer The EASY nLC instrument and the Thermo Scientific mass spectrometer communicate with the data system computer through an Ethernet connection routed through an Ethernet switch connected to the data system computer These are the default IP addresses of the data syst
10. default 11 V Vial Rack area Sample Position Selector dialog box 41 vial rack positions 18 vial tray 16 virus scanner McAfee 5 volume analytical column equilibration 30 column in custom wash table 33 conflicts 45 flush autosampler wash step 33 precolumn equilibration 29 sample loading 24 sample pickup 24 W waiting for contact closure 47 wash autosampler 33 Windows updates automatic viii Thermo Scientific Index T X Xcalibur data system Column Arrangement dialog box Sequence Setup view 40 Instrument Setup window 21 New Sequence Template dialog box 36 Sequence Setup view 35 Status page Information view Home Page window 47 EASY nLC Series User Guide for the Xcalibur Data System 51
11. e Fill out a reader survey online at www surveymonkey com s PQM6P62 e Send an e mail message to the Technical Publications Editor at techpubs lcms thermofisher com X EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific Installing the EASY nLC Control Software This chapter describes how to install the EASY nLC virtual instrument VI control software from the LC Devices DVD It also describes how to ensure communication between the data system computer and the EASY nLC instrument Contents e Checking the System Prerequisites e Removing a Previous Version of the Instrument Control Software e Installing the Current Version of the Instrument Control Software e Excluding the EASY nLC from the VirusScan Enterprise Checking the System Prerequisites The LC MS system consists of the EASY nLC instrument a Thermo Scientific mass spectrometer and a data system computer Before installing the EASY nLC VI control software do the following e Install compatible versions of the Thermo Foundation platform and the Xcalibur data system or equivalent Thermo Scientific data system Ensure that you have administrator rights to install the EASY nLC VI control software for the Xcalibur application e Remove any earlier version of the EASY nLC VI control software from the data system computer see Removing a Previous Version of the Instrument Control Software on page 2 Thermo Scientific EASY nLC Series Us
12. nLC from the VirusScan Enterprise on page 5 6 Check the instrument information If necessary change the instrument information as described in To change the loop volume column setup idle mixture or idle flow rate on page 13 Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System 11 2 Setting Up the Foundation Instrument Configuration Setting Up the EASY nLC Configuration 7 Set up the autosampler configuration in the Autosampler Configuration area as follows a Select the autosampler model Autosampler ASA or Autosampler ASC b Select the plate type 96 or 384 well plates or a 4 x 6 ASA or 6 x 8 ASC vial holder Note All plate formats for the ASC model have six extra vial positions on the side When you set up an Xcalibur sequence these vials are numbered 1 V1 to 1 V6 Figure 6 Thermo EASY nLC Configuration dialog box hermo FASY n onfiguration mesm EASY nLC Network Configuration The IP Address can be found in the IP Address Hostname 172 16 0 103 About box on the EASY nLC instrument ee 3 imi Run samples as this user User must sores exist on the EASY nLC Touch screen software version 3 1 Test Connection Connection OK LC device driver and instrument information LC device driver LCServer 1 4 2 1 LC Instrument Proxeon Biosystems A S EASY nLC HPLC Driver Info 23 141 Serial Number LC 000390 Loop volume 20 0 pl Column setup Two column Idle flow rate
13. of this document even if the information in the document is followed properly This document is not part of any sales contract between Thermo Fisher Scientific Inc and a purchaser This document shall in no way govern or modify any Terms and Conditions of Sale which Terms and Conditions of Sale shall govern all conflicting information between the two documents Release history Revision A July 2012 Revision B January 2013 Revision C November 2013 Software version Foundation platform version 1 0 2 SP2 or later Xcalibur data system version 2 1 or later installed on the Foundation platform EASY nLC device driver version 2 7 or later For Research Use Only Not for use in diagnostic procedures Contents Thermo Scientific Chapter 1 Chapter 2 Chapter 3 Chapter 4 PUCTACGs cuca izainnd a Reeau dian ot tama dane ainda ENEE wx ARAE EE v Related Documentation os aces sucks susunane rruen nerneur vi System Requirement sese ieai sei eee cee hela Oboe i a ee he bee ede bas vii Data System Computer Settings 0 0 eee cee eee eae viii Cautions and Special Notices i680 p tench hed ie eh ed Vee Eee eR eE ix Contacting USi 5 iio ced dag eae dhe hee boa oe eee ea chs nao k x Installing the EASY nLC Control Software c cece eee ees 1 Checking the System Prerequisites 255 5 50ur abi ebyiecadeelebiaore wees 1 Removing a Previous Version of the Instrument Control Software 2 Installing the Current Ver
14. 17 sample layout selecting 16 sample loop size 45 Sample Pickup and Loading page 23 Sample Position Selector dialog box selecting sample layout and start position for the instrument configuration 17 selecting the vial or microplate well positions for a sequence 41 screen saver settings viii Select Vials button New Sequence Template dialog box 41 sequence runs premature termination of v setting up the injection sequence table 35 starting from the Sequence Setup view 43 Sequence Setup view of the Home Page window 36 software requirements vii solvents specifying in solvent reservoir bottles 12 16 wash bottles W1 to W3 32 Source list autosampler wash step 33 Standard option autosampler wash 31 33 Start Position readback 16 Thermo Scientific Status page of the Information view Home Page window 47 survey link x system requirements vii T technical support contacting x Test Connection button 11 15 Thermo EASY nLC Configuration dialog box 10 Thermo Foundation Instrument Configuration window 7 Thermo logo touch screen software 11 touch screen software About box 11 Maintenance gt Devices page 13 selecting the MS detector connected through contact closure to the EASY nLC instrument 47 version 12 tray type 16 35 troubleshooting communication issues viii 7 11 contact closure issues 47 48 data acquisition issues viii run time errors 45 U URLs ordering spare parts x User Name configuration parameter 14 user name
15. 200 nl min IFC Enabled Idle mixture 5 B Max Pressure 280 bar Autosampler Configuration Solvents for LC Pump Autosampler ASC Autosampler rae rte B acetonitrile Sample layout Row first Startpos Al Lx Edit button c Optional Set up the default sample layout and start position to be used when creating a new Xcalibur sequence as described in Setting Up the Sample Layout and Start Position on page 17 8 In the Solvents for LC Pump area type the names of the solvents in the boxes for solvent bottles A and B Solvent bottle A typically contains LC MS grade water with 0 1 formic acid Solvent bottle B typically contains LC MS grade acetonitrile with 0 1 formic acid 12 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific 2 Setting Up the Foundation Instrument Configuration Setting Up the EASY nLC Configuration 9 If the OK button is unavailable click Test Connection to download the new configuration settings to the data system computer 10 Click OK to accept the settings and close the Thermo EASY nLC Configuration dialog box 11 Click Done to close the Thermo Foundation Instrument Configuration window You must close the Foundation application before opening the Xcalibur data system The Xcalibur data system and the Thermo Foundation Instrument Configuration window cannot be open at the same time To change the loop volume column setup idle mixture or idle flow
16. Configuration window click Test Connection Then verify the new settings EASY nLC Configuration Dialog Box Parameters When you establish a connection between the EASY nLC instrument and the data system computer this dialog box displays the user specified loop volume idle flow rate idle mixture and column setup You set these parameters from the EASY nLC touch screen software as described above in To change the loop volume column setup idle mixture or idle flow rate Table 4 describes the parameters in the Thermo EASY nLC Configuration dialog box Table 4 EASY nLC Configuration dialog box parameters Sheet 1 of 3 Parameter Description EASY nLC Network Configuration Use this area to specify the IP address for the EASY nLC instrument IP Address Hostname Specifies the IP address of the EASY nLC instrument This value must match the value stored by the instrument Tip To check the IP address and host name stored by the instrument press the company logo in the upper left corner of the EASY nLC touch screen User Name Specifies the user name Note The user name that you type in this box must match the user name for the person currently logged in to the instrument The user name is listed in the lower left corner of the EASY nLC touch screen For information about logging in to the EASY nLC instrument refer to the EASY nLC Series Getting Started Guide 14 EASY nLC Series User Guide for the Xcalibur Data Syste
17. EASY nLC software from the VirusScan Enterprise 8 7 0i software Log in to the data system computer as an administrator Open the McAfee VirusScan Console application as follows a Do one of the following e On the Windows 7 taskbar right click the McAfee icon U and choose Manage Features gt VirusScan Enterprise e From the Windows 7 desktop choose Start gt All Programs gt McAfee gt VirusScan Console In the Task column right click Access Protection and choose Properties from the shortcut menu The Access Protection Properties dialog box appears To open the Rules dialog box do the following a Under Categories choose Anti virus Standard Protection b Under Rules select Prevent IRC Communication c Click Edit The Rules Details dialog box opens Modify these settings e In the Processes to Include box delete the asterisk Note When the Processes to Include box contains only a single asterisk the virus scanner prevents IRC communication for all processes e In the Processes to Exclude box type Thermo Note When the Processes to Exclude box contains the text Thermo the Access Protection service for Internet Relay Chat IRC communication processes ignores processes that begin with Thermo EASY nLC Series User Guide for the Xcalibur Data System 5 1 Installing the EASY nLC Control Software Excluding the EASY nLC from the VirusScan Enterprise 6 Click OK to close the
18. Number of Samples to Specifies the number of samples to add to the sequence table in addition to the initial vial Select position Clear All button Clears all of the Plate and Vial Rack entries and the Initial Position entry Select button Sequentially selects the number of vial or well positions specified in the Number of Samples to Select box The selected vials or wells are highlighted in green in the Plate Rack and Vial Rack areas The vials or wells are ordered sequentially according to the sample selection layout selection Dialog box buttons OK Accepts the settings and closes the dialog box Cancel Closes the dialog box without accepting the changes Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System 19 __ ____ Creating Xcalibur Instrument Methods This chapter describes how to set up the instrument method parameters for the EASY nLC instrument An Xcalibur instrument method meth contains the chromatographic and data acquisition grap q parameters for your LC MS system When creating new instrument methods set up the parameters for all of the configured devices Contents e Setting Up the EASY nLC Instrument Method Parameters e Saving Instrument Methods Note For information about setting up the instrument method parameters for the mass spectrometer refer to the Help for the mass spectrometer Setting Up the EASY nLC Instrument Method Parameters Use the Instrument Setup w
19. Y nLC icon The Thermo EASY nLC Configuration dialog box opens see Figure 5 Figure 5 Thermo EASY nLC Configuration dialog box Thermo EASY nLC Configuration EASY nLC Network Configuration Add t z The IP Address can be found in the ess Hos z About box on the EASY nLC instrument User name admin Run samples as this user User must exist on the EASY nLC Click Test Connection to test the connection LC device driver and instrument information LC device driver LCServer 1 4 2 1 Autosampler Configuration Solvents for LC Pump Autosampler m A water Plate installed Edit B acetonitrile Sample layout Row first Startpos Al 10 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific 2 Setting Up the Foundation Instrument Configuration Setting Up the EASY nLC Configuration 3 Ensure the following e The EASY nLC instrument is connected to the data system computer by way of an Ethernet switch see To connect the EASY nLC instrument to the data system computer on page 8 e The EASY nLC instrument is turned on and you are logged in as an administrator to the instrument 4 Set up the network configuration for the EASY nLC instrument as follows a Type the IP address of the EASY nLC instrument and your user name in the appropriate boxes You must have an account on the EASY nLC instrument with the same user name The default user name is admin but y
20. are loss of data or invalid test results or might contain information that is critical for optimal performance of the system Note Highlights information of general interest Tip Highlights helpful information that can make a task easier Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System ix Preface Contacting Us There are several ways to contact Thermo Fisher Scientific for the information you need io bX To contact Technical Support for the EASY nLC instrument Phone 800 532 4752 Fax 561 688 8736 Web site www proxeon com E mail North and South America us techsupport analyze thermofisher com Other continents eu techsupport cmf thermofisher com Knowledge base www thermokb com Find software updates and utilities to download at mssupport thermo com To order consumable and spare parts for the EASY nLC instrument For the EASY nLC 1000 instrument go to www proxeon com productrange nano_Ic_easy nlc_1000 accessories_spares index html For the EASY nLC II instrument go to www proxeon com productrange nano_lIc accessories spares index html To get local contact information for sales or service Go to www thermoscientific com wps portal ts contactus To copy manuals from the Internet Go to mssupport thermo com agree to the Terms and Conditions and then click Customer Manuals in the left margin of the window To suggest changes to the documentation or to the Help
21. be used and the injection volume You can specify a different instrument method for each injection Tip You cannot change the tray type for the EASY nLC instrument in the Sequence Setup view of the Xcalibur Home Page If you choose Change gt Tray Name from the menu bar the following Home Page message appears The configured autosampler does not offer any selection of trays To change the tray type close the Xcalibur data system Then open the EASY nLC Configuration dialog box as described in Setting Up the EASY nLC Configuration on page 10 and select the appropriate tray type Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System 35 4 Creating and Running Sequences Creating Sequences To create sequences in the Xcalibur data system 1 Do one of the following to open the Home Page Sequence Setup view mmm e From the Home Page Roadmap view click the Sequence Setup icon on e From any Home Page view choose View gt Sequence Setup View e From the Instrument Setup window click the Home Page icon x and then choose View gt Sequence Setup View 2 To open the New Sequence Template dialog box do one of the following e Choose File gt New or e Click the New Sequence icon on the toolbar The New Sequence Template dialog box opens see Figure 13 36 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific 4 Creating and Running Sequences Creating Se
22. e analytical column equilibration step 30 precolumn equilibration step 29 sample loading step 24 McAfee virus scanner 5 microwell plates 16 MS detector triggering data acquisition 44 47 negative value in the Inj Vol column of the Sequence Setup view 40 network cards vii network configuration EASY nLC 11 network firewall 2 New Sequence Template dialog box Xcalibur 36 P parameter description tables Auto sampler page 33 EASY nLC Configuration dialog box 14 Gradient page 27 Pre column and Analytical Column page 29 Sample Pickup and Loading page 24 Sample Position Selector dialog box 18 plate installed selecting 16 Plate Rack area Sample Position Selector dialog box 42 50 EASY nLC Series User Guide for the Xcalibur Data System plate type 16 port 22 TCP IP traffic 2 Position column Sequence Setup view table 40 power saver settings viii Pre Column and Analytical Column page 28 prerequisites installation 1 pressure maximum See maximum pressure pump solvents Aand B 16 readbacks EASY nLC Configuration dialog box 15 Status page Information view 47 Tray Type box New Sequence Template dialog box 37 release notes vii requirements computer vii software vii Row First option Sample Position Selector dialog box 42 Run Sample icon Sequence Setup view 43 Run Sequence dialog box 43 Run Sequence icon Sequence Setup view 43 run time error messages 45 S sample layout and start position configuration setting for
23. e the autosampler draws solvent from the specified bottle and then ejects the solvent into the needle wash bottle W4 3 Performs a final wash cycle with a preset volume of solvent A from bottle 3 W3 4 Refills pump S with solvent A from bottle 3 W3 to be ready for the next injection e In the Volume uL column type a volume from 0 to the sample loop size plus 8 pL If you enter a volume that is higher than 58 pL an Invalid Method Parameter message box appears and the program resets the volume to the maximum allowed e In the Cycles column type a value from 0 to 10 32 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific 3 Creating Xcalibur Instrument Methods Setting Up the EASY nLC Instrument Method Parameters Auto sampler Page Parameters Table 9 describes the parameters on the Auto sampler page of the EASY nLC view of the Instrument Setup window Table 9 Auto sampler page parameters Sheet 1 of 2 Parameter Standard Description Standard option Selecting this option enables the standard wash option Flush Volume uL Custom Specifies the flush volume that pump S is to push through the sample loop Range for the 20 pL sample loop 30 25 to 100 75 pL Range for the 50 pL sample loop 60 26 to 70 75 pL Custom option Selecting this option enables the custom wash program For information about the steps that the autosampler performs during a custom wash
24. eactivates this feature Tip Maintaining long term emitter stability with glass emitters might require a constant solvent flow through the system Idle Mixture Displays the user specified solvent composition B that the instrument is to pump through the system at the end of an injection sequence Column Setup Displays the user specified selection Displays One Column or Two Column IFC Displays whether the intelligent flow control IFC mode is enabled The IFC mode is a user selectable option in the touch screen software version 2 8 Max Pressure Displays the maximum system pressure Autosampler Configuration Use this area to select the autosampler type plate type sample layout and initial vial position Autosampler Thermo Scientific Specifies the autosampler model e ASC Autosampler with refrigerated compartment and 6 extra vials e ASA Autosampler with a cooled plate and without the 6 extra vials EASY nLC Series User Guide for the Xcalibur Data System 15 2 Setting Up the Foundation Instrument Configuration Setting Up the EASY nLC Configuration Table 4 EASY nLC Configuration dialog box parameters Sheet 3 of 3 Parameter Description Plate Installed Specifies the plate type that you plan to use 96 well plate 384 well plate or 6 x 8 vial plate Sample Layout Displays the default sample layout vial or microplate well positions for an injection sequence Start Position Displa
25. em computer and the EASY nLC instrument Table 3 Default IP addresses for the LC system System module IP address Data system computer 172 16 0 101 EASY nLC instrument 172 16 0 103 Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System 7 2 Setting Up the Foundation Instrument Configuration Adding the System Devices to the Instrument Configuration To connect the EASY nLC instrument to the data system computer 1 Connect a shielded Ethernet cable to the LAN port on the back panel of the EASY nLC instrument 2 Connect the other end of the Ethernet cable to an Ethernet switch 3 Using a second shielded Ethernet cable connect the Ethernet switch to the appropriate network card installed in the data system computer For more information about setting up the communication cables refer to the EASY nLC Series Getting Started Guide IMPORTANT Avoid IP conflicts with other units on the network especially the mass spectrometer Adding the System Devices to the Instrument Configuration Your Thermo Scientific LC MS system consists of a Thermo Scientific mass spectrometer and an EASY nLC instrument To control these instruments from the Xcalibur data system you must add the installed device drivers to the instrument configuration Note The Xcalibur data system and the Foundation Instrument Configuration window cannot be open simultaneously To add devices to the instrument configuration 1 Close
26. er Guide for the Xcalibur Data System 1 1 Installing the EASY nLC Control Software Removing a Previous Version of the Instrument Control Software Before or after installing the EASY nLC VI control software do the following e Connect the EASY nLC instrument to the data system computer through an Ethernet connection see Connecting the EASY nLC Instrument to the Data System Computer on page 7 e Connect the EASY nLC instrument to the Thermo Scientific MS detector with a contact closure cable The data system synchronizes the run timing between the LC system and the mass spectrometer through the cable For information about connecting the contact closure cable refer to the EASY nLC Series Getting Started Guide e Check the laboratory local area network LAN port for Internet access to the remote support server For remote diagnostics the laboratory must have a LAN port with Internet access The network firewall must allow outgoing Transmission Control Protocol Internet Protocol TCP IP traffic from the EASY nLC instrument to the remote support server at IP address 195 41 108 93 port 22 After installing the EASY nLC VI control software exclude the software from the virus scanner as described in Excluding the EASY nLC from the VirusScan Enterprise on page 5 Removing a Previous Version of the Instrument Control Software To remove a previously installed version of the EASY nLC Xcalibur VI 1 Depending on the Windo
27. etting Started Guide using the stand alone touch screen application EASY nLC Series Troubleshooting and Maintenance Guide EASY nLC Series User Guide for the Xcalibur Data System The data system also provides Help for the EASY nLC virtual instrument control software To access the EASY nLC manuals on the data system computer Choose Start gt Programs or All Programs gt Thermo Instruments gt Manuals gt LC Devices gt Thermo To open the Help from the EASY nLC view of the Instrument Setup window To open the Welcome page of the Thermo EASY nLC Help choose Help gt Thermo EASY nLC Help from the menu bar e To open the Help topic for the current page of the Thermo EASY nLC view do one of the following Choose Help gt Help On Current Item F1 from the menu bar Press the F1 key on the computer keyboard gt To open the Help from the Thermo EASY nLC Configuration dialog box of the Foundation Instrument Configuration window Press the F1 key on the computer keyboard vi EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific Preface System Requirements Table 1 lists the data system computer hardware and software requirements for the EASY nLC device driver This device driver is provided on the LC Devices DVD and is designed for use with the Thermo Xcalibur data system or an equivalent Thermo Scientific mass spectrometry application IMPORTANT Before you install the de
28. fic EASY nLC Series User Guide for the Xcalibur Data System 39 4 Creating and Running Sequences Creating Sequences 7 Click OK to create the sample sequence The sequence information appears in the Sequence Setup view see Figure 14 Figure 14 Sequence Setup view on the Xcalibur Home Page lt FileName Path 7 Inst Meth Position Inj Vol 1 Assapl1 C oscalibur D ata calibur methods Protein Assay Al 2 Assayl2 C Xcalibur D ata Xcalibur methods Protein Assay amp 2 3 Assayl3 C Xcalibur D ata Xcalibur methods Protein Assay A3 4 Assayl4 C Xcalibur D ata Xcalibur methods Protein Assay Ad When the Inj Vol column lists a value of 1 the instrument uses the Sample Pickup volume that is specified in the instrument method 8 Check the following e The vial or well values in the Position column Make sure that the entries are valid and use uppercase letters e The numeric value in the Inj Vol column When the value is 1 the data system uses the sample pickup volume specified in the instrument method IMPORTANT The sequence table contains a column called Inj Vol injection volume If the column does not appear choose Change gt Column Arrangement and add the Inj Vol column to the displayed columns To ensure that the injection volume specified in the instrument method file is used as intended the default value in the Inj Vol column is set to 1 which is an invalid i
29. gt Run This Sample or click the Run Sample icon P in the Sequence Editor toolbar e To inject a range of samples select the samples that you want to inject in the sequence table Then choose Actions gt Run Sequence or click the Run Sequence icon in the Sequence Editor toolbar or e To inject the entire sequence choose Actions gt Run Sequence or click the Run Sequence icon in the Sequence Editor toolbar One of the following actions occurs e Ifthe data system detects a new instrument configuration the Change Instruments In Use dialog box opens see Figure 16 Go to step 2 e Ifthe instrument configuration has not changed since the last sequence run the Run Sequence dialog box opens see Figure 17 Go to step 3 Figure 16 Change Instruments In Use dialog box Change Instruments In Use mx Instrument In Use Start Instrument Thermo EASY nLC Yes Yes LTQ Orbitrap Velos MS Yes Geert Gite 2 If the Change Instruments In Use dialog box opens do one of the following e Ifthe EASY nLC instrument is not listed as the Start Instrument click the EASY nLC row in the Start Instrument column and select Yes Then click OK to accept the new setting and close the dialog box e If the EASY nLC instrument is listed as the Start Instrument click OK to close the dialog box The Run Sequence dialog box opens see Figure 17 Thermo Scientific EASY nLC Series User Guide for the Xca
30. indow of the Xcalibur data system to create instrument methods for your LC MS system To create an instrument method enter the data acquisition settings for the mass spectrometer and the injection and chromatographic settings for the LC system To open the Instrument Setup window From the Xcalibur Home Page do one of the following e Choose GoTo gt Instrument Setup oe e Click the Instrument Setup icon in the Roadmap view of the Home Page The Instrument Setup window opens The view bar contains icons for the configured devices Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System 21 3 Creating Xcalibur Instrument Methods Setting Up the EASY nLC Instrument Method Parameters To set up the instrument method parameters for the EASY nLC instrument 1 In the view bar of the Instrument Setup window click the Thermo EASY nLC icon The EASY nLC view opens in the Instrument Setup window with the Sample Pickup and Loading page displayed see Figure 9 At the bottom of the view the Solvents area lists the solvents specified in the instrument configuration To change the names of the mobile phase solvents see Setting Up the EASY nLC Configuration on page 10 2 Enter the appropriate instrument method settings for the EASY nLC instrument Click Next to open the remaining three pages of the EASY nLC view page by page Figure 9 shows the Sample Pickup and Loading page of the EASY nLC view with typical sample picku
31. l the Thermo EASY nLC device driver on a Windows 7 computer exclude the Thermo EASY nLC software from the Prevent IRC Communication list for the computer s virus scanner see Excluding the EASY nLC from the VirusScan Enterprise on page 5 If you do not exclude the Thermo EASY nLC software from the Prevent IRC Communication list the data system computer cannot establish communication with the EASY nLC instrument Table 2 Data system computer settings Category Settings Power saver e Screen saver None e Hibernate Not enabled or Never e Turn off hard disk Never Turn off monitor Never Windows updates If the data system computer is connected to the Internet turn off the automatic Windows updates Virus scanner For the Windows 7 operating system exclude the Thermo EASY nLC software from the virus scanner viii EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific Preface Cautions and Special Notices Make sure you follow the cautions and special notices presented in this guide Cautions and special notices appear in boxes those concerning safety or possible system damage also have corresponding caution symbols This guide uses the following types of cautions and special notices CAUTION Highlights hazards to humans property or the environment Each CAUTION notice is accompanied by an appropriate CAUTION symbol IMPORTANT Highlights information necessary to prevent damage to softw
32. libur Data System 43 4 Creating and Running Sequences Running Sequences Start instrument Figure 17 Run Sequence dialog box iG Acquisition Options User admin Instrument Start Instrument T Thermo EASY nLC Yes LTO Orbitrap Velos MS Run Rows 1 Priority Sequence V Start When Ready p ocessing Actions Instrument Method Start Up Browse Quan Qual Shut Down Browse Reports Programs RE Programs Pre Acquisition Browse EN Create Quan Summary Post Acquisition Browse Run Synchronously v Pre Acquisition V Post Acquisition After Sequence Set System On D Standby Off 3 Check whether the EASY nLC instrument is listed as the start instrument in the Acquisition Options area The Start Instrument column displays Yes for the instrument that triggers data acquisition When the EASY nLC instrument begins its gradient program it signals the MS detector to begin data acquisition This signal is transmitted through the contact closure cable Depending on whether the EASY nLC instrument is listed as the start instrument do one of the following e Ifthe EASY nLC instrument is listed as the start instrument go to step 5 If the EASY nLC instrument is not listed as the start instrument make it the start instrument as follows a Click Change Instruments The Change Instruments In Use dialog box open
33. lowable entry is 137 pL however for Xcalibur sequence runs the sample loading volume plus the precolumn equilibration volume plus the analytical column equilibration volume must be less than 120 pL See Taking Corrective Action for Run Time Errors on page 45 Flow uL min Specifies the flow rate for the analytical column equilibration step If the Flow box is empty the instrument runs at the set pressure Range 0 to 3 pL min Max Pressure bar Specifies the maximum pressure for the analytical column equilibration step EASY nLC II range 0 to 280 bar EASY nLC 1000 range 0 to 980 bar Note If both the Max Pressure and Flow boxes are empty the instrument runs at the maximum instrument pressure Leave these boxes empty if you want the instrument to run at the highest possible flow rate that is allowed under the maximum instrument pressure limit 30 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific 3 Creating Xcalibur Instrument Methods Setting Up the EASY nLC Instrument Method Parameters Auto Sampler Page Use the Auto sampler page to set up the wash step for the needle and sample loop see Figure 12 To open the Auto sampler page 1 Open the EASY nLC view of the Instrument Setup window 2 Click the Auto sampler tab If you do not see the Auto sampler tab click Next Figure 12 Auto sampler page Sample pickup and loading Gradient Pre column and Analytical column Auto sam
34. m Thermo Scientific 2 Setting Up the Foundation Instrument Configuration Setting Up the EASY nLC Configuration Table 4 EASY nLC Configuration dialog box parameters Sheet 2 of 3 Parameter Test Connection button Description Tests the Ethernet connection between the data system computer and the EASY nLC instrument If the connection is established the status box to the right of the button turns green and contains this message Connection OK In addition the LC device driver and instrument information appears in the box below the status bar IMPORTANT Ifthe following message appears exclude the Thermo EASY nLC software from the McAfee virus scanner as described in Excluding the EASY nLC from the VirusScan Enterprise on page 5 An established connection was aborted by the software in your host machine Contact your local IT administrator for assistance LC Device Driver and Instrument Information LC Device Driver Displays the LC device driver version LC Instrument Displays the instrument type Driver Info Displays the instrument s touch screen software version Serial Number Displays the serial number that is listed on the instrument s back panel Loop Volume Displays the user specified loop volume stored by the EASY nLC instrument Idle Flow Rate Displays the user specified idle flow rate that the instrument maintains at the end of an injection sequence Entering a value of 0 d
35. n Selector For information about the parameters in the Sample Position Selector dialog box see Table 5 on page 18 The functionality of the Sample Position Selector dialog box depends on where you access the dialog box e When you open the Sample Position Selector dialog box from the EASY nLC Thermo Scientific Configuration dialog box of the Thermo Foundation gt Instrument Configuration window use the dialog box to set up the sample layout and initial vial or microplate well position for an injection sequence see Setting Up the Sample Layout and Start Position on page 17 When you open the Sample Position Selector dialog box from the New Sequence Template dialog box in the Xcalibur Sequence Setup view use the dialog box to interactively set up the vial or microplate well positions for the injection sequence as described in the following procedure To open the Sample Position Selector dialog box from the New Sequence Template dialog box and set up the sample positions Click Select Vials in the Samples Area see Figure 13 on page 37 The Thermo EASY nLC Sample Position Selector dialog box opens see Figure 15 The rows for the 6 x 8 vial tray are labeled A F and the columns are labeled 1 8 The six additional vials in the Vial Rack area are numbered 1 V1 to 1 V6 Figure 15 Thermo EASY nLC Sample Position Selector dialog box Thermo EASY nLC Sample Position Selector x Plate rack Vial rack 1 2 3 E JE amp w
36. nLC Xcalibur VI or click Uninstall Wait for the program to be removed from the list e For Windows XP click Remove see Figure 2 Wait for the program to be removed from the list Figure 2 Add or Remove Programs window for the Windows XP operating system F Add or Remove Programs mef iy me ji T Currently installed programs C Show updates Sort by Name Change or Remove z z Programs i gt ThermoLC Devices Thermo EASY nLC Xcalibur I Size Click here for support information Used Last Used On To remove this program From your computer click Remove 30 20MB 152 00MB Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System 3 1 Installing the EASY nLC Control Software Installing the Current Version of the Instrument Control Software Installing the Current Version of the Instrument Control Software iM To install the EASY nLC Virtual Instrument software 1 Check the installed versions of the Foundation platform and the Xcalibur data system and make sure that these versions are compatible with the LC Devices version that you are installing see System Requirements on page vii 2 Close all running programs before starting the installation Qe If applicable remove the previously installed version of the EASY nLC VI software see Removing a Previous Version of the Instrument Control Software on page 2 4 Load the LC Devices DVD into the data system comp
37. nformation about the EASY nLC configuration parameters see the next topic Setting Up the EASY nLC Configuration 5 Click Done to close the Thermo Foundation Instrument Configuration window Tip You must close the Instrument Configuration window before you open the Xcalibur data system The two applications cannot be open at the same time Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System 9 2 Setting Up the Foundation Instrument Configuration Setting Up the EASY nLC Configuration Setting Up the EASY nLC Configuration Use the Thermo EASY nLC Configuration dialog box to set up and test the communication connection to the data system computer to specify the autosampler model and the plate type that you are using and to identify the mobile phase solvents Clicking the Edit button in the Autosampler Configuration area opens the Sample Position Selector dialog box where you set up the sample layout row first or column first and the start position for an Xcalibur injection sequence To set up the EASY nLC configuration in the Foundation platform 1 If you have not already added the EASY nLC instrument to the Configured Devices list of the Thermo Foundation Instrument Configuration window add it as described in Adding the System Devices to the Instrument Configuration on page 8 2 In the Configured Devices list of the Thermo Foundation Instrument Configuration window double click the Thermo EAS
38. ng page 1 Open the EASY nLC view of the Instrument Setup window 2 If the Sample Pickup and Loading page is not displayed click the Sample Pickup and Loading tab Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System 23 3 Creating Xcalibur Instrument Methods Setting Up the EASY nLC Instrument Method Parameters Sample Pickup and Loading Page Parameters Table 6 describes the parameters on the Sample Pickup and Loading page of the EASY nLC view of the Instrument Setup window Table 6 Sample Pickup and Loading page parameters Parameter Sample Pickup Description Volume pL Specifies the sample volume that the autosampler withdraws from the vial or well position specified in the Xcalibur sequence Maximum volume sample loop volume 2 pL The graphic on the Home gt Overview page touch screen displays the sample loop size Flow uL min Specifies the flow rate at which pump S draws the sample into the sample loop Range 0 to 40 pL min The flow must be greater than 0 when the sample pickup volume is greater than 0 Tip For aqueous sample matrixes set the flow rate to 20 pL min Sample Loading Volume pL Specifies the volume of solvent A that the instrument uses to push the sample out of the sample loop and onto the precolumn two column setup or the analytical column one column setup Typically two times the sample pickup volume plus 2 pL is sufficient Range
39. njection volume When the Xcalibur data system detects the invalid value during a run it automatically takes the actual injection volume from the corresponding instrument method file overriding the 1 with the correct value During a run the autosampler draws the specified injection volume sample pickup volume into the sample loop The system then flushes the sample out of the sample loop and onto the LC column with the specified sample loading volume Specifying an insufficient loading volume can result in blank injections or poor injection reproducibility because the system is either not loading or only partially loading the sample onto the LC column Changing the sample pickup volume by specifying a different value in the Inj Vol column of the sequence does not change the sample loading volume specified in the instrument method To prevent incompatible settings avoid using the Inj Vol column to vary the injection volume Instead create different instrument methods where each instrument method contains compatible settings for the sample loading and sample pickup volumes see Sample Pickup and Loading Page Parameters on page 24 You can use a different instrument method for each sequence row 40 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific 4 Creating and Running Sequences Setting Up the Injection Positions with the Sample Position Selector Setting Up the Injection Positions with the Sample Positio
40. nt A consumption in the method is greater than 120 0 during precolumn analytical column and sample loading Modify the method so that the total volume for the sample loading the precolumn equilibration and the analytical column equilibration steps is less than 120 uL Gradient Solvent A consumption is greater than 132 pL Check the gradient table Reduce the volume of solvent A that is used to produce the gradient Autosampler wash flush volume out of range 137 30 25 lt gt 100 75 For a 20 pL sample loop enter a volume from 30 25 to 100 75 pL in the Flush Volume box in the Standard Wash area of the Auto sampler page Table 11 lists common run time errors caused by incorrect entries in the Xcalibur sequence Table 11 Run time errors caused by incorrect entries in the sequence table Message EASY nLC devices reported an error during the Prepare for Run command Remedy Reduce the injection volume sample pickup volume The sample pickup volume cannot be greater than the sample loop size minus 2 pL or 18 pL for a 20 pL sample loop EASY nLC Series User Guide for the Xcalibur Data System 45 P Viewing the Run Status in the Information View Use the Status page of the Xcalibur Information view to view the run status of the EASY nLC instrument To monitor the run status for the EASY nLC instrument from the Information view 1 Open the Xcalibur Home Page window as follows From the P
41. on area click Edit The Thermo EASY nLC Sample Position Selector dialog box opens The Number of Samples to Select box is unavailable see Figure 8 Figure 8 Sample Position Selector dialog box Thermo EASY nLC Sample Position Selector x Plate rack Vial rack 1 2 3 4 5 6 7 8 v A 2 3 4 5 6 7 8 1 B 9 10 11 12 13 14 15 16 2 cig 18 19 20 21 22 23 24 3 D 25 26 27 28 29 30 31 32 4 E B 34 35 36 37 38 39 40 5 Fia 42 43 44 45 46 47 48 6 Sample selection layout Sample positions dick on plate Row first Initial position A1 Number of samples to select 0 2 a Select ox Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System 17 2 Setting Up the Foundation Instrument Configuration Setting Up the Sample Layout and Start Position 2 In the Sample Positions area click Clear All 3 In the Plate Rack area select the default initial sample position by clicking a position 4 In the Sample Selection Layout area select the Row First or Column First option 5 Click OK to save your changes and exit the Thermo EASY nLC Sample Position Selector dialog box Note You can also open the Thermo EASY nLC Sample Position Selector dialog box by clicking Select Vials in the Samples area of the Xcalibur New Sequence Template dialog box see Figure 13 on page 37 Sample Position Selector Dialog Box Parameters Table 5 describes the parameters in the Sample Position Selector dialog box When you access
42. ot see the Gradient tab click Next Figure 10 Gradient page Sample pickup and loading Gradient m Gradient 00 00 N A 200 5 10 00 10 00 200 aig 204 203 202 201 200 199 198 196 To set up the gradient program 1 In the Time 0 00 row do the following e In the Flow nL min column type a value from 1 to 2000 e Inthe B column type a value from 0 to 100 2 To add an additional time point table row click Add Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System 25 3 Creating Xcalibur Instrument Methods Setting Up the EASY nLC Instrument Method Parameters 3 In rows 2 or higher of the gradient table do the following a In the Duration column type a value from 0 01 to 9999 49 The value in the Duration column is the length of time that you want the instrument to take to reach the specified flow and B conditions When you type a value in the Duration column the value in the Time column changes to the current Duration value plus the value in the previous row b In the Flow nL min column type a value from 1 to 2000 c Inthe B column type a value from 0 to 100 Both the flow rate and B composition change linearly between time points 4 Edit the table as follows e To delete a row select the row and click Del e To reorder the rows as applicable select the row and click Up or Down 26 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific
43. ou can change this to any existing user account on the EASY nLC instrument Tip The EASY nLC IP address is listed in the About box which you can open by pressing the THERMO logo in the upper left corner of the touch screen The current user is listed in the lower left corner of the touch screen If necessary change the IP address on the Configuration gt Network page of the touch screen software b Click Test Connection to test the connection to the EASY nLC instrument and to download the instrument configuration information to the data system computer 5 Check the message in the status box to the right of the Test Connection button e Ifthe connection is established the status box changes to green see Figure 6 and the information about the LC device driver and instrument appears e Ifa connection is not established the status bar changes to red Check the Ethernet cable connections the IP address of the EASY nLC instrument and the IP address of the network card installed in the data system computer see Connecting the EASY nLC Instrument to the Data System Computer on page 7 e Ifan antivirus program or firewall blocks the connection the application displays this error message An established connection was aborted by the software in your host machine Contact your local IT administrator for assistance For information about excluding the EASY nLC software from the McAfee virus scanner see Excluding the EASY
44. p and loading settings for the EASY nLC 1000 instrument and a sample in an aqueous matrix Figure 9 EASY nLC view of the Instrument Setup window LTO Orbitrap Velos MS Thermo EASY nLC Sample pickup and loading Gradient Pre column and Analytical column Auto sampler m Sample pickup brit Volume 10 ul Max is loop size 2 yi Flow 20 4 min m Sample loading Volume 22 ul Aow ul min Max pressure 800 Bar m Solvents 22 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific 3 Creating Xcalibur Instrument Methods Setting Up the EASY nLC Instrument Method Parameters For information about the pages of the EASY nLC view see these topics e Sample Pickup and Loading Page on page 23 e Gradient Page on page 25 e Pre Column and Analytical Column Page on page 28 e Auto Sampler Page on page 31 Sample Pickup and Loading Page Use the Sample Pickup and Loading page to specify the following e The sample volume that the autosampler withdraws injection volume from the vial or well position in the Xcalibur injection sequence and the flow rate at which the autosampler draws the sample into the sample loop e The solvent volume that pump A uses to push the sample out of the sample loop and onto the column assembly and the flow rate and maximum pressure for the sample loading step To open the Sample Pickup and Loadi
45. pler m Auto sampler wash Standard Flush volume ul Note Max vol is loop size 8 ul Wash bottle is no 4 Solvents A water B acetonitrile ema te To set up the standard autosampler wash step 1 Select the Standard option 2 In the Flush Volume uL box type the volume of solvent A from wash bottle 3 that the autosampler uses to flush the needle and the sample loop The range depends on the sample loop size see Table 9 on page 33 Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System 31 3 Creating Xcalibur Instrument Methods Setting Up the EASY nLC Instrument Method Parameters To set up a custom wash program 1 Select the Custom option The custom wash table becomes available 2 For each row that you want to add to the program click Add You can select a different wash solvent for each row 3 Edit each row as follows e In the Source column select the wash solvent source Bottle 1 Bottle 2 or Bottle 3 IMPORTANT The EASY nLC tray compartment holds up to four wash bottles e Bottles 1 and 2 in positions W1 and W2 can contain custom wash solvents e Bottle 3 in position W3 must contain solvent A e Bottle 4 in position W4 must contain the needle wash insert During a custom wash program the autosampler does the following 1 Empties the contents of pump S into the needle wash bottle W4 2 Performs the steps in the custom wash program During each cycl
46. program see To set up a custom wash program on page 32 Custom wash table columns Order Defines the order of the custom wash steps Source Volume pL Specifies the wash solvent source for the current wash step Selections Bottle 1 Bottle 2 or Bottle 3 Note Bottles 1 and 2 contain custom wash solvents Bottle 3 contains solvent A Specifies the wash solvent volume for each wash cycle Maximum allowed volume at run time Sample loop size plus 8 pL Note The Xcalibur method editor for the EASY nLC instrument does not know the configured sample loop size When you submit a sequence with the instrument method Xcalibur acquisition service validates the custom wash volumes against the configured sample loop size To prevent solvent from being drawn into pump S the program limits the volume to the sample loop size plus 8 pL For information about changing the sample loop size on the Maintenance gt Devices page of the touch screen software see To change the loop volume column setup idle mixture or idle flow rate on page 13 Cycles Specifies the number of wash cycles for the wash step Range 1 to 10 Note For each cycle the autosampler draws the specified volume of wash solvent from the specified source into the needle and then ejects the wash solvent into wash bottle 4 W4 Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System 33 3 Creating Xcalibur Instr
47. quences Figure 13 New Sequence Template dialog box General Base File Name test Starting Number 1 Path C calibur Data Browse Instrument Method C calibur methods easy rOwse Processing Method Browse Calibration File Browse Samples Number of Samples 5 Tray Type 6x8 vials v T Injections per Sample 1 Initial Vial Position 41 Re Use Vial Positions For the EASY nLC Base Sample ID Cancel Selection instrument you cannot change the tray type in the Sequence Setup view Tray Type is a read only parameter Bracket Type None Non Overlapped Overlapped Calibration ac Add Standards Add OCs Number of brackets After First Calibration Only inechorepe lars m After Every Calibration _ Add Blanks Add Blanks Fillin Sample ID for Standards J Fill in Sample ID for OCs Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System 37 4 Creating and Running Sequences Creating Sequences 3 In the General area do the following e Type the base file name for the data files e Type a starting number for the value that the data system appends to the base file name e Browse to select the directory location for the data files in the Path box e Browse to select the instrument method in the Instrument Method box 4 Set up the vial or well positions by doing one of the following e To use the text bo
48. rate 1 From the instrument s touch screen do the following a Open the Maintenance gt Devices page b In the Devices list select EASY nLC HPLC and then press the Properties tab The Properties view for the HPLC device on the Maintenance gt Devices page opens Figure 7 Figure 7 EASY nLC HPLC properties view touch screen software version 3 0 or later About Summary EASY nLC HPLC Laon volume 20 0 ul Cy One column setup _ Load speed protection LSP Idle flow rate 50 nl min Idle mixture 90 B Factory Reset Apply c Press the corresponding box to open the keypad and enter the appropriate numeric values for these parameters e For Loop Volume type the nominal volume of the sample loop installed on valve S 5 20 or 50 pL e For Idle Flow Rate type an appropriate value in nL min When idle the instrument pumps the specified solvent mixture at the specified flow rate e For Idle Mixture B type a value from 0 to 100 Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System 13 2 Setting Up the Foundation Instrument Configuration Setting Up the EASY nLC Configuration d Select or clear these check boxes as appropriate One Column Setup and Load Speed Protection LSP Note The LSP check box is available in the 3 0 or later version of the EASY nLC touch screen software e Click Apply 2 In the EASY nLC Configuration dialog box of the Foundation Instrument
49. rocessing Setup window choose GoTo gt Xcalibur Home Page From the Instrument Setup window click the Return to Home Page icon X on the toolbar Note The Qual and Quan Browser windows open in front of the Home Page window 2 Ifthe Information view is hidden click the Info View icon on the View toolbar see Figure 18 on page 48 3 During a run click the Thermo EASY nLC expand collapse icon to display the EASY nLC run progress and the instrument component readbacks on the bottom portion of the Status page 4 If the MS detector remains in the Waiting for Contact Closure state during a run do the following e Check the contact closure connection between the contact closure pins on the back panel of the EASY nLC instrument and the MS detector s Start In pins e Check the MS detector selection with the part number for the appropriate contact closure cable on the Configuration gt Connections page of the EASY nLC touch screen application Figure 18 shows the Status page for an LC MS system with an EASY nLC instrument and an LTQ XL MS detector The EASY nLC instrument is loading the sample onto the column assembly and the MS detector is waiting for the contact closure signal from the EASY nLC instrument Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System 47 5 Viewing the Run Status in the Information View Figure 18 Status page of the Information view File Actions View Tools
50. rom the same sample vial or well Make sure that the vial or well contains a sufficient volume Clear the Re Use Vial Positions check box if you want the autosampler to make replicate injections from different sample vials or wells Load the autosampler with a sufficient number of replicate samples e In the Initial Vial Position box type the vial position for the first sample injection For alphanumeric entries use uppercase letters see Taking Corrective Action for Run Time Errors on page 45 Note The sequence editor fills in the Positions column of the sequence table with contiguous vial or well locations based on the initial position the number of samples the number of replicate injections and the sample selection layout option selected in the Foundation Instrument Configuration window e Selecting the Row First option in the instrument configuration application prompts the sequence editor to fill in the positions row by row for example A1 A8 B1 B8 and so on e Selecting the Column First option in the instrument configuration application prompts the sequence editor to fill in the positions column by column for example Al F1 A2 B2 and so on e Clearing the Re Use Vials check box prompts the sequence editor to enter an additional vial or well position for each replicate 6 To set up the remaining parameters in the New Sequence Template dialog box refer to the Sequence Setup view Help Thermo Scienti
51. s b Click the Thermo EASY nLC row in the Start Instrument column The blank cell now displays Yes c Click OK to accept the new setting and close the dialog box 44 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific 4 Creating and Running Sequences Taking Corrective Action for Run Time Errors 5 In the Run Sequence dialog box do the following a Make sure that the Start When Ready check box is selected b Check the range in the Run Rows box c Click OK to submit the sample run to the acquisition queue Taking Corrective Action for Run Time Errors Thermo Scientific The method editor in the EASY nLC view of the Instrument Setup window checks each P parameter entry however it does not check for volume conflicts among the entries If you have specified an invalid volume for sample loading column equilibration or the gradient program an error message appears when you attempt to run samples with the instrument method from the Sequence Setup view Table 10 describes the corrective action for common run time errors that are caused by incompatible settings in the Xcalibur instrument method Table 10 Run time errors caused by incompatible settings in the instrument method Message Sample pickup flow must be greater than 0 when pickup volume is not 0 Corrective action Enter a nonzero flow rate in the Flow box in the Sample Pickup area on the Sample Pickup and Loading Page Solve
52. sion of the Instrument Control Software 4 Excluding the EASY nLC from the VirusScan Enterprise 5 Setting Up the Foundation Instrument Configuration 7 Connecting the EASY nLC Instrument to the Data System Computer 7 Adding the System Devices to the Instrument Configuration 8 Setting Up the EASY nLC Configuration pnia snp mene sue a wewee ene 10 Setting Up the Sample Layout and Start Position 000 000 eee 17 Creating Xcalibur Instrument Methods nnn anaana 21 Setting Up the EASY nLC Instrument Method Parameters 21 Sample Pickup and Loading Page i204 bbed cae darederavdscdadvdowses 23 Gradient Page ouro esireenriei ci Re CaS 25 Pre Column and Analytical Column Pave 2 ue cee cee eed pewee cues 28 Auto Sampler Page choice tei cincienny Scene bpeters kee tena kows 31 Saving Instrument Methods 425 u50reiederieseebewneketawdderaredths 34 Creating and Running Sequences 0 eceee eee nann 35 Creating SEQuEenCes nde wee cafe teens sas pee tow ewe los 35 Setting Up the Injection Positions with the Sample Position Selector 4 Rumning Sequences seses sisese koeie eei ea Olas Web ae whet obese ale en eae 43 Taking Corrective Action for Run Time Errors a nus eee eees esa des 45 EASY nLC Series User Guide for the Xcalibur Data System iii Contents Chapter5 Viewing the Run Status in the Information View
53. t Method Parameters Pre Column and Analytical Column Page Use the Pre column and Analytical Column page to set up the column equilibration steps see Figure 11 To open the Pre column and Analytical Column page 1 Open the EASY nLC view of the Instrument Setup window 2 Click the Pre column and Analytical Column tab If you do not see the Pre column and Analytical Column tab click Next Figure 11 Pre column and Analytical Column page Sample pickup and loading Gradient Pre column and Analytical column m Pre column equilibration Volume 4 ui i aap a J Lo ta a oy ff Flow ul min Qe Max pressure 800 00 Bar i O 28 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific 3 Creating Xcalibur Instrument Methods Setting Up the EASY nLC Instrument Method Parameters Pre Column and Analytical Column Page Parameters Table 8 describes the parameters on the Pre column and Analytical Column page of the EASY nLC view of the Instrument Setup window Note For information about determining the appropriate flow rate for the precolumn and analytical column equilibration steps refer to the EASY nLC Series Getting Started Guide Table 8 Pre column and Analytical Column page parameters Sheet 1 of 2 Parameter Pre column Equilibration Description Volume pL Flow L min Specifies the volume of solvent A that pump A pushes through the precolumn to waste d
54. the Xcalibur data system if it is open 2 From the Windows taskbar choose Start gt Programs or All Programs gt Thermo Foundation x x gt Instrument Configuration where x x is the installed version of Thermo Foundation The Thermo Foundation Instrument Configuration window opens see Figure 4 The installed devices are listed in the Available Devices list The configured devices are listed in the Configured Devices list Figure 4 shows an instrument configuration with a Thermo Scientific mass spectrometer added to the Configured Devices list 8 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific 2 Setting Up the Foundation Instrument Configuration Adding the System Devices to the Instrument Configuration Figure 4 Thermo Foundation Instrument Configuration window LTO Orbitrap Velos MS Thermo EASY nLC LTO Orbitrap Velos MS aime a Tip To access Help press F1 or click Help 3 Add the EASY nLC instrument to the instrument configuration as follows a Make sure that the Device Types list is set to All or AS Autosampler If the Device Type list is set to LC the EASY nLC VI for Xcalibur device does not appear in the Available Devices list b In the Available Devices list double click the Thermo EASY nLC icon A copy of the Thermo EASY nLC icon appears in the Configured Devices list 4 Set up the configuration options for each configured device For i
55. ument Methods Saving Instrument Methods Table 9 Auto sampler page parameters Sheet 2 of 2 Parameter Description Buttons Add Adds a row to the wash program Del Deletes the selected row from the wash program Up Moves the selected row up Down Moves the selected row down Saving Instrument Methods After you set up the instrument method parameters for the LC MS system save the instrument method to an appropriate directory To save your current instrument method 1 Choose File gt Save As The Save As dialog box opens 2 In the File Name box type a name for the new instrument method 3 Click Save The File Summary Information dialog box opens 4 Click OK 34 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific Creating and Running Sequences This chapter describes how to create and run sequences in the Xcalibur Sequence Setup view Contents e Creating Sequences e Setting Up the Injection Positions with the Sample Position Selector e Running Sequences e Taking Corrective Action for Run Time Errors Creating Sequences In the Xcalibur data system the table that contains the sample injection information is called a sequence In addition to the data file name for each injection the sequence must contain the vial or microplate well positions of the samples unknowns calibration standards quality control check standards and so on in the sample set the instrument method to
56. uring the precolumn equilibration step Range 0 to 137 pL IMPORTANT The maximum allowable entry is 137 pL however for Xcalibur sequence runs the sample loading volume plus the precolumn equilibration volume plus the analytical column equilibration volume must be less than 120 pL Specifies the flow rate for the precolumn equilibration step If the Flow box is empty the instrument runs at the set pressure Range 0 to 100 pL min Max Pressure bar Specifies the maximum pressure for the precolumn equilibration step If the Max Pressure box is empty the instrument runs at the set flow rate EASY nLC II range 0 to 280 bar EASY nLC 1000 range 0 to 980 bar Tip If both the Max Pressure and Flow boxes are empty the instrument runs at the maximum instrument pressure Leave these boxes empty if you want the instrument to run at the highest possible flow rate that is allowed under the maximum instrument pressure limit Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System 29 3 Creating Xcalibur Instrument Methods Setting Up the EASY nLC Instrument Method Parameters Table 8 Pre column and Analytical Column page parameters Sheet 2 of 2 Parameter Description Analytical Column Equilibration Volume pL Specifies the volume of solvent A that pump A pushes through the analytical column during the analytical column equilibration step Range 0 to 137 pL IMPORTANT The maximum al
57. uter and follow the instructions in the Thermo LC Devices InstallShield Wizard 5 On the Select Features page of the wizard select the Thermo check box on the left and then select the Thermo EASY nLC Xcalibur check box on the right see Figure 3 Figure 3 Select Features page of the Thermo LC Devices InstallShield Wizard Select the features you want to install and deselect the features you do not want to install CTC SSI of Thermo42D OK Surveyor Thermo EASY nLC Xcalibur Thermo Description Thermo EASY nLC Xcalibur Vl Space Required on C OK Space Available on C 406239184 K 6 When the installation finishes restart the data system computer The installation is complete when you log back in 4 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific 1 Installing the EASY nLC Control Software Excluding the EASY nLC from the VirusScan Enterprise Excluding the EASY nLC from the VirusScan Enterprise The McAfee VirusScan Enterprise software blocks the Thermo EASY nLC software when the data system computer uses the Windows 7 operating system IMPORTANT For the Windows 7 operating system you must exclude the EASY nLC VI software from the McAfee blocking list For the Windows XP operating system exclude the EASY nLC VI software from the McAfee blocking list only if the EASY nLC instrument is experiencing communication problems Thermo Scientific To exclude the
58. vice driver provided on the LC Devices DVD ensure that the data system computer has a compatible version of the Foundation platform as noted in the Thermo LC Devices x x x Release Notes where x x x is the version you want to install You can find the Release Notes document which includes the system requirements on the LC Devices DVD Table 1 Data system computer hardware and software requirements Hardware Minimum requirements Data system computer e 2 GHz processor with 1 GB RAM e DVD ROM drive e 80 GB hard drive capacity e Display resolution 1280 x 1024 resolution e 2 Network cards e NTFS format Software Requirements Operating system Microsoft Windows operating system e Windows 7 Professional 32 bit and 64 bit or e Windows XP Workstation SP3 Third party software Adobe Reader 9 0 or later Thermo Scientific software LC Devices Foundation platform EASY nLC device driver 2 8 0 or later 1 0 2 SP2 or later 2 7 0 1 0 2 SP2 or 2 0 0 SP1 LC Devices 2 8 0 and later are compatible with Windows 7 32 bit and 64 bit Thermo Scientific EASY nLC Series User Guide for the Xcalibur Data System vii Preface Data System Computer Settings Table 2 lists the computer settings that are required to avoid the premature termination of data acquisition during sequence runs It also provides the virus scanner setting that is required to establish communication with the EASY nLC instrument IMPORTANT After you instal
59. wer saver settings viii system requirements vii virus scanner setting 5 device driver installing 4 system requirements vii uninstalling 2 Thermo Scientific Device Types list 9 Devices InstallShield Wizard 4 documentation online x survey x Driver Info readback 15 duration gradient program 27 E EASY nLC VI control software Auto sampler page 31 EASY nLC Configuration dialog box 14 EASY nLC view Instrument Setup window 22 Gradient page 25 Pre Column and Analytical Column page 28 Sample Pickup and Loading page 23 Sample Position Selector dialog box 17 41 Edit button EASY nLC Configuration dialog box 16 error messages run time 45 Ethernet connection 8 F F1 key accessing Help with vi firewall network 2 flow rate analytical column equilibration step 30 gradient program 27 precolumn equilibration step 29 sample loading step 24 sample pickup step 24 Foundation platform vii G Gradient page 25 EASY nLC Series User Guide for the Xcalibur Data System 49 Index H H Help accessing vi hibernate setting viii Home Page icon 36 Idle Flow Rate readback 15 Idle Mixture readback 15 Information view Xcalibur data system 47 Initial Position entry 16 injection volume 23 40 Intelligent Flow Control IFC system readback 15 IP address EASY nLC instrument 14 IRC communication 5 L LAN port EASY nLC 8 Loop Volume readback 15 manuals accessing from the data system computer vi maximum pressur
60. ws operating system do one of the following e For Windows 7 choose Control Panel gt Uninstall a Program The list of installed programs appears see Figure 1 e For Windows XP choose Start gt Settings gt Control Panel gt Add or Remove Programs 2 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific 1 Installing the EASY nLC Control Software Removing a Previous Version of the Instrument Control Software Figure 1 Uninstall window for the Windows 7 operating system ce f fa OU a gt Control Panel Programs Programs and Features v 4 Search Programs and Features p Control Panel Home Uninstall or change a program View installed updates To uninstall a program select it from the list and then click Uninstall Change or Repair Turn Windows o gt E gt features on or off l Ki A Install a program from Name Publisher Version eek P Thermo Foundation 2 0 i 200 E Thermo LC Devices Thermo Fisher Scientific Inc 2 6 0 8 Thermo LC Devices LC Shared Library Thermo Fisher Scientific Inc 2 6 0 Bl Thermo LC Devices Thermo EASY nLC Xcalibur VI Thermo Fisher Scientific Inc 2 6 0 p Thermo Fisher Scientific Inc Product version 2 6 0 2 In the list of installed programs locate Thermo LC Devices Thermo EASY nLC Xcalibur VI 3 Depending on the operating system do one of the following For Windows 7 double click Thermo LC Devices Thermo EASY
61. xes in the Samples area of the New Sequence Template dialog box go to step 5 or e To use the Sample Position Selector dialog box go to Setting Up the Injection Positions with the Sample Position Selector on page 41 Tip You can set up the sample positions for a sequence in three ways e Inthe Samples area of the New Template Sequence dialog box type the number of samples in the Number of Samples box and the starting vial position in the Initial Vial Position box e In the Sample Position Selector dialog box do one of the following Type the number of samples in the Number of Samples to Select box and then click Select see Figure 15 on page 41 Click the positions in the Plate Rack and Vial Rack areas Use this method to set up noncontiguous nonsequential or noncontiguous and nonsequential positions 38 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific 4 Creating and Running Sequences Creating Sequences 5 Set up the vial or well positions by using the Samples area of the New Sequence Template as follows e In the Number of Samples box type the number of samples for the injection sequence e In the Injections per Sample box type the number of injections that you want the autosampler to make for each sample e For replicate sample injections do the following Select the Re Use Vial Positions check box if you want the autosampler to make replicate injections f
62. ys the default initial sample position for an injection sequence Edit button Opens the Sample Position Selector dialog box where you can select the sample layout and initial position for an Xcalibur sequence see Setting Up the Sample Layout and Start Position on page 17 Solvents for LC Pump Use this area to describe the solvents that make up the gradient A Specifies the solvent in solvent bottle A B Specifies the solvent in solvent bottle B 16 EASY nLC Series User Guide for the Xcalibur Data System Thermo Scientific 2 Setting Up the Foundation Instrument Configuration Setting Up the Sample Layout and Start Position Setting Up the Sample Layout and Start Position When you open the Sample Position Selector dialog box from the Thermo EASY nLC Configuration dialog box the Number of Samples to Select box and the Select button are unavailable When setting up the configuration options for the EASY nLC instrument use the Sample Position Selector dialog box to set up the sample layout and initial vial or microplate well position for an injection sequence For information about using the Sample Position Selector dialog box to interactively select the vial or microplate well positions for an injection sequence see Setting Up the Injection Positions with the Sample Position Selector on page 41 To set up the sample layout and start position for an Xcalibur injection sequence 1 In the Autosampler Configurati

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