Home

EASY-nLC II 2.8 User Guide Version A

1. Thermo Home Batch Setup Method Editor scripts Category Name Log Book Prepare Flush air Support Description a Output Devices Flush vol threshold pl 1000 Job 00 01 08 24 aug 2010 17 42 admin Schedule Exit Configuration f No graph J Maintenance No graph 7 Press the Parameters tab select the cell next to Flush Vol Threshold ul and type 10 This is the acceptable amount of compressibility that helps ensure good system performance overall 48 EASY nLC II User Guide Thermo Scientific 5 Preparation for Use Purging and Flushing the Pumps 8 Press Schedule 10 11 Thermo Scientific The Home gt Queue page appears Thermo Home Method Editor Maintenance Configuration Bes Auto E Submitter Job name Duration Graphs continue admin Flush air Job 00 00 00 G sep 2010 13 14 admin In the Jobs list make sure the check box in the Auto Continue column is selected for Flush Air Choose Home gt Overview again and press START Default graphs are selected in the two graph windows in Maintenance gt Scripts but you may change the selection from the corresponding lists These graphs help you to monitor the job Wait for the job to end the flush air script continues until the flush air volume values are below the specified threshold Note While a maintenance job or a script is running you cannot start other jobs
2. 2 0 B1 Sample 2 Next BSA 30 min Exit 24 8 19 51 Overhead of Using the EASY nLC II System to Control RePlay For a typical RePlay application injecting and loading 5 pl of sample with a gradient of 60 minutes 30 minutes RePlay the full timing is as follows 2 minutes Refilling pumps at 100 l min 2 minutes Injecting 5 ul at 20 pl min and waiting for pressures to settle 12 minutes Loading the sample onto the analytical column with 15 pl at 1 25 pl min 60 minutes Gradient autosampler wash 30 minutes RePlay while also reequilibrating the analytical column In total you can achieve an MS utilization rate of close to 85 percent 90 106 despite the fact that the EASY nLC II system cannot overlap parts of two runs in parallel Thermo Scientific EASY nLC Il User Guide 131 B Miscellaneous Installing and Using the RePlay External Device Column switching goes one step further in efficiency but at the cost of extra pumps columns and tubing which increases the price of the solution and introduces extra dead volume and delays into the transfer lines Working with nano flow systems you must minimize dead volume wherever possible and limit connections and switching as much as possible 132 EASY nLC II User Guide Thermo Scientific B Miscellaneous Declaration of Contamination Declaration of Contamination Thermo Scientific To return service or repair EASY nLC II equipment you
3. 2010 Thermo Fisher Scientific Inc All rights reserved EASY nLC EASY Column AFC and IFC are trademarks and Microtiter is a registered trademark of Thermo Fisher Scientific Inc PEEKsil is a trademark of SGE International Pty Ltd Corp This product uses FreeRTOS from www freertos org available upon request FreeRTOS is a trademark of Real Time Engineers Ltd The following are registered trademarks in the United States Advion and RePlay are registered trademarks of Advion BioSystems Inc Duran is a registered trademark of Schott AG PhotoMOS is a registered trademark of Panasonic Electric Works Co Ltd VICI and Valco are registered trademarks of Valco Instruments Co Inc Microsoft and Windows are registered trademarks of the Microsoft Corporation in the United States and other countries 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 rece
4. Figure 62 Back Pressure script parameters Thermo Home Batch Setup Method Editor SCIENTIFIC COE Category Name rest Log Book X gum pressure Support Description 500 5 Output Devices Job 00 01 02 24 aug 2010 17 40 eco Maintenance Pump A J Desired flow Real flow Pressure Flow nl min Flow nl min 300 000 0 250 000 0 200 000 0 150 000 0 100 000 0 50 000 0 0 0 Configuration 300 0 250 0 200 0 150 0 100 0 leq ainssaid 50 0 1 2 3 Time min 4 0 0 5 Pump B J Desired flow Real flow Pressure 300 000 0 250 000 0 200 000 0 150 000 0 100 000 0 50 000 0 0 0 50 000 0 100 000 0 300 0 250 0 200 0 150 0 100 0 leq ainssalg 50 0 0 0 1 2 3 Time min 4 5 Thermo Scientific 10 Maintenance Maintenance Scripts Test Autosampler Torque The Autosampler Torque script measures the torque required to move the autosampler on each of its axes Figure 63 Contact your local Thermo Fisher Scientific representative before running this script Figure 63 Autosampler Torque script description Thermo Home Batch Setup Method Editor EMaintenances Configuration f f No graph X Log Book Test Autosampler torque Support J ription Devices Tests the individual torque required for the autosampler manipulator axes a graph J Job 00 01 02 24 a
5. HPLC outlet Pre column Venting Tee Analytical column Note The systems maximum pressure is set at the factory to 300 bar 280 bar with IFC enabled Parameters such as column resin and frit size can vary greatly and generate different back pressure You can perform reequilibration and sample loading quicker at higher flow rates which help to reduce cycle time when analyzing samples Thermo Scientific Preparing the Pre Column 5 Preparation for Use Installing Columns This procedure assumes that IFC is activated the default If IFC is disabled the EASY nLC II application ignores the Max Pressure input field and instead the set flow passively regulates the pump To prepare the pre column 1 Choose Maintenance gt Scripts 2 Select Prepare in the Category list 3 Select Precolumn equilibration in the Name list Thermo ge SCIENTIFIC Support Devices Job 00 00 07 24 aug 2010 Log Book Fs J frm equili J 1725 admin Schedule Exit Configuration ouo A Desired flow Real flow Pressure 300 000 0 300 0 250 000 0 2500 T ER 2 D T 2000000 200 0 E 150 0000 1500 5 E hu 100 0000 100g 50 000 0 50 0 Time min _ Pump B J Desired flow Real flow Pressure 300 000 0 300 0 250 000 0 250 0 a E 2000000 200 0 E 150 0000 1500 5 2 100 0000 1000 F 50 000 0 50 0 0 0 0 0 0 1 2 3 4 5 Time min
6. K keyboard connection 14 L Leaks script 113 leaks checking for 104 lifting instructions 12 low pressure solvent filters 104 maintenance daily 103 quarterly 104 scripts 46 105 yearly 104 Maintenance tab 27 mass spectrometer connection 35 menu structure 21 Method Editor tab 25 method creating 54 136 EASY nLC II User Guide Microtiter plate 60 monitoring the run 63 See also RePlay monitor method mouse connection 14 MS Connection script 111 network access troubleshooting 92 network connection 36 P performance specifications 119 picking up a sample 55 plate formats 75 plates calibrating 78 power requirements 11 pre column equilibration 58 Precolumn Equilibration script 108 pre column preparation 51 preparing autosampler bottles 44 pre column 51 solvent bottles and waste containers 43 waste container 45 well plate 54 product description 2 pump flow control advanced 5 description 5 Intelligent 6 Purge Solvent script 106 purging and flushing pumps 47 Q quarterly maintenance 104 replacing main power fuse 124 RePlay configuring 126 connecting to EASY nLC 125 monitor method 131 overhead of using 131 setup method 128 RePlay external device installing and using 125 RePlay method 131 reset pressure sensor script 118 running batch editing 67 Thermo Scientific S safety standards EMC xiv sample acquisition 63 loading setup 55 pick up check 84 Pickup script 112 run troublesho
7. The options for the remote connection are in the Connect to Proxeon Support area Thermo Scientific EASY nLC Il User Guide 97 9 Remote Support Connecting the EASY nLC II System to the Support Server EASY nLC II User Guide Figure 55 Support page under the Maintenance tab Thermo scieuriric Home Batch Setup Method Editor Configuration Scripts Connectto EASY nLC support Log Book E Remote Port Number 2000 Connect Status Not Connected Message window can be used to chat with support when connected Show f Clear Devices Log file copy 9 To home directory To USB removable storage Copy Log Files Status Contact Job 00 00 00 Thermo Fisher Scientific easysupport proxeon com Edisonsvej 4 http www proxeon com hplic DK 5000 Odense C Phone 45 6557 2300 Denmark Fax 45 6557 2301 30 Aug 2010 14 14 admin Exit 2 Press Connect A pop up box opens f Message to support team Name Email address Phone number Enter message Accept Cancel 3 In the pop up box briefly describe the problem that you are experiencing Provide your contact information and press Accept The Status box on the right side of the Support page changes from Not Connected to Connected Thermo Scientific 9 Remote Support Saving System Files on a USB Removable Storage Device The EASY nLC II system is now connected to the support server
8. calibrating autosampler 71 plates 78 wash waste vial positions 83 change administrator password 39 checking sample pickup 84 solvent levels 103 valves 104 column wash 60 columns installing 50 Thermo Scientific compliance electromagnetic xiv components system 2 troubleshooting 93 Configuration tab 29 configuring devices 40 EASY nLC 32 RePlay 126 connecting add ons 14 mouse and keyboard 14 the mass spectrometer 35 to the network 36 connection instructions 13 consumable parts list 123 contact closure troubleshooting 92 control system advanced 5 Intelligent 6 cooler temperature checking 104 creating a batch with the Microtiter plate 60 amethod 54 new user accounts 40 plate formats 75 daily maintenance 103 data types graphs 65 Declaration of Conformity xiv Declaration of Contamination 133 devices configuring 40 E EASY nLC II description 2 equilibrating the analytical column 52 external connections 13 EASY nLC II User Guide 135 Index F F flow control system 5 flow line diagram 122 flow parts and lines 15 flow sensor calibration 104 Flow Sensors script 117 Flush Air script 107 fuse replacing 124 G gradient building 57 graphical data types 65 H hardware overview 7 Home tab 22 IFC See pump flow control installing columns 50 instrument back pressure checking 104 operation 4 Intelligent flow control system 6 interface layout 20 Isocratic Flow script 110
9. enough for a gt 10 h 0 10096 B 300 nl min gradient e 1 nL min to 533 pL min flow range External pressure sensor Valves VICI Valco rotor stator 6 ports 3 positions 1 6 1 2 or CENTERED Autosampler Flow Line Diagram Legend Material Len cm ID um Material FS Fused silica PS PEEKsil P PEEK TF Teflon Example FS 50 25 Peltier cooled Capacity is maximum 20 C below ambient temperature measured at 6096 RH Expect less if you remove the side panels use an adapter plate in the autosampler other than the standard plate or both Plate holder ejects through spring mounted autosampler door e 4 containers for waste or wash liquids 298000000006 99900000000 9990900090 6090 P5 557150 55 150 299900900000 298080900000 99000000000 9999900000909 8990999000000 122 EASY nLC II User Guide Solvent B TF 15 500 Thermo Scientific LUN Miscellaneous This appendix contains information you might refer to periodically including how to replace the main power fuse how to install an external device such as Advion RePlay a consumable parts list and transport and disposal instructions Contents Cleaning Materials e EASY nLC II Consumable Parts List Replacing the Main Power Fuse Installing and Using the RePlay External Device Declaration of Contamination Transport Instructions Cleaning Materials When cleaning the
10. 195 41 108 93 Defaut 1P Job 00 01 02 Save configuration 24 aug 2010 23 42 admin Exit 2 For Network type select Dynamic addressing DHCP or use a Fixed IP address Your IT administrator knows which option to select If IT asks about a MAC address for the instrument that is the instrument s unique network identifier it is labeled on the connector panel on the back of the instrument The identifier is a series of numbers and letters and might look like this MAC address 00 E0 4B 07 89 65 3 After setting up the network address attach the Ethernet cable and restart the system to get connected See Closing Down the EASY nLC II System on page 33 for information about the power down procedure and then immediately go to Starting the EASY nLC II System on page 19 and Logging In to the EASY nLC II System on page 32 External users can now access the file server in the EASY nLC II system to look at log files for example Microsoft Windows Explorer might display the file server as shown in Figure 22 Thermo Scientific EASY nLC Il User Guide 37 4 Configuring the EASY nLC II System User Permissions Figure 22 File server access through Windows Explorer S T 227750 Badin File Edit View Favorites Tools Help AN Back gt s Search ie Folders E Address E le 4 172 22 50 184admin i gt E Folders X Name Size Type Date Modified Desktop
11. 4 In the Scripts view press the Parameters tab and set the desired flow volume and maximum pressure Thermo Fisher Scientific recommends running the reequilibration at a set pressure rather than a set flow to make sure the column pressure limit and the instrument pressure limit 300 bar are not exceeded If the flow field is left empty the pump will run at the specified pressure For best results use a reequilibration volume of 10 column volumes 5 Press START The default graphs appear in the two graph windows but you may change the selections from their corresponding lists These graphs help you to monitor the progress 6 Monitor the actual flow rate to become familiar with the flow pressure relationship on your particular column Thermo Scientific EASY nLC Il User Guide 51 5 Preparation for Use Installing Columns Equilibrating the Analytical Column This procedure assumes that IFC is activated the default If IFC is disabled the EASY nLC II application ignores the Max Pressure input field and instead the pump is regulated passively by the set flow To equilibrate the analytical column 1 Choose Maintenance gt Scripts see Figure 29 2 Select Prepare in the Category list 3 Select Analytical col equilibration in the Name list Figure 31 Scripts page under Maintenance Thermo SCIENTIFIC Log Book Support Devices Job 00 01 02 24 aug 2010 23 55 admin Exit Home Me
12. Analytical Col Equilibration Prepare Isocratic Flow Test MS Connection e Test Sample Pickup Test Leaks e Test Valve Check Test Back Pressure e Test Autosampler Torque e Calibrate Valve Tune e Calibrate Flow Sensors Calibrate Reset Pressure Sensor Thermo Scientific EASY nLC Il User Guide 105 10 Maintenance Maintenance Scripts Prepare Purge Solvent 106 EASY nLC II User Guide The Purge Solvent script fills the chosen pump or pumps with solvent and then ejects it into the waste container Use the Purge Solvent script when exchanging solvents removing air from solvent lines or filling pumps To set the Purge Solvent script parameters 1 Press Purge Iterations to specify the number of empty fill cycles you want performed 2 Select the check boxes for the appropriate pump or pumps Note Entering a value of zero 0 for purge iterations will just refill the pumps Th emo Home Batch Setup Method Editor Log Book fem X f solvent Support Devices Purge iterations Job 00 00 07 24 aug 2010 17 24 admin Schedule Exit Maintenance Configuration Pump A J Desired flow Real flow Pressure 300 000 0 300 0 250 000 0 2500 m 2000000 200 0 E 1500000 1500 5 aa 2 100 0 5 iz 100 000 0 s 50 000 0 50 0 0 0 0 0 o 1 2 3 4 5 Time min Pump B Desired flow Real flow Pressure 300 000 0 300 0 250 000 0
13. Closing Down the EASY nLC II System Thermo Scientific EASY nLC Il User Guide 17 3 Instrument Control Software Using the Touch Sensitive Screen Using the Touch Sensitive Screen 18 EASY nLC II User Guide The EASY nLC II system has no keyboard or computer mouse Control of the instrument comes directly through the instrument monitor Figure 16 Finger touch control 2 zx EASY alll i The monitor face is a transparent layer of glass that detects finger pressure even while wearing gloves and sends the corresponding commands to the computer In most cases pressing a button displayed on the screen causes the instrument to carry out a certain operation However when the application requires text or numeric input the application displays a keyboard or numeric keypad on top of the main window Figure 17 so that you can enter values on the screen by pressing the appropriate keys Figure 17 Touch keyboards ESC p 1 2 3 4 5 5 7 8 9 0 Home End 4 4 4 4 Am 4 am 4 4 4 4 4 4 4 5 6 Tab q w e r t y u i o p Del 3 4 A 4 4 4 4 Am P 4 4 4 4 4 E 4 f t vw E XT 7 8 g Caps lock a s d t g h j k 2 2 Enter j J da 4 4 4 As 4 4 A 4 4 4 A 4 xt n xr xt Nr xt xr n xr xr xr xr xt D g 6 5 Shift z x c v b n m i i Shift t 0 A A A A A A A A A A As A A 4 q J 3 E E cp Ji 3 Ctrl Alt space Alt Gr
14. EASY nLC II User Guide The Overview page provides a full schematic overview of the system complete with real time updating of all critical component data Figure 39 Figure 39 Overview page under Home Thermo Home BatchSetup Method Editor Maintenance Configuration SCIENTIFIC 142 8 bar i 100 0 ul min 3 4 150 0pl Graphs 29 Solvent A IFC enabled Idle flow 25 5 Queue 547 4 nl min 143 0 bar 100 0 ul min 2 9 150 0pl TIEN 900000 o00000 9 oooooo 147 5 bar 547 4 nl min A 40 0 ul min Waste D 5 0 150 0pl Pickup sample terr Refill A B Solvent B 5 i 1 f oo EM E eme om i 00 00 uM ministrat EREU Job 24 aug 2010 17 35 1 0 A1 Sample 1 admin 20100824 17 OTILIB SA 30 min admin 24 3 B1 Sample 2 EE E sA 30 min Exit 24 8 19 51 The system schematic provides information on all hardware parts in the system Pump position pressure and flow Valve position Percentage of B being delivered Actual flow being measured Samples to be analyzed plus their current status Current sample and job plus the next sample and job The sample analysis process can be divided into different activities such as Pickup sample Load precolumn and so on When executed these activities are highlighted in the activity box in the center of the page and give you a quick overview of the sample s prog
15. It stays connected until you press Disconnect or the network connection is broken Inform Thermo Fisher Scientific if you would like to initiate remote support and the Technical Support team will start to monitor and diagnose your system Saving System Files on a USB Removable Storage Device The EASY nLC II application is designed with network access in mind Through the network you can back up your system export and import batches and methods analyze your system by examining the log files and perform many other tasks However when you are not connected to the network or the network is broken or you do not have a computer with network access that is near the EASY nLC II system you can use the copy function You can copy all the system files on the system to a USB removable storage device such as a USB flash drive or memory stick Before continuing you need a USB storage device formatted with the file system FAT FAT16 and at least 128 MB of free space The EASY nLC II system is shipped with a USB flash drive for your convenience To copy system files to a USB storage device 1 Choose Maintenance Support Thermo Im er M Tre ee Mieres ua Maintenance mereri SCIENTIFIC Scripts Connect to EASY nLC support Log Book C 4 Remote Port Number 2000 Connect Status Not Connected Suppor 4 Message window can be used to chat with support when connected Show Clear Devices Log file copy 9 To home di
16. 0 1000 8 0 0 a 50 000 0 50 0 100 000 0 00 o 1 2 3 4 5 Time min The green START button is active when a job or script is ready to execute During execution you can terminate a running job by pressing the now active red STOP button You can automate the start of certain scripts for example scheduling them for later execution However you can schedule only those scripts where the Schedule button is active You cannot schedule some scripts because they require supervision or hardware changes to the system Thermo Scientific Purging and Flushing the Pumps Because the pumps have been purged with methanol before delivery of the EASY nLC II instrument make sure to purge them thoroughly before doing any analytical runs In addition you must purge the pumps when switching to a new solvent 5 Preparation for Use Purging and Flushing the Pumps The purge pump job fills the chosen pump or pumps with solvent and afterwards ejects it into the waste container You can specify how many iterations of this job you want performed After purging the pumps flush them to remove any air that might be trapped inside You can specify the maximum number of iterations for this job the period of time that the pumps are being pressurized and the success criteria for stopping the operation earlier than scheduled To purge and flush pumps A and B 1 Go to Maintenance gt Scripts to open the Scripts view 2 In the Category list select
17. 0 A1 9 59 09 Thermo Scientific Thermo Scientific 7 Autosampler Calibration Calibrating the Wash Waste Compartment 6 After the EASY nLC II application finishes the script a Eject the tray b Check all wells one by one with a pipette C Check that there are 2 pL left in all wells Ifthere are more than 2 pL left in a wells there might be a problem with the autosampler calibration or the autosampler tray adapter plate is not aligned Calibrate the autosampler again and do the sample pickup check Ifthere are more than 2 pL left in some of the wells there might be air in the system Run the Flush Air script on pump S see Figure 52 and then the sample pickup check Figure 52 Flush Air script under Maintenance tab Category Name Category Name Category Name f Prepare J f Flush air J f Prepare f Flush air f f Flush air from pumps Make sure to check solvent levels before running Parameter Value Flush pump A ts Flush pump B x Flush pump S x Flush vol threshold ul 10 00 Description Parameters Out Flush air started Pump A Iteration 1 Pump B Iteration 1 Pump B Refilling Pump A Refilling Schedule o Schedule o You are finished when there are 2 pL left in all wells Schedule STOP EASY nLC Il User Guide 85 ee ON Troubleshooting This chapter offers help with the EA
18. 2 of 2 Symptom Possible causes Action System is running slower Memory filled with Choose Home Graph and disable and slower graph data from long the graphs by pressing No Graph in gradient run each graph window System overload caused Restart the application or by other reasons power down power up from Maintenance Scheduling Unhandled error or SVG Program error in the In most situations you can continue scalable vector graphics error software release your work by pressing Ignore or OK More serious error situations might require restarting the HPLC To improve on the software quality e mail details to easysupport proxeon com Or connect to the remote support server See Connecting the EASY nLC II System to the Support Server on page 97 EASY nLC II User Guide 95 LLL AM Remote Support The EASY nLC II system is designed with an integrated remote support function While connected Technical Support can follow the processes on your system to better understand any issues that arise Contents Connecting the EASY nLC II System to the Support Server Saving System Files on a USB Removable Storage Device Connecting the EASY nLC II System to the Support Server Check that your EASY nLC II system is connected to the network before you continue For more information see Network Connection on page 36 To connect to the support server 1 Choose Maintenance Support see Figure 55
19. 2010 12 34 st t Login Login and Exit button Begin by choosing one of the top menus The application displays a number of tabs down the left side of the active area Press these tabs to display views related to that topic During a session the application remembers which side tab you selected inside each top menu and shows that tab when you next choose that menu Use the current user area to log off any page in the application Press the logo in the upper left corner to view version information about the currently running software 20 EASY nLC II User Guide Thermo Scientific 3 Instrument Control Software General Interaction Principles General Interaction Principles Press buttons only one time for a specific action If the action is important or irreversible the EASY nLC II application displays a confirmation dialog box so that you can cancel the action You can edit many tables usually when you have entered the data yourself You can also access single cells by touching the cell twice to open a keyboard keypad display for changing the entry Long tables have vertical scroll bars that you move either by pressing the up or down arrow symbols or by dragging the scroll bar and moving it explicitly In some tables you can select multiple rows by pressing check boxes on several rows or complete columns by pressing the table headings If a screen has blank input fields press inside the field to enter data The app
20. 3 Flush pump S x Flush vol threshold ul 10 00 _ No graph J Job 00 01 08 24 aug 2010 17 42 admin Schedule Exit EASY nLC Il User Guide 107 10 Maintenance Maintenance Scripts Prepare Precolumn Equilibration 108 EASY nLC II User Guide The Precolumn Equilibration script equilibrates the pre column with solvent from pump A The script utilizes the IFC system unless it is disabled and the procedure below is based on IFC being active If IFC is disabled the EASY nLC II application ignores the Max Pressure setting and the pump is run passively at the specified flow Use this script to equilibrate flush the pre column determine a suitable pre column reequilibration and load flow for method setup or when initializing a new pre column Tosetthe Precolumn Equilibration script parameters 1 Press Volume to set how much solvent A to use for reequilibration of the pre column For best results use 10 column volumes for reequilibration 2 Press Flow to set the flow rate for performing the reequilibration of the pre column If the flow field is left empty the pump operates at the set pressure 3 Press Max Pressure to set the maximum allowed pressure If the pressure field is left empty the pump operates at the set flow as long as it is below the instrument maximum pressure of 300 bar If both a flow and a max pressure are specified the pump flow is limited by whichever parameter is reached fi
21. A Qallusers File Folder 19 05 2005 11 16 Type 8 My Documents P batches File Folder 30 03 2005 12 51 P a dd ress c 3 My Computer CibatchLog Fie Folder 20 05 2005 14 21 5 amp 9 my Network Places QymaintenanceLog File Folder 20 05 2005 12 33 here Entire Network methods File Folder 30 03 2005 12 51 amp V Microsoft Terminal Services QsystemLog File Folder 19 05 2005 13 53 Microsoft Windows Network 2 statistics_20050519_110358 EA Proxeon S a Hplc 8 Y Hple a 3 Proxeon HPLC 172 22 50 18 5 amp admin 1KB XML Document 19 05 2005 11 04 4 Type the IP address of the instrument for example file 172 22 50 18 and press ENTER You need a valid user name and password to enter the file space Your user name and password are the same as those on the EASY nLC II system User Permissions There are four types of users Guest default Normal Super user and Administrator Each user type has access to different levels of functionality in the EASY nLC II application Table 8 User types and permission levels Type Menu Guest Normal Super user Administrator Home x x x X Batch Setup X X X X Method Editor X X X X Maintenance X X Configuration X 1 X User has read write access X User has read only access Blank Feature is not accessible Having a Guest user account means you Can access the top menu but not enter data or issue commands to the system If a featur
22. BSA 30 min Save Precolumn equilibration Batch Setup Method Edito Maintenance Configuration Volume pl Flow 5 00 yl min Max pressure Bar Analytical column equilibration Job 00 00 07 Volume 4 ul ye X A DE LE _ Flow 0 5 ul min SAY f j IA 24 aug 2010 bd C 17 28 AX Max pressure 280 Bar admin To set up the pre column equilibration 1 In the Precolumn Equilibration area press the Volume box to set the amount of solvent A to use for reequilibration of the pre column For best results use 10 column volumes for reequilibration 2 Press the Flow box to set the flow rate for performing the reequilibration of the pre column If you leave the input box empty the pump will operate at the set pressure 3 Press the Max Pressure box to set the maximum allowed pressure If you leave the input box empty the pump operates at the set flow as long as it is below the instrument pressure of 280 bar Note If you specify both a flow rate and a maximum pressure the pump flow is limited by the parameter that is reached first To equilibrate the analytical column 1 In the Analytical Column Equilibration area press the Volume box to set the amount of solvent A to use for equilibration of the analytical column For best results use 10 column volumes for reequilibration 58 EASY nLC II User Guide Thermo Scientific 6 Running the First Sample Creating a Method 2 Press the
23. Cannot run a batch The following or similar error message pops up when you run the batch Execute queue Execute batch 6 x BSA 30min r configuration nfi st be O Plate vials 6x8 6x8 vials 1 Vial rack 6x1 1x6 FIXED f N Close C Plate format If you want to run the batch you deleted from your must create the plate format again configuration with the Delete button from Maintenance gt Devices gt Autosampler gt Tools The needle cannot reach the bottom of the solvent bottles position 1 3 Thermo Scientific The needle in the original autosampler ASA is not designed to reach the bottom of the solvent bottles None EASY nLC Il User Guide 91 8 Troubleshooting Contact Closure Contact Closure Symptom The MS is waiting for a contact closure signal Or The EASY nLC II system is waiting for a ready signal from the MS Network Access Symptom System is not responding after pressing Save Configuration on the Network page under Configuration Possible causes Wrong contact closure setup Possible causes Length of completion time to update the network settings Action If you are running two way contact closure with feedback from the MS set the contact closure Protocol setting to Two way on the Configuration Connections page If you are running one way contact closure that is the EASY nLC II system sends a start signal to the
24. Ctrl m zs Accept The keys light up when pressed so that you do not need to look at the text or number field where the entry is also shown If preferred you can attach a USB based mouse or keyboard to the instrument s back panel but the user interface has been optimized for touch screen interaction Thermo Scientific 3 Instrument Control Software Starting the EASY nLC II System Starting the EASY nLC II System Press the On Off switch on the back of the instrument Figure 18 The On Off switch on the back panel 220 240 V d LJ The start up procedure takes between 5 and 10 minutes while the instrument does the following 1 Boots the internal computer 2 Initializes all of the necessary software components 3 Checks all the hardware parts pumps valves autosampler plate cooler flow sensor and so on 4 Starts the application During this process the screen shows a status bar to indicate progress EAS Y nLC Please wait SCIEN d ad Ee Thermo Scientific EASY nLC Il User Guide 19 3 Instrument Control Software User Interface Layout User Interface Layout The EASY nLC II user interface provides the following menu structure Figure 19 EASY nLC II user interface layout with menus Top menus e fc E pt eee ET i Thermo Home BatchSetup Method Editor I i g A a ee ee ee ee ee ee Pe J Graphs Queue L ee Job 00 00 00 Current user 23 aug
25. Exit 2 Press Power Down The EASY nLC II application displays a white screen with a small progress bar When the progress bar is filled out and the message appears indicating you can safely turn off the instrument go to the next step 3 After receiving the message that you can safely turn off the instrument turn off the power switch on the back of the instrument EASY nLC The instrument can now be turned off safely SCIENTIFIC EASY nLC Il User Guide 33 Configuring the EASY nLC II System After turning the instrument on and logging on as a system administrator you must configure the instrument for use and define the work environment between the instrument and computer Contents Setting Up the Mass Spectrometer Connection Network Connection User Permissions Setting Up the Mass Spectrometer Connection IMPORTANT Before you connect the HPLC to a mass spectrometer make sure that the protective grounding is shared between the instruments itN To set up the mass spectrometer connection 1 Choose Configuration Connections The application displays a number of options Figure 21 Connections page Thermo Home Batch Setup Method Editor Maintenance Users Mass Spectrometer Connection Instrument SEES Finnigan LTQ OrbiTrap J Network Time Communications Use Contact Closure X Use Ethernet Backup Contact Closure Settings EASY nLC Thermo Finnigan LTQ OrbiTrap Protocol Two way J
26. Flow box to set the flow rate for performing the equilibration of the analytical column If you leave the input box empty the pump will operate at the set pressure 3 Press the Max Pressure box to set the maximum allowed pressure If you leave the input box empty the pump operates at the set flow as long as it is below the instrument pressure of 280 bar Note If you specify both a flow rate and a maximum pressure the pump flow is limited by the parameter that is reached first Page 5 5 Use Page 5 5 for setting up the autosampler washing procedures see Figure 35 The EASY nLC II instrument conducts the wash while the gradient is running Autosampler Wash For most samples in low concentrations flushing the needle and loop with solvent is sufficient You can set the amount of solvent for this action in the Flush Volume box Use 100 pL as the default flush volume Note The minimum flush volume that you can set is the loop volume needle volume 1 yl Figure 35 Page 5 5 Edit view of the Method Editor Thermo Home Batch Setup Method Edito Maintenance Configuration SCIENTIFIC File eae Name BSA 30 min Save Autosampler wash Flush volume ul Custom wash Order Source Volume Cycles Job 00 00 07 24 aug 2010 17 29 4 D Exit amp 55 Thermo Scientific EASY nLC II User Guide 59 6 Running the First Sample Creating a Batch Custom Wash Fo
27. Prepare and in the Name list select Purge Solvent Thermo SCIENTIFIC Log Book Support Devices Job 00 00 07 24 aug 2010 17 24 admin Exit Home Batch Setup Method Editor Name f fr solvent J Category Parameter v Value Purge iterations 10 EJ EJ Schedule Flow nl min z E z E Lu Configuration fr Desired flow Real flow Pressure 300 0 300 000 0 250 000 0 200 000 0 150 000 0 a o o peq ainssald 100 000 0 50 000 0 0 0 09 0 e NS 4 5 Time min fr B J Desired flow Real flow Pressure 300 0 300 000 0 250 000 0 200 000 0 150 000 0 a o o req aunssald 100 000 0 50 000 0 0 0 z 0 0 o 1 2 3 4 5 Time min 3 Press the Parameters tab select the cell next to Purge Iterations and type 10 This is the number of times that the pumps will completely refill and eject their volume Note Bottle three W3 in the autosampler bottle holder fills the S pump and the solvent ejects into bottle four W4 which is always used for waste liquid Thermo Scientific EASY nLC II User Guide 41 5 Preparation for Use Purging and Flushing the Pumps 4 To save this setting and continue press Schedule The Home gt Queue page appears 5 Return to Maintenance gt Scripts 6 In the Category list select Prepare and in the Name list select Flush Air
28. again You might need to calibrate the autosampler for more precise penetration of the Microtiter plate or wash waste containers Repetition of the error without any visible Contact Thermo Fisher Scientific easysupport proxeon com reason Time out on flow sensor Air in the pumps Run the maintenance script Prepare Flush Air on page 107 Sample is not being Air in the pumps On the ASA Check that the solvent picked up level in W3 is more than 2 3 full otherwise refill it Run the maintenance script Prepare Flush Air on page 107 If this does not work try running the Leak Test on pump S see Test Leaks on page 113 and then the Flush Air script Valve unknown position Thermo Scientific Valve trimming needed Run the maintenance script Test Valve Check on page 114 EASY nLC Il User Guide 89 8 Troubleshooting Autosampler Autosampler 90 EASY nLC II User Guide See Table 13 for tips on handling autosampler issues To access the Autosampler control window Figure 54 go to Home gt Overview and press the plate icon Figure 54 Autosampler control window XYZ Robot Cc f Eject Plate Insert Plate 16x24 HT MIP Uncalibrated Goto Calibration Goto Well Heol C 4 Needle Up Needle Down 4 w C C 7 0 C Set Disable Temperature 68 RC Close WA B Cooler Table 13 Autosampler troub
29. all phases of operation service and repair of this instrument Failure to comply with these precautions or with specific warnings elsewhere in this manual violates safety standards of design manufacture and intended use of the instrument and may result in injury or loss of life Special Notices The following two stickers appear on the EASY nLC II HPLC instrument SHARP NEEDLE AND MOVING PARTS WARNINS KEEP HANDS CLEAR AIGUILLE POINTURE ET PARTIES MOUVANTES AVERTISSEMENT TENIR LES MAINS L CART This sticker indicates that care should be taken to prevent personal injury or damage to parts of the EASY nLC II viii EASY nLC II User Guide Thermo Scientific Preface This sticker alerts you to consult this manual for instructions on how to operate the HPLC The safety and special notices in the documentation include the following 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 software loss of data or invalid test results or may 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 Contacting Us There are several ways to contact Thermo Fisher Scientific for the information you need To contact Tech
30. below an air conditioning or heating vent Place the EASY nLC II instrument as close as possible to the detector and mass spectrometer to minimize dead volume in the transfer lines Place the EASY nLC II system away from any detector mass spectrometer vents however to avoid exposure to direct heat For optimal autosampler plate cooling place the unit in an area where the working temperature is within 5 30 C 41 86 F The optimal humidity range is between 20 80 RH Avoid condensing humidity Lifting Instruction The instrument weights 32 kg 71 Ib For safety reasons use two people to move the instrument to a table cart for transport Gloves are recommended Figure 11 Lifting by hand MD Thermo EASY nLC II UH TRA External Connections 12 EASY nLC II User Guide The EASY nLC II instrument has several types of connections to external services or devices that you can make Connecting to the Mass Spectrometer through Contact Closure Connecting to Remote Support Connecting to a Local Area Network for Data Exchange Attaching Mouse and Keyboard to the USB Connections Attaching Add on Products through the P Bus and RS 232 Thermo Scientific 2 Installation External Connections Connecting to the Mass Spectrometer through Contact Closure The EASY nLC II stand alone application contains preconfigured values for most of the standard mass spectrometers used in proteomics For
31. in square brackets The city code or area code is enclosed in parenthesis For countries other than the U S when you are dialing from within the specified country dial the 0 of the city code For countries other than Italy when you are dialing from outside the country do not dial the 0 of the city code x EASY nLC Il User Guide Thermo Scientific Thermo Scientific Preface Table 2 Customer Service information for Europe Sheet 2 of 4 Belgium Phone 32 0 2 482 3030 Fax 32 0 2 482 3031 E mail service sid benelux thermofisher com Address Clintonpark Keppekouter Ninovesteenweg 198 B 9320 ERMEBODEGEM AALST Denmark Phone 45 70 236267 Fax 45 70 236263 E mail service sid dk thermofisher com Address Fruebjergvej 3 2100 K benhavn Finland see Sweden Norway and Finland e French speaking North Africa Algeria Morocco and Tunisia Phone 33 0 1 60 92 49 50 Fax 33 0 1 60 92 48 99 E mail service sid lesulis thermofisher com Address 16 Avenue du Qu bec Silic 765 Z A de Courtaboeuf F 91963 Les Ulis C dex Germany Phone 49 0 6103 408 1050 Fax 49 0 6103 408 1213 E mail service dreieich thermofisher com Address Im Steingrund 4 6 D 63303 Dreieich Italy Phone Numero Verde 800 823 162 Fax 39 02 95320 225 E mail assistenza tecnica it thermofisher com Address Strada Rivoltana I 20090 Rodano Milano EASY nLC II User Guide xi Preface Table 2 Custome
32. instrument setup and manage system configuration Table 7 Configuration pages Sheet 1 of 3 Use the Users page to manage user accounts create edit and delete For more information see Creating New User Accounts on page 40 Thermo SCIENTIFIC Connections Network Time Backup Job 00 00 00 24 aug 2010 17 17 admin Exit Home Editor Mainten Name Administrator Password Re enter Level Norma fF Graphs Pump A E fos 8 a E rasiert p pum Cooling j 1 Use the Connections page to view which mass spectrometer connects to the EASY nLC II system and how the communication is set up For more information see Setting Up the Mass Spectrometer Connection on page 35 Thermo SCIENTIFIC Users Network Time Backup Job 00 00 00 23 aug 2010 1238 admin Home Batc Mass Spectrometer od Editor Maintenance Instrument PROFINET OR Communications Use Contact Closure X Use Ethernet Contact Closure Thermo Finnigan LTQ OrbiTrap Peripheral Control Settings EASY nLc Protocol Two way OUTI State at start Open Ni OUT2 Start at Gradient IN2 OUT3 Signal width Tee TTL GND f f Default Jf EASY nLC Il User Guide 29 3 Instrument Control Software Application Menu Structure Table 7 Configurat
33. mass spectrometer at a given B percentage or when cleaning the instrument columns t hod To set the Isocratic Flow script parameters 1 Press Volume to specify the total volume to be delivered by the pumps 2 Press Flow to specify the flow rate to be used 3 Press AB Mix to specify the solvent composition Th ormo Home Batch Setup Method Editor Log Book pum X f flow J Support Description Devices Parameter v Value 10000 030 AB Mix 96B 30 z Job 00 01 02 24 aug 2010 17 46 admin Schedule Exit Maintenance Configuration Pump A J Desired flow Real flow Pressure 300 000 Somn 250 000 0 2 a 2000000 2000 o 150 000 0 1500 5 a a 1000 5 it 100 000 0 El 50 000 0 50 0 00 0 0 o 1 2 3 4 5 Time min Pump B Desired flow Real flow Pressure 300 000 0 300 0 250 000 0 250 0 200 000 0 i 2000 c E 1500000 a ao 100 000 0 1500 S 2 soo000 S 1000 5 0 0 50 000 0 50 0 100 000 0 1 00 o 1 2 3 4 5 Time min Thermo Scientific 10 Maintenance Maintenance Scripts Test MS Connection The MS Connection script tests the contact closure function between the EASY nLC II system and the mass spectrometer Use this script when testing contact closure There are no parameters associated with the MS Connection script Instead it uses the settings from the Configuration Connection page see Settin
34. must complete and submit a Declaration of Contamination of Equipment to Thermo Fisher Scientific with the returned equipment Qualified personnel must review the declaration Contact Thermo Fisher Scientific for additional copies of this form or if you have any questions regarding the contents of this declaration Description of equipment Equipment type model Serial No Date of receiving purchasing equipment Reason for return Maintenance Repair End of test Describe symptoms and problems Equipment condition Has the equipment been used Yes No Has the equipment been exposed to potentially harmful substances Yes No If yes attach list of all known harmful substances including chemical name and symbol and precautions associated with the substances Were any of the harmful substances Radioactive Yes No Toxic Yes No e Corrosive Yes No Explosive Yes No Has the equipment been properly decontaminated and or cleaned before being returned Yes No Legally binding declaration I hereby declare that the information supplied on this form is accurate and sufficient to judge any contamination level Name Job title Organization Address Telephone Fax E mail Signature EASY nLC Il User Guide 133 B Miscellaneous Transport Instructions Transport Instructions To prepare for transport 1 Open the tray door on the H
35. procedures for keeping the instrument functioning properly and help pinpoint possible problems Select the category and then the individual script After selection the application provides explanatory text when the list of input parameters appears You must enter parameters correctly before starting the script Next to the script control area the application displays two configurable graphs so that you can monitor the process For the complete list of maintenance scripts and how to set them up see Maintenance Scripts on page 105 Thermo SCIENTIFIC Log Book Support Devices Job 00 02 33 24 aug 2010 1722 admin Home Method Editor Batch Setup Category Name Prepare Jf Purge solvent j Purge solvent from punps Make sure to check solvent levels before running Configuration Desired flow Real flow Pressure 300 0000 somno 250 0000 2000000 E 1500000 1000000 50 0000 ur oo 60 000 0 100 000 0 Time min Pump B J Desired flow Real flow Pressure 300 000 0 s 250 0000 2500 2000000 Schedule o E wooon 1000000 sooo oo 50 0000 rt 50 0 100 000 0 o 5 Time min 8 a beal EASY nLC II User Guide 27 3 Instrument Control Software Application Menu Structure 28 EASY nLC II User Guide Table 6 Maintenance pages Sheet 2 of 2 Use the Log Book page
36. right positions position A1 1 and F8 2 in the case of the default 6 x 8 vial adapter shown in ligure 47 2 Go to Maintenance Devices 3 From the Devices list select the autosampler 4 Under the Tools tab in the Plate area press Calibrate see Figure 45 on page 75 The Autosampler Plate Calibration dialog box opens Figure 47 78 EASY nLC II User Guide Thermo Scientific 7 Autosampler Calibration Calibrating Plates 5 Press the green well in the upper left corner of the plate view 1 This moves the needle to the current saved coordinate Figure 47 Plate view in the Autosampler Plate Calibration dialog box Autosampler Plate Calibration Calibrate Plate HT MTP Needle In Out Needle Up Down 3 5 E9 4 Needle Left Right HF ee e TT Note The EASY nLC II application copies the saved coordinate to the left side of the slash except for Needle Up Down which starts with the top position The left side coordinate is the working current coordinate and is not saved yet 6 Center the needle above the well by pressing the Needle In Out 3 and Needle Left Right 4 buttons 7 Lower the needle by pressing the Needle Up Down buttons 5 until the needle reaches a level of 2 3 mm 0 08 0 12 in above the well Start with the big step button WV and change to the small step button V when you are near the well Thermo Scientific EASY nLC Il User Guide 79 7 Autosam
37. the custom wash has ended the autosampler automatically empties the pump and refills with solvent from bottle 3 to be ready for the next injection Creating a Batch 60 EASY nLC II User Guide Use the Edit view under Batch Setup to set up and schedule batches for sample acquisition on the EASY nLC II system See also Preparing a Well Plate on page 54 and Creating a Method on page 54 To create a batch with the Microtiter plate 1 Go to Batch Setup gt Edit If you do not see a plate overview press View Racks see Figure 36 on page 61 Thermo Scientific 6 Running the First Sample Creating a Batch 2 Browse the methods list next to the Injections box and select method1 created in Creating a Method on page 54 3 If the plate format is not set to your installed plate choose an appropriate plate by pressing the plate button The p ate button is the rectangular button imprinted with the plate name in the lower left side The default plate is 6 x p18 vials 4 Press the DOWN ARROW labeled 1 to select all vials wells from the first column A1 to H1 Note You can also select vials wells by pressing them individually You can clear well selections by pressing them again 5 Press Add to link the selected vials wells with the chosen method Your touch screen should look similar to the screen in Figure 36 Figure 36 Vial Well selection in the Edit view under Batch Setup Methods list Thermo Hom
38. to electronically enter all the service Thermo SCIENTIFIC Scripts actions that have been carried out on the instrument There is a E Devices general log where you can enter general comments about all the components and areas for several specific actions that are used actively in other parts of the application primarily statistics admin Exit Method Editor Maintenance Configuration Log record contains one term per line g pi Log book Time From First record Queue job Time To een Last record fy f Search Search result Timestamp Source Summary 05 08 2010 17 52 49 admin Batch admin 20100805 1751 05 08 2010 17 44 34 admin Batch admin 20100805 1742 19 07 2010 10 41 58 admin Batch admin 20100719 1039 12 05 2010 17 03 40 admin Purge solvent 00 00 00 29 04 2010 10 29 34 admin Purge solvent 00 00 00 29 04 2010 10 28 42 Super Purge solvent 00 00 00 R D4 2010 13 43 4 laimin Enter Log Entry lisneratic flaw san an Use the Support page to set up T support from Thermo Fisher Scripts Scientific For a more Log Book comprehensive description see Devices Chapter 9 Remote Support Job 00 00 00 30 Aug 2010 14 14 admin Exit Method Editor Configuration Connect to EASY nLC support Remote Port Number 2000 f Connect fs Status Not Co
39. under Batch Setup tab Thermo Home Baten Setup Method Editor Maintenance Configuration SCIENTIFIC MEE File f J CE Add Injections 1 BSAS30 min RePlay Batch Extensions v i Method Sample 2 BSA 30 min RePlay Sample 4 BSA 30 min RePlay Sample 5 BSA 30 min RePlay Sample 6 BSA 30 min RePlay Job 00 02 46 Sample 00 00 16 Gradient 00 00 25 aug 2010 00 06 admin 3 Edit the batch that is add change or remove samples that are not yet tested 4 Press Reschedule The batch continues running Thermo Scientific 6 Running the First Sample Editing the Running Batch 5 Optional Go to Home gt Queue see Figure 43 check the sample list by pressing Properties and then press Show Samples Figure 43 Sample list on the Queue page of the Home tab Thermo Home BatchSetup Method Editor Maintenance SCIENTIFIC Configuration Overview Braphs piss Submitter Job name Duration 1 C famn famin 201008241 714 11 04 15 Job 00 00 00 24 aug 2010 17 14 admin E Thermo Scientific EASY nLC Il User Guide 69 6 Running the First Sample Troubleshooting a Sample Run The Job Properties box for the Sample list appears see Figure 44 Figure 44 Sample list job properties on the Queue page Thermo 777 SCIENTIFIC Overview Auto continue Submitter Graphs ditor Maintenance Job name admin 20100824 1714 Conf
40. vial positions 1 Press the bottle container in W4 1 as show in Figure 50 This moves the needle to the current saved coordinate Note The EASY nLC II application copies the saved coordinate to the left side of the slash except for Needle Up Down which starts with the top position The left side coordinate is the working current coordinate which is not yet saved Figure 50 Vials Wash view in the Autosampler Vials Wash Calibration dialog box Calibrate Vials Wash Needle Intout 2 Needle UpiDown 4 7 Needle LeftRight 3 ga pa A i 00 00 2 Center the needle above the well by pressing the Needle In Out 2 and Needle Left Right 3 buttons Lower the needle by pressing the Needle Up Down buttons 4 until it reaches a level of 2 3 mm 0 08 0 12 in above the bottle Start with the big step button 4 and change to the small step button 4 when you are close to the well Using the small step button J slowly let the needle enter the hole and lower the needle until it reaches the bottom of the wash insert in the bottle The ASA autosampler has a limit of 850 steps on the z axis vertical This limit is set to avoid bent needles You might be able to go for more than 850 steps during calibration but the needle does not go more than 850 steps during normal work Note The PEEKsil at the top of the needle holder rises a little when the needle reaches the bottom
41. ASY nLC II User Guide Thermo Scientific 6 Running the First Sample Starting Sample Acquisition Starting Sample Acquisition Before starting the sample acquisition make sure that the system is properly set up This includes having contact closure see Connecting to the Mass Spectrometer through Contact Closure on page 13 Also make sure that the mass spectrometer or any other detector has been set up in accordance with the method At this point you might want to run the maintenance script outlined in Test MS Connection on page 111 itN To verify that the mass spectrometer connection contact closure works as expected 1 Choose Home gt Overview 2 Press START to start running the batch A message box opens prompting you to check that various conditions are fulfilled see Figure 38 Figure 38 Start Queue message box EASY nLC Is plate ready Check buffer A and B Check waste container Check autosampler bottles IEEE uu Accept Cancel J J 3 Press Accept The instrument now starts processing the batch Monitoring the Run You can monitor the run and the system status during analysis from two different screens Home gt Overview or Home gt Graphs e Using the Overview Page in the Home Menu e Using the Graphs Page in the Home Menu Thermo Scientific EASY nLC II User Guide 63 6 Running the First Sample Monitoring the Run Using the Overview Page in the Home Menu 64
42. Figure 45 Figure 45 Tools page under Maintenance Devices showing plate format selection to copy Thermo Home Batch Setup Method Editor MMaintenanees Configuration Scripts Devices Log Book Name Type Add Device Support Cooled Autosampler ASC Autosampler Autosampler Devices n EASY nLC HPLC HPLC Piston Pump flow feedback Pump A Piston Pump wi flow fee About Prope Remove Device Jt Cooled Autosampler ASC Autosampler Plate Vials Wash f New f Calibrate Current Vials Calibrate Job 00 00 00 D Manipulator 6x8 Vials X ee 23 aug 2010 Reset 12 38 Copy To Delete SE admin _ s Copy To Calibrate Selected plate format button button ZE 2 In the Devices list select Cooled Autosampler and then press the Tools tab Thermo Scientific EASY nLC II User Guide 75 7 Autosampler Calibration Managing Plate Formats 3 Do one of the following a Press New to open the Create New Plate Format dialog box Figure 46 Figure 46 Create New Plate Format dialog box Create new plate format p Format 16x24 J HT MTP Name P L N N Accept Cancel emmen b Select a format from the Format list and type in a name c Press Accept d Under the Tools tab press Calibrate or On the Tools page select an existing plate format from the Plate list an
43. I system is flushed with methanol CAUTION Methanol CAS number 67 56 1 is highly flammable and toxic by inhalation ingestion or skin absorption Take appropriate measures to protect yourself and your equipment Make sure that the mobile phases are miscible with methanol or start up with an intermediate solvent as mobile phase Thermo Scientific EASY nLC Il User Guide 43 5 Preparation for Use Preparing Solvent Bottles and Waste Containers Solvent A and B Bottles Follow these recommended steps for preparing the two solvent bottles To prepare the solvent bottles 1 Fill the 25 ml Schott Duran blue cap bottles with the mobile phase and degas them either by sonification or by sparging them with helium gas 2 Place the bottles in the holder on top of the instrument solvent A bottle in the front and solvent B bottle in the back position 3 Mount the inline filter assembly on the blue cap lids as shown in Figure 25 and Figure 26 Figure 25 Inline filter assembly exploded view Bu ly Figure 26 Assembled inline filter B IMPORTANT To establish proper surface wetting prime the filters with methanol or acetonitrile Autosampler Bottles The EASY nLC II autosampler holds up to four bottles three for washing solvents and the fourth for ejecting waste and cleaning the outside of the injection needle For the bottle holder positions see Figure 27 on page 45 You can operate the instrument after instal
44. IFC system for reequilibration and loading The EASY nLC system then performs these two steps ignoring any input in the Max Pressure input box of the method editor and regulating the pumps passively by using the specified flow rate To reduce cycle time and aid system robustness keep the IFC system turned on Thermo Scientific 1 Introduction Graphical Hardware Overview Graphical Hardware Overview The following figures illustrate both external and internal features of the EASY nLC II instrument Table 2 describes the elements on the instrument s back panel Figure 6 External system features Solvent bottles gasy nLC M Touch monitor Autosampler with vial or plate adapter Waste Exit for and wash solvents beaker transfer lines Thermo Scientific EASY nLC II User Guide 7 1 Introduction Graphical Hardware Overview EASY nLC II User Guide Figure 7 occi oe sy eee e CX e00000000000000090 000000 ee i RS e This device complies with Part 15 of the FCC Rules Operation is subject to the following two co 1 this device may not cause harmful interference and 2 this device must accept amy interference received including interference that may cause undesired operation This instrument is for research use only DEVICE EASY nLC S N P N LC100 LAN MAC ADDRESS 00 60 E0 45 4E 28 Proxeon Biosystems A S Web www proxeon com Phone 45 6557 2300 Fax 45 6557 2301 External view of instru
45. Include sensor A i3 ES 2 000 0 1000 Include sensor B x T0000 50 0 00 0 0 Inspection only B 0 1 2 3 4 5 Time min Period min 30 Pump B Desired flow Real flow Pressure Job 00 00 09 300 0 300 0 250 0 2500 D E 2000 E 2000 E 25 aug 2010 Feat 1500 5 09 05 oan 100 0 El 0 0 0 0 Exit o 1 eT Time min This script auto zeroes the pressure sensor Contact your local Thermo Fisher Scientific Technical Support representative before running this script 118 EASY nLC II User Guide Thermo Scientific MEN 5 Specifications This appendix contains the following specifications for the EASY nLC II system as well as a flow line schematic Contents Performance Specifications Technical Specifications Flow Line Diagram Performance Specifications See Table 15 for performance specifications Table 15 Performance specification Sheet 1 of 2 Item Specification Flow range gradient 20 2000 nL min Recommended 100 1000 nL min Flow while loading and Up to 100 pL min equilibrating Pressure range 0 300 bar Retention time Recommended flow range 0 1 to 0 496 reproducibility RT RSD Typically better than 1 outside recommended flow range Peak widths Typically 3 5 s fwhm using EASY column nano bore columns as supplied at delivery For more information go to www proxeon com productrange nano lc easy columns Carryover Typically 0 0596 Conditions Injec
46. MEN EM rua em Exit 35 c Use the Edit 4 5 page to control Thermo reequilibration For more information on this page of the Edit view see Page 4 5 on page 58 SCIENTIFIC File Job 00 00 07 24 aug 2010 admin Exit Bai Maintenance Configuration Name BSA 30 min Precolumn equilibration Volume 180 ul x gt i Flow 5 00 pimin Le m d Max pressure 280 Bar o Analytical column equilibration Volume 4 ul Flow 0 5 ul min Max pressure 280 Bar 45 gt Thermo Scientific Maintenance Thermo Scientific Table 5 Method Editor pages Sheet 3 of 3 Use the Edit 5 5 page to control autosampler wash For more information on this page of the Edit view see Page 5 5 on page 59 3 Instrument Control Software Application Menu Structure Thermo SCIENTIFIC File CEN Job 00 00 07 24 aug 2010 admin Exit Home Batch Setup Maintenance Configuration Name BSA 30 min Autosampler wash Flush volume 100 0 yi O Custom wash Order Source Volume Cycles 4 D e 5 5 gt Press the Maintenance tab to perform a number of functions designed to provide and improve system status Table 6 Maintenance pages Sheet 1 of 2 Use the Scripts page to view a collection of useful maintenance
47. MS but feedback from the MS is ignored set the contact closure Protocol setting to One way on the Configuration Connections page Action As indicated allow for wait time while the system changes network settings Cannot access the EASY nLC II system through the network 92 EASY nLC II User Guide Changed network address Go to Configuration Network and press Save Configuration Thermo Scientific Starting Up the System Symptom Dark monitor Possible causes Broken or open main power fuse 8 Troubleshooting Starting Up the System Action Remove the right side panel of the EASY nLC II instrument If there is no light on the pumps switch off the EASY nLC II instrument and check the main fuse Defective monitor Attach an external monitor to the VGA connector at the back of the instruction labeled MONITOR Startup bar is no longer proceeding Damaged system Switch off the EASY nLC II instrument and wait five seconds before switching it on again In some cases this recovers the system Contact Thermo Fisher Scientific if the problem recurs easysupport proxeon com System Component States Valves Pumps Autosampler and Plate Cooler Symptom The LED on the device is blinking red Thermo Scientific Possible causes Device in bootloader mode Action The device stopped working This could also block the function of other devices Contact Thermo Fisher Scie
48. Peripheral Control OUT1 State at start Open J INT 4 Start In OUT2 sReady Out pa o Start at Gradient J N2 Ready in iad OUTS J Start Out Signal width abes TIL GND 23 aug 2010 12 38 Thermo Scientific EASY nLC II User Guide 35 4 Configuring the EASY nLC II System Network Connection 2 From the Instrument list select the correct mass spectrometer see Figure 21 The application comes preconfigured with most of the standard mass spectrometers used in proteomics but if you cannot find your particular mass spectrometer select Generic instead If your mass spectrometer does not appear on the list and you would like to see it supported in a future release of the EASY nLC II application provide this information to Thermo Fisher Scientific Customer Service see Contacting Us on page ix 3 Ensure that the Use Contact Closure option is selected for the HPLC mass spectrometer communications protocol 4 To define contact closure from the Protocol list select One way synchronization see Figure 21 that is a simple signal at the start of sample loading or start of gradient With a two way connection the HPLC waits for an acknowledgement that the mass spectrometer is ready to accept the sample To correctly set the start state and signal width refer to the mass spectrometer documentation for how to set up contact closure at the mass spectrometer end By setting the Start At value to
49. Related Documentation Safety and Special Notices Contacting Us Declaration of Conformity To suggest changes to documentation or to Help Complete a brief survey about this document by clicking the link below Thank you in advance for your help SURVEY Thermo Scientific EASY nLC Il User Guide vii Preface Related Documentation In addition to this guide Thermo Fisher Scientific provides the following documents for the EASY nLC instrument EASY nLC Virtual Instrument for Xcalibur Quick Reference EASY nLC II Preinstallation Requirements Guide Safety and Special Notices Make sure to follow the safety practices presented in this guide and observe the safety and special notices that appear in boxes Safety Practices The following safety practices will help to ensure the safe operation of your instrument Before removing the protective panels disconnect the HPLC from all power sources or risk exposure to dangerous voltages Always replace blown fuses with fuses of the size and rating indicated on the fuse panel See Replacing the Main Power Fuse Repair or replace faulty insulation on power cords Use the EASY nLC II instrument with appliances and power sources that have the proper protective grounding When connected to analytical equipment such as a mass spectrometer also ensure that the protective grounding is shared between the instruments Observe all written safety precautions during
50. SY nLC II system in the form of troubleshooting tips The following tables provide troubleshooting tips in several different areas Contents Running a Batch Autosampler Contact Closure Network Access Starting Up the System System Component States Valves Pumps Autosampler and Plate Cooler Miscellaneous Thermo Scientific EASY nLC Il User Guide 87 8 Troubleshooting Running a Batch Running a Batch For troubleshooting tips when running a batch see Table 12 Figure 53 Waiting for MS contact closure signal on the Overview page Thermo SCIENTIFIC 142 8 bar 100 0 ul min 3 4 150 0pl Graphs Queue Solvent A 143 0 bar 100 0 ul min 2 9 150 0ul Solvent B Batch Setup Method Editor Maintenance Configuration 547 4 nl min 147 5 bar 40 0 ul min 5 0 150 0p 547 4 nl min Init ialize system Refill A B Equilibrate precolum n Equilibrate analytical column utosampler wash refill S Waiting for MS EEEE oooooo oooooo IFC enabled Idle flow 25 5 pr Waste Gradient 00 00 Exit Job 00 00 06 f f Sample 00 00 06 Administrat EREU Job admin 20100824 17 24 aug 2010 1 0 A1 Sample 1 in dro 9I 14 BSA 30 min 6 Samples admin 24 8 18 43 25 8 00 23 2 i Sample 2 BSA 30 min 24 8 19 51 EL Ra AUN NILUM Message in activity box Table 12 Running a batch troubleshooting t
51. ace for batch jobs and methods that other users apart from system administrators cannot see In addition there is a public file space for methods and batch jobs that all users can see and use Only Super users and Administrators can copy files into the public spaces and only Administrators can delete files and directories from the public space To create a new user account 1 Log in as admin otherwise see Logging In to the EASY nLC II System on page 32 2 Choose Configuration gt Users The Users page opens See Figure 23 on page 39 3 Press New Select which privileges to give the new user For user privileges by user type see Table 8 on page 38 If you want the user to carry out instrument maintenance you must select Super user as the user type If you want the user to restore data to the system select Administrator as the user type 4 Create an initial password for the defined user Tip Only certain users have access to the Configuration gt Users screen to change their own password All users however can change their current password from the Logout confirmation box by pressing Change Password dministrator f Logout Change Password e Change password J button Stop Application Power Down Cancel J 4 s 4 Configuring Column Setup Loop Volume Idle Flow and IFC 40 EASY nLC II User Guide Use the Devices page on the Maintenance tab to set global
52. admin account is also admin The first time you start the application it automatically logs you in as a guest Guest appears above the Login button Figure 20 EASY nLC Il Login screen f User gt admin gt Password aca Z1 Accept Cancel IMPORTANT Change the admin password as soon as possible for security reasons To log off the EASY nLC II system 1 Press Exit 2 Press Logout in the confirmation dialog box dministrator f B Logout Change password J Stop Application J Power Down Cancel J d J X An administrator can start a controlled power down or exit the application from this dialog box If you are not an administrator you can only log out or change your password 32 EASY nLC II User Guide Thermo Scientific 3 Instrument Control Software Closing Down the EASY nLC Il System Closing Down the EASY nLC Il System Thermo Scientific Closing down the instrument in a controlled manner is important to allow all the components to shut down in an orderly sequence Using this controlled method saves important data so that the instrument starts with the correct information the next time you use it WARNING If you turn off the power switch during normal operation you risk damaging essential system components Follow the procedure outlined here whenever possible To turn off the EASY nLC Il system 1 Press
53. ady present Thermo Scientific Thermo Scientific B Miscellaneous Installing and Using the RePlay External Device 3 Select the device and press Accept 4 Verify that the RePlay device was added to the system by locating it in the Devices list Selecting the device shows information about the device driver see Figure 68 Figure 68 RePlay About tab under the Devices list Thermo scicvriric ome Batch Setup Method Editor maintenance Configuration Scripts Devices LogBook NETS Type RePlay Fluid Storage m Rotary Valve Valve A Rotary Valve Es Manufacturer Advion BioSciences Inc Job 00 00 09 Product RePlay Type Fluid Storage Driver info 0 1 0 Firmware info N A 25 aug 2010 09 14 Serial Number N A Usages Capture Playback Eluent admin Slaves lt NiA gt EASY nLC II User Guide 127 B Miscellaneous Installing and Using the RePlay External Device 128 EASY nLC II User Guide To set up a method with RePlay 1 To use the RePlay device enable the method extension on page 1 of the Method Editor you can also open an existing method and enable its extension An extra RePlay Method Editor page should now be available Thermo SCIENTIFIC File CE Name BSA 30 min Open Close Save Description Home Batch Setup fMethod Editori Maintenance Configuration Standard 30 min BSA separation wiRePlay Method extensions
54. and columns that it requires You must connect the RePlay device to the EASY nLC II contact closure port by using the OUT3 pins on the back panel see Figure 67 Thermo Scientific EASY nLC Il User Guide 125 B Miscellaneous Installing and Using the RePlay External Device 126 EASY nLC II User Guide Figure 67 RePlay device connection to the EASY nLC contact closure port RePlay Remote cable n connector IN3 OUT3 IN2 OUT2 IN1 OUT1 o o D O EASY nLC contact closure port S5 LCO Fleet LXO or LTO MS Detector right side panel MM S To connect RePlay to the EASY nLC Il system Turn off the instrument Turn on the instrument Connect an EASY nLC II analytical column to RePlay valve position 5 Connect Valve 4 to the RePlay focusing column and MS emitter To configure the RePlay device in the EASY nLC II application in the Devices list If not press Add Device Select a device to add Advion RePlay Autosampler ASA Autosampler ASC In the Select a Device to Add dialog box locate the Advion RePlay device Fluid Storage Autosampler Autosampler EASY nLC Proxeon Thermo Focusing Lens HPLC Thermo Focusing Lens Pump A Accept Piston Pump w flow feedback Cancel Peripheral control port Choose Maintenance Devices and check that the RePlay device is not alre
55. and the autosampler bottle in position W3 and the bottles in position W1 and W2 if used Refill if necessary ASA autosampler only Make sure that the solvent level in the W1 W2 and W3 autosampler bottles never falls under 2 3 of the bottle volume Tip You can also inspect the purity of the solvent to ensure no visible precipitates have formed that might lead to blockages in the pump line 2 Visually inspect the autosampler wash bottle in position W4 and the waste container in the pump compartment and empty if necessary 3 Run the Purge Solvent and Flush Air scripts with two purge iterations or until flush volumes fall below 10 pL Thermo Scientific EASY nLC Il User Guide 103 10 Maintenance Quarterly Maintenance Quarterly Maintenance Perform the following procedures quarterly every three months To check instrument back pressure Run the Back Pressure script for both solvents To check the cooler temperature Check that the plate temperature is at its set point or approximately 18 to 20 C 64 to 68 F below ambient temperature readout on the Home gt Overview page To check autosampler pickup Run the Sample Pickup script to check the autosampler pickup function To check the EASY nLC II system for leaks Run the Leaks script for A B and for System to check for and pinpoint instrument leaks To check the check valves Run the Purge Solvent script while checking that solvents are exiti
56. anufacturers Name EN61010 1 2001 Second edition Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use EN61010 2 81 Part 2 081 Particular requirements for automatic and semi automatic laboratory equipment for analysis and other purposes EN IEC 61326 1 2006 A1 1998 A2 2001 and A3 2003 EMC requirements for electrical equipment for measurement control and laboratory use Proxeon Biosystems A S Manufacturer s Address Edisonsvej 4 DK 5000 Odense Denmark Type of Equipment Laboratory Instrumentation Model Name EASY nLC II Model Numbers LC110 LC111 Serial Number LC 000100 and later Year of Manufacture 2010 I the undersigned hereby declare that the equipment specified above conforms to the above Directive s and Standard s A Ole Vorm Site Manager August 31 2010 EASY nLC II User Guide xv EN E Introduction The EASY nLC II is a compact high performance liquid chromatography system designed for split free flows down to the low nanoLiter min range The system provides ease of use and ease of service without sacrificing functionality or performance Figure 1 EASY nLC Il system gasy nLC II b Contents Product Description Graphical Hardware Overview Thermo Scientific EASY nLC II User Guide 1 1 Introduction Product Description Product Description In the EASY nLC II system all necessary components a
57. be the sample loading start you might be able to acquire data from early eluting peptides that would not ordinarily bind to the column material and thus be missed from the actual gradient For this to be effective you must be running in a one column system configuration mode Note Moving the Start At point to the sample loading start can lead to small retention time variations due to possible differences in pressure buildup before the A B mixing starts in the actual gradient Network Connection 36 You can access the EASY nLC II system from other computers and for remote support if it is connected to your network IMPORTANT Ask your local IT administrator to help you configure the EASY nLC II system on the local network The following information will help you through the process EASY nLC II User Guide Thermo Scientific 4 Configuring the EASY nLC Il System Network Connection To connect the EASY nLC Il system to a local network 1 From the top menu press the Configuration tab and then press the Network tab The Network view displays configuration address information Thermo Home Batch Setup Method Editor Maintenance Users Address E nnections Network 9 Dynamic Fixed mmm Name LC 000125 Domain proxeon com Time IP 9 o o O Subnet 0 0 o o Backup Gateway 0 0 0 0 DNS Ol 0 0 o Support server IP
58. breue acer ty 103 Quarterly Maintenance oe eere ep TRE RI et AG ERE EMEN MP AS 104 Yearly M inte ance v ovs SEE RACE VERREM E RO IER 104 Maintenance Scripts 0 ccc eee eens 105 Prepare Purge Solvent sity Soo s breeders obe M pa os 106 Prepdre Flush Agi uoc dubi ee tct nma APER qr Ra Rr UR 107 Prepare Precolumn Equilibration vig es cheer eme tenete e 108 Prepare Analytical Col Equilibration 231 022 rr E ce ced as 109 Prepare Isocratic Flow ici oes XE oa P aed CAE Pape EE wed 110 Dest MS Connection a d into EHI e aS e ah PME oce poe ood 111 Test Sample Pick Gps i o d crede oeste dee s hood Vac ba d ais 112 Teste Ledkse pr cecidisse Latt cs lr Past extet 113 Teste Vave Check arrere roenan oa a E E AA e Edd 114 Test Back PI SSUEE 2 02 iara osana Seta ok C ahi ia i eiaa pia by 116 Test Autosampler Torque sped tke de die ie eee EE 117 Calibrate Valve Tune i343 tsokt ces Veboa EA der eai qrais dederant ote 117 Calibrate Flow Sensors n n d soc DE por Se RO We ee wee a d 117 Calibrate Reset Pressure Sensor 23 conde ae ce Uo mae ERE EIER 118 EASY nLC II User Guide V Contents vi Appendix A Appendix B EASY nLC II User Guide Specifications qe I 119 Performance Specifications ao dts dou enn o ve oce hea ubt eR TRES Ba 119 Technical Specific tions aner Ee en ear sper We atin an Wadi b de oll 120 Flow bine Dirac rata ocv bred aos a QEON D a ATP Red aua 122 Miscellaneols aia wise t
59. ce 200 0 A T 2000000 CBANS E 2 150 000 0 1500 E 100 0000 1000 50 000 0 50 0 0 0 0 0 o 1 2 3 4 5 Time min Thermo Scientific 10 Maintenance Maintenance Scripts Prepare Flush Air Thermo Scientific The Flush Air script pressurizes the pump and then releases that pressure into the flow lines toward the waste container which is effective for removing air from inside the pump head Pressurization time is calculated dynamically while the script is executed and is based on measurements from previous iterations The pump pressurizes releases pressure and empties for each iteration of the script The pressurization is limited to 200 bar and the script measures the amount of pumping required to reach 200 bar Because the solvents are slightly compressible and the pump itself expands even an air free pump allows some pumping before reaching 200 bar Depending on the solvents a pumped volume less than 10 pL is acceptable To set the Flush Air script parameters Press Flush Vol Threshold to enter the tolerated threshold for an accepted test result Below 10 pL is satisfactory The script terminates after the first iteration that gives a flush volume below the set threshold Thermo Home Batch Setup Method Editor Maintenances Configuration SCIENTIFIC Friese koso C De Y Support Parameters Devices Parameter v Value Flush pump A x Flush pump B
60. d press Copy To to create a new plate format based on your selection see Figure 45 S i d To select a given plate format 1 Go to Maintenance gt Devices see Figure 45 2 In the Devices list select Cooled Autosampler and then press the Tools tab see Figure 45 3 Under Tools select a plate format from the Plate list Press Use Note The plate selection in your MS control software must match the instrument selection S ld To delete a plate format 1 Go to Maintenance gt Devices see Figure 45 2 In the Devices list select Cooled Autosampler and then press the Tools tab Figure 45 3 Under Tools select a plate format from the Plate list 4 Press Delete Note You cannot delete the current plate 76 EASY nLC II User Guide Thermo Scientific 7 Autosampler Calibration Preparation and Basic Principles Preparation and Basic Principles Thermo Scientific Explanation of autosampler coordinates x axis On the Microtiter plate Direction A to H y axis On the Microtiter plate Direction 1 to 12 z axis Needle height CAUTION Calibration of the autosampler requires removal of the side plate and visual inspection of the autosampler needle holder Because the autosampler compartment contains moving parts and sharp needles make sure to keep hands clear when operating the autosampler during calibration A x UU N No 0 d DW To calibrate vials and microtiter plates Ensure that the m
61. d to overpressure for instance stoppage caused by bad samples It also more effectively uses the pressure range of the system 0 300 bar Additionally IFC builds up pressure rapidly Figure b Schematic diagram illustrating the IFC principle Pressure sensor A A B mixing Tee Flowsensor A Cd From pump B For example you can program the system to reequilibrate columns at a pressure of 180 bar at 5 pL min or at both of these settings The setting reached first which becomes the limiting parameter depends on the dimensions of the attached column or columns If the instrument reaches 180 bar pressure before it reaches the 5 pL min set point it adjusts the flow rate to maintain the 180 bar pressure If the 5 nL min is reached before the 180 bar pressure the pump adjusts the flow rate to maintain the 5 pL min through the flow sensor The maximum flow with the IFC algorithm is 100 pL min but the back pressure of the system itself without columns attached reaches maximum pressure between 20 25 pL min The duration of the reequilibration or loading step depends on the total volume of solvent specified and the actual flow rate If the set pressure is the limiting factor the flow rate is variable as a consequence varying the duration of these steps If the duration of the reequilibration and loading steps must be fixed limit the step by flow rather than the pressure Using the EASY nLC II application you can turn off the
62. ddress 81 45 453 9100 81 45 453 9110 analyze jp thermofisher com C 2F 3 9 Moriya cho Kanagawa ku Yokohama 221 0022 P R China Phone Fax E mail Address Thermo Scientific free lines 800 810 5118 400 650 5118 86 10 88370548 analyze cn thermofisher com 7th Floor 7F Tower West Younghe Plaza No 28 Andingmen East Street Beijing 100007 EASY nLC II User Guide xiii Preface Table 3 Customer Service information for Australia and Asia Sheet 2 of 2 P R China Phone free lines 800 810 5118 400 650 5118 E mail analyze cn thermofisher com Address Building 6 No 27 Xin Jin Qiao Road Shanghai 201206 Declaration of Conformity U S Safety and EMC Electromagnetic Compliance Standards Safety This instrument has been reviewed for compliance with standard ANSI UL 3101 1 Electrical Equipment for Laboratory Use Part 1 General Requirements 1st Edition EMC This device complies with Part 15 of the FCC Rules Operation is subject to the following two conditions xiv EASY nLC Il User Guide 1 This device may not cause harmful interference and 2 This device must accept any interference received including interference that may cause undesired operation WARNING Changes or modification to this unit not expressly approved by the party responsible for compliance could void the users authority to operate the equipment Note This equipment has been tested and found to comply wi
63. ds batches instrument configuration and user accounts Data that you restore from a backup includes all of this information You can also remove changes you have made to the system completely or selectively by restoring the factory default settings Performing a system restoration does not impact statistics on pumps valves and so on in the Maintenance Statistics menu Application Menu Structure Thermo Home Batch Setup Method Editor SCIENTIFIC Users Backup Maintenance Connections Location I backupl Network Time Backup acku Factory reset i Reset Job 00 00 00 23 aug 2010 admin Exit Restore IMPORTANT Make sure that no one else is using the EASY nLC II system when restoring a backup because this also influences user accounts EASY nLC Il User Guide 31 3 Instrument Control Software Logging In to the EASY nLC II System Logging In to the EASY nLC II System The EASY nLC II application has a user management system that assigns specific access rights to each user When the instrument ships it contains two predefined users guest and admin The default user account is guest and does not require a password With this account you can see and monitor the system but you cannot enter any new information To start using or configuring the EASY nLC Il system 1 Log in as the admin user see Figure 20 2 Press Login the factory password for the
64. duction go cae on a nns nananana anaana odeag ies UM DEBERE e ME 1 Product Descoplalles eoe had wii EI Repent near non wer apud Rape 2 System Components isle n 2 System Configuration for Two Column Setup 0 0 0 0 ee eens 3 System Configuration for a One Column Setup less 0000000 4 Instrument Operibus ioc Situs ico de s etree che cain 4 Pump Flow Control 134 2i cepo LES DRE ERI bite ties E a ea arg 5 Graphical Hardware Overview 4 cee ces ether ERR CE eee eee 2 Chapter2 Installation cuoco eusccsakope n RUEREEREEEeR nannaa nrnna 11 Placement HMM 11 DROW CL sanera it e a cae E ae deca ILC ILI E a AE 11 Temperature and Humidity c enna uode acer tengo stun lectu a d 12 Lifting Iusttucigl dou doce sew e eke V EU OF Kod ORAE bo dol 12 External Connections iis ck elds acd e Re ICE PS ee ee CER Re 12 Connecting to the Mass Spectrometer through Contact Closure 13 Connecting to Remote Support 0 0 ec eee 13 Connecting to a Local Area Network for Data Exchange 14 Attaching Mouse and Keyboard to the USB Connections 14 Attaching Add on Products through the P Bus and RS 232 14 How Partsand MINES s oye Tat po Sab egies E bra c P aoc Ag 15 Chapter3 Instrument Control Software Lsuuuususuueeeeseeeeeeeeee 17 Using the Touch Sensitive Screen iai epa Re Hee CR cape Ee s 18 Starting the EASY nLC II System oscuras on eto dO elio ope aoa Ds 19 Thermo Scienti
65. e Baten Setup Method Editor Maintenance Configuration SCIENTIFIC File J CE Add Injections 1 EE methods M f sacr Extensions X Unselect 1 2 3 4 5 6 7 8 View racks View list Open Save Job 00 00 00 Close 23 aug 2010 12 35 Schedule admin Exit 6x8 vials Plate button Note To use different methods in the same batch choose another method select new wells and press Add 6 Press Save to save the batch Thermo Scientific EASY nLC Il User Guide 61 6 Running the First Sample Creating a Batch 7 Press Schedule to submit the batch to the queue The application switches to the Home gt Queue page see Figure 37 Figure 37 Queue page under the Home tab S CIEHTIEIC Batch Setup Method Editor Maintenance Configuration Overview Auto iy A Submitter Job name Duration continue 1 C bomn famin 201008241 714 11 04 15 Graphs Job 00 00 00 24 aug 2010 17 14 admin 8 If necessary move any scheduled batches in the job queue by selecting it and pressing Up or Down You can also delete batches from the Queue by selecting the batches and pressing Delete For a batch run to start automatically after the previous batch is finished you must select its corresponding check box in the Auto Continue column In the case in Figure 37 the admin 20100824 1714 job does not auto continue when the 6 x BSA 30min job is done 62 E
66. e center of the well or not all of the liquid is picked up from one or more of the vials For problems with the wash waste containers The needle is not placed in the center of the bottle Contents Replacing the Autosampler Adapter Plate Managing Plate Formats Preparation and Basic Principles e Calibrating Plates e Calibrating the Wash Waste Compartment The calibration covers the following areas of the autosampler Original autosampler ASA Microtiter target Wash waste plate bottles Thermo Scientific EASY nLC Il User Guide 71 7 Autosampler Calibration Replacing the Autosampler Adapter Plate Latest autosampler ASC Microtiter vial plate Extra vials Wash waste bottles Replacing the Autosampler Adapter Plate Depending on your autosampler type follow the appropriate procedure To replace the adapter plate on the ASA autosampler To replace the adapter plate on the ASC autosampler To replace the adapter plate on the ASA autosampler 1 Go to Home Overview to eject the tray 2 Use the 2 5 mm hex key provided with your system to remove the current plate from the autosampler tray Turn the hex screw counterclockwise 72 EASY nLC II User Guide Thermo Scientific 7 Autosampler Calibration Replacing the Autosampler Adapter Plate The adapter plate is mounted with 4 or 6 hex screws 4 hex screws 6 hex screws 4 Fasten the hex screws in the followin
67. e is not available the application grays blanks it out so the user cannot access that function 38 EASY nLC II User Guide Thermo Scientific 4 Configuring the EASY nLC Il System User Permissions Changing the Administrator Password The EASY nLC II application supplies a simple administrator password Change it to a more secure password as soon as possible t To change the administrator password 1 Log in as admin otherwise see Logging In to the EASY nLC II System on page 32 2 In the top menu press the Configuration tab and then press the Users tab The Users page opens Figure 23 Figure 23 Users page under Configuration Thermo SCIENTIFIC Home Batch Setup Method Editor Maintenance uses d User admin Connections A Network EE dministrator IPTE Time Password Backup Re enter Level Job 00 00 00 Graphs f Pump A J eum B J foam J fm Cooling J 23 aug 2o f Open f Close f New 17 17 P admin Exit 3 Press Open and select Admin from the User list You can now enter and confirm a new password 4 Press Save to save the user account information Thermo Scientific EASY nLC Il User Guide 39 4 Configuring the EASY nLC II System User Permissions Creating New User Accounts Use the Users page to create new user accounts for all the people or groups that will use the instrument Each user has a private file sp
68. ent B Autosampler Instrument Operation During sample runs the execution of methods the EASY nLC II instrument goes through a series of predefined steps Pump refilling from solvent bottles Column reequilibration using the A pump Sample pickup where sample is drawn from the autosampler and into the loop Sample loading when the A pump loads the sample from the loop onto the column Gradient formed by the A and B pumps the sample content is eluted off of the column Autosampler wash when the S pump washes the autosampler needle and loop The system performs as many steps in parallel as is physically possible minimizing the cycle time 4 EASY nLC II User Guide Thermo Scientific 1 Introduction Product Description Pump Flow Control The EASY nLC II instrument contains split free high pressure syringe pumps capable of delivering flows between 10 nL min and 300 pL min at different resolutions and with different precision Different flow control systems are active during different method subprocesses reequilibration loading and gradient to optimize the pumping system performance The Advanced Flow Control system AFC accurately controls the flow from each pump A and B pump providing enhanced gradient control and retention time reproducibility During column reequilibration and sample loading it is more important to actively control the pressure that these processes are performed under than
69. ethod Editor Maintenance Configuration appropriate positions on the 6x BSA 30min plate use the arrows to select Open whole rows or columns and add the selection to the batch job Repeat the process and Save Job 00 00 00 Close ma Oe O0COCOOO 23 aug 2010 12 35 either save the batch or r Schedule schedule it directly Ext T When using 384 well plates you can use the arrows near the bottom of the screen to navigate between the currently shown selections In this case pressing the middle square shows a full screen view not active for input and pressing anywhere in the full screen view takes you back to the 8 x12 active selection bi Use the View List view to Thermo Home 0 0 Method Editor Maintenance Configuration see the batch job as a list of m gt z 43 Add Injections 4 BSA30 min j Batch Extensions j samples in the order of feat a Unselect Method processing Each row in the cmd in 1 D GAt Sample t BSA 30 min table holds information about vws Toms eese TRE m the position name and a ect Sampie3 BSA 30 min method for that position TESI 4 eot sample 4 BSA 30 min 5 m 0 E1 Sample 5 BSA 30 min Open For more information see e ort sampies BSA 30 min Save Editing the Running Batch on mo m KS Close page 67 Sched
70. fX RePlay a ie Capture Playback Eluent 24 aug 2010 18 34 admin Exit E 16 Thermo Scientific Thermo Scientific B Miscellaneous Installing and Using the RePlay External Device 2 On page 3 of the Method Editor properly set up the gradient For more information see To build a gradient on page 57 SCIENTIFIC File i cac NN Name BSA 30 min RePlay Save Thermo Home Batc o Method Editor Maintenance Configuration Gradient A o Time Duration Flow B cvi Ts 2750 90 250 0 80 2250 70 gt z ri 60 3 E 1750 3 amp z 1500 50 3 F i 1250 40 m 100 0 30 75 0 Job 00 01 02 20 50 0 25 0 E 10 0 0 o o 10 20 30 40 50 60 Time min 24 aug 2010 EERE pond Exit 3 6 RePlay uses a nanoflow splitter and a valve to split the EASY nLC II eluent to a direct online analysis and to a Capture Cartridge in the RePlay device The RePlay valve switches to direct the captured chromatogram to the MS for a second analysis of the same injection To acquire the second analysis the gradient has to be prolonged EASY nLC Il User Guide 129 B Miscellaneous Installing and Using the RePlay External Device 130 EASY nLC II User Guide 3 On page 6 enter the playback delay relative to the MS START signal which can be the start of either sample loading onto the columns or the start of the gradient and set from the Configuration gt Connecti
71. fic EASY nLC II User Guide iii Contents iv Chapter 4 Chapter 5 Chapter 6 EASY nLC II User Guide User Interface avout Sosa das nate e vete do wes e E Vig xtd V Ns 20 General Interaction Principles oeeuedu a een d acte eoe d estadio ete lined 21 Application Menu Structure d san up ox V rg DPA ace A ERE AR 21 Homo EU pev Burk need eet apes da SUOU p b eM 22 Batch Setup dos bi Rer e EOM RERO OUR ERU PM E ORE Rd e Ped 23 Method Edit r ssie ohne pace Exacetvay Exo o a E d LOMA ures aie OR 25 Maintenance 22x eee a E eS ie esp ile Beate Maa Une UD Re 27 Configuratie o1 oce ace Eo ERE Shoes ac De t RP requies 29 Logging In to the EASY nLCG II System o o siesigre aces beet eae aes vacet 32 Closing Down the EXSY nLC ILSystenm 2102 c poe A mek the icr cede 33 Configuring the EASY nLC Il System LLuuuuuusuuueueeueee 35 Setting Up the Mass Spectrometer Connection s asss 0 000 ee eee 35 Network Connection iese doivtekct arats Tec Tele do UTEM ls DN Te aote 36 User Permissions tyes sa te sed ven PEN Sect rte eoo vA ane s 38 Changing the Administrator Password 0 00 00 eese 39 Creating New User Accounts 0 0 cee cece eens 40 Configuring Column Setup Loop Volume Idle Flow and IFC 40 Preparation for Use otiose Ioh ter fee et UE RES os 43 Preparing Solvent Bottles and Waste Containers 0 000 eee ee eee 43 Solvent A amd B Bottless bs pepe Ey to eR ES de me ER S On eeu 44 Aut
72. g Up the Mass Spectrometer Connection on page 35 For a description of the MS Connection script see Figure 57 Figure 57 MS Connection script description Thermo Home Batch Setup Method Editor Maintenance Configuration SCIENTIFIC Scripts Category Name f f f Pump A Log Book Test vw MS connection Desired flow Real flow Pressure Support 300 000 uM Description Parameters Output SED EE ices 250 000 0 ae Tests the connection to the mass 200 000 0 2000 amp spectrometer using gradient ready and 160 0000 150 0 gradient done events 2 iu 100 0000 100 0 5 50 000 0 50 0 0 0 0 0 o 1 2 3 4 5 Time min Fam B Desired flow Real flow Pressure Job 00 01 02 300 000 0 aooe 250 000 0 250 0 200 000 0 E a E 1500000 i a 100 000 0 1500 S 24 aug 2010 m CES 2 500000 F E E e 1000 3 admin 50 000 0 50 0 e 100 000 0 00 Exit o 1 2 3 4 5 Time min Thermo Scientific EASY nLC II User Guide 111 10 Maintenance Maintenance Scripts Test Sample Pickup 112 EASY nLC II User Guide The Sample Pickup script tests the autosampler pickup function by picking up a set volume from the well plate and ejecting it into waste see Figure 58 Use this script when testing the sample pickup function and determining sample pickup flow rates for method setup Tosetthe Sample Pickup script parameters 1 Press Vo
73. g balanced order a Screw in the upper right corner b Screw in the lower left corner c Screw in the upper left corner d Screw in the lower right corner e Screws in the middle Note Mounting the screws in a balanced order can avoid distortion of the adapter plate The autosampler is now ready for new plate formats First you must calibrate the Microtiter target plates See Managing Plate Formats Preparation and Basic Principles and Calibrating the Wash Waste Compartment Thermo Scientific EASY nLC Il User Guide 73 7 Autosampler Calibration Replacing the Autosampler Adapter Plate KD To replace the adapter plate on the ASC autosampler 1 Use the Overview page of the Home menu to eject the tray 3 L4 i s 3 E j gm ms n ere te PE ETEL PLEZLLL 3 POLLE SOL LII LI 3 Place the new adapter plate in the autosampler The autosampler is now ready for new plate formats First you must calibrate the Microtiter target plates See Managing Plate Formats Preparation and Basic Principles and Calibrating the Wash Waste Compartment 74 EASY nLC II User Guide Thermo Scientific 7 Autosampler Calibration Managing Plate Formats Managing Plate Formats Follow the appropriate procedure to use an existing plate format create your own plate format or delete a format you no longer need To create a new plate format 1 Go to Maintenance Devices See
74. he flow lines between the components see Figure 15 In addition to pumps and valves several filters and check valves have been added The two check valves on lines A and B ensure that solvent is drawn from the solvent bottles and ejected to waste without switching valve positions The filters and check valves are not visible on most schematics because these extra components do not affect the main sample processing logic Figure 15 Complete physical layout and schematics 0009000000000 Pressure Sensor Filter Filter Flow sensor Check valve For complete dimensions see Technical Specifications on page 120 EASY nLC Il User Guide 15 LLL AM Instrument Control Software The EASY nLC II instrument is controlled by software that runs on an integrated computer this eliminates the need for a dedicated computer next to the instrument This chapter provides basic instructions for the reliable and efficient operation of the EASY nLC II system The application presents an easy to use interface for defining methods sample processing protocols scheduling batches for execution on a Microtiter plate basis following the system progress and status managing users and carrying out maintenance and repair work Contents Using the Touch Sensitive Screen Starting the EASY nLC II System User Interface Layout General Interaction Principles Application Menu Structure Logging In to the EASY nLC II System
75. he pump operates at the set flow as long as it is below the instrument pressure of 280 bar Note If you specify both a flow rate and a maximum pressure the pump flow is limited by the parameter that is reached first Page 3 5 is for building a gradient It consists of a schematic view for setting parameters and a graph view showing the gradient as a function of time see Figure 33 You can use the four buttons at the bottom of the page for adding lines to the gradient moving lines up and down and deleting lines To move or delete lines select the line by pressing it The rest of this section guides you through building a gradient for analyzing a BSA digest on a C18 column Because this procedure is intended to help you set up a method make sure to alter the parameters stated to match your own sample and column characteristics Figure 33 Page 3 5 Edit view in the Method Editor Thermo Home Batch Setup Method Edito Maintenance Configuration SCIENTIFIC File cac NN Name BSA 30 min Save Gradient H 0 Duration Flow B En NOB NIA 300 bis 20 250 0 80 300 225 0 70 L 90 a 300 E 1750 a 3 4 c 150 0 50 g 300 z 125 0 40 F 100 0 300 30 750 Job 00 00 00 20 50 00 300 soa 250 a 00 0 5 10 15 20 25 30 35 40 46 50 23 aug 2010 Time min EE fu ee Exit 35 E Thermo Scientific Thermo Scientific 6 Running the First Sample Creat
76. icrotiter plates are in place in the tray Go to Maintenance Devices From the Devices list select the autosampler Press the Tools tab below the Devices list In the Plate area select a plate format from the list or create a new plate format To create and select plate formats see Managing Plate Formats on page 75 Press Calibrate to calibrate the selected plate format The Calibration dialog box opens In the Calibration dialog box press Eject to eject the autosampler tray Check that the correct adapter plate is mounted on the autosampler Place a Microtiter plate on the adapter plate and cover the Microtiter plate with a plastic film or a rubber mat Insert the autosampler tray and press Insert EASY nLC Il User Guide 71 7 Autosampler Calibration Calibrating Plates 11 Remove the left side panel so that you can watch the movement of the needle Use a black marker pen to make a line on top of the PEEKsil tubing e 12 Calibrate the plate See Calibrating Plates next Calibrating Plates For information on selecting an existing plate or creating a new one see Managing Plate Formats on page 75 IMPORTANT When calibrating plates on the latest autosampler ASC make sure to remove the vial caps or plate covers Keeping the caps or covers on makes the needle susceptible to bending To calibrate plates 1 Put empty vials with no caps in the top left and bottom
77. ientific EASY nLC II User Guide 9 1 Introduction Graphical Hardware Overview 10 EASY nLC II User Guide Figure 10 Internal features view 3 i Integrated computer Autosampler ASC Enclosed refrigerated sample compartment Thermo Scientific LLL m Installation The following sections provide important information on installing the Thermo Scientific EASY nLC II instrument Be sure to review the Safety and Special Notices on page viii before proceeding Contents Placement Power Temperature and Humidity Lifting Instruction External Connections Flow Parts and Lines Placement The EASY nLC II instrument weighs approximately 32 kg 71 Ib and its dimensions are approximately w 35 x 438 x h 45 cm w 14xd 15x 18 in Allow for at least 15 cm 6 in of free space at the back of the instrument for proper air circulation Power Use this instrument only with properly grounded appliances and power sources 120 Vac 50 60 Hz 250 W 230 Vac 50 60 Hz 250 W For UPS dimensioning assume 250 W For 120 Vac one T 5 AL 250 V fuse 5 x 20 mm IEC 60127 For 230 Vac one T 2 5 AL 250 V fuse All fuses supplied with the instrument are UL Listed and CSA certified Thermo Scientific EASY nLC Il User Guide 11 2 Installation Temperature and Humidity Temperature and Humidity Avoid locations with high air humidity or extreme changes in temperature such as direct sunlight drafts or directly
78. iguration Duration 11 04 15 admin 20100825 0002 job properties Position Name Method Sample 2 BSA 30 min RePlay BSA 30 min RePlay BSA 30 min RePlay 3 0 D1 Sample 4 Job 00 00 00 4 0 E1 Sample 5 5 0 F1 Sample 6 BSA 30 min RePlay 24 aug 2010 17 14 admin Cen Close Show batch IMPORTANT Reschedule the batch after you complete the batch edit otherwise the instrument stops after the current sample run Troubleshooting a Sample Run Ifa problem occurs during a sample analysis the EASY nLC II instrument displays the error in a pop up box and stops the sample processing In this situation the safest approach is to press Cancel Batch and then go to Maintenance gt Scripts see Executing Maintenance Scripts on page 46 Here you can try to localize fix the error through maintenance scripts jobs see Maintenance Scripts on page 105 When the problem is fixed reschedule the batch possibly changing the number of samples their well positions or both 70 EASY nLC II User Guide Thermo Scientific MEN i Autosampler Calibration This chapter describes how to calibrate the autosampler Use this procedure in the following situations When you want to use a new type of target plate or Microtiter plates When you would like to replace the needle For problems with the sample pickup The needle is not placed in th
79. in f Open f Close f Save Description Standard 30 min BSA separation Method extensions O RePlay 15 c EASY nLC Il User Guide 25 3 Instrument Control Software Application Menu Structure 26 EASY nLC II User Guide Table 5 Method Editor pages Sheet 2 of 3 Use the Edit 2 5 page to control sample pickup For more information on this page of the Edit view see Page 2 5 on page 55 Use the Edit 3 5 page to control the gradient For more information on this page Thermo SCIENTIFIC File Maintenance Configuration Name BSA 30 min Sample pickup SCIENTIFIC File Volume 10 00 pi Flow 20 00 pimin Sample loading icr Volume 30 00 yl NS OFT rm ay m i Flow T pimin Soi Cy 24 aug 2010 O L 4 Max pressure 250 Bar I admin Exit 2 5 c Thermo Maintenance Configuration Name BSA 30 min Gradient of the Edit view see Page 3 5 on Time Duration Flow 48 cu 00 00 N A 300 5 Ss m page 56 Ex 30 00 30 00 300 40 2250 P _ 200 0 a 35 00 05 00 300 100 E wee o 3 100 so 43 00 08 00 300 100 E B 45 00 02 00 300 o paa 30 Job 00 00 00 Taa 2 50 00 05 00 300 0 50 0 25 0 as SJ v 00 bp AT 0 5 10 15 20 25 30 35 40 45 50 29 aug 2010 Time min 4
80. ing a Method To build a gradient 1 Focusing on the first row of the gradient table press the first cell in the Flow column enter 300 nL min in the pop up box and press ACCEPT 2 Press the first cell in the B column enter 10 96 in the pop up box and press ACCEPT The gradient is now set to start at 300 nL min and 10 B 3 Press ADD to add another row Press the cell in the second row under the Duration column and set the duration to 20 min Press the cell in the second row under the Flow column and set it to 300 nL min 5 G Press the cell in the second row under the 96 B column and set it to 40 7 Press ADD to add another row 8 n this new row set duration to 5 min flow to 300 nL min and 96 B to 100 9 Press ADD to add another row 10 In this new row set the duration to 15 min flow to 300 nL min and 96 B to 100 The gradient is now completed Note You do not need to reequilibrate the columns at the end of the gradient The parameters for reequilibration are entered directly and separately as described in the next section The reequilibration runs parallel with the sample pickup at the beginning of a sample execution run EASY nLC Il User Guide 57 6 Running the First Sample Creating a Method Page 4 5 Use page 4 5 to set parameters for the column reequilibrations see Figure 34 Figure 34 Page 4 5 Edit view of the Method Editor Thermo Home SCIENTIFIC File cat Name
81. instructions on setting up the mass spectrometer connection in the application see Setting Up the Mass Spectrometer Connection on page 35 To connect a mass spectrometer see Figure 12 Figure 12 Two way plug configuration for a mass spectrometer EASY nLC II P BUS DA mass spectrometer IN3 OUT3 NP IN1 OUT1 OUTI ic Start In EEEBEEEEEEET MG Q Int ouT2 Ready Out IN2 OUTa TIL f GND t Connecting to Remote Support You can have Thermo Fisher Scientific online support review your EASY nLC II system Using shielded cables connect the LAN port of the instrument to the local network that has Internet capability m If the EASY nLC II system sits on the private network side of the mass spectrometer MS control computer you must move it to the public network permanently or temporarily to access the remote support server See Figure 13 Figure 13 Mass spectrometer control computer from private to public network Private Private network network Ctrl PC Public network Public network Thermo Scientific EASY nLC II User Guide 13 2 Installation External Connections Connecting to a Local Area Network for Data Exchange The EASY nLC II instrument can work in stand alone mode but if you require access to a network ask the local network administrator for guidance see Network Connection on page 36 Figure 14 Connecting the HPLC to a comp
82. instrument configuration settings such as one or two column setup loop size idle flow settings and IFC For parameter descriptions see Table 9 To configure devices 1 Choose Maintenance Devices 2 From the Devices list select EASY nLC II HPLC Thermo Scientific Thermo Scientific 4 Configuring the EASY nLC Il System User Permissions 3 Press the Properties tab to configure your settings Figure 24 Figure 24 Device configuration for HPLC on the Properties tab Thermo Home Batch Setup Method Editor Maintenances Configuration Pe SCIENTIFIC Add Device Remove Device Scripts Devices Log Book Name Type Support Cooled Autosampler ASC Autosampler Autosampler Devices M EASY nLC HPLC HPLC Piston Pump flow feedback Pump A Piston Pump wi flow fee About Summary EASY nLC HPLC ra Loop volume 20 0 ul Ene Seba L___ E Disable IFC ob 0600 Idle flow rate 25 nl min Idle mixture 5 B 24 aug 2010 17 18 a io fa Table 9 HPLC parameter descriptions Sheet 1 of 2 Parameter Description Loop Volume Make sure to configure the loop volume correctly as method input parameters are limited by the loop volume If a smaller loop is physically mounted over what is configured in the EASY nLC II application the instrument might actually draw sample into the pump Idle Flow Rate Maintaining long term emitter stability with glass em
83. inue ale STE AUR Qc inca bap RR RR CR ADAE d 123 Cleaning Materials nsako iet enant doe bti ol eel dus tebe tania A RA 123 EASY nLC II Consumable Parts List 3 5 sa0se0 oe oec s EL ee ad 123 Replacing the Main Power Fuse ose vet oar rou pU da d en 124 Installing and Using the RePlay External Device 00 000000 125 Declaration of Contamination s es Sad sade nis ts nae xta str i o en e 133 Transport Instructions ois o C oppure e Meroe o SA RO sedg 134 US Se ene ce ee ee E A A 135 Thermo Scientific EN i Preface This guide describes how to use the EASY nLC II nano flow liquid chromatography LC system with software version 2 8 The information provided also applies to EASY nLC systems that have been upgraded to version 2 8 regardless of autosampler version This guide is intended for laboratory technicians using the EASY nLC high performance liquid chromatography HPLC system to execute analytical runs It assume basic knowledge of how to use menu driven software and familiarity with standard laboratory practices as well as HPLC terminology and practices Before you turn on the instrument Thermo Fisher Scientific recommends reading Chapter 1 Introduction Chapter 2 Installation and Chapter 3 Instrument Control Software While the instrument is running review Chapter 4 Configuring the EASY nLC II System Chapter 5 Preparation for Use and Chapter 6 Running the First Sample Contents
84. ion pages Sheet 2 of 3 Use the Network page to understand how the EASY nLC II system is identified in a connected computer network Consult your Thermo 30 EASY nLC II User Guide IT administrator before making changes to the system For more information see Network Connection on page 36 Use the Time page to set the internal clock in the EASY nLC II system and the display format used throughout the application to print date and time information tetiteeras Home BatchSetup Method Editor Maintenance Users Address Connections Network 9 Dynamic Fixed network Il Name LC 000125 Domain proxeon com Time IP 0 0 0 O Subnet 0 0 o 0 Backup Gateway 0 0 0 0 ONS 0 0 o 0 Support server IP 195 41 108 93 F Detaut iP Job 00 01 02 Save configuration 24 aug 2010 admin Exit Thermo Home Batch Setup Method Editor Maintenance MED Users Time Settings Connections f jt Network f CERE f Set Time Synchronize NTP sewer clock isc org Backup f f Set Format damm yyyy HH mm ss Presets J Job 00 00 00 23 aug 2010 admin Exit Thermo Scientific Thermo Scientific 3 Instrument Control Software Table 7 Configuration pages Sheet 3 of 3 Use the Backup page to save and restore backups of the system database and configuration A backup includes metho
85. ips Sheet 1 of 2 Symptom Possible causes Gradient does not start Waiting for contact closure see Figure 53 Action Check the contact closure connections Maximum pressure is Possible blocked exceeded during sample column loading or reequilibration IFC disabled or pressure is maintained but the flow is dramatically decreased IFC enabled 88 EASY nLC II User Guide 1 Remove all columns from the system 2 Run the Precolumn Equilibration script with a flow of 10 pL min This should give you a back pressure of approximately 80 bar Thermo Scientific 8 Troubleshooting Running a Batch Table 12 Running a batch troubleshooting tips Sheet 2 of 2 Symptom Max pressure is exceeded during gradient Possible causes Back pressure that is too high on the column for the specified flow Action Reduce the flow in the method and schedule a new batch with the changed method Blocked flow paths Contact Thermo Fisher Scientific for advice easysupport proxeon com It takes extremely long before flow rate stabilizes Air in the system Run the maintenance script Purge Flush All See Purging and Flushing the Pumps on page 47 Leakage in the system Run the maintenance script Test Leaks on page 113 XYZ robot error Possible step loss on one of the motors because of a blockage in the movement of the axis Remove the obstacle and try
86. itters might require constant flow through the system Idle flow is a functionality that activates when the last sample in a batch is done and is at a constant flow through the system at a specified rate and as a percentage of B If you do not want such a flow type zero 0 in the Idle Flow Rate box EASY nLC Il User Guide y 4 Configuring the EASY nLC II System User Permissions Table 9 HPLC parameter descriptions Sheet 2 of 2 Parameter Description One Column Setup You can configure the EASY nLC II instrument as a one column or a two column setup If the system is operated as a one column system you can select its corresponding check box here and the diagram on the Overview page of the Home menu will reflect this The actual execution of the method is unaffected by this setting Disable IFC For a description of IFC see Pump Flow Control on page 5 You can disable it by selecting this check box 42 EASY nLC II User Guide Thermo Scientific LLL AM Preparation for Use This chapter describes how to prepare the EASY nLC II instrument for use Log in to the system with either Super user or Administrator privileges For details see Logging In to the EASY nLC II System on page 32 Contents Preparing Solvent Bottles and Waste Containers Executing Maintenance Scripts e Purging and Flushing the Pumps Installing Columns Preparing Solvent Bottles and Waste Containers Before shipment the EASY nLC I
87. ived 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 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 September 2010 Software version EASY nLC II version 2 8 For Research Use Only Not for use in diagnostic procedures a a E Contents it PE vii Related Documentation odes Sd 0E Rae Pare ARCU a dee CHAR OUR C E viii Safety and Specia Notic s cnn dokinn chau en e do ORO OCA SC Rat ol erra i viii Safety Practic s usa sacr anb e ac ba Ro Rl dee M A aa p Banca da viii Special PIOHOBS etis ubt reo bb be b edt MORE ede tib d dps viii Contacte DS iis cesi etre sak EA tds ba O Pere ove aed qud todas ix Declaration of Conformity caa reet LE ARI ER HERE GE AU races xiv USS Safety and EMC Electromagnetic Compliance Standards xiv Canadian Safety and EMC Electromagnetic Compliance Standards xv European Safety and EMC Electromagnetic Compliance Standards xv Chapter Intro
88. late 96 or 384 wells or one 1 6 x 8 vial adapter for a total of 54 vials e 4 liquid containers for solvents rinse and waste ASA previous model Cooled maximum 20 C 68 F below ambient temperature Can hold a 4 x 6 vial adapter or one 1 Microtiter plate with 96 or 384 wells 4 liquid containers for solvents rinse and waste Computer Embedded computer Thermo Scientific 1 Introduction Product Description Table 1 EASY nLC Il system components Sheet 2 of 2 Monitor Touch sensitive 12 1 in 30 7 cm 800 x 600 pixels that can be used with gloves Total size w 35 cm 14 in 438 cm 15 in 4 45 cm 18 in Total weight 32 kg 71 Ib Utilities Main power 120 230 Vac 50 60 Hz 250 W Software e EASY nLC II stand alone application version 2 8 System Configuration for Two Column Setup For a two column setup the components are connected as shown in Figure 2 Figure 2 HPLC flow paths for a two column setup A B mixing Tee Sample loop Vending Analytical Precolumn Tee column Eu Solvent B Autosampler Waste Thermo Scientific EASY nLC II User Guide 3 1 Introduction Product Description System Configuration for a One Column Setup For a one column setup the components are connected as shown in Figure 3 Figure 3 HPLC flow paths for a one column setup A B mixing Tee Sample loop Vending Analytical column Solvent A Pump B e Solv
89. leak test for pump S as it does not operate under pressure during normal batch execution Figure 59 Leaks script parameters Thermo Home Batch Setup Method Editor Maintenance Configuration SCIENTIFIC COE Category Name Cc s g Pump S J Log Book Test Leaks X Desired flow Pressure Support 300 000 0 300 0 m j Description arameters Output PES mH Devices 2000000 zona Parameter Value E 150 0000 Pi p 100 000 0 Send j i 50 0000 A 0 0 50 0 50 000 0 1 0 0 B oW cT Time min m graph J Job 00 01 02 24 aug 2010 17 48 HD EASY nLC II User Guide 113 10 Maintenance Maintenance Scripts 3 If selecting to perform a System test ensure that the sample column output line Valve S port 3 is connected to waste input Valve W port 2 Figure 60 Figure 60 Column output line to waste input Valve S Port 3 Test Valve Check The Valve Check script performs a self test for the selected valves Use this script when valves begin to move slowly or cannot reach the required positions and after exchanging valve parts rotor stator IMPORTANT Only use this script when instructed to by Thermo Fisher Scientific Make sure to record any numbers that the script generates 114 EASY nLC II User Guide Thermo Scientific 10 Maintenance Maintenance Scripts To set the Valve Check scri
90. lect the autosampler 3 Press the Tools tab below the Devices list About Properties ool Cooled Autosampler ASC Autosampler Plate Vials Wash Cc P New Calibrate Current Vials Calibrate f Manipulator 6x8 Vials J c Reset f Copy To 4 In the Vials Wash area press Calibrate The Autosampler Vials Wash Calibration dialog box opens where you calibrate the wash or vial position see Figure 49 on page 82 Thermo Scientific EASY nLC Il User Guide 81 7 Autosampler Calibration Calibrating the Wash Waste Compartment 82 EASY nLC II User Guide Figure 49 Autosampler Vials Wash Calibration dialog box Autosampler Vials Wash Calibration Calibrate Vials Wash Needle In Out Needle Up Down Prepare the bottle for position 4 used for ejecting waste and cleaning the outside of the injection needle See Autosampler Bottles on page 44 Place the bottle in position W4 Eject the autosampler by pressing Eject and place the vial bottle in V1 Insert the autosampler tray if ejected and press Insert Remove the left side panel so that you can watch the movement of the needle Use a black marker pen to make a line on top of the PEEKsil tubing 10 Continue with calibrating the wash waste vial positions Thermo Scientific Thermo Scientific 7 Autosampler Calibration Calibrating the Wash Waste Compartment To calibrate the wash waste
91. leshooting tips Sheet 1 of 2 Symptom Possible causes Sample pickup fails Bad autosampler calibration or air in the sample S pump Action Fill some of the wells with a given volume of solvent A for example 12 pL Put a plastic film on the Microtiter plate Run the maintenance script Test Sample Pickup on page 112 with a pickup volume of 10 pL and your default flow Run the script for each well Eject the tray and check the wells one by one with a pipette Run the Prepare Flush Air on page 107 script or calibrate the autosampler if the problem still appears Thermo Scientific 8 Troubleshooting Autosampler Table 13 Autosampler troubleshooting tips Sheet 2 of 2 Symptom The needle cannot penetrate the plastic filmon needle tip the Microtiter plate Possible causes Action Damaged or bent 1 Move the autosampler to position A1 by using the Autosampler control window on the Home Overview page See Figure 54 on page 90 2 Remove the left side panel and gently push the PEEKsil cover upwards on the autosampler assembly to expose the PEEKsil needle The needle should not be bent or damaged in any way 3 If there is any residue on the needle tip use tissue with some ethanol to wipe it 4 Let go of the PEEKsil cover again and check if the PEEKsil needle is on level with the cover the PEEKsil cover should completely cover the needle
92. lication displays a keyboard keypad Many button actions provide a confirmation box where the user is prompted to either accept or cancel an action In the rest of this guide we use the menu left side area to refer to a given screen accessed first by pressing the top menu tab and then by pressing the appropriate side tab for example Home Queue Application Menu Structure Thermo Scientific The EASY nLC II application has five top menus Home Batch Setup Method Editor Maintenance Configuration EASY nLC Il User Guide 21 3 Instrument Control Software Application Menu Structure Press the Home tab to view the default menu for the application It provides three pages to view and change information about running samples and instrument components Table 3 Home menu pages Sheet 1 of 2 Use the Overview page to view a graphical layout of the instrument components Graphics of most hardware parts are made touch sensitive so you can control them through the shortcut menu that appears when you touch a specific part This view is dynamically updated For more information see Using the Overview Page in the Home Menu on page 64 4 Graphs Queue Job 00 00 00 23 aug 2010 1234 guest Login 4 1 bar 0 0 nmin E 200 yl IFC enabled Idle flow 2515 Ro CENTER 7 n min Solvent A 8 3 bar 0 0 ni min 4 2 150 0u mur herding 3 2 150 0
93. ling the washing botte for needle cleaning position W4 and the solvent A bottle position W3 t To prepare the autosampler bottles 1 Fill one of the bottles with solvent A and degas it 2 Turn on the system go to Home gt Overview and press Eject Plate 3 Put the bottle containing the needle wash insert into position W4 CAUTION You must place the washing bottle holding the needle wash insert in position W4 Any other position could lead to instrument malfunction and possible breakdown 44 EASY nLC II User Guide Thermo Scientific 5 Preparation for Use Preparing Solvent Bottles and Waste Containers 4 Put another bottle containing solvent A into position W3 The S pump is filled from this position You can place wash solvents for customized washing procedures in position W1 and W2 See Figure 27 on page 45 5 Make sure all the bottles are installed with the lids fitting tightly 6 After positioning the bottles press Insert Plate to move the plate back into the instrument Figure 27 Wash bottle with needle wash insert and view of the four positions on the autosampler bottle holder ASC model Wash bottle with Autosampler needle wash insert bottle holder Waste Container The EASY nLC II waste container sits behind a small door next to the autosampler door All solvent waste except waste from the autosampler goes into this waste container To prepare the waste container 1 Open the autosampler d
94. lts use 10 column volumes for reequilibration 2 Press Flow to set the flow rate for performing the reequilibration of the analytical column If the flow field is left empty the pump operates at the set pressure 3 Press Max Pressure to set the maximum allowed pressure If the pressure field is left empty the pump operates at the set flow as long as it is below the instrument maximum pressure of 300 bar If both a flow and a maximum pressure are specified the pump flow is limited by whichever parameter is reached first Thermo Home Batch Setup Method Editor Maintenance Configuration SCIENTIFIC scripts Category Name s f f Pump A J Log Book Prepare i Analytical col eq Desired flow Real flow Pressure DT En ouo eats me Parameters 250 000 0 zen Devices Bcc 200 0000 ET Parameter v Value a 5 150 000 0 1500 amp olume ul 1000 sous 1000 g Flow l min 5 00 50 000 0 50 0 0 0 00 Max pressure bar 250 00 0 1 2 3 4 5 Time min f _ No graph J Job 00 01 02 24 aug 2010 23 55 admin Schedule Exit Thermo Scientific EASY nLC Il User Guide 109 10 Maintenance Maintenance Scripts Prepare lsocratic Flow 110 EASY nLC II User Guide The Isocratic Flow script runs solvent A and B at a fixed mixture ratio The script utilizes the AFC system to accurately control the flow Use this script to tune the
95. lume to specify the volume to be picked up 2 Press Flow to specify the flow rate used while aspirating the sample 3 Press Position to specify the position of the sample in the autosampler See the Position pop up box in Figure 58 Figure 58 Sample Pickup script parameters Thermo Home Batch Setup Method Editor Maintenance Configuration Scripts l Category Name f f f No graph J Log Book Test Sample pickup Support Description Parameters Output p Devices l Parameter Value olume ul 10 00 Flow l min 1 00 Position Select position v O Plate vials Job 08 53 12 Sample 00 00 42 AIRE Gradient 00 00 bd 1 25 aug 2010 08 56 admin sss Accept Cancel Exit Thermo Scientific Test Leaks Thermo Scientific 10 Maintenance Maintenance Scripts The Leaks script tests for possible leaks on each pump valve and throughout the flow path and can also help determine where a possible leak is located The flow sensors measure the leaks or they are calculated from pump piston movement Use this script when you suspect leak problems in the pumps the system flow path or both To set the Leaks script parameters 1 Press the Parameters tab 2 To select which pumps or system to perform the leak test on press the cell in the Value column next to Subsystem s see Figure 59 Note You cannot perform the
96. ment s back panel PROXEON e00 2 2 0600 00000000000000 oDe OA w IND OUT IN T2 Int OUTS As eeeeeeece ee 2 nditions CE 120230 V 680 Vr 250 w eee fee 0v TSA MeV fFuenun2dV Tsa Nov Table 2 Back panel elements Element Description IN OUT Contact closure primarily for communication with the mass spectrometer P BUS For communication with HPLC add on instruments RS 232 For communication with HPLC add on instruments for example a syringe drive USB Port for keyboard or mouse two 2 provided PS 2 Input for connection of keyboard and mouse LAN For 10 100 Mb sec Ethernet connection MONITOR Output for connection of external display IJO Mains power switch Fuse holder below Replaceable fuse ratings I O switch e For 120 V T5 AL 250 V e For 230 V T 2 5 AL 250 V P N Product order code S N HPLC serial number LAN MAC ADDRESS The MAC address of the embedded computer Gives the EASY nLC II system a fixed IP address on your local network Thermo Scientific 1 Introduction Graphical Hardware Overview Figure 8 Internal features view 1 Well plate 96 or 384 Three 3 positions for Waste bottle with wash Six 6 extra vial positions or vial 6 x 8 adapter wash solvents insert for cleaning of the for standards or regular outside of the needle samples Figure 9 Internal features view 2 Flow Valves sensors Pressure sensors Pumps Thermo Sc
97. n N o Rly m OHS d t 3 I Flow T ul min os t gt 24 aug 2010 oot 4727 G Max E B es ax pressure NEZ ar Exit ext e 2 5 To set up the autosampler to pick up sample 1 In the Sample Pickup area press the Volume box and enter the default amount of sample to be picked up To prevent contamination of sample pump S the maximum volume is set to loop size minus 2 pL 2 Press the Flow box to set the flow rate at which the autosampler picks up sample The autosampler is able to pick up sample with a flow between 0 and 40 pL min Set the speed according to the sample viscosity and accuracy needed however 20 uL min is a good starting point when sample is in an aqueous solution No excess of sample will be picked up just the exact amount of the requested sample Tosetup sample loading 1 In the Sample Loading area press the Volume box to set how much solvent A to use to load the sample from the loop onto the pre column Normally it is sufficient to load the pre column with 2 5 times the amount of sample picked up 2 Press the Flow box to set the flow rate for sample loading onto the pre column If you leave the input box empty the pump operates at the set pressure IFC enabled EASY nLC Il User Guide 55 6 Running the First Sample Creating a Method Page 3 5 56 EASY nLC II User Guide 3 Press the Max Pressure box to set the maximum allowed pressure If you leave the input box empty t
98. nLC II User Guide Thermo Scientific B Miscellaneous Installing and Using the RePlay External Device 5 Replace with the right type of fuse see Table 18 on page 121 You can insert the fuse holder two ways e With the 110 120V showing at the bottom ot e With the 220 240V showing at the bottom Figure 66 Figure 66 Positioning of the 220 240V fuse et AA OZL OLL A057 30 318 04 830 IIAV 1N3W300INn M3AOIIN S3SN4 A06Z HLM AINO 361 220 240 V Y Ll Installing and Using the RePlay External Device Currently the EASY nLC II system supports the Advion RePlay device Thermo Fisher Scientific may choose to support additional external devices for use with the EASY nLC II instrument in future releases The RePlay device was specifically developed for nano LC MS analysis It splits the sample from one injection so a second analysis is provided with no additional sample required RePlay is composed of a primary column a 6 port valve a flow sensor tubing a control panel and a secondary column emitter It performs a second analysis of one sample without a reinjection which allows for twice the analytical possibilities from every nanoLC injection For detailed information visit the Advion BioSciences Web site www advion com biosystems replay index php Disclaimer Thermo Fisher Scientific does not offer support for or is in any way responsible for the functionality of the RePlay device itself and the associated tubing
99. ng the A and B waste tubing and passing into the waste container when the pumps are ejecting Make sure that no solvents are going into the pump through these tubes during refill Yearly Maintenance 104 EASY nLC II User Guide Schedule the following procedures once a year itN v itN To exchange low pressure solvent filters and high pressure inline filters Thoroughly clean new solvent filters before use as these can be a contamination source To check flow sensor calibration Run the Flow Sensor calibration script with the Inspection Only parameter enabled This checks that the flow sensor calibration is working Enter 30 minutes for Period IMPORTANT Check the valve rotor shifts regularly When the valve rotor shifts exceed 6000 Thermo Fisher Scientific recommends checking the system for leaks From this point forward perform leak tests every 1000 shifts and exchange the rotor when the leak test fails Thermo Scientific 10 Maintenance Maintenance Scripts Maintenance Scripts To execute all listed maintenance scripts 1 In the EASY nLC II application choose Maintenance gt Scripts 2 Select a category and then a name for the specific operation You can also schedule some of the scripts for execution using the job queue Each of the following sections combines the category with the actual script name Prepare Purge Solvent Prepare Flush Air Prepare Precolumn Equilibration Prepare
100. nical Support Fax 45 6557 2301 Web site www proxeon com productrange nano_lIc E mail easysupport proxeon com Address Thermo Fisher Scientific Edisonsvej 4 DK 5000 Odense C Find software updates and utilities to download at mssupport thermo com To copy manuals from the Internet Go to mssupport thermo com and click Customer Manuals in the left margin of the window Thermo Scientific EASY nLC II User Guide ix Preface To contact Customer Service for ordering information For North America see Table 1 For Europe see Table 2 For Australia and Asia see Table 3 on page xiii To order consumable and spare parts go to Www proxeon com productrange nano lc spares accessories Table 1 North America United States Phone Fax E mail Address Customer Service information for North America 1 800 532 4752 1 877 373 4006 us customer support analyzegthermofisher com 1400 North Point Pkwy 10 West Palm Beach FL 33407 Canada Phone Fax E mail Address Table 2 Europe Austria Phone Fax E mail Address 1 800 530 8447 1 905 890 9161 us customer support analyzegthermofisher com 2845 Argentia Road Unit 4 Mississauga Ontario L5N 8G6 Customer Service information for Europe Sheet 1 of 4 43 0 1 333 50 34 0 43 0 1 333 50 34 26 service sid austria thermofisher com Wehlistrasse 27b A 1200 Wien In the following tables the country code is enclosed
101. nnected Message window can be used to chat with support when connected Show Clear Log file copy 9 To home directory To USB removable storage f Copy Log Fies Status Idle Contact Thermo Fisher Scientific Edisonsvej 4 DK 5000 Odense C Denmark easysupport proxeon com http Avww proxeon com hple Phone 45 6557 2300 Fax 45 6557 2301 Use the Devices page to view and configure hardware devices including calibrating the autosampler You can add instruments to the HPLC such as the Advion RePlay device see Installing and Using the RePlay Thermo SCIENTIFIC Scripts Log Book Support owes il External Device on page 125 For configuration details and oe parameter descriptions see Configuring Column Setup admin Loop Volume Idle Flow and Exit IFC on page 40 Configuration Devices Cooled Autosampler ASC Autosampler Autosampler p EASY nLC HPLC ms Piston Pump flow feedback Pump A Piston Pump wi flow fee Add Device Remove Device Ir EASY nLC HPLC E One column setup E Disable IFC Loop volume 20 0 pi Idle flow rate 25 nl min Idle mixture 5 B Factory Thermo Scientific Configuration Thermo Scientific 3 Instrument Control Software Application Menu Structure Press the Configuration tab to control the
102. ntific easysupport proxeon com EASY nLC Il User Guide 93 8 Troubleshooting Miscellaneous Miscellaneous For additional issues that might arise while using the EASY nLC II system consult these miscellaneous troubleshooting tips Table 14 Table 14 Miscellaneous troubleshooting tips Sheet 1 of 2 Symptom Copying log files to a USB memory stick failed Possible causes Memory stick in wrong format Action Format the memory stick in FAT FAT16 format Memory stick not recognized by the system Use one of the memory sticks supplied by Thermo Fisher Scientific or try plugging the stick into the other USB port Contact Thermo Fisher Scientific if neither action works easysupport proxeon com Copying to the USB memory stick does not stop Forgot the admin password 94 EASY nLC II User Guide Too much data to be copied Remove the memory stick from the EASY nLC II system and press Ignore when the error message appears Contact Thermo Fisher Scientific for a system check up easysupport proxeon com If you are already logged into the EASY nLC system Thermo Fisher Scientific can extract the password from the system in two ways Ifthe network is set up for remote access choose Maintenance Support and press Connect or Runa factory reset Thermo Scientific Thermo Scientific 8 Troubleshooting Miscellaneous Table 14 Miscellaneous troubleshooting tips Sheet
103. ob 00 00 00 24 aug 2010 admin f Exit f Method Editor Maintenance Configuration Auto Submitter Job name Duration continue Ready jadmin 6 x BSA 30min 110415 4 1 O admin ladmin 20100824 1714 11 04 15 w y mum num pesa fessi Specify and schedule sample processing jobs for execution by using views under the Batch Setup tab Table 4 Batch Setup pages Sheet 1 of 2 Use the File page to view or change a directory tree of saved batch jobs that can be copied deleted or exported You can create or import new jobs and create or delete directories on this page Job 00 00 00 23 aug 2010 1235 admin Exit Home Copy Export Delete Edit Schedule Method Editor Maintenance New Batch Configuration New Folder home amp admin gt public super E user B x BSA 30min EASY nLC Il User Guide 23 3 Instrument Control Software Application Menu Structure Table 4 Batch Setup pages Sheet 2 of 2 Use the Edit page to specify new batch jobs There are two views Use the View Racks view to Thomo SCIENTIFIC see a graphical view of the F f f Add Injections 4 Browse methods v Batch Extensions E chosen plate format Selecta 3 3 method from the list enter _ i 5 4 4 3 5 T z sample volume and number of injections highlight the View ist M
104. of the well EASY nLC Il User Guide 83 7 Autosampler Calibration Calibrating the Wash Waste Compartment 84 EASY nLC II User Guide Wo dM amp Press Capture when you are satisfied with the current xyz coordinate for position W4 The color of the bottle container number changes from green to red If the vial needs to be calibrated press V1 vial 5 to select it If needed repeat step 2 through step 5 for vial A Press Save and then press Close to close the calibration dialog box To check sample pickup Identify the positions of the wells that you have problems with Fill the wells with a given volume of solvent A This example uses 12 pL Puta plastic film on the Microtiter plate Insert the tray On the Maintenance gt Scripts page run the Sample Pickup script with a pickup volume of 10 uL and your default flow see Figure 51 Run the script for each well you pointed out in step 1 Figure 51 Sample Pickup script under Maintenance tab Category Name Category Name Category Name g rest 3 guum pickup 3 test 3 Sample pickup J f f s is X Parameters Output pa cE Description Parameters Test autosampler sample pick up Please Sample pickup started fill plate well with a defined volume Parameter Value Prime pump filling before running test olume pl Max volume Loop volume 2pl ull 10 00 Max flow 40 0 pl min Flow ul min 20 00 Position
105. older to i3 logfiles zip Compressed zippe 24 10 2005 12 23 Share this Folder Q3 maintlog zip Compressed zippe 24 10 2005 12 23 3 My Computer 100 EASY nLC II User Guide Thermo Scientific 9 Remote Support Saving System Files on a USB Removable Storage Device The files are as follows batchlog zip Contains one folder for each batch you have run with information on samples methods and EASY nLC II device performance etc zip Contains system configuration files export zip Contains exported batches and methods per user and system backup logfiles zip Contains system log files with information on overall system performance maintlog zip Contains one folder for each maintenance script you have run with information on EASY nLC II device performance If you have problems with your system forward the files to Thermo Fisher Scientific Technical Support for assistance Thermo Scientific EASY nLC Il User Guide 101 eg Maintenance This chapter provides various maintenance schedules for the EASY nLC II system and describes the system s built in maintenance scripts and when to use them Consistent use of these procedures will help ensure the longevity of your instrument Contents Daily Maintenance Quarterly Maintenance Yearly Maintenance Maintenance Scripts Daily Maintenance Check the solvent levels on a daily basis To check solvent levels 1 Visually inspect solvent bottle A solvent bottle B
106. ome Overview page Press Eject Plate 2 Remove the plates tubes 3 Remove the waste container and other bottles see Autosampler Bottles on page 44 4 Close the tray door on the Home Overview page Press Insert Plate 5 Close down the system properly see Closing Down the EASY nLC II System on page 33 6 Remove the cables on the back side Tip For the original ASA autosampler only Secure the autosampler by using the transport collars see Figure 71 before shipping the instrument This is not necessary on the latest ASC model Figure 71 ASA autosampler only Transport collar location transport collars after closing down the system 7 Fill out the Declaration of Contamination of Equipment in Declaration of Contamination on page 133 8 Place the EASY nLC II instrument in the original shipping container 134 EASY nLC II User Guide Thermo Scientific LLL m ie Index A accounts adding new 40 adapter plate replacing ASA autosampler 72 ASC autosampler 74 admin changing password 39 advanced flow control system 5 AFC See pump flow control analytical Col Equilibration script 109 analytical column equilibrating 52 installing 50 autosampler bottles preparing 44 calibration 71 checking pickup 104 setting to pick up sample 55 Torque script 117 troubleshooting 90 wash 59 Back Pressure script 116 Batch Setup tab 25 batch troubleshooting 88 building a gradient 57 C
107. on 66 EASY nLC II User Guide You can stop the sample acquisition during the run by pressing the red STOP button on the Home Overview page This halts all actions on the HPLC system and the following buttons become active Restart Sample Runs the method from its beginning again cleaning up the system as it does Only press this button if you are sure you have enough sample material in the Microtiter plate Cancel Sample Cancels the current sample and skips to the next sample in the batch job Cancel Batch Cancels the entire current batch job Eject Plate Ejects the plate for your inspection to help you determine how to proceed Thermo Scientific 6 Running the First Sample Editing the Running Batch Editing the Running Batch You can edit the running batch if you want to add sample or remove samples that are not yet tested To edit the running batch 1 Choose Home gt Queue Thermo somes Batch Setup Method Editor Configuration Overview 1 Auto i Submitter Job name Duration Graphs continue aueue Rumning admin admin 20100825 0002 06 55 00 Job 00 02 38 Sample 00 00 08 Gradient 00 00 25 aug 2010 00 05 admin Thermo Scientific EASY nLC II User Guide 67 6 Running the First Sample Editing the Running Batch EASY nLC II User Guide 2 Select the batch job from the list and press Edit The Edit page under the Batch Setup tab appears see Figure 42 Figure 42 Edit page
108. ons page This value as shown in Figure 69 indicates how long into the gradient to switch back to playback mode At the end of the gradient the contact closure control reverts to capture mode Figure 69 RePlay Playback Delay setting Thermo SCIENTIFIC File 1 ca NN Name BSA 30 min RePlay Save RePlay Capture Playback Eluent Timing Home Batch Setup Me Maintenance Configuration Playback delay 35 00 min sec Job 00 01 02 24 aug 2010 19 13 admin Thermo Scientific KD wy B Miscellaneous Installing and Using the RePlay External Device To monitor the run of a RePlay method 1 When running a sample using a method with the RePlay extension open the Overview page under Home 2 View the RePlay RP mode display in the center status box see Figure 70 Figure 70 RePlay method status display Thermo S CIEMTIEIC Batch Setup Method Editor Maintenance Configuration s T 142 8 bar IFC enabled Overview 100 0 ul min Idle flow 25 5 3 4 150 0pl Graphs Queue Solvent A Cancelled 143 0 bar 100 0 ul min 2 9 150 0ul EEEE 900000 900000 147 5 bar h 40 0 ul min Waste 5 0 150 0p Refill AB RP Capture Solvent B ms Job 00 00 06 f f f f Sample 00 00 06 b Gradient 00 00 f f oo Administrat SEU G Jo 24 aug 2010 1 0 A1 Sample 1 admin 20100824 17 1695 O EBSA 30 min 6 Samples m 24 8 18 43 25 8 00 23 D
109. oor and insert the waste container in the compartment see Figure 28 without mounting the yellow lid Figure 28 Waste container compartment 2 Make sure to properly position the waste container 3 Close the door Thermo Scientific EASY nLC Il User Guide 45 5 Preparation for Use Executing Maintenance Scripts Executing Maintenance Scripts 46 EASY nLC II User Guide Note Before proceeding with the preparation for use make sure to perform all tasks described so far in Preparing Solvent Bottles and Waste Containers on page 43 You cannot execute maintenance scripts when the EASY nLC II instrument is running batches Figure 29 Scripts page under Maintenance Thermo Home Batch Setup Method Editor scripts Category Name f 4 Log Book Prepare wj Purge solvent X Support Parameters Output Devices Purge solvent from pumps Make sure to check solvent levels before running Job 00 02 33 24 aug 2010 17 22 admin Schedule Exit Flow nl min Flow nl min Configuration f Pump A J Desired flow Real flow Pressure 300 000 0 300 0 250 000 0 250 0 200 000 0 T 2000 c 150 000 0 A 100 000 0 150 0 S a 50 000 0 1000 0 0 a 50 000 0 50 0 100 000 0 00 o 1 2 3 4 5 Time min Pump B Desired flow Real flow Pressure 300 000 0 300 0 250 000 0 250 0 200 000 0 a 200 0 c 150 000 0 A 100 000 0 150 0 5 a 50 000
110. or scripts EASY nLC Il User Guide 49 5 Preparation for Use Installing Columns Installing Columns 50 EASY nLC II User Guide The EASY nLC II instrument comes with a trap column EASY Column 2 cm ID 100 um 5um C18 A1 an analytical column EASY column 10 cm ID 75 um 3 um C18 A2 and the unions and fittings for connecting 375 um OD and 1 32 in OD columns You can use other types of column ODs Contact Thermo Fisher Scientific for help choosing the appropriate fittings Two 1 32 in OD PEEKsil lines are attached to the instrument one called col out and the other waste in To connect columns 1 Connect the red union SC600 on the col out PEEKsil Read the Instructions for Use note provided with the union 2 Connect the pre column to the other end of the union using the provided sleeves SC603 if it is a 375 um OD column 3 Connect the flow out end of the pre column to the Venting Tee SC601 Again use the provided sleeve SC603 if it is a 375 um OD column 4 Connect the angled end of the Venting Tee with the waste in PEEKsil 5 Connect the straight end of the Venting Tee to the analytical column and use the provided sleeve SC603 if it is a 375 um OD column 6 Connect the analytical column outlet to the transfer line going to the detector Figure 30 FASY nLC Il column setup 375 um OD columns ZDV connector Waste in line Ej noed wr erisva pole war Asya
111. osampler Bottles ed d Cato ero aed Ratha M dosage abt Ak ah ee 44 Waste COH Bebe desde dete deron MAD D ae MeL es MA ots 45 Executing Maintenance Scripts Lue qud E e Vat eC e eat tat te UR tu gn 46 Purging and Flushing the Pumps eaa aes td doe e teach ettet ies 47 Installing Columns cx FECE Ghee wing Exsce obe e Mp ede CL baci da 50 Preparing the Pre Col mfiszo zi iex aper ep ARE Sb EE EURO abd ODU acia 51 Equilibrating the Analytical Colum ies i erre pere Ep eps 52 Running the First Sample Lusuuuseseeeeeeeeeeeeeeeeee 53 Prepared Well Plates iud eae P tira Race S etae S acer d 54 Creatine a Method ss ead oes Sane eae Res dex Qa v utt moet aa aid s 54 Page T S oa bi ha ered V Ead esqerod E entiers 54 Page DID saei ao mist on pud Ra eaa fea toria ea E Read 55 Pape ubt betae Sie ole RM ele Ma etal phys T Ba tho LA 56 Pasco ot shes ag owed er aaa we esa ae cro D EM Cetus 58 Page DI eau d seers a etate d ob he Era Grind ees ch E do ter Re cisco 59 Gieating a Batch i uude PO eai a og itia a esten sp UH omnes aay eae 60 Starting Sample Acquisition cuins eos RUE e Ro ta MN etae sp ER 63 Monitoting the Rutt ise Get theca etna oT A E EEROLEE MET Rd ecl E 63 Using the Overview Page in the Home Menu sees sess 64 Using the Graphs Page in the Home Menus 4 oy ves a ei tented ier ey s 65 Thermo Scientific Thermo Scientific Chapter 7 Chapter 8 Chapter 9 Chapter 10 Contents Stopping Sample Acquisition c
112. oting 70 saving system files on removable storage 99 scripts Analytical Col Equilibration 109 Autosampler Torque 117 Back Pressure 116 Flow Sensors 117 Flush Air 107 Isocratic Flow 110 Leaks 113 Maintenance 105 MS Connection 111 Precolumn Equilibration 108 Purge Solvent 106 Reset Pressure Sensor 118 Sample Pickup 112 Valve Check 114 Valve Tune 117 setup mass spectrometer connection 35 method with RePlay 128 pre column equilibration 58 solvent bottles preparing 43 solvent levels checking 103 specifications performance 119 technical 120 starting EASY nLC 19 stopping sample acquisition 66 system configuration 3 system files saving to removable storage 99 system overview monitoring 64 system startup troubleshooting 93 T technical specifications 120 temperature and humidity 12 touch screen interaction principles 21 using 18 transport instructions 134 troubleshooting autosampler 90 contact closure 92 Thermo Scientific Index S network access 92 running a batch 88 sample run 70 system components 95 system startup 93 turning off EASY nLC 33 U user interface layout 20 user permissions 38 V Valve Check script 114 Valve Tune script 117 verifying mass spectrometer connection 63 W warranty 123 wash column 60 waste container preparing 43 45 well plate preparing 54 EASY nLC II User Guide 137
113. outside of the EASY nLC II instrument use a mild detergent and a clean cloth EASY nLC Il Consumable Parts List These EASY nLC II parts are consumable parts and not covered by the normal first year Limited Warranty offered by Thermo Fisher Scientific or any other service contract agreement containing extended warranty coverage All flow lines including Tee pieces and fittings nuts ferrules sleeves and valve blank stops Check valves Solvent filters inline filters and all associated filter holders Pump piston seals Valve rotors and stators Thermo Scientific EASY nLC Il User Guide 123 B Miscellaneous Replacing the Main Power Fuse All bottles and lids All sample vials microtiter plates and associated lids and mats Columns Fuses For consumable and spare parts descriptions and ordering information go to www proxeon com productrange nano lc spares accessories Replacing the Main Power Fuse The first four numbered steps of this procedure correspond to the series of pictures in Figure 65 Note Before proceeding make sure to turn off the system and remove the power cable To replace the main power fuse 1 Place the EASY nLC II instrument so that you can access the connection panel on the back side 2 Pull out the power plug 3 Place a screwdriver in the top opening of the fuse holder Pull the fuse holder out Figure 65 Replacing the main power fuse 4 124 EASY
114. p1 Solvent 8 Eam z Sample SS OG Use the Graphs page to view up to four graphs each selectable from a list with historic data for the currently active sample For more information see Using the Graphs Page in the Home Menu on page 65 Thermo scicwriric oul Method Editor Pump A J Maintenance 22 EASY nLC II User Guide Overview Pump B d Desired flow Real flow Pressure Desired flow Real flow Pressure 3000 200 0 1 T Queue ba 3 2500 2500 2 cL 2000 g gos a E Eos wont os E 1000 02 02 500 00 a E oo d Joo in uk cp cic nants ti if oth eee he Time min Time min Gradient J Sample Cooling J 10 eet Current temperature Target temperature 900 Job 00 00 00 800 10 zo mo amp EE o8 E 5 g Zas moB H wos Bos 23 aug 2010 aoo B 1235 co 2 200 02 admin nan 00 00 00 f Exit 000 025 050 075 1 00 00 05 10 15 20 25 30 35 40 45 50 iai Time min Time min Configuration Thermo Scientific Batch Setup Thermo Scientific Table 3 Home menu pages Sheet 2 of 2 Use the Queue page to view a list of batch jobs currently executing or waiting You can edit a running batch from this page For more information see Editing the Running Batch on page 67 3 Instrument Control Software Application Menu Structure Overview Graphs CEN J
115. pler Calibration Calibrating Plates 8 Move slowly with the small step button V and lower the needle until it reaches the bottom of the well on the PEEKsil tubing moves up Observe when the black mark a bit Note The PEEKsil at the top of the needle holder rises a little when the needle reaches the bottom of the well You can observe this when the black mark moves 9 When you are satisfied with the current xyz coordinate for the well bottom press Capture The color of the well changes from green to red see Figure 48 Figure 48 Plate calibration captured Autosampler Plate Calibration Calibrate Plate HT MTP Needle In Out Needle Up Down Needle LeftRight J 00 20 10 Press the green well in the lower right corner of the plate view see 2 in Figure 47 which moves the needle to the current saved coordinate 11 Repeat step 6 to step 9 on that well 80 EASY nLC II User Guide Thermo Scientific 7 Autosampler Calibration Calibrating the Wash Waste Compartment 12 Press Save and then press Close to close the calibration dialog box 13 Refit the side plate on the system 14 Replace vial caps and plate covers as necessary Calibrating the Wash Waste Compartment Make sure that you have placed an empty vial in position V1 and a wash bottle with insert in position W4 t To calibrate the wash waste compartment 1 Go to Maintenance Devices 2 From the Devices list se
116. pt parameters Select the check boxes for the valves that you want to test Figure 61 Figure 61 Valve Check script parameters Thermo Home Batch Setup Method Editor Maintenance Configuration CN paika f Pump A X v v Log Book Test Valve check T Desired flow Real flow Pressure Support 300 000 0 eru tion Parameters Output 2600 250 000 0 Devices m mp 200 000 ann e a 150 000 0 1500 5 z e i 100 000 0 100 0 5 50 000 0 50 0 0 0 0 0 o 1 Jo o a ao Time min Pump B Desired flow Real flow Pressure Job 00 01 02 300 000 0 300 250 000 0 250 0 200 0000 T 2000 c E 1500000 D amp 24 su 200 E 100 000 0 1500 47 40 amp 500000 ORE u 0 0 3 admin 50 000 0 50 0 i eas n Exit o 1 2 3 4 5 Time min Thermo Scientific EASY nLC Il User Guide 115 10 Maintenance Maintenance Scripts Test Back Pressure 116 EASY nLC II User Guide The Back Pressure script determines instrument back pressure for solvent A and B The script runs at a preset flow and measures the back pressure on the system Use this script when testing for instrument flow path blockage Before running the script connect column output to waste input as shown in Figure 60 on page 114 it hod To set the Back Pressure script parameters Test solvent A solvent B or both A and B by selecting the corresponding check box or check boxes Figure 62
117. r Service information for Europe Sheet 3 of 4 Netherlands Phone Fax E mail Address 31 0 76 579 55 55 31 0 76 581 09 61 service sid benelux thermofisher com Takkebijsters 1 NL 4817 BL Breda Norway see Sweden Norway and Finland Spain Phone Fax E mail Address 34 914 845 965 34 914 843 598 service sid spain thermofisher com C Valportillo I n 22 1a Planta Edificio Caoba ES 28108 Alcobendas Madrid Sweden Norway and Finland Phone Fax E mail Address 46 0 8 556 468 20 46 0 8 556 468 08 service sid nordic thermofisher com Pyramidbacken 3 S 141 75 Kungens Kurva Stockholm Sweden Switzerland Phone Fax E mail Address xii EASY nLC II User Guide 41 617 16 77 40 41 617 16 77 20 service sid ch thermofisher com Neuhofstrasse 11 4153 Reinach Thermo Scientific Preface Table 2 Customer Service information for Europe Sheet 4 of 4 United Kingdom Phone Fax E mail Address 44 0 870 241 1034 44 0 1442 233 667 service sid hemel thermofisher com Stafford House 1 Boundary Park Boundary Way Hemel Hempstead Hertfordshire HP2 7GE Table 3 Customer Service information for Australia and Asia Sheet 1 of 2 Australia and Asia Australia Phone Fax E mail Address 61 39757 4300 61 9763 1169 analyze au thermofisher com PO Box 9092 5 Caribbean Drive Scoresby VIC 3179 Japan Phone Fax E mail A
118. r a more thorough wash of the needle and loop you can select the Custom Wash check box Use this option to set up a user definable procedure for washing the loop and needle When you use a custom wash the flush volume that you set in the Autosampler Wash area defaults to a preset volume to flush the loop and needle with as much solvent as possible before starting the custom wash The principle for setting the Custom Wash parameters is similar to that for setting up the gradient The Custom Wash table consists of three user editable parameters described in Table 10 Table 10 Custom wash parameters Parameter Description Source Defines which wash bottle is picking up the solvent You can choose between position 1 2 or 3 position 4 is for waste and cleaning of the outside of the needle Position 3 is the default position where the pump is filled and normally contains solvent A For example you can fill position 1 and 2 with other solvents and select them as the Source to better clean the needle and loop Volume Defines how much solvent is picked up To avoid any contamination between the solvent being picked up and the pump the pump is set to pick up 1 pL of air before picking up sample and the volume maximum is set to the following needle volume loop volume 2 pL Cycles Defines how many cycles are going to be performed Every cycle is completed with the volume set under volume being flushed out in wash bottle 4 When
119. re integrated into one simple compact unit making installation easy connect the power cable and a network cable if needed and turn on the power An embedded computer controls the system and provides an intuitive HPLC interface using an integrated touch sensitive monitor The instrument has a fixed configuration that optimizes the sample handling by using in depth knowledge of the instrument design and performance characteristics Instead of having to set up every aspect of a method from timing to individual control commands you can enter the parameters that describe the result you want The software manages the required low level control Furthermore you can connect the instrument and the integrated computer to the major mass spectrometers and include them in IT schedules for backup procedures and other functions System maintenance is provided through a range of controlled software built in diagnostic tests and the internal layout of all instrument components System Components 2 EASY nLC II User Guide The EASY nLC II system consists of the following components Table 1 EASY nLC Il system components Sheet 1 of 2 Hardware Pumps 3 high pressure pumps 1 300 bar with associated pressure sensors Valves 4 high pressure valves Flow sensor 2 nano flow sensors e Autosampler ASC latest model e Cooled maximum 20 C 68 F below ambient temperature Can hold 6 fixed vials plus either one 1 Microtiter p
120. rectory To USB removable storage Copy Log Files Status Contact Job 00 00 00 Thermo Fisher Scientific easysupport proxeon com Edisonsvej 4 http www proxeon com hplc DK 5000 Odense C Phone 45 6557 2300 Denmark Fax 45 6557 2301 30 Aug 2010 14 14 admin Exit Thermo Scientific EASY nLC Il User Guide 99 9 Remote Support Saving System Files on a USB Removable Storage Device 2 Insert the USB storage device into the connector panel on the back of the EASY nLC II instrument Choose between one of two USB ports on the upper left side of the panel 3 In the Log File Copy area select the To USB Removable Storage option 4 Press Copy Log Files When the copying is finished the following confirmation appears Copy To USB Copied successfully It is now safe to remove the USB storage Close 5 Press Close and remove the USB storage device from the connector panel 6 Insert the USB storage device into your computer Five zipped files appear on the USB storage device see Figure 56 Figure 56 Windows XP view of the USB memory stick File Edit View Favorites Tools Help Qe amp JO search 7 Folders EJ Address E File and Folder Tasks Name Type Date Modified i3 batchlog zip Compressed zippe 24 10 2005 12 23 zii ippe 123 a Miks a new folder Betc zip Compressed ppe 24 10 2005 12 s p export zip Compressed zippe 24 10 2005 12 23 e prende f
121. ress When the batch starts the green START button is grayed out and the red STOP button becomes active When the instrument is running three counters measure the time for job sample and gradient Thermo Scientific 6 Running the First Sample Monitoring the Run Using the Graphs Page in the Home Menu The Graphs page displays up to four graphs with historic data for the currently active sample Figure 40 Figure 40 Graphs Page under Home Thermo domes BatchSetup Method Editor Maintenance Configuration i f f Overview l Pump A Pump B be Graphs Desired flow Real flow Pressure Desired flow Real flow Pressure 300 0 300 0 10 eu 10 Queue 250 0 250 0 a d 08 2000 amp gos 2000 c a E a 5 1500 S 5 1500 5 A g s 2 1000 5 100 0 5 02 0 2 50 0 50 0 0 0 0 0 0 0 0 0 0 1 2 3 4 5 o 1 2 3 4 5 Time min Time min f f _ Gradient X Sample Cooling X 10 100 0 Current temperature Target temperature 90 0 Job 00 00 00 20 0 10 z 700 d E soo 3 28 x e Eos 500 amp G a 2 wo 2 E o5 23 aug 2010 TE 30 0 Fa e dom Ci 200 02 admin ee 0 0 0 0 oo Exit 0 00 0 25 0 50 0 75 1 00 00 05 10 15 20 25 30 35 40 45 50 XIT Time min Time min Table 11 describes the different types of graphs that are available To change graphs select from their corresponding lists Table 11 Graphical data types Graph Descrip
122. rst Thermo Home Batch Setup Method Editor MMaintenances Configuration scripts pect pals f f f z Pump A X Log Book Prepare 1 Precolumn equili w Desired flow Real flow Pressure Support 300 0000 S 250 000 0 250 0 Devices 3 m Parameter Value Ea E s 150 000 0 1500 5 0 0 M e 3 0 0 Max pressure bar 25000 o 1 2 3 4 5 Time min f Pump B X Desired flow Real flow Pressure Job 00 00 07 RA 300 0 250 000 0 uh E m 2000000 200 0 s 2 24 aug 2010 z 150 000 0 1500 5 17 25 2 100 000 0 100 0 Ej admin 50 000 0 50 0 Schedule Du loo Exit 0 1 2 3 4 5 Time min Thermo Scientific 10 Maintenance Maintenance Scripts Prepare Analytical Col Equilibration The Analytical Col Equilibration script equilibrates the analytical column with solvent from pump A The script utilizes the IFC system unless it is disabled and the procedure below is based on IFC being active If IFC is disabled the EASY nLC II application ignores the Max Pressure setting and the pump runs passively at the specified flow Use this script to equilibrate flush the analytical column determine suitable analytical reequilibration flow for method setup or initialize a new analytical column To set the Analytical Col Equilibration script parameters 1 Press Volume to set how much solvent A to use for reequilibration of the analytical column For best resu
123. sees dats ate ek tet obe teen dO ete Noe daa 66 Editing the Running Batch sa ciae aeree reete a eti eta ded 67 Troubleshooting a Sample Run i ies ren ee rA er NR ERR 70 Autosampler Calibration 0 00 0 cece eee eee eee eee eens 71 Replacing the Autosampler Adapter Plate 0 0 ee eee eee 72 Managing Plate Formats iu o citeutdored une ve erdt ain uei dod o tci dee 75 Preparation and Basic Principles 25 mus excea exce ace E obe he 77 Calibrating Plates seta est odes presa poo xo bx aer deny a Rer har pt dons 78 Calibrating the Wash Waste Compartment irri key hh ER Rh 81 Troubleshooting 2 ilililku lc xg 3 Seon d eee ie ede ds 87 Running a Bath 2 ucc sea eo va sees ela p ean eda TE CS 88 Jutosatap ler ooo eode he dues Haee noon est ad eel ae Ad 90 Contact Glostte cfr CEPIT Pr RIED ET 92 Network ACCESS ics Jos eee vate weed Ae Ro as EEE E EE E 92 Startine Up the Systemer eie rr i sabes les pie a ERE AREE dS 93 System Component States Valves Pumps Autosampler and Plate E MEE TE E T EE E edo e rate ed os py cd 93 Miscellansous ist nan a a tio c ese pop Ue aie ee ken RES Rc ce DR 94 Remote SupD lt eros ose rin ace teehee IR RA IT MERIT ARARSS 97 Connecting the EASY nLC II System to the Support Server 97 Saving System Files on a USB Removable Storage Device Lus 99 Malliteante c ia ia Rely RM DUO CS a wo e CE Gcr ogee Ka s 103 Daily Maintenances asndan pe dene CP d a UE Geo Sw ola
124. tem If you deactivate IFC the EASY nLC II application ignores the maximum pressure input fields and instead the pumps are regulated passively by the set flow only relevant for sample loading and equilibration steps The following sections describe the five pages and guidelines to creating a method 1 Choose Method Editor gt Edit 2 If you are not already on page 1 5 switch pages by pressing the appropriate arrow near the bottom of the screen Page 1 5 To open existing methods and close or save the currently opened method 1 Press SAVE 2 In the Save File dialog box press the white box beneath the name 3 Type method 1 on the keyboard and press OK 4 Press SAVE in the Save File dialog box You may enter a detailed explanation or description in the white text box 5 Go to page 2 5 by pressing the right arrow 54 EASY nLC II User Guide Thermo Scientific Page 2 5 Thermo Scientific 6 Running the First Sample Creating a Method Use page 2 5 see Figure 32 to set the parameters for picking up the sample from the well plate and loading it onto the pre column Figure 32 Page 2 5 Edit view in the Method Editor Thermo Home Batch Setup Method Edito Maintenance Configuration SCIENTIFIC File CE Name BSA 30 min Save Sample pickup Volume pl Flow 20 00 ul min ue 0000909 990000000000 990099000000 886050000000 Sample loading Job 00 00 07 Volume Ai
125. th the limits for a Class A digital device pursuant to Part 15 of the FCC Rules These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instruction manual may cause harmful interference to radio communications Operation of this equipment in a residential area is likely to cause harmful interference in which case the user will be required to correct the interference at his own expense Note You must use shielded cables with this unit to ensure compliance with the Class A FCC limits Thermo Scientific Preface Canadian Safety and EMC Electromagnetic Compliance Standards Safety This instrument has been reviewed for compliance with standard CAN CSA C22 2 No 61010 1 Second edition Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Part 1 General Requirements EMC This Class A digital apparatus meets all requirements of the Canadian Interference Causing Equipment Regulations European Safety and EMC Electromagnetic Compliance Standards Thermo Scientific Application of Council Directive s 2006 95 EEC Low Voltage Intertek Group plc 89 336 EEC Electromagnetic Compatibility DELTA Denmark Standard s to which conformity is declared M
126. thod Editor Batch Setup Category Name f f Prepare w Analytical col eq Parameter v Value eN 10 00 Flow p min 5 00 Max pressure bar 250 00 Schedule Configuration Pump A J Desired flow Real flow Pressure 300 0000 300 0 250 0000 250 0 zT m f 2000000 200 0 E 150 0000 1500 5 E 100 0000 1000 F 50 000 50 0 0 0 0 0 NP e T Time min f _ No graph X 4 In the Scripts view press the Parameters tab and set the desired flow volume and maximum pressure Thermo Fisher Scientific recommends running the equilibration at a set pressure rather than a set flow to make sure the column pressure limit and the instrument pressure limit 300 bar are not exceeded If the flow field is left empty the pump runs at the specified pressure For best results use an equilibration volume of 10 column volumes 5 Press START The default graphs appear in the two graph windows but you may change the selections from their corresponding lists These graphs help you to monitor the progress 6 Monitor the actual flow rate to become familiar with the flow pressure relationship on your particular column or columns 52 EASY nLC II User Guide Thermo Scientific LLL m Running the First Sample This chapter describes how to perform analysis using the EASY nLC II system Thermo Fisher Scientific recommends that you quickly start
127. tion Pump 3 For each of the three pumps shows desired flow black line real flow blue line and pressure red line over time Valve 4 For each of the four valves shows valve position over time Gradient Shows the theoretical gradient dotted red line and the actual gradient dynamically calculated on the feedback values from the pumps full red line plus the actual flow calculated on the feedback values from the pumps Temperature Shows the measured temperature on the plate cooler over time Thermo Scientific EASY nLC II User Guide 65 6 Running the First Sample Stopping Sample Acquisition To see a more detailed view you can touch the graphs to enlarge them for example the flow graph for Pump B in Figure 41 Touch them again to minimize the view Also by touching the y axis you can cycle through several preset scales Figure 41 Enlarged graph on the Graphs page Thermo iome BatchSetup Method Editor Maintenance Configuration Overview Pump B C Desired flow Real flow Pressure 300 0 Queue 300 000 0 275 0 275 000 0 250 0 250 000 0 225 0 225 000 0 200 0 200 000 0 1750 D E 1750000 n 3 150 0 z 150 000 0 Ex z c 1250 125 000 0 Job 00 00 00 100 000 0 ine Sample 00 00 00 750 Gradient 00 00 75 000 0 50 0 50 000 0 24 aug 2010 17 32 250 25 000 0 admin ao EG E NS pr NI i i 0 0 E 0 0 0 5 10 1 5 20 25 30 35 40 45 50 Exit Time min Stopping Sample Acquisiti
128. tion of 100 fmol tryptic digests of BSA on EASY columns as supplied at delivery and by using the Standard Wash procedure for the autosampler Autosampler pickup 100 18000 nL with standard loop volume range 100 58000 nL with up to 60 pL loop Thermo Scientific EASY nLC Il User Guide 119 A Specifications Technical Specifications Table 15 Performance specification Sheet 2 of 2 Item Specification Injection reproducibility 0 2 at 5 pL pickup injection RSD 3 0 at 100 nL pickup Injection linearity 0 9985 at 0 5 10 pL injection volume Autosampler formats Original autosampler Latest autosampler ASC ASA x 6 Vials 6 x 8 Vials 6 Vials 1x96 well MTP 1x96 well MTP 6 Vials 1 x 384 well MTP 1 x 384 well MTP 6 Vials 2x48 PCR strips 6 Vials 4x24 PCR strips 6 Vials Autosampler cooling 20 C below ambient typical setting 7 C 45 F Technical Specifications Technical specifications are subdivided as follows e General specifications Table 16 Physical specifications Table 17 Electrical specifications Table 18 Communication specifications Table 19 Parts list Table 20 Table 16 General Item Specification Sound pressure level 70 dBA Operating temperature 5 to 30 C Storage temperature 25 to 60 C Humidity 20 to 80 RH non condensing Sample viscosity 0 1 to 5 cP Solvent A Water with 0 1 formic acid Solvent B Acetonitrile with 0 1 formic acid Safet
129. to have precise flow control needed during gradient elution To meet this need you can use the Intelligent Flow Control system IFC to specify an operating pressure rather than a flow Advanced Flow Control System Thermo Scientific The advanced flow control system is active during the gradient step of the method execution and during execution of the isocratic flow maintenance script see Prepare Isocratic Flow on page 110 The AFC system uses the output of the flow sensors to regulate the flow accurately maintaining the desired flow from each channel even during sudden pressure changes that can occur when changing the solvent composition See Figure 4 Figure 4 Schematic diagram of the AFC principle Pressure sensor A A B mixing Tee EASY nLC II User Guide 5 1 Introduction Product Description Intelligent Flow Control System 6 EASY nLC II User Guide The Intelligent Flow Control IFC system is active during reequilibration steps and sample loading during the execution of the precolumn and analytical column reequilibration scripts see Prepare Precolumn Equilibration on page 108 and during pressure buildup just before the gradient proper The IFC system uses output from both the flow sensor and the pressure sensor to regulate the flow see Figure 5 You can specify a desired flow rate or a desired operating pressure This ability reduces the loss of productivity caused by system stoppage relate
130. ug 2010 17 50 excog Calibrate Valve Tune The Valve Tune script automatically retunes the valve Do not run this script unless instructed to by a Thermo Fisher Scientific field service engineer Calibrate Flow Sensors Thermo Scientific The Flow Sensors script calibrates the flow sensors on pumps A and B Use this script when you change the type of solvent in A or B or you suspect the calibration to be wrong Before calibration connect a 10 um ID tubing to the Column Out line to achieve back pressure at approximately 50 bar at 500 nL min EASY nLC Il User Guide 117 10 Maintenance Maintenance Scripts To set the Flow Sensors script parameters 1 Select the check boxes for the flow sensors that you would like to calibrate Figure 64 2 Press Period to specify the amount of time in minutes for stabilization of the flow before the calibration is performed The script makes a 2 point calibration and uses the stabilization period at both calibration points Figure 64 Flow Sensors script parameters Calibrate Reset Pressure Sensor Thermo Home Batch Setup Method Editor Configuration CNN us f 3 lt f Pump A M Log Book Calibrate wj Flow sensors X Desired flow Real flow Pressure Support 5 000 0 eal Description Output son Devices NL z 2000 amp Parameter v Value 30000 a 1500 5
131. ule v admin Exit 6x8 vials Press Schedule to enter the samples for processing This takes you to the Batch Queue tab in the Home menu 24 EASY nLC II User Guide Thermo Scientific Method Editor Thermo Scientific 3 Instrument Control Software Application Menu Structure Press the Method Editor tab to define sample processing methods Table 5 Method Editor pages Sheet 1 of 3 Use the File page to view a directory tree of saved methods that you can copy delete or export You can create or import new methods and create or delete directories from this page Edit Job 23 aug 2010 admin Thermo SCIENTIFIC ru 1238 Home Batch Setup MMethod Edito Maintenance Configuration New Folder f New Method Copy home A lac im in public Export Delete gt super gt user Import i ae 4 Bsa 30 min Standard 30 min BSA separation Use the Edit 1 5 page to view and change the values that make up a method The methods are result oriented so you state the type of performance you require from the instrument Each section shows the flow parts that are affected or controlled by the inputs For more information on this page of the Edit view see Page 1 5 on page 54 Job 23 Jit File aug 2010 12 38 admin Home Batch Setup Maintenance Configuration Name BSA 30 m
132. using the application to run additional analytical tests as practical experience is the most efficient way to understand an instrument such as an HPLC Contents Preparing a Well Plate Creating a Method Creating a Batch Starting Sample Acquisition Monitoring the Run Stopping Sample Acquisition Editing the Running Batch Troubleshooting a Sample Run Thermo Scientific EASY nLC Il User Guide 53 6 Running the First Sample Preparing a Well Plate Preparing a Well Plate If you have not made a plate selection go to To select a given plate format on page 76 The EASY nLC II instrument ships with adaptors for vials 56 vials in a 6x8 6 configuration MTP 96 and 384 well and PCR strips Note The instrument comes preinstalled with a 20 uL loop With this loop size the instrument is capable of picking up sample volumes between 0 and 18 pL To prepare the well plate 1 Load some vials wells with your sample preferably a known standard 2 Make sure that no air is trapped in the sample vials wells and that the sample is in the bottom of the vials wells To do this tap the vial plate gently against a hard surface to have the sample move to the bottom 3 Put on the vial cap or plate mat Creating a Method The Edit view under Method Editor consists of five pages to guide you through all the parameters required for a working LC method The protocol is based on IFC being active the default sys
133. uter network 3 c g CQ The instrument can be connected to ordinary computer networks by using a standard Ethernet cable that has RJ 45 connectors When the instrument connects to a network other computers on the network can access files on the instrument for example logs exports imports reports statistics and backups when they know the IP address of the instrument and have a valid user login account and password a Attaching Mouse and Keyboard to the USB Connections You can use a mouse and keyboard instead of the built in touch screen but Thermo Fisher Scientific recommends using the touch interface For more information see Using the Touch Sensitive Screen on page 18 Connect the USB mouse or keyboard by using the USB connectors on the back of the instrument The EASY nLC II application detects the mouse or keyboard within 30 seconds The application does not recognize all USB based keyboards or mice Attaching Add on Products through the P Bus and RS 232 The add ons are controlled and powered through the P bus and or controlled through the RS 232 interface ee oO CO CSOS SS ESS PRO YEON seeeeeceeeoeoeeoe eccorcocceeece eeeeeeeeoeseeoee 0000000 Seeeoceoeceeeecoese A Cx f oR Q mme o RS 232 P bus 14 EASY nLC II User Guide Thermo Scientific 2 Installation Flow Parts and Lines Flow Parts and Lines Thermo Scientific The pump and valve compartment of the EASY nLC II instrument also contains all t
134. y According to IEC 61010 120 EASY nLC II User Guide Thermo Scientific Table 17 Physical Item Dimensions WxDxH A Specifications Technical Specifications Specification w 35 cm 14 in d 38 cm 15 in 45 cm 18 in Weight 32 kg 71 Ib Weight in the shipping container Table 18 Electrical Item Power requirements 42 kg 93 Ib Specification 120 Vac 50 60 Hz 250 W 230 Vac 50 60 Hz 250 W For UPS dimensioning assume 250 W Fuses Table 19 Communication Item Contact closure IN circuit OUT circuit LAN For 120 Vac one T 5 AL 250 V fuse 5 x 20 mm IEC 60127 For 230 Vac one T 2 5 AL 250 V fuse All fuses are UL Listed and CSA certified Specification 3 outputs 3 inputs and 6 ground pins TTL Level PhotoMOS relay protected against high voltages Continuous switch current from 0 8 to 5 V is 35 mA 10 100 Mb s BaseT Ethernet USB 2 x USB 1 1 for keyboard and mouse RS 232 Reserved for HPLC add ons or high level MS control by using serial communication P Bus Reserved for HPLC add ons using the internal EASY nLC instrument protocol bus 8 wire control and limited power at 9 24V PS 2 Input for connection of keyboard or mouse Monitor Thermo Scientific Output for connection of external display EASY nLC Il User Guide 121 A Specifications Flow Line Diagram Table 20 Parts Item Pumps Specification 140 pL volume

EASY-nLC II 2.8 User Guide Version A

Contents

Download Pdf Manuals

Related Search

Related Contents