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SPE-TT Tutorial Version 2009-08-11. Introduction. This document is a step-by-step guide for the usage of a SPE-TT system that elutes samples from SPE cartridges into the liquid handler. Installation: Software & Hardware installation instructions (under construction) .

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introduction
Introduction
  • This document is a step-by-step guide for the usage of a SPE-TT system that elutes samples from SPE cartridges into the liquid handler.
    • Installation: Software & Hardware installation instructions (under construction).
    • Start-up: Start-up of an already installed instrument.
    • Transfer Process: Step-by-step description of a (series) of transfer.
    • Special Tasks: Insert Racks/ Supplied procedures and simple modifications of procedures/ Pooling of cartridges.
    • Trouble Shooting: Problems/Debug information.
  • This is a Quickstart guide!
    • Read the complete manual for details.
    • Installation & calibration of the Liquid Handlerand Prospekt2must be finished.
  • This manual is based on
    • PrepGilsonST 1.2.77
    • HyStar 3.2 (optionally SR1 or SR2)
spe tt installation

SPE-TTInstallation

Under Construction

software
Software
  • You must install PrepGilsonST and HyStar on the same computer.
    • Typically use the HyStar computer.
  • When using the TopSpin computer note that you
    • must install PrepGilsonST and HyStar.
    • must have a sufficient number of COM ports.(1xLiquid Handler, 1xProspekt2, 2xLink = 4xCOM).
  • If HyStar is already installed
    • do Help/About and check that is HyStar Version 3.2.
    • Do not install the Gilson215 Software from HyStar!!!
    • Open the windows control panel. Do add removesoftware. Select HyStar and do modify.Verify that the Gilson215 option is not selected.
  • If HyStar is not installed
    • Install HyStar according to the generalinstallation instructions.
    • Do not enable the Gilson215 option.
    • No special fraction collector license is required.
data connections
Data Connections
  • Connection of the LiquidHandler through
    • Preferably build in COM1 of the computer.
    • Additional COM-ports supplied by a PCI board mounted in the computer
    • Optionally byUSB-RS232 converter.
    • Not by Ethernet-RS232converter(Moxa NPort Server)
  • Other instrumentscan use any kind ofCOM ports.
prepare the transfer in prepgilsonst
Prepare the Transfer in PrepGilsonST
  • Open PrepGilsonST
  • If the following window is shown,choose SPE-TT.
  • If this windows is not shown
    • Check that the used configuration is SPE-TT.
    • See page 32 Change the configurationof how to select theSPE-TT application.
  • If the liquid handler showsstatusyellow, doService Tools/Initialize
prepare the transfer in prepgilsonst1
Prepare the Transfer in PrepGilsonST
  • For convenient access to instrument + racks press  to move the arm.
  • Connect the Prospekt2 tothe T-piece at the needle.(Note: This connection might have beenremoved for other applications)
  • Open the TubeBlock, andinsert a sufficient number empty tubes.
  • The solvent at the dilutor of the liquid handler should be the same as the solvent used for transfer in the Prospekt2.
  • Note the position(s) of theracks with TubeBlock(s) andthe empty tubes.
  • In CleanProcedures doPurge system to removeair bubbles from dilutor andcapillaries.
check the trays with out barcode reader
Check the Trays – without Barcode reader
  • With barcode reader racksare automatically detected. Skip this page!!!
  • Open the configuration withView|Tray
  • Check if the racks you wantto use are available andcorrectly positioned.
  • Move the mouse over theracks/trays to check fordetails (Note: lower part of thetooltip shows the properties of theselected zone i.e. where you clickedthe last time with the mouse)
  • Details of how to changehe rack positions you find on page 23 Define Rack Positions manually.

Stop

define parameters for transfer in hystar
Define Parameters for Transfer in HyStar
  • In HyStar open the Transfer Settingsfrom the menu(in flow injection) or theProspekt2 context menu (in Acquisition).
  • Set the cartridge drying time
    • 1min for dried cartridges
    • 30min for loaded cartridges.
  • Set Probehead wash&dry to 2min & 150µl
  • Define the volume in the NMR tubeas excess volume.
    • 3mm 40mm 190µl
    • 2.5mm 40mm 140µl
    • 2mm 40mm 80µl
    • SJ 1.7mm 23mm 30µl
    • For precise transfer set flow 100-250µl/min.
  • Activate the option finalize!
special version for hystar 3 1 only define parameters for transfer in hystar
Special Version for HyStar <3.1 onlyDefine Parameters for Transfer in HyStar
  • With HyStar 3.2 and later skipthis page!
  • In HyStar enter FlowInjection.
  • Open the TransferSettings.
  • Set the cartridge drying time
    • 1min for dried cartridges
    • 30min for loaded cartridges.
  • Set Probehead wash&dry to 2min & 150µl.
  • Enter the complete volume(volume to the Needle tip+ volume in the NMR tube)
    • For example volume to needle tip=180µl
    • Volume for 2mm Tube 40mm 80µl
    •  TransferVolume = 260µl
    • For precise transfer set flow 100-250µl/min.
  • Activate the option finalize!
preparation i prepare software
Preparation I – Prepare Software
  • Do Preparation and selectStart Preparation Automation.
  • The Create Orders dialog opens.
  • Check for Created Orders
    • Typically it should be empty.
    • If there are orders left over fromprevious runs, check if you reallywant to execute them. If not …mark and delete them.
  • Check the displayed cartridgetrays.
    • Verify that the right trays aredisplayed. If not use theSelect … tray buttons to load thecorrect trays files

?

preparation ii select procedure cleaning
Preparation II – Select Procedure/Cleaning
  • Select the procedure for the transfer:
    • default_SPETT.gsp:Transfer only, no mixing.HyStar Versions <3.1 must use this procedure.
    • SPETT Mix 2_0mm Tubes.gspSPETT Mix 2_5mm Tubes.gspSPETT Mix 3_0mm Tubes.gsp:Mixing after transfer, liquid volume fromHyStar must lead to 40mm filling height.
    • SPETT Mix 1_7mm 30ul.gsp:Mixing after transfer, liquid volume fromHyStar must lead to 23mm filling height.
  • For the first transfer an empty cartridge should be used for cleaning of the system. Identify a cartridge that can be used.
    • You can perform such a transfer in HyStar (If PrepGilsonST is not used, needle remains in the waste) or …
    • Follow the instructions on the next pages for the automated transfer. Choose only this cartridge, select one tube and press create the order before you continue the selection with the “real” cartridges.
transfer select cartridges
Transfer– Select Cartridges
  • Select*) cartridge(s) with themouse.
    • Selected cartridges are shownin the upper window.
    • For a simple assignment of cartridgeto tube, select one cartridge andfinish the setup as describe below.
    • Cartridges are processed in the orderof their position in the tray. For a userdefined order, finish the setup for the1st cartridge as described below.
  • Click on destination toselect the NMR tube(s).
  • More cartridges can be selectedin a second round.

*) Select cartridges with left click. Multipleselections with CTRL-click or by openinga rectangle around a range of cartridges.

transfer select tubes
Transfer – Select Tubes
  • Select*) a sufficient number of tubes as destination forthe selected cartridges.
  • The selected tubes areassigned to the cartridgesand displayed in the upperwindow.
  • The first selected tube isassigned to the firstcartridge in the list.
  • For each selected cartridgeyou must select a tube.

*) Select cartridges with left click. Multipleselections with CTRL-click or by openinga rectangle around a range of cartridges.

transfer create orders
Transfer – Create Orders
  • Press Create Orders
  • The list of cartridges &tubes in the upper windowis converted into ordersin the right window.
  • PrepGilsonST returnsto the Source selection.
  • Repeat the process untilthe you have defined allcartridges, then leave thewindow with OK.
  • Attention OK will startthe Automation.
    • No further cartridge can beadded to the list, before current cartridges are completely processed.
    • Depending on the drying time this can be a lengthy process!
transfer verifying the orders
Transfer – Verifying the Orders
  • After closing the Create Order Files dialog the automation starts.
  • Orders will be shown as white circles.
  • First step is the test for valid parameters (volumes etc) which changes the color of the circles to purple. In case of problems the color turns to red and the automation stops.

1) The orders are normally sorted by the time of the Create Orders action. If more then one transfer was created at a time, the cartridges are sorted by their position in the tray. Check page 39 Potential problems – Order of Transfer

transfer start the automation run
Transfer – Start the automation run
  • After successful validation the liquid handler starts the automation.
  • If HyStar is ready
    • A message appears.
    • The transfer starts.
    • No further actionsare required.
  • If HyStar is not ready,because thechromatographyis still running
    • A message appears.
    • PrepGilsonST retriesautomatically every 60sec until HyStar is ready for transfer.
    • Ensure that HyStar automatically switches to the flow injection window after the chromatography.
    • When HyStar is in the flow injection window and ready for the transfer, the automation will continue normally.
transfer proceeding
Transfer – Proceeding
  • In the upper window of PrepGilsonST the progress can be observed:
    • Purple=Ready for transfer // Blue=Transfer running // Green=transfer done
  • To remove filledtubes for NMRmeasurements
    • Do notremove tubeswhile the automation isrunning.The arm of the liquid handler can start moving at any time!
    • Do not press the Pause button. HyStar and PrepGilsonST may get out of synchronization.
    • Press the End after current order button, wait that the automation stops, removethe desired tubes, and restart the automation for the next schedules cartridges.
results
Results
  • Do File/Open Print Dialogand Save New … Preview.
  • This will bring up a compactinformation of all orders
  • For each order line you can getdetailed information with theright mouse button.
define rack positions manually
Define Rack Positions manually
  • Open the configuration withView|Configuration
  • Click on the trays
new rack i choose the position of the rack
New Rack I: Choose the position of the rack
  • Check if the displayed racks corresponds to the current situation in the liquid handler If not …
    • Delete any wrong positionedracks with a double click.
    • For racks that contain partsyou must first delete thecontained tube blocks/ wellplate and then delete the rack.
  • To insert the new racks …
    • Click the right mouse buttonat the position where therack is located.
    • DoInsert BarcodeSimulation Data
    • Do not useInsert rack at selected site.With this procedure furtherdefinitions of rack propertiesare required.
new rack ii insert empty racks
New Rack II: Insert empty racks
  • For MATCH tubes(all sizes) choose Rack Code 348B.
  • For SampleJet tubes(all sizes) choose Rack Code 205MI.
  • Enter an uniqueID for the Rack, forexample MatchRack1.All racks in the system should havedifferent names.
  • Leave the rest unchanged and press OK.
  • Repeat the procedure for other racks.
insert tube blocks in empty rack i
Insert Tube Blocks in empty Rack I
  • The previously inserted rack (348B or 205MI) with the twoempty positions for the TubeBlocks is displayed in the tray.
  • Click with the right mouse button the upper empty field and doInsert BarcodeSimulation Data
    • Do not useInsert rack at selected site.With this procedure furtherdefinitions of rack propertiesare required.
insert tube blocks in empty rack ii
Insert Tube Blocks in empty Rack II
  • Possible tube blocks (MATCH or SampleJet) will be shown.
    • Select the block with correct tubing size.
  • Define a unique ID, for exampleTubeBlock1_2mm. All blocks in the system must havedifferent names.
  • Leave all other settings unchanged.
  • Repeat the procedure for the 2nd block.
supplied procedures
Supplied Procedures
  • default_SPETT.gsp: All filling heights
    • Stays indrain during wash+dry of needle and dry of cartridge.
    • Goes to bottom1)of selected source during transfer.
    • Returns to drain for next transfer.
    • No further actions.
  • SPETT_Mix_2.0mm Tubes.gsp : Liquid level 40mm=80µl SPETT_Mix_2.5mm Tubes.gsp : Liquid level 40mm=140µl SPETT_Mix_3.0mm Tubes.gsp : Liquid level 40mm=190µl SPETT_Mix_1.7mm Tubes 30ul.gsp : Liquid level 23mm=30µl1)
    • Stays in drain during wash+dry of needle and dry of cartridge.
    • Goes to bottom1)of selected source during transfer.
    • Performs a mixing of the tube content after the transfer.
    • Returns to drain and cleans the needle, waits for the next transfer.
    • The mixing volume is fixed.Using smaller volumes in HyStar will cause the procedure to fail. Using higher sample volumes may result in inefficient mixing

1) After 10mm the system checks for obstructions. The procedure will work with 1.5mm needle and without needle switch, however the detection only works with 0.5mm needle + needle switch.

2) The 1.7mm procedure works with a lower filling height adapted to the 1.7mm (Cryo)Probe.

test the transfer procedure
Test the Transfer Procedure
  • PrepGilsonST supplies a possibility to check if a transfer is possible.
  • With the selected procedure, the current volumes in HyStar and the type of NMR tubes you can test the orders.
    • Do Preparation and select Start Verify Test all Orders Testloop.
    • Follow the instructions following page 15 Transfer– Select Cartridgesidentical to the normal setup procedure.
    • At the end PrepGilsonST will onlyverify the procedure but not startthe transfer procedure.This allows you to check for errors.
  • By running the standard Start Preparation Automation dialog
    • The above generated test orders can be deleted.
    • The above generated and tested orders can be started.
pooling of cartridges i
Pooling of Cartridges I
  • Pooling means to combine the liquid from several cartridges into one sample tube.
  • Proceed as follows
    • Use CTRL-Click to selectthe cartridges that containthe same compound forexample cartridgesA4,A8,A12.
    • As destination assignthe same tube forall cartridges by usingCTRL-Click on thesame tube.
    • To avoid confusion,do Create ordersbefore you define thenext set of cartridges

CTRL-Click 3x times on the same tube

pooling of cartridges ii
Pooling of Cartridges II
  • Take care, that the selected tubes/containers are large enough to hold the complete volume of all transfer procedures.
  • The mixing procedures are not very efficient in this case. The calculation for the mixing assumes the amountof liquid from one transfer in a tube.
change the configuration
Change the configuration
  • When opening PrepGilsonST a window isshown, where you can choose SPE-TT.
  • If during start-up this windowsis not shown:
    • do edit settings
    • reactivate the window
    • restart PrepGilsonST.
start transfer during chromatography
Start Transfer during Chromatography
  • The SPE-TT system allows you to start PrepGilsonST while HyStar is still running a chromatography. In this case the transfer into the tubes automatically starts after the end of the chromatography.
  • In HyStar …
    • In Acquisition|Shutdown Settings, activateSwitch to Flow Injection window.
    • Define the transfer settings in the iconof the Prospekt2 before you startthe automation in PrepGilsonST.
    • For details check the HyStar documentation.
  • In PrepGilsonST
    • Start the automation while the chromatography runs.
    • Select the cartridges which you expect to be filledduring the chromatography.
    • A message indicates that the transfer is not started.
    • The system will automatically retry every 60sec. OnceHyStar is in the flow injection window, the transfer will start.
potential problem timing volumes in hystar
Potential Problem – Timing/Volumes in HyStar
  • The correct setup of HyStaris essential.
  • Excess volume
    • You must define the volume from cartridgetoneedletip as transfervolume.
    • You must define the liquidinthetube asexcessvolume.
    • Otherwise the mixingprocedure may notbe correctly performed.
  • Finalize
    • You mustactivate the finalizeoption
    • Otherwise the timing calculations forthe movement of the needle isincorrect.The needle may go into the sample tubewhile drying gas is still going through theneedle.

For both settings the same amount of liquid is delivered, but ….

With this setting PrepGilsonST assumes 30µl in the tube and mixing will probably fail.

potential problem rack block definition
Potential Problem – Rack/Block definition
  • Note: Whenever a message like the following appears, you have tried to install a TubeBlock with same identifier two times.
  • The ID must be a unique name!
  • Change the name of the tube block/rack you wantto insert. In this case forexample TubeBlock2_2mm
potential problem tray file selection
Potential Problem – Tray file selection
  • If a error message like the followingappears, you have chosen a tray filefrom a non-standard directory.
  • Use tray files only from the standardHyStar-DirectoryC:\Program Files\Bruker Daltonik\HyStar\Hardware\Prospekt2(or the HyStar on your system, that is shown in the message)
potential problem volume too low
Potential Problem – Volume too low
  • Error during verification indicates wrong volumes.
  • Right click for “Error Info” or “Current Order Info” for details.
  • Example: Volume dispensed into the tube is too low for the mixing. Increase volume or use tube with smaller inner diameter.
  • The minimum filling height formixing is approx. 29mm(=2/3 of the standard volume)
potential problem order of transfer
Potential Problem – Order of Transfer
  • Inside each order cartridges are sorted by the position in the tray.
  • The sorting of the orders is define with
    • This order is defined with Edit/Settings
    • Choose:By Time of the Creation orBy Order Name = Tray positionof the cartridges.
  • Example: To use 1H12 as first cartridge for cleaning of the system.
    • Select 1H12, assign tube 1, press Create Order. Select 1A1, 1A2, 1A1, 1E3, 1B8, assign tube 2..6, press Create order. Leave the Create orders dialog.
    • The transfer is done in the order [1H12], [1A1, 1A2, 1B8, 1E3].
    • If you select 1H12, 1A2, 1A1, 1E3, 1B8, assign tubes 1…6, press Create Order. Leave the Create orders dialog, the order is [1A1, 1A2, 1B8, 1E3, 1H12].
potential problem too much cleaning
Potential Problem – Too much cleaning
  • Each time you start a automation process a cleaning procedure is executed to clean the system but also to ensure that the complete flow path is filled with liquid.
  • If you transfer cartridge individually and not as sequence, you can disable the cleaning to save time and deuterated solvent.
  • Do disable the cleaning completely do Edit/Settings.
    • You can disable the Cleaning procedure at thebeginning and/or endof the transfer.
  • In this case you shouldperform a cleanneedlemanually before the firstcartridge transfer.
potential problem needle does not fit
Potential Problem – Needle does not fit
  • The 0.5mm needle is supplied with a sensor, that detects if the needle inserts correctly into the tube/container.
  • For this purpose the needle moves 20mm down and checks for any obstructions and then goes to the bottom of the tube.
  • If the needle cannot move into the tube
    • The needle moves up and a second attempt is done.
    • If this also fails the automation is aborted.
    • As it is not possible to automatically abort the transfer in HyStar at this stage, the needle remains at the current position (on top of the tube).
    • By this you have a good chance to recover the sample from the cartridge.
    • All further cartridges are not transferred.