1. SPE-TT Tutorial Version 2009-08-11

2. 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 Quick start guide!
Read the complete manual for details.
Installation & calibration of the Liquid Handler and Prospekt2 must be finished.
This manual is based on
PrepGilsonST
HyStar 3.2 (optionally SR1 or SR2)

3. SPE-TT Installation
Under Construction

4. 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 remove software. Select HyStar and do modify. Verify that the Gilson215 option is not selected.
If HyStar is not installed
Install HyStar according to the general installation instructions.
Do not enable the Gilson215 option.
No special fraction collector license is required.

5. 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 by USB-RS232 converter.
Not by Ethernet-RS232 converter (Moxa NPort Server)
Other instruments can use any kind of COM ports.

6. SPE-TT Start-up

7. 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 configuration of how to select the SPE-TT application.
If the liquid handler shows status yellow, do Service Tools/Initialize

8. Prepare the Transfer in PrepGilsonST
For convenient access to instrument + racks press  to move the arm.
Connect the Prospekt2 to the T-piece at the needle. (Note: This connection might have been removed for other applications)
Open the TubeBlock, and insert 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 the racks with TubeBlock(s) and the empty tubes.
In CleanProcedures do Purge system to remove air bubbles from dilutor and capillaries.

9. Check the Trays – without Barcode reader
With barcode reader racks are automatically detected Skip this page!!!
Open the configuration with View|Tray
Check if the racks you want to use are available and correctly positioned.
Move the mouse over the racks/trays to check for details (Note: lower part of the tooltip shows the properties of the selected zone i.e. where you clicked the last time with the mouse)
Details of how to change he rack positions you find on page 23 Define Rack Positions manually.
Stop

10. Define Parameters for Transfer in HyStar
In HyStar open the Transfer Settings from the menu(in flow injection) or the Prospekt2 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 tube as excess volume.
3mm 40mm 190µl
2.5mm 40mm 140µl
2mm 40mm 80µl
SJ 1.7mm 23mm 30µl
For precise transfer set flow µl/min.
Activate the option finalize!

11. Special Version for HyStar <3
Special Version for HyStar <3.1 only Define Parameters for Transfer in HyStar
With HyStar 3.2 and later skip this 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 µl/min.
Activate the option finalize!

12. SPE-TT Transfer Process

13. Preparation I – Prepare Software
Do Preparation and select Start Preparation Automation.
The Create Orders dialog opens.
Check for Created Orders
Typically it should be empty.
If there are orders left over from previous runs, check if you really want to execute them. If not … mark and delete them.
Check the displayed cartridge trays.
Verify that the right trays are displayed. If not use the Select … tray buttons to load the correct trays files ?

14. 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.gsp SPETT Mix 2_5mm Tubes.gsp SPETT Mix 3_0mm Tubes.gsp: Mixing after transfer, liquid volume from HyStar must lead to 40mm filling height.
SPETT Mix 1_7mm 30ul.gsp: Mixing after transfer, liquid volume from HyStar 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.

15. Transfer– Select Cartridges
Select*) cartridge(s) with the mouse.
Selected cartridges are shown in the upper window.
For a simple assignment of cartridge to tube, select one cartridge and finish the setup as describe below.
Cartridges are processed in the order of their position in the tray. For a user defined order, finish the setup for the 1st cartridge as described below.
Click on destination to select the NMR tube(s).
More cartridges can be selected in a second round.
*) Select cartridges with left click. Multiple selections with CTRL-click or by opening a rectangle around a range of cartridges.

16. Transfer – Select Tubes
Select*) a sufficient number of tubes as destination for the selected cartridges.
The selected tubes are assigned to the cartridges and displayed in the upper window.
The first selected tube is assigned to the first cartridge in the list.
For each selected cartridge you must select a tube.
*) Select cartridges with left click. Multiple selections with CTRL-click or by opening a rectangle around a range of cartridges.

17. Transfer – Create Orders
Press Create Orders
The list of cartridges & tubes in the upper window is converted into orders in the right window.
PrepGilsonST returns to the Source selection.
Repeat the process until the you have defined all cartridges, then leave the window with OK.
Attention OK will start the Automation.
No further cartridge can be added to the list, before current cartridges are completely processed.
Depending on the drying time this can be a lengthy process!

18. 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

19. 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 actions are required.
If HyStar is not ready, because the chromatography is still running
PrepGilsonST retries automatically 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.

20. 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 filled tubes for NMR measurements
Do not remove tubes while the automation is running. 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, remove the desired tubes, and restart the automation for the next schedules cartridges.

21. Results Do File/Open Print Dialog and Save New … Preview.
This will bring up a compact information of all orders
For each order line you can get detailed information with the right mouse button.

22. SPE-TT Special Tasks

23. Define Rack Positions manually
Open the configuration with View|Configuration
Click on the trays

24. 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 positioned racks with a double click.
For racks that contain parts you must first delete the contained tube blocks/ well plate and then delete the rack.
To insert the new racks …
Click the right mouse button at the position where the rack is located.
Do Insert Barcode Simulation Data
Do not use Insert rack at selected site. With this procedure further definitions of rack properties are required.

25. 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 unique ID for the Rack, for example MatchRack1. All racks in the system should have different names.
Leave the rest unchanged and press OK.
Repeat the procedure for other racks.

26. Insert Tube Blocks in empty Rack I
The previously inserted rack (348B or 205MI) with the two empty positions for the TubeBlocks is displayed in the tray.
Click with the right mouse button the upper empty field and do Insert Barcode Simulation Data
Do not use Insert rack at selected site. With this procedure further definitions of rack properties are required.

27. 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 example TubeBlock1_2mm. All blocks in the system must have different names.
Leave all other settings unchanged.
Repeat the procedure for the 2nd block.

28. Supplied Procedures default_SPETT.gsp: All filling heights
Stays in drain 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)
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.

29. 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 Cartridges identical to the normal setup procedure.
At the end PrepGilsonST will only verify the procedure but not start the 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.

30. Pooling of Cartridges I
Pooling means to combine the liquid from several cartridges into one sample tube.
Proceed as follows
Use CTRL-Click to select the cartridges that contain the same compound for example cartridges A4,A8,A12.
As destination assign the same tube for all cartridges by using CTRL-Click on the same tube.
To avoid confusion, do Create orders before you define the next set of cartridges
CTRL-Click 3x times on the same tube

31. 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 amount of liquid from one transfer in a tube.

32. Change the configuration
When opening PrepGilsonST a window is shown, where you can choose SPE-TT.
If during start-up this windows is not shown:
do edit settings
reactivate the window
restart PrepGilsonST.

33. 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, activate Switch to Flow Injection window.
Define the transfer settings in the icon of the Prospekt2 before you start the 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 filled during the chromatography.
A message indicates that the transfer is not started.
The system will automatically retry every 60sec. Once HyStar is in the flow injection window, the transfer will start.

34. SPE-TT Trouble Shooting

35. Potential Problem – Timing/Volumes in HyStar
The correct setup of HyStar is essential.
Excess volume
You must define the volume from cartridge to needle tip as transfer volume.
You must define the liquid in the tube as excess volume.
Otherwise the mixing procedure may not be correctly performed.
Finalize
You must activate the finalize option
Otherwise the timing calculations for the movement of the needle is incorrect. The needle may go into the sample tube while drying gas is still going through the needle.
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.

36. 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 want to insert. In this case for example TubeBlock2_2mm

37. Potential Problem – Tray file selection
If a error message like the following appears, you have chosen a tray file from a non-standard directory.
Use tray files only from the standard HyStar-Directory C:\Program Files\Bruker Daltonik\HyStar\Hardware\Prospekt2 (or the HyStar on your system, that is shown in the message)

38. 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 for mixing is approx. 29mm (=2/3 of the standard volume)

39. 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 or By Order Name = Tray position of 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].

40. 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 the beginning and/or end of the transfer.
In this case you should perform a clean needle manually before the first cartridge transfer.

41. 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.