SPE-TT Tutorial Version 2009 -08 -11 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
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 Prospekt 2 must be finished. • This manual is based on - Prep. Gilson. ST 1. 2. 77 - Hy. Star 3. 2 (optionally SR 1 or SR 2) 2 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Un de SPE-TT Installation © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann r. C on s tru cti on
Software • You must install Prep. Gilson. ST and Hy. Star on the same computer. - Typically use the Hy. Star computer. • When using the Top. Spin computer note that you - must install Prep. Gilson. ST and Hy. Star. - must have a sufficient number of COM ports. (1 x. Liquid Handler, 1 x. Prospekt 2, 2 x. Link = 4 x. COM). • If Hy. Star is already installed - do Help/About and check that is Hy. Star Version 3. 2. - Do not install the Gilson 215 Software from Hy. Star!!! - Open the windows control panel. Do add remove software. Select Hy. Star and do modify. Verify that the Gilson 215 option is not selected. • If Hy. Star is not installed - Install Hy. Star according to the general installation instructions. - Do not enable the Gilson 215 option. - No special fraction collector license is required. 4 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Data Connections • Connection of the Liquid. Handler through - Preferably build in COM 1 of the computer. - Additional COM-ports supplied by a PCI board mounted in the computer - Optionally by USB-RS 232 converter. - Not by Ethernet-RS 232 converter (Moxa NPort Server) • Other instruments can use any kind of COM ports. 5 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
SPE-TT Start-up © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Prepare the Transfer in Prep. Gilson. ST • Open Prep. Gilson. ST • 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 7 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Prepare the Transfer in Prep. Gilson. ST • For convenient access to instrument + racks press to move the arm. • Connect the Prospekt 2 to the T-piece at the needle. (Note: This connection might have been removed for other applications) • Open the Tube. Block, 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 Prospekt 2. • Note the position(s) of the racks with Tube. Block(s) and the empty tubes. • In Clean. Procedures do Purge system to remove air bubbles from dilutor and capillaries. 8 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Check the Trays – without Barcode reader • With barcode reader racks are automatically detected. Stop 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. 9 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Define Parameters for Transfer in Hy. Star • In Hy. Star open the Transfer Settings from the menu(in flow injection) or the Prospekt 2 context menu (in Acquisition). • Set the cartridge drying time - 1 min for dried cartridges - 30 min for loaded cartridges. • Set Probehead wash&dry to 2 min & 150µl • Define the volume in the NMR tube as excess volume. - 3 mm 40 mm 190µl - 2. 5 mm 40 mm 140µl - 2 mm 40 mm 80µl - SJ 1. 7 mm 23 mm 30µl - For precise transfer set flow 100 -250µl/min. • Activate the option finalize! 10 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Special Version for Hy. Star <3. 1 only Define Parameters for Transfer in Hy. Star • With Hy. Star 3. 2 and later skip this page! • In Hy. Star enter Flow. Injection. • Open the Transfer. Settings. • Set the cartridge drying time - 1 min for dried cartridges - 30 min for loaded cartridges. • Set Probehead wash&dry to 2 min & 150µl. • Enter the complete volume (volume to the Needle tip + volume in the NMR tube) - For example volume to needle tip=180µl - Volume for 2 mm Tube 40 mm 80µl - Transfer. Volume = 260µl - For precise transfer set flow 100 -250µl/min. • Activate the option finalize! 11 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
SPE-TT Transfer Process © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
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 13 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann ?
Preparation II – Select Procedure/Cleaning • Select the procedure for the transfer: - default_SPETT. gsp: - - Transfer only, no mixing. Hy. Star Versions <3. 1 must use this procedure. SPETT Mix 2_0 mm Tubes. gsp SPETT Mix 2_5 mm Tubes. gsp SPETT Mix 3_0 mm Tubes. gsp: Mixing after transfer, liquid volume from Hy. Star must lead to 40 mm filling height. SPETT Mix 1_7 mm 30 ul. gsp: Mixing after transfer, liquid volume from Hy. Star must lead to 23 mm 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 Hy. Star (If Prep. Gilson. ST 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. 14 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
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 1 st 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. 15 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
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. 16 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Transfer – Create Orders • Press Create Orders • The list of cartridges & tubes in the upper window is converted into orders in the right window. • Prep. Gilson. ST 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! 17 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
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 18 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Transfer – Start the automation run • After successful validation the liquid handler starts the automation. • If Hy. Star is ready - A message appears. - The transfer starts. - No further actions are required. • If Hy. Star is not ready, because the chromatography is still running - A message appears. - Prep. Gilson. ST retries automatically every 60 sec until Hy. Star is ready for transfer. - Ensure that Hy. Star automatically switches to the flow injection window after the chromatography. - When Hy. Star is in the flow injection window and ready for the transfer, the automation will continue normally. 19 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Transfer – Proceeding • In the upper window of Prep. Gilson. ST 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. synchronization. Hy. Star and Prep. Gilson. ST may get out of - 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. 20 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
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. 21 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
SPE-TT Special Tasks © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Define Rack Positions manually • Open the configuration with View|Configuration • Click on the trays 23 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
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. 24 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
New Rack II: Insert empty racks • For MATCH tubes(all sizes) choose Rack Code 348 B. • For Sample. Jet tubes(all sizes) choose Rack Code 205 MI. • Enter an unique ID for the Rack, for example Match. Rack 1. All racks in the system should have different names. • Leave the rest unchanged and press OK. • Repeat the procedure for other racks. 25 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Insert Tube Blocks in empty Rack I • The previously inserted rack (348 B or 205 MI) with the two empty positions for the Tube. Blocks 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. 26 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Insert Tube Blocks in empty Rack II • Possible tube blocks (MATCH or Sample. Jet) will be shown. - Select the block with correct tubing size. • Define a unique ID, for example Tube. Block 1_2 mm. All blocks in the system must have different names. • Leave all other settings unchanged. • Repeat the procedure for the 2 nd block. 27 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Supplied Procedures • default_SPETT. gsp: All filling heights - Stays in drain during wash+dry of needle and dry of cartridge. - Goes to bottom 1) of selected source during transfer. - Returns to drain for next transfer. - No further actions. • SPETT_Mix_2. 0 mm Tubes. gsp : Liquid level 40 mm=80µl SPETT_Mix_2. 5 mm Tubes. gsp : Liquid level 40 mm=140µl SPETT_Mix_3. 0 mm Tubes. gsp : Liquid level 40 mm=190µl SPETT_Mix_1. 7 mm Tubes 30 ul. gsp : Liquid level 23 mm=30µl 1) - Stays in drain during wash+dry of needle and dry of cartridge. - Goes to bottom 1) 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 Hy. Star will cause the procedure to fail. Using higher sample volumes may result in inefficient mixing 1) After 10 mm the system checks for obstructions. The procedure will work with 1. 5 mm needle and without needle switch, however the detection only works with 0. 5 mm needle + needle switch. 2) The 1. 7 mm procedure works with a lower filling height adapted to the 1. 7 mm (Cryo)Probe. 28 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Test the Transfer Procedure • Prep. Gilson. ST supplies a possibility to check if a transfer is possible. • With the selected procedure, the current volumes in Hy. Star 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 Prep. Gilson. ST 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. 29 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
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 A 4, A 8, A 12. 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 3 x times on the same tube 30 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
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. 31 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Change the configuration • When opening Prep. Gilson. ST 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 Prep. Gilson. ST. 32 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Start Transfer during Chromatography • The SPE-TT system allows you to start Prep. Gilson. ST while Hy. Star is still running a chromatography. In this case the transfer into the tubes automatically starts after the end of the chromatography. • In Hy. Star … - In Acquisition|Shutdown Settings, activate - Switch to Flow Injection window. Define the transfer settings in the icon of the Prospekt 2 before you start the automation in Prep. Gilson. ST. For details check the Hy. Star documentation. • In Prep. Gilson. ST - 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 60 sec. Once Hy. Star is in the flow injection window, the transfer will start. 33 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
SPE-TT Trouble Shooting © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Potential Problem – Timing/Volumes in Hy. Star • The correct setup of Hy. Star 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 For both settings the movement of the needle is incorrect. the same amount The needle may go into the sample tube of liquid is while drying gas is still going through the delivered, but …. needle. 35 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann With this setting Prep. Gilson. ST assumes 30µl in the tube and mixing will probably fail.
Potential Problem – Rack/Block definition • Note: Whenever a message like the following appears, you have tried to install a Tube. Block 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 Tube. Block 2_2 mm 36 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
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 Hy. Star-Directory C: Program FilesBruker DaltonikHy. StarHardwareProspekt 2 (or the Hy. Star on your system, that is shown in the message) 37 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
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. 29 mm (=2/3 of the standard volume) 38 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
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 1 H 12 as first cartridge for cleaning of the system. - Select 1 H 12, assign tube 1, press Create Order. Select 1 A 1, 1 A 2, 1 A 1, 1 E 3, 1 B 8, assign tube 2. . 6, press Create order. Leave the Create orders dialog. - The transfer is done in the order [1 H 12], [1 A 1, 1 A 2, 1 B 8, 1 E 3]. - If you select 1 H 12, 1 A 1, 1 E 3, 1 B 8, assign tubes 1… 6, press Create Order. Leave the Create orders dialog, the order is [1 A 1, 1 A 2, 1 B 8, 1 E 3, 1 H 12]. 39 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
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. 40 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
Potential Problem – Needle does not fit • The 0. 5 mm needle is supplied with a sensor, that detects if the needle inserts correctly into the tube/container. • For this purpose the needle moves 20 mm 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 Hy. Star 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. 41 © Bruker Bio. Spin 2008 -2009; File: Tutorial 11 - SPE-TT; Version: 2009 -08 -11; By: Dr. Ulrich Braumann
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