LC-(SPE)-(Cryo)NMR(/MS) as Routine Analysis Technique: Instrumentation and Software

1. LC-(SPE)-(Cryo)NMR(/MS) as Routine Analysis Technique:Instrumentation and Software Dr. Ulrich Braumann Bruker Biospin, Rheinstetten

2. Agenda • Startup SampleTrack • Startup Preparation Program PrepGilsonST • Startup NMR Program TopSpin and ICONNMR • Setup Orders

3. Startup instruments and prepare the System Dr. Ulrich Braumann Bruker Biospin, Rheinstetten

4. Prepare SampleTrack PC • Login to the SampleTrack PC, typically with • User: samtrack • passwd: bsts • Check that the following programsare running • SampleTrack Webservice • SampleTrack Communicator • SampleTrack Client • If not, start the programs

5. Prepare SampleTrack PC • In the SampleTrack Client check go to the page instrument status • Typically all instruments will show“not responding” as the programs onthe other computers are not started • Follow the instructions on the nextpages to startup the instrumentprograms and in parallel observe inthis window that the connectionsare established. • The instrument programs whichcommunicate with SampleTrack are • Compass Sample Track communication • ICONNMR • PrepGilsonST

6. Prepare SampleTrack PC • Clean up Sample orders • Attention: During the next steps the automation programs are started. • If they detected the barcode of samples which have open orders in SampleTrack, the preparation or NMR/MS measurement may start immediately. • Therefore it is recommended to search for open orders from previousruns and –if required-delete them. • Go to the pageSample Orders • Search forNot yet finished Samples • Check if this orders shallbe really executed. If notdelete them.

7. Prepare PrepGilsonST • Start PrepGilsonST, and select the“Profiler” application. • Gilson remain disconnected– This is normal! • A white bar must indicate aof SampleTrack and PrepGilsonST • In SampleTrack Client must change from “not responding”to “preparation loop not started”

8. Start PrepGilsonST • Do Preparation/Start preparation Automation • The program must connect to the liquid handler. • The LiquidHandler must start reading barcodes. • In the SampleTrack Client the status must turn to green

9. Prepare ICONNMR & SampleJet • On the TouchScreen of the SampleJet check if the correct mode is selected • Do not use “manual mode” – switch to the correct shuttle diameter. • Ensure that you have selected the correct diameter. • Remove any sample from the magnet and ensure that the shuttle with the correct diameter is in the magnet. • Open the ICONMR- Configuration • Load the configuration for the SampleJet automation • Check settings for locking, shimming, ATMA. • If shimfile-loading is activated, check that the define shimfile is up-to-date.

10. Start ICONNMR • Start ICONNMR automation, do not define any experiments • Start the automation procedure • Select “SampleTrack(SampleJet). • In SampleTrack the status must turngreen “online waiting for orders”.

11. Prepare HyStar & MS Control • Identify the standard method path ofHyStar (“D:\data” or “D:\methods”). • From this path open the SampleTable“openaccess.xml” – or create it. • The SampleTable should have one lineand the method should be thesame as used during the analysis. • Enter the acquisition window ofHyStar with this line. • The Acquisition window must openand HyStar must show green status. • The MS control software must correctlyopen and show green status. • If you cannot open HyStar/MS control withthe method selected for the measurements,STC will not be able either!

12. Prepare HyStar & MS Control • Start the program SampleTrack Communication. • Start in online mode = yes • HyStar will vanish in the background. • The program must start and show status ready for all parts.

13. Finish • All instruments are now ready to run experiments. • If the any experiment start immediately • you have samples in the Liquid handler, NMR sample changer or LC-MS changer which still have open order in SampleTrack. • Go to page Prepare SampleTrack PC and check if the listed orders are still wanted. Either delete the orders or remove the rack with the corresponding samples from the instruments.

14. Process Samples Dr. Ulrich Braumann Bruker Biospin, Rheinstetten

15. Setup Orders - manual • Have the barcoded rack/wellplate with the samples ready. • In SampleTrack do File/New • Enter the ID of the tay (upper text like “RCK000183”) • Enter the geometry identification of the tray. • Select the sample position in the tray from the displayed layout. • Select the “supermethod” that defines the complete process from the preparation to the measurment in the NMR and LC/MS • If required customize the supermethod, for example to disable certain NMR experiments or disable the NMR or MS measurement part completely. • Note: Disabeling the prepartion is not possible, as this also includes the assigment of the ID of the original sample to the position of the SampleJet tube/LC-MS well.

16. Sample Preparation • Open the door of the hood • Place the tray with the samples into the liquid handler. • Ensure that an unused rack with SampleJet tubes and a unused wellplate for MS aliquot is available. (Note: If a number of samples already were prepared from the source rack, the original destination rack(s) must be available. I.e. you may have to return them from the SampleJet or LC-MS autosampler) • Close the door of the hood • The liquid handler must start scanning the barcodes to identify the new samples. • The liquid handler will look for available destination racks. • The preparation will start automatically.

17. Sample Preparation • The destination for a sample preparation will be automatically determined by sample track. • Samples will be prepared symmetrically from source to destination.Sample #1A1, #2A2, #13 B1 etc (i.e. not submitted sample position will remain empty in the destination). • The destination rack will remain reserved for the source rack. • As consequence. • You can remove a not completely filled destination rack from the liquid handler to start the NMR or LC-MS measurements. • The preparation will continue into another free rack (if a suitable rack is found). • However the empty spaces in both destinations racks are not available for other source racks!

18. NMR Measurement • Remove samples from the liquid hanlder • Open the door of the hood. • Remove the a rack with the SampleJet tubes. • Close the door of the hood. • Note: If the preparation of one rack is not completely finished, the preparation will pause until the original SampleJet rack is back in the liquid handler! • Insert the tray into the SampleJet • Ensure that current shuttle diameter matches the samples tube diameter. • At the SampleJet touchscreen select “Load rack”. • Select the position and place the rack into the caroussel. • Confirm with done. • The SampleJet starts automatically to read the barcode of the rack • ICONNMR will start executing the NMR experiments for the found samples.

19. LC-MS Measurement • Remove samples from the liquid hanlder • Open the door of the hood. • Remove the a rack with the SampleJet tubes. • Close the door of the hood. • Note: If the preparation of one rack is not completely finished, the preparation will pause until the original SampleJet rack is back in the liquid handler! • Insert the tray into the LC-MS autosampler • Submit the ## name of the wellplate. • The orders for this wellplates will be take from SampleTrack and will start automatically. • It is recommended to run 1-3 blank runs before the first real sample, to equilibrate the LC-MS system.