Mass Directed Purification from Teledyne Isco

1. Mass Directed Purification from Teledyne Isco CombiFlash®a Name You Can Rely On

2. What to say when the customer says: “I don’t see my compound” This is NOT the best response: “Let me see if Jack is still in Lincoln”

3. Goals: • Sources of issues • Approaches to resolving problems • Overview of “chemical” issues

4. Troubleshooting:Divide et imperia !

5. I have a problem! I don’t see my peak!! “Chemical” Mass Spectrometer Fluid Interface CombiFlash System “Stupid” items

6. Start with the “stupid” items! • Error Message on Rf? • Carrier solvent bottle full? Primed? • Correct carrier solvent? • MS enabled? • Chemistry • Probe gas lines? • Probe? • Clean cone? • Nitrogen pressure?

7. Flowchart Got spectra ? Check flow into probe “Stupid” things Method Development injection Fixed? Yes Yes • Chemical issue • Clog before or in MRA valve Done!!

8. Inject compound(s) known to work- MS injection • Method development screen • Verifies MS working • Verifies most functions of Fluid Interface • Limits location of clog (if one exists)

9. Trouble shooting flow chart Flow = 0.2 mL/min? Works now? Clean probe and test injection again Check flow into probe Yes No Done!! • Clog between pump and probe • Leak between pump and probe Run known sample on CombiFlash

10. MRA- Continued • Check flow into probe- 0.2 mL/min • Check Pulse Dampener pressure reading- verify flow

11. Mass Spectrometer • Use 0.5 or 1 mL glass syringe • Fill with fluid, push fluid through probe • This cleans clogs, make sure flow is straight (if not, bent tip)

12. Trouble shooting Sample run Ok? Run known sample on CombiFlash Clean Capillary Fixed? No No Call Tech Service Fix CombiFlashissue

13. Run known sample on CombiFlash • Last of the “simple things” • If this works, system working fine • Even Test Mix A verifies the chromatography system works

14. MS- Capillary • Shut down MS completely • Turn off roughing pump • Remove cone- watch o-rings!!! • Use 1/16” HPLC fittings • Use methanol to purge plug • Sonication in MeOH or DCM may be needed

15. CombiFlash Error Messages • The Mass Spectrometer inlet cone may be plugged which could prevent detection of mass signals. Continued operation will not cause damage. Contact a service technician to perform a cleaning procedure. • System sees high vacuum, faster turbo speed • Clean cone • Shut down MS, clean capillary

16. Chemical Issues- weak or no Molecular Ion • Compound doesn’t ionize • Needs different ionization mode • Compound breaks down • Compound forms adduct [M+MeOH + H], [M+Na]

17. Case Study • λmax 200-220 nm • [M+1] = 141 Da expected • Solvent system Hexane/EtOAc • User advised to use a range of masses, would get weak peak @ m/z 123

18. Chemical Case Study Not charged- not seen. Major product (loss of another hydrogen between methyls Very minor product- charged, m/z=122 or 123 (depending on loss of H) H+ binds to non-bonding electrons on oxygen

19. Good leaving groups • -NH2 (leaves as ammonia) • -OH (leaves as water) • COOH (leaves as CO2) • Look for increase in conjugation • Look for easily formed, stable molecules

20. Adducts • Bond to the molecule- usually detected as [M+H+Adduct] • May be more intense than [M+1]

21. Adduct List

22. Adduct example & sources • Solvent • Glassware • Syringe

23. Adducts- potential confusion • Sample dissolved in methanol- use method development, [M+MeOH+H] observed. Purification in hexane/ethyl acetate- will you see adduct? • Sample run on LC-MS mobile phase water/MeCN, [M+MeCN+H] observed. Purification in hexane/ethyl acetate- will you see adduct? • Use a range that covers [M] through [M+adduct]

24. Compound won’t ionize- use different probe?

25. Ionization • Use method development screen, use positive or negative mode • Try APCI (if using electrospray) • Carrier solvent?

26. Routine maintenance • Cleaning cone • Vacuum pump oil • Detector (Tech service task- after at least a year)

27. Cleaning cone • Put MS into Stand-by • Wait 5 minutes for cool-down • Unplug

28. Cleaning Cone • Remove probe • Remove housing

29. Clean the cone • Clean the cone with solvent and a wipe • Note o-ring & plug • Replace housing

30. Replace probe • Replace probe- note switch on top of housing • Replace probe carefully so tip isn’t damaged!

31. Tubing • Desolvation gas • Nebulization gas • APCI gas (APCI probe) • Plug in probe electric • Replace solvent inlet line • What could possibly go wrong?

32. Tuning • Tuning files on CombiFlash system • Contain probe/analyzer/detector parameters for a particular MS unit • MS and CombiFlash are “paired” • Don’t swap an CombiFlash without its MS!

33. Mismatched tune files- one symptom: 239

34. CombiFlash Error Messages • The CPU in the instrument is not powerful enough to support the Mass Spectrometer. The Mass Spectrometer will not be used. • Upgrade to new mother board • Someone “borrowed” the mass spec from another lab? • Please turn the Mass Spectrometer Roughing Pump • Occurs on start-up- pump off, unplugged • Loose/ broken vacuum line • Damaged Pirani probe? (Tech service)

35. CombiFlash Error Messages • Mass Spectrometer Turbo Pump starting • Rebooting when MS connected, spooling up the Turbo pump, pumping down • Should show time remaining • The mass spectrometer has been shutdown it will not be available until the instrument has been rebooted • Someone pressed the “shutdown” menu item for the MS • Unable to communicate with the CMS • USB between MS & MRA unplugged • One of the plugs damaged • Damaged USB cable • Mass spectrometer issue

36. CombiFlash Error Messages • An Unspecified CMS error has occurred. The CMS will be unavailable. • Applies to MS • Check USB plugs, cables • Most likely a Service Call • The MS Tune files must be installed before the MS can be used. The MS will be disabled until the files are installed. • Not using the correct CombiFlash • Files not installed during installation • Unable to communicate with the MRA valve. • Most likely internal to the fluid interface • Check for loose wires inside fluid interface • Check USB cables, plugs

37. CombiFlash Error Messages • Unable to communicate with the CMS fluid interface module. • Usually lost USB connection to fluid interface from CombiFlash • Check plugs, USB cable • The ion source is not connected to the CMS. Please connect the ion source in order to continue. • Probe not plugged in

38. CombiFlash Error Messages • The MRA valve seals have exceeded their recommended life. To prevent potential damage to the instrument, please contact a qualified service technician immediately • Fluid switching valve needs service • A plug has been detected between the fluid interface inject valve and the MS or in the MS nebulizer. The separation can be continued without MS detection, or the plug can be cleared before continuing • Overpressure error • Need to track plug • Make sure inject handle in proper position

39. CombiFlash Error Messages • The Mass Spectrometer inlet cone may be plugged which could prevent detection of mass signals. Continued operation will not cause damage. Contact a service technician to perform a cleaning procedure. • System sees high vacuum, faster turbo speed • Clean cone • Shut down MS, clean capillary

40. CombiFlash Error Messages • MS capillary temperature is [r]C, set point is [r]C. • MS gas temperature is [r]C, set point is [r]C. • Check electrical connection to probe • Tech Service call

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