FC-MS from Teledyne Isco CombiFlash®a Name You Can Rely On
Goals: • Sources of issues • Approaches to resolving problems • Overview of “chemical” issues
I have a problem! I don’t see my peak!! “Chemical” Mass Spectrometer Fluid Interface CombiFlash System “Stupid” items
Start with the simple things! • Carrier solvent bottle full? Primed? • Correct carrier solvent? • MS enabled? • Chemistry • Probe gas lines? • Probe? • Clean cone? • Nitrogen pressure?
Flowchart Got peaks? Check flow into probe “Stupid” things Method Development injection Fixed? Yes Yes • Chemical issue • Clog before or in MRA valve Done!!
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)
Trouble shooting flow chart Flow = 0.5 or 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
MRA- Continued • Check flow into probe- 0.2 mL/min or 0.5 mL/min • Check Pulse Dampener pressure reading- verify flow
Mass Spectrometer • Use 0.5 or 1 mL glass syringe • Full with fluid, push fluid through probe • This cleans clogs, make sure flow is straight (if not, bent tip)
Trouble shooting Sample run Ok? Run known sample on CombiFlash Clean Capillary Fixed? No No Call Tech Service Fix CombiFlashissue
Run known sample on CombiFlash • Last of the “simple things” • If this works, system working fine • Even Test Mix A checks Rf-200
MS- Capillary • Shut down MS completely • Shut 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 too
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]
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
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
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
Adducts • Bond to the molecule- usually detected as [M+H+Adduct] • May be more intense than [M+1]
Adduct example & sources • Solvent • Glassware • Syringe
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]
Ionization • Use method development screen, use positive or negative mode • Try APCI (if using electrospray) • Carrier solvent?
Routine maintenance • Cleaning cone • Vacuum pump oil • Detector (Tech service task- after at least a year)
Cleaning cone • Put MS into Stand-by • Wait 5 minutes for cool-down • Unplug
Cleaning Cone • Remove probe • Remove housing
Clean the cone • Clean the cone with solvent and a wipe • Note o-ring & plug • Replace housing
Replace probe • Replace probe- note switch on top of housing • Replace probe carefully so tip isn’t damaged!
Tubing • Desolvation gas • Nebulization gas • APCI gas (APCI probe) • Plug in probe electric • Replace solvent inlet line • What could possibly go wrong?
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!
Key Markets PharmaceuticalDrug Discovery NaturalProducts AgriculturalChemistry Foodsand Flavors Petrochemicals