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FC-MS from Teledyne Isco. Combi Flash ® a Name You Can Rely On. Goals:. Sources of issues Approaches to resolving problems Overview of “chemical” issues. Troubleshooting: Divide et imperia !. I have a problem!. I don’t see my peak!!. “Chemical”. Mass Spectrometer. Fluid Interface.

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FC-MS from Teledyne Isco

CombiFlash®a Name You Can Rely On


Goals:

  • Sources of issues

  • Approaches to resolving problems

  • Overview of “chemical” issues


Troubleshooting:Divide et imperia !


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 List


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]


Compound won’t ionize


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!


Mismatched tune files- one symptom:

239


Key Markets

PharmaceuticalDrug Discovery

NaturalProducts

AgriculturalChemistry

Foodsand Flavors

Petrochemicals


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