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Mass Directed Purification from Teledyne Isco. Combi Flash ® a Name You Can Rely On. 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”. Goals:. Sources of issues Approaches to resolving problems

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Mass directed purification from teledyne isco

Mass Directed Purification from Teledyne Isco

CombiFlash®a Name You Can Rely On


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”


Goals
Goals:

  • Sources of issues

  • Approaches to resolving problems

  • Overview of “chemical” issues


Troubleshooting divide et imperia
Troubleshooting:Divide et imperia !


I have a problem
I have a problem!

I don’t see my peak!!

“Chemical”

Mass Spectrometer

Fluid Interface

CombiFlash System

“Stupid” items


Start with the stupid items
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?


Flowchart
Flowchart

Got spectra ?

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
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
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


Mra continued
MRA- Continued

  • Check flow into probe- 0.2 mL/min

  • Check Pulse Dampener pressure reading- verify flow


Mass spectrometer
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)


Trouble shooting
Trouble shooting

Sample run Ok?

Run known sample on CombiFlash

Clean Capillary

Fixed?

No

No

Call Tech Service

Fix CombiFlashissue


Run known sample on c ombi flash
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


Ms capillary
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


Combi flash error messages
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


Chemical issues weak or no molecular ion
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
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
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
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
Adducts

  • Bond to the molecule- usually detected as [M+H+Adduct]

  • May be more intense than [M+1]



Adduct example sources
Adduct example & sources

  • Solvent

  • Glassware

  • Syringe


Adducts potential confusion
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
Ionization

  • Use method development screen, use positive or negative mode

  • Try APCI (if using electrospray)

  • Carrier solvent?


Routine maintenance
Routine maintenance

  • Cleaning cone

  • Vacuum pump oil

  • Detector (Tech service task- after at least a year)


Cleaning cone
Cleaning cone

  • Put MS into Stand-by

  • Wait 5 minutes for cool-down

  • Unplug


Cleaning cone1
Cleaning Cone

  • Remove probe

  • Remove housing


Clean the cone
Clean the cone

  • Clean the cone with solvent and a wipe

  • Note o-ring & plug

  • Replace housing


Replace probe
Replace probe

  • Replace probe- note switch on top of housing

  • Replace probe carefully so tip isn’t damaged!


Tubing
Tubing

  • Desolvation gas

  • Nebulization gas

  • APCI gas (APCI probe)

  • Plug in probe electric

  • Replace solvent inlet line

  • What could possibly go wrong?


Tuning
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!



Combi flash error messages1
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)


Combi flash error messages2
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


Combi flash error messages3
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


Combi flash error messages4
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


Combi flash error messages5
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


Combi flash error messages6
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


Combi flash error messages7
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


Key markets
Key Markets

PharmaceuticalDrug Discovery

NaturalProducts

AgriculturalChemistry

Foodsand Flavors

Petrochemicals


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