August 10, 2022, 07:02:03 AM
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Topic: Following reactions via GC/MS or LC/MS vs TLC or spectrophotometry  (Read 355 times)

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Offline Babcock_Hall

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I am interested in how to decide when use GC/MS or LC/MS to follow a reaction, not TLC (spectrophotometry is not always applicable).  Put another way, what can one learn from chromatography coupled to MS that one cannot using TLC.

One advantage I can see is that chromatography plus mass spectrometry identifies compounds, whereas without standards, TLC does not.  A second advantage is that TLC is hard to quantitate.  Quantitation by GC is possible provided that one supplies an internal standard and does a calibration curve.  LC might provide quantitative data, but I am less well read in quantitative LC (most of my LC experience is with modified vs unmodified proteins, and we could simply use peak areas assuming that the molar absorptivity of the protein didn't change).  TLC can run into problems when there is little change in Rf from reactant to product or when unusual stationary phases are needed.

Against these advantages are some disadvantages.  The first is that one needs to have methods for either GC or LC, and one also needs an appropriate column (it is difficult to see how one column fits all).  There is developmental time and material.  One may or may not have the product to use as a standard.  GC may require that compounds be made more volatile by conversion into derivatives.  I imagine that industry could have people and facilities that are dedicated to solving problems of these kinds, and I would be interested in knowing more.  Yet I am also interested in how academic labs deal with these issues.  I realize that this is an open-ended question.
« Last Edit: July 23, 2022, 10:01:31 AM by Babcock_Hall »

Offline kriggy

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I like to use all / more of them and combine the information. I work in an academic synthetic lab doing custom radiolabeling for our institute and others so we commonly move from one project to another and while we are at an acadmic institution our work is more like in a CRO so keep that in mind because other perspective might be different. Also I think you summarized the differences/advantages it really well.

TLC is quick, simple and cheap. No reason not to run it because you at least see if your starting material is consumed and new compounds are formed. By use of various TLC stains you can somehow identify your spots. I could run radio-TLC as well when doing labeling but we tent to not to because of the associated dangers. We prefer to run radio-HPLC because you dont end up with capillary filled with radioactive solution and you can use the vial for HPLC-MS or do scintilation counting.

Because of various works we do we have maybe 20 different LC columns from C4 through C8, phenylhexyl, multiple C18 to hilic therefore HPLC method developement is simple. ALso, each one of my colleagues and me have preffered go-to column when starting new method. Im pretty sure we could run most of our samples on C18 or the phenylhexyl (my fav :D) and the peptides on C4 or C8. We dont have UPLC therefore our runs are like 60 minuets therefore we always do TLC first if possible.
GCMS is for the stuff thats not suitable for LC: to name a few we did: small aliphatic amines,  silyl ethers, benzyl chlorides, ant pheromones.

We dont do calibration curves since we are not an analytical lab but we also developed an analytical method for separation and quantitaion of adenosine phosphates but thats not common. We just integrate the peaks to get rough idea how much stuff is there but if its 60 or 80% is usually not much difference.

I spent some very short time in industry RnD as well. We had an analytical department that developed methods for projects They had maybe 5 GC and LC systems.  I just prepared the sample based on the method - much more rigorous that I dn at my job (=take small aliquot of reaction into ACN/water and dump it into LCMS) - but we got detailed analytical data esp. quantitation of products in the reaction. THe disadvantage was that it took significant time because they were quuite busy therefore I submit sample after 60 minutes of reaction which then asks to either add reagent or quench based on the results but I get results the next day...

TLDR: It all depends on various details but: TLC always then LCMS or GCMS. You can run 90% of analysis on  universal column using maybe 3 universal gradients (slow, normal, fast). Develop specific method based on your needs and situation (developing  method for a day if you have 1 LCMS in lab for 20 ppl wont make you any friends)

Offline wildfyr

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For LC C18 is by far the most common and flexible column, but its not one size fits all.

GCMS only works for lowish MW materials but its pretty fast and not too too hard to sort out the data usually

LCMS is by far the most useful, but always requires the most time and expertise. Its not a job for dribs and drabs. If I understand right, you work with undergraduate researchers and do a lot of work yourself. You will have to be the expert, untangling mass spectra is a very learned skill.

In both cases, you typically develop a few work horse methods that work for 80%+ compounds. Right now I think our analytical chemist uses the same method for like 7 totally different compounds I ask for GCMS on at various times.

Offline rolnor

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We used the same column for all, 99%H2O-1%CH3CN gradient to 1%H2O-99%CH3CN. We used ammoniumformate as buffer.  it was just a C8-column. We ran very diverse compounds. Even triphenylphosphine oxide was detectable. With GC you can not run stuff like carboxylic acids.

Offline MOTOBALL

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With analytical chemistry, these are generally the options:

1) FAST

2) CHEAP

3) RIGHT

but the killer is that you can, GENERALLY, only have any 2 out of 3.

If you run TLC on microscope slides, you can monitor disappearance of starting material or appearance of products (or both if they are both non-volatile) in real-time (FAST & CHEAP).

Depending on the reaction (and the rxn medium) you may miss (volatile) product(s).

TLC on silica gel/microscope slides is wonderful for carbohydrate chemistry.

The generalized analytical probelm that you present  is the equivalent of "how long is a piece of string".
 

Offline rolnor

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Agree.

Offline Babcock_Hall

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Re: Following reactions via GC/MS or LC/MS vs TLC or spectrophotometry
« Reply #6 on: August 08, 2022, 10:33:01 AM »
Let me put a plug in for following reactions spectrophotometrically, especially when the reactions are slow (students are sometimes impatient).  This method can be used qualitatively to assess when the reaction is over (no change in absorbance over time).  Using it quantitatively is a bit tougher, but should be possible under some circumstances.  One can walk right up to a spectrophotometer, and they are generally user-friendly.  Following the disappearance of a TLC spot depends upon the sensitivity of the visualization method.
« Last Edit: August 08, 2022, 10:53:38 AM by Babcock_Hall »

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