Chromatography Forum

Which column would you recommend for the analysis of the following amino acids ?

GABA, Methionine and homoserine ?

Would ideally like an LC/MC compatible method or something I could run at very low wavelength.

TIA

Stanlee

Hi, Stanlee

You can use Primesep 200 with ACN-water-ammonium formate (around 10-30 mmol).

Here are the links
aminobutiric acids (three isomers alfa, beta, gamma)
http://hplcmethods.com/compound_143.php

Methionineand other aminoacids
http://hplcmethods.com/compound_064.php

What are your requirements to analysis (LOD, run time, resolution). I will check if we have homoserine in the lab. If we do we will develop a method for your-free of charge.

Contact us if you have more questions.

Regards,

Vlad

Run time not an issue so LOD and resolution would be the key critieria.

I have contacted a colleague who has a Primesep 200 that I can borrow but any starting point for method development would be great.

Stanlee

Stanlee,

You might need 150 mm column to have more room to play. If you have 50 mm it is fine too.
Starting point ACN-water-ammonium formate, you can either premix solvents or use three channels of your LC system:
A: ACN
B: Water
C: Ammonium formate 100 mmol at pH=3
Start with 20%A, 60% B and 20% C. This will create 20/80 ACN-water with 20 mmol of buffer. Using three channels allow you to create various mobile phase and do method development much faster.

Make sure that you equilibrate column after previous unknown runs. Column has certain buffering capacity. If you provide us with your email I will send you information on equilibration of Primesep columns (or ask your colleague, he might already have one)


Run your samples separately to see where they elute. If you have co-elution or long/short retention, change your mobile phase; you can change amount of ACN, buffer concentration and pH of the buffer. Your compounds will react slightly different to mobile phase changes. Amount of ACN will have same effect as in RP chromatography; if you increase ion strength of the buffer your ionizable compounds will elute faster. You can also play with pH. pH will effect both ionization state of the phase and ionization state of compounds.

Check this link to see how mixed mode chromatography selectivity advantage:

http://www.nexep.com/Technology_2D_Properties.html

Let me know your results after a few runs and I can advise you on further development.

Regards,

Vlad

It would be better if stanlee would report his results here, since it has been made public. That way we all could finally find out whether mixed mode produces mode mix, or not.

Hi Stanlee,

I have GABA and methionine. I did not find homoserine in the lab but I have serine and alfa-methyl-serine. You can consider methylserine as good substitution as it has same amount of carbons and approximately same pKA. I hope that we will develop a method for you-actually I am thinking about two methods one for UV (phosphoric acid or phosphate buffer) and another one LC/MS compatible (ammonium formate or formic acid). I will post the link as soon as we develop these methods.

I hope that this will convince Hans too.

Will let you all know how I get on once the column arrives !

Stanllee,

Here is two methods we developed for you. Homoserine will elute the same way serine and alfa-methyl-serine elutes; you have enough resolution to accommodate slight change in properties (which might exist for homoserine vs. serine)
As you can see retention time can be adjusted by both amount of organic and amount/nature of buffer. One method is UV compatible with ACN-water-phosphoric acid, another one is LC/MS/ELSD compatible with ACN-water-formic acid. You can also use ammonium formate instead of formic acid. With ammonium formate you can create higher ion strength mobile phase and elute your amino acids faster.

Also note that order of elution changes for GABA and methionine when you switch from phosphoric acid to formic acid.

http://hplcmethods.com/application_114.php (if link does not work cut and paste)



P.S. Hans I just wondering if this is enough “…we all could finally find out whether mixed mode produces mode mix, or not.â€

Good, you can do standards . . . ., at what amount/injection? What sort of equilibrium times did you need to get a stable baseline at 200nm? Or are these baselines corrected in some way?

Sielc, why did you change from the 200 to the 100 ?

I have only arranged for the loan of the 200.

Will the separation work on the 200 ?

Thanks
Stanlee

Stanlee,

Will do PS-200 today, I could not find PS-200 (it is like one proverb about shoemaker who never has his own shoes because he is busy fixing other people shoes).

I'll update you soon.

Vlad

Stanlee,

Can you provide me with your email address-I would like to share Primesep 200 results with you.

Regards,

Vlad

Vlad,
answering the questions I asked could be of some help, but if you want to do more, here is a repeat of a former suggestion: Send me your column(s) for testing in this lab.
Or an improvised problem (partially mentioned somewhere else): Dissolve a polar amino acid in a huge excess of bmimOTf (butylmethylimidazolium triflate) and find (separate) the amino acid using only UV detection.
Another idea: Get a bit of plasma from a hospital or a physicians office near you and analyze it for Pistek´s (post Sept. 5) compounds.

That might help to diminish the tarnish created by the impression that mere advertising is behind your posts of disputed relevance.

Hans,

Matrix can have effect on separations and we know that. We developed two methods one UV and another one LC/MS, UV can bi tricky due to matrix effect, quality of solvents and reagents, etc.

If you are analyzing complex matrix ussually some sample prep is required. If you use a guard column for this application (weaker column then PS-100, for example PS-C), you can trap proteins and then wash guard backwards during your analysis, or just descard guards after you plug it

http://www.sielc.com/pdf/SIELC_September_2004.pdf.

We provided a lead for the separation and optimization is up to chemist. There are bunch of tools to decrease effect of matrix (SPE, precipitation, etc.)

We are running in cycles, but, nevertheless, let me repeat: Determining ascorbic acid in tomatoes was a breeze, doing the same in blood produced so many problems that I gave up. Or take a peek at J Chrom B, 678, 137 (1996), even at the third step of chromatography the matrix was so prominent that I am still proud of having, demonstratively, been able to be within about 10% of the true value.
Here another suggestion: Prove that you can determine cortisol in the blood of highly catabolic patients to within 1% of the true value (using your columns and UV detection).

Or, to make it short, good products sell themselves.

(Actually, it would be very pleasing if my questions from above, were answered.)