Chromatography Forum

Hello to everyone.

I'm very new to LC analyzes, and now I'm trying to create a method for the analyzation of protein hydrolysates.

Since I don't have a fluoresence detector, I'm stuck with the PITC-method. I created my very first standards, from a standard solution of 1mM (of each AA) in 6M HCl. I chose this, because I'm going to do the hydrolysis with the HCl method, so I thought the standard should be in the same matrix.

But alas... when I did the first run, I had very strange results (read: only like five clear peaks and the standard should contain 20 aa), and now I don't know where to start the problem solving. I diluted my standard in 1:100. Here are my LC setup and the method I used for the derivatization of the AA standard:

Restek Pinnacle DB c18 - column, in +40c
flowrate 1 ml/min
Detector at 254nm
Buffer gradient from A to B, in 25min

Buffer A:
38g Na-Acetate
2l mQ-H2O
1,1ml TEA (Triethylamine)

Buffer B:
60% Acetonitrile

Derivatization:
Take 25ul of your sample in HCl (standard in this case) and dry it using Nitrogen gas (I don't have a vacuum exicator...). Resuspense the sample into EMT (Ethanol, mQ, TEA 2:2:1 vol/vol/vol) and dry it again. Repeat. Resuspense the sample into freshly made PEMT (PITC, EtOH, mQ, TEA, 1:7:1:1 vol/vol/vol/vol). Let it react in rt for apprx. 20min and dry it again. Resuspense the sample into 400ul of Buffer A and transfer the sample solution into a 1,5ml vial and seal it. Begin HPLC analysis

I'd be very happy if you could point me to my flaw, or to the direction where to look for problems. And what would you recommend for the consentration of the standards?

Thank you in advance

Heikki Laine

Does your buffer A have a pH specification? As described, it will be about pH 10.5, and this is probably not what you want.

You can estimate the concentration of amino acids in your samples from the percent protein, and assume that the average MW of the resulting amino acids is around 120. If you know the expected amino acid profile, you can compute the theoretical amino acid concentrations. Then make your top standard 1.5-2x higher than the highest expected concentration in your samples. Build your calibration set from there down to the lowest expected concentration. You don't need to have all the amino acids the same concentration in the standard; you can approximate the profile of your samples.

Sorry, I forgot to mention. I've adjusted the pH with Acetic Acid down to 5,93.

I'm going to repeat the standard derivatization today, so I'll post if anything new comes up..

It seems that I made a mistake in the derivate process. I got very good results today. Now I'm interested in how to recognize the peaks/amino acids from the chrom.gram.

My supplier for the standard didn't supply me with one, so I tried to find standard retention times for different aa but didn't find anything conclusive. If you have any tips, I'd appreciate it.

Heikki

Heikki,

Here are two references which should help you out.

Journal of Chromatography, Biomedical Applications, Vol. 431,
pp. 271-284 (1988)

Journal of Liquid Chromatography, vol. 11 (3), pp. 613-646 (1988).

You could also try contacting Waters (or other vendors) for an application bulletin for PTC-amino acid analysis. Good luck!

You can identify the peaks from individual standards, but so that you don't have to make 20 derivatizations try this. Lay out your individual standards in a 4x5 grid. Make a mixture from each column and each row; you will have 9 vials. Analyze the vials. Overlay pairs of chromatograms; there will be one common compound in each pair, and the corresponding peaks should overlay each other.

In fact, you will never find asparagine nor glutamine in protein hydrolyzates because they hydrolyze to aspartate and glutamate plus ammonia. Acid hydrolysis also destroys tryptophan; you need a special alkaline hydrolysis for that. It is common to see partial oxidation of methionine to met-sulfoxide and met-sulfone, and sometimes cystine to cysteic acid. It can be hard to decide if that is oxidation of the protein, or an artifact of the hydrolysis. So you can see that the list of amino acids is not the same one from your biology textbook.

@Mark: Thank you for your tipp with the grid. I'll also going to implement a AA method. Maybe I will derivatize with DABSYL.