Chromatography Forum

Hi All,

I have to develop a HPLC method for the analysis of an amino acid (no chromophores) in a peptide sample as an excipient (as a stablizer). If I have to develop a method without any pre-column derivatization, will I be able to use a RI detector?. I know RI detection is great for sugars but will I able to detect and quantify an amino acid based on RI of the amino acid, assuming I can find a RP-HPLC column and m.phase conditions to sepearate the amino acid from the peptide?.

Thanks in advance for your thoughts.

Ananda

How large is your peptide?

Amino acids by RI will be very tough, first off RI is not the most sensitive mode of detection. Second, and probably the bigger issue, your method would have to be an isocratic one. RI detectors don't like gradients much. All of the AA work I have ever done has employed a gradient. You will be way better off doing a simple pre column derivitization and anayze using UV or FLD with a reverse phase gradient. Underivitized amino acids can be done using ELSD or MS if you really have some aversion to making the derivitive.

Thanks for your valuable comments AA. My peptide is ~ 10kD. I dont have much experience in using RI detectors so your comments are great. So you mean RI is not a god option but pre-column derivatization will be better option. Thanks.

Ananda

For this kind of application, RI or ELSD would be fine. The stabilizer is in very high concentration, and derivatization would require a huge dilution. Since you only want one AA, you can find isocratic conditions for it. Just let the peptide build up until it is a nuisance, then flush it out.

By the way, is your amino acid histidine? That seems to be a popular stabilizer for protein drugs.

Hi Mark,

Thanks for your comments. Can you recommend any columns that I can use without derivatization?. AA is I am intersted using is Methionine, an antioxidant.

Ananda

How about ultrafiltration?
Methionine is relatively unpolar, my guess is that it should be retained well in RP.

I was able to retain methionine on our PolarAdvantage or PolarAdvantage-II columns using 5% MeCN + 0.1% TFA. Retention time was 3.4min on a 150mm column. With a gradient, you can do your peptide too!

With mix mode columns you can retain methionine for 10 minutes even with 20-40% ACN. You can use BLIS (Buffer Less Ion Separation) and elute methionine in 8010 minutes with ACN-water. I will work as well as low UV (195-200 nm):

http://www.sielc.com/compound_064.html

The gradient in first method used to elute amino acids with two amino groups. In your case it is easy because you have only one amino acid.

SIELC Tech,

Even if you can detect the AA, at that low wave length, how about the noise level and also the peptide biding to the column?Should I be able to use your column to detect both the peptide and the AA or peptide binds to the column and it will affect the function and the life time of the column?
Thanks
Ananda

Your peptide will bind to the column. You can use the same guard column and trap peptide without effecting determination of you amino acid.
How many amino acids your peptide has? How many basic sites? If it is too big and too basic, you can trap it with the guard and wash the guard during the analysis. PLease see following link about use of the guard for trapping:

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

If you use pure reagents you should not have problem with detection-methionine has some UV acivity.

Thanks a lot for your suggestions. It look like a doable thing.

Ananda