Chromatography Forum

Hello fellow scienterrific researchers,

I'm working on an amino acid analysis method. Currently, I'm running a gradient method with a C-18 column:

Column: Atlantis C-18, 100 x 2.1mm, 5µm
MPA: 0.1 M Ammo acetate Buffer
MPB: 100% MeOH
Detection: UV-Vis (338nm)

and I'm preparing the samples by using automated pre-column derivatization with o-phthalaldehyde (OPA). I've only been looking at alanine and glutamine thus far, but I'm hoping to be able to see all the primary and secondary amino acids for future research needs.

I recently read a paper that discusses all the different AA analysis methods, including AA being derivatized with:

1. Ninhydrin (post-column)
2. OPA + Sodium hypochlorite (post-column)
3. PITC (pre-column)
4. AQC (pre-column)
5. DABS-Cl (pre-column)
6. FMOC-Cl (pre-column)
7. NBD-F (pre-column)

It seems to me that maybe the FMOC-Cl is the best method for HPLC with fluorescence detector capability, would you agree?

In the paper, it also discusses the importance of hydrolysis of the samples in preparation for the analysis, would you please share your thoughts on that?

Thank you!!

Hi and since this appears to be your first post on the forums, welcome.

I've seen this performed on a UPLC with UV detection and it hasn't been an issue. I've also seen this performed many years ago on an HPLC with a fluorescence detector and it worked well too. Just depends on what equipment you have.

As for the hydrolysis, this step is needed when the amino acid analysis method is performed to analyze the molar ratio of amino acids in a peptide or protein sample. The hydrolysis conditions (e.g. 6N HCl, 110°C for 24 hours) break the peptide bonds between the amino acids.

I would suggest in addition to your paper, you take a look at USP <1052> Biotechnology-Derived Articles - Amino Acid Analysis. A lot of good information there along with some different hydrolysis conditions/cocktails for protection of sensitive amino acids (e.g. Cys, Trp).

I would also suggest - depending on your financial situation - that you look into purchasing an amino acid analysis kit from either Agilent or Waters, the Eclipse and AccQ-Tag, respectively. Makes life very simple, in my opinion.

The kits are very good indeed. I've only used AccQ tag, but suspect that other manufacturers' products will also be of high quality. You don't need to use Waters special columns with AccQ tag if you don't want to, or even their buffers, but the derivatised samples are stable almost indefinitely, the derivatisation really works, and although pricey, the whole thing is just a guaranteed solution out of the box.

Thank you both!! your kind guidance has been really helpful!