Chromatography Forum

Hello,

I am searching for help. Could anyone please tell me, if it is possible to detect aminoacids with UV-Vis detector after derivatization with OPA and FMOC and separation on AminoQuant column (Agilent)?
What are the problems of this method?

Thank you very much

Since a molecule has to absorb light (UV here) to fluoresce you can certainly also use absorbance (UV) for detection. Fluorescence will be more sensitive, that´s all.

Dear Tumbir,

I do not quite understand your question. You seem to be using AminoQuant columns and Agilent equipment. The AminoQuant systems was develoiped especifically for Amino Acid analyses. Theerefore, yes, it is possible to do it.

Perhaps your question is more related to the problems of this method. There are many. I used the system and method for over 6 years and I can tell of many difficulties. If you specify more your question(s), perhpas I can be of help.

Let me know,

josebenjamin

It might be instructive to get more details on the problems. We gave up on OPA (a single amino acid in cerebrospinal fluid was supposed to be analyzed, the OPA produced several peaks, among other things which I forgot), even with FMOC we never got rid of one (probably) interference, though that was the way to go (another problem that was not finished, in that case, because I quit that lab).
(This was not based on a manufacturer`s example)

Dear JoseBenjamin and HW Mueller,

Thank you very much for the answers.

The fact is that I am going to introduce amino acid analysis with AminoQuant in our laboratory. We have only limited resources and only UV-Vis detector, not fluorescence. The manufacterers write, of course, that their method is the best one and that I can detect 24 amino acids in urine, plasma and cerebrospinal fluid. But in the internet I can find, that there are problems with the detection of cysteine and thryptophan, and that the actual number of the amino acids detected is 16. And that there are a lot of interferences in urine samples.
Could you please tell me, if you do not work any more with this system, then why?

Sincerely

tumbir

Dear Tumbir,

I do not remember having problems with Trp analyses. For Cysteine and Cystine there are special procedures when you use AminoQuant technology.

If you do not have it, I recommend you get a copy of the AminoQuant Manual. There you will find all the conditions, mobile phases, reagents, some hydrolyses techniques, and the special procedures for S containing aminoacids.

The AminoQuant technology works reasonably well, but it is not a simple one to implement, and is not applicable to all cases. Also, it is not a rugged method, in other words, the separation and analysis is somewhat delicate. Some AAs where not specifically incorporated in the separation, but the retention of many of the unusual ones are studied in the manual.

There are no problems when using Aminoquant with Uv-Vis detection, and just in case you do not know, you have to use preferably Agilent, reagents, and standards.

Good Luck,

Josebenjamin

Hi Tumbir

In the pathology lab I used to work in, we had both a dedicated amino acid analyser (Biochrom - ion exchange, ninhydrin) and a couple of HPLC's running the Waters Picotag method (phenylisothiocyanate, UV). There were always issues with covering all the bases with the Picotag method, citrulline runs with ammonia, homocystine and cysteine-homocysteine disulphide are both poorly resolved from other amino acids. We ran the bulk of our workload by Picotag but any time we were interested in the problem amino acids we ran the samples on the Biochrom.

Also, running urines on any system that uses UV (or, I suspect fluorescence) detection will be a nightmare due to the large number of UV absorbing compounds present in urine. We always ran our urines on the Biochrom.