Chromatography Forum

I am working on another HPLC project to do amino acids and taurine. At the very least I would like to be able to do Arginine, Serine, Threonine and Taurine but doing them all in one shot would be a bonus

I have read a bewildering number of papers and app notes most of them on OPA+FMOC for proline. I am aware of Waters Accutag though that is also special column plus expensive reagents. So far I haven't seen one paper where they are able to get separation of all the amino acids without a special column, the closest I have found is an Agilent App note that uses a Poroshell C18 but a special high pH column because the separation is at pH >8.

I haven't seen really anything on using FMOC alone without OPA.

I am waiting for some 3-mercaptopropionic acid for the OPA method I played with in needle derivatization and OPA (had some) and dithiothreitol (which was a bust I got 2 peaks per amino acid and a mess) and propyl mercaptan I got separation of a lot of them. Does anyone have or seen a protocol that works on a normal C18 column and separates them all?

we have this method on one of our columns, please send me an inquiry to mail(at)helixchrom.com.

Here is our study for these amino acids. It is not RP with UV. This is our mixed-mode HILIC cation- and anion-exchange approach with ELSD. You need CAD, ELSD, MS or RI for taurine since it has almost no UV activity:

https://www.linkedin.com/posts/vlad-orl ... esktop_web

I have pretty good separation on an Agilent Poroshell InfinityLab Poroshell 120 EC-C18 4.6 x 100mm 2.7 micron using OPA and 3-mercaptopropionic acid.

My mobile phase is A 0.1N Na2HPO4 acidified with citric acid to pH 6.38
30 minute gradient of 5%ACN/5%MeOH to 25/25%.

I failed to get anything with cysteine and didn't see much from cystine either. Agilent mentions a dithio-dipropionic acid (DTDPA) reagent for cysteine but doesn't mention anything about what it does nor how to use it. That would be the dimer form of 3-mercapto propionic acid. Waters mentions disulfide interchange so cys and cys-cys forms cys-3-mpa

The literature doesn't do a very good job explaining what to do with cysteine and cystine.

Agilent sells the dithiodipropionic acid DTDPA but doesn't state how to use it. From what I can tell it should be added during an acid hydrolysis and converts both cyseine and cystine to cysteine-s-s-3-mercpato propionic acid.

The other options are hit it with iodoacetate to convert cysteine as s-carboxymethyl cysteine.

The final option is to add an oxidant H2O2 perchloric acid and convert cysteine to cysteic acid.