Chromatography Forum

I am profiling amino acids in hydrolysates. Currently using mobile phase system:
sodium acetate trihydrate (pH 7.2) + TEA + TFA
acetonitrile+methanol+ sodium acetate trihydrate adjusted to pH 7.2(using glacial acetic acid) (10:10:5)

Derivatization using OPA and FMOC (online pre-column derivatization in the autosampler)

column: Zorbax Eclipse rapid resolution column (3.0mmx50mm, 1.8um)
Temp: 40C

Problems:
1) Frequent back pressure issues
2) varying areas when individual amino acids are injected
3) varying resolution times (run-to run) but relative positions of all 17 amino acids (in standard amino acid mix) remain the same.

(checked with the suppliers ...... no concrete solutions offered )

Any suggestions?????

With FMOC the reaction keeps going. So it is necessary to adjust protocol properly. Do you use stop reagent? Can ISTD be used? If 1.8 not crucial there are new columns over 2 and under 3 with excellente resolution. This should help with blocking.

Alexandre, what reaction keeps going with FMOC? are you talking about the small reaction with water (which didn´t bother anything in my case)?
I have always wondered why one would use OPA in conjunction with FMOC.
One can get FMOC amino acids which can be used as standards to work out the HPLC.

if you can afford to, you can change to using a commercial kit. We've used Waters' AccQ tag, which is ludicrously expensive but totally reliable, and very, very easy to use.

Hi everybody,

Replying to your posts:

lmh: I am using a commercial kit (agilent products- amino acid std mix, OPA, FMOC).
Further using the recommended derivatization method too.... (with some washing steps included)

Alexandre: No I have not included any internal stds. Will try it.....
No, I cannot shift to 2um or 3um .... recently purchased the 1.8um assuming it will be good for simultaneous separation of all amino acids (imagine having to run 5-6 runs of the same sample with different mobile phases to separate and quantify 17-18 aminoacids)

HW Mueller: Yes I did try out eliminating the OPA derivatization but I continue to have these problems. I get a lot of unexplained peaks when I use water (instead of OPA) to eliminate the dilution effect as it is a derivatization reaction which goes like :
1ul borate buffer+5ul OPA (I replaced this with water)+1ul sample+1ul FMOC

Any more suggestions???

I hate to do unpaid advertising work for Waters, but the AccQ tag is a different chemistry to FMOC, and is more reliable. Much more reliable. On the other hand, if you've got wandering retention times there may be something more fundamental going wrong.

There seem to be a million reliable FMOC methods out there. As already mentioned, there is also the tremendous advantage of being able to buy derivatzed amino acids.

There seem to be a million reliable FMOC methods out there. As already mentioned, there is also the tremendous advantage of being able to buy derivatzed amino acids.

I have been working with OPA and had trouble with my calibration. I got pretty good separation of all the amino acids though cystine was interfering with valine and tryptophan. It was all over the place. I think I need to increase the OPA amount

Anyways I am now trying FMOC and the derivatization seems to work well but the separation is generally poor. There is only a few literature sources an no app notes but I tried many variations of one listed to go from 10% ACN 40% MeoH 50% acetate buffer to 50%acetate buffer 50%ACN and another that just uses an acetonitrile gradient from 28-60%. So far there are many coelutions the FMOC-OH peak is a taily mess. I am currently using amino methyl propanol as my stop reagent though I may buy ADAM.

Does anyone have a separation protocol that works for FMOC?

So far my observations are
OPA/MPA
-Cannot do Proline, nor cysteine
-Separate fairly well
-calibration issues as I said I probably need to increase reagent and decrease sample ammounts
-Derivatives have short stability life though not an issue with in needle derivatization
-solution not stable either
-Many literature sources and app notes
-I do well with methanol gradient, Na2HPO4/citric acid ph 6.38 buffer. 10cm Poroshell C18

FMOC
-Can do proline cysteine and cystine
-has trouble with tyrosine and histidine
-solution in acetonitrile is stable
-derivatives stable
-derivatives very difficult to separate having issues with taurine and glutamate may have to settle for partial separation
-Not many literature sources have not seen any that get baseline separation of all amino acids.
-I use acetate pH 4.20 buffer with 0.1% triethylamine and acetonitrile gradient

After working with the methods quite a bit the best I have come up with is use OPA/MPA for everything except pro, cys, and cys-cys. Any cystine present can be reduced to cysteine with excess MPA in the sample? so it doesn't interfere with valine/trptophan. Mobile phase is 0.1N Na2HPO4 citric acid to pH 6.38 and methanol gradient 10-60%.

After trying almost everything with the FMOC amino acids on my 10cm 2.7um Poroshell EC C18, the best I got was A=0.1 formic acid/KOH pH 3.0 with 0.1% Et3N 25 deg 32-65% ACN. I have complete coelution of asparagine glutamine and leucine phenylalanine but have the rest including cystine cysteine proline. If anyone has a separation that gets all the FMOC amino acids please share.