So I have a GC method with ethyl chloroformate that works for almost all amino acids except it doesn't do arginine, and serine and threonine smear badly due to the active hydroxyls.
I tried OPA and 3-MPA and using a methanol gradient managed to get all the amino acids and tuarine except cystine coelutes with valine and tryptophan and proline doesn't work. Also I tried calibrating from 5-200ppm and it was very nonlinear I beleive I need to decrease the amino acids and increase the OPA/MPA but all the literature says make a 1mg/ml solution.
I decided to try FMOC amino acids. I am having trouble with coelutions. I'd like to at least get arginine, serine, threonine, taurine and maybe cysteine and cystine. I was doing pretty well with an acetonitrile gradient but taurine and glutamate coelute.
Does anyone have a good method that works for all amino acids on a regular C18 column?
Dear Steven,
we are using HPLC routinely to analyze amino acids. The method includes OPA pre-column derivatization which we do automatically in the autosampler. I also captures arginine. The eky for sepration is the proper combination of column and gradient. The Gemini column is really good and also offers very good longterm stability (where previous column types often failed). If proline is needed, we add a step with FMOC. Details can be found in several of our previous publications, e.g. in Rohles et al. (2016) doi: 10.1186/s12934-016-0553-0.
In short: Amino acids were quantified by HPLC (Agilent 1200 Series, Agilent Technologies) using α-aminobutyrate as internal standard and pre-column derivatization with o-phthaldialdehyde and fluorenylmethyloxycarbonyl chloride . The derivatized analytes were separated on a reverse phase column (Gemini 5µm C18, 150 x 4.6 mm, Phenomenex, Aschaffenburg, Germany) at 40°C and a flow rate of 1 mL min-1 using a gradient of 40 mM NaH2PO4 (0.5 g L-1 sodium azide, pH 7.8) and acetonitrile/methanol/water (45%/45%/10%), and detection was based on fluorescence measurement (340/450 nm).
Best.
Christoph
I am also doing the in needle derivatization for both FMOC and OPA/MPA. OPA MPA don't seem to work for cysteine in addition to proline though I do get a peak for cystine. I am using a 10cm poroshell c18 and a phosphate/citrate pH 6.38 buffer and methanol gradient. I think my biggest problem is I am looking for amino acids ~100ppm so if you have 20 amino acids at >100ppm and the OPA/MPA is 1000ppm it becomes the limiting reagent.
I am getting a good response with just FMOC and an acetonitrile gradient with acetate pH 4.2 buffer I just need to fine tune the separation. I have trouble separating fmoc-taurine and fmoc-glutamate. All the early eluters arg/glu/gln/asn/asp/ser/thr/tau are pretty close together at the start.
Article Microscale analysis of amino acids using gas chromatography-...
Serine and threonine issue is fixed in our paper, but not arginine.
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.
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