For amino acid analysis in HPLC-UV, you need to derivatise your samples.

Robin thanks for reply, I'm agree with you but which method is simple & best because im doing amino acids first time....

HPLC-florascent detection method is the most reliable method. The Amino acid composition is analyzed after sealed tube hydrolysis with 6N HCl for 22 h at 110 C. After the Hydrolysis, the acid is evaporated in vacuum oven and the sample is kept in NaOH dessicator to remove traces of acid. The residue is brought in to 1 ml of Sodium Citrate-Perchloric acid sample diluent (pH 2.20) and filtered through 0.2 µm membrane filter. Separation of amino acids can be done in a column packed with a strongly acidic Na + type cation exchange resin (Styrene-divinyl benzene copolymer with sulfinic group) under gradient elusion at a flow rate of 0.3 ml/minute by using two buffers, (A) sodium citrate-perchoric acid (pH 3.2); (B) boric acid sodium citrate-sodium hydroxide (pH 10.0). The amino acids quantify using a fluorescent detector (FLD-6A) after post column derivitization with O-pthaladehyde and 2-mercaptoethanol. Amino acid standard solution for fluorescent detection is used as external standard.

Yes if you have florescent detector then go with HPLC-florescent detection method as said above by Dr Kalanjiam. If not, then go with derivatisation. That will help you out.

Dear M. RAJAMOHAMED & Robin I have HPLC with UV detector..so im looking for derivatization method....thanks for rply

Recently I was asked if this analysis was possible, not having a fluorescent detector and found very good information by a spanish researcher Isidro Hermosin (Lab. tecnologia de los Alimentos, Faculta de Quimica, Univ. Castilla-la Mancha) using derivatization with DEEMM, a very stable and cheap reagent. However, as with every method, we have to weigh advantages and disadvantages.... I can send you the papers I collected.

Derivatise amino acids with pthalaldehyde (Sigma).

Check this:

http://www.chem.agilent.com/Library/slidepresentation/Public/Amino%20Acid%20Analysis_062410_Rita%20Steed.pdf

Thank you very much Teodoro Kaufman for valuable reference. Also thanks to robin for your valuable comments.Robin im trying with pthalaldehyde only.

revert back to you after fruitful end.

Dear friends,

I have tried to separate glycine (GLY) on uv detector without derivatization and successfully getting sharp peak at RT 2.6 min....of glycine std using water:ACN::50::50,flow 0.8ml,wavelength 215nm,Column C18,250,5um,injection volume 50ul,using standard solution of 1mg/ml. but finished product contain Glycine (GLY) & lysine (LYS) which is not getting resolved....I have tried buffers & shuffled mobile phase ration's but not yet resolved the complex mixture of GLY & LYS...so what shall I do to resolve them without derivatization and shift the peak on extended RT i.e. after 5min.....

suggestions are welcome

regards,

Jag dish

Dear Jagdish,

please refer to Waters guide on column. You may find some suitable column like Symmetry or Xterra for amino acids which is End Pack polar Columns specially used for separation for acids.

Dear Jagdish,

Refer www.merckmillipore.com/chromatography web and click on application finder and specify your analyte as amino acid and get the required separations on ZIC-p HILIC column without derivatisation.

Thanks ravi & sanjay.... I will try shortly

Ortho-phthaldehyde (OPA) , with a dash mercaptoethanol forms a derivative with amino acids that is easily detected in UV detector, 21 amino acids separated in less than 20 mins including Taurine. It takes a bit of fiddling to resolve the methionine and phenylalanine peaks. Changing gradient and adding some THF worked.

Dear friend

Recently we tried with Dansyl chloride, we are getting good results. Even though it is expensive, but also effective.

@ phani, how many amino acids have you separated with densyl chloride???

Hi jagdish. May i know what is you amino acid source. I too am intending to carry out quantitification of amino acid....but my source is gelatin....kindly info the method you used. As what i read the best is by OPA. Tks.

thanks Barry, how do you do derivatization with OPA & mercaptoethanol ????

Hi!! well, i have a procedure, but in spanish, if you want try it...

Rapid assay for amino acids in serum or urine by pre-column derivatization and reversed-phase liquid chromatography.

D C Turnell and J D Cooper

Clinical Chemistry

March 1982 vol. 28 no. 3 527-531

@ Ruth...Gracias querida ... Voy a tratar pronto

muchas gracias por su colaboración operaton.

@ Barry ....heartly thanks....

Ok, no problem :)

choose PDA detector insted of UV detector

Okay pankaj....i will tyry

How to quantify L-Serine Methl ester (Amino acid Methyl ester) by RPHPLC with UV Detector or with GC, Can anyone suggest a good method.

Check Once it may useful

http://iopscience.iop.org/1742-6596/214/1/012122/pdf/1742-6596_214_1_012122.pdf

check this kit. using kit u can analysis amino acid by UV detector. 

Can I use VWD detector instead of DAD  for AA analysis ?

Dear Hai Le Thanh,

you can use DAD detector.

I want to detect glycine by RPHPLC, what is the best method with derivatization technique? i have done derivatization between glycine and 0-phenylenediamine but it doesn't show any peaks when i run in hplc.

You can use precolumn derivazation method either using OPA & FMOC, then measuring on UV detection. Refer to Agilent method.

There are several methods avialble depends on what kind of HPLC system you have. Water's AccQ precolumn derivazation method is good, same as Agilent AAA method. Check both Waters and Agilent website, you will be able to find detailed method.

Try dansyl chloride as derivatising reagent followed by retaining on RP mode in UV region.