I have HPLC with fluorescence detector & I want to quantify amino acids of some cereal grain by RPHPLC, what is the best method with derivatization technique?
You can use pre column or post column ninhydrin derivatization technique, but post column is mostly used technique. Without derivatization it is not possible to analyze amino acids by HPLC.
I remember my early work in the lab when I went home smelling of vinegar and with purple-stained hands.
There are many ways to analyze amino acids with fluorescence. Besides FMOC you can look at derivatization with FITC or OPA. OPA (ortho-Phthaldiamide) is nice because it is non-fluorescent until it reacts with the amino acids.
Many (most?, all?) techniques have some problem with proline. It is never enough to run a single amino acid as a standard. The response is different for all amino acids so you need to run a control of the entire 20 amino acids. It is likely that you may see more than 20 peaks. There are a lot of non-protein amino acids floating around. And even from hydrolyzed proteins, you can run into amino acids that have been modified by post-translation modifications.
The matrix in which the amino acids are found can have a profound effect upon the derivatization and the separation. The best way to manage that is to use the method of standard additions and add a small aliquot of known concentrations of all the amino acids to your sample before derivatization. Then do the derivatization and separation. This should be done with 3 or more different amounts of added amino acids and the curves extrapolated to zero added amino acid. You will see that the amino acids do not all have the same response to derivatization and detection.
If you are just looking at hydrolyzed, purified proteins, you can often forgo the method of standard additions.
First, my fingers were moving faster than my brain. OPA is ortho-phthaldialdehyde.
Second, you may want to look at these resources. Notice that resolution is not always to baseline even with standards. :
http://www.aaccnet.org/publications/cc/backissues/1985/Documents/chem62_97.pdf
http://www.microdialysis.se/Fluorescence%20detection%20of%20Amino%20Acids.pdf?cms_fileid=e71472155b1737f5c964fee675821981&disposition=attachment
http://www.chem.agilent.com/Library/slidepresentation/Public/Amino%20Acid%20Analysis_062410_Rita%20Steed.pdf
http://www.chem.agilent.com/Library/chromatograms/59801193.pdf
http://www.thermoscientific.com/content/dam/tfs/ATG/CMD/CMD%20Documents/114372-PO-ECD-Amino-Acids-70537.pdf
http://www.knauer.net/fileadmin/user_upload/produkte/files/Dokumente/application_notes/vbs0011n_uhplc-pda-fld_determination_aqc_amino_acids_bluespher.pdf
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