Amino acid analysis by HPLC

[vellaikumar]

Dear Members,

I would like to present a seminar on use of HPLC on Amino acid analysis. In this regard, can any one tell me the source from where I can collect information on the above said topics.

If ready ppt slides found in net also be useful for understanding.

Regards

[mbicking]

You may have to look at some individual manufacturers to get application information. For example, the following link on the Agilent site describes a way to generate the derivatives in the autosampler, and complete the analysis all in one step.

http://www.chem.agilent.com/temp/rad5BE23/00000385.PDF

I am sure the other manufacturers have similar applications. I know Phenomenex has both GC and LC applications. You may also want to look at the web sites of the companies that manufacture the reagents (Suepelco, Regis Chemicals, Pickering, etc.), as they may have more information.

[Hfranz]

mbicking: I fear your link does not work, but it is probably about pre-column derivatisation. Further AA analysis methods are:
- Post-column derivatisation (for instance, check http://www.pickeringlabs.com)
- Electrochemical detection (for instance, check http://www1.dionex.com/en-us/ic/cons4776.html)

[Uwe Neue]

I can send you information slides on the AccQ-Tag method: derivatization in seconds, HPLC methods or fast (8 minute) UPLC methods for amino acids from hydrolysates or 33 minutes for 45 physiological amino acids. Contact me at the e-mail address below or at Uwe_Neue@Waters.com.

[mbicking]

I can send you a pdf of the file if you can't find it on the Agilent site. It covers automated pre-column derivatization and separation info.

[oscarBAL]

Dear Vellarkumar.
There are a lot of AAA HPLC method but I just want to remind you that you should not talk about this; with out speak about Sample treatment, because this couls be a very important issue.


Oscar

[Kostas Petritis]

There are a lot of review articles on amino acid analysis and that would be the place to start in my opinion. I have personally introduced several methods for the analysis of underivatized amino acids. There are three problems when analyzing amino acids and I would suggest that you divide your presentation accordingly:

1) Separation of amino acids: These molecules are small, and very polar/ionic and that was one of the drivers for their derivatization. In general, after derivatization, they can be separated in reversed phase chromatographic conditions.

2) Detection of amino acids: With the exception of Phe, Tyr and Trp (among the proteinogenic amino acids) these molecules do not contain a strong chromophore group that makes their detection very difficult (and that was actually the main driver in the past for their derivatization). After derivatization these molecules could easily be detected by UV or fluoresence techniques. There are some references where underivatized amino acids are separated by ion exchange, followed by post-column derivatization and UV/fluoresence detection. In such applications, about 60 amino acids were able to be analyzed simultaneously (Armstead and Ling, J. Chromatogr. 586, 1991, 259-263).

3) Matrix intereferences/applications: Of course no analytical method is of any use if you can not apply it to your application and depending on what you want to analyze some derivatization methods work better than others.

Derivatization methods are good, but they all have their drawbacks. That was one of the main drivers for us to develop methods for the direct analysis of amino acids (i.e. without derivatization). Our papers are also dealing separately with the problem of separation, detection and applications... Below are some of my contributions on the field, you can find plently references therein:

Ion-pairing reversed-phase liquid chromatography/electrospray ionization mass spectrometric analysis of 76 underivatized amino acids of biological interest: a new tool for the diagnosis of inherited disorders of amino acid metabolism M. Piraud, C. Vianey-Saban, K. Petritis, C. Elfakir, J.-P. Steghens, D. Bouchu Rapid Commun. Mass Spectrom. 19 (2005) 1587-1602.

Validation of an ion-interaction chromatographic analysis of underivatized amino acids in commercial preparation using evaporative light scattering detection K. Petritis, M. de Person, C. Elfakir, M. Dreux Chromatographia 60 (2004) 293-298.

Study of selenium distribution in the protein fractions of Brazil Nut Bertholletia escelsa T. Chunhieng, K. Petritis, C. Elfakir, J. Brochier, T. Goli, D. Montet J. Agr. Food Chem. 52 (2004) 4318-4322.

ESI-MS/MS analysis of underivatized amino acids: a new tool for the diagnosis of inherited disorders of amino acid metabolism. Fragmentation study of 79 molecules of biological interest in positive and negative ionization modeM. Piraud, C. Vianey-Saban, K. Petritis, C. Elfakir, J.-P. Steghens, A. Morla, D. Bouchu Rapid Commun. Mass Spectrom. 17 (2003) 1297-1311

The simultaneous determination of 1-aminocyclopropane-1-carboxylic acid and cyclopropane-1-1-dicarboxylic acid in Lycopersicum esculentum by high-performance liquid chromatography-electrospray tandem mass spectrometry K. Petritis, G. Koukaki, E. Koussissi, C. Elfakir, M. Dreux, V. Dourtoglou Phytochem. Anal. 14 (2003) 347-351

A comparative study of liquid chromatographic detectors for the analysis of underivatized amino acids K. Petritis, C. Elfakir, M. Dreux J. Chromatogr. A 961 (2002) 9-21

Evaporative light scattering detector as an in-line monitoring detector for the stop-flow liquid chromatography-nuclear magnetic resonance analysis of compounds with weak/no chromophore group K. Petritis, I. Gillizeau, C. Elfakir, M. Dreux, A. Petit, N. Bongibault, W. Luijten J. Sep. Sci. 25 (2002) 593-600

Ion-pair reversed phase liquid chromatography-indirect conductivity detection and response deviations of underivatized amino acidsK. Petritis, P. Chaimbault, C. Elfakir, M. Dreux J. Sep. Sci. 24 (2001) 397-405
HPLC-CLND for the analysis of underivatized amino acids K. Petritis, C. Elfakir, M. Dreux LC-GC Europe 14 (2001) 389-395.

Simultaneous analysis of underivatized chiral amino acids by liquid chromatography-ionspray tandem mass spectrometry using a teicoplanin chiral stationary phase K. Petritis, A. Valleix, C. Elfakir, M. Dreux J. Chromatogr. A 913 (2001) 331-340.

Determination of 1-aminocyclopropane-1-carboxylic acid and structural analogue by liquid chromatography and ionspray tandem mass spectrometry K. Petritis, V. Dourtoglou, C. Elfakir, M. Dreux J. Chromatogr. A 896 (2000) 335-341.

Parameter optimization for the analysis of underivatized protein amino acids by liquid chromatography and ionspray tandem mass spectrometry
K. Petritis, P. Chaimbault, C. Elfakir, M. Dreux J. Chromatogr. A 896 (2000) 253-263.

Determination of 20 underivatized proteinic amino acids by ion-pairing chromatography and pneumatically assisted electrospray mass spectrometry P. Chaimbault, K. Petritis, C. Elfakir, M. Dreux J. Chromatogr. A 855 (1999) 191-202

Ion-pair reversed-phase liquid chromatography for the determination of polar underivatized amino acids using pefluorinated carboxylic acids as ion pairing agent K. Petritis, P. Chaimbault, C. Elfakir, M. Dreux J. Chromatogr. A 833 (1999), 147-155.