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ANALYSIS OF AMINO ACIDS BY REVERSED PHASE CHROMATOGRAPHY WITH PRECOLUMN DERIVATISATION AND UV/VISIBLE DETECTION


Key words:Amino Acids, RP-HPLC, Precolumn derivatisation, o-Phthalaldehyde (OPA), 2-Mercaptopropionic acid, Ultraviolet detection.


Introduction:
Underivatised amino acids do not significantly absorb light in the UV/visible spectrum. In order to increase sensitivity, their analysis normally includes a derivatisation step in which the amino acids react with a precursor to yield a strong UV/Visible chromophore or a fluorescent compound. Derivatisation can be performed before the chromatographic separation or after the separation step itself.

In this Application Note we describe a procedure which consists in pre-column derivatisation with O-Phthalaldehyde (OPA) in a borate buffer, giving UV absorbing iso-indoles that can be separated by reversed phase chromatography. The derivatisation is performed automatically by the autosampler before injection, and detection is carried out using a UV detector set at λ=338nm.


Sample treatment:
Samples are digested in acid medium to the complete hydrolysis of the protein fraction. After extraction and washing, the hydrolised amino acids are filtered and vialed, ready for analysis.

Derivatisation of the amino acids is performed automatically by the autosampler. This instrument is able to draw reagents from different vials and mix them according to a program.


Chromatographic method:
10L of a standard mix (equivalent to 10pmol of each amino acid) are mixed with the OPA reagent and given sufficient time to react.

The subsequent iso-indole derivatives are injected onto a C18 column and are eluted with a phosphate AcN/MeOH/H2O gradient.


Reagents:
Amino acid standards, OPA (o-Phthalaldehyde), 3-MPA (3-mercaptopropanoic acid), Sodium tetraborate, Sodium h
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Source:


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