These goals are achieved with automated, online derivatization using o-phthalaldehyde (OPA) for primary amino acids and 9fluorenylmethyl chlor of ormate (FMOC) for secondary amino acids; the automated derivatization is then integrated with rugged HPLC analysis.

The complete procedure is rapid, accurate, sensitive, and reproducible using the Agilent 1100 HPLC.

Combining OPA and FMOC chemistries enables fast pre-column derivatization of amino acids (AA) for chromatographic analysis.

The reaction mixture is buffered at a pH of 10.2, which allows direct derivatization of acid hydrolyzed proteinpeptide samples.

The primary AAs are reacted first with OPA using 3-mercaptopropionic acid (3-MPA).

The secondary AAs do not react with the OPA; but are then derivatized using FMOC.

The incorporation of 3-MPA into the indoles decreases their hydrophobicity, and as a result, the OPAderivatives elute chromatographically before the FMOC derivatives.

Excess FMOC and its degradation products elute after the last of the secondary AAs and do not interfere with the analysis.

The derivatization process is fast Rapid, Accurate, Sensitive, and Reproducible HPLC Analysis of Amino Acids Amino Acid Analysis Using Zorbax Eclipse-AAA Columns and the Agilent 1100 HPLC John W.

Ricker, Brian A. Bidlingmeyer, and Cliff Woodward and is easily automated using the Agilent 1313A autosampler.

Because of the advantageous reaction speeds, both derivatizations are complete at room temperature.

The automated procedure provides a high degree of reproducibility.

SEPARATION OPTIONS The Zorbax Eclipse-AAA column contains batch-qualified reversedphase material.

When used according to the protocol described in this Technical Note, the column enables the user to separate the mAU 10 5 0 mAU 15 10 5 0 338 nm 1 2 262 nm 3 9 4 7 8 10 11 6 12 5 amino acids commonly found in proteinpeptide hydrolysates.

The A and B mobile-phase components are easy to prepare, and the gradient consists of linear segments (refer to Experimental Conditions, Mobile Phase section, for details).

This combination contributes to a rugged protocol that can be accomplished on the Agilent 1100 HPLC using either binary or quaternary solventdelivery systems.

The chromatogram in Figure 1 illustrates typical routine sensitivity in high-throughput applications that can be obtained on the Agilent 1100 HPLC binary system using the 1100 Diode Array Detector (DAD).

Separation conditions are listed in 16 17 15 18 14 19 21 13 20 0 2 4 6 8 10 12 Figure 1: Routine Analysis, High-Throughput Separation of 24 Amino Acids Using the Eclipse-AAA Protocol.

The column dimensions are 4.6 x 75 mm, 3.5 m.

See Table 1 for peak identification.

Detection: A. 338 nm (OPA amino acids), B.

FMOC-amino acids).

A B min Page 2 and 3: the Experimental Conditions section Page 4 and 5: from the original AminoQuant method Page 6 and 7: Gradients: For 75 mm column length Page 8 and 9: Table 3: Preparation of Low-Sensiti Page 10: For more information on our product.

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