Practical Protein Chemistry - A. Darbre 1989

Enantiomeric analysis of amino acid mixtures by high-performance liquid chromatography
HPLC
Enantiomeric analysis

An aliquot of each fraction (20 µL) is applied to a stainless steel reversed-phase Column (150×4.6 mm) packed with Spherisorb ODS-18 using a chiral Mobile phase, where individual Amino Acids are separated into their D- and L-isomers. If necessary, acetonitrile is added to the eluent to decrease the retention time. The detection of amino acids in the eluate is performed in the same manner as in the cation-exchange Chromatography step. The retention times are summarized in Table 9.1.

The chromatograms and Separation conditions for the enantiomers of the four amino acid groups are shown in Figs. 9.2–9.5. Using special samples enriched with the L- and D-forms, it was demonstrated that, without exception, the D-isomer elutes from the column before the L-isomer in all cases.

The acidic (Ser, Thr, Cya, Asp, Glu) and basic (Lys, Arg, His) groups are eluted with an aqueous solution containing a chiral additive. For the group comprising neutral amino acids (Gly, Ala, Val, Met, Leu, Ile), acetonitrile (1%) is added to the mobile phase to reduce the retention time. The third group (Phe, Tyr, Trp) is eluted with The addition of 14% acetonitrile.

Complete baseline resolution of the enantiomers for all amino acids is achieved within each group.

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FIG. 9.2. Enantiomeric separation of acidic amino acids (Group 1) on a reversed-phase column. Mobile phase: chiral additive (Cu-DPA) in Water. Room Temperature; flow rate 0.1 mL/min, increasing to 0.2 mL/min after 30 min.

FIG. 9.3. Enantiomeric separation of neutral amino acids (Group 2) on a reversed-phase column. Mobile phase: chiral additive (Cu-DPA) in water and aqueous acetonitrile (1%). Room temperature, flow rate 0.25 mL/min.

FIG. 9.4. Enantiomeric separation of aromatic amino acids (Tyr, Phe, and Trp) on a reversed-phase column. Mobile phase: chiral additive (Cu-DPA) in water and aqueous acetonitrile (14%). Room temperature, flow rate 0.5 mL/min.

FIG. 9.5. Enantiomeric separation of basic amino acids on a reversed-phase column. Mobile phase: chiral additive (Cu-DPA) in water. Room temperature, flow rate 0.2 mL/min.