Amino acid elution volume

Acetate buffer (pH = 8) could not effectively elute amino acids during chromatographic separation therefore, it was necessary to use 1.5 mM glycine solution. Imprinted amino acids eluted between 5 and 8 column volumes, whereas the corresponding enantiomers eluted in 3-4 column volumes. Unfortunately, the imprinted polymers could not achieve baseline separations. The nonimprinted enantiomer was eluted as a sharp peak followed by a broad, overlapping peak, which corresponded to the imprinted enantiomer. Nonimprinted polymers did not show any ability to separate racemic solutions. 

Sulfosalicylic acid has most commonly been used to precipitate proteins prior to ion-exchange amino acid analysis (11). In this mode, SSA allows for a very simple sample preparation that requires only centrifugation of the precipitated sample and then direct injection of the resulting supernatant solution. The supernatant solution is already at an appropriate pH for direct injection. Also, the SSA does not interfere chromatographically since it elutes essentially in the void volume of the column. It has been noted that, if an excessive amount of SSA is employed, resolution of the serine/threonine critical pair can suffer (12). The use of SSA prior to reversed-phase HPLC can be more problematic, since its presence can interfere with precolumn deriva-tization. For example, Cohen and Strydom (13) recommend the separation of the amino acids from the SSA solution on a cation-exchange resin prior to derivatization with phenylisothiocya-nate (PITC). 

A problem with the chromatographic determination of cysteic acid is that there is almost no retention of cysteic acid. For both reversed-phase HPLC and ion-exchange amino acid analyzers (usually employing cation-exchange resins), cysteic acid is essentially eluted within or near the void volume of the column. This makes it more susceptible to unknown chromatographic interferences from various matrices. When cysteine is alkylated by 3-bromopropylamine, the product (S-3-aminopropylcysteine) looks very similar to lysine in structure. Hale et al. (90) show that this alkylated species affords excellent chromatographic separation on four different commercially available amino acid analysis systems and that, indeed, it does elute very near lysine in each case (see Fig. 4). 

Method. A standard amino-acid analyzer (Technicon or an equivalent) may be used. The reagents for development and the buffers are prepared as for analysis of amino acids. The analytical column (24 cm X 0.57 cm) consists of Zeocarb 226-4.5% DVB (average particle diameter, 24 jum). The two buffers are prepared by dissolving 8.74 g of potassium citrate, 60.36 g of potassium chloride, 10 ml of Brij and 100 ml of n-propanol (for the first buffer, 140 ml of n-propanol for the second buffer) in enough water to make a total volume of 11. The pH of each buffer is 7.4. For analysis the sample is adjusted to pH 7.4 and an aliquot portion is applied to the column. The column temperature is maintained at 43 °C for 103 min and is automatically switched to 75 °C for the remainder of the run. The flow-rate of the buffer is 42 ml/h. The first buffer is automatically replaced by the second after 120 min. The second buffer is necessary for the separation of tryptamine and cadaverine. The use of the increased temperature results in a shorter elution time. The retention times of some basic amino acids and amines are listed in Table 4.3. Absorption is monitored at 570 nm with a 1,5-cm flow cell. 

Method. Solutions of amino acids in phosphate buffer (pH 9.3) are mixed with an equal volume of freshly prepared 0.4 M pyridoxal solution (adjusted to pH 9.3) and permitted to stand at 8 °C for 30 min. (The molar ratio of pyridoxal to amino acid should be >75 1.) At this point, 1 ml of sodium tetrahydroborate solution (100 mg/ml in 0.1 N sodium hydroxide) is added and the contents are gently shaken. Excess of sodium tetrahydroborate is destroyed by addition of sufficient hydrochloric acid (pH 1-2) prior to column chromatography. The pyridoxal derivatives are separated on a column (100 X 0.6 cm) of Aminex A-5 ion-exchange resin (Bio-Rad) at a mobile phase flow-rate of 33 ml/h. The eluting solvents consist of 0.2 N buffers at pH 3.40,4.44 and 4.86 and a 0.35 N buffer at pH 5.86 (all of the buffers are sodium citrate). The separation of a number of pyridoxyl-... 

Aspartic acid was separated by chromatography on a calibrated colunn of BioRad AG1-X8, 100-200 mesh, anion exchange resin. Hie resin was regenerated with 4 colunn volumes of 1 M sodium Half of the sanple was applied to the colunn elution was carried out with 1 M acetic acid. The aspartic acid fraction was evaporated in a rotatory evaporator. Hie L-leucyl-DL-aspartic acid dipeptides were synthesized by the procedure of Manning and Moore (14). Hie D/L aspartic acid ratio was determined with a Beckman Model 118 Automatic Amino Acid Analyzer. 

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