Buffer reagents, amino acid separation

Method (manual). An aliquot portion (0.1 ml) of effluent containing the amino acid is mixed with 3 ml of a solution containing 1.5 ml of o-phthalaldehyde (10 mg/ml in ethanol), 90 ml of sodium tetraborate buffer (0.05 M, pH 9.5) and 1.5 ml of a solution of 2-mercaptoethanol (5 mg/ml) in ethanol. The latter reagent should be freshly prepared each day. The reaction mixture is permitted to stand for 5 min and the intensity of fluorescence is measured in a fluorimeter (340-nm excitation, 455-nm emission). The buffered reagent may be also useful as a spray reagent for amino acids separated by TLC, although such an investigation has not been reported. 

CE has been applied extensively for the separation of chiral compounds in chemical and pharmaceutical analysis.First chiral separations were reported by Gozel et al. who separated the enantiomers of some dansylated amino acids by using diastereomeric complex formation with Cu " -aspartame. Later, Tran et al. demonstrated that such a separation was also possible by derivatization of amino acids with L-Marfey s reagent. Nishi et al. were able to separate some chiral pharmaceutical compounds by using bile salts as chiral selectors and as micellar surfactants. However, it was not until Fanali first showed the utilization of cyclodextrins as chiral selectors that a boom in the number of applications was noted. Cyclodextrins are added to the buffer electrolyte and a chiral recognition may... 

Amino acids can be separated without prior derivatization on a cation-exchange resin column. The elution buffers are classically lithium citrate buffers with different pH values and salt concentrations, which are applied stepwise. There is usually a programmed increase in column temperature. Consequently, there are numerous variables affecting the separation of the individual amino acids [6]. For the detection of the amino acids, the column effluent is mixed with the ninhydrin reagent. Nowadays there are only very few manufacturers of AAAs left. The considerable cost of purchase and the operation costs are a potential threat to the widespread application of this technique, although it is still considered to be the definitive method for diagnosing disorders of amino acid metabolism. 

As described above, the amino acids are separated at a constant flow on a high-resolution cation-exchange column using buffer and temperature gradients. The postcolumn reaction with the ninhydrin reagent is carried out at 135°C and the absorbances of the reaction products are read at both 570 and 440 nm. Amino acids are identified by comparing their retention time and 570/440 ratio with that of authentic reference substances (see Fig. 2 .2). 

Fig. 8 Separation of standard mixture employing precolumn derivatization with AQC. Gradient elution with acetonitrile and acetate buffer (pH 5.0) was employed with a C18 column. Standard three-letter abbreviations for amino acids were used also, CA = cysteic acid, AMQ = hydrolyzed excess reagent, and nle = norleucine. Data was supplied by Stephen D. Smith, Ross Products Division of Abbott Laboratories, Columbus, OH.

Fig. 9 Chromatogram of the separation of the dabsyl derivatives of all 20 naturally occurring amino acids on a Cik column. Gradient elution employed acetonitrile, methanol, and acetate buffer (pH 6.5). Note the presence of a large peak due to the hydrolysis product of excess derivatizing reagent. Peaks marked with asterisks have not been identified. (From Ref. 154. Copyright 1991 Elsevier Science.)...

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. 

The three general approaches to enantiomer separation entail a chiral stationary phase, a chiral mobile phase, or a chiral reagent. Tandem columns, with reversed and chiral stationary phases, were used to separate 18 D-L pairs of PTC-amino acids in 150 min. OPA-amino acid enantiomers have been separated on both ion-exchange and reversed-phase columns using a sodium acetate buffer with a L-proline-cupric acetate additive. Chiral reagents, such as Marphey s reagent and OPA/IBLC (A-isobutiril-L cysteine), were successfully used for racemization analysis within 80 min. 

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