Amino acid separations

The most widely appHed colorimetric assay for amino acids rehes upon ninhydrin-mediated color formation (129). Fluorescamine [38183-12-9] and (9-phthalaldehyde [643-79-8] are popular as fluorescence reagents. The latter reagent, ia conjunction with 2-mercaptoethanol, is most often used ia post-column detection of amino acids separated by conventional automated amino acid analysis. More recently, determiaation by capillary 2one electrophoresis has been developed and it is possible to determine attomole quantities of amino acids (130). 

Amino acid separations represent another specific application of the technology. Amino acids are important synthesis precursors - in particular for pharmaceuticals -such as, for example, D-phenylglycine or D-parahydroxyphenylglycine in the preparation of semisynthetic penicillins. They are also used for other chiral fine chemicals and for incorporation into modified biologically active peptides. Since the unnatural amino acids cannot be obtained by fermentation or from natural sources, they must be prepared by conventional synthesis followed by racemate resolution, by asymmetric synthesis, or by biotransformation of chiral or prochiral precursors. Thus, amino acids represent an important class of compounds that can benefit from more efficient separations technology. 

Table 9.1. Retention times and accurate masses for TBDMS derivatized amino acids (separation conditions Chapter 9.1.A)...

A solution of 257 g. (1.05 moles) of barium chloride dihydrate in about 500 cc. of hot water is added to the reaction mixture, with vigorous shaking, and the mixture is heated on a steam bath for one-half hour. A heavy precipitate of the barium salt of the amino acid separates at once. After cooling to room temperature, the barium salt is collected on a suction filter, transferred to a beaker, and washed with two 250-cc. portions of hot water (8o°). After drying at ioo°, the barium salt weighs 225-230 g. (80-82 per cent of the theoretical amount). 

Wu S, Dovichi NJ (1989) High-sensitivity fluorescence detector for fluorescein isothiocyanate derivatives of amino acids separated by capillary zone electrophoresis. J Chromatogr 480 141-155... 

May be useful if amino acid separations are also performed. / F values tend to be low, and glucose and galactose are not resolved. Use second after solvents containing pyridine to remove it (C)... 

A closer examination of the natural GAr sequence reveals that it is an imperfect repeat of single alanine amino acids separated by one to three glycine residues (Baer et al., 1984). Strikingly, the alanine residues are never adjacent to each other and never spaced by more than three glycine residues. To study the constraints of the repeat the iKB-a-GAr chimera were most helpful as short repeats of eight amino acids were sufficient for full... 

Figure 2. Tracing of paper chromatogram showing amino acids separated from an H2SO4 extract of MarceUus shale and Onondaga shale. 1 and 5 are standards 2 and 4 are Marcellus shale 3 is Newton Hamilton (Onondaga) shale...

Table V. Amino Acids Separated Paleozoic Rock Samples...

Formation of Salts. Amino acids have certain characteristics of both organic bases and organic acids because they are amphoteric. As amines, tile amino acids form stable salts, such as hydrochlorides or aromatic sulfonic acid salts. These are used as selective predpitants of certain amino acids. As organic acids, the annuo acids form complex salts with heavy metals, the less soluble salt being used for amino acid separation. 

Ion-exchange Separations. Because amino acids are amphoteric, they behave as adds or bases, depending upon the pH of die solution. This makes it possible to adsorb amino acids dissolved in water on either a strong-acid cation exchange resin or a strong-base anion exchange resin. The affinity varies with the amino acid and the solution pH. Ion-exchange resins are widely used in amino acid separations. 

The residue is dissolved in fifteen to twenty times its weight of methyl alcohol (Note 6), filtered and an excess of pyridine (Note 7) is added. The free amino acid separates on standing overnight. It is collected on a Buchner funnel, washed thoroughly with methyl alcohol and dried. The yield is 92-102 g. (30-33 per cent of the theoretical amount). If a pyridine-free product is desired, it is dissolved in 200 cc. of warm water, filtered and the filtrate poured into 2 1. of methyl alcohol (Note 8). There is less than 10 g. of product in the mother liquors. It may be isolated by evaporating to dryness, washing with methyl alcohol and purifying by reprecipitation in the same way. 

In a i-l. round-bottom flask is placed 760 g. of concentrated ammonium hydroxide (sp. gr. 0.9) and to this is slowly added 150 g. of a-bromocaproic acid (Note 1). The flask is well stoppered and allowed to stand in a warm place (50-55°) for twenty to thirty hours. The amino acid separates and is filtered off with suction and washed with methyl alcohol (Note 2). This crop of crystals weighs 51-56 g. The aqueous filtrate is evaporated nearly to dryness on a steam bath and then treated with about 250 cc. of methyl alcohol. This precipitates a second crop of amino acid contaminated with ammonium bromide. On washing with methyl alcohol and recrystallizing from water, there is obtained 10—15 g. more of pure product. The total yield is 63-68 g. (62-67 per cent of the theoretical amount). 

To obtain free alanine the alanine ester is heated for about 6 hours with 5 times its weight of water on a water bath, until the alkaline reaction has disappeared. The solution is evaporated on a water bath till crystallisation begins. The liquid is allowed to stand at 0°, when about 30 gms. alanine separate optical examination shows this to consist of almost pure d-compound. From the mother liquor a second crop of 20—25 gms. may be obtained, and this still consists of fairly pure active amino-acid, so that the total yields amount to 50—55 gms. The last mother liquor still contains a fair amount of active alanine, but it is mixed with so much racemic substance that it cannot be separated from it by mere recrystallisation from water. The first two crops are dissolved once more in hot water, and the liquid evaporated on the water bath till it begins to crystallise. At 0° a large quantity of the pure, active amino-acid separates out. 

BH Reitsma, ES Yeung. Optical activity and ultraviolet absorbance detection of dansyl L-amino acids separated by gradient liquid chromatography. Anal Chem 59 1059-1061, 1987. 

A mixture of alanine, glutamic acid, and arginine was chromatographed on a weakly basic ion-exchange column (positively charged) at pH 6.1. Predict the order of elution of the amino acids from the ion-exchange column. Are the amino acids separated from each other Explain. 

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. 

The use of ligand exchange has been examined for the analysis of PTH (phenylthio-hydantoin) amino acids separated on silica gel plates [92]. The method is an extension of the procedure developed for organophosphate pesticides [84]. The chromatoplate is sprayed with a solution of palladium(II) chloride and calcein. Palladium complexes with calcein to form a non-fluorescent chelate. However, in the presence of many sulfur-containing compounds, such as PTH-amino acids, the palladium is displaced from the complex liberating free calcein which gives an intense fluorescence. This method is capable of determining 0.1-nmole amounts of PTH-amino acids. 

The use of pH-sensitive fluorescent indicators as spray reagents has been recently examined for the determination of sulphur-containing pesticides and amino acids separated by TLC [158,159]. The procedure is adapted from a ligand-exchange method of Frei and Mallet [160]. The separated pesticides are brominated directly on the TLC plate. This treatment oxidizes the pesticides and liberates hydrobromic acid as a side product. On... 

The ninhydrin test. Heat a solution of the compound with a few drops of a 0.25 per cent aqueous solution of ninhydrin (Expt 5.99). a-Amino acids give a blue-violet coloration. This highly sensitive test is also given by some /5-amino acids and by some peptides and proteins, particularly on warming. The colour test is of great value in the characterisation of the a-amino acids separated by t.l.c., and the Rf values are useful aids to identification. 

Two types of crosslinking domains exist in tropoelastin those rich in alanine (KA) and those rich in proline (KP). Within the KA domains, lysine residues are typically found in clusters of two or three amino acids, separated by two or three alanine residues. These regions are proposed to be Q-helical with 3.6 residues per turn of helix, which has the effect of positioning two lysine sidechains on the same side of the helix, although there is no direct structural evidence (Brown-Augsburger et al., 1995 Sandberg et al, 1971), and facilitating the formation of desmosine crosslinks. Desmosine crosslinks are formed by the condensation of two allysine... 

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