Amino acids chromatographic separation

Direct and indirect chromatographic methods were developed and compared in systematic examinations for the enantioseparation of P-amino acids direct separation of underivatized analytes involved the use of commercially available Crownpak CR(-I-), teicoplanin, and ristocetin A CSPs [148], while indirect separation was based on precolumn derivatization with 2,3,4,6-tetra-G-acetyl-f)-D-glucopyranosyl isothiocyanate (GITC) or A - a-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (EDAA, Marfey s reagent), with subsequent separation on a nonenantioselective column. 

PDCAAS (%) is calculated as the product of the true protein digestibility and the amino acid score (or lowest amino acid ratio) relative to reference protein. Amino acid composition of protein is determined by hydrolyzing the protein into its component amino acids and then separating the amino acids chromatographically as described below. True protein digestibility is determined according to Alternate Protocol 2. 

There are two major categories for amino acid analysis (a) free amino acid analysis and (b) determination of total amino acid content. The total amino acid content includes contributions from the free amino acids and the amino acids that are originally protein bound. These protein-bound amino acids must first be liberated before chromatographic analysis. This necessitates a more extensive, and problematic, sample preparation. Because the sample preparation procedures are so disparate, it is convenient to address these two categories of amino acid analyses separately. It should be noted that while the sample preparations for these analyses are quite different, both utilize essentially the same chromatographic techniques for the second stage of amino acid analysis. 

Figure 2. Chromatographic separation of amino acids after derivatization with phenylisothiocyanate (PITC) A. Separation of 200 picomole standard amino acid mix H containing 18 amino acids. B. Separation of an extended amino acid mix containing 28 amino acids. The standard one-letter abbreviations are used for the usual amino acids. Nonstandard amino acids are Sp, phosphoserine Hp, hydroxyproline Citr, citrulline Tau, taurine aAba, a-amino butyric acid HKl HK2, hyi oxylysines Om, ornithine , artifacts from reagents.

The DNP-amino acids, after separation into individual spots on the chromatographic plate, can be eluted from the scraped off area by adding 4 ml of water to the material in a small tube. The tube is heated at 50° in a water bath for 15 minutes and centrifuged to clear the solution. The color is read against known standards at 360 nm. Direct estimation of DNP-, PTH-, and DANS-amino acids separated on the thin-layer plate can be performed by fluorescence and fluorescence quenching techniques (P8). It is also possible to convert unmodified amino acids, separated on a silica gel G chromatographic plate, into DNP-amino acids by in situ conversion as was described in Section 4.7.18. The DNP-derivatives can then be developed in the second dimension and the spots analyzed quantitatively. 

The dansyl amino acid is separated and identified by chromatographic methods. The dansyl procedure is about 100 times more sensitive than the DNFB method because the dansyl amino acids are highly fluorescent and therefore detectable in minute quantities. 

Fig. 11.2.12. Normal phase separation of amino acids. Chromatographic conditions column, Zorbax NH2 (250 x 4.6 mm I.D.) mobile phase, 10 mM potassium phosphate, pH 4.3 (A), acetonitrile-water 50 7 (v/v) (B) flow rate, 2 ml/min temperature, 35 °C. Peaks 1, phenylalanine 2, leucine 3, isoleucine 4, methionine 5, tyrosine 6, valine 7, proline 8, alanine 9, hypro 10, threonine 11, glycine 12, serine 13, histidine 14, cysteine 15, arginine 16, lysine 17, hydroxylysine 18, glutamic acid 19, aspartic acid. Reproduced from Smolensk et al. (1983), with permission.

The threefold S5nnmetry of qnatemarized amines makes them ideal guests for 18-crown-6 and its derivatives. Consequently, reverse-phase chromatographic colnmn materials modified with chiral 18-crown-6 derivatives have been used to successfully separate racemic mixtures of the common amino acids. The separation coefficients were enhanced at lower temperatnres and at higher loading levels of the crown ether. ... 

Crown ethers can be introduced into the ion chromatographic system via the mobile and/or the stationary phase. Sousa et al [67] used them for the first time as mobile-phase additives describing a separation of amino acid isomers separated with dinaphthal-18-crown-6. But even simple inorganic anions can be separated with this method [68]. However, the number of applicable crown ethers is limited because of their solubilities. Moreover, crown ethers are relatively expensive, so their application at higher concentrations is not justified. 

It was not the aim of this chapter to describe all chromatographic separations of racemic amino acids accomplished so far, but rather demonstrate applicability of these methods to separation of DL-amino acids. Chiral separations, using TLC, enables rapid and inexpensive testing both of optical enantiomers, their derivatives, peptides, and control of enantiomeric purity. 

Melanotropin. [9034-42-8] (18-22 amino acids peptide), amorphous. Extract separated by ion-exchange on carboxymethyl cellulose, desalted, evapd and lyophilised, then chromatographed on Sephadex G-25. [Lande et al. Biochem Prep 13 45 1971.]... 

The great leap forward for chromatography was the seminal work of Martin and Synge (7) who in 1941 replaced countercurrent liquid-liquid extraction by partition chromatography for the analysis of amino acids from wool. Martin also realized that the mobile phase could be a gas rather than a liquid, and with James first developed (8) gas chromatography (GC) in 1951, following the gas-phase adsorption-chromatographic separations of Phillips (9). 

One method that combines the good chromatographic properties with improved limit of detection is the separation of isoindole derivatives of amino acids that may be detected fluorimetrically. This method may be applied to protein hydrolysates, and used in automated format in routine analyses [22]. 

Tasks (1) and (2) are relatively easy to accomplish. The sample is first heated with hydrochloric acid to break all of the peptide linkages (amide bonds) in the protein. The resulting solution is then passed through a chromatographic column (recall the discussion on pp. 5-6 in Chapter 1). This separates the different amino acids and allows you to determine their identities and concentrations. 

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