Chromatographic separation of amino acids

7 Mono- and polysaccharides after aqueous extraction of particulate matter 

The hot water procedure will extract free monosaccharides and soluble polysaccharides, such as starches and reserve cell material. Hydrolysis of more labile a-1,4 linked polysaccharides may occur during the extraction procedure at near-neutral pH and add to the pool of monosaccharides. Estimations of the monosaccharide contents of aqueous extracts follow the method earlier described after suitable dilution to attain the analytical range, which depends on the amount of extracted particulate matter. In regions of high productivity dilution up to 10-fold with distilled water may be necessary. Oligo- and polysaccharide contents of aqueous extracts may be determined after hydrol3rsis as outlined earlier. 

Particulate matter may thus be characterized by three different sugar fractions extractable monosaccharides, extractable polysaccharides Le., starch-like substances) and total sugars after hydrolysis (including monosaccharides, starches and structural polysaccharides). An analogous approach may also be adopted to characterize three different amino acid fractions of particulate matter by employing the common analytical techniques of Section 26.3. 

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Another difficulty in the gas chromatographic separation of amino acids is the choice of detector and it may be necessary to split the gas stream and use two different detectors. The flame ionization detector, which is commonly used, is non-specific and will detect any non-amino acid components of the sample unless purification has been performed prior to derivatization. In addition the relative molar response of the flame ionization detector varies for each amino acid, necessitating the production of separate standard curves. As a consequence, although gas chromatography offers theoretical advantages, its practical application is mainly reserved for special circumstances when a nitrogen detector may be useful to increase the specificity. 

Demonstrating the existence of multiple transaminases in the late 1930s and early 1940s was very difficult. Chromatographic separation of amino acids was not available until the mid-1940s and paper chromatography was not in common use until 1948-1950 when Hird and Rowsell finally showed the existence of the wide range of transaminases and the universality of the transamination process. 

Chromatographic separation of amino acids in the test protein is required for chemical scoring methods. The selectivity and sensitivity of chromatographic methods vary and comparing results of more than one method may be required, particularly when proteins with unusual amino acid profiles are being tested. 

Frank H, G. J. Nicholson and E. Bayer, Rapid gas chromatographic separation of amino acid enantiomers with a novel chiral stationary phase , J. Chromatogr. Sci. 15 174-176(1977). 

Prior to the chromatographic separation of amino acids on Dowex 50 columns, Carsten (C5) first desalts the urine sample on Amberlite IR 100 or Duolite C 3 and removes most of the nitrogenous bases on Amberlite IRA 400. This preliminary treatment allows for amino acid separations at ordinary temperatures using 2M and 4M HC1 on H+ columns for elution, instead of buffer mixtures a single column of 25 g of Dowex 50 is sufficient for all amino acids and 350-375 one-milliliter fractions are collected. The resolving power of this method does not seem to be as satisfactory as Moore and Stein s procedures, and it is not less time nor labor consuming. 

Figure 6. Gel inclusion chromatographic separation of amino acids on a-, 6-, y-cyclodextrin bead polymer columns, and on Sephadex G-25 column (1.6x88 cm, pH 5.0 phosphate buffer, flow rate 10 ml/h, 20 °C).

Such cases can be encountered in the case of thin-layer chromatographic separation of amino acids, using copper complexes of long chain amino acids as chiral additives via a ligand exchange approach. The copper complexes of alkyl amino acid chiral additives are so strongly adsorbed on the RP stationary phase that they act as a chiral stationary phase [154-156]. 

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.

Figure 6. Chromatographic separation of amino acids after derivatization with phenylisothiocyanate (PITC)using physiological conditions A. Free amino acids found in rat brain tissue. B. Free amino acids in ant hemolymph. C. Bone collagen hydrolysate.

Solids that strongly attract water and other polar solvents are the common media for achieving classical column-chromatographic separation of amino acids and peptides, on the basis of the partition principle (Hearn, 1991 Hancock, 1984). Cellulose (i.e. paper in the form of sheets or powder), one of the media of this type used since the earliest days of chromatography, also has the capacity to bind, through adsorbed water, to one enantiomer of certain amino acids, e.g. tryptophan, more strongly than to the opposite enantiomer (chiral or enantioselective separation chromatographic resolution), because cellulose is homochiral (constructed purely of one enantiomer). 

The observation of enantioselective binding properties of polysaccharides dates back to the early SOs. At this time Kotake [95] and Dalgliesh [96] achieved thin layer chromatographic separation of amino acid enantiomers on cellulose carriers. However, the poor chiral recognition capacity of native polysaccharides hampered further developments. 

Kanazawa H, Sunamoto T, Matsushima Y, Kikuchi A, Okano T. Temperature-responsive chromatographic separation of amino acid phenylthiohydantoins using aqueous media as the mobile phase. Anal Chem 2000 72(24) 5961-5966. 

H. Bruckner and M. Wachsmann, Design of chiral monochloro-S-trizine reagents for the liquid chromatographic separation of amino acid enantiomers,/. Chromatogr. A., 2003, 998, 73-82. 

Disz I, Berkeez R, Peter A. Application of chiral derivatizing agents in the high-performance liquid chromatographic separation of amino acid enantiomers a review. J Pharm Biomed Anal 2007 47 1—15. 

For the liquid chromatographic separation of amino acids and amino compounds, indirect separation techniques after precolumn labeling of the amino group are also widely used. By the precolumn derivatization approach, amino compounds are converted into structures that are suitable for separation and suitable for detection by various sensitive detectors. For the separation of the labeled amino compoimds, a wide variety of separation columns, including reversed-phase, can be used. Concerning detection, UV—Vis absorbance, fluorescence, and also MS (MS/MS) detectors are widely used, depending on the properties of the derivatization reagents. 

The chromatographic separation of amino acids labeled with I and of iodides can be performed on paper and on columns for their detection and quantitative determination. Paper chromatography is very convenient for characterization fractionation on columns is better adapted to preparation and determination. 

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