Amino acids separation techniques

Other amino acid separation techniques include ... 

TABLE 13.7 Selected References on Carboxylic and Amino Acids Separations by OHLM Techniques ... 

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. 

Table 5.9 Selected references on carboxylic and amino acids separations by BOHLM techniques... 

In order to calculate amino acid residence times from Equation 21, the ratio of the d to l enantiomers of the various amino acids are required as a function of depth in the oceanic water column. Unfortunately, there have been no investigations of the amino acid enantiomers dissolved in any natural waters. The analyses are difficult because most d- and l-amino acids are not separable by the usual amino acid analytical techniques. One exception is isoleucine, which forms alloisoleucine when it racemizes (Equation 13). Isoleucine and alloisoleucine are separable on the buffered columns of the automatic amino acid analyzer (88). However, as can be seen from Table V, only very small amounts of alloisoleucine would be produced from the racemization of isoleucine, unless... 

Electrophoresis and thin-layer chromatography are analytical separations—small amounts of amino acids are separated for analysis. Preparative separation, in which larger amounts of amino acids are separated for use in subsequent processes, can be achieved using ion-exchange chromatography. This technique employs a column packed with an insoluble resin. A solution of a mixture of amino acids is loaded onto the top of the column and eluted with a buffer. The amino acids separate because they flow through the column at different rates, as explained below. 

Selective Crystal Dissolution. An effective method for distinguishing bulk (lattice trapped) versus surface impurities involves the selective dissolution of a crystal sample while testing the liquid and/or crystalline phases for relative purity. In this technique, a small sample of crystals of a narrow size fraction is washed with successive small amounts of clean solvent until most of the crystalline phase is dissolved. The filtrate and/or crystalline phase are analyzed after each washing to discern whether impurities reside predominantly at the surface, or are more evenly distributed throughout the crystalline phase. The general approach is described by Narang and Sherwood (1978) for quantifying caproic acid incorporation in adipic crystals, and by Addadi et al. (1982) for amino acid separations. 

Peptide chromatography can also be carried out on a reverse-phase column by the ion-pairing technique. The ion pair has increased affinity for the support and allows small hydrophilic peptides to be retained. It also offers a different selectivity and has been reported to improve resolution. Heptane-sulfonic acid and tetrabutylammonium phosphate were used with large peptides (Hancock et al, 1978b), and trifluoroacetic acid was used for both peptides and proteins (Bennett et al, 1979). Amino acid separations have also been carried out on a reverse-phase column using anionic surfactants for ion pairing (Kraak et al, 1977). 

Fluorenylmethylchloroformate (FMOC) was used in the precolumn derivatiza-tion technique for amino acids. Separation of 30 residues was completed in 23 min using a C g column (A = 263 nm, ex 313 nm, em) and an 18/82 -> 99/1 methanol/ water (25 mM ammonium phosphate at pH 6.5) gradient. Amino acid levels down to lOOpmol were readily detected [467]. 

A perusal of the voluminous literature on the analysis of amino acids indicates that most separation procedures resort to automatic amino acid analyzer techniques. Teachers at small colleges or researchers on limited budgets may not have access to an automatic amino acid analyzer. Therefore, these workers must rely more heavily on planar chromatography procedures (both PC and TLC) for amino acid separations. 

Biomolecule Separations. Advances in chemical separation techniques such as capillary zone electrophoresis (cze) and sedimentation field flow fractionation (sfff) allow for the isolation of nanogram quantities of amino acids and proteins, as weU as the characterization of large biomolecules (63—68) (see Biopolymers, analytical techniques). The two aforementioned techniques, as weU as chromatography and centrifugation, ate all based upon the differential migration of materials. Trends in the area of separations are toward the manipulation of smaller sample volumes, more rapid purification and analysis of materials, higher resolution of complex mixtures, milder conditions, and higher recovery (69). 

Electrophoresis is used primarily to analyze mixtures of peptides and proteins, rather than individual amino acids, but analogous principles apply. Because they incorporate different numbers of amino acids and because their side chains are different, two peptides will have slightly different acid-base properties and slightly different net charges at a particular pH. Thus, their mobilities in an electric field will be different, and electrophoresis can be used to separate them. The medium used to separate peptides and proteins is typically a polyacrylamide gel, leading to the term gel electrophoresis for this technique. 

However, it was not until the beginning of 1994 that a rapid (<1.5 h) total resolution of two pairs of racemic amino acid derivatives with a CPC device was published [124]. The chiral selector was A-dodecanoyl-L-proline-3,5-dimethylanilide (1) and the system of solvents used was constituted by a mixture of heptane/ethyl acetate/methanol/water (3 1 3 1). Although the amounts of sample resolved were small (2 ml of a 10 inM solution of the amino acid derivatives), this separation demonstrated the feasibility and the potential of the technique for chiral separations. Thus, a number of publications appeared subsequently. Firstly, the same chiral selector was utilized for the resolution of 1 g of ( )-A-(3,5-dinitrobenzoyl)leucine with a modified system of solvents, where the substitution of water by an acidified solution... 

In the ion-exchange technique, separated amino acids exiting (eluting) from the end of the chromatography column mix with a solution of ninhydrin and undergo a rapid reaction that produces an intense purple color. The color is detected by a spectrometer, and a plot of elution time versus spectrometer absorbance is obtained. 

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