Separation of amino acid mixtures

The identification and quantitation of the individual amino acids in a mixture is often required in metabolic studies and investigations of protein structure. The use of thin-layer chromatography or electrophoresis may be adequate to indicate the relative amounts and number of different amino acids in a sample but the use of gas-liquid chromatography or an amino acid analyser is essential for quantitative analysis. 

Paper chromatography has been used successfully for many years and is still a useful tool despite the fact that thin-layer techniques, especially with readily available commercially prepared plastic or foil-backed plates, offer advantages of speed, resolution and easier handling. Larger volumes of sample can be applied to paper, permitting the subsequent elution of a particular amino 

H-Butanol 7 Very widely used for one-way runs or as a first 

Phenol 160 g Gives a large spread of values for a range of 

Water 40 ml j amino acids. Must always be used second in any 

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Thin-Layer Chromatography (tic). Tic (126) is used widely for quahtative analysis and micro-quantity separation of amino acid mixtures. The amino acids detected are developed by ninhydrin coloring, except for proline and hydroxyproline. Isatia has been recommended for specific coloring of pToline (127). 

The experimental setup for high-speed CZE can be seen in Figure 9.8. Highspeed CZE, or fast CZE (FCZE), yielded 70 000 to 90 000 theoretical plates for the separation of amino acid mixtures. Complete separation was achieved in under 11s, using a capillary length of 4 cm (24). 

Amino acid analysers based on ion exchange resins are available commercially. These achieve good separations of amino acid mixtures. Fluorescent derivatives of separated amino acids constitute a very sensitive means of detecting these compounds in seawater [256,258]. Fluorescent derivatives that have been studied include o-phthalaldehyde [259], dansyl [260], fluo-rescamine [261], and ninhydrin [261]. 

There are two main objectives under this heading (i) to convert amino acids into volatile derivatives for GLC and mass-spectrometric study and (ii) to introduce a group or groups with conveniently measured ultraviolet/visible absorption or fluorescence characteristics, so that TLC, HPLC, circular dichroism and other physicochemical and spectroscopic studies, or analytical separation of amino acid mixtures, may be accomplished. The analytical methods themselves are discussed in Chapter 3 and in later sections of this chapter. 

Farrelly and Watkins (F4) have used high voltage electrophoresis of unmodified urine or deproteinized serum for the rapid separation of fourteen amino acids in one direction on a thin-layer plate. Evered and Dando (E16) have employed low voltage electrophoresis for one way separation of amino acids on Whatman No. 1 paper using various buffer solutions. They stated that only the acidic and basic amino acids, p-amino acids, and cystine could be separated completely from a complex mixture such as blood or urine. Scherr (SIO) and Stevens (S52) have also used low voltage electrophoresis for the unidirectional separation of amino acid mixtures on cellulose acetate strips. 

Silica gel layers were used already in 1946 by Consdek, Gordon and Martin [99] for electrophoretic separation of amino acid mixtures. Pastuska and Trinks [196] and Honegger [123] were the first to draw attention to the advantages in TLC. The combination of normal TLC in one direction and electrophoretic separation in the second (Fig. 69), as described on pp. 106—114 by Hannig and Pascher in the chapter on electrophoresis, is especially interesting. Individual results are thus not discussed here. 

Hong, S. U. and M. L. Bruening. 2006. Separation of amino acid mixtures using multilayer polyelectrolyte nano filtration membranes. /. Memb. Sci. 280 1-5. 

The first of the two experiments given below illustrates the separation of amino-acids, now an almost classic example of the use of paper chromatography the second illustrates the separation of anthranilic acid and iV-methylanthranilic acid. Both experiments show the micro scale of the separation, and also the fact that a mixture of compounds which are chemically closely similar can be readily separated, and also can be identified by the use of controls. 

Fig.7-10. Separation of amino acids after derivatization with OPA and A -isobu-tyryl-L-cysteine. Column Superspher 100 RP-18 (4 pm) LiChroCART 125-4, mobile phase 50 mM sodium acetate buffer pH 7.0/sodium acetate buffer pH 5.3/methanol, flowrate 1.0 ml min temperature 25 °C detection fluorescence, excitation 340 nm/emission 445 nm. Sample amino acid standard mixture. (Merck KGaA Application note W219189 reproduced with permission from H. P. Fitznar, Alfred-Wegener-Institute for Polar and Marine Research.)...

Mainly inorganic, especially mixtures of metals with similar chemical characteristics, e.g. lanthanides separation of amino acids. 

CSPs and chiral mobile phase additives have also been used in the separation of amino acid enantiomers. Another technique that should be mentioned is an analysis system employing column-switching. D-and L- amino acids are first isolated as the racemic mixture by reverse-phase HPLC. The isolated fractions are introduced to a second column (a CSP or a mobile phase containing a chiral selector) for separation of enantiomers. Long et al. (2001) applied this technique to the determination of D- and L-Asp in cell culture medium, within cells and in rat blood. 

Fig. 7 Typical reversed-pbase separation of amino acids. Precolumn derivatization of a standard amino acid mixture was achieved employing FMOC. Resolution was achieved by gradient elution with acetonitrile, methanol, and acetate buffer (pH 4.2) on a C,8 column. Standard three-letter abbreviations for amino acids are used also, CySO H = cysteic acid. (From Ref. 164. Copyright 1983 Elsevier Science.)...

Exercises in thin-layer chromatography. Separation of amino acids. Prepare solutions of DL-alanine, L-leucine and L-lysine hydrochloride by dissolving 5 mg of each separately in 0.33 ml of distilled water, measured with a graduated 1 ml pipette (leucine may require warming to effect solution). Mix one drop of each solution to provide a mixture of the three amino acids and dilute the remainder of each solution to 1 ml to give solutions of the respective amino acids. The latter will contain about 5 pg of each amino acid per pi. Apply approximately 0.5 pi of each of the solutions to a Silica Gel G plate and allow to dry in the air (i.e. until the spots are no longer visible). 

Many different solvent developers have been used in the separation of sugars and related compounds. Three of these, phenol-water, collidine-water, and 1-butanol-acetic acid-water,27 also commonly employed in the resolution of amino acid mixtures, are widely used. Other commonly used solvent developers are 1-butanol-ammonia-water, 1-butanol-ethanol-water,27 1-butanol-pyridine-water,61 ethyl acetate-acetic acid-water, and ethyl acetate-pyridine-water.26... 

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. 

Little has been done with this procedure in the case of urine. Paper electrophoresis under high potential (10,000 volts) has yielded excellent separations of amino acids (W9). It has been applied to urine analyses by Noller et ah (Nl). Biserte et ah have used it for group fragmentation (B30) under potentials of 300 to 400 volts in volatile buffer mixtures at pH 2.4, 3.9, 6.5, and 8.9 and find it suitable as a first step prior to further, more complete separations by paper chromatography. 

As stated above, the utility of silica based stationary phases does not limit its use to organic mobile phases. For many years it has been commonplace in flash chromatography to use aqueous solvents to elute analytes from silica based media. Isocratic elution with mixtures of butanol, acetic acid and water is standard protocol for the separation of amino acids and a carefully prepared combination of methanol, chloroform and water is useful for general organic compounds. Peptides are also readily purified by gradient elution on normal phase silica, moving from acetonitrile to aqueous mobile phase 3,2l This technique is particularly useful for extremely hydrophilic peptides that are not strongly retained on reversed phase media. 

Naturally occurring amino acids can be obtained by hydrolyzing proteins and separating the amino acid mixture. Even so, it is often less expensive to synthesize the pure amino acid. In some cases, an unusual amino acid or an unnatural enantiomer is needed, and it must be synthesized. In this chapter, we consider four methods for making amino acids. All these methods are extensions of reactions we have already studied. 

Another widely used method for qualitative and quantitative analysis of amino acid mixtures is high-performance liquid chromatography (HPLC) (see Experiments 2 and 6). The mixture of amino acids is first subjected to reaction with phenylisothiocyanate (PITC) to convert them to the phenylthiocarbamyl-amino acid derivatives, which are then subjected to chromatographic separation. The derivativatization of the amino acids serves two purposes it attaches a UV-absorbing tag, which makes their quantitative determination easy, and it converts them to a more hydrophobic form, which is necessary for good separation on the reverse-phase system commonly used with this technique. This method of amino acid analysis will be used in Experiment 6. 

The. applications of ion-exchange chromatography are exemplified by the selection shown in table 4.18. Among the most notable are the separation of lanthanides and actinides using a citrate, lactate or EDTA eluting agent the separation of many metals as halide complexes on anionic resins and the separation of amino-acids with citrate buffers. The use of pressurized systems for complex mixtures is likely to become more widespread in the future. 

Finally, libraries aimed to chiral resolution of racemates will be covered here in particular, the use of chiral stationary phases (CSPs) has recently been reported for the identification of materials to be used for chiral separation of racemates by HPLC. The group of Frechet reported the selection of two macroporous poly methacrylate-supported 4-aryl-1,4-dihydropyrimidines (DHPs) as CSPs for the separation of amino acid, anti-inflammatory drugs, and DHP racemates from an 140-member discrete DHP library (214,215) as well as a deconvolutive approach for the identification of the best selector phase from a 36-member pool library of macroporous polymethacrylate-grafted amino acid anilides (216,217). Welch and co-workers (218,219) reported the selection of the best CSP for the separation of a racemic amino acid amide from a 50-member discrete dipeptide iV-3,5-dinitrobenzoyl amide hbrary and the follow-up, focused 71-member library (220). Wang and Li (221) reported the synthesis and the Circular Dichroism- (CD) based screening of a 16-member library of CSPs for the HPLC resolution of a leucine ester. Welch et al. recentiy reviewed the field of combinatorial libraries for the discovery of novel CSPs (222). Dyer et al. (223) reported an automated synthetic and screening procedure based on Differential Scanning Calorimetry (DSC) for the selection of chiral diastereomeric salts to resolve racemic mixtures by crystallization. Clark Still rejxrrted another example which is discussed in detail in Section 9.5.4. 

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