Amino acid analysis gradient system

Phenylisothiocyanate (PITC). The use of PITC (Edman Reagent) to form thiohydantoin (PTH) derivatives of amino acids for protein sequencing is well known. The development of reversed phase systems for the separation of these PTH derivatives has resulted in the introduction of both isocratic and gradient elution systems (Fig. 11.2.11). A microbore HPLC unit for PTH-amino acid analysis is now available along with the recently introduced gas phase sequenator... 

All reverse-phase HPLC systems used for amino acid analysis are very sensitive to minimal changes in buffer concentrations, gradient shape, and temperature. 

Proteins may differ widely in lipophilicity depending on their amino acid composition. In the literature example shown in Figure 12.16, the reproducibility of three batches of a 300 A ODS packing for the separation of a mixture of proteins was studied. The mobile phase used was the popular system for protein analysis utilising gradient elution with aqueous trifluoroacetic acid and acetonitrile with gradually increasing acetonitrile content. Under these conditions the most lipophilic proteins elute last. 

Solvent systems encompass a dizzying array of permutations of organic solvents, buffers, and other mobile-phase additives. However, the most commonly employed solvent systems involve acetonitrile, methanol, and/or tetrahydrofuran. Buffers are typically acetate (pKa 4.8) or phosphate (pKa 1.3 and 6.7) at approximately 100 mM. For the analysis of a small number of free amino acids, isocratic elution is often possible. For the determination of an overall amino acid profile from a hydrolysate sample, complicated ternary gradients are often necessary. 

Gradient elution is also helpful for acids, the pH can be increased during a run, hastening the elution of the stronger acids. Alternatively or in addition, the ionic strength can be increased to promote faster elution. Such a gradient system was used in the analysis of amino acids mentioned earlier (Figure 9.8). 

Anderson et al. (1963) have described a more comprehensive system for nucleotide analysis. Their system has been recommended for adaptations of some amino acid analysers. A single column 0.9 X 160 cm of Dowex-1 ( x8, 200- 00 hydraulically fractionated to about 60 p particle diameter) washed in acid and alkali was packed in sections in 0.15 M sodium acetate pH 4.4. The sample, vol 0.5-1.5 ml in buffer, was eluted by a 1.4-1 linear gradient from 0.15-3 M sodium acetate at a constant pH (4.4), flow-rate (1 ml/min) and temperature (40°C). The first few peaks were extremely sharp and a lower flow rate could be used here. Good separations of quite complex mixtures were obtained in 28 hr. The triphosphates, UTP, ATP, GTP could be separated more quickly (in 6 hr) on a 0.9 x 50 cm column of the same resin eluted with a 1 1 linear gradient from 0.5 M sodium acetate, 0.25 M NaCl to 1.0 M sodium acetate, 0.5 M NaCl pH 3.6 at 1.7 ml/min at room temperature. A freshly packed column was used for each determination in both cases. 

Fig. 3 RP-HPLC analysis of a mixture of dansyl amino acids. Conditions stationary phase 4 /xm Nova Pak Cig mobile phase gradient of methanol and tetrahydrofuran versus aqueous phosphate buffer detection in a fluorescent detector excitation 338 nm, emission 455 nm. Amino acids are abbreviated by the one-letter system. (From Ref. 12.)...

An unusual EC reaction applicable to the detection of most amino acids involves complexation with copper ions in solution. It was later shown that this also occurs at copper electrodes. Although sensitivity limits of 0.5 to 18 ng injected depending on the amino acid have been reported for this system, it is not widely used, probably because the difficulties of resolving 20-I-amino acids on reversed-phase columns. Most workers therefore use derivatisation of the amino acids prior to chromatography for their analysis. Even though many such derivatives, e.g. phenylisothiocyanate (PITC), are electroactive, most analysts favour fluorescence detection since it is more compatible with the necessary solvent gradients as well as being more selective. 

This gradient system has been adapted for the analysis of coastal tind interstitial waters where compounds derived from amino acid degradation such as P-alaavae, taurine or amino butyric acids may occur in addition to the standard amino acids given in Table 26-1. For less complex samples such as, e.g., hydrolysates the gradient run time may be abbreviated and a linear gradient employed. It should, however, be noted that under these conditions glycine and threonine are usually not separated. 

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