Amino Acids, Peptides and Proteins - Dévényi, T., Gergely, J. 1976

Gas chromatographic analysis of amino acid derivatives. Current status. Capabilities and limitations of the method
Challenges in quantitative amino acid analysis

Quantitative analysis of Amino Acids by GC is of unquestionable interest. As a rule, the Quantitative determination of the Amino Acid Composition of a peptide is one of the crucial steps in sequence analysis. Since the degradation of a large protein yields A large number of fragments, it is desirable to spend a minimum amount of time and material on the analysis of each. Employing GC in this case meets these requirements quite well. Numerous studies on GC of Amino acids are ultimately aimed at solving this problem. However, a truly effective quantitative method is subject to incomparably higher demands than a qualitative one. Considering also the difficulties in preparing and separating Amino Acid Derivatives, it becomes clear why a standard method for their quantitative determination by gas Chromatography has not yet been developed. The main difficulties, as emphasized in the section on derivative preparation, are associated with polyfunctional amino acids. A method that ignores their identification can find only limited application. The quantitative analysis of only simple amino acids cannot satisfy the experimentalist [40]. The question of whether all amino acids occurring in Proteins can be determined by GC with sufficient accuracy still remains open. Here, we can only briefly review the existing conditions and possibilities. The Problems associated with the instrumentation required for quantitative GC have already been discussed earlier (see p. 302).

The goal of quantitative Amino acid analysis is to determine the molar composition of a peptide, i.e., the types and amounts of its constituent amino acids. Expressing the molar ratios of amino acids by comparing the content of a given amino acid with the corresponding value of The amino acid present in the mixture in the smallest amount, while providing a correct picture of the overall COMPOSITION OF THE starting compound, does not provide information about the size, i.e., the length of the polypeptide chain. To achieve this, one must determine The amount of at least one of the Amino Acids and relate it to the amount of the peptide used. Naturally, such a determination is meaningful only if all the amino acids making up the peptide are detected with equal accuracy. In this case, one can work with one or more Internal Standards having suitable retention volumes and find individual correction factors specific to the amino acid derivatives being determined, which, as already mentioned, depend heavily on the type of detector [45, 48].

Peaks obtained with so-called specific detectors do not reflect structural differences between components. These detectors respond to only one group present in all derivatives. When detecting DNP-amino acid methyl esters with an electron capture detector, only very small, Structure-dependent signal variations were observed [54]. This detector responds specifically to the DNP group of amino acid derivatives. Another measurement method, available only in exceptional cases, is based on the general conversion of various amino acids into a single compound, such as methane [3].

For the quantitative Determination of Amino acids, unlike conventional quantitative GC, it is the content of the parent amino acid in the mixture that is important, rather than that of the amino acid derivative. Errors in all operations performed prior to Separation and identification are included in the overall error of the final analytical result. Therefore, The conversion of amino acids must be quantitative or at least quantitatively reproducible. Large differences in yields, even if reproducible, will certainly complicate the work and make it extremely laborious. As shown in The Study of The formation of TFA-amino acid butyl esters, the average yields for Certain amino acids differed very little from one another and were about 96% [16, 53]. However, during the preparation of volatile TFA-methyl esters of Ala, Gly, and Val, significant losses of substance occurred even with careful concentration of the samples [19, 53]. If highly volatile derivatives are chosen for amino acid analysis, such operations are best avoided.

Naturally, the method of sample storage and Introduction has a major influence on the analytical results. Since Some amino acids are sensitive to Hydrolysis, special precautions are necessary [19, 53]. However, above all, the analysis of amino acid mixtures requires standardized experimental conditions for amino acid conversion. Judging by the glaring discrepancies in the published data, this problem has not yet been resolved.

Peak reproducibility on a gas chromatogram is determined by The behavior of the components on the Column. Undoubtedly, this is where one of the most serious problems of quantitative GC lies. As is well known, partial decomposition and adsorption phenomena (tailing) have to some extent interfered with the analysis of many polyfunctional Organic compounds [36]. We have already mentioned similar observations regarding amino acid derivatives [19, 53, 78]; the measurements by Cruickshank and Sheehan [16] also did not yield satisfactory results. For all 20 analyzed amino acids, with the exception of His and Cys, the variation in peak size was less than 10%. However, for only 10 amino acids were these values less than 5%. In similar measurements, Lamkin and Gehrke [53] obtained significantly better results, but they studied fewer amino acids and performed fewer experiments. The clear advantage of direct on-column injection used by these authors should be noted. Subsequently, Stalling et al. [83] described the quantitative GC analysis of all Natural Amino Acids.

Finally, it should be emphasized that both for the quantitative conversion and separation of amino acids, and for good signal reproducibility with sufficient accuracy, appropriate calibration is primarily required. In this case, peak areas could be directly and reliably correlated with the micromolar amounts of amino acids. The value of the method can only be judged after its application to the analysis of a suitable peptide hydrolysate and comparison with results obtained by other Methods.