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
Gas chromatography of peptides
Biemann et al. method

Based on Youngs' work [129] on the GC of N-acetyl amino acid esters, it was to be expected that the Separation of the corresponding peptide derivatives would be possible only in exceptional cases. Studies in this direction were carried out by Evans [25]. Biemann et al. [6] studied polyamino alcohols formed from N-acetylpeptide esters upon reduction with LiAlH₄; their preparation had previously been described by Karrer and Nicolaus [50]:

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These compounds are so volatile that even tri- and tetrapeptides can be subjected to GC. Volatility is significantly reduced if hydroxy groups are present in the starting molecule or are formed during reduction. Since separation proved unsatisfactory due to large retention volumes and tailing, it was proposed [7] to eliminate the hydroxy groups by replacing them with chlorine followed by subsequent reduction:

The Polyamines resulting from these reactions exhibited significantly smaller retention volumes and yielded sharper peaks during gas chromatographic separation. Using mass spectrometry, all the Peptides studied could be identified as both polyamino alcohols and polyamines. Certain Amino Acids lose some structural features during double reduction, and their resulting products yield mass spectrometry peaks with the same number of mass units, thus becoming indistinguishable. These include Ala and Ser, Val and Glu, Pro and Orn, as well as α-aminobutyric acid, Thr and Asp. In such cases, they can be distinguished in the mass spectrum if the reduction is carried out in the presence of LiAlD₄, where the reduced groups are labeled with one or more deuterium atoms [7]. To illustrate this using the last Three amino acids as Examples: upon double reduction, the side chain of α-aminobutyric acid (I) remains unchanged, one deuterium atom is incorporated into Thr (II), and three into Asp (III):

Due to the differences in the masses of the corresponding fragments, all three Amino acids can be distinguished. Unfortunately, this rather elegant method has drawbacks, which limit its scope of application. The most serious drawback is that by-products that may form during incomplete reduction cannot be isolated. When dealing with a mixture of peptides of unknown composition, it is difficult to distinguish the true derivative from a by-product, as the latter can also be detected in the gas chromatograph. Another side reaction resulting from peptide bond Cleavage during reduction can lead to the appearance of new compounds (free amines and aldehydes), which further complicate the picture. It is known that during the reduction of a tertiary amide with LiAlH₄, Cleavage of the amide bond occurs, forming aldehydes and amines [104]. Similar cleavage reactions have already been observed for Proline peptides [74]. They also occur during the reduction of peptides with other metal hydrides [63]. We must admit that this method can only be applied to relatively simple mixtures of a few simple amino acids. But even with this limitation, its important advantage is that only a very small amount of substance (a few milligrams) is required for analysis.