GAS CHROMATOGRAPHIC ANALYSIS OF AMINO ACIDS AS ETHYL CHLOROFORMATE DERIVATIVES.
MICHAEL R. SCHILLING, & HERANT P. KHANJIAN
A number of chromatographic procedures have been developed for identification of proteins that are present in egg albumin, blood serum, and skin (Bidlingmeyer et al. 1984; Pickering 1989; Haynes et al. 1991; Hušek 1991). The procedures typically involve hydrolysis of protein by strong aqueous acid to release amino acids, derivatization to convert the amino acids into species amenable to detection, chromatographic separation, and quantitation of the amino acid derivatives. Proteins are identified by interpretation of the amino acid composition data. Because the procedures were developed for analysis of organic materials that are relatively free of inorganic contaminants, they may also be suitable for identification of proteins present in adhesives, sizings, and other conservation materials composed exclusively of organic substances (Sinkai and Sugisita 1990).
However, it is not clear that the same analytical procedures would give reliable results for egg, casein, and animal glue tempera paints that contain inorganic pigments. Some inorganic compounds are known to interfere with the hydrolysis and derivatization reactions, thereby reducing the concentrations of some or all amino acid derivatives (Grzywacz 1994; Bidlingmeyer et al. 1984). The amount of reduction depends upon both the composition of the pigment present and the derivatization procedure.
For example, high-performance liquid chromatography (HPLC) was used to measure the concentrations of amino acids (in the form of phenylthiocarbamyl derivatives) that were pres-ent in acid hydrolysates of glue and egg tempera paints (Halpine 1992). Copper pigments were found to reduce the concentration of all amino acid derivatives, whereas calcium pigments reduced the concentration of aspartic acid and glutamic acid derivatives only. In contrast, White (1984) used a gas chromatographic (GC) procedure (involving derivatization by trifluoroacylation and methylation) to analyze acid hydrolysates of the gypsum ground of a painting. It was found that the concentrations of aspartic acid and glutamic acid derivatives were unaffected by calcium interferences. From these observations it may be concluded that calcium interferes with the formation of phenylthiocarbamyl derivatives of aspartic acid and glutamic acid, not with the liberation of these amino acids from the proteins during acid hydrolysis.
In certain instances, it may be possible to minimize pigment interferences by removing cations from amino acid hydrolysates prior to derivatization. In an HPLC (phenylthiocarbamyl derivatization) procedure used to analyze the proteins present in large samples of plaster (Ronca 1994), pigment interferences were greatly reduced by eliminating calcium from the amino acid hydrolysates using an ion-exchange resin. Aspartic acid and glutamic acid concentrations in acid hydrolysates of protein-containing plaster samples closely matched those of the reference samples of protein. Unfortunately, ion exchange may not be a feasible option for removing inorganic cations from acid hydrolysates of microsamples of paint, due to losses incurred by transfer steps and column holdup. Accordingly, it is important to study the extent to which pigments interfere with the results of any chromatographic procedure used for the identification of proteinaceous binding media.
Physical aging is another factor that may complicate protein identification. It is well documented that proteins are susceptible to the effects of aging and that certain amino acids, such as cysteine and methionine, are more susceptible to chemical alteration and decomposition than others (Karpowicz 1981). However, the impact of physical aging on the chromatographic identification of proteins has yet to be established.
Paint samples that contain two or more proteinaceous binding media may present particular problems to the analyst. In some instances samples of paint may contain more than one proteinaceous medium from the intentional mixing of media by the artist. More com-monly, samples of paint removed for analysis may be unavoidably contaminated with traces of underlying ground layers. The two proteinaceous media most likely to be present together in tempera paint samples are animal glue (from ground layers) and egg. Because mixtures of proteinaceous media differ in composition from unadulterated media, amino acid composition data for mixed media may not correlate well to reference data.
This paper is a continuation of the research reported in a previous article (Schilling et al. 1996) in which a gas chromatographic procedure was developed for analysis of unpigmented, unaged protein. The procedure was a modification of a method originally developed for blood serum analysis (Hušek 1991). One advantage of ethyl chloroformate (ECF) derivatization is that fatty acids, present as triglycerides in drying oils and other lipids, may be converted to ethyl esters and analyzed with the amino acid derivatives. This additional qualitative information may aid in differentiating egg yolk from egg white (Nowik 1995). The modified procedure employed vapor-phase acid hydrolysis of protein, derivatization of the free amino acids using ethyl chloroformate, splitless injection of the N(O,S)-ethoxycarbonyl amino acid ethyl ester derivatives, separation of the esters on an HP-INNOWAX capillary column, and flame ionization detection (FID). Amino acid compositions were reported in terms of moles per 100 moles detected, and the weight percent of protein was estimated from the sum of the weight percentages of all amino acids detected. Sample weights were determined to the nearest 0.1 mg on an ultramicrobalance. Henceforth, this procedure will be referred to as the ECF procedure. A list of pertinent information regarding amino acids appears in appendix 1.