GAS CHROMATOGRAPHIC ANALYSIS OF AMINO ACIDS AS ETHYL CHLOROFORMATE DERIVATIVES.
MICHAEL R. SCHILLING, & HERANT P. KHANJIAN
2 SCOPE OF RESEARCH
2.1 PIGMENT INTERFERENCES IN ECF PROCEDURE
A study was conducted to identify the effects of pigments in the ECF procedure. Gelatin was hydrolyzed with samples of 22 different artists' pigments that were selected on the basis of chemical composition to include most of the common pigments. Samples were prepared using the following procedure. Approximately 100 μg of dry pigment was weighed onto an aluminum pan and transferred to a 1 ml hydrolysis tube, to which was added 100 μl of an aqueous solution of food-grade gelatin (approximately 1 μg of gelatin/μl) and 2 μl of L-norleucine internal standard (1500 μg /μl in 0.1M HCl). After the solution was evaporated to dryness with a stream of nitrogen, the tubes (a maximum of 13) were placed inside a 25 ml hydrolysis chamber. Included with each set of samples were an empty tube that served as a blank and another tube that contained 100 μg of rabbit skin glue to monitor the hydrolysis yield. Half the hydrolysate solution was derivatized, following the ECF procedure (Schilling et al. 1996). To dissolve the derivatives prior to injection, 200 μl of benzene was used. Due to the toxicity of the reagents, a fume hood should be employed during the derivatization step. The concentrations of amino acids in the pigmented gelatin hydrolysates were compared to those of unpigmented gelatin, and the amount of protein in the sample was estimated from the sum of the weight percentages of all ECF-derivatized amino acids.
2.2 EFFECTS OF ACCELERATED AGING ON AMINO ACID COMPOSITION OF TEMPERA PAINT
To ascertain the effects of light and heat aging on proteinaceous paint media, egg yolk paints and rabbit skin glue paints were prepared with selected pigments and known pigment-to-binder composition following traditional procedures (Kay 1983), applied to glass plates, and allowed to dry at room temperature for 9 months. Afterward, one set of all paint samples was aged at 80�C for 6 weeks; an identical set was placed in a Weather-o-meter exposure chamber at 50�C and 50% RH for 500 hours; and a third set was stored at ambient conditions of lighting, humidity, and temperature.
After aging, the paints were sampled for GC analysis. Sample weights were <100 μg for egg paints and <300 μg for glue paints. Half the hydrolysate solution was derivatized, and benzene was used to dissolve the derivatives (from 50 μl to 200 μl was used, depending on the amino acid concentration). The amino acid compositions of the aged paints were compared to those of paints stored in ambient conditions. Changes in amino acid concentration were calculated for each paint mixture, and estimates of total protein content were compared to data that were derived from proximate analysis (Bergquist 1981).
2.3 SCHEME FOR IDENTIFICATION OF PROTEINACEOUS BINDING MEDIA
A scheme for protein identification was developed based on the molar compositions of the amino acids that were essentially unaffected by pigment interferences and aging. The scheme incorporated data analysis techniques employed in other studies: concentration tables (Halpine 1992; Ronca 1994) and correlation matrices, discussed in appendix 2(Sinkai and Sugisita 1990). In addition, estimates of protein content were useful for eliminating erroneous matches from the correlation matrix results (see data for Mantegna's Holy Family, listed in section 3.3).
Reference data used in the identification scheme were taken from the compilation of amino acid compositions reported in Schilling et al. 1996. Multiple sets of composition data for collagen (rabbit skin glue, fish glue, collagen), casein, and egg were reduced to averages for simplicity. The data listed in appendices 3 and 4 were expressed in molar percentage formats consistent with the protein identification scheme.
To more readily identify mixtures of whole egg and rabbit skin glue that may be present in objects, paints consisting of mixtures of these media were prepared. Lead white was ground with whole egg and rabbit skin glue mixed in varying proportions. The paints were applied to glass plates and allowed to dry under ambient lighting, temperature, and relative humidity conditions for four years. Samples were removed for GC analysis, and the concentration data were evaluated using the protein identification scheme. Experimentally determined protein content data were compared to the known composition of the paints to assess the accuracy.
2.4 ANALYSIS OF PAINT SAMPLES REMOVED FROM OBJECTS
To illustrate the utility of the ECF procedure, paint and ground samples from a number of objects were analyzed. From the analytical results, protein content was estimated and the proteinaceous binding media were identified. For most samples, the entire hydrolysate volume was derivatized, and the derivatives were dissolved in 10 μl of benzene prior to injection. Sample weights ranged from 6 μg to 40 μg for most samples; larger samples were analyzed when appropriate, based on the expected protein content.