2 ANALYSIS OF NATURAL POLYSACCHARIDE MATERIALS

2.1 DERIVATIZATION TECHNIQUES FOR GC ANALYSIS OF SUGARS

Chemists have employed a wide variety of analytical techniques for the characterization of carbohydrate compounds, probably the most widespread over recent years being gas chromatography (GC). Since GC is dependent on the volatility of the analytes, it is necessary for the sugars to undergo hydrolysis, followed by some form of derivatization reaction prior to analysis.

Methods involving the formation of fully and partially methylated methyl glycosides, acetates, acetals, trimethylsilyl ethers, and more volatile alditol acetate derivatives of monosaccharides were popular for a time (McInnes et al. 1958; Bishop and Cooper 1960; Bishop 1964; Lehrfeld 1981; Blakeney et al. 1983). Today, however, trimethylsilyl (TMS) derivatives of carbohydrates are among the most widely used, primarily due to their high volatility and ease of preparation (Sweeley et al. 1963; Sullivan and Schewe 1977; Honda et al. 1979; Li et al. 1983; Twilley 1984).

Chromatograms of sugar-based materials can be complicated by the presence of between 1 and 5 peaks for each monosacharide, arising from the existence of the different structural forms: formation of the corresponding oxime-TMS derivative reduces the number of potential derivatives (Decker and Schweer 1982; Al-Hazmi and Stauffer 1986; Long and Chism 1987). Aldononitrile acetate derivatives can also be prepared via synthesis of the oxime intermediate (Dmitriev et al. 1971), the derivatization process being unaffected by the presence of water in the reaction mixture (Churms 1990).

Methods for the separation of neutral sugars in gums have been reported (Al-Hazmi and Stauffer 1986), but the uronic acid components were analyzed using additional spectrophotometric and differential GC techniques (Selvendran et al. 1979; Lehrfeld 1981). Neutral sugars and uronic acids were analyzed simultaneously as trimethylsilyl methyl glycosides: methanolysis of the uronic acids was performed, then the neutral sugars were trimethylsilylated (Ha and Thomas 1988). A similar method was used in a study of Auracaria bidwilli gum (Aspinall and Fairweather 1965; Aspinall and McKenna 1968).

An obvious problem associated with the use of any of these methods for the analysis of gums used in works of art is the sample size. Many of these techniques will not be sufficiently sensitive for the sometimes sub-nanogram samples available to the conservation scientist. Nevertheless, there has been some progress in the analysis of gum media from art objects using GC techniques, usually in conjunction with thin layer chromatography (TLC): TLC itself is suitable for qualitative analysis of larger gum media samples (∼1 mg).

2.2 CHROMATOGRAPHIC ANALYSIS OF GUM MEDIA FROM WORKS OF ART

GC and TLC were utilized to analyze the trimethylsilyl derivatives of sugars resulting from the hydrolysis of samples of the surface coating and paint from a wooden Egyptian sarcophagus, dating from the 21st dynasty. Findings disclosed the presence of gum tragacanth and honey (Masschelein-Kleiner and Tricot-Marckx 1965; Masschelein-Kleiner et al. 1968). Gum arabic was identified as the medium of a 16th-century manuscript by TLC (Flieder 1968), while the use of gum tragacanth in the paint of three ancient Egyptian epitaphal stelae was revealed by TLC (Szyszko 1972).

Birstein (1975) employed GC for his study on the problems associated with the binding media found in Asian wall paintings, and GC was the method chosen for the study of paintings found in the tomb of Nefertari at Luxor (Mora et al. 1990; Palet and Porta, 1990). Twilley (1984) published a report on the analysis and artistic applications of plant gums. Analysis of samples was achieved via a number of techniques, including GC of trimethylsilyl sugar derivatives.

Erhardt et al. (1988) employed GC analysis of TMS-oxime derivatives for their studies of gum media, and, most recently, Bleton et al. (1996) reported on a GC method for the analysis of ink samples from ancient manuscripts.

Derrick and Stulik (1990) used pyrolysis–gas chromatography (Py-GC) to characterize natural gums used in works of art. Gums arabic, tragacanth, guar, ghatti, and karaya all gave distinguishable and reproducible pyrograms, enabling their identification.

As part of a wider project investigating the media used by William Blake and other 19th-century British artists, we have developed a simple derivatization technique for the analysis of extremely small samples of gum media by GC-MS. The method of sample preparation, using HMDS with trifluoroacetic acid in pyridine, was originally employed for the silylation of syrups and concentrated aqueous solutions of sugars and starch hydrolysates (Brobst and Lott 1966) but, when adapted, proved highly satisfactory for the preparation of gum media samples removed from works of art. It was chosen for its relative simplicity, in that no special reaction conditions were required and the derivatization itself was both rapid and repeatable, giving rise to a sugar “fingerprint” for each of the gum materials studied.