CHINESE JOURNAL OF ANALYTICAL CHEMISTRYVolume 41, Issue 11, November 2013 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2013, 41(11), 17731779.
Received 20 May 2013; accepted 12 August 2013 * Corresponding author. Email: email@example.com This work was supported by the National Natural Science Foundation of China (No. 21175104) and the Education Department of Shaanxi Province Foundation, China (Nos. 12JK0618, 13JZ045). Copyright 2013, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(13)60693-3
Application of Gas Chromatography-Mass Spectrometry for the Identification of Organic Compounds in Cultural Relics WU Chen, WANG Li-Qin*, YANG Lu, Ma Zhen-Zhen School of Cultural Heritage, Northwest University, Xian 710069, China
Abstract: As various kinds of organic materials own abundant information, it is significant to identify them in artworks to study the ancient science, technology, economy, culture and the protection of such kind of relics. Gas chromatography-mass spectrometry is an ideal method for the identification of organic residues in artworks due to the combination of high-performance separation with precisely quantitative analysis and micro sampling. This review mainly presents the application of GC-MS analytical technology in the characterization of proteins, lipids and polysaccharides in artworks, including the sample pre-treatment, GC-MS instrumental conditions and the mathematics models for identification. Additionally, some prospects of the development in this field are also discussed. Key Words: Gas chromatography-mass spectrometry; Cultural relics; Organic residue; Review
It is a difficult task to analyze organic residues in artworks in the field of cultural heritage conservation[1,2]. Recently, there have been some reports on analyses of organic residues from the binding media, protective coatings and so on in artworks. As one of the most widely used techniques, gas chromatography-mass spectroscopy (GC-MS) has been applied in the investigation of almost every kind of organic materials in artworks, but there are very few reports in China.
Based on the high-performance separation of gas chromatography and high selectivity of mass spectroscopy, GC-MS is an effective approach for the qualitative and quantitative determination of complex organic materials. As it has the advantage of micro sampling with the detection limit of nanograms, and reduced destruction to the artwork, GC-MS is an incomparable approach for the investigation of organic materials in artworks. However, the sample preparation for GC-MS is quite more complex than that for the spectroscopic techniques. Therefore, selection of effective methods of sample pre-treatment and other experimental conditions would
directly affect the accuracy of the analytical results. To provide scientific basis for the identification of artworks, this review mainly presents the application of GC-MS technique in the characterization of proteins, lipids and polysaccharides in artworks, including the sample pre-treatment, GC-MS experimental conditions and the mathematics models for the identification of artworks.
2 Pre-treatment of samples Most of relic samples are complex organic and inorganic
mixtures, and also include many impurities arose from degradation, environmental pollution, microbe activity and so on. Thus, the pretreatment step is quite necessary prior to the GC-MS analysis. It should be noted that for the GC-MS analysis, a sample should satisfy the following conditions after pretreatments: (1) The sample should be of thermal stability and volatility, and generally can be gasified under 250 C; (2) It does not contain inorganic materials like inorganic salts and acids which may be harmful to the GC column or affect the accuracy of the analytical result. Basically, there are three
WU Chen et al. / Chinese Journal of Analytical Chemistry, 2013, 41(11): 17731779
steps in a pre-treatment process, including purification, hydrolysis and derivatization. The purpose of purification is to eliminate interference from samples, and the purpose of hydrolysis is to decompose macromolecular compounds into small molecular compounds. Meanwhile, to satisfy the requirements of gas chromatography, the derivatization step is usually used to decrease the polarity and gasification temperature of the fractions from hydrolysisand increase the stability of the analytes.
The common organic materials found in artworks include binding media, protective coating and so on. Based on the chemical compositions, the organic materials can be classified into three categories: proteins, lipids and polysaccharides (carbohydrates). Protein materials commonly used in cultural relics include animal glue, egg and milk, etc. Lipids include drying oil, animal lipids, waxes, resins, etc. Carbohydrates include starch, honey and vegetable gum (such as Arabia gum, peach gum, tragacanth), etc. Different pretreatment methods are usually adopted according to the different chemical properties of the components of samples. 2.1 Pre-treatment of proteins
Ammonia extraction assisted by ultrasonication was the
most widely used method for proteinaceous purification[6,7]. In the method, proteins were completely soluble in ammonia solution, while insoluble inorganic pigments could be separated as precipitates after solid-liquid separation step. However, such method was not suitable for artworks containing copper-based pigments, such as malachite (CuCO3Cu(OH)2) and azurite (2CuCO3Cu(OH)2) due to the possible formation of copper complexes with ammonia, which affected the accuracy of the GC-MS result. To solve this problem, Gautier et al used a C18 pipette tip to eliminate the copper ions-based pigment interferences, and successfully analyzed the samples from two Italian wall paintings in the 13th and the 14th centuries. The cation-exchanger was also proposed for protein purification. As the column was loaded with the hydrolysate of proteinaceous sample, amino acids and pigments were retained by the resin, and then the amino acids could be eluted with a concentrated ammonia solution. Based on the principle of adsorption and desorption, this method avoids the dissolution and complexation phenomena and can eliminate almost all inorganic salts and polysacchardes in artworks, which is quite suitable to eliminate interference of cooper-based pigments. Nevertheless, the procedure is complicated, and requires strict operations to avoid introducing impurity.
The traditional hydrolysis process with 6 M HCl for 24 h in vacuum was commonly applied to the protein hydrolysis. However, this method is time-consuming and the hydrolysis is always incomplete. Colombini et al proposed a microwave- assisted digestion method for protein hydrolysis, which could
enable the rapid and complete digestion of protein samples. With this method, the glue proteins were completely hydrolyzed in 1.5 h. Therefore, the method was not only timesaving, but also could prevent the oxidization of amino acid, resulting in improved recovery of amino acids[10,11]. In recent years, enzymatic hydrolysis has also been used for protein hydrolysis, especially for the amino acids which are sensitive to chemical hydrolysis. For example, enzymatic hydrolysis of asparagine and glutamine can prevent the racemization of amino acids. However, this method usually takes long reaction time, which makes it not suitable for the analysis of large amounts of samples. Trypsin is the most frequently used proteolytic enzyme due to its specificity for a given protein[13,14], which is the basis of the protein identification. Overall, microwave-assisted digestion method may replace the traditional hydrolysis method because of its high efficiency. For the enzymatic hydrolysis, further research should be carried out to increase its usefulness for the analyses of artworks.
Currently, the derivatization methods for amino acids mainly include silylation and alkylation. N-methyl-N-(tert- butyldimethylsilyl trifluoroacetamide) (MTBSTFA)[15,16] and N,O-bistrimethylsilyltrifluoroacetamide BSTFA) are the widely used silylation reagents which can react with almost all compounds containing active hydrogencarboxyl, hydroxyl, thiol, amino and imino groups of amino acids to produce silyl ether and silyl ester. In the case of MTBSTFA, the protein derivatization reaction is shown in Fig.1. Meanwhile, ethyl chloroformate (ECF) is the most commonly used reagent for the alkylation of amino acids[19,20]. By comparing the two derivatization methods, silylation reaction is more complete, but the experimental conditions are harsher and the reaction should be performed in anhydrous environment. Alkylation has advantages of high reaction rate, no influence by disturbance, and the direct use of hydrolysis liquid in derivatization, but the derivatization efficiency is less than that of the silylation reagents.
Fig.1 Derivative reaction equation of MTBSTFA and amino acids
WU Chen et al. / Chinese Journal of Analytical Chemistry, 2013, 41(11): 17731779
2.2 Pre-treatment of lipid samples Lipid samples are usually purified based on the principle
that the similar substances are more likely to be dissolved by each other. Experimentally, organic solvents, such as dichloromethane, chloroform, ethyl ethermethanol and others, are used to dissolve the drying oils, animal fats, waxes, resins, etc. in the lipid samples. To obtain high extraction efficiency, mixed solvents are used or fractional extraction processes are adopted with different solvents.
After the extraction proces