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CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 41, Issue 11, November 2013 Online English edition of the Chinese language journal Cite this article as: Chin J Anal Chem, 2013, 41(11), 1773–1779. Received 20 May 2013; accepted 12 August 2013 * Corresponding author. Email: [email protected] 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 REVIEW 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, Xi’an 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 1 Introduction 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 [35] . 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 ºC; (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

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Page 1: Application of Gas Chromatography-Mass Spectrometry for the Identification of Organic Compounds in Cultural Relics

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), 1773–1779.

Received 20 May 2013; accepted 12 August 2013 * Corresponding author. Email: [email protected] 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

REVIEW

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, Xi’an 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

1 Introduction

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[3�5]. 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 ºC; (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

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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 hydrolysis and 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 (CuCO3·Cu(OH)2) and azurite (2CuCO3·Cu(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[8] 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[9]. 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[12]. 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)[17] are the widely used silylation reagents which can react with almost all compounds containing active hydrogen carboxyl, hydroxyl, thiol, amino and imino groups of amino acids to produce silyl ether and silyl ester[18]. 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

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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 ether methanol 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 process, saponification of the lipid extractives is then performed by alkaline alcoholic solutions, and the most widely used reagent is KOH. Generally, lipid samples contain multiple components, such as oils, waxes, resins etc., therefore the hydrolysis products of a lipid sample contains not only fatty acid methyl ester bust also unsaponifiable moieties, such as sterols and neutral organic compositions, etc. The unsaponifiable products have strong polarity, and are not easy to be gasified, so a derivatisation reaction should always be followed prior to GC-MS analysis to increase their volatility. The commonly used derivatization reagents are MTBSTFA[21], BSTFA[22,23], ECF[24,25] and so on. Although the above treatment method of lipid has good derivatization efficiency, it has the drawbacks of high cost, complicated operation and time-consuming etc.

In recent years, the one-step reaction of thermally-assisted hydrolysis-online methylation has been used in lipid analysis in artworks. The commonly used methylation reagents include tetramethylammonium hydroxide (TMAH)[26,27], (m-trifluoro- methylphenyl)trimethylammonium hydroxide (TFTMAH)[28,29]

and trimethyl sulfonium hydroxide (TMSH), etc[30,31]. In this method, the sample and methylation reagents are directly injected into the gas chromatograph inlet, and the esterification reaction between them can occur at high temperature, leading to the selective ester bond breaking. At the same time, the polar groups, such as hydroxyl and carboxyl groups can react with the reagent instantaneously to generate the corresponding methylation products for chromatographic analysis. It can be seen that the method is simple, and does not need sample pretreatment. Moreover, such method can not only improve the analysis efficiency, but also avoid impurities possibly introduced in the multi-step process. Overall, the one-step method is one of the important strategies in the analyses of organic materials of artworks. 2.3 Pre-treatment of polysaccharides

For artwork samples, polysaccharides are firstly pretreated

by hydrolysis, followed by removing the inorganic deposits through centrifugation to avoid their interference. The hydrolysis of polysaccharides includes acidic hydrolysis and methanolysis. Trifluoroacetic aid (TFA) is considered the most suitable reagent for acidic hydrolysis of polysaccharides

into monosaccharides[32]. Bleton et al[33,34] proposed a polysaccharide methanolysis using acetyl chlorure/methanol, and the result showed that such methanolysis permits the best recovery of oses and uronic acid. The method made it possible to simultaneously separate and identify polysaccharides and glycoproteins containing neutral, acidic and alkaline sugar[35].

After centrifuged for purification, the monosaccharides were commonly derivatized with trimethylsilylation[9] and/or acetylation[36]. During hydrolysis, monosaccharides might undergo intramolecular reactions to form cyclic hemiacetals, which made the chromatographic analysis complicated with multiple peaks in chromatograms. This problem was overcome by converting monosaccharides into acyclic oximes or diethyl mercaptal derivatives[36,37]. For example, Bonaduce et al[38] reported the conversion of monosaccharide and uronic acids to diethyl mercaptals and lactones by using ethanethiol- trifluoroacetic acid (2:1, V/V), resulting in one chromate- graphic peak for each monosaccharide and uronic acid. 2.4 Pretreatment of mixed organic samples

Generally, there is a mixture of proteins, lipids and

polysaccharides in real artwork. Theoretically, the organic compounds could be successively treated with different methods described in Section 2.1 and 2.3 for GC-MS analysis. However, such process not only increases the analysis workload, but also requires larger amount of samples and may result in more damage to the cultural relics, which will break the principle of non-destructive or micro-damage analysis. In recent years, determination of several kinds of organic compounds simultaneously has been extensively investigated. Usually, the mixture is first separated, and each component is then pretreated with different methods, such as hydrolysis, derivatization, and chromatographic analysis. For example, to separate lipid and protein components, the sample could be extracted and separated according to their different solubilities in various solvents. Amino acids can be dissolved in aqueous phase, while lipids are extracted into organic phase by organic solvents, such as diethyl ether[39,40]. However, carbohydrates and proteins can not be separated by solvent extraction due to their similar solubilities. The C4 column is quite feasible to separate them. Lluveras et al[41] developed a procedure for the separation and characterization of lipids, proteins and polysaccharides simultaneously by using diethyl ether and C4 column. With the method, lipids, proteins and polysaccharides were fist dissolved in ammonia, and lipids were extracted into diethyl ether, while proteins and polysacchairides were still in water phase. Based on the monolithic sorbent tip technique with a C4 stationary phase, proteins and polysaccharides can be separated and purified before hydrolysis. The separated fractions can be analyzed separately by GC-MS. These studies established the methods for the analyses of mixed organic materials in artwork samples and were applied in real sample characterization[42].

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3 GC-MS analyses of cultural relics Weakly polar columns are generally used for the gas

chromatographic separation of cultural relics samples. Currently, the stationary phase of widely used column is made up of 5% diphenyl-95% dimethylpolysiloxane. Because the complicated compositions in cultural relics samples have different boiling points, the temperature programmed chromatography with the range from 50 ºC to 300 ºC is widely used to separate and detect each component. The mixed compounds can be separated and detected successively according to their boiling points from lower to higher. By adjusting the temperature program, flow rates of carrier gas and other experimental parameters, satisfactorychromatographic separation can be achieved. For example, after optimization experiments, the chromatographic conditions of proteinaceous glue analysis was determined, i.e., the inlet temperature of 280 ºC, constant flow of 1.5 mL min�1. In the temperature program, the initial temperature was kept at 100 ºC for 2 min and the temperature was then increased to 280 ºC at a rate of 6 ºC min�1, and kept for 10 min. Under the above chromatographic conditions, we obtained the GC-MS chromatogram of simulated sample of pig skin glue with cinnabar (Fig.2). It can be seen that the alanine (Ala), glycine (Gly) and other 12 kinds of amino acids have been separated completely with retention time within 10�30 min. For the GC separation of lipids and carbohydrates, the programmed temperature is usually in the range of 50�320 ºC which is wider than that for proteinaceous materials.

4 Mathematics models and applications in

identification of organic residues in artworks Although the qualitative and quantitative data acquired

from GC-MS analysis are the basis of organic material identification in artworks, it is difficult to identify the organic components accurately without the classification and extraction of useful information from the great amount of data. There are four kinds of mathematics models of the identification on the whole.

Fig 2 GC-MS chromatogram of simulated sample of pig skin glue with cinnabar

4.1 Identification based on specific markers Differentiating organic substances based on certain markers

is a simple and convenient method which is suitable for the identification of some samples with simple compositions such as animal proteinaceous glue. 4.1.1 Identification of animal glue

Animal glue can be identified according to the presence or

not of hydroxyproline which only exists in animal tissues. Wei et al[43] analyzed the binding media of the polychrome pottery of Xihan Dynasty at Qingzhou, Xiangshan, Shandong by GC-MS. The existence of hydroxyproline showed the use of animal glue. On the Basis of the presence of hydroxyproline, we inferred the use of animal glue in the wall paintings of the Five North Provinces’ Assembly Hall, at Ziyang, Shanxi China. 4.1.2 Identification of natural resins

The resin categories can be identified based on the different

compositions of the fatty acids. Cartoni et al[44] performed the GC-MS analyses and acquired the GC-MS chart of the raw and aged common terpenoid resins, including turpentine, dammar, copal and elemi. Based on the specific fatty acid composition of each kind of resin, two oil paintings in 17th century were identified to likely contain turpentine. 4.1.3 Identification of waxes

Wax is mainly composed of esters containing long-chain

fatty acids and alcohols. According to carbon chain lengths and the odd/even numbers of carbon, different kinds of waxes can be classified. Regert et al[45] studied the composition features of beeswax, spermaceti, carnauba, Japan wax as well as pine resin using high temperature gas chromatography and mass chromatography (HT GC-MS). Ten samples from a sculpture made by the famous French sculptor Barye in 19th century were investigated, and the results indicated the presence of beeswax in the samples. 4.2 Identification by content ratios

Because the substances consist of same components, the

amount of each component may be different. Thus, it is possible to distinguish the substances according to the content ratios of each composition. This method was successfully applied in the identification of drying oils and proteinaceous materials. 4.2.1 Identification of dying oils

Because the drying oil made of fatty acids, the ratio

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between the stearic and palmitic acid can be commonly used to identify the different types of drying oils Table 1 . The pure linseed oil, poppy oil, sunflower oil and the mixed samples with pigments were investigated by Gimeno- Adelantadoa et al[46]. The results indicated that the drying oil of two Spanish Baroque oil paintings was likely linseed oil according to the ratio between the stearic and palmitic acid. In addition, for the same samples, there was almost no change of the ratio before and after aging. However, the different pigments would induce certain influence on the ratio of stearic acid to palmitic acid. 4.2.2 Identification of proteins

From a lot of experimental data, the content ratio of amino

acids in the common proteinaceous binding media has the following results (Table 2): the content ratio of glutamic acid (Glu) to alanine (Ala) in milk exceeds 3 and the content ratio between aspartic acid Asp and praline Pro in egg is larger than 4, which are much higher than those in the other proteinaceous binding media. Based on the different ratios, the animal glue, milk and egg in proteinaceous binding media can be identified successfully.

As proteins and fats are composed of many kinds of amino acids and fatty acids, various kinds of components should be detected by GC-MS analyses. However, in the above identification model only the data of two constituents were provided and the information of all components could not be covered, leading to the low analysis accuracy, especially for the samples with mixed substances. 4.3 PCA identification

Principal component analysis (PCA) is introduced due to

the limitations of the model described in Section 4.2. PCA can retain the most information of the investigated data and simplify the complicated data. PCA can reduce the dimensions of the multi variables through the linear combination of maximum variance of the multidimensional data and replace the original data with several new variables (the principle component), revealing the inherent connection of the multi variables. PCA was widely used in the identification of proteins and carbohydrates. The amino acid compositions of the commonly used proteinaceous binding media were processed with PCA by Bonaduce. The score plot of the first two principle components is shown in Fig.3[47]. From the figure, it can be seen that different proteins were clearly distinguished and the binding media in Terracotta Warriors were analyzed to contain egg component. Moreover, based on the PCA analysis results of vegetable gum, the organic component of one wall painting was fruit gum, and the other two were the mixture of tragacanth and fruit gum[38].

Table 1 Ratios of stearic acid to palmitic acid in three lipids Drying oil S/P Linseed oil 0.62 ± 0.04 Poppy oil 0.22 ± 0.01 Sunflower oil 0.57 ± 0.01

Table 2 Molar ratios of several amino acids in proteinaceous binders

Proteinaceous binder Glu/Ala Asp/Pro Animal glue ~0.5 ~ 0.3 Egg ~1.3 > 4.0 Milk > 3.0 ~ 1.0

Fig.3 PCA score plot of standard proteinaceous binders ( ) and

samples from the polychromy of Qin Shihuang’s Terra cotta Army (�)[47]

4.4 Identification by discriminant analysis

Discriminant analysis is a multi variable statistical method

that can determine the categories according to the characteristic values of the research object with the categories that have been settled. This analysis method can preserve most of the original information and provide comprehensively accurate result. When using the discriminant analysis, the first step is to establish the corresponding discriminant model based on the multi characteristic observations of the samples whose categories have already been known, and then the unknown samples can be classified with this model. The commonly used discriminant analysis methods include Fisher Discriminant, Bayes Discriminant and Distance Discriminant etc. We established the Bayes Discriminant function on the basis of the amino acid data obtained from the GC-MS analyses of Chinese common proteinaceous binding media in ancient[48]. The models are shown as followings: Fegg yolk = 22.177AAla + 84.683AGly + 8.032AVa l+ 92.115ALeu –

34.761AIle + 62.530APro + 27.254AMet + 25.862APhe + 43.521AAsp + 54.078AHyp + 71.946AGlu – 1801.096;

Fegg white = 30.123AAla + 86.478AGly + 29.041AVal + 67.558ALeu – 39.488AIle + 66.954APro + 63.963AMet + 28.256APhe + 45.750AAsp + 56.155AHyp + 74.565AGlu – 1973.374;

Fskin and bone glue = 56.539AAla + 190.235AGly + 26.343AVal + 102.900ALeu – 87.979AIle + 104.025APro + 53.176AMet

+ 25.844APhe + 44.672AAsp + 129.220AHyp + 139.441AGlu – 5698.781;

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Ficthyocolla = 67.630AAla + 190.265AGly + 35.765AVal + 87.003ALeu

– 84.950AIle + 99.336APro + 57.215AMet + 18.530APhe

+ 36.454AAsp + 135.137AHyp + 140.599AGlu – 5820.685;Fmilk = 19.805AAla + 110.158AGly – 2.980AVal + 122.218ALeu –

32.925AIle + 82.583APro + 51.153AMet + 30.615APhe + 36.939AAsp + 65.579AHyp + 98.226AGlu – 2805.851

where, A is the normalized mole number of the amino acid, for example Aala is the normalized mole number of alanine; F is defined as the Bayes discriminant function of whose highest score is regarded as the most likely type.

The amino acid compositions of the sample from a Yuan dynasty wooden polychrome artwork were analyzed, the results were listed in Table 3. Also the Bayes discriminant scores are shown in Table 4. The results indicate that the binding medium is most likely animal glue. 5 Conclusion and Prospects

Due to the advantages of efficient separation, accurate

quantification and less sample requirement, GC-MS was increasingly applied to the analysis of organic residues in artworks. Researches mainly focus on the pretreatment of the samples (including purification, hydrolysis and derivatization), the selection of GC-MS conditions and construction of models for the identification of proteins, lipids and polysaccharides. However, the GC-MS procedures are still complex and the

analysis results are easily interfered by impurities. With more work carried out on this research field, there appear the following new trends in the characterization of organic materials of artworks: (1) Simplification of sample pretreatments Not only should the analysis time be shortened, but also the impurity introduced in the process should be largely reduced. The related research can refer to the one-step reaction of thermally assisted hydrolysis-online methylation of lipid analysis to simplify the pretreatment steps for organic substance. (2) Determination of complex organic samples simultaneously For example, the separation of lipids and proteins can be realized prior to chromatographic determination by taking advantage of their significantly different solubility in specific solvents. With such process, various organic materials can be identified simultaneously by taking sample only once. Meanwhile, more information can be acquired with reduced sampling of artifacts, obeying the principle of non-destructive or micro-damage analysis of cultural heritage. (3) Elimination of impurities in samples Artwork samples are usually complex with a lot of unknown inorganic and organic components, so how to eliminate the interference and improve the analytical accuracy is one of critical problems in the analyses of artworks. To this end, different methods, such as chemical reagent, extraction, and chromatography have been chosen to remove impurities according to the impurities in different cultural samples.

Table 3 Relative amino acid molar percentages of artwork sample[48]

Ala Gly Val Leu Ile Pro Met Phe Asp Hyp Glu 4.1 8.4 1.8 2.0 1.4 3.4 1.9 1.3 4.0 6.4 49.4

Table 4 Bayes discriminant analysis score of amino acids of artwork sample[48]

Standard binder Yolk Egg white Skin glue Isinglass Milk Score 3531.71 3620.46 4643.45 4601.1 4231.299

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