气质联用技术鉴定颐和园彩画胶料及相关问题

为了鉴定颐和园彩画的有机胶料,利用气相色谱质谱联用技术(GCM S)对3个颐和园建筑彩画YH-1、YH-2、YH-3进行了分析。样品前处理包括氨水萃取蛋白、固相萃取C4柱排杂,微波辅助盐酸水解蛋白质、硅烷试剂进行氨基酸衍生化。以我国古代常用胶料动物胶、蛋、牛奶和不同配比的动物胶与蛋的混合胶作为标准胶料,以正亮氨酸为内标、利用气相色谱质谱联用技术测定胶料中的丙氨酸、甘氨酸等14种氨基酸含量。在分析胶料氨基酸组成特征的基础上结合主成分分析,鉴定出颐和园3个彩画样品均属于动物胶与蛋组成的混合胶,并对混合胶在绘画中的使用及其稳定性等相关问题进行了初步探讨。该研究丰富了颐和园彩画的制作材料,为我国彩绘文物胶料鉴定和为后期"原材料"的修缮和保护提供了科学依据。     

柱前衍生高效液相色谱法对动物组织中胶原蛋白的测定

动物组织中的胶原蛋白经酸水解后生成包括羟脯氨酸在内的氨基酸混合物,用邻苯二甲醛（OPA）与其中的一级氨基酸衍生,再用9-芴基甲氧基羰酰氯（FMOC）与其中的羟脯氨酸衍生,用反相高效液相色谱法测定羟脯氨酸含量。羟脯氨酸是胶原蛋白的特异性氨基酸且含量稳定,因而可通过样品中羟脯氨酸的含量计算胶原蛋白含量。在0.01-50 mg.L^-1范围内,羟脯氨酸的峰面积和质量浓度之间的相关系数为0.9993,保留时间和峰面积的相对标准偏差分别为0.30%和2.9%。该法选用FMOC与氨基酸衍生产物的特征波长检测,能够完全屏蔽一级氨基酸衍生物的干扰,可快速、准确、高效地测定羟脯氨酸及胶原蛋白的含量。     

鱼明胶作为卤化银纳米粒子载体的研究

采用的鱼明胶样品是从深海鱼的鱼皮提取而得,在成份和物理性质方面鱼明胶与通常使用的动物明胶有显著的差别.研究表明,鱼明胶的蛋白成分中α组分占绝对优势,且其中分子量较小的α2组分又比分子量较大的α1组分含量高得多.在鱼明胶中几乎没有分子量最大的γ组分.其氨基酸残基中,脯氨酸和羟脯氨酸的含量比动物胶低,而蛋氨酸含量却明显高于后者.鱼明胶还具有较低的胶凝温度.这些特点可能使之适宜作为分散介质来制备AgBrI纳米粒子乳剂.此外,鱼明胶中含有较多含硫物质,而且杂质铁主要以三价形式存在.并且进一步利用鱼明胶作为保护载体,在广泛的胶银比(即明胶量与银量之比值)范围(从8∶ 1到4∶ 1)获得了平均粒径为14 nm的 AgBrI纳米粒子乳剂.该乳剂具有良好的单分散性和热稳定性.对纳米粒子乳剂进行硫-金协同敏化可以提高其感光度.若适当增加敏化剂用量和适当延长敏化时间这种协同敏化作用的效果更好.     

反相高效液相色谱法测定人肌腱中的胶原蛋白

建立了测定人肌腱中胶原蛋白含量的高效液相色谱法。动物肌腱中的胶原蛋白经酸水解后生成包括羟脯氨酸在内的氨基酸混合物,用2,4-二硝基氯苯对水解产物衍生化,然后以0.01 mol/L乙酸钠-乙酸缓冲液(pH 6.0)-乙腈(体积比为80∶20)为流动相,经反相C18柱分离,紫外检测器于360 nm波长处检测来测定羟脯氨酸的含量。羟脯氨酸是胶原蛋白的特异性氨基酸且含量稳定,因而可通过样品中羟脯氨酸的含量来计算胶原蛋白的含量。该方法对羟脯氨酸的检出限为3 μg/L,羟脯氨酸为3 μg/L~100 mg/L时与峰面积的线性关系良好;样品测定的相对标准偏差为1.95%,加标回收率为98.4%~110.8%。对60份人肌腱样品中胶原蛋白的含量进行了测定。所建立的方法灵敏、准确、干扰少,适用于肌腱中胶原蛋白含量的测定。     

基于分子印迹识别的化学发光传感器测定人血清中的游离羟脯氨酸

羟脯氨酸(Hydroxyproline,HYP)是一种非必需氨基酸,主要存在于胶原组织中,是构成胶原的重要成分之一,作为胶原蛋白中的特异性氨基酸,可以反映机体中胶原蛋白含量的变化,而胶原蛋白含量变化又与许多疾病有关,如烧伤、创伤后的修复、骨质疏松、肝硬化、肺纤维化、肾纤维化、肿瘤等,因此测定血液中游离羟脯氨酸含量有重要临床意义.     

PICO-TAG反相色谱法测定鸡蛋中氨基酸含量

采用PICO-TAG反相色谱法测定了氨基酸,方法的相对标准偏差为0.75％,平均回收率为97.12％。对鸡蛋样品进行了测定,发现不同产地不同皮色鸡蛋中氨基酸含量无显著差异,样品测定相对标准偏差为1.67％。结果表明测定方法快速、灵敏、准确、重现性好。     

PICO－TAG反相色谱法测定鸡蛋中氨基酸含量

采用ＰＩＣＯ－ＴＡＧ反相色谱法测定了氨基酸，方法的相对标准偏差为０．７５％，平均回收率为９７．１２％。对鸡蛋样品进行了测定，发现不同产地不同皮色鸡蛋中氨基酸含量无显著差异，样品测定相对标准偏差为１．６７％。结果表明测定方法快速、灵敏、准确、重现性好。     

明胶大分子的照相性质研究——Ⅰ.明胶中的蛋氨酸砜及亚砜的测定

应用氨基酸分析仪对十六种国外明胶(大部分为IAG明胶)的氨基酸含量进行了测定,其中重点进行了蛋氨酸及其氧化产物含量的测定。测试结果表明:明胶中蛋氨酸含量随不同样品变化较大,亚砜含量一般较少,所有照相胶都含有砜,绝大多数胶样含有亚砜。     

植物形成愈伤组织的激素调节与核酸及氨基酸代谢的关系

本文将绿豆子叶切段培养在Miller培养基上附加或不附加生长素和激动素,研究了受伤和激素诱导形成愈伤组织的作用.实验证明绿豆子叶切段必须有生长素和激动素才能形成愈伤组织.25S和18S的RNA明显增加。对照子叶切段比形成愈伤组织的切段中自由氨基酸的含量多,而结合氨基酸的情况相反.脯氨酸和羟脯氨酸的含量在两种组织中都是随着培养时间的延长而增加,但是形成愈伤组织的切段中羟脯氨酸和脯氨酸含量的比例却大大超过对照。     

鸡蛋和乌鸡蛋中12种金属元素含量的研究

本文利用原子荧光光度计(AFS)和电感耦合等离子体发射光谱仪(ICP)测定普通鸡蛋和乌鸡蛋中的12种金属元素的含量,方法操作简单、数据准确可靠,且具有良好的精密度,样品的回收率为88.0%～116.5%.从样品检测的结果来看,从金属元素的角度来讲乌鸡蛋的营养价值比鸡蛋的营养价值要高.     

GC-MS identification of proteins in wall painting samples: A fast clean-up procedure to remove copper-based pigment interferences

A new approach was explored to purify proteins in a multi-step procedure for the characterisation of proteinaceous materials (casein, animal glue, and egg) in artwork samples by gas chromatography-mass spectrometry. High concentrations of inorganic salts, such as azurite, have been found to impair the determination of protein via amino acid analysis. The effect of varying concentrations of copper-based pigments on the quantification of amino acids was evaluated through the analysis of replica paintings prepared with the three types of proteinaceous materials. Glycine, aspartic and glutamic acids are the amino acids most affected by the presence of copper salts. In the case of high concentration of salts, this interference hampers the correct identification of the proteins. To eliminate the inorganic salts, a C18 pipette tip was used to clean-up the ammonia extracts before the acidic hydrolysis step. The clean-up procedure allows us to prevent the influence of the inorganic salts and thus allows correct protein identification, though the quantitative recovery of proteinaceous material is quite low. The effectiveness of the optimised procedure was evaluated by analysing samples from two Italian wall paintings from the 13th and the 14th centuries. Without the clean-up it would not have been possible to detect the presence of a mixture of egg and animal glue in one case, and that of egg in the other one.     

Animal glues in mixtures of natural binding media used in artistic and historic objects: identification by capillary zone electrophoresis

Animal glues were often used in historic and artistic objects, e.g. as paint ground, as binders for pigments, or as adhesives. The sources were egg, casein, or different collagens. For restoration and conservation purposes it is important to know which kind of animal glue a museum object contains. Capillary electrophoresis can deliver such information, because it enables differentiation among the three proteinaceous glue classes according to their different amino acid patterns after hydrolysis. This work deals with the most relevant problem in practice, whether this identification is obstructed by the presence of other binders, with which they are mixed in many real samples; in particular, interference from plant gums and drying oils was investigated. Capillary electrophoresis of the hydrolysates (after reaction with 6 mol L^–1 HCl) was performed with an acidic background electrolyte consisting of chloroacetic acid (51.9 mmol L^–1) adjusted with LiOH to pH 2.26. The underivatised analytes were detected with a contactless conductivity detector. It was found that the constituents of the plant gums (monosaccharides) or drying oils (long-chain fatty acids and short-chain dicarboxylic acids) never interfered with identification of the animal glues, as shown for artificial mixtures of the different binders even at tenfold excess over the animal glue, and for egg tempera samples. The method was used to identify the filling material from a statue from the eighteenth century.     

Classification of protein binders in artist's paints by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry: an evaluation of principal component analysis (PCA) and soft independent modelling of class analogy (SIMCA)

Proteomics techniques are increasingly applied for the identification of protein binders in historical paints. The complex nature of paint samples, with different kinds of pigments mixed into, and degradation by long term exposure to light, humidity and temperature variations, requires solid analysis and interpretation methods. In this study matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectra of tryptic-digested paint replicas are subjected to principal component analysis (PCA) and soft independent modelling of class analogy (SIMCA) in order to distinguish proteinaceous binders based on animal glues, egg white, egg yolk and milk casein from each other. The most meaningful peptide peaks for a given protein class will be determined, and if possible, annotated with their corresponding amino acid sequence. The methodology was subsequently applied on egg temperas, as well as on animal glues from different species. In the latter small differences in the MALDI-TOF mass spectra can allow the determination of a mammal or sturgeon origin of the glue. Finally, paint samples from the 16(th) century altarpiece of St Margaret of Antioch (Mlynica, Slovakia) were analysed. Several expected peaks are either present in lower abundance or completely missing in these natural aged paints, due to degradation of the paints. In spite of this mammalian glue was identified in the St Margaret samples.     

Characterisation of standard tempera painting layers containing proteinaceous binders by pyrolysis (/methylation)-gas chromatography-mass spectrometry

Summary Thirty standard painting layers were analysed by pyrolysis-gas chromatography/mass spectrometry (Py-GC-MS) and by Py-GC-MS in the presence of tetramethylammonium hydroxide (pyrolysis/methylation). Painting layers were prepared according to Renaissance recipes for tempera, employing proteinaceous binders (egg, glue and casein) and six different pigments. Thermal degradation products of proteins, carbohydrates and lipids were selected for semiquantitative analysis based on single/summed ion monitoring (SIM) mode. The relative distribution of these products was used to characterise binding media for the purpose of their identification in painting layers.     

Analytical study of proteinaceous binding media in works of art by gas chromatography using alkyl chloroformates as derivatising agents

In this work, we present the results obtained in an analytical study of the different types of proteinaceous binding media most commonly used in paintings, using GC-FID as the technique of analysis and GC-MS as a confirmatory technique. The application of this methodology requires prior hydrolysis of the proteins in the binding media to obtain free amino acids and then volatile derivatives, in this case by reaction with chloroformates due to advantages of speed, safety and the aqueous medium in which the reaction occurs. The method proposed for the proteinaceous binding media study is to calculate the proportions of the different amino acids with respect to alanine. This method provided good characterisation of different binding media, such as pork gelatine, beef gelatine, albumin, egg white and casein. The proposed method is used for the identification of binding media (including mixtures of binders) present in real samples from paintings in the city of Valencia, Spain.     

Identification of garlic in old gildings by gas chromatography-mass spectrometry

The proteinaceous content of garlic (Allium sativum) was characterised according to its amino acid composition by using a gas chromatography-mass spectrometry (GC-MS) analytical procedure. The procedure was tested on fresh and aged garlic samples as well as on reference gilding specimens prepared according to old recipes. The proteinaceous pattern showed a characteristic distribution of amino acids with glutamic acid being the major component. The average amino acidic composition was: glutamic acid (Glu; 29%), aspartic acid (Asp; 17%), serine (Ser; 11%), alanine, glycine, valine, leucine, lysine and phenylalanine (Ala, Gly, Val, Leu, Lys and Phe; 5-6%), isoleucine, proline and tyrosine (Ile, Pro and Tyr; 2-3%), methionine and hydroxyproline (Met and Hyp; 0.5%). In order to distinguish this material from animal glue and egg, which are the other proteinaceous media commonly used in gilding techniques, a database of amino acid percentages of the three proteins was built up and submitted to principal component analysis. Three separate clusters were obtained, allowing the protein identification. The application of the procedure on several gilding samples from Italian wall and easel paintings (13th-17th century) permitted to evidence the use of garlic as a gluing agent.     

Identification of proteinaceous binding media of easel paintings by gas chromatography of the amino acid derivatives after catalytic hydrolysis by a protonated cation exchanger

Summary A procedure for the hydrolysis of proteins, based on catalysis by the protonated form of a strong cation exchanger, was established for proteinaceous binding media (e.g. casein, egg, animal collagene) used for objects of art. The experimental parameters for the hydrolysis (temperature, time) were optimized, as well as the conditions for sorption and desorption of the amino acids on the cation exchanger. The results for the identification of proteins by the gas chromatographic pattern of the amino acid derivatives, after hydrolysis by the ion exchanger and by hydrochloric acid were compared. The former method proved to be more efficient due to the mild conditions, avoiding the formation of humins in the presence of carbohydrates, and reducing the dissolution of pigments. The method was applied to identify the proteinaceous vehicles of samples from priming and paint layers of easel and wall paintings from the 16th and 18th century.     

The simultaneous analysis of proteins, lipids, and diterpenoid resins found in cultural objects

We report a GC-MS method for the simultaneous analysis of proteins oil, and diterpenoid resins found in cultural objects. The method was initially designed for protein analysis of protenaceous paints and adhesives and involves acid hydrolysis as the first step. The amino acids in the protein hydrolysates, thus obtained, are treated with propan-l-ol/ hydrogen chloride and then pentafluoropropionic anhydride. The procedure was found also to yield the propyl esters of fatty acids derived from lipids and diterpenoid acids derived from natural resins, and thus allows the choice of a single method for the analysis of artists media which contain either oil s or proteins or mixtures of both proteins and oils or even resins. Thus natural mixtures such as egg yolk and also mixtures made by the artist such as animal glue/seed oil emulsions can be analysed. Coupled with FTIR analysis of paints and the staining of cross sections, to indicate layer structure the method can help to elucidate the paints and adhesives used by artists.     

Direct infusion mass spectrometry as a fingerprint of protein-binding media used in works of art

A direct infusion mass spectrometry method for the characterization of proteinaceous glues from binding media used in pictorial works of art prior to conservation or restoration treatment is proposed. Amino acids are released by acid hydrolysis and dissolved in a mixture of acidic water and ethanol. This mixture is directly infused into a mass spectrometer without any derivatization. The mass spectrometer is operated in positive ion electrospray mode (ESI-MS) to yield [M+H](+) ions for the amino acids. Relative amounts of each amino acid are calculated for each protein (beef and porcine gelatines, albumin, casein and egg). The analyzed proteins were satisfactorily distinguished. The method is easy and fast, and shows good sensitivity and resolution. The proposed method has been successfully applied to artistic samples from items of the cultural heritage of Valencia (Spain).     

The contribution of gas chromatography to the resynthesis of the post-Byzantine artist’s technique

Gas chromatographic analysis of ethyl chloroformate derivatives of samples taken from the paint layers of post-Byzantine panel paintings permitted the successful characterisation of the different binding media used in them. This paper describes an analytical study of various post-Byzantine binding media such as egg yolk and egg/oil emulsion, using gas chromatography. The characterisation of these icons’ binding media is an important task, as it contributes to our understanding of and the reconstruction of the post-Byzantine artists’ palette. It also enables us to investigate the validity of our assumptions about the influences of Venetian style on Greek icon painting techniques from the sixteenth to the early nineteenth century, which up to now have been based on information in artists’ handbooks. The methodology involves two experimental steps: (1) hydrolysis of the proteins and triglycerides in the binding media to obtain free amino acids and fatty acids, and (2) the formation of ethyl chloroformate derivatives via derivatization with ethyl chloroformate (ECF). This methodology is of considerable interest, since it permits the identifcation of the nature of the proteinaceous binders used in these works through the simultaneous derivatization and determination of amino acids and fatty acids. Advantages of this methodology include the small quantity of sample required and the minimum preparation time involved. The proteinaceous media can be determined based on the ratios of seven stable amino acids, while the type of emulsions and drying oils used can be determined from the fatty acid ratio. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.