Identification of lipid binders in old oil paintings by separation of 4-bromomethyl-7-methoxycoumarin derivatives of fatty acids by liquid chromatography with fluorescence detection

A HPLC-fluorescence method for identification of drying oils from binding media or protective film used in pictorial works of art prior to conservation or restoration is proposed. Fluorescence derivatization of fatty acids released by hydrolysis of structural drying oils is studied. The derivatization reagent was 4-(bromomethyl)-7-methoxycoumarin with 18-crown-6 as catalyst. Mobile phase was programmed from methanol-water (90:10 v/v) to methanol-water (100:0 v/v) in 25 min. The excitation and emission wavelengths were 325 and 395 nm, respectively. Under these chromatographic conditions, coumarin derivatives of myristic, palmitic, oleic and stearic acids were satisfactorily resolved. The method shows good sensitivity, with a detection limit of 6.0 x 10(-8) mmol, and good linearity between 1.0 x 10(-7) and 1.8 x 10(-4) mmol of each analyte. Peak area ratios among fatty acids derivatives, especially the stearic acid/palmitic acid peak area ratio, are useful to identify the drying oils. 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.     

Characterization of medieval proteinaceous painting media using gas chromatography and gas chromatography — mass spectrometry

Proteinaceous organic materials used as ancient painting media were investigated by capillary gas chromatography (GC) and capillary gas chromatography — mass spectrometry (GC/MS). Medieval wall paintings made by the tempera technique were considered and their binding media were studied by the characterization of their main chemical components. The basic methodology is based on the determination of amino acids in samples of paint layers after hydrolysis and derivatization and on the comparison with reference proteinaceous materials. Multivariate chemometric techniques were used to facilitate the recognition of the protein source from chromatographic data. To characterize the binders further, a method was developed for the determination of fatty acids, present as minor components, by GC/MS. The use of fused-silica capillary columns coated with selected stationary phases allowed the separation of amino acid and fatty acid derivatives in a single analytical run.     

Composition of the floral waxes ofRosa gallica, Jasminum grandiflorumm, andViola odorata

The chemical compositions of the waxes of the flowers ofRosa gallica L., Jasminum grandiflorum L., andViola odorata L. have been characterized. The waxes of these plants mainly contained hydrocarbons and fatty and cyclic alcohols and their esters with fatty acids. The highest level of free fatty acids (12.4%) was found in theJ. grandiflorum wax. Depending on the plant species, the above-mentioned groups of substances differed in composition and amount, but hydrocarbons predominated.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (371) 120 64 75. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 327–333, May–June, 1999.     

Thin-layer chromatography-pyrolysis-gas chromatography-mass spectrometry: a multidimensional approach to marine lipid class and molecular species analysis

A new multidimensional chromatographic method is described in which material separated into lipid-class bands on silica-coated quartz thin-layer chromatography (TLC) rods (Chromarods) is desorbed using a pyrolysis unit interface and introduced directly into a gas chromatograph-mass spectrometer for molecular species analysis. Steryl esters, wax esters, hydrocarbons, ketones, and fatty-acid methyl esters (FAMEs) are thermally desorbed without pretreatment. In order to desorb free sterols, monoacylglycerols (MAGs), aliphatic alcohols, and free fatty acids, the esters are converted to trimethylsilyl derivatives on the rod. Triacylglycerols and phospholipids are converted to FAMEs by thermochemolysis with tetramethylammonium hydroxide. The method's utility is demonstrated with lipids from seawater particulate matter by first confirming the identity of lipid bands with the appropriate standards. The wax ester-steryl ester TLC band contained no more than 8% steryl esters. Wax esters of up to C42 are detected. In six individual acyl lipid classes, C14-C22 fatty acids are detected with C16 acids predominant in all but wax esters. C16-C22 MAGs are identified in the complex acetone-mobile polar lipid band. The method successfully extends the scope of latroscan TLC-flame-ionization detection on Chromarods, which is a widely used technique for lipid-class analysis. Modification of the pyrolysis probe to handle intact TLC rods is a future objective.     

Identification by gas chromatography and mass spectrometry of lipids from the rat Harderian gland

Lipids from rat Harderian glands were extracted with ethyl acetate, hydrolysed with base and examined by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) as trimethylsilyl (TMS), [2H9]TMS, methyl ester-TMS, picolinyl, nicotinate and nicotinylidene derivatives. The latter three derivatives were used to reveal the structures of the alkyl chains of fatty acids, alcohols and glycerol ethers, respectively. Forty-eight compounds were identified, representing about 97% of the total extracted lipids as measured by GC peak areas. The major constituents were fatty acids with chain lengths from 12 to 22 carbon atoms (mainly C18 and C20) and fatty alcohols (C16 to C26) derived from wax esters. Most of these acids and alcohols were unsaturated in the omega-7 position and were accompanied by smaller amounts of the saturated and omega-5 monounsaturated analogues. glycerol ethers were also identified for the first time in this secretion; the ether chains contained from 14 to 19 carbon atoms (mainly 16) and were straight-chain saturated, unsaturated (omega-5 and omega-7) and branched (iso). The only sterol found was cholesterol amounting to 1.24% of the total extract.     

Analysis of fatty acids by graphite plate laser desorption/ionization time-of-flight mass spectrometry

Fatty acids obtained from triglycerides (trioelin, tripalmitin), foods (milk, corn oil), and phospholipids (phosphotidylcholine, phosphotidylserine, phosphatidic acid) upon alkaline hydrolysis were observed directly without derivatization by graphite plate laser desorption/ionization time-of-flight mass spectrometry (GPLDI-TOFMS). Mass-to-charge ratios predicted for sodium adducts of expected fatty acids (e.g. palmitic, oleic, linoleic and arachidonic acids) were observed without interference. Although at present no quantitation is possible, the graphite plate method enables a simple and rapid qualitative analysis of fatty acids.     

Abnormal composition of commerical waxes revealed by supercritical fluid chromatography

Summary The analysis of natural waxes is a complicated process because they occur as complex mixtures. A study using supercritical fluid chromatography with packed columns made it possible to observe the behavior of each family of hydrocarbons, fatty esters, alcohols, acids and triglycerides. The latter were then separated according to their function type and alkyl chain lengths using octadecyl silica as stationary phase. With a polar modifier added to the supercritical fluid, it was possible to analyse certain waxes. Composition anomalies were observed which related to an overabundance of certain compounds which, in excess, are harmful to the quality of waxes intended for use in cosmetics.     

6-Oxy-(acetyl ethylenediamine) fluorescein, a novel fluorescent derivatization reagent for carboxylic acids and its application in HPLC

A novel fluorescent derivatization reagent for carboxylic acids, 6-oxy-(acetyl ethylenediamine) fluorescein (AEF), was well designed, synthesized, and applied to HPLC. The derivatization reaction with 12 fatty acids, including n-valeric acid (C5), n-hexanoic acid (C6), n-heptanoic acid (C7), n-octanoic acid (C8), n-nonanoic acid (C9), n-decanoic acid (C10), lauric acid (C12), myristic acid (C14), palmitic acid (C16), stearic acid (C18), oleic acid (C18:1), and linoleic acid (C18:2), was completed at 55 degrees C within 40 min. The derivatives of fatty acids were separated on a C18 RP column and detected by fluorescence detection. The LODs attained were 0.4-1.2 nM (S/N of 3). It has been demonstrated that AEF is a prominent derivatization reagent for carboxylic acids which is suitable for HPLC.     

Quantifying instantaneous regeneration rates of plant leaf waxes using stable hydrogen isotope labeling

Leaf waxes protect terrestrial plants from biotic and abiotic stresses and are important sedimentary biomarkers for terrestrial plants. Thus, understanding the production and ablation of leaf waxes is critical in plant physiology and for geochemical studies. However, there have been no accurate approaches to quantify leaf wax production at different time scales. In this study, we demonstrate a novel approach to study leaf wax regeneration by irrigating plants with a pulse of deuterium-enriched water, followed by measurements of leaf wax D/H ratios by gas chromatography/isotope-ratio mass spectrometry (GC/IRMS). We demonstrate the efficacy of this approach using the grass species Phleum pratense in a greenhouse environment. Using a binary isotope mass balance model, we are able to quantify the regeneration rates of the C(16), C(18) acids and leaf waxes (C(23)-C(31) n-alkanes; C(22)-C(30) n-acids) over a diurnal cycle. Our results show that within one day 33-47% of C(16) and C(18) acids are regenerated, and thus the recycling time for these compounds is 2-3 days. For C(22)-C(26) n-alkyl lipids, 7-21% are regenerated within one day and thus they require 5-16 days to recycle. In comparison, the recycling time for long-chain n-alkyl lipids (C(27)-C(31)) is as long as 71-128 days. Our approach can be applied to different plants at shorter or longer time scales by adjusting the degree of isotopic labeling, sampling intervals and the amount of irrigation water.     

Identification of proteins,drying oils,waxes and resins in the works of art micro‐samples by chromatographic and mass spectrometric techniques

Simplified method for simultaneous identification of proteins, drying oils, waxes, and resins in the works‐of‐art samples was developed. Liquid chromatography with mass spectrometry and gas chromatography with mass spectrometry were used to identify natural materials most frequently encountered in historical paintings. Protein binders were extracted with ammonia and purified using miniaturized solid‐phase microextraction (Omix tips) to efficiently suppress matrix interferences. Zwitterionic stationary phase was used for separation of 16 underivatized amino acids analysis with hydrophilic interaction liquid chromatography that was subsequently quantified with liquid chromatography with mass spectrometry. Gas chromatography with mass spectrometry was used to analyze drying oils, waxes, and resins after one‐step saponification/transmethylation with (m‐trifluoromethylphenyl)trimethylammonium hydroxide (Meth‐Prep II). While the drawback of this reagent is low reactivity towards hydroxyl groups, sample pretreatment was much simpler as compared to the other methods. Fatty acids derivatization with the Meth‐Prep II reagent was compared with their silylation using N,O‐bis(trimethylsilyl) trifluoroacetamide/trimethylchlorosilane mixture. It was concluded that fatty acids analysis as their methyl esters instead of trimethylsilyl esters had a minor impact on the method sensitivity. The developed method was used to analyze samples from 16^th and 17^th century historical paintings.     

GC-MS analysis of cuticular lipids in recent and older scavenger insect puparia. An approach to estimate the postmortem interval (PMI)

An analytical method was developed to characterize puparia cuticular lipids (hydrocarbons, waxes) and to compare the molecular distribution patterns in the extracts from either recent or older puparia. Acid-catalyzed transesterification and solvent extraction and purification, followed by combined gas chromatography coupled to mass spectrometry, were optimized for the determination of hydrocarbons and fatty acid ethyl esters from transesterified waxes, extracted from a single species of a fly scavenger (Hydrotaea aenescens Wiedemann, 1830). Comparison between recent (2012) or older (1997) puparia contents has highlighted significant composition differences, in particular, a general decrease of the chain length in the n-alkane distribution pattern and, on the contrary, an increase of the ester chain length. Both extracts contain traces of three hopane hydrocarbon congeners. Preliminary results evidence the change in puparia lipid composition over time, thus potentially providing new indices for estimating postmortem interval.     

Hydropyrolysis as a preparative method for the compound-specific carbon isotope analysis of fatty acids

Compound-specific stable carbon isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry is an effective and risk-free means of investigating fatty acid metabolism. Straightforward analysis, however, leads to poor chromatographic resolution, while derivatization adds carbon thereby corrupting the starting stable isotopic composition. Hydropyrolysis is a new approach which defunctionalizes fatty acids to yield the corresponding n-alkanes thus retaining the carbon skeleton intact and improving chromatography, allowing the faithful measurement of carbon isotope ratios.     

Simultaneous Derivatization and Dispersive Liquid–Liquid Microextraction for Fatty Acid GC Determination in Water

Simultaneous derivatization and dispersive liquid–liquid microextraction technique for gas chromatographic determination of fatty acids in water samples is presented. One hundred microlitre of ethanol:pyridine (4:1) were added to 4 mL aqueous sample. Then a solution containing 0.960 mL of acetone (disperser solvent), 10 μL of carbon tetrachloride (extraction solvent) and 30 μL of ethyl chloroformate (derivatization reagent) were rapidly injected into the aqueous sample. After centrifugation, 1 μL sedimented phase with the analytes was analyzed by gas chromatography. The effects of extraction solvent type, derivatization, extraction, and disperser solvents volume, extraction time were investigated. The calibration graphs were linear up to 10 mg L^?1 for azelaic acid (R ² = 0.998) and up to 1 mg L^?1 for palmitic and stearic acids (R ² = 0.997). The detection limits were 14.5, 0.67 and 1.06 μg L^?1 for azelaic, palmitic, and stearic acids, respectively. Repeatabilities of the results were acceptable with relative standard deviations (RSD) up to 13%. A possibility to apply the proposed method for fatty acids determination in tap, lake, sea, and river water was demonstrated.     

13C nuclear magnetic resonance spectroscopy for determining the different components of epicuticular waxes of olive fruit (Olea europaea) Dritta cultivar

¹³C nuclear magnetic resonance spectroscopy (NMR) was applied to determine the different components of apolar and polar fractions which were isolated by column chromatography from the crude chloroform-soluble waxes of olive fruits (Olea europaea) Dritta cultivar.¹³C NMR enabled the determination in the wax apolar fraction, of aliphatic aldehydes, and of benzyl, alkyl and glyceryl esters. In particular, the fatty acid composition of alkyl esters, comprising saturated and unsaturated oleic and linoleic acids, was determined. Acyl chain composition and the chain composition of 1,3- and 2-glycerol positions were also determined for triacylglycerols of olive fruit waxes.Oleanolic and maslinic acids were confirmed to be the major components of wax polar fraction. Complete assignments of ¹³C NMR chemical shifts of oleanolic and maslinic acids as a mixture were achieved by using homonuclear correlation spectroscopy with gradient (g-COSY), attached proton test (APT), inverse-detected heteronuclear single-quantum coherence with gradient (g-HSQC), high-resolution heteronuclear correlation spectroscopy (HETCOR) for C-H directly attached pairs and C-H long-range-coupled experiments.     

Fatty acid composition of neutral lipid: Classes of Citrus seed oil

The constituents of total lipids and neutral lipid classes, i.e., hydrocarbons, wax esters, sterol esters, triacylglycerols, free fatty acids, 1,3-diacylglycerols, 1,2-diacylglycerols, free sterols, alcohols, and monoacylglycerols of three species of Citrus namely Citrus sinensis, Citrus paradisi, Citrus aurantium were investigated by thin layer and gas chromatography. Palmitic, oleic and linoleic acids were the major components in all the lipids and lipid classes studied.     

Identification of drying oils used in pictorial works of art by liquid chromatography of the 2-nitrophenylhydrazides derivatives of fatty acids

A new HPLC-UV-Vis method for identification of drying oils from binding media or protective film used in pictorial works of art prior to conservation or restoration is proposed. Chromophore derivatization of fatty acids released by hydrolysis of structural drying oils is studied. The derivatization reagent selected was 2-nitrophenylhydrazine with 1-ethyl-3-(3-dimethyl animopropyl)carbodiimide hydrochloride/pyridine as catalyst. This reaction was carried out using microwave heating. Mobile phase was methanol/water/n-propanol/acetic acid (80:14:5:1) running in isocratic mode. Absorbance was measured at 400 nm. In these conditions, hydrazides of myristic, palmitic, oleic, and stearic acids were satisfactorily resolved. Method shows good sensitivity, with a detection limit of 15 μmol l⁻¹, and good linearity between 0.03 and 3 mmol l⁻¹. Peak area ratios among fatty acids derivatives allows identification of the drying oils. The stearic/palmitic ratio is the most important, because it allows to differentiate among the different drying oils. The proposed method has been successfully applied to real samples from items of the cultural heritage of Valencia (Spain).     

Application of triple quadrupole tandem mass spectrometry to the analysis of pyridine-containing derivatives of long-chain acids and alcohols.

An improved derivatization procedure for the preparation of nicotinate and 3-picolinyl esters from mixtures of fatty alcohols and acids has been developed. The derivatives can be analysed by capillary gas chromatography on an SE-54 type column, which affords separation of the acid and alcohol derivatives with the same carbon chain. Detection with tandem mass spectrometric techniques on a triple quadrupole instrument is feasible, and yields informative spectra devoid of coeluting interferences.     

Composition and seasonal variation of soluble cuticular waxes in Actinidia deliciosa leaves

This research examines the annual evolution and composition of soluble cuticular waxes of Actinidia deliciosa Chev. cv Hayward leaves. Soluble cuticular waxes of foliar blade were extracted in chloroform and analysed by GC-MS. The seasonal weighted mean of the wax coverage was about 24 microg cm(-2). The alkyl alkanoates were the main class of components (10 microg cm(-2)) followed by hydrocarbons (6 microg cm(-2)), terpenes (3 microg cm(-2)), alkanols (1 microg cm(-2)), ketones (1 microg cm(-2)), alkanoic acids (1 microg cm(-2)), alkanals (0.7 microg cm(-2)), and sterols (0.6 microg cm(-2)). The concentration of the soluble cuticular components reached a peak (43 microg cm(-2)) on the 83rd day after bud break. Different causes were proposed to explicate the seasonal evolution of the leaf waxes: biosynthesis of the waxes prevalently during rapid leaf growth; natural wax erosion and evaporation; progressive reduction in the extractability of the intracuticular free compounds due to the slow polymerization of the cutin matrix.