H Chromatography

Chromatography is the name for a group of analytical procedures widely used in the identification of many organic materials. The name for the general procedure came from its first application in 1906, when an extract from leaves was flushed through a column packed with calcium carbonate. The extract separated into a number of differently colored bands as it passed through the column. Each band essentially represented a different compound contained in the extract.

All chromatographic techniques share a common purpose. They are designed to separate mixtures of several different compounds. At the beginning of a chromatographic examination one has a mixture of compounds. During the course of the analysis, the mixture is separated into its different parts. Identification of the separated compounds is carried out with reference to standard (known) compounds analyzed under exactly the same conditions. With some modern chromatographic equipment, it is not necessary to actually analyze standards along with an unknown sample—the unknown may be identified with reference to data acquired at a different time, possibly under somewhat different conditions or even on a different instrument.

All chromatographic techniques involve two major components: a carrier or mobile phase, and a stationary material or stationary phase. The mobile phase, usually liquid or gas, carries the sample mixture over or through the stationary phase. There are three principal chromatography techniques that are applied to analysis of binders in paintings. These are thin layer, gas (or gas-liquid, as it is sometimes called), and liquid chromatography, each of which will be briefly discussed below. The mobile phases in these three techniques are liquid, gas, and liquid, respectively.

Chromatography techniques are quite useful for binder identification because, as it turns out, nearly all binders consist of several different compounds, or they consist of material that can be broken down into mixtures of several different compounds. In their natural states, resins and waxes consist of mixtures of different compounds, which can be analyzed by chromatography. For chromatographic analysis, gums, oils, and proteins are usually first chemically broken down into various smaller compounds, which are then analyzed. In the case of gums, oils, and proteins, direct chromatography analysis of the binder, without initial chemical breakdown, is not currently being carried out.

The simplest chromatographic technique is thin layer chromatogra-phy. In this procedure, small spots of a solution of the sample are placed near the bottom edge of a glass or plastic plate that has been coated with a thin layer of a very fine-grained inorganic compound (many types of inorganic compounds can be used). Also, along the bottom edge of the same plate are placed spots of solutions of reference materials. The plate is placed in a container that contains a small amount of solvent. The solvent is absorbed by the thin layer of coating and moves up the plate by capillary action, just as water moves up a paper towel that has been dipped in it. As the solvent moves up and past the spots of samples and reference materials, these materials are carried along. The different components of the unknown or reference travel up the plate at different rates, and thus are separated. When the solvent has nearly reached the top of the plate, the plate is removed and dried. If the separation has been successful, a sample will show a series of spots (which correspond to the different compounds found in the sample mixture). If the pattern of spots of the reference materials match the pattern of the unknown, an identification can be made. The spots often are not visible in normal light, but sometimes can be seen in ultraviolet light, or the plate may need to be treated with a chemical that makes the spots visible. Thin layer chro-

matography is a useful technique, but requires somewhat larger samples than do the instrumental techniques discussed below.

Gas chromatography is currently a common technique that may be utilized to identify nearly all types of organic binders. In a gas chromatograph, a small amount of a solution of the sample is injected into the instrument. The solution is immediately heated to a high temperature, which completely volatilizes it (turns it into a gas). The gaseous sample is then swept on a stream of the carrier gas (usually helium or hydrogen) through a long narrow-diameter column that has been coated on its inside with a solid or a liquid that has a very high boiling point. Typical columns are 10 to 30 or more meters in length; diameters are usually from 0.2 to 0.5 millimeters. The column is inside an oven, and analyses are usually made while the temperature of the oven is increased from a fairly low temperature (perhaps 100°C) to a higher temperature (up to about 300°C). The components of the sample mixture generally travel through the instrument at different rates, and come out the other end of the column at different times after injection. Usually, heavier compounds, with higher boiling points, move more slowly than lighter compounds; the exact structure and chemical composition of the compound and the nature of the material in the column also affect the rate of travel. As the components come out the end of the column, they go into a detector, which produces a signal, or peak, in the resulting chromatogram. Just as thin layer chromatography produces only a pattern of separated compounds, analysis of a sample by gas chromatography produces only a chart showing peaks (due to the individual separated compounds) coming out of the instrument at different times. Identification of the peaks is done with reference to standard materials run under the same conditions. The heights of individual peaks is related to the quantity of a compound going through the detector, so with appropriate standards it is possible to determine relative amounts of the various components of a mixture, something that is much more easily done with a gas chromatograph than with thin layer chromatography.

There are two limitations to standard gas chromatography as just described. First, a material must be able to be dissolved in order to be analyzed, and second, the dissolved compound must be able to be vaporized without disintegrating. Although many of the compounds found in natural binders are not volatile, fortunately these nonvolatile compounds can often be turned into a related compound that is volatile by reacting them with certain chemicals, a process called derivatization. Unfortunately, some natural binders are not soluble. It is possible to analyze solid samples, without solution and derivatization, by gas chro-matography by utilizing a pyrolysis sampling attachment. A sample placed in a pyrolysis attachment can be heated rapidly to a very high set temperature, which will cause it to fragment into smaller pieces that may be small enough to pass through the gas chromatograph. The fragmentation pattern can be very complex, but with careful control of condi tions the pattern is reproducible, and can serve as a sort of fingerprint for a particular binder.

There are many types of detectors that can be attached to a gas chromatograph. A particularly useful one is a mass spectrometer. We will not discuss this type of instrument in this Appendix. However, the mass spectrometer gives more certainty to identification of individual peaks than do most of the other standard types of gas chromatography detectors. A chromatograph coupled with a mass spectrometer is referred to as a gas chromatography/mass spectrometry system (or GC/MS).

High-performance liquid chromatography (HPLC) is currently less widely utilized for binder analysis than gas chromatography, but it too can be used to analyze most of the natural binders. In a liquid chromatograph, a small amount of sample in solution is injected into the instrument. The sample is then carried in a stream of liquid through a column in which separation of the different components of the sample mixture takes place. HPLC columns are typically 15-30 cm long and 1-4 mm in diameter. They are packed with various types of small spherical particles, around which the sample molecules move as they are pushed along in a high-pressure stream of liquid. In the most common variation of liquid chromatography, the liquids used to carry the samples consist of water mixed with certain organic solvents. As in gas chromatography, a detector at the end of the column produces peaks, the heights of which are related to the relative amounts, so this method, too, is easily made quantitative. A limitation of liquid chromatography is that the sample must be soluble. However, it does not need to be volatile, which makes the technique a little more straightforward to apply to some types of compounds than gas chromatography. As with gas chromatography, there are many types of detectors that can be attached to a liquid chromatograph. As with GC, a mass spectrometer can be a very useful detector, but currently LC/MS systems are relatively expensive and have yet to be very widely used in analysis of binders from paintings.

How are different natural binders analyzed by these chromatogra-phy procedures? Protein-containing binders are broken down into amino acids, the small molecules from which they are built up, and the amino acids are then analyzed. A given protein will contain over a dozen easily analyzed amino acids, the identities and proportions of which can permit specific proteins to be distinguished from one another. All three chromatography procedures discussed above have been applied to amino acid analysis.

Gum-containing paints are broken down into monosaccharides (simple sugars), which are then analyzed. Some gums contain two, others as many as five, different simple sugars, so analysis can sometimes permit specific gums or mixtures of gums to be identified. Again, all three chro-matography techniques have been utilized. In addition to monosaccha-rides, most gums also contain uronic (sugar) acids. These are more difficult to analyze than the monosaccharides.

Paint Analysis

Fig. H.4. Chromatogram of the blue-green paint from Arthur Dove's Dancing Willows, 1943.

The sample, whose FTIR spectrum was shown in Figure H.3, was analyzed by gas chromatography/mass spectrometry (GC/MS). Sample preparation involves chemically breaking down the binder and creating derivatives of some of the compounds in the sample (all acidic compounds are converted into methyl esters, which can be much more easily analyzed in a GC than free fatty acids). The major compounds detected, as shown in the chromatogram at the bottom, include saturated fatty acids (mainly palmitic and stearic) and an unsaturated fatty acid (oleic). Also detected are several small dicarboxylic acids (mainly azelaic and suberic). The combination of saturated fatty acids and dicarboxylic acids is typical of paint samples that contain drying oil binders. The upper chromatogram is an expanded part of the overall chro-matogram. It shows a number of compounds present at much lower levels than those just noted. These additional compounds indicate that the binder contains two types of material in addition to drying oil. Beeswax is indicated by the pattern of even-numbered saturated fatty acids containing from 20 through 26 carbon atoms, maximizing with the 24-carbon acid (labeled A24). Also indicating beeswax are odd-numbered straight-chain hydrocarbons containing between about 21 and 31 carbon atoms, maximizing with the 27-carbon hydrocarbon (labeled H27). Beeswax also contains a substantial amount of palmitic acid and a small amount of stearic acid, compounds that are also present in drying oils. The chromatogram contains two very small peaks from compounds that are found in conifer resins, such as pine (the specific compounds are dehydroabietic and 7-oxodehydroabietic acids).

Many natural resins contain a large proportion, or at least some, relatively small molecules that are amenable to analysis by GC. At the moment, gas chromatography (and in particular, GC/MS) is by far the most useful technique for the specific identification of resins in small samples from works of art.

That oil paint samples consist to a great extent of large molecules that cannot be directly analyzed by any chromatography procedure. It turns out that such samples can be chemically broken down, to a minor extent, into small molecules that can be analyzed. GC is the technique by which these fragments are usually analyzed at the moment. Although most oils contain essentially the same types of fragments, the relative amounts of these vary to some extent between different types of oils, so that in certain cases the type of drying oil in a paint sample can be identified with some confidence by GC analysis.

Fig. H.5. Chromatograms of two plant gums and a black paint sample from an ancient Chinese painted wood panel, dated to about a.d. 600. The gums and paint sample were each chemically broken down and derivatized in a manner used to prepare sugar-containing materials for analysis by gas chromatography/mass spectrometry (GC/MS). The analysis detects monosaccharides (simple sugars) that are present in the samples. The result for the paint sample indicates that a plant gum was the binder, although the specific gum cannot be identified. Some contamination from monosac-charides liberated by the breakdown of the wood panel support is also probably present in the sample.

Fig. H.5. Chromatograms of two plant gums and a black paint sample from an ancient Chinese painted wood panel, dated to about a.d. 600. The gums and paint sample were each chemically broken down and derivatized in a manner used to prepare sugar-containing materials for analysis by gas chromatography/mass spectrometry (GC/MS). The analysis detects monosaccharides (simple sugars) that are present in the samples. The result for the paint sample indicates that a plant gum was the binder, although the specific gum cannot be identified. Some contamination from monosac-charides liberated by the breakdown of the wood panel support is also probably present in the sample.

Time (min.) 2 4 6 8 10 12
Time (mill.) 2 4 6 8 10 12
Time (min.) 2 1 B S 10 12

Fig. H.6. Chromatograms of paint samples from Saint Barbara, a painting on paper by Edward Burne-Jones, ca. 1866-1870.

These samples were chemically broken down and derivatized in a manner used to prepare protein-containing samples for analysis by high-performance liquid chro-matography (HPLC). Each sample was initially allowed to sit in water for a short period of time. The water itself (and whatever part of the sample that may have dissolved) was drawn off and prepared for analysis, as was the remaining solid portion of the sample. This particular procedure often makes interpretation of results from samples that contain more than one protein easier to carry out. The top chromatogram shows the result from the water solution of some paper fibers from the support. The middle chromatogram shows the result from the solid sample of the fibers after the water treatment. Both chromatograms indicate the presence of animal glue; the upper one also suggests the presence of some egg white. The lower chromatogram is from the water-soluble part of a blue paint sample. It indicates the presence of egg white. The chromatograms indicate that the artist used egg white as his paint binder, and glue to seal the paper substrate before paint was applied. Analyses of other paint samples indicated that other (nonproteinaceous) binders were used elsewhere in the painting.

Figure H.4 is a chromatogram from GC/MS analysis of the same paint sample whose FTIR spectrum was shown in Figure H.3. It provides much specific information on the paint binder, indicating the presence of drying oil, beeswax, and a little conifer (probably pine) resin.

Figure H.5 shows how GC/MS can be used to identify carbohydrate-containing media, such as plant gums. The monosaccharides (simple sugars) present in two reference gums are shown, along with the chro-matogram of a paint sample from an ancient Chinese painted panel.

Figure H.6 shows chromatograms from analysis of amino acids in samples from a painting on paper. The types and relative amounts of amino acids indicate that the paper substrate was coated (sized) with gelatin (animal glue). The paint layer itself was bound with egg white.

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