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Thin-layer chromatography is a chromatography technique that separates pigments, identifying molecules. While it has many applications in a wide variety of industries, it is a particularly important technique used in forensic labs, helping scientists determine if two pieces of text were written by the same ink, which can often be an indicator of fraud. Image Credit: ggw/Shutterstock.com What is thin-layer chromatography?The technique of chromatography was first used in 1900 by scientist Mikhail Tsvet to separate the pigments of plants. Later, in the 1930s, new chromatography techniques began to emerge, including thin-layer chromatography (TLC) which was also developed for use in separating plant pigments. The method of TLC involves separating non-volatile compounds by their rate of movement through the stationary phase (a thin layer of adsorbent material, usually silica gel, but sometimes aluminum oxide or cellulose) which is coated onto the glass plate where the process takes place. Since its invention, TLC has evolved over the decades for use in numerous applications. One of the most prominent applications of the technology is the separation of multicomponent pharmaceutical formulations. It is also heavily relied on for separating and identifying various colors, preservatives, cosmetic products, and sweetening agents in the food and cosmetic sectors. In addition, forensic science has also adopted TLC that it is used in ink analysis, usually helping to determine if a document has been forged. Ink analysis is an incredibly important analytical technique in forensic crime labs. Most commonly, it is used to determine if more than one ink was used on a document, which can help to detect fraud and forgery. Related StoriesAlthough, it can be used to reveal a wealth of information about the ink used as well as the substrate it was used on, which can provide invaluable clues in criminal cases. Numerous substances are contained in modern inks, improving the quality of the ink; however, these substances can be used to characterize the ink in several ways that TLC can detect. Possibly, the most important of these substances contained in the ink are the pigments and dyes used to produce the color of the ink. While the color of two inks may appear the same to the human eye, this still can provide useful information regarding the document in question. TLC can reveal characteristic differences in their make-up which allows scientists to see exactly what was written in the original document, and what was added later. The dyes used in inks are solvable in the “vehicle” or the body of the ink. Pigments, which are finely ground multi-molecular fragments, are not soluble in the vehicle. As well as dyes and pigments, oils, solvents and resins also exist in the vehicle to dictate how the ink flows and dries. Further to this, driers, detergents, greases, plasticizers, soaps, and waxes are also added into the vehicle for fine-tuning of the ink’s characteristics. Given the complexity of the molecular mixture of inks along with the possibility of the writing substance contaminating these mixtures, it is easy to see how the forensic analysis of ink presents a significant analytical challenge. However, most ink analyses run by forensic labs have the main aim of determining whether two samples of text were written by the same ink source. So, comparing the make-up of two inks is the major aim of analyses run by forensic investigations into inks. Using thin-layer chromatography to analyze inkBoth destructive and non-destructive techniques have been developed for use in ink analysis. However, because of the nature of forensic samples, it is always preferred to use techniques that have a minimal destructive impact on the sample, so it can be retained for possible future testing. A range of non-destructive approaches are suitable for analyzing inks, including IR absorption and luminescence, Raman spectroscopy, surface-enhanced resonance Raman spectroscopy (SERRS), and UV–vis micro-spectrophotometry. However, these methods are not without their limitations. For example, the spectrum quality of UV–vis micro-spectrophotometry is very sample-dependent. Other techniques which require the removal of a small section of the ink are considered to hold benefits over the purely non-destructive methods. Such approaches include high- performance liquid chromatography, infrared spectroscopy (IR), capillary electrophoresis, and TLC. Research has shown that TLC is a particularly useful method for analyzing inks. Its approach of separating the mixtures that make-up inks into their constituent component dyes and pigments could be highly productive in the comparison of ink samples as well as in the matching of inks to chromatogram databases. Because of this, TLC has emerged as a commonly used method in forensic labs around the world. As well as being a robust and reliable approach, it is also fast, inexpensive, and requires minimal destruction to the documents. Sources:
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Chemical and physical analysis of inks on questioned documents provides valuable information regarding their authenticity. Comparison of these chemical and physical properties of two or more inks can determine: (1) if the inks were made by the same manufacturer; (2) in some cases, whether the inks are products of the same production batch; and (3) the first production date of the specific ink formulation involved. When dating tags are detected, it is possible to determine the actual year or years when the ink was manufactured. Dating tags are unique chemicals that have been added to ball-point inks by some ink companies as a way to determine the year the ink was made. Knowledge of the composition of inks is necessary to understand the reasons for the various methods used to analyze inks. Also, knowledge of the first production date for each type of ink or certain ingredients in the inks is useful for dating inks. Carbon (India) inkIn its simplist form carbon inks consist of amorphous carbon shaped into a solid cake with glue. It is made into a liquid for writing by grinding the cake and suspending the particles in a water-glue medium. A pigmented dye may be used to improve the color. Liquid carbon inks are also commercially available. In the liquid carbon inks shellac and borax are used in place of animal glue and a wetting agent is added to aid in the mixing of the shellac and carbon. Carbon inks are insoluble in water, very stable and are not decomposed by air, light, heat, moisture or microbiological organisms. This class of ink has been available for more than 2000 years. Fountain pen inksThere are two types of fountain pen inks: (1) iron-gallotannate type and (2) aqueous solutions of synthetic dyes. Modern inks of type (2) contain synthetic blue dyes to provide an immediate blue color to the ink which gradually turns black after oxidation on paper. This explains the origin of the name blue-black fountain pen ink. This class of ink is also very stable. This ink is insoluble in water and cannot be effectively erased by abrasion. The most popular fountain pen ink (developed in the 1950s) consists of an aqueous solution of synthetic dyes. These inks are bright and attractive in color, but they are not nearly as stable as the carbon or blue-black inks. Some of the synthetic dyes used fade and are soluble in water. The most modern inks of this type contain pigmented dyes, such as copper phthalocyanine (introduced in about 1953) which makes these inks much more permanent. Ballpoint inksThe ballpoint pen was developed in Europe about 1939 and was initially distributed in Argentina about 1943. In 1946, several million Reynolds ballpoint pens reached the market in the United States. Ballpoint inks consist of synthetic dyes (sometimes carbon or graphite is also added for permanence) in various glycol solvents or benzyl alcohol. The dyes in ballpoint inks can consist of up to 50% of the total formulation. Several other ingredients are usually added to the ink to impart specific characteristics. These ingredients consist of fatty acids, resins, surface active agents, corrosion control ingredients and viscosity adjustors. The fatty acids (oleic is the most common) act as lubricants to the ball of the pen and they also help the starting characteristics of the ball point. Ballpoint inks made before about 1950 used oil-based solvents such as mineral oil, linseed oil, reci-noleic acid, methyl and ethyl esters of recinoleic acid, glycerin monoricinoleate, coconut fatty acids, sorbi-tal derivatives, and plasticizers such as tricresylpho-sphate. Modern ballpoint inks (post-1950) are referred to as glycol-based inks, because of the common use of ethylene glycol or glycol derivatives as a solvent for the dyes. Benzyl alcohol is also commonly used as the vehicle (solvent) by some ink manufacturers. Chelated dyes (introduced commercially around 1953) are stable to light. Red, green, yellow and other colored chelated dyes are now used for various colored ballpoint inks. Pressurized ballpoint inks were developed about 1968. These pens contain a pressurized feed system instead of gravity flow. The physical characteristics of these inks are quite different from the standard glycol based ballpoint inks. The composition is basically the same, but this ink does not become fluid until disturbed by the rotation of the ball point in the socket. Cartridges containing this ink are under the pressure of nitrogen or some other inert gas. The positive pressure on the ink allows the pen to write in all positions and in a vacuum. These pens are used by astronauts during space travel. Rolling ball marker inksRolling ball marker inks were introduced in Japan in about 1968 and shortly thereafter in the United States. These inks are water based and usually contain organic liquids such as glycols and formamide to retard the drying of the ball point. The dyes in these inks are water soluble or acidic dye salts. The light fastness of these dyes range from good for the metal-ized acid dyes to poor for some of the basic dye salts. Water fastness is usually poor, except that some of these dyes have an affinity for cellulose fibers in paper which produces a degree of water fastness. Water-resistant rolling ball marker inks are also available. These inks are totally insoluble in water and can only be dissolved in strong organic solvents, such as pyridine or dimethylsulfoxide (DMSO). Fiber or porous tip pen inksThis class of inks was developed in Japan about 1962 and in the United States about 1965. Fiber tip inks are usually water or xylene based and contain dyes and additives similar to those in rolling ball marker inks and fountain pen inks. The water-based inks are obviously water soluble, whereas the xylene-based inks are water resistant and can only be dissolved with strong organic solvents. Formamide or glycol solvents are essential ingredients in fiber tip inks to keep the fiber tip from drying out. Fiber tip inks that contain metalized dyes are light fast. Gel-pen inksThe most recent development in the writing instrument industry is the introduction of the gel-pen by the Japanese. Four brands of gel-pen inks have been introduced: (1) the Uniball Signo by Mitsubishi; (2) the Zebra J-5; (3) the Pentel Hybrid; and (4) the Sakura Gelly Roll pen. These pens have been marketed by the Japanese since the mid-1980s and a limited supply of the pens was sold in the United States about 1993. Two US manufacturers are now producing these pens. Ink Comparisons and IdentificationsInks are usually examined for three reasons: Method of chemical analysisEquipment, materials and solvents• Merck HPTLC plates (silica gel without fluorescent indicator). The plates should be activated at 100°C for 15 min before use. • TLC scanning densitometer • Reagent grade pyridine, ethyl acetate, 1-butanol, ethanol, benzyl alcohol, DMSO, and water • 1 dram (1.8 g) glass vials with screw caps • 10 ul and 4 ul disposable micropipettes • TLC glass developing chamber to accommodate standard 4 in x 8 in (10 cm x 20 cm) TLC plates with cover • 20 guage syringe needle and plunger (the point of the needle must be filed so that the point is flat) • 10 ul and 20 ul automatic pipettes • temperature controlled oven Procedure• Using the syringe needle and plunger, punch out about 10 plugs of ink from the written line. Dating of InksAs mentioned earlier in this article, there is a huge demand for the dating of inks on questioned documents. Any time during an investigation when there is some question about the date of preparation of a document, an ink dating chemist is needed. Over the past 30 years, the ability to perform these examinations has become widely known and recognized among forensic scientists, document examiners and attorneys throughout the world. The ink dating procedures that will be described have passed the Frye and Daubert tests on numerous occasions and are therefore routinely accepted in US courts. Testimony has also been admitted using these techniques in Israel and Australia. First date of production methodAfter the ink is uniquely/positively identified, the first date of production of that ink or certain ingredients in the ink is determined from the manufacturer of that specific ink formulation. If the ink was not made until after the date of the document, then it can be concluded that the document was backdated. If the ink was available on the date of the document, then the document could have been written on that date. Ink tag methodIf an ink tag is identified in an ink, it is possible to determine the actual year or years when an ink was made. Tags have been added to some ballpoint inks by the Formulab Company since before 1970; however, the use of tags in their inks was discontinued in June 1994. Since the tags are considered proprietary information by Formulab, no further information about the tags can be reported here. Formulab should be contacted directly, if this information is needed. Relative age comparison methodsDating inks by this procedure is based on the scientifically proven premise that as ink ages on paper, there are corresponding changes in the solubility properties of the inks. Therefore, by comparing the solubility or extraction properties of questioned inks with known dated inks of the same formula on the same type of paper and stored under the same conditions, it becomes possible to estimate how long the ink has been written on the document. Two or more inks of the same formulation can be compared without known dated writings to determine whether the writings were made at the same or different times. This is only true if the inks being compared are still aging (drying), because after the ink has aged out (completely dry), no differences in solubility properties are expected, even if the inks were written at different times. Typically inks will become totally dry (as measured by these procedures) within 6 years; some inks become dry in less than 6 years. R-Ratio (rate of extraction) method and percent (extent) extraction method• Using the syringe and plunger, remove 10-15 plugs of ink and paper and place them into 1 dram glass vials. Cap and label the vial with the sample number. Repeat for each sample to be analyzed. Figure 1 R-Ratio curves (rates of extraction) for Bic black ballpoint inks written in 1992, 1994, 1996 and 1998. The matching curves for the 1992 and 1994 inks means that the ink became totally dry after 4 years, because there was no change in rate of extraction after this time. Figure 2 Percent extractions for Bic black ballpoint inks written in 1992, 1994, 1996 and 1998. As with the R-ratios in Fig.1, the age of this ink could only be distinguished between 1994 and 1998. Notice more ink was extracted from the older inks than the newer inks. This is an example of a reverse extraction rate and extent, because most inks extract more from newer inks. Figure 3 Dye ratios for Bic black ballpoint inks written in 1992, 1994, 1996 and 1998. This method allowed the age of all inks to be distinguished between 1992 and 1998. This is because the relative concentrations of the dyes in this ink changed with age up to 6 years. For this ink the solubility properties as measured by the R-ratio and percent extraction methods did not change after 4 years. Dye ratio method The same plate used for the R-ratio and percent extraction measurements can be used to calculate the various dye ratios for each ink being compared. • Develop the TLC plate containing all the spots from the R-ratio and percent extraction measurements in a solvent system of ethyl acetate:ethanol: water (70:35:30, by vol.) for 15 min. • Dry the plate in an oven set at 80°C for about 10 min, then allow the plate to cool to room temperature. • Scan each sample in the densitometer along the direction of the dyes separated in each sample and from the densitometer readings calculate all possible dye ratios for each sample. For example, divide dye 3 by dye 1, divide dye 3 by dye 2, and divide dye 2by dye 1. Compare the dye ratios of corresponding pairs of dyes obtained for questioned and known dated inks to estimate the age of the questioned inks (Fig. 3). Since it has been established that these dye ratios change as ink ages, inks with matching dye ratios are consistent with the inks being written at the same time. Inks with dye ratios that do not match generally means that the inks were written at different times, unless one ink had an unusually large batch variation. It is important to know that depending on the ink formulation involved and the paper it is on, each of the methods described above may not all have equal ability to discriminate the age of the ink being analyzed. For example, it is not uncommon for one method to detect differences in age, when one or both of the other procedures fail to detect this difference. This fact does not negate the positive results of the one method. Only if the results of one method conflict with the results of another method are the overall results negated. Accelerated aging In situations where known dated inks are not available for comparison with questioned inks, accelerated aging of a questioned ink can be performed to estimate its age (Fig. 4). The measurement procedures are identical to those described for R-ratios, percent extraction and dye ratios. This test involves just one additional step which is to heat a sample of the questioned ink for 30 min at 100°C, allow it to cool and equilibrate with the temperaure and humidity in the room for 1 h and then compare the results of the various measurements (using any or all of the R-ratio, percent extraction and dye ratio methods) with the results obtained from an unheated sample of the same ink. Figure 4 Artificial aging of Bic black ballpoint inks written in 1992, 1994, 1996 and 1998. Notice that the newer inks changed more with heat than the older inks. Also, notice that after 4 years, this ink did not change with heat as measured by the percent extraction method. With knowledge of how long it took for this ink to become completely dry, it is possible to estimate the age of questioned Bic black ballpoint inks by determining how much the extraction properties change with heat. Significant differences obtained by any one of the methods indicates that the ink is still drying and is therefore less than 6 years old, since no inks have been found to take longer than 6 years to become completely dry using these methods. If it is known that the specific ink in question takes only 3 years to dry, then it can be concluded that the questioned ink is less than 3 years old. This method can also be used to determine which of two or more inks is newer than the other. This is done by observing which ink changes more with heat; the larger the change caused by heat, the newer the ink. This can only be done when all inks compared consist of the same ink formulation on the same type of paper and stored under the same conditions. This statement applies to all of the relative age comparison techniques described here. |