ANALYTICAL SCIENCES APRIL 1998, VOL. 14 269 1998 © The Japan Society for Analytical Chemistry Analysis of Writing Ink Dyestuffs by TLC and FT-IR and Its Application to Forensic Science Kazuhiro TSUTSUMI* and Kazuya OHGA** *Criminal Investigation Laboratory, Oita Prefecture Police Headquarters, Otemachi, Oita 870, Japan **Department of Applied Chemistry, Faculty of Engineering, Oita University, Dannoharu, Oita 870–11, Japan Black, blue and red writing pens were classified into various groups using the Rf values and color tones of dyestuff bands separated by normal-phase thin-layer chromatography (TLC) of their inks. The classification is effective for the prelimi- nary identification of pens used in crime scenes. A microsampling technique was proposed for the TLC analysis of minute quantities of inks on questioned documents. Furthermore, a combination of reflectance-mode microscope/Fourier transform infrared spectroscopy and the pin-point condensation technique was proved to be useful for the precise discrim- ination of trace amounts of analogous water-soluble dyestuffs on TLC plates. Keywords Forensic science, ink, dyestuff, thin-layer chromatography, Fourier transform infrared spectroscopy Various writing implements have often been used at of them were kindly supplied by their manufacturers. crime scenes in Japan. One of the purposes for the Reference dyestuffs were obtained from manufacturers examination of writing ink is to specify writing imple- and the Identification Reference Data Center, National ments. A number of papers have been published Police Agency, Tokyo, Japan. Water was purified by regarding techniques for forensic examinations, includ- reverse osmosis (Millipore Milli RO15) and deioniza- ing visible spectrophotometry1,2, thin-layer chromatog- tion. Developing organic solvents for TLC were of raphy (TLC)1–4, X-ray microanalysis1, microspec- reagent grade, and were distilled before use. All other trophotometry3,5, high-performance liquid chromatogra- chemicals were also of reagent grade and were used phy (HPLC)6, gel electrophoresis7 and capillary zone electrophoresis.8,9 TLC is the simplest of those meth- ods, and is effective for separating dyestuff compo- Table 1 Number of pens used in this work and the number of nents. A systematic TLC study, however, has been pur- groups into which the pens were classified by the present sued only slightly concerning the analysis of Japanese TLC analysis writing inks. In the present paper we describe a prelim- Mnemonic Number inary preparation of a standard TLC library for inks, Pen Number and also discuss the effectiveness and limitation of symbol of group TLC for the identification of writing implements. We Oil-based also report on a microsampling technique, that is often ball-point pen requested in the analysis of inks on questioned docu- Black BP-Bk 16 9 ments, and the application of a pin-point condensation Red BP-R 10 8 technique10 to the identification of dye TLC bands by Blue BP-Bu 9 5 microscope/Fourier transform infrared spectroscopy marking pen (FT-IR). Black OM-Bk 18 11 Red OM-R 16 10 Blue OM-Bu 16 11 Aqueous Experimental roller-ball pen Black WB-Bk 6 3 Pens and reagents Red WB-R 6 5 In this work were used 161 kinds of black, red and Blue WB-Bu 6 5 blue pens, which were classified for convenience into marking pen four groups, as shown in Table 1: oil-based ball-point Black WM-Bk 20 16 and marking pens as well as aqueous roller-ball and Red WM-R 20 14 marking pens. They are now on the market, and some Blue WM-Bu 18 11 270 ANALYTICAL SCIENCES APRIL 1998, VOL. 14 without further purification. was dried up on a 0.1-mm hydrophobic perfluorinated- polymer film, with which a stainless-steel mirror was TLC coated. This type of drying procedure has been named TLC was carried out using a Merck precoated silica- pin-point condensation by Ikeda and Uchihara.10 The gel 60 F254 backed with an aluminum sheet and a RP-18 residue was subjected to microscope/FT-IR in the F254s backed with a glass sheet. Chloroform solutions reflectance mode. The analysis was performed while were applied to the TLC plates for the analyses of oil- receiving the aid of a Spectra-Tech IR-PLAN based ball-point pen inks and oil-soluble dyestuffs. microspectrometer equipped with a mercury-cadmium- Aqueous solutions were used for water-soluble telluride detector. The resolution was 4 cm–1 and the dyestuffs and the other inks were directly spotted with scanning was performed 256 times. The spectra of ref- their refills. All of the spots were 0.5 – 0.8 mm in erence samples were taken without the attachment in diameter, and the amounts of the inks and reference the transmission mode using the KBr tablet. dyestuffs applied were 1.0 – 1.5 mg. The origins were at 1.0 cm from the bases of the plates. The developing HPLC solvents used were ethyl acetate/ethanol/water (14:7:6), HPLC was performed with a Hitachi L-6000 ethyl acetate/methanol/28% aqueous ammonia (5:2:1) equipped with an Ohtsuka Denshi MCPD-3600 photo- and trichloroethylene/1,1,1-trichloroethane/ethyl diode array detector. The column was a Kagakuhin- acetate (10:1:1) for the normal-phase mode chromatog- Kensa Kyokai L-column ODS (4.6mmf´250 mm) and raphy, and acetonitrile/3% aqueous KBr (7:1) for the the eluent was acetonitrile/aqueous 3% KBr (7:1). reversed-phase mode; they were allowed to creep up to the plates a distance of 6.0 cm. The uniform origin position on the plate, proper loading at the origin and Results and Discussion the solvent saturation in the developing tank, which had been recommended by Lewis4, were kept during all of TLC the TLC analyses in order to obtain a high reproducibil- There are many reference books13 and reports1–4 on ity. the TLC of inks and dyes, in which proper developing solvents have been noted, principally for normal-phase Microsampling silica-gel plates. As a result of a thorough examination A procedure devised by Golding and Kokot11,12 for made for most of those solvents with the Merck normal dyestuff extraction from a filament was modified in phase plate, the one reported by Brunelle and Pro2, order to accommodate to an imitative microsampling ethyl acetate/ethanol/water (14:7:6, abbreviated as the experiment in this work. A fraction of a line (ca. 3 mm B.P. solvent hereafter), was found to provide high long) drawn on filter paper (Toyo Roshi #2) was put in degrees of dye separation to most of the present inks. a haematocrit glass capillary (1 mmf´75 mm) together The results obtained for oil-based and aqueous red with 5 mm3 of a given solvent. The capillary was marking pens with the B.P. solvent are typically sum- sealed, and then sonicated in a Yamato 1210 ultrasonic marized in Tables 2 and 3, respectively. The relative bath at 60˚C for 5 to 10 min. The use of filter paper standard deviations for the Rf values were below 3.1% was grounded on the fact that suspicious letters are usu- for 5-times repeated analyses of the same samples. ally written on paper made from wood pulp. An effec- Frequently observed overlapping of dyestuff bands tive extraction solvent was searched by paper chro- was confirmed through their separation with ethyl matography according to the criteria that it should leave acetate/methanol/28% aqueous ammonia (5:2:1) or a no colors due to the dyestuffs at the starting point, give less polar solvent, trichloroethylene/1,1,1-trichloro- large Rf values to the dyestuffs and have a high volatili- ethane/ethyl acetate (10:1:1). Less-resolved bands hav- ty. The solvents examined were methanol, ethanol, 2- ing high Rfs were separated into more bands with the propanol, aqueous ammonia, acetic acid, pyridine, less-polar solvent; for example, the R1 dye band having DMF, THF, acetone, chloroform, acetonitrile, toluene, Rf of 0.96 in Table 2 yielded four bands having Rfs of ethyl acetate and a mixture of two of these. On the nor- 0.53, 0.47, 0.39 and 0.31. Also, the use of aqueous 3% mal-phase TLC plate was applied a dot of an extract in KBr instead of the water appreciably improved the tail- the above-mentioned capillary tube, whose tip had been ing bands, such as the Y24 and Bw4 bands in Table 3, previously stretched with a small flame and cut off. which were probably due to ionic dyes. This modified solvent system only slightly affected the Rfs and color FT-IR tones of the other bands. FT-IR spectra were obtained with a Nicolet System 710 spectrometer. Among the dyestuff bands obtained Classification from aqueous pen inks through the above-mentioned The Rf values and color tones of the bands separated microsampling/TLC procedure, the desired one was by the normal-phase TLC analysis using the B.P. sol- scraped up from the normal-phase TLC plate onto a fil- vent permitted us to classify the writing pens into vari- ter of 0.45 mm pore size (Millipore Sumplep HV4), and ous groups, as listed in Table 1. Thus, standard TLC then extracted with ca. 40 mm3 of water. The extract tables, such as Tables 2 and 3, appear to be available ANALYTICAL SCIENCES APRIL 1998, VOL. 14 271 Table 2 TLC of oil-based red marking pens Dyestuff banda Marking pene b c Number Color Rf 1 2 3 4 5 6 7 8 9 10111213141516 Y1 yellow 0.96 R1 yellowish red 0.96 R2 red 0.91 R3 red 0.91 Y3 yellow 0.89 Y4 yellowF 0.89 Y9 orangeT (0.79)d R9 yellowish red 0.68 R12 yellowish red 0.64 Y15 yellow 0.62 R15 yellowish redF 0.61 R21 yellowish redF 0.46 R22 pinkF 0.46 Y24 yellowT (0.11)d a.