Forensic Analysis of Blue Ballpoint Pen Inks Using Capillary Electrophoresis

Abstract

Ink analysis involving the determination of specific chemicals can be a useful forensic tool in pen formula differentiation. Thin-layer chromatography is the most widely used forensic technique for ballpoint pen ink examinations; however, limited information is obtained. To address this issue, capillary electrophoresis using an ultraviolet-visible photodiode array detector was employed as an alternative analytical tool. Results were compared with thin-layer chromatography experiments to evaluate the sensitivity and usefulness of capillary electrophoresis. Dye components are rapidly separated, and identification is based on comparison of electrophoretic mobility values and ultraviolet-visible spectra with individually run dye standards. A database of ballpoint pen ink analyses and common-dye reference standards has been initiated for future forensic use. Capillary electrophoresis sample-preparation protocols were designed to closely mimic common procedures for handling ink evidence. Because of the small volume necessary for sample injection, the remaining solution could be further processed using current law enforcement procedures to obtain complementary data. Capillary electrophoresis method development, electropherograms, electrophoretic mobility values, ultraviolet-visible spectra, and electrospray ionization mass spectrometry data are reported.

Introduction

Chemical analysis of questioned documents is an important tool available to forensic examiners. Ink-source comparisons are commonly conducted in casework involving such crimes as tax evasion, insurance fraud, and currency counterfeiting. For example, the recent Martha Stewart conviction partially relied on ink-source-comparison evidence regarding a suspected stock worksheet alteration (Associated Press 2004). A questioned entry on the document was compared to the bulk writing. Differences in the ink samplings suggested that the questioned entry was made on a separate occasion, possibly to cover up insider trading violations. The majority of ink comparison analyses focus on colorant determination because ink formulations tend to have unique, organic dye formulations (Fanali and Schudel 1991; Rhode et al. 1997; Tebbett 1991; Xu et al. 1997; Zlotnick and Smith 1998). In addition, the dyes have ultraviolet-visible absorbances that can be measured and used for identification. Separating ink into its components prior to detection is desirable to simplify identification.

Thin-layer chromatography of ink extracts is the most common technique used by law enforcement agencies for ink-source comparisons (Tebbett 1991; Xu et al. 1997). The U.S. Secret Service maintains an ink library that consists of approximately 8,500 ink-standard thin-layer chromatography slides. Thin-layer chromatography is relatively easy to perform; however, it has disadvantages. First, thin-layer chromatography is not typically automated; thus the spotting of a slide and the measuring of retardation factor (R_f) values are difficult to reproduce. In addition, spectroscopic data is not obtained, although densitometry can be used to analyze spots for quantitative data when compared to a standard, assuming that the chemical identity is known (Aginsky 1994). Oftentimes the spots are faint, rendering them hard to see. In this case, the spots can readily disappear in a matter of minutes. If an ink sample is composed of two or more similar dyes, then thin-layer chromatography can result in two or more unresolved spots with nearly identical colors. This scenario requires multiple runs with different solvent systems, which is time-consuming. Thin-layer chromatography reference slides should be stored in a special environment to prevent spot fading, which requires control of such factors as humidity and ultraviolet radiation exposure. In some cases, photographs of slides may alleviate the need to store reference slides.

Capillary electrophoresis addresses many of these concerns. Capillary electrophoresis is a powerful separation technique that produces theoretical plate counts on the order of 10 to 100 times greater than high-performance liquid chromatography (Skoog et al. 1998). The process is completely automated, and method development is rapid. A photodiode array detector can be used to obtain ultraviolet-visible spectra of each component. Capillary electrophoresis methods can be readily developed to baseline separate analytes with nearly identical structures (Lurie et al. 2004). Data is stored electronically, allowing the development of an automated, searchable library of electropherograms and ultraviolet-visible spectra, while eliminating the need for an environmentally controlled storage space. Other detectors are available and often can be swapped in and out with simple modules. Capillary electrophoresis requires an injection volume of only approximately 5–50 nL, which leaves enough sample for analysis by complementary methods because a typical casework ink extract is on the order of 15–50 μL. This is an important feature for real casework samples because multiple confirmatory exams often are necessary. In this study, excess sample was analyzed by direct infusion into an electrospray ionization mass spectrometer.

Electrospray ionization is a soft ionization technique that results in multiply charged species and minimal fragmentation. This can be useful when analyzing mixtures because the mass spectra are simplified. Also, larger masses can be detected with a normal scan range because of the multiple charging. Ng, Lafontaine, and Brazeau published a study on ballpoint pen ink characterization by direct infusion to an electrospray ionization mass spectrometer (Ng et al. 2002). Seventy-seven inks were analyzed and used to create a searchable library. Conneely, McClean, Smyth, and McMullan reported on Phthalocyanine and Azo dyes using electrospray ionization mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry (Conneely et al. 2001). In particular, the behavior of sulphonate groups in Copper Phthalocyanine pigments was of interest because of the presence of these pigments in blue ballpoint pen inks. According to their results, the sulphonate groups are labile, producing some fragmentation in the electrospray ionization mass spectrometry experiment. Huang, Yinon, and Sigman used liquid chromatography with ultraviolet-visible detection coupled to an electrospray ionization mass spectrometer to study extracted textile fiber dyes in forensic size samples (Huang et al. 2004). Six extracted dyes were characterized with comparison to reference standards. As with most mass spectrometer-coupled instruments, extract solvent compatibility remains a concern.

Experimental Samples

Approximately 50 dye compounds encompassing a variety of colors were obtained from Sigma-Aldrich (St. Louis, Missouri) and the U.S. Secret Service. Figure 1 shows the chemical structures of some dyes and additives that were important in this study. Dyes were used as received to prepare 1 mg/mL stock solutions in methanol. Injection solutions for capillary electrophoresis and electrospray ionization mass spectrometry were prepared from stock solutions as described below. Known-dye-compound analyses were conducted to construct a database incorporating general dye information, electropherograms, electrophoretic mobilities (μ_ep), ultraviolet-visible spectra, and electrospray ionization mass spectrometry data. In addition, ultraviolet-visible spectral libraries have been compiled using the software provided with the capillary electrophoresis instruments. The database and spectral libraries were then used for the component determination in ballpoint pen ink samples. Dyes, as well as other ink components, are continuously being added to the database and libraries, particularly when an unknown dye is identified in an ink sample.

Figure 1: Structures of Dyes and Additives Important to Differentiate Among Ink Formulations

Ink samples were prepared by two methods. To generate the first sample set, lines were handwritten on Whatman (Ann Arbor, Michigan) filter paper with blue pens that were obtained from the FBI's collection. A second approach involved using the pen cartridges to saturate the filter paper to create more concentrated samples. An additional series of ink samples was obtained from the U.S. Secret Service ink library. These samples consisted of some handwritten lines on paper and some ink-saturated filter paper. Samples obtained from the U.S. Secret Service were accompanied with formula information, which was used to determine the accuracy of the capillary electrophoresis methods.

Capillary Electrophoresis

The existence of cationic (basic) and anionic (acidic) dyes in ink and their opposite Coulombic interactions with the capillary wall necessitated the development of two separate capillary electrophoresis methods (Egan et al. 2005).

Cationic Dye Capillary Electrophoresis Method

A Beckman Coulter (Fullerton, California) P/ACE MDQ capillary electrophoresis system fitted with a photodiode array module (λ = 200–600 nm) was used for cationic dye separations. A 75 μm inner diameter fused-silica capillary with a length to detector (L_d) of 30 cm and a total capillary length (L_t) of 40 cm was used. Separations were performed at 30°C in reverse-polarity mode at –25 kV after completion of the rinse sequence (Egan et al. 2005). These conditions resulted in electrical currents of ~ –42 μA. The separation buffer consisted of a 70/30 (volume/volume) mixture of sodium acetate buffer/methanol titrated to pH = 4.40 with concentrated glacial acetic acid [Fisher Scientific, Fairlawn, New Jersey; TraceMetal grade (99.5 percent)]. The sodium acetate buffer portion was composed of 25 mM sodium acetate [Fisher, HPLC grade (99.2 percent)], 25 mM glacial acetic acid, and 10 mM CTAB [hexadecyltrimethylammonium bromide, Sigma-Aldrich (99 percent)]. Barnstead (Dubuque, Iowa) NANOpure Infinity ultrapure water (ρ ≥ 18 MΩ•cm) was used to prepare all aqueous solutions. A syringe equipped with a 0.2 μm filter was used to remove particles when transferring the separation buffer to the capillary electrophoresis vial.

Dye stock solutions were diluted with an appropriate amount of methanol followed by two parts by volume of separation buffer for stacking purposes (Shihabi 2000) to form 100 μg/mL dye injection solutions. In order to prepare ink injection solutions, the ink was first extracted from the paper substrate. An Electron Microscopy Sciences (Fort Washington, Pennsylvania) 1.0 mm diameter core sampler was used to remove punches from the filter paper. The punches were then placed in 200 μL vials with 10 μL of methanol. One punch was sufficient for ink-saturated filter-paper samples, whereas handwritten line samples required up to five punches. The vial was placed in an ultrasonic bath for five minutes to assist in ink extraction. Twenty μL of separation buffer was then added to the extract solution and mixed thoroughly. The paper punches were removed by transferring the extraction solution to a new vial. Hydrodynamic sample injection was performed at 0.3 psi for five seconds.

Anionic Dye Capillary Electrophoresis Method

A Hewlett Packard/Agilent 3D (Palo Alto, California) capillary electrophoresis instrument equipped with a photodiode array detector (λ = 190–600 nm) and a 75 μm inner diameter fused-silica capillary of L_d = 50 cm and L_t = 58 cm was used for anionic dye separations at 30°C. Separations took place in normal polarity mode at 25 kV, which produced electrical currents of ~ 19 μA. The buffer consisted of 25 mM CHES [2-(N-Cyclohexylamino)ethanesulfonic acid, Sigma-Aldrich] and 5 mM β-cyclodextrin (Sigma-Aldrich) titrated to pH = 8.80 with 1 M NaOH.

The concentration of anionic dye injection solutions was 100 μg/mL in methanol. Ink solutions were prepared by placing 1.0 mm punches (one to five) in 30 μL of methanol followed by five minutes of ultrasonic agitation. Paper dots were removed by transferring the extract to a new vial. Hydrodynamic sample injection was conducted at 25 mbar for ten seconds for dyes and inks.

Electrospray Ionization Mass Spectrometry

A Finnigan (San Jose, California) MAT LCQ quadrupole ion trap mass spectrometer operated in direct infusion electrospray ionization mode was used for all mass spectrometry analyses. The instrument was calibrated and tuned using the established methods with the manufacturer-supplied tuning solution, which is a mixture of caffeine, MRFA (L-methionyl-arginyl-phenylalanyl-alanine acetate•H₂O), and Ultramark 1621 (Finnigan, San Jose, California). A 5 μg/mL Crystal Violet in methanol solution was used with the auto-tune feature to optimize the instrument in the positive-ion mode. This optimization resulted in a spray voltage of +4.52 kV, a spray current of 0.19 μA, and a capillary voltage of +10.39 V. A 10 μg/mL Metanil Yellow solution in methanol was used to auto-tune the negative-ion mode, which resulted in a spray voltage of +5.83 kV, a spray current of 5.17 μA, and a capillary voltage of –28.55 V. The sheath gas flow was set at 20 (arbitrary units), whereas no auxiliary gas flow was used. The quadrupole ion trap mass analyzer was set to full scan mode to monitor a mass/charge (m/z) range of 150 to 2000 in both positive- and negative-ion modes. Samples were infused into the ionization source using a syringe pump at 20 μL/minute with the capillary temperature set at 200°C. Dye solutions, which were prepared by dilution of stock dye solutions, were composed of 10 μg/μL in methanol. Ink samples were prepared by placing five 1.0 mm punches in 400 μL of methanol followed by five minutes of ultrasonic agitation.

Results and Discussion

Electrospray Ionization Mass Spectrometry as a Complementary Technique

Capillary electrophoresis separations of prepared cationic and anionic dye mixtures were reported in a previous publication (Egan et al. 2005). Figure 2 shows a typical result for a six-cationic-dye mixture using the cationic dye capillary electrophoresis method. The first three peaks (3.14, 3.35, and 3.60 minutes) are assigned to Rhodamine Base B, Rhodamine 6G, and Hexamethylpararosaniline (more commonly referred to as Crystal Violet), respectively. These assignments were made based on μ_ep's and ultraviolet-visible spectra. The last three peaks (3.74, 4.41, and 4.59 minutes) are attributed to Victoria Blue R, Victoria Blue B, and Victoria Pure Blue BO, respectively. Peak assignment using ultraviolet-visible spectra is difficult because these Victoria Blue dyes have similar structures (Figure 1c) and, hence, similar ultraviolet-visible absorbance profiles (Figure 3). Therefore, assignments were made based on μ_ep comparisons of the peaks to the μ_ep's of the individually measured Victoria Blue reference dyes. In this case it was possible to successfully identify the Victoria Blue dyes in the mixture because it was known that all three were present and all that had to be done was to differentiate them. However, the μ_ep's of Victoria Blue B and Victoria Pure Blue BO are similar enough that if an unknown ink sample resulted in a cationic dye peak with an ultraviolet-visible spectrum that is a close match for a Victoria Blue dye, it would only be possible to differentiate Victoria Blue R based on μ_ep. Therefore, electrospray ionization mass spectrometry was employed as a complementary technique to elucidate among closely related structures, as well as to support all peak assignments. Each Victoria Blue dye gives a distinct peak in positive-ion mode electrospray ionization mass spectrometry: Victoria Blue R [M–Cl]⁺ = 422.5 m/z, Victoria Blue B [M–Cl]⁺ = 470.6 m/z, and Victoria Pure Blue BO [M–Cl]⁺ = 478.6 m/z, which allows the assignment of a specific Victoria Blue dye structure.

Figure 2: Cationic Dye Capillary Electrophoresis Method Electropherogram of a Six-Basic-Dye Mixture (λ_observed = 214 nm)

Figure 3: Independently Measured Ultraviolet-Visible Spectra of Victoria Blue B (Red), Victoria Blue R (Green), and Victoria Pure Blue BO (Blue)

Capillary Electrophoresis of Blue Ballpoint Pen Inks

As discussed in the introduction, the goal of ink differentiation is not necessarily to determine the exact formulation of questioned ink(s), but rather to show that the inks under scrutiny are different or similar. To this end, a series of ten blue ballpoint pen inks obtained from the FBI's collection was examined using the cationic and anionic capillary electrophoresis methods that were developed. The inks have been designated a letter (A-J) in order to protect proprietary information.

Figure 4 shows the electropherograms measured for each pen using the cationic dye capillary electrophoresis method. The pens were also analyzed with the anionic dye capillary electrophoresis method; however, the electropherograms were similar for most of the pens and revealed that few, if any, anionic dyes were present. As will be discussed below, the anionic capillary electrophoresis method did provide supportive data for the presence of a specific pigment. It is worth mentioning that in a previous study on black ballpoint pen inks (Egan et al. 2005), it was discovered that very few cationic dyes were present in black inks; thus the anionic dye capillary electrophoresis method proved the most informative. Differences in the pen formulations are evident upon comparing the electropherograms, with the exception of pens D and E. However, when each peak was assigned using ultraviolet-visible absorbance, μ_ep, and electrospray ionization mass spectrometry data, each pen was determined to be unique. The complete analysis of pen A will be discussed to provide an example of the data obtained and the determination of the components present.

Figure 4: Cationic Dye Capillary Electrophoresis Method Electropherograms of Pens A-J Presented on the Same Axes for Comparison Purposes (λ_observed = 214 nm)

Pen A Results

Figure 5 shows the cationic dye method electropherogram obtained for pen A. Three peaks are observed with migration times of 2.93, 3.10, and 3.60 minutes. Based on the neutral marker (labeled "EOF" for electroosmotic flow), the μ_ep's of the species were calculated to be –1.41, –1.56, and –1.92 x 10^–4 cm²•V^–1•s^–1, respectively, where the negative sign indicates a mobility vector opposing the electroosmotic flow direction. In addition to μ_ep values, the ultraviolet-visible spectrum of each peak was recorded and is shown in Figure 6. An ultraviolet-visible spectral library search provided the best-match data reported in the figure caption. The first peak (2.93 minutes) is assigned to Crystal Violet based on the ultraviolet-visible spectrum match of 0.9988, as well as the μ_ep value, which is a close match to the independently measured μ_ep of Crystal Violet = (–1.45 ± .03) x 10^–4 cm²•V^–1•s^–1. Additional evidence is provided by the positive-ion mode electrospray ionization mass spectrometry spectrum (Figure 7), which resulted in a peak at 372.7 m/z; Crystal Violet [M–Cl]⁺ = 372.5 m/z. In addition, there is a peak present at 358.7 m/z, which is indicative of Pentamethylpararosaniline (also known as Methyl Violet) [M–Cl]⁺ = 358.5 m/z. Methyl Violet is a common degradation product of Crystal Violet formed by demethylation, which is initiated by ultraviolet radiation absorption (Grim et al. 2002). Ink manufacturers do not purchase dyes in pure form, and it is common to observe such impurities as Methyl Violet in inks. In fact, multiple Methyl Violet structures (Penta- and Tetra-methylpararosaniline) were observed with capillary electrophoresis and electrospray ionization mass spectrometry in the Crystal Violet dye standard (Sigma-Aldrich, ≥ 90 percent pure) used in this study.

Figure 5: Cationic Dye Capillary Electrophoresis Method Electropherogram of Pen A (λ_observed = 214 nm)

Figure 6a: Ultraviolet-Visible Spectra of the Peaks from the Electropherogram of Pen A (Blue Line) Overlaid with the Ultraviolet-Visible Spectra of the Best Match from the Spectral Library Searches (Red Line): 2.93 minutes; Best Match, Crystal Violet, Similarity = 0.9988

Figure 6b: Ultraviolet-Visible Spectra of the Peaks from the Electropherogram of Pen A (Blue Line) Overlaid with the Ultraviolet-Visible Spectra of the Best Match from the Spectral Library Searches (Red Line): 3.10 minutes; Best Match, Diarylguanidine, Similarity = 0.9693

Figure 6c: Ultraviolet-visible spectra of the peaks from the electropherogram of pen A (blue line) overlaid with the ultraviolet-visible spectra of the best match from the spectral library searches (red line): 3.60 minutes; best match, Victoria Pure Blue BO, similarity = 0.9779. Nearly identical matches were obtained with Victoria Blue B and Victoria Blue R.

Figure 7: Positive-Ion Mode Electrospray Ionization Mass Spectrum of Pen A

The second peak (3.10 minutes) is not a dye, as indicated by the absence of visible absorption (Figure 6b). The μ_ep value (–1.56 x 10^–4 cm²•V^–1•s^–1) is a match with a diarylguanidine component [μ_ep = (–1.56 ± .02) x 10^–4 cm²•V^–1•s^–1] in the database that was observed in several dye standards. Diarylguanidines are used in the dye industry to form salts with acidic dyes or pigments that otherwise would be insoluble. The positive-ion mode electrospray ionization mass spectrometry peak at 240.5 m/z (Figure 7) suggests that the diarylguanidine in pen A is ditolylguanidine (Figure 1d), which would result in a [M+H]⁺ peak = 240.3 m/z. It is suspected that 1,3-di-o-tolylguanidine is used by a particular manufacturer to form salts with sulfonated Copper Phthalocyanine pigments (Figure 1e) (Green 1990). Evidence of Copper Phthalocyanine is provided by the negative-ion mode electrospray ionization mass spectrum shown in Figure 8. The spectrum is noisy because optimal ionization parameters were not explored and Copper Phthalocyanine is insoluble, which resulted in an order of magnitude ion count decrease of the base peak when compared to the positive-ion mode. The base peak (1185.2 m/z) has not been assigned; however, the presence of multiple sulfonated Copper Phthalocyanines is indicated by the m/z peaks of 814.3, Trisulfonated Copper Phthalocyanine [M–3Na+2H]^– (Conneely et al. 2001), and 734.3, Disulfonated Copper Phthalocyanine [M–2Na+H]^– (Ng et al. 2002). In fact, each pen that has been observed to contain diarylguanidine(s) was also shown to possess Copper Phthalocyanine pigments as reflected by the 734 and 814 m/z peaks in the negative-ion mode mass spectra. Sulfonation of Copper Phthalocyanine is reported to lead to multiple sulfonate groups at various ring locations, which explains the observation of multiple peaks (Green 1990). In addition, a weak ultraviolet-visible spectrum believed to be due to Copper Phthalocyanine has been observed with the anionic capillary electrophoresis method in many of the pens containing diarylguanidines. Figure 9 shows an example of the ultraviolet-visible spectrum that is assigned to Copper Phthalocyanine; however, the peak in the electropherogram is often broad and weak owing to the insoluble nature of Copper Phthalocyanine in the buffers used, as well as the multiply sulfonated species present. It was determined that capillary electrophoresis methods alone are not reliable for detecting this pigment. This is another good example of the usefulness of electrospray ionization mass spectrometry as a complementary method.

Figure 8: Negative-Ion Mode Electrospray Ionization Mass Spectrum of Pen A

Figure 9: Ultraviolet-Visible Spectrum of a Peak Obtained from the Anionic Capillary Electrophoresis Method of Pen A Believed to Be Due to Sulfonated Copper Phthalocyanine

The ultraviolet-visible spectrum (Figure 6c) and μ_ep (–1.92 x 10^–4 cm²•V^–1•s^–1) of the 3.60-minute peak exhibited the characteristics of a Victoria Blue dye, but this data was not sufficient to distinguish between Victoria Blue B and Victoria Pure Blue BO. However, the 478.7 m/z peak in the positive-ion mode mass spectrum (Figure 7) allowed the assignment of the peak to Victoria Pure Blue BO, [M–Cl]⁺ = 478.7 m/z.

Based on the data presented, pen A was determined to consist of Crystal Violet and Victoria Pure Blue BO dyes. In addition, pen A was found to also contain ditolylguanidine and sulfonated Copper Phthalocyanine. The same analyses were conducted on the remaining pens in the group. Table 1 is an analysis summary of the pens, presented as a checklist of the dyes and additives that were detected in the pens. As can be seen in the table, the identification of five components (the CuPh column is not necessary) was sufficient to distinguish among the ten pens. Several of the pens contained more than one diarylguanidine compound, which aided in formula differentiation. For example, pen F was observed to possess five diarylguanidines. Capillary electrophoresis of pen F resulted in a cluster of five peaks (Figure 4) with similar μ_ep's (–1.44, –1.49, –1.58, –1.61, and –1.66 x 10^–4 cm²•V^–1•s^–1) and similar ultraviolet-visible spectra (ultraviolet absorption only). In addition, positive-ion mode electrospray ionization mass spectrometry of pen F resulted in peaks at m/z of 212.4, 226.3, 240.5, 254.5, and 268.5, which were assigned to 1,3-diphenylguanidine, 1-tolyl-3-phenylguanidine, 1,3-ditolylguanidine, 1-dimethylphenyl-3-tolylguanidine, and 1,3-bis(dimethylphenyl)guanidine, respectively. A study by Ng, Lafontaine, and Brazeau indicated similar findings (Ng et al. 2002). The number of different diarylguanidines used in a dye formulation appears to be manufacturer-specific. As an example, capillary electrophoresis of several dye standards from a particular manufacturer has resulted in the same characteristic cluster of diarylguanidines as seen in pen F, whereas another manufacturer's dyes contained only ditolylguanidine analogous to pen A. The type and quantity of diarylguanidines in an ink sample provide another degree of formula differentiation, which could be particularly useful if comparing pens that possess the same dyes but from different dye manufacturers.

CV = Crystal Violet; VBB = Victoria Blue B; VBBO = Victoria Pure Blue BO; VBR = Victoria Blue R; Guan = Ditolylguanidine and related diarylguanidine structures (number of peaks detected in parenthesis); CuPh = sulfonated Copper Phthalocyanine pigment

The capillary electrophoresis methods used in this study are not limited to ballpoint pen inks. Experiments have shown that food dyes, textile dyes, and ink-jet dyes can be separated and identified using the anionic and/or cationic dye capillary electrophoresis methods. Acid Yellow 23 (also known as Yellow Food Dye No. 5 or Tartrazine) was identified in a boiled-down sample of Mountain Dew soda (PepsiCo, Chicago, Illinois) using the anionic capillary electrophoresis method (Egan et al. 2005). The μ_ep's of the assigned capillary electrophoresis peak and a separately measured Acid Yellow 23 dye standard were nearly identical, and a good spectral match was obtained between the ultraviolet-visible spectra. Two textile dyes, Basic Red 18 and Basic Red 29, which have similar μ_ep's, were baseline-resolved using the cationic dye capillary electrophoresis method. Dye identification was achieved by comparison of λ_max and the overall ultraviolet-visible absorption spectra. Studies have been initiated recently on ink-jet dyes. Anionic capillary electrophoresis analysis has shown promise on extractions from colored ink-jet samples. Initial findings suggest that ink-jet dyes are different from ballpoint pen ink dyes in that they are multiply charged and have larger absolute μ_ep values.

Conclusion

A capillary electrophoresis buffer system was developed to separate basic, cationic organic dyes for forensic ink analysis applications. The cationic dye capillary electrophoresis method was used to successfully differentiate ten blue ballpoint pen inks that were extracted from mock forensic samples. Identification of five components (dyes and additives) was sufficient to distinguish the inks. Differentiation of ink with the same observed dye formulations was aided by manufacturer-specific additive identification. Direct infusion electrospray ionization mass spectrometry was used to confirm the assignment of capillary electrophoresis peaks. Initial experiments suggest that the combination of the cationic dye capillary electrophoresis method reported here and a separately reported anionic dye capillary electrophoresis method (Egan et al. 2005) can be applied to food dyes, textile dyes, and ink-jet dyes.

Acknowledgments

The authors thank Dr. Gerry LaPorte for generously donating ink and dye samples from the U.S. Secret Service ink library. Funding, in part, was obtained from the Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee, for postdoctoral research projects.

References

Aginsky, V. N. Determination of the age of ballpoint pen ink by gas and densitometric thin-layer chromatography, Journal of Chromatography A (1994) 678:119-125.

Associated Press. Timeline of Martha Stewart scandal, USA Today [Online]. (March 5, 2004). Available: http://www.usatoday.com/money/2004-03-05-stewart-timeline_x.htm.

Conneely, A., McClean, S., Smyth, W. F., and McMullan, G. Study of the mass spectrometric behaviour of phthalocyanine and azo dyes using electrospray ionisation and matrix-assisted laser desorption/ionisation, Rapid Communications in Mass Spectrometry (2001) 15:2076-2084.

Egan, J. M., Hagan, K. A., and Brewer, J. D. Forensic analysis of black ballpoint pen inks using capillary electrophoresis, Forensic Science Communications [online]. (July 2005). Available: http://www.fbi.gov/hq/lab/fsc/current/index.htm.

Fanali, S. and Schudel, M. Some separations of black and red water-soluble fiber-tip pen inks by capillary zone electrophoresis and thin-layer chromatography, Journal of Forensic Sciences (1991) 36:1192-1197.

Green, F. J. Sigma-Aldrich Handbook of Stains, Dyes and Indicators. Aldrich Chemical, Milwaukee, Wisconsin, 1990, pp. 432-435.

Grim, D. M., Siegel, J. S., and Allison, J. A. Evaluation of laser desorption mass spectrometry and UV accelerated aging of dyes on paper as tools for the evaluation of a questioned document, Journal of Forensic Sciences (2002) 47:1265-1273.

Huang, M., Yinon, J., and Sigman, M. E. Forensic identification of dyes extracted from textile fibers by liquid chromatography mass spectrometry (LC-MS), Journal of Forensic Sciences (2004) 49(2):1-12.

Lurie, I. S., Hays, P. A., and Parker, K. Capillary electrophoresis analysis of a wide variety of seized drugs using the same capillary with dynamic coatings, Electrophoresis (2004) 25:1580-1591.

Ng, L. K., Lafontaine, P., and Brazeau, L. Ballpoint pen inks: Characterization by positive and negative ion-electrospray ionization mass spectrometry for the forensic examination of writing inks, Journal of Forensic Sciences (2002) 47:1-10.

Rhode, E., McManus, A. C., Vogt, C., and Heineman, W. R. Separation and comparison of fountain pen inks by capillary zone electrophoresis, Journal of Forensic Sciences (1997) 42:1004-1011.

Shihabi, Z. K. Stacking in capillary zone electrophoresis, Journal of Chromatography A (2000) 902:107-117.

Skoog, D. A., Holler, F. J., and Nieman, T. A. Capillary electrophoresis and capillary electrochromatography. In: Principles of Instrumental Analysis. 5th ed., Harcourt Brace College Publishers, Philadelphia, Pennsylvania, 1998, pp. 778-795.

Tebbett, I. R. Chromatographic analysis of inks for forensic science applications, Forensic Science Review (1991) 3:71-82.

Xu, X., Koeijer, J. A., Moel, J. J. M., and Logtenberg, H. Ink analysis for forensic science applications by micellar electrokinetic capillary chromatography with photodiode array detection, International Journal of Forensic Document Examiners (1997) 3:240-260.

Zlotnick, J. A. and Smith, F. P. Separation of some black rollerball pen inks by capillary electrophoresis: Preliminary data, Forensic Science International (1998) 92:269-280.