International Journal of Forensic Document Examiners

INK ANALYSIS FOR FORENSIC SCIENCE APPLICATIONS BY MICELLAR ELECTROKINETIC CAPILLARY

CHROMATOGRAPHY WITH PHOTO-DIODE ARRAY DETECTION1

·A comprehensive alternative approach to conventional forensic ink analysis. by X. Xu2

, J. A. de Koeije~, J. J. M. de Moel4 and H. Logtenberg5

REFERENCES: Xu, X., de Koeijer, J. A., de Moel, J. J. M., and Logtenberg, H., "Ink Analysis for Forensic Science Applications by Micellar Electrokinetic Capillary Chromatography with Photo-diode· Array Detection", International Journal of Forensic Document Examiners, Vol. 3, No.3, July/Sept 1997, pp. 240-260.

ABSTRACT: The possibility of forensic ink comparisons by micellar electrokinetic capillary chromatography (MECC) has been studied. High­resolution separation of ca 40 (positively and negatively charged, neutral, water­and alcohol-soluble) dyes of different origin has been achieved in a bare fused­silica capillary, using a pH 10, 3-amino-1-propanol/HCl buffer containing 60 rnM SDS and 0.5 mM Brij-35 as micellar agents with an organic modifier of 30% acetonitrile. More than 100 different peaks of those dyes are spread out over a time window of 30 minutes. More than 100 different peaks of those dyes are spread out over a time window of 30 minutes. The separation patterns of individual dyes were COmPared with those obtained by HPLC and TLC, showing a much higher separation efficiency for MECC each time. In particular, many frequently encountered individual dyes show a number of peaks with :MECC, not always with HPLC and TLC. The separation patterns of those individual dyes from different sources are quite different and very useful in comparing inks. The method was successfully applied to more than 50 gylcol-, solvent- and water-based writing or printing inks of different colours extracted from paper. The separation of inks from 27 blue and black ballpoint pens has been compared with those of HPLC and TLC, supplying more information about colorants, as well as non-colorants. Some inks which can not be discriminated by applying HPLC and TLC methods can be definitely distinguished by using :MECC. The somewhat lower sensitivity of the method was compensated by a simple, effective extraction procedure with methoxyethoxyethanol (MEE) and an aqueous solution of2mM Brij-35 in a very small volume of 1-1.5 IJ.l. In this way, the amount of ink needed for the analysis was comparable to that routinely used for HPLC and TLC. The reproducibility and reliability of this method has also been investigated.

KEYWORDS: Ink, forensic ink comparison, dye, capillary electrophoresis, ballpoint pen, roller ball pen, fine liner pen, marker pen, ink jet ink.

1. Introduction

One of the major tasks of a question document examiner is the comparison of inks in order to determine their differences (if any),

. their sources (e.g. pen brands, printers, country of origin), or their dates (first production date or date of placement on a document). The composition of inks can be quite complicated in order to meet various usage requirements. Dyes used as colouring material for inks may include different classes as defined by Colour Index [1], such as: acid dyes, azoic dyes, basic dyes, direct dyes, disperse dyes, reactive dyes, solvent dyes, or even pigments. The solvents (or carriers) represent a major portion of the remaining materials in the ink. Other materials present in inks may be quite different for various types of inks, e.g. fatty acids, resins (naturally occurring or

1Received June 19, 1997, accepted October 3, 1997. 2Ministry of Justice, Forensic Science Laboratory, Volmerlaan 17, 2288 GD Rijswijk, The Netherlands. 3Ibid, corresponding author. 4Ibid. 5Ibid.

synthetic polymers), surface active agents and corrosion control ingredients [2] or conductivity salts [3].

Advancements in the ability to distinguish, identify and date questioned documents through ink and paper examination is strongly dependent on the development of analytical techniques. Due to its simplicity and cost effectiveness, thin layer chromatography (TLC) is still widely being used in forensic science laboratories for ink analyses [4,5]. However, its lower separation efficiency and limited detection possibilities renders it less suitable for distinguishing similar inks and identifying ink components e.g. ·dyes. High performance liquid chromatography (HPLC), which is already applied by forensic laboratories [6, 7 ,8], can offer a better separation power than TI..C and more quantitative information about the components. However, columns in HPLC are (most) often specifically designed to offer a very good separation for a limited group of (similar) compounds.

Capillary electrophoresis (CE) is a relative new separation technique characterized by a high-resolution separation power. This technique has already been applied to the analysis of dyes used as food additives and in the dye-manufacturing and dye-processing industries. Several papers have described the separation of charged dyes by capillary zone electrophoresis (CZE) [9-13] and charged and neutral dyes by micellar electrokinetic capillary chromatography (MECC) [14, 15]. Some of them have compared the analysis of dyes by HPLC and CE and found that CE has advantages over HPLC in terms of efficiency, resolving power and consumption of expensive and often toxic solvents [13,14]. MECC (by adding organic solvent to the pH buffer) enables efficient separation of (water-insoluble) neutral and charged dyes [16,17]. This method has also shown higher separation power than CZE, where separation is solely based on charge [18,19].

In recent years, CE, due to its exceptional separation efficiency and broad analytical spectrum, has received increasing attention from forensic. scientists and found applications in some of the main analytical fields of forensic interest. An excellent introduction to CE and a review of its applications in forensic science is given in ref. [20]. In the field of dye analysis, some authors analysed acid dyes in wool fibres, only not yet on a scale fitted to realistic forensic casework [16]. One paper proposed a CZE method for the separation of water-soluble fibre-tip pen inks [21]. This was however, not in the stage of analysing inks extracted from a document because the dye solubility or adhesion forces between some of the dyes and the cellulose fibres in the paper and the decomposition of inks with time were problematic.

Inks, especially those from a ballpoint pen, may consist of dyes ranging from small to large, (multi-) charged to neutral or even be very hydrophobic. Many of the dyes are composed of several chei_Tlically very similar substances or derivatives. In this paper, a CE method is described which is applicable to the separation of a broad spectrum of dyes and has a better resolving which is applicable to the separation of a broad spectrum of dyes and has a

240 Copyright © 1997 Shunderson Communications All llights of Reproduction Reserved

better resolving power than existing separation methods known by the authors. Hence it has led to an improved ability to distinguish different inks for forensic applications.

2. Experimental

2.1. Apparatus

A Beckman PI ACE 5500 capillary electrophoresis system (Beckman Instruments Inc., Fullerton, CA, USA) was used. Micro­sample-vials of 50 jll held in place by a spring in a glass electrolyte vial of 4.5 ml were used for sample injection. The amount of buffer at both the inlet and the outlet of the capillary was 4 ml. The polyimide-coated fused silica capillary of i.d. 50 jlrn and o.d. 375 jlm was obtained from Polyrnicro Technologies (Phoenix, AZ, USA). The total length of the capillary was 57 (or 47) em and the length to the detector was 50 (or 40) em. The window of the on-column detector cell was created by burning off a small section (ca. 0.4 em) of the polyimide-coating, after which the excess residue was wiped off with methanol. The detection was performed with a diode array detector in the range of 200 to 600 nm at a band width of 4 nm. The electropherogram at each wavelength was recorded and evaluated with a Beckman PlACE station software (Version 1.0).

2.2 Chemicals and Reagents

3-Amino-1-propanol (3-AP), 2-(2-methoxyethoxy)ethanol (MEE), Trimethylene glycol (TMG), Brij-35 and Acetonitrile (HPLC grade) were purchased from Aldrich Chemical Co. (Gillingham-Dorset, England), Sodium dodecyl sulphate (SDS) from Sigma Chemical Co. (Louis, MO, USA). All other chemicals used for method development were of analytical-reagent grade. Dyes were obtained from various sources including BASF (Ludwigsbafen and Rhein, Germany), CAS (Hoechst, Frankfurt, Germany), MRT (Morton International B.V., Amersfoort, The Netherlands), Aldrich (Bomem, Germany), Merck (Darnmstadt, Germany) and LC (=our own lab. collection). Deionized water was used for the preparation of all solutions.

Standard solutions of dye, mixtures were prepared from individual standard stock solutions of 10 jlg/10 jll in methanol, which were stored in the dark at 2°C.

The separation solution was composed of 70% of a solution of SDS 60mM, Brij-35 0.5 mM, 3-amino-1-propanol (p~ 10) 15 mM, HCl 7.5 mM, pH 10 and 30% acetonitrile. It took three days for micelles in the newly prepared solution to reach equilibrium at room temperature. A 2 rnM Brij-35 solution was used for sample dilution. The separation solution was filtered with a Millex-VV syringe filter of 0.1 jlm pore size (Product no. SL VV 025 LS; Millipore, Bedford, MA, USA) before use.

2.3. Procedures

Ink extraction The ink or a carrier, usually on a paper, was punched out with a home-made hollow steel needle of i.d. 0.45 mm and o.d. 0.6 mm and transferred to a micro-vial. For extraction, 0.3-0.5 ~-tl MEE taken by a 1jll syringe (SGE, Australia Pty. Ltd.) was added to the micro-vial containing the micro-dots. The micro-vial was then sealed with parafilm to prevent evaporation and put into an ultrasonic bath, with water at room temperature, for 5 minutes. After sanification, the extract was diluted with 0.6-1 jll2 mM Brij-35 solution as mentioned above. This mixture was then ready for

Vol. 3, No.3, July/Sept 1997

injection (See Section 3.2). Electrophoresis A new capillary was first flushed with 0.1 M

sodium hydroxide solution for 20 minutes, followed by water for 10 minutes and finally with the separation solution for 10 minutes. The latter solution is kept in the capillary overnight. Each sample was introduced into the capillary by applying a pressure of 70 mbar (0.5 psi) for 4 seconds. The components in the injection solution were separated at +30 kV (72 ~-tA) for 30 minutes with a 57 em long capillary, or at +25 kV (76 ~-tA) with a 47 em long capillary. The capillary was maintained at constant temperature (44°C). Separated components were monitored in the range of 200 to 600 nm. To ensure an optimal performance the capillary was flushed between runs with a HCl (0.01 M) -methanol - NaOH (0.1 M) - separation solution cycle. Blank experiments for the analysis of ink on paper were carried out by taking equivalent amounts of blank paper instead of inked paper.

3. Results and Discussion

3.1 Separation of Standard Dyes

The method development for the ink analysis is based on the ink dyes listed in Table 1. The corresponding dye structures which are available are given in Figure 1. These dyes are included in several classes, showing different charges and charge signs, sizes, hydrophobicities and covering all colour ranges. Some of them are well known, or commonly used, or come from different sources, e.g. Methylviolet (29, 41#), Rhodamine B (26, 44#) and Solvent Blue 38 (3,25,37#). We believe that this dye collection is a good representative for colouring material in inks and can therefore be used to evaluate the separation method to be discussed.

3.1.1 Separation and Injection Solutions

In this work a micelle system has been optimised for the separation of the dye mixture shown in Figure 2a. The buffer used was 60 mM SDS, 0.5 mM Brij-35, 15 mM 3-AP, 7.5 mM Hcl, pH 10 and 30% acetonirtile. A good separation is achieved for all components ofthemixture, including a negatively charged dye A.R. 52 (17#), positively charged dyes S.0.3 (40#), M.V. (29#) and V.B.B. (42#), a zwitterionic dye R.A.B. (26#) and neutral solvent dyes S.Y.162 (#6) and S.B.3 (1#). With this separation system some of the dyes (A.R.52 (17#), M.V. (29#), V.B.B. (42#) and S.B.3 (1#)) show more than one peak with a similar spectrum. These peaks are considered to be attributed to "sub-compounds", i.e. substitution derivatives or isomers, of the dye and will be further referred to in this paper as "sub-peaks". As can be seen from the figure, the separation of this mixture is dominated by the micellar effect (not the charge effect), as the migration order of the dyes is mainly determined by the molecular size (or micelle-philicity).

Micelle In a preliminary experiment a similar separation (with in general a lesser resolution) was also obtained for this mixture by using a deoxycholic or cholic acid instead of SDS, but with a much higher concentration of 100 - 140 rnM. Although the bile salts ( deoxy)cholic acids were found to have advantages in the separation of very hydrophobic compounds [23], they showed less efficiency than SDS, especially for M.V. (29#) and S.B. 3 (1#). Furthermore, the higher concentration of the bile salts led to a higher current and a slower EOF, consequently a higher beat effect and a longer analysis time.

Co-solvent By adding acetonitrile to the micellar solution not

Copyright© 1997 Shunderson Communication All Rights ofReproduction Reserved 241

International Journal of Forensic Document Examiners

Table 1 Standard d~es

Lab C.l. General name Commercial name I Source3 C.I. No. b or Chemical class cc Found in Figure

No.

1. Solvent Black 3 Schwarz *60 /BASF 26150 0.27 2a

2. Solvent Black 47 Schwarz *58/BASF azo(l :2 Cr complex d) 2.0 2b,ll

3. Solvent Blue 38 Blau 722/BASF phthalocyanine, see Fig. 1 3.3 3b, ref. 25,37#

4. Solvent B 1 ue 4 Blaubase 672/BASF 44045:1 3.3 3c, ref. 42#

5. Solvent B 1 ue 64 Blau 698 /BASF phthalocyanine0 not sol. in ale.

6. Solvent Yellow 162 Gelb 075/BASF monoazo 0.27 2a

7. Solvent Red 122 Rot 336 /BASF azo (Cr complex~ 2.0 2b, 3d

8. Basic Blue 11 Victoria bleu Rl Adrich 44040 3.3 3c

9. Solvent Black 46 A-schwarz * 17/BASF azod 2.0 2b

10. Acid Yellow 245 Duasyn Luminous Yellow TICAS 2.0 2d

11. Acid Red 18 Duasyn Acid Ponceau 4RC/CAS 16255 2.0 11

12. Solvent Red 122 Duasyn Red A-B VP 356/CAS 2.0 3d, ref. 7#

13. Acid Orange l 0 Duasyn Acid Orange GG/CAS 16230 2.0 2d,11

14. Acid Orange 7 Duasyn Acid Orange PICAS 15510 2.0 2d

15. Acid Blue I Duasyn Acid Blue V 02/CAS 42045 2.0 2d

16. Solvent Yellow 82 Duasyn Yellow AR VP 303/CAS monoazo (metal complexe) 0.65 2c

17. Acid Red 52 Duasyn Acid Rhodamin BO 1/CAS 45100 0.067 2a

18. Reactive Black 31 Duasyn Black RL-SF VP 228/CAS diazo0 2.0 2b

19. Acid Yellow 23 Duasyn Acid Yellow XXJCAS 19140 2.0 11

20. Acid Blue 93:1 Ink Blue R /CAS 42780 3.3, Number of peaks spead over 30 min; not shown; ref. 24, 28 and 32#.

21. Solvent Black 27 Duasyn Black A-lG VP 280/CAS 1.3 2b.6

22. Direct Yellow 157 Duasyn Direct Yellow 6G-SF VP 13965 1.3 2c 216/CAS (struc. not avail.)

23. Reactive Red 180 Duasyn Brilliant Red F3B-SF VP azod 3.3 2c 218/CAS

24. Acid Blue 93 Ink Blue BIT crist. /CAS 42780 3.3 see 20#

25. Solvent Blue 38 Fast Blue MBSN I Adrich see 3# 3.3 3b; see 3#

26. Solvent Red 49 Rhodamin B/Merck 45170:1 0.27 2a; ref 44

27. Reactive Yellow 37 Duasyn Brilliant Yellow GL-SF VP monoazo0 3.3 2c,11 220/CAS

28. Acid Blue 93 Tinten Blau BJT krist./LC 42780 see 20#

29. Solvent Violet 8 Methylviolet/LC 42535 0.27/1.3 2a, 3a; ref. 41#

30. Pigment Green 7 Phtalocyanine green/LC 74260 not sol. in ate.

31. Pigment Blue 15 Phtalocyanine blue/LC 74160 not sol. in ale.

32. Acid Blue 93 MethyiBlau /Merck 42780 3.3 see 20#

33. Solvent Yellow 161 Morfast Yellow 102 /MRT 33, 35,36#; dyes in an unknown oily medium; broad peaks over a few min.

34. Solvent Red 208 Morfast Red 102 /MRT 2.0 2c

35. Solvent Yellow 143 Morfast Yellow 101/MRT see 33#

36. Solvent Brown 52 Morfast Brown 100 /MRT see 33#

37. Solvent blue 38 Morfast Blue 100/MRT 3.3 3b, ref. 3#, 25#

38. Solvent Red 68 Morfast Red 10 1/MRT 2.0 2c

39. Direct Black 168 Direct Black H8F-SF VP132/CAS diazo, see Fig. 1 3.3 2c

40. Solvent Orange 3. Orangebase 206 /BASF 11270:1 0.13 2a,ll

41. Solvent Violet 8 Violettbase 604 Staubarm /BASF 42535:1 1.3 3a, ref. 29#

42. Solvent Blue 4 Victoria Blue B /Adrich 44045:1 0.27/3.3 2a, 3c, ref. 4

43. Solvent Blue 70 Duasyn blue A-G VP 279/CAS phthalocyanine0 3.3; a broad peak over a few min.

44. Solvent Red 49 Rotbase 543 /BASF 45170:1 no diff. in sept. with 26#

a See Experimental. b A dye with C.I. No. has a known structure shown in Fig. l. c concentration (in J.l.g/1 OJ.l.l) in the injection solution.

d [1, V.7]. c [1, V.3].

242 Copyright © 1997 Shunderson Communications All Rights of Reproduction Reserved

Vol. 3, No.3, July/Sept 1997

Solvent Black 3 (1#)

OH

<Q)-N=N<o>

Na03S~ S03Na

Acid Orange 1 0 (13#)

Solvent Blue 38 (3, 25 and 37#)

OH

Nao3s-@-N=N{cj)

Acid Orange 7 (Q) (14#)

(C2Hsh Nif0~=~(C2H5h l8l...c~V

Solvent Red 4©- HCI Rhodamin B 0 co; (26 and 44#)

NaO,/Q2'=. ftso,Na <Q( )95 CH1~~C,@-N(CH1 ),

Acid Blue 93 lJ ¢ Solvent Violet 8 Methyl Blue 0 1 1 Methyl Violet (20,24,28,32#) 1 (29 and 41#)

Z+ N(CH3)z Cl-~ +

Acid Red 18 (11#)

@--so1Na Pigment Blue 15 (31#), (no Cl-) Pigment Green 7 (30#), (Polychloro-)

Direct Black 168 (39#), ale. based ink (Zeneca, prnt & Im. sys.) for industrial inkjet printers [22].

Figure 1: The dyes (in Table 1) whose structures are known [22].

Copyright © 1997 Shunderson Communication All Rights of Reproduction Reserved

Solvent Orange 3 (40#)

243

.J

International Journal of Forensic Document Examiners

14~----~r----1-7--------------------------------------~

~ ~b <( a E 12

10

8

6

4

2

40

5

29c

29d

1a 29b

6

10 T' ( . ) 15 1me m1n

400 nm 600

~·~ 0 200 400 nm 600

215 nm

20 25

Figure 2a: The separation of standard dye mixture (a) consisting of dyes with different charge signs, sizes and hydrophobicities with a 50 em long capillary.

4

400 nm 60

0 5 10 Time (min) 15 20 25

Figure 2b: The separation of dye mixture (I?) consisting mainly of black dyes, which are thought to be relatively large in size with a 50 em long capillary.

244 Copyright © 1997 Shunderson Communications All Rights of Reproduction Reserved

only the sub-compounds of the water insoluble compounds (e.g. S. B.3(1#)), but also the sub-compounds of the water soluble compounds (e.g.M.V. (29#) and V.B.B (24#)) were separated. Acetonitrile has advantages over methanol due to a faster EOF and a better separation in this case. The amount of acetonitrile in the buffer changes the magnitude of the two effects (micelle and charge) which have a direct influence on the separation behaviour. The sub­compounds ofM.V. (29#) and S.B.3 (1#) began to separate when the portion of acetonitrile reached 15-20%, and achieved the best separation at 30%. On increasing the acetonitrile portion to 40%, the micellar effect became very small for the ionic dyes, so that the positively charged ones were almost merged with the EOF peak and the neutral dye S.B.3 (#1) preceded the negative one A.R.52 (17#). EOF Because negative ions and micelles migrate against the EOF, it is important to obtain a fast EOF, in order to bring all dyes present in the ink to the detector in a reasonable migration time. Besides the micelle and co-solvent, pH and temperature are the other two vital factors affecting the EOF. At pH 10, not only the EOF reaches its highest mobility, but most of the amino-groups on the dyes are deprotonated, leading to an increased micelle effect and a hence better separation. With the increase of buffer temperature, the mobility of the EOF increases more rapidly than that of the negative ions and micelles, resulting in general in a shorter analysis time. For example, it took ca. 25 minutes to complete the separation at room temperature and ca. 15 minutes at 44°C.

Brij-35 It is a neutral surfactant with a molecular weight of 1200 and a critical micelle concentration (CMC) of 0.1 mM in water. When analytes are partitioned in the Brij-35 micelles, regardless of their charge, they migrate with the micelle at zero mobility. At a concentration level of 0.5 mM, Brij-35 has hardly any influence on smaller compounds with migration times less than 10 minutes in Figure 2a, but does reduce the migration time of large ones like S.B. 3 (1#) (ca. 4 minutes shorter). Besides reducing the analysis time for a large compound, it can also increase the solubility of insoluble compounds in ink, resulting in a reduction of spikes in the electropherogram.

Injection solution Generally, with the presence of SDS in an injection (sample) solution, dye peaks are broadened and become smaller with increasing sample solution age. The reason for this phenomenon is not known. An injection solution consisting of an aqueous solution of 0.5 - 5 mM Brij-35 can prevent hydrophobic compounds from precipitating, as well as keep a consistent (sharp) peak shape and height. The portion of the organic solvent in the injection solution should not exceed 1/3. When it exceeds 1/2 an obvious general peak broadening and smaller peaks were observed for all kinds of components, especially for neutral and positively charged ions in various extents.

3.1.2. General Evaluation With Respect To Separation

The separation solution developed for the separation of the mixture in Figure 2a was evaluated with all other dyes in Table 1. Except for the alcohol-insoluble dyes (5, 30, 31#) and part of the Morfast dyes (33, 35, 36#), almost all remaining dyes (excluding the recurring ones ) were separated in the different combinations shown iri Figures 2b-c.

As can be seen from Figure 2, general good separation was obtained for all dyes appearing in these figures. The swelled baseline in Figure 2c was caused by the unknown solvent of dye S.R. 208 (34#). It is worth noting that in Figure 2d the triply-charged acids 11 and 19# are missing whereas the (effectively-) singly­charged acids (15, 17 and 14#), doubly-charged acid (13#) and the

Vol. 3, No.3, July/Sept 1997

acid with unknown charge (10#) are all present. This problem will be discussed in a separate section of this paper.

The high resolving power of this system for the sub-compounds of a dye such as M.V. (29#), S.B. 38 (3#), V.B.B. (42#) and S.R. 122 (7#) offers us an opportunity to distinguish between inks with similar dyes from different sources or different batches. The comparison of some of such dyes is given in Figure 3. As will be seen later, the unique peak shape of the important dye S.B. 38 present in many inks allows for an easy identification.

The separation pattern of each individual dye and the day mixture was compared with that obtained by HPLC [6] (column: 5 J..lm C18, eluent: acetonitrile-water in different ratios). Besides the much better column efficiency of the MECC, there were two significant differences between them. First, under optimized constant elution condition, dyes such as 2, 3, 9, 17, 18,20-24,27, 28, 39# show just a single peak in HPLC but show multiple peaks when using MECC; a few cationic dyes ( 4, 8, 41 #) show more (or a comparable number of) peaks when using HPLC. Secondly, a generally good separation of the dye mixture is only possible in HPLC when using a gradient elution.

The wavelength of the supplied one covers the range from 190 to 600 nm. However, some blue and green dyes e.g. S.B. 38 (Figure 3b) have an absorption maximum in the range of 600-800 run. This limits the possibility to recognize these dyes by such a diode array detector.

3.1.3. Analytical Performance

A standard mixture of dyes (the same as in Figure 2a) in a 30 )ll injection solution composed of 30% methanol and 70% 2 mM Brij-35 solution was analysed repeatedly. The results for the reproducibility of the migration times are given in Table 2 and Figure 4. As is clear from the table the reproducibility of the migration times of the dyes is good as the relative standard deviations were found within the range from 0.4 to 2.0% for 10 consecutive injections. As can be seen from Figure 4, the EOF rate is almost c~nstant during all 24 runs, showing a relative standard deviation of 0,1% for the first 10 runs and 0.66% for all 24 runs. However, the migration time of the dyes increases slowly. This can be explained as the result of a change in the anode electrolyte composition, due to the continuous evaporation of the acetonitrile from the buffer [24], which changes the system's selectivity for the separation of the dyes. Therefore, it is recommended to change the anode solution after every 5 hours. The evaporation, however, has only a slight influence on the EOF. The reproducibility of the peak areas (n = 8 for S.B. 3 and n = 10 for others) was also acceptable, with relative standard deviations between 3.1 and 7.7% (Table 3). At a higher level as will be seen later (Table 5), the relative standard deviation of the peak will be much improved.

The migration time of the EOF with the same capillary or different capillaries was repeatedly measured from various experiments performed with standard dye mixtures and inks over a period of time. The reproducibility of the electroosmotic peak migration time, as shown in Table 4, was within 0.69% with one week for 72 runs and 1.75% with five capillaries in three months for 141 runs.

Some dyes are not stable in a solution when they are in contact with air or exposed to light. As shown in Figure 5 (black circle, line 1), S.B. 3 (1#) begins to decompose after nine injections with the standard injection configuration, where the air is allowed to penetrate the rubber cap and come into contact with the injection solution. This process can be slowed down by partially filling the

Copyright © 1997 Shunderson Communication All Rights of Reproduction Reserved 245

International Journal of Forensic Document Examiners

246

11r-r=======~------------------~r-------~~ 12

~ < E

9

7 c

5

3

22b 400 nm

5

9a

34

16 22c

23c

38

39

27a

10 15 Time (min)

~ E

6

~00 400 nm 600

(~ ~00 27a 400 nm 600

27c

I

570 nm

20 25

Figure 2c: The separation of standard dye mixture consisting mainly of red and yellow dyes with a 50 em long capillary.

:J < E

18

14

10

6

2

d

0

{q «

17 14b

15

14a

J ....,

EbF 5

~~ 200 400 nm 600

13 (~ 10

0 200 400 nm 600

215 nm

570 nm

10 . 15 Tame(man)

20 2 5

Figure 2d: The separation of standard dye mixture consisting only of acid dyes with a 50 em long capillary.

Copyright © 1997 Shunderson Communications All Rights of Reproduction Reserved

Vol. 3, No.3, July/Sept 1997

d

c

~'W[] 80 '

c

k/\ d

~00 ~00 600

a

nm

!; ::> < E

b (')

a

i[ b

_l ...,..._

10 15 20

a 12

"l ..AA.--

Time (min)- 215 nm

c a

I 4 8 12

Time (min) - 570 nm

c

il b c

2 b a

:I c( c ~ E 400 nm 600 ~ co

'<t I

::> 80 => < < E b

'• E 0

I '<t '<t • I CD

\ I 40 :I < \~ ~ E

C\1 .,...

::> ~00 400 nm 600 <

~ E 1"'-

C\1 l

::>

d < E

C\1

15

5 10 15 5 10 15 Time (min)- 215 nm Time (min)- 215 nm

Figure 3: Comparison of dyes from different sources. (a) Methylviolet (29, 41#); (b) Solvent Blue 38 (3, 25, 37#); (c) Victoria Blue B (only 4 and 42#); (d) Solvent Red 122 (7, 12#). The dye Victoria BlueR (8#) which was missed in Figure 2 is shown in (c). For other details see Table 1.

Copyright © 1997 Shunderson Communication All Rights of Reproduction Reserved 247

International Journal of Forensic Document Examiners

Table 2: Reproducibility of migration time (min)8•

S.0.3 R.A.B A.R.52 M.V. S.Y.162 V.B.B S.B.3 EOF

(40#) (26#) (17#) (29#) (6#) (42#) (1#)

First 10 runs

x 4.835 6.083 6.391 7.976 8.332 9.740 12.060 3.348

S.D. 0.027 0.075 0.023 0.159 0.087 0.182 0.175 0.003

R.S.D.% 0.55 1.23 0.35 1.99 1.05 1.87 1.55 0.10

All24 runs

x 4.875 6.226 6.420 8.299 8.516 10.11 12.45 3.341

S.D. 0.045 0.15 0.055 0.33 0.19 0.39 0.40 0.022

R.S.D.% 0.93 2.37 0.85 4.02 2.22 3.81 3.23 0.66

a Conditions are the same as in Fig. 2a.

Table 3: Reproducibility of relative peak area at 215 nma.

S.0.3 (1#) A.R.52 (17#) M.V. (29#) V.B.B. (42#) S.B. 3 (1#)

!lgii 0!11 0.13 0.067 0.27 0.27 0.27

n 10 10 10 10 8

x 0.19 0.20 0.33 0.55 0.42

S.D. 0.012 0.0063 0.019 0.042 0.030

R.S.D.% 6.1 3.1 5.7 7.7 7.2

a Conditions are the same as in Fig. 2a.

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Vol. 3, No. 3, July/Sept 1997

14~------------------------------------~

12 tr + + + + + + + + + + + ++++++++++++

c 10 •••••• •••••••• g II••••••••• 8 ~~~~~~~~~~~~RRRR~~~~~~~~ 6~~AAA~~AA~~9gggggoaa~~~~

~······················· 4 . . . . -. . -. . . . . . . . . -. -. . . -2

o+-----~-----4~----+-----~------~--~

+ 8.8.3 {1)

e V.B.B {42)

o S.Y.162 (6)

X M.V. (29)

V A.R.52 (17)

D R.A.B (26)

• 8.0.3 (40)

• EOF

0 100 200 300 400 500 600

Buffer age (min)

Figure 4: The migration time of the dyes and EOF in Figure 2a as a function of the buffer age. Each marker indicates an electrophoretic run.

Table 4: Reproducibility of migration time EOF (min).

5 capillaries in 3 months

Total runs (n) 141

x 3.336

S.D. 0.059

R.S.D.% 1.75

injection bottle surrounding the micro-sample-vial with water. 1bis is probably because the aqueous atmosphere in the bottle expels the oxygen from the air. By isolating the injection bottle from the air S.B. 3 can stay stable for as long as a few days (circle, line 2). S.O. 3 (40#), however, undergoes decomposition even if the injection bottle is airtight and kept in dark (rectangular, line 2). This might be due to the consumption of the dye by bacteria in the solution.

3.2 Ink Analysis for Forensic Applications

The majority of ink analyses in forensic applications are aimed at the chemical composition of ink on paper. fu this paper, therefore, we just report on the analysis of ink on paper.

3.2.1. Sample Preparation

Sampling Depending on the density of the ink line and the type

One capillary in one week

72

3.348

0.023076

0.69

0.5~------------------------------.

0 +-----~~~-+----~------~--~ 0 5 10 15 20 25

Sample age (hr)

Figure 5: A dye in the injection solution might undergo decomposition by atmospheric oxygen or bacteria in the solution. The sample vial was (1) in contact with air, (2) isolated from air. For other details see text.

Copyright © 1997 Shunderson Communication All Rights of Reproduction Reserved 249

International J oumal of Forensic Document Examiners

::s -t E

30

20

10

a

9.300

0

9. 90 9.340

9.292

2 4

Sample age (hour)

9.430 2min

9.400

6 8

::s -t E

30

b

2min

9.383 9.375

0 20 Sample age (hour)

Figure 6: Peak reproducibility of S.B. 27 (21#) in a small volume (1 J.ll) ink extract obtained by different injection configurations: (a) without para:filrn, but with a good cap; (b) with para:film and any cap. Experimental conditions: Capillary length, 47 em. For other details see text.

of inks, 3 to 8 microdots of an ink line on paper were sampled by punching out pieces of the ink line with the hollow steel needle. The volumes of the extract and the total injection solution are 0.3 -0.5 J.ll and 1 - 1.5 J.ll, respectively. Generally, 5 or more microdots are needed for the analysis of ballpoint pen ink (oil-based) whereas 3 microdots are usually sufficient for the water- or solvent-based inks.

Extraction Because of the variety in ink and paper combinations, the different interactions between them and the often limited sample amount, a universally efficient, simple, fast and reproducible extraction of the ink from the paper is of great importance. Furthermore, the compatibility of the extract solution with the separation system and the detection mode have also to be considered. This implies that the extraction solyents are limited to those which are water-miscible and have a small or no absorption in the UV -visible region.

The extraction efficiency of more than 20 solvents, including alcohols, gylcols, pyridine and dimethylforamide (DMF), was tested with 30 strokes of different ballpoint pen inks, drawn on a photocopy paper two years before. Results have shown that the extraction efficiency are in the order of: DMF>MEE (methoxyethoxyethanol) > TMG (trimethylene glycol)> pyridine> methanol > 2,2,2-trifluorethanol. Although the extraction efficiency ofD.MF is slightly better than that of MEE, its high UV absorbance and its interaction with components in the buffer and the capillary results in a baseline disturbance for a few minutes after the EOF peak. MEE has shown a general good extraction ability to inks and a good compatibility to the MECC. For most inks tested the extraction can be (almost) completed within 5 minutes when placed in an ultrasonic bath. After this extraction procedure two parts of an aqueous solution of 2 mM Brij-35 is added to one part of the MEE extract. This mixture is then treated in an ultrasonic bath for

another 5 minutes, so that the compounds solely soluble in water can also be extr.acted. This extraction procedure has proved to be suitable for both oil-based, as well as solvent-based and water-based inks.

Handling a Small Injection Volume In order to use as little of an ink as possible but maintain a high enough concentration in the injection solution, it is necessary to keep the volume of the injection solution to a minimum. This, however, makes the injection solution very vulnerable to evaporation and adhesion to the wall of the sample vial. In addition, as the evaporation rates of solvents in the solution are different, the ratio of water to organic solvent may be changed to beyond the limit of 1/2. The advantage of using MEE as an extraction solvent is that, due to its high surface tension, adherence to the wall of the sample vial is kept to a minimum. Furthermore, due to its high boiling point (198 oq, it has a very low evaporation rate at room temperature. Therefore, the main problem to be focussed on is the change of the water content of the solution.

In a standard injection configuration the 4.5 ml electrolyte vial, within which the 50 j.tl micro-sample-vial is placed on a spring, is closed with a rubber cap. The four flaps of the cap open only during the injection. When there is no water at the bottom of the electrolyte vial, the water in the injection solution ( 1 - 1.5 J.ll) in the micro-vial evaporates quickly, even when the electrolyte vial is sealed. When the vial is filled with ca. 1 ml water, the volume of the injection solution may increase by water condensation on the wall of the micro-vial or decrease by evaporation of the water through the seam. The magnitude of these effects depends on the seam size between the flaps of the rubber cap. ·

In an experiment as shown in Figure 6a, an injection solution containing the extract of the ink of a Waterman Standard Maxima Fine (25#) ballpoint pen was repeatedly injected. The size of the

250 Copyright © 1997 Shunderson Communications All Rights of Reproduction Reserved

I

i I'

0

:J <C E o:t

:J <C E

:J <C E

16

8

~00

Vol. 3, No.3, July/Sept 1997

400 nm 600

b

11.550 /

400 nm 600

a

25 30 35

Time (min) -- 215 nm

Figure 7: Electropherograms of backgrounds from (A) A4 photocopy paper including optical brighteners and (B) ATLANTA company formula paper. Experimental conditions: Capillary length, 57 em. For other details see text.

peaks of the dye S.B. 27 (21#) (which was found in the ink) increased in the first few hours due to a diminishing sample volume by water evaporation. With continuing evaporation of the water, their size decreased and they-became broader, leading to a decrease in resolutiop.. This is due to a change in the ratio of MEE: water (v:v) as it had increased beyond the required 1/2. Generally, by choosing caps with a relative small flap seam and with the configuration mentioned above, a sample solution of 1 j.ll can stay in the sample tray of the auto-sampler for ca. 4 hours without significant loss in peak resolution.

In order to obtain reproducible results for a small sample volume during a longer time span needed for overnight runs, the injection configuration was modified as follows: (a) The micro-vial spring was stretched till the micro-vial gently touched the rubber cap so as to slow down the rate of exchange of water vapour in and out of the micro-vial and consequently avoid water condensation in the micro­vial; (b) The rubber cap was covered (sealed) with a thinned piece of para.film which was stretched by hand to four times its original size in two orthogonal directions. Separations made with this configuration are shown in Figure 6b. In this experiment the sample vial (rubber cap) was quickly sealed with parafilm right after the first injection and re-injected after 20 hours. It is obvious that the results obtained are comparable. By closing the cap, the oxidation

of compounds in the injection solution can also be avoided.

3.2.2 Analysis of Ink on Paper

Paper Background The blanks of two typically used paper samples have been analysed with the method described above, as shown in Figure 7. The A4 (European format) photocopy paper contains optical brighteners, as seen from the peak at 11.675 minutes in Figure 7a. However, this peak is not seen when using methanol or pyridine as an extraction solvent. The compound corresponding to this peak is light and heat sensitive when dissolved in an aqueous solution. Under day light conditions ca. 50% will decompose in half an hour.

The ATLANTA company formula paper (order no. A 5705-01, Netherlands) has a very low background (Figure 7b) and a stable commercial source and is therefore suitable to be used for storing ink lines.

Ink Types Applied The method has been applied to more than 50 ballpoint pen inks in blue, black, green or red colours, and some water- and solvent-based fluid inks such as permanent marker ink, Fineliner (or roller ball) ink and inkjet printing ink. Results show that the method is generally suitable to all those inks (Figure 8).

Ballpoint inks consist of colouring materials, usually alcohol

Copyright © 1997 Shunderson Communication All Rights of Reproduction Reserved 251

International Journal of Forensic Document Examiners

soluble dyes and occasionally an insoluble pigment. The solvents and additives such as fatty acids, resins etc. are also important to the composition of an ink [2]. They can sometimes significantly affect the separation pattern e.g. the baseline shape. Some of the small additives in the ink usually show peaks which have responses merely in the UV range, as those right after the EOF in Figure 8a.

The composition of the fluid ink (Marker or Fineliner) is generally less sophisticated.· Their electropherograms show less "UV" peaks, as can be seen in Figure 8b-c. Furthermore, in water­based inks the presence of ionic dyes can be expected.

The composition of an inkjet (printer) ink is quite complicated, so as to meet the requirements of the specified viscosity, surface tension, conductivity and particle size. However, the solvents are volatile and the conductivity salts are commonly small compounds e.g. lithium nitrate [3]. They are not expected to have impairing effect on the separation and detection of the other compounds present. Figure 8d shows a separation of such an ink.

Ink Comparisons One of the very important applications of ink analysis in forensic science is to find whether inks from different pieces of evidence have the same source or not. However, some dyes, because of their stability, availability and economic usage, are becoming more and more popular with ink manufacturers. For instance, more than 50% of the blue and black ballpoint or roller ball pens analysed contain Methyl violet (M. V.) and/or Solvent Blue 38 (S.B. 38). This sometimes makes the composition of inks quite similar and difficult to discriminate.

Fortunately, as already shown in Figure 3, many dyes including M.V. and S.B. 38 have a number of peaks. They can be made useful in distinguishing between inks from different sources. In addition, even if one can not find any difference in dye composition, it may be possible to find a difference in the UV -absorbing peaks or strange baseline shapes resulting from additives in the inks.

For instance, the blue Parker Medium, Sheaffer Medium and Sheaffer Fine ballpoint pens can not be distinguished by TLC without the aid of a densitometer, as shown in Figure 9. When using HPLC, there is a small difference in the Methylviolet peak ratios but not enough to draw a definite conclusion. Sheaffer Medium and Sheaffer Fine pens are different in mechanism (the tip size of the pen), but, at least we thought, not in chemistry. However, a clear difference has been found by either comparing the sub-peak ratio of M.V. or S.B. 38 (Figure 9b and c). The Parker Medium pen (Figure 9a) can easily be distinguished from the Sheaffer Medium by the peak ratios of S.B. 38 and from the Sheaffer Fine by M.V. Furthermore, it can be uniquely distinguished from the other two inks by the group of the UV -absorbing peaks occurring between 7-8 minutes shown in Figure 9a.

It must be mentioned that differences in peak ratios may be due to batch-to-batch differences but may in some cases also be caused by the age differences or a paper-ink interaction. Further research will be done in the future to determine the extent of these effects.

Another way to differentiate between inks is to compare the peak ratios between different dyes. However, we expect that it is not as reliable as comparing peak ratios between sub-peaks of a dye, especially if ink samples are taken from different papers. Because different dyes may have a different interaction with paper, the extraction rate of a dye on different papers might not be the same. This may also be the case for sub-components of dyes only quite possibly to a lesser extent.

Because an ink line of paper is not homogeneous, absolute concentration values of peaks of different samples can not be used for an ink comparison.

Reproducibility of Extraction Rate and (Sub-) Peak Ratio The

ink comparison made by (sub-) peak ratios rely on a consistent extraction rate for all compounds from different samples of the same ink.

Figure 10 shows the electropherograms of the ink of a black ballpoint pen sampled repeatedly from the same ink line. Comparable sub-peak ratios of a dye and peak ratios of different dyes are obtained. As listed in Table 5, the relative standard deviation of the peak ratios of different dyes are obtained. As listed in Table 5, the relative standard deviation of the peak area percentage of the three sub-peaks of the dye S.B. 27 (21#) is less than 1.4% at 580 nm and 2.7% at 215 run. The larger deviation at lower wavelength is caused by the interference of the background peak which is light-sensitive as discussed before (Figure 7b).

As a comparison, the separation of the same ink by TLC is attached to the figure. The separation carried out by HPLC for this ink is even worse (normally better) than that of TLC (results not shown).

Capillary Condition Maintenance and Restore Some compounds from the ink may adsorb to the capillary wall and change the wall chemistry. This kind of change can be easily restored by the HCl­MeOH-NaOH-H20-buffer flushing cycle. Except for the last step of the cycle, all other flushing steps are carried out by applying a backward pressure. This cleaning cycle is included to the standard measurement procedure for each run. Because the paper :n:llcro-dots are not removed from the injection solution, fibres from the paper may partially or completely block the capillary tip. This can normally be avoided by placing the capillary 1-2 rnm higher than the electrode. Sometimes a gel-like cellulose or polymer which originates from the paper or the ink may stick on the capillary wall within 1 em from the inlet, leading to a decreased EOF rate. This problem can be solved by using hot water (1 00°C) to remove the gel­like material from the capillary, i.e. filling the buffer vial at the inlet · with hot water, meanwhile applying a backward pressure. If the capillary is permanently blocked and more than 1 em must be removed, the 57/50 em capillary configuration can not be used any more in this setup. This is detennined by the specific capillary cartridge design. However, a 47/40 em capillary configuration can still be used with the ·same capillary. Actually, the ·resolution obtained by the combination of 57 em capillary I 30 kV is just slightly better than the 47 em capillary I 25 kV, but the latter combination has an advantage of a shorter analysis time.

3.2.3. Analysis of Acid Dyes Having High Mobilities

As discussed in relation to Figure 2d, acids with triple-charges do not show within 30 minutes. In fact, those acids which have a mobility close to or higher than that of EOF and seldom stay in the micelle will have a very long migration time or will never reach the detector. However, because the acid dyes used for inks are normally large, e.g. A,B, 93 (20#), D.B. 168 (39#), and the micelle effect is usually larger than the charge effect, they still elute in a reasonable time. In practice, among the inks analysed, only in one case was it found that a dye was clearly missing. In spite of this, we still want to get as much information about the acid dyes as possible in one electropherograrn.

In a preliminary experiment, a combination of pressure and voltage was tried, as. was done in reference [13]. The pressure, however, largely impaired the peak resolution. Another way to speed up the migration of the acids to the detector is to increase the micellar effect. By adding more Brij-35 to the separation medium as mentioned above, the acids will stay in the neutral micelle for a longer time and move with it in a zero mobility. In this way, the

252 Copyright © 1997 Shunderson Communications All Rights of Reproduction Reserved

Vol. 3, No.3, July/Sept 1997

a bg

Rd.B (ref. 26#)

8

5 10 15 Time (min)2o 25

Figure Sa: Electropherogram of ink from a Bic E-32 black ballpoint pen on photocopy paper. Experimental conditions: Capillary length 57 em; Sampling: 5 dots of ink line; Detection: 215 nm. For other details see text and Figure 7.

~~----------------~----------------------------------~ <(

E

nm

EOF 4 8 12 Time (min) 16

Figure 8b: Electropherogram of ink from a Schmidt 117EF/502 black roller ball pen on ATLANTA paper. Experimental conditions: Capillary length 57 em; Sampling: 5 dots of ink line; Detection: 215 om. For other details see text and Figure 7.

Copyright © 1997 Shunderson Communication All Rights of Reproduction Reserved 253

International Journal of Forensic Document Examiners

:I ~ E C 3

2

5 10

~ E

nm 600

15Time (min) 20

5

25

Figure 8c: Electropherogram of ink from a red Staedtler permanent marker pen on ATLANTA paper. Experimental conditions: Capillary length 57 em; Sampling: 5 dots of ink line; Detection: 215 nm. For other details see text and Figure 7.

d bg

~ E

600

8

4

10 Time (min) 20 30

Figure 8d: Electropherogram of ink from an inkjet print on printing paper. Experimental conditions: Capillary length 47 em; Sampling: 5 dots of ink line; Detection: 215 nm. For other details see text and Figure 7.

254 Copyright © 1997 Shunderson Conununications All Rights of Reproduction Reserved

Vol. 3, No.3, July/Sept 1997

2

6.5 ..--------'It----------it-----------, il bg E

5.5 c ·~·

4.5 m u2

sb

3.5 u1 bg

2.5

1.5

0.5

0 2 6 8 10 12 14 16 18 nme(mln)

Figure 9: Comparison of blue inks from (a) Parker Ball Pen Refill Medium Din 16 554/2, (b) Sheaffer ballpoint refill medium and (c) Sheaffer ballpoint refill fine. Two year old ink lines were sampled from the same A4 photocopy paper (as in Figure 7). Experimental conditions: Capillary length 57 em; Sampling: 5 dots of ink line. Peaks: m =Methyl violet, sb = Solvent Blue 38, bg = background of paper, u = unidentified compound. The separation result obtained by the TLC method [4] is attached to the corresponding electropherogram for a comparison.

Copyright© 1997 Shunderson Communication All Rights of Reproduction Reserved 255

International Journal of Forensic Document Examiners

C\1 m

:::l 4~~E 24w ~ ~ 4.800 2 .933

~00 400 nm 600 ~00 400 nm600 ~ a:i

#' u.. <0 0 C\1

w g :J

I m <{ .r::. E a: LO

0 2

co 0 C\1 a)

~6

~.308 E .458

3

~00 400 nm 600

6 8 10 12 Time (min)- 215 nm

14

Figure 10: Electropherograms of a black Waterman standard maxima fine ballpoint pen, sampled repeatedly from tl;te same ink line on ATLANTA paper (see Figure 3b). Experimental conditions: Capillary length 47 em; Sampling: 5 dots of two month old ink line. Peak indicators: bg =background of paper. For the TLC method see Figure 9.

256

at580nm

P.A.Pa(X %)

R.S.D.%

at 210 nm

P.A.Pa (X%)

R.S.D.%

Table 5: Reproducibility of sub-peak area ratios of S.B. 27 (21#) in Figure 10, n = 5.

Peak 1 Peak 2 Peak 3

28.51' 42.42 29.08

1.26 0.61 1.39

24.92 41.22 33.85

2.2 1.0 2.7

a Peak area percentage.

Copyright© 1997 Shunderson Communications All Rights of Reproduction Reserved

time that the acids stay outside the micelle and migrate against the EOF will be decreased, leading to a shorter analysis time.

Figure 11 shows a separation of a group of dyes including the two acids ( 11, 19#) which were missing in Figure 2d. In this experiment, the Brij-35 concentration was increased in steps from the standard 0.5 rnM to 10 rnM. With the increase of the Brij-35 concentration, the acid A Y. 23 (19#). Hence, A.R. 18 (11#) is more micelle-philic. Obviously, Brij-35 has a larger effect on peaks with a longer migration time. Although increasing the Brij-35 concentration changes the selectively of the separation for some (sub-) peaks, e.g. S.B. 47 (2#), in general a good separation for all other dyes is maintained.

In the case of the ink analysis as shown in Figure 12a, just one blue dye (Solvent Blue 38 or S.B. 38) was found with the standard micellar solution consisting of 0.5 mM Brij-35. However, the ink is green. According to the colour principle [25], a green colour originates from two absorption maxima at 400-450 nm (yellow) and 580-700 nrn (blue)'. h was certain that a yellow dye was missing and this dye is probably an acid. When increasing the Brij-35 concentration to 10 mM (Figure 12b), a yellow dye appeared which was identified as A.Y. 23 (19#) (reference to Figure 11). It is also observed that some peak shapes are improved. This might be a result of the better solubility of the ink in this solution.

4. Conclusion

The MECC method developed in this paper shows a generally good separation for all kinds of charged and neutral, water- and alcohol-soluble dyes originating from all kinds of writing instruments including ballpoint pens, roller ball pens, fineliner pens, marker pens and inkjet printers. It has a much higher resolving power to dyes containing several chemically very similar substances or derivatives than TLC and HPLC, as well as CZE (to ionic derivatives). Similar inks which could not be differentiated by other methods now can be distinguished (best) by comparing the area ratios of the separated sub-peaks from the same dye in the different inks. Normally, 0.5 mM Brij-35 in the separation solution is fitted to most analysis purposes. However, when an acid dye is suspected but does not appear in a reasonable time, more Brij-35 can be added to the separation solution. The amount of the ink on paper consumed for the analysis is comparable to TLC and HPLC. The reproducibility of the EOF is excellent, with R.S.D. being 0.1% in 5 hours and 0.66% in a week with one capillary, and 0.69% in three months with a few capillaries. The reproducibility of the migration times of dyes are also good with R.S.D. being from 0.55% to 1.99% in 5 hours. The reproducibility of the peak areas of dyes at a relatively low concentration is acceptable, with R.S.D. being from 3.1% to 7.1%. The reproducibility of the sub-peak area ratios of a dye from different samples is found with R.S.D. being from 0.61% to2.7%.

This method is ready to be used, as an alternative, for the analysis of inks e.g. a ballpoint pen, fluid ink pen and inkjet for a realistic forensic application.

References

[1] Colour Index, Vol. 1(1971), Vol. 3(1971), Vol. 5(1987), Vol. 6(1975), Vol. 7(1982), Vol. 8(1987), edited by The Society of Dyes and Colourists and The American Association of Textile Chemists and Colourists, Made and printed by Lund Hwnphries, Bradford and London, Great Briatain.

Vol. 3, No.3, July/Sept 1997

[2] Brunelle, R.L. and Reed, R.W., Forensic Examination of Ink and Paper, Charles C. Thomas Publisher, Dlinois, USA, 1984, pp. 32.

[3] Leach, R.H. and Pierce, R.J., The Printing Ink Manual, Fifth edition, Blueprint, an imprint of Chapman & Hall, UK, 1993, p. 678.

[4] Standard Guide for Test Methods for Forensic Writing Ink Comparison, 1996 Annual Book of ASTM Standards, Section 14 General Methods and Instrumentation, Vol. 14.02, American Society for Testing and Materials, West Conshohocken, PA, USA, 1996, pp. 903-909.

[5] de Koeijer, J.A, Thin Layer Chromatography for Inks, Internal Document, Code: 330503, Ministry of Justice, Forensic Science Laboratory, Rijswijk, The Netherlands, 1995.

[6] Lyter, A.H., "Examination of Ball Pen Ink by High Pressure Liquid Chromatography," Journal of Forensic Science, Vol. 27, No. 1, January 1982, pp. 2-3.

[7] Tibett, I.R., Chen, C., Fitzgerald, M. and Olson, L., "The Use of HPLC with Multiwavelength Detection for the Differentiation on Non Ball Pen Inks," Journal of Forensic Science, Vol. 37, No.4, July, 1992, pp. 1149-1157.

[8] Wannerdam, M., The Characterization of Dyes in Ball-Point Pens With Help of HPLC and a Diode-Array Detector, Internal research report, Ministry of Justice, Forensic Science Laboratory, Rijswijk, The Netherlands, 1994.

[9] Croft, S. and Hinks, D., "Analysis of Dyes by Capillary Electrophoresis," Journal Society Dyes Colour, Vol. 25, No.3, 1993, pp. 47-51.

[10] Shuster, R. and Gratzfeld-Hi.isgen, A., Analysis of Synthetic Dyes in Food Samples by Capillary Zone Electrophoresis, Company report on Food Analysis, Publication nwnber: 12-5964-0280E, Hewlett Packard, 1995.

[11] Liu, H., Zhu, T., Zhang, Y., Qi, S., Huang, A. and Sun, Y., "Determination of Synthetic Colorant Food Additives by Capillary Zone Electrophoresis," Journal of Chromatography A, 718 (1995) pp. 448-453.

[12] Masar, M., Kaniansky, D. and Madajova, V., "Separation of Synthetic Food Colorants by Capillary Zone Electrophoresis in a Hydrodynamically Closed Separation Compartment," Journal of Chromatography A, 724 (1996) pp. 327-336.

[13] Jandera, P., Fischer, J., Staneks, V., Kucerovas, M and Zvonfcek, "Separation of Aromatic Suphonic Acid Dye Intermediates by High Performance Liquid Chromatography and Capillary Zone electrophoresis," Journal of Chromatography A, 738 (1996) pp. 201-213.

[14] Croft, S.N. and Lewis, D.M., "Analysis of Reactive Dyes and Related Derivatives Using High-Performance Capillary Electrophoresis," Dyes and Pigments, 18 (1992) pp. 309-317.

[15] Burkinshaw, S.M. Hinks, D. and Lewis, D.M., "Capillary Zone

Copyright © 1997 Shunderson Communication All Rights of Reproduction Reserved 257

International Journal of Forensic Document Examiners

13

u. @ 2

0.5 mM Brij-35

2

2 11 40

2 27

27

40 2.5 mM rij-35 27

27 2

11

40

27

5.0 mM Brij-35

11

27 19

40 13

16~

t~ CQoo 400 nm soo

27 1 0 mM Brij-35

19

27

Time (min)- 215 nm

0 5 10 15 20 25 30

Figure 11: The effect of Brij-35 concentration on the migration time and the separation of the standard dye mixture. The triple-charged acids 11# and 19# have a short migration time with higher Brij-35 concentration. Experimental conditions: Capillary Length, 47 em; dye concentration (llg/10 Ill), S.O. 3 40#) = 0.26, S. B. 47 (2#) = 2.0, A.O. 10 (13#), R.Y. 37 (17#) = 3.3, A.R. 18 (11#) = 2.0, A.Y. 23 = 2.0; Micellar solution: standard with variable Brij-35 concentration. For other details see text.

258 Copyright© 1997 Shunderson Communications All Rights of Reproduction Reserved

Vol. 3, No.3, July/Sept 1997

7 :J <! 20 E

:I

6 oct E

10

5 3 4 5

Time (min)- 204 nm

4

3

a: 0.5 mM Brij-35

2

1

Time (min)- 204 nm 0

0 5 10 15 20 25 30

7 6 A.Y.23 (ref. 19#) :J 20

<! ~ 4 E :I E c::(

6 E 2 10

400 nm 600

5 / 0 3 4 5 Tlme (min)- 204 nm

4

3 b: 10 mM Brij-35

2

F S.B.38 (ref. 25#)

Time (min) -- 204 nm

5 10 15 20 25 30

Figure 12: The effect ofBrij-35 concentration on the separation of a green Bic G M 10 ballpoint pen ink. Experimental conditions: Capillary length, 47 em; Sampling: 5 dots of a four month old ink line on A1LANTA paper (see Figure 7); Micellar solution: standard with variable Brij-35 concentration. For other details see text.

Copyright © 1997 Shunderson Communication All Rights of Reproduction Reserved 259

International J oumal of Forensic Document Examiners

Electrophoresis in the Analysis of dyes and Other Compounds Employed in the Dye-Manufacturing and Dye-Using Industries," Journal of Chromatography, 640 (1993) pp. 413-417.

[16] Revillar, A.L. and Havel, J., "Capillary Electrophoresis in the Analysis ofBis-Azo and Tetra-Azo dyes and Other Compounds Related with the Dye-Manufacturing and Dye-Using Industries," HPCE 97, January 26-30, Ninth International Symposium on High Peiformance Capillary Electrophoresis and Related Microscale Techniques, Anaheim, CA, USA 1997, p. 208.

[17] Robertson, J., Wells, R.J., Pailthorpe, M.T., Aumatell, A. and Clark, R., "An Assessment of the Use of Capillary Electrophoresis for the Analysis of Acid Dyes in Wool Fibres," Advances in Forensic Sciences, Vol. 4, Forensic Criminalistics 2, Edited by B. Jacob and W. Bonte in co-operation with W. Huckenbeck and P. Pieper, Proceedings of the 13lh meeting of the International Association of Forensic Sciences, DUsseldorf, August 22"d to 28th, 1993: Multiprint, Berlin, Germany, 1995, pp. 247-249.

[18] Burkinshaw, S. M., Hinks, D. and Lewis, D.M., "Capillary Zone Electrophoresis in the Analysis of Dyes and Other Compounds Employed in the Dye-Manufacturing and Dye­Using Industries," Journal of Chromatography, 640 (1993) pp. 413-417.

[19] Suzuki, S., Shirao, M., Aizawa, M., Nakazawa, H., Sasa, K. and Sasagawa, H., "Determination of Synthetic Food Dyes by Capillary Electrophoresis," Journal of Chromatography A, 680 (1994) pp. 541-547.

[20] Tagliaro, F. and Smith, F.P., ''Forensic Capillary Electrophoresis," Trends in Anal. Chern., Vol. 15, No. 10, 1996, pp. 513-525.

[21] Fanali, S. and Schudel, S., "Some Separation of Black and Red Water-Soluble Fiber-Tip Pen Inks by Capillary Zone Electrophoresis and Thin-Layer Chromatography," Journal of Forensic Science, Vol. 36, No.4, 1991, pp. 1192-1197.

[22] Gregory, P. Chemistry and Technology of Printing and Imaging System, Blackie Academic & Professional, an imprint of Chapman & Hall, Glasgow, UK, 1996, p. 130.

[23] Cole, R.O. and Sepaniak, M.J., "Bile Salt Surfactants In Micellar Electrokinetic Capillary Chromatography, Application to · Hydrophobic Molecular Separation," Journal of Chromatography, 557 (1991), pp. 113-123.

[24] Weinberger, R. and Lurie, .I.S., "Micellar Electrokinetic Capillary Chromatography of lllicit Drug Substances," Anal. Chern., 63 (1991), p. 823.

[25] Zollinger, H., Colour Chemistry, Second, revised edition, Published jointly by VCH Verlagsgesellschaft mbH, Winheim, Germany and VCH Publishers, Inc., New York, NY, USA, 1991,p.11.

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