|James M. Egan
Federal Bureau of Investigation
Counterterrorism and Forensic Science Research Unit
Kristin A. Hagan
Oak Ridge Institute for Science and Education
Federal Bureau of Investigation
Counterterrorism and Forensic Science Research Unit
Jason D. Brewer
Federal Bureau of Investigation
Capillary electrophoresis with ultraviolet-visible photodiode
array detection (190600 nm) was studied as an alternative separation
and identification tool for forensic ink examination. Two different
buffer systems were designed to analyze dye compounds in various
black ballpoint pen ink formulations. Results were compared to thin-layer
chromatography experiments to evaluate the sensitivity and performance
of capillary electrophesis. A database of ballpoint pen ink analyses
and common-dye reference standards has been constructed for future
forensic use. Capillary electrophoresis allows ease of sample preparation
with the ability to separate and identify dye compounds based on
a calculated electrophoretic mobility and a characteristic ultraviolet-visible
spectrum. Protocols for capillary electrophoresis sample preparation
were designed to closely mimic procedures already in place for common
ink-evidence analysis. Because of the small volume necessary for
analysis, the remaining solution could be further processed using
current law enforcement procedures for confirmation.
Determining ink sources used on a variety of documents is a key
priority for forensic document examiners. The ability to distinguish
different inks can be quite useful for several reasons. Document
alteration (e.g., at a date later than indicated) by writing with
a pen of similar color but different dye composition is one specific
example when ink differentiation is crucial in criminal cases. Ink
comparison also can determine the relationship between two samples
in a forgery case that involves an original and a copy or two documents
believed to have the same author. Sample authentication also can
be tested based on ink analysis of raw colorant materials available
during a specific historical period. To unambiguously identify specific
inks, data interpretation is simplified if a chemical-separation
step precedes detection of various components in ink formulations.
Inks are complex mixtures of colorants, vehicles, and additives,
which are adjusted in composition to produce the desired writing
characteristics (Industrial Dyes: Chemistry, Property, Applications
2003). Colorants are compounds that give ink the desired color
and can include any or all of the following chemical classifications:
pigments and/or acidic, basic, azoic, direct, disperse, reactive,
and solvent dyes. Colorants are often the focus of ink analysis
because of their light-absorption and emission properties that can
be detected by various analytical methods. Vehicles or carriers
are usually solvents that allow the ink to flow and carry the colorants
to the material surface. Solvents are the typical ingredients analyzed
in date-of-origin investigations because of their gradual evaporation
from a document (Aginsky 1994; Brunelle 1992; Brunelle and Cantu
1987; Brunelle et al. 1987; Cantu 1996; Cantu 1991; Cantu 1988).
Last, additives can serve as flow (viscosity) modifiers, surface
activators, corrosion controllers, solubility enhancers, and preservatives
(Brunelle and Reed 1984; Leach and Pierce 1993). Detection of these
additive compounds can greatly aid forensic examiners because the
compounds can be manufacturer-specific. Their identity is often
a highly guarded secret in ink formulations, as are the colorants
Typical forensic techniques on questioned documents involve ink
extractions followed by thin-layer chromatography analysis (Standard
Guide for Test Methods for Forensic Writing Ink Comparison 1996;
Tebbett 1991). Knowledge gained from this procedure is limited
to colored spots that are correlated to a calculated retardation
factor (Rf). Densitometry also can be used to probe the
concentration of dye on a specific area of the thin-layer chromatography
slide in reflectance mode (Aginsky 1994). Carefully prepared standards
are required for quantitative analysis of an unknown sample. Problems
with thin-layer chromatography reproducibility can be related to
the difficulty in spotting uniform samples and maintaining constant
environmental influences, which lead to changes in Rf
values. Limited sensitivity, sample destruction, and extra processing
required for additional information are also shortcomings of the
method. Evidence handling requires that samples processed by thin-layer
chromatography be placed under environmentally controlled storage
conditions for future reference to prevent against color fading.
Other analytical techniques, such as infrared, mass spectrometry,
gas chromatography, and high-performance liquid chromatography,
have been used to study different ink components for identification
purposes. All of the techniques differ in the sample processing
(destructive versus nondestructive) and information obtained from
ink analysis. Some of these procedures are nondestructive (Harris
1991; Trzcinska 1993; Varlaskin and Low 1986), whereas others are
more destructive than thin-layer chromatography extractions because
they require that more samples be physically removed from the document
(Merrill and Bartick 1992; Trzcinska 1990). Often, only qualitative
information is obtained from these techniques, relying on pattern-recognition
methods to differentiate inks. Infrared spectroscopy has received
attention as an analytical tool to elicit spectral information from
written ballpoint pen ink. A limited sample set of available inks
allows infrared patterns to be distinguished, but as more pens are
added to a database, statistical analysis methods will become less
useful. Laser-induced infrared luminescence has been investigated
as another method to quickly distinguish two inks of the same color
(Horton and Nelson 1991). However, the method can determine only
if components luminesce, and the analysis was again limited to a
small ink population. Microspectrophotometry provides information
about the ink's acting as a chemical mixture, but still does not
provide the necessary detail that is important in component analysis
for ink differentiation (Zeichner et al. 1988). Laser desorption/ionization
mass spectrometry is extremely useful in detecting particular dyes
in inks, but prior knowledge of the ink components is useful to
aid interpretation of the complex mass spectrum that is acquired
with ink mixtures (Grim and Allison 2003; Grim et al. 2002). One
other useful mass spectrometric technique involves positive- and
negative-ion mode electrospray-ionization to create ions through
direct sample infusion to aid in compound identification (Ng et
al. 2002). High-performance liquid chromatography methods also have
been employed to separate different dye compounds with some success,
although the technique has afforded little understanding of different
inks other than the absence or presence of unassigned chromatographic
peaks (Lyter 1982). More thorough analysis is required to provide
unambiguous ink identification based on standard reference comparisons.
Capillary electrophoresis was chosen over high-performance liquid
chromatography for the front-end separation technique because small
sample volumes and on-capillary sample preconcentration are possible.
Capillary electrophoresis is an excellent candidate to separate
charged- and neutral-dye compounds for detection with ultraviolet-visible
absorption spectroscopy. Two characteristic parameters can be obtained
simultaneously: mobility factor and ultraviolet-visible spectrum
(a photodiode array detector must be used). There have been numerous
studies on inks performed on specific compound classifications or
ink formulations (e.g., ballpoint, nonballpoint, fountain pens)
(Burkinshaw et al. 1993; Croft and Lewis 1992; Fanali and Schudel
1991; Jandera et al. 1996; Rhode et al. 1998; Rhode et al. 1997;
Vogt et al. 1999; Vogt et al. 1997; Zlotnick and Smith 1998). Most
of the capillary electrophoresis investigations focus on a qualitative
approach to distinguish among different brands of pens. Qualitative
electropherogram analysis in a specific ink formulation does not
allow identification of compounds resulting from an observed peak.
Problems with this approach are the limited sampling taken from
the general ink population and the high probability that many formulations
are too similar to be differentiated unless specific information
is known prior to analysis. A reason only qualitative information
was investigated was the collection of single-wavelength data. A
more technical approach to ink separation was performed with ultraviolet-visible
spectral collection on reference standards, which resulted in a
separation of many different dye compounds in a single experiment
(Xu et al. 1997). However, implementation of this particular system
into a casework scenario would be extremely difficult because of
the demanding requirements for preparation of sample and buffer
To devise a capillary electrophoresis protocol that is compatible with casework, this investigation focused on the following procedural requirements:
- Minimal sample preparation comparable to thin-layer chromatography extractions.
- A faster method that would collect more useful data with respect to thin-layer chromatography.
- Ease of implementation without using excessive experimental measures.
- The ability to adapt the technique when extra knowledge is gained.
To this end, one buffer was designed to adequately separate cationic dyes
(general class of basic dyes), and another buffer was designed to
separate anionic dyes (general class of acidic dyes). These buffers
were successful in investigating a number of black ballpoint pen
inks and identified specific dye compounds by comparison of electrophoretic
mobility (μep) and absorbance spectra to standard
references. Libraries are now being compiled that consist of dye
reference standards and extracted ink samples with electropherograms
and absorbance spectra. These libraries will be used to compare
and characterize unknown samples in the future.
Various dye compounds and black ballpoint pen inks were investigated
to survey possible samples encountered by forensic ink examiners.
Barnstead (Dubuque, Iowa) NANOpure Infinity ultrapure water (ρ
≥ 18 MΩ-cm) was used for the preparation of all solutions.
All dyes were initially prepared in methanol at 1.0 mg/mL concentrations.
Dye injection solutions were prepared based on the capillary electrophoresis
method performed and will be further explained in the capillary
electrophoresis experimental section below. Table 1 contains all
dye compounds investigated in this study, and Figure 1 provides
some known dye molecular structures. This is not an exhaustive list
of dyes used in ballpoint pen ink formulations, but it is a starting
point for constructing a database of dye standards. Most dyes were
chosen for their presence in multiple ink formulations and will
be validated by the ink analysis performed on ballpoint pen samples.
The first 11 dyes listed in Table 1 correspond to single-dye compounds,
and the remaining compounds may correspond to single-dye molecules
or a combination of dye molecules. These specific industrial samples
were acquired from the U.S. Secret Service and were studied to understand
the complexity of different dye manufacturers' products.
1: Dye Reference Compounds Investigated for
Capillary Electrophoresis, Matrix-Assisted Laser Desorption/Ionization-Mass
Spectrometry, and Thin-Layer Chromatography Analysis
1: Structure of Single-Dye Reference Compounds Purchased from
Ballpoint pen inks are not referenced to either a make or manufacturer because of the sensitive nature of ink formulations. A simple nomenclature is used instead to differentiate various investigated inks. Samples were obtained from a stockpile of pens located at the FBI Laboratory or acquired from the U.S. Secret Service ink library. Ink samples were prepared differently for each analytical method to study the components. Pen lines were drawn on Whatman (Ann Arbor, Michigan) filter paper for extraction with methanol after various drying times. Drawn pen lines were typically used for mock casework samples that are presented in the text. Inks were also directly removed from the ink cartridge with a capillary for laser desorption/ionization-mass spectrometry and thin-layer chromatography purposes to ensure components were not missed because of low dye concentrations. Inks were also soaked onto Whatman filter paper and placed in plastic bags for storage purposes to allow further studies. Ink-soaked filter papers provided the most concentrated sample extracts and were used to confirm that peaks were not missed or assigned improperly when the extraction process was performed.
Two different capillary electrophoresis instruments were used to perform ink and dye separations. A Beckman Coulter (Fullerton, California) P/ACE MDQ Series capillary electrophoresis system equipped with a photodiode array detector measured the basic dye components present in black ink formulations and dye standards. A Hewlett Packard/Agilent 3D (Palo Alto, California) capillary electrophesis system, also equipped with a photodiode array detector, was chosen to perform all of the acidic dye separations. Reasons for choosing two different systems to complete this study included comparing methods on different instruments and working on parallel solutions simultaneously. All buffer and separation methods could be done on either instrument under the employed conditions.
Two unique buffer systems were prepared to separate the distinct classes of basic and acidic dye compounds necessary for dye analysis of the inks. The main reason for the two buffers involves the variable interaction of two different dye classes with the silica capillary wall. Wall effects substantially dominate the separation efficiency and will be discussed further in the results section.
Cationic Dye Method
All cationic dye component analyses were performed at 30°C
on a Beckman P/ACE MDQ capillary electrophoresis system. Glacial
acetic acid (TraceMetal grade, 99.5 percent) and sodium acetate
(high-performance liquid chromatography grade, 99.2 percent) were
purchased from Fisher Scientific (Fairlawn, New Jersey). Hexadecyltrimethylammonium
bromide (CTAB) (~ 99 percent) and methanol (A.C.S. high-performance
liquid chromatography grade, 99.3 percent) were obtained from Sigma-Aldrich
(St. Louis, Missouri). Direct-mode ultraviolet-visible absorbance
spectra were obtained over the wavelength range of 200 to 600 nm
at a data rate of 32 Hz. Separate data channels were employed to
collect electrical current information and a representative electropherogram
at 214 nm. Fused-silica-coated capillaries of 75 μm inner diameter
(i.d.) were purchased from Phenomenex (Torrance, California). The
separation length to the detector (Ld) was 30 cm, and
the total length (Lt) of the capillary was 40 cm. Prior
to each injection, the capillary was rinsed at 29.0 psi with 1 percent
bleach (1.50 minutes), water (1.00 minute), 0.1 M HCl (2.00 minutes),
water (1.00 minute), 0.1 M NaOH (1.50 minutes), water (1.00 minute),
and run buffer (1.50 minutes) to regenerate the desired wall properties.
Hydrodynamic injection was performed at 0.3 psi for five seconds
unless denoted otherwise. A potential of 25 kV was applied in reverse-polarity
mode, which produced an electric field of approximately 625
The separation buffer consisted of 70/30 (v/v) acetate buffer
(25 mM sodium acetate, 25 mM glacial acetic acid, 10 mM CTAB)/methanol,
which was vacuum-filtrated (0.45 μm cellulose acetate filter
system), and then titrated to a pH of 4.45 with glacial acetic acid.
Individual dye standard injection solutions were prepared by dissolving
an appropriate amount of dye in an acetate solution consisting of
2.5 mM sodium acetate and 2.5 mM glacial acetic acid (pH = 4.5).
Methanol was then added to the dye in acetate solution to prepare
70/30 (v/v) or 50/50 (v/v) acetate/methanol injection mixtures.
A six-dye-mixture standard solution (Table 2) was prepared by mixing
aliquots of the individual dye standards in acetate with methanol
to form a 50/50 (v/v) injection solution. Inks were extracted from
filter paper by removing 1.0 mm diameter punches (one to five punches
as necessary) and then placing the punches in 30 μL of methanol.
An ultrasonic cleaner was used to agitate the extraction solution
for two minutes to assist ink removal. An aliquot of 30 μL
of 2.5 mM acetate solution was then added to the extraction solution
to form a 50/50 (v/v) injection solution.
2: Capillary Electrophoresis Results of Individual
and Mixtures of Basic, Cationic Dyes Performed on a Beckman P/ACE
MDQ Capillary Electrophoresis System with the CTAB/Acetate Buffer
Anionic Dye Method
Anionic, acidic dyes were separated using a buffer composed of
25 mM CHES [2-(N-cyclohexylamino)ethanesulfonic acid] (Sigma-Aldrich)
and 5 mM β-cyclodextrin (Sigma-Aldrich) and adjusted to a pH
of 8.80 with 1.0 M NaOH. The effective length (Ld) of
the 75 μm i.d. fused-silica capillary was 50 cm, and the total
length was 58 cm (Lt). Methanol solutions of extracted
and standard samples were injected in the capillary for ten seconds
at a pressure of 25 mbar. Voltage was applied in the normal mode
(with the outlet electrode negative with respect to the inlet electrode)
at 25 kV and resulted in currents of approximately 19 μA (430
V/cm). Capillary temperature was maintained at 30°C with the
Hewlett Packard Chemstation software package. All separations took
place in less than ten minutes. Prior to injection, a series of
rinse solutions was used to regenerate and clean the capillary for
subsequent runs. These rinsing steps included a one-minute flush
with 1.0 percent bleach, two-minute flush cycles with 0.1 M NaOH
and water, and then a two-minute capillary fill with the separation
buffer. Ultraviolet-visible spectra were collected from 190 to 600
nm, and data channels were used to monitor real-time absorbance
at 214 nm and the electrical current trace. Recording the current
during the experiment is a good diagnostic tool to evaluate system
performance. The dye standards were analyzed individually at 100
μg/mL concentrations in methanol. Two different black pen ink
analyses were performed for dye detection and identification purposes.
A single 1 mm punch was removed from ink-soaked Whatman filter paper
and extracted with 30 μL of methanol. The second experimental
test involved a 1 cm drawn line and was completely extracted with
30 μL of methanol to simulate casework examples.
To demonstrate that this technique was amenable to a casework
scenario when destruction of evidence has to be minimal, a casework
protocol was proposed, and samples were analyzed with all of the
procedures mentioned above. To begin, five 1.0 mm hole punches were
removed from a piece of printer paper with written ink lines that
were dried for one month. An additional five 1.0 mm hole punches,
which were blank and spatially located close to ink lines, were
removed simultaneously from the same piece of paper. Extraction
was performed with 10 μL of methanol and ultrasonicated for
Thin-layer chromatography was performed on all black ballpoint
pen inks and dye reference standards to provide information about
the content and color of different components in extracted samples.
All procedures followed for thin-layer chromatography can be found
in the Standard Guide for Test Methods for Forensic Writing
Ink Comparison (ASTM 1996). Minor modifications were made to
some of the procedures to allow for compatible capillary electrophoresis
sample preparation. Methanol, instead of pyridine, was chosen to
perform the ink extraction from paper substrates because of high
ultraviolet-absorbance for pyridine. The extracted ink was spotted
on high-performance thin-layer chromatography slides (Whatman, 10
x 10 cm and 200 μm separation layer) with disposable micropipettes
(VWR Scientific, West Chester, Pennsylvania). Solvent system I was
chosen for all thin-layer chromatography experiments and consisted
of a 70:35:30 mixture of ethyl acetate, ethanol, and water. Separation
time was limited to a solvent front migration distance of 5.0 cm
that required a 20-minute run. Color was noted for each dye compound,
and the respective Rf was calculated.
Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
All laser desorption/ionization-mass spectrometry data was acquired
with an Applied Biosystems Voyager-DE Biospectrometry Workstation
(Foster City, California) benchtop matrix-assisted laser desorption/ionization
time-of-flight mass spectrometer. Both positive- and negative-ion
modes were used to determine the presence of acidic, basic, or neutral
dye compounds. Fifty laser shots were acquired for each spectrum
with a pulsed nitrogen laser (337 nm) intensity varying at attenuator
levels of 20002300 at a repetition rate of 3 Hz. Accelerating
voltage for the positive mode was 20 kV and the negative mode was
15 kV. The extraction delay was set at 100 nsec and the mass/charge
(m/z) range was 50 - 2000. An external calibrant of a saturated
solution of CsI was used for mass assignment using the peaks for
Cs (m/z 132.91), Cs2I (m/z 392.71),
and Cs3I2 (m/z 652.53). Different
sample types were investigated to determine the best method of analysis.
Ink lines on paper were prepared and mounted as 2 x 2 cm strips
with tape to a 64-well sample plate. Pen ink straight from the cartridge
and the extracted ink solution (methanol and ink) were placed in
different sample wells on a matrix-assisted laser desorption/ionization
sample plate, allowed to evaporate, and then probed. No additional
matrix was added to the samples for laser desorption/ionization.
The results from the laser desorption/ionization-mass spectrometry
analyses were used as confirmation for dye assignments made based
on capillary electrophoresis results.
Results and Discussion
Cationic, basic dyes that are used as colorants in ballpoint pen
inks were chosen as analytes for the cationic dye capillary electrophoresis
method development. Initially, an acetate buffer without hexadecyltrimethylammonium
bromide (CTAB) was used as the background electrolyte for separation
in normal polarity (+25 kV). Significant tailing was observed due
to the Coulombic interaction of the positively charged dyes with
the deprotonated silanol groups of the capillary wall (Weinberger
2000). Poor resolution for sample mixtures was also observed for
the simple acetate buffer. Therefore, additives were explored in
an effort to reduce the analyte-wall interaction. CTAB was employed
as a buffer additive to form a bilayer with the capillary wall,
which reversed the effective surface wall charge. CTAB prevents
the cationic species from adhering to the wall and has been shown
to be effective for storing cationic dyes in glassware (Giles and
McKay 1965). Changing the wall charge reversed the electroosmotic
flow and forced experiments to be run in reverse-polarity mode (25
kV). Tailing was eliminated upon addition of CTAB, and better resolution
also was achieved. Complete baseline resolution of the six-dye cationic
mixture was not observed until the buffer was adjusted to 30 percent
methanol to modify specific compound mobilities (Weinberger 2000).
Each dye standard was run, and the migration times (tm)
and ultraviolet-visible spectra were recorded. Table 2 lists the
μep of the individually run dye standards. Electrophoretic
mobilities were calculated by reference to the mobility of a neutral
marker peak and the apparent mobility of the analyte peak.
Capture of ultraviolet-visible spectra with the photodiode array
detector provides full spectral absorbance for identification purposes.
The reference dye ultraviolet-visible spectra were stored in a database
along with tm's to compare with spectra obtained for
ink extractions. Figure 2 illustrates some of the ultraviolet-visible
spectra obtained for standard dyes and represents a small sample
of the current FBI database. Spectral analysis provides complementary
data to peak μep's for dye identification in various
ink formulations. Both the P/ACE MDQ Series capillary electrophoresis
and Agilent Hewlett Packard systems possess software that stores
spectral references and uses search algorithms to quickly identify
possible spectral matches. Many of the commercial dye compounds
contain complex mixtures of simple dye compounds to produce the
desired color and composition. Some of the commercial dyes were
identified as containing dyes that were characterized from chemical
standards, some that can be partially assigned, and still others
that, because of the lack of a reference, cannot be assigned to
a specific compound. Several commercial dyes that were studied will
not be included because of proprietary formulations. However, knowledge
of these specialty formulations is being acquired and can help interpret
results obtained from ink extractions. Rhodamine Base B solution
was used to estimate the concentration limit of detection and mass
limit of detection of the optimized capillary electrophoresis method.
A signal-to-noise ratio of 3:1 was chosen as the detection limit
and corresponded to a Rhodamine Base B concentration limit of detection
of approximately 3.5 μg/mL. Based on the 30.0 nL injection
plug volume, the mass limit of detection was calculated to be ~
2: Ultraviolet-Visible Spectra of Individual
Reference Dye Compounds Collected with a Photodiode Array Detector
During Capillary Electrophoresis Analysis
To test the resolving power of the capillary electrophoresis method,
a six-basic-dye standard mixture was analyzed. Figure 3 displays
an electropherogram of the six-dye mixture. Table 2 lists the concentrations,
tm's, and μep's of the mixture. The μep
values are all negative because the migration of the dye compounds
is slower than neutral analytes and signifies that the dyes are
traveling against the electroosmotic flow. Comparison of the μep's
of the individually run dyes and the peaks from the mixture allowed
the assignment of each peak to its corresponding dye. The separation
was completed in less than five minutes, and each of the dyes was
baseline-resolved, including the Victoria Blue dyes (see Figure
1k for the structural similarity). In addition, the ultraviolet-visible
spectrum for each peak was searched against the spectral reference
library (contains all listed dyes in Table 1) and confirmed the
migration order of the first three peaks with confidences of 99.32
percent, 99.4 percent, and 99.27 percent, respectively. The last
three peaks could not be differentiated because nearly identical
absorption spectra are observed because of the similarity of the
Victoria Blue structures.
3: Six-dye standard mixture of cationic, basic
dye compounds separated on the Beckman P/ACE MDQ system and labeled
with peak migration times (minutes). (See Table 2.) The electropherogram
was recorded at λ = 214 nm.
Although the acidic buffer with the CTAB wall modifier works well
for preventing the cationic species from sticking to the capillary
wall, the anionic dyes are attracted toward the positive bilayer,
resulting in extensive peak tailing for any anionic species that
are analyzed with this method. Therefore, an alternative buffer
had to be chosen to effectively separate acidic dye compounds in
ink formulations. Success of organic buffers with the cationic organic
molecules led to testing other organic salts with basic pKa's
to separate acidic dyes. Figure 4 depicts an electropherogram of
a mixture of a single cationic dye (Crystal Violet) with six anionic
dyes. Crystal Violet was added to the mixture because of the likelihood
that the violet dye would be present in black ballpoint pen inks.
Analyte migration toward the detector is typical for normal polarity
with the cations migrating extremely fast (less than 2.4 minutes),
neutrals migrating next, and the anions opposing the electroosmotic
flow. The rapid electroosmotic flow caused by high pH conditions
will overcome the anionic migration, resulting in anions reaching
the detector based on the molecule's shape/charge. Slower-moving
anions will reach the detector sooner and have a smaller |μep|,
and the faster-moving anions will be detected later. Table 3 lists
the tm and μep values for individual
3: Results of the Seven-Dye Mixture in CHES/β-Cyclodextrin
4: Electropherogram at λ = 214 nm of a seven-dye mixture
separated using the CHES/β-cyclodextrin buffer. Migration times
indicate the main peaks obtained for all seven compounds. (See Table
Other peaks not specifically labeled as dye peaks are present
in the electropherogram and are associated with various impurities
or decomposition products in the dye samples (Table 1). These extra
peaks are important because ink manufacturers do not purify purchased
dyes; thus these additional peaks provide a level of uniqueness
to formulations. Slight tailing is observed for Crystal Violet (2.20
minutes) because of capillary wall interactions, but the rapid mobility
limits the effect. Metanil Yellow (2.91 minutes) and Sulforhodamine
B (2.96 minutes) are not baseline-resolved; however, this is not
considered a problem because Sulforhodamine B is not commonly used
as a dye in black pen ink formulations, but Sulforhodamine B has
been observed for ink-jet printer inks analyzed with the same experimental
setup. Alternative buffer recipes incorporating different micellar
properties were able to resolve these two peaks; however, at this
time the CHES/β-cyclodextrin buffer provides the best separation
power. Fronting for some of the peaks was observed, particularly
for Acid Blue 92 (4.62 minutes) and Tartrazine (5.52 minutes). Two
factors are responsible for this phenomenon. First, both dyes are
multiply charged (Figures 1a and 1h), which leads to electrodispersion
(Weinberger 2000), whereas the second factor is the presence of
unresolvable components, especially with Acid Blue 92 because it
is reported to be only 40 percent pure (Table 1). Again, different
buffer conditions (e.g., buffer concentration and pH) and additives
(e.g., different micelle-forming detergents) allowed resolution
of the Acid Blue 92 peaks. Negative effects caused by buffer alteration
were resolution loss for the other dye peaks, detracting from any
gain in chromatographic resolution of the Acid Blue 92 impurities.
Table 3 presents the individual dye concentrations in the standard
mixture. Dye concentrations listed have been adjusted to the purity
of dye material reported on the chemical container. Most dye concentration
limits of detection were determined to be 2 μg/mL with the
photodiode array under stated experimental conditions. Acid Blue
92 and Tartrazine have higher concentration limits of detection
because of the broader peak shapes. Lower concentrations could be
observed if a longer injection time was used, but extracted ink
analysis did not warrant these procedures. Approximate mass limits
of detection can be calculated from the total injection volume used
for capillary electrophoresis analysis. For a ten-second injection
at 25 mbar, approximately 55.5 nL of sample would be introduced
to the capillary. For a 2 μg/mL concentration limit of detection,
a 110 pg mass detection limit is estimated.
Reliability and Validation for the Anionic Dye Method
Mobility value reproducibility is important for forensic analysis
because an intense ultraviolet-visible spectrum will not always
be obtained for ink extractions in sample-limited scenarios. In
limited sample cases, a reproducible μep value will
lend credibility to the assignment of a particular dye. Reproducibility
was examined by day-to-day experiments with the prepared anionic
buffer and with three standard mixture (Table 3 analytes) injections
without changing the inlet and outlet buffer solutions. Day-to-day
reproducibility reflects the capillary performance, as well as the
stability of the buffer solution over time. Three repeated anionic
dye mixture runs per day over a one-week time span were recorded,
and the calculated μep's were determined to be within
±2 percent for all dye compounds. Running three experiments
on a single set of anionic buffer solutions would be advantageous
for casework to ensure that a sample, a blank, and a reference mixture
are run under identical conditions. Three consecutive runs on the
same buffer with the reference mixture resulted in all μep
values falling within ±1.5 percent.
Ballpoint Pen Inks
Forensic analysis of ballpoint pen inks was the driving force
in developing capillary electrophoresis buffers that adequately
separate dye compounds for identification. Ink extractions were
subjected to capillary electrophoresis buffer methods, as well as
thin-layer chromatography and laser desorption/ionization-mass spectrometry
analyses to characterize the dyes present in the pen inks studied.
Thin-layer chromatography and laser desorption/ionization-mass spectrometry
were used to complement the capillary electrophoresis analysis and
to search for dye components that might not be detected with capillary
electrophoresis. Figures 5 and 6 present two electropherograms of
ink #1 extraction (Table 4). The U.S. Secret Service sample was
prepared by saturating ink on Whatman filter paper, drying, and
then storing the sample in a plastic bag. Because of the large ink
concentration, a single 1.0 mm punch was removed and extracted with
200 μL of methanol for five minutes. Figure 5 shows the presence
of two dye peaks that are close in μep values and
have spectral similarities greater than 90 percent for Crystal Violet
and Rhodamine Base B. The first dye peak (tm = 3.40 min,
μep = 1.08 x 104 cm2V1sec1)
agrees well with the μep and ultraviolet-visible
spectrum of Rhodamine Base B, whereas the second peak (tm
= 3.99 min, μep = 1.41 x 104
cm2V1sec1) agrees with
the μep and spectrum of Crystal Violet. This ink
formulation was reported to possess Crystal Violet, Rhodamine Base
B, and a yellow dye (personal communication with G. LaPorte, U.S.
Secret Service, June 2004). The yellow dye was not detected using
the cationic capillary electrophoresis method because it is an acidic
dye that interacted strongly with the CTAB bilayer. Figure 6 shows
the presence of one cationic (Crystal Violet, μep
= +0.84 x 104 cm2V1sec1)
and one anionic (Metanil Yellow, μep = 1.45
x 104 cm2V1sec1)
dye. Also in Figure 6 is the disturbance of the neutral markers
with absorbance in the visible region indicating the presence of
the third dye compound, Rhodamine Base B, which was already detected
with the cationic dye capillary electrophoresis method. Thin-layer
chromatography results of ink #1 with the three reference dyes (commercial
products of Crystal Violet, Metanil Yellow, and Rhodamine Base B)
confirmed that these are the only dyes that are present in this
4: Results of complete analyses performed on
commercial black ballpoint pen ink extractions. Two or three thin-layer
chromatography spots were observed for Crystal Violet (Methyl Violet)
because of the presence of demethylation products.
5: Ink #1 extraction was run on the P/ACE MDQ
system at 214 nm with CTAB/acetate capillary electrophoresis conditions
to identify possible cationic dye components.
6: Ink #1 extraction was also run on the Agilent
3D capillary electrophoresis system at 214 nm under CHES/β-cyclodextrin
capillary electrophoresis conditions to identify possible anionic
Laser desorption/ionization-mass spectrometry was performed to
provide complementary data for dye components in ink samples. Individual
compounds were studied to determine molecules that would yield ions
that could be correlated to a specific compound. Only a few dyes
resulted in distinct singly charged ions in either positive- or
negative-ion mode. Crystal Violet and Methyl Violet gave characteristic
mass spectral patterns that have been studied previously for ink-dating
purposes (Grim et al. 2002). A peak at m/z 372 due to the
singly charged molecule minus the chlorine anion was present for
inks containing Crystal Violet (data not shown). There were also
peaks for demethylation products of the dye molecule. There is some
variation in the detected m/z for compounds and is a result
of small sample changes with respect to the calibration standard.
All of the other dyes that were separated in the cationic mixture
displayed characteristic peaks indicative of the single-charged
cationic species minus the chlorine anion: Rhodamine 6G = m/z
444, Victoria Blue B = m/z 470, Victoria Pure Blue BO =
m/z 478, and Victoria Blue R = m/z 423. One peak
appeared in each ionization mode for Rhodamine Base B at m/z
443 because it is a neutral molecule without any counterions. Metanil
Yellow was the only anionic dye that had an easily interpretable
laser desorption/ionization-mass spectrometry that could be assigned
to the single-charged molecule at m/z 351. Metanil Yellow
is also the only anionic dye that has a single charge associated
with the parent molecule and should be the only anionic dye expected
to have detectable singly charged molecular species (Karas et al.
2000). Further analysis of the other dyes did not uncover other
spectral peaks that could be quickly identified with a particular
species, and therefore, laser desorption/ionization-mass spectrometry
interpretation was limited to the analysis of the above-mentioned
dyes. Because of the complexity of the mass spectra resulting from
numerous ink components, only specific spectral peaks were evaluated.
The main goal in performing mass spectrometry of standard dyes and
inks was to determine the potential advantages of linking mass spectrometry
as a secondary detector after capillary electrophoresis separation.
Observation of compound-specific masses has proven that compounds
could be identified based on mass spectrometry and ultraviolet-visible
spectra. Mass spectral information would increase the sample information
obtained, although buffer modifications would have to be made for
mass detector interfacing and would result in less efficient separations.
Table 4 presents the complete analyses performed on all of the
black ballpoint pen inks. Table 4 contains only those results obtained
from the anionic capillary electrophoresis method because all of
the black pens studied possess anionic dyes, with some neutral dyes
and a single cationic dye (Crystal Violet/Methyl Violet). Ink samples
were also investigated with the cationic capillary electrophoresis
method but only yielded the presence of Crystal Violet/Methyl Violet.
More interesting results will be obtained for blue ballpoint inks
with the cationic method because Victoria Blue dyes are used to
achieve blue ink colors. Capillary electrophoresis results were
obtained by extracting a single 1.0 mm hole punch from soaked Whatman
filter paper. The hole punch was extracted with 30 μL of methanol
and directly injected for ten seconds at 25 mbar. Comparison between
the hole-punch technique and one in which a 1.0 cm drawn line was
extracted with the same volume of methanol led to comparable peak
signal intensities. A 1.0 cm line was chosen to represent a typical
casework scenario because normally five 1.0 mm punches are taken
from a questioned document. To obtain the most concentrated sample,
hole punches can be taken from pen lines that overlap because these
spots will contain the largest amount of ink and result in minimal
document destruction. Therefore, a 1.0 cm line is roughly equivalent
to the above casework scenario. Table 4 contains the thin-layer
chromatography results, along with dye assignments based on capillary
electrophoresis results, and corroboration by laser desorption/ionization-mass
spectrometry data when possible. For dyes that were assigned by
capillary electrophoesis, a μep value and an ultraviolet-visible
confidence value were calculated. The confidence percentage, computed
by the Agilent ChemStation software, determined how similar an ultraviolet-visible
spectrum is with respect to a best match from a database of reference
chemicals. In some extracted samples, dye identification of detected
peaks could not be made at this time. The lack of a reference standard
to compare with the capillary electrophoresis or thin-layer chromatography
results is responsible for no chemical identification. As more dye
references are obtained and analyzed, more complete details about
ink formulations will be gained.
One advantage of performing capillary electrophoresis separation
on dyes is the ability to effectively narrow the compound candidates
responsible for a particular peak. One example of this intuitive
approach was used to assign the chemical identity of an unknown
yellow dye compound that was present in ink #7. The peak at 3.94
minutes in Figure 7 was not one of the original reference dyes analyzed.
Based on the ultraviolet-visible spectrum obtained for that peak
(shown in the inset of Figure 7) and the presence of a yellow spot
on thin-layer chromatography plates, the search for a possible dye
was narrowed to a yellow dye with the ultraviolet-visible spectrum
depicted. The ultraviolet-visible spectrum obtained for Metanil
Yellow (Figure 2c) is similar; however, the μep
value for the yellow dye present in ink #7 was very different. The
μep value was very useful in predicting the possible
molecular weight and charge of the compound by understanding that
anionic dyes at shorter tm's have large m/z
ratios, whereas dyes appearing at longer times have smaller m/z
ratios. Based on the predictive nature of the technique, a m/z
between 300 and 360 amu was estimated. A search of the Sigma-Aldrich
Handbook of Stains, Dyes and Indicators (Green 1990) gave only
one candidate that had this m/z and the expected ultraviolet-visible
spectrum: Acid Yellow 42. Acid Yellow 42 was purchased and analyzed
with the capillary electrophoresis method and gave identical μep
and ultraviolet-visible spectral results as the dye found in ink
#7. Spotting this dye solution on the same thin-layer chromatography
plate as the ink verified that this dye had the same visual color
and Rf value as the unknown. Based on thin-layer chromatography
results, there is one additional purple dye that is present in this
ink, and the capillary electrophoresis peak at 4.32 minutes has
an ultraviolet-visible spectrum similar to other purple dyes. Identification
of this dye has not yet been determined, but analysis of more standards
will be crucial in this process.
7: Three dyes are observed in the CHES/β-cyclodextrin
capillary electrophoresis separation method of an extracted sample
of ink #7 at 214 nm. Inset is an ultraviolet-visible spectrum observed
at 3.9 minutes.
Further data can be collected on black ballpoint inks with the possibility of extracting further information from the capillary electrophoresis results. As can be observed in Table 4, there are other peaks that correspond to dyes that have not yet been identified in the electropherograms. Further analyses of the electropherograms also indicate peaks that only exhibit absorbance in the ultraviolet region and could be representative of nondye components found in the specific ink, such as solvents or other additives. Only a limited sampling of inks has been investigated, but so far no ink has shown identical characteristics in the acquired electropherogram, and all could be differentiated based solely on the peak patterns observed. Although chemical identification of every peak detected is not possible, particular electropherogram patterns could be used to differentiate among inks until specific information is determined. Peak area analysis could also be used to calculate specific ratios among different detected ink components to increase confidence in ink assessment. The overall potential of this technique can be advanced when more standard dye samples, as well as other components, can be analyzed in tandem.
The overall goal of this technique is facile introduction into
everyday casework protocols. To accomplish this, the sample preparation
had to mimic currently performed extraction procedures (see Experimental
Samples Section). The ink extraction was injected into the capillary
electrophoresis instrument and separated in fresh buffer, and the
result is presented in Figure 8. The presence of two dyes for ink
#6 was observed, and chemical identity was determined by ultraviolet-visible
spectral database searching and μep value comparison
with reference dye compounds. Crystal Violet was the ultraviolet-visible
spectral match (93.4 percent) for the peak with a μep
of +0.80 x 104 cm2V1sec1.
Metanil Yellow was assigned to the second peak,
μep = 0.45 x 104
with an 83.9 percent ultraviolet-visible spectral match. The low
ultraviolet-visible spectral similarity of the Metanil Yellow peak
is due to the reduced absorbance detected because of the lower concentration
of the mock casework sample. The remaining solution was spotted
on a thin-layer chromatography plate and matrix-assisted laser desorption/ionization
sample plate in an effort to confirm the capillary electrophoresis
results. Thin-layer chromatography analysis of the remaining ink
injection solution resulted in faint spots that were hard to discern.
The second run performed on the same capillary electrophoresis buffer
was a blank paper extraction to indicate possible sample carryover
and to observe peaks attributable to the paper material. No interference
peaks were observed for the blank material. The last injection was
a standard mixture containing the two suspected dyes contained in
the unknown ink formulation and mimicked the sample electropherogram.
8: Capillary Electrophoresis Results at 214
nm of a Casework Protocol Used to Extract Five 1 mm Hole Punches
of Ink #6
The above demonstration indicated that capillary electrophoresis analysis could be performed in addition to techniques already used in evidence analysis. No additional material is necessary for increased sensitivity. In fact, if thin-layer chromatography spots can be observed for the extracted solution, capillary electrophoresis will be successful. The lone disadvantage of capillary electrophoresis is the necessity that the analyte be soluble in the separation buffer. Many dyes are at least partially soluble, but some dye classes are fairly insoluble. One investigated ink in this report resulted in particle formation in the injection solution. Although this occurred, the dyes that remained in solution could still be investigated.
The third injection could be modified based on casework requirements. The one above was chosen to demonstrate that reproducible results could be obtained from successive injections, and simple data overlay could produce confirmatory results. However, the absence of alternative dye possibilities in the standard mixture could lead to arguments made for biasing of the comparison standard. In this case, a full standard library could be developed to analyze for comparison, but a much larger database needs to be developed. Another possibility for a confirmation run could be the suspected pen obtained from a crime scene. Matching peaks from the evidence and the suspected source would provide confirmation that the same dyes are present, although this would not necessarily prove that the particular pen was used for the evidence being processed. Exclusion of a pen could also provide strong evidence in the case of inconsistent dye ingredients.
Ballpoint pen ink analysis is not the only area that could be
investigated by capillary electrophoresis (Liu et al. 1995; Masar
et al. 1996). Yellow Food Dye #5 is a purified form of Tartrazine,
one of the yellow dyes successfully separated in the CHES/β-cyclodextrin
capillary electrophoresis buffer for anionic, acidic dyes. This
food coloring is found in Mountain Dew soda (PepsiCo, Chicago, Illinois)
and was examined with the capillary electrophoresis method. A sample
of Mountain Dew was boiled down to a small volume for increased
concentration of the food-coloring additive. The presence of a Tartrazine
peak was observed in the Mountain Dew sample (data not shown). Other
peaks also are detected by the photodiode array, but examination
of the ultraviolet-visible spectra indicates that only the peak
responsible for Tartrazine has significant absorption in the visible
region. Printer inks and dyes used in the textile and currency industries
are also possible analytes for the methods developed in this report.
Capillary electrophoresis has been shown to be a powerful analytical tool for forensic analysis of dyes contained in ballpoint pen inks. Two unique pieces of information (ultraviolet-visible spectrum and μep value) can be gained from the analysis. These chemical characteristics can be used in ink identification or differentiation on a document. Protocols have been developed for casework scenarios while preserving extracted solutions to be further analyzed with other complementary techniques. The anionic capillary electrophoresis buffer was found to effectively separate black ballpoint ink dye components with higher sensitivity, faster analysis time, and more definitive chemical identification than thin-layer chromatography procedures. Capillary electrophoresis also can detect solvents and other ink additives that are not dyes in the same experiment. Electronic data storage also provides the benefit of database and library search algorithms. Although ballpoint pen ink analysis was the primary focus of this report, these formulated buffers could be used in a multitude of other forensic analyses, such as food and textile dyes.
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.
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