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This is to certify that the
thesis entitled
Laser Desorption/Ionization Mass Spectrometric

Analysis of Methyl Violet: A New Approach to Relative Ink !
Age Determination

presented by

Donna M. Grim

has been accepted towards fulfillment
of the requirements for

 

M.S. degree In Criminal Justice

 

/Majoré/ rofessor

Date )/!/0/02

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

 

LIBRARY
Michigan State
University

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

1 I
l
l I i

 

MAR 2 4 2004

 

NOV 2 6 2006

 

APR 1 8 2007

 

 

 

 

 

 

 

 

 

 

 

6/01 c:/CIFiC/DateDue.p65—p. 15

LASER DESORPTION/IONIZATION MASS SPECTROMETRIC
ANALYSIS OF METHYL VIOLET: A NEw APPROACH To RELATIVE INK
AGE DETERMINATION
By

Donna M. Grim

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirement
for the degree of
MASTER OF SCIENCE

School of Criminal Justice

2002

ABSTRACT
LASER DESORPTION/IONIZATION MASS SPECTROMETRIC ANALYSIS OF
METHYL VIOLET: A NEW APPROACH TO RELATIVE INK AGE
DETERMINATION
By

Donna M. Grim

Laser desorption mass Spectrometry (LDMS) was evaluated as an analytical tool
for the characterization of dyes found in inks. Experimentation revealed that colorants
can be easily detected neat and on paper. The resulting mass spectra provide molecular
level information, allowing for the identification of dyes and their degradation products.
Industrial and forensic science applications were explored, however the focus of this
work is on the forensic aspect Of developing a new approach to determining the relative
age of an ink on a questioned document. This method involves using LDMS to monitor
the degradation of methyl Violet, as an ink ages.

Dye degradation is dependent on such factors as ink formulation, paper, and
storage conditions. In order to determine the relative age Of a document in question,
document examiners often resort to accelerated aging methods to create an “old” ink
from a “new” ink. UV irradiation was found to be an effective means to accelerate the
degradation of methyl Violet. The same degradation products were formed as in natural
aging. Natural and accelerated aging curves can be prepared, so that a correlation

between natural aging and UV accelerated aging can be derived.

To my parents, Bruce Grim and Sue Salvadge, for their perseverance in teaching
me the importance Of determination and a strong work ethic. Their continuing love and
support has given me the courage not to shy away from a challenge, and to always strive

to be best that I can.

iii

ACKNOWLEDGEMENTS

I would like to begin by thanking Dr. John Allison, for not only being a wonderful
adviser, but for also being a friend. Pursuing two degrees at one time has not been easy,
and when life sometimes creates an even harder struggle, Dr. Allison has proven to be
someone with whom I could talk to. He has always been very understanding, and has
always somehow managed to push me to work harder, without saying a word.

I would also like to thank Dr. Jay Siegel, for his support and insight into the
research project. Dr. Siegel was always there to share in my excitement when things
went well, and also to cheer me up when the project sometimes disappointed me.

Of course, I can not forget where I came from. I would like to thank Terry
Sherlock and Dr. George Sarkisian of Graphic Controls Inc., for their suggestions
pertaining to the project, as well for their ink sample contributions, which helped to give
the project a more focused direction.

I would also like to acknowledge Erich Speckin and Roger Bolhouse for their
insightful discussions pertaining to my project. I would like to thank them for providing
the controlled ink library samples, which helped to bring an end to my never ending
project.

On a personal note, I would like to thank my fiancee, Mike Mohr, and my
lab/roommate, Jamie Dunn, for their love and encouragement. I know I can get irritable
when I am severely stressed out, and unfortunately I think that described me all of last

year. Thank you, for being there for me.

TABLE OF CONTENTS

List of Tables .................................................................................... vii
List of Figures ................................................................................... viii

Chapter One: Ink

Introduction .............................................................................. 1
A Brief History of Writing Inks ....................................................... 2
Methyl Violet or Crystal Violet? ...................................................... 4
References ................................................................................ 9

Chapter Two: Instrumentation

Introduction to Desorption/Ionization Mass Spectrometric

Techniques ............................................................................... 1 1
Fast Atom Bombardment Mass Spectrometry ....................................... 13
Laser Desorption/Ionization Mass Spectrometry .................................... 18
References ................................................................................. 22

Chapter Three: Industrial Applications

An Introduction to Ink Properties and Testing ...................................... 24
Mass Spectrometric Analysis of Industrial Ink Jet

Printer Inks ............................................................................... 25
FAB MS Analysis of Ink Directly .................................................... 26
LDMS Analysis ofInk Directly ....................................................... 32
References ................................................................................ 34

Chapter Four: Forensic Science Applications

Introduction to Questioned Document Examination ................................ 35
Notable Contributions to the Field .................................................... 38
Accelerated Aging - The Themial Approach ........................................ 50
Summary ................................................................................. 51
References ................................................................................ 53

Chapter Five: LDMS Analysis of Methyl Violet on Paper

Introduction — The Detection of Ink on Paper ....................................... 55
[JV-Accelerated Aging .................................................................. 59
Developing a Relationship Between Natural Aging and
UV-Accelerated Aging .................................................................. 64
A Note on UV-Accelerated Aging .................................................... 69
Testing the Influence of Variables in Natural Ink Aging .......................... 7O
Insight into the Uncontrolled Natural Aging Curve ................................ 75
References ............................................................................... 80
Chapter Six: Ink Aging Inside of a Pen ................................................... 81
References ............................................................................... 88
Chapter Seven: Conclusions & Future Work ........................................... 89
References ............................................................................... 91

vi

LIST OF TABLES

Chapter One: Ink

Table 1.1: Methyl Violet Degradation Products .......................................... 7
Chapter Two: Instrumentation

Table 2.1: Desorption/Ionization Methods Used in Mass Spectrometry .............. 12
Chapter Three: Industrial Applications

Table 3.1: The Chemical Composition of Two Ink Jet Printer Inks:
Ink A and Ink B .................................................................. 26

vii

LIST OF FIGURES
Chapter One: Ink
Figure 1.1: The Positive Ion Field Desorption Mass Spectrum of Methyl
Violet: a) The Structure of Crystal Violet; b) The Structure of
Methyl Violet ...................................................................... 6

Figure 1.2: An Oxidative Demethylation Mechanism of
N,N-Dimethylaniline ............................................................. 8

Chapter Two: Instrumentation

Figure 2.1: Fast Atom Bombardment Double Focusing Magnetic
Sector Mass Spectrometer ....................................................... 16

Figure 2.2: Charge-Exchange FAB Gun ................................................... 17

Figure 2.3: Matrix-Assisted Laser Desorption Time-of-Flight Mass
Spectrometer (Voyager-DE Mass Spectrometer) ........................... 21

Chapter Three: Industrial Applications

Figure 3.1: The positive ion FAB mass spectrum of Ink A: a) low
mass region; b) high mass region ............................................. 28

Figure 3.2: Structure of Solvent Black 29 .................................................. 30

Figure 3.3: Negative ion FAB mass spectrum of Ink B: a) low mass region,
evidence of triethylene glycol (TEG); b) high mass region ............... 31

Figure 3.4: a) Positive ion LD mass spectrum of Ink A; b) Negative
ion LD mass spectrum of Ink B .............................................. 33

Chapter Four: Forensic Science Applications

Figure 4.1: The C1“ Ion Migration Test .................................................... 39
Figure 4.2: Aging of ballpoint pen ink (sequential solvent extraction)

with Anja M-311 ............................................................. 47
Figure 4.3: Aging of ballpoint pen ink (single solvent extraction)

with Anja M-3l l .............................................................. 47
Figure 4.4: Aging of ballpoint pen ink with Forrnulab 587 ............................ 48

viii

Chapter Five: LDMS Analysis of Methyl Violet on Paper

Figure 5.1: a) Partial positive ion LD mass spectrum of Ink A on paper;
b) Positive ion LD mass spectrum of paper; c) Partial negative
ion LD mass spectrum of Ink B on paper ................................. 58

Figure 5.2: Preliminary UV-Accelerated Aging Study: Bic Ballpoint
Pen Ink on Paper ............................................................... 61

Figure 5.3: A portion of the positive ion LD mass spectrum of Bic black
ballpoint pen ink-on-paper: a) ink from a new document; b) a
38-month old controlled, naturally aged document; 0) ink from
a document irradiated for 6.25 hours with UV light ....................... 63

Figure 5.4: UV accelerated aging study data for new Bic black ballpoint
pen ink on printer paper: a plot of the relative intensity of the
m/z 372, 358, and 344 (x2) peaks versus UV irradiation time .......... 66

Figure 5.5: UV accelerated aging study data for new Bic black ballpoint
pen ink on printer paper: a plot of the average molecular
weight of the dye, methyl violet, versus minutes of UV
irradiation ...................................................................... 68

Figure 5.6: Controlled, natural aging study data for Bic black ballpoint
pen ink on printer paper: a plot of the average molecular
weight of the dye, methyl violet, versus the age of the
document ....................................................................... 68

Figure 5.7: Uncontrolled, natural aging study data for various inks on
different types of paper: a plot of the average molecular weight
of the dye, methyl violet, versus the age of the document ............... 71

Figure 5.8: Controlled, natural aging study data for Bic black ballpoint
pen ink on bond paper: a plot of the average molecular weight
of the dye, methyl violet, versus the age of the document ............... 73

Figure 5.9: UV accelerated aging study data for new Bic blue ballpoint

pen ink on printer paper: a plot of the average molecular weight
of the dye, methyl violet, versus minutes of UV irradiation ............ 75

ix

Figure 5.10: UV accelerated aging study data for ink on a (1958) old
document: a plot of the average molecular weight of the
dye, methyl violet versus minutes of UV irradiation ..................... 77

Figure 5.1 1: UV accelerated aging study data for thermally aged 20 year
old ink on printer paper: a plot of the average molecular weight
of the dye, methyl violet, versus minutes of UV irradiation ............ 77

Chapter Six: Ink Aging Inside of a Pen

Figure 6.1: The positive ion LD mass spectrum of ink on paper from
Pen #1 ........................................................................... 83

Figure 6.2: The positive ion LD mass spectra of ink along a written
line from Pen #2 ............................................................... 85

Figure 6.3: The positive ion LD mass spectra of ink within the cartridge
of Pen #2: a) ink removed from open end of cartridge; b) ink
removed from middle of cartridge; c) ink removed from pen
tip ................................................................................ 87

Chapter One: Ink
Introduction .

Questioned document examination encompasses not only handwriting analysis
and typewriter comparisons, but also the study of ink, paper, ink-on-paper, and writing or
printing instruments. As a result, when a document is called into question, numerous
parameters may be subjected to scientific analysis. This work focuses on the chemical
analysis of ink and ink-on-paper. Ink analysis covers a wide field, including inks found
in writing instruments, printers, typewriters, stamp pads, and photocopiers. The category
of writing instruments includes fountain, ballpoint, felt-tip, and rolling ball pens, to name
a few. Currently in an age dominated by technology, a new emphasis on printer inks is
emerging in regards to ink analysis. Laser toners and ink jet inks comprise the main
printer analyses. Ballpoint pen and industrial ink jet printer inks will be the primary
focus of this work, using laser desorption/ionization mass spectrometric techniques to
identify ink components and monitor how they change as an ink ages.

In general, inks consist of a color component, usually a mixture of dyes or
pigments, dissolved or suspended in either a water or glycol based solvent system, termed
the “vehicle”( 1 ). The name is appropriate given that the job of the vehicle is to transport
the solid colorant to a substrate, commonly paper. Once on paper, solvents begin to
evaporate (2). The vehicle also consists of other components, such as natural or synthetic
resins, to increase the viscosity of the ink and to help the ink adhere to the paper (3).
Typical synthetic resins include vinyl chloride or polyvinyl acetate (4). Additional
additives, such as humectants and surfactants, are included in ink formulations to enhance

the ink’s visual and drying properties. Humectants act as wetting agents, by increasing

dye solubility and slowing the evaporation of the ink (5). Likewise, a surfactant's
amphiphilic nature gives them the ability to decrease the surface tension of water-based
inks (6), preventing the ink from drying out at the tip of a pen (I) and decreasing
wettability problems with the substrate on which the ink is dispensed (6). Surfactants
may also serve as defoaming agents (6). Biocides and corrosion inhibitors may be
included as needed (1). Thus, ink formulations may differ significantly based on their
usage. The development of improved ink analysis techniques would benefit industrial

ink chemists and forensic questioned document examiners.

A Brief History of Writing Inks

Having been used for over 1000 years, iron gallotannate inks are perhaps the
oldest known writing inks (4). The production of this ink involved the addition of iron
salts to a fermented infusion of gall nuts (2,4,7). Gum Arabic was usually included in the
suspension. The high cost of production, accompanied by oxidation problems with the
ink, led to a new formulation in the 19th century. The oxidation of ferric tannate was
prevented by the addition of either hydrochloric or sulfuric acid, which consequently
made the ink more corrosive to the writing instrument, as well as the paper on which the
ink was applied. Dyes were also included in the formulation, since the ink was initially
clear when applied to paper. Early dyes included indigo and aniline dyes, such as soluble
blue (4).

Iron gallotannate inks are also associated with fountain pens. As time
progressed, so-called "fountain pen inks" emerged as a modified iron gallotannate ink.

Earlier versions were termed “blue-black permanent” ink and were characterized by a

lower iron content, and a much higher dye concentration. Later versions were dubbed
“non-staining, washable inks”, due to the absence of iron, and the presence of fugitive
dyes, which were easily removed by oxidants (4).

Currently, ballpoint pens are probably the most widely used writing instruments.
In fact, at least 80% of all questioned document examinations requiring ink analysis,
were written with ballpoint pen ink (8). Ballpoint pen inks are cited as appearing on the
European market in 1945, however they existed nearly 50 years before this date (4). The
first ballpoint pen ink was patented in 1895 (US patent 600,299/1895), followed by
another in 1898 (US patent 533,492/1898), which cited the use of lamp black suspended
in castor oil (4). It is noted that there were roughly 30,000 ballpoint pens commercially
available between 1935 and 1945. Due to the minimal quantity and poor quality of these
pens, sources do not cite the emergence of ballpoint pens until 1945. At this time, the ink
formulations were strictly oil-based. Common oils included linseed, castor, and mineral
oils. Ink chemists preferred the use of linseed oil (4). These inks were characteristically
neutral, and were therefore more compatible with acidic dyes, such as nitroso- or nitro-
azo dyes. Around 1950, a historically significant change in ink formulations occurred, in
that ink chemists switched from an oil-based medium to a glycol-based solvent system (4,
8-10). These inks were slightly acidic (pH 5-6), and were more compatible with basic
dyes, including the aniline-type dyes. The introduction of the blue and green chromium
and copper phthalocyanine dyes occurred around this time, in 1954 (4,9-11). Our
experiments in the lab have also demonstrated that methyl violet was also introduced into
ballpoint pen ink formulations around this same time period. Since the introduction of

ballpoint pens, numerous types of pens have emerged, including felt tip pens (1963) (10),

roller ball pens (1970) (1 l), and erasable pens (1978) (1 1) which have characteristically
different ink formulations. For instance, felt tip and rolling ball pen inks, consist of a

water-based solvent system, greatly decreasing the ink's viscosity (1 1).

Methyl Violet or Crystal Violet?

Methyl violet was chosen as the primary focus for determining which desorption /
ionization mass spectrometric technique to pursue in these studies, as well as for
developing an alternative relative ink age determination method. Methyl violet is a
cationic dye and is used commonly as a toner in both writing and printer inks (12). In our
laboratory, the dye’s presence has been detected using mass spectrometry in both blue
and black pens and ink samples dating back to the early 1950’s. Although methyl violet
is mentioned frequently in the literature as a common dye used in inks (8,13-15), no
production entry date is cited, as in the case of other common dyes, such as the blue and
green phthalocyanines. However, we believe that the switch around 1950 from a strictly
oil-based vehicle to a glycol-based medium influenced the types of colorants and
additives, which would be miscible in the new ink formulations. As a result, methyl
violet was now compatible in a glycol-based vehicle, and proved to be extremely cost
effective and versatile.

Figure 1.1 is a positive ion field desorption (FD) mass spectrum of methyl violet
(14). The structure of crystal violet is shown in Figure 1.1a. Crystal violet is a
methylated cationic dye consisting of three phenyl groups, with six methyl (~CH3 )
groups attached to three nitrogen atoms. This structure will be designated as C+Me6,

representing a carbo-cation center (C), with six methyl groups (Me6), and 0 H atoms

attached to the N atoms. The crystal violet cation has a m/z ratio of 372. When crystal
violet is reduced, meaning that one -CH3 group is replaced with a H atom, the new
pentamethylated form of the dye (C+Me5H1) is methyl violet, having a m/z ratio of 358.
The mass difference between crystal violet and methyl violet is 14 amu, which is easily
accounted for by the loss of the -CH3 group (-15 amu) and the gain of one H atom (+1
amu). This becomes important when analyzing an ink, which is purportedly composed of
“methyl violet”, using mass spectrometry. In a positive ion mass spectrum of methyl
violet, such as the FD mass spectrum shown in Figure 1.1 (14), the base (most intense)
peak appears at m/z 372, with a small peak present at m/z 358. Dyes are usually
mixtures, and methyl violet has been described as a mixture of N-hexa-methyl, penta-
methyl, and tetra-methyl derivatives, with the hexa-methyl homolog predominating (14).
However, quality control sets minimal limits for how much of the pure form of the dye
must be present in order for the dye to still achieve its desired properties. For example,
basic fuchsin, the completely demethylated homolog of crystal violet, is described as “a
homologous mixture of dyes, and in any given lot any homolog may be dominant” (12).
In the case of basic fiichsin, 50% of the dye mixture must be basic fuchsin, with
rosaniline, magenta I, and magenta II comprising the remainder of the mixture. There
appears to be no such standard for methyl violet. When methyl violet is purchased, it is
essentially crystal violet. In the world of chemistry, this is very unusual in that, when

you buy a 5 g sample of one compound, you get 5 g of another.

372

c
H3 \N*—c
a) H3

 

 

 

H30
C+Me6
Cl‘b
H3C
339‘ i I l
288 3GB 4GB 588

Figure 1.1: The Positive Ion Field Desorption Mass Spectrum of Methyl Violet:
(a) The Structure of Crystal Violet; (b) The Structure of Methyl Violet

Another important feature of methyl violet, is the fact that it has five more —CH3
groups to lose. As an ink containing methyl violet ages, each methyl group is
successively lost and replaced by a H atom. This process is evident in a mass spectrum,
because peaks are observed beginning at m/z 372, and continue to appear 14 amu lower
than the previous peak, to m/z 288, which represents the completely demethylated form
of the dye (C+H6). The degradation process follows an oxidative demethylation
mechanism (Figure 1.2) (16). For simplicity sake, N,N — Dimethylaniline was used in
place of the crystal violet structure, as a model to demonstrate the process (17,18). It is
important to note that this is only an example of an oxidative demethylation process,

which may have similarities to that which the crystal violet structure could undergo. In

this case, the sample is initially irradiated with UV-light and an electron is lost (Step 1).
The resulting radical cation is attacked by either a water molecule or another base present
in the ink or paper, which abstracts a proton (Step 2). The radical generated is a very
good reducing agent. Consequently, the radical is oxidized forming a cationic immine
complex (Step 3), which is easily hydrolyzed (Step 4). A proton transfer from the
hydroxyl group to the nitrogen (Step 5) produces the final demethylated molecule and
formaldehyde (Step 6). We refer to the demethylated products as “degradation products”
and each are listed in Table 1.1. Again, this isjust an example of an oxidative
demethylation process, and not necessarily the one that crystal violet follows. For
instance, free crystal violet is already a cation, therefore eliminating the need for the
removal of an electron (Step 1). F urtherrnore, C+Me6 is not a radical (Step 2). Therefore,
the initiating steps may be slightly different, however the chemistry of the degradation
may proceed in a similar manner. The degradation mechanism can be catalyzed by the
presence of oxidizers in ink or paper, such as TiOz, a whitener used in paper production
(19-21). The degree of degradation of the dye, represented in a mass spectrum can be

used as a measure of how old the ink is.

 

 

 

 

 

 

 

m/z Structure
372 C+M96
358 C‘Mesl-h
344 C‘Me4H2
330 C‘Me3H3
316 C’Me2H4
302 C‘Me1H5
288 C’He

 

 

 

 

Table 1.1: Methyl Violet Degradation Products

H3C .- CH3 H3C ._‘\ +'/.CH3 H3C \\"/’ 6H2

hv N +1'120

I
.K ,
," .‘ r/"\ ,
, . . ‘ \
I ' ' ‘ '/ \ \\
. , . ’ \ x
‘,l . ., ,1 .7 .
, \"\ 1' “x“
I ‘ \
' I ' I
l u
i i i i e
._ j ‘
.7 ,‘ , ‘ . ,1

'6 -H,0+

(1) (2) \l‘ "

 

(Formaldehyde) 'H+

Figure 1.2: An Oxidative Demethylation Mechanism of N,N-Dimethlyaniline

References

(I) Chadwick BW, Wang A, inventors. US Patent 5,769,931. 1998 June 23.

(2) Cantu AA. A sketch of analytical methods for documenting dating. Part II. The
dynamic approach: Determining age independent analytical profiles. Int J
Forensic Doc Examiners 1996;2:192-208.

(3) Hall IL, inventor. US Patent 4,130,435. 1978 December 19.

(4) Witte AH. The examination and identification of inks. In: Lundquist F, editor.
Methods of forensic science, vol. 2. New York: Interscience, 1963;35-77.

(5) Kang HR. Water-based ink jet. II. Characterization. J. Imaging Sci 1991;35:189-94.
(6) Kunjappu JT. Molecular thinking in ink chemistry. Ink World l999;5:150-3.
(7) http://www.knaw.nl/ecpa/ink/html/ink.html

(8) Andrasko J. HPLC analysis of ballpoint pen inks stored at different light conditions.
J Forensic Sci 2001;46:21-30.

(9) Brunelle RL, Cantu AA. A critical evaluation of current ink dating techniques. J
Forensic Sci 1987;32:1522-1536.

(10) Cantu AA. A sketch of analytical methods for document examination. Part II: The
static approach. Int J Forensic Document Examiners 1995;1:40-51.

(1 1) Brunelle RL. Ink dating - the state ofthe art. J Forensic Sci 1992;37:113-124.

(12) Green FJ. The Sigma-Aldrich handbook of stains, dyes and indicators. Wisconsin:
Aldrich Chemical Company, I990.

(13) Pachuta SJ, Staral J S. Non-destructive analysis of colorants on paper by time-of-
flight secondary ion mass spectrometry. Anal Chem 1994;66:276-284.

(14) Sakayangi M, Komura J, Konda Y, Watanabe R, Harigaya Y. Analysis of
ballpoint pen inks by field desorption mass spectrometry. J Forensic Sci
1999;44:1204-1214.

(15) Aginsky VN. A microspectrophotometric method for dating ballpoint pen inks — a
feasibility study. J Forensic Sci 1995;40:475-478.

(16) Personal communication with Dr. James N. Jackson, Chemistry Department,
Michigan State University, East Lansing, MI.

(l7) Sako M, Shimada K, Hirota K, Maki Y. Photochemical oxygen atom transfer
reaction by heterocycle N-oxides involving a single-electron transfer process:
oxidative demethylation of N,N-Dimethylaniline. J American Chem Soc
1986;108:6039-6041. '

(18) Duxbury DF. The photochemistry and photophysics of triphenylmethane dyes in
solid and liquid media. Chem Rev 1993;93:381-433.

(19) Zu P, Zhao J, Shen T, Hidaka H. TiOz assisted photodegradation of dyes: a study of
two competitive primary processes in the degradation of RB in an aqueous TiOz
colloidal solution. J Molecular Catalysis A: Chemical 1998;129:257-268.

(20) Markheim MC. Photocatalytic properties of oxides. Report of the New England
association of chemistry teachers: 540-543.

(21) Wu T, Lui G, Zhao J, Hidaka H, Serpene N. Evidence for H202 generation during
the TiOz-assisted of photodegradation of dyes in aqueous dispersions under
visible light illumination. J Physical Chem B 1999;103:4862-4867.

Chapter Two: Instrumentation

An Introduction to Desorption/Ionization Mass Spectrometry Techniques

Mass spectrometry has previously been used in the analysis of inks (1,2). A mass
spectrometer is a very powerful detector following separation by gas chromatography
(GC). In the GC-MS experiment, volatile components of an ink sample are separated,
then ionized using electron impact ionization (EI) and subsequently analyzed by the mass
spectrometer. Desorption / ionization (D/I) methods that are used with mass
spectrometry have the advantage of generating ions for MS analysis from
nonvolatile/themally labile analytes. Currently there are a number of D/I methods
available. These are summarized in Table 2.1. For example, field desorption (FD) can
be used to generate ions from analyte molecules with molecular weights greater than
1,000 (3). In this method, analyte molecules are deposited onto a FD "emitter", which
employs a carbon surface. When the surface is heated in the presence of a high electric
field, ions can be generated from the analyte. FD has been used very recently for the
analysis of ballpoint pen inks (4). Secondary ion mass spectrometry (SIMS) and FAB are
two very similar techniques (5). In a SIMS experiment, the analyte is deposited onto a
metal surface, which is inserted into the ion source of the mass spectrometer. This target
is continuously bombarded with ions having several keV of kinetic energy. Secondary
ions representative of species on the target surface are generated and analyzed. In the
FAB experiment, the analyte is dissolved in a liquid matrix such as glycerol. This
viscous solution is deposited on a metal surface, and inserted into the ion source of the
mass spectrometer. This target is continuously bombarded using fast (keV) Xe atoms,

typically. Both are used with magnetic sector mass analyzers. We note that both FAB

and SIMS have been used to obtain spectra of nonvolatile organic dyes (6,7, 8), and there
has been one report of using SIMS to analyze colorants on paper (9). Matrix-assisted
laser desorption/ionization (MALDI) (10) and LD (11) are very similar experiments,
differing only in the presence of a crystalline matrix used to absorb the laser radiation in
MALDI. In both techniques, a pulsed laser ionizes a solid analyte. The ions generated
are separated using time-of-fiight mass spectrometry. In this work, we consider FAB and

LDMS as techniques for the examination of inks.

 

 

 

 

 

 

 

 

 

 

 

 

Method Analyte Mechanism Mass Analyzer Ionization
Form Method
Thermal
Field Adsorbed on a excitation & Magnetic
Desorption carbon surface electric-field Sector Continuous
(FD) induced
emission
Secondary Ion Fast atoms
Mass Adsorbed on a collide with the Magnetic Continuous
Spectrometry metal surface surface and Sector
(SIMS) deposit energy
Fast Atom Fast atoms
Bombardment Dissolved in a collide with the Magnetic Continuous
(FAB) liquid matrix target and Sector
deposit energy
Matrix
Assisted Laser Embedded in Absorb laser Time of F light-
Desorption/ matrix light from a MS Pulsed
Ionization crystals pulsed laser
(MALDI)
Laser Absorb laser
Desorption Adsorbed on a light from a Time of Flight- Pulsed
(LD) metal surface pulsed laser MS

 

Table 2.1: Desorption/Ionization Methods Used in Mass Spectrometry

12

Fast Atom Bombardment Mass Spectrometry

Instrumentation

A JEOL HX-l 10 (J EOL USA, Peabody, MA) double-focusing magnetic sector
mass spectrometer was used to analyze the positive and negative ions formed following
bombardment of samples with 10 KeV Xe atoms. A double focusing instrument consists
of an electric sector and a magnetic sector. The presence of both analyzers significantly
improves the resolution of the experiment. A schematic of the instrument is shown in
Figure 2.1 (12). The FAB experiment uses a direct insertion probe, which introduces a
planar, metal surface on which the sample is placed, into the ion source. The fast Xe
atoms are generated in a JEOL charge-exchange FAB gun (Figure 2.2) (12). Xe gas is
allowed to flow into the gas chamber, where Xe+ ions are formed. The Xe atoms are
ionized so that they can be accelerated by the wire mesh anode. The fast Xe+ ions are
focused through a series of lenses, which must be optimized by the user, into a gas
chamber, where they are neutralized by Xe gas molecules. Bombardment of the sample
with fast Xe atoms, results in desorption and ionization of the analyte. The desorbed
analyte ions leave the ionization source, and enter the electric sector. In this instrument,
the electric sector is placed before the magnetic sector, although the setup can be
reversed. In the electric sector, an electric field (E) is applied across two plates. The
electric field has two functions. The beam of ions will exit the ion source with a
significant energy spread. As the beam passes through the electric sector in a circular
path, stray ions are deflected away, leaving a more focused ion beam, with a smaller
energy spread. Secondly, the electric field disperses ions according to their kinetic

energy, based on the principle that ions of varying energies will travel paths of different

13

radii (13). The focused ion beam then enters the magnetic sector mass analyzer, where
the ions experience a magnetic field. The ions travel circular paths of different radii, and
are separated on the basis of their momentum. Momentum is the product of the mass of
the ion and its velocity. The “double focused” ions reach the detector, which is an
electron multiplier (13)

The electron multiplier is made of a copper-beryllium alloy, which has the ability
to sputter electrons when bombarded with ions. For example, if one fast ion collides with
the copper-beryllium surface, 10 electrons may be sputtered off. The 10 ejected electrons
are then accelerated to another copper-beryllium surface, which is held at a slightly larger
potential, where each one of the 10 electrons sputters off 10 more electrons. The process

continues, until the signal is amplified 105-106 times (14).

Experimental

FAB analyses are usually performed using 1 nmol of analyte dissolved in 1-2
microliters of glycerol, the viscous liquid matrix. Here we will show that inks can be
analyzed directly by FAB. In some cases, viscous solvents in the ink serve the role of
matrix for the FAB experiment. To directly analyze ink on a paper surface target, several
approaches were considered including saturating the paper in a glycerol matrix.
However, we found that FAB will generate ions directly from a dry paper target (“dry-
F AB”). This approach is used here.

The instrument was calibrated using Ultramark 4000 (PCR Incorporated,
Gainesville, FL), a standard calibration compound. Analyses of ink, neat and on paper,

were performed. In the first experiment, ink was applied directly to the probe tip, without

a matrix. The mass spectrometer was set for mid-field positive ion analysis. Fast atoms
were generated from the JEOL fast atom gun using a potential of 12 kV and an emission
current of 5 mA. These conditions produce informative spectra for the determination of
ink composition. In the second experiment, the ink-on-paper sample (prepared as
described in the laser desorption section) was applied to the probe tip using double stick
tape. In these dry-F AB experiments, short signal durations were encountered. When a
paper sample is introduced and bombarded under standard operating conditions, ions are
generated and detected by the mass spectrometer, but only for a period of 3-5 seconds.
Much more time is required to optimize instrument parameters and obtain a spectrum. If
the flux of fast atoms is decreased, the sample is desorbed more slowly from the target,
resulting in more long-lived signals. For this instrument, one way to accomplish this is to
defocus the FAB beam. This decreases the number of fast atoms striking the target per
unit area per second. While this decreases signal intensity, it allows for the continuous
generation of ions for periods of minutes instead of seconds, so that spectra can be

obtained. Both positive and negative ions can be generated and analyzed in FAB MS.

 

JEOL HX-11O

Beta slit

/ Quadrupole lens
/

Magnetic sector
[] oo

    

= =‘_— Alphaslit

Ffi’fi— Collision cell

I - I - ' O

i. .. I ___ I. \ Collector sht De r
Probe

Ion source

Figure 2.1: Fast Atom Bombardment Double Focusing Magnetic Sector Mass
Spectrometer

JEOL Charge Exchange FAB Gun

Xe“) + e' -—‘> Xc+- + 2e'

Ionization chamber \

 

X-Y deflector

Exit nozzle

Gas chamber

6ch Xealoms \ x‘ls) + Xe" “”0 .
Xe+ (slow) "‘ x°(fast)
- / FAB Sample

 

l—

Probe

Figure 2.2: Charge-Exchange FAB Gun

Laser Desorption/Ionization Mass Spectrometry
Instrumentation

Positive and negative ion spectra of ink samples were obtained using a PE
Biosystems Voyager DE instrument (Framingham, MA) (Figure 2.3). LD MS was
performed on an instrument designed for MALDI MS. In the current MALDI
technology, sample plates containing 100-400 "wells" are introduced into the instrument.
One microliter of a sample solution can be placed in a well. The solvent evaporates,
leaving a solid sample target in the well for analysis. The planar target is moved, using
mechanical X-Y positioning devices, so that a pulsed laser can be focused onto various
positions. For our purposes, this plate can be easily modified such that a piece of paper
containing ink is introduced, and the paper moved so that the laser irradiates one or more
locations where ink is present, generating ions for subsequent MS analysis.

Once the sample is introduced into the ion source, the instrument utilizes a pulsed
nitrogen laser (337 nm, 3 ns, 3 Hz) to desorb and ionize the sample. The ionized
molecules are accelerated and analyzed using a linear time-of-flight mass spectrometer.
The guide wire located in the time-of-flight tube attracts the ions to keep them on a
focused route to the detector. In the time-of-flight tube, ions of different masses
accelerated with the same kinetic energy, will travel at different velocities. As a result,
ions with different m/z values will reach the detector at different times. This allows for
separation ofthe ions according to velocity (13). An electron multiplier, which was
described in FAB section, is employed as the detector (14). The following user-selected
parameters were employed: for analysis of the positive (negative) ions formed by LD,
the sample plate was held at a voltage of 20,000 V (-15,000 V), an intermediate

acceleration grid was held at 94.5% (94.5%) ofthe plate voltage, and a delay time of 150

I8

ns was used between laser irradiation and ion acceleration. The manufacturer supplies a
sample plate that does not contain wells, but is machined such that a polyacrylamide gel
can be attached. When paper is taped onto this metal sample plate, and spectra are
generated at a rate of 3 Hz, full resolution of the instrument is realized using the

conditions cited here. Care must be taken to maintain a flat target.

Calibration

For TOF MS experiments, ion flight times are measured, and m/z values must be
computed. To calibrate LD spectra, a known spectrum containing peaks with known m/z
values is first obtained. To do so, a saturated solution of CSI (99.9%; Aldrich,
Milwaukee, WI) was pipetted onto paper and allowed to dry. Laser irradiation of this
target yields positive ions including Cs+, and ions with the formula Cs(n+1)1n+, such as
C321” These were used for calibrating the instrument, so that flight times for other ions
could be converted into m/z values in the mass spectra generated. Similarly, for neat ink
analysis, 2 uL of the same CsI solution were pipetted onto a gold plate containing wells,

allowed to dry, and used for generating spectra for calibration.

Analysis of Ink on Paper

Two industrial ink-jet inks not yet on the market, identified here as Ink A and Ink
B, were supplied by an ink manufacturer, along with their compositions. Ink A and Ink B
were applied to paper using an air brush (Badger Air-Brush Co, Franklin Park, II. model
250). This method allowed for minimal, uniform sample loading. In preliminary

experiments, the ink from a commercially available ballpoint pen was applied to paper by

19

 

drawing lines across the paper as evenly as possible, covering a l in2 surface area.
Minimizing the space between each line ensured that the laser was irradiating dye-
covered paper. With experience, the laser could be focused on regular written pen
strokes, such as signatures, and spectra obtained. In UV accelerated aging studies, the
sample was irradiated using a UV-lamp (254 nm, 760 microwatts/cmz; UVP Inc., San
Gabriel, CA, model UVGL-58) and analyzed at various intervals. For the various
experiments described, the instrument settings remained the same, with moderate changes
to the sample preparation. Any differences in the sample or in its preparation will be

noted when appropriate.

 

20

 

Linear

   

 

 

 

 

  
 
  

 

 

‘ detector
1
l - — ->.
| Ion path
I ------ >
1 Laser path
I
l
l .
Flight

Beam

guide I tube

wire I
l
|
l
l

L l
aser | Video
I I camera
Laser — :_ I Aperture (grounded)
attenuator ; l .
. | Ground grid
Prism V ~ . ‘

§
\

Variable-voltage 21. . t. .. .
grid &

Sample plate Main
source chamber

 

 
 

Sample
loading
chamber

 
   
 

Figure 2.3: Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometer
(Voyager-DE Mass Spectrometer)

21

References

(I) Brunelle RL, Pro MJ. A systematic approach to ink identification. J Official Anal
Chem 1972;55:823-826.

(2) Aginsky VN. Dating and characterizing writing, stamp pad and jet printer inks by gas
chromatography/mass spectrometry. Int J Forensic Doc Examiners 1996;2:103-
l 16.

(3) Giessmann U, Rollgen FW. Electrodynamic effects in field desorption mass
spectrometry. Int J Mass Spectrom Ion Phys 1981;38:267-279.

(4) Sakayanagi M, Komuro J, Konda Y, Watanabe K, Harigaya Y. Analysis of
ballpoint pen inks by field desorption mass spectrometry. J Forensic Sci
1999;44: 1204-1214.

(5) Lyon PA, editor. Desorption mass spectrometry — are SIMS and FAB the same?
Washington, DC: American Chemical Society, 1985.

(6) Monaghan JJ, Barber M, Bordoli RS, Sedgewick RD, Tyler AN. Fast atom
bombardment mass spectra of involatile sulphonated azo dyestuffs. Org Mass
Spectrom 1982;17:569-574.

(7) Scheifers SM, Verma S, Cooks RG. Characterization of organic dyes by secondary
ion mass spectrometry. Anal Chem 1983;55:2260-2265.

(8) Detter-Hoskin LD, Busch KL. SIMS: secondary ion mass spectrometry. In: Conners
TE, Banerjee, editors. Surface analysis of paper. New York: CRC Press,
1995;206-234.

(9) Pachuta SJ, Staral J S. Nondestructive analysis of colorants on paper by time-of-flight
secondary ion mass spectrometry. Anal Chem 1994;66:276-284.

(10) Karas M, Bachmann D, Hillenkamp F. Influence of the wavelength in high-
irradiance ultraviolet-laser desorption mass-spectrometry of organic molecules.
Anal Chem 1985;57:2935-2939.

(l I) Denoyer E, Van Grieken R, Adams F, Natusch DFS. Laser microprobe mass

spectrometry 1: Basic principles and performance characteristics. Anal Chem
1982;54:26-41.

(12) Szekely G. Mechanisms of ion formation and fragmentation by fast-atom
bombardment and matrix-assisted laser desorption ionization mass spectrometry
[dissertation]. East Lansing (MI): Michigan State University, 1997.

22

(l3) McCIoskey JA, ed. Methods in enzymology Volume 193: Mass spectrometry. New
York: Academic Press, I990.

(14) Watson JT. Introduction to mass spectrometry. Philadelphia: Lippincott-Raven,
1997.

23

Chapter Three: Industrial Applications

An Introduction to Ink Properties and Testing

Industrial ink chemists are continuously formulating new inks and refining old
inks, driven by the consumer demand for cheap, but attractive inks. While doing so, they
must also be careful not to sacrifice other important properties, such as drying time and
the shelf-life of the ink. While some changes in formulations may be slight, others may
have a serious impact on the types of constituents that may be used. Writing inks in
particular have experienced a dramatic change since the early twentieth century. Early
writing inks were composed of iron gallotanate dissolved in an oil based medium (1-3).
Mid-century, ink formulators switched to a glycol based vehicle, which completely
changed the types of dyes which could be included in the formulation (1,4-6).

Before an ink can be released on the market, ink chemists perform a rigorous
shelf-life test (7). In this test, the ink’s initial properties are measured and the ink is
swabbed across a piece of paper, for a color sample. The ink is then subjected to a
thermal accelerated aging study. The sample is placed in an oven (140° F) and monitored
weekly for 2 months. At each weekly interval, the ink’s surface tension, density, and
viscosity are remeasured, and the ink is swabbed on the same paper, to detect any fading.
Eight weeks at 140° F, is believed to be equivalent to 2 years of natural aging (8). A two-
year natural aging study is also performed. In this study, the ink is placed on a shelf at
room temperature and the same properties are monitored at monthly intervals. However,
if the ink undergoes the accelerated aging technique with little to no changes in

properties, the ink will be in production before the natural aging study is completed.

24

Li ghtfastness is another crucial property, that concerns ink chemists, as well as
dye and pigment manufacturers. Lightfastness is the ability of ink to withstand fading
when exposed to light. Some ink industries, such as the ink jet business, may not be as
concerned with this property as others. Ink jet ink is frequently used for “junk mail” or
printing on cardboard packages. In this case, ink chemists formulate the least expensive,
fastest drying inks, with little regard to whether or not the ink will fade in a couple of

years.

Mass Spectrometric Analysis of Industrial Ink Jet Printer Inks

When a solid precipitates out of ink after only two weeks into an accelerated
aging study, or if a significant degree of fading is noted following 3 days exposure to
UV-light, what has chemically occurred? Ink formulators are interested in knowing if an
ink’s components are reacting and, if so, what the products are. Desorption/ionization
mass spectrometric techniques have been shown to provide molecular information about
ink components. Specifically, mass spectrometry can be used to detect the initial
components, as well as the products of reactions that occur. Two industrial ink jet printer
inks, designated Ink A and Ink B, were supplied to us by an ink manufacturer (Graphic
Controls, Inc., Cherry Hill, NJ), which were not yet released on the market. The inks
would be used to print labels directly onto cardboard boxes. Consequently, the inks
needed to be extremely fast drying, since an employee would be at the end of the
conveyor belt to handle the boxes as the freshly printed boxes came off. The complete

ink formulations were provided to us and are listed in Table 3.1. Ink A and Ink B were

25

 

analyzed in positive and negative ion modes using fast atom bombardment (FAB) and

direct laser desorption mass spectrometry (LDMS).

 

 

 

 

 

 

 

Ink B Ink A
Solvent Black - 12% Methyl Violet - 5%
Triethylene Glycol - 56% Nigrosine - 7%
Pyrollidone - 20% Oleic Acid - 60%
Alcohol - 2% Diethylene Glycol - 10%
Surfactant - 10% l-Hexanol - 10%
Surfactant - 8%

 

 

 

 

Table 3.1: The Chemical Composition of Two Ink Jet Printer Inks:
Ink A and Ink B

FAB MS of Ink Directly

Positive and negative ion spectra were obtained of Ink A and Ink B using FAB.
In a typical FAB experiment, 1 1L of a viscous, non-volatile liquid, usually glycerol or
nitrobenzyl alcohol, is applied to the probe tip, in addition to 1 1L of the liquid analyte.
The viscous liquid serves as the matrix for the analyte. The matrix absorbs and transfers
the energy from the fast Xe atoms to the analyte, so that it may be desorbed and ionized.
Ink A and Ink B already contain highly viscous solvents, which in a sense, act as a
matrix. Therefore, in these experiments, an additional matrix was not necessary, and the
inks were analyzed directly.

Figure 3.1 shows two portions of the positive ion FAB mass spectrum of Ink A.

From this spectrum, a number of components were identified. The region of the

26

spectrum below m/z 300 (Figure 3.1a) suggests the presence of oleic acid, C17H33COOH.
In positive ion FAB, neutral analyte molecules are frequently desorbed in protonated
form. The neutral molecule M is generated in the gas phase as a protonated molecule,
[M+H]+. The peak at m/z 283 in the spectrum represents these ions. The protonated
molecule efficiently loses water to form a major peak at m/z 265. Lower mass peaks at
m/z 55, 69, 83, etc. are indicative of the hydrocarbon portion of oleic acid. Similar peaks
are observed in El spectra of alkanes and alkenes. A FAB spectrum of pure oleic acid
(not shown) was obtained, which matches this portion of the spectrum. Oleic acid is a
viscous organic acid, which is the primary solvent of Ink A’s vehicle.

Figure 3.1b represents the higher mass region of the positive ion FAB spectrum of
Ink A, which indicates that one of the dye components is Methyl Violet. The dye is
introduces as a salt (CA) The cation is desorbed directly from the sample and C+ is
observed in the spectrum (m/z 358). As discussed in chapter 1, dyes are frequently
mixtures. For this particular dye, the hexamethylated form is more abundant than the
pentamethylated form (9). Note, the manufacturer used methyl violet, but the mass
spectrum shows it was really crystal violet. This becomes evident when examining the
ink’s spectrum. The base peak (largest peak) in Figure 3.1b is seen at m/z 372, which
corresponds to the hexamethylated structure, whereas the peak at m/z 358 represents the
pentamethylated form of the molecule. The homologue with four methyl groups yields
the peak at m/z 344. The peak at m/z 368 remains unassigned. Key at this point is the
fact that the peaks in the region of the spectrum shown in Figure 3.1b are indicative of the

presence of this cationic dye, and that ink components can be detected using FAB.

27

Relative Intensity

Relative Intensity

 

 

 

 

 

 

 

 

 

3)
69
83
309
95 (M + H+ - H20)
265
(M "’ ”*l
283
o 160 1'50 2bo 260 abo
mlz
372 b)
368
358

344

 

 

 

 

 

 

340

350 360 370 380 390 400 Mb

mlz

Figure 3.1: The positive ion FAB mass spectrum of Ink A: a) low mass region;
b) high mass region

28

 

 

 

In contrast, Ink B contains a very different dye, Solvent Black 29. The dye
consists of a Cr” center with two identical ligands, each carrying a 2- charge, attached to
it. The structure (Figure 3.2) carries an overall negative charge, making it anionic. As
with the cationic dye, an anionic dye is introduced as a salt C+A'. When detecting
negative ions, the dye should be detected as A'. In contrast to ionic materials, neutral
analyte molecules are desorbed in deprotonated form in negative ion FAB, meaning that
the neutral molecule M is detected in the gas phase as [M-H]'. Figure 3.3a shows the low
mass region of the negative ion FAB spectrum of Ink B. The primary solvent, triethylene
glycol, is detected in deprotonated form at m/z 149. In Figure 3.3b, a higher m/z portion
of the same spectrum, the peak at m/z 666 corresponds to the major anionic dye
component (A‘), Solvent Black 29.

The FAB spectra, Figures 3.1 and 3.3, show that components of inks, especially
ionic dyes, are easily detected using FAB. Stable aromatic molecular species such as
dyes are desorbed/ionized in a single form — yielding a single, intense peak with no
fragmentation. In contrast, oleic acid, which is made up mostly of single bonds,

fragments extensively.

29

 

OzN ‘

‘/

Figure 3.2: Structure of Solvent Black 29

3O

 

M+

 

 

 

 

 

 

 

 

 

 

 

 

125

149 (rec — H+)- a)
>.
3:
in
c
o
H
E
o
.2
H

g 359
o
I!
385
150 zoo 250 300 350 400
mlz
666 (A-)

b)
>5
3:
(D
C
a:
H
E
o
.2
H
2
0
a:

500 550 600 650 700 750 800 850

mlz

Figure 3.3: Negative ion FAB mass spectrum of Ink B: a) low mass region; evidence
of triethylene glycol (TEG); b) high mass region

31

LDMS of Ink Directly

To evaluate the utility of various D/I methods for this field, the same ink samples
were analyzed using LDMS. As an example, Figure 3.4a shows the positive ion LDMS
spectrum of Ink A. Only peaks representing the dye are observed. If the spectra shown
in Figures 3.1 and 3.4 are compared, it is clear that more components can be identified
using FAB. This is reasonable based on the mechanism of desorption/ionization
mechanism for these methods. In FAB, the mixture is bombarded with fast atoms and all
species present absorb energy, and can be desorbed/ionized. If a species is ionic, it will
be desorbed directly. If it is neutral, it will be detected if chemistry can occur during the
FAB process that will protonate or deprotonated the molecule (resulting in positive or
negative ions). In contrast, LD is more of an optical technique. Here, the sample is
irradiated with 337 nm light from a nitrogen laser. Only those molecules that absorb light
at this wavelength will be affected. In the case of ink, most of the components are
transparent at this wavelength; only the dye absorbs and is transformed into gas phase
ions. Thus, for mixture analysis of this type, FAB would yield information on a larger
number of components. The situation becomes much different when the target for

analysis is ink on paper, since dyes become the main component present.

32

Relative Intensity

Relative Intensity

372 a)

358

 

344

 

 

 

o 160 260 3b0
mlz

666

 

 

L AL A... _ .‘LL ‘ AAA ‘.‘AAA__‘ __AA 4.1 k; m; _ AA. A—A- A;
w... v. .w. '7 w “7—..- 7w
1 F

I

400 500 600

b)

~ jug-‘L -A -‘~ A. 4- -l .42 k - A

 

 

 

260 4610 500 soo
mlz

1ooo 1zoo 1430

Figure 3.4: a) Positive ion LD mass spectrum of Ink A; b) Negative ion LD mass

spectrum of Ink B

33

References

(l) Witte AH. The examination and identification of inks. In: Lundquist F, editor.
Methods of forensic science, vol. 2. New York: Interscience, 1963;35-77.

(2) http://www.knaw.nl/ecpa/inlc/htmI/ink.html
(3) Cantu AA. A sketch of analytical methods for document dating Part II. The dynamic
approach: Determining age independent analytical profiles. Int J Forensic Doc

Examiners 1996;2:192-208.

(4) Brunelle RL, Cantu AA. A critical evaluation of current ink dating techniques. J
Forensic Sci 1987;32:1522-1536.

(5) Cantu AA. A sketch of analytical methods for document dating. Part I. The static
approach: Determining age independent analytical profiles. Int J Forensic Doc
Examiners 1995;1z40-51.

(6) Brunelle RL. Ink-dating — the state ofthe art. J Forensic Sci 1992;37:113-124.

(7) Kang HR. Water-based ink jet. 1]. Characterization. J Imaging Sci 1991;35:189-194.

(8) Donohue J, Spiro A. Predicting shelf-life from accelerated aging data: the D & A and
variable Q10 Techniques. Medical Device & Diagnostic Industry 1998:68-72.

(9) Sakayanagi M, Komuro J, Konda Y, Watanabe K, Harigaya Y. Analysis of

ballpoint pen inks by field desorption mass spectrometry. J Forensic Sci
1999;44:1204-1214.

34

Chapter Four: Forensic Science Applications

Introduction to Questioned Document Examination

"The ability to determine when a document was written would rate as one of the major
breakthroughs in forensic science, having a significant impact on the detection of all
kinds of fraud. This would result in a corresponding financial benefit to both State and

Federal administration."( 1)

Questioned document examination encompasses a vast range of analyses. Wills
and hospital records are perhaps some of the more common types of documents called
into question. In the case of a will being challenged, a handwriting analyst may be
employed to determine if the signature is authentic or a forgery. The examination
requires a trained eye to detect slight differences between the questioned signature and an
examplar provided. Other types of documents require a more technical analysis. A
hospital record may be called into question concerning a dated entry, which introduces
the complex area of relative ink age determination.

Document examiners typically employ two main approaches to determine the
relative age of an ink on paper: the static approach and the dynamic approach (2-4). The
static approach is rather straightforward. It involves identifying inconsistencies in the
composition of the document, including in both the ink and in the paper (2). A document
examiner is searching for components, that should not be included in the formulation,
since they were either not available or not used at the alleged time of production. In a

classic case, the Hitler Diaries were proven to be fake by the identification of a specific

35

whitener in the paper, which was not used in paper production at the alleged date that the
diaries were written (5). Thin layer chromatography (TLC), microscopy, solubility tests,
infrared reflectance and luminescence, and UV fluorescence are commonly employed
analytical techniques used to determine an ink’s chemical and physical properties (6,7).
The ink’s compositional profile is then compared to a reference library. In 1968,
Brunelle initiated a comprehensive ink library at the Bureau of Alcohol, Tobacco, and
Firearms. The collection, which maintains over 5000 different ink formulations and is
complete for inks in production since 195 8, is now maintained by the United States
Secret Service (2,5-7). The “Ink Library” approach is obviously quite limited, since
samples are not easily accessible to examiners Outside of the Secret Service.

A more sophisticated static approach for ink age determination was introduced in
the mid-1970’s, which eliminated the need to identify the ink formula or the
manufacturer. The ink tagging system, initiated in 1975, required ink formulators to
include a marker, which would be unique only to that ink (2,5-7). The tags were changed
yearly, so that the year of manufacture could be positively identified. Samples of the inks
and a record of the included tags were kept in the International Ink Library, which is
maintained by the United States Secret Service. The tags were usually trace quantities of
rare earth organometallic compounds or trace amounts of optical whiteners. The former
could be detected by performing an x-ray optical fluorescence analysis on a prepared
SrWO4 phosphor from an organic extract of the tag. The whiteners could be analyzed
using a TLC method, which would separate the tags, without separating the dyes (2). The
response was initially overwhelming, with 50% of all US writing inks tagged by 1978.

(6). However the large influx of foreign inks, compounded with ink manufacturers’

36

frequent ink formulation modifications, resulted in a rather short lived program.
Consequently, document examiners sought additional ways to identify inks, and to
determine their relative age.

The dynamic approach to relative ink age determination involves the complex
evaluation of the aging process. As ink ages on paper, solvents evaporate, resins
polymerize, and dyes degrade. Numerous methods have been explored which monitor
these different aspects of the ink. Though these methods differ significantly, they all
share a set of common limitations. For instance, in order for two inks to be accurately
compared, the inks must be on the same paper, of the same formula, and stored under the
same environmental conditions (relative humidity, temperature, light conditions, etc.)
(3,6). If the ink samples are on separate pages, the storage history of both documents
must be known. Additionally, the parameter studied should be independent of the
amount of ink sampled (6). Establishing the accuracy of the experiment, is also a key
factor. Ink aging curves behave exponentially, meaning that the initial rate of aging
proceeds very quickly, and then slowly, asymptotically levels off (3,4,6,8). The rate of
aging and point of leveling off are dependent on the ink formulation. Furthermore, the
accuracy of the technique significantly decreases at this stage of the aging process.
Lastly, one of the two inks must be of a known date. Often times, a document examiner
does not have access to a comprehensive ink library, such as the International Ink
Library, to compare his or her questioned sample to inks of known age. Consequently,
accelerated aging techniques have been developed to create old ink from new ink, in a
very short amount of time (9-1 1). The following sections will briefly outline some of the

notable contributions from the veterans in the field of forensic document examination.

37

Notable Contributions to the Field

Early ink dating methods, developed in the 1930’s, were used to determine the
age of iron gallotannate inks. These methods, termed ion migration tests, were credited
to Mitchell (England) and Hess (Germany) (3,5-7,12). Their studies focused on the
migration of ions, specifically chloride (Cl‘) and sulfate (SO42) ions, assumed to be
present in the ink. It was believed that over time these ions diffused into the paper, away
from the initial ink line. However, the ions were not visible, and therefore needed to be
developed chemically to visualize the “ion diffusion picture”. Figure 4.1 illustrates a Cl'
ion migration test. Figure 4.1a represents an ink sample on paper at the initial time the
ink was applied to paper. Cl' ions are assumed to be present in the ink. Over time, the
Cl' ions migrate into the paper, away from the ink line (Figure 4.1b). The ion migration
test begins by removing a small piece of paper (approximately lemz) with ink from the
document and placing it in a vial. Silver nitrate (AgNO3) is added to the vial, and silver
chloride (AgCl) is formed (Figure 4.1c). Addition of a reducing agent, such as formalin,
results in the formation of elemental silver from the AgCl. The Ag metal produces a
“black shadow” on the questioned document (Figure 4.1d). The distance the Cl' ions
migrated is measured as a function of age. The greater the distance traveled, the older the
ink. The damaged sample is placed back into the document. SO47” ions may also be
monitored, however the chemistry of the ion migration test is significantly different,
employing lead perchlorate and potassium permanganate solutions (3,12). Besides the
high destructiveness of this technique, ion migration tests have serious limitations. Cl'
and SO42” ions were believed to cease diffusing into the paper after only one or two

years, which significantly limited the time span for age determination (3,12). The low

38

accuracy of the experiment, combined with the presence of too many assumptions,

prompted document examiners to develop a more scientific approach.

Ion Migration Tests

a.) _—_

Cl- C"
+Time
__—__
b.)
01' Cl' (31' Cl-
+Ag+
C_) I
A901 A901 AgCl AgCl

+ Reducing

d.) __—_ Agent
A90 A90 A90 A90

Figure 4.1: The CI' Ion Migration Test

In the 1950’s, Kikuchi developed the foundation of the well-accepted and
employed solvent extraction methods (3,6,7). Like Mitchell and Hess, Kikuchi studied
the aging of iron gallotannate ink. She noted that following the application of solvents to
a document, older inks took longer to disperse into the paper than newer inks. She
concluded that an ink’s solubility decreases with age, and worked towards using this
parameter as a measurement of the relative age of an ink. In her experiment, a drop of
dilute oxalic acid was placed on an ink stroke. Using a microscope, she watched the ink
disperse into the oxalic acid, and recorded the time at which the ink stopped dispersing

(3). This proved to be the development of a very primitive relative ink aging curve.

39

Kikuchi’s work extended to ballpoint pen ink analysis, using dilute HCI, as in place of
the oxalic acid.

On the same principle as Kikuchi’s work, Witte focused on the disappearance of
the solvent system as a function of age. His work concentrated on using the ink’s
“copying power” as an indication of the ink’s age (6). Newer inks are not completely
dry. When a piece of paper is pressed on top of the questioned sample, some ink will be
transferred to the other paper. Volatile components evaporate quickly as an ink ages on
paper, ultimately decreasing a document’s copying ability. Witte concluded that the
amount of ink transferred will decrease with the age of the document (6).

Quite a few years later in 1984, McNeil extended the ion migration test of
Mitchell and Hess, to include the migration of F 62+ ions (7). Using scanning Auger
microscopy, McNeil was able to detect the extent of the migration of these ions. He
concluded that Fe2+ ions moved 1 micron every 29 years. McNeil had hoped that this
would help to alleviate the short time frame limitation of the Cl' and 8042’ ion migration
tests, however his method proved to be rather inaccurate. Additionally, McNeil was not
able to reproduce his technique using accelerated aging techniques, the importance of
which will be discussed in a later section.

As the scientific field became more technologically advanced, questioned
document examiners sought the use of advanced analytical instrumentation to more
efficiently quantitate ink aging parameters, such as the evaporation of the solvent system
or the degradation of dye components. Fourier transform infrared spectrophotometry
(FTIR), gas chromatography (GC), and microspectrophotometry have been evaluated as

tools for analyzing parameters of the ink aging process.

40

Humecki is given credit for studying ink aging using FTIR (3,6,13). In this
experiment, a 0.25 ink-on-paper sample is removed from the document in question. The
ink is extracted from the paper with pyridine, and the solution is transferred onto a salt
window. Humecki observed differences in the IR spectra of new and old ink.
Specifically, he noted the hydroxyl (OH), methyl (CH), and carbonyl (CO) absorption
bands changing with time, which he related to the solvent evaporating and the nonvolatile
components oxidizing. Humecki concluded that plotting the ratio of the OH absorption
band to the CH absorption band versus time was a fair representation of ink aging. The
resulting aging curve had the characteristic exponential shape, and leveled off after ten
years. The ratio between the OH and CO infrared absorption bands was considered as a
function of age. However, the results were not as consistent as using the ratio of the OH
and CH absorption bands, meaning that the plot OH/CO ratio versus time sometimes
changed direction, while the plot of OI-I/CH ratio did not. A decrease in the ratio was
believed to be related to an older ink. The use of a ratio rendered this technique to be
mass invariant.

In 1985, Stewart utilized GC to study the disappearance of the solvent system as
an ink ages (3,6,7). Generally, solvents are believed to remain in the paper for up to 1 to
two years (6). Some may argue that the solvents are always present, since the volatile
molecules may become trapped in the polymerized resins (14). Based on this premise,
Stewart used strong solvents to extract the volatile components of the solvent system, and
analyzed them using a GC. He reported a significant decrease in the volatile components,

for up to year, as the ink ages (7).

41

Extensive work has been completed by Aginsky in developing
microspectrophotometry for monitoring both the solvent system and the nonvolatile
components during ink aging (3,14-16). One area of interest involved the study of the
sensitivity of inks to the exposure to certain gases, such as benzylamine and piperidine
(3,14). He observed that a number of fresh inks undergo reversible color changes.
Aginsky measured the reflectance values of the inks as they changed color, using a
microspectrophotometer. He then related the rate of color change to the age of the ink,
noting that the rate of color change decreased as a function of age. Aginsky justified the
depressed rates as a result of the decrease in the available molecules to react with the
gases, since many molecules become trapped in polymerized portions of the ink film.

The study of optical surface changes in inks as they age was also pursued using a
polarizing microspectrophotometer (16). During this experiment, Aginsky observed that
the ratio of dyes present in the ink change on the outer surface layers of the ink film, as
the result of oxidation. Specifically, he studied copper phthalocyanine and methyl violet.
Spectral reflectance measurements of the surface layer were first taken under non-
polarized light, followed by diffuse reflectance measurements of the interior layer under
cross polarized light. The cross polarized light prevented the surface spectral reflectance
from interfering with the diffuse reflectance measurements. The measurements were
taken at two different wavelengths, determined by the dyes present in the ink. A surface
reflectance ratio (R1) and a diffuse reflectance ratio (R2) of the interior surface for the two

dyes were computed.

 

 

R : outer reflectance (dye u R : inner diffuse reflectancemlie 1)
l outer reflectance (dye 2) 2 inner diffuse reflectancewye 2)

42

The data analysis is a bit complicated, in that the aging parameter actually studied is a

ratio of the two ratios:

This ratio was observed to decrease with age, leveling off in approximately six years
(16). The technique is mass invariant, however large errors are associated with the aging
curve.

Though all of these techniques have merit, not one has been more embraced by
the field of questioned document examination than the solvent extraction methods
developed by Cantu and Brunelle (3,4,6-8,17). Fairly recently, Brunelle proposed a
sequential multiple approach for determining the relative age of writing inks (17). The
approach involves the three most commonly used techniques, which include the R-Ratio,
the sequential extraction, and the Dye-Ratio methods.

The premise behind the solvent extraction technique lies in the fact that as an ink
ages, the vehicle slowly evaporates. In theory, a relatively new ink sample on paper will
not be completely dry. Consequently, some of the solvent system will still be present in
the paper, making extraction of the ink using weak solvents easier. An older ink sample
on paper will have dried to a greater extent, making the rate and extent of extraction
much lower. Extraction curves are prepared based on two principles: the R-ratio and the
Lth extraction time (percent extraction) (3,6,17). The R-ratio is a measure of the ease and
rate of extraction, whereas the L‘h extraction time is related to the time needed to extract a

certain percentage of the ink. For instance, a value of L = 0.90 is the time at which the

43

extraction is 90% complete. The percent extraction technique has been determined to be

a more efficient means to date ink, as compared to the R-ratio method.

R-Ratio

In 1979, Cantu developed a technique, based on Kikuchi’s work, which could be
used to measure the rate of the extraction of ink into weak solvents. To begin the
technique, samples are obtained from the document using a blunted or hollow 20-gauge
boring hypodermic needle; 10-15 micro-plugs (approximately 1 mm in diameter) are
removed (13,18,19). Alternatively, a 1 cm line can be removed with a scalpel. The
micro-plugs are placed in 3-5 1L of a’ weak solvent, typically isopropanol, toluene, or n-
butanol for ballpoint pen inks, or a 1:] solution of ethanol and water for other pens (18).
When ink on paper is extracted using a weak solvent, the color of the solvent becomes
darker, indicating that the ink is dissolving. The sample is agitated for a couple of
minutes to ensure that the extraction is complete. The solution containing the extracted
ink is spotted on a TLC plate. The concentration of ink present is determined using a
densitometer. Concentration measurements are taken at various points during the
extraction, with the last measurement taken at or near the completion of the extraction.

An alternative method is to dissolve the ink on the punched out discs directly in a
cuvette for either a UV-visible or a fluorimetry experiment (17). The concentration is
calculated using Beer’s Law, A =8 be. A is the maximum absorbance value,e is the
molar absorptivity constant, b is the path length, and c is the concentration of the analyte

(ink).

44

The R-ratio is defined as Cl/C2. C1 is the concentration of the ink in the
extracting solvent in the initial minutes of the extraction, which is representative of the
highest rate of extraction. C2 is the concentration of the ink in the extracting solvent
taken at the completion or near completion of the extraction of the ink from paper. The
“newer” inks will theoretically have larger R-ratios than the older inks, since more ink
can be extracted from an ink when most of the solvent is still present. Thus, the R-ratio is
expected to decrease as the ink ages. Unfortunately, this is not true for every ink
formula. Since it has been observed for the R-ratio of some inks to actually increase over
time, the ink in question must first be tested by artificially aging a portion of the
questioned document to see in which direction the R-ratio moves as a fimction of age
(17).

th

The Sequential Solvent Extraction Method (L Extraction)

The sequential solvent extraction technique is an extended version of the single
extraction (R-ratio) method previously described. The first part of this experiment is
identical to the R-ratio method, in which the sample is obtained and extracted in the same
manner. However, following the first extraction in the weak solvent, the punched out
discs are placed in a stronger solvent, typically pyridine, so that the remainder of the ink
can be extracted. A percent extraction of ink in a particular solvent after t minutes is then
calculated using the following equation:

P =[v1C1/(v1C1+ V2C2)] x 100
C1 and v1 refer to the concentration of ink in the first extracting solvent and the volume of

weak extracting solvent used, respectively. Likewise, C2 and V2 refer to the concentration

45

of ink in the stronger extracting solvent and the volume of that solvent used, respectively.
If equal volumes for solvents l and 2 are used, the equation may be reduced to:

P =[C1/(C1+ C2)] x 100
This equation may be used if TLC-densitometry is used as the analytical technique for
determining concentration. However, if fluorescence is used a modified version of the
equation is considered:

P = [Vi/(V1+V2)l X(F1/F2),
where F is equal to the fluorescence measurements. Figure 4.2 (6) is representative of a
sequential solvent extraction experiment, in which toluene was used as the weak solvent
and benzyl alcohol was used as the stronger second solvent. Figure 4.3 (6) represents
results from two different single extraction experiments using 2-propanol performed on
the same day. The near overlapping curves suggest a high reproducibility factor for the

solvent extraction method.

46

80']

 

 

 

70
o
w
T'-
o 60
<
E a 0
Lu 50-1 0
.—
uzJ SOLVENT 1 - ISOPROPANOL
g ‘0‘ SOLVENT 2 - METHANOL
1.1.1
o.
ISO-I
0 I I W I T I I I _I_ I I I I I r I I I I I
0 36 38 42

12 24
ACE OF INK (MONTHS)

Figure 4.2: Aging of ballpoint pen ink (sequential solvent extraction)

 

with Anja M-3l l

 

 

 

 

1200
O
X 1100
25 1000
SE -—SAME EXPERIMENT DONE
V N IFF R NT AY
m 900‘ 0 0 E E D
0
Z
O
< 800-1
LIJ
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Li’

.I

O 500
’2 g :t
U1
2 500‘
LAJ
O

400 T T rft T T T T T T T TwT—T T TW'fi—T—W

O 12 35 44

24
ACE OF INK (MONTHS)

Figure 4.3: Aging of ballpoint pen ink (single solvent extraction) with
Anja M-3l l

47

Questioned document examinerstend to prefer using a percent extraction value as
opposed to considering only the rate of reaction (R-ratio). However, this method also has
its limitations. For instance choosing the second solvent sometimes can be a troublesome
task. Figure 4.4 (6) represents a sequential solvent extraction curve in which the second

solvent was not a wise choice, since n-butanol and pyridine are not miscible.

DENSITOMETER

g READINGS

T
on

60 d.—

PERCENT EXTRACTED

h

 

b

 

 

40“500 [*T—T I— T T T 1 T T I—I T T T T m I T T
0 - 12 . 24
ACE OF INK (MONTHS)

Figure 4.4: Aging of ballpoint pen ink with Formulab 587

Other disadvantages, as in other ink dating methods, include differences in drying rates of
the inks. Most inks dry completely within two years, with some inks drying in less than
two months. Therefore, this technique could not be applied to cases in which the age of
the ink in question exceeds this time frame. Also, as the aging curve becomes asymptotic

(reaching its leveling off point), the accuracy of the age determination decreases

48

significantly. This leads to the question of whether or not using the disappearance of the
solvent system is the best option for ink age determination.

Limitations exist for both the R-ratio and L‘h extraction time interpretations of the
extraction curve. First of all, numerous points are required on the curve in order to obtain
optimal accuracy. Also, many errors may be introduced during the actual sample
preparation. For instance if TLC is used, extreme caution must be taken to ensure that
equal amounts of sample are spotted on the plate. If fluorimetry is used, the ink must
contain a dye that fluoresces, and if not, a known standard (rhodamine-type dyes) must be
added which is known to fluoresce (17). In this case, a measurement of the fluorescence
“robbed” by the non-fluorescent extracting solvent is taken. The most obvious limitation
deals with the fact that the inks need to be first tested to see in which direction their R-
ratio or Lth extraction values are going to migrate over time. This suggests that a
complete understanding of the chemistry that is occurring between the ink and the paper

over time has not yet been obtained.

Dye-Ratio

The Dye-Ratio method measures the change in the relative concentration of dyes
in the ink extracted in a weak solvent (7). As previously mentioned, inks are usually
composed of a mixture of dyes or pigments. Ink samples are first extracted and spotted
on a TLC plate as previously described. Typical TLC plate developing solutions for
ballpoint pen inks, include ethyl acetate/ethanol/water (70:35:30) and
n-butanol/ethanol/water (50:10:30) (1). Once the dyes are separated, their concentrations

are obtained using a densitometer. All possible dye ratios are considered and monitored

49

over time. In a typical blue ballpoint pen ink, the dye composition consists of Cu
phthalocyanine blue (dye l), as the primary dye, and methyl violet (dye 2), as a toner. It
has been noted that the ratio of dye 2/dye 1 will decrease as an ink ages. Methyl violet is
significantly less stable than Cu phthalocyanine blue, therefore this would be expected. It
has been noted that no work has ever been attempted for the purpose of explaining the
degradation of the dyes or the dynamics of the Dye-Ratio method. Our work explores the
dynamics of methyl violet degradation and how it can be used as an alternative to the

solvent extraction techniques for determining the relative age of questioned ink.

Accelerated Aging — The Thermal Approach

In order to determine the relative age of an unknown ink, there needs to be a
known standard to compare it to. Using artificial aging techniques, a new ink can be
made to appear chemically as an old ink. The artificial aging process is monitored so that
aging curves may be developed. Data from the unknown ink sample is compared to the
artificial aging curve, and its age is subsequently determined. Currently, the most
accepted method for accelerated aging is using heat, which is based on the Q10 theory.
The Q10 theory states that for a 10°C increase in temperature, the rate of a reaction
(decomposition) doubles (20). Interestingly, there does exist a huge disagreement
amongst questioned document examiners as to the correlation between the length of time
the ink sample is heated as a function of its age. For instance, it has been documented
that heating the sample in an oven at 100° C for 4 minutes is equivalent to 8 days of
natural aging at 25° C (21). On the other hand, another source states that exposing the ink
sample to the same conditions is equivalent to 20 days of natural aging at 22° C (11).

Cantu considered both of these claims and proceeded to perform his own accelerated

50

aging analysis. His studies indicated that 90 days of natural aging at 25° C is the same as
heating the sample at 100° C for 4 minutes (1 I). This discrepancy between the
correlation of natural aging and accelerating aging introduces the possibilities of
significant errors when using this artificial aging method to set up standards for
comparison to a questioned ink sample. However, it is important to note that these
accelerated aging correlations are based on paper aging studies (10). Ink, paper, and ink
on paper, all age quite differently. Thus, there exists an absence of a true understanding
of the dynamics of ink aging on paper. Furthermore, it has been cited that many of these
correlations are pertinent only to a defined set of conditions, such as a particular ink on a
specific type of paper (10). We encountered the same discrepancies using an alternative

method of UV-light to accelerate a very different aging parameter — dye decomposition.

Summary

In summary, a “perfect” method for determining the age of a questioned
document may not exist. There are numerous variables involved in this type of analysis,
which can not be controlled. Such variables include the various types of media the inks
are printed on, storage conditions (temperature and the presence or absence of light), the
writing instrument, etc. Another defect is developing the best accelerating aging
technique. Heating definitely has its flaws, and UV irradiation has not yet been proven.
Perhaps, there is a more efficient technique not yet tested, such as creating an oxygen
enriched environment using ozone to accelerate the oxidative demethylation process of
methyl violet. Lastly, it is possible that both the disappearance of the solvent system, as

well as the degradation of the dye components need to be considered concurrently.

51

Studies would need to be performed to determine if these two techniques compliment
each other. Overall. the sequential solvent technique is sufficient for current analyses of
questioned documents, however there is always room for improvements or more efficient

methods.

52

References

(1) Tebbett IR. Chromatographic analysis of inks for forensic science applications.
Forensic Sci Rev 1991;71:72-82.

(2) Cantu AA. A sketch of analytical methods for document dating. Part I. The static
approach: Determining age independent analytical profiles. Int J Forensic Doc
Examiners 1995;] :40-51.

(3) Cantu AA. A sketch of analytical methods for document dating Part II. The dynamic
approach: Determining age independent analytical profiles. Int J Forensic Doc
Examiners 1996;2:192-208.

(4) Cantu AA, Prough RS. On the relative aging of ink — the solvent extraction
technique. J Forensic Sci 1987;32:1151-1174.

(5) Cantu AA. Analytical methods for detecting fraudulent documents. Anal Chem
1991;63:847-854.

(6) Brunelle RL, Cantu AA. A critical evaluation of current ink dating techniques. J
Forensic Sci 1987;32:1522-1536.

(7) Brunelle RL. Ink-dating — the state ofthe art. J Forensic Sci 1992;37:113-124.

(8) Brunelle RL. Determining the relative age of ballpoint ink using a single-solvent
extraction, mass-independent approach. J Forensic Sci 1989;34:1166-1182.

(9) Sodennan H, O’Connell JJ. Modern criminal investigation. New York: Funk &
Wagnalls, 1935.

(10) Stewart LF, F ortunato SL. Distinguishing between relative ink age determinations
and the accelerated aging technique. Int J Forensic Doc Examiners 1996;2:10-15.

(l 1) Cantu AA. Comments ofthe accelerated aging ofink. J Forensic Sci 1988;33:744-
750.

(12) Witte AH. The examination and identification of inks. In: Lundquist F, editor.
Methods of forensic science, vol. 2. New York: Interscience, 1963;35-77.

(l3) Humecki HJ. Experiments in ball point ink aging using infrared spectroscopy.
Proceedings of the International Symposium on Questioned Documents, July 30-
August 1, 1985:131-135.

(l4) Aginsky VN. Some new ideas for dating ballpoint inks — a feasibility study. J
Forensic Sci 1993;38:1134-1150.

53

(15) Aginsky VN. Comparative examination of inks by using instrumental thin-layer
chromatography and microspectrophotometry. J Forensic Sci 1993;38:111-1130.

(l6) Aginsky VN. A microspectrophotometric method for dating ballpoint inks — a
feasibility study. J Forensic Sci 1995;40:475-478.

(17) Brunelle RL. A sequential multiple approach to determining the relative age of
writing inks. Int J Forensic Doc Examiners 1995;]:94-98.

(18) Standard guide for test methods for forensic writing ink comparison. ASTM method
E: 1422-97.

(19) Merrill RA, Bartick EG. Analysis of ballpoint pen inks by diffuse reflectance
infrared spectrometry. J Forensic Sci 1992;37:528-541.

(20) Donohue J, Spiro A. Predicting shelf-life from accelerated aging data: the D & A
and variable Q10 Techniques. Medical Device & Diagnostic Industry 1998:68-72.

(21) Browning BL. Analysis of paper. New York: Marcel Dekker, 1985.

54

Chapter Five: LDMS Analysis of Methyl Violet on Paper

Introduction — The Detection of Ink on Paper

In Chapter 3, the ease of detecting methyl violet neat in FAB MS and LDMS
experiments was demonstrated. While industrial ink chemists would use results from the
analysis of bulk ink, forensic chemists would benefit more from the analysis of ink on
paper. An important question is related to the sample sizes involved. Is there a sufficient
surface concentration of dye deposited on paper during writing or printing to be detected
in a FAB or LD experiment? Consider a typical ballpoint pen cartridge, which contains
about 0.6 g of ink (1). For the pen we used, the written line was 0.36 mm wide. We
assumed that an ink contains 20% dye by weight. In the case of methyl violet (molecular
weight of 372 g/mol), the ink cartridge would contain 0.3 mmol of dye. Also,
considering that one ink cartridge can write a line approximately 3000 m long (1), this
correlates to about 0.3 mmol/m2 of sample, as a molar surface coverage per unit area. In
a FAB experiment, a nanomole of analyte is typically dissolved in microliter of a viscous
liquid matrix and deposited on the end of a direct insertion probe tip. This correlates to
approximately 0.3 mmol/m2 as an analyte surface coverage, suggesting that there will be
enough dye sample present on paper to detect in a FAB experiment. In MALDI/MS,
picomolar amounts of sample are used, corresponding to a molar surface coverage of 0.3
umol/mz, however in the MALDI experiment, the analyte is not absorbing laser energy
directly. Matrix surface concentrations are larger. Considering the fact that laser-based
methods have been cited as having extremely how detection limits, in the range of 10'18
to 10'19 g, it is likely that the surface coverage of dye deposited on paper during writing

and printing will be sufficient for direct laser desorption. Based on the ease of analysis

55

and flexibility of the LDMS experiment, compared to FAB MS, and the simplicity of the
instrumentation, the decision was made to focus on LDMS, rather than FAB, for the
analysis of ink on paper. We note that FAB spectra were successfully obtained, however
short signal durations (see Chapter 2) made the approach less than ideal.

Figure 5.1a shows a portion of the positive ion LD mass spectrum of the positive
ion LD mass spectrum of Ink A on paper. The peaks shown from m/z 344-372, separated
by 14 amu, represent the components of the cationic dye, methyl violet. These are the
dominant peaks in the mass spectrum. This is very similar to field desorption mass
spectrum reported by Sakayanagi, et al. for the same dye (2). To ensure that all of the
peaks present in the spectrum are indeed from the ink and not from the paper, both
positive and negative ion LD mass spectra were taken of the paper itself. The positive
ion LD mass spectrum of the paper substrate used in all of our analyses of ink on paper,
is shown in Figure 5.1b. When paper is subjected to UV laser irradiation in this
experiment, alkali ions are usually observed as intense peaks at m/z 23 (Na+) and m/z 39
(K). Two peaks, at m/z 284 and m/z 575 are consistently formed. One source cited the
peak at m/z 284 as stearic anhydride, an additive used in paper production (3). The peak
at m/z 574 has an isotopic pattern consistent with that of copper phthalocyanine blue.
Sometimes, blue dyes may be added to increase the paper’s brightness, however there has
been no confirmation that copper phthalocyanine is present in the paper. The key point is
that the peaks are not representative of ink components. These two peaks representing
the paper are not always observed when an ink sample is being studied. This may depend
on the extent of coverage of the paper by the dye. For example, in the experiment that

resulted in the spectrum shown in Figure 5.1a, no paper-related peaks were observed.

56

Ink may also be detected on paper in negative ion mode using LDMS. Figure
5.1c shows a portion of the negative ion LD mass spectrum of Ink B on paper. The peak
at m/z 666, the largest peak in the spectrum, shows that Solvent Black 29 can clearly be
detected. Many spectra can be generated at high signal-to-noise ratios, indicating that
LDMS can be used for the direct detection and analysis of inks-on-paper, at the surface
concentration typically encountered in written documents. Obviously, for an ink sample,
an analyst may not know if an anionic or cationic dye may be present. However, it has
been our experience that sufficient dye is present in ink to perform both positive and

negative ion LDMS analyses.

57

 

 

 

 

 

 

 

 

 

 

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'5 358
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320 3'30 3'40 350 350 3"ro 3'80 350 400
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62° 6'40 650 580 700 7‘20 7:40
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Figure 5.1: a) Partial positive ion LD mass spectrum of Ink A on paper; b) Positive ion

LD mass spectrum of paper; c) Partial negative ion LD mass spectrum of
Ink B on paper

58

U V-A ccelerated Aging

Since LDMS can directly detect dyes on paper from ink, the decision was made to
analyze ink from a ballpoint pen. Over time, ink on paper changes in appearance. This
suggests that the chemical composition of the dye molecules is changing. Possibly, the
dye could polymerize. If this occurred, the LD mass spectrum of aged ink on paper
would show new peaks in the higher mass region. Perhaps, there would be a decrease in
the molecular ion peak intensity and the appearance of peaks in the lower mass region,
indicating that the dye is degrading. If the dye is extremely stable, there is the chance
that the spectrum would not change. In an attempt to generate an aged ballpoint pen
sample to compare with a fresh sample, accelerated aging techniques were considered.
Commonly accepted thermal accelerated aging methods were discussed in Chapter 4.
When deciding on which accelerated aging technique to use, one needs to consider what
component of the ink is being measured as a function of the age of the ink. In the case of
the well-accepted solvent-extraction technique, the amount of dye present in solution is
measured using a densitometer, following an extraction with a mixture of weak solvents.
The premise behind this technique is that it is harder to extract ink from older documents,
since the majority of the solvent system has evaporated. Hence, a less concentrated
extraction solution is indicative of an older ink. This method is indirectly measuring the
solvent content left in the paper. Therefore, applying heat to a new sample makes sense,
in order to evaporate the solvent more quickly. Thermal methods were attempted, in
which our ink sample was placed in an oven at 100°C, for several hours. No changes in
the initial spectrum were noted, which was not completely unexpected. Dye degradation

is a chemical change, not a physical change like that of a solvent evaporating. UV

59

irradiation is commonly employed in the dye manufacturing industry to induce fading,
which also occurs with age (4,5). Therefore, UV light was considered as an alternative
accelerated aging method.

A preliminary UV-aging study was performed using Bic black ballpoint pen ink,
containing the dye methyl violet. The ink was applied uniformly to piece of 4 in2 paper.
An initial positive ion LD mass spectrum was obtained. Approximately one third of the
sample was covered and the entire sample was irradiated using a UV lamp, placed
directly on top of the sample for 12 hours. Another third of the sample was masked off
and the sample was irradiated for another 12 hours. This experiment demonstrated that
significant fading was noted following 12 or more hours of intense UV irradiation. Also,
it showed that by using masks, an array of UV aged ink on a single piece of paper could
be easily generated, introduced as a single sample into the mass spectrometer, and spectra
for each region obtained. For this preliminary work, the simplicity of UV aging made it
an ideal choice. We acknowledge that there is currently no accepted irradiation /age
correlation for this experiment.

The preliminary UV accelerated aging data are presented in Figure 5.2. Positive
ion LD mass spectra are shown of methyl violet dye as originally deposited by the
ballpoint pen onto paper, and the same portion of the mass spectrum is shown following
12 and 24 hours of UV irradiation. After irradiating the sample for 12 hours, degradation
product peaks are clearly present — a series of peaks separated by 14 amu. The m/z 372
peak remains the most intense peak. The spectrum shows that six new compounds,
lower-mass forms of Methyl Violet, are now in abundance. Irradiating the sample for

another 12 hours causes the initial base peak to further decrease in intensity, such that the

60

degradation product peaks dominate. Andrasko noted similar results using high
performance liquid chromatography (HPLC) to study ballpoint pen inks stored under

various light conditions (6.)

 

 

 

 

 

372

358
330 344 372

358
31 6
302
284
330 344
358
31 6 372
284 302
280 300 320 340 360 380 400
mlz

 

 

 

Figure 5.2: Preliminary UV-Accelerated Aging Study: Bic Ballpoint Pen Ink on
Paper

When comparing these results to naturally aged samples (shown and discussed in
the following section), we realized that we had artificially aged the samples past that
which occurs naturally. Aginsky stated the importance of ensuring that the accelerated
aging method mimics natural aging as closely as possible, meaning that the technique
should not induce any chemical changes that would not happen naturally (7).
Consequently, the experimental setup was adjusted by elevating the UV lamp 6 cm above

the sample, and by monitoring the irradiated sample at much shorter time intervals (every

61

15 minutes). This allowed for us to visualize the dye degradation process more clearly,
as well as to mimic the natural aging process more effectively.

UV accelerated aging mimics natural aging from a dye perspective, and can be
characterized by LDMS. Figure 5.3a represents a portion of the positive ion LD mass
spectrum of new black Bic ballpoint pen ink on paper. New ink is characterized by a
large peak at m/z 372, representing the non-degraded dye molecule (C+Me6), with a very
small peak at m/z 358 (C+Me5HI). Figure 5.3b is the positive ion LD mass spectrum of a
38-month old naturally aged Bic black ballpoint pen ink on printer paper. As an ink ages,
lower mass peaks appear in the spectrum representing the molecules that are referred to
as degradation products, accompanied by a decrease in the relative intensity of the
original m/z 372 base peak, representing the original intact dye molecule. The number
and amount of degradation products present is a function of the age of the ink, but if this
were ink on a questioned document, how could the age be determined from the spectrum?
As in other ink dating methods, there must either be spectra from naturally aged samples
for comparison purposes, or there must be a calibrated method for accelerating the aging
of a new sample of similar ink that can be used to create a sample that yields the same
spectrum. One goal of this work is to develop a calibrated UV accelerated aging method.
It has previously been demonstrated that this means of artificially aging ink produces the
same chemical changes in terms of the degradation of methyl violet, which occurs
naturally as a document ages. Irradiating Bic black ballpoint pen ink-on-paper, for 6.25
hours with UV light, produces a very similar mass spectrum (Figure 5.3c) as that of the
naturally aged 38-month old document. Based on this data alone, a calibration for the

UV method can be estimated. Irradiation for 6.25 hours produces the same extent of

62

degradation as what occurs naturally over a period of 38 months. Thus, every hour of
UV irradiation accelerates the aging by approximately 182 days. If a different sample
was in question, the ink could be irradiated until the same extent of degradation was
observed, and from the irradiation time required, the corresponding natural age could be
calculated. This approach, along with other insights into the variables that influence the

rates of dye degradation, are studied in this work.

 

 

 

 

 

 

 

 

 

372
a
>. if?"
.4:
m
C b 372
(D
“E
_ 358
(D
.5 344
£5 330
CD .
[I C 372
358
344
330
I I i LI
285 300 315 330 345 360 375
mlz

Figure 5.3: A portion of the positive ion LD mass spectrum of Bic black ballpoint
pen ink-on-paper: a) ink from a new document; b) a 38-month old
controlled, naturally aged document; c) ink from a document irradiated
for 6.25 hours with UV light

63

Developing a Relationship Between Natural Aging and U V-A ccelerated Aging

Methyl violet can be efficiently degraded on paper with UV irradiation. Ink
samples on paper were subjected to UV radiation, followed by LDMS analysis, for
periods of up to 8 hours. In these time periods, more than 50% of the dye molecules can
be converted into degradation products. Figure 5.4 is a plot of the normalized relative
intensities for each of three mass spectral peaks representing the dye or degradation
product as a function of time for the UV accelerated aging study of Bic© black ballpoint
pen ink-on- paper. The relative intensity is a measure of the concentration of a particular
species in comparison to the other components detected in the sample mixture. In Figure
5.4, the relative abundance of the intact dye molecule (m/z 372) decreases as the relative
abundances of the degradation products (m/z 358, 344) increase with irradiation time,
which is an indication of the dye degrading. How can the data be used to develop a
determination of an ink’s age? There are many options. For instance, the age correlation
may involve the relative intensity of the m/z 372 peak, revealing the amount of dye
remaining in its original form. Another possibility would be to take a ratio of two peaks,
such as the ratio of the m/z 358 peak to the m/z 372 peak, since these two peaks are
always present, and seem to experience the most significant changes as the ink ages.
Ideally, a firnction would be identified, in which a single value could be computed that
incorporates information on all of the degradation products present. One way to
accomplish this is to compute the average molecular weight for the dye at each time
interval. Figure 5.5 shows the plot of the average molecular weight versus the time the
document was irradiated in the UV accelerated aging study of Bic black ballpoint pen ink

on paper. The average molecular weight (MWavg) was calculated by multiplying the

64

normalized intensity of each peak by the nominal mass of that peak, summing all of the
products, and dividing by the sum of the relative intensities of all of the peaks:

MWavg = [RI(m/z 372) x 3721+1RIIm/z 358) x 3581+ [RI(m/z 344) x 3441+
RI(m/z372) + RI(m/z358) + RI(m/z344) +

This value was computed for each of the five spectra acquired per sample, and the five
molecular weights were averaged. Scatter in the data is noted, however a distinct trend is
established in which an increase in the degradation of the dye, i.e., a decrease in the
average molecular weight, occurs over time. The use of MWavg lends some physical
meaning to the experiment. For instance, when the dye is initially deposited on paper, its
average molecular weight would be nearly 372 Daltons, since it is essentially only crystal
violet. If a sample is analyzed that shows an average molecular weight of 364 Daltons, it
is understood that the degradation products are dominating the spectrum, indicating that
the dye has degraded. The lower MWavg limit for this experiment is 288 Daltons, which
would occur if all of the C+Me6 were converted to C+H6. We have not yet found any
naturally aged samples in which degradation has been this extensive. Figure 5.5, shows
that the average molecular weight falls to 361 Daltons following 450 minutes of UV

irradiation.

65

 

 

 

mlz 372 (R2=0-9289)

 

 

   

 

 

 

 

   

 

 

I 2,- - l -
0.8 -— __
3: 1
Z; 1
2 .
I_: 0.6 +— —
0
.2
0‘; O
3 . 0
°‘ 04 .___,, -——- —- ~— °
‘ A
mlz 358 (R2=0.9558) - 2)
I
I
02 T I ‘ I ‘
l A
mlz 344 (x2) (R’=o.8287)
o *— ' T fl? 1 r l
o 50 100 150 200 250 300 350 400 450 500

UV Irradiation Tlme (minutes)

Figure 5.4: UV accelerated aging study data for new Bic black ballpoint pen ink on
printer paper: a plot of the relative intensity of the m/z 372, 358, and
344 (x2) peaks versus irradiation time

Similar dye degradations occur naturally. In order to relate the UV accelerated
aging curve in Figure 5.5 to aging which occurs naturally, a natural ink aging curve
(Figure 5.6) was constructed using a set of controlled ink library samples from Speckin
Laboratories, Okemos, MI. The samples were written with the same pen, on the same
paper, and stored under the same conditions. Again, from LDMS spectra, the average
molecular weights were computed. According to the UV accelerated aging curve
prepared under the outlined experimental conditions, 500 minutes (8.3 hrs) of UV
irradiation degraded methyl violet to an average molecular weight of approximately 360
Daltons. Referring to the straight line fit of the controlled natural aging data (Figure 5.6),

an average molecular weight of 360 Daltons is equivalent to 52 months of natural aging

66

for this particular ink and paper. By combining the information in Figures 5.5 and 5.6, it
appears that 500 minutes of UV irradiation (at the conditions used here) would age a
document by approximately 52 months (~1560 days). Therefore, in this particular study,
for every hour of UV irradiation, the document was aged roughly 187 days. This
compares favorably with the initial estimate of 182 days per hour determined from the
initial data shown in Figure 5.3.

A comment should be made concerning the linearity of the two best fit lines in
Figures 5.5 and 5.6. The linear least squares fit to the data n Figure 5.5 has an R2 value
of 0.8845. The R2 value is a measure of the strength of the linear relationship. The
closer the R2 value is to unity, the stronger the relation. The linear regression line for the
data in Figure 5.6 has an R2 value of0.6331, indicating that the data generated during the
UV accelerating aging study correlated better to a straight line fit than the data
accumulated from the controlled natural aging study. This is not unexpected considering
the time frames of the experiments. The UV accelerated aging study was completed in
the matter of hours, whereas the controlled natural aging study occurred over ten years.
During the UV accelerated aging study, variables affecting the aging process, such as

temperature and humidity fluctuations, were minimal, resulting in a more constant rate of

aging.

67

 

 

 

R2 = 0.8845

 

 

 

 

 

 

 

 

 

 

 

 

Average Molecular Weight (Da)

 

 

 

 

 

 

' _ 777" ——1‘——T—‘fi— —l— T T —1

O 50 100 150 200 250 300 350 400 450 500
UV Irradiation Time (minutes)

Figure 5.5: UV accelerated aging study data for new Bic black ballpoint pen ink on
printer paper: a plot of the average molecular weight of the dye,
methyl vio’let, versus minutes of UV irradiation.

 

 

 

- R2 = 0.6331

 

 

 

 

 

 

 

Average Molecular Weight (Da)

 

 

 

 

o 20 40 60 8’0 100 120
Age (months)
Figure 5.6: Controlled, natural aging study data for Bic black ballpoint pen ink

on printer paper: a plot of the average molecular weight of the dye,
methyl violet, versus the age of the document.

68

A Note on U V-Accelerated Aging

UV accelerated aging is more complex to perform than thermal aging. The details
of the UV-aging experimental set-up can influence the resulting aging curve. Although
the exact correlation between natural aging and artificial (thermal) accelerated aging may
still be unknown, a thermal aging technique is very straightforward. For instance, one
source suggests that, if an ink sample placed in an oven at 100° C for 4 minutes, the age
of the sample increases by 3 months (8). UV aging is not as straight forward and easy to
control. The distance between the UV lamp and the sample, as well as the age of the
light source in the lamp affect the flux (# photons / area*time) of UV photons and
consequently, the aging rate. Furthermore, the power of UV lamps may vary the longer
the lamp is in use. In the preliminary UV aging studies, the lamp was placed directly on
top of the paper. As a consequence, the degradation process proceeded at very high rates.
The UV lamp should not be placed too close to the sample, to ensure that the temperature
of the sample does not increase significantly, and to maintain a moderate photon flux
from the UV lamp. To make certain that heat did not influence the rate of degradation,
the samples were not continuously irradiated. Rather, the lamp was turned off at hourly
intervals for 15 minutes throughout the experiment, to ensure that the sample was not
being heated by the lamp to any appreciable extent. After experimenting with distances
between the lamp and the sample, as well as altering the time increments between
acquiring spectra of the samples in order to obtain a clear description of the gradual
degradation of the dye, it was found that having the lamp 6 cm above the sample surface,
allowing the sample to cool every hour for 15 minutes, and taking LDMS spectra at 15

minutes intervals, allows for generation of an acceptable aging curve. Others attempting

69

to use this aging technique would therefore need to first develop their own aging curve,
using their own defined experimental geometry and lamp. In other words, the experiment
cannot be defined as simply as “heat the sample in an oven at 100° C for 4 minutes to age
the sample 3 months”. This method is laboratory dependent. The accelerated aging rate
of 180-190 days/hour is for this particular system, not to be considered as applicable in

general.

Testing the Influence of Variables in Natural Ink Aging

The rate of the natural aging of methyl violet is dependent on many variables.
Certainly, it would be ideal if the chemical degradation of methyl violet was insensitive
to the common variables involved in ink aging. These variables, mentioned previously,
include ink formulations, the type of paper, relative humidity, and environmental storage
conditions. To determine whether dye aging is sensitive to some of these variables,
naturally aged ink samples were obtained from the departmental archives. The samples
gathered were written in both blue and black ink, and were therefore of different ink
formulations, and were obviously not from the same pen. Also, the types of paper
selected varied significantly. Positive ion LD mass spectra were obtained for more than
25 samples, that spanned a 50 year period. The average molecular weight of the dye was
calculated as previously described, and then plotted versus age (Figure 5.7). The graph
shows differences from the aging curves of the artificial aging study (Figure 5.5) and the
controlled ink library study (Figure 5.6). Substantial scatter is evident in the data, and it
appears as though, after roughly 15 years, all of the samples produce very similar spectra.

Therefore, this study suggests that the generally accepted theory that ink aging (from a

70

solvent content standpoint) will asymptotically level off and eventually stop, may be true
in the case of dye degradation, as well. Another interesting observation is that the
leveling off occurs at an average molecular weight of approximately 365 Daltons. It has
already been demonstrated that UV accelerated aging can surpass this value (Figure 5.5).
What is even more surprising is that the natural aging ink library samples had degraded to
lower average molecular weights (Figure 5.6) than what is seen in Figure 5.7. A series of
experiments were designed to test some of the variables, which were thought to be
causing the deviations (scatter) in the “real life” samples (Figure 5.7). There is also
interest in why the naturally aged samples appear to stop aging, while the accelerated
aging and controlled ink library samples have aged to a greater extent and appear to be

still aging significantly.

 

 

 

 

 

 

 

 

 

Average Molecular Weight (Da)

 

 

361 T—* 'TT___ 7 I '_— ‘74 "—_~I l l i‘ -'l__7*7_“' “l1

0 5 10 15 20 25 3O 35 4O 45 '50 55
Age (years)

Figure 5.7 : Uncontrolled, natural aging study data for various inks on different types
of paper: a plot of the average molecular weight of the dye, methyl violet,
versus the age of the document

71

The type of paper influences both natural and accelerated aging of methyl violet.
To test the influence of the paper on the degradation of methyl violet, an additional set of
samples was provided by the ink library of Speckin Forensic Laboratories. This set was
written with the same pen and stored under identical conditions as the first set, however
this second set was written on bond paper as opposed to printer paper. Positive ion LD
mass spectra of the samples were obtained and a natural aging curve of the average
molecular weight of methyl violet versus time was prepared (Figure 5.8). While there is
significant scatter in the data, the dye appears to be aging at a slower rate than that of the
first set of ink library samples (Figure 5.6). For instance in 10 years, dye in the first set
has degraded to an average molecular weight of 347 Daltons, while the second set, the
dye has only degraded to 366 Daltons. In the data from Figure 5.7, spectra from 10 year
old documents led to a dye MWavg of 365 Daltons. So, it is clear that natural aging as
reflected by dye degradation products is paper dependent. It is important to note that the
artificial UV aging study performed in this laboratory was conducted on the same type of
paper as the first set of ink library samples, which is the basis for our initial correlation
that 1 hour of UV irradiation is equivalent to 187 days of naturally aging. However, the
studies were not performed with the same pen, but with the same company’s ink. It is
important to note that most of the samples in documents selected for the study in Figure

5.7 are “professional correspondence”, mostly on letterhead paper.

 

R2 = 0.4153 1

 

 

 

 

 

 

Average Molecular Weight (Da)

 

 

 

0 20 40 60 80 100 120
Age (months)

Figure 5.8: Controlled, natural aging study data for Bic black ballpoint pen ink on

bond paper: a plot of the average molecular weight of the dye, methyl
violet, versus the age of the document.

Having established that the rate of natural degradation of the dye is paper-
dependent, the decision was made to investigate whether accelerated aging is paper
dependent from data generated from non-white paper. A legal pad, which consisted of
tan paper with a green design on each page, was used for this study. Ink was written on
the different colors of the page, and artificially aged under the same conditions previously
described. There are certain inorganic compounds which are known to catalyze the
degradation of methyl violet in solution (i.e., TiO2), which have been used in the
manufacture of white paper. Colored paper, having a different composition (varying
amounts of whiteners and dyes) than typical white paper, may cause the dye to age at a

different rate. The results of this experiment (not shown), suggested that the artificial ink

73

aging process on the colored and white papers proceeded at similar rates. If ranked, they
would fall in the following order: degradation rate: (tan paper) > (green paper) > (white
paper). In other words, the original hypothesis that degradation on non-white paper
would proceed at a slower rate was incorrect, but one can conclude that the type and
composition of the paper certainly influences the aging process.

Methyl violet in blue ink degrades faster than in black ink. One other factor to
consider when attempting to rationalize the scatter appearing in the uncontrolled natural
aging study (Figure 5.7) involves the influence of ink formulations. Certainly, one would
expect the presence of different dye components, and as a result, changes in the vehicle,
for different ink colors. In the data presented to this point, all of the experiments were
conducted using Bic black ballpoint pen ink. Additional accelerated UV aging studies
using Bic blue ballpoint pen ink were performed and the results are presented in Figure
5.9. Comparing this data to that shown in Figure 5.5, it appears as though components
present in the blue ink cause it to degrade much more rapidly. At 250 minutes of UV
irradiation, for example, it has already surpassed the extent of degradation observed for
the black ink at 500 minutes of UV irradiation. Therefore, if there are components in
blue ink, which are not commonly used in black ink, and they cause the methyl violet to
degrade much faster, this would help explain the scatter present in the uncontrolled,
naturally aged samples (Figure 5.7). Further investigations into the other dyes present in
blue, black, and red ballpoint pen ink, are currently being conducted. Evidence has
already been collected which indicates that other dyes undergo similar oxidative alkyl

degradation processes.

74

 

372 v-~-----------——-~- -—-
R2 = 0.7301

 

_‘ ___z

368 1 1‘ , —- .. . . -2 __-_____

Average Molecular Weight (Da)

 

 

 

 

 

360 :

356 ' l

L . -___ ._ .2- a . ______ I.
O 50 100 150 200 250

UV Irradiation Time (minutes)
Figure 5.9: UV accelerated aging study data for new Bic blue ballpoint pen ink on

printer paper: a plot of the average molecular weight of the dye, methyl
violet, versus minutes of UV irradiation.

Insight into the Uncontrolled Natural Aging Curve

Although the extent of dye degradation in samples 20-50 years old are similar,
this does not necessarily suggest that degradation ceases to occur after 15 years. One
explanation for the data in Figure 5.7 is that, after 15 years on paper, dye molecules do
not continue to react and form degradation products. There could be multiple
explanations for the data. First, if the dye reacts with solvent molecules, the
concentration of the solvent in the paper could be sufficiently small after 15 years that the

reaction essentially ceases. Alternatively, the reaction may involve water, which could

75

always be provided by the relative humidity of the environment in which the document is
stored, but the dye molecules may become immobile over time. They may become
pemianently bound to the paper, trapped in hardened resins, and may be unable to diffuse
to reactive molecules and form products. If these possibilities are correct, then old
samples should not age if one were to subject them to UV accelerated aging. To test this,
a 1958 sample from the naturally aged set was taken and exposed to UV irradiation. The
results of this experiment, which was conducted using the same UV accelerated aging
parameters outlined previously, are shown in Figure 5.10. Initially, the average
molecular weight of the 42-year-old sample was roughly 367 Daltons. Irradiating the
sample with UV light decreased the average molecular weight to approximately 363
Daltons. It is obvious that the ink aging process continued. One interpretation is that
methyl violet on documents that are more than 15 years old can and does continue to
form degradation products, but more slowly than on documents created more recently.
Certainly, in the past 50 years there have been considerable changes in both ink
formulations and in paper chemistry. Ink chemists are learning how to create thinner
films, with different vehicle systems. Older vehicles may form more of a non-reactive,
protective coating on the dye molecules than do newer solvent systems. It would
certainly not be surprising that a document written 40 years ago with a pen that contained
methyl violet in the ink would be a very different chemical system than one created

recently.

76

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Average Molecular Weight (Da)

 

 

W Irradiation (minutes)

Figure 5.10: UV accelerated aging study for ink on a(1958) old document: a plot
of the average molecular weight of the dye, methyl violet, versus
minutes UV of irradiation.

 

R2 = 0.6805

 

 

 

 

Average Molecular Weight (Da)

 

UV Irradiation Time (minutes)

Figure 5.11: UV accelerated aging study data for thermally aged 20 year old ink on
printer paper: a plot of the average molecular weight of the dye, methyl
violet, versus minutes of UV irradiation.

77

A second aspect that is appropriate to point out is that, while UV aging forms the same
degradation products for methyl violet (and other dyes) as natural aging, the mechanisms
are likely not the same. UV radiation forms electronic excited states that may be much
more reactive than ground state molecules. Certainly, between and even within natural
and accelerated aging studies, there may be multiple mechanisms that can lead to the
same set of degradation products.

Solvent molecules from the ink are not the only possible compounds that react
with dye molecules to form degradation products. An additional experiment was
conducted to test the solvent dependency of the dye degradation process. A new sample
was aged in the oven at 100° C for 320 minutes, which is equivalent to 20 years of natural
aging according to a thermal method of aging. Most of the ink solvents should have
evaporated. LDMS spectra of the sample were taken following this aging process. The
spectra showed that the sample was still “new” from a dye degradation standpoint. This
sample was then exposed to the UV accelerated aging experiment. If most of the solvent
was gone, and the degradation of methyl violet was solvent-dependent, then no further
aging would take place. The results of this experiment are shown in Figure 5.11, and
they clearly demonstrate that the aging process can still occur. In fact, the aging process
continues at a rate similar to that observed in Figure 5.4. This may suggest the —H donor
that reacts with the dye molecules may not be a volatile ink component, but may be a
component of the paper or even another dye molecule. Another possibility is that, since
paper is hydrophilic, it quickly absorbs some amount of water vapor from the room, and
can quickly restore sufficient amounts of reactive water molecules in the paper to allow

reaction to occur. Clearly, the chemistry of dye molecules on paper is complex, and

78

much work remains to determine the actual mechanisms through which degradation

OCCUI'S.

79

References

(l) Witte AH. The examination and identification of inks. In: Lundquist F, editor.
Methods of forensic science, vol. 2. New York: Interscience, 1963;35-77.

(2) Sakayanagi M, Komuro J, Konda Y, Watanabe K, Harigaya Y. Analysis of
ballpoint pen inks by field desorption mass spectrometry. J Forensic Sci
1999;44:1204-1214.

(3) Brinen J. The observation and distribution of organic additives on paper surfaces
using surface spectroscopic techniques. Nordic Pulp and Paper 1993;l:123-129.

(4) Csepregi ZS, Aranyosi P, Rusznak I, Toke, L, Frankl, J, Vig A. The light stability of
azo dyes and azo dyeings 1. Light stability of dyeings with reactive and non-
reactive derivatives, respectively, of two selected azochromophores. Dyes and
Pigments 1998;37:1-14.

(5) Galindo C, Kalt A. UV/H2O2 oxidation of azodyes in aqueous media: evidence of a
structure-degradability relationship. Dyes and Pigments 1999;42:199-207.

(6) Andrasko J. HPLC analysis of ballpoint pen inks stored at different light conditions. J
Forensic Sci 2001;46:21-30.

(7) Aginsky VN. Dating and characterizing writing, stamp pad and jet printer inks by gas
chromatography/mass spectrometry. Int J Forensic Doc Examiners. 1996;2:103-

116.

(8) Cantu AA. Comments on the accelerated aging of ink. J Forensic Sci
1988;33:744-50

80

Chapter Six: Ink Aging Inside of a Pen

Aging inside of a pen could contribute to a significant error in relative ink age
determination. A common assumption amongst questioned document examiners is that
ink does not begin the aging process until it is applied to paper. The pen cartridge is
considered a “closed system”, while the ink-on-paper is considered an “open system” (1).
Many ink dating methods are somehow related to the evaporation of the solvent present
in ink, as a function of age (1-1 1). Consequently, the hypothesis is that only a negligible
amount of ink is evaporated from the open ends of the pen cartridge over time. However,
there is a notable absence of any published scientific studies supporting this hypothesis.
Dye degradation is very different than solvent evaporation. Therefore, ink inside of old
pens were subjected to laser desorption/mass spectrometric analysis.

In respect to analyzing the dye component to determine the age of an ink, the
difference between a “new” or fresh ink, and an “old” or aged ink, in terms of mass
spectral data, has been established in the Chapter 5. Figure 6.1 shows the positive ion LD
mass spectrum of ink on paper from a 20-year-old pen (pen #1). The base peak at m/z
372 represents the cationic dye, crystal violet. Thus, the mass spectrum shown if Figure
1 is typical of what would be obtained for a new ink, and is characterized by a large peak
at m/z 372, with a small or absent peak at m/z 358. This suggests that these dye
molecules are stable in this pen, and have not “aged”.

For these experiments, a variety of old pens were gathered which were known to
be 5 to 20 years old. Positive ion LD mass spectra were obtained of the pen lines which
were drawn using them. Pen #1, used to obtain the data in Figure l, was roughly 20

years old, and was known not to have been used for years. The positive ion LD mass

81

spectra obtained from any point along the pen lines were consistent with the spectrum in
Figure 6.1, indicative of new ink. Other pens tested generated the same results, lending
support to the assumption that ink does not age inside of the pen. However, some of the

old pens produced spectra indicative of old ink, which required a more in depth analysis.

82

/

Q/ __.

 

 

 

 

 

 

 

.2: Hie
2 O
a
s
5’ , CH:
N
3 H30
358
2‘. A“ . . . '
350 356 362 368 374 380
mlz

 

 

Figure 6.1: The positive ion LD mass spectrum of ink on paper from Pen #1.

83

A second old pen, pen #2, yielded ink that was clearly "aged" ink. Figure 6.2
shows the positive ion LD mass spectra of the ink from pen #2, which were taken at
various points along the length of the pen line. As the dye degrades, the crystal violet dye
molecules continue to lose -CH3 groups. Evidence for this process appears in each of the
mass spectra of Figure 6.2. A significant increase in the intensity of the m/z 358 peak,
and the appearance of a small peak at m/z 344 are noted. The presence of the degradation
products is an indication that pen #2's ink has been aging in the pen. All three spectra are
very similar and the dyes have degraded, but to what extent have they degraded, when
compared to naturally aged ink-on-paper samples?

The calculation of the average molecular weight of the dye and its degradation
products from LD mass spectral data was recently proposed as an effective way to
determine the extent of dye degradation. Furthermore, a controlled natural aging curve
was created from ink library samples (Speckin Forensic Laboratories, Okemos, MI),
which related the actual age of the ink to the average molecular weight of the dye
computed from mass spectral data. From the mass spectral data in Figure 6.2, the
average molecular weight of the dye from each spectrum was approximately 363 amu. It
is important to note that the visual variations among the three spectra did not have a
significant effect on the calculation of the average molecular weight. Compared to the
natural aging of ink on paper, a dye with an average molecular weight of 363 corresponds
to roughly 35-ycar-old ink. So, in this particular pen, the ink aged faster than if it had

been on paper.

84

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

g. 358 372
2
a
_=.
o
I 5 344
G
L '- 3 Al- -h‘ M
5 ‘ 330 340 350 360 370 380
I m/z
. ‘ 372 1
~ .2‘
. g 358
a
s
9 344
25
i 1 IL
0: . - - ,
330 340 350 360 370 380
mlz A
S a 353 372
' ' 2
“.‘I ll
5
" o
‘, 2 344 l
: 11. IL
1 a A A
330 340 350 360 370 380
| I mlz

 

 

Figure 6.2: The positive ion LD mass spectra of ink along a written line from Pen #2.

85

Considering that a pen cartridge holds enough ink to write a line approximately
3000 m long (1), the sample quantity of ink analyzed in the experiment is not a fair
representation of the ink throughout the entire pen cartridge. The ink may only have
degraded near the tip of the pen, since it is more exposed to atmospheric gases and light,
which may catalyze the oxidative demethylation process. Thus, some pens were
disassembled to investigate the issue further.

Ink from pen #1, taken from the region closest to the metal pen tip, the middle
region of the cartridge, and the open, far end of the cartridge, generated LD mass spectra
indicative of new ink (Identical to that shown in Figure 6.1). The data indicate that the
ink remained stable and intact throughout the entire ink cartridge. Figure 6.3 shows the
positive ion LD mass spectra of ink from pen #2, taken from three regions inside of the
ink cartridge. As in Figure 6.2, the spectra in Figure 6.3 are all very similar, and
representative of a dye with an average molecular weight of 363 amu. The slight
differences realized within the cartridge itself are small. The significance arises when
trying to determine the relative age of a questioned document, when the pen used

contained dye that, from a degradation standpoint, is already 35 years old.

86

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

> a 358 372
.Ԥ
Ink at open end :3
2
> E 344
b 358 372
Ink in cartridge "5
' E
é
a: 344 “I
372
C 358
Ink in pen tip g
' E
g 344
C!

 

 

 

 

 

Figure 6.3: The positive ion LD mass spectra of ink within the cartridge of Pen #2 :
a) ink removed from open end of cartridge; b) ink removed from middle of
cartridge; c) ink removed from pen tip.

87

References

(1) Cantu AA. A sketch of analytical methods for documenting dating. Part II. The
dynamic approach: Determining age independent analytical profiles. Int J
Forensic Doc Examiners 1996;2:192-208.

(2) Brunelle RL, Pro MJ. A systematic approach to ink identification. J Official Anal
Chem 1972;55:823-26.

(3) Brunelle RL. Determining the relative age of ballpoint ink using a single-solvent
extraction, mass-independent approach. J Forensic Sci 1989;3411 166-82.

(4) Brunelle RL. Ink-dating — the state of the art. J Forensic Sci 1992;37:113-24.

(5) Brunelle RL. A sequential multiple approach to determining the relative age of
writing inks. Int J Forensic Doc Examiners 1995;1294-98

(6) Cantu AA, Prough RS. On the relative aging of ink — the solvent extraction
technique. J Forensic Sci 1987;32:1151-74.

(7) Cantu AA. Comments on the accelerated aging of ink. J Forensic Sci
1988;33:744-50

(8) Cantu AA. Analytical methods for detecting fraudulent documents. Anal Chem
1991 ;63:847-54.

(9) Kikuchi Y. Estimation of the age of the writing in blue black ink (first report).
Japanese Police Science Laboratory Report 1959;12:379-86.

(10) Aginsky VN. Dating and characterizing writing, stamp pad and jet printer inks by
gas chromatography/mass spectrometry. Int J Forensic Doc Examiners
1996;2:103-116.

(l 1) Stewart LF. Ballpoint ink age determination by volatile component comparison -
a preliminary study. J Forensic Sci 1985:30;405-411.

88

 

Chapter Seven: Conclusions & Future Work

Laser desorption mass spectrometry (LDMS) is a sensitive analytical tool for the
detection of intact dye molecules and their degradation products directly from a paper
substrate. This molecular level information can be used for simply identifying a specific
dye component present in the ink, or possibly for determining the relative age of an ink
on a questioned document, based on the extent a dye has degraded. As many other
relative ink age determination techniques, our method based on the analysis of dye
degradation products, also has its limitations. All ink dating methods are in some way
dependent on uncontrollable variables, such as ink formulation, type of paper, and
storage conditions. In a typical questioned document forensic science case, storage
conditions most often need to be inferred, and the original pen used to write the document
will most likely never be recovered. Furthermore, documents aged in the “real world”
under natural conditions are very different from those produced artificially in the
laboratory. Although great measures have been taken to make the two conditions the
same, there is no exact correlation between the two, and perhaps only approximations can
be derived. These limitations have driven some skeptics to propose that relative ink age
determination by any method is impossible (1,2). Up to this point, the emphasis has been
on solvent extraction methods, with minimal research into the different possibilities
available from the analysis of dyes (3). Perhaps, the solution to deve10ping the “perfect”
relative ink age determination involves a combination of solvent extraction methods and
dye degradation product analyses.

While this work focused on one dye, methyl violet, there are other common dyes,

such as the rhodamine-type dyes, found in inks. In the case of Rhodamine B and

89

Rhodamine 6G, both dyes have the same molecular weight, but one has four ethyl groups
attached to N atoms, while the other has two ethyl groups, respectively. When exposed
to a light source, the ethyl groups are lost and replaced by H atoms, similar to what
occurs with methyl violet. LDMS can be used to distinguish between the two dyes, by
identifying the number of peaks present in the mass spectrum resulting from the loss of
ethyl groups. Thus, accelerated aging is being explored as a means to degrade dyes for
characterization purposes, as opposed to ink dating purposes. Other light sources, such as
incandescent light bulbs and fluorescent lamps, are also being explored as an alternative
to UV light. Overall, the full potential of using LDMS for the analysis of questioned

documents has not yet been obtained.

90

References

(l) Aginsky VN. Measuring ink extractability as a function of age - why the relative
aging approach is unreliable and why it is more correct to measure ink volatile
components than dyes. Int J Forensic Doc Examiners 1998;4z214-230.

(2) White PC. Is it really possible to determine the age of an ink? Proceedings of the
54th Annual Meeting, American Academy of Forensic Sciences; 2002 Feb 11-16;
Altanta, Georgia.

(3) Andrasko J. HPLC analysis of ballpoint pen inks stored at different light conditions. J
Forensic Sci 2001;46:21-30.

(4) Dunn JD, Grim DM, Allison J, Siegel JA. Molecular level information on the aging
of dyes from ink on paper: What's there and how does it change? Proceedings of
the 54th Annual Meeting, American Academy of Forensic Sciences; 2002 Feb 1 l-
16; Altanta, Georgia.

91