EXPERIM E NT

a Forensic Chemistry

L Crime Scene lnvestigotion

i

Purpose to assemble the pieces of a puzzle-these are all

C . Experience some of the challenges inherent
aspects of forensic science.
Forensic science spans most of the natural sci-
( in the field of forensic science, including careful ences, including chemistry, biochemistry, biology,
observation and thorough record-keeping. physics, geology, and medicine. The field extends
into engineering, anthropology, and psychology as
L . Explore the laboratory techniques involved in
chromatography to illustrate the utility of multi- well. By far, the most common techniques are
component analysis and identification. those of chemists: chromatography and other tech-
niques for analysis of inks, toxins, adulterants,
o Demonstrate the utility of chemical spot tests, fibers, drugs, explosives, gunshot residues, and
a form of qualitative analysis, for revealing the
L invisible and identifying the presence of previously
flammable materials. Biochemical analyses have
recently become important forensic tools, includ-
unknown substances. ing the identification of blood, semen, and DNA.
Fascinating examples of biological applications to
forensics include the entomology and microbiol-
lntroduction
L Forensic science (science applied to legal settings)
ogy of decay processes. Fingerprint analysis, the
study of patterns of blood spatter or glass frag-
is a popular topic of the day. Television series like mentation, and bullet trajectories are examples in
L CSI: Crime Scene Investigation, Law and Order, which the physics and physical properties of mate-
Forensic Files, and Bones were for years on the list rials are studied. The investigation of computer
L of most-watched shows. But in previous genera-
tions, the topic was just as popular, evidenced by
crimes, forgeries, sound recordings, and serial
crimes require techniques from a variety of other
Sir Arthur Conan Doyle's works featuring Sherlock disciplines. It's no wonder, then, that forensic sci-
\- Holmes in the late nineteenth century and Agatha entists typically specialize in only one aspect of
I- Christie's famous investigators, Hercule Poirot and this broad field and are usually trained in an
Miss Jane Marple, solving crimes of the twentieth apprentice setting after obtaining degrees in chem-
century. One of the earliest stories in the history of istry, biochemistry, or biology.
science, Archimedes deducing the essential con' The training of all scientists includes emphasis
cept of buoyancy, was occasioned by a possible on the need for accurate records, but the demands of
crime: whether the king's crown had been adulter- the courtroom and the high stakes of legal proceed-
ated with metals less valuable (and less dense) than ings place extra demands on forensic scientists for
gold. systematic record-keeping and sample storage.
In these earlier eras as well as ours, the fictional These urgent duties also dictate rigorous attention to
investigators seem to require much less time and instrument calibration, blank and standard samples,
t effort to solve their crimes than real-life forensic sci- and sources of possible contaminants or interfer-
entists do. Reading about or watching forensic scien- ences. The "chain of custody" requirement not only
tists at work may be more enjoyable than performing demands that the laboratory personnel keep thor-
the careful collection and analysis procedures that ough records but that they document the entire his-
are required. But the reward of setting an innocent tory of the sample from collection through trial (and
person free or helping to convict a guilty party can preserve the samples in long-term storage in case
be quite motivating, as is the satisfaction of helping further investigation is necessary). Custody issues

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 (*
3-2 Forensic Chemistry

also demand that a sample be protected from con- The word chromatography-formed from the
tamination or degradation, a special challenge for Greek words khroma, meaning "color," and graphein,
biological samples. meaning "to draw or to write"-was coined by the
Russian botanist M. S. Tswett around 1906 to
describe his process of separating mixtures of plant
Pre-lob Preporofion pigments. He allowed a solution of plant pigments to
percolate down a glass column of absorbent powder,
Observotions ond record-keeping
where they separated into colored bands-literally, a
Making observations is the starting point of any sci- "color drawing" on the powder. Even though most
entific enterprise, even if it isn't always the first step chromatographic methods are now used on solutes
chronologically. This is certainly true in the case of that do not produce bands of color visible to the
forensic science, where the preservation of a crime human eye, the term chromatography is applied to
scene is second in priority only to the preservation
\-
any separation procedure employing the same prin-
of human life. Careful recording of observations is ciple as the method described by Tswett. Chromato- 1

essential in any lab setting, but especially so in the graphy is widely used for qualitative or quantitative
context of forensic science where a person's life or analysis or for isolation and recovery of the compo-
freedom may be at stake. Accurate and understand- nents of a mixture.
able records are also necessary in criminal or civil All forms of chromatography employ the same
proceedings because so many different participants general principle: A mixture of solutes in a moving
need to access and process the information: police phase passes over a selectively absorbing medium,
officers, detectives, crime scene investigators, t-he stqtionqry phase. Separation occurs because the
lawyers for all parties involved, and judges. solutes have different affinities for the stationary
Many of you may use the Report form pages of and moving phases. Solutes that have a greater affin-
this lab book to record your observations, make your ity for the moving phase will spend more time in the
calculations, and state your conclusions. In real labo- moving phase and therefore will move along faster
ratory settings, especially in a forensic laboratory, than solutes that spend more time in the stationary
this would not suffice as a lab notebook. A perma- phase.
nently bound (not spiral bound or glued) notebook There are several ways to achieve a flow around l

would be required; data would be written in ink, on or through a stationary phase. ln column chromato-
dated pages that include all original measurements, graphy, small particles of the stationary phase are
notes, calculations, comments, and conclusions. packed in a tube; the moving phase (a liquid or gas)
Electronic equivalents of this type of record are now flows around the particles confined in the tube. In
accepted, but not in a simple document or spread- thin-layer chromatography (TLC), the stationary
sheet form, as these are easily modified. phase is a thin layer of fine particles spread on a
In this experiment, we will explore your powers glass or plastic plate. In pqper chromatography, the
of observation and record-keeping in the laboratory paper itself forms the stationary phase, along with
as a pair of necessary, but not sufficient, skills to per- any solvent adsorbed to the paper. Figure 3-1 illus-
form forensic analysis. trates the separation of a sample mixture as a mov-
ing liquid phase flows through a stationary phase
(such as paper).
Chromotogrophy
In this experiment, we will employ paper chro-
Separating two substances that have quite different matography. The cellulose structure of paper (shown
physical properties is usually a simple and straight- in Figure 3-2) contains a large number of hydroxyl
forward task. A mixture of sand and salt can easily be groups, that can form weak bonds (called hydro-
-OH,
separated by adding water to dissolve the salt, filter- gen bonds) to water molecules. So the stationary phase
ing the mixture to remove the sand, and recovering can be regarded as a layer of water, hydrogen-bonded
the salt by evaporating the water. But what if the sub- to cellulose. If the solvent is water, the moving phase
stances to be separated have similar physical and is also aqueous; but if a mixture of an organic solvent
chemical properties? Separation of a mixture of sev- is used with water, the moving phase is apt to contain
eral closely related substances presents a more diffi- a high proportion of the organic solvent.
cult problem, but the problem has been solved by The solvents employed in chromatography must
the development, over the past I00 years, of a family wet the stationary phase so that the mobile phase
of powerful separation techniques that collectively will move through the paper fibers by capillary
are called chromatography. attraction. A solute in the mobile phase moves along
L Forensic Chemistry 3-3

a
E= t
p
f
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l.t, I

C o
()
.9
( o E
.9,
i5 o

C A B D
Concentration
E

t
FIGURE 3-l ! Schematic illustration of the separation of a three-component mixture by differential
migration on a strip of paper. Parts (A) through (D) represent a time sequence showing how the
e components move along at different rates, (A) At the beginning of a separation, the three components
are clustered together at the point of sample application. (B-D) As the sample migrates along the paper
strip, the three sample components are gradually separated, the fastest-moving component spending
the most time in the moving liquid phase. (D) The final distribution of substances. (E) A smoothed
plot of the concentration of each substance as a function of the distance from the origin, for the
distribution shown in (D).
L
with the solvent during the chromatographic devel- of its time in the stationary phase, it will stay near
\- opment (the term used to describe the process that the point of application.
occurs as the solute moves along and is partitioned After the chromatogram has been developed and
between two phases). As the solute moves, it under- the solutes on the paper have been located, the
goes many successive distributions between the movement of the solute on the paper is expressed by
mobile and stationary phases. The fraction of time it the Rlvalue (called the retention factor), where
(_ spends in the mobile phase determines how fast it
moves along. If the solute spends all of its time in . distance traveled by the solute
^r-
! the moving phase, it will move along with the solvent
front (the leading edge of the solvent moving The distances used in calculating R/ values
(_ through the stationary phase). If it spends nearly all are measured as shown in Figure 3-3. The distance

zOH H CH zOH
HO
H
OH H OH
OH
HH H

ti

FIGURE 3-2 t fne structure of cellulose. Carbon atoms are located also at the intersections of
the lines. The wavy lines indicate that the overall structure consists of multiple chains of these
units. Note the many polar -OH groups on the cellulose. Such groups have an affinity for water
i molecules,

L
3-4 Forensic Chemistry

used, they are perfectly designed for placing small

r Solvent Iront
amounts of ink on the paper. Then the spots are
allowed to dry, and the filter paper is placed in a
tightly closed receptacle containing the developing
solvent, whose vapor saturates the atmosphere of
the container. The paper is supported so that the
0 developing solvent moves through the spots that
Distance trave led have been applied. The point of application must not
by the solvent be immersed in the developing solvent; otherwise,
front
Drstance traveled the solute spots will diffuse into the solvent and be
by the solute greatly diluted.
The solvent flow is allowed to continue for a fixed \-
time or until the solvent front reaches a specified
point on the paper. Then the paper is removed, the
solvent front is marked with a pencil, and the paper
Point of is dried; the result is that the separated solute spots
application
are fixed in their position at the end of development.
If the solutes are colored, they can be readily
! seen on the paper. Colorless spots are located by var-
FIGURE 3-3 | ffre movement of the solute on the ious means. Often the paper is exposed to the vapor
paper chromatogram is expressed by the Rlvalue,
the distance traveled by the solute divided by the of a reagent (such as iodine, which makes organic
distance traveled by the solvent front, compounds visible), or it can be dipped in or sprayed
with a reagent that reacts with the colorless sub-
stances to form a visible spot. When placed under an
traveled by the solute is measured from the point of ultraviolet light, many substances can be located by
application to the center or densest part of the spot; their fluorescent glow.
the distance traveled by the solvent front is mea- After the spots are located and their R1 values
sured from the point of application to the limit of measured, they are compared with standard known
movement of the solvent front (which must be materials run under the same conditions. If possible,
marked immediately after the paper is removed from known and unknown substances are run on the same
the developing chamber because it may be invisible paper. If the known and unknown compounds have
after the solvent evaporates). the same Rp values in different solvent systems and
If all conditions could be maintained constant, R, give the same reactions with various chromogenic
values would be constant. However, variations in (color-forming) reagents, they are probably identical.
temperature or in the composition of the solvent
phase or changes in the paper can alter the R1 value. Experimentol Procedure
The R1 value is useful mainly for expressing the rela-
tive mobility of two or more solutes in a particular Special Supplies and Equipmenti Part l: Pennies
chromatographic system. The absolute Rlvalues may or other coins (at least one per student); 5-I0X magni-
change from day to day, but their values in relation fiers (plastic magnifiers, loupes, and low-power micro-
!
to each other remain nearly constant. scopes are available from Edmund Scientifics, http://
Paper chromatography can be performed on scientificsonline.com, or Educational Innovations, Inc.,
strips or sheets of paper, with the solvent being http://www.teachersource.com). Optional procedure:
allowed to flow either upward or downward. Hori- 1.5 x 1.5 in. x 2 mil ziplock poly bags, http://discount-
plasticbags.com; or coin envelopes.
zontal development may also be used, in which case
the solvent is allowed to flow radially outward from Part 2: Assorted blue or black ink pens; Whatman
the center. Two-dimensional chromatography can be No. I filter paper, 46 x 57 cm sheets (pre-cut each sheet
used to separate complex mixtures by applying two into twelve 11.5 x 19 cm pieces); 30-cm ruler; soft lead
different solvent systems. The paper is first devel- pencils; plastic food wrap; Size 33 rubber bands; scis-
oped in one direction, then turned by 90'and devel- sors; stapler. Optional procedure: 9-in. Pasteur capillary
pipets; ultraviolet lamp (UV light).
oped again in a second solvent.
Development is accomplished in the following Chemicals: Separation of pen inks: The developing
way. First, the solution containing the solutes to be solvent solution is "rubbing alcohol" (70% isopropyl
separated is spotted on the dry paper. If ink pens are alcohol,/water, v/v). Optional procedure: methanol.
(*
Forensic Chemistry 3-5

2. Chromatography of Pen Inks lf pre-cut sheets

I are not already prepared for you, cut an 11.5 x
The degree of difficulty of part I can be increased by
19 cm piece of Whatman No. I filter paper. Using a
supplying extra coins to the "evidence" pool or limit-
soft lead pencil (not an ink pen), draw a line parallel
a ing the selection of coins to a single type, a small
number of mint marks or dates, or by withholding
to the long dimension about 2 cm from the edge. Still
using a pencil, put five small X's on the line, begin-
from students information about the range of the
L coin characteristics (e.g., year and mint). The degree
ning 5 cm from the edge of the paper and spacing the
marks about 2 cm apart. (The X's locate the points
of forensic realism can be increased by instructing
where you will later spot the inks.) Put 70 mL of the
C students in appropriate sample-handling proce-
developing solvent (70% isopropyl alcohol in water)
dures, such as wearing gloves and using plastic
in the bottom of a clean, dry 800- or I000-mL beaker
tweezers (to avoid fingerprints and other contamina-
I
and cover the beaker with plastic wrap held in place
tion) and sample storage (a small ziplock bag or coin
by a rubber band-that way, the enclosed air will
envelope).
become saturated with the vapors of isopropyl alco-
C' hol and water.
I
Using a scrap of filter paper, practice applying
7, Observations and Record-Keeping Choose a small (2-3 mm diameter) ink spots on the paper. For
coin of your own or one supplied by your instructor. ballpoint pens, this will involve marking a small circle
L Handle the coin carefully from this point forward so
that you do not contaminate the "evidence." If your
of ink, going over it several times without enlarging it
beyond 3 mm; for felt-tip pens, you should hold the
instructor directs, obtain an evidence container pen to the paper until the ink has spread to form a 2-3
(paper envelope or plastic bag) and mark it with mm circle. Allow the paper to dry for a minute, then
appropriate identifiers. repeat thls process five or six times. When you are
Make as many observations of your coin as you confident you have mastered the technique of apply-
can and record them on the Data Sheet of your labo- ing the spots, lay the 11.5 x 19 cm piece of paper
L ratory notebook. At a minimum, you will record the
date and mint mark of your coin, but your goal is to
down on a clean paper towel or sheet of clean paper
and carefully apply a small (2-3 mm) spot of ink on
note as many unique aspects of your coin as possi- the first X. Continue spotting the different inks, using
ble so that you can distinguish it from all other a different pen on each X and writing in pencil near
coins in your laboratory. You may wish to use a each X a letter symbol to identify the pen spotted
L magnifying glass or loupe or other available labora- there. Record the identity of each spot. Allow the
spots to dry by gently waving the paper in the air.
tory equipment.
L When you have finished with your observations
and recording tasks, return the coin to your instruc-
After the spots have dried, gently curve the paper
into a cylinder shape, as shown in Figure 3-4, and
tor. Your instructor will return a coin to you. Observe staple the edges together, leaving a gap so that the
( this coin and compare it to the description in your edges do not quite meet. Put the paper cylinder in
lab notebook. Determine whether you believe this is the beaker containing the developing solvent, plac-
L the same coin you originally studied or a different
coin.
ing the spots at the bottom and taking care that the
paper does not touch the wall of the beaker. The
L Form groups of four or five students. One at a
time, attempt to convince your group that this is or
spots of ink must be above the surface of the solvent
in the beaker. Immediately seal the beaker with plas-
I
is not your original coin. The only evidence you may tic wrap secured with a rubber band and allow the
cite is that in your laboratory records. The members solvent to rise to within I cm of the top of the paper.
of your group may challenge you to show them your (This usually takes 30-40 minutes.) Note the position
L recorded observations.
At the end of your presentation, pass a slip of
of the solvent front (the wet-looking leading edge of
the solvent) every few minutes so that you do not let

t paper to each of the group members and ask them to

render their verdict; they should write "is" or "not" on
the solvent front rise all the way to the top of the
paper. Also note how the spots begin to separate into
the ballot, indicating whether they are convinced bands of color as they move along.
L "beyond a reasonable doubt" that your coin is or is
not your original coin. You may interview the mem-
When development is finished, remove the paper
from the beaker and immediately mark the solvent
L bers of your jury group to help you determine which
of your observations were critical to the case and
front with a pencil line before the solvent evapo-
rates. Then dry the paper thoroughly, preferably in
(* which were not. the hood.

L
3-6 Forensic Chemistry

Optional: For an even more realistic test of your

\-..-_ -<-
forensic skills, have your instructor sign a piece of
ordinary paper; you can think of it as an important
letter or a will that might have been forged. (Or your
instructor could sign two documents with the same
pen or two different pens.) Carefully cut the signa-
ture from the document and then cut the signature
Staples into small pieces, small enough to fit into a 20 x
I50 mm test tube. (lf this were a real-life situation, a
forensic laboratory technician would use a syringe
A B needle to punch small samples of ink-laden paper to
be extracted. The original document needs to be pre-
served for courtroom display and further analyses if
i the judicial process so requires.)
FIGURE 3-4 i Gently curve the chromatographic Add 2-3 mL of methanol to the test tube, enough
paper into a cylinder shape and staple as shown
in (A). Put the paper cylinder carefully into the to cover the pieces. Heat the test tube in a water bath
beaker containing the solvent, then cover with ifneeded to increase and hasten the dissolution of
plastic frlm held in place by a rubber band, as the ink, but do not get the tube too hot or else the
shown in (B), methanol will evaporate. You can also use a Pasteur
pipet to squirt the hot methanol over the pieces of
paper. When most of the ink has been extracted from
After the paper has dried, locate the densest part the paper, use a transfer pipet to transfer the
of the band for each color and draw a pencil line methanol solution to another small test tube. Heat
through it. Measure the distance (in millimeters) this test tube until most, but not all, of the methanol
from the pencil line at the point of application to the has evaporated. Then use a transfer pipet to spot
densest part of the band of color, as well as the dis-
this sample onto your chromatograph paper. Add
tance from the point of application to the solvent spots from the suspect pens to the chromatograph
front. Record the values in your report. Calculate and and develop as before.
record the Rf values for every colored spot on the
paper. For each original spot of ink, describe the
number of components you find and their colors.
Can you identify each of the inks by its chro-
matogram? If your neighbor used the same set of
SAFETY PRECAUTIONS:
pens, can you match them by comparing your tables
of R1 values without comparing the chromatograms?
I The isopropyl alcohol and
(Note that this is how chromatographic analyses are methanol used in part 2 are flammable. No
open flames (e.g., Bunsen burners) should
typically compared.)
be used in the laboratory during this
Pencil your name and the date on the chro-
experiment. Dilute solutions of the i

matogram and turn it in with your lab report if your

alcohols are not flammable and readily
instructor so directs.
biodegrade in the wastewater disposal
Matching the Ink: If you wish to test your forensic process of the community.
skills, select four pens with the same ink color; have
your instructor select one of the pens and mark one
spot on a new piece of chromatograph paper (set up
as previously,
with the pencil line and five X's). Next,
make a spot with each of the four pens on the chro-
matograph paper. Develop the chromatogram

When you are finished developing all of your

chromatograms, add water to the beaker until it is
as
before. Determine the identity of the instructor's pen. A WASTE COTLECTION:
The alcohol solvents used in this
experiment are ordinary household
chemicals and can be disposed of in the
nearly full. This diluted solution can be poured diluted form described at the end of the
down the drain unless your lab instructor directs experimental procedure.
otherwise,
(*
Forensic Chemistry 3-7

coNstDER rHIs Bibliogrophy

Are any of the pigments used in the pen inks fluores- Bergslien, E., "Teaching to Avoid the'CSI Effect':
cent? If an ultraviolet light is available in the labora- Keeping the Science in Forensic Science," J. Chem.
tory, test for this property. Are the fluorescent pig- Educ. 2OO6, 83(5), 690-69 1.
ments in addition to the ones you saw in the Berry K., Ishii, G. G., "Forensic Chemistry: An Introduction
chromatogram or are some of the pigments both col- to the Profession," J. Chem. Educ. 1985, 62(12), 1043.
ored and fluorescent? Why might a manufacturer add Breedlove, C. H., "The Analysis of Ball-Point Inks for
fluorescent pigments to its inks? Forensic Purposes," J. Chem. Educ.1989, 66(2),
l. Can you distinguish between an original signa- t70-17 L.
ture and a copy made on a color photocopier or
I
printer? Can you distinguish an inkjet-printed copy Quigley, M. N., Qr, H., "A Chemistry Whodunit," J. Chem.
Educ. 1991, 68(7), 596-597.
from a conventional photocopy?
(_ Experiment with other developing solvents, Strain, H. H., Sherma, J., "Michael Tswett's Contributions
such as 5% acetic acid (household vinegar). Does an to Sixty Years of Chromatography," l. Chem. Educ.
ink developed with rubbing alcohol give the same 1967, 44,235. This article is immediately followed
number of spots as the same ink developed with on page 238 by a translation of Michael Tswett's
I

vinegar? Do they give the same R1 values? Why or article, "Adsorption Analysis and Chromatographic
L why not? Methods," published in I906 in Berichte der
d euts chen b ot an is c hen G esells chaft.

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t RE PO RT 3
Forensic Chemistry Name

Crime Scene Investigation Date Section

t I. Observotions ond record-keeping

Sample Identification:
(a) Describe the key identifying marks of your sample evidence.
]
(-_

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!-\
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I
(b) Do you believe that the coin returned by your instructor was the same as the coin that you initially
studied? What were the key identifiers?
\.-

t
\._
(c) Did you persuade the jury? (Remember, in a criminal trial you must obtain a unanimous verdict; in a
civil trial, only 3/n of the jury usually needs to agree.)
(_

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(d) Which of your observations proved crucial to your case? Which were not needed?
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3-9
 I

2. Chromotogrophy of pen inks

Identity of Pens: 1 2

J 4 5

Pen: I 2 3 4 5

Component I
Color of spot
Distance traveled by spot _mm mm mm mm _mm
Distance traveled by
solvent front _mm _mm mm mm mm

R1 value

Component 2
Color of spot
Distance traveled by spot _mm _mm mm mm mm
Distance traveled by
solvent front mm _mm _mm _mm mm

R1 value
\

Component 3
Color of spot
Distance traveled by spot mm mm mm mm mm
Distance traveled by
solvent front mm _mm mm mm mm

R; value

Component 4
Color of spot
Distance traveled by spot mm _mm mm mm mm
Distance traveled by
solvent front mm _mm _mm _mm mm

R, value

3-ro
\_
Question
l. Does your neighbor's chromatogram have an ink that shares the same set of R1 values as one or more of
your inks?
l

Motching rhe ink

Identity of Pens: I Instructor's 2

(.--
J 4. 5

t
L Component I
Pen: 2 3 4 5

Color of spot
Distance traveled by spot mm _mm mm mm mm
(
Distance traveled by
solvent front mm _mm _mm _mm mm

R1 value

L Component 2
Color of spot
Distance traveled by spot mm mm mm mm
(_- Distance traveled by -mm
solvent front _mm mm mm mm mm
(-
R; value

L Component 3
Color of spot
L Distance traveled by spot mm mm _mm mm _mm
Distance traveled by
L solvent front _mm _mm _mm _mm _mm
i
Rp value

Component 4
Color of spot
t Distance traveled by spot mm mm _mm _mm mm
Distance traveled by
solvent front mm mm _mm _mm mm

\ R1 value

Identity of the Instructor's Pen:

\*
3-r r
Are any of the pigments used in the pen inks fluorescent? If an ultraviolet light is available in the laboratory,
test for this property. Are the fluorescent pigments in addition to the ones you saw in the chromatogram or are
some of the pigments both colored and fluorescent? Why might a manufacturer add fluorescent pigments to its
inks?

Can you distinguish between an original signature and a copy made on a color photocopier or printer? Can you
distinguish an inkjet-printed copy from a conventional photocopy?
Experiment with other developing solvents, such as 5% acetic acid (household vinegar). Does an ink developed
with rubbing alcohol give the same number of spots as the same ink developed with vinegar? Do they give the
same Rlvalues? Why or why not?

3-12