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April 1999 Volume 1
Number 1
Chapter 4 of Forensic
Fiber Examination Guidelines
1.0. Scope
Metameric coloration of fibers
can be detected using UV/visible spectroscopy. If spectroscopy
is restricted to the visible spectral range, differences in dye
components may remain undetected. One method of detecting additional
components is to use thin-layer chromatography (TLC). TLC is
an inexpensive, simple, well-documented technique that can be
used, under certain conditions, to complement the use of visible
spectroscopy in comparisons of fiber colorants. The principle
of the method is that the dye components are separated by their
differential migration caused by a mobile phase flowing through
a porous, adsorptive medium.
2.0.
Reference Documents
SWGMAT Quality Assurance
Guidelines
SWGMAT Trace Evidence Handling Guidelines
ASTM E1492-92 Practice for Receiving, Documenting, Storing, and
Retrieving Evidence in a Forensic Laboratory
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3.0. Terminology
Activation: The heating of the adsorbent layer
on a plate to dry out the moisture and maximize its attraction
and retention power.
Adsorbent: The stationary phase for adsorption
TLC.
Adsorption: The attraction between the surface
atoms of a solid and an external molecule by intermolecular forces.
Chamber: A glass chamber in which TLC development
is carried out.
Thin-Layer Chromatogram: The series of spots visible on the
adsorbent layer after development.
Chromatography: A method of analysis in which substances
are separated by their differential migration in a mobile phase
flowing through a porous, adsorptive medium.
Development: The movement of the mobile phase
through the adsorbent layer to form a chromatogram.
Dye Extraction: The removal of the dye from a fiber
by incubating it in an appropriate solvent.
Eluent: The solvent mixture that acts as
the mobile phase in TLC.
Extractant:
Metameric Pair: Two colors that appear the same
under one illumination but different under another illumination.
Mobile Phase: The moving liquid phase used for
development.
Normal-Phase Chromatogram: Adsorption in which the stationary
phase is polar in relation to the mobile phase.
Origin: The location of the applied sample
or the starting point for the chromatographic development of
the applied sample.
Resolution: The ability to visually separate
two spots.
Retardation Factor (RF): The ratio of the distance traveled
by the solute spot's center divided by the distance traveled
by the solvent front, both measured from the origin.
Saturation: Chamber equilibration with mobile-phase
solvent vapor prior to chromatography.
Solute: In TLC, a mixture of components
to be separated.
Solvent Front: The final point reached by the mobile
phase as it flows up or across the TLC plate during development
of the chromatogram.
Spot: A round zone of sample application
at the origin, or in a chromatogram, a round zone caused by migration
of a component of the solute.
Spotting: Applying a solute sample at the
origin of the TLC plate.
Stationary Phase: The solid adsorbent coating layer
of a TLC plate.
Tailing: A spot distorted during development
into an elongated streak.
Thin-Layer Chromatography
(TLC): A separation
technique in which the flow of solvent causes the components
of a mixture to migrate differentially from a narrow initial
zone in a thinly applied porous adsorptive medium.
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4.0.
Summary of Guidelines
These guidelines are intended
to advise and assist individuals and laboratories that conduct
forensic fiber examinations and comparisons in their effective
application of TLC to the analysis of fiber evidence.
These guidelines are concerned
with the extraction of dyes from single fibers and from bulk
material, classification of the dye or colorant, application
and development of the extractants on TLC plates using an optimal
elution system, and evaluation and interpretation of the resulting
chromatograms. The protocols and equipment mentioned in this
document are not meant to be totally inclusive or exclusive.
Not all fiber type or dye
class combinations are covered in these guidelines.
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5.0.
Significance And Use
5.1. Forensic Analysis
of Fiber Colorants Using TLC
Forensic analysis of fiber colorants using TLC should be considered
for single-fiber comparisons only when it is not possible to
discriminate between the fibers of interest using other techniques,
such as comparison microscopy (brightfield and fluorescence)
and microspectrophotometry in the visible range.
5.2. Extraction Procedures
Carried Out Prior to TLC Analysis
The extraction procedures carried out prior to TLC analysis can
provide useful information about dye classification. TLC can
provide useful qualitative information about dye components.
Similar colors made up of different dye components can be differentiated
using this technique. The application of TLC may serve to discriminate
between fibers, or it may confirm their similarity.
5.3. Situations That Preclude
TLC
TLC is not possible in some circumstances: Short lengths of fibers
or pale colored fibers do not have an adequate concentration
of colorant present to be examined, dye extraction from some
fibers is impossible, or desire to preserve evidence for possible
analysis by another examiner precludes removing the color for
analysis.
5.4. Characterizing Dyes
and Evaluating Eluent Systems
Dye from the known material should first be characterized and
eluent systems evaluated to achieve optimum separation of the
extract. Dye is then extracted from single known and questioned
fibers, using an equivalent amount of material.
5.5. Standard
Dye Mixtures Compensate for TLC's Nonreproducibility
TLC is inherently nonreproducible because of variance in eluents
and temperature conditions. Standard dye mixtures (16) should
therefore be used to check eluent performance.
Examples for the preparation
of standard dye mixtures are given in Appendix
A.
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6.0.
Sample Handling
6.1. Using ASTM 1492-94
Standards
The general handling and tracking of the samples should meet
or exceed the requirements of ASTM 1492-94.
6.2. Pretreatment and
Sample Preparation
Pretreatment (e.g., mounting medium and washing solvent) and
sample preparation must be identical for all known and questioned
fibers being compared on one TLC plate. For removing single fibers
from slide preparations, the following procedure is recommended:
6.2.1. Clean the Coverslip. Any traces of marker pen ink should
be cleaned from the coverslip using an appropriate solvent (e.g.,
acetone);
6.2.2. Crack the Coverslip. The coverslip should be cracked
all around the fiber, and an appropriate solvent, which will
dissolve the mountant but not affect the fiber or the colorant,
should be used; and
6.2.3. Remove and Clean
the Fiber. The
fiber should be removed and washed in the solvent.
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7.0. Analysis
The ease of dye extraction
and the particular extractant required will depend on the generic
class of the fiber and the type of dye present. The generic class
of the known and questioned fibers must be determined prior to
TLC analysis.
Dye classes are classified
into broad groups on the basis of their chemical properties or
method of application. The determination of the dye class of
the known fibers can be helpful in establishing the best extractant,
as well as to assist in the subsequent selection of the most
efficient eluent system.
Documented extraction schemes
(see Appendix B) can be used to determine
the dye class of fibers of known generic classes and, thus, the
optimum extractant. Dye classification is performed on single
fibers or tufts of fiber removed from the known item. A new fiber
or tuft can be used for each classification stage.
7.1. Dye Extraction
Known and questioned fibers must be extracted at the same time under the
same conditions. Single fibers can be extracted in a short length (about
25 mm) of fine capillary tube (internal diameter of about 1.5 mm) that
is sealed at one end. A fine wire can be useful in pushing the fiber down
the tube. The tube must be appropriately labeled.
About 10 µL of the
appropriate extractant (as recommended in Appendix
C and Appendix D) should be introduced
into the tube to cover the fiber sample. A fine glass pipette
or syringe can be used for this. The tube should be heat sealed
to avoid evaporation and incubated for a constant time and temperature
(as recommended in Appendix B), preferably in an oven. Periodic
checks for dye extraction should be made every 15 minutes for
up to 1 hour.
7.2. Dye Extraction for
Bulk Material
Larger fiber tufts (e.g., known samples) can be extracted in
a Durham tube or other suitable small stoppered glass tube, using
about 100 µL of solvent in a sand bath or oven heated to
100°C. Periodic checks should be made every 15 minutes for
up to 1 hour.
7.3. Nonextractable Dyes
If classification indicates that a nonextractable dye or pigment
other than a reactive dye is present, then place one known and
one questioned fiber in labeled capillary tubes. Add approximately
10 µL pyridine/water (4:3) and attempt to extract at about
100°C for one hour. If neither fiber extracts, a positive
association is noted. If the questioned extracts and the known
does not (or vice versa), it is negative. If both questioned
and known bleed dye into solution, there can be sufficient dye
for analysis.
7.4. Elution
Aluminum-backed silica gel 60F 254 plates measuring 5 cm ×
7.5 cm are recommended for normal-phase TLC of fiber dyes (16).
Plates should be stored in a desiccator. If this is not possible,
they should be heat activated before use.
Both known and questioned
dyes to be compared must be applied to the same plate. The extract
should be spotted onto the plate about 1 cm from the lower edge.
This can be done using a double-drawn capillary tube or other
suitable device. Spots should not be too near the edge of the
plate or to each other. Care should be taken to avoid scratching
the adsorbent coating layer.
Spots should be dried using
a hair dryer or hot plate, and repeated applications should be
made until the spot is strongly colored. The spot size should
be uniform and not exceed about 2 mm in size.
At least two (preferably
more) known spots should be included on each plate, on both sides
of the questioned sample or samples. It is advisable to include
a standard dye spot. A note must be made of the sample order
on the plate itself. Plates must be thoroughly dried before developing.
7.5. Development Chamber
Chromatograms can be developed vertically in a glass chamber,
which can be as simple as a covered glass beaker. Commercial
tanks are available (16). Twin trough tanks allow the solvent
to be transferred to the plate side without removing the cover,
but extreme care must be taken when doing this.
The eluent should be added
to the tank and allowed to stand in the closed container for
a few minutes before development, which allows the chamber to
be saturated with the solvent vapor. (This will not be complete
if a beaker is used, but equilibration is not critical when sample
size is very small and the elution time is short.)
The level of the eluent in
a vertical tank should be at least 0.5 cm below the origin or
application spots on the TLC plate. The plate should be eluted
until good resolution is achieved (normally 2 cm from the origin)
but not so far as to allow the spots to become diffuse, which
makes visualization difficult. The plate should be removed, and
the position of the solvent front marked. The plate should be
dried in a hot air stream. The eluent should be discarded.
7.5.1. Selecting the
Eluent. Five
parameters must be considered when selecting the optimum eluent:
7.5.1.1. Separation of component dyes;
7.5.1.2. Sharpness of bands;
7.5.1.3. Movement from the origin;
7.5.1.4. Components traveling at or close to solvent front; and
7.5.1.5. Strength of dye extract from questioned fibers.
There are numerous published
TLC solvent systems that can be applied to the development of
particular fiber and dye class combinations (see Appendixes C
and D).
Two or more systems should
be assessed with the known fibers to determine the optimum eluent
system that can be used for comparison with the questioned fibers.
Equivalent lengths of fiber
should be used for pale fibers or short sample lengths. The extract
from known material should be applied to the TLC plate and developed
in the trial eluents as previously described.
If the eluents produce poor
separation, other eluents appropriate to the dye class are evaluated.
In exceptional circumstances, eluents appropriate to other dye
classes can be used.
After a suitable eluent system
has been found, comparison of known and questioned fibers can
be carried out. Co-chromatography can be carried out for bulk
samples.
After drying, plates should
be examined immediately in visible and in longwave ultraviolet
light. Band positions and colors should be noted.
The color and fluorescence
of the spots and the distance from the center of each spot to
the origin can be measured and recorded. The method of documentation
is a matter of individual laboratory preference.
Plates and samples must be
identifiable. Plates must be either documented by photography
or retained and stored out of direct sunlight in a manner designed
to minimize fading or both.
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8.0.
Report Documentation
Chromatograms of dyes from
the same fibers run in different eluent systems or on different
plate types are considered mutually exclusive. The spot colors,
fluorescence, sequence, and position of the spots obtained from
the dye of the questioned fibers are compared to those from the
corresponding known fibers.
A positive association occurs
when the band colors, fluorescence, sequence, and positions are
consistent between questioned and known fibers. A negative (exclusion)
association is noted when either the questioned or known patterns
show no similarities, or where there are a number of coincident
bands, but one or more bands are missing from the questioned
or known. An inconclusive association is noted when there are
no bands on the TLC plate because insufficient colorant is present
in the extract. In cases where the amount of extract is very
small, the distance traveled by the eluent is very small, and
in some cases the spots may not be well-defined. In these circumstances,
attempts to calculate the Retardation Factor (RF) values can
easily be inaccurate and therefore meaningless.
The TLC methods applied to
the forensic comparison of fiber colorants must have been published
in a recognized forensic journal, forensically relevant textbook,
or in an accredited forensic laboratory manual.
Plates must be identifiable
with respect to case number, sample source, examiner, and date.
Case documentation on TLC must include the source of the samples;
method of dye classification; details of extractants or eluent
systems tested, used, or both; and the results. The use of standard
dye mixtures as system performance checks is strongly recommended.
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9.0. References
(1) Fried, B. and Sherma,
J. Thin-Layer Chromatography: Techniques and Applications.
2nd ed. M. Dekker, New York, 1986.
(2) Geiss, F. Fundamentals
of Thin-Layer Chromatography. Huethig, Heidelberg, Germany,
1987.
(3) Hamilton, R. and Hamilton,
S. Thin-Layer Chromatography. John Wiley, Chichester,
United Kingdom, 1987
(4) Sherma, J. and Fried,
B. (eds.). Handbook of Thin-Layer Chromatography. M. Dekker,
New York, 1990.
(5) Stahl, E. Thin-Layer
Chromatography. Spring-Verlag, New York, 1969.
(6) Schweppe, H. Thin-layer
chromatography in Venkataraman, K. In: Änalytical Chemistry
of Synthetic Dyes. John Wiley, New York, 1977, pp. 23-56.
(7) Beattie, I. B., Dudley,
R. J., and Smalldon, K. W. The extraction and classification
of dyes on single nylon, polyacrylonitrile, and polyester fibres,
Journal of the Society of Dyers and Colourists (1979)
95:295-302.
(8) Beattie, I. B., Roberts,
H. L., and Dudley, R. J. Thin-layer chromatography of dyes extracted
from polyester, nylon, and polyacrylonitrile fibres, Forensic
Science International (1981) 17:57-69.
(9) Beattie, B., Roberts,
H., and Dudley, R. J. The extraction and classification of dyes
from cellulose acetate fibres, Journal of the Forensic Science
Society (1981) 21:233-237.
(10) Grieve, M. C. Forensic
examination of fibres. In: Forensic Science Progress.
(Vol. 4). Springer-Verlag, Heidelberg, Germany, 1990, pp. 41-125.
(11) Hartshorne, A. W. and
Laing, D. K. The dye classification and discrimination of coloured
polypropylene fibres, Forensic Science International (1984)
25:133-141.
(12) Home, J. M. and Dudley,
R. J. Thin-layer chromatography of dyes extracted from cellulosic
fibres, Forensic Science International (1981) 17:71-78
(13) Home, J. M. and Dudley,
R. J. Revision of the scheme for the extraction and classification
of dyes from polyacrylonitrile fibres, Journal of the Society
of Dyers and Colourists (1981) 97:17-19.
(14) Laing, D. K., et al.
Thin-layer chromatography of azoic dyes extracted from cotton
fibres, Journal of the Forensic Science Society (1990)
30:309-315.
(15) Laing, D. K., Dudley,
R. J., Hartshorne, A. W., Home, J. M., Rickard, R. A., and Bennett,
D. C. The extraction and classification of dyes from cotton and
viscose fibres, Forensic Science International (1991)
50:23-35.
(16) Laing, D. K., Boughey,
L., and Hartshorne, A. W. The standardisation of thin-layer chromatographic
systems for comparison of fibre dyes, Journal of the Canadian
Society of Forensic Science (1990) 30:299-307.
(17) Macrae, R. and Smalldon,
K. W. The extraction of dyestuffs from single wool fibres, Journal
of Forensic Sciences (1979) 24:109-116.
(18) Macrae, R., Dudley,
R. J., and Smalldon, K. W. The characterization of dyestuffs
on wool fibres with special reference to microspectrophotometry,
Journal of Forensic Sciences (1979), pp. 117-129.
(19) Rendle, D. F. and Wiggins,
K. G. Forensic analysis of textile fibre dyes, Review of Progress
in Coloration and Related Topics (1995) 25:29-34.
(20) Resua, R. A semi-micro
technique for the extraction and comparison of dyes in textile
fibers, Journal of Forensic Sciences (1980) 25:168-173.
(21) Resua, R., DeForest,
P., and Harris, H. The evaluation and selection of uncorrelated
paired solvent systems for use in the comparison of textile dyes
by thin-layer chromatography, Journal of Forensic Sciences
(1981) 26:515-534.
(22) Robertson J. (ed.) Forensic
Examination of Fibres. Chichester, United Kingdom, Ellis
Horwood, 1992.
(23) The Colour Index
(Vols. 1-6, 4th ed.). Bradford, United Kingdom, Society of Dyers
and Colourists (AATCC, South Carolina) 1985.
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FORENSIC SCIENCE COMMUNICATIONS APRIL 1999 VOLUME 1 NUMBER 1 |
