ASTM E2227-02

Standard Guide for Forensic Examination of Non-Reactive Dyes in Textile Fibers by Thin-Layer Chromatography

SIGNIFICANCE AND USE
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.
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.
TLC may be prohibitively difficult or undesirable in some circumstances. Short lengths of fibers or pale colored fibers may not have an adequate concentration of colorant present to be examined, dye extraction from some fibers may be impossible. The desire to preserve evidence for possible analysis by another examiner may preclude removing the color for analysis.
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.
The development of each individual TLC plate will show some variability as a result of the coating and conditioning of the plate, solvent condition, and temperature. It is important to evaluate the performance of each TLC plate by spotting known materials along with the questioned samples. See Ref (16).
Examples for the preparation of Standard dye mixtures are given in Appendix X1.
SCOPE
1.1 Metameric coloration of fibers can be detected using UV/visible spectrophotometry. If spectrophotometry is restricted to the visible spectral range only, 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, under certain conditions, can be used 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.

General Information

• Status: Historical
• Publication Date: 09-Aug-2002
• ICS: 71.040.50 - Physicochemical methods of analysis
• Technical Committee: E30 - Forensic Sciences
• Drafting Committee: E30.01 - Criminalistics

Relations

• Replaced By: ASTM E2227-02(2008) - Standard Guide for Forensic Examination of Non-Reactive Dyes in Textile Fibers by Thin-Layer Chromatography
• Effective Date: 15-Mar-2008
• Referred By: ASTM E2225-23 - Standard Guide for Forensic Examination of Fabrics and Cordage
• Effective Date: 10-Aug-2002
• Referred By: ASTM E1732-22 - Standard Terminology Relating to Forensic Science
• Effective Date: 10-Aug-2002

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation:E2227–02
Standard Guide for
Forensic Examination of Non-Reactive Dyes in Textile
Fibers by Thin-Layer Chromatography
This standard is issued under the fixed designation E 2227; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope 3.1.6 development—the movement of the mobile phase
through the adsorbent layer to form a chromatogram.
1.1 Metameric coloration of fibers can be detected using
3.1.7 dye extraction—theremovalofthedyefromafiberby
UV/visible spectrophotometry. If spectrophotometry is re-
incubating it in an appropriate solvent.
stricted to the visible spectral range only, differences in dye
3.1.8 eluent—the solvent mixture that acts as the mobile
components may remain undetected. One method of detecting
phase in TLC.
additional components is to use thin-layer chromatography
3.1.9 metameric pair—two colors that appear the same
(TLC). TLC is an inexpensive, simple, well-documented tech-
under one illumination, but different under other illumination.
nique that, under certain conditions, can be used to comple-
3.1.10 mobile phase—the moving liquid phase used for
ment the use of visible spectroscopy in comparisons of fiber
development.
colorants. The principle of the method is that the dye compo-
3.1.11 normal-phase chromatogram—adsorption in which
nents are separated by their differential migration caused by a
the stationary phase is polar in relation to the mobile phase.
mobile phase flowing through a porous, adsorptive medium.
3.1.12 origin—the location of the applied sample or the
2. Referenced Documents starting point for the chromatographic development of the
applied sample.
2.1 ASTM Standards:
3.1.13 resolution—theabilitytovisuallyseparatetwospots.
E 1188 Practice for Collection and Preservation of Informa-
3.1.14 retardation factor (RF)—the ratio of the distance
tion and Physical Items by a Physical Investigator
traveled by the solute spot’s center divided by the distance
E 1459 Guide for Physical Evidence Handling and Related
traveled by the solvent front, both measured from the origin.
Documentation
3.1.15 saturation chamber—equilibration with mobile
E 1492 Practice for Receiving, Documenting, Storing, and
phase solvent vapor prior to chromatography.
Retrieving Evidence in a Forensic Laboratory
3.1.16 solute—in TLC, a mixture of components to be
3. Terminology
separated.
3.1.17 solvent front—the final point reached by the mobile
3.1 Definitions of Terms Specific to This Standard:
phase as it flows up or across the TLC plate during develop-
3.1.1 activation—the heating of the adsorbent layer on a
ment of the chromatogram.
plate to dry out the moisture and maximize its adsorptive
3.1.18 spot—a round zone of sample application at the
power.
origin, or in a chromatogram, a round zone caused by migra-
3.1.2 adsorbent—the stationary phase for adsorption TLC.
tion of a separated component of the solute. The sharpness of
3.1.3 adsorption—the attraction between the surface atoms
the spot relates to the efficiency of the chromatographic band.
of a solid and an external molecule by intermolecular forces.
3.1.19 spotting—applying a solute sample at the origin of
3.1.4 chamber—a glass chamber in which TLC develop-
the TLC plate.
ment is carried out.
3.1.20 stationary phase—the solid adsorbent coating layer
3.1.5 chromatography—a method of analysis in which sub-
of a TLC plate.
stances are separated by their differential migration in a mobile
3.1.21 tailing—a spot distorted during development into an
phase flowing through or past a stationary phase.
elongated streak.
3.1.22 thin-layer chromatogram—the series of spots visible
This guide is under the jurisdiction of ASTM Committee E30 on Forensic
on the adsorbent layer after development.
Sciences and is the direct responsibility of Subcommittee E30.01 on Criminalistics.
Current edition approved August 10, 2002. Published October 2002.
Annual Book of ASTM Standards, Vol 14.02.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
E2227–02
3.1.23 thin-layer chromatography (TLC)—a separation 6.2 Pre-treatment(mountingmedium,washingsolvent,etc.)
technique in which the flow of solvent causes the components and sample preparation must be identical for all known and
ofamixturetomigratedifferentiallyfromanarrowinitialzone questioned fibers being compared on one TLC plate. For
over a planar, thinly-applied porous adsorptive medium. removing single fibers from slide preparations the following
procedure is recommended.
4. Summary of Guide
6.2.1 Any traces of marker pen ink should be cleaned from
the coverslip using an appropriate solvent, for example, ac-
4.1 This guide is intended to advise and to assist individuals
etone.
and laboratories that conduct forensic fiber examinations and
comparisons in their effective application of TLC to the
6.2.2 The coverslip should be cracked all around the fiber
analysis of fiber evidence. and an appropriate solvent, that will dissolve the mountant, but
4.2 The guide is concerned with the extraction of dyes from not affect the fiber or the colorant, should be used.
singlefibersandfrombulkmaterial,classificationofthedyeor
6.2.3 The fiber is removed and extracted in an appropriate
colorant, application and development of the extractants on
solvent. Appropriate solvent selection will depend on the
TLC plates using an optimal elution system, and evaluation
mountant and the sample.
and interpretation of the resulting chromatograms. The proto-
cols and equipment mentioned in this document are not meant
7. Analysis
to be totally inclusive or exclusive.
7.1 The ease of dye extraction and the particular extractant
4.3 Not all fiber type/dye class combinations are covered in
required will depend on the generic class of the fiber and the
this guide.
type of dye present. The generic class of the known and
questioned fibers must be determined prior to TLC analysis.
5. Significance and Use
7.2 Dye classes are classified into broad groups based on
5.1 ForensicanalysisoffibercolorantsusingTLCshouldbe
their chemical properties or method of application. The deter-
considered for single fiber comparisons only when it is not
mination of the dye class of the known fibers can be helpful in
possible to discriminate between the fibers of interest using
establishing the best extractant, as well as to assist in the
other techniques, such as comparison microscopy (brightfield
subsequent selection of the most efficient eluent system.
and fluorescence) and microspectrophotometry in the visible
7.3 Documented extraction schemes (seeAppendix X2) can
range.
be used to determine the dye class of fibers of known generic
5.2 The extraction procedures carried out prior to TLC
classes, and thus the optimum extractant. Dye classification is
analysis can provide useful information about dye classifica-
done on single fibers or tufts of fiber removed from the known
tion.TLC can provide useful qualitative information about dye
item. A new fiber or tuft can be used for each classification
components. Similar colors made up of different dye compo-
stage.
nents can be differentiated using this technique. The applica-
7.4 Dye Extraction—Known and questioned fibers must be
tionofTLCmayservetodiscriminatebetweenfibers,oritmay
extracted at the same time under the same conditions. Single
confirm their similarity.
fibers can be extracted in a short length (about 25 mm) of fine
5.3 TLC may be prohibitively difficult or undesirable in
capillary tube (internal diameter of about 1.5 mm), sealed at
some circumstances. Short lengths of fibers or pale colored
oneend.Afinewirecanbeusefulinpushingthefiberdownthe
fibers may not have an adequate concentration of colorant
tube. The tube must be appropriately labeled.
present to be examined, dye extraction from some fibers may
7.4.1 About 10 µl of the appropriate extractant (as recom-
be impossible. The desire to preserve evidence for possible
analysis by another examiner may preclude removing the color mended inAppendix X3) should be introduced into the tube to
cover the fiber sample. A fine glass pipette or syringe can be
for analysis.
5.4 Dye from the known material should first be character- usedforthisprocedure.Thetubeshouldbeheatsealedtoavoid
evaporation and incubated for a constant time and temperature
ized and eluent systems evaluated to achieve optimum separa-
tion of the extract. Dye is then extracted from single known (as recommended in Appendix X2), preferably in an oven.
Periodic checks for dye extraction should be made every 15
and questioned fibers, using an equivalent amount of material.
5.5 The development of each individual TLC plate will min for up to 1 h.
show some variability as a result of the coating and condition-
7.5 Dye Extraction: Bulk Material—Larger fiber tufts can
ing of the plate, solvent condition, and temperature. It is
beextractedinaDurhamtubeorothersuitablesmallstoppered
important to evaluate the performance of each TLC plate by
glass tube, using about 100 µl of solvent in a sand bath or oven
spotting known materials along with the questioned samples.
heatedto100°C.Periodicchecksshouldbemadeevery15min
See Ref (16).
for up to 1 h.
5.6 Examples for the preparation of Standard dye mixtures
7.6 Nonextractable Dyes—If classification indicates that a
are given in Appendix X1.
nonextractable dye or pigment other than a reactive dye is
present, then place one known and one questioned fiber in
6. Sample Handling
labeled capillary tubes. Add approximately 10 µl pyridine/
6.1 Thegeneralhandlingandtrackingofthesamplesshould water (4:3) and attempt to extract at about 100°C for one hour.
meet or exceed the requirements of Practice E 1492 and Guide If neither fiber extracts, a positive association is noted. If the
E 1459. questioned fiber extracts and the known fiber does not (or vice
E2227–02
versa), there is no association. If both questioned and known 7.9 Two or more eluent systems should be assessed with the
fibers “bleed” dye into solution, there may be sufficient dye for known fibers to determine the optimum eluent system that can
analysis. be used for comparison with the questioned fibers.
7.10 Equivalent lengths of fiber should be used for pale
7.7 Elution—Aluminum backed silica gel plates, with
fibers or short sample lengths. The extract from known
nominal particle size of 60 microns and incorporating a
material should be applied to the TLC plate and developed in
fluorphore excited at 254 nm, such as silica gel 60F 254,
the trial eluents as previously described. If the eluents produce
measuring 5 3 7.5 cm are recommended for normal-phase
poor separation, others appropriate to the dye class are evalu-
TLC of fiber dyes (16). Plates should be stored in a desiccator;
ated. In exceptional circumstances, eluents appropriate to other
if this is not possible, they should be heat activated before use.
dye classes can be used.
7.7.1 Both known dyes and questioned dyes to be compared
7.10.1 After a suitable eluent system has been found,
must be applied to the same plate. The extract should be
comparison of known and questioned fibers can be carried out.
spottedontotheplateabout1cmfromtheloweredge.Thiscan
Co-chromatography can be carried out for bulk samples.After
be done using a double drawn capillary tube or other suitable
drying, plates should be examined immediately in visible and
device. Spots should not be too near to the edge of the plate or
in longwave ultraviolet light. Band position(s) and color(s)
to each other. Care should be taken to avoid scratching the
should be noted.
adsorbent coating layer during spot application.
7.10.2 If the spots don’t move from the origin, a more polar
7.7.2 Spots should be dried using a hair dryer or hot plate,
solvent system should be chosen. If the spots move with the
with repeat spot applications made until the spot is strongly
solvent front, a less polar solvent system should be chosen.
colored. The spot size should be uniform and not exceed about
7.11 Determineandrecordthecolor/fluorescenceandtheRf
2 mm in size.
value of the spots.
7.7.3 At least two (preferably more) known dye spots
7.12 Plates and samples must be identifiable. Plates must be
should be included on each plate, on both sides of the
either documented by photography and/or retained and stored
questioned sample(s). It is advisable to include a standard dye
out of direct sunlight in a manner designed to minimize fading.
spot. A note must be made of the sample order on the plate
8. Report Documentation
itself in pencil, well below the sample spots. Plates must be
thoroughly dried before developing. 8.1 Different solvent systems or stationary phases may
provide additional discriminating power. The spot colors/
7.8 Development Chambers—Chromatograms can be de-
fluorescence,sequence,andpositionofthespotsobtainedfrom
veloped vertically in a glass chamber that may be as simple as
the dye of the questioned fibers are compared to those from the
a covered glass beaker. Commercial tanks are available (16).
corresponding known fibers analyzed under the same condi-
Twin trough tanks allow the solvent to be transferred to the
tions.
plate side without removing the cover, but extreme care must
8.2 A positive association occurs when the colors/
be taken when doing this not to contact the side of the TLC
fluorescence, sequence, and positions of the spots are consis-
plate.
tent between questioned and known fibers. A negative (exclu-
7.8.1 The eluent should be added to the tank and allowed to
sion) association is noted when either the questioned and
stand in the closed container for a few minutes before
known patterns show no similarities, or there are a number of
development, so that the chamber will be saturated with the
coincident bands but one or more bands are missing from the
solvent vapor.
questioned or known pattern. An inconclusive association is
7.8.2 The level of the eluent in a vertical tank should be at
noted when there are no bands on the TLC plate because
least 0.5 cm below the origin/application spots on the TLC
insufficientcolorantispresentintheextract.Incaseswherethe
plate. The plate should be eluted until good resolution is
amount of extract is very small, the distance traveled by the
achieved (normally 2 cm from the origin), but not so far as to
eluent is very small and in some cases the spots may not be
allow the spots to become diffuse, making visualization diffi-
well defined, calculation of the Retardation Factor (RF) values
cult. The plate should be removed and the positio
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