ASTM E2227-02(2008)
(Guide)Standard Guide for Forensic Examination of Non-Reactive Dyes in Textile Fibers by Thin-Layer Chromatography
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.
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
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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(Reapproved 2008)
Standard Guide for
Forensic Examination of Non-Reactive Dyes in Textile
Fibers by Thin-Layer Chromatography
This standard is issued under the fixed designation E2227; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3.1.5 chromatography—a method of analysis in which sub-
stancesareseparatedbytheirdifferentialmigrationinamobile
1.1 Metameric coloration of fibers can be detected using
phase flowing through or past a stationary phase.
UV/visible spectrophotometry. If spectrophotometry is re-
stricted to the visible spectral range only, differences in dye
3.1.6 development—the movement of the mobile phase
components may remain undetected. One method of detecting through the adsorbent layer to form a chromatogram.
additional components is to use thin-layer chromatography
3.1.7 dye extraction—theremovalofthedyefromafiberby
(TLC). TLC is an inexpensive, simple, well-documented tech-
incubating it in an appropriate solvent.
nique that, under certain conditions, can be used to comple-
3.1.8 eluent—the solvent mixture that acts as the mobile
ment the use of visible spectroscopy in comparisons of fiber
phase in TLC.
colorants. The principle of the method is that the dye compo-
nents are separated by their differential migration caused by a
3.1.9 metameric pair—two colors that appear the same
mobile phase flowing through a porous, adsorptive medium.
under one illumination, but different under other illumination.
1.2 The values stated in SI units are to be regarded as
3.1.10 mobile phase—the moving liquid phase used for
standard. No other units of measurement are included in this
development.
standard.
3.1.11 normal-phase chromatogram—adsorption in which
2. Referenced Documents the stationary phase is polar in relation to the mobile phase.
2.1 ASTM Standards:
3.1.12 origin—the location of the applied sample or the
E1459Guide for Physical Evidence Labeling and Related
starting point for the chromatographic development of the
Documentation applied sample.
E1492Practice for Receiving, Documenting, Storing, and
3.1.13 resolution—theabilitytovisuallyseparatetwospots.
Retrieving Evidence in a Forensic Science Laboratory
3.1.14 retardation factor (RF)—the ratio of the distance
3. Terminology
traveled by the solute spot’s center divided by the distance
traveled by the solvent front, both measured from the origin.
3.1 Definitions of Terms Specific to This Standard:
3.1.1 activation—the heating of the adsorbent layer on a
3.1.15 saturation chamber—equilibration with mobile
plate to dry out the moisture and maximize its adsorptive
phase solvent vapor prior to chromatography.
power.
3.1.16 solute—in TLC, a mixture of components to be
3.1.2 adsorbent—the stationary phase for adsorption TLC.
separated.
3.1.3 adsorption—the attraction between the surface atoms
3.1.17 solvent front—the final point reached by the mobile
of a solid and an external molecule by intermolecular forces.
phase as it flows up or across the TLC plate during develop-
3.1.4 chamber—a glass chamber in which TLC develop-
ment of the chromatogram.
ment is carried out.
3.1.18 spot—a round zone of sample application at the
origin, or in a chromatogram, a round zone caused by migra-
This guide is under the jurisdiction of ASTM Committee E30 on Forensic
tion of a separated component of the solute. The sharpness of
Sciences and is the direct responsibility of Subcommittee E30.01 on Criminalistics.
the spot relates to the efficiency of the chromatographic band.
Current edition approved March 15, 2008. Published July 2008. Originally
approved in 2002. Last previous edition approved in 2002 as E2227–02. DOI:
3.1.19 spotting—applying a solute sample at the origin of
10.1520/E2227-02R08.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or the TLC plate.
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
3.1.20 stationary phase—the solid adsorbent coating layer
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. of a TLC plate.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2227 − 02 (2008)
3.1.21 tailing—a spot distorted during development into an important to evaluate the performance of each TLC plate by
elongated streak. spotting known materials along with the questioned samples.
See Ref (16).
3.1.22 thin-layer chromatogram—the series of spots visible
5.6 Examples for the preparation of Standard dye mixtures
on the adsorbent layer after development.
are given in Appendix X1.
3.1.23 thin-layer chromatography (TLC)—a separation
technique in which the flow of solvent causes the components
6. Sample Handling
ofamixturetomigratedifferentiallyfromanarrowinitialzone
6.1 Thegeneralhandlingandtrackingofthesamplesshould
over a planar, thinly-applied porous adsorptive medium.
meet or exceed the requirements of Practice E1492 and Guide
E1459.
4. Summary of Guide
6.2 Pre-treatment(mountingmedium,washingsolvent,etc.)
4.1 Thisguideisintendedtoadviseandtoassistindividuals
and sample preparation must be identical for all known and
and laboratories that conduct forensic fiber examinations and
questioned fibers being compared on one TLC plate. For
comparisons in their effective application of TLC to the
removing single fibers from slide preparations the following
analysis of fiber evidence.
procedure is recommended.
6.2.1 Any traces of marker pen ink should be cleaned from
4.2 Theguideisconcernedwiththeextractionofdyesfrom
the coverslip using an appropriate solvent, for example, ac-
singlefibersandfrombulkmaterial,classificationofthedyeor
etone.
colorant, application and development of the extractants on
6.2.2 The coverslip should be cracked all around the fiber
TLC plates using an optimal elution system, and evaluation
andanappropriatesolvent,thatwilldissolvethemountant,but
and interpretation of the resulting chromatograms. The proto-
not affect the fiber or the colorant, should be used.
cols and equipment mentioned in this document are not meant
6.2.3 The fiber is removed and extracted in an appropriate
to be totally inclusive or exclusive.
solvent. Appropriate solvent selection will depend on the
mountant and the sample.
4.3 Not all fiber type/dye class combinations are covered in
this guide.
7. Analysis
5. Significance and Use
7.1 The ease of dye extraction and the particular extractant
required will depend on the generic class of the fiber and the
5.1 ForensicanalysisoffibercolorantsusingTLCshouldbe
type of dye present. The generic class of the known and
considered for single fiber comparisons only when it is not
questioned fibers must be determined prior to TLC analysis.
possible to discriminate between the fibers of interest using
other techniques, such as comparison microscopy (brightfield 7.2 Dye classes are classified into broad groups based on
their chemical properties or method of application. The deter-
and fluorescence) and microspectrophotometry in the visible
mination of the dye class of the known fibers can be helpful in
range.
establishing the best extractant, as well as to assist in the
5.2 The extraction procedures carried out prior to TLC
subsequent selection of the most efficient eluent system.
analysis can provide useful information about dye classifica-
7.3 Documentedextractionschemes(seeAppendixX2)can
tion.TLCcanprovideusefulqualitativeinformationaboutdye
be used to determine the dye class of fibers of known generic
components. Similar colors made up of different dye compo-
classes, and thus the optimum extractant. Dye classification is
nents can be differentiated using this technique. The applica-
done on single fibers or tufts of fiber removed from the known
tionofTLCmayservetodiscriminatebetweenfibers,oritmay
item. A new fiber or tuft can be used for each classification
confirm their similarity.
stage.
5.3 TLC may be prohibitively difficult or undesirable in
7.4 Dye Extraction—Known and questioned fibers must be
some circumstances. Short lengths of fibers or pale colored
extracted at the same time under the same conditions. Single
fibers may not have an adequate concentration of colorant
fibers can be extracted in a short length (about 25 mm) of fine
present to be examined, dye extraction from some fibers may
capillary tube (internal diameter of about 1.5 mm), sealed at
be impossible. The desire to preserve evidence for possible
oneend.Afinewirecanbeusefulinpushingthefiberdownthe
analysisbyanotherexaminermayprecluderemovingthecolor
tube. The tube must be appropriately labeled.
for analysis.
7.4.1 About 10 µl of the appropriate extractant (as recom-
mendedinAppendixX3)shouldbeintroducedintothetubeto
5.4 Dye from the known material should first be character-
cover the fiber sample. A fine glass pipette or syringe can be
ized and eluent systems evaluated to achieve optimum separa-
usedforthisprocedure.Thetubeshouldbeheatsealedtoavoid
tion of the extract. Dye is then extracted from single known
evaporation and incubated for a constant time and temperature
and questioned fibers, using an equivalent amount of material.
5.5 The development of each individual TLC plate will
show some variability as a result of the coating and condition- 3
The boldface numbers in parentheses refer to a list of references at the end of
ing of the plate, solvent condition, and temperature. It is this standard.
E2227 − 02 (2008)
(as recommended in Appendix X2), preferably in an oven. cult. The plate should be removed and the position of the
Periodic checks for dye extraction should be made every 15 solvent front marked in pencil. The plate should be dried in a
min for up to 1 h. hot air stream. The eluent should be appropriately discarded.
7.8.3 Five parameters must be considered when selecting
7.5 Dye Extraction: Bulk Material—Larger fiber tufts can
the optimum eluent:
beextractedinaDurhamtubeorothersuitablesmallstoppered
7.8.3.1 Separation of component dyes,
glasstube,usingabout100µlofsolventinasandbathoroven
7.8.3.2 Sharpness of bands,
heatedto100°C.Periodicchecksshouldbemadeevery15min
7.8.3.3 Movement of the eluted spots from the origin,
for up to 1 h.
7.8.3.4 Components traveling at or close to solvent front,
7.6 Nonextractable Dyes—If classification indicates that a
and
nonextractable dye or pigment other than a reactive dye is
7.8.3.5 Strength of dye extract from questioned fibers.
present, then place one known and one questioned fiber in
7.8.4 There are numerous published TLC solvent systems
labeled capillary tubes. Add approximately 10 µl pyridine/
that can be applied to the development of particular fiber/dye
water (4:3) and attempt to extract at about 100°C for one hour.
class combinations (see Appendix X3).
If neither fiber extracts, a positive association is noted. If the
7.9 Twoormoreeluentsystemsshouldbeassessedwiththe
questioned fiber extracts and the known fiber does not (or vice
known fibers to determine the optimum eluent system that can
versa), there is no association. If both questioned and known
be used for comparison with the questioned fibers.
fibers“bleed”dyeintosolution,theremaybesufficientdyefor
7.10 Equivalent lengths of fiber should be used for pale
analysis.
fibers or short sample lengths. The extract from known
7.7 Elution—Aluminumbackedsilicagelplates,withnomi-
material should be applied to the TLC plate and developed in
nal particle size of 60 microns and incorporating a fluorphore
the trial eluents as previously described. If the eluents produce
excited at 254 nm, such as silica gel 60F 254, measuring 5 ×
poor separation, others appropriate to the dye class are evalu-
7.5 cm are recommended for normal-phase TLC of fiber dyes
ated.Inexceptionalcircumstances,eluentsappropriatetoother
(16). Plates should be stored in a desiccator; if this is not
dye classes can be used.
possible, they should be heat activated before use.
7.10.1 After a suitable eluent system has been found,
7.7.1 Bothknowndyesandquestioneddyestobecompared
comparisonofknownandquestionedfiberscanbecarriedout.
must be applied to the same plate. The extract should be
Co-chromatography can be carried out for bulk samples.After
spottedontotheplateabout1cmfromtheloweredge.Thiscan
drying, plates should be examined immediately in visible and
be done using a double drawn capillary tube or other suitable
in longwave ultraviolet light. Band position(s) and color(s)
device. Spots should not be too near to the edge of the plate or
should be noted.
to each other. Care should be taken to avoid scratching the
7.10.2 Ifthespotsdon’tmovefromtheorigin,amorepolar
adsorbent coating layer during spot application.
solvent system should be chosen. If the spots move with the
7.7.2 Spots should be dried using a hair dryer or hot plate,
solvent front, a less polar solvent system should be chosen.
with repeat spot applications made until the spot is strongly
7.11 Determineandrecordthecolor/fluorescenceandtheRf
colored.Thespotsizeshouldbeuniformandnotexceedabout
value of the spots.
2 mm in size.
7.7.3 At least two (preferably more) known dye spots
7.12 Platesandsamplesmustbeidentifiable.Platesmustbe
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
outofdirectsunlightinamannerdesignedtominimizefading.
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—Chromatogramscanbedevel-
fluorescence,sequence,andpositionofthespotsobtainedfrom
oped vertically in a glass chamber that may be as simple as a
thedyeofthequestionedfibersarecomparedtothosefromthe
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
be taken when doing this not to contact the side of the TLC
8.2 A positive association occurs when the colors/
plate.
fluorescence, sequence, and positions of the spots are consis-
7.8.1 Theeluentshouldbeaddedtothetankandallowedto tent between questioned and known fibers. A negative (exclu-
stand in the closed container for a few minutes before
sion) association is noted when either the questione
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