ASTM E2227-23e1

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
´1
Designation: E2227 − 23 An American National Standard
Standard Guide for
Forensic Examination of 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. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
ε NOTE—Editorially updated 6.2.1 in June 2023.
1. Scope 2. Referenced Documents
2.1 ASTM Standards:
1.1 This guide is intended as an overview of the Thin-Layer
E620 Practice for Reporting Opinions of Scientific or Tech-
Chromatography (TLC) of fiber colorants (or individual dye
nical Experts
components) present in dyed fibers. It is intended to be applied
E1459 Guide for Physical Evidence Labeling and Related
within the scope of a broader analytical scheme for the forensic
Documentation
analysis of fiber samples. TLC could provide information that
E1492 Practice for Receiving, Documenting, Storing, and
cannot be obtained through other color analyses (such as
Retrieving Evidence in a Forensic Science Laboratory
microspectrophotometry (MSP)) (1).
E1732 Terminology Relating to Forensic Science
1.2 This standard is intended for use by competent forensic
E2224 Guide for Forensic Analysis of Fibers by Infrared
science practitioners with the requisite formal education,
Spectroscopy
discipline-specific training (see Practice E2917), and demon-
E2228 Guide for Microscopical Examination of Textile Fi-
strated proficiency to perform forensic casework (see Practice
bers
E3255).
E2917 Practice for Forensic Science Practitioner Training,
1.3 The values stated in SI units are to be regarded as
Continuing Education, and Professional Development
standard. No other units of measurement are included in this
Programs
standard.
E3255 Practice for Quality Assurance of Forensic Science
Service Providers Performing Forensic Chemical Analysis
1.4 This standard does not purport to address all of the
2.2 Other Standards:
safety concerns, if any, associated with its use. It is the
ISO/IEC 17025 General Requirements for the Competence
responsibility of the user of this standard to establish appro-
of Testing and Calibration Laboratories
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
3. Terminology
1.5 This international standard was developed in accor-
3.1 Definitions—For definitions of terms used in this guide,
dance with internationally recognized principles on standard-
refer to Terminology E1732.
ization established in the Decision on Principles for the
3.2 Definitions of Terms Specific to This Standard:
Development of International Standards, Guides and Recom-
3.2.1 adsorbent, n—the stationary phase for adsorption
mendations issued by the World Trade Organization Technical
TLC.
Barriers to Trade (TBT) Committee.
3.2.2 band, n—one or more colored areas (circular to
elongated shape) on a TLC plate produced by the separation of
This guide is under the jurisdiction of ASTM Committee E30 on Forensic
Sciences and is the direct responsibility of Subcommittee E30.01 on Criminalistics. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved May 1, 2023. Published May 2023. Originally contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
approved in 2002. Last previous edition approved in 2013 as E2227 – 13, which was Standards volume information, refer to the standard’s Document Summary page on
withdrawn in January 2022 and reinstated in May 2023. DOI: 10.1520/E2227- the ASTM website.
23E01. Available from International Organization for Standardization (ISO), ISO
The boldface numbers in parentheses refer to a list of references at the end of Central Secretariat, Chemin de Blandonnet 8, CP 401, 1214 Vernier, Geneva,
this standard. Switzerland, https://www.iso.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
´1
E2227 − 23
the dye components for a particular combination of solvent and and interpretation of the resulting chromatograms. The proto-
stationary phase. Bands are created as the solvent (mobile cols and equipment mentioned in this document are not meant
phase) moves past and reacts with the solute, migrating from to be totally inclusive or exclusive.
the origin.
4.3 Not all fiber type/dye class combinations are covered in
3.2.2.1 Discussion—“Spot” may also be used to describe
this guide.
this area.
5. Significance and Use
3.2.3 chamber, n—a glass enclosure in which TLC develop-
5.1 TLC is an inexpensive and simple technique that could
ment is carried out.
be used to complement other analytical techniques within a
3.2.4 chromatogram, thin layer, n—the series of bands
general analytical scheme related to forensic fiber examination.
visible on the adsorbent layer after development.
5.2 Consider the forensic analysis of fiber colorants using
3.2.5 chromatography, thin layer (TLC), n—a separation
TLC for single fiber comparisons only when the sample size is
technique in which the flow of solvent causes the components
adequate (that is, enough colorant can be extracted for analy-
of a mixture to migrate differentially from a narrow initial zone
sis) and it is not possible to discriminate between the fibers of
over a planar, thinly-applied porous adsorptive medium.
interest using other techniques, such as comparison micros-
3.2.6 development, n—the movement of the mobile phase
copy and MSP. Larger fibrous units (for example, thread or
through the adsorbent layer to form a chromatogram.
tuft) can be treated as an individual sample if determined to be
3.2.7 dye extraction, n—the removal of the dye from a fiber
homogeneous. Do not treat fibers that cannot be directly related
by incubating the fiber(s) in an appropriate solvent.
to each other as a collective sample for the purposes of TLC.
3.2.8 eluent, n—the solvent mixture that acts as the mobile
5.3 The extraction procedures carried out prior to TLC
phase in TLC.
analysis can provide useful information about dye classifica-
3.2.9 exclusionary difference, n—a difference in a feature or
tion. TLC can provide qualitative information about dye
property between compared items that is substantial enough to
components. Similar colors made up of different dye compo-
determine that they did not originate from the same source.
nents can be differentiated using this technique. The applica-
tion of TLC may serve to discriminate between fibers or it may
3.2.10 mobile phase, n—the moving liquid phase used for
support the possibility of fibers sharing a common source.
development.
5.4 TLC can be prohibitively difficult or undesirable in
3.2.11 normal-phase chromatogram, n—adsorption in
some circumstances. Short lengths of fibers or pale-colored
which the stationary phase is polar in relation to the mobile
fibers can lack adequate amounts of colorant necessary to be
phase.
examined by TLC. Dye extraction from some fibers can be
3.2.12 origin, n—the location of the applied sample or the
impossible (2, 3). Some fiber types do not truly extract, but
starting point for the chromatographic development of the
change or lose color. Reactive dyes are covalently bonded to
applied sample.
the fiber and typically cannot be removed by conventional
3.2.13 resolution, n—the ability to visually separate two
extraction methods, but can be released from cotton and wool
bands.
by disrupting the fiber by enzymatic or chemical digestion,
3.2.14 retardation factor (R ), n—the ratio of the distance
f respectively (1). The desire to preserve evidence from delete-
traveled by the solute band’s center divided by the distance
rious change or for possible analysis by another examiner can
traveled by the solvent front, both measured from the origin.
preclude removing the color or employing a destructive
method for analysis.
3.2.15 solute, n—in TLC, a mixture of components to be
separated.
6. Sample Handling
3.2.16 solvent front, n—the final point reached by the
6.1 The general handling and tracking of the samples should
mobile phase as it flows up or across the TLC plate during
meet or exceed the requirements of Practice E1492 and Guide
development of the chromatogram.
E1459.
3.2.17 spot, n—a visible concentration of sample applied to
6.2 Generally, only a single fiber is necessary for extraction
the TLC plate; also known as the origin.
(except for cotton and viscose classification).
3.2.18 stationary phase, n—the solid adsorbent coating
6.2.1 For very pale fibers, a small tuft is necessary.
layer of a TLC plate.
6.3 Before working with the unknown, the dye from the
known material should first be characterized and eluent sys-
4. Summary of Guide
tems evaluated to achieve optimum separation of the extract.
4.1 This guide is intended to advise and to assist individuals
Dye is then extracted from both known and questioned fibers,
and laboratories that conduct forensic fiber examinations and
using an equivalent amount of material, including similar
comparisons in the effective application of TLC.
length and depth of dyeing.
4.2 This guide is concerned with the extraction of dyes from 6.3.1 If sufficient known sample exists, different fibers may
single fibers and from bulk material, classification of the dye or be used for each part of the classification. However, best
colorant, application and development of the extractants on practice is to use one known fiber cut into smaller pieces to
TLC plates using an optimal elution system, and evaluation carry out classification.
´1
E2227 − 23
6.3.1.1 A “blank” can be helpful as a means of easy temperature, preferably in an oven. Periodic checks for dye
comparison to see if extraction has occurred. A fiber/piece of extraction should be made every 15 minutes for up to 1 hour.
fiber is placed in a glass tube with water and heated in the same
7.5.2 Fiber disruption can be used for wool and cotton fibers
way as the test piece/fiber. that are reactively dyed. The colored solutions released are not
6.3.2 If the questioned fiber is short or pale in color, there true dye extracts but are nonetheless amenable to separation
and analysis by TLC ((1), pp. 239-240).
can be insufficient dye for TLC. In such instances, an amount
of known sample (equivalent to the amount of questioned
7.6 Non-extractable Dyes—An extraction with pyridine/
sample that is able to be tested) is extracted to determine (a) the
water (4:3) at 100 °C for one hour using additional sample
efficacy of the extraction solvent and (b) the minimum length
should be attempted if the classification indicates that a
or amount of fiber needed to obtain a useful quantity of
non-extractable dye other than a reactive dye is present. If both
extracted colorant. Utilize the smallest possible volume of
questioned and known fibers “bleed” dye into solution, there
extraction solvent (4).
may be sufficient dye for analysis. Dyes that typically do not
extract are ingrain, sulfur, and vat dyes.
6.4 Ensure that all pre-treatments (mounting medium, wash-
ing solvent, etc.) and sample preparation techniques are iden-
7.7 Elution—Silica gel plates, with nominal particle size of
tical for all known and questioned fibers being compared on
60 microns and incorporating a fluorophore excited at 254 nm
one TLC plate. The following procedure is recommended for
(for example, silica gel 60 F , measuring 5 cm by 7.5 cm),
removing single fibers from slide preparations.
are recommended for normal-phase TLC of fiber dyes (6).
6.4.1 Clean any traces of marker pen ink from the coverslip
Plates should be stored in a desiccator; if this is not possible,
using a solvent (for example, acetone).
they should be heat-activated before use.
6.4.2 Remove or crack the coverslip all around the fiber.
7.7.1 Both known dyes and questioned dyes to be compared
Apply a solvent that will dissolve or rinse away the mountant,
are applied to the same plate. The extracts should be spotted
but not affect the fiber or the colorant. Remove the fiber from
onto the plate about 1 cm from the lower edge. This can be
the mount.
done using a double-drawn capillary tube or other suitable
6.4.2.1 Discussion—It is recommended that the suitability
device. Spots should not be too near to the edge of the plate or
of the solvent be evaluated with the mounted known fiber
to each other. Take care to avoid scratching the adsorbent
before it is applied to the mounted questioned fiber.
coating layer during spot application.
7.7.1.1 Questioned and known samples are applied to the
7. Analysis
same plate because the development of each individual TLC
plate can show some variability as a result of the coating and
7.1 The ease of dye extraction and the particular extractant
conditioning of the plate, solvent condition, and temperature
required will depend on the generic class of the fiber and the
(6).
type of dye present. The generic class of the known and
7.7.2 Include a solvent blank spot (extractant alone) on the
questioned fibers is determined prior to TLC analysis. See
same plate as the questioned and known sample(s). A standard
Guides E2224 and E2228.
dye spot, if used, would also be included on the same plate.
7.2 Dye classes are classified into broad groups based on
7.7.3 Spots should be dried (for example, using a hair dryer
their chemical properties or method of application. The deter-
or hot plate) with repeat spot applications made until the spot
mination of the dye class of the known fibers can be helpful in
is strongly colored. The spot size should be uniform and not
establishing the best extractant, as well as to assist in the
exceed about 2 mm in size.
subsequent selection of the most efficient eluent system.
7.7.4 Notes are made of the sample order on the plate itself
7.3 Standard dyes can be used as a control to check eluent in pencil in a position that will not interfere with the sample
spots (for example, at the top of the plate or well below the
performance. Examples for the preparation of standard dye
mixtures are given in Appendix X1 and (5). origin). Plates shall be thoroughly dried before developing.
7.8 Development Chambers—Chromatograms are devel-
7.4 Documented extraction schemes (see Appendix X2),
oped vertically, typically in a glass chamber.
based on generic fiber types, can be used to determine the class
7.8.1 The eluent is added to the tank and allowed to stand in
of dyes present in the fiber. Dye classification is performed on
the closed container before development so that the chamber
a sample removed from the known item. A new sample can be
used for each classification stage. has enough time to become saturated with the eluent vapor
(about 30 min). Filter paper can be added to the perimeter of
7.5 Dye Extraction—Known and questioned fibers are ex-
the container to assist in equilibration of the eluent and its
tracted under the same conditions (time and temperature) using
vapor.
an appropriate solvent ((4), Appendix X2). Fibers can be
7.8.2 The level of the eluent in a vertical tank should be at
extracted in a sealable vessel of appropriate size relative to the
least 0.5 cm below the origin/application spots on the TLC
sample (for example, a short length of a fine capillary tube or
plate.
a conical vial).
7.9 Eluent:
7.5.1 The extractant is introduced ((4), Appendix X2) into
the tube in a sufficient volume to immerse the sample. A glass 7.9.1 Consider the following five parameters when selecting
the optimum eluent:
pipette or syringe can be used for this procedure. The vessel is
sealed to avoid evaporation and then incubated at a constant 7.9.1.1 Separation of component dyes,
´1
E2227 − 23
7.9.1.2 Sharpness of bands, between known and questioned samples should be carefully
7.9.1.3 Movement of the eluted bands from the origin, considered. Additional sampling of areas of the known textile
7.9.1.4 Components traveling at or close to solvent front, may be necessary (for example, dye batch variation between
and back and sleeves of a shirt).
7.9.1.5 Strength of dye extract from questioned fibers.
8.1.2 Possible reasons for chromatographic differences in-
7.9.2 Two or more eluent systems should be assessed with
clude dissimilar sample characteristics (for example, amount of
the known fibers to determine the optimum eluent system. If
dye, color, dye components, dye class), heterogeneity, contri-
none of the systems described within this document result in
bution from extraneous materials, or origination from different
adequate separation, other eluents can be evaluated.
source materials. Additional samples can provide supplemental
7.9.3 There are numerous published TLC eluent systems
data to assist in assessing such differences.
that can be applied to the development of particular fiber/dye
8.1.2.1 In regards to UV examination of plates, “extra”
class combinations (see Appendix X3).
bands may result not from dye components, but from fiber
finishing agents or contaminants (such as grease or oil).
7.10 The extract from the known material is applied to the
Therefore, use caution when interpreting the significance of a
TLC plate and developed in the trial eluents as previously
band seen in one sample but not the other.
described. The plate is eluted until good resolution is achieved
(normally 2 cm from the origin), but not so long as to allow the 8.1.3 If suitable chromatographic separation is obtained,
comparisons of chromatograms can provide information re-
bands to become diffuse, making visualization difficult. The
plate is removed from the eluent and the position of the solvent garding the potential relationship between the sources of the
samples.
front is marked in pencil. The plate can be evaluated before or
after being dried. If the eluents produce poor separation, other
8.1.3.1 When exclusionary differences are observed be-
eluents appropriate to the dye class should be evaluated. tween compared chromatograms, the sources of the sa
...