Fiber Guidelines, Chapter 4 (FSC, April 1999)
Thin-Layer Chromatography of Nonreactive Dyes in Textile Fibers
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.
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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.
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.
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.
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.
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.
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.
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:
18.104.22.168. Separation of component dyes;
22.214.171.124. Sharpness of bands;
126.96.36.199. Movement from the origin;
188.8.131.52. Components traveling at or close to solvent front; and
184.108.40.206. 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.
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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FORENSIC SCIENCE COMMUNICATIONS APRIL 1999 VOLUME 1 NUMBER 1