1、Designation: E 2227 02Standard Guide forForensic Examination of Non-Reactive Dyes in TextileFibers by Thin-Layer Chromatography1This standard is issued under the fixed designation E 2227; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revis
2、ion, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 Metameric coloration of fibers can be detected usingUV/visible spectrophotometry. If spectrophotomet
3、ry is re-stricted to the visible spectral range only, differences in dyecomponents may remain undetected. One method of detectingadditional components is to use thin-layer chromatography(TLC). TLC is an inexpensive, simple, well-documented tech-nique that, under certain conditions, can be used to co
4、mple-ment the use of visible spectroscopy in comparisons of fibercolorants. The principle of the method is that the dye compo-nents are separated by their differential migration caused by amobile phase flowing through a porous, adsorptive medium.2. Referenced Documents2.1 ASTM Standards:E 1188 Pract
5、ice for Collection and Preservation of Informa-tion and Physical Items by a Physical Investigator2E 1459 Guide for Physical Evidence Handling and RelatedDocumentation2E 1492 Practice for Receiving, Documenting, Storing, andRetrieving Evidence in a Forensic Laboratory23. Terminology3.1 Definitions of
6、 Terms Specific to This Standard:3.1.1 activationthe heating of the adsorbent layer on aplate to dry out the moisture and maximize its adsorptivepower.3.1.2 adsorbentthe stationary phase for adsorption TLC.3.1.3 adsorptionthe attraction between the surface atomsof a solid and an external molecule by
7、 intermolecular forces.3.1.4 chambera glass chamber in which TLC develop-ment is carried out.3.1.5 chromatographya method of analysis in which sub-stances are separated by their differential migration in a mobilephase flowing through or past a stationary phase.3.1.6 developmentthe movement of the mo
8、bile phasethrough the adsorbent layer to form a chromatogram.3.1.7 dye extractionthe removal of the dye from a fiber byincubating it in an appropriate solvent.3.1.8 eluentthe solvent mixture that acts as the mobilephase in TLC.3.1.9 metameric pairtwo colors that appear the sameunder one illumination
9、, but different under other illumination.3.1.10 mobile phasethe moving liquid phase used fordevelopment.3.1.11 normal-phase chromatogramadsorption in whichthe stationary phase is polar in relation to the mobile phase.3.1.12 originthe location of the applied sample or thestarting point for the chroma
10、tographic development of theapplied sample.3.1.13 resolutionthe ability to visually separate two spots.3.1.14 retardation factor (RF)the ratio of the distancetraveled by the solute spots center divided by the distancetraveled by the solvent front, both measured from the origin.3.1.15 saturation cham
11、berequilibration with mobilephase solvent vapor prior to chromatography.3.1.16 solutein TLC, a mixture of components to beseparated.3.1.17 solvent frontthe final point reached by the mobilephase as it flows up or across the TLC plate during develop-ment of the chromatogram.3.1.18 spota round zone of
12、 sample application at theorigin, or in a chromatogram, a round zone caused by migra-tion of a separated component of the solute. The sharpness ofthe spot relates to the efficiency of the chromatographic band.3.1.19 spottingapplying a solute sample at the origin ofthe TLC plate.3.1.20 stationary pha
13、sethe solid adsorbent coating layerof a TLC plate.3.1.21 tailinga spot distorted during development into anelongated streak.3.1.22 thin-layer chromatogramthe series of spots visibleon the adsorbent layer after development.3.1.23 thin-layer chromatography (TLC)a separationtechnique in which the flow
14、of solvent causes the componentsof a mixture to migrate differentially from a narrow initial zoneover a planar, thinly-applied porous adsorptive medium.4. Summary of Guide4.1 This guide is intended to advise and to assist individualsand laboratories that conduct forensic fiber examinations and1This
15、guide is under the jurisdiction of ASTM Committee E30 on ForensicSciences and is the direct responsibility of Subcommittee E30.01 on Criminalistics.Current edition approved August 10, 2002. Published October 2002.2Annual Book of ASTM Standards, Vol 14.02.1Copyright ASTM International, 100 Barr Harbo
16、r Drive, PO Box C700, West Conshohocken, PA 19428-2959, United Sparisons in their effective application of TLC to theanalysis of fiber evidence.4.2 The guide is concerned with the extraction of dyes fromsingle fibers and from bulk material, classification of the dye orcolorant, application and devel
17、opment of the extractants onTLC plates using an optimal elution system, and evaluationand interpretation of the resulting chromatograms. The proto-cols and equipment mentioned in this document are not meantto be totally inclusive or exclusive.4.3 Not all fiber type/dye class combinations are covered
18、 inthis guide.5. Significance and Use5.1 Forensic analysis of fiber colorants using TLC should beconsidered for single fiber comparisons only when it is notpossible to discriminate between the fibers of interest usingother techniques, such as comparison microscopy (brightfieldand fluorescence) and m
19、icrospectrophotometry in the visiblerange.5.2 The extraction procedures carried out prior to TLCanalysis can provide useful information about dye classifica-tion. TLC can provide useful qualitative information about dyecomponents. Similar colors made up of different dye compo-nents can be differenti
20、ated using this technique. The applica-tion of TLC may serve to discriminate between fibers, or it mayconfirm their similarity.5.3 TLC may be prohibitively difficult or undesirable insome circumstances. Short lengths of fibers or pale coloredfibers may not have an adequate concentration of colorantp
21、resent to be examined, dye extraction from some fibers maybe impossible. The desire to preserve evidence for possibleanalysis by another examiner may preclude removing the colorfor analysis.5.4 Dye from the known material should first be character-ized and eluent systems evaluated to achieve optimum
22、 separa-tion of the extract. Dye is then extracted from single knownand questioned fibers, using an equivalent amount of material.5.5 The development of each individual TLC plate willshow some variability as a result of the coating and condition-ing of the plate, solvent condition, and temperature.
23、It isimportant to evaluate the performance of each TLC plate byspotting known materials along with the questioned samples.See Ref (16).5.6 Examples for the preparation of Standard dye mixturesare given in Appendix X1.6. Sample Handling6.1 The general handling and tracking of the samples shouldmeet o
24、r exceed the requirements of Practice E 1492 and GuideE 1459.6.2 Pre-treatment (mounting medium, washing solvent, etc.)and sample preparation must be identical for all known andquestioned fibers being compared on one TLC plate. Forremoving single fibers from slide preparations the followingprocedure
25、 is recommended.6.2.1 Any traces of marker pen ink should be cleaned fromthe coverslip using an appropriate solvent, for example, ac-etone.6.2.2 The coverslip should be cracked all around the fiberand an appropriate solvent, that will dissolve the mountant, butnot affect the fiber or the colorant, s
26、hould be used.6.2.3 The fiber is removed and extracted in an appropriatesolvent. Appropriate solvent selection will depend on themountant and the sample.7. Analysis7.1 The ease of dye extraction and the particular extractantrequired will depend on the generic class of the fiber and thetype of dye pr
27、esent. The generic class of the known andquestioned fibers must be determined prior to TLC analysis.7.2 Dye classes are classified into broad groups based ontheir chemical properties or method of application. The deter-mination of the dye class of the known fibers can be helpful inestablishing the b
28、est extractant, as well as to assist in thesubsequent selection of the most efficient eluent system.7.3 Documented extraction schemes (see Appendix X2) canbe used to determine the dye class of fibers of known genericclasses, and thus the optimum extractant. Dye classification isdone on single fibers
29、 or tufts of fiber removed from the knownitem. A new fiber or tuft can be used for each classificationstage.7.4 Dye ExtractionKnown and questioned fibers must beextracted at the same time under the same conditions. Singlefibers can be extracted in a short length (about 25 mm) of finecapillary tube (
30、internal diameter of about 1.5 mm), sealed atone end. A fine wire can be useful in pushing the fiber down thetube. The tube must be appropriately labeled.7.4.1 About 10 l of the appropriate extractant (as recom-mended in Appendix X3) should be introduced into the tube tocover the fiber sample. A fin
31、e glass pipette or syringe can beused for this procedure. The tube should be heat sealed to avoidevaporation and incubated for a constant time and temperature(as recommended in Appendix X2), preferably in an oven.Periodic checks for dye extraction should be made every 15min for up to 1 h.7.5 Dye Ext
32、raction: Bulk MaterialLarger fiber tufts canbe extracted in a Durham tube or other suitable small stopperedglass tube, using about 100 l of solvent in a sand bath or ovenheated to 100C. Periodic checks should be made every 15 minfor up to 1 h.7.6 Nonextractable DyesIf classification indicates that a
33、nonextractable dye or pigment other than a reactive dye ispresent, then place one known and one questioned fiber inlabeled capillary tubes. Add approximately 10 l pyridine/water (4:3) and attempt to extract at about 100C for one hour.If neither fiber extracts, a positive association is noted. If the
34、questioned fiber extracts and the known fiber does not (or viceversa), there is no association. If both questioned and knownfibers “bleed” dye into solution, there may be sufficient dye foranalysis.7.7 ElutionAluminum backed silica gel plates, withnominal particle size of 60 microns and incorporatin
35、g afluorphore excited at 254 nm, such as silica gel 60F 254,measuring 5 3 7.5 cm are recommended for normal-phaseTLC of fiber dyes (16). Plates should be stored in a desiccator;if this is not possible, they should be heat activated before use.7.7.1 Both known dyes and questioned dyes to be comparedm
36、ust be applied to the same plate. The extract should beE2227022spotted onto the plate about 1 cm from the lower edge. This canbe done using a double drawn capillary tube or other suitabledevice. Spots should not be too near to the edge of the plate orto each other. Care should be taken to avoid scra
37、tching theadsorbent coating layer during spot application.7.7.2 Spots should be dried using a hair dryer or hot plate,with repeat spot applications made until the spot is stronglycolored. The spot size should be uniform and not exceed about2 mm in size.7.7.3 At least two (preferably more) known dye
38、spotsshould be included on each plate, on both sides of thequestioned sample(s). It is advisable to include a standard dyespot. A note must be made of the sample order on the plateitself in pencil, well below the sample spots. Plates must bethoroughly dried before developing.7.8 Development Chambers
39、Chromatograms can be de-veloped vertically in a glass chamber that may be as simple asa covered glass beaker. Commercial tanks are available (16).Twin trough tanks allow the solvent to be transferred to theplate side without removing the cover, but extreme care mustbe taken when doing this not to co
40、ntact the side of the TLCplate.7.8.1 The eluent should be added to the tank and allowed tostand in the closed container for a few minutes beforedevelopment, so that the chamber will be saturated with thesolvent vapor.7.8.2 The level of the eluent in a vertical tank should be atleast 0.5 cm below the
41、 origin/application spots on the TLCplate. The plate should be eluted until good resolution isachieved (normally 2 cm from the origin), but not so far as toallow the spots to become diffuse, making visualization diffi-cult. The plate should be removed and the position of thesolvent front marked in p
42、encil. The plate should be dried in ahot air stream. The eluent should be appropriately discarded.7.8.3 Five parameters must be considered when selectingthe optimum eluent:7.8.3.1 Separation of component dyes,7.8.3.2 Sharpness of bands,7.8.3.3 Movement of the eluted spots from the origin,7.8.3.4 Com
43、ponents traveling at or close to solvent front,and7.8.3.5 Strength of dye extract from questioned fibers.7.8.4 There are numerous published TLC solvent systemsthat can be applied to the development of particular fiber/dyeclass combinations (see Appendix X3).7.9 Two or more eluent systems should be a
44、ssessed with theknown fibers to determine the optimum eluent system that canbe used for comparison with the questioned fibers.7.10 Equivalent lengths of fiber should be used for palefibers or short sample lengths. The extract from knownmaterial should be applied to the TLC plate and developed inthe
45、trial eluents as previously described. If the eluents producepoor separation, others appropriate to the dye class are evalu-ated. In exceptional circumstances, eluents appropriate to otherdye classes can be used.7.10.1 After a suitable eluent system has been found,comparison of known and questioned
46、fibers can be carried out.Co-chromatography can be carried out for bulk samples. Afterdrying, plates should be examined immediately in visible andin longwave ultraviolet light. Band position(s) and color(s)should be noted.7.10.2 If the spots dont move from the origin, a more polarsolvent system shou
47、ld be chosen. If the spots move with thesolvent front, a less polar solvent system should be chosen.7.11 Determine and record the color/fluorescence and the Rfvalue of the spots.7.12 Plates and samples must be identifiable. Plates must beeither documented by photography and/or retained and storedout
48、 of direct sunlight in a manner designed to minimize fading.8. Report Documentation8.1 Different solvent systems or stationary phases mayprovide additional discriminating power. The spot colors/fluorescence, sequence, and position of the spots obtained fromthe dye of the questioned fibers are compar
49、ed to those from thecorresponding known fibers analyzed under the same condi-tions.8.2 A positive association occurs when the colors/fluorescence, sequence, and positions of the spots are consis-tent between questioned and known fibers. A negative (exclu-sion) association is noted when either the questioned andknown patterns show no similarities, or there are a number ofcoincident bands but one or more bands are missing from thequestioned or known pattern. An inconclusive association isnoted when there are no bands on the TLC plate becauseinsuff
