1、Designation: E 2227 02 (Reapproved 2008)Standard 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
2、 the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 Metameric coloration of fibers can be detected usingUV/visible spectrophotometry. I
3、f spectrophotometry 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,
4、can be used to comple-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.1.2 The values stated in SI units are
5、 to be regarded asstandard. No other units of measurement are included in thisstandard.2. Referenced Documents2.1 ASTM Standards:2E 1459 Guide for Physical Evidence Labeling and RelatedDocumentationE 1492 Practice for Receiving, Documenting, Storing, andRetrieving Evidence in a Forensic Science Labo
6、ratory3. Terminology3.1 Definitions of 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 atoms
7、of a solid and an external molecule by 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
8、.1.6 developmentthe movement of the mobile 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
9、 appear the sameunder one illumination, 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 samp
10、le or thestarting point for the chromatographic 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
11、from the origin.3.1.15 saturation chamberequilibration 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 c
12、hromatogram.3.1.18 spota round zone of 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
13、 ofthe TLC plate.3.1.20 stationary phasethe 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.1This guide is under the jurisdiction of
14、 ASTM Committee E30 on ForensicSciences and is the direct responsibility of Subcommittee E30.01 on Criminalistics.Current edition approved March 15, 2008. Published July 2008. Originallyapproved in 2002. Last previous edition approved in 2002 as E 222702.2For referenced ASTM standards, visit the AST
15、M website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, Unite
16、d States.3.1.23 thin-layer chromatography (TLC)a separationtechnique in which the flow 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 t
17、o assist individualsand laboratories that conduct forensic fiber examinations andcomparisons 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, a
18、pplication and development 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 comb
19、inations are covered 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
20、 fluorescence) and microspectrophotometry 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-nen
21、ts can be differentiated 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 concen
22、tration of colorantpresent 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 evaluate
23、d to achieve optimum 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 conditio
24、n, and temperature. 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
25、samples shouldmeet or 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 th
26、e followingprocedure 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 fibe
27、r or the colorant, should 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 a
28、nd thetype of dye present. 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
29、inestablishing the best 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 isd
30、one on single fibers 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
31、finecapillary tube (internal diameter of about 1.5 mm), sealed atone end.Afine 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 f
32、iber sample. A fine 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
33、to 1 h.7.5 Dye Extraction: 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 classificatio
34、n indicates that anonextractable 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 associatio
35、n is noted. If thequestioned fiber extracts and the known fiber does not (or viceE 2227 02 (2008)2versa), 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
36、size of 60 microns and incorporating 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
37、and questioned dyes to be comparedmust be applied to the same plate. The extract should bespotted 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 sh
38、ould be taken to avoid scratching 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 (
39、preferably more) known dye 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 developi
40、ng.7.8 Development ChambersChromatograms 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 take
41、n when doing this not to contact 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
42、be atleast 0.5 cm below the 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 t
43、hesolvent front marked in pencil. 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
44、from the origin,7.8.3.4 Components 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
45、 eluent systems should be assessed 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
46、 plate and developed inthe 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,comparis
47、on of known and questioned 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 mo
48、re polarsolvent system should 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 an
49、d/or retained and storedout 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 compared 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
