1、Designation: E2227 13Standard Guide forForensic Examination of Non-Reactive Dyes in TextileFibers by Thin-Layer Chromatography1This standard is issued under the fixed designation E2227; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revisio
2、n, 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. If spectrophotometry
3、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 compl
4、e-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 This standard does not replace knowledge, skill, abil
5、ity,experience, education, or training and should be used inconjunction with professional judgment.1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.4 This standard does not purport to address all of thesafety concerns, if a
6、ny, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E1459 Guide for Physical Evidence Labeling and Re
7、latedDocumentationE1492 Practice for Receiving, Documenting, Storing, andRetrieving Evidence in a Forensic Science LaboratoryE2224 Guide for Forensic Analysis of Fibers by InfraredSpectroscopyE2228 Guide for Microscopical Examination of Textile Fi-bers3. Terminology3.1 Definitions of Terms Specific
8、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 intermolecular
9、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 mobile phasethroug
10、h 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, but different
11、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 chromatographic develo
12、pment 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 chamberequilibration
13、 with mobilephase solvent vapor prior to chromatography.3.1.16 solutein TLC, a mixture of components to beseparated.1This 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 Sept.
14、 1, 2013. Published September 2013. Originallyapproved in 2002. Last previous edition approved in 2008 as E2227 02 (2008).DOI: 10.1520/E2227-13.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards v
15、olume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.1.17 solvent frontthe final point reached by the mobilephase as it flows up or across the TLC plate d
16、uring develop-ment of the chromatogram.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 where the sharp-ness of the spot relates to the efficiency of the chromatographicband.3.1.19 spottingapply
17、ing a solute sample at the origin 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.3.1.23
18、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 to assist individ
19、ualsand 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, application and d
20、evelopment 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 cov
21、ered 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) a
22、nd 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-nents can be differ
23、entiated using this technique. The applica-tion ofTLC 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 colora
24、ntpresent 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 opti
25、mum 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 temperatur
26、e. It isimportant to evaluate the performance of each TLC plate byspotting known materials along with the questioned samples.See Ref (1).35.6 Examples for the preparation of Standard dye mixturesare given in Appendix X1.6. Sample Handling6.1 The general handling and tracking of the samples shouldmee
27、t or exceed the requirements of Practice E1492 and GuideE1459.6.2 Pre-treatment (mounting medium, washing solvent, etc.)and sample preparation shall be identical for all known andquestioned fibers being compared on one TLC plate. Forremoving single fibers from slide preparations the followingprocedu
28、re 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,
29、 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 and thetype of dye
30、present. The generic class of the known andquestioned fibers shall be determined prior to TLC analysis.See Guides E2224 and E2228.7.2 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 b
31、e helpful 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 classifi
32、cation isdone on single fibers or tufts of fiber removed from the knownitem. A new fiber or tuft can be used for each classificationstage.3The boldface numbers in parentheses refer to a list of references at the end ofthis standard.E2227 1327.4 Dye ExtractionKnown and questioned fibers shall beextra
33、cted at the same time under the same conditions. Singlefibers can be extracted in a short length (about 25 mm) of 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 shall be appropriately labeled.7.4.1 About 1
34、0 l of the appropriate extractant (as recom-mended in Appendix X2) should be introduced into the tube tocover the fiber 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 recomm
35、ended 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 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 ov
36、enheated to 100C. Periodic checks should be made every 15 minfor up to 1 h.7.6 Nonextractable DyesIf classification 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 pyrid
37、ine/water (4:3) and attempt to extract at about 100C for one hour.If neither fiber extracts, a positive association is noted. If thequestioned 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
38、be sufficient dye foranalysis.7.7 ElutionAluminum backed silica gel plates, with nomi-nal particle size of 60 microns and incorporating a fluorphoreexcited at 254 nm, such as silica gel 60F 254, measuring 5 7.5 cm are recommended for normal-phase TLC of fiber dyes(1). Plates should be stored in a de
39、siccator; if this is notpossible, they should be heat activated before use.7.7.1 Both known dyes and questioned dyes to be comparedshall be applied to the same plate.The extract should be spottedonto the plate about 1 cm from the lower edge. This can bedone using a double drawn capillary tube or oth
40、er suitabledevice. Spots should not be too near to the edge of the plate orto each other. Care should 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 stron
41、glycolored. The spot size should be uniform and not exceed about2 mm in size.7.7.3 At least two (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 shall be made of the sample order on the
42、plateitself in pencil, well below the sample spots. Plates shall bethoroughly dried before developing.7.8 Development ChambersChromatograms can be devel-oped vertically in a glass chamber that may be as simple as acovered glass beaker. Commercial tanks are available (1). Twintrough tanks allow the e
43、luent to be transferred to the plate sidewithout removing the cover, but extreme care shall be takenwhen doing this not to contact the side of the TLC plate.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 chamb
44、er will be saturated with theeluent vapor.7.8.2 The level of the eluent in a vertical tank should be atleast 0.5 cm below the origin/application spots on the TLCplate.7.9 Eluent:7.9.1 Five parameters shall be considered when selectingthe optimum eluent:7.9.1.1 Separation of component dyes,7.9.1.2 Sh
45、arpness of bands,7.9.1.3 Movement of the eluted spots from the origin,7.9.1.4 Components traveling at or close to solvent front,and7.9.1.5 Strength of dye extract from questioned fibers.7.9.2 Two or more eluent systems should be assessed withthe known fibers to determine the optimum eluent system th
46、atcan be used for comparison with the questioned fibers.7.9.3 There are numerous published TLC eluent systemsthat can be applied to the development of particular fiber/dyeclass combinations (see Appendix X3).7.10 Equivalent lengths of fiber should be used for palefibers or short sample lengths. The
47、extract from knownmaterial should be applied to the TLC plate and developed inthe trial eluents as previously described. The plate should beeluted until good resolution is achieved (normally 2 cm fromthe origin), but not so far as to allow the spots to becomediffuse, making visualization difficult.
48、The plate should beremoved and the position of the solvent front marked in pencil.The plate should be dried in a hot air stream. The eluent shouldbe appropriately discarded. If the eluents produce poorseparation, others appropriate to the dye class are evaluated. Inexceptional circumstances, eluents
49、 appropriate to other dyeclasses can be used.7.10.1 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. 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 do not move from the origin, a more polareluent system should be chosen. If the spots move with thesolvent front, a less polar eluent system should be chosen.7.11 Determine and re
