forensic significance of fluorescent brighteners

8/13/2019 Forensic Significance of Fluorescent Brighteners

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J. Forens. Sci. Soc. (1977), 17 145

Forensic Significance of Fluorescent BrightenersTheir Qualitative TL Characterisation in SmallQuantities of Fibre and Detergents

J B. F. LLOYDHome Ofice Forensic Science Laboratory, Gooch Street North,Birmingham, England B5 6Q

Fluorescent brighteners derived from detergents or introduced during manufacture arecharacterised in small samples offibre, down to 5pg in mass, by the described thin layerchromatography techniques. The results provide a new way o discriminating betweenfibres mainly in terms o the detergent environment to which they have been exposed.

IntroductionWe were recently asked to determine whether some items of clothing hadbeen washed in a particular brand of detergent powder. As the powder con-tained a fluorescent brightener present in only ca. 5 of the total market, thesubsequent detection of the brightener on the clothing was of appreciableevidential significance. Although the value of this particular result dependedon the fortuitously infrequent occurrence of powders of this type, it becameapparent from other results obtained in connection with the case that sufficientvariation occurs in brighteners present on fibres to justify analysis of brightenersin case-work generally. This is especially so because, at least for qualitativepurposes, the analysis is made by straightforward modifications of thin layerchromatography (TLC) techniques currently used for the characterisation offibre dyes e.g., Garner (1967) both dyes and brighteners can often be charac-terised on the same chromatogram. Even though a fibre may not be coloured,characteristic brighteners introduced during manufacture are likely to bepresent apart from those due to detergents.

Commercial Usage of rightenersFluorescent brighteners (also referred to as fluorescent whiteners and asoptical brightening agents) are present as deliberate additions, and sometimesadventitiously, in many artefacts. The list includes washing powders, fabricconditioners, toilet soaps, textiles, toothpastes, dentures, paper (includingdocument and postage stamp identification tags), printing inks, engine oils,paints, lacquers, photographic media, plastics, cosmetics, and foodstuffs(Sarkar, 197 1 Anders et al., 197 1 Porter, 1974). For textiles applications alone2,000 formulations are commercially available (Barton and Davidson, 1974),

but this belies the relatively restricted range of compounds involved. Gold(1973) gives the following data representing the overall usage in 25 countries:Annual consumption (tons) 35,000Number of commercial products 4,000Trade marks 250Individual compounds 50The distribution amongst the various classes of product is given as:Detergents 57Paper 26Textiles 11

Plastics 6


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The basic structures of brighteners are described in a number of reviews, towhich a selection of references is given (Sarkar, 1971; Porter, 1974; Gold,1973; di Giovancel and von Rutte, 1972; Phillips, 1974; Petersen, 1975).Manufacturers generally name their products arbitrarily; hence, chemicallyindistinguishable brighteners varying in manufacture will have a number ofbrand names differentiating them in manufacture as well as in formulation.In the present work, the 81 commercial products examined, which represent across-section of manufacture and use in Great Britain, yielded 34 distinguishablegroups.Experimental

The following method is suitable for the analysis of brighteners in smallsamples of fibre and washing powders.Examine the sample in ultraviolet light (366nm). Brightened, undyed fibresfluoresce; dyed fibres may not because of quenching and self-absorptioneffects. Nonfluorescent, undyed fibres, which from many types of textiles arerare, are not further examined.Push the fibre, optimally 10-100pg in mass (for smaller amounts see below),into the taper of a Drummond 100pl Microcap pulled down to an internaldiameter of about 50pm. Seal the tip of the pipette, inject 1 .5 ~1f solvent,usually dimethylformamide (but see below), onto the sample from a Hamiltonsyringe, the needle of which must be removed from the pipette immediately toprevent the solvent from running back between the needle and the pipette wall,and seal off the shank. These operations should be performed in subdued light.From this stage the analysis must be completed under a photographic safelight or, for micromanipulation, in light passed through a strong yellow filter.Put the pipette into a protecting length of glass tube, sealed at one end, wrapthe tube in aluminium foil if the extraction cannot be done in a darkroom, andheat the package in an oven at 140 for half an hour with the pipette positionedupright (tip lowermost). Break the seal on the shank when the release of aslight vacuum will push some of the extract into the capillary. Attach thepipette to a length of plastic tubing fitted with a mouthpiece, break off the sealon the tip, and blow the extract in ca 50nl portions, dried intermittently, ontothe origin of a thin layer chromatogram (Camlab Polygram Sil G, stored oversaturated aqueous potassium acetate). Spot diameters are restricted to lessthan 1.5mm. This is effected under a dissecting microscope to which a No. 1hypodermic needle connected to a rubber blowball is fixed so that the (levelled)needle tip is just above the point of focus. fine stream of air from the needlerapidly removes the solvent after each addition. standard mixture ofbrighteners, each at 0.2mg ml-l in dimethylformamide, and a reagent blankare included in the chromatogram.Recondition the chromatogram over potassium acetate for half an hour;transfer it to a lined tank and develop without pre-equilibration in either of,or in each sequentially in the given order: (A) spectroscopic (free from ethanol)chloroform and isopropanol, 100 1.5 vol.; (B) acetone, 5M ammonia aq.,and n-heptane, 45 5 10 vol.; (C) methyl ethyl ketone (redistilled), 5Mammonia aq., and methanol, 40 15 5 vol. Development times are 40minutes, resulting in 15cm movements of the solvent fronts of A and B, andlOcm in the case of C. Examine the completed air-dried chromatogram inultraviolet light (366nm) when the positions of the separated compounds arecorrelated to the standards. Table 1 lists the standards used. If the chromato-gram is to be developed repeatedly, exposure to ultraviolet light between thedevelopments must be kept to a minimum.Washing powders should be extracted in the cold, under a safe light, with amixture of acetone and 5M ammonia aq. (45 5) (Schulze t al., 1974). Whenthe proportion of powder to solvent is 50mg ml-l only a single (50nl) appli-cation of the extract to the chromatogram is necessary. Fabric conditioners are


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T A B L ERf V LUES OF SOME ST ND RD COMPOUNDS

Compound1-(p-carbomethoxypheny1)-3-(p-chlorophenyl)-A~-pyrazolinc.2,5-bis(benzoxazo1-2-yl)thiophene.4-ethyl-7-dimethy aminocoumarin.1-(p-m~lphonamidopheny1)-3-(p-chlorophenyl)-~~-p~razoline.sodium 2-(stilbyl-4")-(naphth0-1',2',4,5)-1,2,3-triazole-2"-sulphonate.disodium 4,4'-bis(4,6-dianilino-s-tria~in-2-~lamino)-2,2'-stilbene isulphonate.disodium 4,4'-bis(4-anilino-6-morpholino-s-triazin-2-ylamino)-2,2'-stilbenediiulphonate.disodium 4,4'-bis~4-anilino-6-(N-2-hydroxyethyl-N-methylamino)-s-triazin-2-ylaminol-2,2 -stilbenedisulphonate.disodium biphenyl-4,4'-distyryl-p-sulphonate.*The solvents are given in the Experimental section.**For the stilbene derivatives the Rf values are of the trans isomers.

Solvent0 34 0.780.17 0.82

soaked into filter paper, which is then washed, dried, and extracted withdimethylfonnamide as in the case of fibres.Some examples of the chromatograms obtained are shown in Figure 1. Thef alues of standard compounds in solvents A and are listed in Table 1.Solvent C is used on the infrequent occasions (for fibres) when brightenersimmobile in and are present.Results and iscussionQuantities of jibre usedIn detergent powders individual brighteners are present at concentrationsranging from 0.02 to 0.5 . At a 20:l wash-load to powder ratio, if thebrighteners are completely adsorbed, concentrations on a fabric after a singlewash will be 0.001 to 0.025 . Hence, in 50pg of fibre the quantities ofbrighteners present will be about 0.5 to 12-5ng. These values tend to increasewith the number of washes, but levels of this order are indicated when thechromatograms are calibrated with known quantities of brighteners.The amounts of fibre used for analysis are equivalent to a small wisp of cottonat the 10pg level, and to a 50mm length ofwool fibre at IOOpg. Major brightenercomponents are readily detected in 10pg of cotton, but minor components maynot be. At the 100pg level the chromatograms are liable to be overloaded andconsequently distorted.For quantities of fibre below lOpg, the volume of extractant is reduced to0.5~1, he spot diameter a t the origin restricted to less than lmm, and thedevelopment time reduced to 15 minutes. Under these conditions the majorcomponents in 5pg samples can be characterised without undue sacrifice ofresolution. An example is shown in Figure 2.Extraction conditionsFor many fibres dimethylfonnamide is a satisfactory extractant; but if thefibre is likely to dissolve, other extractants must be used because deposition ofthe dissolved material at the origin of a chromatogram will cause gross dis-tortion and may bind brighteners irretrievably. Pyridine is a suitable extractantfor acrylic fibres. Other solvents that are useful on occasions are methanol,aqueous methanol, methyl cellosolve, ammoniacal methyl cellosolve andaqueous pyridine (Thiedel and Schmitz, 1967). However, none of thesecompares in efficiency with dimethylformamide for insoluble fibres. Because ofthe sealed tube technique, low-boiling extractants may be used at elevatedtemperatures. Hopefully, more volatile alternatives to dimethylformamide willbe found, as the removal of this solvent from chromatograms is time-consuming.The cold air evaporation technique is preferred to the usual hot air stream,


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Figure 1. Chromatograms of extracts Figure 2. Chromatograms of the samefrom 4 different samples of fibre 50pg) standard mixture as in Figure outerand, on the far right, the standard com- most), and of 5 5 and 10pg samples ofpounds given in Table together with an fibre. These are of the same source as theunidentified compound 6th from the sample on the far left of Figure 1. Theorigin). The solvent is mixtu:e_B. solvent was mixture B.

which can give rise to large local variations in adsorbent activity. After thepipette has been discharged, some extract remains on the fibre, and may beremoved either with dimethylformamide or with the more readily volatilisedpyridine. Usually, small amounts of brightener remain adsorbed. I n the case ofdetergent brighteners any variation in selectivity with which different com-pounds are retained is insufficient to cause qualitative variation in chromato-graphy patterns: the same patterns are obtained when, given sufficient fibre,all the brighteners are removed by Soxhlet extraction.Effects o ultraviolet lightIn the presence of ultraviolet light, brighteners in the dissolved state undergophotodegradation and photoisomerism Kurz and Schuierer, 1967). Photo-degradation is severe in dilute solutions, e.g. 1pg ml-l, the fluorescence ofwhich can disappear within a few minutes on exposure to daylight. Photo-isomerism results in the production of a photo-equilibrated mixture of cis andtrans isomers from the stilbene-derived brighteners, the commonest type, whichare manufactured in and adsorbed on fabric as the trans forms. The cis formsare not adsorbed. Only the trans forms fluoresce, but the two are separatedfrom one another by TLC, and in ultraviolet light the cis spot becomesfluorescent because of the photo-equilibration. At the same time the fluorescenceof the trans form decays within a few seconds to the photo-equilibrium value.Although extracts that have been exposed to unfiltered light produce chromato-grams that are impressively complex the number of separated compounds isdoubled), their use must be avoided: nothing is contributed evidentiallybecause in R pvalues the cis and trans isomers are strongly correlated on silicagel the cis isomer is the more strongly adsorbed) nd, because photo-equilibrium


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constants under typical conditions are of the order of unity, the fluorescence ofeach brightener is divided approximately equally between two spots, whichreduces sensitivity by half.Chromatography conditionsA considerable variety of solvents and adsorbents for the chromatographyof brighteners has been proposed. Apart from the previously mentioned, otherreferences pertinent in the present context are reviews by Longman (1975) andby Shroder and Hagen (1968), and reports of separations on silica gel (Latinak,1964; Figge, 1968;Ganz et al., 1975), on paper and on silica gel (Brown, 1964),on paper/reversed phase (Gasparic, 1969), and on silica gel or alumina(Schlegelmilch et al., 1971). Basic solvents are the most frequently used.The adsorbent and solvents recommended in the present work are theresults of experiments with well over 150 different sets of conditions. All of thecommon detergent brighteners (generally sulphonic acid derivatives) areseparated by the arnmoniacal solvent B, in which the presence of n-heptanestrikingly improves resolution and selectivity (other nonpolar compounds dolikewise). Some weakly retained nonionic brighteners are not separated in B.For these the chloroform/isopropanol mixture A is usually satisfactory. Whenboth types of brightener are present, or when no prior information is available,the solvent mixtures are used sequentially. Because in the adsorbed state onsilica gel the photoisomerism is not inhibited, the brief exposure to ultravioletlight of the chromatogram after each development unavoidably results in someisomerisation and loss of sensitivity.The considerable changes that occur in the adsorbent's activity on contactwith vapour from the ammoniacal solvents render the chromatograms excep-tionally susceptible to distortions caused by poor developing-tank geometry.As a general rule, all points on a chromatogram should be equidistant from thenearest vertical surface in the tank. Pre-equilibration of the chromatographystrip in the tank is deleterious to the separation.Some brighteners are immobile in both of solvents A and B. A reduction inammonia concentration from 5M to 1M can produce some mobility; and allbrighteners examined are mobile in solvent C. However, discrimination withinthis particular group at present remains rather low.Brighteners present in washing powders and related productsTable gives the Market Research GB (1974) data on various brands ofclothes washing materials. The data are in broad agreement with the IPCConsumers' Marketing Manual (1975). Approximate market shares at thetime of these surveys are hence: Proctor and Gamble, 48% ;Lever Bros., 43% ;and shop brands 3%. The remaining materials used for clothes washinginclude the products of small-scale manufacturers, and toilet soaps andwashing-up liquids.

TABLE 2MARKET DISTRIBUTION OF VARIOUS WASHING POWDERS

Proctor and Gamble h v e r Bros. Shop BrandsAriel 20 Persil 16 overall 3Daz 13 Persil Auto. 6Fairy Snow 7 OmoTide 65 Radiant 6DreftBold* i surfux ; O, Stergem** 2Drive* 1*From IPCConswncrs har ket ing Manual (1975). The rest of the data arefrom Market Research Great Britain (1974), which includes a 6 others/don't lu~owigure.**Liquid detergent.


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Fabric conditioners, some of which contain brighteners, are used by 39 ofhousewives (IPC Consumers' Marketing Manual, 1975). In 1973 80 of thismarket was taken by Levers' Comfort and the remainder largely by Boots'Soft Rinse (Financial Times, 1973). Since this date Lenor has been stronglypromoted by Proctor and Gamble, and other brands have appeared. Otherpossible origins of detergent brighteners on fibres are some toilet soaps andnappy-soaking products.Samples of all detergent powders listed in Table 2 were examined by theabove-described techniques. The brighteners found are arbitrarily labelled1-5 in order of their increasing n alues in solvent B These particular valuesare not significantly different from the last five of Table 1. No brightener wasfound in DreJt and Lux. Ariel, Tide, Persil, Omo and Drive contained only 3. Allof the others contained 3, together with 2 in the Proctor and Gamble products,4 in Lever Bros.' products, and 2 usually with 5 in the chain store (shop brand)products with one exception containing 3 and 1From these results and the data in Table 2, on fibres to which detergentbrighteners are substantive (principally cellulosics, wool, polyamides) andwhen the fibre has been washed in only one brand of powder, the distributionof fibres carrying the indicated brighteners will be as follows :

components 3, 4 173 483 2 233 , 2 , 5 3

Fibres washed repeatedly in the common detergents will accumulate compar-able amounts of 3, 4 and 2. At an intermediate degree of randomisation thesecond group will merge with the first or third.Of the fabric conditioners examined, only Soft Rime contained a brightener( I) . Although recent figures are not available a usage of this conditioner in5-10 of domestic clothes washing seems probable.Brightenersfound on jbresSamples (60-9OPg) of fibres from 6 1 articles differing in origin were examinedfor the presence of brighteners. Of these articles, 4 were known not to have beenwashed, the rest were selected because of the likelihood that they had beenwashed.I n 12 of the samples no brighteners were found. With the exception of acarpet fibre, all of these were dyed clothing fibres. They included 2 acrylics,to which detergent brighteners are not substantive. The remaining 9 (cotton,cotton-polyester, nylon and wool) had evidently not been washed and wouldnot be brightened during manufacture (because they were coloured).The numbers of the remaining samples attributable to the various washingpowder groups (excluding any differentiation due to brighteners not attribut-able to detergents) are:

(a) components 3, 4 6(b) 3 7ici 3, 6(d) 5, 2 (includes a sample from (a) and anotherfrom (e\'l\ ~ /(el 3 , 4 , 2 17(f) 9 I 5 (includes 2 samples in (c), and 1 in (a))These results are collectively from the various types of fibre to whichdetergent-derived brighteners are substantive. No variation according to fibretype within these results was apparent; and considerations of colour so far asthis class of brighteners is concerned are, of course, irrelevant except to theextent that lightly coloured clothing will be the most frequently washed.Apart from the degree of randomness implied by (e) with which the powders


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used in any one household are selected the figures are consistent with themarket distribution data. Hence it is suggested that the described techniquesmay be used to qualitatively evaluate the detergent environment to which asusceptible fibre has been exposed. The evidential significance of the result willbe determined by the pertinent marketing statistics and by the composition ofwashing powders at the time concerned given substantivity.As well as detergent brighteners others added during manufacture arepresent on undyed fibres. The samples examined included 27 of undyed cotton.Each of a group of 9 of these contained only the three common detergentbrighteners i .e. there was no discrimination within the group. Similarly therewas no discrimination within another group of 4 and another of 3. But eachof the remaining 11 samples was discriminated from all of the others largelybecause of the presence of additional brighteners. The corresponding prob-ability of a randomly selected matched pair within these 27 samples is 0.13.As this result is from fibres of the same type between which the usual colour andthin layer chromatographic comparisons of dyes are not available even the

modest degree of discrimination obtained is not without forensic significance.Amongst the other samples examined were 4 of white acrylic fibre each ofwhich was differentiated from the others.The chromatograms from the coloured fibres often contained separated dyecomponents as well as brighteners and both types of compound were thereforecharacterised simultaneously. Although none of the objects of this work was todevelop the already well-established techniques used in the forensic charac-terisation of fibre dyes it is obviously most important to the economy of casework material that in any further developments the analysis of dyes andbrighteners should be considered conjointly.

General DiscussionThe principal aim of this work was to understand the extent to whichdetergent traces on small amounts of fibre might be characterised. To this endvarious other materials could be sought: for instance the softening additivesand substantive antimicrobial compounds used in some preparations couldprobably be detected fairly readily in small samples; and perfume and tracesof borates phosphates and detergents remaining on incompletely rinsed fabric

might be of evidential value. However the techniques described will probablyfind most use in the correlation of transferred fibres with possible points oforigin although in the future the significance of this type of comparison willundoubtedly be enhanced by the introduction of the liquid column chromato-graphy and HPTLC techniques presently under development. Apart frombrighteners dyes are potentially characterisable by these techniques at a farhigher level of specificity than is possible a t present.There are also obvious applications in the characterisation of plastics papersand other materials; and in the detection of contact traces despite the strengthwith which a brightener is generally held within a material. For instance thebacks of some of the photographs prepared in connection with this papercarried fluorescent marks left by transfer of brighteners between other photo-graphs with which contact had occurred during processing. In one case theoriginating photograph was clearly identifiable from the transferred pattern ofits uneven margin.

cknowledgementsI am very grateful to various members of the dyestuffs and detergents

industries for their help in many ways in connection with this work.


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eferencesANDERS, ., ANLIKER, . and VEENEMANS,. J., 1971, The Secret o White,(Ciba-Geigy Ltd.)BARTON,. and DAVIDSON,., 1974, Rev. Prog. Coloration, 5 3.BROWN,. C., 1964,J Soc. Dyers Colours, 80 185.FIGGE,K., 1968, Fette Seifen Anstr Mittel, 70 680.DI GIOVANCEL,. and VON RUTTE,R., 1972, Invest. Inform. Text. Tensioactivos,15 189.Financial Times, 1973, 1 February.GANZ, . R., SCHULZE,. STENSBY,. S., LYMAN,. L. and MACEK, ., 1975,Environ. Sci. Tech., 9 738.GARNER,W., 1967, Textile Laboratory Manual, 4 chapter 6 (Heywood BooksLtd.).GASPARIC,. 1969, Chemicke Listy, 63 1363.GOLD,H., 1973, MVC-Rep. Miljoevardrcentum (Stockholm), 2, 23.IPC Consumers Marketing Manual UK 1975, Section 5.6.KURZ, . and SCHUIERER,., 1967, Fette Seifen Anstr Mittel, 69 24.LATINAK,., 1964,J. Chromatogr., 14 482.LONGMAN,. F., 1975, Talanta, 62 1.Market Research Great Britain, 1974, 14 (9), 19.PETERSEN,., 1975, Angew. Chemie (German ed.), 87 693.PHILLIPS, ., 1974, Photochemistry (Chem. Soc., London), 5, 758.PORTER, . J. 1974, Optical Brightening Agents, DSIR New Zealand, Rep. C.D.2188SARKAR, K., 1971, Fluorescent Whitening Agents (Merrow Publishing Co. Ltd.).SCHLEGELMILCH,., ABDELKADER,. and ECKELT,M., 1971, Textilindustrie,73 274SCHULZE,., POLCARO,. and STENSBY,., 1974, Soap, Cosmetics and ChemicalSpecialities, November.SHRODER,. and HAGEN, ., 1968, Plaste Kautsch., 15 625.THIEDEL, . and SCHMITZ,., 1967,J. Chromatogr., 27 413.

forensic significance of fluorescent brighteners

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