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J. Forens. Sci. Soc.

(1977), 17, 145

Forensic Significance of Fluorescent Brighteners: Their Qualitative TLC Characterisation in Small Quantities of Fibre and Detergents
J. B. F. LLOYD Home Ofice Forensic Science Laboratory, Gooch Street North, Q & Birmingham, England B5 6
Fluorescent brighteners derived from detergents or introduced during manufacture are y the described thin layer characterised in small samples offibre, down to 5pg in mass, b f discriminating between chromatography techniques. The results provide a new way o fibres mainly in terms o f the detergent environment to which they have been exposed.
Introduction We were recently asked to determine whether some items of clothing had been washed in a particular brand of detergent powder. As the powder contained a fluorescent brightener present in only ca. 5% of the total market, the subsequent detection of the brightener on the clothing was of appreciable evidential significance. Although the value of this particular result depended on the fortuitously infrequent occurrence of powders of this type, it became apparent from other results obtained in connection with the case that sufficient variation occurs in brighteners present on fibres to justify analysis of brighteners in case-work generally. This is especially so because, at least for qualitative purposes, the analysis is made by straightforward modifications of thin layer chromatography (TLC) techniques currently used for the characterisation of fibre dyes e.g., Garner (1967) : both dyes and brighteners can often be characterised on the same chromatogram. Even though a fibre may not be coloured, characteristic brighteners introduced during manufacture are likely to be present apart from those due to detergents.

Commercial Usage of Brighteners Fluorescent brighteners (also referred to as fluorescent whiteners and as optical brightening agents) are present as deliberate additions, and sometimes adventitiously, in many artefacts. The list includes washing powders, fabric conditioners, toilet soaps, textiles, toothpastes, dentures, paper (including document and postage stamp identification tags), printing inks, engine oils, paints, lacquers, photographic media, plastics, cosmetics, and foodstuffs (Sarkar, 1971;Anders et al., 1971; Porter, 1974). For textiles applications alone 2,000 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) 35,000 4,000 Number of commercial products Trade marks 250 Individual compounds 50 The distribution amongst the various classes of product is given as: Detergents 57% Paper 26% Textiles 11% Plastics 6%

in light passed through a strong yellow filter. Petersen. Phillips. dyed fibres may not because of quenching and self-absorption effects. and a reagent blank are included in the chromatogram. chloroform and isopropanol. 5 ~ 1 usually dimethylformamide (but see below).5 vol.2mg ml-l in dimethylformamide. and blow the extract in ca. inject 1 .. yielded 34 distinguishable groups. to which a selection of references is given (Sarkar. 50nl portions. wrap the tube in aluminium foil if the extraction cannot be done in a darkroom. and seal off the shank. onto the sample from a Hamilton syringe. di Giovancel and von Rutte. Push the fibre. 1974. Break the seal on the shank when the release of a slight vacuum will push some of the extract into the capillary. optimally 10-100pg in mass (for smaller amounts see below). 5M and n-heptane. 5M ammonia aq. In the present work. 1971. Manufacturers generally name their products arbitrarily. Nonfluorescent. Brightened. are not further examined. When the proportion of powder to solvent is 50mg ml-l only a single (50nl) application of the extract to the chromatogram is necessary. 45 15 5 vol. diameter of about 50pm. (45 5) (Schulze et al. This is effected under a dissecting microscope to which a No. and heat the package in an oven at 140' for half an hour with the pipette positioned upright (tip lowermost). These operations should be performed in subdued light. resulting in 15cm movements of the solvent fronts of A and B. or in each sequentially in the given order: (A) spectroscopic (free from ethanol) 1.The basic structures of brighteners are described in a number of reviews. If the chromatogram is to be developed repeatedly. the needle of which must be removed from the pipette immediately to prevent the solvent from running back between the needle and the pipette wall. Spot diameters are restricted to less than 1. Recondition the chromatogram over potassium acetate for half an hour. stored over saturated aqueous potassium acetate). 1972. Table 1 lists the standards used. each at 0. dried intermittently. transfer it to a lined tank and develop without pre-equilibration in either of. 1973. Gold. sealed at one end.. for micromanipulation. 40 minutes. hence. Examine the completed air-dried chromatogram in ultraviolet light (366nm) when the positions of the separated compounds are correlated to the standards. (B) acetone. Washing powders should be extracted in the cold. chemically indistinguishable brighteners varying in manufacture will have a number of brand names differentiating them in manufacture as well as in formulation. 1974. and methanol. exposure to ultraviolet light between the developments must be kept to a minimum.5mm. into the taper of a Drummond 100pl Microcap pulled down to an internal of solvent. Porter. which represent a cross-section of manufacture and use in Great Britain.. undyed fibres fluoresce. 1 hypodermic needle connected to a rubber blowball is fixed so that the (levelled) needle tip is just above the point of focus. Attach the pipette to a length of plastic tubing fitted with a mouthpiece. Development times are 40 ammonia aq. undyed fibres. the 81 commercial products examined.. Fabric conditioners are + + + + + + . 1974). break off the seal on the tip.. Examine the sample in ultraviolet light (366nm). From this stage the analysis must be completed under a photographic safe light or. 100 5 10 vol. onto the origin of a thin layer chromatogram (Camlab Polygram Sil G. A fine stream of air from the needle rapidly removes the solvent after each addition. 1975). (C) methyl ethyl ketone (redistilled). and lOcm in the case of C. under a safe light. Experimental The following method is suitable for the analysis of brighteners in small samples of fibre and washing powders. Put the pipette into a protecting length of glass tube. with a mixture of acetone and 5M ammonia aq. Seal the tip of the pipette. which from many types of textiles are rare. A standard mixture of brighteners.

2'stilbenediiulphonate. These values tend to increase with the number of washes. disodium 4. 1-(p-m~lphonamidopheny1)-3-(p-chlorophenyl)-~~-p~razoline. 1967).001 to 0.TABLE 1 Rf VALUES OF SOME STANDARD COMPOUNDS Compound * * 1-(p-carbomethoxypheny1)-3-(p-chlorophenyl)-A~-pyrazolinc. and extracted with dimethylfonnamide as in the case of fibres. if the brighteners are completely adsorbed.4'-distyryl-p-sulphonate.4. The cold air evaporation technique is preferred to the usual hot air stream.3-triazole-2"-sulphonate. but if the fibre is likely to dissolve. For quantities of fibre below lOpg. concentrations on a fabric after a single wash will be 0. Results and Discussion Quantities of jibre used I n detergent powders individual brighteners are present at concentrations ranging from 0. none of these compares in efficiency with dimethylformamide for insoluble fibres. . 0.5)-1. dried. At the 100pg level the chromatograms are liable to be overloaded and consequently distorted. Solvent* A B 0. However. disodium 4. Pyridine is a suitable extractant for acrylic fibres.the spot diameter at the origin restricted to less than lmm. The amounts of fibre used for analysis are equivalent to a small wisp of cotton at the 10pg level.5-bis(benzoxazo1-2-yl) -thiophene. Major brightener components are readily detected in 10pg of cotton. in 50pg of fibre the quantities of brighteners present will be about 0. but minor components may not be. aqueous methanol. Some examples of the chromatograms obtained are shown in Figure 1. Under these conditions the major components in 5pg samples can be characterised without undue sacrifice of resolution. methyl cellosolve.025%.5%. Because of the sealed tube technique. the volume of extractant is reduced to 0.5~1.78 0. and the development time reduced to 15 minutes. Hence. and to a 50mm length ofwool fibre at I OOpg. low-boiling extractants may be used at elevated temperatures. The Rfvalues of standard compounds in solvents A and B are listed in Table 1.4'-bis(4. 2. as the removal of this solvent from chromatograms is time-consuming. Other solvents that are useful on occasions are methanol. which is then washed.17 4-ethyl-7-dimethy!aminocoumarin.2 -stilbenedisulphonate. Hopefully. Extraction conditions For many fibres dimethylfonnamide is a satisfactory extractant.02 to 0.5 to 12-5ng. disodium 4. but levels of this order are indicated when the chromatograms are calibrated with known quantities of brighteners. other extractants must be used because deposition of the dissolved material at the origin of a chromatogram will cause gross distortion and may bind brighteners irretrievably. Solvent C is used on the infrequent occasions (for fibres) when brighteners immobile in A and B are present. **For the stilbene derivatives the Rf values are of the trans isomers.2'-stilbene disulphonate. more volatile alternatives to dimethylformamide will be found.82 soaked into filter paper.6-dianilino-s-tria~in-2-~lamino)-2.2. disodium biphenyl-4. ammoniacal methyl cellosolve and aqueous pyridine (Thiedel and Schmitz.2'.34 0.4'-bis~4-anilino-6-(N-2-hydroxyethyl-N-methylamino)-s-triazin-2ylaminol-2. *The solvents are given i n the Experimental section. An example is shown in Figure 2. At a 20:l wash-load to powder ratio. sodium 2-(stilbyl-4")-(naphth0-1'.4'-bis(4-anilino-6-morpholino-s-triazin-2-ylamino)-2.

1967). which are manufactured in and adsorbed on fabric as the trans forms. and of 5. some extract remains on the fibre. and may be removed either with dimethylformamide or with the more readily volatilised pyridine. small amounts of brightener remain adsorbed. These are of the same source as the sample on the far left of Figure 1. Chromatograms of the same standard mixture as in Figure 1 (outer most). brighteners in the dissolved state undergo photodegradation and photoisomerism (Kurz and Schuierer.g. the standard compounds given in Table 1 together with an unidentified compound (6th from the origin). given sufficient fibre. the commonest type. After the pipette has been discharged. Photodegradation is severe in dilute solutions. The solvent was mixture B. Effects o f ultraviolet light I n the presence of ultraviolet light. all the brighteners are removed by Soxhlet extraction. Chromatograms of extracts from 4 different samples of fibre (50pg) and. The cis forms are not adsorbed. Figure 2. At the same time the fluorescence of the trans form decays within a few seconds to the photo-equilibrium value. Although extracts that have been exposed to unfiltered light produce chromatograms that are impressively complex (the number of separated compounds is doubled). 1pg ml-l. which can give rise to large local variations in adsorbent activity. on the far right. and in ultraviolet light the cis spot becomes fluorescent because of the photo-equilibration. Only the trans forms fluoresce. Usually. because photo-equilibrium . The solvent is mixtu:e_B.Figure 1. e.and. Photoisomerism results in the production of a photo-equilibrated mixture of cis and trans isomers from the stilbene-derived brighteners. 5 and 10pg samples of fibre. their use must be avoided: nothing is contributed evidentially because in Rpvalues the cis and trans isomers are strongly correlated (on silica gel the cis isomer is the more strongly adsorbed) . but the two are separated from one another by TLC. the fluorescence of which can disappear within a few minutes on exposure to daylight.. I n the case of detergent brighteners any variation in selectivity with which different compounds are retained is insufficient to cause qualitative variation in chromatography patterns: the same patterns are obtained when.

" h v e r Bros. For these the chloroform/isopropanol mixture A is usually satisfactory. on paper and on silica gel (Brown. When both types of brightener are present. the brief exposure to ultraviolet light of the chromatogram after each development unavoidably results in some isomerisation and loss of sensitivity. which reduces sensitivity by half. Persil Persil Auto. i Lux surf . the solvent mixtures are used sequentially. Basic solvents are the most frequently used.. A reduction in ammonia concentration from 5M to 1M can produce some mobility. 1968. 1971). and "shop brands" 3%. Apart from the previously mentioned. The rest of the data are from Market Research Great Britain (1974). Some brighteners are immobile in both of solvents A and B. and all brighteners examined are mobile in solvent C. Because in the adsorbed state on silica gel the photoisomerism is not inhibited. The remaining materials used for clothes washing include the products of small-scale manufacturers. 1964. The adsorbent and solvents recommended in the present work are the results of experiments with well over 150 different sets of conditions. TABLE 2 MARKET DISTRIBUTION OF VARIOUS WASHING POWDERS Proctor and Gamble Ariel 20% Daz 13 % Fairy Snow 7% 5% Tide Dreft Bold* O % L . All of the common detergent brighteners (generally sulphonic acid derivatives) are separated by the arnmoniacal solvent B. Some weakly retained nonionic brighteners are not separated in B. However. discrimination within this particular group at present remains rather low. The data are in broad agreement with the IPC Consumers' Marketing Manual (1975). Lever Bros. Omo Radiant 16 % 6% 6% 6% Shop Brands overall 3% Stergem** 2 1% Drive* *From IPCConswncrs' harketing Manual (1975). 43% . Pre-equilibration of the chromatography strip in the tank is deleterious to the separation. the fluorescence of each brightener is divided approximately equally between two spots. Chromatography conditions A considerable variety of solvents and adsorbents for the chromatography of brighteners has been proposed.. As a general rule. 1969). on paper/reversed phase (Gasparic. **Liquid detergent.constants under typical conditions are of the order of unity.. and on silica gel or alumina (Schlegelmilch et al. Brighteners present in washing powders and related products Table 2 gives the Market Research GB (1974) data on various brands of clothes washing materials. Approximate market shares at the time of these surveys are hence: Proctor and Gamble. 1964). 1975).% O/. Figge. or when no prior information is available. and reports of separations on silica gel (Latinak. . Ganz et al. and toilet soaps and washing-up liquids. The considerable changes that occur in the adsorbent's activity on contact with vapour from the ammoniacal solvents render the chromatograms exceptionally susceptible to distortions caused by poor developing-tank geometry. other references pertinent in the present context are reviews by Longman (1975) and by Shroder and Hagen (1968). which includes a 6 % others/ don't lu~ow figure. 48% . all points on a chromatogram should be equidistant from the nearest vertical surface in the tank. in which the presence of n-heptane strikingly improves resolution and selectivity (other nonpolar compounds do likewise).

. Brightenersfound o n jbres Samples (60-9OPg) of fibres from 6 1 articles differing in origin were examined for the presence of brighteners. I 5 (includes 2 samples in (c). Samples of all detergent powders listed in Table 2 were examined by the above-described techniques. together with 2 in the Proctor and Gamble products. 2 6 (d) . The numbers of the remaining samples attributable to the various washing powder groups (excluding any differentiation due to brighteners not attributable to detergents) are: 6 (a) components 3. 2 17 (f) 9 . 3. Persil. Apart from the degree of randomness implied by (e) with which the powders \ ~ . 3 7 ici . nylon and wool) had evidently not been washed and would not be brightened during manufacture (because they were coloured). 3 48% . From these results and the data in Table 2. 2 23% . and 1 in (a)) These results are collectively from the various types of fibre to which detergent-derived brighteners are substantive. only Soft Rime contained a brightener ( I ) .. are used by 39% of housewives (IPC Consumers' Marketing Manual. No brightener was found in DreJt and Lux.. / . Although recent figures are not available a usage of this conditioner in 5-10% of domestic clothes washing seems probable. Of these articles. Ariel. 3 . Since this date Lenor has been strongly promoted by Proctor and Gamble. cotton-polyester. 4 in Lever Bros. All of the others contained 3. to which detergent brighteners are not substantive. and considerations of colour so far as this class of brighteners is concerned are. 3. 4 17% . and other brands have appeared. all of these were dyed clothing fibres. 1973). Of the fabric conditioners examined. 4 . 4 were known not to have been washed. I n 1973 80% of this market was taken by Levers' Comfort and the remainder largely by Boots' Soft Rinse (Financial Times..' products. At a n intermediate degree of randomisation the second group will merge with the first or third. on fibres to which detergent brighteners are substantive (principally cellulosics. of course. 4 (b) . Omo and Drive contained only 3. Other possible origins of detergent brighteners on fibres are some toilet soaps and nappy-soaking products. I n 12 of the samples no brighteners were found. The brighteners found are arbitrarily labelled 1-5 in order of their increasing Rnvalues in solvent B. No variation according to fibre type within these results was apparent. irrelevant except to the extent that lightly coloured clothing will be the most frequently washed. polyamides) and when the fibre has been washed in only one brand of powder. Tide. The remaining 9 (cotton. 3 . 2 (includes a sample from (a) and another from (e\'l (el . and 2 usually with 5 in the chain store (shop brand) products with one exception containing 3 and 1. . These particular values are not significantly different from the last five of Table 1. 1975). 4 and 2. 2 . the distribution of fibres carrying the indicated brighteners will be as follows : components 3.Fabric conditioners.. 5.. wool. With the exception of a carpet fibre. the rest were selected because of the likelihood that they had been washed. 5 3% Fibres washed repeatedly in the common detergents will accumulate comparable amounts of 3. some of which contain brighteners. They included 2 acrylics.

For instance. various other materials could be sought: for instance. as well as brighteners. To this end. and another of 3. General Discussion The principal aim of this work was to understand the extent to which detergent traces on small amounts of fibre might be characterised. As this result is from fibres of the same type between which the usual colour and thin layer chromatographic comparisons of dyes are not available. Each of a group of 9 of these contained only the three common detergent brighteners. Amongst the other samples examined were 4 of white acrylic fibre. The evidential significance of the result will be determined by the pertinent marketing statistics and by the composition of washing powders at the time concerned. largely because of the presence of additional brighteners. However. dyes are potentially characterisable by these techniques at a far higher level of specificity than is possible at present. The corresponding probability of a randomly selected matched pair within these 27 samples is 0. The samples examined included 27 of undyed cotton. and traces of borates.e. even the modest degree of discrimination obtained is not without forensic significance.. Hence. I n one case the originating photograph was clearly identifiable from the transferred pattern of its uneven margin.used in any one household are selected. But each of the remaining 11 samples was discriminated from all of the others. Although none of the objects of this work was to develop the already well-established techniques used in the forensic characterisation of fibre dyes. phosphates and detergents remaining on incompletely rinsed fabric might be of evidential value. As well as detergent brighteners. it is suggested that the described techniques may be used to qualitatively evaluate the detergent environment to which a susceptible fibre has been exposed. Apart from brighteners. although in the future the significance of this type of comparison will undoubtedly be enhanced by the introduction of the liquid column chromatography and HPTLC techniques presently under development.13. the figures are consistent with the market distribution data. the techniques described will probably find most use in the correlation of transferred fibres with possible points of origin. i. There are also obvious applications in the characterisation of plastics. papers and other materials. others added during manufacture are present on undyed fibres. and perfume. there was no discrimination within the group. The chromatograms from the coloured fibres often contained separated dye components. and both types of compound were therefore characterised simultaneously. the softening additives and substantive antimicrobial compounds used in some preparations could probably be detected fairly readily in small samples. there was no discrimination within another group of 4. it is obviously most important to the economy of case work material that in any further developments the analysis of dyes and brighteners should be considered conjointly. given substantivity. Acknowledgements I am very grateful to various members of the dyestuffs and detergents industries for their help in many ways in connection with this work. Similarly. and in the detection of contact traces despite the strength with which a brightener is generally held within a material. . each of which was differentiated from the others. the backs of some of the photographs prepared in connection with this paper carried fluorescent marks left by transfer of brighteners between other photographs with which contact had occurred during processing.

. 758. 625. Prog.. F. Chromatogr. 482.). H. H. K. Dyers Colours. W.. 23. 1975. G. J.. 1975. K. THIEDEL. P... 1 February. J. 9. Tech. Cosmetics and Chemical SCHULZE. K. E. J.. M.. J. 1969. G. 1964. 189.J. 1974. 274 J. (Ciba-Geigy Ltd. M.Fluorescent Whitening Agents (Merrow Publishing Co. LONGMAN. 14 (9). Chemie (German ed.. SHRODER. 1967. 1967. LATINAK. Talanta. SCHULZE. Angew. SCHLEGELMILCH. 1.. G. Environ.. and VON RUTTE. 70. 1974. 1971. Textilindustrie. T. F. 1974.. STENSBY. and DAVIDSON. 1974. DSIR New Zealand. Rep. J. R. L. Sci. 63. 19.D.. ABDELKADER. C. Inform. Section B 5. The Secret o f White. 1975. Miljoevardrcentum (Stockholm). LYMAN. 80. 2. BROWN.. J.). R. P. Text. E. 413. PORTER. H. Rev. R. chapter 6 (Heywood Books GARNER.. 4.. November. and SCHUIERER. 185. L. Chromatogr. H. 1975. Coloration.. 1974.References ANDERS. 1968. 1973. London).. Textile Laboratory Manual. Specialities. Ltd. 62. C. 1968. Soc.. 5. and HAGEN.. Fette Seifen Anstr Mittel. 1972.) BARTON. GASPARIC. Fette Seifen Anstr Mittel.. D. 1363. . GANZ. 87.. DI GIOVANCEL. and ECKELT.. and MACEK. 15. Tensioactivos. G. PHILLIPS. 1971. KURZ. 1971. 738. J. and SCHMITZ. 1967.. Photochemistry (Chem. Plaste Kautsch. Market Research Great Britain. S.. . 14. PETERSEN. GOLD. POLCARO. 27. 69. and STENSBY. C. 73. FIGGE. 1964. 2188 SARKAR. Soap.. 15. 680. Chemicke Listy. MVC-Rep. G. 1973.). Financial Times. Ltd. J. 24. J..6.... 5. F. Invest. ANLIKER. Optical Brightening Agents. 693. Soc.. and VEENEMANS. A. D. S. 3. IPC Consumers' Marketing Manual UK.