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%

The basic structures of brighteners are described in a number of reviews, to which 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, chemically indistinguishable brighteners varying in manufacture will have a number of brand names differentiating them in manufacture as well as in formulation. In the present work, the 81 commercial products examined, which represent a cross-section of manufacture and use in Great Britain, yielded 34 distinguishable groups.
Experimental The following method is suitable for the analysis of brighteners in small samples of fibre and washing powders. Examine the sample in ultraviolet light (366nm). Brightened, undyed fibres fluoresce; dyed fibres may not because of quenching and self-absorption effects. Nonfluorescent, undyed fibres, which from many types of textiles are rare, 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 internal of solvent, diameter of about 50pm. Seal the tip of the pipette, inject 1 . 5 ~ 1 usually dimethylformamide (but see below), onto the sample from a Hamilton syringe, 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, and seal off the shank. These operations should be performed in subdued light. From this stage the analysis must be completed under a photographic safe light 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, wrap the tube in aluminium foil if the extraction cannot be done in a darkroom, and heat the package in an oven at 140' for half an hour with the pipette positioned upright (tip lowermost). Break the seal on the shank when the release of a slight vacuum will push some of the extract into the capillary. Attach the pipette to a length of plastic tubing fitted with a mouthpiece, break off the seal on the tip, and blow the extract in ca. 50nl portions, dried intermittently, onto the origin of a thin layer chromatogram (Camlab Polygram Sil G, stored over saturated aqueous potassium acetate). Spot diameters are restricted to less than 1.5mm. This is effected under a dissecting microscope to which a No. 1 hypodermic needle connected to a rubber blowball is fixed so that the (levelled) needle tip is just above the point of focus. A fine stream of air from the needle rapidly removes the solvent after each addition. A standard mixture of brighteners, each at 0.2mg ml-l in dimethylformamide, and a reagent blank are 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) 1.5 vol.; (B) acetone, 5M ammonia aq., chloroform and isopropanol, 100 5 10 vol.; (C) methyl ethyl ketone (redistilled), 5M and n-heptane, 45 15 5 vol. Development times are 40 ammonia aq., and methanol, 40 minutes, resulting in 15cm movements of the solvent fronts of A and B, and lOcm in the case of C. Examine the completed air-dried chromatogram in ultraviolet light (366nm) when the positions of the separated compounds are correlated to the standards. Table 1 lists the standards used. If the chromatogram is to be developed repeatedly, exposure to ultraviolet light between the developments must be kept to a minimum. Washing powders should be extracted in the cold, under a safe light, with a mixture of acetone and 5M ammonia aq. (45 5) (Schulze et al., 1974). When the proportion of powder to solvent is 50mg ml-l only a single (50nl) application of the extract to the chromatogram is necessary. Fabric conditioners are

+ +

+ +

TABLE 1

Rf VALUES OF SOME STANDARD COMPOUNDS

Compound * *
1-(p-carbomethoxypheny1)-3-(p-chlorophenyl)-A~-pyrazolinc. 2,5-bis(benzoxazo1-2-yl) -thiophene. 0.17 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 disulphonate. 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-2ylaminol-2,2 -stilbenedisulphonate. disodium biphenyl-4,4'-distyryl-p-sulphonate. *The solvents are given i n the Experimental section. **For the stilbene derivatives the Rf values are of the trans isomers.

Solvent* A B 0.34 0.78

0.82

soaked into filter paper, which is then washed, dried, and extracted with dimethylfonnamide as in the case of fibres. Some examples of the chromatograms obtained are shown in Figure 1. The Rfvalues of standard compounds in solvents A and B are listed in Table 1. Solvent C is used on the infrequent occasions (for fibres) when brighteners immobile in A and B are present. Results and Discussion Quantities of jibre used I n detergent powders individual brighteners are present at concentrations ranging from 0.02 to 0.5%. At a 20:l wash-load to powder ratio, if the brighteners are completely adsorbed, concentrations on a fabric after a single wash will be 0.001 to 0.025%. Hence, in 50pg of fibre the quantities of brighteners present will be about 0.5 to 12-5ng. These values tend to increase with the number of washes, but levels of this order are indicated when the chromatograms are calibrated with known quantities of brighteners. The amounts of fibre used for analysis are equivalent to a small wisp of cotton at the 10pg level, and to a 50mm length ofwool fibre at I OOpg. Major brightener components are readily detected in 10pg of cotton, but minor components may not be. At the 100pg level the chromatograms are liable to be overloaded and consequently distorted. For quantities of fibre below lOpg, the volume of extractant is reduced to 0.5~1,the spot diameter at the origin restricted to less than lmm, and the development time reduced to 15 minutes. Under these conditions the major components in 5pg samples can be characterised without undue sacrifice of resolution. An example is shown in Figure 2.

Extraction conditions For many fibres dimethylfonnamide is a satisfactory extractant; but if the fibre is likely to dissolve, 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. Pyridine is a suitable extractant for acrylic fibres. Other solvents that are useful on occasions are methanol, aqueous methanol, methyl cellosolve, ammoniacal methyl cellosolve and aqueous pyridine (Thiedel and Schmitz, 1967). However, none of these compares in efficiency with dimethylformamide for insoluble fibres. Because of the sealed tube technique, low-boiling extractants may be used at elevated temperatures. Hopefully, more volatile alternatives to dimethylformamide will be 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,

Figure 1. Chromatograms of extracts from 4 different samples of fibre (50pg) and, on the far right, the standard compounds given in Table 1 together with an unidentified compound (6th from the origin). The solvent is mixtu:e_B.

Figure 2. Chromatograms of the same standard mixture as in Figure 1 (outer most), and of 5, 5 and 10pg samples of fibre. These are of the same source as the sample on the far left of Figure 1. The solvent was mixture B.

which can give rise to large local variations in adsorbent activity. After the pipette has been discharged, some extract remains on the fibre, and may be removed either with dimethylformamide or with the more readily volatilised pyridine. Usually, small amounts of brightener remain adsorbed. 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, given sufficient fibre, all the brighteners are removed by Soxhlet extraction.

Effects o f ultraviolet light I n the presence of ultraviolet light, brighteners in the dissolved state undergo photodegradation and photoisomerism (Kurz and Schuierer, 1967). Photodegradation is severe in dilute solutions, e.g., 1pg ml-l, the fluorescence of which can disappear within a few minutes on exposure to daylight. Photoisomerism results in the production of a photo-equilibrated mixture of cis and trans isomers from the stilbene-derived brighteners, the commonest type, which are manufactured in and adsorbed on fabric as the trans forms. The cis forms are not adsorbed. Only the trans forms fluoresce, but the two are separated from one another by TLC, and in ultraviolet light the cis spot becomes fluorescent because of the photo-equilibration. 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), 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) ;and, because photo-equilibrium

constants under typical conditions are of the order of unity, the fluorescence of each brightener is divided approximately equally between two spots, which reduces sensitivity by half.

Chromatography conditions A considerable variety of solvents and adsorbents for the chromatography of brighteners has been proposed. Apart from the previously mentioned, other references pertinent in the present context are reviews by Longman (1975) and by 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 the results of experiments with well over 150 different sets of conditions. All of the common detergent brighteners (generally sulphonic acid derivatives) are separated by the arnmoniacal solvent B, in which the presence of n-heptane strikingly improves resolution and selectivity (other nonpolar compounds do likewise). Some weakly retained nonionic brighteners are not separated in B. For these the chloroform/isopropanol mixture A is usually satisfactory. When both types of brightener are present, or when no prior information is available, the solvent mixtures are used sequentially. Because in the adsorbed state on silica gel the photoisomerism is not inhibited, the brief exposure to ultraviolet light of the chromatogram after each development unavoidably results in some isomerisation and loss of sensitivity. 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. As a general rule, all points on a chromatogram should be equidistant from the nearest vertical surface in the tank. Pre-equilibration of the chromatography strip in the tank is deleterious to the separation. Some brighteners are immobile in both of solvents A and B. A reduction in ammonia concentration from 5M to 1M can produce some mobility; and all brighteners examined are mobile in solvent C. However, discrimination within this particular group at present remains rather low. Brighteners present in washing powders and related products Table 2 gives the Market Research GB (1974) data on various brands of clothes washing materials. The data are in broad agreement with the IPC Consumers' Marketing Manual (1975). Approximate market shares at the time of these surveys are hence: Proctor and Gamble, 48% ; Lever Bros., 43% ; and "shop brands" 3%. The remaining materials used for clothes washing include the products of small-scale manufacturers, and toilet soaps and washing-up liquids.
TABLE 2 MARKET DISTRIBUTION OF VARIOUS WASHING POWDERS

Proctor and Gamble Ariel 20% Daz 13 % Fairy Snow 7% 5% Tide Dreft Bold* O % L ,"

h v e r Bros. Persil Persil Auto. Omo Radiant

16 % 6% 6% 6%

Shop Brands overall

3%

Stergem** 2 1% Drive* *From IPCConswncrs' harketing Manual (1975). The rest of the data are from Market Research Great Britain (1974), which includes a 6 % others/ don't lu~ow figure. **Liquid detergent.

Lux surf

;%
O/,

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

used in any one household are selected, the figures are consistent with the market distribution data. Hence, it is suggested that the described techniques may be used to qualitatively evaluate the detergent environment to which a susceptible fibre has been exposed. 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, given substantivity. As well as detergent brighteners, others added during manufacture are present on undyed fibres. The samples examined included 27 of undyed cotton. Each of a group of 9 of these contained only the three common detergent brighteners, i.e., there was no discrimination within the group. Similarly, there was no discrimination within another group of 4, and another of 3. But each of the remaining 11 samples was discriminated from all of the others, largely because of the presence of additional brighteners. The corresponding probability 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 and thin 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 of which was differentiated from the others. The chromatograms from the coloured fibres often contained separated dye components, as well as brighteners, and both types of compound were therefore characterised simultaneously. Although none of the objects of this work was to develop the already well-established techniques used in the forensic characterisation of fibre dyes, 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.

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. To this end, various other materials could be sought: for instance, the softening additives and substantive antimicrobial compounds used in some preparations could probably be detected fairly readily in small samples; and perfume, and traces of borates, phosphates and detergents remaining on incompletely rinsed fabric might be of evidential value. However, the techniques described will probably find most use in the correlation of transferred fibres with possible points of origin, 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. Apart from brighteners, dyes are potentially characterisable by these techniques at a far higher level of specificity than is possible at present. There are also obvious applications in the characterisation of plastics, papers and other materials; and in the detection of contact traces despite the strength with which a brightener is generally held within a material. For instance, 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. I n one case the originating photograph was clearly identifiable from the transferred pattern of its uneven margin.

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.

References ANDERS, G., ANLIKER, R. and VEENEMANS, G. J., 1971, The Secret o f White, (Ciba-Geigy Ltd.) BARTON, D. and DAVIDSON, H., 1974, Rev. Prog. Coloration, 5, 3. J. C., 1964, J. Soc. Dyers Colours, 80, 185. BROWN, K., 1968, Fette Seifen Anstr Mittel, 70, 680. FIGGE, DI GIOVANCEL, G. and VON RUTTE, R., 1972, Invest. Inform. Text. Tensioactivos, 15, 189. Financial Times, 1973, 1 February. J., STENSBY, P. S., LYMAN, F. L. and MACEK, K., 1975, GANZ, C. R., SCHULZE, Environ. Sci. Tech., 9, 738. W., 1967, Textile Laboratory Manual, 4, chapter 6 (Heywood Books GARNER, Ltd.). GASPARIC, J., 1969, Chemicke Listy, 63, 1363. GOLD, H., 1973, MVC-Rep. Miljoevardrcentum (Stockholm), 2, 23. IPC Consumers' Marketing Manual UK, 1975, Section B 5.6. KURZ, J. and SCHUIERER, M., 1967, Fette Seifen Anstr Mittel, 69, 24. LATINAK, J., 1964, J. Chromatogr., 14, 482. LONGMAN, G. F., 1975, Talanta, 62, 1. Market Research Great Britain, 1974, 14 (9), 19. PETERSEN, S., 1975, Angew. Chemie (German ed.), 87, 693. PHILLIPS, D., 1974, Photochemistry (Chem. Soc., London), 5, 758. PORTER, L. J., 1974, Optical Brightening Agents, DSIR New Zealand, Rep. C.D. 2188 SARKAR, A. K., 1971,Fluorescent Whitening Agents (Merrow Publishing Co. Ltd.). H. and ECKELT, M., 1971, Textilindustrie, SCHLEGELMILCH, F., ABDELKADER, 73, 274 J., POLCARO, T. and STENSBY, P., 1974, Soap, Cosmetics and Chemical SCHULZE, Specialities, November. E. and HAGEN, E., 1968, Plaste Kautsch., 15, 625. SHRODER, THIEDEL, H. and SCHMITZ, G., 1967,J. Chromatogr., 27, 413.