Forensic Science International, 51 (1991) 239 - 250 Elsevier Scientific Publishers Ireland Ltd.

239

MICROSPECTROFLUORIMETRY OF FLUORESCENT DYES AND BRIGHTENERS ON SINGLE TEXTILE FIBRES: PART 3 - FLUORESCENCE DECAY PHENOMENA

ANDREW W. HARTSHORNE and DAVID K. LAING

Central Research and Support Establishment, Home Office Forensic Science Service, Aldermaston, Reading, Berkshire, RG7 4PN (U.K.)

(Received January llth, 1991) (Accepted August 23rd, 1991)

Summary

Microspectrofluorimetry is used to examine the decay in emission intensity of a range of fluores- cent materials. The suitability of decay parameters as a means of discrimination, and the effects of decay on the comparison and specification of fluorescence colours are investigated.

Key words: Microspectrofluorimetry; Single fibres; Dyes; Brighteners; Fluorescence decay

Introduction

A previous paper [l] presented a method of obtaining fluorescence emission spectra from single textile fibres. During the course of this work, it was noted that for some materials, the intensity of fluorescence emission decreased with time, particularly for fluorescent brightening agents. This decay is due to the conversion of the fluorescent species into an inactive form [2]. The rate of decay, monitored at a particular emission wavelength, may therefore provide an addi- tional point of discrimination for fluorescent materials.

The decay of fluorescence may also have an effect on the chromaticity coor- dinates calculated from emission spectra [3]. If the rate of decay is not constant at each emitted wavelength, then changes in chromaticity coordinates will occur. This is likely to occur with mixtures of fluorescent compounds which have dif- ferent decay rates. It could also be a problem with a single species, where the measurement scan time is slow compared to the rate of decay of fluorescence. For a system exhibiting a slow decay and where the rate of decay at each wavelength is constant, chromaticity coordinates should be invariant.

This paper examines the decay characteristics of a number of fluorescent dyestuffs and brightening agents. The effect of decay on chromaticity coor- dinates is also assessed.

Printed and Published in Ireland

240

Theory

During the process of excitation, a fluorescent material is raised from its elec- tronic ground state into the first excited electronic state. Once in this excited state, four processes resulting in deactivation may occur [4].

The most readily apparent process is that of fluorescence, the emission characteristics being recorded by the microspectrofluorimeter. During this pro- cess, the excited molecule is eventually returned to its original state. This also occurs with two of the alternative processes, where deactivation is by thermal means or through phosphorescence. By all these processes, the molecule is made available for re-excitation. For a given set of conditions the relative contribution to deactivation of each of these processes should remain constant, with no effect on the intensity of fluorescence emission.

The fourth process, photochemical reaction, forms a new species. This has the effect of decreasing the number of molecules available for excitation, with a con- sequent decrease in fluorescence emission intensity. The truns-to-& isomerisa- tion of stilbene-based fluorescent brightening agents [2] is one example of such a reaction.

The rate of photochemical reaction will depend on the number of molecules in the excited state, which itself depends on the number of molecules in the ground state and the intensity of the exciting radiation. However, as the intensity of ex- citing radiation is effectively constant under the conditions employed for microspectrofluorimetry, the rate of photochemical reaction should depend only on the number of molecules in the ground state. A first-order rate law [5] should therefore apply, for which the logarithm of the concentration of the reacting spe- cies is proportional to time.

Assuming that fluorescence emission intensity is proportional to the number of molecules in the ground state, then a plot of the logarithm of fluorescence in- tensity against time should be a straight line. From the gradient of the line, the half-life of the decay process can be calculated. For a first-order process, this parameter is constant and should therefore be an additional point of comparison for fluorescent compounds.

Experimental

Measurewwnt of decay curves Ten fibre samples dyed with fluorescent dyestuffs or brighteners were selected

for examination, as shown in Table 1. Well-separated fibres were mounted on glass microscope slides, using XAM neutral medium improved white (BDH, Poole, Dorset, U.K.) and a coverslip.

For each sample, the decay of fluorescence intensity with time was obtained. This was achieved by determination of the corrected fluorescence emission spec- trum, using the NanoSpec microspectrofluorimeter as previously described [l], from which was ascertained the wavelength of maximum emission. A second fibre in the sample was then excited and the fluorescence intensity at the wavelength of maximum emission monitored, for a period of either 15 or 60 min,

241

TABLE 1

FLUORESCENT DYES AND BRIGHTENING AGENTS EXAMINED BY MEASUREMENT OF EMISSION INTENSITY DECAY

Sample identity Dye cont. Decay time % (min)

Cubes used and monitored wavelength (nm)

Number of runs

Dyes 1 Acidol Brilliant Yellow 8G 2.5 2 Acid01 Brilliant Yellow 8G 2.5 3 Foron Brilliant Yellow 2.5

SE-6GFL 4 Dispersol Orange D-G 5.5 5 Dispersol Red C-B 7.0 6 Dispersol Red C-B 7.0 7 Yellow Viscose *

8 Yellow Viscose *

9 Pink Viscose *

10 Pink Viscose *

Brightening agents 11 Leucophor EFG 12 Leucophor EFG 13 Leucophor EFA 14 Leucophor EFA 15 Leucophor EFR 16 Nylon Baby Knitting

Yarn

0.4 60 A 0.4 60 H2 0.8 60 A 0.8 60 H2 0.8 60 A * 60 A

15 15 15

15 H2 592 15 H2 593 15 H2 618 60 A 517 60 H2 521 60 A 440 60 H2 584

A 495 H2 520 H2 520

440 520 440 520 440 440

3 3 2

*Dye concentration unknown.

as noted in Table 1. Intensity values were measured every 5 s during these periods.

For samples giving appreciable fluorescence emission intensity under both cube A and H2 excitation [l], a decay curve was obtained for each. Replicate de- cay curves, using a new fibre for each measurement, were obtained for some col- orants, so that the reproducibility of the method could be assessed. For Dispersol Red C-B, which gave two fluorescence emission maxima, decay was monitored at each wavelength.

The effect of decay on chrmaticity coordinates The effect of fluorescence decay on chromaticity coordinates was assessed for

the three colorants shown in Table 2. These were chosen to represent a fluores- cent brightening agent (Leucophor EFA), a dyestuff whose fluorescence is stable (Dispersol Red C-B) and a dye which decays appreciably (Acid01 Brilliant Yellow 8G). For each sample, ten emission spectra from the same position on a single fibre were obtained, the spectra being captured at 5-min intervals from the start of excitation. Cube H2 excitation was used for all three samples, with the addi-

242

TABLE 2

CHROMATICITY VALUES OBTAINED FROM FLUORESCENT DYESTUFFS UNDERGOING DECAY

Time (min)

Cube HZ Cube A chromaticity coordinates chromaticity coordinates

X Y X Y

Dispersol Red C-B 7.0%a 0 0.6128 5 0.6124

10 0.6130 15 0.6125 20 0.6128 25 0.6128 30 0.6129 35 0.6126 40 0.6126 45 0.6127

Acid01 Brilliant Yellow 8G 2.5% 0 0.2973 5 0.3234

10 0.3294 15 0.3326 20 0.3317 25 0.3331 30 0.3338 35 0.3305 40 0.3336 45 0.3348

Leucophor EFA 0.8% 0 0.3215 5 0.3296

10 0.3271 15 0.3263 20 0.3240 25 0.3228 30 0.3174 35 0.3177 40 0.3084 45 0.3088

0.3867 0.3871 0.3865 0.3869 0.3867 0.3867 0.3866 0.3869 0.3869 0.3868

0.6758 0.6537 0.6486 0.6458 0.6468 0.6455 0.6450 0.6482 0.6454 0.6444

0.6549 0.1489 0.0577 0.6479 0.1489 0.0637 0.6502 0.1480 0.0590 0.6509 0.1482 0.0634 0.6530 0.1482 0.0627 0.6541 0.1482 0.0631 0.6589 0.1483 0.0626 0.6587 0.1480 0.0625 0.6672 0.1482 0.0634 0.6669 0.1484 0.0622

BError ellipse parameters for this sample: mean x = 0.6127, mean y = 0.3868; major axis length = 0.0010, minor axis length = 0.0001; angle of inclination to x-axis = -43.36’.

243

15

3

5

0 15

7

\

t

2

0 15

. 6

4 0 15

8

0 60

Time (min>

Fig. 1. Fluorescence’intensity decay curves of selected fluorescent dyes (for sample identification, see Table 1).

244

60 0 60

11 12

0 60 0 60

14

I 1 0 60

15

0 60

0 60 0 60

10 I

,

I I

Time(min>

Fig. 2. Fluorescence intensity decay curves of selected fluorescent dyes and brightening agents (for sample identification, see Table 1).

245

tional use of cube A excitation for Leucophor EFA, which gave strong fluorescence with both cubes.

Results and Discussion

Decay curves Typical decay curves for each colorant under both cube A and H2 excitation

are shown in Figs. 1 and 2, from which several patterns of decay are evident. Of the various dyeings examined, Dispersol Red C-B (Fig. 1, curves 5 and 6)

shows no decrease in fluorescence intensity over a period of 15 min. Foron Brilliant Yellow SE-6GFL (Fig. 1, curve 3) and Dispersol Orange D-G (Fig. 1, curve 4), both exhibit slow fluorescence decay, indicating that photochemical reaction is relatively unfavoured. Previous work [3] noted that these dyeings in- corporated a fluorescent brightening agent, but this is not apparent from the de- cay curves observed. However, the emission maxima of dyestuff and brightener are at very different wavelengths. At the wavelength monitored, the latter spe- cies can make only a small contribution to the fluorescence intensity observed, hence its decay is undetected.

The decay curves for the yellow (Fig. 1, curves ‘7 and 8) and pink (Fig. 2, curves 9 and 10) viscose samples show a rapid initial decay, followed by a slow decrease in intensity. This rapid initial decay could be due to the presence and decay of

TABLE 3

DECAY PARAMETERS OF FLUORESCENT DYES AND BRIGHTENING AGENTS

Sample Cube Run First-order decay Second-order decay

Slope Znter- Corre- Slope Inter- Corre- wt lation cept lation

(x1000) coefficient (x1000) (x10) coefficient

Acidol Brilliant Yellow 8G 2.5%

Leucophor EFR 0.8%

Leucophor EFG 0.4%

Leucophor EFA 0.8%

Nylon Knitting Yarn

A 1 -0.9030 1.2840 0.9746 0.3656 0.2628 0.9946 A 2 -0.9154 1.1860 0.9719 0.4090 0.2904 0.9935 A 3 -0.8887 1.3420 0.9713 0.3357 0.2495 0.9927 H2 1 -2.5200 3.4550 0.9291 0.2322 0.0146 0.9994 H2 2 -2.3350 3.5640 0.9353 0.1775 0.0178 0.9994 H2 3 - 2.6370 3.3700 0.9302 0.2804 0.0126 0.9993

A 1 -0.4058 2.2870 0.9962 0.0874 0.0724 0.9949

A 1 H2 1

A 1 -0.3684 2.7770 0.9982 0.0459 0.0471 0.9900 H2 1 -0.1266 -0.0213 0.9740 0.1598 1.0070 0.9859

A 1 A 2 A 3

- 0.3466 2.2200 0.9646 0.0685 0.0962 0.9966 - 0.2053 0.3789 0.9731 0.1982 0.6568 0.9909

- 0.8305 0.8027 0.9332 -0.4795 1.4350 0.9300 - 0.6957 0.6080 0.9405

1.6010 - 0.1630 0.9974 0.2486 0.1877 0.9931 1.2670 0.1100 0.9990

246

a fluorescent brightening agent, the monitored wavelength being close to the maximum emission wavelength typically observed for fluorescent brighteners. At the monitored wavelength therefore, this species is making a large contribu- tion to the observed fluorescence.

The remaining samples, Acid01 Brilliant Yellow 8G (Fig. 1, curves 1 and 2) and the four fluorescent brightening agents (Fig. 2, curves 11 - 16), all gave a rapid decay of fluorescence intensity. This is a permanent effect; the area of fibre which had been irradiated shows a marked decrease in fluorescence intensity upon re-excitation, compared with areas irradiated for the first time.

As these five samples were known to contain only one fluorescent species, removing any problems associated with multiple fluorescence decay, cor- respondence to a first-order decay rate equation was assessed. This was achieved by finding the regression line [6] between the log (intensity) and time values, followed by calculation of the line’s coefficient of correlation. These results are shown in Table 3. An acceptable fit to a straight line was inferred with a correla- tion coefficient greater than 0.99.

From Table 3, it can be seen that only two decay curves, Leucophor EFR (cube A excitation) and Leucophor EFA (cube A excitation), gave an acceptable fit to a first-order rate equation. This indicates that the assumption of first-order kinetics may be invalid in the other cases. As a check, the correspondence to a second-order process [5] was ascertained. For this the reciprocal of the concen- tration of the reacting species is proportional to time. A plot of the reciprocal of emission intensity against time should therefore be a straight line.

Table 3 shows that all the decay curves gave correlation coefficients greater than 0.99 when fitted to a second-order rate equation. The reason for this result

015 035.40

0.3868 045

00,20.25

030

010

03864 1 0.6120 0.6124 0.6128 x 0.6132

Fig. 3. Decay chromaticity diagram for Dispersol Red C-B (cube H2 excitation).

247

is not clear. However, if decay is truly a second-order process, then more than one molecule is involved in the photochemical reaction step. It may well be necessary for the excited species to collide with another dye molecule before reaction can take place.

For such a second-order process, the half-life depends on the initial concentra- tion of the reacting species. Because of this, the shape of the decay curve will vary with both dye concentration and the time since the decay process began, which is not the same as the time since the start of measurement. Conversion of the fluorescent species to an inactive form will have been taking place since manufacture, whenever radiation of the correct wavelength was available for ex- citation. The calculation of half-life as a discrimination parameter in this case is therefore valueless. For a similar reason, reproducibility of the regression line equations should be poor. Reference to Table 3, for samples from which replicate decay curves were obtained shows that this is indeed the case.

The effect of decay on chromaticity coordinates The chromaticity coordinates obtained from single fibres after various periods

of excitation are shown in Table 2. The coordinates are plotted in the chromatici- ty diagrams of Figs. 3-6. It should be remembered when interpreting these results that the coordinates obtained by cube H2 excitation are greatly restricted [3], so any variation may be limited.

For Dispersol Red C-B (Fig. 3), the variation in coordinates is very small and there appears to be no correlation with time. This is to be expected, as this dyestuff was shown not to decay. As this set of coordinates were obtained from the same place on a single fibre, they provide an assessment of the instrumental

00

Fig. 4. Decay chromaticity diagram for Acid01 Brilliant Yellow 8G (cube H2 excitation).

248

errors of microspectrofluorimetry. Calculation of a chromaticity error ellipse [7] gives a major axis length of 0.0010, which is comparable to the instrumental er- ror obtained for microspectrophotometry [8].

Figures 4-6 show that for the remaining dyes, which do exhibit decay, chromaticity coordinates do change radically with time. The reason for this behaviour is not clear. As decay is due to the formation of another species, it is possible that this product may itself fluoresce, but with a different emission spec- trum. Another possibility is that during the time taken for measurement of a

0.06E i-

0.064

Y 05

015.40 025

0.062

0.06C

,.

I-

3-

3,

010

0.0s

0.0s

00

0.146 0*146 )( 0.150 0.152

Fig. 5. Decay chromaticity diagram for Leucophor EFA (cube A excitation).

249

O-67

Y

0.66

0.65

0.64

30

2

00 025

020

015 010

0.30 0.31 )( 0.32 033

Fig. 6. Decay chromaticity diagram for Leucophor EFA (cube H2 excitation).

complete spectrum, a relatively greater decay in emission intensity can occur for red wavelengths. Emission intensity values at the blue end of the spectrum would be measured accurately, but those at longer wavelengths would appear to be less than their true values. This would be most marked when emission intensi- ty was decaying rapidly, in the first ten or so minutes of the decay process. The overall effect should be to cause a shift in coordinates away from the red area of the chromaticity diagram. However, in most cases, this did not occur, with er- ratic movements towards both red and green being observed.

Conclusions

It was originally thought that the calculation of the half-life of the fluorescence emission decay process would be a valuable parameter for the discrimination of fluorescent fibres. However, the complications ensuing from the second-order kinetics of the process mean that this is not so. As the half-life depends on the initial concentration of the reacting species, then each fibre in a given sample will have a different half-life, dependent upon concentration at the time of dyeing, and subsequent exposure to exciting radiation. For fibres dyed with more than one fluorescent species, the situation is even more complex, as the active com- pounds will almost certainly have different decay rates. For these reasons,

250

fluorescence decay phenomena are of little use in the discrimination of single tex- tile fibres.

For a fluorescent material which exhibits decay, the effect on the observed emission colour, as represented by its chromaticity coordinates, is also difficult to interpret. Because of decay, the emission spectra and chromaticity coor- dinates obtained from these materials must be treated with caution. On a similar theme, the comparison of fibres by fluorescence microscopy must also be rather subjective in nature.

References

A.W. Hartshorne and D.K. Laing, Microspectrofluorimetry of fluorescent dyes and brighteners on single textile fibres: Part 1 - fluorescence emission spectra. Fore&c Sei. Znt., 51 (1991) 203-220. J.B.F. Lloyd, Forensic significance of fluorescent brighteners: their qualitative TLC characterisation in small quantities of fibre and detergents. J. Forensic Sci. Sot., 17 (1977) 145 - 152. A.W. Hartshorne and D.K. Laing, Microspectrofluorimetry of fluorescent dyes and brighteners on single textile fibres: Part 2 - colour measurements. Forensic 5%. Znt., 51(1991) 221- 237. A. Streitwieser and C.H. Heathcock, Introduction to Organic Chemist?, Macmillan, New York, 1976. W.J. Moore, Physical Chemistry , (5th edn.), Longman, London, 1972. L. Poole and M. Borchers, Some Common Basic Programs, Adam Osborne Associates, Berke- ley, California, 1978. D.K. Laing, A.W. Hartshorne and R.J. Harwood, Colour measurements on single textile fibres. Fmm.sic Sci. Znt., 30 (1986) 65 - 77. A.W. Hartshorne and D.K. Laing, The definition of colour for single textile fibres by microspec- trophotometry. Foremic Sci. Znt., 34 (1987) 107- 129.