Forensic Science International, 14 (1979) 215 - 219 0 Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
215
VISUALIZATION OF DYES ON THIN-LAYER CHROMATOGRAPHY PLATES BY MEANS OF THE ARGON ION LASER
JOHN I. THORNTON and PAUL J. CASALE Forensic Science Group, Department of Biomedical and Environmental Health Sciences, School of Public Health, University of California, Berkeley, California 94720 (U.S.A.) (Received November 3, 1978; in revised form March 1, 1979; accepted April 15, 1979)
Summary A simple method for the detection of nanogram amounts of dyes by means of their fluorescence in the visible region of the spectrum is described. To stimulate the fluorescence, an argon ion laser with principal lines of 514.5 nm and 488.0 nm is used. The sensitivity of the technique allows the visualization of an amount of dye smaller than could be detected with either visible absorbance or ultraviolet stimulated fluorescence.
Introduction The characterization and identification of dyes in cosmetics, inks, drugs, foodstuffs, and other materials is generally based on analytical techniques exploiting the strong absorption of energy in the visible regionof the spectrum shown by the dye molecule. In addition, many dyes fluoresce strongly when excited by energy in the ultraviolet, and many crime laboratories utilize ultraviolet fluorescence in addition to visible absorption in the characterization of the dye by chromatographic means. We wish now to describe a sensitive method for the visualization of dyes based upon the fluorescence stimulated by high-energy visible light delivered by an argon ion laser. The phenomenon of fluorescence is well understood and has found much application in crime laboratories. However, most forensic applications of 3uorescence involve the excitation of materials in the ultraviolet region, with the emitted radiation being observed in the visible region of the spectrum. An inherent limitation to this mode of operation is the power delivered by the source. Most common low-energy sources of ultraviolet energy are quite weak, delivering on the order of a few hundred microwatts per square centimeter. Although this is sufficient for many purposes, the power output of the ultraviolet source may be insufficient to record, either visually or photographically, the small fluorescence yield from nanogram amounts of dye. By Stokes’ Law, the fluorescence emission will be at the exciting wavelength, or, more commonly, at a longer wavelength than the exciting wavelength. Depending on the nature of the particular dye molecule, it may be possible to excite the dye with visible energy and observe fluorescence which is also in the visible region of the spectrum. This has rarely been attempted
216
successfully in the crime laboratory, however, because of the inherent nature of energy sources for the visible region. Incandescent sources of whatever power operating at a color temperature of approximately 2850 K emit the bulk of the radiation in the infrared region, which is, for all practical purposes, useless for the purpose of stimulating fluorescence. The argon ion laser, on the other hand, represents an efficient means of delivering intense energy in the visible region. The amount of power that can be delivered, in a practical sense, to a dye molecule greatly exceeds that which can be delivered by either an incandescent lamp or a low-energy mercury discharge lamp emitting in the ultraviolet region of the spectrum. Consequently the emitted radiation will be greater, allowing for the visualization of a smaller amount of dye material. This has been demonstrated in the present study by the detection and visualization of cosmetic dyes, and is applicable to dyes in inks and in soliddosage drug preparations. The thrust of this work, however, is not the characterization of dyes, but rather the visualization of them.
Materials and methods A Spectra-Physics Model 164 Argon Ion Laser was utilized in this study. This type of laser was introduced into the forensic field by Menzel and coworkers [l] for the visualization of latent fingerprints by means of the inherent fluorescence of dermal secretions. This is a high-power, continuous wave laser capable of emitting at a number of wavelengths, with the lines at 514.5 nm and 488.0 nm representing the strongest lines. The laser delivers a beam of 1.5 mm diameter with a divergence of 0.5 milliradians, necessitating further divergence of the beam to cover a thin-layer chromatography plate. This was achieved by placing an inexpensive glass negative lens with a focal length of -24 mm in the beam path immediately forward of the laser head. This resulted in a beam diameter of 10 cm at a distance of 100 cm from the diverging lens. A 40X, 0.65NA microscope objective placed in the beam path was found to give a comparable divergence of the beam. Figure 1 represents a diagram of the equipment configuration. A barrier filter is needed
barrier filter
\
Fig. 1. Equipment
diverging lens
configuration
for visualizing
dyes on thin-layer
plates.
217
to separate the excitation energy from the emitted energy. This was achieved by means of a Fish-Schurman AL-515-7 laser safety goggle filter, having a sharp cutoff below 560 nm with an optical density of 7 at 515 nm. The method of Cotsis and Garey [2] was used for the separation of lipstick dyes by thin-layer chromatography. The writers do not contend that lipstick dyes represent the most important class of dyes to a forensic laboratory, but for purposes of this study they are more easily quantitated than some other types of dyes. Lipsticks were dissolved in cyclohexane and spotted on a 0.25 mm thick silica gel thin-layer chromatography plate. The plate was then developed using benzene-n-amyl alcohol-cont. HCl (65:30:5) as a developing solvent. The dyes were visualized by placing the thin-layer plate in the laser beam as illustrated in Figure 1. A 35 mm camera with Kodak Tri-X film (ASA 400) was used to record the emitted fluorescence from the dyes examined in this study. With 4 watts of power being delivered by the laser to the diverging lens, the exposure time at f4.5 was on the order of l/8 seconds. The exposure time is, of course, a function of distance, laser power, amount of dye present, and whether the laser is being operated in a monochromatic mode or is emitting all lines.
Fig. 2. Photograph of cigarette taken with visible light; no lipstick dye is apparent. Fig. 3. Photograph of the same cigarette under the laser beam. The photographic emulsion is exposed entirely by the intense fluorescence emitted by the lipstick dye; a barrier filter has eliminated the exciting energy.
218
Results and discussion
Serial dilution of precisely weighed amounts of various lipsticks were made, using cyclohexane as a solvent. The limit of sensitivity for the visualization of the cosmetic dyes comprising the lipsticks was found to be on the order of 2 to 8 X lo-’ grams of lipstick. The amount of dye corresponding to this amount of lipstick was not critically determined, but lipsticks generally contain solid dyes in the range of 6-15%, and stain dyes in the range of l-3% [ 31. At the level of 2-8 nanograms of lipstick, the dye cannot be seen on the thin-layer plate with normal incandescent light or under a low-energy ultraviolet lamp. Figure 2 illustrates a cigarette on which a small quantity of lipstick is smeared. The lipstick dye cannot be observed with visible light, nor can it be observed with ultraviolet light. Figure 3 illustrates the same cigarette in the laser beam, the laser delivering 4 watts of power to the diverging lens.
Fig. 4. Laser excited fluorescence of lipstick dyes, as observed on a thin-layer chromatographic plate. The spots represent the dye present in approximately 50 nanograms of lipstick, or approximately 5 nanograms of dye.
219
The radiant energy exposing the film emulsion is the fluorescence emission from the lipstick dye. Figure 4 illustrates a thin-layer plate on which lipstick extracts were chromatographed. The spots represent the dye extracted from 50 nanograms of lipstick, or approximately 5 nanograms of dye. Although the laser is capable of delivering energy of shorter wavelength for excitation of the dyes, in the present study of cosmetic dyes it was found that the emission maxima of the dyes were above the 514.5 nm line. Consequently, the laser was operated with all lines emitting. The writers consider this visualization technique to be attractive from the standpoint of sensitivity. Nanogram quantities of many dyes may be visualized by the stimulated fluorescence, allowing the detection of trace quantities of materials which could not otherwise be detected by visual examination or ultraviolet fluorescence. Although the present study was confined to cosmetic dyes, the writers have observed intense fluorescence with textile dyes and inks, and laser visualization would seem applicable to these materials as well.
References 1 B. E. Dalrymple, J. M. Duff and E. R. Menzel, Inherent fingerprint luminescence detection by laser. J. Forensic Sci., 22 (1977) 106 - 115. 2 T. P. Cotsis and J. C. Garey, Determination of dyes in lipsticks by thin layer chromatography. Proc. Toilet Goods Ass. Sci. Sec., 41 (1964) 3 - 11. 3 L. P. Torry, Lipstick, rouge, eye make-up and manicure preparations, in H. W. Hibbot (ed.), Handbook of Cosmetic Science, Macmillan, New York, 1963, pp. 295 - 329.
215
VISUALIZATION OF DYES ON THIN-LAYER CHROMATOGRAPHY PLATES BY MEANS OF THE ARGON ION LASER
JOHN I. THORNTON and PAUL J. CASALE Forensic Science Group, Department of Biomedical and Environmental Health Sciences, School of Public Health, University of California, Berkeley, California 94720 (U.S.A.) (Received November 3, 1978; in revised form March 1, 1979; accepted April 15, 1979)
Summary A simple method for the detection of nanogram amounts of dyes by means of their fluorescence in the visible region of the spectrum is described. To stimulate the fluorescence, an argon ion laser with principal lines of 514.5 nm and 488.0 nm is used. The sensitivity of the technique allows the visualization of an amount of dye smaller than could be detected with either visible absorbance or ultraviolet stimulated fluorescence.
Introduction The characterization and identification of dyes in cosmetics, inks, drugs, foodstuffs, and other materials is generally based on analytical techniques exploiting the strong absorption of energy in the visible regionof the spectrum shown by the dye molecule. In addition, many dyes fluoresce strongly when excited by energy in the ultraviolet, and many crime laboratories utilize ultraviolet fluorescence in addition to visible absorption in the characterization of the dye by chromatographic means. We wish now to describe a sensitive method for the visualization of dyes based upon the fluorescence stimulated by high-energy visible light delivered by an argon ion laser. The phenomenon of fluorescence is well understood and has found much application in crime laboratories. However, most forensic applications of 3uorescence involve the excitation of materials in the ultraviolet region, with the emitted radiation being observed in the visible region of the spectrum. An inherent limitation to this mode of operation is the power delivered by the source. Most common low-energy sources of ultraviolet energy are quite weak, delivering on the order of a few hundred microwatts per square centimeter. Although this is sufficient for many purposes, the power output of the ultraviolet source may be insufficient to record, either visually or photographically, the small fluorescence yield from nanogram amounts of dye. By Stokes’ Law, the fluorescence emission will be at the exciting wavelength, or, more commonly, at a longer wavelength than the exciting wavelength. Depending on the nature of the particular dye molecule, it may be possible to excite the dye with visible energy and observe fluorescence which is also in the visible region of the spectrum. This has rarely been attempted
216
successfully in the crime laboratory, however, because of the inherent nature of energy sources for the visible region. Incandescent sources of whatever power operating at a color temperature of approximately 2850 K emit the bulk of the radiation in the infrared region, which is, for all practical purposes, useless for the purpose of stimulating fluorescence. The argon ion laser, on the other hand, represents an efficient means of delivering intense energy in the visible region. The amount of power that can be delivered, in a practical sense, to a dye molecule greatly exceeds that which can be delivered by either an incandescent lamp or a low-energy mercury discharge lamp emitting in the ultraviolet region of the spectrum. Consequently the emitted radiation will be greater, allowing for the visualization of a smaller amount of dye material. This has been demonstrated in the present study by the detection and visualization of cosmetic dyes, and is applicable to dyes in inks and in soliddosage drug preparations. The thrust of this work, however, is not the characterization of dyes, but rather the visualization of them.
Materials and methods A Spectra-Physics Model 164 Argon Ion Laser was utilized in this study. This type of laser was introduced into the forensic field by Menzel and coworkers [l] for the visualization of latent fingerprints by means of the inherent fluorescence of dermal secretions. This is a high-power, continuous wave laser capable of emitting at a number of wavelengths, with the lines at 514.5 nm and 488.0 nm representing the strongest lines. The laser delivers a beam of 1.5 mm diameter with a divergence of 0.5 milliradians, necessitating further divergence of the beam to cover a thin-layer chromatography plate. This was achieved by placing an inexpensive glass negative lens with a focal length of -24 mm in the beam path immediately forward of the laser head. This resulted in a beam diameter of 10 cm at a distance of 100 cm from the diverging lens. A 40X, 0.65NA microscope objective placed in the beam path was found to give a comparable divergence of the beam. Figure 1 represents a diagram of the equipment configuration. A barrier filter is needed
barrier filter
\
Fig. 1. Equipment
diverging lens
configuration
for visualizing
dyes on thin-layer
plates.
217
to separate the excitation energy from the emitted energy. This was achieved by means of a Fish-Schurman AL-515-7 laser safety goggle filter, having a sharp cutoff below 560 nm with an optical density of 7 at 515 nm. The method of Cotsis and Garey [2] was used for the separation of lipstick dyes by thin-layer chromatography. The writers do not contend that lipstick dyes represent the most important class of dyes to a forensic laboratory, but for purposes of this study they are more easily quantitated than some other types of dyes. Lipsticks were dissolved in cyclohexane and spotted on a 0.25 mm thick silica gel thin-layer chromatography plate. The plate was then developed using benzene-n-amyl alcohol-cont. HCl (65:30:5) as a developing solvent. The dyes were visualized by placing the thin-layer plate in the laser beam as illustrated in Figure 1. A 35 mm camera with Kodak Tri-X film (ASA 400) was used to record the emitted fluorescence from the dyes examined in this study. With 4 watts of power being delivered by the laser to the diverging lens, the exposure time at f4.5 was on the order of l/8 seconds. The exposure time is, of course, a function of distance, laser power, amount of dye present, and whether the laser is being operated in a monochromatic mode or is emitting all lines.
Fig. 2. Photograph of cigarette taken with visible light; no lipstick dye is apparent. Fig. 3. Photograph of the same cigarette under the laser beam. The photographic emulsion is exposed entirely by the intense fluorescence emitted by the lipstick dye; a barrier filter has eliminated the exciting energy.
218
Results and discussion
Serial dilution of precisely weighed amounts of various lipsticks were made, using cyclohexane as a solvent. The limit of sensitivity for the visualization of the cosmetic dyes comprising the lipsticks was found to be on the order of 2 to 8 X lo-’ grams of lipstick. The amount of dye corresponding to this amount of lipstick was not critically determined, but lipsticks generally contain solid dyes in the range of 6-15%, and stain dyes in the range of l-3% [ 31. At the level of 2-8 nanograms of lipstick, the dye cannot be seen on the thin-layer plate with normal incandescent light or under a low-energy ultraviolet lamp. Figure 2 illustrates a cigarette on which a small quantity of lipstick is smeared. The lipstick dye cannot be observed with visible light, nor can it be observed with ultraviolet light. Figure 3 illustrates the same cigarette in the laser beam, the laser delivering 4 watts of power to the diverging lens.
Fig. 4. Laser excited fluorescence of lipstick dyes, as observed on a thin-layer chromatographic plate. The spots represent the dye present in approximately 50 nanograms of lipstick, or approximately 5 nanograms of dye.
219
The radiant energy exposing the film emulsion is the fluorescence emission from the lipstick dye. Figure 4 illustrates a thin-layer plate on which lipstick extracts were chromatographed. The spots represent the dye extracted from 50 nanograms of lipstick, or approximately 5 nanograms of dye. Although the laser is capable of delivering energy of shorter wavelength for excitation of the dyes, in the present study of cosmetic dyes it was found that the emission maxima of the dyes were above the 514.5 nm line. Consequently, the laser was operated with all lines emitting. The writers consider this visualization technique to be attractive from the standpoint of sensitivity. Nanogram quantities of many dyes may be visualized by the stimulated fluorescence, allowing the detection of trace quantities of materials which could not otherwise be detected by visual examination or ultraviolet fluorescence. Although the present study was confined to cosmetic dyes, the writers have observed intense fluorescence with textile dyes and inks, and laser visualization would seem applicable to these materials as well.
References 1 B. E. Dalrymple, J. M. Duff and E. R. Menzel, Inherent fingerprint luminescence detection by laser. J. Forensic Sci., 22 (1977) 106 - 115. 2 T. P. Cotsis and J. C. Garey, Determination of dyes in lipsticks by thin layer chromatography. Proc. Toilet Goods Ass. Sci. Sec., 41 (1964) 3 - 11. 3 L. P. Torry, Lipstick, rouge, eye make-up and manicure preparations, in H. W. Hibbot (ed.), Handbook of Cosmetic Science, Macmillan, New York, 1963, pp. 295 - 329.
