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Solvent Enhancement Effects in Thin-Layer Phosphorimetry J.
N. Miller," D. L.
Phillipps, and D. Thorburn Burns'
Department of Chemistry, Loughborough University, Loughborough, Leicestershire LE 1 1 3TU. U.K.
J. W. Bridges Department of Biochemistry, University of Surrey, Guildford, Surrey, GU2 5XH. U.K.
The construction and use of an improved thin-layer phosphorimeter is described. The device permits flexible chromatography media to be scanned at 77 K, and also allows a complete characterization of the luminescence properties of the chromatographically-separatedsolutes. It is shown that the phosphorescence intensities of a variety of adsorbed materials are greatly increased by spraying the chromatography medium with a suitable solvent immediately before examination. The magnitude of the effect depends on the stationary phase, the structure of the adsorbed material, and the solvent sprayed. With the aid of the enhancement effect, nanogram quantities of separated solutes can be analyzed. The instrument Is also suitable for examining the luminescence of adsorbed molecules at ambient temperatures.
Combinations of chromatographic methods and fluorescence spectrometry a t room temperature have been repeatedly shown t o be powerful analytical techniques which combine t h e selectivity of chromatographic separations with t h e sensitivity of fluorimetry. T h e use of fluorimetry to locate and quantitate the components of complex mixtures separated by thin-layer chromatography (TLC) is a particularly convenient approach (e.g., Ref. 1) which permits the simultaneous study of several very small samples, and instruments designed for the quantitative fluorimetric scanning of TLC plates are commercially available. Although recent studies have amply demonstrated t h e analytical value of phosphorimetry ( 2 ) ,a technique normally performed a t 77 K, combinations of TLC and phosphorimetry have so far been little used. Rinefordner a n d co-workers determined biphenyl in oranges ( 3 ) , 4nitrophenol in urine ( 4 ) , and alkaloids in tobacco ( 5 ) by separating the samples on TLC plates, eluting the separated fractions with a suitable solvent, and determining them using conventional phosphorimetric sampling techniques. Studies have also been described in which t h e phosphorescence of samples separated by TLC have been detected visually by dipping the chromatography plates into liquid nitrogen and observing them under UV illumination. Sawicki et al. (6) investigated a series of aromatic atmospheric pollutants using this approach, and more recently de Silva and Strojny ( 7 )have detected nanogram quantities of various drugs. In the authors' laboratory a thin layer phosphorimeter has been developed (8) as a n accessory suitable for commercially-available spectrofluorimeters. This device permits the in situ quantitative determination and spectroscopic characterization, a t 77 K, of solutes separated on flexible TLC media. The present paper describes the construction and evaluation of a n improved thin-layer phosphorimeter. I n particular it is demonstrated t h a t the phosphorescence of solutes adsorbed on TLC plates can sometimes be greatly enhanced by spraying t h e plates with organic solvents before scanning, a procedure which permits t h e determination of nanogram quantities of such solutes. 'Present address, Department of Chemistry, The Queen's University, Belfast, Northern Ireland. 0003-2700/78/0350-0613$01,00/0
EXPERIMENTAL Construction of t h e Thin-Layer Phosphorimeter. The thin-layer phosphorimeter is designed to fit the sample compartment of the Baird-Atomic (Braintree, Essex, U.K.) "Fluoricord" spectrofluorimeter, and is illustrated in Figures 1 and 2. The TLC plate is affixed with elastic bands to the outside of a hollow copper sample drum (diameter 6.5 cm) which can be filled with liquid nitrogen through a narrower upper cylinder. The bottom of the drum is lipped to allow accurate positioning on the turntable in the holder compartment. This turntable is driven by a 12-\' motor (Maxon 2126-912, Trident Engineering Ltd., Wokingham, U.K.) via a reduction gearbox and intermediate gear. The rate of rotation of the turntable is controlled by a variable output transformer and provides a scanning rate of 3-40 cm m i d . The outer cylinder of the sample holder compartment is pierced to permit two silica windows to be fitted. These windows allow incident light to reach the sample on the TLC plate at 45' to the normal, and the emitted light (observed at 45' to the normal) to reach the detector. Slots are provided on the cylinder which hold fixed slits. An annular space approximately 7 mm across separates the surface of the TLC plate from the inner surface of the cylinder and this space, and the outer surfaces of the silica windows, are continuously swept by a stream of dry oxygen-free nitrogen to prevent the formation of ice and to minimize luminescence quenching by oxygen. Phosphorescence and other long-lived luminescence phenomena are distinguished from prompt emissions and scattered light using a single disc phosphoroscope (9). The disc, 65 mm in diameter, has three equally spaced slots 13 X 16 mm long cut in it and is driven a t speeds of up to 10500 rpm by a 12-V electric motor (Faulhaber 26 PC.210, Portescap U.K. Ltd., Reading, U.K.). The phosphoroscope assemblq is painted matt black, and is fitted with a light baffle; it can be removed completely to permit observations of total luminescence. Small modifications to the sample chamber door on the spectrofluorimeter are necessary to accommodate the phosphorimeter assembly. Operation of t h e Thin-Layer Phosphorimeter. Chromatographic separations were normally performed on silica gel TLC plates with aluminum foil backing (E. G. Merck, obtained through British Drug Houses, Poole, U.K.); the layer thickness was 250 pm and the plates supplied were cut into strips 20 cm long and 5 cm wide. (In a few experiments cellulose (Merck) or alumina plates prepared in this laboratory were used.) The luminescence background signal of the silica gel stationary phase was reduced by developing the plates in a chromatography tank with ethanol. Luminescent impurities were carried to one end of the thin layers and were removed by cutting the top 2 cm off the plates. The plates were then dried thoroughly before the chromatography proper began. Samples, 5 pL in volume were applied to the TLC plates using an Arnold microapplicator (Burkard Instruments, Rickmansworth, C.K.). Test experiments showed that the relative standard deviation of the sample spot diameter obtained using this method was as low as 0.8%. The precision obtained using disposable micropipettes (Corning) was inferior (relative standard deviation 2.570) but still adequate for many analyses. Chromatographic separations were carried out in a Shandon chromatographic tank lined with Whatman No. 1 filter paper; all experiments were carried out in the dark to minimize the possibility of photodecomposition. The drugs studied, their sources and luminescence characteristics and, where applicable, the solvent systems used and the R, values obtained, are shown in Table I. After development of a chromatogram, the plate was dried and wrapped C 1978 American Chemical Society
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Table I. Sources and Properties of Compounds Studied
Compound Benzophenone 4-Aminobenzoic acid Phenobarbitone Phenobarbitone sodium N-Methylphenobarbitone 5-Phenyl-5-methylharbituric acid
Pericyazine Chlorpromazine-HCI Methotrimeprazine maleate Procaine-HC1 Sulfanilamide N-4-Acetylsulfanilamide Sulfadiazine Sulfamerazine Sui f ame thaz ine Sulfamethoxazole
Source
Excitation wavelength, nm
Phosphorescence wavelength, nm
B.D.H. Ltd. B.D.H. Ltd. May and Baker Ltd. May and Baker Ltd. Winthrop Labs. May and Baker Ltd. May and Baker Ltd. May and Baker Ltd. May and Baker Ltd. B.D.H. Ltd. May and Baker Ltd. See Ref. 1 0 May and Baker Ltd. May and Baker Ltd. May and Baker Ltd. May and Baker Ltd.
350 305 266 266 260 266 315 310 305 310 305 290 310 310 310 310
445 425 395 395 395 395 540 490 485 430 405 410 420 41 2 410 4 12
Solvent system for TLCa
Rf value
1
0.57
2 3 5
0.52 0.28 0.61 0.40
1
0.55
4
a Solvent systems: (1) Ch1oroform:butanol:acetic acid, 15:1:1, v/v/v. ( 2 ) Methanol. ( 3 ) Chloroform:methanol, 9:1, v/v. (4) Chloroform:butanol:ethanol:25% ammonia, 15:5:5:1 v/v/v/v. ( 5 ) Ethyl acetate:methanol:25% ammonia, 17:6:5, v/v/v.
Figure 1. The thin-layer phosphorimeter attachment. (a) An exploded view of the sample drum (top), outer cylinder and turntable assembly (bottom), and single disc phosphoroscope (left). (b) Assembled, showing the fluorimeter sample compartment door and tubes for the dry nitrogen supply
--
Figure 2. Sectional diagram of the thin-layer phosphorimeter attachment, showing the light baffle (A), slits (B,C), disc phosphoroscope (D), phosphoroscope motor (E), turntable (F), intermediate gear (G), turntable driving motor (H), and outer cylinder (J). The sample drum is omitted for the sake of clarity
round the sample holder drum. It was then sprayed with ethanol or another solvent (see below) until just wet and the drum inserted into its compartment and filled with liquid nitrogen. After being allowed to cool for 2 min, the TLC plate was scanned at the rate of ca. 5 cm min-' and the results were displayed on the chartrecorder attached to the fluorimeter. The detection limit of a compound was taken to be that concentration giving a luminescence signal equal to twice the standard deviation of the background signal. Phosphorescence lifetimes were determined using a chart recorder as previously described (10). In experiments to test the precision of thin-layer phosphorimetry, and in studies of the effects of spraying solvents on to phosphorescent spots, 5-pL samples of ethanolic solutions of various compounds were applied to TLC plates and studied directly, Le., without a prior chromatographic step. All solvents and other chemicals were of AR or equivalent grade.
RESULTS U'hen a single 5-wL sample of benzophenone on a silica gel layer was scanned repeatedly using the thin-layer phosphorimeter, the measured peak intensities showed a relative standard deviation of 4%. When samples that had been separated by TLC before scanning were studied, reproducible results were obtained only when the TLC plate was sprayed with a solvent (see below) and when an internal standard was used. In these circumstances the relative standard deviation for several solutes was ca. 8%. Preliminary experiments showed that the phosphorescence intensity of adsorbed solutes was often dramatically enhanced by spraying the developed plate with a solvent before scanning; thus, the phosphorescence of chlorpromazine hydrochloride on silica gel was enhanced 100-fold by spraying the silica gel with ethanol from a distance of 20 cm for about 30 s. T h e background phosphorescence was enhanced only 10-fold. An extensive study of this phenomenon was undertaken, with the following results. I t was found that the measured phosphorescence intensity was maximized by spraying the plate until it had just acquired a wet appearance, Le., there was a continuous film of solvent on the stationary phase. Spraying for longer periods produced no extra enhancement but sometimes caused the disruption of the stationary phase when t h e plate was subsequently cooled. In practice, no difficulty was experienced in assessing the optimal spraying time. Table I1 shows the enhancement of phosphorescence of four drugs, adsorbed on silica gel layers, by a variety of solvents. The effects of a series of alcohols were investigated, to study the influence of hydrocarbon chain length on enhancement.
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Table 11. Enhancement of Phosphorescence on Silica Gel by Spraying with Various Solvents
Solvent Methanol Ethanol 1-Propanol 2-Propanol 1-Butanol 2-Methyl- Z-propanol
1-Pentanol 1-Hexanol Triethylamine Dimethylformamide Formamide Diethylke tone Ethanol/potassium iodide Methyl iodide Dimethylsulfoxide Hexane Water
Dielectric constant ( 2 0 C)“
Refractive Index ( 2 5 C)“
32.8 24.3 20.1 18.3 17.1 10.9 13.9 13.3 2.4 37.6 109.5 17.0
1.326 1.359 1.383 1.375 1.397 1.383 1.408 1.416 1.398 1.427 1.446 1.392
7.0 46.7 1.89 80.4
Sulfadiazine
Sulfamethazine
Phenobarbitone
Methotrimeprazine maleate
65 91
25 1 240 250 270 240 60 136 70 6 152
15 20 20 34 35
96 80 75 100 100
16 9 2 9 5
66 50
30
75
120
112 100
30 45 21 5 108
1.530 1.477 1.372 1.333
21 4
18
205 2 137 11 11
288 8 3 00 31 19
18
53 45 32
12 1
1
13
86 12 50
11
7
“ From “Handbook of Chemistry and Physics,” 55th ed., 1975. ____-
Table 111. Enhancing Effect of Ethanol on the Phosphorescence of Compounds Adsorbed onto Silica Gel Compound N-Methylphenobarbitone Phenobarbitone Phenobarbitone sodium 5-Phenyl-5-methyl barbituric acid Sulfadiazine Sulfamerazine Sulfamethazine Methotrimeprazine maleate Pericyazine Chlorpromazine-HC1
Table IV. Detection Limits of Compounds on TLC Plates (ng/spot)
Enhancement factor 16 20 20 18
91 165 240 80 55 52
Further solvents with diverse dielectric constants were examined, and methyl iodide and ethanol saturated with potassium iodide were used to ascertain whether a useful external heavy atom effect could be observed. In all cases ethanol, which was readily available in a state of high purity, produced a substantial enhancement of phosphorescence, and this solvent was used in subsequent studies involving other chromatographic media and samples. Phosphorescence enhancements were also observed on cellulose and alumina layers, but these effects were much smaller than those on silica gel layers; thus the enhancement factor for methotrimeprazine maleate sprayed with ethanol was 80 on silica gel, 2.8 on alumina, and 2.2 on cellulose. The effects of ethanol spraying on 10 drugs (including those listed in Table 11) adsorbed on silica gel are shown in Table I11 - substantial enhancements were observed in all cases. T h e phosphorescence lifetimes a t 77 K of sulfadiazine and sulfamethazine adsorbed on silica gel were determined, and compared with the values obtained in a n ethanol glass. I n all cases the values obtained were 0.7 f 0.1 s, in excellent agreement with previous determinations (10). Table IV shows t h e limits of detection of six compounds determined using the thin-layer phosphorimeter a t 77 K; the results are compared with recent estimates of the visual detection limits, also a t 77 K, given by d e Silva and Strojny (7).
DISCUSSION I t is apparent from the results t h a t thin-layer phosphorimetry, in conjunction with the solvent enhancement technique, offers a new and powerful approach to the analysis of complex mixtures of luminescent materials. Nanogram
Compound Sulfanilamide Sulfadiazine Sulfamethoxazole N-4-Acetylsulfanilamide 4-Aminobenzoic acid Procaine hydrochloride
TLC phosphorimetry, 77K 0.5 2 3 7 0.6 2
Visual detection limits, native phosphorescence 77K (Ref. 7 ) 100 1000 1000 100 100
quantities of phosphorescent compounds can be determined using a relatively simple accessory fitted to a fluorimeter, and the reproducibility, which is similar to that obtained in t h e fluorimetric scanning of TLC plates at room temperature (II), is quite adequate for most applications. The method will be expected to be of the greatest value in the analysis of mixtures of closely-similar materials such as a drug and its metabolites. Thus, nanogram quantities of thioridazine and five of its metabolites have been successfully separated and analyzed in recent experiments (12). The development of a thin-layer phosphorimeter also foreshadows the introduction of phosphorescent label molecules which, like the currently-used fluorescence labels, form strongly phosphorescent derivatives with specific functional groups of non- or feebly-luminescent molecules. Phosphorescent labels for phenols are under study in this laboratory (E. U. Akusoba and J. N. Miller, unpublished work). The apparatus described can also be used without liquid nitrogen for analyses by the newly-developed technique of room-temperature phosphorimetry (13). The data cited may provide some evidence on the nature of the phosphorescence enhancement effect. Lawrence and F r e i showed ( 1 4 ) t h a t spraying d i m e t h y l a m i n o naphthalenesulfonyl (“dansyl’‘) derivatives of primary amines on TLC plates with a triethanolamine-isopropanol mixture stabilized their fluorescence, and that other spray-reagents, including dioxan and various aqueous buffers, produced increases in the fluorescence signals. These effects were ascribed to the desorption of the sample molecules from the chromatographic medium into a thin surface layer of solvent. The enhancements of phosphorescence found in the present work, however, are far larger, and seem to depend on a number of factors, including the sprayed solvent, the chromatographic
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stationary phase, and the type of compound under study. Consideration of Table I1 fails to reveal any obvious correlations between the observed magnitudes of the enhancement and the physical properties of the solvents; this is probably because the refractive index and dielectric constant data cited were mostly determined a t room temperatures rather than 77 K. Among the eight alcohols used, 1-propanol and 2propanol generally gave the best enhancements (although ethanol has been used in routine analysis, see above) and 1-pentanol and 1-hexanol gave poor results. The attempt to utilize the heavy atom effect by adding potassium iodide to the ethanol spray proved useful in the case of sulfadiazine but was of little value in the other cases: similarly uneven results have been observed in ethanol glasses a t 77 K ( I 5 ) ,and in room temperature phosphorimetry (16). Table I11 shows that different types of sample molecules will show widely different enhancement effects: the four oxybarbiturates showed similar but very moderate enhancements, the three sulfonamides all showed very substantial enhancements, and the three phenothiazines exhibited intermediate enhancement values. In contrast to the situation in room temperature phosphorescence (19,there is little evidence that the number of charged groups on the molecule has an appreciable effect on the observed enhancement; on the other hand the results in the case of sulfadiazine, sulfamerazine, and sulfamethazine indicate the possible importance of non-polar substituents. I t thus seems likely that no single factor can account for the enhancement phenomenon which is of such importance in thin-layer phosphorimetry.
ACKNOWLEDGMENT We thank L. A. Gifford and M. J. Jaycock for many valuable discussions, and A. Stevens for expert technical assistance.
LITERATURE CITED (1) R. W. Frei, J. F. Lawrence, and P. E. Belliveau, Fresenius Z. Anal. Chem.. 254, 271 (1971). (2) C. M. O'Donnell and J. D. Winefordner. Clin. Chem. ( Winston-Salem, N.C.). 21, 285 (1975). (3) W. J. McCarthy and J. D. Winefordner, J . Assoc. Off. Agric. Chem.. 48, 915 (1965). (4) W. J. McCarthy and J. D. Winefordner. Anal. Chim. Acta, 35. 120 (1966). (5) J. D. Winefordner and H. A. Moye, Anal. Chim. Acta, 32, 278 (1965). (6)E. Sawicki and H. Johnson, Microchem J., 8, 85 (1964). (7) J. A. F. desilva and N. Strojny, Anal. Chem., 47, 714 (1975). (8) L. A. Gifford, J. N. Miller, D. T. Burns, and J. W. Bridges J . Chromatcgr.. 103, 15 (1975). (9) L. Langouet, Appl. Opt., 11, 2358 (1972). (10) J. W Bridges, L. A. Gifford, W. P. Hayes, J. N. Miller, and D. T. Burns, Anal. Chem., 468 1010 (1974). (1 1) D. E. Janchen, in "Quantitative Paper and Thin Layer Chromatography", E. J. Shellard, Ed., Academic Press, London, 1968. (12) D. L. Phillipps. J. N. Miller, and J. W. Bridges, in preparation. (13) R. A. Paynter, S. L. Wellons, and J. D. Winefordner, Anal. Chem., 46, 736 (1974). (14) J. F. Lawrence and R. W. Frei, J . Chromatogr., 66, 93 (1972). (15) W. J. McCarthy and J. D. Winefordner, Talanta, 78, 305 (1970). (16) T. Vo Dinh, E. Lue Yen, and J. D. Winefordner, Anal. Chem., 48, 1186 (1976). (17) S. L.Wellons, R. A. Paynter, and J. D. Winefordner, Spectrochern. Acta. Part A , 30, 2133 (1974).
RECEIVED for review November 29, 1977. Accepted December 23, 1977. We are grateful to the Medical Research Council for their continuing support of this work through the award of a Project Grant (G973/349/C).
Comparison of Different Experimental Configurations in Pulsed Laser Induced Molecular Fluorescence J. H. Richardson* and S. M. George' Lawrence Livermore Laboratory, General Chemistry Division, University of California, Livermore, California 94550
Three laser excitation sources (nitrogen pumped dye laser, cavity dumped argon ion laser, and externally pulse-picked mode-locked argon ion laser) are compared using rhodamine B as an ideal fluorophor. The major variable which was compared is peak power vs. repetition rate, complementary characteristics in current lasers. Boxcar detection is used with the first two sources, while photon counting techniques are used with the latter source. The limits of detection achieved were 1.0, 0.5 and 15 parts-per-trillion, respectively. For many analytical problems, the nitrogen pumped dye laser appears to be the most flexible and generally applicable laser excitation source.
T h e recent development of lasers has awakened a renaissance in many disciplines of chemistry. Analytical chemistry has been somewhat reluctant to embrace this new technology, but within the past several years there have been many reports of laser technology being applied advantageously to various analytical problems: (1) gas phase detection of atoms and molecules by absorption ( I , 2), fluorescence (3-91, Present address, University of California, Chemistry Department, Berkeley, Calif. 94720. 0003-2700/78/0350-0616$0 1.OO/O
electrical conductivity ( I O ) , and ionization ( 2 1 ) ; (2) optoacoustic techniques for gas (12) and condensed phases ( 1 3 , 14); (3) detectors for thin-layer chromatography (25) and high pressure liquid chromatography (16,177);(4) detection of trace contaminates in solids (18,19);(5) detection of trace species in solution by fluorescence techniques (20-26). The many capabilities offered by laser technology invariably give rise to confusion as to which laser is most appropriate for a given application. For example, for trace detection in solution, linewidth is obviously a minor consideration but tunability is just as obviously a major consideration. Less obvious are the relative merits of laser excitation pulse width. A narrow pulse width is desirable for temporal discrimination (23) against background fluorescence, but how significant the temporal resolution can be, will depend on the relative decay times of the laser excitation source, the background fluorescence, and the signal fluorescence. Other factors are repetition rate and peak power. With current laser technology, these two factors can be treated simultaneously, as they inevitably involve a trade-off; high peak powers entail low repetition rates, and high repetition rates entail low peak powers. The relative merits of these latter two factors with respect to trace detection in solution have not been examined experimentally. There have been several theoretical papers 1978 American Chemical Society
