tern similar to phenformin was observed. The molecule forms a molecular ion a t mle 251 and a t mle 253 in a ratio of 3:l indicating the presence of a single chlorine atom. The molecular ion through beta cleavage forms the most abundant ion, a t mle 208 and mle 210, identical to that formed from phenformin. The GCIMS for metformin after extraction and derivatization is shown in 'Figure 5; as expected, the molecular ion a t mle 223 and mle 225 is also the most abundant species formed. The contribution by the even electron species a t rnle 208 and m/e 210 is considerably less in the case of metformin. T o show the clinical usefulness of the developed method, a diabetic patient was administered a therapeutic dose of 100 mg sustained release preparation of phenformin orally. The plasma levels achieved are shown in Figure 7. The curve shows that it took 2 to 3 hours to achieve the peak plasma level of 150 nglml. Plasma concentration then declined with an initial half-life of 4.5 hours (ie., rate constant = 0.15) followed by a terminal half-life of 13 hr (rate constant = 0.05 hr). The urinary excretion rate plot for the same patient is shown in Figure 8. The cumulative excretion of unchanged phenformin a t 36 hr was 48% of the dose and excretion into urine could be detected up to 60 hr after the administration of the dose.
ACKNOWLEDGMENT The authors thank A. N. Wick for providing the sample of l4C-phenformin used in this study. LITERATURE CITED (1)A. Loubatieres, "Oral Hypoglycemic Agents," G. D. Campbell, Ed., Academic Press, New York. N.Y., 1969,Chap. 1. (2)F. Davidoff, N. fng.J. Med., 289, 141 (1973). (3) R. Beckmann and G. Herbner, Aezneim. Forsch., 15, 765 (1965). (4) L. Freedman, M. Blitz, E. Gunsberg, and S. Zk, J. Lab. Clin. Med., 58, 662 (1961). (5)R. E. Bailey, Clin. Biocbem., 3, 23 (1970). (6)H. Hall, G. Ramachander, and J. M. Glassman. Ann N.Y. Acad. Sci,, 148,601 (1968). (7N.A. F. Wickramasingh and S. R. Shaw, J. Chromatogr., 71, 265 (1972). (8) E. R. Garrett and J. Tsau. J. Pbarm. Sci., 61, 1404 (1972). (9)P. J. Murphy and A. N. Wick, J. Pharm. Sci., 57, 1125 (1968). (10) A. Weissberger. Ed "The Chemistry of Heterocyclic Compounds Striazines and Derivatives," Interscience, New York, N.Y., 1959,p 225.
RECEIVEDfor review August 19, 1974. Accepted October 31, 1974. A portion of this paper was presented a t the annual APhA Meeting, in the Pharmaceutical Analysis Section held a t San Diego, in November, 1973. The authors acknowledge the support of Ciba-Geigy Corporation Arsdley, N.Y., in making this study possible.
Thin-Layer Chromatography and Photoacoustic Spectrometry Allan Rosencwaig Bell Laboratories, Murray Hill, N.J. 07974
Stan S. Hall Deparfment of Chemistry, Rutgers University, Newark, N.J. 07 102
Thin-layer chromatography (TLC) is a widely used and highly effective technique for the separation of mixtures into their constituent compounds ( 1). The identification of the separated compounds on the TLC plate, can, however, be a fairly difficult procedure, particularly if the TLC spots are not visible, and the use of coloring reagents is undesirable. Although conventional spectrometric methods can often be used to locate, and in some cases identify, the separated compounds directly on the TLC plate, they have not proved to be very reliable, especially in the UV wavelength region. This is so because the adsorbent material is usually too opaque to perform transmission spectrometry and too highly scattering to perform reflection spectrometry on the separated compounds. In most cases, compound identification is performed after extraction from the adsorbent layer. The spots are first detected-visually if they are colored, by UV radiation if they fluoresce, by iodine vapor localization, or by densitometric techniques. If none of these procedures is possible, then reference chromatographs can be used in conjunction with color producing reagents. After the spots have been located, they are scraped off, and the compound is extracted from the adsorbent with a suitable eluent. Compound identification can then proceed by performing conventional absorption spectrometry on the eluent solution. However, even with the eluent solution, severe difficulties can be experienced in obtaining absorption spectra in the ultraviolet due to excessive light scattering from solid adsorbent particles suspended in the solution. 548
A N A L Y T I C A L C H E M I S T R Y , VOL
47, NO 3, M A R C H 1975
It would be of considerable value if the location and identification of the TLC-separated compounds could be performed in a nondestructive fashion directly on the TLC plates. Photoacoustic spectrometry offers a simple and sensitive technique for performing this very function. In photoacoustic spectrometry (2, 31, a solid sample is placed inside a closed cell containing air and a sensitive microphone. The solid is then illuminated with chopped monochromatic light. Any light absorbed by the solid is converted, in part or in whole, into heat by nonradiative transition processes. The resultant periodic heat flow from the solid absorber to the surrounding gas creates pressure fluctuations in the cell that are detected by the microphone. There is a close correspondence between the amount of light absorbed by the solid sample and the magnitude of the acoustic signal. A photoacoustic spectrum thus corresponds, qualitatively a t least, to an optical absorption spectrum, provided that the non-radiative processes dominate in the dissipation of the absorbed light energy. Photoacoustic spectrometry enables one to obtain spectra similar to optical absorption spectra on any type of solid or semisolid material, whether it be crystalline, powder, amorphous, gel, etc. Furthermore, since only the absorbed light is converted into sound, light scattering presents no problems. This technique has been used to obtain optical data on inorganic materials, ( 2 ) , on hemoproteins, ( 3 ) ,and on plant matter such as marine algae ( 4 ) .I t should prove possible, therefore, both to locate the TLC-separated
A1
949,LLq 9 49,LLq
SENSITIVITY s:i
5.3
I
Figure 1. ( a ) Photoacoustic spectra on TLC plates and ( b ) UV absorption spectra on solutions of (I) p-nitroaniline, (11) benzylidene acetone, (111) salicyclaldehyde,(IV) 1-tetralone,and (V) fluorenone
compounds and to identify them directly on the TLC plates by obtaining their optical absorption spectra with the photoacoustic technique. T u Figure 1 ( a ), we show the photoacoustic spectra taken in t h e spectral range of 200 to 400 nm on five compounds that were developed as spots on TLC plates. The TLC plates used were commercial pre-coated plastic sheets with 0.25 rnm of F-254 silica gel adsorbent. The compounds, p nitroaniline, benzylidene acetone, salicylaldehyde, l-tetralone, arid fluorenone, were developed using either a 3 or 5% m e t h a r d in benzene solvent. The sheets were then baked a t 125 "C for one to two minutes to remove any excess solvent. Photoacoustic spectra were run on both intact sections of 1he TLC plates and on material scraped from the plates with similar results. In Figure 1 ( b ) we show, for comparison, the published UV absorption spectra of these. 5 compounds in solution (5 1. We note that the photoacoustic spectra are qualitatively very similar to the solution spectra. Full quantitative agreement cannot be expected since the photoacoustic technique mtlasures only the amount of absorbed optical energy de-excited through the nonradiative or heating processes, and several of these compounds fluoresce under ultraviolet irradiation, that is de-excite partially through radiative processes. Nevertheless the strong qualitative similarity between the photoacoustic spectra and the optical absorption spectra permits a rapid and unambiguous identification of The compounds. To determine Ihe sensitivity of the photoacoustic technique for this application, we show in Figure 2, photoacoustic spectra taken o n a TLC plate on which benzylidene acetone spots of different concentration were developed. The top spectrum was taken on a spot developed from a starting
203
I
Mo
I
400
nrn
Figure 2. Photoacoustic spectra on spots of benzylidene acetone on a TLC plate. The spots were developed from starting drops containing 9.49 pg, 1.11 pg, and 0.095pg of benzylidene acetone drop containing 9.49 pg of benzylidene acetone. The middle spectrum contained 1.11 pg and the bottom spectrum 0.095 pg in the starting drop. The spectra were taken on the developed spots, and these can be expected to contain a smaller quantity of the compound thzn is present in the starting drops. We see that the top two spectra are readily identifiable as that of benzylidene acetone, while the bottom spectrum is not nearly as well defined, though the main absorption band is still visible. It is interesting to note that since the developed spot for the bottom spectrum contains less than 0.1 pg of benzylidene acetone and is -0.3 cm2 in area, this spot then consists roughly of one monolayer of compound adsorbed onto the silica gel. This experiment indicates the possibility that under certain conditions, photoacoustic spectrometry may be sensitive enough to detect a monolayer of an adsorbed compound. Photoacoustic spectrometry can thus be readily used as a simple and highly sensitive technique for locating and nondestructively identifying compounds separated by thinlayer chromatography, directly on the TLC plates. LITERATURE CITED (1) E. Stahl, "Thin-Layer Chromatography," 2nd ed., Springer-Verlag, New York, N.Y., 1969. (2) A. Rosencwaig, Opt. Commun., 7, 305 (1973). (3) A. Rosencwaig, Science, 181, 657 (1973). (4) A. Rosencwaig and S. S. Hall. Proceedings Fourth Food-Drugs From the Sea Conference. Mayaquez, Puerto Rico, 1974, to be published. (5) 'DMS UV Atlas of Organic Compounds," Vol. I-V, H H Perkampus, I Sandeman. and C. J. Timmons, Ed.. Plenum Press, New York, N.Y., 1966-1971.
RECEIVEDfor review July 25, 1974. Accepted November 4, 1974.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 3, M A R C H 1975
549
