This is the same as the result of a previous calculation. Thus one obtains the predicted number of structural formulas, i.e., 5 5 , for the combinations along C , and C3, which must be multiplied by the 6 possibilities available for the combinations along C2, or a total of 330 structural formulas. Also found is the predicted total number of combinations pn+ n’+n” . , or 3* = 6561 combinations. This is because
there are 729 possible combinations for the plane G C i , 0-C3alone, so that to obtain the total number of combinations this number must be multiplied by the number of combinations along the axis 0-CZ, i.e., 9 combinations. This finally gives 729 x 9 = 6561 possible combinations. RECEIVED for review February 1, 1971. Accepted October 10, 1972.
Digital Integration Method for Fluorimetric Studies of Photochemically Unstable Compounds R. J. Lukasiewicz’ and J. M. Fitzgerald Department of Chemistry, Unicersity of Houston, Houston, Texas 77004 Fluorescence sensitivity can be improved by increasing the intensity of exciting radiation; however, the sample often photolyzes under such conditions. A method for assay of fluorescent compounds under these conditions is reported. A single source of polychromatic ultraviolet radiation is used to excite fluorescence and to initiate photochemical reaction. In dilute solution, both fluorescence intensity and rate of photochemical reaction are linearly dependent on the incident intensity. Pseudo first-order decay curves of fluorescence signal V S . time are obtained. Initial fluorophor concentration can be measured either by extrapolation to the initial rate of decay or by digital integration of the fluorescence signal for a fixed time. Instrumentation was constructed and evaluated using quinine sulfate; both data readout systems are discussed. Low ppb concentrations of d,l-tryptophan, d,l-5-hydroxytryptophan, tryptamine, 5-hydroxytryptamine, d,l-dopa, dopamine, quinine sulfate, and norepinephrine can be measured using digital readout with relative standard deviations of about 0.5%. Chemical pretreatment of the sample i s not required; native fluorescence of fluorophor is used. The high precision and increased sensitivity make it possible to distinguish between very small concentration differences at ppb levels.
PHOTOCHEMICAL DECOMPOSITION can often compete with solution luminescence when a compound is subjected to ultraviolet radiation. Photolysis of the sample has been previously regarded as a serious problem since the fluorescence intensity changes during the time of the experiment (1-4). Photolysis can be prevented or retarded by using a monochromator or other intensity attenuation so as to limit the power and wavelengths in the excitation beam. Unfortunately, such attenuation causes a reduction in emitted fluorescence intensity and the sensitivity of the method is decreased. Reviews of simultaneous photolysis-fluorescence are available ( 1 , 2 ) ; reports Union Oil Company of California, Research Department, P. 0. Box 76, Brea. Calif. 92621. Address all correspondence to this author. ( 1 ) I . B. Berlman, “Handbook of Fluorescence Spectra of Aromatic
Molecules,” Academic Press, New York, N.Y., 1965. (2) S . Udenfriend, “Fluorescence Assay in Biology and Medicine,” Vol. I-Vol. 11, Academic Press, New York, N.Y., 1962, 1969. (3) D. W. Fink and W. R. Koehler. ANAL.CHEM., 42,990 (1970). (4) A. M . Gillespie, A M I / .Lett., 2 , 609 (1969).
concerning determination of umbelliferone (3) and hallucinogens ( 4 ) are typical examples. Photosensitive compounds can be treated with reagents which shift the excitation spectra to longer wavelengths, thus eliminating the need to expose the sample to short wavelengths ( 2 ) . Such pretreatment increases the time and effort required for a determination. Several photokinetic analytical methods have been developed recently (5,6). Direct photochemical titration of quinine can be accomplished using a single UV source for both photolysis and fluorescence excitation (7). However, this titration is time-consuming because exhaustive photolysis of the sample is required. Photochemical kinetics are experimentally rapid and convenient and provide useful analytical data. Fluorescence is a particularly suitable method for following the rate of photolysis. In dilute solution (absorbance
