O'Haver (34) demonstrated that de termination of pyrene in the presence of excess anthracene was greatly im proved by use of the second derivative spectrum. A related but more powerful tech nique, called selective modulation, has also been introduced by O'Haver and Parks (35). Here, the excitation mono chromator is wavelength modulated as the emission monochromator is scanned. The resulting ac photocurrent is detected with a lock-in amplifi er. The central excitation wavelength is chosen so that wavelength modula tion will cross a maximum or mini mum in the excitation spectrum of the first component, while the modulation crosses a sloping portion of the second component's excitation spectrum. In this case, the fluorescence intensity of the first component will be modulated at twice the fundamental frequency, while the emission of the second com ponent will be modulated exclusively at the fundamental frequency. Thus, scanning the emission monochromator with the detection system locked onto the proper frequency produces the emission spectrum of the desired com ponent exclusively. The use of the two wavelength parameters in a derivative scheme can be quite useful, since a fingerprint spectrum can be deter
mined. The importance of this point was illustrated by IR analysis of three components. The major difficulty with derivative and modulation techniques is that they must find spectral regions where the derivative forms of the spectra for each component satisfy specific crite ria with regard to the form of the spec tral overlap and S/N, and thus the ap plicability is rather limited. In con trast, our approach, which simply uses the linear additivity of the spectra of the individual components, is always valid and is limited by the more fun damental considerations of signal-tonoise ratio of the measurement. Also, our system has the potential to com pute derivative, difference, and modu lation spectra in both wavelength di mensions simultaneously. Synchronous Excitation Tech nique. Lloyd (36) has introduced a novel technique for qualitative and quantitative analysis of mixtures, called the synchronous excitation technique. The synchronous spectra are obtained by locking the excitation and emission monochromator drives together at a fixed wavelength inter val, usually on the order of 20 nm, and then scanning them over a fixed inter val. The resulting spectrum usually contains only a few peaks which then
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754 A · ANALYTICAL CHEMISTRY, VOL. 49, NO. 8, JULY 1977
Perylene-
Anthracene Fluoronthane
Pyrene Chrysene
Figure 6. EEM of mixture of anthracene, perylene, pyrene, fluoranthene, and chrysene
form a unique fingerprint for each compound. This technique has been applied to the identification and quantification of crude oils (37) and their partially fractionated compo nents. The synchronous scanning protocol is easily visualized on the EEM; it rep resents the fluorescence intensity measured along a line defined by \em = Xex + Λ, where A is the number of wavelength units difference in the set tings of the monochromators. Essen tially, the synchronous scan concept simultaneously takes advantage of variability of both the excitation and emission spectra of fluorescent species. However, although this tech nique works well in cases where welldefined vibronic bands exist, as in the example mixture of Figure 1, it is blind to other regions of the EEM where better separation might be ob tained. Time-Resolved Spectroscopy. A potentially very powerful technique in multicomponent luminescence uses the fact that the lifetime of an excited state can be a useful distinguishing feature. Most of the effort here has been in phosphorescence lifetimes be cause the lifetimes of triplet states (10 MS to 10 s) are much longer than those of excited singlet (fluorescent) states ( 100 ps to 500 ns). Instrumentation for time-resolved phosphorescence has been described by Callis et al. (38), St. John and
