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The advantage of depth-correlated 2D IR PAS over conventional ATR analysis is the ability to analyze systems comprising multiple layers. As demonstrated earlier in this chapter, 2D PAS provides the depth sequence of multiple layers. At this stage, however, 2D PAS generates only qualitative information about the depth distribution of chemical moieties. The quantitative depth profiling must wait for
Repetitive Perturbations Beyond DIRLD
(A) Transmission (Bulk) (®) ATR (Surface)
1800 800 1800 800
Wavenumber, v Wavenumber, v
Figure 8.42 Comparison of IR spectra of a SHEL film: (A) transmission spectrum probing the entire sample and (B) ATR spectrum probing primarily the layer near the surface
the development of more rigorous derivation of thermal wave propagation in the system. Unfortunately, such derivation in turn requires a priori knowledge of the composition-sensitive thermal diffusivity of the system. Additional complications arises when separate layers having similar compositions, such as adhesive layers, are present in the system. In PAS analysis, such multiple layers with identical compositions must be represented as a fictitious single layer corresponding to the spatial linear combination of individual layers. Finally, unlike DIRLD spectroscopy, the phase delay of individual PAS signals can exceed well over n/2. Some signals originating from a very deep layer can be delayed by several cycles compared to those coming from much shallower layers. There is no way of distinguishing sinusoidal signals spread apart by the phase difference of a multiple of n.
8.3.3 DYNAMIC FLUORESCENCE SPECTROSCOPY
Analytical techniques based on periodic modulations of fluorescence signals with external repetitive perturbations have been known for some time. As early as in 1970, for example, Vesolova et al. carried out the detection of sinusoidal fluorescence response signals arising from the sample excitation using modulated light.33
Dynamic 2D Correlation Spectroscopy Based on Periodic Perturbations
Importantly, they noted that different fluorescing components of the sample responded to the excitation with different phase angles. McGown and coworkers reported a series of work on similar phase-resolved excitation-emission fluorescence experiments with modulated excitation.34-36 The phase shift of the dynamic fluorescence response is related to the fluorescence lifetime, which provides additional selectivity to the origin of the signal. The effect of modulation frequencies and associated phase shifts of fluorescence signals were examined with multiway curve resolution analysis of dynamic excitation-emission data.
Although such phase-resolved fluorescence data are well suited for straightforward dynamic 2D correlation analysis, little attempt was made to exploit the 2D technique in the fluorescence field until a much later date. The first application of generalized 2D correlation analysis to fluorescence spectroscopy was carried out by Rosselli et al., using the wavelength of excitation as a perturbation variable.25 Nakashima et al. also reported work on 2D fluorescence correlation spectroscopy based on steady changes in external variables, such as excitation wavelength and sample composition.37 Specific applications of generalized 2D correlation to fluorescence spectroscopy using such stationary perturbation will be discussed separately in Chapter 9.
Dynamic 2D fluorescence correlation spectroscopy based on phase-sensitive detection of sinusoidally modulated signals was put forward by Geng and coworkers.38-40 Sinusoidal modulation of laser light field with a frequency range of 5-30 MHz was used to generate fluorescence signals, which can be separated into the in-phase and quadrature components. In a dynamic 2D fluorescence experiment, one can actually generate three different types of 2D correlation spectra: excitation-excitation spectra, emission-emission spectra, and excitation-emission spectra. The combined use of these 2D correlation spectra with associated phase maps provides unprecedented spectral resolution advantage to the identification of fine vibronic structures of highly overlapped fluorescence spectra. For example, the resolution of different microenvironments of a probe molecule in a biological system has become possible without relying on statistical fitting of multi-exponential fluorescence decay curves. The high sensitivity of a fluorescence probe is a major advantage over other optical probes such as IR in the detection of low-concentration samples, if combined with the 2D correlation approach.
The feasibility of extending the 2D correlation technique beyond the analysis of DIRLD data has been demonstrated. It was shown that new types of information, quite different from those obtained by dynamic 2D IR experiments, could be generated. 2D SAXS spectra, obtained for the structural reorganization of a microphase-separated block copolymer system undergoing dynamic deformation, show the deformation-induced spreading of interdomain Bragg distances