Phase Angle and Modulation Spectra

The fractional contritmtiem of each flucM Ophore to the total intrasity of the sample is given by 

Resolution of the Two Emission Spectra of Tiyptophan Using Phase-Modulation Spectra 

As described in Chapto 17 on time-resolved protein fluorescence, tryptophan at pH 7 displays a double-exponential 

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In recent years, the use of phase-senative detection has diminished, probably because of the realization that the phase-sensitive spectra contain less information than the phase-angle and modulation spectra. In the time ittakes to record the phase-sensitive spectnim, one can now record the phase angle and/ca modulation across the emission spectnntL From tiiese phase-modulation spectra, one can compute the phase-resolved spectra. It should be... 

Another example of die resolution of qiectra from die phase-angle and modulation spectra is shown in Figure 22.15 for die DNA probe DAPI dissolved in water at pH 7.1. As described in Section 5.6.C DAM displays a multiexponential decay in water widi decay times of 0.15 and 2.50 ns. The fractional coiriribution of each decay fime... 

An example of the use of apparent phase and modulation lifetimes is given in Figure 535. for the mixture of ACF and AFA. This figure shows die diase-angle and modulation spectra in terms of xjf and xj i. The fact that xjf < xj for a hetero oeous deci is evident by comparison die iqiper and low panels. Also, one immediately notices fiiat the lif me by phase or... 

Figure 10. Phase-resolved fluorescence emission spectra for the same sample shown in Figure 9 with a narrow detector phase angle region scanned (0 to 14 ). Note that the emission maxima shifts with detector phase angle and the sign of the phase-resolved intensity changes (curve 9.0°). Both these observations are Indicative of the presence of two components. Laser excitation at 457.9 nmj modulation frequency is 100 MHz.

Theory. If two or more fluorophores with different emission lifetimes contribute to the same broad, unresolved emission spectrum, their separate emission spectra often can be resolved by the technique of phase-resolved fluorometry. In this method the excitation light is modulated sinusoidally, usually in the radio-frequency range, and the emission is analyzed with a phase sensitive detector. The emission appears as a sinusoidally modulated signal, shifted in phase from the excitation modulation and partially demodulated by an amount dependent on the lifetime of the fluorophore excited state (5, Chapter 4). The detector phase can be adjusted to be exactly out-of-phase with the emission from any one fluorophore, so that the contribution to the total spectrum from that fluorophore is suppressed. For a sample with two fluorophores, suppressing the emission from one fluorophore leaves a spectrum caused only by the other, which then can be directly recorded. With more than two flurophores the problem is more complicated but a number of techniques for deconvoluting the complex emission curve have been developed making use of several modulation frequencies and measurement phase angles (79). 

The effect of the stmctural modulation present in the INC phase on the NMR spectra can be visualized as a spatial variation in the rotation of the Si[0]4-tetrahedron away from its orientation in the a-phase (Fig. 20). In the orthorhombic (a) phase, the Si-position has point symmetry m and one edge of the Si-tetrahedron is perpendicular to c, whereas this edge is rotated by about 10° in the monoclinic ( 3) phase. One view of the INC phase involves a variation of this rotation angle along the crystallographic b direction giving a modulation wavelength of approximately 3b. This rotation of the tetrahedron away from its position in the a-phase is reflected in the Si chemical shift. 

Spectra relaxation of indole has also been observed using the FD method. In this case the frequency responses were dependent on the observation wavelength. The phase angles were larger and the modulations waller... 

In this expression, the 7 (2) are the individual emission spectra the/- ate the fractional contributions to the total steady-state intensity, s 1.0 and nij is the modulation of the ith component and is its phase angle. Dqtending upon the needs of the experiment, the steaify-state qwe-ttum of each species, can be replaced by the steady-... 

Solid-state C variable-amplitude cross polarization magic-angle spinning (VACP/MAS) nuclear magnetic resonance (NMR) spectra were acquired for the sorbitol samples. Proton decoupling was achieved by a two-pulse phase modulation (TPPM) sequence. Identical C spectra were measured for the y-form sorbitol samples, and a representative spectrum is shown in Figure 9. 

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