Measurement methods fluorescence

The continuous methods combine sample collection and the measurement technique in one automated process. The measurement methods used for continuous analyzers include conductometric, colorimetric, coulometric, and amperometric techniques for the determination of SO2 collected in a liquid medium (7). Other continuous methods utilize physicochemical techniques for detection of SO2 in a gas stream. These include flame photometric detection (described earlier) and fluorescence spectroscopy (8). Instruments based on all of these principles are available which meet standard performance specifications. 

Functions of Standards. Fluorescent standards can be used for three basic functions calibration, standardization, and measurement method assessment. In calibration, the standard is used to check or calibrate Instrument characteristics and perturbations on true spectra. For standardization, standards are used to determine the function that relates chemical concentration to Instrument response. This latter use has been expanded from pure materials to quite complex standards that are carried through the total chemical measurement process (10). These more complex standards are now used to assess the precision and accuracy of measurement procedures. 

FBAs can also be estimated quantitatively by fluorescence spectroscopy, which is much more sensitive than the ultraviolet method but tends to be prone to error and is less convenient to use. Small quantities of impurities may lead to serious distortions of both emission and excitation spectra. Indeed, a comparison of ultraviolet absorption and fluorescence excitation spectra can yield useful information on the purity of an FBA. Different samples of an analytically pure FBA will show identical absorption and excitation spectra. Nevertheless, an on-line fluorescence spectroscopic method of analysis has been developed for the quantitative estimation of FBAs and other fluorescent additives present on a textile substrate. The procedure was demonstrated by measuring the fluorescence intensity at various excitation wavelengths of moving nylon woven fabrics treated with various concentrations of an FBA and an anionic sizing agent. It is possible to detect remarkably small differences in concentrations of the absorbed materials present [67]. 

Just as above, we can derive expressions for any fluorescence lifetime for any number of pathways. In this chapter we limit our discussion to cases where the excited molecules have relaxed to their lowest excited-state vibrational level by internal conversion (ic) before pursuing any other de-excitation pathway (see the Perrin-Jablonski diagram in Fig. 1.4). This means we do not consider coherent effects whereby the molecule decays, or transfers energy, from a higher excited state, or from a non-Boltzmann distribution of vibrational levels, before coming to steady-state equilibrium in its ground electronic state (see Section 1.2.2). Internal conversion only takes a few picoseconds, or less [82-84, 106]. In the case of incoherent decay, the method of excitation does not play a role in the decay by any of the pathways from the excited state the excitation scheme is only peculiar to the method we choose to measure the fluorescence (Sections 1.7-1.11). 

At present, two main streams of techniques exist for the measurement of fluorescence lifetimes, time domain based methods, and frequency domain methods. In the frequency domain, the fluorescence lifetime is derived from the phase shift and demodulation of the fluorescent light with respect to the phase and the modulation depth of a modulated excitation source. Measurements in the time domain are generally performed by recording the fluorescence intensity decay after exciting the specimen with a short excitation pulse. 

Intramolecular charge transfer in p-anthracene-(CH2)3-p-Ar,Af-dimethylaniline (61) has been observed174 in non-polar solvents. Measurements of fluorescence-decay (by the picosecond laser method) allow some conclusions about charge-transfer dynamics in solution internal rotation is required to reach a favourable geometry for the formation of intramolecular charge-transfer between the donor (aniline) and the acceptor (anthracene). 

Many probes are now known that display changes in fluorescence lifetime on complexation of the analyte, photophysical properties some of them are summarized in Table 10.2. While we have listed the lifetimes of the free and the bound forms of the probes, there is no straightforward equation to calculate the analyte concentration using the mean lifetime as was in the case of the absorbance and intensity (Eqs. (10.14) and (10.15)). The mean lifetime depends not only on relative concentration of the probe species (free and complexed) but also on their decay times, quantum yields, and to some extent on the measurement (method or conditions). While the mean lifetime is independent of total probe concentration, this value generally depends not only on analyte concentration but also on excitation and observation wavelengths.03 ... 

The fluorescence lifetime can be measured by time-resolved methods after excitation of the fluorophore with a light pulse of brief duration. The lifetime is then measured as the elapsed time for the fluorescence emission intensity to decay to 1/e of the initial intensity. Commonly used fluorophores have lifetimes of a few nanoseconds, whereas the longer-lived chelates of europium(III) and terbium(III) have lifetimes of about 10-1000 /tsec (Table 14.1). Chapter 10 (this volume) describes the advantages of phase-modulation fluorometers for sensing applications, as a method to measure the fluorescence lifetime. Phase-modulation immunoassays have been reported (see Section 14.5.4.3.), and they are in fact based on lifetime changes. 

M. G. Badea and L. Brand, Time-resolved fluorescence measurements, Methods Enzymol. 61, 378-425 (1979). 

Contrary to the above-described detection methods, fluorescence up-conversion and optical Kerr gate techniques readily achieve picosecond/femtosecond time resolution (Ippen and Shank 1975 Shah 1988 Takeuchi and Tahara 1998), because they are in the pump-probe measurement, in principle. 

The fluidity (nanoviscosity) in an organized surfactant assembly on soUds can be substantially different from that in the bulk aqueous phase and hence, the diffusional resistance experienced by the probe in the micelle will be considerably different from that faced in the bulk solution [ 145]. Measurement of the viscosity or fluidity of the interior of a micelle is based on measurement of fluorescence properties that depend on the mobihty of the probe in the interior. A commonly used method for such studies involves the intramoleciflar... 

Fluorescence is the basis of a number of measurement methods for atmospheric gases. In the case of SOz, for example, a Zn (213.8 nm) or Cd (228.8 nm) lamp is used to excite the SOz and the fluorescence in the 200- to 400-nm region is monitored (Okabe et al., 1973 Schwarz et al., 1974). Again, this technique works for those species whose excited states are sufficiently... 

There are some methods that are specific to HCHO. For example, the Hantzsch reaction of HCHO, collected with a diffusion scrubber, with ammonium acetate, acetic acid, and acetylacetone to form diacetyldihydrolutidine, which is measured using its fluorescence at 470 nm, has been applied to air measurements (Dasgupta et al., 1988, 1990 Kleindienst et al., 1988a,b Lawson et al., 1990 Khare et al., 1997). Reaction with 1,3-cyclohexanedione and ammonium acetate to form a dihydropyridine derivative that is measured by fluorescence has been used in conjunction with a diffusion scrubber (Fan and Dasgupta, 1994). Enzymatic methods have been used in which formaldehyde dehydrogenase catalyzes the oxidation of HCHO to HCOOH in the presence of -nicotinamide adenine dinucleotide, NAD+, which is reduced to NADH. The latter is measured by fluorescence at 450 nm (Lazrus et al., 1988 Ho and Richards, 1990). 

This paper reports on research involved the design, construction, and evaluation of a portable instrument, a "luminoscope", for detecting skin contamination by coal tars via induced fluorescence. The instrument has been used in the laboratory to measure the fluorescence of various coal tars and recycle solvents from liquefaction processes spotted on filter paper on rat and on hamster skin. The practical use of the devices in field test measurements to monitor skin contamination of workers at coal gasifier is discussed. The paper also discusses the practicality and usefulness of the luminescence method for detecting skin contamination. 

A simplified instrument for the measurement of fluorescent lifetimes using the stroboscopic method has been described by Brown (67). The major virtue of this system is that it makes use of a Tektronix oscilloscope to obtain all the necessary trigger pulses, including a trigger of continuously variable delay. Since most laboratories are equipped with a good oscilloscope, the need to purchase expensive trigger-delay apparatus is thus eliminated. 

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