Fluorometry technique, measuring

The brief history, operation principle, and applications of the above-mentioned techniques are described in this chapter. There are several other measuring techniques, such as the fluorometry technique. Scanning Acoustic Microscopy, Laser Doppler Vibrometer, and Time-of-flight Secondary Ion Mass Spectroscopy, which are successfully applied in micro/nanotribology, are introduced in this chapter, too. 

Global planeness and large scale scratches are usually evaluated by HDI instruments as shown in Fig. 3(a) [8], which is a surface reflectance analyzer to measure flatness, waviness, roughness of a surface, and observe scratches (Fig. 3(h)), pits (Fig. 3(c)), particles (Fig. 3(d)) on a global surface. These surface defects can also be observed by SEM, TEM, and AFM. Shapes of slurry particles can be observed by SEM and TEM, and their movement in liquid by the fluorometry technique as shown in Chapter2. 

Riboflavin can be assayed by chemical, en2ymatic, and microbiological methods. The most commonly used chemical method is fluorometry, which involves the measurement of intense yeUow-green fluorescence with a maximum at 565 nm in neutral aqueous solutions. The fluorometric deterrninations of flavins can be carried out by measuring the intensity of either the natural fluorescence of flavins or the fluorescence of lumiflavin formed by the irradiation of flavin in alkaline solution (68). The later development of a laser—fluorescence technique has extended the limits of detection for riboflavin by two orders of magnitude (69,70). 

This chapter presents new information about the physical properties of humic acid fractions from the Okefenokee Swamp, Georgia. Specialized techniques of fluorescence depolarization spectroscopy and phase-shift fluorometry allow the nondestructive determination of molar volume and shape in aqueous solutions. The techniques also provide sufficient data to make a reliable estimate of the number of different fluorophores in the molecule their respective excitation and emission spectra, and their phase-resolved emission spectra. These measurements are possible even in instances where two fluorophores have nearly identical emission specta. The general theoretical background of each method is presented first, followed by the specific results of our measurements. Parts of the theoretical treatment of depolarization and phase-shift fluorometry given here are more fully expanded upon in (5,9-ll). Recent work and reviews of these techniques are given by Warner and McGown (72). 

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 development of hydrodynamic techniques which allow the direct measurement of interfacial fluxes and interfacial concentrations is likely to be a key trend of future work in this area. Suitable detectors for local interfacial or near-interfacial measurements include spectroscopic probes, such as total internal reflection fluorometry [88-90], surface second-harmonic generation [91], probe beam deflection [92], and spatially resolved UV-visible absorption spectroscopy [93]. Additionally, building on the ideas in MEMED, submicrometer or nanometer scale electrodes may prove to be relatively noninvasive probes of interfacial concentrations in other hydrodynamic systems. The construction and application of electrodes of this size is now becoming more widespread and general [94-96]. 

In the mechanism of an interfacial catalysis, the structure and reactivity of the interfacial complex is very important, as well as those of the ligand itself. Recently, a powerful technique to measure the dynamic property of the interfacial complex was developed time resolved total reflection fluorometry. This technique was applied for the detection of the interfacial complex of Eu(lII), which was formed at the evanescent region of the interface when bathophenanthroline sulfate (bps) was added to the Eu(lII) with 2-thenoyl-trifuluoroacetone (Htta) extraction system [11]. The experimental observation of the double component luminescence decay profile showed the presence of dinuclear complex at the interface as illustrated in Scheme 5. The lifetime (31 /as) of the dinuclear complex was much shorter than the lifetime (98 /as) for an aqua-Eu(III) ion which has nine co-ordinating water molecules, because of a charge transfer deactivation. 

Consider one small molecule, phenylalanine. It is an essential amino acid in our diet and is important in protein synthesis (a component of protein), as well as a precursor to tyrosine and neurotransmitters. Phenylalanine is one of several amino acids that are measured in a variety of clinical methods, which include immunoassay, fluorometry, high performance liquid chromatography (HPLC see Section 4.1.2) and most recently MS/MS (see Chapter 3). Historically, screening labs utilized immunoassays or fluorimetric analysis. Diagnostic metabolic labs used the amino acid analyzer, which was a form of HPLC. Most recently, the tandem mass spectrometer has been used extensively in screening labs to analyze amino acids or in diagnostic labs as a universal detector for GC and LC techniques. Why did MS/MS replace older technological systems The answer to this question lies in the power of mass spectrometer. 

Fluorometry is a very sensitive technique - up to 1000 times more sensitive than spectrophotometry. This is because the fluorescence intensity is measured above a... 

The time of data collection depends on the complexity of the (5-pulse response. For a single exponential decay phase fluorometry is more rapid. For complex 5-pulse responses, the time of data collection is about the same for the two techniques in pulse fluorometry, a large number of photon events is necessary, and in phase fluorometry, a large number of frequencies has to be selected. It should be emphasized that the short acquisition time for phase shift and modulation ratio measurements at a given frequency is a distinct advantage in several situations, especially for lifetime-imaging spectroscopy. 

It will be seen that, as in the case of the LED, control of the bias voltage gives simple modulation of the laser output intensity. This is particularly useful in phase-modulation fluorometry. However, a measure of the late awareness of the advantages of IR techniques in fluorescence is that only recently has this approach been applied to the study of aromatic fluorophores. Thompson et al.(51) have combined modulated diode laser excitation at 670 and 791 nm with a commercial fluorimeter in order to measure the fluorescence lifetimes of some common carbocyanine dyes. Modulation frequencies up to 300 MHz were used in conjunction with a Hamamatsu R928 photomultipler for detecting the fluorescence. Figure 12.18 shows typical phase-modulation data taken from their work, the form of the frequency response curves is as shown in Figure 12.2 which describes the response to a monoexponential fluorescence decay. 

Fluorometry is a superior optical technique in terms of sensitivity and specificity. Merits of fluoroimmunoassays (FIAs) and fluoroimmuno-like assays (FILAs) include the stability and freedom from hazards of fluorescent labels compared to radioactive tracers, the moderate cost of analysis, the wide availability of the equipment needed, and the potential high sensitivity. In general, the sensitivity of fluorescence measurements is 10- to 1,000-fold higher than the absorption counterparts. 

An indication of the potential sensitivity of fluorometry is that single-molecule detection has been based almost exclusively on the use of fluorescent labeled compounds. In addition, fluorometric determinations can combine several parameters simultaneously ( multidimensional techniques), such as excitation and emission wavelengths, fluorescence lifetime, and polarization, providing additional specificity and versatility to the analytical measurements. 

Two techniques, phase and pulse fluorometry, are used for the direct measurement of fluorescence decay rates, and their principles are described by Birks and Munro (1967), Parker (1968), and Birks (1970). The photon sampling method has proved useful and versatile. This is an iterative technique in which single photons are counted as a function of the time at which they appear after excitation and a complete decay curve is built up. (For recent references see e.g. Zimmerman et al., 1973, 1974). Wider use of the photon sampling technique will increase the precision of lifetimes obtained and extend the range of compounds studied to those with shorter lifetimes or very low fluorescence yields. 

It has long been recognized that both the diffuse spectra and quenching problems can be alleviated by performing the fluorescence measurement in a low-temperature solid matrix, rather than in a fluid solution. The most common low-temperature matrices used in molecular fluorometric analysis are frozen liquid solutions the analytical characteristics of frozen-solution luminescence spectrometry have been discussed extensively in the literature (2-10). Obviously, MI represents an alternative technique to use of frozen liquid solutions for low-temperature fluorometric analysis. There are two principal advantages of MI over frozen-solution fluorometry. First, in MI, any material which has an appreciable vapor pressure at room temperature can be used as a matrix one is not limited by the... 

Several methods that do not require chemical separation are available for measuring uranium in urine (in units of total mass or total activity). These methods include spectrophotometric (total mass), fluorometric (total mass), kinetic phosphorescence analysis (KPA) (total mass), and gross alpha (total activity) analyses (Wessman 1984). The most widely used methods for routine uranium analysis are a-spectrometry and liquid scintillation spectrometry. These methods utilize the natural radioactivity of uranium and are sensitive and require little sample preparation. Photometric techniques such as fluorometry and phosphorometry are less widely used, but kinetic phosphorescence analysis is becoming more widely used. Measurements of total uranium do not provide the relative isotopic abundance of the uranium isotopes, but this may only be important when converting between activity and mass when the isotopic ratios are uncertain. 

In spite of the demonstration that at least the receptor assays using receptor from bovine lung measure very specifically /-propranolol (62), most other workers appear to have used the racemic material as a standard and to have recorded assay results in terms of racemic substance. That this procedure can give usable results is supported by the generally good correlation between the receptor assay methodology and other nonenan-tioselective techniques such as GLC or fluorometry. However, it would seem that given the enantioselective properties of the receptor system, it would behoove researchers to analyze and report /-propranolol or /-propranolol equivalents whenever possible. 

Serum magnesium has been measured by various techniques including fluorometry, flame emission spectroscopy, and atomic absorption spectrometry Today, pho-... 

The analytic methods generally fall into two groups (1) those that do not require the destruction of organic materials in the sample and (2) those that require the elimination of interfering matter before the selenium content can be measured. X-ray fluorescence and some of the neutron activation analysis techniques do not require sample destruction, whereas spectrophotometry, GC, atomic absorption spectrometry, polarography, titration, spark source, MS, fluorometry, and other neutron activation analysis techniques require some degree of sample destruction. Fluorometry, atomic absorption spectrometry, and neutron activation analysis are the most frequently used methods. 

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