Fluorometry

Because fluorometry is subject to many interferences, its main application to surfactant analysis has been use as an HPLC detector for quantification of APE or LAS. There has been some use as a stand-alone technique. 

LAS has relatively weak fluorescence compared to multi-ring compounds, so that there is no advantage to using fluorescence rather than UV absorbance in terms of sensitivity. However, fluorescence may be used for increased selectivity. 

Direct fluorescence measurement has been used to determine the approximate level of lignin sulfonate in natural waters polluted by paper mill waste. In this case, the choice of excitation and emission wavelengths is made to minimize interference from naturally occurring fluorescent compounds, such as humic acids (119,120). 

Anionic surfactants can be determined at low concentrations by extraction of the ion pair with a cationic fluorescent molecule, such as Rhodamine B or Safranine-T (121-123). This method is entirely analogous to the ion-pair extractions, such as the methylene blue method, discussed in Chapter 12. The fluorescent method has no greater selectivity, but is somewhat more sensitive. Because of fluorescence quenching effects, this approach can be 

If certain anionic fluorescent molecules are immobilized in a matrix such as cross-linked polyvinylalcohol or a modified glass surface, they show an increase in fluorescent intensity and a decrease in emission wavelength in the presence of a cationic surfactant. Although not suitable for trace analysis, this phenomenon is proportional to concentration over a range of about 30, and is insensitive to anionic and nonionic surfactants (126). 

Fluorescence occurs when a molecule absorbs light at one wavelength and reemits light at a longer wavelength. An atom or molecule that fluoresces is termed a fluorophore. Fluorometry is defined as the measurement of the emitted fluorescence Hglit. Fluorometric analysis is a widely used method of quantitative analysis in the chemical and biological sciences it is accurate and very sensitive. 

Obviously, it is advantageous to make the measurement on an absorption peak whenever possible, in order to minimize this curvature, as well as to obtain maximum sensitivity. Because a band of wavelengths is passed, the absorptivity at a given wavelength may vary somewhat from one instrument to another, depending on the resolution, slit width, and sharpness of the absorption maximum. Therefore, you should check the absorptivity and linearity on your instrument rather than relying on reported absorptivities. It is common practice to prepare calibration curves of absorbance versus concentration rather than to rely on direct calculations of concentration from Beer s law. 

If there is a second (interfering) absorbing species whose spectrum overlaps with that of the test substance, nonlinearity of the total absorbance as a function of the test substance concentration will result. It may be possible to account for this in preparation of the calibration curve by adding the interfering compound to standards at the same concentration as in the samples. This will obviously work only if tbe concentration of the interfering compound is essentially constant, and the concentration should be relatively small. Otherwise, simultaneous analysis as described earlier will be required. 

Other chemical and instrumental sources of nonlinearity in absorbance measurements may include hydrogen bonding, interaction with the solvent, nonlinear detector, nonlinear electronics, noncollimated radiation, and high signal levels (saturation). 

Nonuniform cell thickness can affect a quantitative analysis. This is potentially a problem, especially in infrared spectrometry, where cell spacers are used. Air bubbles can affect the pathlength and stray tight, and it is important to eliminate these bubbles, again especially in the infrared cells. 

The absoiptivity at a given wavelength may vary from instrument to instrument. Therefore, always run a standard. 

FIGURE 8.11 An energy level diagram depicting the phenomenon of fluorescence in a molecule or complex ion. 

When the concentration of a fluorescing analyte is small so as to result in a small absorbance value, the intensity of the resulting fluorescence is proportional to concentration and is therefore measured for quantitative analysis. Thus the usual procedure for quantitative analysis consists of the measurement of a series of standard solutions of the fluorescing analyte or other species proportional to the analyte. A graph of fluorescence intensity vs. concentration is expected to be linear in the concentration range studied. 

Fluorometry and absorption spectrophotometry are competing techniques in the sense that both analyze for molecular species and complex ions. Each offers its own advantages and disadvantages. As stated above, the number of chemical species that exhibit fluorescence is very limited. However, for those species that do fluoresce, the fluorescence is generally very intense. Thus we can say that while absorption spectrophotometry is much more universally applicable, fluorometry suffers less from interferences and 

FIGURE 8.14 The IR spectrum of toluene. Note that the x-axis is the wavenumber. Also note the sharpness of the 

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Organic Analytes As noted earlier, organic compounds containing aromatic rings generally are fluorescent, but aromatic heterocycles are often phosphorescent. Many important biochemical, pharmaceutical, and environmental compounds are aromatic and, therefore, can be analyzed quantitatively by fluorometry... 

Fluorometry and Phosphorimetry. Modem spectrofluorometers can record both fluorescence and excitation spectra. Excitation is furnished by a broad-band xenon arc lamp foUowed by a grating monochromator. The selected excitation frequency, is focused on the sample the emission is coUected at usuaUy 90° from the probe beam and passed through a second monochromator to a photomultiplier detector. Scan control of both monochromators yields either the fluorescence spectmm, ie, emission intensity as a function of wavelength X for a fixed X, or the excitation spectmm, ie, emission intensity at a fixed X as a function of X. Fluorescence and phosphorescence can be distinguished from the temporal decay of the emission. 

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). 

PL is often referred to as fluorescence spectrometry or fluorometry, especially when applied to molecular systems. Uses for PL are found in many fields, including... 

In 1944, Lewis and Kasha (52) identified phosphorescence as a forbidden" transition from an excited triplet state to the ground singlet state and suggested the use of phosphorescence spectra to identify molecules. Since then, phosphorimetry has developed into a popular method of analysis that, when compared with fluorometry, is more sensitive for some organic molecules and often provides complimentary information about structure, reactivity, and environmental conditions (53). 

Figure 5B. Correlation of right-angle light scatter measured by fluorometry and flow cytometry. The top panel shows flow-cytometric data of side scatter of fixed, stained cells during the time course of stimulation by 1-nM (solid line, solid circles) or 0.01-nH (dashed line, open circle) FLPEP. The bottom panel shows the corresponding right-angle light-scatter data acquired pseudo-simultaneously on live cells in the fluorometer. The flow-cytometric data have been averaged, but the fluorometry data are plotted for both duplicates from one donor. Reproduced with permission from Ref. 27. Copyright 1985 Rockefeller University Press.

Rabbit peritoneal neutrophils were harvested and their release of p-glucuronidase was measured at 37°C, as described previously (13). For indo-1, neutrophils were washed twice in a calcium-free buffer, then loaded with 15 indo-1 acetoxymethyl ester (24) for 40 min at 37 C at a density of 5 x 10 cells/ml. The cells were then washed twice more in calcium-free buffer, resuspended to a density of 1 X 10 cells/mL, and kept on ice. Prior to fluorometry, cells were diluted 4x with the appropriate buffer at 37 C. For CTC, neutrophils were incubated with 20 pH CTC at 37°C in the spectrofluorometer cuvette. All measurements were carried out using an SLH-Aminco SPF 500C fluorospectrometer interfaced with an IBM PC microcomputer. 

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 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. 

Tan, Y.A., Low, K.S., and Chong, C.L., Rapid determination of chlorophylls in vegetable oils by laser-based fluorometry, J. Sci. Food Agric., 66, 479, 1994. Bhattacharya, D. and Medlin, L., Algal phylogeny and the origin of land plants, Plant Physiol., 116, 9, 1998. 

In situ densitometry has been the most preferred method for quantitative analysis of substances. The important applications of densitometry in inorganic PLC include the determination of boron in water and soil samples [38], N03 and FefCNfg in molasses [56], Se in food and biological samples [28,30], rare earths in lanthanum, glass, and monazite sand [22], Mg in aluminum alloys [57], metallic complexes in ground water and electroplating waste water [58], and the bromate ion in bread [59]. TLC in combination with in situ fluorometry has been used for the isolation and determination of zirconium in bauxite and almnimun alloys [34]. The chromatographic system was silica gel as the stationary phase and butanol + methanol + HCl -H water -n HF (30 15 30 10 7) as the mobile phase. 

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]. 

A high specific interfacial area and a direct spectroscopic observation of the interface were attained by the centrifugal liquid membrane (CLM) method shown in Fig. 2. A two-phase system of about 100/rL in each volume is introduced into a cylindrical glass cell with a diameter of 19 mm. The cell is rotated at a speed of 5000-10,000 rpm. By this procedure, a two-phase liquid membrane with a thickness of 50-100 fim. is produced inside the cell wall which attains the specific interfacial area over 100 cm. UV/VIS spectrometry, spectro-fluorometry, and other spectroscopic methods can be used for the measurement of the interfacial species and its concentration as well as those in the thin bulk phases. This is an excellent method for determining interfacial reaction rates on the order of seconds. 

By the total internal reflection condition at the liquid-liquid interface, one can observe interfacial reaction in the evanescent layer, a very thin layer of a ca. 100 nm thickness. Fluorometry is an effective method for a sensitive detection of interfacial species and their dynamics [10]. Time-resolved laser spectrofluorometry is a powerful tool for the elucidation of rapid dynamic phenomena at the interface [11]. Time-resolved total reflection fluorometry can be used for the evaluation of rotational relaxation time and the viscosity of the interface [12]. Laser excitation can produce excited states of adsorbed compound. Thus, the triplet-triplet absorption of interfacial species was observed at the interface [13]. 

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. 

The effect of PCP-like drugs was also studied in vivo. In these experiments, the ability of these drugs to enhance haloperidol-induced DA metabolism was assessed by measuring DA and homovanil-11c acid (HVA) concentrations in the striatum. In this paradigm, saline or drug was administered (SC) immediately after the administration of 0.1. mg/kg ha 1 operido 1. The rats were killed 45 minutes later, and DA and HVA levels were estimated fluorometri-tal ly (Snel 1 et al. 1984). 

Delori FC (2004), Autofluorescence method to measure macular pigment optical densities Fluorometry and autofluorescence imaging, Arch. Biochem. Biophys. 430 156-162. 

Szmacinski, H. Lacowicz, J. R. Lifetime-based Sensing Using Phase-Modulation Fluorometry. In Fluorescent Chemosensor for Ion and Molecule Recognition. ACS Symposium Series 538, 1993. 

Beechem, J. M., Knutson, J. R., Ross, B. A., Turner, B. W. and Brand, L. (1983). Global resolution of heterogeneous decay by phase/modulation fluorometry Mixtures and proteins. Biochemistry 22, 6054-8. 

Karvinen, J., Laitala, V., Makinen, M. L., Mulari, O., Tamminen, J., Hermonen, J., Hurskainen, P. and Hemmila, I. (2004). Fluorescence quenching-based assays for hydrolyzing enzymes. Application of time-resolved fluorometry in assays for caspase, helicase, and phosphatase. Anal. Chem. 76, 1429-1436. 

Diamandis, E.P. (1993) Time-resolved fluorometry in nucleic acid hybridization and Western blotting techniques (Review). Electrophoresis 14, 866-875. 

Nithipatikom, K., and McGown, L.B. (1987) Homogeneous immunochemical technique for determination of human lactoferrin using excitation tranfer and phase-resolved fluorometry. Anal. Chem. 59, 423. 

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