Fluorometry technique

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. 

The advantages and disadvantages of the fluorometry technique in general hold true here. The fluorescence detector is not universal (it will give a peak only for fluorescing species), but it is thus very selective (almost no possibility for interference) and very sensitive. 

The absorption of light is proportional to the scalar product of the incident electric field and of a molecular vector named the transition moment. Thus, excitation of an isotropic population of fluorescent species by polarized light generally creates a temporary anisotropic population of excited molecules. Molecular motions progressively destroy this anisotropy, and affect the polarization of the reemitted fluorescence light which can be studied using pulse fluorometry techniques. 

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. 

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. 

Boto and Bunt [465] used thin-layer chromatography for the preliminary separation of chlorophylls and phaeophytins from seawater, and combined this with selective excitation fluorometry for the determination of the separated chlorophylls a, b, and c, and their corresponding phaeophytin components. An advantage of the latter technique is that appropriate selection of excitation and emission wavelengths reduces the overlap among the emission spectra of the various pigments to a greater extent than is possible with broadband excitation and the use of relatively broadband filters for emission. 

CL as an analytical tool has several advantages over other analytical techniques that involve light (mainly absorption spectroscopy and fluorometry) high detectability, high selectivity, wide dynamic range, and relatively inexpensive instrumentation. 

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 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... 

The fact that very few chemical species fluoresce works to both the advantage and disadvantage of fluorometry as a quantitative technique. Explain this. 

The basic theory, principles, sensitivity, and application of fluorescence spectrometry (fluorometry) were discussed in Chapter 8. Like the UV absorption detector described above, the HPLC fluorescence detector is based on the design and application of its parent instrument, in this case the fluorometer. You should review Section 8.5 for more information about the fundamentals of the fluorescence technique. 

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

Knowledge of the dynamics of excited states is of major importance in understanding photophysical, photochemical and photobiological processes. Two time-resolved techniques, pulse fluorometry and phase-modulation fluorometry, are commonly used to recover the lifetimes, or more generally the parameters characterizing the S-pulse response of a fluorescent sample (i.e. the response to an infinitely short pulse of light expressed as the Dirac function S). 

Pulse fluorometry uses a short exciting pulse of light and gives the d-pulse response of the sample, convoluted by the instrument response. Phase-modulation fluorometry uses modulated light at variable frequency and gives the harmonic response of the sample, which is the Fourier transform of the d-pulse response. The first technique works in the time domain, and the second in the frequency domain. Pulse fluorometry and phase-modulation fluorometry are theoretically equivalent, but the principles of the instruments are different. Each technique will now be presented and then compared. 

When the number of data points is large (i.e. in the single-photon timing technique, or in phase fluorometry when using a large number of modulation frequencies), the autocorrelation function of the residuals, defined as... 

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. 

The use of high-speed modulated excitation (f> kr + knr) combined with coherent detection methods has resulted in the popular techniques of frequency domain fluorometry, also known as phase-modulation fluorometry. These techniques can be used to determine the temporal characteristics of both fluorescence and phosphorescence and will also be addressed later in this chapter. 

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. 

It was known that the intracellular concentrations of the reduced and oxidized forms of the pyridine nucleotides vary in different cell types and under different cell culture conditions.(17) Harrison and Chance applied the NAD(P)H fluorescence technique and found that culture fluorescence can be related to the metabolic state of the cells. 18,19) Since then, more than a hundred papers on NAD(P)H fluorometry have been published. However, they are primarily divided into three major categories ... 

The mathematical basis for the exponential series method is Eq. (5.3), the use of which has recently been criticized by Phillips and Lyke.(19) Based on their analysis of the one-sided Laplace transform of model excited-state distribution functions, it is concluded that a small, finite series of decay constants cannot be used to represent a continuous distribution. Livesey and Brouchon(20) described a method of analysis using pulse fluorometry which determines a distribution using a maximum entropy method. Similarly to Phillips and Lyke, they viewed the determination of the distribution function as a problem related to the inversion of the Laplace transform of the distribution function convoluted with the excitation pulse. Since Laplace transform inversion is very sensitive to errors in experimental data,(21) physically and nonphysically realistic distributions can result from the same data. The latter technique provides for the exclusion of nonrealistic trial solutions and the determination of a physically realistic solution. These authors noted that this technique should be easily extendable to data from phase-modulation fluorometry. 

Two example-determinations of TTX using fluorometry coupled with high-performance liquid chromatography (HPLC) are introduced here, along with some other promising assay techniques such as mass spectrometry. 

---