Excitation-emission fluorescence measurements

Trilinearity in the underlying model can be assumed, e.g., when the output from a chromatograph is subjected to excitation-emission fluorescence measurement in the low-concentration range. In this case, the underlying model is often low-rank trilinear, the data produced for one sample are trilinear and a PARAFAC model separates the data uniquely into unit chromatograms, unit excitation spectra and unit emission spectra as A-, B- and C-loadings. 

The physical basis of spectroscopy is the interaction of light with matter. The main types of interaction of electromagnetic radiation with matter are absorption, reflection, excitation-emission (fluorescence, phosphorescence, luminescence), scattering, diffraction, and photochemical reaction (absorbance and bond breaking). Radiation damage may occur. Traditionally, spectroscopy is the measurement of light intensity... 

Emissions from both the and the previously unreported lli states of the IF molecule have been observed in the gas-phase reaction of L with F2 at low pressure a four-centre complex has been proposed as the reaction intermediate. A combined theoretical-experimental programme has been conducted to establish techniques for the study of excited-state transitions in Ij and IC1. Experimental techniques based on two-step excitation using two synchronized, tunable lasers have been developed, and successfully applied to excited-state fluorescence measurements on ICl. lodine(i) chloride adsorbed on silica gives the same Raman spectrum as that obtained from adsorbed l2. ... 

Raman scatter, and excitation emission fluorescence spectroscopy (EEFS). They use interaction with radiation from different regions of the electromagnetic spectrum to identify the chemical nature of molecules. For example, absorption of UV and VIS radiation causes valence electron transitions in molecules which can be used to measure species down to parts per million concentrations for fluorophores (i.e., EEFS) determination can even go down to parts per billion levels. Whereas UV, VIS, and EEFS are limited to a smaller, select group of molecules, the NIR, IR, and Raman scatter spectroscopy techniques are probing molecular vibrations present in almost any species their quantification limits are somewhat higher but can still be impressive. The reader is referred to textbooks for further details on basic principles of these spectroscopic techniques [3]. 

Finally, an example should be provided about a class of methods, which have also explorative purposes, which will be discussed with more detail and theoretical depth in Chapter 7, that is multiway analysis methods [81]. These methods, among which parallel factor analysis (PARAFAC) will be shown here in action compared to PCA, are to some extent referred to as the conceptual (and mathematical) extension of PCA to arrays of order higher than two. They show their potentiality when the variability of a data set is related to different sources, or conditions, at which a full set of properties for each sample is measured [17-21]. An example, quite common in the food science analysis, is the excitation-emission fluorescence landscape, where, for each sample, an emission spectrum is recorded at each wavelength of the excitation signal. 

As we will see further in this chapter, knowing the structure of the data plays a fundamental role when applying any multiway technique. For illustrating this, we will comment on the data collected from the two most popular instrumentations used in food sciences nowadays that are able to produce multiway data Excitation-emission fluorescence spectroscopy (EEM) and hyphenated chromatographic systems (i.e. gas chromatography connected to mass spectrometry—GC-MS). The benefit and drawbacks of both techniques in the framework of food analysis will be discussed in successive chapters. Here we will just focus on the stmcture of the three-way array. Figure 1 shows the final three-way structure that is obtained when several samples are analysed by both EEM and hyphenated chromatography. However, the inner structure of this tensor varies due to the different nature of the measurement... 

Better detection limits are obtained using fluorescence, particularly when using a laser as an excitation source. When using fluorescence detection, a small portion of the capillary s protective coating is removed and the laser beam is focused on the inner portion of the capillary tubing. Emission is measured at an angle of 90° to the laser. Because the laser provides an intense source of radiation that can be focused to a narrow spot, detection limits are as low as 10 M. 

Fluorescence. The fluorescence detection technique is often used in clinical chemistry analyzers for analyte concentrations that are too low for the simpler absorbance method to be appHed. Fluorescence measurements can be categorized into steady-state and dynamic techniques. Included in the former are the conventional simultaneous excitation-emission method and fluorescence polarization. 

Fig. 6.2.5 Fluorescence spectra of pholasin after treatment with 5M guanidine hydrochloride. Left, excitation spectrum measured at 460 nm right, emission spectrum measured with excitation at 360 nm. From Henry et al., 1973, with permission from Elsevier.

Any factor that affects the size or shape of a molecule, the hindered movement of a fluorophore within a molecule, or the energy transfer within the molecule will affect the measured depolarization of its fluorescence emission. Therefore, the conformation of humic fractions in solution can be studied as a function of pH, ionic strength, temperature, and other factors by depolarization measurements. The principle of the method is that excitation of fluorescent samples with polarized light stimulates... 

In situ quantitation For fluorimetric evaluation there was excitation at = 313 nm and the fluorescence emission was measured at = 365 nm (monochromatic filter M 365). This arrangement yielded the most intense signals. (The emission beam at X, = 365 nm is appreciably more intense than the visible yellow fluorescence.) Further treatment of the chromatogram with liquid paraffin - -hexane (1+2) is not to be recommended. 

In situ quantitation Fluorimetric evaluation was carried out with excitation at exc = 365 nm and the fluorescence emission was measured at > 400 nm (cut off filter K 400). 

The fluorescence depolarization technique excites a fluorescent dye by linearly polarized light and measures the polarization anisotropy of the fluorescence emission. The fluorescence anisotropy, r, is defined as... 

The simplest fluorescence measurement is that of intensity of emission, and most on-line detectors are restricted to this capability. Fluorescence, however, has been used to measure a number of molecular properties. Shifts in the fluorescence spectrum may indicate changes in the hydrophobicity of the fluorophore environment. The lifetime of a fluorescent state is often related to the mobility of the fluorophore. If a polarized light source is used, the emitted light may retain some degree of polarization. If the molecular rotation is far faster than the lifetime of the excited state, all polarization will be lost. If rotation is slow, however, some polarization may be retained. The polarization can be related to the rate of macromolecular tumbling, which, in turn, is related to the molecular size. Time-resolved and polarized fluorescence detectors require special excitation systems and highly sensitive detection systems and have not been commonly adapted for on-line use. 

Square-650-pH having a pKa in the physiological pH range (pKa = 7.1 for free dye and the pKa 6.1 when labeled to an antibody) was recently introduced by SETA BioMedicals [119]. This dye is commercially available as a free carboxylic acid and a mono-NHS ester. Square-650-pH has spectral properties similar to those of the CypHer dyes but is fluorescent in both the protonated and deproto-nated forms. This dye displays reasonable molar absorptivities (135,000 and 48,000 M-1cm-1) and quantum yields (16% and 9%) for the protonated and deprotonated forms, an extremely large Stokes shift of more than 100 nm for the deprotonated form, and enables excitation and emission ratiometric measurement... 

Fluorescence measurements are carried out in 50 mM Hepes/KOH (pH 7.2), 100 mMKCl, 0.5 mMEDTA, and 2 mMDTT at 20°. To 0.1 or 0.05 /iM mouse eIF4E(28-217) (i.e., the first 27 amino acid residues are truncated) (Niedzwiecka et al., 2002) are added solutions of increasing cap analog concentrations. For this protein—ligand association, an excitation wavelength of 280 nm and an emission wavelength of 337 nm are used. 

More fluorescence features than just the emission intensity can be used to develop luminescent optosensors with enhanced selectivity and longer operational lifetime. The wavelength dependence of the luminescence (emission spectmm) and of the luminophore absorption (excitation spectrum) is a source of specificity. For instance, the excitation-emission matrix has shown to be a powerful tool to analyze complex mixtures of fluorescent species and fiber-optic devices for in-situ measurements (e.g. 

Figure 9.2 Typical spectral scan of a fluorescent compound showing its absorbance peak or wavelengths of most efficient excitation and its emission peak or wavelengths where light emission occurs. The Stoke s shift is the distance in nanometers between the absorbance peak and the emission peak. The larger the Stoke s shift, the less interference that will occur from excitation light when measuring fluorescence emission.

Fluorescence Measurement Fluorescence spectra were measured on a Spex Fluorolog 212 spectrofluorometer equipped with a 450 W xenon arc lamp and a Spex DM1B data acquisition station. Spectra were recorded in the front-face illumination mode using 343 nm as the excitation wavelength. Single scans were performed using a slit width of 1.0 mm. PDA fluorescence emission spectra were recorded from 360 to 600 nm, with the monomer and excimer fluorescence measured at 376.5 and 485 nm, respectively. Monomer and excimer peak heights were used in the calculation of the ratio of excimer to monomer emission intensities (Ie/Im). Excitation spectra were recorded from 300 nm to 360 nm and monitored at 376.5 and 500 nm for the monomer and excimer excitation, respectively. 

The OPA reaction proceeds rapidly at room temperature and the post-column fluidics are simplified by replacing the existing reaction module and photometer with a fluorescence detector. Excitation occurs at 330nm, and emission is measured at a wavelength of 430nm or above. 

The film reacted with adipoyl chloride followed by coupling with 7-hydroxycoumarin was subjected to methanolysis at 1 N HC1 and 60°C. The regenerated coumarin was assayed at pH 10 by fluorescence spectroscopy at an excitation wavelength of 329 nm and an emission wavelength of 455 nm. A Hitachi MPF-4 Fluorescence Spectrophotometer was used for all fluorescence measurements. 

Hg(II), it is also sufficiendy protonated to display some background fluorescence (Figure 3.7). Excitation was at 335 3 nm, emission was measured at the emission maximum centered near 416 nm. (Reproduced from Ref. 6. Copyright 1990 American Chemical Society.)... 

Native fluorescence of a protein is due largely to the presence of the aromatic amino acids tryptophan and tyrosine. Tryptophan has an excitation maximum at 280 nm and emits at 340 to 350 nm. The amino acid composition of the target protein is one factor that determines if the direct measurement of a protein s native fluorescence is feasible. Another consideration is the protein s conformation, which directly affects its fluorescence spectrum. As the protein changes conformation, the emission maximum shifts to another wavelength. Thus, native fluorescence may be used to monitor protein unfolding or interactions. The conformation-dependent nature of native fluorescence results in measurements specific for the protein in a buffer system or pH. Consequently, protein denatur-ation may be used to generate more reproducible fluorescence measurements. 

Fluorescence measurements of the compoimd were measirred freshly, after an incubation of one day and two months in various solvents such as 1.5 x lO M, 1.5 X lO" M and 1.5 x 10 M of MeOH, CHCI, THF, DMF and DMSO at room temperature. In the experiments, all the sample solutiorrs were excited at 337 nm. As shown in Figs. 49.7-49.9, when the fluorescence spectra of the compound was irrvestigated, the same peculiarities were observed as mentioned above. Optimrrm fluorescence yield was obtained in 1.5 x 10M of MeOH, therefore, we decided to measure fluorescence in it (Fig. 49.9). Maximum fluorescence emission intensity was observed at 398 rrm in the fluorescence spectra. The results showed that the fluorescence intensities increased by time. These observatiorrs are compatible with the other results obtained from UVATS spectrophotometer to clarify the behaviour of the molecule in solution state. 

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