Quantitative Fluorescence Spectroscopy

Zheng, J. (2006). Spectroscopy-based quantitative fluorescence resonance energy transfer analysis. Methods Mol. Biol. 337, 65-77. 

The iron content of the produced samples was determined by X-ray fluorescence spectroscopy. Quantitative analysis was performed with the help of analytictJ standard and the results are listed in the second column of Table 1. 

Wang, Y.-L, and Taylor, D.L. (1989b). Fluorescence Microscopy of Living Cells in Culture. Part B Quantitative Fluorescence Microscopy- Imaging and Spectroscopy. Academic Press, New York. 

Fluorescence Microscopy of Living Cells in Culture, Part B Quantitative Fluorescence Microscopy—Imaging and Spectroscopy... 

Durkin AJ (1995) Quantitative fluorescence spectroscopy of turbid samples, PhD Dissertation, University of Texas at Austin. 

Wang YL and Taylor LD (1989) Methods in Cell Biology part A Fluorescent analogs, labeling cells and basic microscopy. 29 and part B quantitative fluorescence microscopy imaging and spectroscopy 30 Fluorescence microscopy of living cells in culture. Part A and B, (Eds), Academic Press, Inc., San Diego, New York, Berkely, 498 pp. 

Fries J R, Brand L, Eggeling C, Kdllner M and Seidel CAM 1998 Quantitative identification of different single molecules by selective time-resolved confocal fluorescence spectroscopy J. Phys. Chem. A 102 6602-13... 

Quantitative aluminum deterrninations in aluminum and aluminum base alloys is rarely done. The aluminum content is generally inferred as the balance after determining alloying additions and tramp elements. When aluminum is present as an alloying component in alternative alloy systems it is commonly deterrnined by some form of spectroscopy (qv) spark source emission, x-ray fluorescence, plasma emission (both inductively coupled and d-c plasmas), or atomic absorption using a nitrous oxide acetylene flame. 

In addition to the spark emission methods, quantitative analysis directly on soHds can be accompHshed using x-ray fluorescence, or, after sample dissolution, accurate analyses can be made using plasma emission or atomic absorption spectroscopy (37). 

Measuring Protein Sta.bihty, Protein stabihty is usually measured quantitatively as the difference in free energy between the folded and unfolded states of the protein. These states are most commonly measured using spectroscopic techniques, such as circular dichroic spectroscopy, fluorescence (generally tryptophan fluorescence) spectroscopy, nmr spectroscopy, and absorbance spectroscopy (10). For most monomeric proteins, the two-state model of protein folding can be invoked. This model states that under equihbrium conditions, the vast majority of the protein molecules in a solution exist in either the folded (native) or unfolded (denatured) state. Any kinetic intermediates that might exist on the pathway between folded and unfolded states do not accumulate to any significant extent under equihbrium conditions (39). In other words, under any set of solution conditions, at equihbrium the entire population of protein molecules can be accounted for by the mole fraction of denatured protein, and the mole fraction of native protein,, ie. 

Zirconium is often deterniined gravimetrically. The most common procedure utilizes mandelic acid (81) which is fairly specific for zirconium plus hafnium. Other precipitants, including nine inorganic and 42 organic reagents, are Hsted in Reference 82. Volumetric procedures for zirconium, which also include hafnium as zirconium, are limited to either EDTA titrations (83) or indirect procedures (84). X-ray fluorescence spectroscopy gives quantitative results for zirconium, without including hafnium, for concentrations from 0.1 to 50% (85). Atomic absorption determines zirconium in aluminum in the presence of hafnium at concentrations of 0.1—3% (86). 

X-Ray Fluorescence analysis (XRF) is a well-established instrumental technique for quantitative analysis of the composition of solids. It is basically a bulk evaluation method, its analytical depth being determined by the penetration depth of the impinging X-ray radiation and the escape depth of the characteristic fluorescence quanta. Sensitivities in the ppma range are obtained, and the analysis of the emitted radiation is mosdy performed using crystal spectrometers, i.e., by wavelength-dispersive spectroscopy. XRF is applied to a wide range of materials, among them metals, alloys, minerals, and ceramics. 

The experimental principle is illustrated in Fig. 3. The interaction of the polymer with the liposomal membranes causes the perturbation of the bilayer. This perturbation follows the leakage of calcein from the liposome. Calcein in high concentration in the liposome is self-quenched, but has strong fluorescence intensity by the leak from the liposome. Therefore, the extent of the membrane interaction can be estimated quantitatively from the fluorescence spectroscopy. 

Csilibrsition. Quantitative binding analysis requires knowing the concentration of FLPEP, which can be determined for a stock solution of FLPEP by absorption spectroscopy. The quenching by the antibody is essentially quantitative, and the relative amounts of free and bound ligand are calculated from the relative fluorescence intensity. 

Fluorescence intensity detected with a confocal microscope for the small area of diluted solution temporally fluctuates in sync with (i) motions of solute molecules going in/out of the confocal volume, (ii) intersystem crossing in the solute, and (hi) quenching by molecular interactions. The degree of fluctuation is also dependent on the number of dye molecules in the confocal area (concentration) with an increase in the concentration of the dye, the degree of fluctuation decreases. The autocorrelation function (ACF) of the time profile of the fluorescence fluctuation provides quantitative information on the dynamics of molecules. This method of measurement is well known as fluorescence correlation spectroscopy (FCS) [8, 9]. 

It is of interest to examine the development of the analytical toolbox for rubber deformulation over the last two decades and the role of emerging technologies (Table 2.9). Bayer technology (1981) for the qualitative and quantitative analysis of rubbers and elastomers consisted of a multitechnique approach comprising extraction (Soxhlet, DIN 53 553), wet chemistry (colour reactions, photometry), electrochemistry (polarography, conductometry), various forms of chromatography (PC, GC, off-line PyGC, TLC), spectroscopy (UV, IR, off-line PylR), and microscopy (OM, SEM, TEM, fluorescence) [10]. Reported applications concerned the identification of plasticisers, fatty acids, stabilisers, antioxidants, vulcanisation accelerators, free/total/bound sulfur, minerals and CB. Monsanto (1983) used direct-probe MS for in situ quantitative analysis of additives and rubber and made use of 31P NMR [69]. 

Stabilisers are usually determined by a time-consuming extraction from the polymer, followed by an IR or UV spectrophotometric measurement on the extract. Most stabilisers are complex aromatic compounds which exhibit intense UV absorption and therefore should show luminescence in many cases. The fluorescence emission spectra of Irgafos 168 and its phosphate degradation product, recorded in hexane at an excitation wavelength of 270 nm, are not spectrally distinct. However, the fluorescence quantum yield of the phosphate greatly exceeds that of the phosphite and this difference may enable quantitation of the phosphate concentration [150]. The application of emission spectroscopy to additive analysis was illustrated for Nonox Cl (/V./V -di-/i-naphthyl-p-phcnylene-diamine) [149] with fluorescence ex/em peaks at 392/490 nm and phosphorescence ex/em at 382/516 nm. Parker and Barnes [151] have reported the use of fluorescence for the determination of V-phenyl-l-naphthylamine and N-phenyl-2-naphthylamine in extracted vulcanised rubber. While pine tar and other additives in the rubber seriously interfered with the absorption spectrophotometric method this was not the case with the fluoromet-ric method. 

Somsen et al. [796] have reported the use of SERR spectroscopy for the in situ selective determination and semi-quantitative analysis of structurally similar dyes separated by TLC. The limits of identification of the TLC-SERRS method (ca. 5ng applied) were sufficient for acquisition of spectra of impurities present in the certified dye standards. SERRS may also be used for in situ identification of highly fluorescent molecules on HPTLC plates. 

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