Fluorescence method

Note that in liquid phase chromatography there are no detectors that are both sensitive and universal, that is, which respond linearly to solute concentration regardless of its chemical nature. In fact, the refractometer detects all solutes but it is not very sensitive its response depends evidently on the difference in refractive indices between solvent and solute whereas absorption and UV fluorescence methods respond only to aromatics, an advantage in numerous applications. Unfortunately, their coefficient of response (in ultraviolet, absorptivity is the term used) is highly variable among individual components. 

Zare R N and Dagdigian P J 1974 Tunable laser fluorescence method for product state analysis Science 185 739-46... 

Accuracy The accuracy of a fluorescence method is generally 1-5% when spectral and chemical interferences are insignificant. Accuracy is limited by the same types of problems affecting other spectroscopic methods. In addition, accuracy is affected by interferences influencing the fluorescent quantum yield. The accuracy of phosphorescence is somewhat greater than that for fluorescence. 

Chemical Properties. Elemental analysis, impurity content, and stoichiometry are determined by chemical or iastmmental analysis. The use of iastmmental analytical methods (qv) is increasing because these ate usually faster, can be automated, and can be used to determine very small concentrations of elements (see Trace AND RESIDUE ANALYSIS). Atomic absorption spectroscopy and x-ray fluorescence methods are the most useful iastmmental techniques ia determining chemical compositions of inorganic pigments. Chemical analysis of principal components is carried out to determine pigment stoichiometry. Analysis of trace elements is important. The presence of undesirable elements, such as heavy metals, even in small amounts, can make the pigment unusable for environmental reasons. 

In this work the results of reseai ch common sorbtion-X-Ray-fluorescence analysis of Pb(II), Cd(II), Zn(II) and Mo(VI) with preconcentration on complexing chemical silica gel modified with mercaptane groups and modified with 8-hydroxyquinoline were described. The conditions and limits of determination of the X-Ray-fluorescence method in the thin lawyers ai e discussion. 

It was shown that X-Ray-fluorescence method do possible to separate metals in the multycomponents samples by different methods of synthesis chemical modified silica and different ways of coordination of ion metals on the surface. 

Okabe H., P. L. Splitstone, and J. J., Ball. Ambient and Source SOt Detector Based on, i Fluorescence Method. Air Pollution Control Assoc. 23 (1973), pp. 514-516. 

More sensitive detection methods and more objective recording methods (e g the employment of scanners) are constantly been striven for m order to overcome this illusion It IS for this reason too that fluorescent methods have been introduced to an increasing extent on account of their higher detection sensitivity This allows an appreciable reduction in the amount of sample applied, so that possible interfering substances are also present m smaller quantibes This increases the quality of the chromatographic separation and the subsequent m situ analysis... 

It is instructive to compare the sensitivity which may be achieved by absorption and fluorescence methods. The overall precision with which absorbance can be measured is certainly not better than 0.001 units using a 1 cm cell. Since for most molecules the value of emax is rarely greater than 105, then on the basis of the Beer-Lambert Law the minimum detectable concentration is given by cmin> 10 3/105= 10 8M. 

Fluorescence Methods for the Analysis of Nucleic Acids in Recombinant Biological Products... 

The theory and application of this fluorescence method have been discussed in detail by LePecq and others (3,8). The assay requires that there is sufficient ionic strength to minimize ionic binding (e.g., O.IM sodium chloride), that the pH is 4-10, that no heavy metals are present, that the fluorescence is not enhanced on binding to other excipients (e.g., proteins) and that at least portions of the nucleic acids are not complexed. These requirements can usually he met when dealing with recombinant products in some cases the samples must he manipulated to create the appropriate conditions. In the intercalative method of dye binding, proteins rarely interfere with the assay, and procedures have been developed to remove the few interferences they may cause (e.g., the use of heparin or enzymatic digestion of the protein 9). 

The sensitivity of the fluorescence methods varies considerably with the instrument used. Advances in modern instrumentation and the power of today s computers allow for a much improved sensitivity. Using commercially available instruments and modern computers equipped with appropriate software, detection limits down to 10 pg of calf thymus DNA can be achieved using ethidium bromide. (We have achieved such levels using several Perkin-Elmer MPF66 Instruments at various locations.)... 

In current practice the fluorescence assay is often followed by the use of hybridization techniques when more selectivity is required. We have for instance used the fluorescence techniques to obtain data on the nucleic acid content of malaria vaccine proteins produced in Escherichia coli. The rapid turnaround time of the fluorescence assay is particularly useful during the early stages of purification to determine the optimal process conditions. After the final process has been arrived at and a variety of methods used to assess the nucleic acid content (including the hybridization techniques), the fluorescence method can be developed for routine quality-control purposes. In certain cases, particularly at high protein concentrations, the dye may bind to the protein with... 

In order to further extend the utility of fluorescence methods the use of time-resolution methods, fluorescence polarization, and laser techniques should be explored. The addition of other dyes with enhanced fluorescence properties on binding and increased selectivity to various types of nucleic acids will be necessary to further develop more useful analytical methods. 

Applications of the oxalate-hydrogen peroxide chemiluminescence-based and fluorescence-based assays with NDA/CN derivatives to the analysis of amino acids and peptides are included. The sensitivity of the chemiluminescence and fluorescence methods is compared for several analytes. In general, peroxyoxalate chemiluminescence-based methods are 10 to 100 times more sensitive than their fluorescence-based counterparts. The chief limitation of chemiluminescence is that chemical excitation of the fluorophore apparently depends on its structure and oxidation potential. 

Particular attention has been devoted to the fluorescence methods, which are now of such topicality, and to methods of increasing and stabilizing the fluorescence emissions. Nowhere else in the literature is there so much detailed information to be found as in the first part of this book, whose more than 600 literature references may serve to stimulate the reader to enlarge his or her own knowledge. 

Franco, C. M., Fente, C. A., Vazquez, B., Cepeda, A., Lallaoui, L., Prognon, P., and Mahuzier, G., Simple and sensitive high-performance liquid chromatography-fluorescence method for the determination of citrinin. Application to the analysis of fungal cultures and cheese extracts, /. Chromatogr. A, 723, 69, 1996. 

Fluorescence method This method for nonconducting liquids uses fluorescein dye. The blue light activates fluorescence, and the green light, which has intensity directly proportional to the film thickness (Hewitt, 1969-1970), is emitted. 

This method is perfectly suitable for low concentrations of fluorescent materials. However, in order to study factors which affect the fluorescence quantum yield, such as molecular association or photochemical reactions, much higher concentrations than can be used in the right-angle fluorescence method are required. This follows from the fact that the 0 - 0 vibrational bands in the absorption and emission spectra often overlap. Therefore at relatively high concentrations light emitted at these overlapping wavelengths will be reabsorbed. 

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