Spectrofluorometer types

For the determination of supplemental vitamin E in infant formulas, Woollard and Blott (222) employed a radially compressed Radial-PAK cartridge. This enabled lipid material to be rapidly cleared by stepping up the mobile-phase flow rate from 2 ml/min to 10 ml/min after elution of the a-tocopheryl acetate. Fluorescence detection, using a filter-type fluorometer, allowed the indigenous a-tocopherol to be conveniently estimated, while UV absorbance detection was used to quantify the a-tocopheryl acetate. Supplemental retinyl acetate could be assayed simultaneously with either added or indigenous vitamin E using the appropriate detection mode. With the aid of a dual-monochromator spectrofluorometer, a-tocopheryl acetate and a-tocopherol could be determined simultaneously with wavelengths of 280 nm (excitation) and 335 nm (emission), but the increased selectivity eliminated detection of the vitamin A esters (233). 

In addition, to the basic spectrofluorometer discussed earlier (see Figure 3-17), other types of fluorometric instruments include a ratio-referencing spectrofluorometer, time-resolved fluorometer, flow cytometer, and hematofluorometer. 

Instruments that measure fluorescence are termed eitherfluorometers or spec-trofluorometers. The distinction between the two types is based on the approaches that are used to separate the excitation and emission light into monochromatic light. Fluorometers use interference or glass filters, and spectrofluorometers use gratings or prisms. 

Medium-priced uncorrected spectrofluorometers, inexpensive enough to be used for routine analytical work. (2) Uncorrected research spectrofluorometers, more expensive and adaptable for many different types of investigation one such instrument, for example, can be fitted with a phosphorescence attachment (including a rotating can) that converts it into a spectrophosphorimeter. (3) Corrected, or absolute, spectrofluorometers that directly record fluorescence excitation and emission... 

As an example of the first technique, consider the determination of the aromatic hydrocarbon, anthracene, in the presence of its isomer, phenanthrene. Phenan-threne does not absorb in the ultraviolet at wavelengths longer than 360 nm. Since anthracene has an excitation band above 360 nm, it is possible to excite only anthracene. In an experimental study [24] of this type of mixture, the actual wavelength used was 365 nm. As can be seen from Figure 9.8, the best wavelength at which to measure the fluorescence of anthracene on an uncorrected spectrofluorometer would be about 400 nm. It is also possible to determine phenanthrene by use of the second technique. Phenanthrene and anthracene are excited intensely at 265 nm, but phenanthrene fluoresces at 350 nm where anthracene does not (Fig. 9.8). 

D-Fructose, anhydrous dioxane, anhydrous pyridine, anhydrous THF, CH3CN, HEMA, EDMA, AIBN, CuCl, 2,2 -bypyridine, anhydrous DMF, ethyl 2-bromoisobutyrate, MeOH, 0.1 N HCl, 50% Me0H/H20 (v/v) phosphate (0.05 M) buffer (pH 7.4). Thermolyn vortex mixer (Type 16700), INSTECT Model 1060 stirrer, quartz cuvette, spectrofluorometer. 

Thermolyn vortex mixer (Type 16700), INSTECT Model 1060 stirrer, quartz cuvette, Shimadzu RF-5301PC spectrofluorometer. 

In luminescence spectroscopy the emission of the luminophore is monitored. There are two different types of luminescence spectra that can be recorded with modern spectrofluorometers, emission and excitation spectra. Note that although the name implies that these instruments measure only fluorescence, spectrofluorometers can also measure phosphorescence, especially when special accessories are added. Spectrofluorometers contain both an excitation monochromator and an emission monochromator. In emission spectra, the excitation wavelength is fixed and the emission monochromator is scanned. The excitation is usually fixed at a wavelength at which the sample has significant absorbance. In excitation spectra, on the other hand, the emission wavelength is fixed and the excitation monochromator is scanned. The emission is normally fixed at a... 

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