Fluorescence detectors programmable

TABLE 10.12 Concentrations of 10 PAHs in National Institute of Standards and Technology (NIST) Standard Reference Material SRM 1649 Air Particles Determined by Dual Programmable Fluorescence Detector Method... 

The use of the HPLC technique with a programmable fluorescence detector was described for FLU analysis (201). This method was compared with on-line microdialysis used as the cleanup step for a chicken liver sample. After the on-line microdialysis sample cleanup, the resultant HPLC chromatograms were free of background interference, enabling the programmable detector to optimize the quantitation of the three analytes in a single run. The limit of quantitation was 1 yug/ml. 

Liquid Chromatograph. The liquid chromatograph was comprised of a Waters 660 Solvent Programmer, two Waters 6000A pumps, a Waters U6-K Injector and a Waters 440 absorbance detector (254 nm). Whatman micro-capillary tubing (0.007" ID) was used to transfer the HPLC column effluent from the 254 nm absorption detector to the fluorescence detector. 

The analysis of PAHs by fluorescence detection HPLC is often the water analysts first introduction to HPLC. The analysis of the WHO six PAHs (section 11.8.1.1) namely fluoranthene, benzo[6]fluoranthene, henzo[k]fluoranthene, benzo[a]pyrene, benzo[g/j/ perylene and indeno[l,2,3-cd]pyrene was carried out using fixed excitation and emission E wavelengths. With the advent of relatively cheap variable wavelength programmable fluorescence detectors, the detectors can be optimised for each separate PAH with a resultant lowering of detection limit. Ultratrace determination of PAHs down to 180fg of benzo[a]pyrene was reported as early as 1983. 

With the vast development of technology, fluorescence detectors have become programmable. Optimization of wavelength-pair maxima for each analyte can be time programmed during the chromatographic run. 

Figure 5.14. Schematic diagram of a fluorescence detector with rapid scanning monochromators for programmable selection of excitation and emission wavelengths.

All participants used fluorescence detection with programmable excitation and emission wavelengths. Three of them used diode-array absorption detection in addition depending on the PAH, the signal from either the fluorescence or the absorption detector was selected. 

Estimation of true vitamin E in foods requires quantitative determination of all its components since they vary in their biological potency. This vitamin consists of four tocopherols (a, jS, y, and 6) and four tocotrienols (a, jS, y, and d), but the three major constituents responsible for vitamin E activity are the a-, jS-, and y-tocopherols. While these compounds are fluorescent, their esters must be reduced to free alcohols for total tocopherol assays. Total vitamin E can be directly obtained through fluorimetry, but the determination of individual components is carried out using LC with fluorimetric detection. This procedure has been used to determine the composition of vitamin E in seed oils from maize, olives, soya beans, sesame, safflower, and sunflower by measuring the content of all the four tocopherols plus a-tocotrienol. The simultaneous determination of tocopherols, carotenes, and retinol in cheese has been carried out using LC with two programmable detectors coimected in series, a spectrophotometer and a fluorimeter. Carotenes have been determined photometrically, and fluorimetric measurements have been obtained for tocopherol and retinol. 

The highest sensitivity and selectivity in vitamin E LC assays are obtained by using fluorescence or electrochemical detection. In the former, excitation at the low wavelength (205 nm) leads to improved detection limits but at the expense of selectivity, compared with the use of 295 nm. Electrochemical detection in the oxidation mode (amperometry or coulometry) is another factor 20 times more sensitive. In routine practice, however, most vitamin E assays employ the less sensitive absorbance detection at 292-295 nm (variable wavelength instrument) or 280 nm (fixed wavelength detectors). If retinol and carotenoids are included, a programmable multichannel detector, preferably a diode array instrument, is needed. As noted previously, combined LC assays for vitamins A, E, and carotenoids are now in common use for clinical chemistry and can measure about a dozen components within a 10 min run. The NIST and UK EQAS external quality assurance schemes permit interlaboratory comparisons of performance for these assays. 

The chromatographie system eonsisted of a quaternary pump, an automatic sample injector (Hewlett-Packard 1050 Series, Palo Alto, CA, USA) and a programmable fluorimetric detector (Hewlett-Packard, 1046 Series). The detector was linked to a data system (Hewlett-Paekard, HPLC ChemStation) for data acquisition and calculation. According to the results of preliminary experiments obtained by injecting directly into the analytical column standard solutions of PAHs, the deteetor was programmed to measure the fluorescence intensity at the exeitation/emission wavelengths pairs listed in Table 1. 

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