Fluorometric detector

A typical set-up for measurement of residual oxygen in a closed vessel, such as food packs is shown in Figure l5, 24. The system consists of a benchtop phase-fluorometric detector (Luxcel Biosciences) built on the basis of low-cost optoelectronic components, which operates with a flexible fibre-... 

In this connection, it is interesting to note that Gardner [263] isolated free amino acids at the 20 nmol/1 level from as little as 5 ml of sample by cation exchange, and measured concentrations on a sensitive amino acid analyser equipped with a fluorometric detector. 

Depending on the derivatizing agent used, spectrophotometric or fluorometric detectors are usually employed. Electrochemical detection of underivatized amino acids is limited to amino acids possessing aromatic or sulfur-containing side-chain, even if derivatization procedmes to attach electrochemical active moieties to the amino acids can be employed, as well as other approaches... 

Selectivity and sensitivity were increased by Rodrignez-Delgado et al. [369] using a fluorometric detector, and 17 phenolic snbstances were separated and their concentration was measured in about 35 min of chromatographic rnn. 

Detection can be performed using UV, UV-Vis, DAD, fluorometric detector (ELD) or ED. Gatti et al. [444] report an on-line photochemical derivatization to enhance the selectivity. A photo reactor system was inserted on-line between the column and the ELD. The eluate was irradiated at 254 and 366 nm inducing alterations that modified spectral properties of the analytes. Recently an increasing number of studies have reported the use of LC-MS for the identification of both retinoids and carotenoids [426,433,445-447],... 

Because of their high sensitivity, fluorescence detectors are particularly useful in trace analysis when either tire sample size is small or the analyte concentration is extremely low. Although fluorescence detectors can become markedly nonlinear at concentrations where absorption detectors are still linear in response, their linear dynamic range is more than adequate for most trace analysis applications. Unfortunately, fluorometric detectors are often susceptible to background fluorescence and quenching effects that can plague all fluorescence measurements. 

In liquid chromatographic analysis of quinolone antibacterials, most popular is the fluorometric detector due to the inherent fluorescence of these drugs and the advantages in terms of selectivity and sensitivity that this detector offers (Table 29.6). Fluorometric detection after postcolumn derivatization with sulfuric acid has also been reported (203). However, quinolones exhibit also remarkable ultraviolet absorption and are therefore ideal for direct determination without derivatization. Detection can be performed in the wavelength range of 254-295 nm. 

The most common detectors used for the determination of SPA are the detectors that operate at the UV wavelength of 233 or 280 nm (104,111,113,115-119,126,144,71). Fluorometric detectors are used mainly for the determination of ethoxyquin (133-135), and it is also possible to use electrochemical detectors (123,137). A very progressive electrochemical detection system is based on measuring the array potential of SPA, with the previous oxidative removal of impurities (128). 

Fluorometric detector Electrochemical detectors 1 x 10-11 g 1 x 105 For fluorescent species with conjugated bonding and aromaticity Relatively insensitive to temperature and flow fluctuations nondestructive can be used with gradient elution often, chemical derivatization is done on analytes to form fluorescent species uses deuteriem lamp for 190-400 nm or tungsten lamp for 350-600 nm... 

Fluorometric detectors are sensitive to compounds that are inherently fluorescent or that can be converted to fluorescent derivatives either by chemical transformation of the compound or by coupling with fluorescent reagents at specific functional groups. If derivatization is required, it can be done before chromatographic separation or, alternatively, the reagent can be introduced into the mobile phase just before its entering the detector. 

In protein chromatography, two main types of detector have been used, the UV detector and the fluorometric detector. 

Levels of IAA were analyzed by HPLC with a fluorometric detector and indole-propionic acid as an internal standard. Levels of ABA were analyzed by GLC with an ECD and [14C]ABA as an internal standard. Means of results of 3 experiments and standard errors are given (n=3). Reproduced and revised with permission from ref. 8. 

Determination of human plasma levels of aloe-emodin and rhein using HPLC with a fluorometric detector has been described (Krumbiegel and Schulz, 1993). 

The separation power and sensitivity of CE is illustrated in Figure 21.23. A mixture of 18 amino acids is separated in 30 min, at quantities ranging from 2 to 7 attomoles. The amino acids were derivatized with fluorescein isothiocyanate (Fl i C) to form fluorescent derivatives, and a fluorometric detector system was employed. The detection limits ranged down to 10""2o mol in 1 nL, corresponding to 10 " M and 6000 molecules ... 

The dansyl derivatives, which have a napthtalene structure, are excellent derivatives for primary amines (Mietz and Karmas, 1978). They are easily formed and detected by a uv (HPLC) detector in amounts as little as 10 ng, therefore the method does not require high sensitivity or fluorometric detectors. Gradient elution improves the separation, allowing for a broad range of derivatives to be separated in a relatively short time (40 min). Use of reverse-phase microparticular (5-10 /u,m) columns can further improve the HPLC separation (Gouygou et al, 1992). Desiderio et al (1987) described the use of a reversed-phase HPLC method for the quantification of putrescine, cadaverine, spermidine, and spermine from brain extracts as the dansyl derivatives. 

Various analytical techniques such as spectrophotometry/colorimetry, FL and infrared spectrometry, voltammetry, thin-layer chromatography (TLC), gas chromatography (GC), and HPLC based on ultraviolet, diode array, or fluorometric detectors have been reported in the literature for analysis of vitamin E. Various critical and comprehensive reviews are available on vitamin E quantification in food and clinical samples (Ball, 1988, 1998 Lumley, 1993). The AOAC International Official Methods of Analysis (1995) provides several methods based on older, chemical approaches. The applications of these analytical techniques are briefly summarized below. 

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