Spectrofluorometric detection

Figure 13 illustrates the use of spectrofluorometric detection in the reversed-phase chromatographic analysis of the control serum sample originally shown in Fig. 10. The advantages of fluorometric detection in eliminating background and increasing selectivity are obvious.

Fio. 13. Chromatogram of the control serum sample using spectrofluorometric detection (285 nin excitation. 320 nm cutoff). Conditions are the same as in Fig. 10. Reprinted with permission from Hartwick et at. (H7). Copyright by Elsevier Scientific Publishing Company, Amsterdam. 

Pena A, Lino CM, Alonso R, Barcelo D, Determination of tetracycline antibiotic residues in edible swine tissues by liquid chromatography with spectrofluorometric detection and confirmation by mass spectrometry, J. Agric. Food Chem. 2007 55(13) 4973-4979. 

Ensafi, A. Dehaghi, G.B. (2000). Flow-Injection Simultaneous Determination Of lodate And Periodate By Spectrophotometric And Spectrofluorometric Detection. Anal. Sci. Vol.16, pp 61-64, ISSN 09106340... 

The absence of a chromophore for sensitive detection is a problem which is met with HPLC determination of these thiol analytes. However, two approaches are possible to resolve this point 1) direct detection using electrochemistry, either amperometry on gold/mercury amalgamated electrodes or coulometry on porous graphite electrodes 2) derivatization coupled with UV or spectrofluorometric detection. The derivatization reactions, either precolumn or postcolumn, involve different thiol selective reagents ammonium 7-fluorobenzo-2-oxa-l,3-diazole-4-sulphonate (SBD-F), 0-phthalaldehyde (OPA), etc. 

The antioxidant concentration of the fish was determined on its TL. The antioxidant in TL was extracted by shaking with acetonitrile and allowing the phases to separate. The upper acetonitrile layer was separated and filtered using a Millipore filter, then injected to an HPLC equipped with a spectrofluorometric detection (Yankah et al submitted for publication). 

Rezaei, B., A. A. Ensafi, and S. Nouroozi. 2005. Flow-injection determination of ascorbic acid and cysteine simultaneously with spectrofluorometric detection. Anal. Sci. 21 1067-1071. 

When one is deciding what column geometry is optimal for trace analysis with unlimited sample volume, two additional points should be evaluated. First, to what extent does the analysis require accurate and reproducible injections Strict performance specifications may eliminate microbore columns from consideration. The accuracy and reproducibility of injection systems that deliver 0.1-, 0.2-, and 0.5-/xL samples have not been adequately characterized. Second, if the analyte of interest requires postcolumn derivatization, construction of a postcolumn reaction system that is compatible with the exceedingly small band volumes characteristic of microbore columns may be extremely difficult, but not impossible. Apffel et al. (28) developed and evaluated both packed-bed and open tubular postcolumn reactors for use with 1-mm i.d. analytical columns. Catecholamines were postcolumn derivatized with o-phthal-aldehyde and detected spectrofluorometrically. The 5-/zm particle... 

Cholerton S, Idle ME, Vas A, et al. Comparison of a novel thin-layer chromatographic-fluorescence detection method with a spectrofluorometric method for the determination of 7-hydroxycoumarin in human urine. J Chromatogr 1992 575 325-330. 

Thiamine, a soluble vitamin, is an essential nutrient for humans and is important in carbohydrate metabolism, maintaining normal neural activity and preventing beriberi. Various analytical techniques have been reported for the determination of thiamine in pure form, in pharmaceutical preparations, or in biological fluids. Spectrophotometric methods suffer from poor sensitivity (mg/L detection limit). Spectrofluorometric methods usually involve the conversion of thiamine to thiochrome.2 High performance liquid chromatography requires a post-column derivatization step3 and instrumentation for electrophoresis-based methods is expensive.4... 

This behavior is not surprising in that many investigators have detected photoeffects under conventional spectrofluorometric conditions. Studies conducted on the UV-irradiation of lysozyme (13) and L-glutamate dehydrogenase (14) revealed a concomitant loss in enzyme activity and fluorescence emission with irradiation time. The primary photoeffects were destruc-... 

Methods of analysis. The total protein concentration of crude solutions, including cellular proteins, was determin by the biuret method (72). With dilute xanthan solutions, this method was not sensitive enough, and a spectrofluorometric assay was carried out. This method consisted of an acid hydrolysis of the samples followed by derivatization with o-phthaldialdehyde and HPLC analysis on a Cl8 reverse column of the amino acids released, using spectrofiuorescence as the detection nKxie. 

A highly specific detection of folic acid in very low concentrations and in the presence of all other vitamins is accomphshed by spraying with permanganate (Rgt. No. 199) [70, 77]. Even 0.02 [xg can be detected through its blue fiuorescence in UV radiation of 365 nm. In this way, quantitative determination appears possible directly on the chromatogram or after elution of the spots we hope to report on this soon. Quantitative determination should likewise be possible with the help of the sensitive spectrofluorometric method [32]. 

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