Fluorescence spectrofluorimetry

Fluorescence spectrofluorimetry is a type of electromagnetic spectroscopy that generates absorbance and emission fluorescent spectra, giving insights into biomolecular structure. 

Equation (2) also shows how the intensity of fluorescence varies when the frequency of the exciting light varies. For a given solution the fluorescence intensity is proportional to 7oe0 and for many substances in solution the fluorescence efficiency () is approximately independent of the excitation frequency. Thus, if the intensity of exciting light is kept constant as the frequency is varied, the fluorescence intensity will be proportional to e, the molecular extinction coefficient of the solute. Hence the true excitation spectrum frequently corresponds closely to the absorption spectrum of the compound (see Fig. 2). Spectrofluorimetry can thus be used to measure the absorption spectra of fluorescent solutes, but at concentrations far lower than could be measured directly with an absorption spectrophotometer. It has the further advantage that the... 

Methylcarbamate insecticides have been recently labeled with DNS-C1 [145]. The procedure involves the hydrolysis of the carbamates with 0.1 M sodium carbonate to form a phenol and methylamine [166]. The two hydrolysis products are labeled with DNS-C1 and subsequently detected and determined quantitatively by TLC on silica gel layers by scanning spectrofluorimetry in situ. The reaction conditions were examined, and optimum conditions for hydrolysis and labeling were established [167]. The overall reaction scheme is shown in Fig. 4.62. The phenol derivatives of a number of N-methylcarbamates have been separated by one- and two-dimensional TLC [168], and the fluorescence behaviour and stability of the derivatives have been examined [169]. Most of the DNS derivatives fluoresce at similar wavelengths (excitation, ca. 365 nm emission, ca. 520 nm). The fluorescence spectrum of a typical DNS derivative is shown in Fig. 4.63. The method has been applied successfully to the analysis of low concentrations of carbamates in water and in soil samples with little or no clean-up being required [170,171]. Amounts as low as 1 ng of insecticide can be detected instrumentally. Visual limits of detection are ca. 5-10 ng per spot. 

Membrane permeabilization activity of peptides is currently measured by the use of artificial membrane bilayers, such as liposomes or erythrocytes. The hposome leakage assay can be performed by using spectrofluorimetry with a concentration-dependent quenching of a dye (calcein, carboxyfluorescein) encapsulated in liposomes. Disruption of hposomes in the presence of peptide-inducing leakage will lead to an increase in the fluorescence intensity of the liposome solution. Erythrocyte lysis assay is based on the absorption of hemoglobin, which can be measured once released into the extracellular medium upon erythrocyte lysis in the presence of peptide. 

Navalon et al. developed a method for the determination of trace amounts of ciprofloxacin, based on solid-phase spectrofluorimetry [9]. The relative fluorescence intensity of ciprofloxacin fixed on Sephadex SP C-25 gel was measured directly after packing the gel beads in a 1 mm silica cell, using a solid-phase attachment. The wavelengths of excitation and emission were 272 and 448 nm, respectively. The linear concentration range of the compound was 0.3-10 ng/mL, with a RSD of 1.2% (for a level of 4 ng/mL) and a detection limit of 0.1 ng/mL. This method was used for the determination of ciprofloxacin in human urine and serum samples with a recovery of 100% in all cases. 

Spectrofluorimetry differs from absorption spectrophotometry in not yielding an absolute scale of values. For this reason it is essential to employ a reference standard for quantitative measurements. For example, some pharmacopoeial tests, such as the test for uniformity of content for digitoxin tablets, employ a spectrofluorimefric assay and comparison wi an ofticial reference standard. Quantitative Spectrofluorimetry has been proposed for a munber of naturally fluorescent compoimds, including ergometrine, riboflavine, tiie catechol-amines, phenothiazines, the barbiturates (at pH 13), and certain antibiotics such as chlortetracycline and oxytetracycline. 

Fluorescent systems spectrofluorimetry spectrophotometry inorganic and organic fluors... 

Kingsley, G. R., Clinical chemistry. Anal. Chem. 43, 15R-41R (1971). Kirkbright, G. F., The application of non-flame atom cells in atomic absorption and atomic fluorescence spectroscopy. Analyst London) 96, 609-623 (1971). Kirkbright, G. F., Saw, C. G., and West, T. S., Determination of trace amounts of tellurium by inorganic spectrofluorimetry at liquid nitrogen temperature. Analyst London) 94, 457-460 (1969). 

A fluorometric method was developed for determination of the level of (20) in biological fluids <89MI 823-04). The 8-carboxy-2-methoxy-l 1-0X0-11// derivative of the 2,3-benzologue of (12) was analyzed by laser induced spectrofluorimetry <80AC1554>. Fluorescence data of pyoverdine II metal complexes (containing a pyrimido[l,2-a]quinoline moiety) were measured at different pH <87T226i>. 

Analysis of carboxylic acids of free porphyrins in urine, by means of HPLC/FD. The chemical structure of the porphyrins presents the natural property of being fluorescent compounds, which makes them detectable by spectrofluorimetry. 

The use of the mustard oil reaction prior to separation by TLC is mentioned in a few publications [54, 59]. One of these deals with the detection of 3,4-dimethoxy-phenylethylamine, the dimethyl ether of dopamine, in urine samples. The compound is converted to the isothiocyanate and separated on silica gel plates. These are then sprayed with a mixture of equal volumes of sulfuric acid and methanol and irradiated with UV light. An intense fluorescence develops and permits the detection of ng amounts of 3,4-dimethoxyphenylethylamine [54]. The derivative can be extracted with methanol for quantitative detetmination by spectrofluorimetry (excitation at 365 nm, fluorescence at 465 nm). The only other isothiocyanates which yielded a similar fluorescence were those of 3-methoxytyramine and 4-O-methyldopamine. Silica gel thin-layer plates have also been used to separate norephedrine from norpseudo-ephedrine as isothiocyanates [59]. 

Molybdate/rhodamine B Spectrofluorimetry 0.0003-0.095mgPr Quenching of fluorescence... 

Molybdate/thiamine Spectrofluorimetry 0.007-6.6mgP - Generation of fluorescent... 

Spectrofluorimetry Fluorescence measurements of humic substances can provide criteria for the differentiation and classification of substances of various origins. Humic substance groups are excited at 350 nm and emit at 450mn, a spectral region relatively free from interference by other groups. Measurement of the fluorescence is a relatively selective method of analysis. Fluorescence caimot be used for the direct determination of functionality. 

Shpol skii spectrofluorimetry has been used for the determination of PAHs in crude enviromnental sample extracts with minimum sample cleanup. This technique gives a vibrationally resolved fluorescence spectrum of samples dissolved in a suitable solvent (usually an -alkane) at cryogenic temperatures, e.g., 26 K. It combines the selectivity of an infrared spectrum with the sensitivity of fluorimetry, though the sensitivity suffers considerably from the presence of large amounts of interfering substances such as fatty components in crude extracts since these give a poor-quality matrix with a high sample absorbance. 

Yu FS, Li L, Chen F (2008) Determination of adenosine disodium triphosphate using pruh-floxacin-terbium (III) as a fluorescence probe by spectrofluorimetry. Anal Chim Acta 610 257-262... 

CPE followed by spectrofluorimetry was applied to analyse the concentration of vitamin Bi in samples of urine (Tabrizi 2006). In this method, Triton-XI14 surfactant was used for CPE. The procedure was accomplished by adding an aqueous solution of thiamin, ferricyanide and Triton-X114 in an alkaline medium. The surfactant-rich turbid phase and diluted aqueous phase were attained after centrifugation of the mixture. The surfactant-rich phase was collected and diluted in an ethanol water mixture prior to measurement of the fluorescence excitation and emission intensity. Thiamin was oxidized by ferricyanide under alkaline conditions to form thiochrome, which is a fluorescent species. During the extraction procedure, thiochrome was entrapped in surfactant micelles. Thus, thiamin was separated from the biological matrix after derivatization. The fluorescence intensity responded linearly with the concentration of thiamin under optimized conditions. 

In a FI photochemical technique coupled to spectrofluorimetry for the determination of vitamin Bi, thiamin was converted to thiochrome by a photochemical reaction (Chen et al. 1998). The photochemical reaction was conducted in continuous flow mood. Acetone was used as s sensitizer to enhance the fluorescence intensity. The obtained LOD was at microgram scale. Another photochemical procedure coupled with synchronous fluori-metry has been reported for the selective determination of vitamin Bi, B2 and Bg (Guo et al. 1993). In case of vitamin Bi, the LOD was obtained in nanogram scale. 

Most of the analytical techniques based on the spectrofluorimetry involve the generation of fluorescent thiochrome from thiamin via an oxidative or photochemical reaction in the absence or presence of catalyst. 

This phenomenon is known as fluorescence. The intensity of fluorescence produced from a sample molecule can be measured and termed in a technique known as spectrofluorimetry. 

We have seen the relationship between absorption spectrophotometry and spectrofluorometry. A similar relationship exists between atomic absorption spectrophotometry and atomic fluorescence spectrophotometry. In atomic fluorescence, the flame retains its role as a source of atoms these atoms, however, are excited by an intense source of radiation and their fluorescent emission is assayed at an angle of 90° in a manner similar to that of spectrofluorimetry. Lack of sufficiently intense source for many elements has been the limitation of this technique, however, with time instrumental developments are overcoming this problem. High intensity hollow-cathode lamps, or xenon or mercury discharge lamps are used. 

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