With native fluorescence

Sluszny, C., Yeung, E.S. (2004). One-and two-dimensional miniaturized electrophoresis of proteins with native fluorescence detection. Anal. Chem. 76, 1359-1365. 

Huorescence Specific compounds with native fluorescence or with fluorescent tag fg-pg... 

Fluorescence Assay for Analytes with Native Fluorescence... 

Wu, N., Li, B. H., and Sweedler, J. V., Recent developments in porphyrin separations using capillary electrophoresis with native fluorescence detection, J. Liquid Chromatogr., 17, 1917,1994. 

It was realized early on that the fluorescence exhibited by certain substances was a valuable property for their analysis because it was measurable at lower concentrations than optical absorbance, because it was linear with concentration over a wide concentration range, and because its relatively high specificity permitted determination even in the presence of other substances. Numerous analytical methods for the determination of compounds with native fluorescence have therefore been developed. Many methods were also developed where a compound of interest was converted to a fluorescent product by a specially devised chemical reaction to enable it to be determined by the sensitive technique of fluorimetry. Our main concern is with the application of reagents designed to make fluorescent derivatives of compounds of interest, in order to permit their determination or detection by fluorimetric methods allied to chromatographic (or electrophoretic) separation processes. 

The small inner diameter of the separation capillary used in CE implies a short optical pathway, and the consequent poor concentration sensitivity when conventional UV detectiOTi is used. To overcome this drawback several techniques have been developed some of them consist in application of general approaches that are not specifically addressed to CE analysis of alkaloids. One is the use of LIF detector for analysis of alkaloids with native fluorescence [68, 69] or after their off-line derivatization [64, 88, 112]. Sample pretreatment, a second major approach, is popularly employed in combination with sample extraction and can be conveniently applied in analysis of alkaloids because they can be easily retained in cationic-exchange sorbents in solid-phase extraction (SPE) mode [113, 114]. It may be interesting to focus on more specific aspects to detect very low levels of analytes using limited amoimts of samples to this regard chemiluminescence reactions and the use of online preconcentration methods will be considered. 

Solubilization of an active H,K-ATPase is also a prerequisite for reconstitution of the enzyme into liposomes. With these H,K-ATPase proteoliposomes it is then possible to study the transport characteristics of pure H,K-ATPase, without the interference of residual protein contamination that is usually present in native vesicular H,K-ATPase preparations. Rabon et al. [118] first reported the reconstitution of choleate or n-octylglucoside solubilized H,K-ATPase into phosphatidylcholine-cholesterol liposomes. The enzyme was reconstituted asymmetrically into the proteoliposomes with 70% of the pump molecules having the cytoplasmic side extravesicular. In the presence of intravesicular K, the proteoliposomes exhibited an Mg-ATP-dependent H transport, as monitored by acridine orange fluorescence quenching. Moreover, as seen with native H,K-ATPase vesicles, reconstituted H,K-... 

Khashaba et al. [34] suggested the use of sample spectrophotometric and spectrofluorimetric methods for the determination of miconazole and other antifungal drugs in different pharmaceutical formulations. The spectrophotometric method depend on the interaction between imidazole antifungal drugs as -electron donor with the pi-acceptor 2,3-dichloro-5,6-dicyano-l,4-benzoquinone, in methanol or with p-chloranilic acid in acetonitrile. The produced chromogens obey Beer s law at Amax 460 and 520 nm in the concentration range 22.5-200 and 7.9-280 pg/mL for 2,3-dichloro-5,6-dicyano-l,4-benzoquinone and p-chloranilic acid, respectively. Spectrofluorimetric method is based on the measurement of the native fluorescence of ketoconazole at 375 nm with excitation at 288 nm and/or fluorescence intensity versus concentration is linear for ketoconazole at 49.7-800 ng/mL. The methods... 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

Fluorescence detection, alone or with the aid of derivatizing reagents to enhance detector responses and improve the chromatographic resolution, has also been used for the determination of biogenic amines. Lakshmana and Trichur (1997) used native fluorescence to analyze NE, DA, and 5HT in rat brain utilizing an isocratic separation on an ODS CIS column. The detection limits reported were 100-250 pg on column. 

Lakshmana MK, Trichur TR. 1997. An isocratic assay for norepinephrine, dopamine and 5-hydroxytryptamine using their native fluorescence by high-performance liquid chromatography with fluorescence detection in discrete brain areas of rat. Anal Biochem 246 166-170. 

Lee, T. T., Lillard, S. J., and Yeung, E. S. (1993). Screening and characterization of biopharmaceuticals by high-performance capillary electrophoresis with laser-induced native fluorescence detection. Electrophoresis 14, 429—438. 

Since the PSP toxins lack native fluorescence, useful UV absorption or adequate volatility, more traditional analytical procedures such as gas chromatography or spectrometry have proven ineffective in assaying for the toxins. A number of chemical assays for the toxins have been developed though with the fluorometric method of Bates and Rapoport (3 ) proving to be the most useful to date. This assay is based on oxidation of the PSP toxins under alkaline conditions to fluorescent derivatives. The assay is highly sensitive, fairly specific for the PSP toxins and was incorporated into a detection method in the column chromatographic separation of the toxins described by Buckley et al (4 ). 

Hupka et al. [29] developed a method for the determination of morphine and its phase II metabolites, morphine-3-beta-D-glucuronide and morphine-6-beta-D-glucuronide in the blood of heroin victims. The method is based on immunoaffinity SPE, RP-HPLC isocratic separation (mobile phase 90% lOmmol KH2PO4, 2mmol 1-heptanesulfonic acid, adjusted to pH 2.5 with H3PO4 and 10% acetonitrile flow rate 1.5 mL/min), and laser-induced native fluorescence detection. 

This type of derivatization seems promising for the detection of drugs and metabolites with phenolic groups. A recent application on the determination of chlorophenols in surface water showed that the dansyl derivatives of phenols are readily convertible to the highly fluorescent dansyl-OH and dansyl-OCHs products after postchromatographic irradiation (281). Fluorescence gain factors of up to 8000-fold were obtained for chlorophenol derivatives with a low native fluorescence. 

Retinol and its esters and unesterified tocopherols and tocotrienols possess strong native fluorescence, but neither vitamin D nor vitamin K fluoresce. The carotenoids commonly associated with foods do not fluoresce to any significant extent, except notably phytofluene, which is found in considerable amounts in tomatoes (22) and in smaller amounts in carrots (130) and which fluoresces six times more intensely than retinyl acetate (131). 

For coumarins in orange fruits (115), the HPLC used a Zorbax Rx C8 (250-mm X 4.6-mm ID, 5 fim) column maintained at 25°C, and analysis was performed by binary-gradient elution using 0.1% HOAc in acetonitrile (eluent A) and 0.1% HOAc in HzO (eluent B). In the author s lab, standard coumarins could be separated by isocratic elution on Zorbax Rx C8 column with acetonitrile-0.1% HOAc in water (35 65) at 1.0 ml/min, as presented in previous work (1). The eluate from the column was passed to a UV detector (UV 330 nm) and then into a fluorescence detector (excitation at 340 nm, emission at 425 nm). As for the specificity, some of the coumarins do not have native fluorescence. Nine coumarins are separated under UV 330 nm, and three coumarins could not be detected with fluorescence detection. Detailed conditions for coumarin analysis in foods and absorption spectra of coumarins obtained by online diode array detector with HPLC were presented by Lee and Widmer (1). Since coumarins exhibit strong absorption in the ultraviolet region, absorption at approximately 313 nm has been used to estimate the dilution of cold-pressed lemon oil with distilled oil (12). Analysis of umbelliferone (7-hydroxy-coumarin) and scopoletin (6-methoxy-7-hydroxycoumarin) in citrus fruits was performed using... 

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