Fluorescence measurement procedure

Definition and Uses of Standards. In the context of this paper, the term "standard" denotes a well-characterized material for which a physical parameter or concentration of chemical constituent has been determined with a known precision and accuracy. These standards can be used to check or determine (a) instrumental parameters such as wavelength accuracy, detection-system spectral responsivity, and stability (b) the instrument response to specific fluorescent species and (c) the accuracy of measurements made by specific Instruments or measurement procedures (assess whether the analytical measurement process is in statistical control and whether it exhibits bias). Once the luminescence instrumentation has been calibrated, it can be used to measure the luminescence characteristics of chemical systems, including corrected excitation and emission spectra, quantum yields, decay times, emission anisotropies, energy transfer, and, with appropriate standards, the concentrations of chemical constituents in complex S2unples. 

Functions of Standards. Fluorescent standards can be used for three basic functions calibration, standardization, and measurement method assessment. In calibration, the standard is used to check or calibrate Instrument characteristics and perturbations on true spectra. For standardization, standards are used to determine the function that relates chemical concentration to Instrument response. This latter use has been expanded from pure materials to quite complex standards that are carried through the total chemical measurement process (10). These more complex standards are now used to assess the precision and accuracy of measurement procedures. 

Howard [27] determined dissolved aluminium in seawater by the micelle-enhanced fluorescence of its lumogallion complex. Several surfactants (to enhance fluorescence and minimise interferences), used for the determination of aluminium at very low concentrations (below 0.5 pg/1) in seawaters, were compared. The surfactants tested in preliminary studies were anionic (sodium lauryl sulfate), non-ionic (Triton X-100, Nonidet P42, NOPCO, and Tergital XD), and cationic (cetyltrimethylammonium bromide). Based on the degree of fluorescence enhancement and ease of use, Triton X-100 was selected for further study. Sample solutions (25 ml) in polyethylene bottles were mixed with acetate buffer (pH 4.7, 2 ml) lumogallion solution (0.02%, 0.3 ml) and 1,10-phenanthroline (1.0 ml to mask interferences from iron). Samples were heated to 80 °C for 1.5 h, cooled, and shaken with neat surfactant (0.15 ml) before fluorescence measurements were made. This procedure had a detection limit at the 0.02 pg/1 level. The method was independent of salinity and could therefore be used for both freshwater and seawater samples. 

HPLC-based electrochemical detection (HPLC-ECD) is very sensitive for those compounds that can be oxidized or reduced at low voltage potentials. Spectrophotometric-based HPLC techniques (UV absorption, fluorescence) measure a physical property of the molecule. Electrochemical detection, however, measures a compound by actually changing it chemically. The electrochemical detector (ECD) is becoming increasingly important for the determination of very small amounts of phenolics, for it provides enhanced sensitivity and selectivity. It has been applied in the detection of phenolic compounds in beer (28-30), wine (31), beverages (32), and olive oils (33). This procedure involves the separation of sample constituents by liquid chromatography prior to their oxidation at a glassy carbon electrode in a thin-layer electrochemical cell. 

In this section an overview of the numerous methods and principles for the discrimination of enantiomers is given. First, the interaction principles of the polymer-based methods adapted from chromatographic procedures are illustrated. The discrimination of enantiomers was achieved some decades ago by using different types of stationary materials, like cyclodextrins or polymer-bonded amide selectors. These stationary-phase materials have successfully been appointed for label-free optical sensing methods like surface plasmon resonance (SPR) or reflectometric interference spectroscopy (RIfS). Furthermore, various successful applications to optical spectroscopy of the well-established method of fluorescence measurements for the discrimination of enantiomers are described. 

The main drawback with pesticides is that only a limited number are naturally fluorescent. The procedure is usually straightforward for fluorescent pesticides and measurements can be made immediately after separation. But for those that are not fluorescent, physical, chemical or biochemical treatments are required. 

FDNB reacts with all free -NH2 groups, including e-amino groups. Following acid hydrolysis of the dinitrophenylated protein, a separation of the dinitrophenylated N-terminal amino acid from the rest of the hydrolysis products can be achieved by organic solvent extraction of the hydrolysate. A more modern procedure utilizes dansyl chloride instead of FDNB. This method requires less protein, because dansylated amino acids can be identified via fluorescence measurements. 

If possible, make more accurate measurements of the wavelengths using a high-resolution grating spectrometer as indicated in Fig. 2. The instructor will advise on the most appropriate measurement procedure for the particular setup employed. The spectrometer should have 0.1-nm resolution, or better if possible, and should be equipped with a photomultiplier or charge-coupled detector (CCD). An instrument used for Raman or fluorescence measurements would be quite suitable. 

The measuring procedure can be optimized without further chromatography. It can be changed as required, for instance by choosing different measuring modes, such as reflectance or fluorescence mode of scanning. 

General procedure. The excitation and emission spectra of fluorescence were measured at room temperature and optimum excitation and emission wavelengths were selected from these spectra. Three mL of rutin and 1 mL of lxl0-4 mol/L Fe(III) were taken into a vial, mixed well and stood for 5 min. The content of the vial was made up to 10 mL with distilled water. The fluorescence intensity of the system was then measured in a 1 cm quartz cell. The fluorescence intensity of the solution was measured at 506 nm with excitation at 491 nm. All fluorescence measurements were made using 1 nm increment, 1 s integration time, S acquisition mode and YES auto zero. 

Basic procedure. Carbaryl standard stock solution was prepared in distilled water through ultrasonication for 6 h at 40 to 50°C. Carbaryl solution (0.1 pmol/L - 0.1 mmol/L) was diluted with distilled water in a 10 mL calibrated flask containing 2 mL phosphate buffer solution (pH 7), 2 mL ethanol (20%) and 2 mL SDS solution (0.1 mmol/L). The fluorescence measurements were performed at Xcm = 349 nm and Xex = 281 nm. 

First attempts to measure electron transfer reactions in these maquettes focused on the Ru -P -V system. All ruthenium-modified complexes were prepared according to standard procedures (4, 23). Time-resolved fluorescence measurements of Ru(II )-Pyj revealed lifetimes of about 490 ns, consistent with those of Ru(2,2 -bipyiidine)3 (24). Based on this lifetime, the only ET-rates that would be directly measurable by this technique are those faster than about 2 X 10 s. A possible ET reaction takes place as shown in Scheme I. 

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