Fluorescence filter fluorimeters

The development of photodetectors enabled the human eye to be replaced by a much more sensitive detector of light intensity. The evolution of modem colorimeters and of spectrophotometers capable of operation in both the ultraviolet and visible regions of the spectrum has been discussed.217,218 The phenomenon of fluorescence was first employed for quantitative analysis in the 1930s, when the first filter fluorimeters were constructed. An article has outlined the development of fluorescence analysis up to 1980.219 Lasers have now been employed long enough in analytical chemistry for a historical account to be given.220... 

The more sensitive method of Rees et al. (R6) requires about 7 jul plasma per ml dye solution (6 mg l-anilinonaphthalene-8-sulfonic acid per liter phosphate buffer, pH 7.6). The fluorescence intensity of the plasma-dye mixture is measured (activation peak 370 nm, fluorescent peak 485 nm) and the albumin concentration obtained from a standard curve. The dye is stable and has low blank fluorescence a filter fluorimeter is satisfactory for the determination. Bile pigments in excess of about 5 mg/100 ml plasma interfere with the method low results are presumably caused by competition between bilirubin and dye for binding. Bovine albumin has been stated to produce more intense fluorescence with anilinonaphthalene-sulfonate than human albumin (R6). This requires confirmation, since dt... 

The fluorescence detector is available as either a filter fluorimeter or as a continuous wavelength fluorimeter. The filter fluorimeters are less expensive, but in most cases low wavelength excitation is not possible with these instruments, and this makes, e.g., the determination of indoles and catecholamines by their native fluorescence impossible. The selectivity of the fluorescence detector is much better than that of the ultraviolet detector, and for favorable compounds the sensitivity may also be better. 

The unfiltered seawater sample is mixed with the reagent by means of a peristaltic pump with air segmentation in a sample-and-wash routine based on the autoanalyser principle (Fig. 1). After a reaction time of approximately 2 min the reaction stream is degassed and passed through a filter fluorimeter where the fluorescence is monitored (Xe 340 nm, Xem 455 nm). 

The wavelengths in fluorescence spectrophotometers are generally selected with the aid of monochromators line emitters are predominantly used in simple filter fluorimeters, but continuum radiators can also be employed in conjunction with cut-off filters. Fig. 32 clearly shows the basic principle... 

There are different constructions of fluorescence detectors, filter fluorimeters, and spectrofluorimeters with different hght sources, the most common one is the deuterium lamp. 

Fluorescence detectors are based on filter-fluorimeters or spectrofluori-meters. They are more selective and can be up to three orders of magnitude more sensitive than UV absorbance detectors. The detector responds selectively to naturally fluorescing solutes such as polynuclear aromatics, quinolines, steroids and alkaloids, and to fluorescing derivatives of amines, amino acids and phenols with fluorogenic reagents such as dansyl chloride (5-(dimethylamino)-l-naphthalene sulfonic acid). 

Flnorescence spectroscopy, although not a new teclmique, is stiU, compared to other analytical methods, relatively immature in terms of standardization of measurement. As mentioned in Chapter 1, the birth of fluorescence spectroscopy was marked by the work of Sir George Gabriel Stokes, who in 1852 reported his studies on quinine bisulfate using what today would be considered a filter fluorimeter arrangement, as shown in Figure 5.1. 

Instruments for the measurement of fluorescence are known as fluorimeters or spectrofluorimeters. The essential parts of a simple fluorimeter are shown in Fig. 18.1. The light from a mercury-vapour lamp (or other source of ultraviolet light) is passed through a condensing lens, a primary filter (to permit the light band required for excitation to pass), a sample container, a secondary filter (selected to absorb the primary radiant energy but transmit the fluorescent... 

Procedure. Measure the fluorescence of each of the above solutions at 445 nm, using that containing 62.0 mL of the dilute quinine solution as standard for the fluorimeter. Use LF2 or an equivalent primary filter (/cx = 350 nm) and gelatin as the secondary filter if using a simple fluorimeter. 

Surface fluorescence of NADH/NADPH can be recorded continuously with a DC fluorimeter and correlated with changes in experimental conditions. A mercury arc lamp (with a 340-375 nm filter in front) is used as a hght source for fluorescence excitation. The fluorescence response of reduced NADH/NADPH was measured at 450-510 nm. The DC fluorimeter and the Hg arc lamp are connected to the kidney by a trifurcated fiber optics light guide. NADH/NADPH fluorescence emission can be corrected for changes in tissue opacity by a 1 1 subtraction of reflectance changes at 340-375 nm from the fluorescence. To determine NADH/NADPH redox state of the total surface area of kidney cortex and to evaluate whether certain areas were insufficiently perfused, fluorescence photographs of the total surface area were taken. The study demonstrated that the surface fluorescence method is simple and provides specific information about the mitochondrial oxidation-reduction state. 

Fluorimeter (also Fluorometer or Fluorophotometer). In this instrument, the sample is excited by a light beam of suitable short wavelength. The remitted fluorescent lighl is picked up by a photometer, usually placed 90 degrees from incidence, A filter or monochromator is provided which excludes the exciting waveband and transmits the fluorescent light, See also Fluorometers. 

Thacker [24] reported the design of a miniature flow fluorimeter for liquid chromatography. The body of the fluorimeter was machined from a block of aluminium and contained a low-pressure mercury lamp, an excitation filter, a quartz flow cell, an emission filter, a photomultiplier tube and a photoconducter in order to compensate for fluctuations in lamp intensity. Fluorescence was examined at a direction perpendicular to that of the excitation light. The cell was small enough for it to be attached directly to the end of the column with a minimum dead volume. 

A fluorescence spectrometer (or spectrofluorimeter) is provided with two monochromators to study both the excitation and fluorescent spectra. The two spectra are used in the elucidation of structure and identification of the molecule, as well as in defining the optimum conditions for quantitative determination. A fluorimeter uses filters in each beam. For the observation of luminescence decay, shutters are interposed alternately in the primary and secondary beams. 

Luminescence affords a very sensitive means of detection in flowing systems such as HPLC, electrophoresis, flow injection, and flow cytometry. HPLC fluorescence detectors are similar in operation to conventional fluorimeters. Most fluorescence detectors use filters for crude monochromation. Filters pass light in a wider band than do monochromators. This favors spectral sensitivity because more light excites the sample and is collected by the detector. Grating monochromators, on the other hand, favor selectivity. The fluorimetric detector is susceptible to the usual interferences that hinder fluorescence measurements, namely, background fluorescence and quenching. 

Figure 3.12. Corrected fluorescence spectra of 4 29 SSB. Data were obtained at a fina concentration of 8 pM in bufTei- B at 2S C in the absence thick curves) and presence thin curves) of saturating amounts of ssDNA (300 pM poIy(dT). The excitation (21 - 308 nm) and emission spectra (A 276 nm) were corrected for background emission, inner filter effect of DNA absorption and wavelength dependence of the fluorimeter response. Source Soengas, M. S., Reyes Mateo, C Salas, M., Ulises Acuna, A and Gutidrrez, C 1997, J. Biol Chan. 272. 29S-302. Autborlnation of reprint accorded by the American Society for Biochemistiy and Molecular Biok). ...

Laser Fluorimeter As 2i source of biological information we propose the use of a multi-station (up to 12 sampling locations) towed sea water laser fluorimeter for water quality analysis specific to selected hydrocarbons which might be present in the area. The laser excites elements of the plankton population and that of calibrated hydrocarbons (e.g. breakdown products of munitions contents) present in the water. The fluorescent spectra are received through a fibre optic cable, split and counted through specific filters. From this data a direct correlation of the effects of pollution on the plankton population can be made. The system would be towed in conjunction with the multi-sensor towed array. 

In simple fluorimeters where a limited number of analytes are measured a bandpass or cut-olf filter may be used to. select the fluorescence photons. If a... 

Dilute or extract the sample with 0 1N sulphuric acid to give a final concentration of quinine suitable for the fluorimeter being used usually about 0 5 //g per ml is satisfactory. The excitation radiation must be within the range 300 to 400 mju and is most conveniently obtained by the use of a high-pressure mercury vapour lamp and a filter to isolate the line at 355 m. A secondary filter transmitting light between 420 and 450 m/z is used between the fluorescent solution and the detector. Calibrate the instrument with a standard solution or solutions of quinine in 0 1 N sulphuric acid and measure the fluorescence intensity of the sample. 

Using a primary filter 0X1 and an OB2 in conjunction with an OY13 as a secondary filter calibrate the fluorimeter with one of the standard solutions. Measure the fluorescence of the sample solutions and the remaining standard solutions. 

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