Filter fluorimeters

In the filter fluorimeters, the excitation light is passed through a (interference) filter of a suitable bandwidth. With a band-pass filter, the intensity of emitted light that passes through a specified wavelength is measured. 

Since the fluorescence is a factor of the intensity of the excitation light, lasers that are the light sources of highest intensity are preferred. However, since low-wavelength lasers are very expensive, the mostly used lasers are diode lasers at high wavelength, for small flow cells. 

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Schematic diagram of a simple dual-filter fluorimeter. Excitation using a xenon lamp. Filters used to select the wavelength in both the excitation and emission beams.

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... 

These are highly selective and among the most sensitive of detectors. They are based on filter fluorimeters or spectrofluorimeters (p. 376) but are usually purpose-designed for hplc or capillary electrophoresis (p. 168). The optical arrangement of a typical detector using filters is shown in figure 4.30. Excitation and emission wavelengths are selected by narrow bandpass filters. 

Figure 8. Manifold for the determination of total amino acids by FIA (29). Key S, an automatic sample changer F, a filter fluorimeter (Turner Model 111) and R, the 1,2-henzenedicarbaldehyde reagent used as the carrier stream.

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 direct determination of amino acids using ion-exchange analysers. Any standard amino-acid analyser may be modified by the addition of a sensitive filter fluorimeter to allow the determination of amino acids down to a few picomoles. The reagent employed, o-phthalaldehyde, is best prepared as given in section 2.1.3 and mixed 1 1 with the column effluent, allowing 2 min reaction time at room temperature before passing the reacted stream through the fluorimeter cell. 

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... 

The model LS-2B is a low-cost, easy to operate, filter fluorimeter that scans emission spectra over the wavelength range 390-700 nm (scanning) or 220-650 nm (individual... 

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

Generally, filter fluorimeters are more sensitive than those with monochromators. However, they lack scaiming options and hence cannot be used for investigations related to molecular structure. Nevertheless, for a number of routine analyses filter instruments are quite satisfactory. If a filter fluorimeter is used, the excitation filter should be a narrow band filter (+5nm) centred at 325 nm. On the emission side, a wide band pass filter ( 10-20 nm) centred at 420 nm should be used. Scanning fluorimeters should have excitation and emission monochromators with slit widths of 5 nm or smaller. 

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. 

Figure 5.1. Sir George Gabriel Stokes (1852) and his filter fluorimeter.

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