Filter fluorometer

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

In the laboratory, solutions of analytes that fluoresce are tested by measuring the intensity of the light emitted. The instrument for measuring fluorescence intensity is called a fluorometer. Inexpensive instruments used for routine work utilize absorption filters similar to what was described previously for absorption spectrophotometers (see Figure 8.2 and accompanying discussion) and are called filter fluorometers. Two such filters are needed—one to isolate the wavelength from the source to be absorbed, the wavelength... 

Spectrofluorometer or filter fluorometer with an excitation cutoff filter <285 nm and an emission filter >320 nm... 

Alternatively, a measured volume of air drawn through an impinger containing ammonium acetate and 2,4-pentanedione formaldehyde forms a fluorescence derivative, 3,5-diacetyl-l,4-dihydrolutidine fluorescence of the solution measured by a filter fluorometer (Dong and Dasgupta, 1987). 

In vivo fluorescence Turner Designs/Model 10 Double-beam filter fluorometer 1.0% 1 0.02 s to 63% 4 1.0 s to 98% Better than 5 ppt of detection Method Ref. 14... 

Fluorescence detectors that use filters to select excitation and emission wavelengths are called filter fluorometers. This type of detector is the most sensitive, yet the simplest and least expensive. A diagram of this simple form of fluorescence detector is shown in Fig. 2. Usually, in order to enhance the fluorescence collected from the flow cell, lenses are employed along with filters. The lenses are positioned before the excitation filter and after the flow cell to focus and collect the light. 

Fluorescence was measured with a Turner model 111 filter fluorometer. The excitation filter was a Corning 7-60 (365 nm primary wavelength). The emission filters were Wratten 65-A (495 nm primary wavelength) and 2-A (sharp-cutoff below 415 nm). A digital multimeter was connected to the recorder terminals of the fluorometer to provide digital readout. Fluorescence-quenching (FQ) titrations were performed in batches. Preliminary experiments indicated that quenching was independent of time (at least 26 hours) after 30 minutes. Equilibration times of 60 minutes were used. 

The two types of fluorescence instruments are the filter fluorometer and the spectro-fluorometer the principal type of phosphorescence instrument is the spectrophospho-rimeter. 

Figure 9.3. Schematic diagram (top view) of the components of a fluorometer (filter fluorometer or spectrofluorometer). The source is a mercury-arc or xenon-arc lamp. The excitation grating or primary filter transmits only a portion of the radiation emitted by the source. Most of the exciting radiation passes through the sample cell without being absorbed. The radiation absorbed causes the sample to fluoresce in all directions, but only the emission that passes through the aperture or slit and through the secondary filter or fluorescence grating is measured by the phototube, or photomultiplier. The output of the detector is either measured on a meter or plotted on a recorder. From G. H. Schenk, Absorption of Light and Ultraviolet Radiation, Boston Allyn and Bacon, 1973, p 260, by permission of the publisher.

The usual source employed in filter fluorometers is a 4-watt mercury-arc lamp, either with a clear quartz envelope (emitting primarily 254-nm radiation) or one... 

The detector in some inexpensive filter fluorometers is a phototube, but in most of the better filter fluorometers and in all spectrofluorometers a high-gain photomultiplier tube is used. The photomultiplier is far more sensitive to low radiation levels and is therefore recommended for trace analysis. 

In a filter fluorometer the output of the detector is usually displayed on a meter. In a spectrofluorometer, the output is displayed on a recorder to give the excitation and emission spectra. An oscilloscope may also be used, particularly when decay rates are to be measured. 

Filter fluorometers can be used in automated analytical systems, for example the Technicon AutoAnalyzer . 

Components of an Uncorrected Spectrofluorometer. The design of filter fluorometers and spectrofluorometers has already been discussed here, we shall describe in more detail the components of an uncorrected spectrofluorometer (see Fig. 9.7). 

Obviously, a medium-priced spectrofluorometer could have been equipped with a xenon-arc source and used for the analysis, but the filter fluorometer is usually preferred for routine work. In addition, the latter is more sensitive for example, the detection limit for ASA on the Turner spectrofluorometer is 10 M, whereas the detection limit using the Turner filter fluorometer is lO" M. 

In routine analysis for salicylic acid, a medium-priced spectrofluorometer again could have been used. Since the xenon arc has a higher intensity at 308 nm than at 254, a spectrofluorometer with a xenon arc could give a lower detection limit for salicylic acid than would a filter fluorometer. In the analysis for salicylic acid in aspirin tablets, however, it did not prove necessary. The filter fluorometer thus provided a cheaper, more convenient, approach to routine analysis. 

Mercury or xenon arc lamps are used. A schematic of a xenon arc lamp is given in Fig. 5.42. The quartz envelope is filled with xenon gas, and an electrical discharge through the gas causes excitation and emission of light. This lamp emits a continuum from 200 nm into the IR. The emission spectrum of a xenon arc lamp is shown in Fig. 5.43. Mercury lamps under high pressure can be used to provide a continuum, but low-pressure Hg lamps, which emit a line spectrum, are often used with filter fluorometers. The spectrum of a low-pressure Hg lamp is presented in Fig. 5.44. 

Arriaga, E., Chen, D. Y, Cheng, X. L., and Dovichi, N. J., High-efficiency filter fluorometer for capillary electrophoresis and its application to fluorescein thiocarbamyl amino-acids, J. Chmmatogr. A, 652, 347, 1993. 

Filter fluorometers provide a relatively simple, low-cost way of performing quantitative fluorescence analyses. As noted earlier, either absorption or interference filters are used to limit the wavelengths of the excitation and emitted radiation. Generally, fluorome-ters are compaet, rugged, and easy to use. 

Lamps. The most common source for filter fluorometers is a low-pressure mercury vapor lamp equipped w ith a fused silica window. This source produces useful lines for exciting fluorescence at 254,302,313,546,578, 6hl, and 773 nm. fndividual lines can be isolated with suitable absorption or interference filters. Because fluorescence can be induced in most fluorescing compounds by a variety of wavelengths, at least one of the mercury lines ordinarily proves suitable. 

See Figure 27-8. A filter fluorometer usually consists of a light source, a filter for selecting the excitation wavelength, a sample container, an emission filter and a transducer/readout device. A spectrofluorometer has two monochromators that are the wavelength selectors. 

---