Fluorimeters fluorescence detector

Certain compounds, whether present in solution or in solid state (as molecular or ionic crystals) emit light when they are excited by photons in the visible or near ultraviolet domain of the spectrum. This phenomenon, called luminescence, is the basis of fluorimetry, a very selective and sensitive analysis technique. The corresponding measurements are made with fluorimeters or spectrofluorimeters and, for chromatographic applications, with fluorescence detectors. 

Useful features in a fluorescence detector include a 96-well plate format, internal thermostatted chamber, variable wavelengths for excitation and emission, and user-friendly software for analysis of kinetic assays. For some applications, an older-style, single-cuvet fluorimeter can be used, but this is completely impractical when... 

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. 

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 post-column configuration has also been used in conjunction with fluorescence. Typically, this involves depositing the eluent on a membrane, or collecting fractions, as is commonly done in HLPC. The resulting spots or fractions can then be imaged with an appropriate fluorimeter, fluorescence microscope, or other suitable fluorescence detector. 

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). 

Fig. 7. Fluorescence polarization (FP). (a) The formation of the large FITC—protein A—IgG complex which leads to a net increase in plane polarized light transmitted from the solution. Molecular weights of the protein A-FITC, IgG, and complex are ca 43,000, 150,000, and 343,000, respectively, (b) Detection of IgG by fluorescence polarization immunoassay using A, a laboratory fluorimeter where (O) represents AP = change in polarization, and B, a portable detection unit where (D) is —fiV = change in voltage (27). The field detector proved to be more sensitive than the fluorimeter.

Once the analyte has been separated from the matrix in LC, the best approach to the detection of the molecule must be determined. This section will discuss the detection techniques of ultraviolet/visible (UVA IS), fluorescence (FL), and electrochemical (EC) detection, with MS being addressed separately in Section 4.2. When deciding on the most appropriate detector for an LC separation, the appropriate chemical data on the analyte should be collected by using a spectrophotometer, fluorimeter, and potentiometer. 

The first measurement we make when starting a fluorescence study is not usually a fluorescence measurement at all but the determination of the sample s absorption spectrum. Dual-beam differential spectrophotometers which can record up to 3 absorbance units with a spectral range of 200-1100 nm are now readily available at low cost in comparison to fluorimeters. The wide spectral response of silicon photodiode detectors has made them preeminent over photomultipliers in this area with scan speeds of a few tens of seconds over the whole spectral range being achieved, even without the use of diode array detection. 

Cassidy and Frei [23] designed a microflow cell for the Turner Assoc. Model III fluorimeter for use with HPLC. Nanogram quantities of fluorescent materials could be detected. The volume of the flow cell was only 7.5 jul. The detector was unaffected by the flow-rate or composition of the solvent. This gives this detector a decided advantage over refractive-index or UV detectors. The peak shapes were symmetrical and the linear range of response was 2-3 orders of magnitude. 

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... 

Fig. 7. Effect of immunoaffinity chromatography on HPLC-fluorescence analysis of lAA in an extract from Pinus sylvestris shoots. Column 250 X 5.0 Iran i.d. 5 p.m ODS Hypersil. Mobile phase 25 min, 25-75% gradient of methanol in I % aqueous acetic acid. Flow rate 1 ml min . Detector fluorimeter, excitation 280 nm, emission 350 nm. Samples A. acidic, diethylether extract from 0.3 g fresh weight tissue B. as A but extract subjected to immunoaffinity chromatography [60,93].

The optical layout (Figure 6.21) is similar to the conventional fluorimeter in that the detector is placed at right angles to the primary incident beam which is supplied from a xenon or deuterium source. However, due to the flow-cell dimensions and orientation only a small amount of the fluorescent energy will fall on the photodetector. Increased sensitivity is provided by the... 

For the reasons pointed out in (Banishev et al., 2008a), the same laser fluorimeter has been optimized for measuring the nanosecond fluorescence decay (the kinetic mode of the fluorimeter operation). The curve represents the dependence of the number NnfW of fluorescence photons in the detector gate (with wide tg) on the gate delay time tad with respect to a laser pulse. For the model (la) an expression for kinetic curve can be written as ... 

Estimation of true vitamin E in foods requires quantitative determination of all its components since they vary in their biological potency. This vitamin consists of four tocopherols (a, jS, y, and 6) and four tocotrienols (a, jS, y, and d), but the three major constituents responsible for vitamin E activity are the a-, jS-, and y-tocopherols. While these compounds are fluorescent, their esters must be reduced to free alcohols for total tocopherol assays. Total vitamin E can be directly obtained through fluorimetry, but the determination of individual components is carried out using LC with fluorimetric detection. This procedure has been used to determine the composition of vitamin E in seed oils from maize, olives, soya beans, sesame, safflower, and sunflower by measuring the content of all the four tocopherols plus a-tocotrienol. The simultaneous determination of tocopherols, carotenes, and retinol in cheese has been carried out using LC with two programmable detectors coimected in series, a spectrophotometer and a fluorimeter. Carotenes have been determined photometrically, and fluorimetric measurements have been obtained for tocopherol and retinol. 

Other, faster detectors that can be used in place of a conventional PMT include microchannel plate photomultiplier tubes (MCP-PMTs) (31) and streak cameras (37). Because of their expense, the use of these devices is usually confined to home built fluorimeters foimd in dedicated fluorescence laboratories, and is therefore not discussed here. 

Optical spectroscopy requires either spectrophotometers, to measure absorbance, fluorimeters, to measure fluorescence, or microscopes, which can measure fluorescence or absorbance of single cells or small groups of cells. Fluorimeters and spectrophotometers usually require solutions or suspensions of material in conventional cuvettes microscopes provide two-dimensional images from smears, slices or siufaces. Other devices that record signals resolved in two-dimensions include gel scanners and microplate readers. Essentially these devices sample the object in an organized manner (detectors can be set up to record absorbance or fluorescence) and information is stored in an electronic array that maps precisely the physical layout of the original object. 

FIA-fluorescence manifolds for the determination of acid dissociable cyanide, (a) Conventional approach, (b) Introduction of an enrichment step. Detector fluorimeter (Xex/Xem = 331/379 nm) IV injection valve OPA o-phthalaldehyde RC reaction coil. 

---