Fluorimeters

Some features of the spectra obtained with conventional equipment are illustrated with unsaturated hydrocarbons as fluorophores that have been adsorbed from gas or liquid phase on highly porous metal oxide powders as scattering substrates. 

The weak 5i-band dominates and the strong 52-band is completely misrepresented. All these facts will be discussed more detailed in Section 8.6 on quantitative fluorimetry. 

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Larger molecules generally caimot be studied in quite the same way, as an electric discharge merely breaks them up into smaller molecules or atoms. In such a case excited states are usually produced by optical excitation using light of the same or higher energy. Many modem fluorimeters are made with two... 

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.

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... 

The simpler fluorimeters are manual instruments operating only at a single selected wavelength at any one time. Despite this they are perfectly suitable for quantitative measurements, as these are almost always carried out at a fixed wavelength. The experiments listed at the end of this chapter have all been carried out at single fixed wavelengths. 

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. 

Calcein solution. Dissolve sufficient calcein, or its disodium salt, in the minimum amount of 0.40M potassium hydroxide solution and dilute with water to give a concentration of 60 mg L"1 in a graduated flask. A small amount of EDTA solution (about 1.0 mL of 0.03M for every 100 mL calcein solution) may be needed in the calcein solution to achieve balancing of the blank on the fluorimeter. This is only necessary in those cases in which the potassium hydroxide used is found to contain a small amount of calcium impurity. 

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. 

For FL detection, maximum emission and excitation wavelengths are determined using a fluorimeter. Stoev used fluorescence detection to analyze for closantel (excitation at 335 nm, emission at 510 nm) residues in animal tissue. 

Colored and fluorescent dyes have the advantage of being relatively cheap and easy to use. Standard procedures are available for detection of the dyes using colorimeters and fluorimeters. Some of these instruments can be used in the field to analyze samples as they are collected following exposure to the dyes. Fluorescein has been widely used for studying spray deposition within and outside canopies. ... 

Many different detectors are used in RPLC, including ultraviolet-visible spectrophotometers (UV-VIS), refractive index (RI) detectors, electrochemical (EC) detectors, evaporative light-scattering detectors, fluorimeters, and... 

For the investigation of triplet state properties a laser flash photolysis apparatus was used. The excitation source was a Lambda Physik 1 M 50A nitrogen laser which furnished pulses of 3.5 ns half-width and 2 mJ energy. The fluorescence decay times were measured with the phase fluorimeter developed by Hauser et al. (11). 

Fluorescence Lifetimes. Fluorescence lifetimes were determined by the phase shift method, utilizing a previously-described phase fluorimeter. The emission from an argon laser was frequency doubled to provide a 257 nm band for excitation. Fluorescence lifetimes of anisole and polymer 1 in dichloro-methane solution were 2.2 and 1.4 nsec, respectively. Fluorescence lifetimes of polymer films decreased monotonically with increasing DHB concentration from 1.8 (0) to 0.7 nsec (9.2 x 10 3 MDHB). Since fluorescence lifetimes (in contrast to fluorescence intensities) are unaffected by absorption effects of the stabilizer, these results provide direct evidence in support of the intensity measurements for RET from polymer to stabilizer. 

On continued excitation in the phase fluorimeter, the fluorescence lifetime of polymer 1 films also decreased with time. The lifetime decrease was exponential with an average loss constant of 8.2 1.2 x 10 lf sec-1 (1.5 pm thick film) from measurements at different sites on the film. These findings constitute direct evidence for RET from the polymer to a photoproduct(s) in support of the fluorescence intensity measurements. 

Fluorescence Studies. Fluorescence spectra of films on glass plates were obtained with a Perkin-Elmer MPF-3 spectro-fluorimeter. A previously-described phase fluorimeter was utilized for fluorescence lifetime determinations. 

The first photoelectric fhiorimeter was described by Jette and West in 1928. The instrument, which used two photoemissive cells, was employed for studying the quantitative effects of electrolytes upon the fluorescence of a series of substances, including quinine sulfate [5], In 1935, Cohen provides a review of the first photoelectric fluorimeters developed until then and describes his own apparatus using a very simple scheme. With the latter he obtained a typical analytical calibration curve, thus confirming the findings of Desha [33], The sensitivity of these photoelectric instruments was limited, and as a result utilization of the photomultiplier tube, invented by Zworykin and Rajchman in 1939 [34], was an important step forward in the development of suitable and more sensitive fluorometers. The pulse fhiorimeter, which can be used for direct measurements of fluorescence decay times and polarization, was developed around 1950, and was initiated by the commercialization of an adequate photomultiplier [35]. 

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.

Fluorimeters have similar components to UV/visible spectrometers but differ in the geometry of the radiation beams and in the need to be able to select or scan both excitation and emission wavelengths. 

Luminescence instrument LS-3B luminescence instrument LS-5B Accessories low flow cell, cell holders, bioluminescence spectroscopy, fluorescence spectro scopy, recorder/printers, low-temperature luminescence, fluorescence plate reader, polarization accessory, microfilm fluorimeter LS-2B... 

Fluorimeters Microfilm fluorimeter LS-2B Perkin-Elmer Corporation Analytical Instruments Division 761 Main Avenue... 

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