**3. Applications**

224 Macro to Nano Spectroscopy

Cuvettes may be circular, square or rectangular (the latter being uncommon), and must be constructed of a material that will transmit both the incident and emitted light. Square cuvettes or cells will be found to be most precise since the parameters of path length and parallelism are easier to maintain during manufacture. However, round cuvettes are suitable for many more routine applications and have the advantage of being less expensive. The cuvette is placed normal to the incident beam. The resulting fluorescence is given off equally in all directions, and may be collected from either the front surface of the cell, at right angles to the incident beam, or in line with the incident beam. Some instruments will provide the option of choosing which collecting method should be employed, a choice based upon the characteristics of the sample. A very dilute solution will produce fluorescence equally from any point along the path taken by the incident beam through the sample. Under these conditions, the right-angled collection method should be used since it has the benefit of minimizing the effect of light scattering by the solution and cell. This is the usual

Fluorescence is a very sensitive technique. This is the one criterion that makes it a viable replacement to many radioisotope-labeling procedures. However, it is extremely susceptible to interference by contamination of trace levels of organic chemicals. Potential sources of contamination are ubiquitous since any aromatic organic compound can be a possible source of fluorescence signal. For example, the researcher is a possible source of this type of contamination since oils secreted by the skin are fluorescent. Good laboratory procedure is essential in preventing solvents and chemicals from becoming contaminated with high background fluorescence that could hinder low-level measurements. Solvents should be of the highest level purity obtainable commercially. In addition, care must be taken to eliminate all forms of solid interference (suspended particulates such as dust and fibers). These will float in and out of the sampling area of the cuvette via convection currents, and cause false signals due to light scattering while they remain in the instrument's beam.

Fluorescence spectra and quantum yields are generally more dependent on the environment than absorption spectra and extinction coefficients. For example, coupling a single fluorescein label to a protein reduces fluorescein's quantum yields ~60% but only decreases its molar extinction coefficient by ~10%. Interactions either between two adjacent fluorophores or between a fluorophore and other species in the surrounding environment

Many environmental factors exert influences on fluorescence properties. All fluorophores are subject to intensity variations as a function of temperature, pH of the aqueous medium, and solvents polarity. In general fluorescence intensity decreases with increasing temperature due to increased molecular collisions that occur more frequently at higher temperatures. These collisions bleed energy from the excited state that produces fluorescence. The degree of response of an individual compound to temperature variations is unique to each compound. While many commercially available dyes are selected for their temperature stability, care should be taken to eliminate exposure of samples to drastic temperature changes during measurement. If possible, the temperature of instrument's sample compartment should be regulated via a circulating water bath. At lower assay temperatures, higher fluorescence signal will be generated. It has been found that a 50%

measuring condition in analytical procedures.

can produce environment-sensitive fluorescence.

**2.3 Sample preparation** 

In this section, the applications of fluorescence spectrophotometry as a powerful tool for quantitative analysis, characterization, and quality control in different fields will be reviewed and discussed in details. This section will include the use of fluorescence spectrophotometry as a powerful spectroscopic tool in several fields of science.
