Chapter 2:
Advantages of Fluorescence

Chapter 3:
Instrumentation

Chapter 4:
Variables of Fluorescence

Chapter 5:
Calibration and Standards

An Introduction to
Fluorescence Measurements

Fluorescence is the molecular absorption of light energy at one wavelength and its nearly instantaneous re-emission at another, usually longer, wavelength. Some molecules fluoresce naturally and others can be modified to make fluorescent compounds. 

Fluorescent compounds have two characteristic spectra: an excitation spectrum (the wavelength and amount of light absorbed) and an emission spectrum (the wavelength and amount of light emitted). These spectra are often referred to as a compound's fluorescence signature or fingerprint. No two compounds have the same fluorescence signature. It is this principle that makes fluorometry a highly specific analytical technique. 

Fluorometry is the measurement of fluorescence. The instrument used to measure fluorescence is called a fluorometer or fluorimeter. A fluorometer generates the wavelength of light required to excite the analyte of interest; it selectively transmits the wavelength of light emitted, then it measures the intensity of the emitted light. The emitted light is proportional to the concentration of the analyte being measured (up to a maximum concentration). Fluorometers employ monochromators (a spectrofluorometer), optical filters (a filter fluorometer), or narrow band light sources like LED’s or lasers to select excitation and emission wavelengths.

Fluorometry is chosen for its extraordinary sensitivity, high specificity, simplicity, and low cost as compared to other analytical techniques. Fluorometry is ordinarily 1000-fold more sensitive than absorbance measurements. It is a widely accepted and powerful technique that is used for a variety of environmental, industrial, and biotechnology applications. It is a valuable analytical tool for both quantitative and qualitative analysis.

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