Principles of Spectrophotometry

Another very common analytical laboratory technique is the measurement of the concentration of a compound, reaction component, or reaction product (or its rate of appearance or disappearance) by measuring its absorbance of a specific wavelength of light. A spec-trophotometer is an instrument that can produce light at a selected wavelength, direct it through a sample (typically held in a cuvette), and measure the intensity of light transmitted by the sample. Many types of spectrophotometers exist. A UV/vis spectrophotometer, a type found in most laboratories, can produce light in both the ultraviolet (UV) spectrum (~190-340 nm) and the visible (vis) spectrum (340-800 nm).

The absorbance of a particular compound in a solution can be detected using a spectro-photometer. For example, NADPH2, a co-factor produced by activity of the enzyme glucose-6-phosphate dehydrogenase, absorbs light at the wavelength of 340 nm. Thus, an increase in NADPH2 of a solution can be detected as an increase in the absorbance of the solution at 340 nm. The peak absorbance wavelength has been determined for many substances.

The actual concentration of a chemical can be determined by its absorbance at a given wavelength, such as 340 nm for NADPH2, using the Beer-Lambert Law represented by the mathematical equation A = s l c. In this equation, A is the absorbance of a solution at a given wavelength of light. The term s is a proportionality constant that defines the extent of absorption. It is called the molar extinction coefficient or the molar absorption coefficient, and is typically given in the units cm1 and M"1. The term l is the path length of light through the sample (usually 1 cm), and the term c is the concentration of absorbing material in the sample (usually in moles/liter, or M). Molar extinction coefficients have been determined for a great many compounds at specific wavelengths and environmental temperatures. The chromato-graphic technique HPLC combines high-pressure column chromatography with a spectro-photometer so as to detect specific chemicals by their characteristic absorption at given light wavelengths as they are eluted from the column and pass through the light beam.

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