Use of a Spectrophotometer

USE OF THE SPECTROPHOTOMETER 2

INTRODUCTION

In addition to the light microscope, the spectrophotometer is usually found in the biological laboratory. This instrument allows the investigator to identify compounds of biological interest and quantify them. Identification is determined by producing an absorption spectrum of a compound (see the laboratory exercise on plant pigments, Exercise 10). Quantifying the amount of the material present, usually in solution, is done: (a) directly if the substance is a strong absorber of a measurable wavelength (8) of light; (b) indirectly by chemically modifying the compound so that it is a strong absorber at a measurable wavelength of light; or (c) indirectly by stoichiometrically coupling its reaction to the formation of another light absorbing compound.

MATERIALS

2,6-dichlorophenol indophenol, graduated cylinders, flasks, stir plates, stir bars, micropipettors, spectrophotometer tubes, rulers, spectrophotometer

A. BEER-LAMBERT LAW

Spectrophotometry, or spectrophotometric analysis, refers to the quantitative determination of the radiant energy ratio of incident to transmitted light beams at a given wavelength. Spectrophotometers are instruments that allow for the determination of this ratio. Spectrophotometers are designed to make these measurements over a given range of wavelengths of the electromagnetic spectrum. Usually, in the biological laboratory a spectrophotometer will allow for measurements in the UV and visible wavelengths.

If Pi is the intensity of the incident beam of light and P t is the intensity of the transmitted light, then, by definition, the ratio Pt/Pi equals the transmittance, T, and log Pi/Pt equals the absorbance, A. Thus:

A = -log T . . . . . . . . . . . . . . . . . . . . . . . . . . . (1)

A fundamental law of spectrophotometry is the Beer-Lambert law, or simply Beer’s Law, that states that the amount of radiant energy absorbed (log Pi/Pt) by a...