The Basics of Spectrophotometry

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The Basics of Spectrophotometry

Color is everywhere in our daily lives. Did you know you can actually measure color? The spectrophotometer is an essential tool for biologists and technicians when analyzing chemical and biological samples.

In simple terms, colors are dependent on light. We do not actually see colors rather, what we see as color is the effect of light shining on an object. When white light shines on an object, it may be reflected, absorbed, or transmitted. Glass transmits most of the light that comes into contact with it, thus it appears colorless. Snow reflects all of the light and appears white. A black cloth absorbs all light, and so appears black. A red piece of paper reflects red light better than it reflects other colors. Most objects appear colored because their chemical structure absorbs certain wavelengths of light and reflects others.

When discussing light, we are usually referring to white light. A thin line of light is called a ray; a beam is made up of many rays of light. When white light passes through a prism (a triangular transparent object) the colors that make up white light disperse into seven bands of color. These bands of color are called a spectrum. Seven colors constitute white light: red, orange, yellow, green, blue, indigo, and violet. In any spectrum, the bands of color are always organized in this order from left to right.

Suppose we shine a beam of white light at a substance that absorbs blue light. Since the blue component of the white light gets absorbed by the substance, the light that is transmitted is mostly yellow, the complementary color of blue. This yellow light reaches our eyes, and we “see” the substance as a yellow colored substance.

The color variation of a system that undergoes a change in concentration of some component is the basis of colorimetric analysis.

What is Colorimetery?

Colorimetry is simply the measurement of color. Colorimetry is the determination of the concentration of a substance by measurement of the relative absorption of light with respect to a known concentration of the substance. In visual colorimetry, natural or artificial white light is generally used as a light source and determinations are usually made with a simple instrument termed a colorimeter or color comparator. When the eye is replaced by a photoelectric cell, the instrument is termed a photoelectric colorimeter.

A colorimetric analysis is based on the principle that many substances react with each other and form a color which can indicate the concentration of the substance to be measured. When a substance is exposed to a beam of light intensity (Iâ‚€) a portion of the radiation is absorbed by the substance’s molecules and a radiation of intensity (I) is emitted. This difference in intensity is used for the colorimetric determination.

The quantity of radiation absorbed is given by the Beer-Lambert Law:

 

A = Ɛ · l · C

 

A    is absorbance

Ɛ   is the molar extinction coefficient [L/(mol·cm)]

l     is path length (cm)

C     is concentration (mol/liter)