Department of Biology
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Theory of Spectrophotometry
Many compounds absorb light, but not all absorb the same amount, or the same colors (wavelength). Some absorb mainly in the visible spectrum (see below) while others absorb mainly in the ultraviolet or infrared range. Here we are mainly interested in measuring absorbance in the visible range.
When light passes through a solution some of it will strike molecules and be absorbed, thus the amount coming out the other side is
less than the amount that entered. Note that there are two ways of looking at this: you can either think about how much light makes it
through the solution (transmittance) or how much gets absorbed by the molecules (absorbance). Clearly the two are related, the
transmittance is simply the amount not absorbed and vv. However, the two are measured differently: transmittance is expressed at the
percentage of light that passes through, while absorbance is expressed as:
There are three sources of absorbance, the container (usually a clear glass test-tube or cuvette), the solvent which is nearly always water, and the dissolved molecules. In most cases it is only the last that is of interest. To ensure that the container and solvent do not interfere with our readings we use a ‘blank’ which is a tube containing only solvent as a reference point. By defining the amount of light that passes through the blank as 100% transmission we can assume that any reduction in the sample tube is due to the dissolved molecules.
Each type of molecule absorbs each wavelength of light in a characteristic pattern. In general you can make a good guess about a solution's absorbance pattern by looking at the color of the solution: the color it appears is the color that is NOT absorbed. So a blue solution is absorbing all the colors except blue. As you will find out, this does not mean that all blues are identical, nor that the absorbance of other colors is necessarily complete.
Two other factors determine the amount of light absorbed, the length of the light path through the solution and the concentration of molecules in the solution. The first is seldom a factor, since we always use the same size tubes, the length of the light path is always the same. The effect of concentration is usually what we are looking at in the lab. Suppose you dissolve different amounts of a red dye and measure the amount of blue light that passes through, or is absorbed. Typical results are shown below.
Note that the absorbance rises linearly with concentration, while transmittance decreases in a non-linear fashion. The linear relationship between absorption and concentration is described mathematically by the Lambert-Beer law: A = Ecl, where A = absorbance, E = extinction coefficient (inverse of the slope of the line), and l = light path, in cm (usually = 1 cm). It is this linear relationship between absorbance and concentration that make absorbance useful in the lab; if one solution shows twice the absorbance of another, then its concentration is twice as high.