Principle of Spectrophotometer

The purpose of spectrophotometer is to detect the quantity of the samples such as DNA, RNA and proteins. It involves passing of the monochromatic light (light of single wavelength) through the sample. The reason for this is Nucleic acids and proteins absorb light at specific wavelength alone. For example

DNA - 260nm
RNA - 260nm
Proteins - 280nm

There are certain other compounds that absorb light of 230 nm. They are Guanidine and Phenol. 

In order to calculate the concentration of nucleic acids and proteins, a formula is deduced from the law called Beer lamberts law. According to this law...

The amount of radiation absorbed may be measured in a number of ways:

Transmittance, T = P / P₀

% Transmittance, %T = 100 T
 
Absorbance,

A = log₁₀ P₀ / P
A = log₁₀ 1 / T
A = log₁₀ 100 / %T
A = 2 - log₁₀ %T

The absorbance can also be formulated as below 

A=ebc

Where A is absorbance (no units, since A = log₁₀ P₀ / P )
e is the molar absorbtivity with units of L mol^-1 cm^{-1
b is the path length of the sample - that is, the path length of the cuvette in which the sample is contained. We will express this measurement in centimetres.
c is the concentration of the compound in solution, expressed in mol L-1}

The reason why we prefer to express the law with this equation is because absorbance is directly proportional to the other parameters, as long as the law is obeyed.