PerkinElmer 200 UV/VIS Series, User Manual

Page: 159 / 177

Theory of Operation Principles of Operation
8 - 3
Figure 8-1. Diagram showing Beer’s Law.
Beer’s Law can be expressed in mathematical form as follows:
Absorbance = log (I
o
/I) = abc
I
o
= Intensity of light beam at entrance of flowcell
I = Intensity of light at exit of flowcell
a = Molar absorptivity
b = Cell pathlength
c = Solute concentration
When a and b are held constant (which is the case in most HPLC
applications), the absorbance measured should be directly propor-
tional to solute concentration in the cell. The linear relationships pre-
dicted by Beer’s Law remain strictly true only for monochromatic
(single wavelength) light beam. Also, the linear relationship between
absorbance and concentration holds only at low absorbances; at high
absorbance levels, this relationship usually becomes nonlinear. Fur-
thermore, in the chromatographic process, there are other factors
that contribute to nonlinearity. These include sample preparation,
injection, loading factors, reversible and nonreversible adsorption on
the column, wide spectral bandwidth and high background absor-
bance of the mobile phase. The chromatographer should therefore
control these parameters as carefully as possible.