PerkinElmer 200 UV/VIS Series, User Manual

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Principles of Operation Theory of Operation
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Theory of Operation
Beer’s Law
The Series 200 UV/VIS LC Detector belongs to a class of analytical instruments known as
spectrophotometers. This class of optical instruments measures the transmittance of light
through a cell containing a solution of chemical compounds. The light which has passed
through the cell is collected and converted to an electrical current by a silicon photodiode.
The amount of current produced by the photodiode is directly proportional (within a well-
defined and specified range) to the amount of light hitting the photodiode. The spectropho-
tometer then internally converts the current to a voltage corresponding to absorbance, which
may then output to a recording device, computer, meter or display on the front panel so that
the user may monitor the instrument activity.
The Series 200 UV/VIS LC Detector, like other spectrophotometers, relies on the principle
that certain solutions absorb light in an amount directly proportional to the concentration of
solute presented to the light. Bouger and Lambert discovered that when light was passed
through a thin layer of material, a portion of that light energy was absorbed and the remain-
der was transmitted. The amount of light transmitted was proportional to the thickness
(pathlength) of the material and was dependent on the material composition and the wave-
length of light. An exponential relationship was developed between transmitted light and
thickness. Later on, Beer extended this principle to solutions and demonstrated that the
transmission of light in a solution is proportional to the concentration of solute and the dis-
tance the light travels within the solution (pathlength). Beer's Law states that the absor-
bance of light by a chemical compound in solution is directly proportional to:
• The concentration of the solute
• The light pathlength of the cell containing the solution
• The molar absorbtivity of the solute (i.e. the amount of light that may be absorbed by one
mole of the solute; this property is specific to the material itself)