PerkinElmer LAMBDA 265, User Manual

Page: 9 / 61

Introduction. 9
Once a sample absorbs light energy, the intensity of incoming light P
0
(the energy from the
light source) reduces to P by losing some of its energy; therefore, we define the
transmittance of a sample as follows:
Figure 3. Sample Absorption
As a result of absorption by a sample, transmittance is defined as;
T = P / P
0
And Absorbance is defined as:
A = -log T = log( P
0
/ P )
Absorbance is related to c (concentration) and b (pathlength of light) according to the
Lambert-Beer law and the equation;
A=
εbc
Where,
ε, the molar absorption coefficient of a particular molecule, varies with the
wavelength.
Although Beer's law has several limitations, it is widely applied to quantitative analysis.
Figure 4. Transmittance and Absorbance
PDA UV-Visible Spectrophotometer
The PDA Spectrophotometer has a multi-channel detector controlled by a microprocessor
which collects spectral data for several wavelengths simultaneously. In contrast, a PMT
Spectrophotometer has a single channel detector which collects data for each specific
wavelength in turn. Therefore the PDA Spectrophotometer is much faster and gives more
reproducible results than the PMT Spectrophotometer which depends on a rotating grating or
prism operated by a stepper motor to obtain individual wavelengths.
One feature of a PDA is an integrating photo-detector, which integrates charge depending on
the light intensity. The advantage of the integrating function is named Felgette's S/N
advantage or Multi-channel advantage.