PerkinElmer 200 UV/VIS Series, User Manual

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Principles of Operation Theory of Operation
8 - 4
Optical Design
In order to make an absorbance determination, two measurements must be made, I
0
and I.
The ratio I/I
0
defines transmittance. In a single beam absorption detector, the measurement
of I
0
is made with no sample in the light beam, and I is measured after the sample is placed
in the beam. This is a less than ideal situation for the continuous flow detectors used in
HPLC. First, it requires periodic removal of the flowcell in order to measure I
0.
Second, it is
not possible to correct for any instantaneous change in I
0
when I is being measured. To
account for these problems, the Series 200 UV/VIS LC Detector uses a dual-beam design. A
reference beam is used to account for any changes in intensity independent of the sample in
the flowcell, and a sample beam is used to measure intensity changes that result from
absorption by the flowcell contents. The following equations result:
Absorbance =- log (Transmittance)
=- log (I/I
0
)
= log (I
0
/I)
The Series 200 UV/VIS LC Detector's dual-beam optical design consists of either a deute-
rium (for 190-360 nm) or tungsten (for 360-700 nm) light source, a patented monochromator,
a flowcell and reference cell, and a pair of matched silicon photodiodes. The dual-beam
design is a primary reason for the very high stability of the detector.
Light from the source lamp passes into the monochromator through an entrance slit, which
directs the energy onto a pair of curved collimating mirrors. Each is adjusted to reflect at a
slightly different angle so that two independent beams are created as the light from the
lamp (source) strikes the mirrors (see Figure 3-2).
The two beams are next reflected onto a diffraction grating, which reflects and disperses the
polychromatic light of each beam into its component wavelengths. The grating is rotated by
a mechanism linked to the stepper motor drive. Different grating angles reflect different
wavelengths through the monochromator exit windows. When a wavelength value is
entered, the stepper motor moves to adjust the grating angle so that the desired wavelength
is reflected to the exit windows and then passed through the sample and reference cells.