64
Circular Dichroism in Protein Analysis
light,
I
0
, does not appear for calculation
of
absorbance,
so
there
is
no
need
for a reference beam in measurements.
However, a new double-beam instrument
made by OLIS using sample and reference
beams permits direct calculation of the
CD signal. This technique reduces drift
over
time
and
factors
such
as
lamp
fluctuations are negated, since they affect
both beams equally.
The light source is usually a 150 or 450 W
xenon arc, which provides a continuous
spectrum from the edge of the vacuum
UV into the infrared region. Linearly
polarized light from a monochromator
passes through a modulating device, a
photoelastic modulator, which converts
the
plane-polarized
light
to
circularly
polarized light, alternating between left-
and right-circularly polarized light at the
modulation frequency. In the detection
end of the spectrometer, the light intensity
is also modulated at the same frequency as
the incident light.
A different modulation method, digital
subtraction, is used by OLIS. The unique
aspect of this method is that both of the two
diverging orthogonally polarized beams
are rotated by 180
. Then, the digitized
signals from the two photomultiplier tubes
are combined in such a way as to yield
the CD signal directly and to cancel
the contributions of noise. So, in this
system, the absolute CD is measured and
no calibration is required. However, one
shortcoming is that the two beams pass
through different parts of the sample and
the cuvette; the signiFcance of this is
still uncertain.
The cooling system of the instruments
is usually water or a narrow bore central
heating system pump with car radiator
cooling fluid. If it is possible, an air-
cooled lamp may give better performance.
Nitrogen flow is needed to avoid the
creation
of
ozone,
which
reacts
with
surfaces of the mirrors. Nitrogen may
also
keep
oxygen
from
entering
the
sample compartment, as it absorbs the
incident light.
High quality quartz cuvettes, which
transmit the full wavelength range of
UV–vis light, are required for CD ex-
periments. Cuvettes have rectangular and
cylindrical shapes. A cylindrical cuvette
has lower birefringence than a rectangular
shaped one. However, rectangular cells are
more often used especially in titration ex-
periments. Different pathlength cuvettes
are used for measurements at different
wavelengths. Ten and 1 mm pathlengths
cannot be used for wavelengths under 200
and 190 nm respectively. ±or shorter wave-
lengths, and for more strongly absorbing
solvents at longer wavelengths, cells of
0.1 or 0.05 mm are necessary. ±or these
cells, as the total volume is small, a higher
concentration is demanded to keep ab-
sorbance of more than 1.0.
5.2
Measurements
±or measurements in the far UV, solvent
absorbance is not negligible, even with
aqueous
solutions.
It
is
necessary
to
check
the
CD
signal
as
a
function
of concentration to have a maximum
absorbance of less than 1.5. If a linear
relationship of absorbance as a function of
concentration is not there, then there are
solute–solute interactions in the sample.
Abase
l
inespec
t
rumshou
ldbeco
l
lec
ted
of solvent and buffer under the same
conditions as the sample spectrum.
The spectrometer scans from longer to
shorter wavelengths. Initially, the examin-
ing wavelength should be set 20 nm longer
than that demanded by the experiment and
it should be established that in this region
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