Agilent Technologies Performing the EP stray User Manual
Technical Note
Performing the EP stray-light test
with potassium chloride on
UV-visible spectrophotometers
Introduction
Measured stray light has two components:
• light coming from the light
source of the instrument that
lies outside of the bandwidth
of the selected wavelength, λ
n
• ambient light that reaches the
detector either directly or by
simple reflections, see figure 1
For instruments with reversed
optics such as the Agilent 8453
UV-visible spectrophotometer
1
ambient light can not reach the
detector, see figure 2. The equation used to calculate transmittance and thereby absorbance is:
T = (I + Is)/(I0+ Is)
Where T is transmittance, I0is
intensity of incident light, I is
intensity of transmitted light and
Isis intensity of stray light. Stray
light has an increasing influence
on spectroscopic measurements at
low levels of intensity, that is, high
absorbances.
The result is that stray light causes a negative bias in instrument
response and eventually becomes
the limiting factor for absorbance
and therefore concentration that
can be measured. The effect of
various levels of stray light on
measured absorbance compared
with actual absorbance is shown
in Figure 3.
This Technical Note examines the
influence of sample composition,
sample temperature, bandwidth
and wavelength accuracy on the
result of the stray-light test using
potassium chloride as described in
the European Pharmacopoeia
(EP).
2
Figure 1
Terminology used when describing stray light
Ambient light
λ
n-1
λ
n
λ
n+1
Sample
Source
Ambient light
Detector
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Measurement of stray light
To measure stray light, a filter is
required that absorbs all light of
the wavelength at which the measurement is to be made and transmits higher and lower wavelengths. Figure 4 shows this ideal
stray light filter. At the measured
wavelength (200 nm) the transmission is 0% whereas at all other
wavelengths it is 100%. Such filters
do not exist in practice, so cut-off
filters are used which transmit all
light above a certain wavelength
and block all light at lower wavelengths.
Salt solutions, for example, potassium chloride (12 g/l), sodium
iodide (10 g/l) and sodium nitrite
(50 g/l) in water, can be used as
standard stray-light filters at 200,
220 and 340 nm respectively (see
figure 5).
The user should keep in mind that
all 3 filters are only approaches to
the ideal test filter. The slope of
the absorption edge shows no infinite value like the ideal filter does.
In addition the used filters block
all light from wavelengths shorter
than the measured one. The contribution of stray light that might
result from these wavelengths is
thus eliminated. This leads to
smaller stray light values than
would be expected from the ideal
filter. The user should be aware of
this systematic deviation as a consequence of using non-ideal stray
light filters.
Source
Ambient light
Detector
Grating
Entrance slit
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
True absorbance [AU]
Measured absorbance
[AU]
0% Stray light
0.01% Stray light
0.1% Stray light
1% Stray light
Figure 3
The effect of stray light on measured sample absorbance
Figure 2
Simplified schematic of a diode-array spectrophotometer
The EP stray-light test
Stray-light measurement with the
Agilent 8453 UV-visible spectrophotometer comprises the
three test described above. The
potassium chloride test is speci-
fied in the EP2:
“…Stray light may be detected at
a given wavelength with suitable
filters or solutions: for example
the absorbance of a 12 g/l solution of potassium chloride in a 1
cm cell should be greater than
two at 200 nm when compared
with water as the compensation
liquid.”
An absorbance of greater than two
means a transmittance of less than
one percent. For a number of reasons the potassium chloride test is
the most critical of the three straylight tests. As shown in figure 5,
this is the only test in which the
measured wavelength is situated
very close to the cut-off slope. As
a consequence the test is extremely sensitive to the wavelength
accuracy of the spectrophotometer. Even small deviations will
result in test failure. For this reason a wavelength recalibration
should be performed before each
stray-light test.
Further, the measurement time is
also relevant. When measuring the
transmittance with the test solution in the light path the intensity
at the detector is very low. The
signal-to-noise ratio can be
improved by increasing the integration time of the spectrophotometer. All three stray-light tests
with the Agilent 8453 spectophotometer were performed using
integration times of 5 s.
0
20
40
60
80
100
100 200 300 400 500 600 700 800 900 1000
Wavelength [nm]
Transmittance
[%]
0
20
40
60
80
100
200 250 300 350 400 450 500 550 600
Wavelength [nm]
Transmittance
[%T]
NaNO
2
NaIKCl
Figure 5
The spectra of potassium chloride (12 g/l), sodium iodide (10 g/l) and
sodium nitrite (50 g/l) in water
Figure 4
The ideal spectrum of a stray-light filter at 200 nm
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