Measuring the Stray Light Performance

of UV-visible Spectrophotometers
Technical Note
Typically the intensity of stray
light is not dependent upon the
intensity of the transmitted light,
however if it remains near
constant it becomes the dominant
term at low levels of I. The result
is that, at high absorptions, stray
light causes a negative bias in
instrument response and
eventually is the limiting factor for
absorbance and therefore
concentration that can be
measured. This compromises the
photometric accuracy of the
instrument. The effect of various
levels of stray light on measured
absorbance compared with actual
absorbance is shown in Figure 1
(page 2).
Introduction
Stray light is defined as detected
light of any wavelength that is out-
side the bandwidth of the selected
wavelength. The equation used to
calculate transmittance and there-
fore absorbance is:
T = (I + I
s
)/(I
o
+ I
s
)
where:
T = transmittance
I
o
= intensity of incident light
I = intensity of transmitted light
I
s
= intensity of stray light
To measure stray light, some kind
of filter is required that absorbs all
light of the wavelength at which
the measurement is to be made
and transmits higher and lower
wavelengths (the sources of the
stray light) as shown in Figure 2
(page 2). In practice such filters
do not exist, so cut-off filters
which transmit all light above or
below a certain wavelength and
block all light in the wavelength
range, are used. For example, fig-
ure 3 (page 3) shows the spectrum
of NaNO
2
.
This Technical Note examines
some aspects of the methods for
assessing stray light for UV-visible
spectrophotometers.
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Measurement Procedures
In practice there is no standard
procedure for measuring stray
light. However, the following
procedures are frequently used.
Procedure A
Procedure A is the one specified
by the ASTM.
1
It is used by several
UV-visible spectrophotometer
manufacturers.
1. Measure reference/balance with
nothing in the sample area
(that is, on air).
2. Block the light beam path and
measure % transmittance at the
test wavelength.
3. Insert cell with appropriate test
solution and measure transmit-
tance at the test wavelength.
4. Subtract the % transmittance
value measured for the blocked
beam from the measured value
for the test solution. This is the
amount of stray light.
Procedure B
Procedure B is the one most com-
monly applied by instrument
users.
1. Measure reference/balance with
nothing in the sample area
(that is, on air).
2. Insert cell with appropriate test
solution and measure transmi-
tance at the test wavelength.
This is the amount of stray
light.
Procedure C
Procedure C is the one prescribed
by the European Pharmacopoeia
and the procedure that Agilent
uses to specify stray light of spec-
trophotometers. This is because it
is the closest procedure to actual
sample measurements and gives
the most relevant stray light
values.
1. Insert cell with appropriate
solvent (usually water) and
measure reference/balance.
2. Insert cell with appropriate test
solution and measure transmit-
tance at the test wavelength.
This is the amount of stray
light.
The stringency of the three proce-
dures is such that:
A < B < C
Figure 1
The effect of stray light on measured sample absorbance
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
0.01% Stray ligh
1% Stray light
0% Stray light
0.1% Stray light
Measured
Absorbance
Figure 2
The ideal spectrum of a stray light filter
Wavelength [nm]
200 250 300 350 400 450 500
0
20
40
60
80
100
600 700
Transmittance
[%]
This is due to the fact that stray
light is a relative measurement of
the intensity measured with the
test sample in place compared to
the reference measurement with-
out the test sample. Measuring the
reference on cuvette/solvent
results in a lower reference
intensity than measuring on air.
Thus procedure C will give a
higher stray light than procedures
A and B. Secondly, measuring and
subtracting the apparent
transmission when the light beam
is blocked will reduce the
measured stray light for any
electronic bias which may be
present. Thus A will give a lower
stray light result than B or C.
It should also be noted that the
time of the measurement is also
relevant. When measuring the
transmittance with the test
solution in place the signal is very
low and the signal-to-noise ratio
will be poor. When using a short
measurement time the results may
vary significantly due to the noise.
Some manufacturers recommend
taking an average value measured
over as much as 60 seconds.
Agilents stray light measurement
is made using a time of 5 seconds.
Measured Values
To illustrate the effect of these
different procedures stray light
was measured at 340 nm using
NaNO
2
solution. Table 1 shows the
results of measuring stray light on
a typical Agilent 8453 diode-array
spectrophotometer and a similar
specification conventional
scanning spectrophotometer using
the above procedures.
The results show firstly that
Agilent's specification is very con-
servative in comparison to typical
performance. Secondly it shows
that when comparing specifica-
tions between different instru-
ments it is important to know how
the measurements were made. In
this case, Agilents specification
for stray light is apparently worse
than for the other instruments but,
when identical measurement
methods are used, its stray light
performance is, for this sample,
better.
A simple comparison of instru-
mental specifications can be very
misleading.
It should also be noted that the
amount of stray light is wave-
length and sample dependent.
Thus stray light measurements
made at one wavelength using a
stray light standard can only be
used for comparative purposes.
References
1
ASTM E 387-84, Standard Test
Method for Estimating Stray
Radiant Power Ratio of
Spectrophotometers by the
Opaque Filter Method.
Cell Heading Agilent 8453 Instrument A
Specified stray light < 0.05 % < 0.02 %
Measurement procedure C A
Measured stray light
Using procedure A 0.0059 0.0079
Using procedure B 0.0136 0.0174
Using procedure C 0.0126 0.0250
Table 1
Specifications and actual measured stray light at 340 nm for the Agilent 8453 diode-array
spectrophotometer and a conventional scanning spectrophotometer
Wavelength [nm]
200
300 400 500 600
700
Transmittance
[%]
0
20
40
60
80
100
Figure 3
The spectrum of NaNO
2
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Publication Number 5965-9503E