Sc.
Instrumentation M. R. Sharpe, Pye Unicam Ltd. Cambridge CB1 2PX
ife.
United Kingdom
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&
Stray
Light in UVSpectrophotonv Ultraviolet-visible (UV-VIS) spectrophotometers have been used as a tool for chemical analysis for many years. They find application in many fields, including industry, medicine, and university research. It is therefore important that such instruments be reliable in operation, convenient to use, and very accurate. Since spectrophotometers were first produced commercially their performance has steadily improved. Most currently available instruments now meet the above requirements, so that analysts can apply them to their work with confidence. In recent years one particularly noticeable change in these instruments has been the use of microprocessors to give much greater flexibility and con-
venience of operation, as well as allowing automation of many analyses. Less noticeably, there have been significant improvements in optical components, which have resulted in longer instrument life and greatly improved photometric accuracy, particularly because the effects of “stray light” have been significantly reduced. Not many years ago the UV stray-light specification for a spectrophotometer was usually about 1%. Now, however, figures of less than 0.01% are often quoted, 0003-2700/84/0351-339A S01.50/0 ic) 1984 American Chemical Society
which is such a low level of stray light that its effects can be ignored for most analyses. Not all analysts, however, have access to the latest instruments, and consequently there are in use a wide variety of spectrophotometers which vary greatly in age and performance. All users of spectrophotometers should be aware of the practical limitations of their instruments, even if they are the latest models. This article will show how stray light can affect analytical accuracy and will discuss the sources of stray light and how recent improvements in optical components have benefited instrument performance and life.
Stray Light and Its Effects on Measurement Accuracy Figure la shows the basic compoa simple single-beam spectrophotometer. The monochromator entrance slit is illuminated by a light source. The monochromator is adjusted to the analytical wavelength, and the light emerging from the exit slit is passed through a sample. The light flux transmitted by the sample is measured by a detector, usually a photocell, photodiode, or photomultiplier. If I„ light flux incident on sample and / light flux transmitted by the nents of
=
=
x
,
/
x X
\\\
'j
sample, then the transmittance T at the analytical wavelength is
T
I_
(1)
lo
in terms of absorbance units, which used because the absorbance A is proportional to the product of the sample path length and concentration, or
are
A
=
-login?’
(2)
If measurements are made over a range of wavelengths, then the absorbance spectrum of the sample can be obtained. The monochromator acts as a filter
ANALYTICAL CHEMISTRY, VOL. 56. NO. 2,
FEBRUARY 1984
•
339 A
(a)
o
I
Monochromator
©
Light Source
Detector
Sample Transmittance T
bandwidth, centered at the analytical wavelength, and a proportion a of the stray light from outside the spectral bandwidth, then the total transmitted light flux is TI0 + als. The measured transmittance TM of the sample at the analytical wavelength is then rr
TI0 T
_
~
Otlg
,n
,3)
/() + /s
Putting the fractional stray light S S
=
Is
as
(4)
Iq + Is
gives
T + S(a
T) (5) It is thus clear that stray light can give Tm
=
-
rise to a difference between the true sample transmittance T and the measured transmittance TmTo illustrate the practical effects of
Figure 1, (a) A single-beam spectrophotometer; (b) spectral bandwidth
for the light source and passes light in a small band of wavelengths centered at the required wavelength. This small range of wavelengths is determined by the monochromator spectral bandwidth, which is proportional to the monochromator slit widths. When the entrance and exit slits are of equal width, the bandwidth has approximately a triangular energy profile. The spectral bandwidth is customarily defined as the width of the triangular profile at half maximum energy, as shown in Figure lb. In practice a monochromator is not a perfect device, and it can transmit a small flux of light over the entire wavelength range of the light source.
This “unwanted” component of light flux outside the spectral bandwidth is known as stray light, and it can cause serious measurement errors for the unwary analyst. Because the light transmitted by most samples varies with wavelength, the proportion of stray light transmitted by a sample will not be equal to the sample transmittance T at the analytical wavelength. If the “wanted” light flux incident on the sample within the monochromator spectral bandwidth is /„ and the unwanted stray light flux is Is, then the total light flux incident on the sample is /„ + Is. If the sample transmits a proportion T of the light within the spectral
Figure 2, The effect of stray light on absorbance ments for several stray-light levels 340 A
•
measure-
Figure 3. Absorbance
ANALYTICAL CHEMISTRY, VOL. 56, NO. 2, FEBRUARY 1984
stray light some particular cases will be examined. a > T. This is the most common case in practice. In quantitative analysis one usually measures the absorbance of a sample at a peak of absorbance, when the sample transmittance is comparatively small. In conseT in Equation 5 is posiquence « tive, most of the stray light being transmitted. If one takes the rather ideal case when « 1 and all the stray light is transmitted by the sample, then Fig-
=
ure 2
shows the measured absorbance
function of the true absorbance, for several values of stray light. In the absence of stray light, the concentration of a sample is proportional to the measured absorbance (Beer-Lambert law). However, an instrument which, for example, has 0.1% stray light shows measurable deviations from linearity at only 1.5 A, where A absorbance units. At higher concentrations there is an increasing loss of senas a
=
error
for several stray-light levels
^
Microbore sample injection poses new problems in HPLjC. And Rheodyne solves them. People using microbore columns want to inject miniscule samples-typically only a fraction of a microliter. It’s not easy to form a sample that small with high precision. And it’s even harder to convey it to the column with low dispersion. Rheodyne solved these problems with the micro sample injection valve pictured below. The sample holding chamber is a tiny hole bored through the valve's rotor. You load the sample through a built-in
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sitivity and, consequently, measurement precision, until at 3 A increasing
sample concentration produces no further increase in absorbance. To show these effects more clearly Figure 3 shows the absorbance error as a function of the true absorbance for several values of stray light. a < T. This is a more unusual example and corresponds to a sample that absorbs relatively little light at the measured wavelength, but absorbs most of the stray light. This results in ft T being negative, and consequently the measured absorbance is higher than the true absorbance. A practical example is the spectrum of benzene vapor at about 250 nm, which is sometimes used as a spectral resolution test for instruments. Stray light can cause the absorbance minima between the benzene absorption peaks to be partially “filled in” by the positive absorbance error, apparently degrading the spectral resolution. a =si T. If the sample has the same transmittance outside the spectral bandwidth as at the analytical wavelength, then (a T) ^ 0, and there is no absorbance error. Calibrated neutral glass filters, used to measure absorbance accuracy of spectrophotometers, are an example of such a material, which has been deliberately chosen to eliminate the effects of stray light when checking photometric accuracy
From Light Source
,
Collimating Mirror
—
Focusing Mirror
from Other Orders
Figure 4. A diffraction grating monochromator, illustrating the
sources
of stray light
—
of an instrument. In conclusion, it should be noted from these examples and from Equation 5 that the effective amount of stray light is highly dependent on the absorption spectrum of the sample being measured. Instrument manufacturers usually specify the stray light where its effects are greatest in the UV, using a particular form of test sample to be discussed later. The effective stray light with other samples will usually be less than the specification figure, hut the analyst should be aware that this depends on the sample spectrum (1,2).
Origins of Stray Light Figure 4 shows a typical diffraction grating monochromator. The entrance slit is illuminated by the light source, and light from this slit is focused to a parallel beam by the collimating mirror, this beam being incident on the grating. The grating is rotated to diffract light of the required wavelength onto the focusing mirror, which in turn focuses it onto the exit slit. The main source of stray light in most spectrophotometers is usually the dispersing element in the monochromator, either a prism or a diffraction grating. Scattering of light and unwanted reflections from other optical elements can also contribute significantly to the stray light, depending 342 A
•
the relative quality of the dispersing element and how carefully the monochromator has been designed and internally baffled. The strong zero-order spectrum can be particularly troublesome when reflected and scattered at walls and mirrors. These various sources of stray light are indicated in Figure 4. Good monochromator design depends on ensuring that the mirrors and dispersing element are of high quality with little scatter and that scattered light is minimized by baffling so that it cannot reach the exit slit. In addition, light can he diffracted twice or more by the grating on reflection from the mirrors. This can also be avoided by careful design and baffling. Higher order spectra are usually removed by suitable filters. It is also possible for stray light to arise outside the monochromator, such as from light leaks in the instrument, allowing some light directly to the sample or detector from outside the instrument or directly from the light source. However, in a well-designed, well-constructed instrument this latter source of stray light should be completely negligible and will not be further considered. Most modern instruments now use a diffraction grating as a dispersing element in the monochromator, as prisms in general have a poorer stray light performance and require complicated precision cams to give a linear wavelength scale. Replica gratings can now be produced more cheaply than prisms and require only a simple sine bar mechanism for the wavelength on
scale.
ANALYTICAL CHEMISTRY, VOL. 56, NO. 2, FEBRUARY 1984
A diffraction grating of the type a UV-VIS spectrophotometer consists of a glass or silica substrate on which there is a layer of resin for a ruled replica grating or a layer of photoresist for a holographic (or interference) grating. This surface is covered with fine parallel grooves produced by the relevant manufacturing process and is finished with a reflecting layer of aluminum, which in modern instruments is also covered with a transmitting protective film to prevent oxidation or contamination of the aluminum. The profile of the grating grooves is usually a shallow triangle, with the wide faces of each groove tilted at an angle known as the blaze angle, which results in the grating having maximum diffraction efficiency at a certain wavelength, usually in the UV. Figure 5 shows electron micrographs of some gratings, and the slightly inclined wide groove faces can be clearly seen. A typical reflection grating in a UV-VIS spectrophotometer may have 1200 grooves/mm, which means the grooves are spaced at about 800-nm intervals. The grating may have a width of 20 mm or more, giving a total of at least 24 000 grooves. To obtain constructive interference across this number of grooves with little light scattering, the spacing and form of the grooves must be accurate to within a few nanometers to give a high-quality grating. Mechanical diamond ruling of gratings to near this accuracy is one of the marvels of modern technology. Holographic gratings, generated from a laser interference pattern, have exused in
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riDrinr?
lb
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(a)
Figure 5, (a) A conventional ruled grating; (b) a blazed holographic grating
tremely precise groove spacings and smoother groove surfaces, resulting in an order of magnitude or more lower stray light than can be achieved by mechanical ruling. In consequence, most modern instruments now use holographic gratings in preference to ruled gratings. For further information on gratings the reader will find a wealth of literature on the subject— for example, Reference 3.
Quantitative Aspects of Stray Light If the entrance slit of a monochromator is illuminated with monochromatic light, for example by a laser,
then as the instrument wavelength setting \j is scanned through the monochromatic wavelength Ac the energy from the exit slit appears as shown in Figure 6. This shows the output peaking at both the first and second orders of diffraction, the first order being that normally used. For a perfect monochromator one would expect the curve to be the triangular bandwidth function shown previously as Figure lb. This is shown as a dashed line. The excess energy outside the bandwidth function is the small fraction of the monochromatic light scattered in the monochromator to
give stray light at other wavelength settings. It is convenient to consider the energy from the monochromator shown as Figure 6 as a function R(A/,AC), the ratio of the energy at wavelength setting A/ to that when set to the first-order wavelength Ac, The
function R(A/,Af)
can
therefore be put
as
R(A/,Ar)
=
R„(A/,AC) + R,(A/,At.) (6)
where R0(Xi,Xc) energy within triangular bandwidth and = ft.s (A/,A(.) stray light energy =
Curves of the type shown as Figure of monochromatic wavelengths, can be used to assess the stray-light, performance of a monochromator when it is used with a
6, obtained for a series
continuum light source, as is normally the case in a spectrophotometer. In practice, however, it is difficult to relate these results to practical measurements of transmittance or absorbance on a sample, as so many other factors are involved in a complete spectrophotometer. In addition, as discussed earlier, the stray-light errors depend very much on the absorption spectrum of the sample. Figure 7 illustrates how the various components of a spectrophotometer influence its performance. If
/(A) = source spectrum E( A) grating efficiency =
M(A) = mirror reflectance P( A) detector sensitivity then the output voltage from the detector is proportional to the product of all these factors, so that at a wavelength setting A/ the detector output =
D(A/)
D(Xi)
Figure 6. Monochromator energy output when illuminated by light source 344 A
•
a
monochromatic
ANALYTICAL CHEMISTRY, VOL. 56, NO. 2, FEBRUARY 1984
is given by
J(A/)£i(A/)M"(A/)P(A/) (7) where n is the number of reflecting surfaces in the light beam. {continued on p. 348 A) =
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/(X)
Source
Energy
E(X)
Figure 8. The effect of aging flectance at 200 nm
Grating
Efficiency
on
the reflectance of aluminum at 200 nm. R
Figure 7 shows these factors for typical components: tungsten lamp and deuterium arc sources, a grating blazed in the UV, an aluminum mirror, and an S20 photocathode. The detector output D{\) can be obtained from a single-beam instrument if one takes a series of readings at wavelengths over the instrument range, without altering the zero control. Alternatively, for a double-beam instrument the same measurements have to be made with it working in a single-beam mode. Using Equations 6 and 7 it is possible to calculate how the transmittance of a sample is affected by stray light (4). If
M(\)
Mirror
Reflectance
Tm(X)
=
P{\)
Detector
T(X)
=
B
=
measured sample
transmittance true sample transmittance instrument spectral bandwidth
Sensitivity
then at wavelength X/
Tm(Xj)
=
T(X/)
+1
J_J7’(XC)
-T(X/))Ks(X/,Xc)|^jdXc D(X)
Detector
Response
Instrument
Figure 7. Performance of spectrophotometer components 348 A
•
(8)
This equation is equivalent to the simpler Equation 5 given earlier. Thus, to predict the stray-light error of a transmittance or absorbance measurement from the monochromator stray-light function R(X/,XC), a very laborious calculation is required based on difficult measurements. This emphasizes why the effect of stray light cannot be easily specified by instrument manufacturers for practical measurements on particular samples. What else can we learn from Equation 8? As in Equation 5 it shows that the transmittance error depends on
ANALYTICAL CHEMISTRY, VOL. 56, NO. 2, FEBRUARY 1984
=
re-
the sample transmittance T(X/) at the analytical wavelength and on the transmittance T(XC) of the sample over the whole instrument range. In addition it shows how the instrument response D(X) can magnify the straylight error if the analytical wavelength X/ is chosen to be where D{X]) is small, for example near the end of the usable range of a particular light source, when the stray light from other wavelengths where D(XC) is large is greatly magnified in effect by the ratio of the values of D(X). This type of effect becomes very evident when, for example, the light output of the deuterium arc source falls off with age or mirrors become
dirty or
con-
taminated, both effects resulting in D(X) decreasing in the UV and, in consequence, the UV stray light increasing in effect. It will be noticed from Equation 8 that the stray-light error is apparently inversely proportional to the spectral bandwidth B. However, because the stray-light function Rs(Xj,Xc) is also proportional to the bandwidth, as it depends on the area of slit into which light is scattered, the stray-light error in practice is largely independent of the bandwidth when a continuum light source is used. It should also be noted that the height of the monochromator slits should be kept to a minimum compatible with other requirements, such as allowing enough energy for adequate signal-to-noise ratio, so that the slit area into which stray light can be scattered is kept to a minimum (2, 4).
Recent Improvements in Stray-Light Performance In recent years there have been very significant reductions in stray-light
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by about 5% per year and at a faster rate when exposed to a flux of UV radiation similar to that from a deuteri100
Reflectance
80
-
60
-
40
-
R°/o
20
-
J_i_i-1-1—i—i—L
200
300
600
400
800 1000
\(nm)
Figure 9. The effect of UV irradiation and subsequent washing of aluminum levels quoted by the manufacturers of most commercial spectrophotometers. The two main reasons for this are the
introduction of holographic diffraction gratings and the use of mirrors coated with a protective film. Coated optics. The mirrors in a spectrophotometer are normally coated with aluminum, the metal with the
on
the reflectance
highest reflectance over the required wavelength range. Freshly deposited aluminum slowly grows a layer of aluminum oxide on its surface, which results in a progressive loss of reflectance at UV wavelengths, which is accelerated by exposure to UV radiation. Figure 8 shows how at 200 nm the reflectance of aluminum in air decreases
Figure 10. The effect of UV irradiation and subsequent washing on the reflectance of silica-coated aluminum 350 A
•
ANALYTICAL CHEMISTRY, VOL. 56, NO. 2, FEBRUARY 1984
in a spectrophotometer. instruments there are up to 12 mirror reflections before the light beam reaches the detector, so that even a 5% loss of reflectance at each mirror can halve the energy. The oxide layer growth can be prevented by overcoating the aluminum with a thin transparent layer. Some manufacturers use a layer of magnesium fluoride for this purpose, but it is not very satisfactory as it is relatively soft and has poor chemical resistance, and thus cannot be easily cleaned. A better solution to the problem is to use a silica or synthetic quartz coating, which is hard and chemically resistant. The correct coating thickness will also enhance the reflectance at UV wavelengths by constructive interference effects within the thin film. Aluminum mirrors coated with silica do not age like bare aluminum. If they become dirty, they can be washed with a mild detergent and distilled water to restore the original high reflectance. Figure 9 shows that washing an uncoated mirror after it has been exposed to a deuterium arc for 170 days um
arc
In
some
source
does not restore its reflectance. Contrast this with Figure 10 for a silicacoated mirror; after 530 days’ exposure to the same environment, wash-
ing fully restores the original reflectance. The use of silica-coated aluminum mirrors thus ensures long mirror life with enhanced reflectance in the UV and minimum deterioration of stray-light performance.
Holographic diffraction gratings. In recent years a new process for making diffraction gratings has been developed: the holographic or interference method. The grating is made by first coating a glass substrate with a layer of photoresist, which is then exposed to interference fringes generated by the intersection of two collimated beams of laser light. When the photoresist is developed it yields a surface pattern of parallel grooves. When coated with aluminum this becomes a diffraction grating. Compared with a ruled grating, the grooves of a holographic grating are much more uniformly spaced, smooth, and uniformly shaped, resulting in much lower stray light levels. The diamond ruling process requires days to rule one grating, whereas a holographic grating requires only a few minutes’ exposure time. Simple holographic gratings have a sinusoidal groove profile with no welldefined blaze wavelength and a maximum diffraction efficiency of 40%. With suitable interferometer geometry it is possible to produce blazed (continued on p. 356 A)
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•
•
Independent detector, injection temperature control Quick change of analytical conditions Features on-column, on-detector fullyglass-lined FID, FTD, ECD and FPD. CIRCLE 346
GC-9A/9AM keyboard. Sample volume is selectable in four steps. Repeat sampling for up to a maximum of 10 samples. Sample vial detection eliminates the need for batch step sample placement. CIRCLE 347
Shimadzu’s High Performance-Low cost Gas Chromatograph Famllv. Dedicated detector low cost
means
V GC-8APr Gas Chromatograph
Microprocessor version of Shimadzu compact GC A GC-mini3 Gas Chromatograph GC-mini3 offers multi-stage tem-
Unbelievable low-cost
isothermal GC
Shimadzu’s GC-8APr Gas Chromatograph is dedicated to high-performance rapid analysis in isothermal or temperature-programmed mode. Lets you choose from FID, TCD, FPD and ECD models. And it can be fully automated for high-volume dedicated uses. Other features include automatic repeat temperature programmed gas chromatography. And a cryogenic valve unit that permits temperature controlled GC to the subambient temperature range.
GC-8AI Gas Chromatograph The GC-8AI Gas Chromatograph features dual-column dual-flow TCD and FID. Single-column dedicated ECD for the best results in electroncapture detection. CIRCLE 352
CIRCLE 350
perature-program capability, an oncolumn on-detector system and near ambient column temperature even with high injection port temperature. CIRCLE 348
Low-cost temperature programmable GC A GC-8AP Gas Chromatograph
Shimadzu’s well-known
compact GC A GC-mini2 Gas Chromatograph
Compact and reliable, Shimadzu’s popular GC-mini2 offers outstanding repeatability in resetting initial column temperature. FID and ECD models are available. CIRCLE 349
The GC-8AP Gas Chromatograph is a high-quality low-cost dedicated detector design. Digital settings and readouts, and you can choose FID, constant current TCD, FPD and ECD models. CIRCLE 351
For automatic
sampling
liquid
AOC-8 Syringe-Type Automatic Sampler
This accessory boosts capability of GC-8A Gas Chromatographs through automatic repeative injection of the same sample up to three times as well as automatic successive injection of 50 samples—100 samples if syringe is washed by next sample. r CIRCLE 353
iSSSf"-*'-'
SHIMADZU SCIENTIFIC INSTRUMENTS, INC. 7102 Riverwood Drive, Columbia, Maryland 21046, U.S.A. Phone: (301) 997-1227 SHIMADZU (EUROPA) GMBH Acker Strasse 111, 4000 Dusseidorf, F.R. Germany- Phone (0211) 666371 Telex: 08586839 SHIMADZU CORPORATION INTERNATIONAL MARKETING DIV. Shinjuku-Mitsui Building, 1-1, Nishishinjuku 2-chome, Shinjuku-ku. Tokyo 160, Japan. Phone: Tokyo 03-346-5641 Telex:0232-3291 SHMDT J.
Fully-automated HPLC system A LC-4A Liquid Chromatograph System A cost-efficient ternary gradient, low-pressure mixing chromatograph with microprocessor control of all system functions. Complete safety and self-diagnostic checks. Standard UV detector with fluorescent refractive index and diode array detectors. Expandable by Chromatopac C-R2AX option. Two-channel recording, auto zero adjust programmable in BASIC. Main frame interface. CIRCLE 360 ON READER SERVICE CARD
Flow rate variable demand from 1 ~
on
9,90Ofjl/minute without changing the pump head LC-5A Microbore Column Liquid Chromatograph
Compared to conventional HPLC, the LC-5A cuts solvent consumption up to 95 percent with theoretical plate numbers exceeding 200,000. The total system dead volume including flow cell is only 2u,\. CIRCLE 361 ON READER SERVICE CARD
Shimadzu’s Liquid Chromatograph Familv.
Double beam for drift-free
operation A SPD-2A UV Spectrophotometric Detector The SPD-2A features a versatile high-performance grating monochromator having a wavelength range from 195 to 350nm. The double beam optical system cancels drifts caused by fluctuation of the light source to ensure high stability. 8,ul and 0.5jul flow cells are available.
A three-dimensional spectro-chromatogram by SPD-M1A
CIRCLE 362 ON READER SERVICE CARD
3-dimensional output is standard. Full spectrum from 200 to 699nm with 1nm resolution
The only energy-corrected high-sensitivity
fluorescence HPLC detector with the following features A RF-530 Fluorescence HPLC Monitor
Xenon source—concave holographic grating for both excitation and emission—12/il square cuvette fits all 1/16" O.D. tubing.
A SPD-M1A Photodiode Array Spectrophotometric Detector
With functions for realtime processing as standard. Shimadzu delivers the advantage of wide-ranging LC in modular designs. The SPD-M1A Photodiode Array Spectrophotometric Detector for LC enables three dimensional spectro-chromato-
grams and single and double-wavelength range chromatograms in realtime. Spectrum memory on-flow is available. No computer knowledge is needed for operation. The photodiode array sensor covers the entire UV and VIS spectrum range and all detector signals in the UV/VIS wavelength range are processed by dedicated microprocessor combined with a dialog system on the CRT. CIRCLE 364 ON READER SERVICE CARD
CIRCLE 363 ON READER SERVICE CARD
SHIMADZU SCIENTIFIC INSTRUMENTS, INC. 7102 Riverwood Drive, Columbia, Maryland 21046, USA. Phone: (301) 997-1227 SHIMADZU (EUROPA) GMBH Acker Strasse 111, 4000 Dusseldorl. F.R. Germany. Phone: (0211) 666371 Telex: 08586839 SHIMADZU CORPORATION INTERNATIONAL MARKETING DIV. Shinjuku-Mitsui Building, 1-1, Nishishinjuku 2-chome. Shinjuku-ku, Tokyo 160. Japan, Phone:Tokyo03-346-5641 Telex:0232-3291 SHMDT J.
gratings with the required triangular groove profile and high blaze efficiency of the order of 80%. Figure 5 shows electron micrographs of a conventional ruled grating and of a blazed holographic grating. It is evident that the holographic process produces grooves that are an order of magnitude smoother and more regular. Holographic gratings used in commercial spectrophotometers are either original master gratings produced directly by an interferometer or replica gratings, which are reproduced from a master holographic grating by molding its grooves onto a resin surface on a glass or silica substrate. The replication process can produce gratings that are almost as good as master gratings. Both types of gratings are coated with an aluminum reflecting surface and usually also with a protective layer of silica or magnesium fluoride, as described previously for mirrors. Referring back to Figure 6 for a typical UV-VIS grating 20 mm wide with 1200 grooves/mm, the theoretical minimum value of R(\tX) for monochromatic light of 600 nm, with a spectral bandwidth of 3 nm, is of the order of 10-7 between diffracted orders (4), because of limiting diffraction effects. A ruled grating of this type usually has a corresponding minimum of between 10-5 and 10-6, whereas holographic gratings can have minima of less than 10“6 and sometimes as low as 3 X 10"7. The holographic process is thus capable of producing gratings that almost reach the theoretical stray-light minimum.
Standard Tests for Stray Light To compare the stray-light performance of spectrophotometers, standard tests are recommended by the American Society for Testing and Materials (ASTM Designation E387-72). These tests have been almost universally adopted by spectrophotometer manufacturers. The tests are based on the principle of measuring the transmittance of a sample that has virtually zero transmittance at the wavelength at which the stray light is to be measured and a high transmittance at wavelengths from which the stray light originates. The measured transmittance of the test sample is then a measure of the stray light, as only stray light is transmitted by the sample. This can also be deduced from Equation 5. If the sample transmittance T at the set wavelength is zero, then the measured transmittance Tm = aS. If a, the proportion of stray light transmitted, is 1, then the measured transmittance Tm = S, the total stray light. For most test samples a can differ appreciably from 1, and therefore the standard tests are really only compar356 A
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ative. Thus, for many practical samples the effective stray light can differ significantly from that given by the test. Stray light is usually at a maximum where the instrument energy is at a minimum. For this reason most manufacturers quote stray light at 220 nm, where the deuterium arc lamp energy is small, and at 340 nm, close to a minimum of the tungsten lamp energy, as shown in Figure 7. Also, 340 nm is chosen as it is an important and widely used biochemical wavelength. At 220 nm the ASTM test measures the transmittance of a 10-g/L solution of Nal in a 10-mm path length cell. This solution has a transmittance of less than 10-1° at 220 nm, but transmits most of the energy at wavelengths longer than 265 nm. At 340 nm a 50-g/L solution of NaN02 in a 10-mm path length cell is used. Some doubts bave been expressed about this test at 340 nm (5), because most spectrophotometers use a bandpass filter around 340 nm to reduce stray light. Consequently, the ASTM test can give an optimistic measurement because the test solution only measures a small proportion of the stray light in the filter passband. Unless the test is modified it is to be expected that manufacturers will continue to use the recommended test. A word of warning on the test solutions given above: It is preferable to use fresh solutions, particularly when the stray-light levels being measured are very low (for example,
