M. R. Sharpe, Pye Unicam Ltd. Cambridge CB1 2PX United Kingdom
Light in uv-vi,. Spectmphot6meL Ultraviolet-visible (UV-VIS) spectrophotometers have been used as a tool for chemical analysis for many years. They find application in many fieids, including industry, medicine, and university research. I t is therefore important that such instruments he 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 convenience of operation, as well as allowing automation of many analyses. Less noticeably, there have heen 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 ahont 1%.Now, however, figures of less than 0.01% are often quoted, 0003-2700/84/0351-339AS01.50/0 c 1984 American Cnemical Society
which is such a low level of stray light that its effects can he 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 he 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 Effectson
Measurement Accuracy Figure l a shows the basic components of a simple single-beam spectroen. photometer. The trance slit is illuminated hy 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 = light flux incident on sample and I = light flux transmitted by the
%I
\
sample, then the transmittance T at the analytical wavelength is
T = i
I”
(1
or in terms of absorbance units, which are used because the absorbance A is Proportional to the product ofthe sample path length and concentration,
A = -logloT
(2)
If measurements are made over a range of wavelengths, then the ahsorhance spectrum of the sample can be obtained. The monochromator acts as a filter
ANALYTICAL CHEMISTRY. VOL. 56, NO. 2. FEBRUARY 1984 * 339 A
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 TIo + a18. The measured transmittance TMof the sample at the analytical wavelength is then
Putting the fractional stray light S as
I
t l
gives
dgure 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 mmwhromari,r slit widths. When the entrance nnd exit slits are of equal width. the hnndwidth hasapproximately a triangular energy profile. The spectral bandwidth is customarily defined as the width of rhe triangular orofile at half maximum enerev. ... . as shown in Figure I b. In practice a monuchrumator is not a perfect device, and it can transmit a small flux nf 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 a t the analytical wavelength. If the “wanted” light flux incident on the sample within the monochromator soectral bandwidth is I, and the unwanted stray light flux is I,, then the total light flux incident on the sample is I, t I,. If the sample transmirs a proportinn T of the light within the spectral
TM = T + S(a- T) (5) I t is thus clear that stray light can give rise to a difference between the true sample transmittance T and the measured transmittance TM. To illustrate the practical effects of 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 ahsorhance of a sample at a peak of absorhance, when the sample transmittance is comparatively small. In consequence a - T in Equation 5 is positive, most of the stray light being transmitted. If one takes the rather ideal case when (Y = 1 and all the stray light is transmitted by the sample, then Figure 2 shows the measured absorbance as a 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 1.5A, where A = absorbance units. At higher concentrations there is an increasing loss of sen-
I 2.0
3.0
”” Figure 2. The effectof stray light on absorbance measure-
ments for several stray-light levels
Figure 3. Absorbanc:e error for several stray-light levels
340A * ANALYTICAL CHEMISTRY, VOL. 56. NO. 2. FEBRUARY 1984
Microbore sample injection poses new problems in HPK. And Rheodyne solves them. People using microbore columns want to inject niniscule samples-typically only a fraction of I 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. T o show these effects more clearly Figure 3 shows the absorbance error as a function of the true absorbance for several values of stray light. LY < T. This is a more unusual example and corresponds to a sample that absorbs relatively little light at the measured wavelength, hut absorbs most of the stray light. This results in a - 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 hetween the benzene absorption peaks to be partially “filled in” by the positive absorbance error, apparently degrading the spectral resolution. a .= 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 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 he discussed later. The effective stray light with other samples will usually be less than the specification figure, but the analyst should be aware that this depends on the sample spectrum ( 1 , Z ) . 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
*
Figure 4. A diffraction grating monochromator, illustrating the sources of stray light
on 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 be 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.de. signed, well-constructed instrument this latter source of stray light should be completely negligible and will not he 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 scale.
ANALYTICAL CHEMISTRY, VOL. 56. NO. 2. FEBRUARY 1984
A diffraction grating of the type used in 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 grooveslmm, 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 ex-
,
., .
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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 he achieved hy mechanical ruling. In consequence, most modern instruments now use bolographic gratings in preference to ruled gratings. For further information on gratings the reader will find a wealth of literature on the subjectfor example, Reference 3.
Quant"ative As*s 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 XI is scanned through the monochromatic wavelength X, the energy from the exit slit appears as shown in Figure 6. This shows the output peaking at both the fmt and second orders of diffraction, the first order being that normally used. For a perfect monochromator one would expect the curve to he the triangular bandwidth function shown previously as Figure lh. 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
Relative
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(X[,&), the ratio of the energy at wavelength setting XI to that when set to the first-order wavelength A,. The function R(XI,&) can therefore he put as
R(Xr,)L) =R0(Xr,)L) +R.(XI,)L) (6)
where R,(X,,X,) = energy within triangular bandwidth and R.(X~,L)=straylighteners~ Curves of the type shown as Figure 6, obtained for a series of monochromatic wavelengths, can he used to assess the stray-light performance of a monochromator when it is used with a continuum light source, as is normally the case in a spectrophotometer. In practice, however, it is difficult to relate these results to practical measurementa 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
Wavelength Setting A,
I ( X ) = source spectrum E(X) = grating efficiency M(X) = mirror reflectance P(X) = detector sensitivity then the output voltage from the detector is proportional to the product of all these factors, so that at a wavelength setting Xr the detector output D(Xr) is given by
D(Xr) = I(XI)E(hr)M"(XI)P(Xr) (7) where n is the number of reflecting surfaces in the light heam. (continued on p . 348 A ) SUA
ANALYTICAL CHEMISTRY, VOL. 56.
NO. 2,
FEBRUARY 1984
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Figure 8. The effect of aging on the reflectance of aluminum at 200 nm. R = re flectanceat 200 nm 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(X) can be obtained from a single-beam instrnment if one takes a series of readings a t 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
1oc
.
?
TdX) = measured sample transmittance T(X) = true sample transmittance B = instrument spectral bandwidth
.
then a t wavelength Xr
Tnn(Xr)= T ( x I )
+
1m
[T(Xc)
- ~ ( b ) I & ( ~ r A *D(L) )A c
Figure c. Performance of spectrophotometer components 348A
(8)
This equation is equivalent to the simpler Equation 5 given earlier. Thus, to predict the stray-light erroi of a transmittance or absorbance measurement from the monochromator strhy-light function R(Xr,X,), 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
the sample transmittance “(XI) a t the analytical wavelength and on the transmittance T(X,) 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 XI is chosen to be where D h )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(X,) 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 contaminated, 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(Xr,L)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-LigM Performance In recent years there have been very significant reductions in stray-light
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4
umIHuwI
I AI -
Fresh
- U V Irradiated for 170 Days
I
-Then Washed
I
Figure 9. The effect of UV ihadiation and subsequent washing on the reflectance of aluminum levels quoted by the manufacturers of most commercial spectrophotometers. The two main reasons for this me 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
-
AI
highest reflectance over the required wavelength range. Freshly deposited aluminum slowly grows a layer of alu. minum oxide on its surface, which results in a progressive loss of reflectance a t UV wavelengths, which is accelerated by exposure to UV radiation. Figure 8 shows bow a t 200 nm the reflectance of aluminum in air decreases
+ SOr - Fresh
I
- UV Irradiated for 530 Days
I
-Then Washed
I
Figure 10. The effect of UV irradiation and subsequent washing on the reflect.,,of silica-coated aluminum 3501
ANALYTICAL CHEMISTRY, VOL. 56, NO 2, FEBRUARY 1984
by about 5%per year and at a faster rate when exposed to a flux of UV radiation similar to that from a deuterium arc source in a spectrophotometer. In some instruments there are up to 12 mirror reflections before the light beam reaches the detector, so that even a 5%loss of reflectance a t 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 he 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 a t 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 does not restore its reflectance. Contrast this with Figure 10 for a silicacoated mirror; after 530 days’ exposure to the same environment, washing 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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1
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--3-dlmen8ional outaut is standard. Full sp&tnrm from Po0 to 699nm with lnm resolution
A SPD-MIA Phdodiode Anay Spectrophotometric Detector
The only energy-corrected high-sensltivity fluorescence HPLC detector with the following festures
A RF-530 fluomence HPLC Monitor Xenon source-concave holographic grating for both excitation and emission-lZFI square cuvette fits all l/lWO.D. tubing. CIRCLE 363 ON READER SERVICE CARD
With functions for realtime processing as standard. Shimadzu delivers the advantage of wideranging LC in modular designs. The SPDMlA Photodiode Array Spectrophotometric Detector for LC enables three dimensional spectro-chromato-
grams and single and doublewave length range chromatograms in realiime. 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 REAOER SERVICE CARD
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 hy 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 hack to Figure 6 for a typical UV-VIS grating 20 mm wide with 1200 grooveslmm, the theoretical minimum value of R(X,,X,) for monochromatic light of 600 nm, with a spectral bandwidth of 3 nm, is of the order of between diffracted orders (4). he. cause of limiting diffraction effects. A ruled grating of this type usually has a corresponding minimum of between and whereas holographic gratings can have minima of less than 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 T o 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 a t 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 he deduced from Equation 5. If the sample transmittance T a t 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 (Y can differ appreciably from 1,and therefore the standard tests are really only compar356A
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 a t 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 NaI in a IO-mm path length cell. This solution has a transmittance of less than 10-’0at 220 nm, hut 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 have been expressed about this test a t 340 nm ( 5 ) , because most spectrophotometers use a handpass 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 passhand. Unless the test is modified i t is to he 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,
