Light in uv-vi,. Spectmphot6meL

Ultraviolet-visible (UV-VIS) spec- trophotometers 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 spectro- photometers were first produced com- mercially their performance has stead- ily improved. Most currently avail- able instruments now meet the above requirements, so that analysts can apply them to their work with confi- dence.

In recent years one particularly no- ticeable change in these instruments has been the use of microprocessors to give much greater flexibility and con- venience of operation, as well as allow- ing automation of many analyses. Less noticeably, there have heen significant improvements in optical components, which have resulted in longer instru- ment life and greatly improved photo- metric 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,

M. R. Sharpe, Pye Unicam Ltd. Cambridge CB1 2PX United Kingdom

%- 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

which vary greatly in age and perfor- mance. All users of spectrophotome- ters 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

I

wide variety of spectrophotometers \

optical components have benefited in- strument performance and life. Stray Light and Its Effects on Measurement Accuracy

sample, then the transmittance T at the analytical wavelength is

T = i (1 I” Figure l a shows the basic compo- nents of a simple single-beam spectro- photometer. The en. or in terms of absorbance units, which trance slit is illuminated hy a light are used because the absorbance A is source. The monochromator is adjust- Proportional to the product of the ed to the analytical wavelength, and sample path length and concentration,

A = -logloT (2) the light emerging from the exit slit is passed through a sample. The light flux transmitted by the sample is mea- sured by a detector, usually a photo- cell, photodiode, or photomultiplier. If = light flux incident on sample obtained. and I = light flux transmitted by the

If measurements are made over a range of wavelengths, then the ahsorh- ance spectrum of the sample can be

The monochromator acts as a filter

ANALYTICAL CHEMISTRY. VOL. 56, NO. 2. FEBRUARY 1984 * 339 A 0003-2700/84/035 1-339AS0 1.50/0 c 1984 American Cnemical Society

I t l

dgure 1. (a) A single-beam spectrophotometer; (b) spectral bandwidth

for the light source and passes light in a small band of wavelengths centered

This “unwanted” component of light flux outside the spectral bandwidth is

at the required wavelength. This small range of wavelengths is determined by the monochromator spectral band- width, which is. proportional to the mmwhromari,r slit widths. When the entrance nnd exit slits are of equal width. the hnndwidth hasapproxi- mately a triangular energy profile. The spectral bandwidth is customarily defined as the width of rhe triangular orofile at half maximum enerev. as

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 transmit- ted by a sample will not be equal to the sample transmittance Tat the an- alytical wavelength. If the “wanted” light flux incident on the sample with- in the monochromator soectral band- ... .

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.

width is I, and the unwanted stray light f lux is I,, then the total light f l u x incident on the sample is I, t I,.

If the sample transmirs a proportinn T of the light within the spectral

I

2.0 3.0

Figure 2. The effect of stray light on absorbance measure- ments for several stray-light levels

Figure 3. Absorbanc

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 TM of the sample at the analytical wave- length is then

Putting the fractional stray light S as

gives

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 mea- sured 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 analy- sis one usually measures the ahsorh- ance of a sample at a peak of absorh- ance, when the sample transmittance is comparatively small. In conse- quence a - T in Equation 5 is posi- tive, 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 Fig- ure 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 concentra- tion 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 lin- earity a t only 1.5 A, where A = ab- sorbance units. At higher concentra- tions there is an increasing loss of sen-

””

:e error for several stray-light levels

340A * ANALYTICAL CHEMISTRY, VOL. 56. NO. 2. FEBRUARY 1984

Advertising removed from this page

sitivity and, consequently, measure- ment precision, until at 3 A increasing sample concentration produces no fur- ther increase in absorbance. To show these effects more clearly Figure 3 shows the absorbance error as a func- tion of the true absorbance for several values of stray light.

LY < T. This is a more unusual ex- ample 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 conse- quently the measured absorbance is higher than the true absorbance. A practical example is the spectrum of benzene vapor a t about 250 nm, which is sometimes used as a spectral resolu- tion test for instruments. Stray light can cause the absorbance minima he- tween the benzene absorption peaks to be partially “filled in” by the positive absorbance error, apparently degrad- ing the spectral resolution. a .= T. If the sample has the same

transmittance outside the spectral bandwidth as at the analytical wave- length, then (a - T ) = 0, and there is no absorbance error. Calibrated neu- tral glass filters, used to measure ab- sorbance accuracy of spectrophotome- ters, are an example of such a materi- al, 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 Equa- tion 5 that the effective amount of stray light is highly dependent on the absorption spectrum of the sample being measured. Instrument manufac- turers 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 ef- fective stray light with other samples will usually be less than the specifica- tion 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 mir- ror, this beam being incident on the grating. The grating is rotated to dif- fract 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 mono- chromator, either a prism or a diffrac- tion grating. Scattering of light and unwanted reflections from other opti- cal elements can also contribute sig- nificantly to the stray light, depending

Figure 4. A diffraction grating monochromator, illustrating the sources of stray light

on the relative quality of the dispers- ing element and how carefully the monochromator has been designed and internally baffled. The strong zero-order spectrum can be particular- ly troublesome when reflected and scattered at walls and mirrors. These various sources of stray light are indi- cated in Figure 4. Good monochroma- tor 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 dif- fracted 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.

I t is also possible for stray light to arise outside the monochromator, such as from light leaks in the instru- ment, 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 ele- ment in the monochromator, as prisms in general have a poorer stray light performance and require complicated precision cams to give a linear wave- length scale. Replica gratings can now be produced more cheaply than prisms and require only a simple sine bar mechanism for the wavelength scale.

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 pho- toresist for a holographic (or interfer- ence) 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 instru- ments is also covered with a trans- mitting protective film to prevent oxi- dation or contamination of the alumi- num. The profile of the grating grooves is usually a shallow triangle, with the wide faces of each groove tilt- ed at an angle known as the blaze angle, which results in the grating having maximum diffraction efficien- cy at a certain wavelength, usually in the UV. Figure 5 shows electron mi- crographs 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 a t 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-

342 A * ANALYTICAL CHEMISTRY, VOL. 56. NO. 2. FEBRUARY 1984

Advertising removed from this page

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 bo- lographic gratings in preference to ruled gratings. For further informa- tion on gratings the reader will find a wealth of literature on the subject- for example, Reference 3.

then as the instrument wavelength setting XI is scanned through the monochromatic wavelength X, the en- ergy from the exit slit appears as shown in Figure 6. This shows the out- put peaking at both the fmt and sec- ond orders of diffraction, the first order being that normally used. For a perfect monochromator one would ex- pect 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

Quant"ative As*s Of Stray Light If the entrance slit of a monochro-

mator is illuminated with monochro- matic light, for example by a laser,

fraction of the monochromatic light scattered in the monochromator to

Relative

Wavelength Setting A,

give stray light a t 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 X I to that when set to the first-order wavelength A,. The function R(XI,&) can therefore he put

R(Xr,)L) =R0(Xr,)L) +R.(XI,)L)

where R,(X,,X,) = energy within tri- angular bandwidth and

as

(6)

R.(X~,L)=straylighteners~ Curves of the type shown as Figure

6, obtained for a series of monochro- matic wavelengths, can he used to as- sess 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 re- late these results to practical measure- menta of transmittance or absorbance on a sample, as so many other factors are involved in a complete spectropho- tometer. In addition, as discussed ear- lier, 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

I ( X ) = source spectrum E(X) = grating efficiency M(X) = mirror reflectance P(X) = detector sensitivity

then the output voltage from the de- tector is proportional to the product of all these factors, so that a t a wave- length 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 )

S U A ANALYTICAL CHEMISTRY, VOL. 56. NO. 2, FEBRUARY 1984

Advertising removed from this page

Advertising removed from this page

Advertising removed from this page

1 oc

? . .

Figure c. Performance of spectropho- tometer components

348A ANALYTICAL CHEMISTRY, VOL. 56

- Anathar Caw

~l D%%atw

I Figure 8. The effect of aging on the reflectance of aluminum at 200 nm. R = re flectance at 200 nm

Figure 7 shows these factors for typ- 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 stray- light error if the analytical wavelength XI is chosen to be where D h ) is

ical components: tungsten lamp and deuterium arc sources, a grating blazed in the UV, an aluminum mir- ror, and an S20 photocathode.

The detector output D(X) can be obtained from a single-beam instrn- ment if one takes a series of readings a t wavelengths over the instrument range, without altering the zero con- trol. 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 possi- ble to calculate how the transmittance of a sample is affected by stray light (4 ) . If

TdX) = measured sample transmittance

B = instrument spectral T(X) = true sample transmittance

bandwidth

then a t wavelength X r

Tnn(Xr) = T ( x I ) + 1- m [T(Xc)

D(L) - ~ ( b ) I & ( ~ r A ) * A c (8)

This equation is equivalent to the simpler Equation 5 given earlier. Thus, to predict the stray-light erroi of a transmittance or absorbance mea- surement from the monochromator strhy-light function R(Xr,X,), a very laborious calculation is required based on difficult measurements. This em- phasizes why the effect of stray light cannot be easily specified by instru-

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 con- taminated, both effects resulting in D(X) decreasing in the UV and, in consequence, the UV stray light in- creasing 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 mono- chromator slits should be kept to a minimum compatible with other re- quirements, 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

ment manufacturers for practical measurements on particular samples.

What else can we learn from Equa- tion 8? As in Equation 5 it shows that the transmittance error depends on

In recent years there have been very significant reductions in stray-light

, NO. 2. FEBRUARY 1984

Advertising removed from this page

by about 5% per year and at a faster rate when exposed to a flux of UV ra- diation similar to that from a deuteri- um 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 pre- vented by overcoating the aluminum with a thin transparent layer. Some manufacturers use a layer of magne- sium 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 coat- ing, which is hard and chemically re- sistant. The correct coating thickness will also enhance the reflectance a t UV wavelengths by constructive inter- ference effects within the thin film.

Aluminum mirrors coated with sili- ca 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 re- flectance.

Figure 9 shows that washing an un- coated mirror after it has been ex- posed to a deuterium arc for 170 days does not restore its reflectance. Con- trast this with Figure 10 for a silica- coated mirror; after 530 days’ expo- sure to the same environment, wash- ing fully restores the original reflec- tance. The use of silica-coated alumi- num mirrors thus ensures long mirror life with enhanced reflectance in the UV and minimum deterioration of stray-light performance.

Holographic diffraction grat- ings. In recent years a new process for making diffraction gratings has been developed the holographic or interfer- ence method. The grating is made by first coating a glass substrate with a layer of photoresist, which is then ex- posed to interference fringes generat- ed by the intersection of two collimat- ed beams of laser light. When the pho- toresist 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 dia- mond ruling process requires days to rule one grating, whereas a holograph- ic grating requires only a few minutes’ exposure time.

Simple holographic gratings have a sinusoidal groove profile with no well- defined blaze wavelength and a maxi- mum diffraction efficiency of 40%. With suitable interferometer geome- try it is possible to produce blazed

(continued on p . 356 A )

I I AI - Fresh I - UV Irradiated for 170 Days

-Then Washed

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 diffrac- tion gratings and the use of mirrors coated with a protective film.

Coated optics. The mirrors in a spectrophotometer are normally coat- ed with aluminum, the metal with the

highest reflectance over the required wavelength range. Freshly deposited aluminum slowly grows a layer of alu. minum oxide on its surface, which re- sults in a progressive loss of reflec- tance a t UV wavelengths, which is ac- celerated by exposure to UV radiation. Figure 8 shows bow a t 200 nm the re- flectance of aluminum in air decreases

- AI + SOr - Fresh I

I -Then Washed I - UV Irradiated for 530 Days

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

Advertising removed from this page

Advertising removed from this page

Advertising removed from this page

Advertising removed from this page

Advertising removed from this page

gratings with the required triangular groove profile and high blaze efficien- cy of the order of 80%. Figure 5 shows electron micrographs of a convention- al ruled grating and of a blazed holo- graphic grating. I t is evident that the holographic process produces grooves that are an order of magnitude smoother and more regular.

Holographic gratings used in com- mercial spectrophotometers are either original master gratings produced di- rectly hy an interferometer or replica gratings, which are reproduced from a master holographic grating by mold- ing its grooves onto a resin surface on a glass or silica substrate. The replica- tion 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 de- scribed previously for mirrors.

Referring hack to Figure 6 for a typ- ical UV-VIS grating 20 mm wide with 1200 grooveslmm, the theoretical min- imum value of R(X,,X,) for monochro- matic 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 al- most reach the theoretical stray-light minimum.

Standard Tests for Stray Light

mance of spectrophotometers, stan- dard tests are recommended by the American Society for Testing and Ma- terials (ASTM Designation E387-72). These tests have been almost univer- sally adopted by spectrophotometer manufacturers.

of measuring the transmittance of a sample that has virtually zero trans- mittance 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 trans- mitted by the sample. This can also he deduced from Equation 5. If the sam- ple transmittance Tat the set wave- length is zero, then the measured transmittance TM = as. If a, the pro- portion 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 compar-

To compare the stray-light perfor-

The tests are based on the principle

ative. Thus, for many practical sam- ples 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 manu- facturers quote stray light a t 220 nm, where the deuterium arc lamp energy is small, and at 340 nm, close to a min- imum of the tungsten lamp energy, as shown in Figure 7. Also, 340 nm is chosen as it is an important and wide- ly 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 trans- mits most of the energy at wave- lengths 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 ex- pressed about this test a t 340 nm (5 ) , because most spectrophotometers use a handpass filter around 340 nm to re- duce stray light. Consequently, the ASTM test can give an optimistic measurement because the test solu- tion only measures a small proportion of the stray light in the filter pass- hand. 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 solu- tions given above: I t is preferable to use fresh solutions, particularly when the stray-light levels being measured are very low (for example, <0.1%).

The advantage of using these test solutions for comparative tests is that the materials are readily available in pure form to most analysts, and hence truly comparative measurements can be made. I t might be thought that some glass high-pass cutoff filters could serve the same purpose as the solutions, but in practice the transmit- tance of such filters can vary from one to another, and in some cases they also exhibit some fluorescence, which can contribute to the apparent stray light.

Conclusion

trophotometers, in particular the in- troduction of holographic diffraction gratings and coated mirrors, have re- sulted in stray-light errors in most modern spectrophotometers being greatly reduced. There is, however, a need for analysts to appreciate how stray light can affect measurements so that they can ensure its effects are negligible, particularly when using older instruments or when working with highly absorbing samples. The analyst can, if necessary, either use appropriate calibration techniques to allow for the nonlinear effects of stray light on absorbance measurements or,

Improvements to the optics of spec-

alternatively. adjust the measurement conditions (sample concentration or path length. for example) to ensure that stray-light effects are negligible.

The standard ASTM stray-light tests can be used to estimate the ef- fects to he expected from stray light, but the limitations of these tests should be understood.

The practical effects of stray light on a spectrophotometric measurement are complex because they are in- fluenced by the quality of all the opti- cal components in the instrument. as well as by the absorption spectrum of the sample. Thus, there is no entirely satisfactory method for correcting for stray-light errors in practice, except hy complex measurements on the in- strument, which an analyst would not normally wish to pursue. The best method is to ensure that stray-light effects are negligible.

References (1) “Standards in AbsorDtion Soectrome-

try. Ultraviolet Spectrbmetry Group”; Burgees, C.: Knowles. A., as.: Chapman and Hall: London. U.K. and New York. %I ” tu01 I . . & _ . 1.7”s.

(2) “Standard Method for &timatin Stray Radiant Ener American loci- ety for Testing and f&?rials, Designa- tion E387-72.

(3) Hutley. M. C. “Diffraction Gratings? Academic Press: London, U.K., 1982

(4) Sharpe, M. R.; Irish, D. Opt. Acto 1978.25(9), 861-93.

(5) Kaye. W. Anal. Chem. 1901.53, 2201-6.

Michael Sharpe received A.R.C.S and BSc. degrees with honors in physics from Imperial College, Lon- don, in 1954. He was elected a Fellow of the Institute of Physics in 1980. From 1954 to 1967 he worked for Im- perial Chemical Industries, Ltd., Bil- lingham-on-Tees, on instrumentation projects and on the properties of ma- terials. Since 1967 he has worked for Pye Unicam, Ltd., Cambridge, in the spectrophotometry deuelopment de- partment, where he is now principal scientist. (Pye Unicam is a scientific instrument company of the interna- tional Philips Group. Pye Unicam’s spectrophotometers are distributed in the US. by Sargent-Welch Scien- tific).

356A ANALYTICAL CHEMISTRY. VOL. 56. NO. 2, FEBRUARY 1984