Instrumentation Wilbur Kaye Duane Barber Robert Marasco Beckman Instruments, Inc. Irvine, Calif. 92713
Design of o Microcomputer-Controlled UV-VIS Spectrophotometer When first introduced into spectrophotometry, the microcomputer was used to manipulate controls and reduce data. More recently it has been used to perform certain logic functions such as spectral comparisons. These functions have been accomplished largely by adapting the microcomputer to existing spectrophotometers. T h e next step in this evolutionary process involves modification of the spectrophotometer to a d a p t it better to the microcomputer. Advantages in-
mount the drift problems t h a t have always plagued the single-beam mode. Let us begin by considering the major factors t h a t control accuracy in absorption spectroscopy—signal-tonoise ratio (S/N), linearity, resolution, stray light, polarization and sample thickness. T h e first two interact most strongly with the optical mode and the microcomputer. Noise can be subdivided into rapid and slow variations in a signal produced by some mechanism other than
quired in the double-beam mode. It is not uncommon to experience a 30% signal loss at every reflection. Noise arising from electrical contacts and mechanical vibration is lower in the single-beam mode, because there is no need to modulate the beam rapidjy and demodulate the signal. In the context of this article, it is presumed t h a t single-beam refers to use of an unmodulated beam. It is common for double-beam instruments to be operable in a so-called single-
elude more versatility, higher accuracy, lower cost and less operator attention. When designing such an instrument, a reconsideration of all the basic principles of spectrophotometry may be necessary. Little is more basic to the design of a spectrophotometer than the optical mode, i.e., single-beam or doublebeam. With few exceptions, manufacturers of automatic spectrophotometers abandoned the single-beam approach a decade ago. Why even reconsider it? Five reasons are advanced here: (1) simplicity of the optics, (2) superior signal-to-noise ratio (S/N), (3) versatility, (4) adaptability to the microcomputer and (5) unobstructed sample compartment. However, none of these reasons would be worth considering if not for the improvements in electronics t h a t allow the instrument designer to sur-
a change in sample absorption. T h e former will continue to be called noise and the latter drift. Those factors strongly affecting noise are outlined in Table I. Four of these factors are identified as increasing S/N when operating in the single-beam mode. Why? Consider the number of photons. In double-beam operations, approximately half the photons are discarded while the modulator or beam splitter blocks the beam to establish t h e zero or dark-current level. In a well-designed system, this zero level will change very slowly with either time or wavelength. Of the remaining photons, half are utilized in establishing the reference level which also may change slowly with time. Another important reason for conservation of photons in the singlebeam mode involves losses at optical elements. More reflections are re-
beam mode wherein a single modulated beam is used. This mode does not have the noise reduction possibilities of the true single-beam mode, at least when using photomultiplier detectors. It is no simple matter to design a rotating beam splitter t h a t is perfectly reproducible in the displacem e n t of the beam. Rotating elements are often the least reliable parts of an instrument. Varying magnetic fields, particularly from electric motors may modulate multiplier gain and lead to a beat with a modulated double-beam signal. In the single-beam mode, fewer motors are needed and no beating can occur. Noise arising from Schlieren, i.e., beam fluctuations introduced by thermally produced refractive index gradients and from atmospheric dust, is often ignored. This noise is worst when using narrow slits. Hence, it fre-
0003-2700/80/0351 -437 A$01.00/0 © 1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 3, MARCH 1980 · 437 A
Table 1. Factors Affecting S/N
Table II. Relation between Signal and Noise Signal
SB DB 1. 2. 3. 4. 5. 6.
Available photons Detector quantum efficiency Integration time Amplifier noise Electrical contacts Mechanical stability
7. Magnetic fields 8. Schlieren 9. Dust
+ + + + _ + - +
SB = single beam, DB = double beam, φ = increases S/N, Q = decreases S/N
quently is confused with photon or shot noise. However, when striving for a maximum S/N, these factors become important. T h e additional slit image usually formed at the beam splitter of a double-beam instrument aggravates this type of noise. An experimental prototype of a sin gle-beam microcomputer-controlled instrument was used in this study. It utilized a 50 W tungsten-halogen bulb and a 30 W deuterium lamp. T h e monochromator was of conventional f/10 Littrow grating design with slits passing bands of 0.1 to 5 nm width. A number of different photomultiplier detectors were employed. Figure 1 shows expanded scale ana log single-beam recordings of the sig nal at different levels of 546 nm radia tion with a 5 nm band-pass. T h e dif ferent light levels were obtained with neutral density filters of the indicated absorbance. Unusually high S/N ratios were recorded with a half-second inte gration period. T h e S/N of the unattenuated signal is approximately 50 000, which is one to two orders of magnitude better than t h a t usually achieved with commercial spectropho tometers. Note also t h a t the scale has been expanded in the figure. In the re cording with the A = 3.07 filter, noise is only 0.00002% of full scale. Figure 2 shows the detector output at very low light level and at zero light (dark current). For the bottom record ing, the dynode voltage was reduced to zero, and the recording displayed the amplifier noise. T h e detector used here had the lowest dark current of eight available R375 photomultipliers. T h e dark current was 0.4 nA at a pho tomultiplier cathode voltage (E c ) of 1000 V and one-fifth the manufactur er's "typical" value. This type of re cording clearly shows the advantage of a low dark current in measuring highly absorbing samples. However, dark
1.05 8.3 5.5 1.6
1 χ χ χ χ 0
10" 2 10-" 10- 8 10~8
Noise 2.0 9 2.2 2.2 1.6 8
x x x χ x χ
H/rt
S/N 5
4
ΙΟ" ΙΟ" 7 10~7 ΙΟ""9 ΙΟ" 9 10- 1 0
5 χ 10 1.1 χ 104 3.8 x 103 25 10 0
E c = photomultiplier cathode voltage
2 9 8 9 1.3
χ χ x χ x
Ec(v) 5
1010-« 10- $ 10"β ΙΟ- 5
80 315 644 700 700 0
BHUKHHHHHHSBB
current has little influence on S/N at high light levels. Photon or shot noise theoretically varies with the square root of the sig nal, and a deviation from this relation ship may reveal other sources of noise. Data from Figures 1 and 2 are summa rized in Table II. T h e anode current signal has been normalized so t h a t sig nal = 1 when the beam is unattenuated. T h e dark current is equated to a theoretical transmittance signal for a detector having zero dark current. The ratio, N / V S is reasonably constant ex cept at the signal extremes. T h e devia tion at the dark current level is under standable because half of the noise is attributable to amplifier noise. At the high end, the increased noise probably can be attributed to residual Schli eren. Drift of the signal can occur for many reasons; the major ones are identified in Table III. Here we find the major advantage of the doublebeam mode. T h e maximum drift seen in Figure 1 is only 0.02% of the signal over a period of 6 min. Since only a few seconds are required to complete a cycle of sample and reference read ings, the S/N ratios listed in Table II are pertinent for transmittances mea sured in the single-beam mode. T h e above S/N levels indicate the sensitivity one could hope to achieve in the measurement of sample absorp
tion, b u t many other factors must be considered before comparable accura cy is achieved. Probably the greatest hurdle to be overcome in the singlebeam mode is linearity. T h e major fac tors affecting linearity are identified in Table IV. Only three of these fac tors are discussed here: multiplier hys teresis and beam displacement, be cause they are influenced by the opti cal mode; and A/D counts, because they are of primary concern to t h e mi crocomputer. Detector linearity is probably the primary reason for the present un popularity of the single-beam mode. B u t why is detector linearity depen d e n t on the optical mode? T h e reason is multiplier hysteresis which is a vari ation in multiplier gain caused by the accumulation of electrostatic charge on insulators in the vicinity of the dynodes. This charge distorts the elec tron trajectory between dynodes. It varies with multiplier current and, therefore, with light level and dynode voltage. A signal-dependent gain is t a n t a m o u n t to a nonlinear response in the single-beam mode. T h e charge usually does not decay rapidly, so gain does not vary much between sample and reference determinations in the double-beam mode, and a more linear response is obtained. There has been considerable reduc tion in hysteresis in photomultiplier
Table III. Factors Affecting Signal Stability
Table IV. Factors Affecting Photometric Linearity
1. Electronic stability 2. Thermal coefficient of detector 3. Thermal coefficient of source 4. Atmospheric absorption 5. Mechanical stability 6. Magnetic fields
SB
DB
-
+
_
+
-
+ + + +
SB m single beam, DB = double beam, ® = increases stability (decreases drift), θ « decreases stability (increases drift)
438 A · ANALYTICAL CHEMISTRY, VOL. 52, NO. 3, MARCH 1980
1. 2. 3. 4.
Multiplier hysteresis Dielectric hysteresis Beam displacement Anode c u r r e n t / dynode current 5. Internal reflections 6. Sample scatter 7. A/D counts
SB
OB
+
+ -
— -
-· -'
SB m single beam, DB = double beam, © - increases linearity, Q = decreases linearity
Figure 2. Signal, noise, and stability at different light levels. E c = photomultiplier cathode voltage. I a = anode current
Table V. Spatial Variation of Transmittances, NBS 930 Filter
ΔΤ
1-522 2-522 3-522 1-359
0.11% 0.55 0.22 0.06 0.05 0.15 0.12 0.15 0.10
2-359 3-359 1-148 2-148 3-148
detectors in the past few years. How ever, we have yet to see a totally hys teresis-free photomultiplier. Figure 3 shows the test we use to identify hys teresis. An expanded scale is used to simplify observation of the small timedependent change in signal accompa nying a change in light level and/or dynode voltage. An overshoot in the recorded signal indicates t h a t multi plier gain decreases as charge devel ops. If gain increases with charge, it is likely to be lower at lower light levels, and transmittance will read low. T h e detector used here is relatively free of hysteresis. It is not unusual to find tubes of even the "reduced hysteresis" type with 20 times higher hysteresis. T h e optical beam in a spectropho tometer is easily displaced by a slight wedge in the sample or reference cell. This displaces the beam to a different area of the detector cathode. Photomultipliers, particularly of the sidewindow variety, are very sensitive to
beam position. In the double-beam mode, it is not possible to use t h e same cell simultaneously for both ref erence and sample. Matched cells m u s t be used even for 2% accuracy. For greater accuracy, the same cell must be used sequentially to hold sample and reference materials. There is then no improvement in total time required to obtain an accurate spec t r u m in double-beam relative t o sin gle-beam operation. In the singlebeam mode the limitation of sequen tial scanning has always been recog nized. A further difficulty accompanies the defocusing of the beam by the sample. Beam size on the detector is in fluenced by sample thickness and re fractive index. In thick cells, even the small refractive index differences be tween solvents and solutions can raise problems, and accuracy can be im proved only by reducing the defocus ing effect. If sample and reference beams do not approach the photocathode from the same direction, as in the popular arrow system, and a side-win dow detector is used, it is not possible for two equally defocused beams to fall on identical areas of the photocathode. Such a problem need not exist in the single-beam mode. Microcomputers must first convert the detector's analog signal to digital form. T h e ADC circuits used for this function have a finite resolution. T e n to twenty thousand counts commonly are used, and linearity is obviously limited to one count or more. I t is pos sible to average several counts and surmount this linearity limit, but only if a noise greater t h a n half a count is present. Here is a case where noise im
440 A · ANALYTICAL CHEMISTRY, VOL. 52, NO. 3, MARCH 1980
proves accuracy. For the results re ported below, averages of eight such counts are used, and the transmit tance readings are significant to 0.001%. Proof of linearity at the level at t e m p t e d here is extremely difficult. T h e aperture addition a n d square law methods of measuring linearity are not easily adapted to the instrumenta tion used. Transfer standards such as the N B S 930 neutral density filters could be used if calibrated with cus tom instrumentation of the requisite accuracy. N B S reports its transmit tance to 0.01%, but does not certify ac curacy to better than 0.5%. Our experience has indicated t h a t accuracy is not limited to the stability or cleanability of these filters, b u t to their spatial variation in transmit tance. Table V itemizes the differ ences in transmittance extremes when scanning from one end of these filters to the other using a beam of approxi mately 1 x 2 mm cross-section. N B S specifies t h e beam size and filter loca tion used for its measurements, b u t it is virtually impossible to reproduce an identical beam size in commercial in struments. While it is recognized t h a t absolute and independent measurements are presently unavailable, there is still ev idence of a very high degree of lineari ty in the measurements reported here. I t is possible to control most of t h e factors outlined in Table IV with the exception of multiplier hysteresis, and hysteresis is the only source of nonlinearity we have identified definitely within photomultiplier detectors. T h e test identified in Figure 3 can be used to predict the approximate magnitude
OOVV
=
c
Mill •
Table VI. Averages of Transmittances Measured with Six Photomultipliers
τ
SSW/Fllter
.3A
3-522
1-522
2A
3A
55.795% 55.748 55.736 55.699 55.605
30.884%
9.867% 9.858 9.853 9.852 9.842 9.85
1.050% 1.047 1.047 1.046 1.044
0.085% 0.083 0.085 0.086 0.083
0.2%
±
5 rati 2 1 0.5 0.1 NBS
30.869 30.849 30.851 30.823 30.89
SSW = spectral slit width
Table VII. Standard Deviations among Transmittances Measured with Six Photomultipliers SSW/Fllter
.3 A
3-522
1-522
2A
3A
0.004% 0.004 0.004 0.004
0.004%
5 nm
0.042%
0.033%
0.014%
2 1
0.026
0.022
0.014
0.037
0.023
0.5 0.1
0.024
0.017
0.036
0.025
0.011 0.011 0.017
0.004
0.002 0.002 0.002 0.004
SSW = spectral slit width
Figure 3. Hysteresis test. SSW = spec tral slit width
and sign of the nonlinearity. If the de tector is the major source of nonlin earity, as suspected, then the standard deviation among transmittance of t h e same filters measured with different detectors should provide an indication of the level of achievable linearity. Five Schott neutral density filters were used for t h e linearity test. T w o of these filters identified by numbers 3-522 a n d 1-522 were N B S 930c stan dards. All filters were cleaned careful ly prior to the test a n d were located where the beam measured approxi mately 5 X 5 mm. T h e transmittances reported in Table VI are averages of readings taken with six different photomultipliers and at five different spectral slit widths. Different slits were used because hysteresis was known t o vary with light level. T h e r e is some evidence t h a t transmittance varies with slit width, a n d the small spectral variation in transmittance of the filters may explain this deviation. Table VII lists t h e standard devia tions among transmittances measured with t h e six photomultipliers. I t is un derstandable t h a t standard deviation would increase at narrow slits because of an increased noise level. At the wid-
est slits the standard deviation in creases, because hysteresis increases as dynode voltage decreases. At inter mediate slits the standard deviations obtained with t h e single-beam instru m e n t described above are considera bly smaller than those achievable with existing commercial instruments. In summary, it has been shown t h a t there is considerable room for further improvement in sensitivity and accu racy in absorption spectrophotometry. S/N levels as high as 50 000 and repro
ducibilities of 0.017% at 30% Τ have been achieved. These values have been obtained with instrumentation oper ated in t h e single-beam mode. Im provements in electronic circuitry have permitted the requisite stability. Multiplier hysteresis is no longer the forbidding factor for excellent lineari ty in the single-beam mode. T h e mi crocomputer provides all t h e instru mentation required to automate sin gle-beam operation.
Wilbur Kaye (left) received his B.S. in chemistry from J. B. Stetson University in DeLand, Fla., in 1942 and a Ph.D. in chemistry from the University of Illinois at Urbana in 1945. Now principal staff scientist at Beckman Instruments' Scientific In struments Division in Irvine, Calif., Kaye specializes in ultraviolet and infrared spectroscopy, molecular structure, X-ray diffraction, gas chro matography, electron microscopy and light scattering.
Duane Barber (center) received his B.S. degree in mechanical engineer ing from California State Polytechnic University in Pomona, Calif., in 1967 and his M.B.A. degree from Califor nia State University in Fullerton in 1977. He is project engineer at Beckman's Clinical Instruments Division. Robert Marasco, an engineer at the Scientific Instruments Division of Beckman, is responsible for the-design of optics and accessories on spec trophotometers.
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