Anal. Chem. 1981, 53.366-369 Naranjk, D.; Thomassen, Y.; Van Loon, J. C. Anal. Chim. Acta 1979, 770, 307. Miyazaki, A.; Barnes, R. M. Anal. Chem., companion paper in this issue. Colella, M. B.; Siggia, S.; Barnes, R. M. Anal. Chem. 1980, 52, 967-972. Annu. Book ASTM Stand. 1978, Part 37 (Water), 315. Kitagawa, K.; Yanagisawa. M.; Takeguchi, T. Anal. Chim. Acta 1980, 775,121. Winge, R. K.; Peterson, V. J.; Fassel, V. A. Appl. Spectrosc. 1979, 33, 206-219. Barnes, R. M.; Genna, J. S. Anal. Chem., 1978, 51, 1065-1070. Hackett, D. S.; Siggia, S. I n “Environmental Analysis”; Ewing, G. W., Ed.; Academic Press: New York, 1977; p 253. Fukamachi, K.; Morimoto, M.; Yanagisawa, M. Bunseki Kagaku 1972, 27, 26-31.
(13) Fukai, R. Nature (London) 1967, 273, 901. (14) Thompson, D. A,, Ann. Clln. Blochem. 1980, 77, 44.
RECEIVED for review July 28,1980. Accepted November 14, 1980. This work was supported by the Department of Energy (Office of Health and Environmental Research) Contract DE-AC02-77EV-04320. Results were presented in part a t the 6th Annual Meeting of the Federation of Analytical Chemistry and Spectroscopy Societies and a t the 29th Annual Meeting of the Japan Society for Analytical Chemistry. A. M. acknowledges the Science and Technology Agency of Japan for travel support.
Noise and Digital Resolution in a Microprocessor-Controlled Spectrophotometer Wilbur Kaye‘ and Duane Barber Beckman Instruments, Inc., Imine, California 927 13
Until recently most UV-VIS spectrophotometers portrayed spectra in the analogue domain. The trend is now to digitize the signal prior to display. This facilitates data storage and signal manipulations such as integration, differentiation, scale expansion, conversion to concentration, spectral comparisions, etc. Digitization also facilitates, but is not essential, for microprocessor control of spectrophotometer functions. However, it is possible to lose information, i.e., increase the uncertainty or reduce signal-to-noise (S/N) in the digitization process, and an understanding of the problem is desirable to obtain optimum performance from a digital instrument. Of fundamental importance is the digital resolution or reciprocal of the pertinent digitizing counts. Digitization of transmittance in spectrophotometers is usually accomplished by means of an analogue-to-digital converter (ADC). In principle it is possible to operate in the digital domain by photon counting, but the light levels (and consequent S/N) in most absorption instruments is so high that this method is undesirable (1). Digital resolution is here defined as the reciprocal of the ADC counts corresponding to one unit of the recorded signal. In double-beam instruments digitization may occur either before or after taking the ratio I/Z@ In single-beam instruments each signal is digitized. Similarly the log conversion required for absorbance readout may be located before or after the ADC. The influence of digitization on noise is strongly influenced by these design details. Analyses of the digital problems are here illustrated with a Beckman DU-8 prototype spectrophotometer. The analogue noise characteristics of this instrument have been reported elsewhere (2).
EXPERIMENTAL SECTION Apparatus. The DU-8 is a single-beam, digital, microprocessor-controlled spectrophotometer. The prototype used here employed a conventional Littrow grating monochromator with stepped slits having spectral slit widths (SSW) of 0.1, 0.2, 0.4, 1, 2, and 4 nm. A 50-W tungsten-halogen source and R928HA and R375 photomultiplier detectors were employed. For purposes of this study the dynode power supply was removed from its normal microprocessor control and varied manually. Voltage at the preamp output was monitored with a Systron-Donner Model 7110 digital voltmeter (DVM). Ordinate operation is illustrated by the simplified schematic shown in Figure 1. When operating in the transmittance mode the shutter, Sh, may be considered open and the power supply, PS, providing a minimum voltage to the detector. Presumably wavelength has been set to the desired value and a reference or blank has been inserted into the beam. A “gain-set’’ command institues the following sequence. The anode signal is amplified by the operational amplifier, OA, and a capacitor, C, across the 0003-2700/81/0353-0366$01 .OO/O
feedback resistor, R, integrates the signal with a 0.5-5 period. Switches S1, S6, and S7 are closed by the microprocessor (pP). This applies a signal to the ADC resulting in a finite count proceeding to the pP. The ADC can deliver up to 20000 counts. If the counts do not fall between 7500 and 9000, the pP signals the PS to change voltage by small steps and thereby iteratively changes gain of the photomultiplier. When the appropriate count is reached, Sh is closed, S6 opens, and S5 closes. This effectively amplifies the anode dark signal by 5X. If this new signal differs from zero, the pP influences the digital-to-analogue converter (DAC) to change the bias applied to OA in iterated steps and thereby compensates for detector dark current. The pP then commands the shutter and switch S5 to open and switch S6 to close. A “run” command now causes the ADC count, proportional to the blank or reference signal Io,to be stored. A second “run” command causes the pP to place a sample in the beam changing the light level on the detector and the counts out of the ADC. The pP takes the ratio of this count and displays the resulting transmittance on LED, tape, or plotter readouts. The pP cycles every half-second called an “update” and all the above iterative processes occur at half-second intervals. S/N is improved by a “boxcar” or running average method. The number of updates averaged is called a “read average” (RA) and this value is entered via the keyboard. Both Io and I values are averaged. In the absorbance mode the “gain-set’’command establishes dynode voltage and dark current compensations in the same manner described above. The switch S8 closes (S7 open) applying to the ADC a signal proportional to the log of the anode current. Switches Sl-S4 are sequentially closed, and the pP is calibrated for absorbances of 0, 1, 2, and 3. Typically each signal decade produces 4300 counts. The first “run” command replaces the blank with a sample and the difference between the new ADC count and the stored count is read out as sample absorbance. When performing a wavelength scan, the pP is capable of storing 280 descrete Io or log Io counts along with the wavelength correspondingto each Io value. From the programmed wavelength interval and the scan speed, the pP distributes the wavelengths at which data are to be stored. A “background” scan (Io versus A) is made with a blank or reference cell in the beam and this is followed by a sample (I vs. A) scan. Only the background data are stored while the T or A values are calculated and plotted during the sample scan. Sample and background scans can be run at different speeds. If the sample scan takes longer than 140 s, a linear interpolation between stored Io values is taken to provide an Io value at the wavelength of each Z update.
RESULTS AND DISCUSSION Signal Stability. Any analysis of noise in a single-betun instrument requires a knowledge of the signal stability or drift. The drift characteristics of the instrument used here were studied by using a series of 24-h scans. Ambient temperature was monitored during these scans. Proper equilibration of 0 1961 American Chemical Society
ANALYTICAL CHEMISTRY. VOL. 53, NO. 2. FEBRUARY 1981 * 367 %T
99 9
100.00
i
Simplified circuit diagram of the DU-8.
the instrument was found necessary to obtain the low drift rates used in this study. Both the source and detector had to he powered for a t least 2 h before emission intensity and detector sensitivity stabilized. Source equilibration appears to be a purely thermal effect. Detector sensitivity changes appear to he related to the secondary electron emission properties of the dynodes. The gain slowly and exponentially changes when first exposing a powered dark adapted photomultiplier to light. This effect is independent of the small and rapid charge hysteresis effect reported elsewhere (2). The magnitude and sign of the gain change varies from tube to tube. It has been observed to range from 4 . 9 % to +0.8% in R928HA detectors. The observed time constants for this gain change ranged from 29 to 60 min. Fortunately gain returns to its dark adapted level very slowly and nonexponentially so that gain change of equilibrated detectors need not affect Tr or A measurements. The major factors influencing drift after instrument equilibration were temperature, wavelength, and source aging. Photomultipliers are known to have a temperature coefficient which varies with wavelength. Surprisingly it was found that the temperature coefficient of the instrument was only ahout one-fifth that given for the detector by the manufacturer (Hamamatsu Corp.). The coefficients were observed to he - 0 . 1 2 % / T a t 600 nm and -0.37%/"C a t the worst wavelength, 780 nm. No instrument components having a positive coefficient were found. Considerable thermal inertia was observed. The tungsten-halogen lamp was found to age with a steady reduction in emission at 600 nm at a rate of 0.01% /h. No fluctuation with line voltage was detected. Measurement of Noise. Noise in spectrophotometry is usually expressed in terms of either peak-to-peak (P-P) or RMS values. The RMS values are less dependent on measurement details; however, both values obtained on digital instruments are inaccurate when analogue noise is lower than the digital resolution. It is customary to make both measurements hy subdividing an extended record into short time intervals and averaging the results of these intervals. Noise expressed in P-P terms increases with the length of the interval. RMS measurements are little influenced by the interval. When obtaining data from analogue recordings, the interval is usually 10 periods (40 time constants) long. In this study the interval corresponded to 10 printouts. The exact time interval then depended upon the RA and print time. Figure 2 illustrates the noise problem in the DUM. A 50Ox ordinate scale expansion was used. "Tic" marksidentify 30-8 intervals on the abscissa. The DU-8 was equilibrated prior to obtaining all the noise data reported here. The ordinate is labeled ' IT"', hut actually monitors only the "l" values since a constant 1, was introduced with the first "run" operation.
I
I
I
Figure 1.
5
I
I
READ AVERAGE 7
I'
Flgure 2. Transmittance vs. time at two read averages: wavelength 650 nm, spectral slit width 1 nm. RC period 0.17 s. %T
lwo
99 9
I I _. ...
; ,
1
~
1
READ AVERAGE 1 100 w
i
22% and in the A mode when T < 22%. Wavelength Scanning. Both I and Io change when scanning wavelength even though the ratio Illo may remain constant in regions where the sample does not absorb. This still does not eliminate the possibility of observing intervals of apparent infinite S/N, particularly when highly absorbing
380
Anal. Chem. 1981. 53. 369-374 A
,
I
I
I
I
I
I
'
I
SLIT 1.0 RA 1 SCAN 2 nmlmln.
0.079
0.083
'
-
094.00
0.10
%f
Flgve 5. Absorbance spectrum of a blue filter under condkions similar to Figure 4.
samples are recorded in the T mode. Since analogue noise varies in proportion to the square root of the signal, the Z signal analogue noise may be considerably smaller than the digital resolution at all slit widths. This situation is illustrated in Figure 4. A portion of the spectrum of a blue glass filter is shown under three instrumental conditions. The ordinate has
been expanded lOOOX and the digital resolution steps are clearly evident. The wavelength interval within which noise causes the signal to dither between discrete counts is much smaller than the apparently noiseless intervals. Consequently averaging does little to improve accuracy. Neither the analogue noise nor digital resolution of the Io signal can be discerned in Figure 4 since these correspond to less than 0.1% of the T value. However, the Io signal changes with wavelength; hence the ADC counts of Io increase with decreasing wavelength. Transmittance then appears to change within the noise-free intervals. Now consider this same spectral region recorded in the A mode as shown in Figure 5. Some noise is detectable, but ADC steps cannot be identified. Averaging can be reliably used to improve readability. From eq 1the digital resolution is expected to be more than 200X better in Figure 5 than in Figure 4.
LITERATURE CITED (1) Ingle, J. D., Jr.; Crouch, S. R. Anal. Chem. 1972, 44, 1375. (2) Kaye. W.; Barber, D.; Marasco, R. Anal. Chem. 1880, 52, 437A.
RECEIVED for review May 1,1980. Accepted October 10,1980.
Low Absorbance Spectrophotometry Wilbur Kaye Beckman Instruments, Inc., Imine, Callfornia 927 13
Recently there has been a resurgence of interest in lowabsorbance UV-VIS spectrophotometry stimulated largely by developments in optoacoustics (I). Any improvement in low-absorbance capability can be utilized in lowering analytical detection limits, improving analytical accuracy, reducing sample volume, and permitting the study of weak transitions. For many years the generally accepted lower limit of reliable absorbance measurements by conventional spectrophotometry has been 0.002 A (2). This absorbance level is considerably higher than the noise level specified for many high-quality spectrophotometers and warrants reassessment. The instrument used here was a modified Beckman DU-8capable of being operated with a noise of 9 X lo4 A RMS at 0 A (3). This rivals noise in laser lensing, optoacoustics, and fluorescence excitation techniques. However, a number of problems have had to be resolved before this excellent S / N could be converted into useful absorbance data.
EXPERIMENTAL SECTION Apparatus. The spectrophotometer used here was a prototype Beckman DU-8 modified by replacing the side-window R928HA photomultiplier and diffuser with an R375 end-window photomultiplier mounted within a Halon (Diano Corp., Woburn, MA) lined enclosure. The argument for use of such an optical integrator has been given elsewhere (4). The source for the spectra shown below was a 50-W tungsten-halogen lamp. It was totally enclosed in such a way as to minimize Schlieren (5). The monochromator consisted of a conventional Littrow mounted holographic system with fixed slits having spectral slit widths of 0.1,0.2,0.4, 1,2, and 4 nm. The measured half-bandwidth with the narrowest slit was 0.18 nm. The novel portion of the optical system allowing the accurate measurement of low-absorbance spectra is identified in Figure 1. The exit slit, S, is imaged 1 in. from the right-hand side of the sample compartment by transfer mirrors M1 and M2. The sample compartment is isolated from the remainder of the 0003-2700/81/0353-0369$01.00/0
Table I. Sources of Error at Low Absorbance 1. S/Nand drift 2. sample defocusing/displacement
3. internal reflections 4. cell scatter 5. sample scatter/Schlieren 6. hysteresis 7. tracking error purgeable optical train by windows W1 and W2. A large concave mirror M3 reimages the beam on the entry port of the optical integrator and detector D. Either an aperature at A may block all but the specularly transmitted rays or a flag at A may block these rays and permit 30' of the forward scattered rays to be detected. Alternately both transmitted and scattered rays may be detected. The size of the entry port of the optical integrator is dictated by the size of the beam with the widest slit after being defocused by a 10-cm cell filled with CSz, a material of high refractive index. It should be apparent that rays scattered from points nonconjugate to the integrator port will not be efficiently collected by mirror M3. A quantitative estimate of the collector efficiency is given in Fi@;ure2. This graph was obtained by placing a thin transluent Mylar film in the beam and measuring the detected signal as a function of the distance of the film from the W2 toward W1. The signal has been arbitrarily normalized on its maximum value.
RESULTS AND DISCUSSION Many studies have addressed the numerous sources of error in spectrophotometry (6). Those factors which have a special significance for low-absorbance measurements are identified in Table I. These will be analyzed briefly and their solution will be illustrated with some demanding low-absorbance spectra. Noise and Drift. Noise in this instrument is a function of analogue (shot and Schlieren) noise, read average, and 0 1981 American Chemical Soclety
