1458
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978
Performance Characteristics of a Vidicon-Based Spectrometer with an Autoranging Amplifier Ronald M. Hoffman and Harry L. Pardue" Department of Chemistry, Purdue University, West Lafayette, Indiana 47907
An autoranging amplifier has been adapted to a vidicon-based spectrometer and the performance of the system is evaluated for scan times of 10 ms and scan repetition rates of 100 Hz. For detector currents less than about 70 nA, the uncertainty is reduced to that imposed by the dark current noise that results in standard deviations of about 0.06 nA. The linear dynamic range for the system with autoranging approaches four orders of magnitude while that for the system without autoranging is limited to less than three orders of magnitude by the ten-bit analog-to-digital converter used with the system. Photometric error curves are presented to show the uncertainties expected at different absorbance levels with and without autoranging, and spectral data for a slow reaction involving alkaline phosphatase and a fast reaction involving the formation of azobilirubin are presented to demonstrate applications for chemical studies. An apparent first-order rate constant determined for the latter reaction agrees well with values determined using conventional instrumentation.
Recent reviews have called attention to the widespread applications of imaging detectors for analytical spectrometry (1,2). In early work from this laboratory, it was shown that in some situations a silicon target vidicon exhibits a linear dynamic range of a t least four orders of magnitude (3-5). However, it was also shown that with existing systems, it is difficult to take full advantage of the intrinsic dynamic range capabilities of such a detector in rapid scanning experiments because of quantization errors that result when a fixed range analog-to-digital converter is used to sample signal amplitudes that vary with wavelength and/or time. It was suggested that an autoranging amplifier system used with a vidicon-based spectrometer could improve signal-to-noise characteristics for spectra that produce large variations in signal amplitude (3). In this work, we have adapted an autoranging amplifier system siniilar to the one described earlier (6) to a vidiconbased spectrometer and evaluated the performance of the system for fixed absorbance measurements and for slow and fast kinetic processes. The system limits the photometric error to that imposed by source and detector noise for scan rates u p to 100 Hz.
EXPERIMENTAL The vidicon circuitry is an improved version of that described earlier (3)that permits computer control of horizontal and vertical scan formats. The autoranging amplifier is inserted between the vidicon output circuitry and the ADC input, and the operation of the system is discussed with the aid of the block diagram in Figure 1. In order to provide common mode noise rejection, the signal voltage from the vidicon amplifiers is sampled at the input of the autoranging system by a differential amplifier with unity gain. The output from the differential amplifier goes into a two-stage amplifier with programmable gain stages in steps differing by factors of 2 from I X to 5lPX. The [Jutput from the programtnable amplifier is sampled by absolute value circuitry and passed to three comparators set to turn on aiid off at about 9, 6. and 4 V. 0003-2700/78/0350-1458$01 O O / O
respectively. The comparator outputs are used by the digital control logic to program the gain setting to maintain output A (Figure 1)within the desired range. Output A is also connected to a + 5 amplifier with voltage offset capability and a voltage follower at output B. Output B was used in most of this work, and the autoranging amplifier was used to keep the output at B between 35 and 95% of the full scale range of -1 to f l V of the 10-bit, analog-to-digital converter (ADC) at the input of the PDP-12 comput,er (Digital Equipment Corporation, Waltham, Mass.) used in this work. The computer monitors the gain setting for each signal sampled by the ADC. The autoranging amplifier includes optional modes for manual or computer controlled gain settings, but these were not used in this work. The autoranging amplifier used in this work was similar to that described earlier (6) except that a digital-to-analog converter resistor ladder network (Anolog Devices AD75201 was used in place of the ladder network uf diodes gates and resistors in the feedback loop of the operational amplifier.
RESULTS AND DISCUSSION Unless stated otherwise, all spectral data reported below were obtained with a 100-Hz repetition rate and all uncertainties are reported a t the one standard deviation level ( f l e). Amplifier Characteristics. Fixed voltage levels from a precision voltage source were used to evaluate the precision, linearity, accuracy, and settling time for different gain settings of the autoranging amplifier. Results reported here are based upon five measurements a t each gain setting for each of ten voltage levels ranging from 0.01 to 10.0 V. Results that illustrate the accuracy, precision, and linearity of the gain sett,ings are presented in Table 1. Expected vs. observed gains are summarized by the linear regression equations included in the table. Slopes for both outputs are less than unity a t the 95% confidence level, but intercepts do not differ from zero. Although these equations could be used t o convert measured voltages to actual values, in this work the average gain values determined for each gain setting were used t o convert measured values to actual values. Settling times were estimated by observing the outputs on a storage oscilloscope. Settling times were less than 5 ps a t all gain settings for 2X changes for both outputs. However, these settling times increased gradually with the magnitude of the gain change up to a maximum value of 20 ps for a 512X gain change. In all subsequent studies, a clock frequency of 150 kHz or period of 6.67 ps was used as the maximum rate (or minimum time) for range changes. This allows ample settling time for all 2X gain changes. We estimate the maximum signal slew rate the system can handle is about 62 Vjms which probably exceeds the response speed of the silicon vidicon. Peak-to-peak noise levels for the €3 output ranged from a low of 1mV ( l x to 64X) to a high of 5 mV ( @ 512X). Values for the A output were about the same as low gain settings but were up by a factor of 5 at the four highest gains. Because the noise levels for gain ranges used in this work were below the ADC quantization error, and because efforts to reduce noise levels resulted in longer settling times, the faster response speed was chosen at the expense of the noise levels cited above. c 1978 American Chernlcal Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11,
SEPTEMBER 1978
1459
Table I. Accuracy and Precision of Gain Settings expected gaina 1
2 4 8 16 32 64 128 256 512
observed gainb A
1.01 2.01 4.00 8.00
15.9 31.8 63.4 126 253 505
gain error, %b
relative standard deviation, %
B
A
B
AC
BC
Ab
Bb
0.203 0.403 0.803 1.61 3.21 6.40 12.7 25.4 51.0 101.5
t1.0 +0.5 0.0 0.0 - 0.6 - 0.6 - 0.9 - 1.6 - 1.2 - 1.4
t 1.5 t 0.7
0.1
t0.4 +0.6 + 0.3 0.0 -0.8 -0.8 -0.4 -0.9
0.3 0.3 0.3 0.2 0.1
0.4 0.0 0.1
2.3 1.2 0.7 0.6 0.3 0.3 0.4 0.1
1.7 0.6 0.3 0.4 0.4 0.3 0.3 0.2 0.0 0.0
0.4
0.3
0.1 0.1 0.4 0.0 0.0 0.0
0.0 0.0 0.0
0.0 0.0
Values based upon average of five measurements of each of ten voltage a Expected gain for output B = Value given + 5. levels. Linear regression equations, measured gain ( y ) vs. expected gain ( x ) . Y A = (0.986 i 0 . 0 0 0 4 ) ~t ~0 . 1 f O.08;Se = Values for five measurements on a 0.21; r = 1.00000. Y B = (0.992 -i 0 . 0 0 0 8 ) ~t ~0.03 -r 0.03; S , = 0.08; r = 1.00000, single voltage level; worst case for ten data sets.
INPUT
SIGNAL
DIFFERENTIAL AMPLIFIER
A
I1 I
1
----
ABSOLUTE
VALUE^
I
OUTPUT A
AMPLIFIER
L 7 -
I
COMPARATOR
Figure 1. Block diagram of autoranging amplifier system
S p e c t r a l Data. A combination of colored glass filters was used to produce a peak spectral response near 570 nm. The resulting signal vs. wavelength profile is shown in Figure 2 where each volt on the signal axis corresponds to approximately 690 nA from the detector. As an initial test of the system, 56 spectra were recorded for the dark current and 100% T signal, and the 56 data values a t each of the several wavelengths were used t o compute the average responses plotted in Figure 2 a n d standard deviations of repeated measurements a t each wavelength. Data were recorded with and without the autoranging amplifier; and those data obtained without the autoranging amplifier were obtained with a fixed gain setting to give near full scale ADC response a t the peak current. Transmittance errors for the two types of experiments were computed from the sum of variances of one dark current and two 100% T measurements (56 points each) so t h a t the values are representative of the three measurements t h a t would be required for an absorbance determination. The resulting transmittance errors with and without the autoranging amplifier are plotted in Figure 2 along with the f 1 / 2 LSB quantization error and detector response. The errors observed without autoranging closely parallel the f l/ 2 LSB from the ADC quantization error. It is clear t h a t the autoranging amplifier improves the accuracy significantly at the longer and shorter wavelengths where the detector signal is lowest. T h e error plots in Figure 2 represent the combined effects of two 100% T measurements and one dark current measurement a t each point and do not indicate relative contributions of the different measurements to the total error. Repeated measurements of dark current gave standard deviations ranging from 0.03 to 0.08 nA with an average value
Figure 2. Spectral response and photometric errors for system with and without autoranging. (0)Spectral signal adjusted with filter combination (690 nA/V) (A)Error without autoranging. ( 0 ) Error with autorange (0)ADC quantization error
of 0.06 f 0.015 nA. Standard deviations of 100% T signals depended upon the signal amplitude, and ranged from lows 390 nm, t o highs of 0.6 of 0.12 nA @ 687 n m and 0.2 nA nA between 450 and 640 nA. Even for the lowest signals 687 nm), the variance of the dark current signal ((0.06 nA)2 or 0.0036 nA2)contributes only about 11% of the total variance ((0.06 nAI2 + 2 (0.12)2or (0.0324 mA2) at 100% T . Neutral density filters and a linearity and transmission standard (L/TS Model 2001, Technometrics, Lafayette, Ind. 47907) were used to evaluate the linear dynamic range of the vidicon-based spectrometer with and without the autoranging amplifier. For each combination of filters and L / T S settings, 15 scans a t 10 ms/scan were recorded for dark current, 100% T , and the filter combination and the absorbance and relative absorbance error were computed from the average values. Data were evaluated a t 450, 500, and 550 nm. Data obtained a t 550 nm with and without autoranging are presented in Figure 3. There is virtual overlap in the two data sets for absorbances up to about 2.0; however, above 2.0, the data for the system without autoranging begin t o deviate from the expected line with the scatter being very bad above 2.5, while the data for the system with autoranging agree well with expected values up to 3.5 and deviate only slightly at 4.0. Thus the autoranging amplifier extends the dynamic range by between 1 and 1.5 absorbance units. Linear regression data for this and the other data set at 450 and 500 nm are included in Table 11. It should be noted that regression statistics for the system without autoranging are comparable to those for the system with autoranging only when the absorbance range
(a
1460
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978 ~
Table 11. Linear Regression of Observed vs. Expected Absorbance for Vidicon Spectrometer with and without Autoranging autowavelength, nm
absorbance range
I,angi ng
550
0.2 to 4.0 0.2 t o 2.1 0.2 to 4.0 0.2 t o 4 . 0 0.2 to 2.7 0.2 to 4.0 0.2 t o 4.0 0.2 to 2.7 0.2 to 2.2 0.2 t o 4.0 0.2 to 3.3
no no
550 550
500 500 500
450 450 450 450 450
slope
amp.
Yes
no no Yes no
*
1.15 c 1.04 c 1.02 i 1.23z
0.05 0.016 0.01
1.04 i 1.21 * 1.25 i 1.13 2 1.03 i
0.02 0.03 0.03
0.05 1.06 I 0.01
no
no Yes Yes
intercept
s
0.008
0.03
1.02 * 0.01
-0.15
3
standard error
s
0.214 0.05 0.06 0.192 0.03
0.10 0.03 i 0.03 - 0 . 2 3 1 0.095 -0.06 i 0.02 -0.06 * 0.04 - - O . l l i 0.05 -0.12 i 0.05 -0.02 t 0.009 -0.051 i 0.068 -t 0.07 I 0.03 -0.04 -0.04
12;
1
5
d.
'
:err. coef.
(
0.984 0.998 0.998 0.987 0.999 0.998 0.997 0.996 0.9998 0.992 0.999
0.08
0.09 0.09 0.013 0.151 0.06
'
" " " "
" '
'
""
'
" " " "
I
1
""""
I
A
c
c A A
A A
3 '
c
3
A
c 4
c
A
A
A
L
*
c
A
A
.L
L L 4
L -
-
L
,
,
,
' A
1 1 . 1 , 1 .
01
00
I0
I
1000
SIGNAL CURRENT ( N A )
Figure 3. Comparison of expected and measured absorbance (A) Without autoranging ( 0 ) With autoranging I O -
for which data are processed is a t least one absorbance unit smaller for the former than for the latter. Standard deviations of repeated measurements a t each intensity level were computed and results are presented for the system with and without autoranging in Figure 4A. While t h e d a t a for the system without autoranging show a slight trend toward increased noise with increased signal amplitude for most of the range examined, the data for the system with autoranging exhibit a fixed noise level for low signal levels, and then a rapidly rising noise level with increased signal amplitude a t higher signal levels. The intercept on the noise axis for a least squares fit of data between 0.1 and 50 nA for the system with autoranging is 0.08 f 0.02 nA. This probably represents an upper limit for the detector dark current noise contribution to the system noise. Figure 4B shows the effects of these errors on the relative absorbance errors for both systems. The narrow valley in the photometric error curve for the system without autoranging is characteristic of systems with large error components that are independent of transmittance while the broad minimum in the other curve IS characteristic of systems in which the major noise components are proportional to T o r t o TI'* ( 7 ) . We have used equations (7)and procedures developed earlier (8) to evaluate error coefficients for the error components that are independent of and proportional to transmittance. Least squares values of independent and proportional errors are 8.3 X and 4.5 X respectively, for the system without autoranging and 1.3 X f 4.5 X and 1.3 X respectively, for the system with autoranging. Although the uncertainty on the independent error coefficient for the autoranging system is large, it still shows an order of magnitude improvement compared to the value without autoranging. This reflects the fact that ADC quantization errors
P
E
0 8-
0 6-
04-
/
A
/
4
3 01
00
0 5
I 0
5
2 0
2 5
3 0
ABSORBANCE
Figure 4. (A) Effect of signal amplitude on imprecision. (A)Without autoranging. ( 0 )With autoranging. (B) Relative absorbance error vs. absorbance for neutral density filters. (A)Without autoranging. ( 0 ) With autoranging. RSA = Relative absorbance error
have been minimized. The 100% current on which these calculations were based was 993 nA, so that the independent error coefficient for the system with autoranging corresponds to 0.013 f 0.04 nA. The 95% confidence limit (0.08 nA) includes the value of 0.06 nA measured for the dark current. I t is reasonable t o assume that most of t h e uncertainty represented by the flat portion of the lower curve in Figure 4A results from dark current noise. Chemical Applications. Two chemical systems, one involving a relatively slow reaction and the other involving a relatively fast reaction, were used to compare performance with and without the autoranging amplifier. The slow reaction involved the alkaline phosphatase catalyzed hydrolysis of
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978
A
B
Figure 5 . kbsoi-d:ioit spa. rra fo: alkalirie phosphatase catalyzed hydrolysis of phenolptirhalein inomphosphate. Ordinate. absorbance: abscissa. waveleng:h ( n m j c,4) Without autoranginc (spectra at 0.34, 1.68. 3.39. 4 7 4 , and ti 75 mi (tottuiii tfi tap)) (B) N i t h autoranginGI (same as A ) phenolphth&in i i , ~ ~ i ~ ( i ~ , ~ i:isini: ~ ~ ~ rconditions ) l l ~ i t ~ described earlier 1 9 . This react io;; i\ slow ctnOugh that, mulriple scans can be averaged t o i-va!iiaie pliotornet ric errors as functions of absorharicr. Fig,urtlq 5.A and 5H show results obtained without and with :he autclra!:ping amplifier. For the data in Figure SB there w r e b t > \era1 range c!ianges as functions of' wavelength. and a t I t (ill? rarige csharige because of the 11 timc: at t h e absorption maximum. change in absor1,anc.r T h e scatter a t highrr sorbaiicr; in Figure 5'4 is a consequence of the AI-)(.' quaritizarioii e l r u i and this scatter is virtually eliminatecl i i i 1 1 1 b~ ill Figiire 5B. Each curve :ihitted) is the average of in each p!ot (ailti f!tirer L . t i ~ ' \ five runs and the iepetiriie I. r e i i d TO evaluate relative absorbance errcirs f o r ~ t ? etw, .:ystt!ns. Photometric errors for both reactiori: \fi P R i irtual!: idriitic,ai Lo those presented for neutral tiensit!, f'iltetFigure 4B. l e m exainint-(l *as the reac~ionof unconiazc~t)enzriies~ilfoiiate at p H jugated bilirubili ( I '('I31!\,i1li, 4.8 in the presenw ( ~ 1,.at't'eine hiid I)eiizoic acid. Unpublished work in this latjc,r
