Advisory Panel Jonathan W. Amy Glenn L. Booman Robert L Bowman
Jack W . Frazer Howard V. Malmstadt William F. Ulrich
INSTRUMENTATION
Instrumentation of a Spectrophotometric System Designed for Kinetic Methods of Analyses Theodore E. Weichselbaum and William H. Plumpe, Jr. Sherwood Medical Industries, Inc. Subsidiary of Brunswick Corp. St. Louis, Mo. 63103
T
H E CONCEPT of kinetic-based analytical methods has been of interest to analytical chemists for many years. Initially, catalytic reactions, enzyme reactions especially, were recognized as useful analytical tools because of their sensitivity and selectivity. In recent years, t h e measurement of the rates of competitive reactions has been used for the in situ simultaneous determination of closely related mixtures. In spite of these and other advantages, the actual application of kinetic techniques to routine practical analysis has not been extensive. T h e reason for this is primarily instrumental. T h e commercial instrumentation readily available u p t o the present, although accurate, reliable, and sophisticated, has been designed primarily for equilibrium measurements. T h e first problem in kinetic analysis is t h a t t h e measurem e n t s m u s t be made with respect to an accurately known time reference. Also, the modes of taking and presenting d a t a in useful forms for a technician are not convenient or built-in a n equilibrium instrument. For practical routine kinetic analysis, instrument design m u s t take into consideration the specific problems associated with measurements on a dynamic system. The subject of the recent 21st Annual S u m m e r Symposium on Analytical Chemistry (Pennsylvania S t a t e University, July. 1968) was the role of computers in analytical chemistry. Most of the discussion centered around the applications of computers for d a t a reduction. However, as was pointed out, the computer itself can do little to improve poor d a t a . This is especially true with kinetic methods. For example, on paper, one can do a simultaneous differential kinetic determination of a mixture of manyunknowns. In practice, two or three components are the maximum, and this limitation is purely instrumental. R e cently, the Digccon instrument (Sher-
Harry B. Mark, Jr. Department of Chemistry University of Michigan Ann Arbor, Mich. 48104
wood Medical Industries, Inc., St. Louis, Mo.) has been designed and built, with modern computer electronic technology and components, t o meet the requirem e n t s for use in kinetic-based analysis. This paper discusses the "philosophy" behind the criteria chosen as a requirem e n t for practical kinetic measurements, the special instrumental problems, and t h e design solutions of these problems t h a t were necessary t o meet these criteria. Design Criteria T h e first general requirement was t h a t this instrument be a very stable noisefree spectrophotometer. T h e transmittance range should be 0 t o 100% (the actual i n s t r u m e n t provides an overrun to 110%) with a linearity of 0 . 1 % in the readout (this does not include nonlinearity introduced b y t h e photocell which would be added to this tolerance). T h e absorbance range should be 0 to 2.0 absorbance units. Again, t h e actual instrument with overrun can read to 2.1 absorbance units in two ranges with an accuracy of 1 % of the reading plus 0.001 absorbance unit. T h e reason one increment on the readout must be added t o the tolerance is t h a t 1 % of the reading becomes vanishingly small a t low a b sorbances and leaves no room for the inevitable small variations in the servo potentiometer and gear train. Secondly, because m a n y of the imp o r t a n t analytical kinetic reactions, such as enzyme assays, catalyst determinations, etc., are measured during their initial rate period (pseudo-zero order conditions), where the initial reaction rate is the p a r a m e t e r proportional to concentration, it was decided t h a t the ins t r u m e n t should be capable of giving a direct readout of the derivative of the a b s o r b a n c e - t i m e curve (which gives t h e rate of t h e reaction a s a function of time). I t was also decided t h a t t h e
derivative readout be continuous rather than a two (or even multiple) fixed-point method, because valuable information such as induction period variation with concentration, maximum reaction rates, etc., can be lost in the dead periods between measurements (actually a continuous readout can be considered to be an infinite fixed-point method) a n d , because, multiple fixed-point sampling is electronically more complex than continuous measurement. I t was felt t h a t direct electronic differentiation of t h e signal should be employed rather than the more complex comparison-integrator technique. Both methods perform the identical operation on the signal and, hence, a r e equally sensitive t o input noise. W i t h careful design component choices, the stability of a direct differentiator is comparable with the comparisonintegrator technique and has one less operational amplifier to serve as a possible source of trouble. T h e range of reaction times of most kinetic methods over which the rates are to be measured is from 20 seconds to 10 minutes. T h u s , the range of rates to be measured b y this instrument must be between 0.001 to l.Oabsorbance unit per minute. No a t t e m p t was made to handle rates of very fast reactions, because these require specialized mixing and readout methods beyond the scope of a general purpose instrument. T h e rate function should be usable over the entire 0 to 2 absorbance-unit range of the instrument. T h e rate accuracy should be a t least 1 % + 0.001 unit from 0 to 1 absorbance unit; progressively deteriorating to about 5 % a t an absorbance of 2. T h e last design criteria was digital readout t o 0.001 absorbance unit with a variable scale factor (or coefficient multiplier) which allows the readout t o be obtained directly in concentration units. T h e instrument was designed so t h a t the absorbance reading could be VOL. 4 1 , NO. 3, MARCH 1969
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INSTRUMENTATION
SPECTROSCOPY
multiplied from 0.000 to 5.998 times and t h e rate reading could be multiplied from 0.000 to 11.996. As will be seen below, the digital readout is incorporated in a very simple and straightforward manner in the logarithm conversion operation of t h e circuit. A permanent record should be pro vided, also. This system incorporates a digital printout and can give a continuous analog o u t p u t for a pen recorder etc., by means of a repeater potentiometer on the servo. Instrument Design
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General Considerations. A general block diagram of the rate instrument is given in Figure 1. T h e i m p o r t a n t feature is the digital logic sequencer module which is programmed by the operator to control sequentially all operations (sample handling, stirring, washing, in duction, measurement, etc.) of a par ticular analytical method. Once set for a particular analysis, the timing of the reaction and its measurement are auto matically controlled and, hence, known to a high degree of accuracy. This module is constructed with conventional digital logic circuitry. T h e design and operation of the control sequencer will be discussed in detail in a subsequent paper and, hence will not be discussed here. It should be noted, however, t h a t it is designed in a flexible manner with extra unused logic circuits so t h a t it can be programmed for any future analysis sequence t h a t is developed. Also, the details of sample handling, reaction chambers, and cuvette will be discussed elsewhere. Only the unique design fea tures of accurate measurement of the reaction course will be discussed here.
For operation as a spectrophotometer in transmittance or absorbance (a non linear element converts transmittance to absorbance) as function of time mode, the basic operational amplifier circuitry is conventional for a single-beam spectro photometer and the servo system acts only as a digital voltmeter. Extreme care must be taken to eliminate noise and drift in the light source and the photocell. This is especially important when operating in a rate mode, because the derivative of a signal containing noise and drift is impossible to handle. It was decided to use a single beam rather than dual beam, because excessive noise problems and both mechanical and electronic complications arise from the dual-beam system. Also, with care in design and the use of quality operational amplifiers, the long term drift of the single-beam system is less than 0.003 absorbance unit per hour. Thus, no significant improvement could be ob tained with a dual-beam system. Noise. T o start with, 60 cycle line noise and transient pulses from switches, etc., must be eliminated by careful at tention to shielding, line filtering, and ground paths. Light source, photocell, and amplifier noises are more difficult to deal with because they contain low frequency components, some of which have frequencies of the same magnitude as the period of the signals to be mea sured. This, of course, limits the use of filtering techniques, and these low fre quency noises must be eliminated a t their source. First, the light source must be stabi lized. Because typical line voltage fluc tuations have periods on the same order of magnitude of the rates to be measured, a system with poor line voltage regu lation might give the same light intensity a t the beginning and end of an hour's operation, b u t will have short term fluc tuations which seriously interfere with rate readings. For example, if a light
Sample Reaction Chambers
Constant Temperature Regulator
Cuvette
Separate Analog Output to Plotter, Recorder, or Outside Digital Computer
Analog Printing Module
Spectrophotometer
Sample Handling Digital Logic Control, Stir, Wash, Sequencer Induction, etc.
Wash Water
Spectrophotometer Light Regulator
Vacuum and Pressure Pumps
Circle No. 92 on Readers' Service Card
Figure 1. 104 A ·
ANALYTICAL CHEMISTRY
Block diagram of the Digecon system
0.001 Abs. unit
INSTRUMENTATION
intensity fluctuation caused a 0.001 a b sorbance unit fluctuation every 15 seconds (Figure 2), a variation which would be considered negligible in straight absorbance measurement, the a\-erage rate observed between points A and Β would be + 0.008 unit per min, and a corre sponding negative rate would be observed between Β and C. T h u s , the reading would show a rate variation in excess of 0.016 unit per min, which is a higher value t h a n some of the rates measured in certain determinations. These re quirements virtually dictate a high g a i n low noise electronic regulator. A con s t a n t voltage regulator such as a cell storage b a t t e r y is not satisfactory be cause a tungsten light bulb will give a decreasing light o u t p u t for a t least one hour after being turned on (apparently the resistance changes on warming of the bulb and socket). It is necessary to use a regulator which samples both voltage and current, in effect monitoring the bulb resistance and compensating the applied voltage accordingly. Even with a perfectly regulated a n d compensated lamp power supply used with a mechanically rigid optical system, serious light level variation was still found. These variations arise from heat convection flow around the light bulb itself. I n s t r u m e n t s with the lamp in a chimney with vents at both the top and b o t t o m are especially bad in this respect. T h e bottom trace in Figure 3 is a noise level recording of the o u t p u t (at the differentiator o u t p u t ) of an old style Beckman DU lamp in its original form. T h e middle trace shows the effect of blowing air across the bulb. T h e top trace shows the same unit with baffling installed to prevent convection currents across the light p a t h . N o t e the accept able rate noise level of 0.001 unit per min indicated on the figure. T h e proper choice of photocells is equally i m p o r t a n t from a noise point of view. Some solid s t a t e photocells exhibit good signal-to-noise ratios and allow low impedance circuitry to be used. How ever, to d a t e no suitable solid s t a t e device has been found for operation a t wavelengths under 4000 A. Some photomultipliers have good wavelength re sponse and high sensitivity, b u t all have signal-to-noise ratios of 1 to 2 orders of magnitude greater than a photodiode tube as shown in Figure 4. Cooling photomultipliers primarily reduces only the d a r k current noise which is only a small p a r t of t h e total current and noise and, consequently, has little value. Re ducing the n u m b e r of dynodes used gives an improvement, b u t even connecting typical photomultipliers to utilize only
Figure 2.
Hypothetical effect of variation of lamp line voltage 0.001lAbs./Min
0.001 Min
Figure 3. Noise level of lamp output. Recording of the differentiator output with filter time constants of 1 second (left hand side) and 10 seconds (right hand side). Bottom trace: Lamp in chimney; Middle trace: Forced circulation of air around lamp; Upper trace: Baffled lamp
0.001Abs./Min
0.001 Min
Figure 4. Noise level of phototube output. Recording of the differentiator output with filter time constants of 1 second (left hand side) and 10 seconds (right hand side). Light adjusted for equal anode currents. Bottom trace: 5 dynode photomultiplier; Middle trace: Gas diode phototube; Upper: Vacuum diode phototube VOL. 4 1 , NO. 3, MARCH 1969
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INSTRUMENTATION
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106 A · ANALYTICAL CHEMISTRY
one dynode still gives a much poorer signal-to-noise ratio t h a n t w o element tubes, without giving a significant im provement in sensitivity. G a s diodes were found to give poorer signal-to-noise ratios t h a n vacuum tubes (see Figure 4). Statistical theory states t h a t the signalto-noise ratio will vary with t h e square root of t h e number of photoelectrons captured. As the use of gas or multiplier electrodes m a y give larger electrical out p u t s b u t d o n o t change t h e number originally emitted from t h e cathode, it is generally better to use photodiodes and depend upon t h e excellent solid state amplifiers available to supply t h e required gain. However, it w a s found t h a t it is necessary to use a gas o photocell when operating below 3800 A because a vacuum photocell has too low a signal in this range. Photocell amplifiers and load resistors must be carefully chosen. Wirewound resistors would be desirable b u t a r e practically unobtainable in t h e light values required here. Vapor deposited resistors work well b u t must be tested for noise level. Field effect transistor input stages a r e used in all operational amplifiers and the finished amplifiers are tested a n d graded to utilize only t h e best in t h e photocell circuits. Differentiator Circuit (Rate Mode). T h e o u t p u t of the absorbance circuit (a non linear element is used in t h e direct a b sorbance readout mode) could simply be coupled to an active differentiator t o give rate readings, b u t this approach tends to magnify errors in t h e absorb ance circuit. T h e nonlinear element does not have a sufficiently accurate log out p u t to be used in this way. Figure 5 shows t h e characteristics of a nonlinear element absorbance conversion circuit. Although t h e deviations from ideal be-
1.000 0.900 0.800 0.700 0.600 0.500 0.400, 0.300 0.200 0.100 0 Ο 10 20 30 40 50 60 70 80 90 100 Figure 5. Simulated characteristics of a nonlinear element used in a log con verter
INSTRUMENTATION
havior stayed within a 1 % envelope, exaggerated here for clarity, this error would give much more t h a n 1 % error in r a t e reading. In this example, the o u t p u t is 1 % high a t an absorbance of 0.5, a t A and 1 % low a t 0.84 a t C. W i t h a reaction changing between A and C the o u t p u t would appear to swing 0.327 absorbance unit instead of 0.340, an error of nearly 4 % . Therefore, the re quirements for a log converter are much more stringent when used in rate deter minations t h a n for equilibrium m e t h o d s or direct absorbance measurements. F o r t u n a t e l y this problem can be avoid ed. T h e derivative of absorbance with respect to time is equivalent to -pf( —log™ T r a n s ) α ί = -log10e(—^—J/Trans
(1)
This can be accomplished electrically by differentiating t r a n s m i t t a n c e and read ing the result with a servo system with t r a n s m i t t a n c e as a reference rather t h a n a fixed voltage as shown in Figure 6. Since this is a true electrical analog of the above mathematical function, the accuracy and absorbance range are now primarily limited only b y noise level a n d the limits of the photocell and amplifiers. T h r e e decades have been covered experi mentally b u t the range was reduced to two decades to allow t h e use of concen tration multipliers a t up to twelve times the numerical rate value, while staying
within t h e voltage range of readily avail able amplifiers. T h e blank rate control shown provides an offsetting 0 to allow direct concen tration readings on reactions whose con centration versus r a t e lines do not inter cept zero. T h e servo operates as a null seeker, driving the servo potentiometer to equal the differentiator o u t p u t and displaying the potentiometer setting on a mechani cal counter dial (the digital p r i n t o u t also arises directly from the position of this mechanical counter dial). As the transm i t t a n c e value varies, t h e voltage per increment on the potentiometer changes proportionately, and over a two decade range will vary over a 100:1 ratio. T o maintain good servo action, the servo amplifier gain should also vary accordingly. Several a u t o m a t i c gain control systems were tested. T h e ob vious approach would be to a t t e m p t to maintain the motor driving power pro portional to t h e rotational distance off null. These systems all introduced addi tional problems such as disturbance of the amplifier null point, so a method was devised in which the motor drive was proportional to the length of time off null, regardless of voltage. This is shown in Figure 7. W h e n an error is introduced the drive to the motor will slowly increase and the motor will start to move. After a b o u t two seconds the drive will reach m a x i m u m and the motor will run a t full speed. Upon reaching the new setting the drive level will remain a t full for about 100 ms to allow the application of full reverse drive to stop overshoot and then be reduced below the minimum required to move the motor. If a small change is introduced
Photocell Differentiator
Photocell Amplifier
Concentration Multiplier
Blank Rate
3
6
Figure 6.
Servo Amp
7
Readout
Motor Speed
Seconds Figure 7. Servo startup response to a step input
such as would be encountered when tracking a changing rate, the servo will reach the new reading while the motor is still turning slowly and will remove drive almost instantaneously. In prac tice this gives smooth following of a varying reading regardless of the voltage applied to the servo pot as long as the servo amplifier gain is sufficient to sense the smallest voltage increments required. Short duration noise peaks are a t t e n uated since the servo will not build u p drive rapidly enough to follow them. W h e n resting on a reading with a high voltage applied to the servo potentiom eter, as when working a t low absorbances, the exact null will often lie between two adjacent wires on the potentiometer winding. T h e servo will then h u n t back and forth between the two wires, b u t since this m o v e m e n t will be m a d e as soon as the drive exceeds the minimum a m o u n t required to move the motor, t h e drive will not be high enough to sustain the t y p e of oscillation usually encoun tered under these conditions. Since this m o v e m e n t will be a small fraction of the accuracy tolerance of the i n s t r u m e n t it has no practical significance. Suggested Reading
Servo Pot
0
Driving Voltage
Motor
Basic diagram of the absorbance derivative circuit
W. J. Blaedel and G. P. Hicks, "Advances in Analytical Chemistry and Instrumen tation," Vol. 3. C. N. Reilley, Ed., Inter science, New York, 1964. H. B. Mark, Jr., L. J. Papa, and C. N. Reilley, ' 'Advances in Analytical Chemis try and Instrumentation," Vol. 2. C. N. Reilley, Ed., Interscience, New York, 1963. H. B. Mark, Jr., G. A. Rechnitz, and R. A. Greinke, "Kinetics in Analytical Chem istry," Interscience, New York, 1968. Κ. Β. Yatsimerskii, "Kinetic Methods of Analysis," Pergamon Press, New York, 1966. "Model 1011 Digecon System, A SemiAutomatic Spec trophotometric System for Chemical Analysis," Bulletin No. 10V-87, Sherwood Medical Industries, Inc., Brunswick Corp., 1831 Olive St., St. Louis, Mo. 63103. VOL. 4 1 , NO. 3, MARCH 1969
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