A Continuous Semi-Automatic Reaction Rate Measuring Instrument for Kinetic Based Analyses Theodore E. Weichselbaum, William H . Plumpe, Jr., Raymond E. Adams, and John C. Hagerty
Sherwood Medical Industries Inc., Subsidiary of Brunswick Corporation, St. Louis, Mo. 63103 Harry B. Mark, Jr.
Department of Chemistry, The University of Michigan, Ann Arbor, Mich. 48104 A semi-automatic modular instrument system for chemical analyses by both kinetic-based or equilibrium methods is described. This system is a high stability low noise spectrophotometer that is primarily designed to measure continuously the rates of chemical reactions. However, absorbance and transmittance can also be measured as a function of time. Furthermore, a coefficient multiplier is built into the servo system so that concentration in appropriate units is read out directly. Sample handling, certain sequential operations in the initiation of the reactions, readout printing, etc., are controlled automatically with respect to time by a solid state digital logic system. Factors controlling the accuracy and linearity of the instrument electronic response as well as example results of the analysis of several species are reported.
THE DESIGN of direct reaction rate readout instruments falls into two general categories. In the first category are the fixed point instruments which simply measure either the time required for the experimentally measured parameter, such as absorbance, to change between two predetermined points, variable time procedure (1-3), or the magnitude of the change in the measured parameter over a fixed time interval, fixed time procedure (3, 4 ) . The second category includes instruments which make a continuous reading of the course of the reaction as a function of time; either recording absorbance-time curves or the rate of change of absorbance as a function of time (5-7). This paper describes an instrument which can be operated in both the fixed point and continuous readout mode. Major emphasis, however, has been given to the continuous rate of change mode of operation as it was felt that most of the chemical reactions employed for kinetic based analytical methods are run under pseudo zero order conditions (initial reaction rate methods) wherever possible (3, 8-10> as the kinetics of the chemical reactions will, in general, be well behaved (no inter-
ference from side reactions, back reactions, etc.) (3). Under these reaction conditions, the rate of the chemical reaction is the parameter that is proportional to concentration of the species to be determined (3, 4 , 9), thus it was decided to again place emphasis on a continuous rate of change (derivative) rather than absorbance-time mode of operaton. Of course, a continuous rate is advantageous for other reasons, both in routine work and research. Some of the kinetic reactions of analytical interest can not be run under pseudo-zero order conditions (3, 8), have induction periods which vary with concentration of the species to be determined ( 1 1 ) , or may be competitive reactions such as those employed in differential kinetic analysis (3, 8). One could use a multiple fixed point sampling of the reaction data also, but it was felt that a direct continuous derivative was superior from a measurement point of view (as a continuous system is in reality an infinite fixed point system) and is electronically far simpler and less costly. Comparison integrator techniques of performing the derivative operation on an input signal have previously been described (5-7). However, it was felt that direct differentiation, using the technique described in this paper is more straight forward. Both methods perform the same operation and are equally prone to “noise amplification” and, therefore, the electronically simplest technique seemed best for a practical instrument. This paper describes in detail the design of an extremely stable-low noise spectrophotometer module and its derivative circuitry, the digital logic control of the time sequence, sample handling, etc., and reports the analytical results obtained under three different modes of operation with three different types of chemical reaction. These results are compared with those obtained for the same samples using the standard clinical methods of analysis for these species. GENERAL CONSIDERATIONS
(1) H. V. Malmstadt, and G. P. Hicks, ANAL. CHEM., 32, 394
(1960). (2) H. V. Malmstadt and H. L. Pardue, ibid., 33, 1040 (1961). (3) H. B. Mark, Jr., G. A. Rechnitz, and R. A. Grienke, “Kinetics
In Analytical Chemistry,” Interscience, New York, 1968. (4) W. J. Blaedel and G. P. Hicks, ANAL. CHEM., 34, 388 (1962). (5) H. L. Pardue, ibid., 36, 633 (1964).
(6) H. L. Pardue and W. E. Dahl, J. Elecfroatial. Chem., 8, 268 ( 1964). (7) H. V. Malmstadt and S. R. Crouch, J. Chem. Edirc., 43, 340 (1966). (8) H. B. Mark, Jr., L. J. Papa, and C. N. Reilley, “Advances in Analytical Chemistry and Instrumentation,” Vol. 2, C. N. Reilley, Ed., Interscience, New York, 1963. (9) W. J. Blaedel and G. P. Hicks, “Advances in Analytical Chemistry and Instrumentation,” Vol. 3, C. N. Reilley, Ed., Interscience, New York, 1964. (10) K. B. Yatsirnerskii, “Kinetic Methods of Analysis,” Pergamon Press, Oxford, 1966.
A block diagram of the system is shown in Figure 1. The single most important module of the instrument is, of course, the stabilized spectrophotometer. In order to be able to do quantitative analysis utilizing dynamic analytical procedures, it was felt that the spectrophotometer-light regulator system must have a drift stability of at least 0.003 Abs unit per hour, a low noise level to produce a rate (derivative) error of less than 0.001 Abs unit/min, a photometric accuracy of 0.01 Abs unit at 1.0 and 0.001 Abs unit near zero, a photometric linearity of 10.001 Abs unit over the 0 to 1.0 Abs unit range, and a continuous rate measurement (deriva-
(11) T. E. Weichselbaum, J. C. Hagerty, and H. B. Mark, Jr., ANAL.CHEM., 41, (3) 103A (1969). VOL. 41, NO. 6, MAY 1969
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Figure 1. Schematic flow diagram of reaction rate system tive) mode with an accuracy of =tl.Ozplus 0.001 Abs/min. (explained below) (12- 17). The control module (or “mind”) of the system is the digital logic sequencer. This unit is a completely solid state device which allows accurate and semiautomatic programming of the sequencial operations involved in performing a particular analysis. For example, the manual switches of the digital logic sequencer (see Figure 2 for a switch panel layout) provide for the programming of the exact timing of the introduction of samples, the exact timing of the reaction period, the printing interval of the parameter following the course of the reaction, the discharge of the sample from the cuvette on completion of a n analysis, and subsequent washing-drying of the cuvette and transfer of sample in preparation, for the next determination (vacuum and pressure pumps). There are actually two constant temperature reaction chanbers (as described below) which are sequentially controlled with respect to triggering of the reaction, mixing of reactants, and incubation for exact time by the digital logic sequencer prior to introduction into the cuvette and subsequent measurement (also controlled by this module). It is important t o note that the logic capabilities built into this are beyond those necessary for carrying out the semi-automated analyses presently available (11, 12, 17). Thus, a program for a future undeveloped analysis can be readily devised and, more important, the unit can be programmed, with minor modifications, to control an automated sample preparation, dilution, procedure change, sample number, and other identification control, etc. (17, 18). This module is (12) “Operational Manual for the Model 1011 Digecon System,” Sherwood Medical Industries, Inc., Subsidiary of Brunswick Corporation, 1831 Olive Street, St., Louis, Mo. 63103. (13) K. Linhardt and C. Walter, “Methods of Enzymatic Analysis,” 2nd Ed., H . 4 . Bergmeyer, Ed., Academic Press, New York, 1965,p 799. (14) R. J. Henry, “Clinical Chemistry, Principles and Technics,” Harper and Row, New York, 1965. (15) T. E. Weichselbaum, Amer. J. Cliti. Pathol., 16, Tech. Sec., 40 (1946). (16) Kaplan, A., “Standard Methods of Clinical Chemistry,” Vol. 5, S. Meites, Ed., Academic Press, New York, 1965, p 245. (17) T. E. Weichselbaum, W. H. Plumpe, Jr., and H. B. Mark, Jr., Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1968. (18) T. E. Weichselbaum and W. H. Plumpe, Jr., unpublished results, 1968. 726
ANALYTICAL CHEMISTRY
completely flexible. The instrument is also designed to give a digital readout, as only in this manner can sufficient resolution be displayed (analog display is also possible, however). The unique design features and operation of each module and the total integrated function of the total system are described in detail below. A complete circuit diagram of the spectrophotometer (and digital readout circuit) is shown in Figure 3. SPECTROPHOTOMETER MODULE
Transmittance Mode. In making transmittance readings, the photocell is connected directly to a current to voltage transducer with a 10 or 100 meg ohm load or feedback resistor as shown in Figure 4. This circuit presents an effectively zero load impedance to the photocell and, thus, maintains a constant voltage across the photocell regardless of the intensity of light on the photocathode. At 0.0 absorbance the photocell current is set a t 0.1 pA which develops a 1.0-volt signal on the 10-meg load resistor. In a conventional circuit, this 1 volt would be subtracted as a voltage drop across the load resistor from the 91 volts from the photocell supply, reducing the photocell voltage to a n effective 90 volts. As the gas phototube is not a linear transducer with respect to voltage changes, the transmittance reading would be, thus, non-linear under this condition. The current to voltage transducer circuit, however, eliminates this problem (19-21) and supplies two selectable outputs of 1 volt or 5 volts a t 100% T . This I-volt output is read as 100.0% T o n a self-balancing servo potentiometer with a Veeder-Root-type digital readout dial. (This servo also derives a repeater potentiometer to provide a recorder or computer output.) The readout is in units of 0.1 % T with a n accuracy of 0 . 2 x T. The servo also drives a printer system to provide a permanent record of the reading. This printer utilizes a set of counter wheels which have raised numbers and which are driven by the same servo shaft as the visual readout wheels. Pressure sensitive paper that needs (19) “Applications Manual for Philbrick Octal Plug-In Computing Amplifiers,” G . A. Philbrick Researchers, Inc., Needham, Mass., 1956, (20) “Handbook of Operational Amplifier Applications,” BurrBrown Research Corp., Tuscon, Ariz., 1963. (21) C . N. Reilley, J. Chem. Educ., 39, A853, A933 (1962).
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Figure 2. Panel layout of digital logic sequencer module control switches
no ink ribbon is fed above the wheels and is struck by magnetically activated reeds, as shown in Figure 5. The digital logic sequencer module activates the magnetic reeds as described below. The servo will actually resolve 0 . 0 2 2 T which can be read on the graduations between digits on the final 0.1 % dial, but the printer is constructed in a manner such that it must print digits ( I @ , therefore, the printer has an alignment mechanism that moves the wheel to the nearest digit during printing. The servo uses a reversible shaded pole motor controlled by two PNP transistors, Q15 and 4 1 6 (see Figure 3), that selectively short pairs of windings. The bias to these transistors is controlled by two NPN transistors, 4 6 and Q7, which cut off all current to the bases of Q15 and 4 1 6 when drive is not required. When drive is applied, 4 6 and 4 7 supply a negative bias during the negative half cycle of the ac from the shading winding and a path to ground during the positive half cycle, allowing Q15 and Q16 to operate as bipolar transistors, thus controlling each pair of windings with a single transistor. The amplifier, amp 5, driving 4 6 and 4 7 is a dual FET input differential in differential out amplifier. One FET input is connected to the signal and the other to the rate feedback amplifier, amp 4, which modifies the signal from the feedback potentiometer. The rate feedback operational amplifier, amp 4 (Sherwood Model DlOO), is a 1 :1 follower with the derivative of its input signal superimposed on its output. This gives a similar action to a tachometer gener-
ator. For example, if the servo potentiometer is approaching null in a positive going direction d V / n will be positive and, as its value is added to the voltage from the potentiometer wiper, the output voltage of the rate feedback amplifier will equal the input signal before the potentiometer wiper has advanced to null. Consequently, the servo motor drive will be reversed and will decelerate the motor before actual null is reached, preventing overshoot and oscillation. The servo also has an automatic gain control which will be discussed later in relation to rate measurements. Absorbance Mode. When operating in the absorbance mode the photocell amplifier operates as a current to current amplifier with a gain of approximately 1600 when the 10meg load resistor is used (see Figure 5). (The current gain will be 16,000 with the 100-meg load.) A silicon carbide varistor (nonlinear element) is inserted in the current output path. After appropriate curve-shaping adjustments (18) are made, the voltage across the varistor will be a good approximation of the logloof the current through the varistor. As the voltage across the varistor will be 0 at 0% T and about - 10 volts at 100% T , an offset voltage of f 1 0 volts must be introduced to make the voltage read at *W (Figure 3) be 0 at 100% T, corresponding to 0 absorbance. This voltage is supplied by R15 on Figure 3. R13, a 300-K resistor in parallel with the varistor, aids in curve shaping. The diode in parallel with the varistor prevents amplifier “latch up” if the operating range is exceeded. Because the varistor reVOL. 41, NO. 6. MAY 1969
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Figure 4. Schematic diagram of transmittance measuring circuit quires approximately a 2 0 : l current ratio to develop the absorbance r,ange from 0.0 to 1.0, a current is introduced through R8 which cancels about 5 % of the photocell current. As this 5 % is subtracted at all transmittances, the current range through the varistor will correspond to 5 T to 95 T over an actual 10% to 100% T range. The varistor voltage is coupled to amplifier 2 which functions in this mode as a high input impedance 1 :1 follower. The output of amplifier 2 is connected to R35, the concentration control (or coefficient) potentiometer, allowing scale expansion on the readout for direct readout in concentration units. The wiper voltage from this potentiometer is read by the servo in the same manner as transmittance. The readout covers a range of 0 to 1.O absorbance in 0.001 absorbance unit. The second range from 1.0 to 2.0 absorbance is obtained by switching to the 100-meg load. Continuous Rate (Derivative) Readout Mode. To obtain rate of change of absorbance (derivative of absorbance with respect to time) readings, the output of the absorbance conversion amplifier could be fed to an analog differentiator (19). However, this type of differentiator tends to magnify (19, 20) errors (18) and noise in the absorbance conversion circuit and also it is desirable to be able to continuously follow reaction rates over an absorbance range greater than the 1 absorbance range of the varistor circuit of Figure 6. Therefore, a different system is used wherein all non-linear elements are eliminated. When a logarithmic function is differentiated with respect to time, the logarithm term is actually eliminated and a linear term is produced. Thus, d( - Log,, trans.) d Time
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As -Logl0e is a constant, the electrical analog of this term requires only that the proper calibration and its polarity are observed, and the designer need only concern himself with producing the derivative of transmittance and dividing this derivative by the instantaneous value of transmittance. The derivative of transmittance is produced by a standard differentiator (19, 20) with its band pass restricted to attenuate noise and to improve its stability (19, 20). In the rate mode the photocell amplifier, amp 1, operates as a current to voltage transducer as in the transmittance mode except that the 5-volt output is used instead of the I-volt output, and various filtering capacitors can be selected to reduce photocell and "reaction" noise (19, 20). (A wide range of time constants is supplied to tailor the instrument characteristics to a particular reaction. Obviously, if a long time constant were used on a high rate that lasted for only a short time, the instrument would not settle on a reading before the reaction reached completion. Conversely, when reading a very slow reaction, noise would be much more significant than with a fast reaction, and a long time constant would be used.) The derivative output from the differentiator, amp 2, is connected to the concentration control potentiometer and the VOL. 41, NO. 6, MAY 1969
729
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voltage on the wiper is fed to the servo input as shown in Figure 7. To divide the derivative of transmittance by the instantaneous value of transmittance, the transmittance signal is fed to an inverter whose output is applied to the servo potentiometer network. This network allows the polarity of the servo potentiometer to be reversed for positive and negative rates without losing the zero point or calibration. As d Trans./d Time is read in reference to the transmittance signal, the equation is satisfied by this electronic analog. The 730
ANALYTICAL CHEMISTRY
only limitations on the absorbance range over which rates can be read are the voltage limits of the amplifiers and the noise level of the system. In practice a range of 1.0 absorbance can be covered with linearity of better than 1%, and usable readings can be made to 2.0 absorbance (approximately 5 % error). A blank rate control (see Figure 3) is provided that offsets the differentiator output by an adjustable fraction of the transmittance voltage. This control can be used to correct for
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Figure 8. Servo startup response to a step input
reactions whose rate US. concentration plots do not intercept zero. As the transmittance value varies, the voltage per increment on the servo potentiometer changes proportionately and over a two-decade range will vary over a 1OO:l ratio. To maintain good servo action, the servo amplifier gain should also vary accordingly. Several automatic gain control systems were tested. The obvious and conventional approach would be to attempt to maintain the motor driving power proportional to the rotational distance off null. These systems, however, all introduced additional 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 (the AGC circuit of Figure 3). When an error is introduced, the drive to the motor will slowly increase and the motor will start to move. After about two seconds, the drive will reach maximum and the motor will run at full speed. This action is illustrated in Figure 8. Upon reaching the new setting, the drive level will remain at full for about 100 milliseconds 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 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 practice this gives smooth following of a varying reading regardless of the voltage applied to the servo potentiometer, as long as the servo amplifier gain is sufficient to sense the smallest voltage increments required. When resting on a
reading with a high voltage applied to the servo potentiometer, as would be encountered when working at low absorbances, the exact null will often lie between two adjacent wires on the potentiometer winding. The servo will then hunt back and forth between the two wires, but since this movement will be made as soon as the drive exceeds the minimum amount required to move the motor, the drive will not be high enough to sustain the type of oscillation usually encountered under these conditions. As this movement will typically be a small fraction of the accuracy tolerance of the instrument, it has no practical significance. Regulated Light Source. One of the most difficult technical problems in rate measurements is noise elimination. First, 60-cycle noise and transient pulse (from switches, etc.) interference must be eliminated by careful attention to shielding, line filtering, and to ground paths. Light source, photocell, and amplifier noise 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 measured. This, of course, limits the use of filtering techniques, and these low frequency noises must be eliminated at their source. First, the light source must be stabilized. As typical line voltage fluctuations have periods on the same order of magnitude as the rates to be measured, a system with poor line voltage regulation might give the same light intensity at the beginning and end of an hour’s operation, but will have short term fluctuations which seriously interfere with rate readings. For example, if a light intensity fluctuation caused a 0.001 absorbance unit fluctuation every 15 seconds as shown in Figure 9, a variation which would be considered negligible in straight absorbance measurement, the average rate observed between points A and B would be plus 0.008 unit/minute and a corresponding negative rate would be observed between Band C. Thus, the reading would show a rate variation in excess of 0.016 unit/minute which is a higher value than some of the rates being measured in certain determinations. These requirements virtually dictate a high gain-low noise electronic regulator. A constant voltage regulator as a wet
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Figure 10. Schematic diagram of regulated lamp power supply circuit VOL. 41, NO. 6,MAY 1969
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cell storage battery is not satisfactory because a tungsten light bulb will give a decreasing light oufput for at least one hour after being turned on. Apparently, because the resistance changes because of 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. The circuit employed is shown in Figure 10. The voltage to the inverting input of the amplifier will be the sum of the voltage across R2 and R3. The voltage across R2 will be a fraction of the voltage across the bulb and the voltage across R 3 will be proportional to the current through the bulb. By varying the ratios of resistors, the weight given to current and voltage feedback can be adjusted to compensate the characteristic variations in the bulb resistance. Even with a perfectly regulated and compensated power supply used with a mechanically rigid optical system, serious light level variations were still found, These variations arise from heat convection flow around the light bulb itself. Instruments with the lamp in a chimney with vents at both the top and bottom are especially bad in this respect. The bottom trace in Figure 11 is a noise level recording of the output 732
ANALYTICAL CHEMISTRY
(at the differentiator output) of a n old style Beckman DU lamp in its original form. The middle trace shows the effect of blowing air across the bulb. The top trace shows the same unit with baffling installed to prevent convection currents across the light path. Photocell. The proper choice of photocells is equally important from a noise point of view. Some solid state photocells exhibit good signal to noise ratios and allow low impedance circuitry to be used. However, to date no suitable solid state dtvice has been found for operation at wavelengths under 4000 A. Some photomultipliers have good wavelength response and high sensitivity but all have signal-to-noise ratios of 1 to 2 orders of magnitude greater than a photodiode tube as shown in Figure 12. Cooling photomultipliers primarily reduce the dark current noise which is only a small part of the total current and noise, and, consequently has little value. Reducing the number of dynodes used gives a n improvement but even connecting typical photomultipliers to utilize only one dynode still gives a much poorer signal-tonoise ratio than two element tubes without giving a significant improvement in sensitivity. Gas diodes gave poorer signal to noise ratio than vacuum tubes (see Figure 12). Statistical
Figure 12. Noise level of phototube output Recording of differentiatoroutput with filter time constants of 1 second (left hand side) and 10 seconds (right hand side). Light adjusted for equal anode currents Top trace: Vacuum diode phototube. Middle trace: Gas diode phototube. Bottom trace: 5-dynode photomultiplier theory states that the signal to noise ratio will vary with the square root of the number of photoelectrons captured (22). As the use of gas or multiplier electrodes may give larger electrical outputs but does not change the number originally emitted from the cathode, it is generally better to use photodiodes and depend on the excellent solid state components available to supply the required gain. It should be pointed out, however, for measurements at wavelengths less than 3800 A, the output signal from a vacuum diode is too low. Thus, gas-filled diodes were chosen for this instrument. As can be seen in Figure 12, the noise characteristics, although greater than the vacuum diode, are below the required noise level necessary to obtain a *0.001 absorbance unit/minute error in the rate readings. Photocell amplifiers and load resistors must be carefully chosen, of course. Vapor deposited resistors are employed as the photocell load resistors but must be tested for noise level. Field effect transistor input stages are used in all operational amplifiers, and the finished amplifiers are tested and graded to utilize only the best in the photocell circuits. DIGITAL LOGIC SEQUENCER The basic logic circuitry of this control module is shown in Figure 13. The operation of this circuit is explained below. (22) D. DeVault, “Rapid Mixing and Sampling Techniques in Biochemistry,” B. Chance, R. H. Eisenhardt, 0. H. Gibson, and K. K. Lonborg-Holm, Editors, Academic Press, New York, 1964, p 167.
Timers. The control has two 6-minute, 50-second timers, and two 60-second timers. The two 60-second timers are merely the seconds counter of the 7-minute timers gated and reset at preset times of 10 to 60 seconds. Each timer is made up of three counters in series; one feeding pulses to the other. The first counter, a Mode 10 counter, counts the one-hertz pulses, resets on 10 seconds, and transfers a unit pulse to the next counter. This counter is a Mode 6 counter, which resets on the sixth pulse or 60 seconds and feeds a unit pulse to the following or minutes counter. The minutes counter can count to six before resetting. The resetting of each counter is controlled by clock pulses generated in the one-hertz clock (described below). Digit Coincidence Gate. The outputs of the 10-second counter and the minutes counter are fed into coincidence gates. These gates give a positive pulse when the output of the counter and the output of the switches coincide. This positive pulse in conjunction with the proper clock pulse gives the print command, and puts a count into the print counter. Light Coincidence Gate. The output of the timer is also fed into a second set of coincidence gates which turns on the ready lights in front of the sample chambers at a preset time. These gates are controlled and constructed in the same manner as the digital coincidence gates. Digital Clock. A 60-hertz sine wave from the ac line is fed into a counter and is then divided down to produce a 1-hertz pulse output. This counter consists of 7 flip-flops in series, gated to reset on the 60th positive excursion of the ac line. The output of each flip-flop is fed into a decoder and the pulses VOL. 41, NO. 6, MAY 1969
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from the decoder are gated into the counter to control and reset as well as into the print sequencer to properly time and sequence each print event. Print Counter. Three flip-flops are connected in series to form this counter. It counts print commands generated by timers and resets these timers. It is also reset itself when the preset number is up, or the count of seven has been reached. This counter uses a coincidence gate to determine when the proper count has been reached. The reset pulse fed to timer number 1 can also be switched off so that timer number 1 can order a continuous print. Servo Motor Logic. The servo motor can be set to operate in two different modes: (i) The servo reads continuously; turns off only when a print command is given, and only turns back on when the print sequence is complete; and (ii) the servo is initially off and is activated only when either timer starts. It reads until the print command is received and then turns off. The servo will turn back on again if more than one print has been programmed, or if the second timer is also running. 734
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Print Sequence Logic. Each event in the print sequence is controlled by a latch. The latches are turned on and off sequentially by clock pulses generated in the digital clock. Power Supplies, A 3.8-volt regulated power supply is used for the logic circuits and a 160-volt unregulated supply is used for leadout tube anodes. Circuits in Console Not Controlled by Timers. The following circuits are not controlled automatically and must be activated by the operator using the appropriate switches on the digital logic sequencer panel (see Figure 2): (a) Wash vacuum control switch, (b) Wash inject switch, (c) Wash select switch, (d) Stir switch for reaction chamber No. 1 and stir switch for reaction chamber No. 2, (e) Stopcock position control, and (f) Sample vacuum control for removing sample and/or wash water from the cuvette. A Typical Sequence of Operation. (See Figure 2 for the program controls.) 1 . Set minutes and seconds switches for desired initial print interval. 2. Set print count switch.
3. Set print interval switch. 4. Set ready light switch. The ready light interval has to be, of course, equal to or less than the initial print interval. 5 . Simultaneously with the initiation of a reaction in the reaction chamber (see Figure 14), the start switch is pushed and a 1-hertz pulse from the clock is gated into the counter. The output of the counter is monitored by the digit coincidence gate and the ready light coincidence gate. When thecounter reaches the time set by the ready light switch, the ready light coincidence gate flips a latch which turns on the ready light. This indicates that the reaction can be introduced into the cuvette and the stopcock switch is pressed. The next event is coincidence between the initial print interval switches and the digit coincidence gate. This produces a pulse which resets the timer, stores a pulse in the print counter, gates out the initial print interval switches, gatesin the print interval switch, and starts the print sequencer. This takes place in much less than one second; and the next 1-hertz pulse fed to the counter will be the first second of the first print interval. When coincidence has been reached between the print interval switch and the counter, a second pulse is produced which resets the timer, stores a second pulse in the print counter, and starts the print sequence. One to seven prints can be selected. When the preset number has been reached, the print counter gives a reset pulse which resets all counters and gates stopping the sequence. The sample is then removed from the cuvette by pressing the vacuum switch. After washing and drying of the cuvette, another sample is ready to be determined in the same manner. SAMPLE REACTION CHAMBER-CUVETTE SYSTEM
A schematic diagram of the reaction chamber-cuvette system is shown in Figure 14. The reagents and sample are inserted manually into the reaction chamber. The washing, drying, and transfer of sample from reaction chamber to the cuvette, are mechanically operated by the appropriate switches on the digital logic sequencer panel (Figure 2). There are two identical reaction chambers so that a second determination can be in the process of being prepared while the first determination is being run in the cuvette. Rapid even stirring is accomplished by means of a plastic coated spiralwire magnet (indicated by the number 3 in Figure 14) which is spun by the alternating field of the induction motor (number 6 in Figure 14). As temperature control for accurate kinetic measurement is an important factor, a “proportional” thermistor bridge temperature controller system is employed. The principle and design of the proportional temperature controller will be reported in the near future. ANALYTICAL RESULTS
Four different kinetic reactions were chosen to illustrate the applicability, accuracy, and precision of the instrument. These analyses illustrate also three modes of operation of this instrument. The reactions were (i) the assay of the alkaline phosphatase enzyme, (ii) the assay of the lactic dehydrogenase enzyme, (iii) the Biuret reaction for the determination of total protein in blood, and (iv) the Berthelot or phenolindophenol reaction for blood urea nitrogen (BUN). Alkaline Phosphatase. This reaction involves the direct hydrolysis of the p-nitrophenylphosphate reagent to form p nitrophenol as the product. The production of the highly colored p-nitrophenol is used to follow the reaction. As the initial rate of this reaction is pseudo-zero order after a brief
Figure 14. Schematic diagram of sample reaction chamber-cuvette system 1. and 2. Reaction chambers Plastic coated magnet Cuvette Washed inject nozzle Stir magnet motor Connection to stopcock motor Sample vacuum inlet Cuvette water jacket 10. Reaction chamber water jacket 11. Cuvette light opening 12. Wash vacuum connection 3. 4. 5. 6. 7. 8. 9.
induction period under the conditions used (12, 13), the system was used in the rate (derivative) mode and the rate of the reaction was read at any time during a period between 3.0 and 7 minutes. The sample handling and reagent preparations, concentrations, and sequence of additions were a slightly modified version (12) of the standard clinical fixed-time (30-minute) method (13). The results of several determinations are shown in Table I. Lactic Dehydrogenase. In this reaction, the lactic dehydrogenase catalyzes the reaction of pyruvic acid with a reduced form of nicotinamide adenine dinucleotide (NADH) to form lactic acid and the oxidized form of NADH (NAD). The NADH absorbs at 3400A, and the measurement of its change in concentration is used to follow the reaction as a function of time. The initial rate of this reaction is also pseudo-zero order under the conditions employed (12, 14). Again the experimental procedure was essentially identical with the Henry, Chiamori, Golub, and Berkman method VOL. 41, NO. 6, MAY 1969
735
Table I. Comparative Data Obtained for Assay of Alkaline Phosphatase in Human Serumsa and Standard Bessey-LowryBrock Fixed Time Methodb All values expressed in Bessey-Lowry-BrockunitsSb Temperature 32 "C Serum 1
2
3
Initial rate method
Values reportedc by Hyland
2.1 2.1 2.6 2.5 2.5 4.9 5.2 4.9 5.2 5.2 8.2 8.4 8.3 8.3 8.3
3.0
Table 111. Comparative Data Obtained for Assay of Total Protein in Human Seruma Using the Above Fixed Time Kinetic Method and the Kjeldahl Method. Temperature 30 "C Fixed time method, Kjeldahlbmethod, Serum g/100 ml g/100 ml 1 6.51, 6.59 6.6 2 3 4 5
5.0d
7.8
Table IV. Comparative Data Obtained for BUN Analysis in Human Serum. Using the Above Maximum Rate Method and Modified Chaney-Marbach, Berthelot Equilibrium Method* Kinetic method Average Chaney-Marb ach Serum Mg BUNa,c deviation method) Mg ZBUN 1 1.02 f0.08 6.5, 7.5 2 3
Table 11. Comparative Data Obtained for Assay of Lactic Dehydrogenase in Human Serum. Using the Above Initial Reaction Rate Method. Temperature was 30 "C Sample Takenb Found 506 349 259 100
Hyland Clinical Chemistry Control Serum, Hyland, Division of Travenol Laboratories, Inc., Los Angeles, Calif. b All solutions were prepared by dilution of a sample of Hyland control serum which contained 506 international LDH units.
(14, 15). The instrument was used in the continuous rate mode. The initial rate was found to be constant for a period from one to six minutes after starting the reaction. The results of a series of determinations are shown in Table 11. Total Protein. The Weichselbaum modification of the Biuret method for total blood protein was used (15). However, instead of using the normal equilibrium procedure (color fully developed after 30 minutes), a fixed time kinetic procedure was used. The absorbance change during the first 2 minutes of the reaction is measured. The change of absorbance was directly proportional to concentration of protein.
ANALYTICAL CHEMISTRY
1.4 6.5 6.4 6.4
Human serum from St. Luke's Hospital, St. Louis, Mo.
Hyland Clinical Chemistry Control Serum, Hyland, Division of Travenol Laboratories, Inc., Los Angles, Calif. * Reference (13). c The values reported by Hyland appear in literature accompanying the Standard Control Serum. The method is the BesseyLowry-Brock fixed-time 30-minute procedure. d The concentration factor converting rate (absorbance units/ minute to Bessey-Lowry-Brock units) was calculated using the 5.0-unit Hyland standard serum.
736
1.50 6.43 6.44 6.41
* The Van Slyke of 6.25 is used to convert nitrogen to protein.
4 5 6
a
506 354 253 101
1.51, 6.53, 6.39, 6.52,
1 a
14.18 52.90 10.74 12.40 24.02 12.70
& O . 18
f0.28 f0.21 f0.26 f O .84 f0.2
14.4, 52.5, 10.4, 11.7, 23.2, 13.3,
14.8 51.5 11.0 11.9 21.6 12.5
The serum used was supplied by St. Luke's Hospital, St. Louis,
Mo.
Reference (16). Each value for the kinetic method is an average of five determinations.
The initial rates of the Biuret reaction were too fast, under any concentration variation, to measure with this instrument, and it was necessary to go to the fixed time direct absorbance reading mode of operation. Example results are shown in Table 111.
Blood Urea Nitrogen (BUN). A recently reported kinetic modification (11) of the standard Berthelot (or endophenol) equilibrium method (20 minutes for full color development) (16) was used. As previously reported ( I I ) , no conditions could be found under which the complex series of reactions involved in this method would follow pseudo-zero order kinetics (or any straight forward order) (11) so an initial reaction rate approach could not be used. Furthermore, the reaction was found to have a n induction period which varied with concentration of BUN which made fixed point methods impossible. However, it was found that the reaction rate reached a maximum value a few minutes after initiation of the reaction and that the magnitude of this maximum rate was directly proportional to concentration of BUN. Thus, the system was used in the rate mode in conjunction with the peak hold circuit (11). A few examples of BUN determinations are shown in Table IV.
RECEIVED for review November 14,1968. Accepted February 6, 1969. Presented at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 1968.
