Automated Measurement of Reaction Rates with Digital Readout in Concentration Units SIR: In recent years there has been a growing interest in the use of reaction rate measurements for quantitative chemical analyses (2, 3, 5, 7, 14). A significant amount of effort is being directed toward the development of improved methods for measuring reaction rates (1, 3, 6, 9). Major objectives of this work are to automate the analytical procedure and to provide direct readout of concentration data. We wish to report the adaptation of an electrical divider (12) (for the continuous computation of reciprocal time) in combination with automatic control equipment to provide digital readout of concentration data. The characteristics of the method have been evaluated by utilizing enzyme reactions. PRINCIPLES OF THE METHOD
Reaction conditions are adjusted to achieve a first order dependence between reaction rate and the concentration of the sought-for constituent. The reaction rate is measured near zero reaction time before the concentrations of reactants change significantly. Under these conditions,
C=k-
1
(1)
At
where C is the concentration of soughtfor constituent, At is the measured time interval, and k is a rate constant dependent upon reaction conditions. The measurement system is represented by Figure 1. The signal system provides an output voltage which is dependent upon the concentration of some reactant or product in the reaction cell. The bias voltage, Ea, is selected to represent the
desired change in concentration over which the reaction time is to be measured. SI and Sz are relay contacts operated by the control system. M is a synchronous motor which is attached to the shaft of the ten-turn potentiometer represented by Rsn. O A l is an operational amplifier operating as an inverter amplifier. At the beginning of each run SI is closed, S pis open and the potentiometer is set so that R,, is zero. As the reaction proceeds, the output from the signal system changes continuously a t a rate dependent upon the reaction velocity. When the output signal reaches a preset level the control system simultaneously opens S1 and closes 82. Opening S1 applies the bias voltage, Ea, in opposition to the voltage from the signal system. Closing S2 starts the synchronous motor operating. The motor continues to operate until the voltage from the signal system increases by the amount Eh. When this happens the control system opens S2, deactivating the motor. The result is that the motor operates during the time required for the reaction to proceed to the extent represented by Eb. As the motor operates, it rotates the shaft of the ten-turn potentiometer a t a constant rate. At the end of the sequence described above the input resistance is given by
where k' is a constant representing the product of the rotational speed of the motor (rev./min.) and the resistance per revolution of the potentiometer (ohms/ rev.), The output voltage, E,, from the inverter amplifier is given by Equation 3.
,-REACTION
Substituting for Rim from Equation 2 above gives
SYSTEM
&>
r---------
E, 115V AC
I
=
E.R 1 -(%')z
which permits calibration of this voltage in concentration units. The output is read on a digital voltmeter. In practice plots of reciprocal time or output voltage from the divider us. concentration frequently have non-zero intercepts. The source, E,, in Figure 1 is a zero adjust to compensate for such intercepts. EXPERIMENTAL
Signal Systems. The amperometric signal system is identical to that described earlier (8, 13). The transistor stabilized Spectronic 20 spectrophotometer (Bausch & Lomb, Inc., Rochester, N. Y.) is modified and utilized for transmittancy measurements. The modified circuit is illustrated in Figure 2. The output from the sample phototube is connected to the input of a stabilized operational amplifier operating as a current to voltage converter. A Heath operational amplifier stabilized by a Philbrick K2P chopper amplifier (9) is used in this work. An increase in transmittance results in a positive increase in current a t the input to this amplifier. The amplifier employs a 5-megohm feedback resistor and has an output of 5 volts per pa. input. A convenient operating range for the phototube is 0.1 pa. which results in a 100% T output of 0.5 volt. To observe small changes in transmittance on a sensitive scale of a recorder or other detection device it is necessary to buck out all or most of this 0.5-volt output. This is accomplished by summing an opposing 0.1 pa. current (0.03 volt across 300 kn.) with the phototube current. The 100% T setting is made by adjusting the Spectronic 20 intensity control until an output of zero volt is obtained. Small variations can then be observed on any sensitivity scale of the measuring instrument. To permit time for mixing and for temperature equilibrium to be established, the reaction is permitted to proceed for a few seconds before the analytical data are collected. This is accomplished by introducing a small bias voltage which must be overcome
(4) 0.04
Combining Equations 1 and 4 gives the result
10
RLUY
etTO COYPARATOR 3WK
A
+---
Figure 1. Combined block-schematic diagram of the automatic reaction-rate instrument
1426
ANALYTICAL CHEMISTRY
where the quantity in parentheses is held constant from one run to the next. Since E;, is negative, Equation 5 predicts a positive output voltage with magnitude proportional to the concentration of sought-for constituent. R, in Figure 1 is a sensitivity control
-Figure 2. Detector circuit for transmittance measurements
by the signal systelm before the measurement sequence outlined above is initiated. The pre-measurement signal is developed across the 6-ohm resistor in the voltage divider. This resistor, in conjunction with S3, provides a displacement of 6 >: 10-3 pa. from the 100% T condition. The direction of this displacement depends upon whether an increase or decrease in transmittance is to be measured. For the galactose reaction involving tt decrease in transmittance, the loo%;, T setting is made with Sa open. Then Sa is closed to establish the initial1 condition for the rate measurement. In this initial condition the phototube current is 6 X 10-3 pa. larger than the opposing current. The control system introduces Ea into the circuit and starts M only when the phototube current becomes equal t3 the opposing current and the output from OA2 is zero. The 0.047 pf. capacitor in the feedback loop of OA2 provides filtering to reduce 120-cycle noise superimposed on the d.c. output from the Spectronic 20. To accommodate the circuitry described above the following changes are made in the Spectronic 20. The meter leads are disconnected from the circuit. The lead from pin 8 of the sample phototube is disconinected and a single conductor shielded lead is soldered to this phototube pin. This lead serves as the input to OAf!. The shielding of the signal lead is connected to the grounds of the Spectronic 20 and the operational amplifier circuits. These changes inactivate the dark current control on the Spiectronic 20. It is observed that the dark current from the phototube is less than 1 X lo-" ampere. The control system is that described earlier consisting of the Sargent Concentration Comparator (6) and modified auxiliary relay system (8). Connections for the amperometric determination of glucose and glucose oxidase have been described (8, I S ) . Connections for the spectrophotometric determination of galactose are shown in Figure 2. The operational ainplifier (OA1) used in the divider is an unstabilized amplifier with input drift in the range of a few millivolts per day. It is desirable that the input source, E,,, be a t least 100 times larger than the maximum amplifier drift. Also it is desirable that the source have very low internal resistance. Appreciable resistance in the source results in large errors in computed values of the reciprocals of short times. A mercury cell with 1.34 volts output and low internal resistance is used. The input resistance, R,", is a 100-kQ. 0.25% linearity 100-kn. ten-turn potentiometer. The feedback resistor, RP, has a value of 40 ka. The synchronous motor, M , has a rotation speed of four revolutions per minute. Substituting these quantities into Equation 4, it is seen that the divider has a sensitivity of 80.4 volt-sec. The motor used (Hurst Synchronous Motor, Series AR-SM-4 r.p.m., Princeton, Ind.) has
a clutch which releases the potentiometer shaft from the motor when power is removed, thus permitting the potentiometer to be reset to zero a t the end of each run. The motor is plugged into P5 of the auxiliary relay system (6). Details of the zero compensation system have been described ( 1 1 ) . The compensated signal for a standard is applied across the sensitivity potentiometer (100 ko., one turn) shown in Figure 1. The slider is adjusted until the meter reads the concentration of the standard. I n this work, concentration data are read from a Digitek digital voltmeter (United Systems Corp., Dayton, Ohio). Procedure. Procedures for the automatic measurement of rates of the glucose and galactose reactions have been described (4, 8, I S ) . Only those modifications required to provide direct readout of concentration data are described here for glucose, glucose oxidase, and galactose. A detailed procedure is given for the assay of galactose oxidase preparations. The general procedure for each determination is as follows. At the beginning of each run, R,, in Figure l is set at zero ohms. The reagents and sample (or standard) are added to the sample compartment. The start switch on the comparator is closed momentarily, The remainder of the measurement including analog conversion to reciprocal time is completed automatically. Details of the calibration procedures are given below. GLUCOSE.The rate us. concentration curve for glucose has a non-zero intercept. Zero compensation and calibration are accomplished as follows. The divider outputs for solutions containing 20 and 40 pap.m. glucose are stored in positions 1 and 2, respectively, of the zero compensation system. Then the zero compensation system is switched to position 3. The balance potentiometer on the follower amplifier is adjusted until the follower output is zero. Then the zero compensation system is switched to position 4 and R, is adjusted until the digital voltmeter reads 200 mv. The system is now calibrated so that each 100-mp. output represents te? parts per million of glucose. After the calibration step is completed, the zero compensation system is switched to position 5 and glucose unknowns are run. GALACTOSE.Plugs P2E and P 3 of the auxiliarv relav svstem (6) are connected to afpropr
