An Automated Instrumental System for Fundamental Characterization of Chemical Reactions Stanley N. Deming’ and H. L. Pardue Department of Chemistry, Purdue Uniuersity, Lufuyette, Ind. A computer-controlled instrumental system was developed for the characterization of chemical reactions. Routine procedures as well as the decision-making procedures of data interpretation and experimental design have been automated. The system was evaluated by characterizing several aspects of the alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate. The studies verified the ability of the instrumental system to interpret data accurately, to design experiments properly, and to achieve the goal of a characterization in a rapid and efficient manner. DURING THE PAST DECADE, great emphasis has been placed o n the automation of instrumentation for routine analyses (1-4). Less emphasis has been placed on the automation of instrumentation for the fundamental characterization of chemical systems (5). This is largely because the required experiments in such characterizations are investigative or nonroutine in nature and include widely varying parameters that are usually defined o n the basis of results from preceding experiments. However, if the conditions for a particular investigative experiment can be established, then the actual execution of that experiment may well involve routine procedures. In our view, many routine procedures can be performed by electronic operations, by electrically-controlled mechanical operations, or by both. These operations, and thus the procedures, can and should be completely automated. Other procedures associated with these experiments involve thought and decision-making processes and can be automated if sufficient information is or can be made available. If certain aspects of the investigation can be interpreted on the basis of established chemical behavior and if the investigation has a stated goal, then in many cases the definition of conditions for necessary future experiments is reduced to a well defined decision-making process based o n the results of previous experiments. Figure 1 is a pictorial representation of the procedures conventionally employed for the characterization of a chemical system. Data interpretation, experimental design, and the information base all require or make use of thought or decision-making processes. Initiation and control of the experiment, data acquisition, and data processing and display are routine procedures that can be performed manually but have been automated to a greater or lesser extent over the past decade. It is the experimenter’s responsibility to provide the information base-what relevant information is already known about the chemical system, what information about the chemical system is desired, a method of obtaining the desired Present address, Department of Chemistry, Emory University, Atlanta, Ga. (1) H. L. Pardue, Rec. Chem. Progr., 27, 151 (1966). ( 2 ) W. J. Blaedel and G . P. Hicks, Advan. Anal. Chem. Instrum., 3, 105 (1964). (3) Technicon Instruments Company, Ed., “Automation in Analytical Chemistry,” Mediad, White Plains, N. Y.,1967. (4) G. A. Rechnitz, ANAL.CHEM.,40, 455R (1968). (5) G. P. Hicks, A. A. Eggert, and E. C. Toren, ibid., 42, 729 ( 1970).
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information, and criteria for determining when the desired information has been obtained or when to stop trying to obtain it. The experimenter then draws upon the information base to design the investigative experiments by interpreting the available data in its relationship to attaining the desired goal. As the characterization proceeds, the experimenter constantly updates the information that is known about the chemical system. Thus, the information base grows as the characterization progresses and allows experiments to be designed with greater degrees of sophistication. Once the conditions for a particular experiment have been defined, it is then a matter of executing the experiment using more or less routine procedures. Of the three routine procedures shown in Figure 1 , the experimenter is probably most intimately involved with the initiation and control of the experiment. Although work has been done on the real-time automated control of experiments (6), very little effort has been expended on the automation of experiment initiation in its broadest sense, that of preparing the actual chemical system from its components and presetting as many other variables as necessary. Specifically, very little work has been done t o automate the reagent addition aspect of experimentation. Automated data acquisition is well developed. Similarly with the introduction of the electronic desk calculator, computers, and associated peripherals, data processing and display has achieved a high degree of automation. After a sufficient number of investigative experiment: have been executed, the characterization is considered complete : either the desired information has been obtained or it is determined t o be unwise for one reason or another t o pursue it further. At this point the experimenter has available in the information base the results of all of the experiments. He can consider the characterization complete or perhaps initiate another round of experiments in which he desires new or different information about the chemical system. Figure 2 is a pictorial representation of the procedures conceived for the automated characterization of a chemical system. In this situation, it is still the experimenter’s responsibility to provide the information base. Once provided, however, the experimenter is free to undertake other tasks. The instrumental system will automatically design experiments by interpreting data obtained from the information base and execute the experiments with automated initiation and control of the experiments, data acquisition, and data processing and display until the characterization is considered complete. At this point, the experimenter can draw upon the information base to retrieve the results of the characterization. If he desires, he can initiate another round of experiments or he can consider the characterization complete. The heart of any automated instrumental system is the controller. Controllers used in instrumentation for routine analyses assume repetitive and invariant procedures for the initiation and control of each analysis, a well defined and often (6) S. P. Perone, D. 0. Jones, and W. F. Gutknecht, ANAL.CHEM., 41, 1154 (1969).
ANALYTICAL C H E M I S T R Y , VOL. 43, NO. 2, F E B R U A R Y 1971
INTERPRETATION
DESIGN
A
CONTROLLER
L
1
INITIATION CONTROL
I ”ITGoN1 I -;’IT1
PROCESSING ACOUl SI TlON DISPLAY
CONTROL
DATA ACOUlSlTlON
DISPLAY
Figure 1. Procedures conventionally employed for characterization of a chemical system
Figure 2. Procedures conceived for automated Characterization of a chemical system
specific procedure for acquiring data, and a single method of processing and displaying the data that is usually autonomous except for a triggering signal from the controller. Rudimentary types of “experimental design” and “information base” exist in these controllers; the “data interpretation” function is not necessary for the control of routine analyses. If it is necessary t o change analyses, the controller is adjusted manually or in some instances it is replaced with a new one that is pre-programmed for the new analysis. The automation of instrumentation for nonroutine analyses can make no assumptions of constancy. The controller must be capable of initiation and control for a wide variety of experimental conditions, of data acquisition that may need to be performed at different rates or even by totally different techniques, and of data processing and display by any of many possible methods. In addition, the controller must be capable of interpreting data, of designing experiments, and of communicating with the information base. Above all else, it must be versatile and adaptable to adequately handle the many types of characterizations for the variety of chemical systems that might possibly be encountered. An obvious and unifying choice for the controller is a small digital computer. It is versatile, adaptable, and with suitable peripherals, capable of executing all of the control functions required of a controller for a n automated instrumental system. The programmable memory of the computer is ideal as a variable and expandable information base. Not only can it store relevant information already known about the system, the goal of the characterization, and criteria for determining when the characterization is complete, but through its programming capability it can also store a method of obtaining the desired information. The logic and decision-making capabilities of a digital computer are well suited for data interpretation and experimental design. Arithmetic operations combined in various patterns provide a versatile method of data treatment. The input-output (Z/O) functions coupled to appropriate peripherals or directly to the experiment itself make possible the complete automation of initiation and control, of data acquisition, and of data display. This report describes the results of a project designed to develop and evaluate an instrumental system suitable for the automated characterization of chemical systems. The complete instrumental system can be divided into four distinct elements : a programmable digital computer (controller), a n analytical instrument that transduces chemical properties into electrical information, a procedure for acquiring that information, and procedures for initiation and control of the experi-
ment. The analytical instrument used to monitor the reaction is a stabilized visible spectrometer (7). Data acquisition is accomplished with a modified analog-to-digital converter (8). Procedures for initiation and control of the experiments involve a n electro-mechanical reagent addition system. CONTROLLER
Computer and Standard Peripherals. The digital computer used in the automated instrumental system is a Model H P 21 15A (Hewlett-Packard, Palo Alto, Calif.), a 16-bit machine with a machine cycle time of 2.0 Fsec. The particular configuration used in this work has 8192 words of core memory and a n extended arithmetic unit. Each of its eight individually-addressable Z/O channels can be interfaced to a peripheral device through a standard microcircuit card and a connecting cable. Relevant standard peripherals include a 10-bit unipolar analog-to-digital converter (ADC) (Model C002, Digital Equipment Corporation, Waltham, Mass.), a Model H P 2737A high-speed paper tape reader, an H P 2752A teleprinter, a Model H P 6933A 16-bit bipolar digital-to-analog converter (DAC), and a digital storage oscilloscope (Model 601, Tektronix, Inc., Beaverton, Ore.). An HP 12553A 16-bit Duplex Register microcircuit card is used to interface the computer t o the electro-mechanical reagent addition system. Programming. I n keeping with the requirements of versatility and adaptability, Hewlett-Packard BASIC (9) was chosen as the macro programming language. The key feature of H P BASIC is the CALL statement that allows the transfer of parameters and control to and from user-written subroutines. It is an on-line, interpretive, conversational version that is dedicated to one user. The ease and simplicity with which a program can be entered, modified, and executed make it a n ideal macro programming language for automated instrumental systems. The version of BASIC used in this work does not contain matrix operations and thus leaves more core space available for the user’s programs. Input-output and specialpurpose routines are efficiently written as machine language subroutines that can be accessed through CALL statements in the BASIC program. The 12-machine language subroutines used in this work require 45 base page locations and 523 locations in higher core. The interrupt capability of BASIC allows the experimenter to regain control of the system, should (7) H. L. Pardue and S. N. Deming, ANAL.CHEM., 41,986 (1969). (8) S. N. Deming and H. L. Pardue, ibid.. 42, 1466 (1970). (9) R. M. Moley, Hewlett-Packard J . , 20(3), 1968, pp 9-13.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 2, FEBRUARY 1 9 7 1
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K
Figure 3. Diagram of system for initiation and control it be necessary at any time, simply by striking any key on the teletype keyboard. Because only one 16-bit output word is used for the initiation and control functions and because that word contains information for 11 devices that d o not all change at the same time, a logical A N D routine is used to prepare the output word. Individual bit patterns for the coding of information on the current status of each of the output devices are stored in separate memory locations. If the status of a particular device is to be changed, the bit pattern in the corresponding core location is changed. The output routine then performs a logical A N D on each of the coding words to generate the composite output word that is finally sent to the interface. Initiation and Control. Initiation and control of the experiments in this work are accomplished using the electromechanical system diagrammed in Figure 3. Single lines denote electrical connections, heavy black lines represent mechanical coupling, and the double lines at the right indicate Teflon (Du Pont) tubing. All electronics are modular with separate chassis and circuit grounds. The modules (with the exception of the computer) are contained in a standard rack mount. All mechanical components are mounted on thick aluminum plates for proper alignment and rigidity,. Cables are used to connect the electronics to the electromechanical components. The mechanical components are connected t o the analytical instrument through Teflon tubing. A square Plexiglas cap was fitted to the top of the spectrometer cell. Six small holes were drilled in it near the edges to accept tubing from each of the four reagent addition syringes, from the rinse valve, and from the apparatus used to remove solution from the cell. A larger hole was drilled near the center to accommodate the stirring rod. The cap was painted black t o minimize problems associated with stray light. All tubing entering the cell was wrapped with black tape to prevent the conduction of light into the cell. For the same reason, the stirring rod was painted black above the cell. Reagent Addition. In this work, electrical pulses from the computer are eventually converted to mechanical motion that is used to deliver up to four different solutions to the spectrometer cell. If a constant total volume is to be added to the spectrometer cell in each experiment of a characterization, then the concentrations of up to three components may be varied independently (within limits). The fourth solution must act as diluent to obtain the required volume. LEVELCONVERTERS. Compatibility and additional buffering between the computer and reagent addition system was accomplished by using level converters. Level converters used are Fairchild 9109 hex inverters (Fairchild Semiconductor, Mountain View, Calif.) with 1.5-K and 330-ohm 194
pullup resistors added at the +12-V input and $6-V output, respectively. All 16 bits of the computer output word are inverted. Logical “1” is +6 V and logical “0” is 0 V. Eight of the bits go to the stepping motor drivers. Seven bits are used to switch relays. STEPPING MOTORDRIVERS. Type A202-2 Series 18 stepping motor drivers (Sigma Instruments, Ind., Braintree, Mass.) rated a t +28 V and 2 A are used to decode forward and reverse +6 V to 0 V (logical “1” to logical “0”) pulses into four current patterns for proper bidirectional rotation of each of the four stepping motors. By decoding in this manner, it is possible to use only eight bits of information t o drive the four stepping motors (forward and reverse pulses for each of four stepping motors). Setting up the proper sequential current patterns within the computer and outputting these directly to the stepping motors would have required 16 bits of information (four current patterns for each of four stepping motors) and more machine language programming but would have resulted in less complex and expensive hardware. The stepping motor drivers are powered from a 28-V, 8-A supply. STEPPING MOTORS. Sigma Instruments Series 18 bidirectional stepping motors (Model 18-2537d16-18) are used to drive the micrometer syringes that ultimately add the reagents to the spectrometer cell. They are compact, two-phase, inductor-type motors with 16 steps per revolution (22.5 degrees/step). The stepping motors are connected to the micrometer syringes by means of flexible mechanical linkages incorporating splined shafts. MICROMETER SYRINGES.Each of the four Gilmont ultraprecision micrometer syringes (Catalog No. 7874, ColeParmer Instrument Co., Chicago, Ill.) has a 2.5-ml capacity with a quoted accuracy of 0.02 (0.5 pl). Calibration in this laboratory of one of the syringes showed a n accuracy of 0.9966 (volume delivered/volume indicated) and deviations from linearity not exceeding 0.3 p1. Each revolution of the micrometer head dispenses 50 pl of solution. Thus, each step of the stepping motors delivers 3.125 pl of solution and 320 steps are required to deliver 1.000 ml. Delivery is accomplished at a rate of approximately 0.31 nil/sec. It was necessary to modify the micrometer syringes a t the swivel joint between the micrometer spindle and the glass syringe plunger because of excessive wear at the original bearing surfaces. The modification was accomplished by machining the original swivel joint into two separate pieces, shaping them to fit a miniature ball bearing race, and bonding them t o the race. This arrangement eliminates binding and wear a t the swivel. However, the linear hysteresis in the modified mechanism is increased to be equivalent to about 0.05 ml. The syringes must be returned beyond the position for starting forward motion and then driven forward to the starting position to eliminate this play before adding reagents. RELAYS. In-line dry reed normally-open SPST relays are used to isolate electrical circuits for mechanical operations from the more delicate electronic control circuitry. Each relay coil is wired into the collector circuit of a switching transistor powered by a separate f5.9-V power supply. Thus, 110 VAC can be switched on or off by the computer for control functions. SOLENOIDS.Split ring devices attached to six linear-action 110-VAC solenoids (Model 4-2-04001, Stearns Electric Corp., Milwaukee, Wis.) are used to operate switching valves for control of solution flow and control of rinse and vacuum operations. The solenoids are normally in the relaxed state. A logical “1” from the computer will close a relay that energizes a given solenoid.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 2, FEBRUARY 1971
VALVES. Hamilton miniature valves (Type 3LLL2, Hamilton Co., Whittier, Calif.) are used for switching the flow of solutions from reservoir to syringe to cell. The common port of the T-type configuration is connected to the syringe. The port corresponding to the relaxed state of the solenoid is attached t o Teflon tubing (KF24TF, Hamilton Co.) that leads t o the spectrometer cell. The port corresponding t o the energized state of the solenoid leads t o the stock reagent solution reservoir. Luer-type connectors are used to attach the tubing t o the syringes and valves. Additional Operations. Additional operations provided by the initiation and control system include a rinse operation to automatically add rinse water to the spectrometer cell, a vacuum operation to automatically remove the contents of the cell, and a stirrer operation to turn a stirring motor on o r off for proper mixing within the cell. RINSE. A solenoid-activated Hamilton valve is used to allow deionized distilled water to flow from a n elevated reservoir into the spectrometer cell. The length of time the valve remains open determines the amount of water that flows into the cell. In this work, the flow rate was 1.25 mljsec. VACUUM. Removal of solution from the spectrometer cell is accomplished by a vacuum operation. The design that was adopted makes use of a mechanism that translates rotational motion from a synchronous motor to linear motion. A simple linkage pushes the tip of a Teflon suction tube (KF24TF, Hamilton Co.) from the top of the spectrometer cell to the bottom of the cell. Vacuum is then applied to the tube until the cell is empty. The vacuum is turned off and the tube pulled t o the top of the cell by further rotation of the synchronous motor. Limit switches on the drive linkage are used to assure consistent positioning of the suction tube. STIRRING.A 110-VAC synchronous motor connected to a stirring rod that was prepared by grinding three angular grooves on the closed tip and approximately 0.25 inch u p the side of a thick-walled nuclear magnetic resonance ( N M R ) tube is positioned above the spectrometer cell. The stirring rod is connected to the shaft of the stirring motor by means of a brass adapter with nylon set screws. The motor is controlled by the computer through one of the programmable relays. It is normally on and usually turned off only when the suction tube is being raised o r lowered. Evaluation of Automated Instrumental System. The evaluation of the automated instrumental system was separated into three phases. The first phase demonstrates that the total instrumental system can operate as an integrated whole when performing routine procedures. Experiments were preprogrammed into the information base. The date interpretation and experimental design procedures were not used. The second phase shows o n a modest scale the ability of the controller to design experiments if provided sufticient information. Data interpretation procedures were not used in this phase. The third and final phase verifies the ability of the instrumental system t o perform autonomously all of the decision-making and routine procedures represented in Figure 2. The system was given relevant information already known about the chemical system to be characterized, the goal of the characterization, a method for trying t o achieve that goal, and criteria for determining when the characterization was complete. Acting entirely o n its own, the instrumental system was then able to complete the characterization and report the results to the experimenter. EXPERIMENTAL
The instrumental system was evaluated by characterizing various aspects of the reaction in which p-nitrophenyl phos-
phate is hydrolyzed to p-nitrophenol and inorganic phosphate by the enzyme alkaline phosphatase in basic solution. The substrate is colorless in the visible region of the spectrum but the highly-colored yellow product p-nitrophenol provides a means of following the reaction photometrically. In all of the experiments described in this work, the total cell volume was 6.000 ml. All reactions were carried out a t 25.0 "C. The points plotted in the figures that follow are individual pieces of data and d o not represent the averages of several runs. REAGENTS. All solutions were prepared using reagent grade chemicals and distilled water that was passed through a mixed anion-cation exchange resin (Amberlite MB-3). ENZYME.The programs within this laboratory are directed a t clinically important enzymes and their determination in human serum. F o r this reason, alkaline phosphatase solutions were prepared from Versatol-E elevated serum calibration reference (Lot No. 2713109, Warner-Chilcott Laboratories, Morris Plains, N. J.) diluted to volume. If not used immediately, the resulting enzyme solution was stored in a refrigerator at 0 "C until further use (never more than 36 hours). Approximately 2 hours before use, the enzyme solution was placed in a water bath at 25 "C. After reaching maximum activity, the solutions were stable at room temperature for at least 6 hours. BUFFER. 2-Amino-2-methyl-1-propanol (2A2MlP) (Sigma Chemical Co., St. Louis, Mo.) was used to buffer the chemical system. Buffer solutions were prepared by mixing the required amount of 2A2MlP with water in a large beaker, adding concentrated HCI dropwise until the desired pH was obtained (the solution had to be cooled to room temperature frequently for proper pH measurement), and making up to final volume with water. SUBSTRATE.Disodium p-nitrophenyl phosphate (PNPP) was used as substrate in this study (Sigma 104 Phosphatase Substrate, Sigma Chemical Co.). Solutions were prepared by weighing a n appropriate amount of PNPP, transferring it t o a volumetric flask, dissolving it in water, and making the solution up t o volume with water. The containers were covered with aluminum foil to prevent photodecomposition of the substrate. PRODUCT. A knowledge of the molar absorptivity ( e ) of the product p-nitrophenol (PNP) is necessary to convert changes in absorbance to changes in concentration for the calculation of reaction rates. Spectrophotometer standard P N P (Stock No. 104-8, Sigma Chemical Co.) was used to prepare a series of solutions of known P N P concentration in 0.7M 2A2MlP buffer a t p H 10.30. Transmittance values were taken a t 404 nm with the modified spectrometer (7). A nonweighted linear least squares treatment of the data yields a slope corresponding to eb of 22.903 X l o 3 liter mole-'. INHIBITOR.Solutions of inorganic phosphate were prepared by dissolving Na2HPOI.7 H 2 0in water, adjusting to the proper p H with a concentrated aqueous NaOH solution, and making up to volume with water. METHODS.The syringes are connected to and filled from reservoirs of solutions necessary to carry out a given characterization ( e . g . , enzyme, substrate, buffer, and water for a substrate dependence study). Care is taken to exclude air bubbles both in the syringes themselves and in the connecting Teflon tubing. The micrometers are set to zero (syringes full). The BASIC compiler and subroutine overlay tapes are loaded into the computer. An initiation subroutine is used to initialize relevant parameters ( e . g . , turn off rinse water and vacuum) before the interface is turned on. The appropriate BASIC language program is then loaded and executed. There is usually an initial dialog in which the controller requests necessary information ( e . g . ,concentrations of reagents and total cell volume). This is followed by the autonomous characterization during which appropriate data are displayed on the digital storage oscilloscope and teletype.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 2, FEBRUARY 1971
195
0
1 2 E H Z Y N E ADDED, NL
Figure 4. Reaction rate us. amount of enzyme added. [ZAZMlP] = 0.75M [PNPP] = 3.00mM @moleml-2 sec-' Slope = 3.485 X Intercept = 0.027 X @moleml-I sec-l Standard error of estimate = 0.030 X sec-I Correlationcoefficient = 0.99997
pmole ml-1
The procedure can be repeated for more refined characterizations or the system can be changed for an entirely different characterization. T o accomplish the latter only the solutions and computer program need be changed. PHASE ONE-ROUTINE
OPERATIONS
T o demonstrate that the total instrumental system can operate as a n integrated whole when performing routine procedures, several characterizations were carried out by preprogramming parameters for the individual experiments into the information base. The data interpretation and experimental design capabilities of the system were not utilized for this part of the study. All of the characterizations in this phase used the following syringe assignments. Channel 1: water to act as diluent. Channel 2: 2A2MlP buffer. Channel 3: a solution of alkaline phosphatase enzyme. Channel 4: PNPP substrate. Linearity of Reaction. The linearity of the pseudo-zeroorder hydrolysis of p-nitrophenyl phosphate by alkaline phosphatase enzyme was determined by making five preprogrammed kinetic runs. Two of the runs were observed for 250 sec, two were followed for 500 sec, and one was monitored for 1250 sec. The enzyme was prepared by adding 3.00 ml of reconstituted serum and 25 ml of 1.57M, p H 10.30 2 A 2 M l P buffer to a 50.0-ml volumetric flask and making up to volume with water. The concentration of buffer in the enzyme solution was 0,785111. All five experiments were identical except for the data acquisition rate: 250 data points were taken at rates of 1 sec/ point, 2 sec/point, or 5 sec/point. Initially, 1.516 ml of water was added as diluent t o make u p the necessary 6.00-ml total cell volume. Next, 2.366 ml of 1.57M, pH 10.30 2 A 2 M l P buffer was added. The water and buffer were stirred for 15 196
sec t o assure complete mixing. One milliliter of enzymebuffer solution was then added and allowed to mix for 1 min: The total buffer concentration in 6.00 ml of solution was 0.75M. Finally, 1.119 ml of 1.607 X 10-2M PNPP substrate was added to provide a concentration of 3.00mM P N P P in the cell and to initiate the reaction. After waiting for 15 sec t o allow complete mixing, the data acquisition was begun. Oscilloscope plots of absorbance as a function of time were found t o be linear within the resolution of the oscilloscope (0.417). The maximum change of absorbance was from 0.34 absorbance unit to 1.29 absorbance units for the 1250-sec reaction. A least squares analysis of rate as a function of minutes followed showed a slight decrease (-0.26 %/min). This might be attributed in part to the slight decrease in concentration of PNPP (-1.4% after 21 minutes) but is probably not highly significant. This was confirmed by the low correlation coefficient (-0.85). Buffer Dependence. Three preprogrammed characterizations were made of the dependence of the enzyme reaction rate on buffer concentration. The enzyme solution was prepared as described in the Linearity of Reaction section but with 1.57M, p H 10.31 2A2MlP buffer. The final cell concentration of substrate was 3.00mM. The same solutions were used for all three characterizations. Each reaction was followed for 256 sec. The reaction was found to achieve a maximum rate at approximately 0.50M buffer. This finding is consistent with the work of Bowers and McComb ( I O ) . In the first and third characterizations, replicate runs were made a t 0.75M buffer. The per cent standard deviation (% std dev) of the five replicates in the first study was 0.54. In the third study, the % std dev of the three replicates was 0.68. Incubation Dependence. A preprogrammed characterization of the influence of incubation period upon reaction rate was carried out. Water, buffer, and enzyme solutions were added to the spectrometer cell and stirred for varying lengths of time. Substrate was added at the end of the incubation period and data acquisition was immediately initiated. Each reaction was followed for 256 sec. The enzyme solution was prepared by reconstituting Versatol-E with 3.00 ml of 0.9% NaCl solution and diluting to 50 ml with the saline solution. N o buffer was added t o the solution. Concentrations of reactants in the spectrometer cell were 3.00mM P N P P substrate and 0.75M p H 10.31 2A2MlP buffer. A slight increase of rate with time was found but is probably not significant (correlation coefficient = 0.49). This behavior is in agreement with the work of Bowers and McComb (10). Enzyme Dependence. An important relationship for ana-
lytical purposes is the dependence of the reaction rate upon the amount of enzyme added. In this preprogrammed characterization, the concentration of PNPP substrate in the cell was 3.00mM and the p H 10.30 2A2MlP buffer was 0.75M. The enzyme solution was prepared as described in the Linearity of Reaction section. The results are shown in Figure 4. It is t o be emphasized that the data points shown are the results of individual runs. They are not averages. All data points in the characterization are included. None were eliminated. The high degree of linearity is revealed in the correlation coefficient of 0.99997 and ___~
-~
(10) G. N. Bowers, Jr., and R. B. McComb, Clin. Chem., 12, 71 (1966).
ANALYTICAL CHEMISTRY, VOL. 43, NO. 2, FEBRUARY 1971
v)
.4 v)
I
I 0 4
I
w
ln I
-1
x \
v)
-1 wc4
0
3 w t-
utY
0
0
2
4
6
C P N P P I , MMOLRR
0
10
0
Figure 5. Reaction rate us. concentration of p nitrophenyl phosphate
i
CPNPPI
3
t
MMOLRR
4
Figure 6. Reaction rate us. concentration of substrate at different inhibitor concentrations
[ZAZMlP] = 0.75M V,,, = (4.291 f 0.029) X pmole mi-’ sec-1 K , = (6.25 f 0.15) X lO-4M pmole mi-’ Standard error or estimate = 0.043 X sec-l
Phosphate concentrations: 0 = 1.009 X 10-ZM 0 = 3.242 X 10VM A = 5.494 X 10-2M = 7.745 x 10-2M x = 9.997 x 10-2M
+
is indicative of the reliability and precision attainable with the total system. Substrate Dependence. The final characterization of the first phase of the evaluation of the automated instrumental system was a study of the substrate dependence of the enzyme reaction. The concentration of pH 10.30 2A2MlP in the spectrometer cell was 0.75M. The enzyme solution was the same solution used in the preceding Enzyme Dependence study. The results of 20 runs are shown in Figure 5. The line through the experimental points was calculated using the Michaelis-Menton equation. Values of 4.29 X pmole ml-I sec-l for V,,, and 6.2 X 10-4M for K, were obtained using the weighted least squares technique of Wilkinson (11). The point corresponding t o 0 mM substrate could not be included in the least squares treatment. The % std dev of the four points corresponding to 3.00 mM P N P P is 1.30%. The precision of the data is further reflected by the standard error of estimate that is 1.OO% of V,,,. PHASE TWO-EXPERIMENTAL DESIGN
This phase of the evaluation of the automated instrumental system makes use of a single characterization to demonstrate on a modest scale the ability of the controller to design experiments. In this phase, data interpretation procedures were not used. The goal of the characterization was to obtain information that would indicate the type of inhibition produced by the product inorganic phosphate. In studies of this type, reaction rate ( u ) is determined for several levels of substrate concentration (s) at each of several levels of inhibitor concentration (i). ( 1 1 ) G. N. Wilkinson, Biochem. J., 80, 324 (1961).
1
Linear plots of l / c cs. 11s for each i and of l / u us. i for each s can be used to determine the type of inhibition ( 1 2 ) . The controller was given the concentrations of the stock reagent solutions and the limits over which the concentrations of substrate and inhibitor were to be varied. The experimental design procedure consisted of creating a matrix of 25 experiments (five levels of substrate and five levels of inhibitor) and executing them in a random manner. The enzyme solution was prepared by diluting 3.00 ml of reconstituted Versatol-E and 3.09 ml of 1.57M, pH 10.31 2 A 2 M l P buffer t o 50.0 ml with deionized distilled water. The final concentration of 2 A 2 M l P buffer in the spectrometer cell was 0.0157M. However, this was supplemented by the buffering action of the p H 10.30 phosphate inhibitor. Four syringes were used and contained water, phosphate inhibitor, enzyme-buffer solution, and PNPP substrate. The results of the characterization are shown in Figure 6 where reaction rate is plotted as a function of substrate concentration for each of five inhibitor concentrations. The lines connecting the points were computed using the MichaelisMenton equation. Values of V,,, and K , for each of the inhibitor levels were obtained by weighed least squares method (11). The data points shown are the results of individual kinetic runs. They are not averaged data. All data from the characterization are shown. No points were eliminated. Figure 7 contains weighted least squares plots (IZ) of l / u us. l/s(so-called double-reciprocal or Lineweaver-Burk plots) for each of the five inhibitor concentrations. Values of V,,, and K , are given in Table I .
(12) H. R . Mahler and E. H. Cordes, “Biological Chemistry,” Harper and Row, New York, N. Y . , 1966, pp 25Ck256. ANALYTICAL CHEMISTRY, VOL. 43, NO. 2, FEBRUARY 1971
197
0
4
600
1200
1800
[PNPPI, L I T E R S / M O L E
0
24
Figure 7. Lineweaver-Burk plots of phosphate inhibition data Phosphate concentrations:
-30
n = 5.494 x 0 = 7.745 x 0 = 9.997 x
3
PHASE THREE-DATA
INTERPRETATION
This third and final phase in the evaluation of the automated instrumental system makes use of all of the routine and decision-making procedures outlined in Figure 2. The results of previous experiments are interpreted as they relate to achieving the stated goal, and new experiments are designed to complete the characterization in a rapid and efficient manner. The goal of many chemical characterizations is to acquire sufficient data so that the relationship between a dependent variable and one or more independent variables may be determined. The range of each independent variable is usually divided into a number of intervals, and the value of the dependent variable is determined for each interval in the range. An example of this type of experimentation is the substrateinhibitor characterization shown in Figure 6. If, as is often the case, the relationship between the dependent variable and an independent variable is not linear, the results of experiments executed at equal intervals of the inde198
e
90
120
=
0.5010 X 10-3M
0 = 1.622 X 10-3M
A
2.749 X 10-3M x 10-3~ = 4.997 x 1 0 - 3 ~
=
+ = 3.876 x
Figure 8 shows l / o us. i for each of the five substrate concentrations. The straight lines in this figure are the results of unweighted least squares analyses. This type of behavior is indicative of competitive inhibition (12).
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Substrate (PNPP) concentrations:
10-*M 10-2M 10-*M
Table 1. V,,, and K,, from Phosphate Inhibition Study V,,,, pmole ml-1 sec-1 X lo6 poi, M x 102 K,, M x 103 1.009 2.71 1.91 3.242 3.68 8.3 5.494 2.61 8.3 7.745 1.97 8.2 9.997 2.03 10.9
30
C P O 4 3 , HHOLAR
Figure 8. Reciprocal rate us. phosphate concentration at different substrate concentrations
X = 1.009 X 10-2M = 3.242 X 10-*M
+
0
pendent variable will yield a curve that is “overdetermined” in one region and “underdetermined” in another. Reference to Figure 5 will clarify this point. If six experiments had been performed at equal intervals of the independent variable, one each at 0, 2, 4, 6, 8, and lOmM PNPP, then the resulting values for the dependent variable would have fallen between 3.25 and 4.25 X 10-6 pmole ml-1 sec-I. The upper portion of the curve would have been adequately determined at the expense of having obtained very little information about the sharply-rising portion of the curve. This is often undesirable. One technique that avoids the problem of underdetermined curves is t o make the intervals of the independent variable small enough that the required resolution for the dependent variable will be assured. In Figure 5 , a resolution of 0.2mM PNPP for the independent variable would have resulted in a very well defined curve. However, the price paid for using this technique is wasteful inefficiency because the upper portion of the curve would then be overdetermined. A more efficient procedure is t o carry out a low-resolution study and use it as a basis for a future characterization in which well-chosen values of the independent variable will yield values for the dependent variable that more evenly define the curve. While the example cited above made use of the experimenter for interpreting the data and designing new experiments, the following sections show that such characterizations can be completely automated. Phosphate Inhibition. The relationship between reaction rate and phosphate concentration is nonlinear. One set of criteria that will assure sufficient properly-spaced experimental points to yield a well-defined curve showing this nonlinear relationship is the following. For any two contiguous experimentally-determined data points, the difference between
ANALYTICAL CHEMISTRY, VOL. 43, NO. 2, FEBRUARY 1971
0
40
80 [PO41 t flMOLAR
120
1 1
0
2
4
[PO41
Figure 9. Reaction rate us. phosphate concentration
t
6 RflOLflR
8
10
Figure 10. Reaction rate us. phosphate concentration
Numbers beside data points refer to order in which points were obtained [2A2MlP] = 0.262M [PNPP] = 3.00mM
Numbers beside the data points refer to the order in which the points were obtained [2A2MlP] = 0.262M [PNPP] = 3.00mM
the values of the independent variable cannot exceed a specified amount ( A X ); similarly, the difference between the values of the dependent variable cannot be greater than a predetermined value (A Y). Clearly, the criterion of maximum AX can be used t o establish the conditions for the initial set of experiments. The criterion of maximal A Ycan be used in designing the later, more refined experiments. These criteria were used both as the goal and as conditions for determining when the goal had been reached in two studies of phosphate inhibition. The reagent addition system was prepared as described in the substrate-inhibitor study (Phase Two). Substrate concentration in the cell was 3.00mM PNPP. The pH 10.30, 2A2MlP buffer concentration in the cell was 0.262M. The results of the first study are shown in Figure 9 in which reaction rate is plotted as a function of phosphate concentration. The numbers beside the experimental points indicate the order in which the points were obtained. Initially, six experiments were carried out t o satisfy the A X requirement (30.0mM phosphate). These six data points were then scanned t o detect AY's greater than the specified limit (10% of the maximum reaction rate in the set). If a pair of data points exceeded this limit ( e . g . ,points 1 and 2), a new experiment was carried out (experiment 7) in which the phosphate concentration was set equal t o the midpoint (15mM) between the two values of interest (OmM and 30mM). After obtaining the reaction rate for this concentration of phosphate, the expanded set of seven data points was examined for the A Y criterion, and so on. After 15 experiments that took a total of approximately 21/2hours, the characterization was considered complete. The line drawn in Figure 9 was calculated from parameters obtained from a nonweighted least squares treatment of l / u GS. i for points 7-15. Points 1-6 were not included because the enzyme solution had not reached maximum activity at the start of the characterization.
Figure 10 shows the results of a similar study in which the range of phosphate concentration was much smaller. A more concentrated enzyme solution was used. The AX requirement was 5.0mM phosphate. The A Y requirement was 5 % of the maximum rate in the set. The line was calculated using all 15 data points. pH Dependence. In some instances, the experimenter may be interested in finding the value of the independent variable that yields a maximum or minimum value for the dependent variable. Again, an initial set of experiments at equal intervals throughout the range of the independent variable can serve as a basis for future, more refined experiments. To demonstrate again the ability of the automated instrumental system to interpret data and design experiment, a characterization was carried out to determine the pH at which the enzyme reaction rate was maximal. The enzyme solution was prepared by reconstituting two vials of Versatol-E with 3.00 ml of 0.9% NaCl each and diluting to 50.0 ml with saline solution. The pH was varied by adding a constant total amount, but different relative amounts, of pH 8.72 and pH 10.72, 0.837M 2A2MlP buffers. The contents of the four syringes were pH 8.72 buffer, pH 10.72 buffer, enzyme solution, and substrate. The concentration of substrate in the cell was 3.00mM PNPP. The buffer concentration in the cell was 0.49M 2A2MlP. The algorithm for determining the pH maximum required five initial experiments in which the ratio of buffer volumes was varied in four increments to cover the range of interest of the independent variable (pH 8.72 to pH 10.72). These data were examined to find the maximum rate in the set. Two new experiments were designed and carried out at intervals equal to one half the original interval on either side of the volume ratio corresponding to the maximum rate. The procedure was repeated until the resolution of the independent variable (buffer ratio) was of the range. This required 11 experiments to attain this resolution at the pH maximum.
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(D
0
8.5
1
9.0
9.5
PH
10.0
10.5
1
3
Figure 11. Reaction rate us. pH Numbers beside the data points indicate the order in which the points were obtained [2.42MlP] = 0.49M [PNPP] = 3.00mM
The results of the determination are shown in Figure 1 1 . Enzyme reaction rate is plotted as a function of calculated pH. Volume ratios were converted to pH assuming a pK, of 9.72 for the buffer (13, 14). After each experiment, the solution was collected and its pH measured immediately. The agreement between measured pH values and calculated pH values was never worse than 0.10 pH unit and averaged 0.05 pH unit. The numbers beside each point indicate the order in which the points were obtained. A replicate characterization yielded identical results. It can be seen that the instrumental system found the pH maximum in a straightforward and efficient manner. CONCLUSIONS
The primary advantage of using an automated instrumental system is the increased efficiency of the experimenter. Most obvious is the fact that he has more free time to accomplish other tasks that d o not lend themselves as readily to automation. The rapid availability of the results of a characterization is another means of increasing the efficiency of the experimenter. The slow steps of experiment preparation have been
(13) S. Glasstone and A. F. Schram. J. Amer. Chem. SOC..69. 1213 (1947). (14) C . E. O’Rourke, L. B. Clapp and J. 0. Edwards, ibid.,78, 2159 (1956).
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greatly speeded up. The progress of a project can be measured in hours rather than in days. This is especially important if the results of a characterization are invalid for one reason or another (poorly designed experiments, a n insufficient number of controlled parameters, etc.) The “wasted” time with the automated instrumental system would be only a fraction of what it would be with conventional approaches. Thus, the “setbacks” are also measured in hours rather than days. Another advantage of the instrumental system is the accurate and repeatable initiation of experiments. Uncertainties in reagent concentration and amount of time between operations in setting up a n experiment can be minimized. The result of this close control of parameters is a repeatability of better than 1 in many cases. A further advantage of the automated instrumental system is the ease of drastically changing the type of experiment used to characterize a chemical system. Replacing the analytical instrument with a polarograph, a liquid-solid chromatograph, or a n atomic absorption spectrophotometer would cause no serious problems. One criticism might be that the preparation and debugging of suitable BASIC language computer programs could more than offset any efficiency gained during the actual characterization. This is doubtful for three reasons. BASIC is extremely easy t o debug. Simply retyping a statement changes the program. There is no time-consuming off-line translation and compilation of the whole program. Second, the procedures used in the characterizations are routine in nature and tend to be the same from characterization to characterization. Thus, the only parts of a program that take time to write are the sections that deal with experimental design and data interpretation. The routine procedures are programmed using BASIC language subroutines. Finally, many characterizations are repetitive. For example, the determination of pH optimum could be used for several chemical systems or for diferent buffers with one chemical system. Only one program need be written for the characterizations. This study has shown the feasibility of using a completely automated computer-controlled instrumental system for the fundamental characterization of a chemical reaction. The resulting instrumental system has been applied primarily to the study of solution kinetics. However, it is applicable to other types of chemical investigations as well. ACKNOWLEDGMENT
The authors are grateful to Craven Smith for his technical assistance in the mechanical design. RECEIVED for review September 17, 1970. Accepted November 9, 1970. This investigation was supported in part by PHS Research Grant No. GM13326 from the National Institutes of Health and in part by a grant from the Indiana Elks Association. One of us (SND) is grateful for a predoctoral fellowship from the Ethyl Corporation.
ANALYTICAL CHEMISTRY, VOL. 43, NO. 2 , FEBRUARY 1971
