vided the substituent has an adequate effect on ring electron density. Systems containing two more more rings can be distinguished from tetra-alkyl or pyridinium compounds. The large shift observed for the introduction of an oxygen atom in the indolenine ring (compound No. 43, cf. No. 42) makes distinguishing between these closely related structures easy. Clearly one could distinguish free amines from quaternaries. ESCA has the advantage that the information can be obtained nondestructively on a small amount of sample-considerably less than a milligram here, and as little as a few micrograms if vacuum deposition techniques are used. It is obvious, however, from the better than 5-eV spread observed for quaternary compounds that single-atom correlations will be of limited value. This becomes clear when the spread is compared to the total spread of some 10 eV for a variety of nitrogen functional groups as reported in a preliminary correlation chart ( I ) . The large anion effects require that the counterion must be determined to correct for its effect to some standard counterion-e.g., Br-. Some adjustment must also be made for substituent effects. This points out that a multiple-atom correlation must be the approach used. The binding energies of anions and possible substituents can be easily determined by ESCA.
The ESCA technique will be of significant value in structural determinations once a sufficiently large body of information is available to do multiple-atom correlations. In this sense it is similar to IR, NMR, and mass spectrometry which depend on empirical information. Indeed it will be a complementary tool to these techniques. Meanwhile ESCA can still be used in specific cases to distinguish between alternative structures. ACKNOWLEDGMENT
We would like to acknowledge the assistance of William Swartz in many aspects of our work. We also thank the following for their help in starting our ESCA program: A. Waraksa, S. Hagstrom, R. Nordberg, A. Fahlman, C. Nordling, and K. Siegbahn.
RECEIVED for review November 30,1970. Accepted February 19, 1971. One of us (J.J.J.) would like to thank the National Institutes of Health for a pre-doctoral fellowship during the term of this research. Work supported in part through funds provided by the U. S. Atomic Energy Commission under Contract AT-(40-1)-4043.
ELLA (The Experimental LINC-L,aboratory Analytical System) Applied to Experimental Control A. A. Eggert,' G . P. Hicks,2 and J. E. Davis3 Department of Medicine and Department of Chemistry, University of Wisconsin, Madison, Wis. 53706
Traditionally, the steps in a classical analytical experiment are performed by the experimenter. Advances in on-line computer technology, digital implementation, and mechanical hardware now make it feasible for an on-line computer to control the performing of an experiment and make modifications in the procedure based on data gathered earlier in the experiment. To this end, a computer system has been designed which will completely perform a kinetic experiment to a desired end point, making all necessary decisions and controlling all the instrumentation by means of a special purpose time-sharing system. In addition, the system can present the data it gathers in various ways to the experimenter via teletype, oscilloscope, and incremental plotter. This development makes it possible for the system to perform experiments and produce the data in acceptable form for publication, with the experimenter supplying only the reagents for the experiment and the labels for the graphs. THE NECESSITY of producing chemical analyses rapidly and accurately has led to a revolution in chemical instrumentation. Yet the large quantities of data generated by these instruments can raise more questions than they answer unless the data can be analyzed and reduced to a form which human
Present address, Department of Chemistry, Duke University, Durham, N. C . 27706. Present address, Laboratory Computing, Inc., 4915 Monona Drive, Madison, Wis. 53716; author to whom correspondence should be addressed. Present address, Department of Chemistry, Purdue University, Lafayette, Ind. 736
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ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
beings can understand. In chemical research the decision of what to do next in an experiment often depends upon data already gathered, but which will take hours or days to analyze. Valuable observations are frequently lost or obtained only by repetition of experiments because data could not be analyzed quickly enough when the experiment was first performed. Decisions made during experiments are frequently based on partially analyzed data and mental sketches of what the results should mean. Then too, errors in manual reagent manipulation can frequently sidetrack an investigation while a nonexistent phenomenon is sought. To solve these problems, many have recently introduced digital laboratory computers into their research (1-4). Considerable justification for the use of such computers (4) and the analysis of the areas in which they may be applied to analytical chemistry (5)have previously been presented in the literature. Applications of laboratory computers previously reported have primarily stressed their use in the gathering and reducing of data, although Perone et a/. have reported feedback control (3) and Hicks et al. (5) have reported some online analysis and work on hard-copy data presentation. (1) G. P. Hicks and A. A. Eggert, Clin. Chem., 14,798 (1968). (2) J. W. Frazer, ANAL.CHEM., 40 (8), 26A (1968). (3) S. P. Perone, J. E. Harrar, F. B. Stephens, and R. E. Anderson, ibid., p 899. (4) G. E. James and H. L. Pardue, ibid., 41,1618 (1969). ( 5 ) G. P. Hicks, A. A. Eggert, and E. C. Toren, Jr., ibid., 42, 729 (1970).
~~
Recently, a system was reported which used a laboratory computer to mix reagents and choose more data points based on simple statistical criteria (6). Two significant requirements still remain, however, before the computer has truly reached its potential. First reagent mixing, reaction measuring, and decision-making must be done as simultaneous rather than sequential actions. Second, decisions regarding new data points must be based on chemical rather than just statistical procedures. The early development of ELLA (Experimental, LINC-Laboratory Analytical System) has previously been reported ( I , 5). ELLA was primarily concerned with three areas of the analytical experimental process: data acquisition and reduction, analysis, and presentation of data. ELLA was originally designed to read any instrument which produced a voltage ramp proportional to the rate of a reaction. Using techniques developed by Cordos et al. (7), the rate was converted to a voltage level. This voltage level was then read periodically by a LINC (Laboratory ZNstrument Computer) (8) which reduced the data to rate information and saved it for future analysis. Various data analysis options which could be applied to both off-line and on-line data were present in the system. These produced printouts of data and constants as well as oscilloscope displays of the analyzed data. Information could be plotted on a digital incremental plotter. Other features such as graph labelling, magnetic tape files, simulated desk calculators, and bar and broken line graphs were also available in the software system to aid the experimenter in understanding the meaning of his experimental results (5) To solve the problems discussed initially, it is necessary to have a system which can perform a significant part of the mechanics of an experiment and make intermediate decisions based on chemical principles to obtain acceptable experimental results. ELLA was designed to permit such additions because the computer was not dedicated to reading the analytical instrument, but needed only to reference the hardware at most every two seconds to get the rate information. Consequently, it was possible to expand ELLA to do a complete experiment. The expansion of ELLA has been accomplished by extending the principles of hardware-software interaction already developed. An extensive digital control system was built which synchronized the operation of external equipment to the programming of the computer. In addition a reagent thermostating and mixing system was designed to permit the automatic handling of all solutions. Special programming was added to ELLA to allow the system to control external equipment, read data, and make decisions in a special purpose time-sharing system. Consequently, ELLA could make up the reagent mixtures to be run, move them into the spectrophotometer, read the rate of the reactions, analyze the data from the previous mixtures, and decide where to take more points based on chemical considerations, all at the same time. This whole system operated in only 2K of memory and used magnetic tape storage in such a manner as not to consume experimental time. The expanded ELLA has been used to study two substrate curves. The first was the nearly ideal system of alkaline phosphatase-p-nitrophenolphosphate, and the second a highly nonideal system purposely obtained by improperly (6) S . N. Deming and H. L. Pardue, ANAL.CHEM., 43, 192 (1971). (7) E. M. Cordos, S. R. Crouch, and H. V. Malmstadt, ibid., 40, 1812 (1968). (8) W. A. Clark and C. E. Molnar, Ann. N . Y . Acad. Sci., 115, 653 (1964).
COMPUTER
It
UQ ACCUMULATOR BUFFER
SEQUENCE TIMER
RATE METER
HOTOMETER
OLENOID VALVES
WASTE FLASK
MIXING CHAMBER
Figure 1. Interrelationship of mechanical hardware and interface All physical and electrical connections between hardware are shown
adjusting the reagent concentrations of the lactic dehydrogenase-lactate reaction. In the former case, the reaction constants were determined with a reproducibility of 5-7 % relative while in the latter case the reproducibility was 7-26 % relative. These were as good as the results obtained by human experimenters working under the same conditions. HARDWARE
All the equipment used in ELLA, except the computer IjO devices, is shown in the block diagram in Figure 1. The spectrophotometer is built around a modified Bausch and Lomb Spectronic 20. A third photocell and a logarithmic amplifier have been installed in the case to give a stable, linear signal of 1.OO-volt output change for an absorbance change of 1.00 unit. An offset control provides for suppressing up to three decades of absorbance and for convenient recorder scale expansion at any given absorbance. The short-term drift of the spectrophotometer-logarithmic amplifier combination is less than 1 mV/min (0.001 absorbance unit/min). A thermostated flow-through cell was installed in the spectrophotometer to allow solution to be transferred into the spectrophotometer automatically. The temperature was maintained at 37.0 & 0.1 “C by water pumped from a thermostatic bath (Yellowstone Instruments Model 72). The digital computer used was a p-LINC-100 (2K memory, 8-psec memory cycle time) manufactured by Spear, Inc. [LINC computers are manufactured by both Spear, Inc., Watham, Mass. (JL-LINC)and Digital Equipment Corporation, Maynard, Mass. (PDP-12A)I (8, 9). The standard LINC configuration includes an oscilloscope, keyboard, teletype, two magnetic tape units, 6 DPDT relays, and a 16channel, 9-bit analog-to-digital converter (24 psec conversion time). The data terminal panel has been modified in the authors’ laboratory to include an interrupt clock (0.1, 1, 10, and 60 Hz pulses) and an interface to drive an incremental plotter (Houston Instruments, COMPLOT Model DP-1-1). Plugs have also been added to bring out the accumulator, clock pulses, operate pulse lines, and level lines on the data terminal panel. With these modifications, the LINC can be (9) “Small Computer Handbook,” Digital Equipment Corporation, Maynard, Mass., 1970. ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
737
toon
Figure 2. Schematic diagram of reaction rate interface Resistors are 1% li2 watt, unless otherwise specified. Resistors marked * are closely matched with other similarly marked resistors in that part of the circuit. Operational amplifiers OAl and OA3 are Burr Brown, Model 3112/12C. OA2 and OA4 are Burr Brown, Model 3115/12C. OA6 and OA7 are Analog Devices, Model 111. OA5 is Analog Devices, Model 118A. The feedback capacitors are 200 V polycarbonate dielectric. Relays are Magnecreaft reed relays, ca. 1-msec operation time. NAND gates are Texas Instrument SN7400N and SN7420N. The flip-flops are Texas Instrument SN7472N and SN7576N. The 5-volt supply is Computer Products, Model MC411. The =tl5-volt supply is Philbrick, Model PR-300
readily connected to the experimental equipment. All voltage levels are compatible with TTL logic. The digital pipet (on loan from the DuPont Company), has the capability of picking up 5 ml in 5-pl steps. The amount of reagent to be picked up or delivered is determined by the number of clock pulses from the accumulator buffer. The pipet is thermostated by water from the heating bath. The pipet draws reagents into a 7-ml holding coil which is filled with buffer and thermostated in the heating bath. The reagents are drawn into the holding coil through a steel tube in the arm of a turntable. The turntable has 25 positions for 5-6 cups and turns at the rate of 2 cups per second while the arm is raised and lowered for each cup. The interface initiates turntable movement by an electronic switch which can only be turned off when a cup is in position. The reagents are expelled from the pipetting system into a thermostated mixing tube which is stirred by a Magnestir Model 52617 manufactured by the Chicago Apparatus Company. The mixing tube was constructed by cutting a polypropylene test tube in half and implanting two small metal tubes into the bottom. The tube was then wrapped with Calorex heat transfer tubing which was connected to the heating bath. The sequence timer controls values which sample the reaction mixture and drain and wash the mixing chamber. One port of the mixing chamber is connected to the thermostated flow-through cell of the spectrophotometer and then through a solenoid valve to a waste flask. This valve is activated first and draws new sample through the flow-through cell for 10 seconds. The next valve activated is in the tubing between the other port of the mixing tube and the waste flask. This valve drains the mixing chamber and remains on until the next time the mixing chamber is needed. The final valve 738
ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
connects a flask of wash solution with the mixing cell. After the cell is drained, this valve is opened for 5 seconds to allow wash solution to enter the mixing chamber by gravity flow. All valves are model C2d X 143 manufactured by Skinner Electric. The vacuum pump is a Model 2 Neptune DynaPump manufactured by Universal Electric Company. INTERFACE
This section contains a detailed description of the electronics which were built and need not be read to understand the operation of ELLA. Reaction Rate Interface. The reaction rate interface (RRI) has been described (5). It was designed using a method suggested by Cordos et al. (7) and modified by Toren et al. (IO) in which a voltage level proportional to a voltage ramp can be obtained by integrating for a fixed time with one polarity, then reversing the polarity and integrating for the same amount of time. Several improvements have been made in the RRI since its construction was first reported. These changes have greatly improved the stability of the instrument and virtually eliminated calibration adjustments. The new configuration is shown in Figure 2. The logic design is the same as reported previously. The logic and the analog circuitry are hard-wired on four modular cards as indicated by the dashed lines. The circuitry between operational amplifier OAl, the sample and hold amplifier, and OA4, the integrating amplifier, has been modified to improve the stability. The third card contains the autoscaling circuit which has been (10) E. C. Toren, Jr., A. A. Eggert, A. E. Sherry, and G. P. Hicks, Clin. Chem., 16, 215-21 (1970).
moved after the integration amplifier to permit scaling without having to reintegrate. Relay R7 is manually controlled and selects the source of the input. Relays R8 and R9 are for scale selection and are controlled by the computer. OA5 is used in scale selection. OA6 changes the sign of the voltage level if negative, OA7 outputs the sign as a separate voltage level. R2, R3, R8, and R9 correspond to computer relays 3, 2, 1, and 0, respectively. These relays are duplicated in the interface to avoid transmitting low signal levels in long leads through a noisy external environment. Accumulator Buffer. The turntable and the pipet are operated by an accumulator buffer (Figure 3). To understand the operation of this buffer, it is necessary to be familiar with the external control lines of the LINC. The LINC has 16 lines called operate lines numbered in octal 0-17 (8). Operate line n (0 5 n 5 179) will transmit a 4-microsecond logic 1 pulse when an “OPR n” instruction is executed. Operate line m (0 5 m 5 138) will transmit arbitrarily long pulses when an “OPRi m” is executed. The pulse in the latter case is terminated when a logic 1 level ($5 volts) appears on the external level (XL) line m. Each operate line between 0 and 138 is paired with an XL line, and these may be used together or independently. XL lines may also be read by an “SXL m” instruction which causes a skip in the programming when the XL line m is at logic 1. Each operate line pulse is followed by a 0.4 microsecond pulse on each of three lines called 2.1, 2.2, and 2.3. These pulses are spaced by 2 microseconds and appear on the lines in the order given. The accumulator can be read by external devices at any time by their initiation. The computer will read into its accumulator external information when its read line is pulsed with a logic 1. The LINC produces the 60, 10, and 1 Hz pulses which have been used in ELLA. The accumulator buffer consists of two parts. The first is an up counter which is gated by device selection flip-flops. After this part of the accumulator buffer has been loaded and a device selection flip-flop has been set, the lowest flipflop in the buffer is toggled by a clock whose speed is determined by the device selected. The lowest flip-flop toggles the next, and this process continues until the tenth flip-flop in the buffer. When the toggling process changes all the flip-flops to logic l’s, the buffer, through a NAND gate network, produces a logic 0 (0 volts) which is used to clear all the device selection flip-flops. The other part of the accumulator buffer consists of one flip-flop which stores the direction in which the devices (presently, only the pipet) are to run. It is not connected to the rest of the flip-flops in the buffer and is not affected by the counting that occurs in them. During the experiment, information necessary to control equipment is transferred into the accumulator buffer, after which the computer is free to do other operations. All eleven flip-flops in the accumulator buffer behave in the same manner during transfer. To transfer a number to the accumulator buffer, it must first be loaded into the LINC accumulator. An OPRi 1 instruction is then executed. This effects the transfer in the following manner: The OPRi 1 instruction causes a logic 1 pulse to be sent on operate line 1 which continues until a logic 1 level appears on external level line 1. The pulse on operate line 1 is applied through a NAND gate to the SET of the transfer control flip-flop, causing a logic 1 to appear on external level line 1 and the operate level to cease on line 1. After each operate pulse, the computer sends out pulses on the 2.1, 2.2, and 2.3 lines. The transfer control flip-flop prevents these pulses from ac-
Figure 3. Schematic drawing of the accumulator buffer The flip-flops are Texas Instrument SN7476N. The NAND gates are Texas Instrument SN7400N (2 input) and SN7420N (four input). The power supply is Computer Products, Model MC411. Lines marked “A” are from the LINC accumulator. Lines marked “I” go to the accumulator
tivating except after an OPRi 1 has been executed. Line 2.1 is nanded with the transfer control flip-flop, which produces a logic 0 only when both of them are logic 1. This logic 0 is applied to the CLEAR of each flip-flop in the accumulator buffer. The accumulator buffer must be cleared before transfer to remove the previous contents which would interact with the new information to produce an erroneous value. After the 2.1 pulse has been used to clear the buffer, the 2.2 pulse is nanded with the transfer control flip-flop and this result is inverted to give the right logic pulse, namely, logic 1 . This is then nanded with the individual bits of the accumulator, and the result is placed gainst the SET’S of the flip-flops of the accumulator buffer. The flip-flops are set to logic 1 which corresponds to the logic 1 bits in the accumulator. Pulse 2.3 is nanded with the transfer control flip-flop and then the logic 0 is applied to the CLEAR of the transfer control flip-flop to shut it off. This must be done to prevent unwanted transfers by other OPR instructions. After the transfer is complete, the program immediately executes an OPR 6. Since the operate 6 line is externally connected to the accumulator read line, this pulse causes the information in the accumulator buffer to be reread into the accumulator by a process inside the computer similar to the one just described. The accumulator is then checked by the program against the information that was originally sent to the buffer. If the information is the same, the program continues; if not, the accumulator is reloaded with the ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
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+5
2
A
120 n r CLOCK
.
+24
3 AMPS
Figure 4. Schematic diagram of control logic for stepping motor of digital pipet Stepping motor can be driven in either direction at 120 steps per second. The flip-flops are Texas Instrument SN7472N. The NAND gates are Texas Instrument SN7400N. The 5-volt power supply is Computer Products, Model MC411. The 24-volt supply is a Kepco, Model PRM24.5
correct value and the transfer executed again until it is successful. Device Selection. Once the correct number has been transferred to the accumulator buffer, a device must be selected. The pipet is selected by executing an OPRi 4 instruction. The pulse generated is nanded with the clock to prevent the pipet flip-flop from being set while the clock is sending a pulse which might cause a counting error. When the pulse does pass through the NAND gate, it sets the pipet flip-flop and a logic 1 level appears on external level line 4, causing the pulse on operate line 4 to terminate and the computer to proceed to its next instruction. Meanwhile, the pipet flip-flop begins to allow the clock pulse through. These pulses cause the accumulator buffer to count up and enable the movement of the pipet. This process continues until the acchmulator buffer flip-flops all reach logic 1, at which time the pipet flip-flop is cleared. The clock is constructed around a uni-junction transistor and gives short logic 1 pulses at approximately 120 Hz. The turntable is controlled in the same manner as the pipet. Since the turntable continues to run until its control flip-flop is turned off and does not rely on a specific number of pulses, it is not necessary to nand the clock into the setting circuit of the turntable flip-flop. The 60-Hz computer clock is used for the timing. The turntable control flip-flop is set with an OPRi 3 instruction. Digital Pipet and Turntable. The pipet control logic is composed of two parts (Figure 4). First is a directional control which allows the pipet to operate in either direction. The second part is the pattern-making circuitry which drives the stepping motor. The directional control circuitry consists of two cross-coupled NAND gates which are connected to the Q and outputs of the directional control flip-flop of the accumulator buffer. These NAND gates act as a setreset flip-flop, the output of which determines which set of the counting NAND gates to be active. When one set is active, the flip-flops which control the stepping motor position are moved through their cycle in one direction, while if the other set of NAND gates is enabled, the flip-flops are moved through their cycle in the other direction. Micro-switches are attached to the ends of the pipet lead screw to send an
a
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immediate stop pulse back to the device control flip-flop to prevent damage to the equipment if the plunger reaches the end of the pipet barrel. The turntable logic is relatively simple, consisting of a relay controlling a triac. A manual push button and a camcontrolled switch which prevents the table from stopping between positions are in parallel with the relay. Since the table requires second for each step, the accumulator buffer is set to allow 30 counts of the 60-Hz clock to move one table position. Valving System. A third logic system controls the valves which allow solutions to move into and out of the mixing chamber. This logic, shown in Figure 5 , is activated when the pipet begins to expel reagents. By nanding the output signal with the gated clock for the pipet, this logic is designed to trigger a monostable circuit which delays until all the reagents have been expelled into the mixing tube and the mixing period is over. This time is fixed at about 15 seconds (10 for the monostable and 5 for the counting circuit). At the same time the monostable is triggered, the drain control flip-flop which keeps the solenoid valve to the waste container open is cleared. Therefore, as the pipet starts pushing out the solution, the mixing tube has all its ports closed to receive the solution, When the monostable fires, the master control flip-flop gates the 1-Hz clock from the computer. The counting circuit counts to 5 , generates a pulse, and clears itself so it can repeat the cycle. The first pulse generated by the counter causes the flip-flop which controls the movement of solution into the spectrophotometer to be toggled on. Five seconds later, the second pulse causes the delay flip-flop to come on and allow the sampling to continue for 5 more seconds. Consequently, for 10 seconds the solution is moved through the spectrophotometer cell to flush out and replace the last sample. At the end of the next 5-second period, the delay flip-flop is toggled off, which then turns the drain control flip-flop on. When the drain control flip-flop comes on, it clears the sampling control flip-flop and prevents it from switching again by applying a logic 0 to its J input. The drain control flip-flop opens the drain solenoid, which allows the excess reagents to be removed from the mixing tube. It also removes the logic 0 being applied against
I NIT1ALlZATlON ROUTINE
1“ ON-LINE OPERATING SYSTEM
[
CLEAN-UP ROUTINE
[
IUP-DATING SYSTEM
1
+
DONE
Figure 6. Flow chart, the experimental control and decision-making portion of
ELLA
Figure 5. Schematic diagram of valve sequence logic The monostable gives a delay time. The five-second counter is the time base for valve sequencing. The flip-flops are,Texas Instrument SN7472N and SN7476N. The NAND gates are Texas Instrument SN7400N. The unijunction transistor is General Electric D13T1
by means of series resistors and by pass capacitors to suppress voltage spikes caused by flip-flops changing state. All unused inputs are tied to the + 5 volt supply. Cables are shielded when necessary. For flip-flops driving distant circuits, a 5000-ohm series resistance is added at the source to reduce capacitive loading on the flip-flops, thereby preventing them from switching. Capacitors are used extensively to suppress transients in the power switching circuits. The above precautions should not be regarded as a reflection of the quality of the TTL logic, but as a consequence of its wide bandwith (a result of being fast) (12).
the wash control flip-flop’s J input, preventing it from coming on. With the fourth 5-second pulse, the wash control flipflop comes on, and wash solution begins running through the wash control solenoid valve into the mixing tube. In addition, the logic 0 against its K input which was keeping the master control flip-flop on is removed. With the fifth clock pulse, the master control flip-flop and wash control flip-flop are turned off, leaving the system in the same state as before the monostable was triggered, with only the drain control flip-flop on. In addition to control of the valves, this logic must also trigger the computer to start reading the rate of the reaction. To do this, the third clock pulse, the one which causes the draining system to be activated, also causes a flip-flop to be cleared which holds external level line 10 at logic 1. This flip-flop remains off until the fourth clock pulse causes it to be reset to logic 1. The computer detects the signal on XL line 10 by an SXL 10 instruction in the program. Logic Design. All logic circuits were developed and tested on the Heath EU-801A Analog-Digital Designer (11). Only after the circuit design had been proved were the components hard-wired. To eliminate noise, all logic cards are surrounded by grounded metal strips which function as a ground plane. The accumulator buffer card is laced with these interconnected strips to reduce radiated noise. All analog and digital grounds are kept separate. Power and ground connections to individual logic packages are made in parallel from metal strips to prevent ground loops. The logic packages are grouped as to type, when necessary, and these are decoupled
The software of ELLA has five important tasks: initializing and calibrating the system; reading the spectrophotometer by means of the reaction rate interface and reducing the data; controlling the reagent preparation equipment; making decisions as to what to do next; and doing the system bookkeeping including saving the data. These tasks are overlapping in time and often require several programs to be present at once in the system. This section explains how the softwear is set up to accomplish these tasks. The overall flow diagram of ELLA is given in Figure 6 . Programming Sections. For programming purposes, the software of ELLA is broken into four parts called the initialization routine, the on-line operating system, the updating system, and the clean-up routine. These four sections are first discussed from the aspect of what tasks are being performed at what times. Then the important subroutines are discussed from the aspect of how they accomplish their objectives. The first subprogram in ELLA, called the initialization program, obtains necessary information from the experimenter, sets up the standard tubes, calibrates the reading system, and prepares the blank reading tube. The program requests the experimenter to enter the volume of enzyme to be used and the time to wait between the start signal and when readings are taken. This is all the experimenter is required to do; all other operations described are automatic. The
(11) Heath Company, “Heath Analog Digital Designer, EU-801 Series,” Benton Harbor, Mich., 1968.
(12) H. V. Malmstadt and C. G. Enke, “Digital Electronics for Scientists,” W. A. Benjamin, Inc., New York, N. Y . , 1969, p 163.
SOFT WARE
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CALCULATE
v &;&----q OR)
ANOTHER
TUBE ABORT
SET FLAG FOR DONE
I
RATION NO I1
I
I
REJECT POINT
CALCULATE RATE PREPARATION OPERATION
T1
PRINT RESULTS
RETURN WHERE INTERRUPTED
TO UP-DATE
SYSTEM
TO ON-LINE ROUTINE
Figure 7. Flow chart of on-line operating system
Figure 8. Flow chart of up-dating system
enzyme volume is requested for the convenience of the experimenter and could be determined by convention if a standard amount of enzyme could be decided upon. The delay time gives the system a means of compensating for incubation time and system stabilization. Both of these pieces of information could be determined by the system, but since the experimenter generally knows this information, considerable time is saved by informing the system rather than making it discover facts already known. The program uses the enzyme volume to set up the first five tubes at arbitrary values of 0.660, 0.315, 0.150, 0.070, and 1.385-m1 substrate. These values were chosen because, when rearranged, they represent a progression which is almost geometric, and thus, permit a good initial estimate of the K M(Michaelis constant) (13) to be obtained if it is within the dynamic volume range between 0.05 and 2.00 ml. The volumes of the other reagents are fixed by system convention. The program checks that the reaction rate interface is functioning within tolerance by running a calibration routine. The input to the reading hardware is first shorted, and a hardware zero reading determined. The track and hold circuitry (Figure 2) is then used to generate a ramp to calibrate the scale setting at a fixed point on the scale. At the same time, the pipet and turntable are indexed through the procedure of making up a blank tube (no substrate) for a zero reaction rate correction. When the calibration is finished and the reagents have been picked up by the pipet, data storage areas are initialized, and the pipet is cycled to release the reagents. The reagents are then automatically mixed and transferred to the flow-through cell of the spectrophotometer.
The on-line operating system performs many tasks simultaneously (Figure 7). First it takes readings of the rate of the reaction occurring in the spectrophotometer and controls the reaction rate interface (RRI). The data gathered from the RRI must be reduced and stored. At the same time, the pipet must be cycled to prepare the next tube. After at least three tubes have been run, the optimum volume of substrate to put in the next tube (the actual decision-making) and an up-date of the reaction constants must be calculated. Finally, during this whole process, the program must set flags indicating any error which might be present, such as too much enzyme or an experimenter request to abort. The up-dating program in the system runs after each tube to process the information from the previous tube and set up for the next (Figure 8). First the pipet is discharged and the timing circuitry activated to set up the tube just prepared for reading and reinitializes the hardware so it can prepare another tube. Next the flags which were set by the on-line operating program are examined to determine if the program has finished its analysis or if there is some reason to abort the experiment. If the experiment is aborted, the reason is printed. Various position pointers also are up-dated. Aside from the purely bookkeeping tasks, the program calculates the fit of the rate points. The calculated initial rate is corrected for the blank and printed along with other relevant information shown in Figure 9A. Finally the system performs bookkeeping and initializes all the routines for the next tube. If the experiment is finished or aborted, the clean-up routine is called; if not, control is returned to the on-line operating program. The clean-up program stores the data in the file on magnetic tape for successive experiments. The data from aborted experiments can also be saved on request. The system then
(13) L. Michaelis and M. L. Menton, Biochem. Z . , 49, 333 (1913). 742
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prints out the material saved along with its file name and the date (Figure 9B). Special Routines. Several of the individual software routines are important and distinct enough in design to be discussed apart from the program sections of which they are part. Often routines are split between sections for convenience, but function as a unit in actual operation. Four routines will be discussed : on-line monitor system, rate reading system, reagent preparation system, and the decisionmaking system. The most complex of the program sections is the on-line operating system because this is the only section in which more than one program is operating at a time. To control this functional time-sharing, a monitor system was developed to keep the reading, reagent preparation, and decision-making systems from interfering with each other. The monitor assumes overall control of the on-line operating system as soon as that system is loaded. It gives up that control to programs which will end by again returning control to the monitor or to programs from which control can be retaken by means of the 1 Hz interrupt clock. When the on-line operating system is started, the monitor starts the decision-making program and looks for the signal from the interface to indicate that the reaction mixture is in the spectrophotometer. Having received this signal, the monitor waits the specific time before starting the reading system. The reading system and the reagent preparation system are started at the same time. Since the decision-making system may still be running, the monitor keeps a record of the states of all three systems. When all systems have finished, the monitor yields control to the up-date system. The monitor responds only to two commands from the experimenter while it is in control: Z to abort the experiment and A to abort the reading of the particular tube in the spectrophotometer. Even these will not cause the monitor to give up control until it has moved the hardware through its complete cycling. This is done to guarantee the hardware will be ready to run another experiment without manual initiation being required. The rate reading system has been described in detail previously (5). The program sends out a pulse on operate line 5 which starts the reaction rate interface. Pulses are sent out on operate line 17 after one second and two seconds. The rate is read on A/D channel 12 and its sign on channel 13. If the value is less than one quarter of full scale, it is amplified in the hardware by 4 or 16 according to the setting of computer relays, If the value is still less than a quarter scale, the integration time base is changed from 2 to 8 seconds and a reintegration is performed. When the value is finally put on the proper scale, it is multiplied by scaling factors and saved. Once the time base is changed, it remains at 8 seconds, but the rest of the scaling is done each time for all points. The number of points gathered is dependent upon the rate of the reaction. After enough points have been gathered, they are fitted by a straight line, and those whose error exceeds a fixed limit are rejected. The process is repeated until only a 2 error level is allowed. The rate is then taken to be the t = 0 intercept of the fitted line. The first tube is used as a blank correction for the rest of the tubes. The control of reagent preparation is comparatively simple due to the accumulator buffer. A table prepared by the program tells how many clock pulses the accumulator buffer should count and which instrument should be pulsed. The program checks if any device is active and if there is anything left to do to prepare the present tube in the table. If no
z
0.6599 0.3149 0.1500 0.0700 1.3850 0.2049 0.4099
0.2011 0.1747 0.1147 0.0636 0.2398 0.1406 0.1913
00 00 00
03 03 04 05
1.00
1.00 1.00 0.03 0.03 0.03 0.02
0.0000 0.0000 0.0000
0.2047 0.2047 0.2055 0.2024
A
THE EXPERIMENT CODE IS El-059 6/29/70 X VALUES
Y VALUES
0.6599 0.3149
0.2011 0.1747 0.1147 0.2398 0.1406 0.1913
0.1500
1.3850 0.2049 0.4099
B
Figure 9. Printout of ELLA Part A : On-line printout, one line printed after each tube. Columns in order are: substrate volume in ml, rate in o.d./min, points in current model, relative error of estimation, and K,w in ml Part B: Printout of the experiment after it has been stored on tape with only those points kept by the system included
device is active and if the table is not exhausted, the number of clock pulses to permit (taken from the table) is transmitted to the accumulator buffer and the correct device selection flip-flop is set. The whole operation of checking and transmitting takes only two dozen machine language instructions. The reagent preparation system is programmed to do any substrate curve which can be run by adding a constant amount of enzyme to a variable amount of substrate and a constant amount of some third reagent or combination of reagents. The reagents are picked up in the order buffer, enzyme, other reagents, substrate, and buffer. The first buffer is needed to prevent diffusion of reagents into the digital pipet. The second increases the volume in the mixing tube when the reagents are first expelled by the pipet, thus preventing rapid enzyme-substrate reaction in a small volume before dilution by the first portion of buffer. The reagent preparation routine can make up tubes with as many as 7 different reagents, although the substrate curve is set up to use only 5, 2 of which are buffer. The decision-making system is a critical part of ELLA’S programming. When 3 initial points have been obtained experimentally, a line is fitted to them by least squares to determine the constants of the enzyme-substrate system (14). The error of estimation is then compared with an error discriminator (chosen as 3 for this application). If the error of estimation is below the discriminator, all the points are kept; if not, a routine which rejects the worst point by a suitable criterion is called. It places the worst point at the end of the data set, reduces the number of active points by one, and causes a recycling of the whole procedure. If 6 or more points are left in the system when the error of estimation falls below the discriminator, a flag is set indicating the experiment is done. If less than 6 points remain (3 must always remain), then a new substrate volume is computed by an appropriate formula based on the constants of the equation as they have been estimated by the statistical fit. The necessary values are computed and stored in the preparation table. This procedure is repeated after every tube is run. Decision-Making. In order to program a computer to make a decision, it is necessary to know the parameters on which
z
(14) “Handbook of Tables for Mathematics,” The Chemical Rubber Co., Cleveland, Ohio, 1967, p 850. ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
743
be found. For the enzyme substrate studies, this is accomplished by taking five points spread over the range of the system in approximately a geometric series. When three points are gathered, they are fitted with a Lineweaver-Burk plot (Equation l), -1= - -K n r l u v s
1
f-
v
(1)
(Vis the maximum possible velocity, u is the measured velocity, s is the substrate concentration, and K.M is the Michaelis
[SUBSTRATE] Figure 10. Relative informational content of rate measurement us. substrate concentration The weighting factor gives the relative worth of measurements at different substrate values in the determination of the constants
the decision is based and what results are expected from every possible set of values for those parameters. In other words, a computer cannot make a decision unless that decision is so well defined that a properly trained human can always make the decision correctly when given the same information (15). For example, one decision fitting these criteria which ELLA makes is whether the system is reading on the correct scale. The “correct scale” can be defined as the one on which the reading is the nearest to full scale without being greater than full scale. The computer can be programmed to make this decision by having it compare the reading it takes with upper and lower thresholds. If the reading is between the thresholds, it is accepted; if not, then scaling is indicated. If the reading on the least sensitive scale is above the upper threshold then no correct scale exists. Most decisions are not so clear cut. Very often the rcgion defined by the possible parameter values must be mapped into a series of points which represent the possible decisions. Too many sets of values for the parameters exist to define for each the correct decision it should induce. In such cases it is necessary to formulate rules for the mapping which may be subject to change as more information is gathered. By properly formulating these rules it is possible to reflect the chemistry of the system and, as a consequence, to gain more information than can be obtained by using only statistical considerations. One example of a significant decision is that of optimum experimental conditions for subsequent measurements to determine the curve of a function for which some points have already been taken. This decision-making ability is programmed into ELLA, Other requirements to solve this type of problem include taking points that are far enough separated to minimize fitting errors and deciding when to end the data gathering process because sufficient information has already been gathered. All three of these requirements are met in the decision-making portion of ELLA. Where to take the points to get the best fit of the data depends on the values of the constants which are to be determined. Since these are obviously not known when the experiment begins, a way of initially estimating them must
constant) (la), and the constants are calculated. Using these initial estimates of the constants, it is possible to decide where to choose new points. If the new points are to add maximum information with minimum noise, then the weighting factors for the informational content of the rate values must be high at the positions picked. To determine these weighting factors, the derivatives of the equation to be fitted (Michaelis-Menton equation, Equation 2 ) (13) were taken with respect to the constants to be determined (Equations 3 and 4) (17).
(3)
(4) These equations give the sensitivity of the reaction rate to the values of the constants as functions of the substrate concentration. Equations 3 and 4 are plotted in Figure 10, along with their sum. This sum represents the informational content of the measurements for values of substrate concentrations that could be choser.. The amount of noise in the measurements is assumed to remain constant. The best position to measure is somewhere above the K.Mvalue. This plot is the basis of the decision-making procedure for finding new points. A second consideration arises because if the new point were always chosen at the best possible position, then most of the points on the line would be highly concentrated and a slight error in placing the line through them would yield large errors. Even considering the fact that the estimates of the constants are calculated after every new experimental point, the amount the best new point will move is small and will get smaller as more points are taken. Therefore, it is necessary not to take the best point every time, but rather to take points which are close to the best. For this reason the points at 1, 2 , and 3 times K M are chosen as the points to make the new measurements. The multiplier factor is chosen in a cyclic fashion. After each new point is run, the constants are recalculated, the K M is multiplied by the correct factor, and the multiplier factor is stepped to the next value. The new point calculated will be the next one made up and run by the hardware, provided that the termination condition has not been reached. The end condition is set as an error of estimation below some threshold with a certain number of points in the estimation. Three per cent is used as the error of estimation threshold to allow for any nonreproducibility in the me(16) H. Lineweaver and D. Burk, J. Amer. Chem. Soc., 56, 658
(15) A. Newel1 and H. A. Simon, “Computers and Thought,” McGraw-Hill, New York, N. Y., 1963, p 279. 744
ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
(1934).
(17) G. E. P. Box, Ann. N . Y. Acad. Sci.,86, 792(1960).
chanical hardware. For nearly ideal enzyme systems, it is possible to use a lower value for the threshold of the error of estimation. Six points are set as the minimum number of points ‘necessary. This is twice the number that ELLA needs to start the decision-making process, and it requires that at least one point must be chosen by ELLA to complete the curve after the initial set of tubes is run. Changes in the equipment or use in other applications might indicate that either or both of these numbers be changed. One problem which can occur is that points with a low signal/noise level may be gathered in the early part of the experiment while the system is scanning to make an estimation of the constants. To remove the disproportionate amount of error contributed by these points, which are at the low substrate levels, a cut OR of points is made below K M / 2 . These points are thrown away as soon as there are enough other points to allow reliable estimates of the constants. Further improvement in the quality of the data is obtained by discarding points which add large amounts of error to the data. Several approaches were used to find these data points, but the one which is most effective is to reject the data points which deviate most from the fitted line. As in the case discussed in the previous paragraph, after each point is rejected, the line is refitted before any more points are tested for rejection. When the error of estimation falls below the threshold used to determine the completion of the experiment, no more points are rejected. All rejected points are simply moved to the end of the list of points and are used again after each new point is added experimentally to the data set. This prevents good points from being rejected from the system as soon as they are gathered because a bad point was taken early in the experiment. Since the good points are not permanently removed from the system, they will even eventually cause the bad point to be rejected. Failure to implement this last provision causes the system to wander and not converge. RESULTS AND DISCUSSION Substrate Curve Studies. Two enzyme systems were studied. The alkaline phosphatase system was studied because it is a nearly ideal system. The lactic dehydrogenase (LDH) system was studied because it can be made very nonideal and difficult for the human experimenter to study. In order to determine the reproducibility of ELLA, a substrate curve was made for a system which is close to ideal. The alkaline phosphatase-p-nitrophenyl phosphate (PNPP) reaction is given in Equation 5 (18).
+ OH- alk. p-nitrophenolate (yellow) + HP042-
p-nitrophenyl phosphate (colorless)
phos
L
(5)
A pool of high clinical samples was used as a source of the enzyme and PNPP (Aldrich Chemical Co., Milwaukee, Wis.) as the substrate. These were mixed in pH 10.15, 0.50M AMP-HCl (2-amino-2-methyl-1-propanol) buffer. The reactions were run at 37 O C , and the appearance of the p-nitrophenolate anion was measured at 404 nm. The reagents were placed on the turntable in the order buffer, enzyme, buffer, substrate, buffer. The volume of enzyme to be used, (0.2 ml) and the incubation delay time before reading (15 seconds) were entered in response to the system’s programmed
l/CSUBSTRRTEl Figure 11. Lineweaver-Burk plot of duplicate substrate curve determinations of alkaline phosphatase
Pool of high clinical samples was used as the enzyme source. A first data set. * Second data set. Computer drawn and labelled
request for this information. The system proceeded to determine the substrate curve by itself, with the experimenter only having to add reagents to keep the turntable filled. Duplicate substrate studies on one pool are shown in Figure 11. For five substrate studies on the same enzyme pool, the relative standard deviation of the KM’s was 7.2% and that of the V‘s was 6.1 %. The values are of the same quality as humanly performed experiments under the same conditions which have a standard deviation of 5-1Oz. Better reproducibility could be obtained by reducing the rejection threshold of the error of estimation, increasing the number of points necessary for a successful fit, or by fitting the data points with a better chemical model. Tightening the parameters might require improvements in the reagent transfer system, however. The second system chosen for study was the LDH reaction system (Equation 6) (19).
NAD (nicotinamide adanine dinucleotide) (Boehringer Mannheim) was used as the co-enzyme and sodium lactate (Sigma Chemical Company, St. Louis) was used as the substrate. The reaction was run at pH 7.8 at 37 “C in 0.15M phosphate buffer. The appearance of the reduced form of NAD was measured at 340 nm. Human serum was again used as the enzyme source. The reagent positioning on the turntable was buffer, enzyme, co-enzyme, substrate, buffer. Since it was desirable to test the ability of ELLA to determine substrate curves under adverse conditions, two steps were taken to make the study of the LDH reaction more difficult. A less than optimum amount of NAD was added to the reaction mixture. As the reaction proceeded, the NAD was used up and the rate of the reaction decayed rapidly. This made the tracings on the strip chart recorder difficult to measure manually. Second, a concentrated lactate stock solution was used, so that only small volumes of solution were pipetted. By these means the limitations of the system were tested.
(18) “Manual of Methods,” Vol. 2, DuPont Instrument Products
Division, Wilmington, Del., 1968.
(19) S. Passen and W. Gennaro, Amer. J. Clin. Path., 46,69 (1966). ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
745
0
2
7
= t
1
0
-= I -
0
i
0.5
G
1 .G
1.5
CSUBSTRRTEI Figure 12. Four substrate curves of the same LDH sample
+
L' = Vs/(K.w S) Pool of high clinical samples was used as the enzyme source. V and KM were calculated by Lineweaver-Burk plot. A First data set.
* Second data set.
0 Third data set. OFourth data set. Computer drawn and labelled
No changes were necessary in the programming t o run this system because the substrate curve program was set up to mix three components. This was done because most enzyme reactions require either a dye or a coenzyme if they are to be read spectrophotometrically. The reagents were placed in the turntable and the volume of enzyme (0.2 ml) and the incubation delay time (20 seconds) entered. ELLA then performed the experiment as before without any further human intervention. The results of the experiment are shown in Figure 12. The reproducibility of the K.w's (relative standard deviation = 26.6%) is, as expected, poor, while that of the V's (7.9%) is only slightly worse than that for alkaline phosphatase. The rapid decay in the reaction rate was primarily responsible for the poor reproducibility. While ELLA was programmed to compensate for slowly decreasing rates (3, the correction procedure was overwhelmed by the rapid decay of the rates of the LDH reaction. For some mixtures the rate decreased to less than 4 0 z of its initial value during the period it was being read by the system. Since the initial rate is needed to use Equations 1 and 2, these equations could be expected to be inaccurate whcn used to fit the data measured from the curved tracings of the LDH reaction. An improvement in the compensation routine could potentially improve the reproducibility with which the reaction constants can be measured, but reactions which deviate significantly from theory will always be difficult to handle. The second factor, pipetting small volumes, also affected the reproducibility, but not nearly to the same extent as did the nonlinear reaction rates. Its effects were probably more serious for the nonlinear reaction rates than they would have been for a more ideal system. When the effects of the two factors were combined, it became likely that ELLA would not be able to converge to a value within the threshold for the error of estimation. The fact that ELLA was able to converge consistently within the required error of estimation is much more significant than that the standard deviation of the KM'Swas not very good. Manually performed studies on the LDH system run under the same conditions gave worse reproducibility than those performed by ELLA,
3z
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Evaluation of ELLA. The overall system performed more satisfactorily than was expected. The proper interaction between the software, electronics, and mechanical hardware was obtained and yielded the desired results. The final ELLA configuration was stable and could be applied to numerous enzyme systems without modification. Limitations were a result of lack of experience and materials available and not inherent theoretical limitations of the approach. More work in perfecting the equipment and software are sure to produce significant improvement in the results that can be obtained. Two important limitations exist in the softwear of ELLA. ELLA is first limited by the statistical fit used (the LineweaverBurk Plot). This plot is frequently used in enzyme studies and was therefore adopted for this project. It has several significant limitations due to the way the points are distributed relative to the intercepts. Better results could be obtained by statistically fitting a more stable theoretical function and by using a better chemical model. Second, tightening the requirements of an acceptable point of completion would also improve the results. Hardware improvements are needed, however, before such software changes would be justified. Although the hardware performed adequately for the experiments performed, several improvements would significantly improve the results. The first of these is to stabilize the A-D channels which are being pushed to their stability limit. Second, the turntable proved to be a poor device to use for reagent pick-up. It is easy to contaminate, hard to thermostat, and requires periodic filling. Replacing this device with a multi-port value would increase the reproducibility of solution preparation. Third, thermostating could be improved by building the whole system into a block, which would also eliminate the fourth problem, the long distances solution must be transferred. These hardware improvements would greatly increase the quality of the results without requiring redesign of ELLA. The complete time required to prepare a reaction mixture, read it, print the results, and use them to make a decision concerning what to do next required from 3 to 71/* minutes, depending on the rate of reaction. Since, however, three mixtures were at various stages of processing, another data point was being added to the set approximately every 1 to 2 '/z minutes. The overall time for a substrate curve was from 20 to 30 minutes. This time could be reduced by making the system more compact. The minimum time per point added could be reduced to 45-90 seconds (depending on rate of reaction) which would be limited by the speed at which the hardware can perform and the incubation time of the reaction. At that speed, the constants for a serum enzyme sample could be determined in 10-15 minutes. CONCLUSIONS Based on the development of ELLA as described above, previously reported work with ELLA (9,and other recently reported work (6), it is possible to draw four significant conclusions about the automation of analytical experiments. These conclusions, coupled with those made based on the first parts of ELLA as reported (3, strongly support the development of more ELLA-like systems in other areas of analytical research. The principle of virtually complete automation of an analytical experiment under computer control is workable. Three complete subsystems-data acquisition, reagent mixing, and reagent transfer-operate independently and simultane-
ously under computer control. Such computer control removes timing and transfer errors of a human experimenter, yet does not take more than a small fraction of the running time of the computer. The computer can control the peripheral hardware without being,totally dedicated to it. Hardware to perform chemical experiments automatically is presently feasible. Although much of the equipment of ELLA is not standard, it can be built. By improving present instruments and building good transfer systems, the analytical chemist can build automated equipment to improve the speed and reliability with which he can perform his work. Hardware improvements are needed, however, to realize the full potential of automated systems. Well defined chemical decisions can be made by an on-line computer in a research environment. The decisions made by ELLA and similar systems appear relatively simple in nature but they are sophisticated enough to produce acceptable chemical data. The quality of the decision-making can be improved and new decisions can be implemented based upon what has been learned. The use of computer decisionmaking can greatly increase the speed at which knowledgeable decisions of how to proceed during an experiment can be made and can remove human error and bias from the decisions. In order to make decisions on-line by computer it is necessary to define the decisions carefully so that the computer can be programmed to make them. This, too, promises to add to the knowledge of chemical systems by forcing chemists to weigh variables more carefully and to define processes in greater detail. Computer decision-making may help to standardize research approaches and make laboratory procedures and equipment more universal in nature.
The ability of the laboratory oriented computer to d o sophisticated data analysis is established. A system which, both on-line and off-line, produces the type of analysis that the chemist needs to conduct informative experiments has been constructed. Powerful routines manipulate data into forms for visual presentation. The basis for an even more extended data analysis system exists. Moreover, the laboratory machine is able to run a special purpose time-sharing system with data analysis operating in the foreground and hardware oriented routines running in the background. The further applications of computerized research to analytical chemistry seem unlimited. Any set of decisions that can be completely defined can be programmed and, therefore, can be made automatically and without the bias of the human observer. The development of such systems will allow the chemist more time to devote to the theoretical aspects of chemistry, since the laboratory work necessary for hlm to draw his conclusion will be performed rapidly and thoroughly by automated systems. ACKNOWLEDGMENT
The authors wish to thank the Du Pont Company for the use of a modified Spectronic 20 and the digital pipet. They also wish to acknowledge Arletta E. Sherry for her technical assistance and Walter J. Blaedel for his counsel.
RECEIVED for review December 30, 1970. Accepted February 22,1971. This work was supported by the National Institutes of Health, Grant No. G M 10978, and the Graduate Fellowship Program of the National Science Foundation.
Determination of Titanium by Controlled-Potential Coulometry L. P. Rigdon and J. E. Harrar Chemistry Department, Lawrence Radiation Laboratory, University of California, Livermore, Calif. 94550
A procedure has been developed for the determination of titanium by means of controlled-potential coulometry. The method is based on the reduction of Ti(IV) to Ti(lll) at -0.20 V VI. SCE in 9M H2S04. Background corrections are low, and samples containing 0.1-10 mg Ti per ml can be analyzed with an accuracy and precision of 0.1-0.2%. The Ti(lV)-Ti(lll) couple is reversible, thus by reoxidizing the Ti(lll) to Ti(IV) at +0.22 V, the quantity of each oxidation state can be determined and certain interferences can be avoided. Principal interferences are As(lll), Bi(lll), Cu(ll), Mo(VI), Se(VI), and Te(lV). Five mg of Ti can be determined accurately in the presence of >5 mg Nb, >4 mg Zr, 5 mg V, 0.2 mg W, 2 mg Fe, or 0.5 mg CI-. The method has been applied to the analysis of Ti-W alloys of high tungsten content, after separation of the titanium by hydrolytic precipitation. ALTHOUGH EXISTING DATA on the electrochemical behavior of titanium suggest that this element could probably be determined by controlled-potential coulometry, no such methods have been reported. This technique would afford an accurate and precise alternative to the titrimetric and gravimetric methods presently used for the analysis of titanium metal, its alloys, and compounds ( I , 2). (1) E. R. Scheffer in “Treatise on Analytical Chemistry,” Part 11, Vol. 5 , I. M. Kolthoff and P. J. Elving, Ed., Interscience, New York, N. Y.,1961, pp 1-60. (2) R. Z . Bachman and C. V. Banks in “Progress in Nuclear Energy. Analytical Chemistry,” Vol. 3, Part 4,C. E. Crouthamel, Ed., Pergamon Press, New York, N. Y ., 1963, pp 95-104.
For constant-current coulometry, Ti(II1) can be electrogenerated from Ti(IV) with 100% current efficiency at platinum electrodes in strong mineral acid media (3). However, preliminary investigations showed that these conditions are not directly applicable to coulometry at controlled potential. The concentrations of titanium in the electrolyzed solutions are some one-hundred times lower in controlled-potential coulometry, and the background current from hydrogen evolution at the platinum electrode is relatively large at the applied potentials needed for quantitative Ti(1V) reduction. A mercury working electrode, on the other hand, appears to be ideally suited to the determination of titanium. Polarographic literature (4) indicates that the electrochemistry of the Ti(IVkTi(II1) system is well defined in several supporting electrolytes. Of these, strong sulfuric acid (5-8) was chosen for development of the controlled-potential coulometric procedure. This electrolyte is compatible with most (3) J. J. Lingane, “Electroanalytical Chemistry,” 2nd ed., Interscience,New York, N. Y., 1958, pp 588-598. (4) “Handbook of Analytical Chemistry,” L. Meites, Ed., McGraw-Hill, New York, N. Y., 1963, Sec. 5 , pp 55-97. (5) J. J. Lingane and J. H. Kennedy, Anal. Chim. Acta, 15, 294
(1956). (6) D. K. Banerjee, C. C. Budke, and F. D. Miller, ANAL.CHEM., 31,1836 (1959). (7) G. M. Habashy,Z. Anorg. Allg. Chem.,306,312 (1960). (8) G. M. Habashy, Collect. Czech. Chem. Commun., 25, 3166 (1960). ANALYTICAL CHEMISTRY, VOL. 43, NO. 6, MAY 1971
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