Anal. Chem. 1980, 52, 139-142
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Dual Microcomputer-Controlled Stopped-Flow Spectrometer Israel R. Bonnell and James D. Defreese' Department of Chemistry, University of Kansas, Lawrence, Kansas 66045
A commercial stopped-flow spectrometer has been automated using two microcomputers, one controlling the spectrometer and acquiring the rate data via assembly language programs and the other providing operator Interaction and sophisticated calculations via BASIC programs. The computers operate independently of one another except during brief transfers of programs or data over a high speed serial link. Such parallel processing provides a simple and inexpensive method for overcoming the limitations associated with the low speed of microcomputers for measurement applications which also involve significant computation, e.g., reaction rate methods. The performance and flexibility of the automated system is demonstrated by its application to the determination of phosphate and alkaline phosphatase activity by reaction rate methods and to fundamental studies of bilirubin kinetics.
Reaction rate methods of analysis are attractive compared
to equilibrium methods because of their greater speed, relative freedom from side reactions, ability to analyze mixtures of closely related compounds without physical separation, and nature as a relative measurement technique ( 1 ) . For enzyme activity determinations, it is the rate itself that is measured. Recent advances in instrumentation have made it possible to perform analysis by reaction rate methods on a routine basis (2,3). Reaction times may range from milliseconds to several minutes. For reaction times in the millisecond range, a rapid mixing and fast detection system are necessary. This is usually accomplished with stopped-flow mixing and spectrometric detection. The stopped-flow unit is a versatile device in that it is not only a rapid mixer, but also a precise aliquoter and diluter which is well suited for automated sample handling. The data acquisition system can be a storage oscilloscope, rate meter, or sophisticated computer system ( 3 ) . An on-line computer to control the stopped-flow system, acquire data, process them, and print out the results makes the system applicable to a variety of reaction rate analysis and fundamental kinetic studies. In the totally automated systems ( 4 , 5 ) even the sample preparation is under computer control and a complete series of experiments can be done without operator interaction. With the advances in large scale integrated circuit (LSl) technology, microcomputers have become available at low cost. In addition, a variety of support circuits which can be software-programmed to perform different functions are available to make interfacing instruments to microcomputers very easy. Despite these developments, microcomputer systems have not been used extensively in automated stopped-flow systems. The limitations often cited are low speed and limited computing power. One way to overcome these limitations is to use more than one microcomputer to control the system. This paper describes an automated stopped-flow system controlled by two microcomputers linked by a simple serial interface. The general considerations involved in the configuration of the system are discussed and data are presented to show the versatility of the system for reaction rate methods of analysis, for the study of the kinetics of fast reactions, and for equilibrium absorbance measurements. 0003-2700/80/0352-0139$01OO/O
INSTRUMENTATION A block diagram of the stopped-flow spectrometer system is given in Figure 1. I t basically consists of a commercial stopped-flow spectrometer controlled by two microcomputers. Details of these units, including interfaces and software, are discussed in the following sections. Stopped-Flow Spectrometer. The stopped-flow spectrometer is a GCA/McPherson (Acton, Mass.) 730-S. It consists of a regulated light source (701-50), filter grating double monochromator (700 with 700-56), stopped-flow module (730-ll),and photomultiplier module (701-30). The syringe plungers are pneumatically driven and pressure-actuated valves are used to control the flow of solutions. The observation cell is 20 mm long with quartz windows. The stopped-flow module has a built-in sequencer which can be controlled from the front panel or remotell with T T L level signals. TJpon initiation of a cycle, the reagent, sample, and stopping syringes are emptied; reagent and sample are drawn into the respective syringes; and the plungers are pushed forward to mix the sample and reagent. The sequencer gives a T T L level trigger signal when the flow stops. Constant temperature is maintained by circulating thermostated water through copper tubing in contact with the metal support block of the stopped-flow head. The temperature of the solution in the stopped-flow cell can be monitored with a thermistor probe. Crouch et al. (3) have compared this stopped-flow system with some of the other commonly used systems. Computer Systems. Altair 8800B (MITS Inc., now Pertec model 1100) and ADD8080 (6) microcomputers are used to control the system and perform the necessary calculations. Both systems are based on the 8080A microprocessor. The Altair 8800B with 36 kilobytes of semiconductor read/write memory, dual floppy disks (North Star MDS), CRT terminal (Heath H9), line printer (Centronics 306C), and 4 serial 1/0 ports (MITS 88-2SIO) handles operator interaction, data and program storage, data reduction, and control of the ADD8080. All circuit boards are commercial units which are compatible with the SlOO bus of the Altair 8800B. The ADD8080 system consists of a set of standard cards (6) and custom designed circuits assembled on wire-wrap boards, all of which fit into an Analog Digital Designer (ADD8000, E and I, instruments, Derby, Conn.). Different systems to suit the user's needs can be set up very easily by connecting the appropriate cards to the ccimputer bus. In addition to the central processing unit, our ADD8080 system has 2 kilobytes of erasable programmable read only memory (EPROM), 4 kilobytes of semiconductor read/write memory, one serial I/O port, one 8-bit parallel input/output port, a 12-bit analog-to-digital converter (ADC), two 12-bit digitalto-analog converters (DACs) and counter/timers. It controls the operation of the stopped-flow spectrometer, acquires the data, and transmits them to the Altair for further processing. I t also displays either the raw or processed data on an X-Y oscilloscope display. Data Acquisition Interface. A block diagram of the data acquisition interface is shown in Figure 2. It consists of a variable gain instrumentation amplifier (LF152, National Semiconductor),sample and hold (SHM-IC1, Datel Systems Inc.) and a 12-bit successive approximation analog-to-digital converter (ADC-HX12BGC, Datel) with a conversion time of C 1979 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 1, JANUARY 1980
Table I. Configuration of Counters in 8253 Timer counter # mode 0
3
1
2
2
0
input Q, clock of ADD8080, 2.048 MHz 20.08 kHz from counter X O 20.08 kHz from counter $0
output
function
frequency, 20.08 kHz
-5 V
frequency divider
variable frequency
READY signal from stopped flow READY signal from stopped flow
clock for data acquisition software-controlled time delay
pulse
k L DISKS
AFi , ADD8080
Figure 1. Block diagram of stopped-flow spectrometer system
PUT
gate
0
ADD8080 BUS
Figure 2. Data acquisition system block diagram
20 p s . At the detector module, the current output from the photomultiplier tube (PMT) is converted to a voltage by an operational amplifier current-to-voltage converter and filtered by a three-pole active filter with a cutoff frequency of 20 kHz (7). This voltage signal is fed by shielded cable to the instrumentation amplifier on the analog-to-digital converter card. When an analog-to-digital conversion is initiated by the ADD8080, the sample and hold goes to the hold mode, and the ADC starts a conversion after 2-ps delay to allow the sample and hold output to settle. The DATA VALID status signal goes to a “0” state during conversion time. The control circuitry is designed such that if an attempt is made to read the ADC output before conversion is complete, the processor goes into a wait state until conversion is complete. The 8080A microprocessor uses an 8-bit data bus, and the output of a 12-bit ADC has to be read as two bytes. Reading the ADC output using input/output instructions is slow and not very flexible. However, if the ADC output is addressed in memory space (memory mapped 1/01 a variety of instructions for accessing memory are available in the 8080A instruction set to read in and store data at high speeds under software control (8, 9). The ADC output is connected to the data bus using inverting tristate buffers (74367). The 8 least significant bits of the ADC are assigned to a memory address, and the 4 most significant bits to the next. When these memory locations are addressed, the buffers are enabled and the ADC output is available on the data bus to be read by the processor. Data acquisition systems similar to the one described above are now available in a single package from several manufacturers.
Timing. Timing signals for control of and data acquisition from the spectrometer are provided by an Intel 8253 programmable interval timer in the ADD8080. This integrated circuit consists of three presettable 16-bit down counters which can be independently programmed in 6 different modes (IO, 11). The configuration of the counters for the present work is shown in Table I. Counter p0 is used to divide the computer clock frequency of 2.048 MHz to 20.08 kHz. The output of counter it0 is divided by counter #1 to set the data acquisition rate which can range from 0.3 Hz to 20 kHz. This range can be increased further by varying the output frequency of counter 70. Counter a2 is used as a software-controlled time delay. Typical uses are setting premeasurement delay time, e.g., for reactions which have an induction period, time delay between last point on the rate curve and “infinity” readings, and time delay between successive trigger pulses to the stopped-flow system for flushing it with new solutions or wash solutions. A unique feature of this system is the option to gradually increase the time interval between data points during the acquisition of a rate curve. Since the rate of change in signal is maximum a t the start of the reaction and decreases as equilibrium is approached, an excess of points may be acquired when the signal is not changing appreciably if data points are taken at constant time intervals. For multicomponent analysis by kinetic methods, the data acquisition rate often has to be varied such that approximately the same number of data points per four half lives of each component are acquired for good accuracy and precision (12). Use of the 8253 timer in the ADD8080 allows the data acquisition rate to be varied easily under software control. The period between one output pulse to the next from counter P 1 is proportional to a 16-bit number loaded in the count register. If the count register is reloaded between output pulses, the present period is not affected, but the next period will be proportional to the new number. The counter is loaded with a larger number after acquiring each data point to increase the time delay before acquiring the next point. The operation of the timer is such that no timing errors are introduced because of reloading the counter or the cycle time of the computer (11). Stopped-Flow Control Interface. The sequencer in the stopped-flow module can be controlled remotely with T T L level signals. A schematic of the stopped-flow control interface is shown in Figure 3. The ADD8080 computer gives a 500-11s trigger pulse (CYCLE) to monostable MS1 which provides a 100-ps 0 1 pulse. This pulse causes transistor Q2 to turn “ON” for 100 p s , starting the stopped-flow cycle. The (CYCLE) pulse also resets the READY status flag to “0”. When the flow stops, the 1 0 transition of (READY) triggers monostable MS2 to provide a 100-ps 0 1 pulse, causing the (READY) status flag to be set to “1”. The READY and CYCLE lines are optically isolated to prevent voltage transients from reaching the ADD8080 when the stopped-flow is triggered. Photomultiplier Gain Programming. I t is desirable to adjust the gain of the P M T so that the signal a t 100% transmittance gives full scale output on the ADC. A tracking analog-to-digital converter (TADC) is used to program the
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-
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 1, JANUARY 1980 CI
47
I I
f
5v
! AOD8083
I
-I l-dL& a3
47
!
4 *5v
STOPPED-FLOW
Figure 3. Control interface between ADD8080 and stopped-flow spectrometer. All resistances are given in ohms and capacitances in microfarads. Q1, Q2, and Q3 are 2N2222's. MS1 and MS2 are 74121's. FF is a 7476
gain of the PMT to get the desired output current. The details of this circuit are discussed elsewhere (13). Communication Interface. Two Universal Synchronous/ Asychronous Receiver/Transmitters (USARTs) (Motorola 6850 in the Altair and Intel 8251 in the ADD8080) are used to establish an RS 232C compatible high speed serial link between the two computers. This interface makes the system very versatile as the ADD8080 can be connected to any ASCII encoded keyboard (e.g., CRT terminal, Teletype, etc.) for stand-alone operation or to another computer (Altair in this case) for more complex tasks without any modifications to hardware or software. The baud rate for communication is switch selectable, with 4800 baud being used between the Altair and the ADDSOSO. ADD8080 Software. The EPROM in the ADD8080 contains a monitor with powerful debugging capabilities similar to those described by Hughes and Sawin (14). Routines for serial communication with another computer and for display of data on an oscilloscope are included as part of the monitor. A listing of the monitor program will be sent to interested readers upon request. The programs for controlling the stopped-flow spectrometer and acquiring data are stored on the disk and are down-loaded to the ADD8080 from the Altair during system startup. Parameters for data acquisition, viz., data acquisition rate, number of data points, premeasurement delay time, time delay between the last data point and infinity readings, number of ADC conversions to be averaged for each data point, number of cycles to be ensemble averaged, and number of blank cycles before measurements are made on new samples, are stored in a table which is transmitted to the ADD8080 from the Altair. Using the information in this table, the ADD8080 acquires and stores a rate curve without intervention by the Altair. When the parameters are changed, a new table is sent to the ADD8080. All the ADD8080 programs are written in assembly language to increase speed and reduce memory requirements. Altair Software. The communication routine which controls ADD8080 consists of two modules. Module I is basically a keyboard monitor routine which reads in commands and addresses entered at the Altair console, verifies and stores them in a table in memory. Module I1 handles the communication using the information stored in the table. The communication routine is designed to detect any errors that may occur during transmission and storage a t the receiving end. Most of the ADD8080 programs are developed on the Altair, and the communication routine is used to transfer them for testing and debugging purposes. During routine operation of the system, module I is not used and module I1 becomes an assembly language subroutine cAI.Led by the BASIC main program with the necessary information entered in the table
141
using FILL (or POKE) commands. The main program (12.5K) which controls the whole system is written in North Star BASIC,version 6. It interacts with the ADD8080 through the communication routine. Upon startup, it downloads the data acquisition routine to the ADD8080. The stopped-flow spectrometer is flushed with distilled water or blank solution, and the gain of P M T is adjusted by the TADC to produce a fullscale output from the system ADC. Once the 100% transmittance value has been recorded, the system is ready to take rate data on the sample. The program requests that the operator enter the parameters for data acquisition, initializes the parameter table for the data acquisition routine, and transmits it to the ADD8080. Next, the operation(s) to be performed on the data, viz., reaction rate analysis by measuring slope of initial rate curve, fixed time or variable time methods, calculation of first-order rate constants, smoothing of data ( 1 5 ) ,and equilibrium absorbance measurements, are entered. A rate curve is acquired and displayed on the oscilloscope for examination by the operator. If the data are acceptable, further calculations will be performed and the results printed out. When optimum values of the parameters for data acquisition have been selected, the interactive mode can be exited and the system will make the measurements in rapid succession and print out the results. The system software has been designed such that while the Altair is performing calculations on one set of data, the ADD8080 will be acquiring the next set of data. This mode of operation in which two computers share the work makes efficient use of the capabilities of both for increased speed and flexibility. Copies of these programs can be supplied upon request. Data Handling. Since the Altair 8800B computer is almost completely dedicated to performing calculations, the system allows different types of analysis of the data in real time. Most reaction rate analyses are performed by measuring the slope of the initial rate curve. The slopes of overlapping portions of the rate curve are calculated by the method of least squares, and the linear portion is used to calculate the rate (16). Many reactions have induction periods, and this method ensures that rate measurements are not made until pseudo-zero-order conditions exist. First-order rate constants are calculated from the slope of -In (A, - A,) vs. time. The slope and intercept and their standard deviations as calculated by least squares analysis are printed out along with the standard error of estimate and correlation coefficient tu enable the user to assess the significance of the results (17). The user can also select the fixed time or variable time methods of analysis, but the data often must be smoothed using the software smoothing routine for good precision ( 1 5 ) .
RESULTS AND DISCUSSION Determination of Phosphate. Phosphate determination is based on the method of Javier et al. (18). The reagent is 0.074 M Na2Mo04.2H20in 0.6 M nitric acid (19). Phosphate reacts with Mo(V1) to form 12-molybdophosphoricacid (12MPA) which is monitored at 430 nm. First-order kinetics are observed with half life in the range of 600 ms. The fixed time method is used as it has been shown to give better dynamic range than initial rate or variable time methods (20, 21). Calibration data for the determination of phosphate are given in the range of 1 to 15 ppm phosphorous in Table 11. The slope, intercept, correlation coefficient, and standard error of estimate are (8.40 0.03) X (-1.8 f 0.4) X W3, 0,9999, and 3.5 X respectively. The poor precision a t low concentration may be due to the fact that a high intensity light source and scale expansion techniques were not used in acquiring these data ( 2 2 ) . Determination of Alkaline Phosphatase Activity. The measurement of alkaline phosphatase activity is based on the
*
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 1, JANUARY 1980
Table 11. Determination of Phosphate
Table IV. Effect of Caffeine on
concn. of P, ppm
AA/At
RSD, %a
0.97 2.90 4.83 6.76 9.66 11.6 14.5
0.006 0.023 0.039 0.055 0.079 0.096 0.1 20
23 4 1.7 2.1 2.3 1.3 1.1
Seven determinations.
1 2
4 8
AA/min 0.248 0.1 29 0.0667 0.0339
caffeine concn., mM
first-order rate constant, s - '
4.20 20.1 39.9 59.4 75.8
5.50 6.49 5.80 5.59 4.77
RSD,
%b
1.4 2.5 0.6 0.2 0.8
Experimental conditions were as reported in ref. 25. Eieht redicate runs.
a
Table 111. Determination of Alkaline Phosphatase Activitya relative dilution factor
Bilirubin-p-DiazobenzenesulfonicAcid Reactiona
activity,b U/L RSD, %c 138 72.4 37.3 18.9
1.0 1.1 1.0
1.8
a Temperature = 30.0 i 0.1 "C. Molar absorptivity of p-nitrophenol at 410 nm in pH 10.3 buffer = (1.789 3 0.007) X lo4. Four replicate measurements.
hydrolysis of p-nitrophenyl phosphate to p-nitrophenolate ion a t p H 10.3 using conditions similar to those described by Bowers and McComb (23). The formation of p-nitrophenolate ion is monitored a t 410 nm. Measurements are made under pseudo-zero-order conditions and the rates calculated by the least squares method. This is a relatively slow reaction with measurement times exceeding 60 s. Results of the determination of alkaline phosphatase activity in dilutions of ENZA-TROL (Dade Division, American Hospital Supply Corp., Miami, Fla.) are given in Table 111. In order to calculate the enzyme activity in international units, it is necessary to know the apparent molar absorptivity of p-nitrophenolate ion a t 410 nm. The stopped-flow spectrometer itself is used to determine the absorbances of standard p-nitrophenol solutions in p H 10.3 buffer from which the apparent molar absorptivity is calculated. Fundamental Kinetic Studies of Bilirubin. The stopped-flow spectrometer is well-suited for studying the kinetics of fast reactions with half lives in the range of milliseconds. Bilirubin is a yellow bile pigment, and its determination in blood serum is very important for the diagnosis of hepatic disorder ( 2 4 ) . It is most often determined by coupling with p-diazobenzenesulfonic acid to form an azodipyrrole which absorbs at 530 nm. First-order conditions are observed at high concentrations of p-diazobenzenesulfonic acid, with half lives ranging from 0.002 to 0.400 s depending on reaction conditions (25). A mixture of caffeine and sodium benzoate is used to solubilize the pigment, which is insoluble in water and strongly bound to serum albumin. Caffeine forms soluble complexes with bilirubin and decreases the rate of the reaction. The effects of caffeine on the pseudo-first-order rate constant are given in Table IV. The results are similar to those reported by Landis and Pardue (25). However, a decrease in the rate constant is observed a t low caffeine concentrations, possibly due to the effect of bilirubin solubility. Further studies on the effect of these complexation processes on the reactivity of bilirubin will be presented later.
CONCLUSION The experiments described above illustrate the versatility of the automated stopped-flow system for a variety of chemical studies. Two microcomputers working in parallel overcome, to a considerable extent, the limitations imposed by their
relatively low speed for measurement applications involving extensive computation. Such a system can be easily expanded to take on additional control and computational tasks. If such increases in system capabilities begin to result in decreased speed and/or complicated implementation, it is now economically attractive, both in terms of cost and time, to use additional microcomputers instead of pushing the capabilities of a given system to its limit. Here a serial link between the computers was chosen as hardware and software requirements were minimal. This approach works quite efficiently when each computer performs a well-defined task and interaction between the computers is minimal.
LITERATURE CITED (1) Malmstadt, Howard V.; Cordos, Emil A,; Delaney, Collene J. Anal. Chem. 1972, 44, 26A-41A. (2) Malmstadt, Howard V.; Dehney, Collene J.; Cordos. Emil A. Anal. Chem. 1972, 44, 79A-89A. (3) Crouch, S. R.; Holler, F. J.; Notz, P. K.; Beckwith, P. M. Appl. Spechosc. Rev. 1977, 73(2). 165-259. (4) Mieling, Glen E.; Taylor, Richard W.; Hargis, Larry G.; English, James; Pardue, Harry L. Anal. Chem. 1976, 48, 1666-1693. (5) Krottinger, D. L.; McCracken, M. S.; Malmstadt, H. V. Am. Lab. 1977, 9(3), 51-59. (6) Avery, James; Lovse, Daniel "The ADD-8080 Microprocessor Manual"; Department of Chemistry, University of Illinois, Urbana, Ill., 1977. (7) Avery, J. P.; Malmstadt, H. V. Anal. Chem. 1976, 48, 1306-1313. (8) Rony, Peter R.; Titus, Jonathan A,; Larsen, David G. Am. Lab. 1976, 8(2), 119-120. (9) "8080 Microcomputer Systems User's Manual"; Intel Corp., Santa Clara. Calif., 1975; Chapter 3, p 8. (IO) Carlin, Fred H.; Howard, James A. Comput. Design, 1979, 78(5), 213-21 7. ( 1 1) "Comoonent Data Catalog"; Intel Corp., Santa Clara, Calif., 1976; Chapter 12, p 65. (12) Ridder, Gregg M.; Margerum, Dale W. Anal. Chem. 1977, 49, 2090-2098. (13) Dalle-Molle, Rich; Defreese, James D. Anal. Chem. 1979, 51, 1755-1 759. (14) Hughes, T. P.; Sawin, D. H., I11 Comput. Design, 1978, 77(11), 99-107. (15) Savitzky, Abraham; Golay, Marcel J. E. Anal. Chem. 1964, 36, 1627-1639. (16) Pardue, Harry L. Cliri. Chem. 1977, 23. 2189-2201. (17) Davis, Robert B.; Thompson, James E.; Pardue, Harry L. Clin. Chem. 1978, 24, 611-620. (18) Javier. A. C.; Crouch, S. R.; Malmstadt, H. V. Anal. Chem. 1969, 47, 239-243. (19) Beckwith. P. M.: Scheeline. Alexander; Crouch, S. R. Anal. Chem. 1975, 47, 1930-1936. (20) Carr, Peter W. Anal. Chem. 1978, 50, 1602-1607. (21) Crouch, S. R. In "Computers in Chemistry and Instrumentation", Vol. 3, Mattson, James S., Mark, Harry B., Jr., MacDonald, Hubert C., Jr., Eds.; Marcel Dekker: New York, 1973; Chapter 3. (22) Ref. 3; p 181. (23) Bowers, George N., Jr.; McComb, Robert B. Clin. Chem. 1966, 72, 70-89. (24) Routh, Joseph I . In "Fundamentals of Clinical Chemistry", 2nd ed.,Tietz, Norbert W., Ed.; W. 8 . Saunders: Philadelphia, Pa., 1976; Chapter 19. (25) Landis, John B.; Pardue, Harry L. Clin. Chem. 1978, 24, 1690-1699. ~I
RECEIVED for review June 25, 1979. Accepted September 12, 1979. Presented in part a t the Sixth Annual Meeting of the Federation of Analytical Chemistry and Spectroscopy Societies, Philadelphia, Pa., September 1979. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for partial support of this research. This investigation was also partially supported by the University of Kansas General Research allocation ~3095-XO-0038.
