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amount of acetic acid giving a positive test in 15-min reaction time was 30 micrograms. This spot test has the advantage over others for carboxylic acids in being directly applicable to aqueous solutions of carboxylic acids or carboxylate salts. Kinetic Determination of Carboxylic Acids. Despite the catalytic effect of nickel(I1) and a high reaction temperature, the absolute rate of hydroxamic acid formation from carboxylic acids is low. The extent of reaction is, moreover, equilibrium-limited. For these reasons a “total change” method of analysis is not feasible, and instead an initial rate
(25) assay was developed. The procedure described above M solution of acetic is planned for an approximately acid, but by suitably modifying the reaction time and spectrophotometer path length a wide variation in concentrations and sample acids could be accommodated. The technique is probably limited to aliphatic acids. The relative rate data of Table I11 suggest that some selectivity or freedom from interference may be achieved in mixtures of aliphatic and aromatic acids. Figure 2 shows absorbance-time data for several concentrations of acetic acid covering a 100-fold range. The initial slope is a linear function of concentration, and this relationship provides a working curve alternative to the “onepoint” procedure that is described. The reproducibility of these measurements is indicated by replicate determinations of initial slope dAsoo/dt for 5 solutions containing 2.20 X 10-2M acetic acid; the mean slope value was 3.15 X sec-l, with standard deviation 0.026 X sec-l and range sec-l. 0.09 X Though it is clearly limited by slow reaction rates and by interference (for example, from carboxylic acid derivatives), the method suggested here has the desirable feature of being a spectrophotometric procedure for aliphatic carboxylic acids that is directly applicable to their aqueous solutions, in the to 10-1M range, regardless of ionic state. RECEIVED for review August 12, 1971. Accepted September 29, 1971. Financial support by the National Institute of Dental Research Training Program (DE-171) and National Institutes of Health General Research Support Grant R R 05456 is gratefully acknowledged. (25) H. B. Mark, Jr., and G. A. Rechnitz, “Kinetics in Analytical Chemistry,” Interscience Publishers, New York, N. Y., 1968, Chap. 3.4.
LABTRAN-A Language and System for Programming Chemical Experiments E. C. Toren,”Jr., R. N. Carey,’ A. E. Sherry, and J. E. Davis2 Departments of Medicine and Pathology, University of Wisconsin, Madison, Wis. 53706 A new interpretive language demonstrates the principles of the manipulation of laboratory instrumentation in a real-time, on-line system. Although specifically developed to carry out chemical experiments using a p-LINC computer and specially designed hardware, these principles can be extended to other types of experiments and computers. Each one-letter code either calls a calculation routine or operates a specific device (pipet, valve, or rate measuring interface). Using the interactive editor, programs can be written by laboratory personnel totally unfamiliar with computers and the usual programming languages. Operating programs are stored on magnetic tape and can be readily recalled for execution. A test result exceeding some threshold value can cause immediate, further, on-line testing of the same sample for different constituents.
COMPUTERIZED AUTOMATION in chemistry and clinical laboratories is developing at a rapid rate; the recent reports are I n absentia from Department of Chemistry, Duke University, Durham, N.C. Present address, Department of Chemistry, Purdue University, Lafayetie, Ind.
becoming too numerous to cite completely. From this laboratory, we have reported the development of a computer interface ( I ) based on the double-integration principle of Cordos, Crouch, and Malmstadt ( 2 ) and the development of a system, ELLA (3),to acquire, reduce, and analyze laboratory data. Many on-line and off-line plotting and numerical analysis features were added and, finally, the resulting ELLA system ( 4 ) was used in a closed-loop control situation t o perform the kinetic characterization of enzymes (with respect to K.M,the Michaelis constant, and Vw,the maximumvelocity). In the ELLA system, operator control was used only to load the turntable and control the plotter operation after completion of the experiment. Deming and Pardue (5), however, (1) E. C. Toren, Jr., A. A. Eggert, A. E. Sherry, and G. P. Hicks, Clin. Clzem., 16, 215 (1970). (2) E. M. Cordos, S . R. Crouch, and H. V. Malmstadt, ANAL. CHEM.. 40.796 (1968). (3) G. P: Hicks, A. A.’ Eggert, and E. C. Toren, Jr., ibid., 42, 729 (1970). ( 4 ) A. A. Eggert, G. P. Hicks, and J. E. Davis, ibid., 43,736 (1971). ( 5 ) S. N. Derning and H. L. Pardue, ibid., p 192.
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Figure 1. LABTRAN instrumentation configuration. Dashed lines indicate computer controlled mechanical connections
were first to demonstrate the on-line characterization of an enzyme, using a different computer system. On-line decision making and reagent mixing principles were also applied by Mueller and Burke (6) to the titration process. Recently, Perone and Eagleston (7) described a computerized hardware and software system using the BASIC language which employed subroutine calls to external interfaces. Although that system was designed to demonstrate the utility of the computer in the undergraduate teaching laboratory, the adaptation of a relatively simple language (BASIC) to the operation of standard interfaces to laboratory equipment is a major step toward making the computer an acceptable tool to students and laboratory scientists who do not wish to be burdened with the tedium of complex programming. It is with these considerations that LABTRAN was developed. Simple instructions which require virtually no knowledge of computers or computer languages are used. A six-port valve which is connected to reagent reservoirs and other devices, a digital pipet which picks up and delivers programmed amounts of samples and reagents, the rate of absorbance change from a modified Spectronic 20 spectrophotometer, and other features to be described in the Instrumentation section are readily operated by this system. The experimenter or technologist need only break down the experimental procedure into the discrete operations which the external devices can implement, input these statements to the computer, load the reagent reservoirs and sample turntable, and begin the experiment. Changes can be readily made by using the interactive editing capabilities. Previous programs can be called by name from the magnetic tape files. Decision programs have been developed to call for further analyses based on current results. One such decision has been demonstrated in this paper. The system is currently being used for spectrophotometric analyses of biological materials, primarily enzymes, by utilizing rate methods. Enzymatic rate methods are used as examples for the purposes of this paper because they are more difficult to implement than colorimetric analyses and because they provide an excellent decision-making example which is of medical diagnostic significance. INSTRUMENTATION
Figure 1 illustrates the LABTRAN system configuration. Much of the interfacing and external hardware design has (6) K . A. Mueller and M. F. Burke, ANAL.CHEM., 43, 641 (1971). (7) S. P. Perone and J. F. Eagleston, J. Chem. Educ., 48, 317
(1971). 340
Figure 2. Sample LABTRAN program to mix two reagents and read the resulting rate of change of absorbance
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been described earlier (3, 4). The LABTRAN system was made possible by the addition of a 6-port fluid valve (Chromatronix, Inc., Model R60V6, Berkeley, Calif.) to the ELLA system (3, 4). This effectively removed the need for a vacuum pump and solenoid valve arrangement and permitted the positive displacement of fluid to all devices from the digital pipet. This valve permits the pumping of at least three reagents and a sample, and frees the turntable for sample use entirely. The flow-through cell of the modified Spectronic 20 spectrophotometer (8), the holding coil, the mixing chamber and reagents (if temperature stable) are all thermostatted using a Tecam TU8 temperature bath and pump (Techne, Ltd., Princeton, N. J.) and a YSI model 63 temperature controller (Yellow Springs Instrument Co., Yellow Springs, Ohio). The control logic for the Slo-Syn stepping motor driving the 6-port valve is identical to the pipet logic described earlier (4). The pulse lines, to the external equipment from the computer (OPR lines), which activate the pipet, valve, and turntable are Exclusive-ORed in the Accumulator Buffer module ( 4 ) so that the same circuitry can be used to control (8) J. E. Davis, Chem. Instrum., 3, 239 (1971).
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the valve and pipet and so that no two devices can operate simultaneously. Further details will be supplied on request. The operation of all the devices, under computer control, is directed by subroutines that drive these devices to the position, or to the amount specified by the calling LABTRAN program, e.g., the LABTRAN statement “V5” would be interpreted by the computer to transmit the required number of pulses to move the valve from its present position to position 5 . From new programs entered at the keyboard, or from old programs on the magnetic tape file, the computer interprets and executes these LABTRAN statements line by line to operate the external hardware of Figure 1. The Accumulator Buffer previously described (3, 4 ) is an interface which supplies the required number and series of pulses to operate the turntable, pipet, and valve. Absorbance or rate of change of absorbance readings are made via the Reaction Rate Interface, previously described ( 3 , 4 ) . By withdrawing the pipet, solutions are removed from the sampler, mixing chamber, or reagent reservoirs to the holding coil. The solutions contained in the holding coil can then be delivered to the mixing chamber or spectrophotometer through ports 3 and 6 of the valve, as shown in Figure 1. Most conceivable combinations of mixing three reagents for spectrophotometric reaction rate and equilibrium analyses can therefore be obtained with the LABTRAN system. PROGRAMMING FEATURES
The LINC-class computer used in this project is well suited for interfacing laboratory instrumentation. LINC (Laboratory INstrument Computer) computers are manufactured by Spear Inc., Waltham, Mass., and by Digital Equipment Corp., Maynard, Mass. (LINC-8 and PDP-12). Magnetic tape orientation and oscilloscope display features permit new program entry and rapid editing in an interactive mode. The built-in programmable relay register, operate lines (OPR), external level lines, and multiplexed analogto-digital channels provide easy communication to external equipment and interfaces, thus greatly simplifying the systems programming. Although the system has been implemented on a p-LINC, the programming principles established in this work can be extended to other types of computers. The LABTRAN instructions presently available are listed in Table 1. Additional instructions are easily inserted into the system as new equipment, calculations, and other features are added. Figure 2 demonstrates a sample LABTRAN program. The action of the various instructions is best understood by further reference to Figure 1 . This program adds 0.5 ml of sample to 4 ml of reagent, mixes, and measures the resulting reaction rate. The steps are interpreted and executed line by line. In step 1, the valve is indexed to position 1 (sampler). The sample turntable is then advanced to step 2 to set up for the next sample. Next, a 0.5-ml (100 X 5 pl) sample is drawn into the holding coil. In step 4, the valve is set to the substrate reagent port and then 4 ml of substrate are drawn into the holding coil. The valve is driven to the mixing chamber port in step 5 after which 4.5 ml of solution, now in the holding coil, are expelled into the mixing chamber and are withdrawn back into the holding coil in the next step. The valve is advanced to the spectrophotometer flow cell port (6) and 3 ml are expelled through the flow cell. A rate (absorbance change per minute) reading is taken, and its value is listed on the Teletype. In the next step, the last 1.5 ml of mixture is expelled through the flow cell (to waste). The XXX command checks for an operator override, increments the sample counter, and returns to the first step of the program. Sample analysis continues repeatedly until such an override occurs. An override consists of an instruction from the keyboard to deviate from the normal routine. Certain necessary steps such as washing
Table I. List of LABTRAN Commands Action Command Decide whether to continue analysis program on this D sample using first analysis value as criterion. (Threshold determined by initial interview with experimeter). List sample number and analysis value on Teletype. L Normalize result of second test to result of first on N same sample. Pipet (+ = Draw in; - = Push out); the volume P is indicated by three characters following sign. Each unit = 5 pl. Measure rate. R Advance sampler turntable to next cup. T Index valve to new position indicated by next digit. V Wait for time in seconds indicated by next three W digits. xxx End of program; return to first step after incrernenting sample counter and checking for operator overrides.
cycles (using buffer or water) have been omitted here for clarity, but are used in actual analyses. Pre-programmed, 3-second pauses are made after each pipet, turntable, and valve operation, to allow fluid pressures to equalize. The experimenter may add other waiting periods using the “W” instruction to allow for mixing and induction periods before the rate determinations are made. Two separate programs may be joined by insertion of a “D” statement between them. When the “D” statement is reached, a subroutine decides whether the computer should continue into the second program (to complete a second analysis on the same sample) or return to the first step of the first program (to analyze a new sample). This decision is based on the relationship of the value obtained from the first analysis to the threshold level given the system by the experimenter during the initiation procedure described below. The program is displayed on the LINC oscilloscope as it is entered. Editing is accomplished by using the delete key and re-entering the correction for the character(s) deleted. Entering a program requires only 5 to 10 minutes. An option display on the oscilloscope is used to initiate the on-line procedure. The options include : (1) initialize, (2) calibrate rate measuring apparatus, (3) set analog base line, (4) execute a program, or ( 5 ) exit. If option 4 is chosen (the main feature described herein), the computer asks which program, whether to report values relative to standards or absolute rates, and under what threshold conditions to execute decision steps. After these questions are answered, the system executes the program on all samples, after which the system returns to the original option display. RESULTS AND DISCUSSION
To demonstrate experimentally the general applicability of the system, an example of enzymatic analysis in a clinical laboratory setting was chosen. Analyses of alkaline phosphatase (ALKPTSE), lactate dehydrogenase (LDH), and a-hydroxybutyrate dehydrogenase (HBDH) are described. The ALKPTSE assay is relatively simple because few reagents are required and because zero order reaction kinetics are readily obtained. It tends best to reflect the accuracy and precision of the system. The LDH and HBDH analyses are more difficult owing to problems in obtaining linear absorbance-time curves (due to non-zero order kinetics), and they require more complicated procedures. The average analysis time was ca. 4 minutes per sample. To demonstrate the on-line decision-making capabilities, an elevated (abnormal) LDH value can be used to set a threshold
ANALYTICAL CHEMISTRY, VOL. 44, NO. 2, FEBRUARY 1972
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Figure 3. Comparison of LABTRAN alkaline phosphatase results with results from the Clinical Laboratories, University of Wisconsin Hospitals The A points represent results from the ACA instrument and * points represent results from the SMA 12/60. The least-squares linear regression line is the one obtained for the ACA points. Slopes, errors of estimations, and intercepts are 0.94 and 0.95, 0.40 and 0.39, and 0.93 and 0.88 for the ACA and SMA 12/60, respectively. The graph was drawn and the points were fitted using the ELLA system (394)
above which the HBDH/LDH activity ratio can be computed by automatically assaying HBDH for those samples with elevated LDH. High ratios are expected for heart disorders and low ratios are expected for liver disease; normal serum ratios are located in between (9). Alkaline Phosphatase. Alkaline phosphatase was determined by a modified Bessey, Lowery, Brock (IO) procedure in which 0.2-ml samples were mixed by the LABTRAN system with 3.9 ml of buffer (0.5M 2-amino-2-methyl-lpropanol, pH 10.15 at 37 “C), and 0.4 ml of 13.3 mg/ml p-nitrophenylphosphate substrate. The resulting reaction rates were read at 405 nm using the modified Spectronic 20 (8) and Reaction Rate Interface (4). A pooled serum sample whose mean activity was 92 IUB as determined on the Automatic Clinical Analyzer, ACA (DuPont Instrument Products Division, Wilmington, Del.), was diluted to 75, 50, 25, and 0 % of its original activity for a linearity check. Each dilution was run in at least pentuplicate in a random order. A correlation factor of 0.96 with a relative standard error of estimation of 1.8% for 27 samples was obtained. The system was calibrated once with the undiluted serum pool. Serum samples were obtained from the Clinical Laboratories of the University Hospitals. These sera had been analyzed on the ACA and on the SMA 12160 (Technicon, Tarrytown, N.Y.), respectively. A comparison of the ALKPTSE values obtained by LABTRAN and those obtained on the ACA and SMA 12/60 is shown in Figure 3. (9) D. T. Plurnmer and J. H. Wilkinson, Biochem. J . , 84, 423 I1 963). (lO~-O~’A. Bessey, 0. H. Lowery, and M. J. Brock, J. BioL. Chem., 164, 321 (1964). 342
A high serum was used to calibrate the system. Correlation factors of 0.94 and 0.95, intercepts of 0.93 and 0.88 IUB, and standard errors of estimation of 0.40 and 0.39 were obtained from comparisons with the ACA and SMA 12/60, respectively. These data indicate that results agree within experimental error when it is considered that the relative standard deviation at the levels of these samples for the ACA and SMA 12/60 are 3.0 and 5.6%, respectively. There are several factors which contribute to the intercepts. There is a time delay of 3 to 4 hours between LABTRAN and routine laboratory analysis. The analysis temperatures were different; 37 “C in the routine laboratory and 30 “C in the LABTRAN system. Finally, the possibility of carryover or buildup in the holding coil cannot be ruled out. A new configuration which eliminates the holding coil and minimizes the possibility of carryover is presently under development. Lactate-Hydroxybutyrate Dehydrogenase. Similar results were obtained for LDH using a modified Wacker (11) procedure in which 0.4-ml samples were mixed with 0.8-ml composite substrate reagent (10 mg of NAD, 0.1 ml of 60W lactate/ml) and 3.3 ml of 0.15M phosphate buffer (pH = 7.8). The rate of increase of absorbance of NADH was measured at 340 nm. A correlation coefficient of 0.98 with a relative standard error of estimation of 1.0% was obtained in dilution studies for 15 triplicate samples run in random order. These values were obtained with a pool serum whose mean activity was 680 IUB as determined by the ACA. The activity of a-hydroxybutyrate dehydrogenase corresponding mainly to the “heart fractions” (LDH4 and LDH, isoenzymes) was determined by substituting a 100-mg a-hydroxybutyrate/ml solution for the 0.1-ml lactate solution in the above procedure. An LDH activity of 210 DuPont IUB (the upper limit of normal for the ACA) was used as a decision-making threshold ( I 2 ) , above which an HBDH analysis was made automatically and on-line. Isoenzyme fractions of LDHs (heart) and LDHl (liver), separated on Sephadex DEAE-A50 ion exchange resin (13) (Pharmacia, Inc., Uppsala, Sweden) were used as samples. The LDHs tissue fraction yielded an HBDH/LDH ratio of 0.59; LDHl yielded a ratio of 0.22, and a pooled serum sample gave an average ratio of 0.40. The results were in qualitative agreement with Plummer and Wilkinson (9). The HBDHiLDH determination illustrates the principle of sequential analysis based on prior results. In clinical laboratories in which screening programs are used, an abnormal screening result could be used to trigger an on-line sequence of second and third generation tests designed to refine further the diagnosis. Presently, the system is somewhat limited by several instrumental problems. In order to wash the sample lines, it is necessary to place a water cup immediately after the sample cup in the turntable. Carryover problems occur as a result of the relatively large volume of the holding coil and associated plumbing, It has been difficult to maintain the entire instrumental apparatus at a constant temperature; especially at the elevated temperature of 37 “C, commonly used for enzymatic analysis, These problems are currently under study.
(11) W. E. C . Wacker, D. D. Ulmer, and B. L. Vallee, New Englund J. Med., 255, 449 (1956). (12) J. 0. Westgard and B. L. Lahmeyer, Abstracts. 23rd National Meeting of the American Association of Clinical Chemists, Seattle, Wash., August 1971, No. 144. (13) G. P. Hicks and G. N. Nalevac, Anal. Biochem., 13, 199 (1965).
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CONCLUSIONS
In the past, computerized chemical analysis systems have not been widely accepted in the laboratory because an easy, conversationql programming language has not been available. Although LABTRAN is not as flexible as other higher level languages, e.g., BASIC, its simplicity and interactive editing features greatly speed the programming of chemical analysis procedures. The sequential testing principle, although described in a clinical laboratory setting, has applicability in other testing areas where one must first discern a malfunction and then pin-point its cause through analytical procedures. A new configuration using a 24-port valve with the elimination of the holding coil is presently being incorporated into the system. The possibility of carryover will therefore be greatly minimized. The addition of a computer-operated, spectrophotometer wavelength drive together with other improvements will provide for the simultaneous automation of chemical analysis experiments requiring a wide variety of reagents and wavelengths. Other readout devices, e.g., electrodes, thermal conductivity detectors, etc., can be easily added to the system.
Finally, the adaptation of the external control interfaces from the ELLA system (4) permits even a small computer like the p-LINC to be used in a time-sharing mode to control and monitor data from several sets of instruments simultaneously. Therefore, the completely automated analytical laboratory becomes a definite possibility. ACKNOWLEDGMENT
The authors are indebted to Walt Pankonin for technical assistance in the construction of equipment and to Arthur A. Eggert for constructive criticism in the preparation of this paper. RECEIVED for review July 6, 1971. Accepted September 22, 1971. This work was supported by grants from the National Institutes of Health, G M 10978, the National Science Foundation, G P 26505, the University of Wisconsin Medical School, and an institutional grant from the American Cancer Society. One of us, R.N.C., gratefully acknowledges an NDEA Title IV Traineeship administered by Duke University.
A New Machine for Automated Solid Phase Peptide Synthesis Victor J. Hruby, Leon E. Barstow, and The0 Linhart Department of Chemistry, University of Arizona, Tucson, Ariz. 85721 A versatile, fail-safe, and simple machine for automated solid phase peptide synthesis was designed and constructed. A dual programming scheme was used. The variable operations such as kind and quantity of solvent or reagent, reaction or wash times, and other variables, are controlled by a Drum Programmer. The repetitive operations needed to get the solvent or reagent into and out of the reaction vessel are controlled by a Card Programmer. A number of failsafe devices were built into the machine to shut it off if electrical or mechanical failures occur. The operation is also monitored with a strip chart recorder which gives a permanent record of a synthesis. The machine can be operated in both automatic and manual modes.
IN 1963, R . B. Merrifield (1) introduced a new approach to polypeptide synthesis called solid phase peptide synthesis (SPPS). The large body of literature on this method (2, 3) attests to its utility and potential. For example, Gutte and Merrifield have used the method for the total synthesis of enzymatically active ribonuclease A (4,5). The same principle has also been applied to other synthetic and degradative problems, and this aspect is receiving increasing attention and investigation (2). The solid phase method can readily be automated (6) so that much of the routine work of solid phase polypeptide and protein syntheses can be done by a machine. (1) R. B. Merrifield, J. Amer. Chem. Soc., 85, 2149 (1963). (2) R. B. Merrifield, Aduan. Enzymology, 32,221 (1969). (3) J. M. Stewart and J. D. Young, “Solid Phase Peptide Synthesis,” W. H. Freeman and Co., San Francisco, Calif., 1969. (4) B. Gutte and R. B. Merrifield, J . Amer. Chem. SOC.,91, 501 (1969). (5) B. Gutte and R. B. Merrifield, J. B i d . Chem., 246, 1922 (1971). (6) R. B. Merrifield, J. M. Stewart, and N. Jernberg, ANAL.CHEM., 38, 1905 (1966).
An examination of the needs of solid phase chemistry and of existing machines (6-11) uncovered no automated instrument with all the requirements we felt to be important. Hence we designed and constructed a machine for automated solid phase synthesis which includes many new desirable features (12). GENERAL DESIGN OF THE INSTRUMENT
Simplicity, versatility, fail-safe operation, and easy maintenance were primary requirements in our design considerations. Standard and easily repaired circuitry was used so that the machine can readily be maintained and modified to suit particular needs. Solid phase peptide synthesis usually consists of the following kinds of methodology. An amino acid is attached to an insoluble, unreactive polymer support and the reactions needed to form a peptide bond are performed on this resin. After each reaction, a series of solvent washes are used to remove unreacted reagent(s) and side product(s) of the re(7) A. B. Robinson, Dissertation, University of California, San Diego, Calif., 1967. (8) A. Loffet and J. Close, in “Peptides, 1968,” E. Bricas, Ed., North-Holland Publishing Co., Amsterdam, Holland, 1968, p 189. (9) K. Brunfeldt, J. Halstrsm, and P. Poepstorff, Acta Chem. Scand., 23, 2830 (1969). (10) G. W. H. A. Mansveld, H. Hindriks, and H. C. Beyerman, in “Peptides, 1968,” E. Bricas, Ed., North-Holland Publishing Company, Amsterdam, Holland, 1968, p 197. (11) A. M. Tometsko, J. Garden, 11, and J. Tischio, Reu. Sci. Instrum., 42, 331 (1971). (12) V. J. Hruby, L. E. Barstow, and T. Linhart, Abstracts, 60th
National Meeting of the American Chemical Society, Chicago, Ill., Sept. 1970, No. 114 (Org.).
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