1902
Anal. Chem. 1982, 5 4 , 1902-1904
Microcomputer-Controlled Hot-Cell Pipetting System Douglas E. Goeringer and Leon N. Klatt *. Analytical Chemistry Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830
The chemical analysis of highly radioactive samples requires a variety of remote operations and equipment. A remote pipetter is a crucial component in numerous analytical procedures. After evaluating several commercial pipetters, we found these units unsuited for hot-cell operations. Many materials are incompatible with the high y radiation field (1-1000 rd/h) and the corrosive chemical environment, e.g., most synthetic polymers and steel. Solid-state electronics have a limited service life due to the absorbed radiation. Remote maintenance of the mechanical system requires a modular design, and all fasteners should have hexagonal surfaces to facilitate servicing with master-slave manipulators. The facility in which a remote pipetter is t o be used significantly influences its design. Consequently, a variety of custom pipetting devices is used in the nuclear industry. Maddox ( I ) reviewed the development of remote pipetting systems through the mid-1960s. Orsello et al. (2) have adapted the Eppendorf pipet t o remote operation through use of a mechanical lever-screw system to operate the plunger. Fortsch and Wade (3) developed a similar system which uses pneumatic actuators. Ochsenfeld e t al. ( 4 ) adapted a commercial plunger-type pipetter to remote operation. The plunger unit, located in the personnel area, is connected to the pipet tip assembly, located in the hot-cell, by a mercury-filled capillary tube. Dykes et al. (5) have developed a remote plunger pipetting system which uses a microcomputer as a control unit, but electronic feedback of the plunger position is not provided. With the increasing emphasis upon the safeguarding of nuclear materials and the introduction of more stringent regulations regarding personnel radiation exposure, improved precision and accuracy in all analyical procedures and improved documentation of the work performed in the hot-cell analytical laboratories are required. Since numerous analytical procedures require a precise and accurate sample aliquot, an improved remote pipetting system is only one of many instrumental developments that will be required to meet these needs. This report describes a high-precision, high-accuracy, microcomputer-controlled hot-cell pipetting system. Major features include electronic sensing of the plunger position, stand alone operation, a software driven self-test of the electronic and mechanical systems, hard copy output of pertinent operational information directly on the analyst's work control sheet, and the capability to implement direct communication with a time-shared data management system.
EXPERIMENTAL SECTION The general arrangement of the remote pipetter system is shown schematically in Figure 1. The fundamental units comprising the system are the mechanical pipetting assembly, the digital control logic, the signal processing electronics, and the microcomputer controller. Detailed features of the individual sections are described below. Mechanical System. A photograph of the mechanical assembly for the pipetter is shown in Figure 2. The metal components consist of anodized aluminum, stainless steel, and gold-plated brass. Configuration of the stainless steel plunger, the Teflon plunger cavity, and the coupling of the plunger to the drive screw are from the original ORNL design (6). This mechanical arrangement facilitates remote assembly and service, and long-term usage has proven it to be a reliable design. The operating principle of the pipetter is the plunger displacement of liquid. The plunger is dimensioned to deliver 1mL over a linear travel of 2.540 cm. Thus, with the combination of an accurately machined 1/4-20 drive screw and 1.8" stepping
motor, a volume resolution of 0.25 pL (incremental resolution of 6.35 wm) is achievable. The previous design's stringent requirements for accurate linear and angular alignment of the motor shaft and drive screw are eliminated by attaching the lead screw directly to the motor shaft. The motor bearings support the thrust load. An adapter suited for the use of disposablepipet tips (Model CT200, Rainin Instruments Co., Inc., Woburn, MA) is threaded into the bottom of the plunger cavity. The tip is changed manually with the master-slave manipulator. The plunger is coupled by a lateral arm to a linear variable differential transformer (LVDT) (Model D5/1000, Robinson-Halpern, Plymouth Meeting, PA). The LVDT is a better method of position sensing in this application compared with shaft encoders and multiturn potentiometers; translational motion is measured directly, and errors due to slippage and backlash in gear trains are eliminated. Note that the stepping motor and LVDT are the only electrical devices located within the remote area. Electronic System. A 1.8" stepping motor (Superior Electric Company, Bristol, CT) having 0.25 N m (35 oz in.) torque is used t o rotate the drive screw. The stepping motor is driven by an STM103 translator module (Superior Electric Co.) which furnishes step sequencing logic and current switching capability. Digital inputs are available for determining the direction of motor rotation and for gating an on-board variable frequency oscillator which determines the motor stepping rate. These digital inputs are controlled by the custom interface described below. Data acquisition and control for the remote pipetter are initiated through the general purpose digital input/output (I/O) board. This digital 1 / 0 board communicates with a customdesigned interface board. The purpose of the custom interface is to interpret binary-coded control instructions from the digital 1/0board, implement those commands, and provide digital data to the digital 1/0 board based on those instructions. Figure 3 shows the various modules and outlines signal flow for the custom interface. The LVDT consists of a primary and two identical secondaries, which are wound on a cylindrical support and connected in a series opposed arrangement. Inside the cylindrical support assembly is a movable core which magnetically couples the excitation signal from the primary to the secondaries. The excitation voltage is generated by an oscillator. A null point position of the magnetic core occurs when it provides equal coupling from the primary to each secondary. Signal output from the LVDT is zero when the core is at the null point. Movement of the core from the null position produces a net secondary output signal. The magnitude of the signal is proportional to the displacement away from null. The secondary output signal is demodulated by a phase detector circuit, which yields an output voltage that is directly related to the position of the core in the LVDT assembly. The output polarity is determined by the direction of displacement from null. This output voltage is adjusted to give a signal, with a -3 dB point of 34 Hz, whose dc amplitude varies from +4 V to -4 V over the full range of plunger travel. No additional signal processing other than a voltage follower between the demodulator and the ADC is used. The output signal from the demodulator is converted to a signed 12-bit number by means of an AD7550 ADC (Analog Devices, Norwood, MA). Increased rejection of noise harmonically related to line frequency is gained by using an ADC clock frequency (261.120 kHz) that is synchronized with the 60-Hz power line via a phase-locked loop frequency multiplier circuit. The controller for the remote pipetter system is a microNOVA MP/ 100 microcomputer (Data General Corp., Westboro, MA). The 16-bit system processing unit with an integral asynchronous serial communicationsport is located on a single board. Additional boards include the memory card with 16K words of random-access memory (RAM) and 16K words of erasable-programmable-read only memory (EPROM),the digital 1/0 board, the custom-designed interface, and another asynchronous serial communications
0003-2700/82/0354-1902$01.25/00 1982 American Chemical Society
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Flpun 1. Block diagram of remote pipener system.
npue 2.
Photograph of mechanical assembly for remote plpener.
F l p n 3. Block &gam of arstan interface fa opsralion ofstepphg mta. analogto-digltal convefter. and so!em!d valve.
port The eontroller power supply and boards are housed in the MP/100 chassis. Operator interaction with the controller is by CRT terminal. A ticket printer i s used to obtain hard copy documentationof pipetter operations. A separate chassis houses the demodulator. solid-state relay, translator module, and stepping motor power supply. Aardware/Software Development. The general projectplan for remote instrumentation development in the Analytical Chemistry Division at ORNL dictates that all newly developed
microcomputer-hased instrumentation should be hardware and software mmpatihle. Future instrument needs also demand more hardwarejsoftware computer power than needed for the initial remote pipetting system. Therefore. the microNOVA M P J I I K l Ifi-hit microcomputer was selected to satisfy these requirementn at a competitive prire and to ensure future hardware, acifrware availibility and support. Software development for the remote pipetter was done with the I h t a General mirroproducts operating Pystem l.UP/OS). A stand-alone subset of M P I O S was generated and stored in EP. ROM along with the FORTRAN I V rnntrol programs and assemhly lanauage drivers. 'lhe mmplete run-time operating sptem and FOKI"AN/assembly languag~firmware wcupy approximately I2K words of memory. The approach taken throughout the control p r w a m is to present t u the operator a list nf rurrentlv availahle options and necessary system status. System options include the ahility to perform a self-mt, to fill the pipet. to empty the pipet. to pipet vanahly s l w l aliquotn. or to output a report for each sample. Fnch eample repnn include output of date. time. operator name. sample identification. aliquot number, and corresponding aliquot size. A l l inputn are preceded by a prompt message. At each point requiring an operator decision only those options consi*tent with a logical operating sequenre will he rerognizrd Cnntnd is then automatically routpd through the proper segment of the p r w a m to the next input pnint. The operator 19 never f o r d to speculate about the prnper sequence of operation. F:mr rhecking and error message output are also present at necemipnry piints throughnut the program. The p r w a m prevents the uperstr,r frnm filling the pipet with a new sample before documenting prior operations on the previous sample. The pipet option calls a plunger p i t i n n i n g suhroutine which uses a stepping mutor rotation-t01inear.travel equation to corn. pute the numher of steps required to deliver a desired volume. Prior calibration of the L V D T in terms of millivnlw per step enables confirmation of p l u n g e r m i t i o n to be made. l l y reading the LVDT output before and after plunger movement OXUR. m e can avoid anv errors awxiarad with long-term simal drift. Note that the L V W is used only for verification of plunger movement l edRedm .l p f m l h a r k p a i t i o n within a working range and not for cw control. This operating mode was chosen because of the m a l l but inherent nonlinearity of the LVDT-demodulatorADC combination rompared to that attainable with the drive screw.
RESULTS AND DISCUSSION Elertronic noise w i t h i n the system. estimated as the standard deviation of the residuals from a least-squares analysis of 82 ADC readings vs. time, is about O.fi mV. Therefore. linear motion corresponding to one step can he resolved. Since. analog signal linea from the L W are located in ronduits housing ac power lines where noise pickup is highly probable, a n experiment was performed to determine the
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Anal. Chem. 1982, 54, 1904-1906
Table I. Accuracy and Precision of Plunger Movement in Millimeters
\n
nominal plunger travel
mean
std dev
2.540 5.080 12.700
2.539 5.078 12.690
0.003 0.003
obsd
0.007
Table 11. Accuracy and Precision of Aliquot Delivery
IOo
IO‘
4 02
1G3
104
FREQUENCY (Hz)
Figure 4. Plot of standard deviation of 2 4 peak-to-peak sinusoidal signal vs. signal frequency attained with analog-to-dlgital converter/ phase-locked loop noise rejection circuit.
efficiency of noise rejection at frequencies harmonically related to 60 Hz. A 2-V peak-to-peak sinusoidal “noise” signal was electronically summed with the demodulator output. Twenty-five ADC readings each were taken at selected “noise” frequencies ranging from 1 to 10 kHz. The standard deviation of the ADC readings at each frequency was calculated and plotted as a function of frequency; the data are summarized in Figure 4. The plot rolls off a t about 10 Hz with a slope of -20 dB decade-l. These results suggest that noise rejection at harmonics of 60 Hz is approximately 45 d B without any signal averaging. The long-term stability of the LVDT-demodulator was measured under actual hot-cell operating conditions by recording voltage readings corresponding to the plunger position after delivery of a 1000-pLaliquot. For a period spanning 99 days (52 data points) a mean of +3.868 with a standard deviation of 0.004 V (i.e., 0.6 p L ) was obtained; the nominal value is 3.864 V. Because the reliability of solution pipetting is directly related tQ the accuracy and precision of the plunger movement, data related to these variables were collected. Fifteen measurements each were taken with a dial micrometer for linear distances corresponding to 100.0, 200.0, and 500.0 p L of plunger displacement. Each measurement was associated with a slightly different region of the drive screw, so that the results suggest pipetter performance over the entire operating range rather than only a small portion of it. Accuracy and precision for plunger motion obtained from these experiments are presented in Table I. Data for the actual delivery of variable sized aliquots of water were also obtained. Prior to obtaining these data, the system was calibrated. A slope of 0.9972 and intercept of -0.42 were used in the equation relating aliquot volume to steps of motor rotation. These variables are entered only during the power-up initialization dialogue. Each aliquot was delivered using a secondary displacement technique; i.e., the pipetter cavity was filled with colored mineral oil and only the tip
100
300
500
P L
IIL
PE
700 ALL
av of delivered 100.5 300.9 501.2 701.9
aliquot, p L relstd dev, % re1 error, %
0.4
0.2
0.1
0.5
0.3
0.2
0.06 0.3
1000 M L 1002.1
0.02 0.2
contained solution to be aliquoted. This technique is routinely used to minimize cross contamination of samples. The volume of each aliquot was determined from its mass and the density of water. A summary of the accuracy and precision for these aliquot deliveries is given in Table 11. The remote pipetting system described herein enables sample aliquots to be accurately and precisely delivered in remote environments. The highly reliable, independent control afforded by a microcomputer facilitates operator interaction and provides accurate documentation of operations. The firmware-resident control programs allow quick instrument modifications to be made for changing applications at minimum cost. Although this system was developed for use in a hot-cell environment, it is equally suited for use in a glovebox environment; as such it is applicable in any situation where hazardous materials, chemical or biological, must be isolated from operating personnel. Details of the instrument may be obtained from the Technology Utilization Office, Oak Ridge National Laboratory, Oak Ridge, T N 37830, Remote Pipetter, Model Q-5780.
LITERATURE CITED (1) Maddox, W. L. In “Progress in Nuclear Energy, Series I X ” ; Stewart, D. C., Elion, H. A., Eds.; Pergamon Press: New York, 1970; Chapter
4. (2) Orseilo, S.; Pozzi, F.; Todini, M. Comltato Nazionole per I’Energia Nucleare Report RT CHI-(71)7, Rome, Italy, 1971. (3) Fortsch, E. M.; Wade, M. A. Anal. Chern. 1974, 4 6 , 2065-2067. (4) Ochsenfeld, W.; Welnlander, W.; Ertel, D. Proc. Remote Systems Techn., 25th, 1077. (5) Dykes, F. W.; Shurtliff, R. M.; Henscheid, J. P.; Baldwin, J. M. Proc. Remote Systems Techn., 27th, 1979. (6) Maddox, W. L.; Haga, F. E.; Fisher, D. J. Proc. Remote Systems Techn., 13th, 1965.
RECEIVED for review May 22, 1981. Resubmitted March 3, 1982. Accepted May 26, 1982. Research sponsored by the Nuclear Fuel Cycle Division, U.S. Department of Energy, under Contract W-7405-eng-26 with Union Carbide Corp. Presented in part a t the 25th Conference on Analytical Chemistry in Energy Technology, Gatlinburg, TN, Oct 1981.
Isocratic Reversed-Phase Liquid Chromatography for Assay of 5’-Nucleotidase with Phosphoric Acid for Reaction Quenching LI-Wen Yu and Roger S. Fager” Department of Physiology, University of Virginia, School of Medicine, Charlottesville, Virginia 22908
The most commonly used methods for preparing tissue extracts of nucleotides or enzyme assays for nucleotidases for high-performance liquid chromatography involve stopping the 0003-2700/82/0354-1904$01.25/0
enzyme reactions with perchloric or trichloroacetic acid, neutralizing, removing the acid by precipitation or extraction, and filtering (I,2). For this reason, the sample handling often 0 1982 American Chemical Soclety
