Digital Cary 14 Henry

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system. The pipets were calibrated by pipetting distilledwater into stoppered t est tubes and weighing. The resultsof these calibrations are given in Table I. These data showexcellent agreement between the two methods. Th e preci-sion of bo th th e hand and remote systems is about 0.1% rel-ative standard deviation for the 200- an d 500-11 pipets . Inthe case of the 100-p1 pipets, t he precision is 0.2% an d 0.6%relative standard deviation for hand an d remote pipetting,respectively. The data in Table I also show th at t he statedvalue of t he pipet may be slightly different than t he actualvalue. These three pipets were within 1% of the statedvalues; an accuracy good enough for most work. For bestaccuracy, however, each pipet shoud be calibrated beforeuse.The Model B remote pipets (2) which have been used atthe Idaho Chemical Processing Plan t for many years haveproved their value for routine measurements. They havebeen reasonably maintenance-free and have proved to bequite reliable. However, solutions such as aqua regia, hy-drolluoric acid, and any other material which corrodesstainless steel could not be pipetted. In addition, organicsolutions could not be pipetted. In some applications,cross-contamination could not be avoided. The remote E p-pendorf system does not suffer from these disadvantagesand has expanded pipetting capabilities. In addition, it ischeaper to fabricate, install, and maintain. A comparison ofthe data obtained by the two methods of remote pipettingis presented in Table 11. These data were obtained over along period of time by a large number of different operatorsand they show the remote Eppendorf system to be bothmore accurate and more precise than the Model B pipettor.At the 500-p1 level, th e Model B system has a n average rel-ative standard deviation of about 0.8% compared to 0.4%for the remote Eppendorf system.The Eppendorf pipets are not trouble-free and do re-quire periodic maintenance. Removing t he pipets at aboutone-month intervals and cleaning and lubricating accord-ing to manufacturers' directions will maintain a constantdelivery. Although this paper describes the installation of asingle pipet, multiple unit s can be installed. A two-unit sys-

Henry Longerich andLouis Ramaley'Department of Chemistry, Dalhousie University, Halifax, N.S. , Canada

Digitization of spectral data followed by numerical dataanaly5is can make possible operations which would be ex-tremely difficult or impossible by manual methods. Exam-ples of such operations are base-line correction; conversionfrom wavelength to wavenumber presentation; smoothing,differentiation, and peak finding ( I ) ; and resolution ofcomplex spectra into component peaks (2).In addition, forinstrument. in which th e readout device, usually a meter orrecorder, is the limiting part of the system, digitization canresuit in improvements in accuracy and precision.' ? iUt i ? (Jr I O whom correspondence should be directed.

(1 ) A . Savirzky and M. J. E. Golay, Anal. Chern..36, 627 (1964)( 2 ) L . M. Schwartz, Anal. Chern.,43, 1336 (1971).

Table I. Calibration of Eppendorf Pipets by B othHand and Remote ProcedurePipet delivery, p l aPipet size,

Pl Hand Remote100 100.2 f .0 8 99.8 * 0 . 2200 200.5 f . 1 200 .7 5 0.08500 503.9 f . 3 5 0 3 . 8 * 0 . 2

a Each value is the average of 6 determinations and the i alue is thestandard deviation of the mean ( S D i d n ) . ~-Table 11. Comparison of th e Model B an dEppendorf Remote Pipetting Systems

Found, plQStated value, pl Model B" Eppendorf

500 495 .2 f . 1 5 0 0 . 6 =k 2 . 0502.0 i . 5503.6 k 3 . 1

a The i alue is the standard deviation of a single analysis. * The valuesfor three different Model B Pipettors are given.

tem operating from a single selector valve is in use; moreunits can be installed if desired.CONCLUSIONS

The pipetting system described in this paper provides asimple, convenient, and inexpensive method for operatingthe Eppendorf pipet remotely. The results obtained in th euse of this system confirm that while high precision pipet-ting may not be accomplished, precision satisfactory formost applications is attained. The system is versatile andcan be used as a permanen t or temporary adj unct to exist-ing equipment.RECEIVED or review March 18, 1974. Accepted June 4,1974.

Digital interlace for a Cary 14 Spectrophotometer

ANALYTICAL CHEMISTRY, VOL. 46, NO. 13, NOVEMBER 1974 2067

Many laboratories today possess minicomputers or com-puting calculators and high qualit> scanning spectropho-tometers such as the Cary 11 (Varian Instruments, PaloAlto, Calif.). The advantages mentioned above can readilybe obtained by providing an interface between the instru-ment and t he computer. This situation has existed for sometime; however, the authors have been unable to find muchin the literature describing the intt.rfacicg of commercialspectrophotometers with digital computers. The one excep-tion to this is a paper by Anderson (3 )which contains a sec-tion briefly describing. in general terms, an interface for a(3) R. E. Anderson, Preprin?UCRL-72039, Lawrence Radiation Lab., Liver-more, Calif.. Dec. 1969.


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1 CARY 14 1

C O W T R O L

PERIPHERALS COMPUTER

INTERFACE FLAG (2' ; INTERFACECONTROL I31

I TACHOMETER 1 ~ 7 4 3 8 I IIU

ita1 information to the computer, and registers for receiptof up to 72 bits of information from the computer. This isaccomplished through one 16-bit computer input-outputchannel. The compute r interface transfers wavelength andtransmittance data to the computer and outputs controlsignals to the spectrophotometer interface. The spectro-photometer interface also contains circuitry for operatingthe spectrophotometer scan and chart motors under com-puter control. The computer system consists of a Model2114B 16-bit computer with an 8K memory (Hewlett-Pack-ard, Palo Alto, Calif.) with magnetic and paper tape pe-ripherals.If a computer interface of the type previously describedis not available. then certain of its functions such as digi-

2.2K Q LOADI I COUNT

.- -.-P

* 5 vL i2...4* 5 v

CARY 14Figure2. Wavelength encoder wiringCary 14 equipped with a Datex scanning attachment. Un-fortunately this article is not in the widely circulated litera-ture.This Aid presents a description of circuitry specificallydesigned to connect a Cary 14 spectrophotometer with ageneral purpose computer interface (4 ) .With minor modi-fications, the circuitry could be used with any similar dual-beam spectrophotometer having a single photodetector. Itis compatible with any computer system that incorporatesfeatures of the general purpose computer interface ( 4 ) .

SPECTROPHOTOMETER INTERFACEA block diagram of the entire system is shown in Figure1. The Cary 14 spectrophotometer interface receives foursignals from the spectrophotometer. The signal from thephotodetector is demodulated with the aid of a rc!ay drivesignal for synchronization. Two signals from an optical La-chometer are used to encode the wavelength. T he demodu-lated signals are digitized in a genera! purpose compui.erinterface similar to one described by Ramaley and M'ilson(4) ontaining a fast, 12-bit analog-to-digital converter(ADC) with an eight-channel analog input multiplexer. adigital input multiplexer for transfer of up to I C : hits o f dig-(4 ) L. Ramaley and G.s. Wilson, Anal. Chern.,42, 606 1970).

18 LINES

I I -8 LINESIB I LINELA G >

CARY INTERFACE

tization must be included in the spectrophotometer inter-face. In addi tion, the use of more th an one computer input-output channel may be required.The spectrophotometer interface is conveniently dis-cussed in three separate and independent sections.Wavelength Encoding. This section is shown in Figure

2. 4 Model TB/C5QO/ES5volt/LO/SN/XG optical tachom-eter (H H Controls Co., Inc., Arlington, Mass.) supplied byC'arian had been attached to the wavelength drive mecha-!:ism of the spectrophotometer by inserting a small shaftinto the gear mechanism on the drive shaft that enters themonochromator. This small shaft was coupled to the shafton the tachometer h y a flexible joint t o alleviate strain. Thegear mechanism of the spectrophotometer had alreadyheen drilled for such a shaft, and no modifications werema d e IO the bpecrrophotometer except to cut a small holein th e front panel to accommodate the tachometer. The ta-chometer produces two square wave outputs when rotating.' I 'nne " H " (,utpuS either leads or lags the "A" output by 90'depending on the rotation (scan) direction. One cycle of ei-t her signal is equivalent t o 0.04 nm.A counter is used to convert the tachometer signals intoa \iawlengti? reading. T h e positive edge of the "A" signaltriggers munostabie inultivibrator (Mono) 11and the nega-tive edge triggers Mono 12 . Th e "B" signal is NANDed

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+ 2 4 V

CONTROL

ON-OFF

1FROMCOMPUTERINTERFACE

Figure 3. Scan and chart control wiring

with the monostable outpu ts so as to direct t he pulses to ei-ther the count-up or count-down input of the counter ac-cording to the scan direction of the spectrophotometer.Each count pulse triggers Mono 1 providing a flag to thecomputer. This allows the computer to avoid interrogatingthe counter during a counting transition by waiting untilafter a count pulse. Before a scan, the computer obtains thepresent spectrophotometer wavelength from the operatorand presets the wavelength counter accordingly. From thistime on, the wavelength counter contains a number fiftytimes th e actual spectrophotometer wavelength in nanome-ters, regardless of scan direction or number of scans.An absolute encoder would remove the need for thecounter at a n increase in cost. Th e computer itself could beused as the wavelength counter by reading the A and/orB signals directly. However, th is decreases programmingflexibility.Spectrophotometer Control. Any of th e functions con-trolled by the electrical front panel switches on the spectro-photometer could also be controlled by the computerthrough relay closures. It was decided tha t it would be use-ful for the computer to control the scan and chart motors.All other func tions were left under manual control.Figure 3 diagrams the wiring of this section of the spec-trophotometer interface. Access to the Cary 14 front panelswitches was made through the repetitive scan (RS) con-nector. A new plug was wired for th e RS socket as shown inFigure 3. Two switches, SP DT an d 3P DT, were used in theinterface to assign control of the c hart and scan motors to

F;:,I2

E;::INS ONCONNECTORREPETITIVE SCAN ->either the Cary 14 front panel or the computer, allowingmaximum flexibility of operation.Chart motor control is achieved through one signal linefrom the computer which operates a SPDT relay (No.JMF-1160-61, Potter and Brumfield) through a high-volt-age inverter.Scan control is somewhat more complicated, requiringone bit to turn the motor on and one bit to set the direc-tion. The spectrophotometer switch that performs this op-eration is SPDT-center off. To duplicate this requires a3PDT relay and SPST relay (No. R40-E3-X4 and No.JMF-1160-61, Pot ter and Brumfield) as well as a circuit t oprevent th e possibility of reversing th e scan motor withoutfirst turning it off when a change in scan direction is re-quested. Any change in the direction signal causes eitherMono 2 or Mono 3 to trigger which in tu rn triggers Mono 4and Mono 5. Mono 5 holds the scan motcr off for 0.17 sec-ond while Mono 4 strobes the new scan direction into thepositive-edge-triggered D flip flop after a delay of 0.09 sec-ond. A logical 1 on the appropriate line turns the scanmotor on. Both on-off relays have mercury-wetted con tactsto suppress switching noise. In addition, it was found nec-essary to bypass th e contacts of t he scan motor relay in theCary 14 with 0.01-pf capacitors to supress switching tran-sients which caused malfunctions in the interface.For instruments that do not have an RS connector, thecontrol relays described above can be wired directly intothe Cary 14 front panel assembly. The authors will supplyinformation on this procedure on request.

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REFPLATEOF T21

*5v t

120K

CARY P.S.I 5vIN7473

PIN W OFDRIVER AMP+ 1 5 V

CARY 14Figure 4. Demodulator wiring

CARY INTERFACE

Signal Processing. The o utput of the photodetector ofthe spectrophotometer is a modulated signal derived froma light beam synchronously chopped a t th e line frequency.This signal repeats itself 30 times a second with equal por-tions of the signal representing the reference energy, thedark current, the sample energy, and the dark current. Ifthe sample and reference energies are equal, the signal ap-proximates a 60-Hz square wave. This signal is synchro-nously demodulated in the spectrophotometer interface bycircuitry shown in Figure 4.Synchronization is partially obtained from the line volt-age. A 20-V, 60-Hz signal is taken from th e transformer ofthe 24-V relay power supply, squared-up by the Zenerdiode-Q1-inverter network and used to trigger Mono 6 and7 . The NANDed output of these monostables, a train of120 pulses per second, is used as a clock signal for a 2-bitcounter and a trigger signal for Mono 8. Mono 9 triggersafter Mono 8s period is finished. The 2-bit counter drives a4-bit data distributer. The pulse from Mono 9 successivelyappears at the output of each of the four NAND gates ofthe dat a distributer. The first three gates drive level shift-ers Q3-Q5 which in tu rn drive three of the four switches ina COS-MOS quad bilateral switch (QBS) (No. CD6014A,RCA). These bilateral switches connect sampling capaci-tors, C,, to the detector signal for demodulating. The peri-ods of Mono 6 and Mono 7 are very shor t, and th e pulsesthey produce may be considered to occur a t the zero cross-ing of the 60-Hz line signal. Mono 8 controls th e delay timebetween zero crossing and sampling, and Mono 9 sets thesampling time. The periods of both monostables are adjust -able up t o 6 msec. Nominal periods of 2.3 msec and 3.3

+ -

->FLAG

msec were found satisfactory for Mono 8 and Mono 9, re-spectively. Th e demodulated signals from the sampling ca-pacitors are output to the analog multiplexer of t he com-puter interface by FET-input operational amplifiers A2-A4 (No. 435, Analog Devices, Cambridge, Mass.).In order to ensure tha t amplifier A2 always samples thereference energy etc . , the timing signals must be synchro-nized with th e chopper in the spectrophotometer. This isdone by taking a 30-Hz spectrophotometer relay drive sig-nal from the plate of tu be T21 in th e Cary 14, level shiftingand squaring this signal, and producing pulses from it withMono 10 which reset t he 2-bit counter. Once the counter isproperly phased with the optical chopper, synchronizationshould be maintained; however, the 30-Hz signal is alwayspresent to ensure this.The fourth signal from th e dat a distributer correspondsto the second dark current period in a full cycle of the de-tector signal. Since it is unnecessary to sample the darkcurrent more than once during each complete cycle, thissignal is used as a computer flag to indicate completion of acycle. The demodulated detector signals can thus be sam-pled afte r every complete cycle if desired. The three signalsfrom A2-A4 corresponding to the reference energy, darkcurrent, and sample energy are multiplexed in turn to theADC, digitized, and inpu t to the computer which computestransmit tance or absorbance from the signals.Some signal conditioning is performed on the detectorsignal before it is demodulated. This signal is taken frompin W of the driver amplifier in the Cary 14 after severalstages of amplification. In order to take advantage of thefull scale ranges of the ADC (2 0 V) and the bilateral

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switches (15 V), amplifier A1 (AD512K, Analog Devices)amplifies and offsets t he detector signal to produce a signalfrom -6 to approximately +5.5 V. The maximum positivevoltage depends on the spectrophotomete r slit width whichis controlled by t he sli t servo. This servo is highly damped ,resulting in the positive voltage varying between +5 and+6 V. Amplifier A1 is bounded between f6.8 V to ensurethat its output does not exceed the power supply voltagesof f 7 .5 V of the bilateral switches.It is possible with a fast ADC to obtain the necessarydata without demodulating the detector signal, simply bysampling the signal a t the appropr iate times. However, thissignal is noisy and the noise would be converted and de-grade the accuracy of th e measurement. Th e demodulatingcircuits average the signal over the period of Mono 9 with atime constant of R,C,, thus eliminating the high frequencynoise on the detector signal. The time constant is conve-niently varied by changing R, according to the amount offiltering, scan rate, and resolution desired. Nominal valuesof R, and C, are 18K and 1 pf, respectively. The exactvalues and stabilit ies of these and other circuit componentsare not critical.If a n analog multiplexer is not available, the dark currentmay be directly subtracted from the reference and samplesignals using differential amplifiers. The resulting two sig-nals may be digitized by a ratioing digital voltmeter to pro-duce transmittance directly and this result input to thecomputer.All logic elements shown in the figures are Texas Instru-ments SSI and MSI TTL integrated circuits. The unla-beled logic gates are either SN7400, SN7404, or SN7410and the monostables are either SN74121 or SN74123. Ex-cept as noted previously for Monos 4, 5 , 8, and 9, all othermonostables have periods of 10 psec. All transis tors are2N3646 and all signal diodes are 1N4148. The logic sectionsof the spectrophotometer interface and the tachometer arepowered by a single +5-V power supply. A f15-V powersupply is used for the analog circuitry, and the relays arepowered by a +24-V Zener regulated supply.

INSTRUMENT PERFORMANCETests of the wavelength encoding circuitry were made byobtaining spectra of the 248.08-nm peak of benzene vaporat 0.04-nm increments. This peak is very narrow, allowingprecise determination of any wavelength error. In a seriesof 16 spect ra in which the wavelength counter was notreset , no loss of counts was observed.It is more difficult to test the performance of the instru-ment with regard to absorbance readings since no good ab-solute standards are available over a wide range of absor-bances. One can only prepare a series of nearly ideal solu-tions and test their adherence to Beers law. Sixteen solu-tions of p-terphenyl (99.9+ % pure, Aldrich Chemical Co.Inc.) in spectral grade isooctane (Fisher Scientific Co.)were prepared over a concentration range 4 X to 9 X1W5F y gravimetric dilution of a 1.632 X 10-4F stock so-

lution. The stock solution was obtained by dissolving 18.79mg of p -terphenyl in 500.0 ml of isooctane. The solute waschosen for its high purity, broad adsorption band whichshould reduce inst rumental deviations from Beers law, andhigh molar absorptivity which allows use of dilute, nearlyideal solutions. The solvent was chosen for its spectraltransparency, its relatively low vapor pressure which helpsto reduce evaporative effects, and it s ability to form a near-ly ideal solute-solvent pair with the solute.These solutions were equilibrated for four hours in thesame room as the instrument and then the absorbanceswere measured at 276 nm in a 1.00-cm cell. Absorbancereadings were taken in two ways, from the Cary 14 chartand by the computer system described above using a pro-gram which measured and averaged 1000 data points. Thedata from both sources were analyzed using a computerprogram to fit th e dat a to a straight line by the method ofleast squares and to compute the slope (molar absorptivity)of the line and its estimated standard deviation.For the twelve solutions of absorbance less than 2.0, themolar absorptivity of p-terphenyl calculated from the chartdata was 30260 f .39% and from the computer da ta it was30750 f 0.27%. Using four additional solutions with absor-bances up to 2.9, the computer data gave 31010 f 0.75%.These last four solutions could not be analyzed from thechart since the recorder of our instrument would not recordabsorbances above 2.0.Beers law is well obeyed up to absorbances of 2.9, indi-cating satisfactory instrumental performance. The comput-er measurements were somewhat more precise than thosetaken from the chart, as would be expected. The method ofpreparation and handling of the solutions should allow aprecision of 0.1% in their concentrations in the worst case.Thus, the standard deviations of the absorptivities shouldreflect instrumental effects almost entirely.The absolute accuracy of the ADC used to digitize thephotodetector signal is hlh least significant bit (f 2. 5 mV),whereas the noise level of t he ADC is f O . l LSB (0.5 mV). Ifthe ADC were the only source of determinate error, themaximum error in absorbance would be f0.0004 at zero ab-sorbance, f0.0 02 at one, f0.02 at two, and f 0. 2 at three.Experimental measurements indicate tha t t he uncertaintyin absorbance caused by noise on the analog signals is con-siderably less than the errors listed above at absorbancesabove one and approach the above values at absorbancesbelow one. Thus, if no determinate errors occur before digi-tization, the total uncertainty in absorbance is caused al-most entirely by the ADC and is approximately that pre-viously stated. Th e difference in absorptivities between t hechart and computer da ta is small but significant. It is diffi-cult to draw conclusions as to which is the more accuratewithout proper absolute absorbance standards.RECEIVEDor review November 9, 1973. Accepted May 6,1974. This work was supported by grants from the Nat ionalResearch Council of Canada and Dalhousie University.

ANALYTICAL CHEMISTRY, VOL. 46, NO. 13 , NOVEMBER 1974 2071

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Digital Cary 14 Henry