Universal Titration Console EDWARD L. D U G G A N and VINCENT L. STEVENS Department of Physiologicol Chemistry, School of Medicine, Universiiy of Colifornia, Berkeley, Colif.
b The console titrator consists of
o
portable potentiometric titrator and a pH stat. It provides the necessary automatic recording of titrations under specific conditions of mixing, buret delivery, and constant temperature. Attention has been given to compactness and routine efficiency in the mechanical and electronic design. The console may b e treoted as a single dependable unit. The titrator yields occurate results with sample volumes of 1 to 5 ml. at sample concentrations of 10 pmoles per ml. This provision permits examination of rare, viscous, or slightly soluble compounds.
inch span. Regular chart paper provides 100 markings over this range. It may be used for a record of titrant volume os. time, disregarding the printed p H numbers, or special blank paper may be used. The curve subtraction device indicated on the diagram (8)uses the blank chart paper, with arbitrary ordinate and abscissa values. The three signals enter the Brown Instrument Co. recorder by way of a 10-position switch, so that the recorder is
rapidly converted from one use to another. The major components used in the two types of titration nre listed in Table I. The provisions for stirring are unique and useful. A constant-speed, brushless motor (1/12 hp.) is fastened by a vibrationless mount to a shelf within the console, in the lower area behind the control panel. The motor shaft drives the transmission (Graham llanufac-
A
titration devices have been developed for p H titration ( 5 , 6 )or for volume recording at constant p H (3, 7). Interest in the physicalchemical properties of the nucleic acids led to the manufacture of the present device. The titration console has advantages over previous designs m convenience of operation, rapidity of accumulation of corrected titration data, and routine use of small sample volumes. The console provides for ordinary p H titrations and for titrations a t constant p H (pH statting). UTOMATIC
APPARATUS
I n over-all design, the console is a Formica-covered table built around the desired amplifier and recorder units, Figure 1 shows the complete titration assembly except for the titrant syringe. All necessary switches are brought out to the panel hoard. A few switches have been left free for future use. The various switches are connected t o the components via an internal panel which permits individual fusing of all circuits. The panel is exposed for fuse-check by removal of the left metal wall of the console. The circuits are under the control of one master switch, or of a program timer (Sangamo Electric Co. Type SR). The timer provides for a n amplifier warm-up period. Accessory switches and outlets are provided so that the table-top assemhlies may he energized through a particular switch on the panel. The relationship among the various units of the console is shown in Figure 2. The pathways of various signals into the chart recorder are shown without the complexity of the power circuits to the various units. The recorder provides a range of pH 2 t o 12 over an 111076
ANALYTICAL CHEMISTRY
Figure 1 .
Universal titration console
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Amplifier Model W
/
*
control Panel I I 0 V.
5, 0, -5 mv. 2 *' Pen Speed
Subtroction Device
(IO-point switch)
AC.
Stirrer 0-2500 RPM.
Volume Signal Motorized Syringe Legend
--* Power. I + S,"""l - > h
I
Amplifier Electrodes
Figure 2.
Model G
pn stat Control (meter reloyl
I
Botlery 7aoeroted
Schematic diagram of titration assembly
Table I.
Major Components of Titration Console
Componen t
Source For Titrat>ions Beckman 8990-80 or 290-lx (filled for use n.ith Beckman Model \V amplifier) Beckman Model \T3 pH 2 to 12, automatic temperature compensation Byon-n Electronik Model T153 X liT’, 2-second pen, =k 5-mv. span Aminco 5-8094, n-ith added micrometer xrew 1/12-hp. shaded pole motor, transmission (Graham 156M).geared to provide0 to 2300 r,p.m.
Glass electrodes
Amplifier Recorder Buret dtirrcr (magnetic.,
F o r pH Statting Beckman 290-1 Beckman Model G , with double uole, double throv switch arid uhone iack conriection for meter relay in place of null met& Custom-built by LIont Cannona Custom-built by Mont Cannon Built by Mont Cannon. Includes Broim pen-drive motor as syringe drive, and proportional volume signal for recorder
Glass electrode; .Amplifier pH stat contiol
Recorder
pH stat drive unit
i
International Inbtrument Co., Canyon, Calif.
- I8
- 12 -9
I
I
3
11
I
4
I
l!
I
ll
I
6
5
I
I
I
7
I
8
PH Figure 3. Titration of acetic acid
tming C o . . In(..). The output from the transmission i b transferred by a clutch niechanisiii :ind belt drive to a vertical shaft of atljustable height. The shaft rotates from 0 to 2600 r.p.ni., under tlie control of a transniibsion setting indeued n.itli a Be( knian Instruments. Inca Duodial. The shaft provides taper fit of a holder for beaker rotation, a dynaniic illy b:ilnnced Alnico U-magnet, 01 a n attachment \!-hich provides electrotlp iotatioii for :iiiiperonietric titrations. Since eutended electrode lead. are used in the console, the Becknian electrode lends are extended by a speciallj built connector and shielded cable to the Inpiit terininnl of the nniplifier in use The calomel and glasq electrode lends for encall nniplifier are further shielcled by metal brnid grounded to the console skeleton. To prevent electrostatic effects through fleuure or vibration, the leads are mounted in a length of largediameter polyeth\ lene tubing. During operation of the Beckmail
Model 11- amplifier and recorder, no fluctuation of p H reading occurs nhich could be ascribed to static charge effects or to magnetic stirring. The differential pH change due to stirred zs. still solutions is cancelledbystandardization of the :tniplifier-recorder with knon n buffer< at the nornial stirring late. ORDINARY pH TITRATION
The titration assembly consists of electrodes, Beckman Model 11- aniplifier, recorder, and motorized buret (Table I). A sinall Becknian calomel electrode n i t h extended lend i h used, its conducting tip immersed in the reservoir of a saturated potassium chloride hridge. The salt bridge ib a polyethylene vial filled with the electrolyte and fitted with a flexible bridge of thinn-alled polvetli ylene tubing (Clay-Adams
Co., Iiic. PE 190, 0.05 inch in inside diameter), The tubing is terminated with a filter paper plug 5 mm. long. Connection between the salt bridge and titration sample is made by the slow diffusion of electrolyte through the filter paper. If the paper plug acts clogged (unstable pH reading), a thin transverse cut through the tubing and paper exposes a free surface of the paper. The buret assembly is an Xniinco compensator (American Instrument Co., Inc.), modified t o accept a second synchronous mobor. The screw advance of the compensator has been discarded in favor of a micrometer he:id. tlie old scren- shaft serving to link the micronieter to the gear drive. The assembly is shon-n in position on a n adjustable base (Figure 1). The micrometer head bears directly on the plunger of a Hecton-Dickinson & Co. syringe (Yale Insulin, 1 ml.). Several syringes were calibrated by weight of water delirereti for 13 interrals of micrometer setting. Syringes having constants of 33.4 pl. per nini. =t0.37, (standard deviation from the mean) \!-ere used as matched reservoirs of alkali and acid titrants. The syringes were proritled with individual polyethylene tips by forcing the heated end of t’he plastic tube over the glass syringe tip. The terminal end of the plastic tubing was bent a t right angles and dran-n to a fine capillary. Titrant may be added :it one of six rates provided by three gear ratios and the two motors. The titrant enters belonthe surface of the saiiiale solution froin the plastic tip. Reserve solutions of XI-hydrochloric acid and sodium h!-droside iT-ere prepwed and standardized. The :iccurately diluted 0.5-1- solutions ivere used as stock titrants. The alkali solutions n-ere stored in paraffined flnslis under sodalime tubes. The passage of nioist nitrogen gss through Tygon tuhing into the corered sample chamber (Figure 1) extracted volatile acids which could acidify a u--ater sample to about pH 3 . Gum rubber tubing did not yield such volatile acids. The nitrogen gas enters the smiple chamber through a coarse VOL. 29, NO. 7, JULY 1957
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irittetl g1:tss plate, without actu:il bubbling through the solution. Temperature control of the s:iniple was provided by a constant teniperature bath (E. H. Sargent &- Co.) n-it'li water circulation by a small centrifugal pump. Temperature constancy was +0.05". The thermocompensator of the Beckman Model W amplifier \\-as immersed in the bath; it provided the desired automatic adjustment of p H range in t,he interval of 23" to 60" C. .4n example of t.he titration curves rapidly obtained on the instrument is shown in Figure 3. The sample was 1 nil. of O.O15*Y sodium acetate in 0.LV sodium chloride. Hydrochloric acid was added to p H 2.3; the titrant was O.05N sodium hydroxide. The curves arc. :IS follows: I, the blank curve for t'he solvent; 11, the interpolated difference curve (I11 minus I); and 111, t,lie uncorrected titration curve for the sample. The offset distance from one curve to another is arbitrary. The pK' value found is 4.66, compared to a published value of 4.57 ( 1 ) . The experimental curves were traced on the blank chart paper on which the differential curl-e was plotted, using t'he curve subbraction device (8). Preliminary results of a broad titrimetric investigation of deoxyribonucleic acid have been reported ( 9 ) . To investigate such labile macromolecules or protein it is convenient to divide the p H range into three sections: p H 2 to 5 , 5 to 9, and 9 to 12. Titrations should commonly span two sections or less, so that contact of the material with acid or with alkali is controlled in time and temperature by the titration conditions. Thus denaturation by storage a t extreme p H values is eliminated, except for the brief interval of the titration. CONSTANT pH TITRATIONS
The p H stat comprises four units, ns s h o a n on the schematic diagram in Figure 2. The pH control assembly uses a contact meter relay and a relnyactuated motor drive similar to those used by Neilands and Cannon ( 7 ) . This p H stat differs in the use of the modified Beckman Model G p H meter as the sensing device, with independence of caircuitry for the two types of p H titration. The p H stat control unit is mounted on and behind the upper half of the console panel. Switches, controls, and the mercury dry cell which provides the voltage source of the volume signal are panel-mounted. The interior assembly includes the necessary rectifier, relays, time-delay condensers, and other parts of the syringe drive control. I t s circuitry is almost identical with that al-
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ANALYTICAL CHEMISTRY
ready described ( 7 ) , The Bronn pendrive motor, the Greiiier Glass Industries syringe buret and the signal Helipot are located in a table-top unit. Connection is made by shielded cables from the control unit to the drive unit.
A battery-operated amplifier-potentiometer (Beckman hIodel G) is used in place of the line-operated amplifiers used in the instruments of Jacobsen and Leonis (3) or of Neilands and Cannon ( 7 ) . While battery operation is a defect, the control function of such a p H meter is superior in sensitivity to that of line-operated models. The performance of the latter is modified to soine degree hy the addition of the meter relay in the output circuit. Certain difficulties ivere apparent in the tests applied to this p H stat. Gross loss of sensitivity of the Beckman Model G amplifier was apparent until the following steps were taken: isolation of the amplifier and electrode shields from the common console ground, use of a choke in the line from the amplifier output to the meter relay, electrical isolation of the dip-tip buret from the metal case of the niotordrive unit, and adequate grounding of the thermostat bath units and centrifugal pump. At this point a sensitivity of 12 contact meter units per p H unit was obtained. The maximal sensitivity of the amplifier with shorting strap in place (electrodes out of the circuit) was 25 units per p H unit. This maximal sensitivity could be approached, n ith electrodes functioning, on careful isolation of the salt bridge from the glass electrode surfaces, in such a way that surface conduction from the salt bridge to the glass electrode shield could not occur. Teflon film in the form of spacer disks or wrapping film separated the salt bridge tubing from the glass electrode surface, especially in passage through the Lucite cover of the titration chamber Under conditions of maximal sensitivity, minimal zero drift of the amplifier occurred. Alkali addition under these conditions is actuated by a pH change of less than 0.03 unit, and the zero drift is less than 0.02 p H unit per hour. The follodring advantages are anticipated for the use of the Beckman Model G amplifier as the sensing element in the p H stat. 1. Any p H value may be chosen, even after the experiment begins, by appropriate setting of the slide-wire. 2 . The actual p H value may be read, using some point on the contact meter as the null point. This point is choqen away from the contacts. 3. The
functioning bensitivity of the a--embly may be measured at any time 4. The p H stat is operating a t three- to fourfold greater sensitivity of control, compared to a p H stat which projects the \\hole p H range of 2 t o 12 on a meter-relay scale. The last advantage is of special importance for pH-statting a t the extremes of buffer capacity. Sensitivity of control prevents excess of alkali addition at minimal buffer le\-el, while such control provides response to the slight p H changes which occur a t high buffer concentration. The use of p H stats iq limited t o the p H range of 3 to 11, since the aqueous solvent gets to minimize p H change per equivalent of titrant outside of this range. The application of the p H stat to enzyme chemistry has been discussed by Neilands and Cannon ( 7 ) . Volume recording a t constant p H can provide kinetic data for hydrolytic and redox enzymatic reactions. The instrument may also be used to follow derivative formation from amino acids (4) or proteins 19), provided the reactions occur in a p H region where the stat has adequate sensitirity of control. ACKNOWLEDGMENT
The authors express their appreciation of the continual cooperation of Joseph Laviolette and Kathan Benetlict of the Engineering Shop, Berkeley campus. The p H stat design :tnd assembly were the responsibilitJ- of Llont, Cannon of International Instrument Co., Canyon, Calif. This research has been supported 1,grants G3291 (C2) and C2287 (C3) of the National Institutes of Health. I-.S. Public Health Service. LITERATURE CITED
Bell, R. P., "Acids and Bases," p. 46, Methuen, London, 1952. Duggan, E. L., Stevens, V. L., Feiferation Proc. 15, 245 (1956). Jacobsen, C. F., Leonis, J., C o m p t .
rend. trav. lab. Carlsberg, SBr. chim. 27, 333 (1951). Levy, ,4.L., Li, C . H., J. Biol. Chent.
213, 487 (1955). Lingane, J. J., ANAL. CHEY.20, 285 (1948). Ibid., 21, 497 (1949). Neilrtnds, J. B., Cannon, 11,D., Ibid., 27, 29 (1955) Stevens, V. L., Duggan, E. I,., Ibid., 29, 10T3 (1957). Wilcox, P. E., University of Washington, Seattle, Wash., personal communication, 1956. RECEIVEDfor review October 12, 1956. Accepted \larch 4. 1957.
