Convenient pH-Stat Assembly K. I. W O O D Division of Protein Chemistry, C.S.I.R.O. Wool Research Loborafories, Parkville,
b A highly accurate and stable pHstat (0.03 pH unit over 3 hours) is described. The use of a null method provides a calibration accuracy which is dependent only on stable components. A 50 to 1 change in the rate of addition of reagent may b e made during an experiment without gear changing. The instrument may also b e used as a manual pH meter or as a 0- to 1300-mv. electrometer input voltmeter. It is cheap to construct and gives convenient troublefree operation.
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HE kinetics ' . ' of a chemical reaction involving the production or consumption of hydrogen ions are most conveniently studied by the use of a pH-stat. This apparatus maintains constant hydrogen ion concentration and continuously records the amount of acid or alkali required to maintain this condition as a function of time. Assemblies described (1-4) have used appropriate equipment with commercial pH meters. This paper describes the construction and operation of a complete p H s t a t having very high accuracy and stability which is cheap to construct and convenient to operate. A recent paper (6) describes a complete recording titrator for which similar claims are made but which employs a d i e r e n t principle from the apparatus described here.
TITRATION VESSEL
The reaction under investigation iS ".A ;,. -~.+ 1.1:" LL" Y2"Y - I-1L I I . U"",,,-w,L,,d A^..Li^ ..."11^. b*LllGU glass vessel, maintained at constant temperature by water which is circulated from a thermostated bath through the jacket surrounding the vessel. A Colora Ultrathermostat is suitable for this purpose. The pH electrodes, thermometer, glass stirring propeller, and the capillary tube through which the reagent is added pass through a polyethylene cap fitted to the titration vessel as shown in Figure 1. Access is also for the introduction .. provided ' ieriments where the quired. ""Y
METER
:ntly accurate values I and pK values the aintain the pH conIan 0.01 pH unit of
a predetermined value for periods up to 2 or 3 hours. This necessitates the use
of an electrometer having high sensitivity and a long-term stability better than 600 pv. With the circuit arrangement due to Scroggie (5) and using regulated voltages and selected tubes, the zero setting WBS sufficiently stable but the input impedance was too low to allow accnrate measurement of voltage through the high internal resistance of a glass pH electrode. Input tubes of the type used had grid currents on the order of lO-'O ampere under these conditions. The circuit was modified to ooerate
screen voltage of t h e EF37A's was reduced by decreasing the resistance in the voltage divider between the screens and the negative high tension line. When this was done, a condition could he found where the bias equaled the floating grid potential but the circuit would "block" if overloaded by a positive-going transient. This would require the input terminals to be short-
circuited to regain normal operating conditions. Returning the lower end of the screen voltage divider to the common cathode connection of the EF37A's prevented this tendency to block and allowed easier setting of the hias. Although the zero grid current point is fairly critical to set and the slope of the Ig/Eg curve is steep through this uoint. it has been found with aeed tubes and regulated voltage supgies that the setting is sufficiently stable to introduce negligible error and has required only infrequent adjustment over a period of 2 years. A selection was made from ten EF37A's and six 12AT7's on hand for use in the final eauiument. Different combinations of 'tubes were tested, noting output stability as the main voltage to the apparatus was varied by a Variac. Some of the EF37A's tested produced a shift of a few millivolts in the output zero setting when the tube envelope was jarred. The two input tubes selected for use were the best match consistent with good mechanical properties. A thermal time delay switch is included in the high tension supply to this circuit which prevents overload of the zero indicating meter under warm-up conditions. This delay in applying the VOl. 32, NO. 4, APRIL 1960
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ANALYTICAL CHEMISTRY
Figure 3.
high tension until the cathodes are heated is also desirable when using electrometer tubes where low drift conditions are important. Means for regulating the voltages supplied to the heaters and high tension are not included in the circuit, as the entire equipment is operated from a 230volt alternating current electronic voltage stabilizer, Type 52500, made by Stabilac Pty., Ltd., Sydney. This unit restricts voltage fluctuations to =tO.5% for mains voltage variations between 190 and 260 volts. In construction, care was taken to esclude light, to avoid undue heating of components, and to provide resilient mountings. Hand-picked and accurately adjusted wire-wound resistors of lon- temperature coefficient n ere used for the circuitry associated with the slide-wire potentiometer (S. TT., Figure 2 ) and high stability carbon and m-irewound components \yere used elsewhere. A Leeds &- Korthrup potentiometer ?‘J pe K2 m s used for calibration of the slide-n ire and temperature scales. To shield and insulate the conncctions between the glass electrode and the input tube, polyethylene-insulated coa\ial cable and connwtors, a shielded ceramic switch wafer, S l a , and a polystyrene dielectric capacitor were used. The circuit (Figure 2) is used as a null detector of balance between the voltage developed by the p H electrodes in the solution under test and a voltage equivalent to the desired pH control value preset on a calibrated slide-wire. The slide-wire scale may be used for direct voltage measurement and is calibrated from 0 to 1300 mv. against a Keston standard cell. With switch S2 in the “pH” position the scale may be calibrated 0 to 13 p H units over a range of temperatures from 0 ” to 60’ C. A n ide range of asymmetrical potential (AP) adjustment is available to allov; the use of electrode systems developing zcro e.m.f. between p H 1.5 and 9.5. A satisfactory glass electrode for this work is a shielded lowresistance type, designed to give low sodium ion corrections a t high pH values and listed as Tvpe B by N. L. Jones, Sandringham, Victoria. This glass electrode and a szturated calomel half cell produce zero e.m.f. a t about pH 1.9.
Graph of pH meter stability
To indicate out-of-balance with the electrometer input null detector a center zero meter is connected across the cathodes of the txin triode, V3. This zero indicator is used initially to standardize the instrument. To set the input tube to floating grid potential the glass electrode lead is disconnected and thp input plug shielded from electrostatic pickup. The screen resistor and the ZERO SET control are then successively adjusted until the output remains a t zero with the input switch SI in both the OFF and TEST positions. Three operations are required to calibrate the slide-wire for pH measurement. The output is set to zero by the ZERO SET control with input smitch 81 in OFF position; by the STD control with S1 in the STD position having first set the T E M P control to the desired value; and by the AP control with S1 in the TEST position when the slidewire is set to the pH of a knon-n buffer solution being measured by the electrodes. p H CONTROLLER
For control purposes an additional meter (supplied by R. G. Holloway, Melbourne) having a light metal vane fitted to its pointer is switched across the zero indicator. Movement of the pointer causes a change in capacitance between this movable vane and a fixed plate ivhich operates a capacity-sensitive device. The capacitance change is used here to control the amplitude of oscillation of a Hartley oscillator. The oscillator is adjusted so that a small increase in capacitance between grid and ground will cause oscillation to cease. The plate of the oscillator tube, T’4a, is fed Lyith 150-volt 50-cycle alternatingcurrent and the modulated output is amplified by the triode, V5a, and applied to the grid of V4b. This signal and a 6.3-volt 50-cycle signal injected into the cathode of V4b are arranged to be opposing and the relative levels of these two signals determine the magnitude and phase of the output from V4b. Output from this stage is transformer-coupled to the control wind-
ing of a small servomotor (Plessey Type CP88208), the direction of rotation and speed within narrow limits thus being determined by the amplitude of oscillation of T’4a. The motor shaft is fitted with a 100 to 1 step-domn gearbox coupled to the micrometer head of a 0.5-ml. Agla all-glass microsyringe combination containing the neutralizing reagent. Provisions for manually driving the motor in a forward or reverse direction for convenience in setting up, together with microsrt-itches to limit microsyringe travel, are included in the circuit details. I n the AUTO position, to eliminate the possibility of overshoot and reversal in direction of rotation of the microsyringe drive, an electromechanical brake operates, clamping the motor shaft abruptly when the pH control point is reached. This brake also is convenient for altering the average speed in the forward direction. The grid of the thyratron Type 2D21 is fed from the plate of V5u and in the absence of a signal is prevented from conducting by a positive direct current voltage a t the cathode. When V4a oscillates, causing an amplified signal to appear a t the thyratron grid sufficient to overcome the voltage at the cathode, conduction occurs actuating the solenoid which releases the brake. During conduction the thyratron cathode becomes more positive and further charges the 32-pf. condenser until plate current is prevented from flowing. The position of the 32-pf. condenser on the 5000-ohm potentiometer in the cathode circuit determines the cycling rate and period of conduction of this stage and thus controls the duty cycle 3f brake application. Controlling the motor by this “pecking” brake results in stepmise rotation of the motor shaft and causes the neutralizing reagent to be added in exceedingly small discrete amounts which are reduced in volume to 0.005 to 0.1 pl. as the RATE control is decreased. This pulsed addition of reagent is desirable, and particularly so, when working in unbuffered solutions, as there is time betiyeen additions for mixing and pH detection. With the component values shown, the forward VOL. 32, NO. 4, APRll 1960
539
rotation of the motor can be controlled to provide a continuously variable rate of reagent addition from 250 t o 5 pl, per minute. The speed of the microsyringe drive may be manuall>- adjusted during the progress of an experiment-under conditions of widely varying reaction rate, or when an experiment requires exceptional precision. By this means the neutralizing rate may be kept optimal and errors due to the stirring of the solution and time lags in the system are minimized. This type of operation using high p H sensitivity, deadbeat control of the microsyringe, and adjustable control rate with negligible proportional control range, has proved to be the best approach for precise work. RECORDER
The microsyringe combination requires 50 turns for complete travel and to it is coupled a 5 to 1 step-down gear for driving a 10-turn 200-ohm helical potentiometer. This potentiometer provides a voltage proportional to the microsyringe position for deflecting a 0- to 10-mv. pen recorder of the selfbalancing potentiometer type. The 3volt battery used for the slide-wire circuit also supplies the voltage across this potentiometer. The voltage avail-
able to the recorder is adjustable by the RANGE control to enable various amounts of microsyringe travel to occupy a desired chart width and a ZERO OFFSET control is provided to allow the zero chart position t o be set for different starting positions of the microsyringe.
Over a period of 2 years the above assembly has been used by Hugh Lindley of this laboratory for following the kinetics of the reaction of chloracetamide with various mercaptans. The results of this ITork will be reported elsewhere.
PERFORMANCE
ACKNOWLEDGMENT
The long-term stability was measured by recording the output variation of the p H meter which constitutes the only significant source of drift. A 108-ohm resistor was inserted between the input and ground to simulate the internal resistance of the glass electrode so that instability caused by input tube grid current changes 1%-ouldbe included. The output from the cathodes of the twin triode, V3, was connected to the 0 to 10 mv. through a n R C filter and a stable source of 5 mv. was placed in series to allow zero output to correspond to the center of the chart. Figure 3 represents rather better than arerage performance of the instrument; nevertheless, numerous similar records taken a t different times have shown that the drift over 24 hours is unlikely to exceed 0.01 p H unit, while for any 3-hour period a maximum drift of 0.003 pH unit can be expected.
The author thanks IT’. J. Sutherland for the mechanical design and construction of the microsyringe drive unit and J. P. E. Human for the design and construction of the titration vessel and stand. LITERATURE CITED
(1) Duggan, E. L., CHEM.
Stevens, \-. L., Ax.4~.
29,1076 (1957).
(2) Jacobsen, C. J., Leonis, J., Compt. rend. trav. lab. Carlsberg. Ski. chim. 27, 333 (1951). (3) Lingane, J. J.. ASAL. CHEM.21, 497 (1949). (4) Kielands. J. B.. Cannon, AI. D., Ibid., 27, 29 (1955). (5) Scroggie, &I. G., TT7Lreless World 58(1), 14 (1952). (6) Williams) R. C., Ruffin, R. S., Mounter, L. A,, A 4 ~CHEX ~ ~31,. 611 (1959).
RECEIVED for review September 21, 1959. rlccepted January 4, 1960.
Determination of Free Fatty Acids in Fat IRWIN HORNSTEIN, JOHN A. ALFORD, 1. E. ELLIOTT,’ and P. F. CROWE Meat laboratory, Eastern Utilization Research and Development Division, Agricultural Research Service, U. S. Department o f Agriculture, Beltsville, Md.
b Naturally occurring free fatty acids may b e determined in the presence of large amounts of unsaponified fat by adsorbing the free fatty acids on a strong anion base exchange resin, washing the resin free of fat with petroleum ether, and converting the free fatty acids directly on the resin to their methyl esters with anhydrous methanol-hydrochloric acid. The nature and concentration of the fatty acids are determined by gas chromotography.
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in this laboratory are concerned with the nature and concentration of free fatty acids present in cured and cooked meats and with their possible effect on flavor. This work describes a method for the separation of the free fatty acidsfromunsaponified fat by adsorption of the fatty acids EVERAL STUDIES
1
Deceased.
540
ANALYTICAL CHEMISTRY
on a strong anion base exchange resin, converting the fatty acids to theirmethyl esters without prior elution from the adsorbent, and the eventual separation of these esters by gas chromatography on a poly(viny1 acetate) column. The fatty acids present are quantitatively estimated, using n-heptadecanoic acid as a n internal standard. METHOD
Reagents. Amberlite IRA-400 (Rohm & Haas Co., Philadelphia, Pa.). Heptadecanoic acid (Eastman Kodak, Rochester, K. Y.) recrystallized twice from methanol. Methanol-hydrochloric acid, anhydrous, 5 to 10% acid. Poly(viny1 acetate), grade AYAC (Bakelite Division. Union Carbide Corp., S e w York). Chromosorb R, 30- to 60- mesh (Celite Division, Johns-Manville, New York). Fatty acid methyl esters (Hormel Foundation, Austin, l h n . ) .
Apparatus. GC-2 gas chroniatograph, equipped with four-filament thermal conductivity detector (Beckman Instrument Co., Fullerton, Calif.). Recorder, 1 my. (Brown Instrument Division. Minnemolis-Honeyjvell, . Philadelphia, Pa.). Pretreatment of Resin. Ten grams of Amberlite IRA400 are stirred n i t h 25 ml. of 1 S sodium hydroxide for 5 minutes. The resin is allowed to settle and the supernatant liquid is discarded. The resin is successively washed with several portions of distilled water to remove free alkali, then with three 25-ml. portions of anhydrous ethyl alcohol to remove water, and finally with three 25-ml. portions of petroleum ether to displace the ethyl alcohol. Separation of Fatty Acids from Fat. If fat emulsions are analyzed, a uolume of emulsion containing 0.1 t o 1.0 gram of f a t is diluted in a 125-ml. separatory funnel with an equal volume of ethyl alcohol and made acid to bromophenol blue with several drops of 11V sulfuric acid. The mixture is extracted mith two 20-ml. portions of petroleum ~
