V O L U M E 2 7 , NO. 1, J A N U A R Y 1 9 5 5
123
Titration of Phosphate in Standards Sample COmPn., NO.of Found, A v . Precision,
Table I. UOr+7 P o d - - - 0.24 0.24 0.24 0.28
0.277 0.139 0.208 0.416
sod-...
0.6 0.5 0.5
XOS3.2 3.2 3.2 3.3
Detns. A4 of Poi--13 7 8 9
0.282 0.145 0.202 0.408
electrodes must be freed of silver bromide. Use hydrogen iodide (specific gravity, l.?), followed by acetone, then rinse with water. CALCULATIONS
M 10.013 0.004 0.005 0.016
Grams per liter of orthophosphate =
i X t X 6 0 X
96,500 103 - X PO4
S
PROCEDURE
To exchange metallic cations for hydrogen ion: Pipette sample onto a filter holding 1 ml. of resin covered with carbon dioxide-free water. Hold in the filter 5 to 7 minutes. The sample must be 1 to 2N mineral acid. It should contain 0.005 to 0.05 millimole of phosphate. Slowly pass treated sample into titration vessel. Rinse with 5 ml. of carbon dioxide-free water. Rinse with 1% hydrochloric acid if much iron or aluminum is present to prevent precipitation of basic phosphates. To provide electrolyte for the base generation: Add 500 p l . of 500 grams per liter of potassium bromide solution to the vessel. .4dd 100 pl. 0.1% methyl red indicator. To preneutralie the strong acids: -4dd 0.LV sodium hydroxide dropm7ise until the methyl red end point is passed. Add 0.01N nitric or hydrochloric acid until the end point is reached, then about 10 to 25 pl. more. The sodium hydroxide must be carbon dioxide-free. Prkpare from 50% sodium hydroxide and carbon dioxide-free water daily. To achieve precise adjustment of strong acid neutralization: insert electrodes and titrate to the methyl red end point. A buffer solution with indicator can be used to match the end point. See (8). Titration step: Reset the clock. 4 d d 100 pl. of 0.1% thymol blue indicator. Titrate to the blue end point. The end point can be matched as indicated above. At the completion of the titration: Record the time. Clean the assembly. Clean the electrodes. Replace the resin. The
59.054
i X t S
i = current, ma.
t = time, minutes
S
=
sample size, pl. RESULTS
Table I gives the results of a series of runs made on samples containing uranium, orthophosphate, sulfate, and nitratrs. The precision is the standard deviation of a single value. Sample size was 25 pl. The samples were made up from aliquots of standardized solutions to give the indicated solution composition. The phosphate solution was standardized by precipitation as ammonium phosphomolybdate and titration with a base. These results show that the method is practical on the micro scale and that orthophosphate determination in the presence of uranium is feasible. LITERATURE CITED
(1) Carson, W. N., Jr., and KO,Roy, ANAL.CHEM.,23, 1019 (1951). (2) Helrich, K., and Rieman, W., ISD.ENG.CHEM.,ANAL.ED., 19, 651 (1947). RECEIVED for review June 22, 1954. Accepted September 21, 1954 Presented at the Northwest Regional Meeting of the AMERTCANC H F U I C A L SOCIETY,B.ichhnd, Wash., June 11 and 12, 1954.
Automatic Titrating and Recording Apparatus For Microbiological Assays CHARLES H. EADES, JR., B. P. MCKAY, W. E. ROMANS,
and
G. P. RUFFIN
Department o f Biochemistry, University o f Tennessee, Memphis, Tenn.
A n automatic titrating and recording apparatus for microbiological assat s has been developed to facilitate the handling of the vast numbers of titrations that must be performed in running routine assays. This instrument can automatically titrate and record results on 225 samples consecutively with a high degree of precision (less than 1% standard error) and an accuracy of better than 97%. The precision and accuracy of the instrument are much better than the limits of error normally experienced in the microbiologicaI procedures themselves. BJ suitable modification, the principles involved can be adapted to assays of vitamins and amino acids, acid base titrations, oxidation and reduction titrations; and similar procedures.
I
N ROUTINE analytical procedures the trend toward time-
saving, automatic instruments with recording attachments has gained momentum in recent years. One of the simple operations which has been notably improved with respect to the time factor is volumetric titrations. Great strides have been taken to remove the buret from the hands of the chemist and technician and place it in the steel clasp of an automatic titrator. Various techniques are used for adding titrant and measuring its volume (1, I , 5-8, 10, If). Lingane (8) pointed out that the
motor-driven, screw-pushed syringe could be timed to mcnsure the volume delivered. The Coleman and Beckman titrat,ors use burets; the Precision t,itratoruses a syringe; whereas Cannon in his dispenser-titrat,or (International Instrument Co., Los Angles, Calif.) uses a timed rate of flow of liquid through a calibrated orifice, the liquid being under a controlled constant head of pressure. Hox-ever, none of the present devices incorporntw a mechanism for automatically recording the titration value, noi' provides for mechanically and automatically changing the sample. APPARATUS
The instrument (Figure 1) is an automatic titrating and recording apparatus designed primarily for use in microbiological assays but offering, through suitable modifications, several other applications. The apparatus consists of: a pH-sensitive electronic controller, a glass-calomel electrode assembly, a sample carriage and automatic sample changer, a solenoid-operated polystyrene valve (9), an automatic recording interval timer, and e!ectronic manometers for maintenance of constant pressure head (of nitrogen) on the alkali supply for titration, and the nitrogen gas supply for stirring samples during titration. The pH-sensitive electronic controller was designed and built to include the following features: Sensitivity to inputs of 2 to 3 mv. (less than 0.1 pH unit) with input electrode currents of the order of IO-" ampere at normal
ANALYTICAL CHEMISTRY
124
pH equivalent potentials-i.e., 5.8 mv. per pH unit from n glasscalomel electrode svstem. Oneration with dower from 117 volts alternating current.
{“off” to “on” action heidg achicveb with B change of DISlittle as 5.8 mv.). Provision for output control action of: a solenoid-operated polystyrene titration valve to open when the sample pH iu below that set in the controller and to close when the desired oH is reached; an automatic sample changer which control: the sample change action following completion of a titration; and the starting of an automatic recording interval timer when the t,itratian valve is open and the stopping of the timer when the valve is closed.
+*so
Figure 2.
Schematic Diagram of pH-Sensitive E l e c t r i d Cont.oller
KCI BRIDGE TO
Figure 1. The Apparatus Left. Rishf.
Front view
Back
view
A sehematio diagram of this controller is shown in Ir’igurc2. Gapns and Pool ( 4 ) and Wu and Rainwater (le) have shown that it is not difficult to select standard Type 954 tubes for use in electrometer service. The authors’ results confirm their findings with regard to the use of these tubes in measuring currents as low as lU-“ ampere. Therefore, the 954 was chosen for the input tube and wired for electrometer service, using the suppreasor grid as the control element, and tying the control and screen grids together to a positive potential. The 954 tuhe is followed by a balanced direct current amplifier utilizing a 12AX7; B 12AU7 operates a plate current relay for output control. Eleetronically regulated direct current is used to provide filament current as well as high voltage and bias potentials. The Virtoreen 5800 tube can serve as well as the 954 in a similar circuit. The Glass-Calomel Electrode Assembly is shown in Figure 3.
This assembly include8 a glass electrode (prepxed by Milton
T. Bush, Department of Pharmacology, Vmderbilt University, Nashville, Tenn.) and three Dlastic tubes. The dass electrode makes junction with the calomel electrode by means of a satu-
rated potassium chloride bridge carried in one of the 1-mm. diameter plastic tubes. Another of the tubes conveys the alkali from the polystyrene valve to t h e sample being titrated: the
Figure 3.
Glass-Calomel Electrode Assembly
third tuhe admits bubbles of nitrogen gas in a continuous stream to provide stirring of the sample during the titration. This assemhlv. of neeessitv. is small in its cros*sectional men in order t6;t the valu& i t will disolace within t h e samole tube may he a t a minimum. Actually,the volume of titrant %hatcan he added is of the order of 10 to 15 ml. in 18 X 150 mm. culture tubes containing 2 to 5 ml. of sample. Obviously, the ooncentration of the titrant must be adiusted to accommodate tho sample and the volume of the sample tuhe. The authors’ experience hits involGed the use of 0.04N sodium hydroxide with a &nd. culture or 0.02N sodium hydroxide with a Zml. culture. The automatic sample changer system and sample tube earriage are shown in Figures 4 and 5, respectively.
A supply of 225 test tubes to b e titrated may h e loaded in the test tube carriage. This consists of a rectangular cart divided by polystyrene separators into 15 rows capable of holding 15 tubes each. Test tubes from the carriage are presented to a rotating turnstile a t the top of t h e changer mechanism (Figure 6 ) , by weigh& actuated sliding push plates in each. row of the carriage (Figure
V O L U M E 7.1, NO. I, J A N U A R Y 1 9 5 s 7). A s the pH-mnsitive controller calls for a change, one tube a t a time is removed from the carriage for each quarter turn of the turnstile. A tube moved through 180‘ of the turnstile is presented to a rack and pinion-lifting mechanism, and raised into the electrode assembly. The lower limit of the lifting rack’s travel actuates a switch for reversing the motor drive. As the lifting rackrises, aGeneva motion is engaged to give the turnstile another quarter turn. When the last test tube from one row of the carriage is delivered, the push pl,ate of that row engage8 a trip lever, releasing the test tube carriage. This allows the carriage to move by weieht actuation to B new row of tubes. The action is reDeated
125
when open, permits alklkali to flow from the constant pressurehead reservoir to the sample. The flow is a t a eonstttnt rate and is timed by the interval timer. Whenever the valve is closed the timer is also stopped. The use of this type of valve, rather than the pinching of a rubber tube, eliminates lag in the beginning of flow and “squirt” of alkali when flow is cut off. The plastic tubing which conveys the alkali from the valve to the sample is not elastic enough to permit “ballooning.” The use of this valve
. . . ~ . ~ ~ ~ ~ ~ from the last row into a stop pisition.
The polystyrene valve has been described (9). This valve,
Fizure 6 .
A u t o m a t i c Sample Changer System
Figure 4.
Figure 5 .
S a m p l e Tube Carriage
Sample Changer T Mechanism
affords the system a constant volume, and a6 a result, the small error due to the lag in flow is eliminated, The solenoid which opens and closes the valve is operated by the pH-sensitive controller in response to the pH sensed by the electrode assembly. The automatic recording interval timer (Simplex Time Recorder Co., Gardner, Mass.) is a commercially available instrument modified by installing auxiliary relays to m a k e t h e timer start and stop with the opening and closing of the polystyrene valve and to print on the recording tape the total time the valve was open. Also, the reset to aero time is automatically performed after the titration time. The electric manometers used in controlling the pressure bead of nitrogen on the alkali and in regulating the flow of nitrogen to the sample for stirring are similar t o those used by Cannon. The principle of the electronic manometer is well known, involving simply a mercury manometer, one side of which is connected to the gas pressure system, the other side m k i n g contact vith a carbon electrode. The elpctrical contact makes or breaks an electronic circuit that operates a release valve in the gas pressure system. In operation, the pressure is maintained a t a eonstant value within a few tenths of a millimeter of mercury. This constant pressure on the alkali reservoir permits the expression of the flow of alkali in terms of time, the volume being directly proportional to time under constant rate of flow. Thus, only time of flow must be recorded. Actual volumes of alkali
126
ANALYTICAL CHEMISTRY
delivered o m he obtained by knowing the rate of flow in milliliters perminute, hut this is unnecessary in microhiologioal assays. Calibration of the instrument is accomplished daily by timing the flow of a definite quantity of titrant in order that the desired volume to be delivered per unit of time may he obtained.
Table I. P r e d s i o n of Titrations
12.6
1
24.0 30.4
2 3 4
OPERATION
35.5 41.7
5
The principles of operation and sequence of events are given in Figure 8 in block disgram.
6 7 3 9
46.7
The sample carriage is loaded with samoles to he titrated in the order in which tLe titration is to proo'eed. The carriage is shifted until the first tube i8 directly in contact with the Geneva. controlled turnstile. The tuhes m e. ~... moved t.n ~. ~ ~ ..._ . . t.he ...- d m t r o d assembly hy suocemive 9O0 turns of the turnstile. The first tube is raised by the lifting mechanism, SO that the sample comes in contact with the glass electrode assembly. If the pH is below 7 (assuming that 7 is the value present on the electronic controller), the electronic controller causes the polystyrene valve to open (permitting alkali to flow) and the recording interval timer to start. When the pH reaches 7, the controller stops the flow
10 11 12
72.0
~
53.2 59.1 66.4
0.1 0.1
0.1 0.0 0.1 0.2 0.2 0.2
78.1 35.5
~~
hack into the tuenstile. The delay period may he vaned, d e p e d ing on time required for the sample to he mixed to a uniform pH throughout. Five to 10 seconds have been found stttisfactory for this work. After the sample is lowered, the time of titrant flow in units of seconds and tenths of seconds is minted on a tane.
~
~
Table 11. Aocuraoy of T i t r a t i o n s Millienniual~ntanf Arid -
coup* of 4
retia1
200 300 350 400 500 GOO
r
~~
trodes. Again, if the pH is gelow 7, the titritition proceeds automatically and is repeated until all samples in the raok are titrated.
rate of alkali flow. PRECISION AND ACCURACY
. . . .. operation, the instrument yieldB reproducible results
Figure 7.
Weight-Actuated Posh Plates Moving T u b e s i n t o T u r n s t i l e
The titrated sample is disposed of dawn a chute into a contamer of washing solution. If the pH of any sample is %hovethat set on the pH controller as the desired end point, the sample will remain around the electrodes only the preset time (5 to 10 sew onds) and be ohmged. A recording of zero will he made for such
place more samples in the machine and start the &c& over. The time consumed in titrating a Sam le is governed by the rate of the BOW of the alkali throughe!t Leur-Lok needle on the outlet side of the polystyrene valve, which is controlled by the pressure head on the alkali and the size opening of the needle. The volume of titrant to be added is governed by the amount of acid to be titrated and the concentration of alkali used a8 the titrant. The alkali flow under the authors' conditions of operation gave approximately 10 ml. per minute. One minute, or
In well within the limits of desired accuracy obtainable in routine assays. Table I summarizes the results of 144 titrations. The standard error of precision does not exceed 1%and in most cases is less than 0.5%. Table I1 shows the data. from a recovery experiment to determine the accuracy that could he expected with the use of the instrument. The accuracy is well within the limits of error inherent in the microbiological assay techniques. Differences observed in this recovery experiment may actually he reflectiiig, in part, a certain amount of error involved in pipee ting the 68hmples. The ins trument can he modified for other routine amsays by -:-~~1~~~~~~ -~ .,. . . , , . xechnique , relatively almnp~e cnltngea. m y ma-vase utrauon can he used, whether it he for microbiological amay of vitamins, amino acids, or other essential nutrients or for the acidity from 8ome other source. By using a different set of electrodes. oxid*-
.~
V O L U M E 2 7 , NO. 1, J A N U A R Y 1 9 5 5 and receiving the samples into a collecting rack instead of disposing of the tubes. There are many possibilities in which the sample changer and principles involved in this instrument could be incorporated to reduce the time which is used in routine analytical procedures. ACKNOWLEDGMENT
This instrument was built for use in work supported in part by a grant from the American Cancer Society as recommended by the National Research Council through the Committee on Growth. Part of the cost of the instrument was defrayed by the University of Tennessee Reserve for Research. LITERATURE CITED
(1) Austin, R. R., Am. Gas Assoc. Proc., 31, 505 (1949).
(2) Dunn, E. B., Melpolder, F. W., Taylor, R. C., and Young, W. S.,Proc. M i d - Y e a r Meeting Am. Petroleum Inst., JOMIII, 45 (1950).
127 Eades, C . If.,J r . , AIcKay, €3. P., and Romans, W. E., Federation Proc., 11, 205 (1952). Gapus, G . H., and Pool, M. I., Review of Scientific Instruments, 8, 197 (1937). Hawes, R . C., Strickler, A., and Petterson, T. H., Elec. Mjg., 4 7 , 76, 212 (1951). Jacobsen, C. F., and Lkonis, JosB, Compt. rend. trav. lab. Carlsberg, SBr. chim., 27, 333 (1951). Juliard, A , , and Cakenberghe, J. van, A n a l . Chim. Acta, 2 , 542 (1948).
Lingane, J. J., - ~ A L CHEM., . 20, 285 (1948). McKay, B. P., and Eades, C. H., Jr., Ibid., 27, 123 (1950). Shaffer, P. A,, Jr., Briglio, A,, Jr., and Brockman, J. -4., Jr., Ibid., 2 0 , 1008 (1948). Wise, E. X , , Ibid., 23, 1479 (1951). Wu. C. S.,and Rainwater, James, U. S. Atomic Energy Commission, A E C Document, MDDC-1671 (1944). R E C E I V Efor D review December 31, 1952. Accepted September 29, 1954. A preliminary report ( 5 ) of a n earlier model of this apparatus mas made before t h e Federation Meetings in New York, April 14 t o 18, 1852.
Paper Chromatography of Cobalt(lll), Coppedll), and
Nickel(l1) Acetylacetonates E U G E N E W. BERG and J A C O B E. STRASSNER Coates Chemical Laboratories, Louisiana State University, Baton Rouge, f a .
Acetylacetone, one of the simplest fi-diketones, was selected for study because its availability and low cost make it a desirable reagent for chromatographic separations. Cobalt(III), copper(II), and nickel(I1) acetylacetonates were separated using mixtures of cyclohexane, dioxane, and methanol as the developing solvent. A mixture of 847” cyclohexane, 10% dioxane, and 670 methanol gave good separations. The mean R J values were reproducible to A0.02. Solubilities of the metal chelates were measured in the developing solvents. Relative adsorption affinities were obtained from dielectric constant measurements. A qualitative relation was found to exist between the relative sequence of R / values and the relative solubility and adsorption affinity (polarization) of the metal chelates.
A
LTHOUGH a large number of metal p-diketone complexes are well known (10, 16), these complexes have not been extensively used for chromatographic separations. Increasing interest in the use of chelating agents for inorganic chromatographic separations has been shown in the appearance of a number of recent articles (1-7, 9, 11-15). Acetylacetone, one of the simplest 8-diketones, was selected for this study because its availability and low cost make it a desirable reagent. Burstall et al. ( 5 ) and Pollard et al. (11) have used solvent systems containing acetylacetone in the chromatographic separation of some inorganic ions. +4number of factors may influence this type of separation-namely, the rate of chelate formation, the presence of excess chelating agent, and the possibility of chelate hydrolysis in the presence of strong acids. In order to avoid these factors the authors have preferred to spot the paper with the preformed metal acetylacetonates and to develop the chromatogram with a solvent mixture in which the chelates are stable. REAGENTS
Acetylacetone (Matheson Co.), redistilled. Methanol, C.P. Cyclohexane (practical grade), redistilled.
Dioxane (technical grade), redistilled. ilqueous solutions, 1%, of the metal ions prepared from: C.P. cobalt(I1) nitrate, C.P. copper(I1) nitrate, and C.P. nickel(I1) nitrate. Solution of dimethylglyoxime in ethyl alcohol, 1%. Solution of dithio-oxamide in ethyl alcohol, 0.3%. PROCEDURE
Acetylacetonates of cobalt(III), copper(II), and nickel(I1) were prepared by shaking 1% solutions of the ions, adjusted approximately to a pH of 7 with sodium acetate, with acetylacetone. The nickel acetylacetonate was extracted with n-butyl alcohol and shaken with distilled water to remove nickel ions. The copper and cobalt acetylacetonates were extracted with methyl isopropyl ketone and shaken with water to remove any ions. In the extraction of the cobalt acetylacetonate, the methyl isopropyl ketone was kept in contact with the original solution until the ketone layer developed a dark green color. This chelate corresponded to the cobalt(II1) acetylacetonate described by Gach (8). The oxidation of the cobalt(I1) to cobalt(II1) was probably due to impurities in the methyl isopropyl ketone. Final colors of the extracted and washed solutions of cobalt, copper, and nickel acetylacetonates were dark green, blue-green and yellow-green, respectively. Hydrometer cylinders, 43 cm. tall and 7 cm. in diameter, served as chromatographic chambers. A part of the cylinder was lined with filter paper soaked with the solvent in order to saturate the chamber more efficiently. Twelve hours were then alloxed for complete saturation of the chamber. Whatman No. 1 filter paper strips 2.5 inches wide were spotted with the extracted solutions of the metal acetylacetonates and dried in air for 1 hour. The strips were then placed in the chamber saturated with vapor and equilibrated for 1 hour before immersion in the solvent. The chromatograms were developed completely in 3 hours, the solvent front having ascended approximately 25 cm. Preliminary u ork indicated that methanol and cyclohexane would be desirable solvents for this study. The cobalt(III), copper(II), and nickel(I1) acetylacetonates all moved with rather large R f values in methanol, whereas only the cobalt acetylacetonate moved in cyclohexane. An appreciable amount of methanol was not soluble in cyclohexane; therefore, a third component, dioxane, was used to form a completely miscible solvent.
