shown in Table I. The salt is dissolved in a nitric-sulfuric acid medium with the addition of a little boric acid and the nitric acid is later fumed away. Since the only possible cationic interference of U(IV) has been converted to V(VI) during the dissolution and no anionic interferences are present, the titration for zirconium can be made directly after the removal of the nitrate. The titration of 5 mg. of zirconium takes about 5 minutes using the equipment and reagents that have been described. The entire procedure including the makeup of the solution, deoxygenating, and the titration takes about 20 minutes. The 1.5% precision for de-
termining zirconium a t this level is within the limitations imposed upon this analysis. If necessary, the precision can be improved by taking larger samples or, to a limited extent, using more dilute titrant. The reproducibility in taking samples seems t o be the chief cause for the lower precision of the analysis in the hot cell.
(4) Fri+bz, J. S., Johnson, Marlene, Ibid., 27, 1653 (1955). ( 5 ) Go ldstein, Gerald, hIanning, D. L., Zittel, H. E., Ibid., 35, 17 (1963). ( f j) Koltoff. I. 11..Liberti. A,.’ J . Am. Chem. Soc. 70, 1885 (1948). (7) Lundell, G. E. F., Hoffman, J. I., “Outlines of Methods of Chemical Analysis,” p. 117, Wiley, Kew York, 14.18
(8)”6i$’on, E. C., Elving, P. J., ANAL. CHEW26, 1747 (1954).
LITERATURE CITED
. .
C H E M . ’ 1206 ~ ~ , (1954):
RECEIVEDfor review May 20, 1963. Accepted July 12, 1963. Division of Analytical Chemistry, 144th Meeting, ACS, Los Angeles, Calif., April 1963. Oak Ridge Xational Laboratory is operated by Union Carbide Corp. for the U. S. Atomic Energy Commission.
Hydrogen Cell Assembly for Standard Electromotive Force Measurements PETER G. SIBBALD’ and GEORGE MATSUYAMA Scientific and Process Instruments Division, Beckman Instruments, Inc., Fullerton, Calif.
b A simple hydrogen cell assembly for the calibration of buffer solutions, glass electrodes, and reference electrodes is described. The design of the cell permits such calibrations to b e made in 20 to 30 minutes with a maximum standard deviation of *0.6 mv. (kO.01 pH).
M
p H instrumentation now permits refinement of the readout to 0.001 pH. Such a high degree of precision, for example, is desired in the measurement of the p H of blood and other body fluids where small changes of p H may have significance. The importance of high precision p H measurements has further been recognized by the assignment of revised standard p H values, given to three decimal places, for the seven rational Bureau of Standards (NBS) reference solutions which define the p H scale ( 5 ) . For very accurate p H measurements, frequent calibrations of a p H cell containing a glass electrode are necessary. This requires a reliable reference buffer, the p H of which has been determined previously with great accuracy. Second, the p H response of the glass electrode must be checked, preferably in the p H range in which it is to be used. It is known that even the best modern electrodes show small variations in response t o p H with time. Third, the ODERN
ELECTRONIC
1 Present address, Research and Development Division, Richfield Oil Corp., Anaheim, Calif.
1718
e
ANALYTICAL CHEMISTRY
stability of the reference electrode with time must be verified. This paper describes a simple hydrogen cell assembly in which standardization of buffers, glass electrodes, and reference electrodes can be made accurately and with relative ease. The hydrogen cell assembly is similar in principle to that described by Bates, Pinching, and Smith (6), modified to obtain more rapid thermal and chemical equilibrium and designed to fit the Beckman Model 28505 Thermomatic constant temperature block. The standardization of a buffer solution against a NBS primary standard is accomplished by measuring the e.m.f of cells with liquid junctions of the following type : Pt; HZ(gas), Primary Standard/Saturated KCl/Buffer X, Hz (gas); Pt The design of the cell assembly permits the formation of physically well defined liquid junctions. The residual liquid junction potential-Le., the difference of the two liquid junction potentials formed between the saturated potassium chloride solution and the primary standard buffer on one side and the test solution on the other-manifests itself as an indeterminate error in the derived pI-1. I n the ideal case, where the liquid junction potentials are numerically identical and opposite in sign, the residual liquid junction potential equals zero. It is, therefore, of great importance to be able to produce and reproduce closely identical liquid junctions at both sides of the salt bridge. h minimum of two buffer standards is needed for the calibration of a glass elec-
trode. Its p H response can be determined by measuring the e.m.f. of cells without liquid junctions of the type Pt, 1 x 2 (gas), Buffer Soln./Glass Electrode
Similarly, the stability of a reference electrode can be verified by measuring the e.m.f. of the cells: PtJ Hz (gas),
Buffer Soln./Reference Electrode EXPERIMENTAL
Apparatus. A sketch of the hydrogen cell assembly is given in Figure 1. It consists of two electrode compartments, a and a’, connected by a saturated KCl bridge, b. Each electrode compartment holds approximately 3 ml. of solution. The liquid junctions are formed at c and e’ and protected from turbulence caused by the bubbling hydrogen gas, by stainless steel balls, d and d’, that imperfectly separate the standard and test solutions from the KC1 solution. The bottom of each electrode compartment is ground a t c and e’ to match the contour of the stainless steel balls. Compartment e holds a stopper, f, that prevents displacement and mixing of the saturated KC1 solution in the capillary bridge with the solutions of the two electrode compartments. Stopper j is ground in place at g. The presence of saturated KC1 in this area maintains electrical continuity. Purified hydrogen gas enters the system through h and h’; i and i’ are platinum electrodes, and j and j ’ are outlets for hydrogen gas. Copper mire leads covered with phone tips, k and k’, are soldered t o the platinum electrodes for electrical connections.
743,
170 Average pH 1 6 7 9 Stand Dev i o 0 0 5
169
Y
168
b
167 a.NBS Standard Sample 189
j'
I
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1
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9
I 10
1
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CELL NUMBER
CELL NUMBER
Average pH 4 025 Stand Dev t o 0 0 9
Figure 1. a, a'
b c c'
d , d' e
f h, h' i, i' j , i'
k, k'
Hydrogen cell assembly
electrode compartments capillary bridge liquid junction area stainless steel balls compartment for saturated KCI stopper, grouid in place a t g inlets for hydtogen gas platinized plotinum electrodes outlets for hydrogen gas phone tips
4 01
ma7 Packaged p h
b -8eckman Packaged pH 4 01
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CELL NUMBER
6.87
5
9 18
Average pH 6.858 Stand Dev t0.006
Average pH 10 005 Stand Dev t o 0 0 7
10 02
10 01
10 00 c.Beckman Packaged pH 6 8 7
6 84
The preparation of platinizing and palladizing solutions and the electroplating of the platinum electrodes were carried out in the manner described by Bates (9). The plattld electrodes were stored in distilled witer when not in use. Before entering the cell assembly, the hydrogen gas was passed through a Deoxo purifier (Engelhard Industries, Inc.), a safety flash, a wash bottle containing distilled water, and another safety flask, respectively. Electromotive force measurements were made with the Beckman Model 1019 research p H mei,er. All measurements were carried out a t 25" C. ==I 0.Ol0 c. Reagents. Reagent grade potassium chloride and N13S standard samples were used for the salt bridge and primary standard buffer solutions, respectively. All solutions were prepared with doubly distilled water. Procedure. A. Standardization of Buffer. Thoroughlj clean and dry the cell vessel. Carefully introduce saturated potassium chloride solution into compartment e until the capillary bridge is filled to c and e'. Insert stopper f into tube e and seat it in ground joint g. Seat the stainless steel balls d and d' in place. Make sure no air bubbles are trapped in the salt bridge. Remove any excess saturated potassium chloride solution in the electrode compartmerts a and a' with absorbent tissue. Fill the electrode compartments a and a' with KBS primary standard buffer and the buffer solution to be standardized, respectively, to a level slightly below outlets j and i'. Place the cell vemel in a Thermo-
p-Beckrnan Solution pH 1 0 0 0
9 99
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7 02 Average pH 7 003
12 47
h Beckman Packaged pH 12 4 5
d . Beckman Solution pH 7 GO
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Figure 2. Performance of hydrogen electrode cell assembly in standardization of buffers a t 25" C.
matic constant temperature block or any other constant temperature bath. Rinse the hydrogen electrodes h, i, h', and i' with distilled water and carefully dry the glass parts with a clean soft tissue. Insert the electrodes firmly in place. -Make gas and electrical connections. Bubble purified hydrogen into the assembly a t a rate of about three bubbles per second. Allow the system 20 to 30 minutes to reach thermal and chemical equilibrium. Measure the e.m.f. of the cell. Compute the pH difference of the two buffers by dividing the measured e.m.f. by the appropriate Nernst coefficient (4). B. Standardization of Glass Electrode. Clean and dry the cell vessel. Fill compartments a, a', and e and capillary bridge b half way with a KBS primary standard buffer or pre-
viously calibrated buffer. Insert stopper e to seat in ground joint g . Fill compartment a with buffer to a level slightly below outlet j. Place the cell assembly in a constant temperature bath. Rinse the hydrogen electrode h, i with distilled water and carefully dry the glass parts. Insert the electrode in compartment a. Make gas and electrical connections. Place the glass electrode to be standardized into compartment a' in such a position that it does not rest on the bottom of a'. Allow 20 to 30 minutes for the system to reach equilibrium, then measure the e.m.f. Calculate the standard potential of the glass electrode using the equation E'GE 2 8 . X H E = EGEus. HE Corr. where E'GE us. "E = standard PO-
f in compartment
+
VOL. 35, NO. 1 1 , OCTOBER 1963
a
1719
.-.-.- p H 4.01,
E O A "
pH 6.07. ED*" p H 9.18.
EOA"
=
C 6 5 1 . 5 2 m v , Stand. D c v . t 6 5 1 . 9 7 m v , Stand. D s v .
= =
20.54 mv
= =
t b 5 2 . 3 5 m y , Stend. De".
=
20.37 mv
2 0 . 2 4 mv
EaAv
653 Y
0
w
652
651
-1
2
3
4
5
8
-
"..
NHE =
ERE..s
HE
+ Corr. 2.3026 RT F pH
in which Eo,, e,. "E = standard POtential of the reference electrode, E R B ~ , . E=E measured potential of the reference electrode, Corr. = hy-
242
u u 1 2 3 4 5 8 9 101112 TIME (DAYS) Figure 4. Eo of No. 39071 saturated catomel reference electrode measured with standard hydrogen cell assembly
The results of the standardizations of commercially available Beckman buffers against an NBS equimolar phosphate buffer (pH. = 6.865 a t 25' C.) are presented in Figure 2. Also included in Figure 2 are results obtained in standardizing a sample of NBS tetroxalate buffer. Ten successive measurements of each buffer were made on 10 different cells. Maximum standard deviat,ions of 0.006 pH units were observed in the intermediate pH range. I n the high acidity and high alkalinity regions, higher maximum standard deviations may be anticipated. The reproducibility of the e.m.f. measurements in these regions is not as good as in the intermediate pH range. This probably results from greater varia-
1
tions in the residual liquid junction potential due to the abnormal mobilities of hydrogen and hydroxide ions. The exact nature of liquid junction potentials is not known, although this has been the subject of many studies through the years. An extensive discussion of the characteristics of several types of liquid junction is given by Guggenheim (7). Bates (3) also gives an excellent summary on this subject. An additional problem of a different nature enters into the picture in the standardization of a pH 4.01 phthalate buffer solution. Here, the platinum black coating of the hydrogen electrode acts as an active catalyst in the hydrogenation of potassium hydrogen phthalate and stable potentials cannot be obtained. As recommended in the literature, palladium black, a less active catalyst, was used for these measurements. However, the difficulty is believed to be not completely remedied. The Beckman No. 41263 glass elec-
E"A" Stond.Dev.
= t243.33 = 2 0.14
mv
mv
t i ! M E (i?r.YS)
Figure 5. assembly
1720
Modified hydrogen cell
ANALYTICAL CHEMISTRY
?0.25 m v
.
RESULTS AND DISCUSSION
242
0
243.
drogen pressure correction factor ( I ) , 2.3026 RT/F = Nernst coefficient (4), and pH = pH of the buffer solution used.
244
Stond. De".
W
9 1 0 1 1 1 2
Figure 3. Eo of No. 41 2 6 3 glass electrode measured in pH 4.01, pH 6.87, and pH 9.1 8 buffers
E'RE
t 2 4 2 . 9 0 mv
244.
TIME (DAYS)
tential of the glass electrode, E G E *,. HE = measured cell potential, and Corr. = hydrogen pressure correction factor (1). C. Standardization of Reference Electrode. The same sequence of operation as in B is followed here. Calculate the standard potential of the reference electrode using the equation
=
Figure 6. E" of No. 39071 saturated calomel reference electrode measured with modified hydrogen cell assembly
trode is recommended for use with the pH meter. The variatisn with time and buffer solution of the standard potential (EO) of such a n electrode is shown in Figure 3. Ten successive measurements in pH 4.01, p H e.87, and pH 9.18 buffer solutions were miide over a period of 2 weeks. Again, the measurements in pH 4.01 buffer show the largest standard deviation although palladized electrodes were used. Moreover, a slightly lower value for the Eo was observed. Similarly, 10 successive measurements were made on the KO. 39071 saturated calomel reference electrode. Figure 4 shows the stability of such an electrode in p H 6.87 buffer over a time interval of 2 weeks. The standard
deviation obtained verifies the good reproducibility and stability of the carborundum frit junction of the reference electrode. The standard potential of the reference electrode could not be determined accurately in the hydrogen electrode cell assembly since the electrode compartment was too short to accommodate the entire electrode. This caused temperature gradients to exist in the reference electrode internal and its electrolyte. To overcome this diificulty, a modified cell assembly as shown in Figure 5 was used. This cell assembly was immersed in a deeper constant temperature bath to keep the entire electrode at the desired temperature. The results
obtained with this modified cell assembly are presented in Figure 6. The average E ” value is 0.43 mv. higher than the corresponding potential of Figure 4. LITERATURE CITED
(1) Bates, R. G., “Electrometric pH Determinations,” p. 160, Wiley, New York,
1954.
(2) Bates, R. G., Ibid., p. 166. (3) Bates, R. G., Ibid., p. 190. (4) Bates, It. G., Ibid., p. 313.
(5) Bates, R. G., J. Research N B S 66A,
179 (1962). (6) Bates, R. G., Pinching, G. D., Smith, E. R., Ibid.,. 45, 418 (1950). (7) Guggenheim, E. A,, J. Am. Chem. Soc. 5 2 , 1315 (1930). RECEIVEDfor review March 25, 1963. Accepted July 11, 1963.
r
Sensitive Method for the Determination ot Submicrogram Quantities of Manganese and its Application to Human Serum ALBERT0 A. FERNANDEZ, CHARLES SOBEL, and S. L. JACOBS Bio-Science laboratories, 7 2330 Santa Monica Blvd., Los Angeles 25, Calif.
b Oxidation of leucomalachite green with periodate in the presence of manganese i s a catalytic type reaction which has been used for the quantitation of submicrogrcim amounts of manganese. There is not, however, linear proportionality between the absorbance at any given time during this reaction and the content of manganese in the system. Experimental data indicated that this reaction i s complicated b y a sutlsequent reaction which destroys the dye. Oxidation of malachite green with periodate in the presence of manganese follows a first order reaction catalyzed b y manganese. This subsequent reaction explains the behavior of the leucomalachite green oxidation curves. A method for determination of manganese in the milliniicrograrn range based on the oxidation of malachite green i s proposed and applied to human serum. The normal level of serum manganese was found to be 0.36 to 0.90 rnHg. per rnl.
T
HE O C C U R R E N C E of minute amounts of manganese in certain biological materials has prompted the development of a variety of procedures (4-10, 12). The most sensitive of these employs neutron activation analysis, a procedure not practical for most analytical laboratories (2,R, IO). An e vcellent review by Cotzias (S) provides a comprehensive background for this su‘sject. The more sensitive of the chemical
methods involve oxidation of colorless Ieucomalachite green (LMG) to the dye, malachite green (MG). Manganous ions are converted by periodate to a higher state of oxidation and then oxidize the leuco base to the dye. Many times the manganese present would necessarily be consumed if the reaction proceeded through etoichiometric reactions involving Mn+2 or its oxidation products. This then would imply a catalytic effect of manganese upon the reactions which may be written Mn+’> -.
NaIO4
MnOX
LAlG
Mnox -+ Mn+l
and the observed effect would be appearance of RIG. If periodate and LMG are in large excess, the reaction should be zero order and, if Beer’s law is obeyed, absorbance plotted us. time should yield a straight line. This, however, is not the case (1%’). The reaction, therefore, seems t o be somewhat complicated and provided the impetus for this investigation leading to the development of a new method for determination of trace amounts of manganese of the order of 1 to I5 mpg. A simple test with this sensitivity would be desirable for the determination of manganese in human blood serum. EXPERIMENTAL
Reagents. All reagents were rragent grade or the purest commercially available. Water used in preparing all reagents was redistilled from all-
glass apparatus. T h e solutions were stored in acid-washed polyethylene bottles. PHOSPHATE ACETATEBUFFER. NaH2P04.H20(27.6 grams) in water and 12.0 ml. of glacial acetic acid were diluted to 100 ml. with water. Then 21.0 ml. of 1.ON NaOH was added. The pH of a 1 to 4 dilution of this solution was 3.6 to 3.7. LEUCOMALACHITEGREEN (p,p’-
tetramethyldiaminotriphenylmethane), obtained from Hopkins and Williams, Ltd. Fifty milligrams was dissolved in 100 ml. of 0.5% (v./v.) HCI. The reagent was stored in a refrigerator and discarded after one week. MALACHITE GREEN, obtained from Matheson, Coleman and Bell. Ten milligrams was dissolved in 100 ml. of water, and discarded after one week.
MANGANESE STANDARD
SOLUTION,
5 pg. per 100 ml. MnS01.H20 (30.8 mg.) was dissolved in 1 liter of 0.1N H2S04; 0.5 ml. of this solution plus 0.5 ml. of 0.1N H2S04 were freshly diluted to 100 ml. with water. Methods. All glassware used was treated with 6N HC1 a t 60’ C. for about 30 minutes and then washed with redistilled water. Experiments designed t o study the kinetics of t h e reaction were performed as follows. Test tubes were set up to contain 0, 0.02, 0.05, and 0.10 pg. of manganese. The folloning then were added in order: 1 ml. of 1N HC1, 7.5 ml. of phosphateacetate buffer, a volume of 1 N NaOH determined to give a final pH of 3.6, and 1.0ml. of leuco-base or dye. Water was added to 14.5 ml. and then 0.5 ml. of 1% sodium periodate solution was added to start the reaction. The tubes VOL 35, NO. 11, OCTOBER 1963
1721
