1304
ANALYTICAL CHEMISTRY
would be highly desirable to have an independent analytical method for asparagine of comparable specificity to the glutaminase method for glutamine. The standard errors for the paper and glutaminase methods are of the same order for the determination of glutamine. For asparagine, the standard errors for the paper method are considerably smaller than those for the hydrolytic method. The error would be diminished by the use of paper with a lower blank correction. No significant error appeared to result from the extra manipulative steps required in the paper method between the extraction of the tissue and application of the concentrate to the paper. If the rigorous standards of cleanliness necessary for a microchemical technique are observed, errors from contamination can be avoided. An important advantage of the paper method is the ancillary information it yields regarding the distribution of amino acids and fluorescing substances in the extract. Its major disadvantage is that the analysis is spread over 4 days. The actual manipulative time amounts to 8 hours per extract, done in triplicate by one operator and including the tissue extraction step. On the other hand, a number of analyses can be made a t the same time, dependingonly on the capacity of the freeze-dryer and chromatography apparatus. The rectangular strips of paper cut from the chromatograms can be stored without deterioration in a desiccator containing sulfuric acid until it is convenient to commence the final step. The “cold alcohol” extraction procedure of Bathurst and ,411ison (8)was found excellent for quantitative paper chromatographic purposes. The use of alcohol has the advantage that the extract is free of peptides and proteins and has a low concentration of inorganic salts. On removal of the alcohol, waxes separate out, leaving a solution very suitable for paper chromatography. I n the experimental method outlined, an extraction macroprocedure has been followed by a microchemical estimation. Where the supply of biological material is limited, the extraction and concentration steps could be adapted to a micro scale. The recoveries of glutamine and asparagine from paper chromatograms are interesting in the light of the observations of Woiwod (!No),who found, using Whatman No. 4 paper, that the recoveries obtained for glycine, valine, and leucine decreases with increasing RFvalue and with increasing distance of migration down the paper. This effect was not apparent in the present study, although it may be a partial explanation of the consistently low recoveries from pure solutions. The drying of the chromatograms at low temperatures is important because of the interaction of phenol with amino acids above 50’ C., as pointed
out by Fowden and Penney ( 7 ) , and also because of the instability of glutamine. One of the factors possibly contributing to the success of the proposed method is that the hydrolysis of the amides is performed on the paper, using a reagent which mercerizes the cellulose and is likely to reach all the amide molecules inside the cellulose fibers. The necessity for an elution step is avoided, with a saving of manipulative time. ACKNOWLEDGRIENT
The author is deeply indebted t o his colleague, J. L. Mangan, who, ITith the technical assistance of J. F. Fisher, conducted analyses for the comparative lvork by two standard methods. Thanks are also due to J. G. Fraser for the preparation of the glutamine and asparagine samples used. Finally, he wishes to acknowledge the invaluable assistance of James Melville, both in the preparation of the manuscript and from stimulating discussions held during the course of the investigation. LITERATURE CITED
(1) Archibald, R. hl., J . Bid. Chem., 154, 643 (1944). (2) Bathurst, N. O., and Allison, R. M., N . 2. J. Sci. Tech., in press. (3) Chibnall, 8. C., and Westall, R. G., Biochem. J., 26, 122 (1932). (4) Consden, R., Gordon, 8 . H., and Martin, A. J. P., Ibid., 38, 224 (1944). (5) Conway, E. J., “Microdiffusion Analysis and Volumetric Error,” rev. ed., London, Crosby Lockwood & Sons, 1947. (6) Dent, C. E., Stepka, W., and Steward, F. C., Nature, 160, 682 (1947). (7) Fowden, L., and Penney, J. R., Ibid., 165, 846 (1950). (8) Hamilton, P., J . Bid. Chem., 158, 375 (1945). (9) Hughes, D. E., and Williamson, D. H., Biochem. J . , 43, xlv (1948). (10) Krebs, H. A,, Ibid., 43, 51 (1948). (11) Martin, A. J. P., and Mittelmann, R., Ibid., 43, 353 (1948). (12) Seuberger, A., and Sanger, F., Ibid., 36, 662 (1942). (13) Pucher, G. W., and Vickery, H. B., IND. ENG.CHEJI.,SNAL. ED., 12, 27 (1940). (14) Reifer, I., and Melville, J., Trans. XIth Intern. Conf. Pure and Applied Chem. (Supplement to Chemistry and Industry, 1948). (15) Vickery, H. B., Pucher, G. W., and Clark, H. E., J . Biol. Chem., 109, 39 (1935). (16) Vickery, H. B., Pucher, G. W.,Clark, H. E., Chibnall, A. C., and Westall, R. G., Biochem. J., 29, 2710 (1935). (17) Vickery, H. B., Pucher, G. W.,and Deuber, C. G., J . Bid. Chem., 145, 45 (1942). (18) Vickery, H. B., Pucher, G. W., Leavenworth, C. S.,and Wakeman, A. J., Conn. Agr. Expt. Sta., Bull. 374, (1935). (19) Vickery, H. B., Pucher, G. W.,Wakeman, A. J., and Leavenworth, C. s.,Ibid., 424 (1939). (20) Woiwod, A. J., Biochem. J.,45, 412 (1949). RECEIVED October 13, 1950.
Determination of Traces of Chloride Potentiometric Titration to the Apparent Equivalence Potential I. M. KOLTHOFF AND P. K. KURODA School of Chemistry, University of Minnesota, Minneapolis, .Minn.
I
N T H E classical method of potentiometric titrations the end point is taken at the location of the maximum in the A E / A c curve. When the equilibrium constant of the reaction is unfavorable and the dilution is high, either no maximum occurs or A E / A c changes so little at the end point that it cannot be determined with a n y degree of accuracy. Under such conditions a potentiometric titration still can yield rapid and accurate results when reagent is added until the a p parent equivalence potential is attained. From the practical point of view a serious limitation of this method is that the
equivalence potential, in general, changes with the ionic strength and the kind of electrolyte in the solution titrated. Moreover, the exact determination of the equivalence potential is impossible, owing to a n unknown liquid junction potential. From an analytical viewpoint these difficulties can be almost completely eliminated by carrying out the titration in a “supporting electrolyte” of high ionic strength and suitable composition. The “apparent” equivalence potential in such a medium can be found b y classical methods. For practical purposes it may be desirable to use various supporting electrolytes.
V O L U M E 23, NO. 9, S E P T E M B E R 1 9 5 1
1305
The potentiometric titration of chloride is never applied to the determination of traces of chloride because the break in potential at the end point is not pronounced. Theoretically it should be possible to determine traces of chloride by potentiometric titration to the equivalence potential. A simple and rapid procedure has been developed using a “supporting electrolyte” which is 0.5 iV in potassium (or sodium) nitrate and 0.1 N in nitric acid. Solutions which are 5 X N in chloride can be titrated rapidly with an accuracy and precision to P 7 c . The accuracy increases in more concentrated chloride solutions. The method should find wide application in the determination of traces of chlorides in varying electrolytes, in potable water, in rocks, etc.
The present paper presents methods for the simple, rapid, and accurate determination of traces of chloride in various materials by titration to the “apparent” equivalence potential. For general purposes-e.g., for the determination of chloride in 0.1 AY solution of most electrolytes--a supporting electrolyte is iecommended which is 0.5 3- in potassium nitrate and 0.1 S in nitric acid. The assumption is made that the liquid junction potential is unaffected when this dectrolyte is made 0.1 S in :my other electrolyte. This has been found to be true within 0.001 volt with several el~ctrolytesinvestigated. For the determination of traces of chlorides in rocks, a fusion F5ith sodium carbonate is necessary. For such a determination a supporting electrolyte which is 0.5 to 1 S in sodium nitrate and 0.1 S in nitric acid is recommended. For the determination of chloride in solutions which are very dilute in electrolytes-e.g., in potable waters, etc.-it is satisfactory to use 0.1 N potassium nitrate as the supporting electrolyte. Furman and Low ( 1 ) proposed an exact method in which small amounts of chloride are determined potentiometrically by measuring the e.m.f. of a concentration cell with two silversilver chloride electrodes. The reference half-cell is composed of the unknown to which a known amount of chloride is added. In this way a liquid junction potential is eliminated. However, it is necessary to know or to determine the solubility product of the silver chloride in the particular electrolyte and to use a quadratic equation in the calculation of the chloride content of the unknown from the e.m.f. of the cell. The proposed type of titration to the equivalence potential is simpler, does not require a knowledge of the solubility product, and gives directly the amount of silver equivalent to the chloride present. In the present work the saturated calomel electrode is used as reference electrode and the supporting electrolyte as the salt bridge. I t is also possible to use as reference electrode a silversilver chloride electrode in the supporting electrolyte saturated with silver chloride. Under such conditions the titration is carried out until the e.m.f. of the cell is equal to zero. EXPERIMENTAL
Materials Used. All salts used in this investigation were C.P. products and were recrystallized until entirely free of chloride. The sodium carbonate used in the fusion of rocks was a C.P. product. I t contained a trace of chloride which was determined by the method proposed in this paper. The nitric acid was free of chloride. Several products of silver chloride were prepared b tion of 0.1 ,V sodium chloride with an escess of 0.1 trate or vice versa. The precipitates were washed with water and then shaken for 10 day- n-ith conductivity water, x-hich was refreshed every day. Suspensionq of the product*, kept in brown bottles, were used in the determination of the equivalence potential. All preparations gave the same equivalence potential. Aliquots of the suspension containing from 10 to 30 mg. of silver chloride were added to the wpporting electrolyte in a final volume of 50 ml. Titration Cell. The titration cell was composed of a saturated calomel electrode of the bottle type and a silver-silver chloride electrode &s indicator electrode in the unknown. The salt bridge was of the U type and contained the supporting electrolyte solidified with agar (2).
In order to test the reproducibility of the indicator electrode, fourteen silversilver chloride electrodes were prepared by electroplating platinum wire electrodes in 5% potassium cyano argentate solutions for 10 minutes, the current being 0.020 ampere. The electrodes were then coated anodically with silver chloride in 1% sodium chloride solutions for 5 minutes (current 0.01 ampere). They were washed repeatedly and stored in conductivity water. In agreement with Taylor and Smith ( 4 ) it was found that it takes a few days before such electrodes attain equilibrium. After 3 to 4 days of standing in distilled water with occasional refreshing of the water, all the electrodes gave the same equivalence potential within 0.001 volt. The indicator electrodes could be used without change for several months. The e.m.f. measurements were made with a Leeds & Northrup student’s potentiometer. In general, the titrations were carried out at room temperature ( 2 5 ” i.2 “ C.).
Q29
-
0.28
-
0
1
2
3
4.
5
6
7
8
9
10
10-3 N SILVER NITRATE, ML.
Figure 1. Titration Curve 5 X 10 -5
N KCl i n 0.5 N KNOs, 0.1 N “01,w i t h 1 X 10 - 8 1. 2.
Y
AgNOa Endpoint Equivalence potential
Apparent Equiyalence Potential. After the silver chloride suspension in the supporting electrolyte had been stirred for 1 minute, constant values of the apparent equivalence potential were attained. This potential was determined a t temperatures between 20’ and 30’ C., the entire cell being a t the desired temperature. From a series of some one hundred measurements, using various electrodes, the equivalence potential in a medium composed of 0.5 S potassium nitrate and 0.1 AT nitric acid was found to be: 0.2700 (&0.0005)
+ 0.0004 ( t - ‘ 2 5 “ C.) volt
This potential was found to be the same when the supporting electrolyte was made 0.1 S in potassium or sodium sulfate, sulfuric acid, or calcium or magnesium nitrate, or 0.15 N in potassium aluminum sulfate. Using a supporting electrolyte composed of 0.5 11’ sodium nitrate and 0.1 N nitric acid, an apparent equivalence potential of 0.2710 volt was measured a t 25’ C. ANALYTICAL RESULTS
The titrations can be carried out rapidly, even when the chloride concentration is of the order of 10-6 A-. The accuracy
ANALYTICAL CHEMISTRY
1306 .
~
Table I.
~~
Comparison of Potentiometric and Photonietric ' Methods for Chloride in Rocks Chloride Content, % ~
Sample Granite Diabase Feldspar Clay Granite Hornblende gneiss
Photometric
Potentiometric
0 . 0 0 8 , 0.009
0.0079, 0 , 0 0 8 4
0.022.0.022 0.007, (0.010) 0 . 0 0 6 , 0.008 0.004 0.023
0.021,0.020 0.0057, 0.0064 0.0057. 0,0062 0.0038 0.021 ~-
and precision depcnd upoii the rcproducitility of thr e.ni.f of the cell and the accuracy with which the e.1n.f. is measured. Under the experimental conditions described aliove. 10-4 X chloride solutions were titrat.ed \vit,li an accuracy and p i ~ i s i o n better than 1%, 5 X 10-5 -Ychlol,ide solutions with a precision of 275, and 10-5 N solutiolis with a precision of 10%. As an example, the change of t8hee.1n.f. during the titration of 5 x 10-6 .Y chloride in the recommended supporting electrolyt,e is shown in Figure 1. The change of the e.1ii.f. from the beginning of the tit,ration until 100% excess of silver was present \vas only 45 rnv. Locating the end point from the iiiaximuni in A E / A c is not possihle in this instance. Traces of chloridc were determined in 0.1 S solut,ions of a nuinher of elect,rolytes wit,h the ahove accur:rcy. Thc support,ing electro1yt.e was 0.5 ATin potassium nitrate and 0.1 S in nitric acid. In general, it is recomniended that each worker cletermiiie bhe apparent equivalence potential hiinself under the selected experimental conditions. The chloride content o f IIississippi River water (almut 5 X 10-5 .\-in chloride) was dt~tcwnincdiiy making 100 nil. of water 0.5 S in potassium nitrat,e and 0.1 .\-in nitric acid (0.1 .\*potassium nitrate alone can also be uwd as supporting electrolyte). The average of 10 determinations indicated a chloride content of 1.83 i 0.03 nig. per liter. I n orc1r.r to test the accuracy, two 2lit,er samples of R-at,cr were evaporated t,o 100 nil., supporting
electrolyte was added, :ind the vhloride was titrated. This concentrate was about 10-8 AYin chloride, which can be titrtaed with a few tenths p e r cent accuracy. In this way a chloride content of 1.81 mg. per liter mas found.
Chloride in Rocks. 1 finely powdered sample (0.5 to 1 grain 1 in a platinum crucible with five times its weight sodium carbonate. After cooling, the melt is extracted with hot water, the suspension is filtered, antl the residue is washed several times with hot ~ a t e r . .I drop of nicthvl orange and chloride-free 7 .T nitric acid is added t o the filtratr until the solution is red and so much more nitric acid that the acid concentrati:n is about 0.1 S after solution to 100 1111 -After cooline to 25 . the solution is titrated with silver nitrate to :in equiva'ience potential of 0.2710 + 0.0004 ( f - 2 5 O C.). . Even C.P. aotliuni r:iri)oiintc rontains t,races of chloride; therefore, it hlank with thc renKc~ntis run by the above procedure.. The difference Iwtween thc timount of silver used by the sample and the hlank gives thc chloride content. Obviously, an exact knowledge of the apparent equivalence potential is of no consequence in this a n a l j , as long as the temperature is the same (preferably within a few tcnths of a degree) in the t,itration of the sainplc and the hlank. IS fused
In T:iI)I(>T sonw rrrults :ire (lonlp:+redwith those ohtainrd by the photometric method rrrciitly described by Iluroda and Sandell (5). Both nir~tliotlsgive results of the same order of accuracy. ACKNOWLEDGMENT
Acknowledgment is made to the Graduate School of the University of Minnesota for a grant which enabled the authors to carry out t.his work. LITERATURE CITED (1) Furman, N. H., antl Low, 0. IT.,Jr., J . .4m. Chem. Soc., 57, 1585 (19 3 5 ) . (2) Kolthoff. I. Ji.,and Laitinrn. H. .I.,"pH and Electrotitrations," 2nd ed., Ten, Tork, John T i l e y t Sons, 1941. (3) Kuroda, P. K., and Sandell. E. B., AXAL.CHEY.,22, 1144 (1950). (4) Taylor, J. K., and Smith. E. R., J.Research Natl. Bur. Stnndnids, 22, 307 (1939).
RWEIVED December 22, 1950:
Argentometric Amperometric Titration of Traces of Chloride With the Rotated 'Platinum Electrode as Indicator Electrode I. 51. KOLTIIOFF AND P. K. KURODA School of Chemistry, t?'nit.ersity of Minnesota, Minneapolis M i n n .
I
N PRELIMIKART work 1,witiiien and Rolthoff (3) s h o ~ e d that good results could he obtained in the amperonietric titration of silvw with chloride or vice versa with the rotated platinum electrode as indirntor clrctrode, if some gelatin \vas added to prevent depolarization of the clertrotlr h?- thc colloidal silver chloride particles arid to ohtniri a smooth deposit of silver on the electrode in the presence of a n escess of silver salt. Laitinen et al. (Z) reportcd Inter thkt cliloridtr in concentrations greater than 0.005 A' rould be titrated with :in accuracy of 0.5% or hetter. However, with 0.001 S chloride solutions they found results which were 5 to 9% low antl in 0.0005 .Y solutions almut 15% low. A considerable improvement was ohtained in these dilute chloride solutions when the medium was composed of a 50-50 inisture of water and acetone. A great practical advantage of the reaction between chloride
and silver is that piecipitation equilibrium is attained very rapidly and hardly :my tendrnrv for the formation of supersaturated solutions is notired during the titration. This is true even in the piesence of small aiiiounts of gelatin. Therefore, on the baris of the solubilitv product, satisfactory results should be obtained in aqueous nirtl~uiii,even a t chloride concentrations as small as 0.0001 S,proyided that the reagent (excess silver) line is not drawn until aftri the addition of such an excess of silver that the solubility of silvei chloride becomes negligibly small. If this precaution is not considered, the reagent line has the arong dope and lon rwults are found, as reported by Laitinen et 01.
As an illustration, Ict u s con~idcrtitration curve I ohtained in the titration at rooin temperature of 0.0001 N chloride in 0.1 N potassium nitrate Qolution containing 0.02% gelatin with 0.01 N silver nitrate (Figure 1 ).
