966
ANALYTICAL CHEMISTRY
amounts of nitrate were saturated with chloroform and analyzed. No interference due to chloroform could be detected even after standing a t room temperature for several days. For other work it was necessary to determine nitrate in the presence of widely varying amounts of phosphate ion, to learn the effect, if any, of such variations on the polarographic nitrate analysis. It was found that the phosphate ion (KH,POI) does not interfere in concentrations up to 25 p.p.m. Higher concentrations were not studied. Accuracy of Polarographic Method. Although concentraions of 0.10 p.p.m. or less nitrate nitrogen may be determined by the polarographic method, more consistent results were obtained by adding to the sample sufficient nitrate to bring the concentration to about 2.0 p.p.m. when testing samples known to contain 0.5 p,p.m. of nitrate nitrogen or less.
Within the range of 0.1 to 10 p.p.m. determination of known nitrate concentrations in distilled water gave results which averaged less than 2% error. Results in filtered Androscoggin River water were in error by less than 3% of a known nitrate increment. Water samples which required flocculation treatment averaged less than 5% error. LITERATURE CITED
(1) Am. Public Health Assoc., New York, “Standard Methods for
the Examination of Water and Sewage,” 9th ed., pp. 69-71, 1946. (2) Lawrance,W. A , , Sewage andInd. Wustes, 22,820 (1950). (3) Rand, AI. C., and Heukelekian, H., ANAL.CHEM.,25, 878 (1953). RECEIVED for review liovember 1 3 , lQ52. Accepted March 12, 1953. This investigation was made possible by a grant from the National Council for Stream Improvement.
Titration of Calcium and Magnesium in Milk and Milk Fractions with Ethylenediamine Tetraacetate ROBERT JENNESS D e p a r t m e n t of Agricultural Biochemistry, Unicersity of Minnesota, S t . Paul, Minn.
method of Schwarzenbach and coworkers ( 4 , for Ttitrating . calcium and magnesium with ethylenediamine 15) 16)
HE
tetraacetate and suitable indicators offers the advantages of rapidity, simplicity, and accuracy. It has found wide application in the determination of hardness in water ( 2 , 3, 8, 9 ) and in determination of calcium and magnesium in limestone and soils ( 1 , 6, 7 ) . I t has been employed successfully for titrating calcium in blood serum, urine, and spinal fluid ( 1 1 , 18, 1 7 ) . Attempts to use it for plant materials (6, 10, 1 8 ) have not been entirely suceessful because orthophosphate interferes with the end point. Obviously, milk, in which the atomic ratio of calcium to phosphorus is approximately 1 to 1, cannot be analyzed by this technique without precautions to eliminate the interference of phosphate. The present paper describes a simple anion exchange technique for overcoming this difficulty by removing phosphate. Furthermore, several alternative methods of preparing milk samples for the analysis have been studied and shown to be satisfactory. APPARATUS AND REAGENTS
Porcelain casseroles, 2-inch and 3-inch diameter Muffle furnace Electric heaters, such as Precision Scientific Co.’s type R H or Ful-Kontrol, with refractory having hole 1 inch in diameter Kjeldahl flasks, 100 ml. Volumetric flasks, 50 and 100 ml. Anion exchange columns. Place 3 grams of the resin Duolite A-4 (Chemical Process Co., Redwood City, Calif.) in a column 7 X 250 mm., with a reservoir 15 X 100 mm. a t the top and a capillary 1 X 25 mm. a t the bottom. Attach a short length of rubber tubing to the capillary with pinch clamp. Prepare the columns for use by backwashing with water to stratify the resin particles and eliminate air, passing several portions of 1 N sodium acetate, and rinsing with distilled water. Microburet, 5-ml. capacity, calibrated in 0.01-ml. divisions Hydrochloric acid, 1 N Nitric acid, concentrated, reagent grade Perchloric acid, 72%) double distilled Sodium hydroxide, 0.5 N and 1.5 N Standard titrating solution. Dissolve 10 grams of disodium dihydrogen ethylenediamine tetraacetate dihydrate (Versene from Bersworth Chemical Co., Framingham, Mass., or Sequestrene from Alrose Chemical Co., Providence, R. I.) and 2 grams of sodium hydroxide pellets in water, and make up to 1 liter. Standardize this solution, which has a titer of approximately 1.0 mg. of calcium or 0.6 mg. of magnesium per milliliter, by titrating standard calcium chloride and magnesium chloride solutions prepared by dissolving respectively calcium carbonate or metallic magnesium in hydrochloric acid and making up to the desired volume.
Calcium indicator. Prepare indicator by grinding 0.2 grams of ammonium purpurate and 100 grams of sodium chloride to an intimate mixture ( 2 ) . Calcium and magnesium indicator. Dissolve 1 gram of Eriochrome Black T in a mixture of 30 ml. of distilled water and 1 ml. of 1 A’ sodium carbonate, and make up to 100 ml. with 2propanol ( 2 ) . Buffer solution 1. Dissolve 4 grams of C.P. sodium tetraborate decahydrate in approximately 80 ml. of distilled water. Buffer solution 2. Dissolve 1 gram of C.P. sodium hydroxide and 0.5 gram of C.P. sodium sulfide in 10 ml. of distilled water. Cool. Mix the two solutions and make to 100 ml. PROCEDURE
Preparation of Samples. Milk samples are prepared for analysis by dry ashing, wet digestion, or acid precipitation of the casein. In dry ashing, a 5-ml. sample in a porcelain dish of 2-inch diameter is evaporated to dryness on the steam bath and incinerated in a muffle furnace a t 600” C. overnight. The ash is moistened with a little distilled water, dissolved with 1 ml. of 1 N hydrochloric acid, transferred quantitatively to a 50-ml. volumetric flask, and made to volume. In wet digestion a 5-ml. sample is digested in a 100-ml. Kjeldahl flask on an electric heater with 5 ml. of concentrated nitric acid until copious brown fumes cease to be evolved. The flask is cooled, 2 ml. of 72% perchloric acid is added, and the digestion is continued until heavy white fumes appear. The digest is transferred to a 50-ml. volumetric flask and made to volume. After some experimentation, the following method was developed for acid precipitation of the casein to yield a filtrate with a maximum calcium content. Ten milliliters of milk are placed in a 100-ml. volumetric flask and diluted with 20 ml. of distilled water. Two milliliters of 1 N hydrochloric acid are then added, and the sample is allowed to stand for 10 minutes, after which 2.5 ml. of 0.5 N sodium hydroxide are added. The acid dissolves colloidal calcium salts and disperses the casein on the acid side of its isoelectric point. Addition of alkali brings the pH to 4.0 to 4.1, whereupon casein is precipitated, and the calcium remains in solution. The contents of the flask are made to volume and, after thorough mixing, the precipitate is filtered off. The filtrate should be water clear. A few determinations were made on rennet whey and on milk dialyzate. Whey is prepared by treating 500-ml. of skim milk a t 35’ C. with 0.5 ml. of commercial rennet extract. After 20 minutes the firm curd is cut into small cubes, and the whey exuded by syneresis is collected and filtered. Dialyzate is prepared by equilibration of 80 ml. of distilled water enclosed in a Visking sausage casing against 4 liters of skim milk for 48 hours a t 5’ C. Anion Exchange. An aliquot (usually 10 ml.) of solution prepared from milk is passed through a column, followed by two 10-ml. portions of distilled water to rinse the column. Air is prevented from entering the column by not allowing the liquid
967
V O L U M E 25, N O . 6, J U N E 1 9 5 3 to fall below the top of the resin. The entire effluent is collected in a 3-inch porcelain casserole. Calcium Determination. To approximately 30-ml. of ionexchanged solution in a casserole (representing an original 10-ml. aliquot), sufficient (1 to 2 ml.) 1.5 N sodium hydroxide is added to bring the pH above 10 as determined with Universal indicator paper. Then 1 scoop (0.2 gram) of the prepared calcium indicator is added, and the solution is titrated, with stirring, with ethylenediamine tetraacetate solution to a purple color that does not change on addition of another drop of titrant. Calcium and Magnesium Determination. To approximately 30-ml. of ion-exchanged solution in a casserole, 0.5 ml. of the borate buffer solution is added, and, if necessary, sufficient 1.5 '1sodium hydroxide (usually 1 to 2 drops) to bring the pH in the range of 8 to 10 as shown by Universal indicator paper. Then 3 drops of the Eriochrome black T indicator solution is added, and the solution is titrated, Kith stirring, with the ethylenediamine tetraacetate solution until the color changes permanently from pink to blue.
also agree satisfactorily with oxalate-permanganate analysis of the solution of dry ash. Table I1 shows the results of magnesium analyses on nine samples by the titration method and the method based on precipitation of magnesium ammonium phosphate and determination of phosphate in the precipitate. The agreement between results by the two methods is reasonably satisfactory m-hen it is considered that both deal with very small amounts of magnesium and that, in the titration method, the titer due to magnesium represents a small difference between two large titers. Table I11 shows the results of recovery tests in which either 0.92 mg. of calcium, or 0.40 mg. of magnesium, or both were added as solutions of the chlorides to 10-ml. portions of milk before dry ashing. The recovery of 101 to 103% of the added octlcium is reasonably satisfactory, and the magnesium recovery of 90 to 95% is probably within the range of accuracy of the magnesium determination.
STANDARD iMETHODS USED FOR COMPARISOh
For comparative purposes calcium was determined by the oxalate-permanganate method of Morris e t al. (13)and magnesium by the magnesium ammonium phosphate method of Bushill et al. ( 5 ) . The latter procedure was slightly modified in that the precipitation of magnesium ammoniuni phosphate was performed in 15-ml. pointed centrifuge tubes, and the precipitate was collected and washed by the technique described by Pyne ( 1 4 ) . Phoaphorus was determined by the Fiske-Subbarow ( I S ) procedure.
Table 11. Analyses of Magnesium i n M i l k s Mg. of Magnesium per 100 MI. of Milk5 Titration method Precipitation method 10.2 10.9 10.7 10.6 11.2 10.5 11.4 10.9 10.7 10.1 11.6 10.3 10.7 10.7 11.0 11.6 10.9 10.1
RESULTS
I t was first ascertained that the anion exchange and titration method would satisfactorily determine calcium in standard solutions containing phosphate. solution was prepared containing 9.2 mg. of calcium, 11.75 mg. of phosphorus (as orthophosphate), and 30 meq. of hydrochloric acid per 100 ml. Tenmilliliter aliquots could not be titrated satisfactorily because of fading end point. However, when such aliquots were passed through the exchanger, the calcium could be titrated quantitatively, and phosphorus determinations showed that the phosphate had been removed quantitatively. Next the new method was compared with the oxalate-permanganate method on a single sample of milk ash. The dry ash representing 50 ml. of skim milk was dissolved and made to 500 ml. of solution containing 0.30 meq. of hydrochloric acid per milliliter. Fifteen 10-ml. aliquots of this solution, titrated by the new method averaged 120.6 zk 0.2 mg. (standard error) of calcium per 100 ml. of milk, a good degree of reproducibility. Fourteen 10-ml. aliquots analyzed by the oxalate-permanganate method averaged 120.8 Z!Z 0.2 mg. (standard error) of calcium per 100 ml. of milk. These two means were found by Student's t test not to differ significantly. In Table I are shown results of calcium analyses on twelve samples of cows' milk. The three methods of preparing the samples for the titration yield closely comparable results which
Table I.
Analyses of Calcium in Milks
iii:4 124.0 122.0 118.8 127.3 120.4
D r y ash 117.0 122.6 137.0 132.5 129.6 131,s 132.2 122.9 121.7 119.6 128.6 121.2
Titration Method Wet ash Noncasein filtrate 118.2 117.1 123.4 122.3 137.2 136.7 133.1 133.4 130.0 129.0 132.7 131.6 130: 5
....
Each value is the average of duplicate determinations.
Table 111. Recovery by the T i t r a t i o n M e t h o d of Calcium and M a g n e s i u m Added t o illilk" Mg. of Calcium per 100 MI." -4dded Found 0 119.6 0 119.5 129.1 9.2 9.2 128.9 0
Rfg. of .Magnesium per 100 M1." Added Found 10.8 0 14.6 4.0 11.0 0 14.6 4.0
Each value is the average of quadruplicate titrations.
The anion exchange titration method is directly applicable to rennet whey and milk dialyzate without incineration or wet digestion; identical results are obtained on unashed and ashed samples. Thus, the method is admirably suited to following changes in dissolved calcium and magnesium which may occur in milk as a result of treatment. Aliquots of 5-ml. of whey or dialyzate are convenient. However, in cases in which it is desired to determine dissolved calcium and magnesium shortly after a treatment of milk (such as heat treatment) only a small portion ( a t most 1ml.) of ultrafiltrate may be available. For such a small amount, a column consisting of 0.5 gram of resin can be used through which the 1-ml. aliquot is passed. This modification proved to be entirely satisfactory, yielding results for dialyzate identical to those obtained with 5 ml. passed through a 3-gram column of resin. DISCUSSION
M g . of Calcium per 100 M I . of Milka
Oxalate-KMn04 dry ash 117.0 123.0 138.0 133.5
Each value is the average of duplicate determinations on dry ash solutions.
.... ... ...
...
...
The removal of phosphate by anion exchange makes possible the application of the titration of Schwarzenbach and coworkers to milk and milk fractions. The titration is of comparable accuracy to the classical methods for determining calcium and magnesium but the entire procedure including ion exchange is much less time consuming. Doubtless the method would be useful for calcium and magnesium analyses of other biological materials, such as certain plant tissues ( 7 , 10, 18), in which phosphate interferes with the titration. The three methods of preparing milk samples for the determination of total calcium and magnesium appear to yield identi-
968
ANALYTICAL CHEMISTRY
cal results; the choice of procedure will depend on individual preference. The dry ashing procedure is the most time consuming of the three. Wet ashing is rapid, but the anion exchange columns require regeneration more frequently when this teohnique is used because the resin becomes saturated with perchlorate ions. I n fact, only one 10-ml. aliquot of wet digest can be passed through a 3-gram column while as many as 15 aliquots of dry ash solution (prepared as described above) can be passed through before regeneration is necessary. Acid precipitation of casein affords a quick and simple means of preparing a clear solution containing all of the milk calcium and magnesium. One slight drawback of this method is the fact that the somewhat uncertain vdume of the casein precipitate must be taken into account in calculating results. However, this point is of minor consideration in most work.
Biedermann, W., and Schwarzenbach, G., Chimia Prague, 2, 56-9 (1948).
Bushill, J. H., Lampitt, L. H., and Filmer, D. F., J . SOC.Chem. Ind. (London),56, 411T413T (1937).
Cheng, K. L., and Bray, R. H., Soil Sei., 72,449-58 (1951). Cheng, K. L., Kurtz, T., and Bray, R. H., AXAL.CHEM.,24, 1640-1 (1952).
Connors, J. J., J . Am. W a t e r W o r k s Assoc., 42, 33-9 (1950). Diehl, H., Goetr, C. A., and Hach, C. C., I b i d . , pp. 40-8. Gastler, G. F., Proc. S. Dakota A c a d . Sci.,28, 77-81 (1949). Greenblatt, I. J . , and Hartman, S., A~YAL. CHEM.,23, 1708-9 (1951).
Kibrick, A. C., Ross, M.,and Rogers, H. E., Proc. SOC.E x p . Bid. Med., 81, 353-5 (1952).
Morris, H. P., Nelson, J. W., and Palmer, L. S., IKD. EXG. CHEM.,ANAL.ED.,3, 164--7 (1931). Pyne, G . T., Analyst, 68, 330 (1943). Schwarzenbach, G., and Ackermann, H., Helu. Chim. A c t a , 30, 1798-1804 (1947).
Schwarrenbach, G., Biedermann, W., and Bangerter, F., Ibid., LITERATURE CITED
(1) Banewicz, J. J., and Kenner, C. T., ANAL.CHEII., 24, 1186-7 (1952). (2) Betr, J. D., and Noll, C. 8.,J. Am. W a t e r W o r k s Assoc., 42,4956 (1950). (3) Ibid., pp. 749-54.
29,811-18 (1946).
Sobel, A. E., and Hanok, -4., Proc. SOC.E x p . Bid. X e d . , 77, 73740 (1951).
Willson, A. E., AXAL.CHEM.,22, 1571-2 (1950). RECEIVED for review Sovember 7 , 1952.
Accepted February 11, 1953. Paper 2931, Scientific Journal Series, Minnesota dgricultural Experimental Station.
Rapid Modified Procedure for Determination of Kjeldahl Nitrogen C. H. PERRIN, Canada Packers, Ltd., Toronto, Ontario, Canada the spectacular success of Wilfarth and of Gunning in reS ducing the time required for Kjeldahl digestions, many chemists have attempted to increase the speed of the reactions still INCE
further. Typical efforts are described by Shedd ( 6 ) , Gerritz and S t . John (2), Lauro ( d ) , and Stubblefield and DeTurk ( 7 ) . Digestion mixtures such as those cited are more rapid in their action than conventional mixtures when used to digest samples such as animal feeds. However, the author thought i t would be interesting to compare the reaction speeds of such forniulas on nicotinic acid, a very refractory heterocyclic nitrogen compound. Kicotinic acid has been included in a number of recent studies of Kjeldahl digestion mixtures (6, 8, 9). Table I gives a comparison of rates of the digestion of nicotinic acid when an extremely brief reaction period is used. Methods making use of oxidizing agents such as perchloric acid, selenium, persulfates, and hydrogen peroxide, which introduce uncertainties, were not included in this study, The “proposed” method referred to in Table I is the subject of this paper.
Table I.
Comparison Speeds of Nicotinic Acid Digestion
Nicotinic Acid. 11.38% N (theoretical). 1!.26% N obtained by KjeldahlWilfarth-Gunning method with 3 hours’ digestion Digestion. 0.5-gram sample, 12 minutes using 550-watt heaters, starting hot. 4.33-minute boil test Method Nitrogen Found. % Kjeldahl-Filfarth-Gunning 1.66 With HgO 1.34 With CuSO4 ( I ) 4.63 Gerritz and St. John ( 8 ) 2 .00 Stubblefield and DeTurk ( 7 ) 11.29 Proposed
believed to be the most rapid macrotechnique yet described. Although no use is made of selenium, phosphates, and other controversial additives, or of unusually intense heat, a complete nitrogen macrodetermination can be made in about 50 minutes. I n the case of all materials investigated to date, the point at which the digestion is complete can be determined visually. The small quantity of acid and alkali required reduces the violence of the reactions, contributes to accuracy, safety, speed of cooling, and makes possible the use of caustic pellets in the distillation step. (The preparation and handling of large quantities of liquid caustic are, perhaps, the most hazardous and unpleasant features of the Kjeldahl determination.) Bumping and foaming in the distillation are virtually eliminated. This permits a more rapid boiling. The chief disadvantage of the proposed method is that it mill stand less abuse than the method of the Association of Official Agricultural Chemists ( I ) . It requires more careful control of heat during digestion and more care in measuring chemicals. With some samples gentle heating during the first few minutes is particularly important because of the high boiling point of the digestion mixture. RE4GENTS
Silica, smooth boiling granules, not selenized (Hengar Co., Philadelphia, Pa.) Mercuric oxide, red, X.F. grade or Ion- in nitrogen. Sodium hydroxide pellets or flakes, low in nitrogen. Boric acid solution. Dissolve 4 grams of C . P . crystalline H3B03 in 100 ml. of distilled nater. Methyl red-bromorresol green indicator. Mix 5 parts of O.2Y0 bromocresol green solution with 1 part of 0.2% methyl red solution, both in alcohol. PROCEDURE
An investigation of a wide variety of catalysts and digestion mixtures led to the conclusion that the efficiency of the KjeldahlWilfarth-Gunning acid-salt-mercury combination ( I ) can be increased to the point where additional oxidizing agents become superfluous. This new combination permitted the development of a rapid Kjeldahl method which offers the following advantages. Except for methods making use of added oxidizing agents, it is
Place a 0.5- or 1.0-gram sample in a dry 500-ml. Kjeldahl digestion flask and add approximately six smooth boiling silica granules, 1.3 to 1.5 grams of mercuric oxide, and 12 =t0.5 grams of potassium sulfate. (Add no filter paper or other matter.) Swirl flask to mix, add 15 f 0.5 ml. of concentrated sulfuric acid, and mix again. Digest for 5 minutes (or until frothing ceases) a t low heat, then boil a t “full heat” until digestion is complete. (By full heat is meant a heating intensity sufficient to bring to a rolling boil 250 ml. of water in a 500-ml. Kjeldahl flask in from 4 to 5 minutes. This is called the boil test.)
