titration of calcium and magnesium in milk and milk fractions with ethylenediamine tetra acetate

8/4/2019 Titration of Calcium and Magnesium in Milk and Milk Fractions With Ethylenediamine Tetra Acetate

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966 A N A L Y T I C A L C H E M I S T R YWithin the range of 0.1 to 10 p.p.m . determination of known

nitrate concentrations in distilled water gave results whichaveraged less than 2% error. Results in filtered AndroscogginRiver water were in error by less tha n 3% of a known ni trateincrement. Water samples which required flocculation tre atmentaveraged less than 5% error.

LITERATURE CITED(1) Am. Public Health Assoc., New York, Standard Methods forthe 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. , an d Heukelekian, H.,ANAL.CHEM. , 5, 878 (1953).RECEIVEDor review liovember 1 3 , lQ52. Accepted Marc h 12, 1953. Thisinvestigation was made possible by a grant from the National Council forStream Improvement.

amounts of nitr ate were saturat ed with chloroform and analyzed.No interference due to chloroform could be detected even afterstanding a t room temperature for several days.

For other work it was necessary to determine nitrate in thepresence of widely varying amounts of phosphate ion, to learnthe effect, if any, of such variations on the polarographic nit rat eanalysis. It was found that the phosphate ion (KH,POI) doesnot interfere in concentrations up to 25 p.p.m. Higher concen-trations were not studied.

Accuracy of Polarographic Method. Although concentra-ions of 0.10 p.p.m. or less nit rate nitrogen ma y be determined bythe polarographic method, more consistent results were obtainedby adding to the sample sufficient nitr ate to bring the concentra-tion to about 2.0 p.p.m. when testing samples known to contain0.5 p,p.m. of nitrate nitrogen or less.

Titration of Calcium and Magnesium in Milk and Milk Fractions with EthylenediamineTetraace ateROBERT JENNESS

Department of Agricultural Biochemistry, Unicersity of Minnesota, S t . Paul, Minn.

HE method of Schwarzenbach and coworkers ( 4 , 15)16) fortetraacetate and suitable indicators offers the advantages ofrapidity, simplicity, and accuracy. It has found wide applica-tion in the determination of hardness in water ( 2 ,3, 8, 9) and indetermination of calcium and magnesium in limestone and soils(1,6, 7 ) . I t has been employed successfully for tit ratin g calciumin blood serum, urine, and spinal fluid ( 1 1 , 18, 1 7 ) . Attempts touse it for plant materials (6, 10, 18)have not been entirely suc-eessful because orthophosphate interferes with the end point.Obviously, milk, in which th e atomic ra tio of calcium to phos-phorus is approximately 1 to 1, cannot be analyzed by this tech-nique without precautions to eliminate the interference of phos-phate. The present paper describes a simple anion exchangetechnique for overcoming this difficulty by removing phosphate.Furthermore, several alternative methods of preparing milksamples for the analysis have been studied and shown to be satis-factory.Porcelain casseroles, 2-inch and 3-inch diameterMuffle furnaceElectric heaters, such as Precision Scientific Co.s type R H orFul-Kontrol, with refractory having hole 1 inch in diameterKjeldahl 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 column7 X 250 mm., with a reservoir 15 X 100 mm. at the top and acapillary 1 X 25 mm. at the bottom. Attach a short length ofrubber tubing to the capillary with pinch clamp. Prepare thecolumns for use by backwashing with water to stratify the resin

particles and eliminate air, passing several portions of 1 Nsodium acetate, and rinsing with distilled water.

T .itrat ing calcium and magnesium with ethylenediamine

APPARATUS AND REAGENTS

Microburet, 5-ml. capacity, calibrated in 0.01-ml. divisionsHydrochloric acid, 1 NNitric acid, concentrated, reagent gradePerchloric acid, 72%) double distilledSodium hydroxide, 0.5 N and 1.5NStand ard titr ating solution. Dissolve 10 grams of d isodium di-hydrogen ethylenediamine tetraacetate dihydrate (Versenefrom Bersworth Chemical Co., Frami ngham, Mass., or Seques-trene from Alrose Chemical Co., Providence, R. I. ) and 2grams of sodium hydroxide pellets in water, and make up to 1liter. Standardize this solution, which has a titer of approxi-mately 1.0 mg. of calcium or 0.6 mg. of magnesium per milli-liter, by titrating standard calcium chloride and magnesiumchloride solutions prepared by dissolving respectively calciumcarbonate or metallic magnesium in hydrochloric acid andmaking up to the desired volume.

Calcium indicator. Prepare indicator by grinding 0.2 grams ofammonium purpurate and 100 grams of sodium chloride to anintimate mixture (2 ) . Dissolve 1 gram of Erio-chrome Black T in a mixture of 30 ml. of distilled water and 1ml. of 1 A sodium carbonate, and make up to 100 ml. with 2-propanol ( 2 ) .Buffer solution 1. Dissolve 4 grams of C.P. sodium tetraboratedecahydrate in approximately 80 ml. of distilled water.Buffer solution 2. Dissolve 1 gram of C.P. sodium hydroxide and0.5 gram of C.P. sodium sulfide in 10 ml. of distilled water.Cool. Mix the two solutions and make to 100 ml.

Calcium and magnesium indicator.

PROCEDUREPreparation of Samples. Milk samples are prepared foranalysis by dry ashing, wet digestion, or acid precipitation of the

casein.In dr y ashing, a 5-ml. sample in a porcelain dish of 2-inch di-ameter is evaporated t o dryness on the steam bath and incineratedin a muffle furnace a t 600 C. overnight. The ash is moistenedwith a little distilled water, dissolved with 1 ml. of 1 N hydro-chloric acid, transferred quantitatively to a 50-ml. volumetricflask, and made to volume.In wet digestion a 5-ml. sample is digested in a 100-ml. Kjeldahlflask on an electric heater with 5 ml. of concentrated nitric aciduntil copious brown fumes cease to be evolved. The flask iscooled, 2 ml. of 72% perchloric acid is added, and the digestion iscontinued until heavy white fumes appear. The digest is trans-ferred to a 50-ml. volumetric flask and made to volume.After some experimentation, the following method was de-veloped for acid precipitation of th e casein to yield a filtrate witha maximum calcium content. Ten milliliters of milk are placed ina 100-ml. volumetric flask and diluted with 20 ml. of distilledwater . Two milliliters of 1N hydrochloric acid are then added,and the sample is allowed to s tand for 10 minutes, after which 2.5ml. of 0.5 N sodium hydroxide are added. The acid dissolvescolloidal calcium salts and disperses the casein on the acid side ofits isoelectric point. Addition of alkali brings the pH to 4.0 to4.1, whereupon casein is precipitated, and the calcium remains insolution. The contents of the flask are made to volume and, afterthorough mixing, the precipitate is filtered off. The filtrate shouldbe water clear.A few determinations were made on rennet whey and on milkdialyzate. Whey is prepared by treating 500-ml. of skim milk at35 C. with 0.5 ml. of commercial renne t extract. After 20minutes the firm curd is cu t into small cubes, and the whey exudedby syneresis is collected and filtered. Dialyzate is prepared byequilibration of 80 ml. of distilled water enclosed in a Viskingsausage casing against 4 lit ers of skim milk for 48 hours a t 5 C.Anion Exchange. An aliquot (usually 10 ml.) of solutionprepared from milk is passed through a column, followed by two10-ml. portions of distilled water to rinse th e column. Air isprevented from entering the column by not allowing the liquid


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V O L U M E 25, NO. 6, J U N E 1 9 5 3to fall below the top of the resin. The entire effluent is collectedin a 3-inch porcelain casserole.Calcium Determination. To approximately 30-ml. of ion-exchanged solution in a casserole (representing an original 10-ml.aliquot), sufficient (1 to 2 ml.) 1.5 N sodium hydroxide is addedto bring the pH above 10 as determined with Universal indicatorpaper. Then 1scoop (0.2 gram) of the prepared calcium indicatoris added, and the solution is titrated , with stirring, with ethylene-diamine tetraacetate solution to a purple color that does notchange on addition of another drop of titrant.Calcium and Magnesium Determination. To approximately30-ml. of ion-exchanged solution in a casserole, 0.5 ml. of theborate buffer solution is added, and, if necessary, sufficient 1.5 '1-sodium hydroxide (usually 1 to 2 drops) to bring the pH in therange of 8 to 10 as shown by Universal indicator paper. Then 3drops of t he Eriochrome black T indicator solution is added, a ndthe solution is titrated, Kith stirring, with the ethylenediaminetetraacetate solution until the color changes permanently frompink to blue.

967

STANDARD iMETHODS USED FOR COMPARISOhFor comparative purposes calcium was determined by the

oxalate-permanganate method of Morris e t al. (13)and magnesiumby the magnesium ammonium phosphate method of Bushill et al.( 5 ) . The l atter procedure was slightly modified in th at the pre-cipitation of magnesium ammoniuni phosphate was performed in15-ml. pointed centrifuge tubes, and the precipitate was collectedand washed by the technique described by Pyne ( 1 4 ) . Phoa-phorus was determined by the Fiske-Subbarow ( I S ) procedure.

RESULTSI t was first ascertained that t he anion exchange and ti trati on

method would satisfactorily determine calcium in standardsolutions containing phosphate. solution was prepared con-taining 9.2 mg. of calcium, 11.75 mg. of phosphorus (as ortho-phosphate), and 30 meq. of hydrochloric acid per 100 ml. Ten-milliliter aliquots could not be titrated satisfactorily because offading end point. However, when such aliquots were passedthrough the exchanger, the calcium could be titrated quantita-tively, and phosphorus determinations showed that the phosphatehad been removed quantitatively.

Next th e new method was compared with the oxalate-perman-ganate method on a single sample of milk ash. The dry ashrepresenting 50 ml. of skim milk was dissolved and made to 500ml. of solution containing 0.30 meq. of hydrochloric acid permilliliter. Fifteen 10-ml. aliquots of this solution, tit rat ed by thenew method averaged 120.6 zk 0.2 mg. (standard error) ofcalcium per 100 ml. of milk, a good degree of reproducibil ity.Fourteen 10-ml. aliquots analyzed by the oxalate-permanganatemethod averaged 120.8 Z!Z 0.2 mg. (standard error) of calcium per100 ml. of milk. These two means were found by Student 's t testnot to differ significantly.

In Table I are shown results of calcium analyses on twelvesamples of cows' milk. Th e three methods of preparing thesamples fo r the titration yield closely comparable results which

Table I. Analyses of Calcium in MilksM g . of Cal cium per 100 M I . of Milka

Oxalate-KMn04 Titra tion Methoddry ash Dry ash Wet ash Noncasein filtrate

117.0123.0138.0133 .5i i i : 4124 .0122.0118 .8127 .3120 .4

117.0122.61 3 7 . 01 3 2 . 5129 .61 3 1 , s132.2122.9121.7119.6128.6121.2

118.2123.4137.2133.11 3 0 . 0132.7131.6. . . .

117.1122.3136.7133.4129.0130: 5. . . .. . .. . .. . .. . .

Each value is th e average of duplicate determinations.

also agree satisfactorily with oxalate-permanganate analysis ofthe solution of dry ash. Tab le I1 shows the results of magnesiumanalyses on nine samples by the ti trati on method and the methodbased on precipitation of magnesium ammonium phosphate anddetermination of phosphate in the precipitate. The agreementbetween results by the two methods is reasonably satisfactorym-hen it is considered that both deal with very small amounts ofmagnesium and that, in the titration method, the titer due tomagnesium represents a small difference between two large titers.Table I11 shows the results of recovery tests in which either 0.92mg. of calcium, or 0.40 mg. of magnesium, or both were added assolutions of the chlorides to 10-ml. portions of milk before dryashing. Th e recovery of 101 to 103% of th e added octlcium isreasonably satisfactory , and t he magnesium recovery of 90 to 95%is probably within the range of accuracy of the magnesium deter-mination.

Table 11. Analyses of Magnesium i n M i l ksMg. of Magnesium per 100 M I. of Milk5

Titration method Precipitation method10 .21 0 . 710 .510.910 .711 .610 .71 1 . 010 .9

1 0 . 91 0 . 611.211 .410.110 .310.711 .610 .1Each value is the average of duplicate determinations on dry ashsolutions.

Table 111. Recovery by the Ti t ra t ion Method of CalciumRfg. of .Magnesium per 100 M1."

an d Magne sium Added to illilk"-4dded Fo und Added Found

Mg. of Calcium per 100 MI."009 .29 . 2

119 .6119.5129.1128.904 . 004 . 0

0 Each value is the average of quadruplicate titrations.

10 .814.611 .01 4 . 6

The anion exchange titration method is directly applicable torennet whey and milk dialyzate without incineration or wet diges-tion; identical results are obtained on unashed and ashed sam-ples. Thus , the method is admirably suited to following changesin dissolved calcium and magnesium which may occur in milk as aresult of treatmen t. Aliquots of 5-ml. of whey or dialyzate areconvenient. However, in cases in which it is desired to deter-mine dissolved calcium and magnesium shortly after a treat mentof milk (such as heat trea tment ) only a small portion ( at most1ml.) of ultrafiltrate may be available. For such a small amount,a column consisting of 0.5 gram of resin can be used through whichthe 1-ml. aliquot is passed. Thi s modification proved to beentirely satisfactory, yielding results for dialyzate identical tothose obtained with 5 ml. passed through a 3-gram column ofresin.

DISCUSSIONThe removal of phosphate by anion exchange makes possible

the application of t he tit rat ion of Schwarzenbach and coworkersto milk and milk fractions. The titration is of comparable ac-curacy to the classical methods for determining calcium and mag-nesium b ut the entire procedure including ion exchange is muchless time consuming. Doubtless the method would be usefulfo r calcium and magnesium analyses of other biological materials,such as certain plant tissues (7 , 10, 18), n which phosphate inter-feres with the titration.

The three methods of preparing milk samples for the deter-mination of total calcium and magnesium appear t o yield identi-


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968 A N A L Y T I C A L C H E M I S T R Ycal results; the choice of procedure will depend on individualpreference. The dry ashing procedure is the most time consum-ing of the three. Wet ashing is rapid, but t he anion exchangecolumns require regeneration more frequently when this teoh-nique is used because the resin becomes saturated with perchlo-ra te ions. In fact, only one 10-ml. aliquot of wet digest can bepassed through a 3-gram column while as many as 15 aliquots ofdry ash solution (prepared as described above) can be passedthrough before regenerat ion is necessary. Acid precipitation ofcasein affords a quick and simple means of prepar ing a clear solu-tion containing all of the milk calcium and magnesium. Oneslight drawback of this method is the fact t ha t the somewhat un-certain vd um e of the casein precipitate must be taken into ac-coun t in calculating results. However, this point is of minorconsideration in most work.

LITERATURE CITED( 1 ) Banewicz, J. J. , and Kenner, C. T., ANAL.CHEII.,24, 1186-7(2 ) Betr, J. D., and Noll, C. 8., .Am. W a t e r W o r k s Assoc., 42,49-(1952).56 (1950).(3) I b id . , pp . 749-54.

Biedermann, W., and Schwarzenbach, G., Chimia Prague, 2,Bushill, J. H., Lampitt, L. H., and Filmer, D . F. , J . SOC.Chem.Cheng, K. L. , and Bray, R. H., Soil Sei., 72,449-58 (1951).Cheng, K. L., Kurtz, T. , and Bray, R. H. , AXAL .CHEM., 4,Connors, J. J., J . Am . W a t e r Wor ks As s oc . , 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. D a k o t a A c a d . Sci., 8, 77-81 (194 9).Greenblatt, I. J . , and Hartman, S., A~YAL.HEM., 3, 1708-9

(1951).Kibrick, A. C., Ross, M.,nd Rogers, H. E. , Pr oc . SOC.E x p .Bid. Med . , 81, 353-5 (1952).Morris, H. P., Nelson, J. W., and Palmer, L. S., IKD.EXG.CHEM., NAL. D.,3, 164--7 (1931 ).Pyne, G . T. , Analyst, 6 8 , 330 (1943).Schwarzenbach, G., and Ackermann, H., Helu. Chim. A c t a , 30,Schwarrenbach, G., Biedermann, W., and Bangerter, F. , I b id . ,Sobel,A. E., and Hanok, -4.,Proc. SOC. x p . Bid. X e d . , 77, 737-Willson, A. E. , AXAL.CHEM., 2, 1571-2 (1950).

56-9 (1948).Ind. (London), 6, 411T41 3T (1937).

1640-1 (1952).

1798-1804 (1947).29,811-18 (1946).40 (1951).

RECEIVEDo r review Sovember 7 , 1952. Accepted February 11, 1953.Paper 2931, Scientific Journal Series, Minnesota dgricultural ExperimentalStation.

Rapid Modified Procedure for Determination of Kjeldahl NitrogenC. H . PERRIN, Canada Packers, Ltd ., Toronto, Ontario, Canada

INCE he spectacular success of Wilfarth and of Gunning in re-S ducing the time required fo r Kjeldahl digestions, many chem-ists have attempted to increase the speed of t he reactions stillfurther. Typical efforts are described by Shedd (6) ,Gerritz andS t . John (2),Lauro ( d ) , and Stubblefield and DeTurk ( 7 ) .

Digestion mixtures such as those cited are more rapid in theiraction than conventional mixtures when used to digest samplessuch as animal feeds. However, the author thought i t would beinteres ting to compare th e reaction speeds of such forniulas onnicotinic acid, a very refractory heterocyclic nitrogen compound.Kicotinic acid has been included in a number of recent studiesof Kjeldahl digestion mixtures (6, 8, 9).

Table I gives a comparison of rates of the digestion of nicotinicacid when an extremely brief reaction period is used. Methodsmaking use of oxidizing agents such as perchloric acid, selenium,persulfates, and hydrogen peroxide, which introduce uncertain-ties, were not included in this study, The proposed methodreferred to in Table I is th e subject of th is paper.

Table I . Comparison Speeds of Nicotinic Acid DigestionNicotinic Acid. 11.38% N (theoretical). 1!.26% N obtained by Kjeldahl-Wilfarth-Gunning method with 3 hours digestionDigestion. 0.5-gram sample, 12 minutes using 550-watt heaters, startinghot. 4.33-minute boil test

Method Nitrogen Found. %Kjeldahl-Filfarth-GunningGerritz and St. John (8)Stubblefield and DeTurk ( 7 )Proposed

With HgOWith CuSO4 ( I ) 1 . 6 61 .344.632.0011.29

An investigation of a wide variety of cataly sts and digestionmixtures led to the conclusion th at t he efficiency of the Kjeldahl-Wilfarth-Gunning acid-salt-mercury combination ( I ) can beincreased t o the point where additional oxidizing agents becomesuperfluous. This new combination permit ted the developmentof a rapid Kjeldahl method which offers the following advantages.

Except for methods making use of added oxidizing agents, i t is

believed to be the most rapid macrotechnique yet described.Although no use is made of selenium, phosphates, and othercontroversial additives, or of unusually intense heat, a completenitrogen macrodetermination can be made in abou t 50 minutes.In the case of all materials investigated to date, the point atwhich the digestion is complete can be determined visually.The small quantity of acid and alkali required reduces theviolence of the reactions, contributes to accuracy, safety, speed ofcooling, and makes possible the use of caustic pellets in the dis-tillation step. (T he preparation and handling of large quanti tiesof liquid caustic are, perhaps, the most hazardous and un-pleasant features of the Kjeldahl determination.)Bumping and foaming in the distillation are virtually elim-inated. This permits a more rapid boiling.The chief disadvantage of t he proposed method is that i t mill

stand less abuse than t he method of the Association of OfficialAgricultural Chemists ( I ) . It requires more careful control ofheat during digestion and more care in measuring chemicals.With some samples gentle heating during the first few minutes isparticularly important because of the high boiling point of thedigestion mixture.

RE4GENTSSilica, smooth boiling granules, not selenized (Hengar Co.,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.2Y0bromocresol green solution with 1 part of 0.2% methyl red solu-tion, both in alcohol.

Philadelphia, Pa.)

PROCEDUREPlace a 0.5- or 1.0-gram sample in a dry 500-ml. Kjeldahl diges-tion flask and add approximately six smooth boiling silicagranules, 1. 3 to 1.5 grams of mercuric oxide, and 12 =t .5 gramsof potassium sulfate. (Add no filter paper or other matter.)Swirl flask to mix, add 15f .5 ml. of concentrated sulfuric acid,and mix again. Digest for 5 minutes (o r until frothing ceases) a tlow heat, then boil at full heat until digestion is complete.(By full heat is meant a heating intensity sufficient to bring to arolling boil 250 ml. of water in a 500-ml. Kjeldahl flask in from 4 to

5 minutes. This is called the boil test .)

titration of calcium and magnesium in milk and milk fractions with ethylenediamine tetra acetate

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