Refractometric Determination of Casein in Skim Milk JOHN G . BRERETON AND PAUL F. SHARP Department of Dairy Industry, Cornel1 University, Ithaca, N. Y.
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ASEIN is the most important industrial by-product of milk. It is used in coating paper for fine halftone re-
cultural Chemists’ method (1) involving precipitating the casein, washing, and determining the nitrogen in the precipitate are shown by the points marked X in Figure 1. To serve as criteria of reliability in developing a method for milk, highly purified moisture-free casein (ash 0.016 per cent, 90) was added in varying amounts to 0.1 N sodium hydroxide and the refractometer readings were made a t 25’ C. The values obtained are represented by the circles in Figure 1. The nitrogen content of the purified casein, as determined by Kjeldahl method, ranged from 15.36 to 15.46 per cent, with an average value of 15.41 for 7 determinations. This value for nitrogen is slightly lower than the 15.67 usually given. Other preparations of purified casein also gave low values. Since the objective was to make the refractometric method agree with the A. O.A. C. method, the A. 0. A. C. nitrogen factor of 6.38 was used in calculating casein. The increase in refractive index resulting from the solution of 1 gram of casein in 100 ml. of 0.1 N sodium hydroxide was 0.00181, as shown in Table I. Robertson (18) used the value 0.00152, which he determined after extracting and drying some reagent casein (19). He gave no analytical data relative to the casein which he used. The value of 0.00152 is in fair agreement with the X values in Figure 1. The refraction of a number of samples of casein was determined and the values are presented in Table 11. The refractive index of the purer caseins approaches the limiting value of 0.00181.
production, on washable wall paper, and in water-soluble paints, glues, plastics, and wool-like fibers. Skim milk from which casein is precipitated is usually purchased in tank car or tank truck lots from butter and cream plants. It has usually been bought a t a flat price irrespective of the casein content, the only information as to the composition of the milk being that obtained from a not-toosensitive hydrometer, less than one third of which value is attributable to casein. With the increase in the price of skim milk, a simple, accurate method for the quantitative determination of casein became of increasing importance. Methods previously suggested or tried may be grouped, on the basis of the principles involved, as follows : Preci itating, washing, drying, and weighing the casein. Xjelghl nitrogen determination. The nitrogen content before and after precipitation or in the washed casein precipitate is determined and converted to protein by using the fartor 6.38 (I,%.
Volume of precipitate. The casein is precipitated Kith acid and the volume of the centrifugally packed precipitate determined (4). Formol titration. Formol titration of milk times a factor is used to indicate t’hecasein content (3,10,11, IS,14, 16,21,23). Alkali-binding power of casein. Since casein possesses appreciable alkali-neutralizing power, titrations made before and after the precipitation and removal of casein are used to indicate casein content (6, 8, 16, 28). Difference in specific gravity before and after removal of casein (gj
7 , 18).
IO I
Refractometric method. Casein is precipitated with acid, the precipitate is washed and dissolved in alkali, and the increased refraction of the solution is determined ( 1 7 ) . The refractometric method was selected because of the general satisfaction which refractometric methods have given in this type of determination, because of the high degree of accuracy which can be obtained on medium-sized samples, and because neither accurately standardized solutions nor accurate adjustment to a single specific temperature is necessary. This paper describes a technique which yields reliable results.
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X ROBERTSON METHOD OPURlFlED C A SElN
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Robertson (17) proposed a refractometric method for the determination of casein in milk, but apparently examined only one sample of milk. The authors applied his procedure to 32 samples of skim milk, using a dipping refractometer. The casein in 50 ml. of milk was Precipitated with 0.1 N acetic acid, and the casein was washed, filtered, and allowed t o drain 30 minutes. The casein and paper were then treated with 100 ml. of 0.1 N sodium hydroxide to dissolve the casein. The difference between the refraction of the casein solution and 0.1 N sodium hydroxide, divided by a constant, gives the casein content of the milk. Robertson stated that the dilution of the solution by the water retained by the casein and paper caused an error which he did not consider great enough to be of significance.
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The results which the authors obtained with Robertson’s method were encouraging, but the water held by the casein and paper led to errors larger than should be tolerated in refractometric determinations. The increase in refractometer scale reading per unit of casein varied by 12 per cent in the different samples. Data obtained by the original Robertson method compared with the Association of Official Agri-
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CASEIN
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FIGURE 1. DETERMINATION OF CASEIK Relation between increase in dipping refractometer scale reading of casein dissolved in 0.1 N sodium hydroxide and amount of casein as determined by method of Association of O5cial Agricultural Chemists.
8?2
November 15, 1942
TABLEI.
ANALYTICAL EDITION
INCREASE IN IMMERSION
REFRACTOMETER READING
urified moisture-free casein in 0.1 N sodium fydroxide solution) Increase in Refraction I~~~~~~~ Produced by 1 Gram of casein in scale Caseinin 100 M l , of NaOH ~~~i~ of 25Reading Immersion Gram D u e to refractometer Sample Casein scale Refractive of Milk a t 25" C . reading index
With varying amounts of Casein Made t o 50 111. with 0.1 N NaOH
a73
Method Applied to Skim Milk The procedure for Obtaining casein from milk gave values which agreed well with those obtained with purified casein:
To 25 grams of skim milk (the authors calibrated a ipet to deliver this amount based on a specific gravity of 1.0367 at 40" to 42" C. add with stirring 225 ml. of tap water at 40" to 42" C., containing 3.8 ml. of a solution of 10 per cent acetic acid. Gram % Let stand for 3 minutes and decant through a 12.5-cm. No. 4 0.414 1.66 3.90 4.71 0,00182 Whatman filter pa er. Wash the casein in the beaker twice by 0.461 1.84 4.30 4.66 0.00180 decantation with T5-ml. portions of water acidulated to pH 0.507 2.03 4.76 4.69 0.00181 0.553 2.21 5.16 4.67 0.00181 4.6 (0.2 ml. of glacial acetic acid per liter of water). Transfer 0.599 2.40 5.59 4.67 0.00181 the casein to the filter and then refilter the entire filtrate. When 0.646 2.58 6.05 4.69 0.00181 all the free liquid has passed through, transfer the casein and filter 0.691 2.76 6 .45 4.67 0,00181 paper to a test tube 25 mm. in diameter and capable of con0.736 2.94 6.93 4.71 0.00182 0.783 3.13 7.31 4.67 0.00181 t'aining a volume of about 70 ml. The tube should be marked 7.85 4.73 0.00183 0.829 3.32 accurately to contain 51.2 ml. Add 25 ml. of 0.2 N sodium 4.67 0.00181 0.876 3.50 8.18 0.921 3.68 8.68 4.71 0.00182 hydroxide and mash the filter paper and casein with a rubber0.921 3.68 8.73 4.74 0.00183 tipped stirrin rod, rinse the rod with distilled water, and make up to the vofume of 51.2 ml. with distilled water. The filter Av. 4.69 0.00181 paper occupies a volume of about 1.2 ml. and consequently the casein is dissolved and made up to a volume of 50 ml. of 0.1 A: sodium hydroxide. TABLE 11. INCREASE IN REFRACTIVE INDEX AT 25" C. PRODCCED Care must be taken to work out all the air bubbles before R Y DISSOLVING 1 GRAMOF CASEINin 100 ML. OF 0.1 N SODIUM adjusting to volume. Because the filter paper can be disinHYDROXIDE tegrated and the air bubbles worked out more readily in a test tube than in a volumetric flask, a test tube was used for adjustIncrease in Refractive Index ment to final volume, in spite of the disadvantage of its large Moisture Corrected cross section at the meniscus. Description of Sample Content Uncorrected for moisture After making up to volume, continue the disintegration of the 7% filter paper with the glass rod. Stir occasionally during 30 Ash-free 6.3 0.00168 0.00179 minutes and filter through a KO,3 Whatman filter paper. DeterGrain curd 1 8.8 0.00166 0.00182 mine the refraction of the solution a t approximately 25" C. Grain curd 2 5.1 0.00170 0,00179 with a dipping refractometer. Perform a control determination Crude, herd milk 8.5 0.00155 0.00169 Crude, Jersey milk 10.6 0.00153 0,0017 1 by adding only filter pa er to the test tube and otherwise follow Crude, Guernsey milk 10.6 0.00151 0.00169 the procedure describe8. Subtract the scale reading obtained Crude, Holstein milk 8.1 0.00152 0.00166 with the control solution from that obtained with the casein Crude, commercial 1 6.6 0,00167 0,00179 Crude, commercial 3 6.7 0.00167 0.00179 solution and divide the difference by 2.37 to obtain the perCrude, commercial 5 7.2 0.00161 0.00174 centage of casein in the milk. Purified moisture- and ash-free, Table I
0,00181
The approximate increases in refractive index resulting from 1 per cent of the following substances dissolved in water were sodium chloride, 0.00177; potassium chloride, 0.00140; calcium chloride, 0.00203; disodium phosphate, 0.00244; lactose, 0.00148; and sucrose, 0.00149. These results indicate that, depending on the substances present as impurities, the refraction of solutions of crude casein might show a variation through a considerable range.
This method has the very great advantage that if a control is run a t the same time, the temperature a t which both are determined may vary from 20" to 30" C. and the alkali added need be only approximately 0.2 N. Robertson (18) and others (6) have shown that the difference in refraction is relatively independent of these two variables. The authors have confirmed this observation. The results obtained on applying this method to a series of different samples of skim milk are represented by the black dots in Figure 1. The method of least squares applied to the "ash-free" casein solutions gave Equation 1.
TABLE111. DETERMINATION OF CASEIX IN SKIM MILK
Sample No. 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
19 20 21 0
(Refractometric method applied t o skim milk from individual cows, white light, 25O C.) Difference in Scale Casein, Casein Calculated from Refraction Reading A . 0. A. C. Equation 1 Equation 2 Breed a t 25' C. Method Casein Error Casein Error Holstein Holstein Holstein Holstein Holstein Holstein Holstein Holstein Holstein Guernsey Jersey Mixed Ayrshire Ayrshire Jersey Jersey Jersey Jersey JersesJersey Jersey
Single determination.
4.705 5.17 5.25 5.28 5.370 5.480 5.55 5.99 6.30 6.26 6,840 6.89 7.12 7.540 7.54 7.58 7.74 8.03" 8.63 9.246 9,li
%
%
%
%
%
1.98 2.22 2.24 2.25 2.290 2.30 2.37 2.48 2.62 2.69 2.84 2.87 3.03 3.130 3.15 3.16 3.21 3.420 3.67 3.846 3.92
2.00 2.20 2.23 2.25 2.28 2.33 2 36 2.55 2.68 2.66 2.91 2.93 3.03 3.21 3.21 3.23 3.29 3.42 3.67 3 93 3.90
+0.02 -0.02 -0.01
1.98 2.18 2.21 2.23 2.26 2.31 2.34 2.52 2.66 2.64 2.88 2.90 3.00 3.18 3.19 3.20 3.26 3.39 3.64 3.90 3.87
0.00 -0.04 -0.03 -0.02 -0.03 +O.Ol -0.03 4-0.04 +0.04 -0.05 4-0 04 +0.03 -0.03 4-0.05 4-0.04 4-0.04 +0.05 -0.03 -0.03
0.00
-0.01 -I-0.03 -0.01 1-0.07 f0.06 -0.03 t0.07 t0.06 0.00 +0.08 +0.06
f0.07 +0.08 0.00 0.00 fO.09 -0.02
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an
INDUSTRIAL AND ENGINEERING CHEMISTRY
TABLEIV. SUMMARY OF DEVIATIONS IN DETERMINATION OF CASEINBY REFRACTOMETRIC AND A. 0. A. C. METHODS Equation Deviations Used in from A. 0. A. C. Number CalculaMethod, of tions % Samples Samples
8
1 2
t0.07-0.09 tO.04-0.06 *0.00-0.03 *O .07-0.09 +0.04-0.06 +0.00-0.03
Casein % ' =
6 3 12 0
11 10
29 14 57 0 62 48
Deviation
Av. deviation
braic
60.037
+0.028
1.0.035
0.00
difference in scale reading 2.348
Av. alge-
- 0.005
(1)
and to the solutions obtained from the casein precipitated from milk the equation: difference in scale reading - 0.003 Casein yo = (2) 2.371
Vol. 14, No. 11
Experiments were made in which the original first filtrate wa8 refiltered before adding the wash water and casein, and after the first, and after the second wash water had been filtered, as well as after the casein had been transferred to the filter. Results of a typical experiment are presented in Table V. The filtrates were clearer, more uniform, and lower in nitrogen and more of the small particles of casein were retained on the filter if the last-mentioned procedure was used. This type of filtration procedure is also employed in the A. 0. A. C. method. Although refiltering earlier in the procedure may save time and may occasionally give fairly good results with some samples of milk, yet milk is encountered frequently where the shortened procedure gives turbid filtrates and leads to error. Therefore, it is recommended that filtration of all samples be carried out as described in the method.
Summary
The casein in 25 grams of skim milk is precipitated with dilute acetic acid a t 40" to 42" C., the precipitate is washed and dissolved in 0.1 N sodium hydroxide, and the refraction is compared with a 0.1 N sodium hydroxide solution containing no casein, The refractive index of the solution is increased by 0.00181 when 1 Pram of Durified casein is dissolved . .. in 10Cml. of 6.1 N sodium hydroxide. TABLEv. EFFECTOF INTRODUCIiYG REFILTRATION STEP AT VARIOUS ST.4GES This method has the distinct advan(High-protein milk, total protein 4.76%) tage that if a control solution containing Difference no casein is run for comparison, the conTotal in Scale Difference Stage of Condition Prptein Protein Reading of i 2.371 centration of alkali need be known only Refiltering of in Less Step Filtrate Filtrate Filtrate Precipitate Casein approximately and the temperature may % % range from about 20" to 30" C., proAfter first filtration Turbid 1.03 3.73 9.08 3 83 vided that the unknown and the control After first solution contain the same amount of wash water Turbid 1.01 3.75 9.04 3.81 After second Faintly alkali and the refraction of both is de0.95 3.81 9.25 3 90 turbid wash water After transfer Faintly termined a t the same temperature. 0.96 3.80 9.24 3 89 turbid of casein The average deviation between results A. 0. A. C. .... 0.92 3.84 obtained with the refractometer procedure and the A. 0. A. C. method was 0.04 per cent of casein, and the maximum error was 0.06 per cent when applied to 21 samples of skim milk in the casein. From the practical analytical standpoint, of a wide range of composition. the extra effort required to lower the impurities further is not justified, since Figure 1 clearly indicates the close agreement between the results obtained with milk and with the pure Literature Cited casein. Table I11 presents data from the milk of individual cows. Assoc. Official Agr. Chem., Official and Tentative Methods of Such milk is more variable than mixed milk and presents Analysis, 5th ed., pp. 270-1 (1940). greater difficulties in analysis. This table gives the difference Brereton, J. G., Baldwin, K. M., and Sharp, P. F., unpublished data. in refractometer scale reading, casein determined by the Burg, B. van der, and Habers, L., "De Waarde van de FormolA. 0. A. C. method, casein content m calculated from the retitratie voor de Berekening van hat Eiwitgehalte der Melk", fractometer reading by Equations 1 and 2, and deviations Handel Genootschap., Bevord Melkkunde, 1935. between the calculated and the casein by the A. 0. A. C. Hart, E. B., Wisconsin Agr. Expt. Sta., Bull. 156 (1907). Herlitzka, A,, Kolloid Z., 7, 251-6 (1910). method. The differences are small as can be expected, conHerrington, B. L., and MacAllister, E., "A Simplified Casein sidering that slight errors are involved in both methods. Determination" (unpublished). The samples are arranged in order of increasing casein content Lindet, L., Ann. chim. anaZ., 7, 361-3 (1902). and the deviations seem to be about equally distributed Matthaiopoulos, G. T., 2. anal. Chem., 47,492-501 (1908). Moir. G. M.. A m l u s t . 56. 147-9 (1931). through the series. The agreement with Equation 2 is Pyne; G. T.,'B i o c L k . J.,26, 1006-14 (1932). slightly better than with Equation 1 and this would be exIbid.,27, 915-17 (1933). pected since Equation 2 was being applied to the data from Richmond, H . D., Analyst, 28, 138-40 (1903). which it was derived. The fact that the agreement between Ibid., 33, 113-17 (1908). Ibid., 36, 9-12 (1911). the equation derived from the purified casein and the casein Richmond, H . D., and Miller, E . H., Analyst, 31, 224-6 (1906). from the milk is good indicates that the amount of contamiRobertson, T. B.. J . Biol. Chem., 2,317-83 (1906): of. OD. 328-34. nating material was small. The deviations summarized in Robertson, T. B., J. IND.ENO.CHEM.,1, 723-5 (1909); Table IV show that with Equation 2 no error greater than Robertson, T . B., J . Phys. C h a . , 13,469-89 (1909). Ibid., 14, 528-68 (1910). 0.06 was encountered. Sharp, P. F., and Struble, E. B. (unpublished). The warming prior to precipitation and the filtration Steinegger, R., 2. Nahrungs u. Genussm., 10, 659-71 (1905). procedure were developed only after numerous attempts to Van Slyke, L. L., and Bosworth, A. W., N. Y. (Geneva) Agr. obtain clear filtrates of uniform nitrogen content and reproExpt. Sta., Tech. BUZZ. 10 (1909). Walker, W. O., J. IND. ENQ.CHEM.,6, 131-3 (1914). ducible refraction of the solution on dissolving the precipitate.
The intercepts are negligibly small and can be disregarded. The relation is linear. Equation 2 differs from Equation 1 by representing a line slightly steeper in slope. This would be expected because of traces of impurities still remaining
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