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T H E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
STUDIES ON TECHNICAL CASEIN. 111-METHODS ANALYSISI By Roscoe H. Shaw
OF
RESEARCH LABORATORIES O F THE DAIRYDIVISION, BUREAUOF ANIMAL INDUSTRY, U S. DEPARTMENT OB AGRICULTURE, WASHINGTON, D. C. Received June 28, 1920
T h e Forest Products Laboratory, which was charged with the supervision of t h e manufacture of glue from casein, issued a set of methods covering t h e testing of casein. These methods embodied t h e best information available a t t h e time and were officially adopted by t h e Office of Aircraft Production. It was soon found, however, t h a t in some cases wide discrepancies arose among t h e results of different analysts, and it therefore became a matter of vital importance t o study analytical methods i n order t h a t the extent t o which grain-curd casein met t h e specifications might be determined with greater certainty. As methods of analysis have since been extensively reported from the Forest Products Laboratory12only a brief summary of our experience will be given.
Vol.
12,
No.
12
TABLE I-RESULTS ON MOISTUREOBTAINED B Y DIFFERENTANALYSTS Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Analyst Per cent Per cent Per cent Per cent Per cent 1 8.96 9.34 9.16 8.44 8.15 2 3 4 5
8.75 7.55 9 01 8.63
8.79 7.80 8.80 8.42
7.74 7.80 8.42 8.16
8.00 6 99 7.89 7.77
7.55 7.48 8.05 7.87
have passed i t , 8 per cent water being allowed as t h e maximum, while t h e other four would have rejected it. T h e range is from 7 . j5 t o 9.01 per cent, or a difference of 1.46 per cent between t h e highest a n d lowest figures. I n Sample 2 , t h e range is from 7 . 8 0 t o 9 . 3 4 per cent. This sample would also have been passed by one analyst and rejected b y t h e others. Substantially t h e same discrepancies are found in t h e other cases. It requires no further discussion t o show t h a t a method yielding such widely different figures i n t h e hands of different analysts will not meet t h e requirements. It is also evident t h a t t h e open-dish method yields results t h a t are too low. For reliable results in moisture t h e standard method of drying t o a constant weight i n partial vacuum a t t h e boiling point of water should be used.
MOISTURE
ASH
The tentative method upon which t h e specification was based called for drying a 3-8. sample of casein for 5 hrs. a t 98’ C. An electric oven was first used, b u t it was impossible t o maintain all parts of t h e oven a t t h e same temperature, and unless t h e dishes were placed side by side t h e checks did not agree. Better results were obtained i n a large double-walled water oven. T h e temperature around t h e center remained quite constant, about 98’ C., and very satisfactory checks were obtained. As a part of t h e regular routine i n t h e determination of f a t by t h e ether-extraction method, t h e Bureau of Chemistry determined t h e moisture in 79 samples of casein submitted b y us. These determinations were made b y drying t o constant weight i n partial vacuum at t h e boiling point of water. Moisture determinations were also made on t h e same samples by t h e tentative method mentioned above, using t h e double-walled water oven. Space does not permit t h e publishing of these figures i n detail. T h e average per cent of moisture for 79 samples, as determined i n t h e open dish a t 98’ C., was 7.44. The corresponding per cent determined by t h e partial-vacuum method was 8.21,showing a difference of 0.77 per cent in favor of t h e latter method. I n connection with t h e ash method, i t was thought desirable t o see how closely several different analysts would agree when following t h e tentative method independently. Five samples of casein, representing rather a wide range, .were placed in wide-mouthed, glass-stoppered bottles, and submitted t o five analysts. I n four cases t h e analysts were allowed t o t a k e their own subsamples from t h e bottles. T h e per cents of moisture obtained b y t h e different analysts are given in Table I. A study of t h e figures reveals some interesting points. I n t h e case of Sample I , one of t h e analysts would
The tentative method for ash, in t h e hands of different analysts, yielded discrepancies even greater t h a n in t h e case of t h e moisture. These discrepancies, however, were limited largely t o low-ash caseins. This is well illustrated i n Table 11, which gives the results of t h e determination of ash by five analysts who included this work with t h e moisture determination discussed i n a preceding paragraph.
1 2
Published b y permission of the Secretary of Agriculture. F. L. Browne, THISJOURNAL, 11 (1919), 1019.
TABLE11-RESULTS Sample 1 Analyst Per cent 1 1.70 2 3 4 5
AVERAGE
1.90 1 20 1.14 1.53 1.49
ASH OBTAINED B Y DIBBERENT ANALYSTS Sample 2 Sample 3 Sample 4 Sample 5 Per cent Per cent Per cent Per cent
ON
3.46 3.61 3.20 3.14 3.36 3.39
4.66 4.74 4.53 4.37 4.38 4.54
4.05 4.12 3.48 3.67 3.58 3.78
3.31 3.68 3.35 3.37 3.43 3.43
Sample I is a low-ash casein, t h e others are medium and high. Considering t h e average figure for each sample as being t h e correct figure, it is found i n Sample I t h a t t h e individual determinations vary as much a s 2 7 per cent from t h a t figure, while i n t h e others t h e largest variation is b u t 9 per cent. I n endeavoring t o explain t h e discrepancies i n t h e ash determination on low-ash casein, t h e thought a t once occurred t h a t there was too little base present t o hold back t h e organic phosphorus, and t h a t i n consequence t h e phosphorus not fixed was volatilized t o a small or large extent, depending on t h e temperature and length of time of ignition. To find whether this was t h e case, a solution of calcium acetate was prepared by dissolving calcium carbonate in acetic acid according t o t h e methods of t h e A. 0. A. C.l Three grams of t h e casein were weighed into a quartz dish a n d j cc. of t h e calcium acetate solution added. Two different solutions were used, one yielding 0.0761 g. CaO and t h e other 0.1523 g. CaO per 5 cc. on ignition. The dishes were allowed t o stand several minutes until t h e solution was ab1 Association of OfficialAgricultural Chemists, “Official and Provisional Methods of Analysis,” 1908; U. S. Dept of Agriculture, Bureau of Chemistry, Bulletin 107 (revised), 21.
Dec.,
1920
T H E J O U R N A L OP I N D U S T R I A L A N D ENGINEERING CHEMISTRY
sorbed, then dried in a drying oven, carefully charred over a small flame, and finally ignited in an electric muffle furnace maintained a t low redness until a white or nearly white ash was obtained. The amount of CaO introduced by t h e calcium acetate solution was subtracted from t h e weight of t h e ash. Early in t h e work, until quartz dishes were obtainable, porcelain dishes were used. It was noted t h a t t h e glazing on these dishes soon became roughened, and i t seemed possible t h a t a base might be supplied t o t h e phosphorus by t h e glazing. T o prove this point, determinations were run in both quartz and porcelain dishes. The results are included with those from t h e added base experiment in Table 111. The phosphorus was determined in t h e ash as follows: The ash was dissolved in 5 t o I O cc. of concentrated nitric acid on t h e stearn b a t h ; washed into a small beaker, and boiled t o oxidize t h e phosphorus; neutralized with ammonia; made faintly acid with nitric acid; and t h e determination completed i n t h e usual manner, using ammonium molybdate and magnesia mixture.
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FAT
According t o t h e tentative methods of t h e Forest Products Laboratory, this determination is made by extracting with ether in t h e usual way in a continuous extractor. The method is time-consuming, and in an effort t o reduce this time t h e Roese-Gottlieb method was modified t o apply t o casein, as follows: Weigh out a I-g. charge of t h e casein into t h e Roehrig tube, and add I O cc. of water. Shake vigorously, b u t not so as t o carry particles of casein near t h e top of t h e tube. Let soak for a t least 15 min.; a longer time is advisable if t h e sample is not finely ground. Add 2 cc. of strong ammonia water, and shake vigorously, again taking care not t o carry particles oE casein near t h e top of t h e tube. Let stand I O min. with occasional shaking. Add I O cc. of 95 per cent alcohol and shake until t h e casein is completely dissolved. From this point proceed as usual with t h e Roese-Gottlieb method. The signing of t h e armistice made t h e work on casein less pressing, and this phase of t h e work was not carried so far as might be desired. Table I V gives TABLE111-EFFECT OF ADDEDCALCIUM ACETATE ON THE DETERMINATIONt h e results on fat in casein both by t h e ether-extracOF ASH CONSTITUENTS tion method and by t h e modified Roese-Gottlieb HighLow- Medium- MediumAsh Ash, Ash Ash method. The determinations b y t h e ether-extraction Casein Casem Casein Casein METHOD Per cent Per cent Per cent Per cent method were made by t h e Bureau of Chemistry. Ash, untreated sample, porcelain dish .......................... Ash, untreated sample, quartz dish. Ash 5 cc Ca(CzHaOz)z solution (6,1523g: CaO added). . . . . . . . . . Ash 5 cc. Ca(CzHaOz)z solution (6.0761 R. CaO added) P untreated sample.. . . . . . . . . . . . . P: 5 cc. Ca(CzHa0z)zsolution (0.1523 g. CaO added). . . . . . . . . . . . . . . . . P,5 cc. Ca(CzH3Oz)zsolution (0.0761 g. CaO added).
..........
................
1.51 1.68
2.41 2.45
3.09 3.22
4.13 4.20
2.25
2.66
3.23
4.60
2.23 0.51
2.54 0.77
3.26 0.74
4.52 1.05
0.80
0.80
0.88
1.04
0.80
0.80
0.89
1.04
It will be noted t h a t about 2 5 per cent more ash was obtained i n t h e case of t h e low-ash casein when a base was added than was obtained b y plain ignition; also t h a t this ash contained about 35 per cent more phosphorus t h a n did t h e ash from t h e untreated casein. This is not t r u e of t h e medium- and high-ash caseins, which of course would follow on t h e assumption t h a t these caseins contained sufficient calcium. Calcium determinations were made on two of these samples by t h e method described in another paragraph. The low-ash casein contained 0 . 2 8 per cent CaO, while t h e high-ash casein contained 1.68 per cent CaO. These results indicate t h a t an added base is necessary in low-ash caseins if accurate results are desired. It is not necessary in medium- or high-ash caseins. There seems t o be little, if any, difference in t h e results obtained in porcelain and quartz dishes. Those made of quartz are in every way preferable, however. Quartz evaporating dishes 2 in. in diameter are a convenient size; t h e porcelain dish will not stand more t h a n a few ignitions before cracking. T h e best results in ash work are obtained by first heating over a very low flame and taking care t h a t t h e contents of t h e dishes do not burst into flame. This operation should take about an hour. After t h e casein has become thoroughly charred, t h e dishes are placed in a muffle furnace (preferably electric) and t h e ignition completed a t as low temperature as is consistent with obtaining a white or nearly white ash.
TABLEIV-COMPARATIVE FIGURESFOR FAT OBTAINED B Y THE ETHEREXTRACTION METHODAND THE MODIFIED ROES$-GOTTLIEB METHOD
__
W - st
SAMPLE Grain-curd casein. HzSO4 cooked-curd casein. Buttermilk casein..
....................
Pat
by the by the-Modified Ether-Extraction Roese-Gottlieb Method Method Per cent Per cent 0.08 1.50 0.20 2.35 3.79 3.92
............. ...................
It will be noted t h a t t h e results by t h e Roese-Gottlieb method are higher t h a n those obtained b y t h e regular extraction method. Two explanations naturally suggest themselves: ( I ) T h e ether-extraction method does not remove all, t h e f a t ; and ( 2 ) t h e extract in t h e Roese-Gottleib method contains other material t h a n fat. We believe t h e first explanation is t h e t r u e one. The grains of casein are hard and not easily penetrated by t h e solvent. This is shown very well in a later table giving t h e results on sugar extracted with 5 0 per cent alcohol from casein of different sized granules. I n t h e modified Roese-Gottlieb method t h e casein goes into solution, and it is impossible for any € a t t o remain locked u p in t h e casein granules. More work is needed along this line, b u t there is certainly good evidence for believing t h a t t h e ether-extraction method fails t o remove all t h e fat, except perhaps when t h e casein is ground t o impalpable powder. SUGAR
Sugar in casein was determined b y adapting t h e method for determining sugar in cattle food. Ten grams of t h e casein were weighed into a 500-cc., widemouthed bottle, and 2 5 0 cc. of 5 0 per cent alcohol added. The bottle was shaken for 4 hrs. in a motor-driven shaking machine, then allowed t o stand for several hours, and t h e solution decanted. This solution was then centrifuged, although this step may not have been absolutely necessary. One hundred cc. more or less, according t o t h e quantity of sugar pres-
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T H E J O U R N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y
e n t , of this nearly transparent solution were taken for t h e determination, which from this point was conducted according t o t h e method of Bryan, Given, a n d Straughn.l Results are given in Table V. TABLE V-SHOWING
PER CENT OF
SUGAR I N
SAMPLES ANALYZED~
SUGAR
SAMPLE
Per cent Natural-sour casein. Trace HzSOa cooked-curd casein.. 1.13 Grain-curd buttermilk casein. 0.95 HzSOi cooked-curd buttermilk casein. 0.62 0.33 uncooked-curd buttermilk casein. .. Grain-curd casein not washed. 3.96 Grain-curd casein’ washed well.. .............................. 1.43 5.43 Grain-curd casein’from partly sour skim milk not washed.. 0.56 Grain-curd casein from partly sour skim milk,’ washed well 1 For description of various manufacturing types of casein see bulletin cited. The so-called grain-curd casein listed in this and other tables was made accordina t o a urocess develoued - bv - the Dairv Division. described in P a r t I1 of this series. Arnold 0 Dahlberg “The Manufacture of Casein from Buttermilk or Skim Milk,” U: S. Dept. hf Agriculture, Bulletin 661 (1918).
......................................... .................................. ................................ ....
............................... ...... .......
I n t h e sugar determination t h e identity of the casein granule is not lost, a n d in order t o learn whether t h e size of t h e granule has a h influence on t h e quantity of sugar extracted by t h e 50 per cent alcohol, two samples of casein were separated by means of sieves into lots of different-sized granules. Table VI gives t h e per cents of sugar obtained on t h e different lots. TABLE VI-EFFECT OF SIZE OF GRANULES ON DETERMINATION OF Sample 1 2
Original Sample Per cent 0.56 2.09
Less than 100 Mesh Per cent 0.90 3.09
40 and 100 20 and 40 Mesh Mesh Per cent Per cent 0.65 0.57 2.01 1.53
SUQAR
Over 20 Mesh Per cent 0.50
CALCIUMOXIDE -CaO------.
.
.....................
PHOSPHORUSIN ASH
-PHOSPHORUS-
....
MagIn Aquanesiurn Ash Regia Nitrate Per Per Per cent cent cent 1.08 0.95 1.16 0.85 0.73 1.00 1.18 1.04 1.09 1.08 1.08 1.25
none 2.43
0.22 1.37
0.76 0.94
0.87 1.34
0.32
0.59
0.77
0.89
1.12 0.08
........
1.03 0.79
Aqua In Regia Ash Per Per cent cent 0.90 0.94 0.54 0.53 1.59 1.76 1.96
Ash Per DESCRIPTIONOF SAMPLB cent Argentinecasein 3.68 Natural-sour casein ............ 2.63 HzS04 cooked-curd casein.. . . . . 4.41 H ~ S O uncooked-curd I casein. . . . 5 21 Grain-curd casein, excessively washed 0 . 3 8 none HCl cooked-curdcasein ...... ~.5.95 2.50 Grain-curd casein made in Dairy Division . . I . . . . . . . . . . . 1.57 0.24 Grain-curd casein, made in factory ....................... 3 . 6 3 1.26 Ejector buttermilk casein,. ..... 1.27 0.12
..............
AND
........
The determination of calcium offers no difficulty, since all of i t is found in t h e ash after ignition. It can be determined also by digesting t h e casein with “Extraction of Grains and Cattle Foods for the Determination of Sugars: A Comparison of the Alcohol and the Sodium Carbonate Digestions,” U. S. Dept. of Agriculture, Bureau of Chemistry, Circular 7 1 (191 1). a Association of Official Agricultural Chemists, “Official and Provisional Methods of Analysis,” 1908; U. s. Dept. of Agriculture, Bureau of Chemistry, Bulletin 107 (revised), 2. 1
No.
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aqua regia. The custamary procedure of precipitating with ammonium oxalate a n d igniting t o calcium oxide was followed. Table VI1 gives t h e results of t h e determination of phosphorus a n d calcium i n t h e samples of casein analyzed. The figures are all based on the moisturefree condition. Attention may again be called t o the behavior of t h e low-ash casein when ignited. I n t h e case of t h e grain-curd casein which was excessively washed, there was no calcipm found i n t h e ash. The true per cent of phosphorus as found by treatment with magnesium nitrate was 0.87, while t h a t found in the ash was b u t 0 . 2 2 , showing t h a t nearly 7 5 per cent of the phosphorus was lost while ashing, since there was no calcium present t o combine with it. I n t h e case of t h e grain-curd casein made i n t h e Dairy Division, which has a low calcium content, t h e t r u e per cent of phosphorus was 0.89, while t h a t remaining in the ash was b u t 0.50 per cent. ACKNOWLEDGMENT
T h e author wishes t o make acknowledgment t o Messrs. F. C. Lamb and A. Biddle who were detailed by t h e Bureau of Aircraft Production t o assist in t h e analytical work. SUPPLEMENTARY NOTE ON THE “FREE ACIDITY” NICAL CASEIN
Reference has already been made t o t h e determination of phosphorus and t h e method given t h a t was used in its determination i n t h e ash. Several methods were tried for determining t h e phosphorus in casein without ashing i t , among them being digestion with aqua regia. This method gave low results. The method which was finally adopted, and which gave very satisfactory results, was t h e official method for determining total phosphorus i n fertilizers, using Method ( a ) z for oxidizing and getting t h e casein into solution.2 It was not found necessary t o evaporate after adding the magnesium nitrate solution, since b y careful manipulation the mixture could be dried and ignited over a Bunsen burner in one operation. CENT OF
12,
....
P K O S P H O R U S AKD CALCIUM
TABLE VII-PER
Vol.
OF TECII-
By W.M.Clark The Office of Aircraft Production specified that all casein, to enter into glue for aircraft should have not more than a certain quantity of “free acid.” The fact that grain-curd casein was to be washed with acidified water seemed to defy the intention of the specifications and caused some surprise. It may be well, therefore, to add a word of explanation. Theoretically a t least, casein when precipitated a t its isoelectric point should consist chiefly though not wholly of free casein uncombined with acid or alkali. The isoelectric point represents only a minimum €or both alkali and acid caseinates, so that there must remain some of each which would doubtless be carried down together with some of the milk salts. In the graincurd process, however, the isoelectric point is somewhat overstepped, so that a little acid caseinate may be produced. This, however, tends to release the casein from its basic combinations. As mentioned before, complete equilibrium is not attained, but the tendency is in the desired direction. Now, on washing with water held just a trifle on the alkaline side of the isoelectric point, there is a continuation of the environment which alone is conducive to the separation of pure casein. At that reaction the strong acid, hydrochloric, can exist “free” only to a very minute extent. If chlorides enter into the commercial product it is only because the washing has not been extensive. Extensive washing would finally require only an indetectable quantity of acid, and a t last when free casein alone was present its own acidity would bring the acidity of pure distilled water to the isoelectric point of pH 4.6. Pure water would require only to be about O.OOOOI N with hydrochloric acid to be a t the isoelectric point of casein. This is an extreme case, but it represents the direction toward which the logic of the method points. To differentiate the chloride content due to the natural salts of the milk and that due to the absorbed acid added during the process of manufacture would be a difficult task. And to separate titrable acidity due to acidic salts from that due to absorbed acid is equally difficult.
,
