February, 1927
. ISD CSTRIAL A N D En'GINEERING CHEMISTRY
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Determination of Gelatin in Ice Cream',' By Roe E. Remington and L. H. McRoberts NORTHDAXOTA FOOD COMMISSIOS, BISMARCK, N. D.
In reaching his ralue for the specific rotation of coinniercial gelatin (-122), Ferris \vas guided by data on four samples, the origin of which he does not give, and by the work of Smith, who used only six samples, mostly of imported gelatin. From his observed rotation of -123", Smith calculated that the rotation on moisture and ash-free basis should be -141", assuming for this purpose 11.4 per ~ cent moisture and 1.6 per cent ash. B ~ g u e ,however, reports that the moisture content varies with the humidity and with the grade of the material, higher grade products b e i n g inore hygroscopic. For medium humidity and best grades he finds 15 to Changes in the Ferris method for the estimation of Ferris Method 16 per cent of moisture. If gelatin in ice cream are suggested to make the method -141" is the correct rotamore accurate and better adapted to use in inspection Ferris' procedure in detail tion for pure dry gelatin, (1) smaller sample, (2) weight inanalysis. These are is as follows: then -123" is too high f o r stead of volume dilution, (3) control of the casein pregelatins containing over 15 cipitation by means of hematoxylin indicator, (4) the Weigh 450 grams of i c e per cent of moisture and ash. cream into a 500-cc. flask, use of alum to assure complete precipitation of the I n c a l c u l a t i n g gelatin warm t o 35', and dilute t o gelatin, and (5) the correction of the nitrogen found t h e mark. Transfer t o a from the nitrogen deterfor that from the milk which comes through into the larger flask and add 10 per mination Ferris made use of cent acetic acid until the filtrate. the nitrogen values of the casein is coagulated, observNitrogen determinations and specific rotation values individual samples useding the volume of acetic acid have been obtained for twenty-eight samples of comused. Calculate t h e volume 15.7 per cent for three sammercial ice cream gelatin, the average value of the nitroof the fat and casein (volume ples and 14.8 per cent for of fat = weight of fat in samgen being 14.84 and that for the specific rotation 117, the fourth. I n his method, ple X 1.075, volume of casein corresponding to 17.53 and 139 on the dry, ash-free however, he directs the use = weight of casein in sambasis. These values are recommended for calculating ple X 0.73) and determine the of the factor 5.56, which gelatin determinations in ice cream. volume of serum by subtractcorresponds to 18 per cent inn this from 500 DlUS the of nitrogen, the value which nimber of cc. of acetic acid was formerly assigned to added. If the curd does not settle readily, shake with a small amount of carbon tetrachloride, dry, ash-free gelatin. Sinit'h6 prepared ash-free gelatin centrifuge, and decant the supernatant serum. Measure 100 cc. and found it to contain 17.53 per cent of nitrogen on the of the serum into a 400-cc. lipped beaker and while still warm dry basis, which is comparable with the results of Halla7 add slowly, with stirring, 200 cc. of 95 per cent alcohol. Cool (17.61) and of Bogue8 (17.50). But if 17.50 is the correct in ice water and let stand until the precipitate settles, leaving the supernatant liquid clear. At this point the precipitate may value, the factor should be 5.71, and if it is desired to calcube allowed t o settle overnight in a n ice box a t as near zero late gelatin on the basis of the original commercial material Centigrade as possible. Filter on a large hardened paper in a Biichner funnel and wash with cold alcohol, two volumes of 95 a still higher factor must be employed, perhaps 6.7 or 6.8.
ERRIS3 has published a method for the estimation of gelatin in ice cream and dairy products, based in part on Smith's work on the optical rotation of gelatin.4 Ferris reports very satisfactory results on a number of ice cream mixes containing known quantities of gelatin. The writers undertook a study of this method, with the thought that it might be suitable for use in food inspection laboratories. An accurate method for the estimation of gelatin, in conjunction with the determination of total nitrogen, would make it possible to determine the milk proteins and gain an idea as to total milk solids.
F
per cent alcohol, and one of water. Drain the precipitate, remove t o a small beaker, and rub up with 50 cc. of water. Allow to soak a t room temperature until the gelatin swells, then heat t o about 90" C. in a hot water bath, filter, and wash with hot water. Make up to 100 cc. a t 35" C. and polarize. Calculate the percentage of gelatin in the sample from the reading a t 35" from the following formula: R X 0.346 X V X 100 when Percentage of gelatin = Rr x 2 x specific rotation' I.V .= weight of sample, V = volume of acetic acid serum, and R = polariscope reading in Ventzke degrees in a 2-decimeter tube. Determine nitrogen in a n aliquot of the gelatin solution and calculate the percentage of gelatin in the ice cream as follows: N X V X 5.55 X 100, when Percentage of gelatin = a X W 65' = weight of sample, 'L = volume of acetic acid serum, a = volume of aliquot of gelatin solution, and N = weight of nitrogen in aliquot of gelatin solution. , 1 Presented before the Diviqion of Agricultural and Food Chemistry a t the i2nd Meeting of the American Chemical Society, Philadelphia, Pa., September 5 to 11, 1926. 2 Published by permission of R. 0. Baird, State Food Commissioner and Chemist of North Dakota. 3 J. Dairy Sci., 6, 555 (1922). 4 J . A m . Chem. S O C ,41, 135 (1919).
Analysis of Gelatin
Obviously, if the method is to be applied in inspection work, where the analyst has no means of knowing the constants on the particular lot of gelatin used in a commercial ice cream, it cannot succeed unless it is possible to deduce average values for the rotation and for nitrogen which can be applied without too great an error to any grade of gelatin likely to be encountered. I n order to obtain such average values, if possible, we have procured samples of gelatin from ice cream makers in North Dakota and also from manufacturers catering to the ice cream trade. The moisture, ash, nitrogen, and specific rotation of these samples as determined by us are given in Tables I and 11. The results given in Table I1 are doubtless more reliable as truly representing ice cream gelatin than are those in Table I, as the samples came directly from makers of gelatin, 5 "Chemistry and Technology of Gelatin and Glue," p. 429, McGrawHill Book Co., 1922. 6 J . A m . Chem. Soc.. 43, 1350 (1921). 7 Z . n n g m . Chem., 20, 24 (190i). 8 Chem. & Met. Eng., 23,105 (1920).
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thus avoiding the possibility of contamination which exists when the packages are opened in ice cream establishments. From the data here presented, 14.84 per cent may be set up as the nitrogen value, corresponding to a factor of 6.75, and 117 may be set up as the specific rotation, of ordinary ice cream gelatin. The maximum error due to differences in composition, using these values, will be 3 per cent of the amount of gelatin present, and the probable error very much less than 3 per cent. Table I-Composition
SAMPLE MOISTCRE ASH
Per cent Per cent 6054 6055 6056 6057 6058 6059 6062 6070 6071 6072 6073 6074 6075 High
Low Av.
ROTATION Ox D R Y , A S H - F R E E BASIS IN 1 % SOLN. A T 350 c. Nitrogen Rotation
P e r cent
13.03 14.48 14.50 14.31 12.76 14.74 13.81 15.25 13.17 12.73 16.53 15.58 14.68
1.61 1.47 1.58 0.86 1.97 2.51 1.00 1.58 1.53 0.98 2.05 3.12 2.34
15.26 14.95 14.87 15.28 15.26 14.61 15.23 14.50 14,96 15.30 14.20 14.27 14.67
123 114 114 121 118 116 118 119 122 124 114 108 111
15.58 12.73 14.20
3.12 0.86 1.74
15.30 14.20 14.81
124 108 117
Per cent
Per cent
18.02 17.23 17.69
144 133 139
Commercial ice cream weighs from 500 to 600 grams to the frozen quart, with an average weight of possibly 550 grams. As a quart makes a convenient sample, it appeared desirable to so modify the method as to permit the use of a quantity smaller than 450 grams, thus making sure that enough material remains for the other determinations to be made, as well as for a reserve supply. Further, ice cream contains a large amount of air incorporated through the mass in finely divided form, and on account of the colloids present this air escapes very slowly, even when the sample is melted. Many samples, particularly those which contain milk powder, in which the casein is but imperfectly brought into solution, will on melting separate a frothy curd from which all the air cannot be removed. A method involving dilution to definite volume is thus open to criticism when applied to commercial ice cream. We have avoided this difficulty by using weight instead of volume dilutions. By so doing we also avoid the rather inconvenient procedure of making to volume and pipetting a t 35". Precipitation of Casein
Ferris directs to "add acetic acid until the casein coagulates." This is rather indefinite. I n practice we find that the quantity of acid required to produce a visible flocculum varies with the temperature, the rate of stirring, and the rate at which the acid is added. Casein is least soluble at its isoelectric point (pH about 4.6), and if this point be not reached, or exceeded, the precipitation is not complete. Clark9 and his associates have suggested the use of methyl red as an indicator. They, however, applied the indicator t o the whey. If methyl red be added to the ice cream mix it dissolves in the fat with yellow color, which is not affected by acid. Hematoxylin gives a much better end point under these conditions, but its p H range (5.0 to 6.0) is somewhat on the alkaline side of the point desired. The change in milk is from pink to yellow as the acid side is approached. Practically all commercial ice creams contain enough fat to carry the curd to the top and keep i t there, nor could we obtain satisfactory settling by adding carbon tetrachloride. 9
THISJ O U R N A L , 12, 1163 (1920).
Vol. 19, No. 2
We have therefore adopted the method of filtering through a small bag of cotton cloth placed in a beaker or funnel. The serum or whey is not clear, nor need it be, as the albumin and traces of fat remaining are removed by subsequent treatment. A saturated solution of alum can be used instead of the acetic acid. The curd in this case is very fine in grain and does not coagulate into lumps nor settle. It can, however, be rapidly filtered out by paper. The use of alum has other advantages to be mentioned later.
of Gelatin Used b y Ice Cream Makers h-ITROGEN
.
Precipitation of Gelatin
In our earlier trials by the Ferris method we always obtained low results, usually recovering about 90 per cent of the gelatin added. I n casting about for an explanation, it was noted that if 2 volumes of alcohol be added to a gelatin solution, no definite precipitate is produced, even a t 0" C., if the gelatin solution is of 0.5 per cent concentration, and at 1 per cent only a part of the gelatin is precipitated in visible form and the precipitate is not filterable. As in ice cream filtrates the precipitate is filterable and the liquid is clear, it must be the albumin which drags the gelatin down with it. We have, however, recovered traces of gelatin from the clear alcdholic liquors after filtration. Apparently then some different method is needed to insure complete p r e cipitation of the gelatin. We should expect that isoelectric gelatin would be precipitated by alcohol. Perhaps the difficulty is due to B slight deviation from the isoelectric point, which cannot be judged a t all sharply by indicators in a milky solution. If this be so, the addition of a polyvalent cation should help. Actually, a few cubic centimeters of a saturated solution of alum, with the alcohol, precipitates the gelatin promptly and completely a t room temperature. Alum alone does not precipitate isoelectric gelatin, nor does alcohol alone, but the two together work beautifully. We have recovered gelatin quantitatively from 0.5 per cent solution by this method. The precipitate swells and clears when placed in water and dissolves when warmed, and apparently the rotation is not affected. Table 11-Composition
of Ice Cream Gelatins f r o m Manufacturers
ROTATION ON DRYA S H - F R E E SAMPLEMOISTUREASH NITROGEN BASIS
.$Gi.%T 35" C .
P e r cent 6065 6066 6067 6068 6069 6076 6077 6078 6079 6080 8081 6082 6083 6084 6085 High
Low -4-2.
Per cent Per cent
Nitrogen
Rotation
P n cent
141 136 139
12.45 11.71 13.30 14.96 14.69 13.93 13.23 13.01 13.17 14.64 15.33 13.73 14.08 13.40 13.20
0.74 0.75 0.97 2.11 2.24 2.60 2.31 2.02 2.16 2.25 1.12 1.02 1.10 1.35 2.21
15.36 15.20 15.20 14.59 14.66 14.64 14.65 15.02 14.74 14.39 14.65 14.93 14.99 15.02 14.60
122 119 119 114 114 115 116 119 119 115 117 118 117 118 118
Per cent 17.69 17.37 17.73 17.59 17.64 17.54 17.35 17.67 17.40 17.31 17.53 17.63 17.67 17.62 17.26
15.33 11.71 13.66
2.60 0.74 1.66
15.36 14.39 14.84
122 114 117
17.73 17.26 17.53
141 136 139 138 138 138 137 140 140 139 140 138 138 138 139
Heating to 90" C. after treatment with water is not only to dissolve the gelatin, for which no such high heat is needed or desired, but to coagulate the albumin. Consequently, we favor bringing the beaker up to this temperature as rapidly as possible, and filtering a t once'as soon as the albumin is coagulated. For extreme accuracy the final dilution should be made at the temperature a t which the specific rotation of gelatin has been determined and the temperature at which the solution is to be polarized. Practically, making
February, 1927
INDUSTRIAL AND ENGINEERING CHEMISTRY
to volume a t 20" C. introduces an error of only 0.4 per cent of the gelatin present. To make sure that the gelatin is all in the soluble form we hold a t a temperature slightly above 35" C. for some time before polarizing. By adhering strictly to the quantities given in our modification of the Ferris method the calculation becomes very much simplified. The percentage of gelatin is obtained by simply substituting the weight of the serum for the volume in the original formula. But as the weight of sample is 200 grams and the specific rotation of commercial gelatin is 117, the only variables become the polariscope reading ( R ) and the weight of serum (bV), hence: Per cent gelatin = 0.00074 X R X W
For the estimation of gelatin by nitrogen, in the final solution, the procedure is similar, using the Ferris formula, but the factor 6.75 instead of 5.55. Ferris does not direct any correction for the nitrogen not due to gelatin in the filtrate. We have found from 2 to 3 mg. of nitrogen in 25 cc. of the final solution in blank determinations containing no gelatin. That the filtrate actually does contain proteins other than gelatin is shown by the invariable appearance of a beautiful violet color (tryptophane reaction: as soon as the sulfuric acid is added for digestion. Pure gelatins get yellow, but never show violet. Per cent gelatin = 0.135 X N X W
in which N is mg. of nitrogen in 25 cc. of the solution, and W the weight of serum.
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Modified M e t h o d Into a tared 400-cc. beaker weigh 200 grams of the melted and thoroughly mixed sample. Add 25 cc. of water and enough hematoxylin indicator to color the sample distinctly pink. Heat t o 40" C. and titrate with 10 per cent acetic acid until the pink color has completely disappeared. Return the beaker to the balance and add water until the weight of the contents reaches 250 grams. Accuracy of 0.5 gram is sufficient. Mix thoroughly and return to the 40" C. water bath until the curd has fully separated. Filter through a small bag of cotton or linen. Weigh out 100 grams of the filtrate, add 3 cc. of a saturated solution of potassium alum, then 200 cc. of 95 per cent alcohol, with stirring. Cooling hastens the separation of the precipitate. Filter by suction on a 9-cm. filter, using a Biichner funnel. Tear up the paper and place in water in a small beaker. Soak until the gelatin has an opportunity to swell. Then place the beaker in warm water for half an hour, first adding water t o approximately 50 cc. Bring rapidly to incipient boiling and filter a t once into a 100-cc. volumetric flask, washing with hot water until the flask is nearly full. Allow the flask to cool to near room temperature, make t o the mark, and mix. The polarization can be made a t once, using a jacketed tube. Circulate water, which has been heated, through the jacket until a thermometer placed in the tubulature reads 40". Then shut off the water, allow the tube to cool to 35", and read. If the flasks are not read a t once they should be heated in a water bath a t 35-40" C. for half an hour or more before placing in the polariscope tube. The weight of serum to be used as a basis in calculating is obtained by subtracting the weight of fat and casein in 200 grams of the sample from 250 grams. Using a 200-mm. tube for polarizing, the per cent of gelatin is given by 0.00074 X R X W. Determine nitrogen in 25 cc. of the solution, deducting 2 mg. for the blank, and expressing the result in milligrams of nitrogen in the 25 cc. The percentage of gelatin is then found by 0.135 X N X W.
Determination of Mineral Nitrogen in Fertilizers' By C . H. Jones VERMONT AGRICULTURAL EXPERIMENT STATION, BURLINGTON, VT.
HE present official methods used in fertilizer analysis
T
was present. The following procedure for the determination of ammoniacal and nitrate nitrogen in fertilizers containing calcium cyanamide, urea, etc., has given very satisfactory results thus far in this laboratory. Method
to determine mineral nitrogen do not give true values when certain organic ammoniates are present. This condition is most marked when cyanamide is a component of the mixture. The value of calcium cyanamide as a source of nitrogen for plant growth is well recognized and its use in the trade well established. It is classed as organic nitrogen and if used by the manufacturer of commercial fertilizers it is but fair to all concerned that the analyst have at his disposal methods that will enable him to distinguish between mineral and organic sources of nitrogen. When the present official methods for nitrate nitrogen mere formulated, cyanamide and urea were not commercial sources of fertilizer nitrogen and their effect on the procedures adopted mas not considered. This may be of little moment where no attention is paid to the different forms of nitrogen present in a fertilizer, but if the manufacturer is required by law to guarantee mineral and organic nitrogen percentages, confusion may arise if the true nitrate content is not determined. Many states place a varying value on the nitrogen in fertilizers based on the form in which it is present. As a rule organic nitrogen is given a higher valuation than the mineral forms. These considerations prompted the writer to attempt to modify the existing official methods so that reliable results for nitrate nitrogen might be secured in present-day fertilizer mixtures, particularly when calcium cyanamide
Test the fertilizer qualitatively for nitrates. If present, proceed as follows: (I) PREPARATION OF ~OLUTIoN-~~ace 4 grams of the sample in a small beaker (150 cc.), add about 40 cc. of water, stir, filter by decantation, and after all the residue is transferred t o the filter wash to just under bulk of 200 cc., letting each washing run through before another is added. Make to bulk of 200 cc., mix, and treat three aliquots of 50 cc. each a s directed under (11) and (111). Or Place 4 grams of the sample in a 200-cc. flask. Add 160 cc. of water, shake thoroughly for 5 minutes, make to bulk of 200 cc., mix, and filter. (11) A>IMONIACAL NITROGEN-PlBCe 50 CC. (eqtlivalent to 1 gram) of the solution prepared according to (I) in a 500-600-cc. Kjeldahl distillation flask, add 150 cc. of water and 5 grams of magnesium oxide (heavy). Connect with an upright condenser, distil 100 cc. of the liquid into a measured quantity of standard acid, and titrate with standard alkali using cochineal or methyl red indicator. (111) NITRATEKITROGEN-(A) Place 50 cc. of the sample prepared according to (I) in a 500-600-cc. Kjeldahl distillation flask, add 10 to 12 perforated glass beads (3 to 5 mm.), 2 grams of reduced iron, and 10 cc. of dilute sulfuric acid (1:l). Rotate slowly and when any violence of reaction has moderated, place on hot plate and boil gently for 5 minutes. Remove, add 40 cc. of water, and cool. Add 100 cc. of strong sodium hydroxide solution. * Connect at once with a n upright condenser by means
1 Presented before the Division of Fertillzer Chemistry at the 72nd Meeting of the American Chemical Society, Philadelphia, Pa., September 5 t o 11, 1926.
The concentrated soda solution used is prepared by dissolving 525 grams 01 flake sodium hydroxide in 1 quart of water. I t should test close to 42' Be. a t room temperature.
*
