440
INDUSTRIAL AND ENGINEERING CHEMISTRY
Vol. 17, No. 7
The authors wish to express their thanks to George M. List for his cooperation in certain phases of this work.
(3) Hall, S.A,, Schechter, M. S., and Fleck, E. E., U. S. Dept. .%gr.? Bur. Entomol. Plant Quarantine, Bull. ET-211 (1944). (4) Neal et aZ., Publ. Health Repts., Supplement 177,2-32 (1944). (5) Nissen, H. B., and Peterson, R. B., Am. SOC.Brewing Chem., Proc. 6th Ann. Meeting, 1942,77-9. (6) Schechter, M. S., and Haller, H. L., J . Am. Chem. Soc., 66, 2129 (1944). (7) Winter, P. K., IND. ESG. CHEX.,ANAL.ED.,15, 571-4 (1943).
LITERATURE CITED ( I ) Fahey, J. E., J . Assoc. Oficial Agr. Chem.,28,152-8 (1945). (2) Gunther, F. .4., IND.ENG.CAEM., ANAL.ED.,17,149 (1945).
PUBLISHED with the approval of the director as Paper 192, Scientific Journal Series, Colorado Agricultural Experiment Station.
the standard curve, gives the concentration of DDT. The use of an alternate solvent, ethylene chloride, having a peak absorption of 530 mp is described. ACKNOWLEDGMENT
Determination of Glycerol In the Presence of Large Concentrations of Gelatin CARL J. WESSEL, STANLEY W. DRIGOT, AND GEORGE W. BEACH, Gelatin Products Corporation, Detroit, Mich. In a convenient method for the determination of glycerol in gelatin the gelatin i s precipitated with sodium tungstate in an acid medium and the filtrate analyzed for glycerol by the official method of the A.O.A.C. A n arithmetic mean of 99.69% recovery and a standard deviation of *0.43 were found for representative determinations.
Diphenylamine indicator. Dissolve 1 gram of diphenylamine i n 100 ml. of concentrated sulfuric acid (I). Retarder. Dilute 150 ml. of sirupy phosphoric. ' cid with 600 ml. of distilled water and add 250 rnl. of concenLlated sulfuric acid ( I ) . PROCEDURE
T
HE common gelatin capsule shell is composed of only a few
constituents-gelatin, glycerol, dyes, water, and a preservative. I n some instances, not considered in this report, plasticisera other than glycerol may be present. Since the concentration of preservative and dye is usually so small as to be negligible, the presence of these substances may be disregarded. It is, therefore, necessary only to remove the gelatin and measure the glycerol by one of the many quantitative methods in the literature. Many of the available procedures for removing protein from aqueous preparations were tried-namely, extractions with acetone, alcohol, and water, precipitation with a petroleum etheracetone mixture, metaphosphoric acid, saturated ammonium sulfate, heavy metal salts, and 10% sodium tungstate. All were unsatisfactory, with the exception of sodium tungstate, which was most efficient in removing the gelatin. The filtrate was then analyzed for glycerol by the method of the A.O.A.C. (1). The biuret test was used to determine completeness of precipitation with colorless solutions, whereas with cdored solutions it was necesbary to add an excess of sodium tungstate to the filtrate. REAGENTS
Ether, reagent grade, 10 N sulfuric acid, and concentrated sulfuric acid. Sodium tungstate solution, 100 grams dissolved in sufficient forms on standdistilled water to make 1 liter. If a -mecbitate in i t m a y be filtered off. 8trong potassium dichromate solution. Dissolve 74.55 grams of dry, recrystallized potassium dichromate in water, add 150 ml. of concentrated sulfuric acid, coql, and dilute with water to 1 liter a t 20" C. One milliliter of ths solution is equivalent to 0.01 gram of glycerol (I). Owing to its h g h coefficient of expansion, i t is necessary to make all volumetric measurements of the solution a t the same temperature a t which it is diluted to volume. Dilute otassium dichromate solution. Measure 50 ml. of the strong diclromate solution into a 1-liter volumetric flask a t 20" C. and dilute to the mark with water a t room temperature (1). Ferrous ammonium sulfate solution. Dissolve 30 grams of recrystallized ferrous ammonium sulfate in water, add 50 ml. of concentrated sulfuric acid, copl,, and dilute with water to 1 liter at room temperature. One milliliter of this solution is'equivalent to approximately 1 ml. of dilute potassium dichromate. The value changes slightly from day to day, SO that it must be standardized daily against the dilute potassium dichromate (1).
It was found that pH values were not a simple guide to the precipitation limits of gelatin with sodium tungstate. Therefore these limits were determined empirically by the quantities of sulfuric acid and sodium tungstate necessary to effect complete precipitation. The formula presented is adequate for removing gelatin in concentrations as high as 70% in the gelatin-glycerol test material: Test mat.erial (glycerol content 25%) 10 N sulfuric acid 10% sodium tungstate Distilled water q.s.
1 . 4 grams 14 ml.
32 ml. 100 ml.
When the test materials are gelatin capsules, a suitable number are opened, the contents removed, and the shells washed with ether until all foreign matter is eliminated, Enough capsule material to furnish a glycerol equivalent of 0.35 g m . is weighed into a 100-ml. volumetric flask, and distilled water is added to dissolve it, followed by 10 N sulfurlc acid and tungstate in the order mentioned. (If the character of the sample is such that it may be difficult to weigh directly into a v o l v e t r i c flask, it is expedient to make a stock solution and take ahquots therefrom.) After diluting to the mark with distilled water, the flask is shaken by hand for 5 minutes to break up large lumps of prefipitate, then placed on a shaking machine for 5 minutes (270 oscillations per minute), transferred to a 250-ml. centrifuge tube, and centrifuged for 5 minutes a t 2000 r.p.m. The supernatant is filtered through Whatman No. 4 filter paper and a 20-ml. aliquot taken for the oxidation. Twelve milliliters of stTong dichromate,. messured a t 20' C., are added to the aliquot in the 100-ml. oxidation flask, followed by 10 ml. of concentrated sulfuric acid. The flask is placed in a boiling water ok steam bath for 20 minutes, removed, cooled to 20" C., and diluted to the mark with distilled water (solution S). The ferrous ammonium sulfate solution is standardized by pipetting 20 ml. into a 250-ml. beaker, and adding 20 ml. of retarder, 4 drops of indicator, and approximately 100.ml. of diatilled water. This solution is titrated rapidly with dilute potassium dichromate.solution until a dark green color is produced. The dichromate is then added dropwise until the color changes from green to blue-gray to deep violet. This is the a titer, made daily to standardize the ferrous ammonium sulfate solution. To measure the amounf of excess dichromate left in solution S a similar ferrous ammonium sulfate solution is made up and titrated to the diphenylamine end point with solution S. This titer is called b. A small bull's-eye lamp focused on the solution is an aid in determining color changes in colored solutions.
ANALYTICAL EDITION
July, 1945 Table
1. Glycerol Loss after Precipitation and during Oxidation
Glycerol Presenb before I'recipitation of Gelatin Gram 0,0000
Stock Glycerol Added after Precipitation of Gelatin Gram
0.2015 0.1612 0.1209 0.0806 0.0403 0.0000 0.0604 0.1209 0.1813
0.0385" 0.0771" 0.1156' 0.1542' 0.1927a 0.1153b 0.1153b 0.1153b
Recover,.
% 99.1 99.2 99.4 99.5 99.3 99.4 100.4 99.6 99.4
n I n gelatin-glycerol test preparation. b I n capsule-shell solution.
The amount of glycerol present in the 20-ml. aliquot taken for oxidation is calculated by the following formula:
G = grams of glycerol = ml. of strong dichromate used
D
I n order to determine the amount of glycerol present in the original 100-ml. sample, multiply the answer by 5. RELIABILITY
OF M E T H O D
The A.O.A.C. method ( I ) gave satisfactory results with known solutions of glycerol, whose concentrations were determined refractometrically. I n a series of seven determinations, the per cent recovery average was 100.1 and standard deviation *0.16. Since there is a possibility of losing glycerol during the oxidation process, it was decided to add known quantities of a stock glycerol solution to the regular test preparation after precipitation of the gelatin to determine if recoveries are quantitative. Identical recovery experiments were also performed with gelatin-glycerol solutions obtained from gelatin capsules. The results are summarized in Table I. The gelatin was precipitated from the gelatin-glycerol solutions and aliquots corresponding to the various weights of glycerol listed in the first column were taken for oxidation. A stock glycerol solution, composition determined refractometrically, corresponding to one prepared for oxidation by removal of gelatin, was added in varying amounts to the filtrates of the gelatin-free solutions. These quantities are indicated in the second column. The recoveries of Table I are very good. The results compare favorably with the arithmetic mean and standard deviation data cited a t the end of-this paper. Recovery experiments were carried out with glycerol addition before precipitation to determine if any glycerol is carried down by the precipitation of gelatin with sodium tungstate (Table 11). I t was necessary to add increased quantities of strong dichromate solution to maintain the proper excess during oxidation of the larger amounts of glycerol. Table I1 indicates that the recovery of glycerol is good when the added glycerol is carried through the precipitation stage. Tests were performed to determine if any glycerol loss occurs when t h e centrifugate, containing suspended particles of the precipitate, is filtered through Whatman KO.4 filter paper prior to oxidation. The first two 50-ml. portions of the filtrate from a gelatin capsule solution gave respectively, 29.4, 29.3, and 29.2, 29.2%. The tests were performed in duplicate. There was no significant change in glycerol content in the two portions, indicating that glycerol loss due to adsorption on Whatman No. 4 filter paper is negligible. The A.O.A.C. procedure specifically recommends a 74% excess of strong dichromate in the solution to be oxidized for best results with diphenylamine indicator. This point w m investigated further to see if it is applicable to the problem.
44 1
The glycerol content of a series of stock solutions was determined, utilizing various amounts of strong dichromate solution. I n addition, the effect of a one-drop excess a t the end point was noted (Table 111). PROCEDURE AND DISCUSSION
The composition of the glycerol solution was determined refractometrically . The glycerol and strong dichromate were memured by weight and the volume of the strong dichromate was determined from its specific gravity a t 20' C. Calibration corrections on burets were used; corrections on pipets and volumetric flasks were insignificant. The b titer is the amount of solution, after oxidation, containing the excess dichromate. This solution was used to titrate the ferrous ammonium sulfate. The b 0.05 column represents the b titer plus a one-drop excess. Column seven represents the increase in per cent glycerol calculated on the basis of the one-drop excess a t the end point; it shows the effect of a larger excess of strong dichromate on the accuracy of the results.
+
The A.0.A.C.-recommended excess of 74% is satisfactory, but an excess as high as 120% will not seriously affect the accuracy of the results, whereas a 40% excess is too low, as a large titer is required and the end point is not sharp. Details regarding the indicator are given by Kolthoff and coworkers ( 3 , 4 , 5 ) . Table I11 also clarifies the effect of the last drop in the titration on the accuracy of the method, especially in the case of an unusually large excess of strong dichromate solution. The A.O.A.C. method indicated an exact 20-minute interval for immersion of the oxidation flask and contents. It was found that an interval varying from 10 to 30 minutes gives equally satisfactory results. The 20-minute period is, however, recommended as a safe value. PRECISION OF M E T H O D
Representative results of determinations made with pure gelatin-glycerol solutions and those obtained from capsules are: 100.7, 99.9, 99.4,, 99.3, 99.5, 99.4, 99.2, and 99.1% with pure gelatin-glycerol solutions.
Table II.
Recovery Experiments with Glycerol A d d e d before Precipitation
Glycerol Present" before Precipitation of Gelatin Gram
Glycerol Added before Precipitation of Gelatin Gram
0.0000 0.0000 0.1129 0.1129 0.1129 0.1129 0.1137 0.1140 0.1140
0.1712 0.1712 0.0000 0.0632 0.1264 0.1895 0.1141 0.0570 0.1712
a
Strong Dichromate Added
Recover>-
MI. 30.0 30.0 25.0 31.0 37 .O 43.5 36.0 30.0 41.0
100.0 100.0 100.0 98.9 100.0 100.5 99.7 99.3 99.2
%
From capsule shells.
Table 111.
Sample 1
2 3 4 5 6 7 8 9 10 11
Effect of Excess Dichromate on End Point and Accuracy of Results
Glycerol Used Recovery Gram %
0,0674 0.0688 0.0668 0.0869 0.0667 0.0670 0.0667 0.0872 0.0672 0.0671 0.0672
100.1 99.7 100.4 100.1 100.3 99.5 105.3 106.5 107.5 106.4 108.9
Excesa Strong Dichromate Used
b
b
+ 0.05
% 10 20 40 74 80 120 160 200 240 280 320
166.68 81.98 38.70 20.52 18.74 12.65 9.73 7.81 6.57 5.48 4.66
166.73 82.03 38.75 20.57 18.79 12.70 9.78 7.86 6.62 5.53 4.71
Incfease In Glycerol
% 0 0
0.1 0.1 0.1
0.5 0.9 1.3 1.8 2.6 3.4
442
INDUSTRIAL AND ENGINEERING CHEMISTRY
99.9, 99.6, 100.4, 99.4, 99.3, 99.8, and 99.3% with gelatinglycerol solutions obtained from gelatin capsules. The arithmetic mean of all these determinations is 99.62% (2). The standard deviation is *0.43. SUMMARY
Val. 17, No. 9
consistent and duplicable. The arithmetic mean of representative determinations is 99.62%, while the standard deviation is *0.43. LITERATURE CITED (1) Assoc. Official Agr. Chem., Official and Tentative Methods of
Analysis, 5th ed., p. 480, 1940.
A method for the determination of glycerol in the presence of large quantities of gelatin has been developed to determine the glycerol content of gelatin capsules that also contain water and insignificant amounts of preservative and dye which do not affect the results of the analysis. After removal of the gelatin by precipitation as the tungstate. the glycerol content is determined by the official method of the .4.0..1.C. The results obtained are
(2) Crumpler, T.B.,and Yoe, J. H., “Chemical Computations and Errors”, pp. 125-34, New York, John Wiley & Sons, 1940.
(3) Kolhhoff, I. M.,and Sarver, L. A., J . Am. Chem. SOC.,52, 4179 (1930). (4) Kolthoff. I. hl., and Sarver, L. A., 2.Elektrochem., 36, 139 (1930). (5) Kolthoff, I. M., and Stenger, V. A., “Volumetric Analysis”, Vol. I, p. 133, New York, Interscience Publishers, 1942. PREBEKTED before the Division of Analytical and Micro Chemistry at the 108th Meeting of the AMERICAN CHEMICAL SOCIETY, New York, N . Y.
Determination of the Capacity of Shallow Jars EARLE R. CALEY Wallace Laboratories, Inc., N e w Brunswick,
N. J.
With jars having a smooth flat rim, this point may also be deH E accurate and rapid determination of the true capacity termined by following the decrease in the weight of the system of shallow jars of small volume commonly used as containers with time, rehtive constancy of weight being m,.ntained for a for certain cosmetic and pharmaceutical products is not so simple considerable interval after evaporation of the last portions of as may first appear. In contrast to volumetric flasks or specific water from the meniscus. Illustrative of this are the curves in gravity bottles, the orifice of such containers is great as compared Figure 1, which show the loss in weight during the first two to their volumetric capacity, and thus errors due to meniscus trials with jar I taken a t 5-minute intervals. Determination of effects are greatly exaggerated when standard calibrating liquids the end point from the appearance of the first minute bubble of such as water or mercury are used. Moreover, it is not easy to air is less laborious, however, and a t least equally accurate. It determine experimentally R ith accuracy the exact point a t which may, indeed, be slightly more accurate, since the volume of water such vessels are truly filled-the point a t which the level of the displaced by the air bubble is partly compensated by the volume calibrating liquid corresponds to that of an ideal plane deterof the remnants of the film of water that ordinarily still remains mined by the top surface of the rim. between the top of the rim and the glass plate a t the time the An obvious method of restricting the level when water is used bubble appears. At any rate, the results of determination of as a calibrating liquid is to fill the jar to overflowing, and to capacity with water shown by Table I are much more precise slide onto the top of the jar a square of plate glass in such a way if time is allowed for evaporation of water from the outside, and as to exclude air bubbles. Excess water may then be removed they are undoubtedly much more accurate. This is evident both from the outside of the jar and underside of the plate by careful from the above discussion and from a comparison with the rewiping, and the capacity determined immediately from the sults of the determination of the capacities of the same jars with difference in weight between the empty and filled system. H oWever, this simple and relatively quick procedure, which is often used in practice, gives noticeably high results, 3.510 in part because of the inclusion of the weight of water contained in a meniscus that forms between the outer edge of the rim and the glass plate. 3.500 More accurate results are obtained if the weight of the filled system is determined after standing long enough to allow the water in this outside meniscus 3.490 to evaporate. A positive error may still remain, however, because of inclusion of the weight of water that remains between the top and the rim and the glass 3.480 plate, If the rim is flat and wide on top, a common form, this error may be of appreciable magnitude. The results of trial determinations on two commer3.410 cial jars by this method are shown in Table I. The averages shown and the average deviations are based upon six successive trials. I was a glass jar with a 3.460 slightly rounded rim of ordinary width; I1 was a plastic jar with a flat rim of more than ordinary width. All the volumetric capacities shown in the table are uni3.450 formly based on 20” C. as the standard temperature. It will be seen that the initially determined capacities are appreciably higher than those found after allowing 3.440 the water in the outer meniscus to evaporate. I n these experiments the appearance of. the first minute bubble 0 PO 40 60 80 100 120 140 of air under the glass plate next to the rim was taken Time In minutes as the indication of complete evaporation of this Figure 1. Loss in Weight with Time and Final Temporary Constancy before water. Appearance of Bubble, B
T
