346
ANALYTICAL CHEMISTRY
The oxidizables as carbon were determined by total wet reduction of potassium dichromate by the sample. The hydrolysis numbers are the milligrams of sodium hydroxide needed to neutralize the acid formed by hydrolysis of 1 gram of acid sample. These hydrolysis numbers could be calculated to percentage of alkyl sulfate. Known increments of cr-ater were analyzed as follows: The acid was analyzed for the original water content. Another sample of the acid was taken and a weighed amount of water was added after the ammonium chloride was mixed with the acid sample. The total water was determined and this value corrected for the amount of water originally present in the sample. The difference was taken as the water recovered. The total percentages contain cr-ater, actual sulfuric acid, butyl hydrogen sulfate, and oxidizables as carbon. The total percentages do not include the ash (normally 0.3 to 0.5%) or take into account the fact that some carbon is totaled twice, in both the butyl hydrogen sulfate and the oxidizables as carbon. *
the white acid analyses w'as indicated as within 0.1% absolute. The reproducibility was good on alkylation acids. The water recovered as compared to a known amount added was in close agreement. The range covered waa from 98.5% to about 30% in a white acid; the black acid from 98% to about 85% titratable acidity as sulfuric acid. LITERATURE CITED
(1) A l m y , Griffin, and Wilcox, ISD. ENG.CHEM.,AN.41,. E D . , 12, 392 (1940). (2) Fischer, K.,Angew. Chem., 48,394(1935). (3) McKinney, C. D.,Jr., and Hall, R. T., IND. ENG.CHEM.,ANAL. ED., 15,460 (1943). (4) Roby, R.F.,Ind. Eng. Chem., 33,1976 (1941). (5) Smith, D.M., and Bryant, W. M.D., J . Am. Chem. Soc., 57, 61 (1935). (6) Toennies,G., andElliot, M., Ibid., 57, 2136 (1935). (7) Wernimont, G.,and Hopkinson, F. J., IXD. ENG.CHEM.,ANAL. ED.,15, 272 (1943).
DISCUSSION
No method of known accuracy and acceptable convenience was available for comparison with this method. The accuracy of
RECEIVEDAugust 1, 1947. Presented before Petroleum Group Sesslons, Fifteenth hIidwest Regional Meeting, .4VERICAN CHEVICAL SOCIETY,Kansas City, N o . , J u n e 1947.
Determination of the Gamma-Isomer Content of Benzene Hexachloride C. V. BOWEN AND MILTON A. POGORELSKIN' Bureau of Entomology a n d Plant Quarantine, United States Department of Agriculture, Beltsville, M d . The gamma isomer of benzene hexachloride has recently become the object of considerable interest because of its outstanding effectiveness as an insecticide. Technical benzene hexachloride contains the gamma isomer, usually to the extent of about 10 to 12940, along with varying proportions of at least four other less active isomers and small amounts of other compounds. A cryoscopic method
B
ENZENE hexachloride has recently been the subject of
extensive investigations as an insecticide and is now being produced in considerable quantities for that purpose. The production of a new material for large-scale use soon necessitates the development of an analytical procedure for its determination. Technical benzene hexachloride, or 1,2,3,4,5,6-hexachlorocyclohexane, is composed of at least five isomers, which differ in their toxicity to insects. The gamma isomer has a much greater insecticidal value than any of the others. Consequently, the determination of this isomer is of primary importance in analysis of the technical product. Several methods for determining the percentage of the isomers in benzene hexachloride have been described, including weighing them after separation by fractional extraction and selective precipitation (Y),weighing after separation by partition chromatography ( 6 ) ,bioassay (4), and infrared spectroscopy ( 1 ) . This paper describes a cryoscopic method of determining the gamma isomer in technical benzene hexachloride. If a mixture of isomers is dissolved in a much larger quantity of one of the isomers, a depression in freezing point of the solvent isomer will be caused by the other isomers. This method has been applied by Haller et al. (3) to the determination of the o,p'- and p,p'-DDT content of a D D T oil. It is also applicable to the determination of the isomers of benzene hexachloride, but is most useful when the 1
Present address, National Cancer Institute, Bethesda, Md.
has now been developed for the determination of the gamma isomer content of mixtures of the isomers or of technical benzene hexachloride. This can be carried out with equipment available to any laboratory and requires a relatively short time. It involves measurement of the depression which a sample of the material to be analyzed produces in the freezing point of pure gamma benzene hexachloride.
isomers are present in substantial amounts. For the D D T analyses Haller et al. used a solvent unrelated to DDT. For the analysis for ybenzene hexachloride this method has been modified in order to eliminate the unrelated solvent. If a known weight of a mixture of the.benzene hexachloride isomers is dissolved in a known weight of pure gamma isomer, a freezing-point depression results, which is dependent upon the content of the other isomers. If a depression for the alpha isomer in the pure gamma isomer is obtained, the amount of gamma isomer present in the mixture may be calculated from the difference betn-een the apparent numbers of moles in 1 gram of the solute in the two cases. Suppose that the adding of 1 gram of the pure alpha compound to 10 grams of the pure gamma compound depresses the freezin point of the gamma compound by 6 C. The other isomers woul! give the same depression when present in the same amounts, with the exception of the gamma isomer, which would give no depression. If 1 gram of an unknown mixture, known to be made up of the isomers, is added to 10 grams of the gamma isomer, any effect on the freezing point of the solvent will be due only to the alpha, beta, delta, an$ epsilon isomers in the mixture. Suppose the depression to be 4 ; approximately one third of the mixture would then consist of the gamma compound. O
PROCEDURE FOR A CRYOSCOPIC METHOD
Apparatus. An oil bath that can be maintained at 120' to 130" C., consisting of a 1000-ml. beaker in which the oil should
V O L U M E 20, NO. 4, A P R I L 1 9 4 8
347 hexachloride, and the alpha isomer in the -,-benzene hexachloride, From the plateaus of the curves the freezing-point depressions caused by the alpha isomer and the sample are obtained, The total number of moles per gram of alpha isomer, designated as a, is obtained from the equation
w
At 7n = ____
1000 wh,
w I
I
I
10
20
30
Figure 1.
3
I
I
40 50 60 70 PERCENT GAMMA CALCULATED
I
I
80
90
I
100
Correction Curve for Cryoscopic Analysis of Benzene Hexachloride
Table I. Determination of Gamma Isomer in Known Mixtures of Benzene Hexachloride Isomers Gamma Found, 70 6.2 10.3 3 10.1 15.6 4 19.3 19.4 25.9 k 29.7 9 50.0 49.5 10 70.1 70.8 11c 70.0 70.3 126 80.3 80.0 13 85.8 85.0 89.8 90.0 14 Cnless otherwise indicated, all samples contain alpha isomer. Contains 70Y0 alpha, 10% beta, 10% delta. Contains 20% alpha, 10% delta. Contains 20y0 delta. Sample No.0 1 2b
a
b c
d
Gamma Present, % 6.3 10.0 10.1 15.1 19.2 20.1 25.0 30.0
have a depth of about 110 mm. A freezing tube, of heat-resistant glass, with external dimensions 20 X 150 mm. A freezing-tube jacket, of heatrresistant glass, with external dimensions 38 X 150 mm. A thermometer, mercury in glass, graduated to 0.1 O, with a range of +70" t o +120° C. The thermometer is inserted through a rubber stopper to hold it in position in the freezing tube. A spiral stirrer made from a 3-mm. glass rod or a 2-mm. metal rod such as Monel metal or Xichrome. The stopper for holding the thermometer has a longitudinal groove on one side to allow for free operation of the stirrer. Reagents. Gamma isomer of benzene hexachloride. The isomer should be recrystallized until the melting point obtained by the conventional capillary-tube method is 112.4-113' C. and the freezing point obtained by a cooling curve is 112.5O C. or higher. Alpha isomer of benzene hexachloride known to contain no gamma isomer. Determination. A 0.5000-gram sample is introduced into the bottom of the freezing tube, and 10.000 grams of pure ybenzene hexachloride are then placed on top of the sample. The freezing tube is daced in the oil bath a t 120' to 130 O C. M7henthe r-benzene hLxachloride and sample are melted, the stirrer and the thermometer are put in position, so that the thermometer reaches to about 0.5 cm. from the bottom of the tube, and the freezing tube is removed from the oil bath and placed in the freezing-tube jacket, which is also being kept warm in the oil bath. The rubber stopper around the freezing tube should be tight enough to create a w a r h dead-air space. The stirrer is worked slowly so as to intimate1 mix the sample throughout the ?-benzene hexachloride. When the temperature of the melt reaches 113" C., the freezing-point apparatus is raised until it is about 1 cm. above the surface of the oil bath. The stirrer is manually operated a t the rate of about 100 complete strokes per minute. The temperature continues to rise for a few minutes before cooling begins. When the temperature reads 114" C., the temperature is recorded, with the aid of a good hand lens to permit interpolating to 0.01 C. (if possible), a t 15-second intervals until the stirrer freezes. The freezing tube is removed from the jacket and the solution remelted in the oil bath. The melting-tube jacket is warmed and the operation repeated as described above, until consistent results are obtained. Freezing-point data for the ybenzene hexachloride alone are obtained in the same manner. Freezing-point data for 0.5000 gram of the a-benzene hexachloride (known to contain no gamma isomer) in 10.000 grams of the gamma isomer are obtained in the same manner.
Calculation. Freezing-point curves are plotted for the ybenzene hexachloride, the sample dissolved in the y-benzene
where vz = moles of material other than solvent in 1 gram of solute TV = weight of solvent in grams w = weight of sample in grams At = freezing-point depression in C. Kj = cryoscopic constant for solvent (gamma isomer) = 16.8 From the same equation the apparent number of moles per gram of unknown mixture are calculated and designated as b. (a - b) X 290.8 X 100 = apparent % gamma isomer in sample The gamma isomer present in the sample acts as an addition to the gamma isomer used as the solvent. Consequently, the results obtained on calculation are high, and a correction may be made by either of two methods. The apparent calculated amount of gamma isomer may be added to the weight of the solvent actually used and the results recalculated. A much simpler method, however, is to use a table or a curve. Figure 1 shows such a curve prepared by calculating the differences in the apparent and the corrected percentages, in n-hich the ordinate gives the percentage to be deducted from the apparent calculated perrentage of gamma isomer in the sample, which is the abscissa. Theoretically a is the number of moles per gram of alpha isomer and may be obtained by simply dividing one by the molecular a eight of benzene hexachloride-namely, 290.8. This theoretical value cannot, however, be used for a in the calculations, bemuse a small amount of other isomers may be present in the gamma isomer that is used as the solyent in the determination. The cryoscopic constant may vary with different samples of the gamma isomer, and must also be determined. The simplified calrulation may then be performed. The freezing-point and meltingpoint specifications for the gamma isomer are the minimum values for materials sufficiently pure to be used in the determination. ANALYSIS OF KNOWN MIXTURES
In order to prove the method, knovin mixtures of the benzene hexachloride isomers were analyzed by the foregoing procedure. The isomers used in these determhations had the following melting points, which were used as criteria of their purity:
c. 157.5-158 289-304 112.4-1 13 138-139.5
Alphe Beta Gamma Delta
The composition of the mixtures and results of the determinations are shown in Table I. The agreement of determinations on two samples of technical benzene hexachloride a.nalyzed by the cryoscopic method and the infrared spectrometer is indicated below: Method Infrared Spectrometer Cryosoopic
Sample 1, % 14.8 15.0
Sample 2, 12.8 12.3
70
DISCUSSION OF METHOD
The cryoscopic constant for the gamma isomer, obtained as an average of 20 determinations with the alpha isomer, was 16.8. Consequently, this value is given for use in the above calculation. Each experimenter should, however, calculate this constant from the data a t hand when the depression caused by the alpha isomer has been obtained. The constant calculated from the depressions caused by the delta and epsilon isomers is in agreement with that obtained by use of the alpha isomer. The alpha isomer is used in this determination because it is the main component of
348
ANALYTICAL CHEMISTRY
technical benzene hexachloride and is easily obtained in a pure form. Cooling curves of paired mixtures of the various isomers iridicate that no association or compound formation occurs on heating them together. There are three points in the manipulation that require close attent,ion. (1) Parallax must be avoided in reading t,he thermometer; an error of 0.05 a C. may easily occur when a ha#ndlens is used. (2) The sample must be dry, free of organic solvents, and completely dissolved and evenly distributed throughout the solvent (gamma isomer). The spiral stirrer (5) has been found niorc effective in accomplishing this than an ordinary ring stirrer. The freezing points will be successively higher until a homogeneous solution is obtained. (3) The supercooling must be reduced to a minimum if the correct freezing point is to be obtained. The method presented here has the advantage over the usual rrposcopic method, in which an unrelated solvent is used and the total number of moles of the solute (sample) in grams is calculated, in that vihen the value for a has been obtained it may be used repeatedly without redetermination for each sample. This is also true for the determination of the freezing point of the gamma isomer that is used as the solvent. .lis long as gamma isomer of the same lot and the same thermometer are available, only one freezing-point determination is necessary for the analysis of each sample. After several readings of the points on the cooling curve have been made and the freezing-point curves plotted, it becomes evident that when supercooling occurs, the high point, or plateau, may be observed direct,ly without malting a time-temperature curve. For the rapid determination of an approximate gamma-isomer content, a temperature depression-gamma content curve may be prepared by the use of known mixtures of the benzene hexachloride isomers or by calculation. Percentages read from such a curve do not require any correction for the gamma cont,ent, as the values on the curve have been obtained from actual readings instead of being calculated. The accuracy of the cryoscopic method depends upon the care with which the temperature readings are made and the precision of the instrument used to make the readings. A Beckmann ther-
mometer permits readings with interpolation to 0.001” C. A 1 to 20 ratio of sample to solvent is equivalent to 0.03% in the calculations. The use of a Beckmann thermometer has one iniport’ant disedva,ntage-a much larger quantity of solvent is necessary than when a thermometer n-ith a smaller mercury content is used. Each 0.01” C., as in the case of the method presented, is equivalent to 0.3%. Other methods for determining the freezing points, such as the use of a platinum resistance thermometer ( 8 ) , offer means for increased precision in reading the trnipera tures. The gamma isomer is a t present available only in limited quantities, but the production of this material in a reagent grade for this determination is practical. The development of a micro- or semimicromethod will obviate to some extent this disadvantage. The cryoscopic constant of 16.8 for the gamma isomer v,-ould indicate that such modifications are practical. Other sources of error ordinarily present in freezing-point determinations ( 2 , 6 ) may Le disregarded in this method for determining the gammi isomer of benzene hexachloride, since they fall outside of the apparent pwcision. LITER4TURE CITED
Daauch, L. R., IND.ESG.CHEM.,AKAL.ED.,19, 779-85 (1947). Glasstone, S., “Textbook of Physical Chemist1J-,”2nd ed.. New
York, D. Tan Nostrand Co., 1946. Haller, H. L., Bartlett, P. D., et al., J . Am. Chem. Soe., 67, 1591602 (1945). HoskinA, W.’M., and Caldwell, A. H., Jr., Soap Sanit. Chemicals, 23(4), 143-5 (1947). Mair, B. J., Glasgow, A. R., and Rousini, F. D., J . Research Natl. Bur. Standards, 26, 591-620 (1941). Ramsey, L. L., and Patterson, W. I., J . Assoc. Oficial Agr. Chem., 29, 337-46 (1946). Slade, R. E., Chemistry & I n d u s t r y , 64, 314-19 (1946). Streiff, A. J., and Rossini, F. D., J . Research Natl. Bur. Standards, 32, 185-95 (1944). RECEIVED Xovrmber 22, 1847. Presented before the Division of Aprioultural and Food Chemistry, Symposium on Insecticides in Food Production, at the 112th Meeting of the AMERICAX CHEMICAL SOCIETY, New York, N. Y, Other papers in this aympoaium were published in the April issue of Indur trial and E n g i n e w i n g C h e m i n t r y , pages 678 t o 717.
Evaluating Fungal Amylolytic Materials for Saccharifying Fermentation Mashes H. D. REESE’, E. I. FULhlER,
B
AND
L. A. UXDERKOFLEK, Zown Stute College, Ames, Iowa
IOLOGICAL saccharifying agents such as barley malt and mold bran vary more or less in enzymic potency from one batch to another, depending upon raw materials and processing variables. Saccharifying agents are much more expensive than the starchy materials processed; hence, for their most efficient and economical use a means must be afforded for testing the enzymic activity, thus making it possible to adjust the proportions to be employed for maximum yield and minimum expense. Physical and chemical tests have been devised for determining the amylase content of amylolytic agents-for example, polarimetric measurements ( 7 ) , viscometric measurements (6),iodometric methods such as those of Wohlgemuth (19),Windisch and Kohlbach (10, 17, 18), and Sandstedt, Kneen, and Blish (12,IS), and reducing sugar methods such as the Lintner determination ( I , 4, 11). Methods for estimating a-amylase and &amylase individually have also been devised by Sandstedt, Kneen, and Blish (19, IS),andKneen and Sandstedt ( 9 ) ,and have been tested and modified by others (5, 6, 8). 1
Present address, Oregon State College, Corvallis, Ore.
.llthough these piocedures afford relatively rapid measure of the amylase potency of saccharifying materials, no exact correlation can be found between any of these physical and chemical tests and actual alcohol yields obtained from the saccharified fermentation mashe.. For instance, chemical tests give neither accurate inforniatiou as to the suitability of a malt for fermentation mashes nor any indication of the required levels for most efficient use. Thorne, Emerson, Olson, and Peterson (14) made extensive tests with a large number of malts and came to the conclusion that “probably only fermentation tests can give an accurate evaluation.” They found that malts which would normally be rejected as distillers’ malts on the basis of Lintner values frequently gave better alcohol yields from wheat mashes than malts with very high Lintner values. Evidently other factors besides those measured by chemical tests for amylase enter into the effectiveness of malt for saccharifying fermentation mashes. The situation as regards correlation of chemical tests for amylase activity with value for saccharifying fermentation mashes is even less favorable with mold bran than with malt. With the
