ANALYTICAL CHEMISTRY
344
LITERATURE CITED
Table IV.
Recovery of Aspartic Acid Added to Hydrolyzed Proteins of
Aspartic Acid Present In sample Added Total
Total Aspartic Acid ReFound covery hfg. %
Protein
Protein Sample Mg.
.tfg.
.Mg.
.Mg.
Crystalline egg albumin
48.2 46.1
4.04 3.87
2.00 4.00
6.04 7.87
6.34 7.82
104.9 99.4
Bovine serum albumin
19.9 27.9
1.93
2.71
2.00 4.00
3.93 6.71
3.80 6.53
96.4 97.3
110
6.93 3 90
2.00
8.93 7.90
8.80 8 00
98.8 101.2
Bacto
- isoelec-
tric casein
62
4.00
Arhimo, A. A., Soumen Kemistilehti, 12B, 6 (1939). Bailey, K., Chibnall, A. C., Rees, M. W., and Williams, E. F., Biochem. J . , 37, 360 (1943). Block, R. J., and Boll$g, D., “Amino Acid Composition of Proteins and Foods, pp. 251-6, Springfield, Ill., Charles C Thomas Publishers, 1945. Braunshtein, E., Nemchinskaya, V. L., and Vilenkina, G. J., Biokhimiyu, 11, 501 (1946). Cannan, R. K., J . Bid. Chem., 152, 401 (1944). Chibnall, A. C., Proc. Roy. SOC.( L o n d o n ) ,B131, 136 (1942). Dakin, H. D., J . B i d . Chem., 141, 946 (1941). Darling, S., Acta Physiol. Scand., 10, 91 (1945). Engeland, R., Ber., 42, 2962 (1909). Foreman, F. W., Biochem. J . , 8 , 4 6 3 (1914). Gordon, A. H., Martin, A. J. P., and Synge, R. L. M.,Zbid., 37,
’
hv. 9 9 5
313 (1943).
the literature for the same proteins using other previously described methods. Recovery experiments, in n-hich known amounts of aspartic acid were added to a sample of three of the proteins mentioned in the preceding paragraph and hydrolyzed for 24 hours with 6 A‘ hydrochloric acid, were also performed. I n these experiments, an average recovery of 99.5% of the total aspartic acid content was obtained (Table IV). Typical polarograms obtained in the studies with casein and casein pluq added aspartic acid are shown in Figure 3.
i15) (16) (17) (18) (19) (20) (21) (22) (23)
Hac, L. R., and Snell, E. E.. J . Biol. Chem., 159,291 (1945). Herasymenko, P., Z. E l e c t r o c h a . , 34, 74 (1928). Kibrick. A. C.. J . BioZ. Chem., 152.411 (1944). Lamanna, C., unpublished data. Semerano, G., and Rao, I. S., Mikrochemie, 23, 9 (1937). Shemin, D., J . Biol. Chem., 1 5 9 , 4 3 9 (1945). Stokes, J. L., and Gunness, hl., Ibid., 157, 651 (1945). Suomalainen, H., and Arhimo, E., ANAL.CREM.,19, 207 (1947) Tarte, P., Rev.can. biol., 4, 477 (1945). Turbe, F., and Richter, bl., Ber., 75, 340 (1942). Warshomsky, B., and Elving, P. J.,BNAL. CHEX,19, 161 (1947). Weiland, T., and Wirth, L., Ber., 7 6 , 8 2 3 (1943) I
RECEIVED September 10, 1947.
Determination of Water in Alkylation Sulfuric Acid WILLIAM H. GOFF, WALTER SCOTT PALMER, AND ROLAR’D F. HUHNDORFF The Texas Company, P o r t A r t h u r , Texas The Karl Fischer method for determination of water has been adapted to determination of water in alkylation sulfuric acid. ‘Pretreatment of sample with anhydrous ammonium chloride prevented decomposition of Fischer reagent and an electrometric technique permitted end-point detection. Accuracy on chemically pure acid solutions was sufficiently good to show average deviation from known water content of less than 0.1% absolute. Reproducibility on alkylation acid was good, as was the reco+eryof known increments of water.
A
.
LTHOUGH many tests have been proposed for determining water by physical properties, only three chemical methods of test of relative importance have been proposed in recenk years: the acetyl chloride-pyridine ( 5 ) , the acetic anhydride-pyridine ( 6 ) , and the Fischer reagent ( 2 ) methods. The first two determine water by acidimetric titrations and require corrections for acidic or basic constituents in the sample. The Fischer reagent method is highly specific for water and operates independently of the acidity of the sample. The method of detecting the end point is described by .41niy, Griffin, and Wilcox ( 1 ) .
h 1-liter bottle reservoir for the Fischer reagent is fitted with a standard-taper pump from a gas-washing bottle. The aspirator and reservoir are separated by a tube of Drierite. The top openings of the buret are protected by similar tubes. A weighing pipet of the Smith or Lunge type. Other end-point
APPARATUS
9 length of Bakelite rod turned t o the size of a So. 6 rubber stopper is bored with four holes t o fit two electrodes, a stirrer, and a buret tip (Figure 1). An end-point detection apparatus consists of a laboratory
model Beckman p H meter or other suitable vacuum tube millivoltmeter, two electrodes, and shielded copper leads from the electrodes to the input terminals of the p H meter. The two electrodes are clean pieces of platinum and tungsten wire, S o . 10 B. &: S. gage, each about 19 cm. in length. Daily polishing with steel wool cleans the electrodes sufficiently t o keep them in working order. 4 variable-speed stirring motor with glass stirrer. Two SO-ml. burets, one automatic, the other offset to permit the tip to pass through the Bakelite stopper. A number of 150-ml. extraction flasks are used for titrating flasks.
DISPENSER
v
Figure 1. Titration Equipment
345
V O L U M E 20, NO. 4, A P R I L 1 9 4 8 detection devices are described by aernimont and Hopkinson ( 7 ) and McKinney and Hall ( 3 ) . Complete titration assemblies for Fischer reagent are commercially available.
In preparation, storage, and subsequent use, the two solutions must always be protected from atmospheric moisture, especially in locations with highly humid atmospheres.
REAGENTS
PROCEDURE
The chemicals used to prepare the Fischer reagent must be of suitable quality in regard to their water cont,ent,. Any water contained in the ingredients will consume the reagent itself, with a correspondingly low water equivalence for the final solution. The Fischer reagent' used contained a ratio of 1 mole of iodine to 2 moles of sulfur dioxide to 8 moles of pyridine and was dissolved in about an equal weight of methanol. This formula gives an equivalence of about 5 mg. of water per ml. of Fischer reagent and is prepared as follows: A stock solution of sulfur dioxide-pyridine is prepared by weighing about a gallon of pyridine into a flask, adding 203 grams of liquid sulfur dioxide per kilogram of pyridine, and storing in a glass-stoppered bottle. To 1359 grams of t>hestock sulfur dioxide-pyridine solution are added 1786 grams of methanol and 453.6 grams (1 pound) of iodine slowly and with frequent coolings, and the solution is swirled to prevent caking in the bottom. If allowed to heat up, the reaction may become explosively violent,. It is allowed to stand for a t least 24 hours before standardization. The standard wat,er in methanol solution is prepared by adding 5 ml. of water to 1 liter of anhydrous alcohol. This gives about 5 mg. per ml. and approximately matches the Fischer reagent. The water equivalents of the solutions are determined as described by illmy, Griffin, and Kilcox ( 1 ) .
The acid to be analyzed is weighed quickly to the nearest milligram into a clean, dry titrating flask and stoppered. Enough acid is used to give 0.04 to 0.10 gram of water; over 2 grams of acid are not used because there would not be an excess of ammonium chloride, which is added as the next step. Five grams of ammonium chloride, which has been dried overnight a t 105" C.. are added to the acid in the flask. This should all be done as quickly as possible and R-ith the care necessary to prevent picking up moisture from the air. The ammonium chloride and acid are mixed with a dry flattened stirring rod, and any soft lumps which form are crushed. Then 20 ml. of anhydrous methanol are pipetted into the flask and used to rinse the stirring rod. The ammonium chloride-sulfate mix will not go into solution but will remain as a finely divided solid which does not interfere with the titration, if it is not allowed to cake a t the bottom of the flask. A 2- or 3-ml. excess of Fischer reagent is added, the flask is placed upon the titration assembly, and thp excess Fischer reagent titrated with the standard watar in methanol solution. A blank determination is made, using the 5 grams of ammonium chloride and 20 ml. of methyl alcohol. The water content is calculated-as follows:
7cwater where 4 B
Table 1. Weirlit of
Sample, Grams 2.055 2.029 2.067 2.026
Fischer Reagent for Blank, 111. 5.11 5.11 5.20 5 20
Water Ratio of Equivalent Fischer of Fischer Reagent to Reagent, Methanol G. HzO/Ml. Solution 0.00428 1.212 0.00428 1.212 0.00415 1.250 0.00415 1,250
H20.
%
1.92 1.92 2.47 2.46
Check Average HZSOi HzSO4
'70
100
= ml. of Fischer reagent added
= ml. of water in methanol solution used in back-titra-
EXP1,ORATORY WORK ON WHITE ACIDS
98.14 96.26 93.72 93.31 93.26 79.47 36.40
70
:;:,9 93.72 93.32 93.24 79.47 36.42
Hz0
Check Hz0
%
",G
6.34 6.81 6.83 20.54 63.47
6.42 6.73 6.55 20.47 63 51
i:;:
Total Deviation Average of Averfrom Hz0 ages 100.00
%
%
%
6.38 6.77 6.84 20.51 63.49
100.09 100.08 99.98 89.91
+0.09
AI-erage deviation
k0.07
$;:
i:;; 1l!E:E; 7:;;; 00.09 +O.Og +0.08
-0.02 -0.09
\$'bite acids (c.P. sulfuric acid and water) were used in the
initial work, because the water content could be determined by subtracting the acidity as sulfuric acid from 100. The results o f t h e acidity as sulfuric acid plus the water by this method gave 100.0 + 9.1%. Total impurities on the white acids were less than 0.01%. The reproducibility of the test is shown in Table 11, ANALYSES OF ALKYLATION ACID SAMPLES
Table 111. Analyses of .illiylation Acid Samples HzO
Titratable Acid as HzSO4
Bctual His04
o/c
R
7%
%
81.00
4.38
6.37 6.33
87.91
3.14
5.06
9.1
0.0616
0,0612
99.13
88.31
1.93
5.0
0,0624
0,0632
99.22
88.44
3.46
5.87 5.47 5.48 6.65
8.7
0.0641
0,0640
100.42
87.47
2.44
c.69
5.8
0.0619
0.0625
99.69
87.47
2.32
6.0
0.0711
0.0706
99.7s
86.73
2.69
7.6
0.0616
0.0625
99,57
96.78
0.99
7.27 7.21 7.21 0.53 0.54 0.68 0.68 4.61 4.59 3.16 3.08
2.8
0.0636
0.0643
1 0.161
9 9 . 74a
Sample
0
x
W
Typical data collected on the duplicate determination of water -. in two samples are given in Table I,
XTe thod
So.
+ C)] x F
lnalysis of White 4cids b? Fischer Reagent
Tahle 11.
88.16 96.24 93.71 93.32 93.22 79,47 36,43
[9 - ( B
tion X R tl = ratio, ml. of Fischer reagent per ml. of water 111 methanol solution C = ml. of Fischer reagent for blank F = grams of water equivalent to 1 ml. of Fischer reagent lli = weight of sample in grams
Tgpical Data
Water in E'iacher Methanol Reagent, Solution, MI. MI. 18.70 3.60 19.00 3.96 21.62 3.35 21.86 3.73
=
2.73 2.78 3.03 3.00 3 3.11 3 3.17 4 3.01 45 3.06 3.13 3 3.15 6 2.75 6 2.81 7 2.95 7 2.93 8 1.92 8 1.92 9 0.79 9 0.82 10 2.46 10 2.47 11 1.85 11 1.89 Fresh acids.
88,39 88.39 888.90 8.91 88.90 88.94 89.54 89.55 88.17 88.23 88.20 87.61 87.58 91.08 97.03 97.94 98.01 91.46 91.45 93.65 93.67
Oxidizablev as HydrolyBuHSOi Carbon sis No.
97.84
0.43
91.00
1.45
93.20
1.41
HzO Added
HrO Recovered
Total Percentage
Gram 0,0641
100.49
Ma.
5%
iVaOH/g. Gram 11.4 0.0652
1.15
1.1
0.0640
0.0628
4.1
0.0637
0.0627
99.52
3.7
0.0622
0 0624
99.60
Analyses of some alkylation acids are shown in Table 111. The percentage of Jvater was determined by this method. The titratable acidity as sulfuric acid was determined by immediate titration at room temperature of a weighed diluted sample with 0.25 W c a r b o n a t e - f r e e sodium hydroxide. The butyl hydrogen sulfate was determined by the difference between the tit'ratable acidity and the total acidity after a diluted portion of the sample had been refluxed 24 hours. The increase in acidity was calculated as one half of the butyl hydrogen sulfate present. This, of course, assumed no dibutyl sulfate to be present as was indicated by Roby (4). The actual percentage of sulfuric acid was calculated by correcting the titratable acidity for the butyl hydrogen sulfate present.
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) Almy, 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
