directly to the reaction mixture, or the bromide ion was removed as follows: The solution n-as adjusted to p H 10 to 11 with sodium hydroxide solution and excess barium chloride solution added. The resulting precipitate of barium phosphate was centrifuged off, ivashed three times with water, and dissolved in the minimum amount of 2 N perchloric acid. The clear solution was run through a column of Dowex 50X4(H+) cation exchanger and the effluent treated with silver perchlorate solution as described above. The results of the phosphate and water (1) analyses are given in Table I.
The results following the barium phosphate procedure appear slightly better, although the number of samples analyzed is too small to allow significance to be attached to the results. However, in the presence of interfering ions, and in particular in the analysis of biological reaction mixtures, the barium phosphate procedure is advisable. The reproducibility of the method was checked by dividing a sample of trisilver phosphate into three portions which were decomposed and analyzed individually. Results were 0.412,0.414, ' of oxygen-18. and 0.415 atom %
LITERATURE CITED
(1) Anbar, M., Intern. J . Appl. Radiation and Isotopes 3, 131 (1958). (2) Bunton, C. A., Llewellyn, D. R.,
Oldham, K. G., Vernon, C. A., J . Chem. SOC.1958, 3574. (3) Cohn, M., J . Biol. Chem. 180, 771 (1949); 201,735 (1953). (4) Rittenberg, D., Ponticorvo, L., Intern. J . Appl. Radiation and Isotopes 1 , 208 (1956). (5) Williams, F. R., Hager, L. P., Science 128,1434 (1958). RECEIVED for review September 14, 1959. Accepted March 14, 1960. Investigations supported in part by a research grant (RG 5842) from the Division of Research Grants, U. S. Public Health Service.
Rapid Colorimetic Determination of 1 P. P.M. of Thiophene in Benzene Use of Permanent Color Standards H. A. BARNETT, C. E. BOLE, and C. F. GLICK Applied Research laboratory, U.
S. Sfeel Corp., Monroeville, Pa.
b The isatin method has been modified to permit rapid colorimetric determination of 1 p.p.m. of thiophene in benzene. A simple block comparator containing combinations of artificial color standards eliminates the need for a spectrophotometer. An unskilled operator can determine thiophene (down to 0.8 p.p.m.) in a sample of refined benzene in 10 minutes in the plant with very simple equipment. By comparison with a variation of ASTM D 1685-59T, the error of the modified procedure was -0.06 and - 0 . 0 9 p.p.m. of thiophene when the artificial color standards were 1 and 2 months old, respectively. The maximum statistical range of any set of determinations, by several operators, on a standard sample of benzene containing approximately 1 p.p.m. of thiophene was 0.04 p.p.m.; the average statistical range was 0.02 p.p.m.
C
benzene-toluene-xylene mixture from coal-tar light oil is usually washed Li-ith 66" Baume sulfuric acid and neutralized before distillation. The distilled benzene normally contains between 100 and 400 p.p.m. of thiophene. The thiophene content can be reduced to a very low level by rewashing with oleum(fuming sulfuric acid) and then with a strong caustic. A method 842
RUDE
0
ANALYTICAL CHEMISTRY
was required to control the washing with oleum prior to mashing with caustic to produce a product with a maximum thiophene concentration of 1 p.p.m. The most important level was from 0.8 to 1.1 p.p.m. of thiophene. The method should be as rapid as possible, yet yield results with a n accuracy within +0.05 p.p.m. of thiophene. The classical method for determining thiophene in benzene depends on reaction of the thiophene with isatin in the presence of concentrated sulfuric acid and an oxidizing agent to form indophenin, the concentration of which is measured colorimetrically (4). I n a standard version of this method (1) the color of the indophenin solution is compared with the colors from standard solutions of thiophene in thiophene-free benzene treated in the same way. These standards must be prepared aneiv for each test, because their colors are fugitive. The lowest concentration of a n y standard is 0.001 gram of thiophene per 100 ml. of benzene (approximately 11 p.p.m. of thiophene by weight). I n most of the modifications of the isatin method used industrially to measure less than 11 p.p.m. of thiophene in benzene the absorbance of the indophenin solution is measured with a spectrophotometer ( 2 ) . French described a method based on the use of a Lovibond Tintometer for the colorimetric determination of less than 25
p.p.m. of thiophene ( 3 ) . I n general, these methods are not convenient for plant use, because they are slow and require skilled operators and expensive equipment. With the method described, l p.p.m. of thiophene can be measured and these disadvantages are largely eliminated. REAGENTS
All chemicals were reagent grade unless otherwise specified. Thiophene-free benzene, prepared as described (8). Isatin Reagent, prepared as described (2). Allow the solution t o stand for a t least 72 hours before use. A small amount of isatin may subsequently crystallize from the solution, but quality is not impaired. After 2 months the reagent deteriorates and must be discaraed. Ferric Sulfate Reagent. Mull 0.020 gram of ferric sulfate, Fe2(S04h.zH2Or Gith approximately 15 ml.' of concentrated sulfuric acid (specific gravity 1.84) in an agate or mullite mortar until all traces of the sulfate have dissolved. (Use acid from a full bottle or one which has not been permitted to stand uncapped in moist air.) Transfer the solution to a 100-ml. volumetric flask, rinse the mortar with several small portions of concentrated sulfuric acid, add the rinsings t o the flask, and dilute to volume with acid. Store in a glassstoppered bottle and minimize contact with moist air.
Clark 8: Lubs pH 8.0 Buffer. Add 3.97 ml. of 0.1N sodium hydroxide (prepared carbonatefree and standardized volumetrically) to 50 ml. of 0.1M boric acid (0.1M solution of boric acid in 0.1M potassium chloride solution) and dilute to 100 ml. with distilled water. Gum Arabic Solution. Dissolve 0.5 gram of gum arabic (acacia, powder, U. S. P.) in 100 ml. of distilled water. Filter through a very coarse paper (Whatman 41H). Stock Chlorophenol Red Solution. Dilute 1 ml. of chlorouhenol red indicator solution (Parsiains, HartmanLeddon Co., Philadelphia, Pa.) to 25 ml. with Clark & Lubs buffer. Stock India Ink Solution. Dilute 1.00 gram of Higgins India ink (from a fresh bottle with no sediment) t o 1 liter with the gum arabic solution. Discard this solution after 1month. APPARATUS
Both the Beckman Model D U and Model B spectrophotometers were used with Corex cells of I-em. path length.
Table I. Proaortions of Solutions of Isatin. Chloroohcnol Red. and India Ink to ~. ,... .................................. .............................. Color-Match lndophenin Solutions from Oleum-Washed Benzenes Containing 0.8. 0.9, 1.0, and 1.1 P.P.M. of Thiophene ~
~
~~~
~
0.8P.P.M. 0.9 P.P.M. M1. Hole M1. 9 0.75 1.00 9.00 7.00
Hole 3
Isatin reagent U. S. P. chloroform Stock chlorophenol red 2 solution Clark & Lubs pH 8.0 buffer StackIndiainksolution 1 Gum arabic solution
4.05 6.00 2.60 4.50
7
14 13
3.69 3.00 2.35 3.50
20
3.70 2.70 3.90 4.00
19
Thiophene Contents of Standard Benzenes, P.P.M.
Age of Color Standards, Days
6 22 31
50
Because of the initial neutralization and distillation of the crude benzenetoluene-xylene mixture, the benzene to he analyzed was free of hydrogen sulfide. The following modified isatin method met the analytical requirements.
64
Figure 1. Schematic plan view of block comparator
8
8.00
1.1 P.P.M. Hole MI. 21 1.21 13.10
Table II. Visual Aging Data for Artificial Color Standards
PROCEDURE
Thirty milliliters of the benzene sample, 5 ml. of ferric sulfate reagent, and 1 ml. of isatin reagent were pipetted in that order into a 2-ounce glassstoppered bottle. The bottle was shaken vigorously by hand for exactly 1 minute and allowed to stand for exactly 5 minutes t o permit the two phases t o separate, and the lower indophenin-acid layer was pipetted offand measured at
3.45 7.00 3.65 7.00
1.0 P.P.M. Hole MI. 15 1.15
Known& 1 1.04 0.97 0.97 1.00 1.04 0.95 0.97 0.88 0.99 0.90
1.03 0.96 0.90 0.98 0.99 0.59 0.88
2
Observer 3 4 Found
1.05 . . . 0.98 . . . ... 0.90 ... 0.98 0.98 . . . 0.59 . . .
5
6
. . . . . . . . . . . . . . . . . . 0.90 0.95
. . .
. . .
. . . . . . 0.87
0.83 0.85 . . . . . . 0.900.91 0.91 0.90 0.51 0.83 0.79 0.83
Range
0.02 0.02 . . . . . . 0.00 . . . . . . 0.03 . . . . . . 0.01 . . . . . . 0.00 . . . . . . 0.01 0.86 . . . 0.03 . . . 0.90 0.01 ... 0.82 0.04
Av. Error 0.00 0.00 -0.07 -0.03 -0.05 -0.06 -0.09 -0.03 -0.09 -0.08
.
O.O0
-0'05 -''06 -o'06
-'O9
Ana3yzed by variation of (2).
once. (As the pipet was lowered through the upper benzene layer, a slow stream of air bubbles was expelled through the tip by a rubber bulb on the pipet, so that no benzene entered the pipet.) The absorbance of this solution was measured immediately (8). The concentration of thiophene was calculated from a calibration curve prepared by treating samples of benzene containing known concentrations of thiophene in the same way. These samples had previously been analyzed for thiophene by a variation of (B), which consisted of mechanical agitation of the test mixture in a n extraction cylinder for a specified time, rather than manual shaking. Both methods produce virtually identical results. Because spectrophotometers and the skilled personnel t o use them may not be available where the oleum washing is performed, any procedure for process control should include a simplified method for color measurement. A
block comparator containing combinationa of a,rtificial color standards was designed for this purpose.
This comparator was a block of opaque black plastic containing 21 vertical holes, arranged in seven rows of three each (Figure 1). Holes 4, 10, and 16 were normally empty. The remaining holes contained sealed glass ampoules, 14 mm. in outside diameter and 90 mm. long, each containing approximately 5 ml. of liquid. Ampoules in boles 5, 6, 11, 12, 17, and 18 contained water to equalize the number of air-glass interfaces, the depth of solution viewed, and the light refraction. The compositions of the liquids in the remaining ampoules are shown in Table I. A General Electric Deluxe cool white fluorescent lamp in a lamp house attached t o the back of the block diffusely illuminated these solutions through horizontal slots intersecting the rows of holes. The solutions in ampoules 1, 2, and 3 were of such concentrat.ions that their phenin-acid solution produced when a fied isitin procedure; iolutions in ampoules 7, 8, and 9 duplicated that from 0.9 u.u.m. of t,bionhene. etc.
VOL. 32,
NO. 7, JUNE 1960
843
turn. The openings of the vertical holes and of the horizontal slots were closed by a lid and cover glass, respectively, t o exclude dirt and extraneous light. The front face of the comparator (Figure 2) was painted a flat, light, neutral gray to facilitate color matching. RESULTS AND DISCUSSION
A series of standard samples of benzene, the thiophene contents of which had previously been determined by the variation of ( d ) , was analyzed for thiophene by several operators using the modified isatin procedure and the comparator. These analyses were repeated at intervals over 9 weeks (Table 11). The visual colorimetric method yields results which are in error [by comparison with the variation of ( d ) ] by -0.06 p.p.m. of thiophene when the artificial color standards are 1 month old, and by -0.09 p.p.m. of thiophene when the standards are 2 months old. The maximum error observed was hO.1 p.p.m., or less, when the standards were less than 2 months old. The maximum
statistical range of any set of ratings of a standard benzene sample by several operators was 0.04 p.p.m. of thiophene; the average statistical range was approximately 0.02 p.p.m. of thiophene. The artificial color standards should be replaced after 1 month. The India ink is probably the least stable standard, and some more stable substitute (a black dye ?) might be found. As suggested by French ( S ) , colored glass or plastic standards would be more stable but also more difficult to prepare and to change in hue. Standards for the analysis of benzene samples from a particular source should be prepared individually to match the indophenin-acid solutions from similar samples. Slight variations must be anticipated in the colors of the indophenin solutions from benzene samples produced at one plant from time to time, or a t different plants. If desired, samples of benzene containing somewhat less than 0.8 p.p.m. of thiophene can be handled by increasing the sample size; samples containing more than 1.1 p.p.m. of thiophene can
be handled by decreasing the sample size or diluting with thiophene-free benzene. Although only the application of the modified procedure to oleum-washed benzene has been demonstrated here, the principle of the method should apply equally well to benzene refined by other processrs. LITERATURE CITED
(1) Am. SOC. Testing Materials, Philadelphia, Pa., “1955 Book of ASTM
Standards,” Part 5, BSTM D 931-50.
( 2 ) Am. Soc. Testing Materials, “Tenta-
tive Method of Test for Traces of Thiophene in Benzene Using Isatin-and Spectrophotometry,’’ ASTPll D 168559T, 1959. (3) French, K. H. V., A N . ~ L .CHEM.20, 301-3 (1948). (4) Meyer, V., “Die Thiophengruppe,” Fnedrich Vieweer und Sohn. Braunschweig, 1888. RECEIVEDfor review March 31, 1959. Resubmitted February 16, 1960. .Accepted February 16, 1960. Tenth Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, March 2, 1959.
-
Determination of Trisatu rated Glycerides in Fats with Mercaptoacetic Acid 1. R. ESHELMAN, E. Y. MANZO, S. J. MARCUS, A. E. DECOTEAU, and E. G. HAMMOND Department o f Dairy and Food Industry, Iowa State University, Ames, Iowa
b A method for the determination of trisaturated glycerides in fats is based on the reaction of the unsaturated glycerides wth mercaptoacetic acid. The mercaptoacetic-glycerides that are formed are separated from the neutral trisaturated glycerides by extraction of the ammonium salts and ion exchange treatment. The method has been applied to a number of commercial fats and oils. The results are reproducible and recovery experiments indicate that the recovery of the trisaturated glyceride i s nearly quantitative.
A
trisaturated glycerides occur in many fats and oils, isolation from their host of structurally similar compounds is difficult. Three methods have been reported for the determination of the trisaturated glyceride fraction in fats. The first developed by Hilditch and Lea (IO) depends on oxidation of the fat with potassium permanganate and the subsequent sepa844
LTHOUGH
ANALYTICAL CHEMISTRY
ration of the neutral trisaturated glycerides from the acidic azelaoglycerides formed in the oxidation of the unsaturated portion of the fat. The second method developed by Hilditch and coworkers (9) depends on the great difference in the solubility of long-chain saturated and unsaturated glycerides. The method of Reiser and Dieckert (20) is based on isotope dilution. The potassium permanganate method has several disadvantages as a quantitative analytical technique. Hilditch ( 8 ) has pointed out the difficulties with emulsions in the separation of the neutral and acidic glycerides. Ting (22) attempted to use this method with butterfat and concluded it was not reproducible. Kartha ( I S ) has claimed that considerable hydrolysis of the ester groups of a fat occurs during potassium permanganate oxidation. He recommended carrying out the oxidation in the presence of acetic acid to neutralize the alkaline materials that are generated. Recently Eshelman and Hammond (6) have confirmed that ester hydrolysis may occur. They also
showed that if the oxidation was carried out in the presence of acetic acid, synthesis of new ester groups occurred and a neutral side product was formed. This has also been confirmed by Lakshminarayana and Rebello ( I @ , n-ho believed the neutral product mas an acetic acid ester of a keto1 formed from partial oxidation of the double bonds. Thus it appears that the permanganate oxidation technique is liable to sereral sources of error and map not give an accurate estimate of the trisaturated glycerides in fat. The crystallization method appears to give reliable results with some fats (5). However, it cannot be applied to the analysis of fats such as butterfat in which there is a great variation in the chain length of the saturated fatty acids that are present. Also the trisaturated glycerides cannot be completely isolated by this method. It is necessary to assume that any unsaturated material concentrated with the trisaturated glycerides is monounsaturated-disaturated glyceride, and that the saturated acids of the trisaturated glycerides are present
