760
ANALYTICAL CHEMISTRY
There is evidence in the literature that barium chromate is quantitatively precipitated a t pH 4.6 ( 1 ) . However, barium chromate is not quantitatively precipitated a t this pH under the conditions employed in the present separation. At p H 4.6 a sizable portion of barium remains in solution. Quantitative precipitation does not occur until the pH range 5.5 to 6.0 is reached.
Table 111. Determination of Barium in the Presence of Strontium PH
Barium Found, Gram
Barium Taken, 0.0972 Gram. 5.0 0.0971 5.3 0.0971
5.4
:.: 5.6 5.8 6 0
Table 11. Comparison of Volumetric and Gravimetric Results" (Barium taken. PH 5.1 5.4 5.4 5,s .5 , 7 6.4
0.0972 gram = 0.1792 grain of BaCrOi)
BaCrOh Found, Gram Gravimetric Volumetric 0.1760 0.1771 0.1781 0.1777 0.1778 0.1790
Ratio,
0.1757 0.1773 0.1776 0.1781 0.1784 0.1785
Barium Taken, 0.0972 Gram.
Strontium Taken, 0.097 Gram
c%?2L
-0.3 0 9 0.5 1.3 1.2 0.4 0.8 0.3 1.2
Vol.
1,002 0.999 1.003 0,998 0.996 1.003 .iv,
-0.1 -0.1 0.0 -0.3 0.1 0.4 0.3 0.2 0.1
0.0972 0.0969 0.0973 0.0976 0.0975 0.0974 0.0973
5.5
6 1
0 0984
1 000
Upon Reprecipitation
a Described procedure was followed, except t h a t solutions were cooled and allowed to stand several hours before filtering.
0.0971 0.0968 0.0970 a , b,
ilttempts to obtain bettrr buffering action with reagents other than acetate were limited by the strong oxidizing properties of the chromate ion as vel1 as by the complexing and precipitating properties of the possible buffers. Quantities ot Reagents Selected. , The various amounts of reagents were chosen because they produced the largest crystals, gave a reasonable time for hydrolysis, and gave the most reproducible results. DISCUSSION OF RESULTS
Determination of Barium. The results obtained isith the recommended procedure are given in Table I. Low results were obtained where the final p H was too lo^. The higher the p H the more quantitative the precipitation of barium, but unfortunately the tendency for strontium to coprecipitate also increases. -1pH of about 5.7 is most suitable. At this recommended pH, about 0.1 to 0.2 mg. of barium appears to remain in solution. An attempt mas made t o determine the composition of the precipitate, since slight fluctuations in the weight of the precipitate were obtained. Barium chromate precipitates obtained in different experiments from the same quantity of barium chloride stock solution were dissolved and the resulting solutions \$ere heated to boiling with ammonium pereulfate and titrated potentiometrically with standard ferrous ammonium sulfate. The results given in Table I1 indicate that the barium found gravimetrically is equal to the chromium found volumetrically. P Determination of Barium in Presence of Strontium. T ~ data
Difference, hlg.
Strontium Taken, 0.039 Gram
-0 1 -0.4 -0.2
Samples reprecipitated.
obtained using the recommended procedure are shoFT n in Table 111. Approximately 100 mg. of barium can be separated in a *ingle precipitation from about 40 mg. of strontium; 100 mg. of Strontium require a double precipitation. Determination of Barium in Presence of Calcium. .1 good separation of barium and calcium is indicated by the results given in Table IV. The pH does not have to be controlled as closely as is the case hen strontium is present. A complrte separation can be obtained n ith a single precipitation.
Table IV.
Determination of Barium in Presence of Calcium
(Bariuin taken, 0.0972 gram. Calcium taken, 0.1 gram) Barinin Found, Grain Difference, AIg. 5.6 0.0972 0.0 pH
6.6 7.0
0.0972 0.0974
0 0 0.2
LITERATURE CITED
(1) Beyer, G. L., and Rieman, W.. A S ~ LCHEY., . 19, 35 (194T). (2) Skrabal, A., and Seustadtl, L., 2 . anal. C h e m . , 44, 742 (1905) (3) Willard, H. H., . ~ K . % L ,C H E x , 22, 1 3 i 2 (1950). RECEIVED for review October 10, 1953.
.Iccepted January 2 5 , 1954.
Chromatographic Separation of Acid Obtained from Oxidation of Alpha-Pinene DORIS E. BALDWIN, VIRGINIA M. LOEBLICH, and RAY V. LAWRENCE N a v a l Stores Research Division, Naval Stores Station, Olustee, F / a ,
I
S CONSECTIOS with the oxidation of a-pinene, a constituent of turpentine, to pinonic and pinic acids (1, 4 ) , a satis-
factory method of qualitative and quantitative analysis was needed. The crude oxidation products obtained contain a number of mono- and dicarboxylic acids. Most of the acids involved are cyclic compounds, including cyclobutane and cyclopentane derivatives as well as acids containing ylactone structures. Some of these acids are produced as by-products and some are formed from impurities present in commercial a-pinene.
Marvel and Rands ($I, Ranisey and Patterson (5, 6 ) , Higuchi, Hill, and Corcoran (Z), and others have reported simultaneous qualitative and quantitative analysis of certain straight-chain acids by partition chromatograph>-. The insolubility of the acids under consideration in aliphatic hydrocarbons prevented use of the procedure presented by Ramsey and Patterson (6) for acids of comparable molecular vieight. Data from chronlatograms employing the method presented by Marvel and Rands (3) are reported as well as the results of a
761
V O L U M E 26, NO. 4, A P R I L 1 9 5 4 modification of their procedure and one other procedure employing benzene and benzene-1-butanol as the developing solvents. Some of the monocarboxylic acids (especially phonic) were not developed enough for separation or positive identification on columns employing chloroform as the mobile phase. h substantial improvement in the separation and identification of these acids was obtained on columns employing benzene as the mobile phase. APPARATUS
1 borosilicate glass chromatographic column 60 em. long and 18 mm. in inside diameter was used. A glass wool plug was used to support the immobile phase and a disk of filter paper was placed on top. REAGENTS
Kater-saturated chloroform, technical grade. Water-saturated benzene, ACS or C.P. grade. 1-Butanol redistilled over potassium carbonate. Silicic acid, Rlallinckrodt chromatographic grade. Distilled xmter. Phenol red indicator. Aqueous sodium hydroxide solution, 0.01 or 0.02-\-. PROCEDURE
Method I. Marvel-Rands (3). Samples of 10 to 30 mg. of the pure acids were developed on columns following the standard procedure presented by Marvel and Rands-namely, increasing the polarity of the eluting solvent in increments of 5% 1-butanol. Method 11. Modified Marvel-Rands. Columns and samples were prepared according to the procedure of llarvel-Rands. The polarity of the developing solvent was increased in increments of 1cc 1-hutanol. To ensure water saturation the 1-butanol-chloroform solutions were shaken with 1 ml. of water per 100 ml. of solution and were separated. Method 111. Benzene. This method emploj-s Rater on silicic acid as the immobile phase and benzene, benzene-1-butanol as the mobile phase. PREPAR~TIOS OF C o L n m AND SOLUTIOSS.The silicic acidwater mixture was slurried with about 100 ml. of benzene and poured into the column. The mixture was stirred to prevent the trapping of air bubbles in the silicic acid. The column rws settled with 5 pounds of air pressure and then packed firmly by hand n i t h a close-fitting plunger. The flow rate of these benzene columns was much faster than the flow rate of chloroform columns. From 7 to 10 ml. were eluted per minute when the columns were run under 1 pound of air pressure. KOchange in eah effluent volumes was found when the columns were run witEou; an>-pressure. From 0.1 to 0.5 meq. of the sample to be chromatographed was made to a volume of 5 ml. .I 2-ml. aliquot was carefully placed on top of the column and forced into the column with a little pressure. The sides of the column were then rinsed with another 2-ml. aliquot of benzene. The 4 ml. of eluant collected were discarded and the collection of IO-ml. fractions x a s begun. Developing solutions used were benzene and mixtures of benzene and 1-butanol of the following compositions: 100 ml. of benzene; 100 ml. of 1% l-butanol-99~obenzene v./v.; 100 ml. of 27, l-butanol-98% benzene v./v.; 100 ml. of 3% l-butanol97yobenzene v./v.; 100 ml. of 40/, l-butano1-96% benzene v./ v . ; 100 ml. of 5y0l-butanol-95yo benzene v./v.; and 100 ml. of 10% l-butano1-90% benzene v./v. To ensure water saturation the 1-butanol-benzene solutions were shaken with 1 ml. of water per 100 ml. of solution and were sep:irated. The developing solutions were added to the column in 30-ml. portions, each portion being added when 10 ml. of the preceding solution was still above the column, X 2-mi. aliquot of the remaining sample solution was titrated, with 0.01S or 0 . 0 2 5 aqueous sodium hydroxide using 2 ml. of 0.003% phenol red indicator, t o determine the total amount of acid added t o the column. The uer cent of the total acid Drestitration minus blank X 100 ent in each fraction = . Theperaliauot titration centages of the individual acids present in the sample are calculated from the amount of acid in each fraction forming the peak. I n cases where the separation of the acids is not complete, the value of the minimum point b e b e e n the curves is divided equally between the two acids.
STASDARDIZATION OF SILICIC ACID. The peak effluent volumes obtained with these columns are sensitive to changes in the water content of the silicic acid. Different batches of silicic acid used in this work were found to vary greatly in the amount of 13-ater required to form a satisfactory slurry, so that each batch had to be standardized in order t o duplicate the peak effluent volumes. The problem was approached from the standpoint of a constant amount of water, and the amount of silicic acid was varied until the peak effluent volume of a particular acid was reproduced. The criterion for a “standard” column was: 14 ml. of water ground with that amount of silicic acid which. when slurried with benzene and packed into a column, a-ould give a peak effluent volume of 200 for propionic acid. Propionic acid was chosen as a standard for t x o reasons: It is readily available in pure form and its peak effluent volume of 200 is such that it is sufficiently wnsitive to minor variations in the composition of the column. The variation in the initial composition of different batches of silicic acid is illustrated by the four different batches of silicic acid used in this work. Peak effluent volumes were reproduced when 20, 18, 15, and 20 grams, respectivply, of the four different lots of silicic acid were ground with 14 ml. of water. Silicic acid sitting open in the laboratory i d 1 gain or lose water rapidly as the room temperature and humidity change. Care should be taken t o reseal bottles of silicic acid once they are opened. Once a batch has been standardized, portions can be n-eighed out into bottles, stoppered tightly, and stored in a desiccator; or these portions can be ground with 14 ml. of water, placed in evaporating dishes, and stored in a desiccator over water until they are used. I n the search for a suitable acid to use for standardization of the columns, the following peak effluent volumes were observed: benzoic acid, 40; m-hydrosybenzoic acid (this acid had to be dissolved in 10% 1-butanol-benzene), 290; butyric acid, 90; propionic acid, 200; and acetic arid, 440.
E
ml
ELUATE
Figure 1. Chromatogram of Acid 3Iixture by Marvel-Rands 3Iethod
Table I. Peak Effluent T‘olumes of Acids Obtained by Marvel-Rands, Modified JIarvel-Rands, and Benzene Methods 31olecular Peak Effluent Volume Acid Formula Method I a Method 110 Method IIIb d-Camphoric CioHisOa 30C 30C 300 Pinonic ClOHl6OJ 40 40 80 Pinononic CsHirOa 50 30 180 Nopinic CioHiiOs 60 60 150 Pinolio CiaHisOs 70 70 200 Homoterpenylic CsHlrOa 70 70 280 Terpenylic C8HlzOa 110, 140 110 340 Terebic ClHio01 14Oc 160C 350d Pinic CsHiaOa 140 280 300 Sorpinic CsHiaOa 17OC 350 380 Hydroxypinic CsHlaOa 330c 750d Values obtained with one batch of silicic acid. b Values obtained with four different batches of silicic acid. C Dissolved in 10% 1-butanol-chloroform. d Dissolved in 10% 1-butanol-benzene.
ANALYTICAL CHEMISTRY
162
I
Table 11. Analysis of a Sample of Crude Pinic Acid Acid Unidentified Pinonio Unidentified Pinic Unidentified % recovery from column
Peak Effluent Volume 30
80 240 300 445 95.2
W
2.00 2 .00 -
'1
\
z
A
Found, % 1.6 11.9 9.8
70.9 1.0
EXPERIMENTAL RESULTS AND APPLICATION
The peak effluent volumes of pure acids that would be expected as products of the oxidation of commercial a-pinene using the three methods described above are tabulated in Table I. Unless i t is so noted in the footnotes, the samples were dissolved in water-saturated chloroform or benzene, depending on the type of column. The recovery of pure acids was 99 to 100% for all columns. Recovery on samples of pilot plant batches of crude pinic acid was from 95 to 100%.
W
2.00
v)
a
e
0;
1.50
2u
$ 1.00 aL I-\ v,
0.50
0
IO0
200
300
Chromatogram of Acid Mixture by Benzene Method
Homoterpenylic, terpenylic, and terebic acids would be likely to occur together in some samples produced under acidic oxidation conditions. The terebic acid is so insoluble in benzene or chloroform that it can be filtered out of the sample solutions and d e veloped separately. Homoterpenylic and terpenylic acids are separated to the same degree with either the modified MarvelRands procedure or the benzene method. Terpenylic acid is developed as two peaks with the Marvel-Rands method. This benzene method has been successfully applied to the analysis of crude samples obtained by the oxidation of a-pinene. A sample of crude pinic acid (prepared in a pilot plant by oxidation of commercial a-pinene with potassium permanganate to pinonic acid followed by the oxidation of pinonic acid with a deficiency of calcium hypochlorite to pink acid) gave the results tabulated in Table 11.
400
ml. E L U A T E Figure 2.
ml. ELUATE Figure 3.
Chromatogram of Acid Mixture by Modified Marvel-Rands Method
Figures 1,2, and 3 show the separation of a synthetic mixture of pure acids using each of the three methods. The Marvel-Rands and modified Marvel-Rands procedures do not separate this mixture of the monocarboxylic acids (pinonic, pinononic, and nopinic). However, in the absence of pinononic acid, pinonic and nopinic acids are almost completely separated. The only real advantage of the modified Marvel-Rands technique is the complete separation of the dicarboxylic acids present (pinic and norpinic). The good separation of both the monocarboxylic and the dicarboxylic acids using benzene ax the mobile phase is illustrated in Figure 3.
ACKNOWLEDGMENT
The authors wish to thank Richard N. Moore of the Naval Stores Research Division for providing some of the pure samples of acids used in this work. LITERATURE CITED (1) Delepine, M., Bull. SOC. chim., [5], 3,1369-82 (1936).
(2) Higuchi, T.,Hill, N. C., and Corcoran, G. B., ANAL. CHEW, 24,491-3 (1952). (3) Marvel, C.S.,and Fbnds, R. D., J . Am. Chem. SOC.,72,2642-6 (1950). (4) Mirphy, C. M.,O'Rear, J. G., and Zisman, W. A.,Ind. Eng. Chem., 45,119-30 (1953). (5) Ramsey, L. L.,and Patterson, W. I., J . Assoc. Oflc. Agr. Chemists, 28,644-56 (1945). (6) Ibid., 31,139-50 (1948). RECEIVED for review October 7, 1953. Accepted November 27, 1953.
Improved Colorimetric Determination of Urinary 17-Ketosteroids HAROLD WERBIN and SlEW ONG Argonne Cancer Research Hospital, The University o f Chicago, Chicago,
A
RECENT paper by hlasuda and Thuline (6) describing a method for estimating 17-ketosteroids in urine prompts a report on a similar modification which has proved to be of value in this laboratory for some time. Several investigators (3, 6, 7, 8) have attempted to improve the colorimetric determination of 17-ketosteroids by employing organic solvents to extract the pink chromogens formed in the Zimmermann reaction. Aqueous potassium hydroxide was used to develop the color of the steroid with dinitrobenzene and chloroform, ether or amyl acetate to
111.
extract it from 60% ( 3 ) or 68y0(5, 7 , 8) alcohol. The procedure described here differs in that: the absolute alcohol technique of Callow et al. ( 4 ) was adopted for the color formation, the alcohol concentration of the pink solution was made to 37% before extraction, and a novel apparatus was introduced to extract the steroid chromogens. These modifications practically eliminated the interfering substances that absorbed strongly a t 400 mp and permitted an average recovery of 96% of the crystalline steroid which was added to urine extracts. The apparatus described
