566
ANALYTICAL CHEMISTRY
methanol, 60% glycerol, and 5 M sodium acetate system, respectively. The slopes of the linear portion of the correlation between the increase in absorbance a t 430 mp and time were used as a direct measure of the initial reaction velocities. The reason for the initial increase in reaction rate which occurs in the 50y0 methanol and the 60% glycerol systems is not known. Experimental evidence indicates that it is not due to the presence of a trace of impurity in the methanol or glycerol which might initially react with the oxidized guaiacol. Further, making up enzyme solution in 50% methanol before addition to the 50% methanol substrate solution did not eliminate the initial increase which occurs before the steady state reaction. The fact that a good linear correlation can be established during the first 3 minutes of the enzymic reaction indicates that this method provides a very accurate determination of initial reaction velocities. Reaction velocities determined by this technique have proved useful in determining activation energies a t low temperatures. This technique should also make possible the determination of accurate initial reaction velocities which are necessary in kinetic studies of activation, inhibition. p H optima, Michaelis constants, etc.. of enzymic reactions at low temperatures.
LITERATURE CITED
Balls, A. K., Ice and Cold Storage, 41, 101 (1938). Balls, A. K., and Lineweaver, H., Food Research, 3 , 57 (1938). Balls, A. K., and Tucker, I. W.,IXD. EXG.CHEM.,30, 415 (1938). Beers, R. F., and Sieer, I. W., J . Biol. Chem., 195,133 (1952). Devik, O., doctoral thesis, University of Minnesota, 1949. Euler, H. von, and Josephson, K., Ann., 452, 158 (1927). Hofstee, B. H. J., Science, 114,128 (1951). Huggins, C., and Talalay, P., J . Biol. Chem., 159, 399 (1945). Jensen, L. B., "Meat and l l e a t Foods," p. 22, New York, Ronald Press, 1949. Joslyn, A f . A , Advances in Enzymol., 9, 613 (1949). Joslyn, 11. A, J . Sei. Food Agr., 1951, 289. Kertesz, 2. I., J . Am. Chem. Soc., 64,2577 (1942). Lineweaver, H., Ibid.,61,403 (1939). Ponting, J. D., and Joslyn, AI. d.,Arch. Biochem., 19,47 (1948). Rudorff, F., Ann. phgsiiz. Chem., 116,55 (1862). St. John, J . L., J . Am. Chem. S x . , 53,4014 (1931). Siser, I. W., Advances in EnzymoZ., 3, 35 (1943). Sieer, I. W., and Josephson, E. S., Food Reseawh, 7 , 201 (1942). Stearn, A. E., Adnances in Enzymol., 9, 25 (1949). Tappel, 8.L., Lundberg, 1%'. O., and Boyer, P. D., Arch. Biochem., 42,293 (1953). '
RECEIVED for review September 8, 1953. Accepted October
24, 1953.
Peak Vo Iume-Conce nt ration Relationships with ProgressiveIy Changing Solvents in Organic Acid Chromatography CHARLES MADER Department of Agricultural Chemistry Research, Oklahoma A . and
et ( 2 ) and RIarshall et a2. (6) have described a D chromatographic system for separation of organic acids using progressively changing solvents. A useful peak volume-conONALDSOX
a2.
centration concept has been developed using the procedure described. APPARATUS AND PROCEDURE
The apparatus used was the closed system s h o m in Figure 1. The long mixing chamber, M , and the tube with a small orifice connecting it to the upper chamber, U , gave complete mixing. Chloroform ( c.P., previously washed, and redistilled) was placed in the mixing chamber. The mixing chamber was connected by means of the side arm t o a pressure unit, brought to the desired pressure and sealed by means of the stopcock on the side arm. The upper chamber was filled with 1-butanol ( c.P., previously distilled over potassium carbonate) in chloroform of the successive varying concentrations discussed below. This chamber was then connected to the constant pressure device of Rlader and Mader (6) at the pressure already present in the mixing chamber. The connecting stopcock was then opened slightly and the mixing was automatic thereafter. T h e c o l l e c t i o n mechanism used was that of Mader and Mader (3). The conductance siphon pipet of Mader and Rlader ( 4 ) was used to prevent evaporation. The column was prepared with v a r i o u s w e i g h t s of silicic a c i d (Mallinckrodt's: for chromatographic analysis). I n all experiments 0.7 ml. of 0.5N sulfuric acid per gram of silicic acid was used. This mixture was slurried with chloroform and c o m p r e s s e d into a solid column. Figure 1. ApparaTwo grams of pure silicic acid were tus
M . College, Stillwater, O k l a .
then slurried on top of this column and compressed to a solid layer. A 2-ml. aliquot of sorghum juice or sirup (previously adjusted t o 0.5N acidity to liberate the acids from their naturally occurring salts, and taken to the volume desired by dilution or evaporation over phosphoric anhydride using vacuum) was forced into the upper layer. After a few milliliters of chloroform had been placed on top of this prepared column it was ready for development. The collected 5-ml. fractions were titrated by means of a microburet with 0.01X sodium hydroxide with phenol red as the indicator. PEAK VOLUME-COSCENTRATION RELATIONSHIPS
If
A
= volume decimal fraction of butanol in chloroform in the upper chamber, Y = volume decimal fraction of butanol in chloroform passing into column, X = volume of eluent in milliliters, and B = volume of mixing chamber in milliliters the generalized volume-concentration equation is:
X =Bln-
A A - Y
This equation, which is mathematically identical with the one described bg Cherkin et al. (I), permits a straight-line plot by plotting log ( A - Y ) against X for a particular A and E . Chromatograms of sorghum juice and sirup were run using columns of 1-cm. diameter weighing from 6 to 12 grams, and A values of 0.35, 0.50, 0.60, and 0.70. The peak volume-concentrations of the acids were not changed by these various column weights except where separation did not occur, as for citric and tartaric acids on a 6-gram column. -4plot of the peak volume-concentrations of the five major acids for the various A values, keeping B constant a t 250, is shown in Figure 2. This graph shows that the peak volumeconcentration for each acid may be expressed by the general equation log ( A - Y ) = mX
+b
V O L U M E 2 6 , NO. 3, M A R C H 1 9 5 4
.4 0
I
I
I
i
567 I
I
-
+ACONITIC +OXALIC +MALIC +CITRIC +TARTARIC
1
-
-7
t
*20
I
*05L
‘A.
.35 -
I
I 10 0
Figure 2.
I 200
I
300
x -
I
I
I
400
500
600
ML.
Peak Volume-Concentration Relationships
The values of m and b were determined from the experimental points by the method of least squares. The peak volume-concentration equations for the five acids are‘ Acid Aconitic Oxalic Malic Citric Tartaric
log ( 4 - Y ) - Y) - Y) log ( A - Y ) log fA - Y ) log (a log ( A
= -0.00513X = -0.00103.Y = -0.00308X = -0.00294X = - 0 00270S
++ f ++
0.268 0.225 0.0786 0.162
0.153
Each equation is characteristic of the particular acid and is useful in identification of the acid. The following opposed factors influence the choice of a solvent system for a particular separation:
1. Peaks are higher ( a ) the shorter the column; ( b ) the less the eluent volume required. 2. Separation is better ( a ) the longer the column; ( b ) the larger the eluent volume.
The desired system is the one which will separate the acids and give the highest peaks. A 10-gram, 1-cm. column will separate all the sorghum acids. Since citric and tartaric acids do not separate on a 6-gram column and do separate on a 10-gram column, columns of less than 10 grams would be preferable only if the acids to be separated do not include citric and tartaric. A graph such a3 that of Figure 2 may be used to determine the least volume a t which good separation will occur. The A = 0.iO system, although it requires the least volume, does not separate malic and oxalic acids, as their peak volume-concentrations are too close to each other to allow satisfactory fractionation. The d = 0.35 system gives the best separation, but peaks are flat and spread out. This makes it difficult to determine where elution of the acids began and increases the blank error. -4s the graph indicates, the A = 0.60 system provides separation {rith the least volume and was chosen for the separation of these acids. Such graphs and characteristic volume-concentration equationq can be found for other acids and other systems, thus aiding the investigator in identification of the acids and affording a means of predicting the best system to try. The system of progressively changing solvent concexitration and the resulting graphical and mathematical use of results can be applied to many other separations previously using stepniqe change of eluent. LITERATURE CITED
(1) Cherkin, A,, Martinez, F. E., and Dunn, 11, S., J . Am. Chem. SOC.,75, 1244 (1953). (2) Donaldson, K. O., Tulane, V. J., and Marshall, L. RI., .%NAL. CHEM.,24, 185 (1952). (3) hlader, C., and hlader, G., Ihid., 25, 1423 (1953). (4) I b i d . , p. 1556. (5) Mader, C., and Rlader. G., Proc. Oklahoma Acad. Sci., in press. (6) Marshall, L. AI., Donaldson, K. O., and Friedberg, F., ANAL. CHEM.,24, 773 (1952). RECEIVED for review July 18, 1953. Accepted October 30, 1953. PreSOCIETY, sented before the Trisectional hleeting of the .kMERICAN CHEXICAL Bartlesville, Okla , October 16. 1953.
Spectrochemical Determination of Copper in Turbine Oils J. E. BARNEY, II Research Department, Standard O i l Co. (Indiana), Whiting, Ind.
TUDIES
of the influence of copper on the oxidation of tur-
S bine oils require the determination of microgram amounts of
that element in the oxidized oil. Although the dissolved copper may be present in concentrations up to several thousand parts per million, the usual range of interest is from 0.1 to 50 p.p.m. The American Society for Testing Materiala method ( 1 ) has been standardized as a laboratory procedure for studying the oxidation characteristics of turbine oils. This test, which is also used on white oils, employs copper and iron as oxidation catalysts. Accelerated oxidation procedures for studying turbine-oil life necessitate the analysis of oil samples for copper content as a function of time. Because the life test must be run on a limited amount of oil, often only a few milliliters will be available for each analysis. The problem has been met by extracting the copper with dilute sulfuric acid ( 5 )or an ion exchange resin ( 3 ) and determining it colorimetrically as the diethyldithiocarbamate complex. These techniques are time-consuming and often intro-
duce enough copper from the reagents to require correction for a large blank. Spectrochemical methods are particularly suited for this kind of analysis. Copper in very low concentrations can be detected by the spectrograph, only a few tenths of a milliliter of sample are required, and the analysis can be made rapidly with reasonable accuracy and precision. Because the samples are organic liquids containing minute amounts of just one or two metals, the absence of undesirable matrix effects greatly simplifies the selection of a spectrochemical procedure. A quantitative spectrochemical method, made possible by the simplicity of the system, has been developed in this laboratory. PROCEDURE
Spectrographic equipment includes a Bausch and Lomb Littrow spectrograph, an A.R.L.-Dietert Multisource, and an A.R.L. -Dietert comparator-densitometer. Electrically heated coils
