1as
V O L U M E 2 4 , NO. 1, J A N U A R Y 1 9 5 2 relative error was 1.0%. The standard deviation of a single value was 3 parts per thousand. Advantages. The proposed method of analysis possesses the follou ing advantages over currently available procedures applicable to the same concentration range: freedom from coprecipitation or occlusion of boric acid; freedom from a blank correction; no requirement of boron-free glassware, because only acid solutions are boiled and then only for a fel\, minutes; minimuin opportunity for loss of boron by volatilization because lengthy evaporation is not necessary; simple equipment; and increased rapiditv and good accuracy.
LITERATURE CITED (1) Foote, F. J., IND.ESG. CHEM.,* 4 ~ 4ED., ~ .4 , 3 9 (1932). (2) Furman, N. H., “Scott’s Standard Methods of Chemical iln:ilYsis,” p. 179, New York, D. Van Yostrand Co., 1939. (3) Kolthoff, I. M., and Stenger, V. A , , “Volumetric Analysis.’ p. 70, New York, Interscience Publishers, 1947. (4) I b i d . , p,114. (5) Mylius, W., Chenl.-Ztg , 57, 195 (19333. (6) Schafer, H., and Sleverts, A.9 2’. anal. Chenl., 121,161 (1941). (7) Ibid., 121, 170 (1941). 18, 607 (1926). (8) TschischeTVskl, s , ,~ ~ d cjLern,, .
RECEIVED April 21, 1951.
Automatically Increasing Solvent Polarity in Chromatography KENNETH 0. DONALDSON, VICTOR J. TULANE, AND LIWRENCE 11. 3IARSfIALL Howard L’niaersity, Washington, D. C . The determination of organic acids separated on silica gel columns is limited in sensitivity by the reduced resolution experienced for the more watersoluble acids. Although the literature contains procedures for improving resolution by manipulation of the solvent, the changes in polarity which accomplish this end arc abrupt and are obtained by frequent manual operations. The widespread use of
I
X SEPARATIOSS employing partition chromatography for organic acids, the resolving power of silica gel systems decrease8 with increasing water solubility of the solutes ( 2 , 4, 5 ) . Isherwood ( 2 ) proposed the use of several solvents, but was aware that no one, or even two, ITould suitably resolve an organic acid mixture of the complexity used in his study. I n a procedure for the determination of furmaric acid ( 4 ) , this was again recognized
5 4.
1
acetic + fumaric
P 7)
L x
E32
-0
si
620
*
9
oconitic
oxalic
10 20 30 4050 60 70 80 9 0 fraction r J r b e r
fraction nJrnber
Figure 1. Chromatograms Typical curve (left) resulting when chromatographic solvent progressively increases in n-butyl alcohol content. Typical results ( r i g h t ) , when a eolvent of fixed ooinposition (10% n-butyl alcohol in chloroform, v / v ) is employed. T h e difference between t h e character of t h e curves as well BS t h e resolving power of the two systems with respect t o fumaric a n d acetic acids contrasts here t h e two types of chromatographic influent. Fraction number X 2 equals ml. of effluent.
apd effluent fractions on the chromatogram were collected by geometrically increasing volumes. I n effect, such a procedure gradually increased the volume of polar mobile phase in each effluent fraction. Marvel and Rands ( 5 ) successfully employed a series
mechanical apparatus for the collection of chromatographic fractions necessitates a procedure that requires no attention once the separation has begun. A simple device permits the delivery of a solvent automatically but gradually increasing in polarity. Recovery studies indicate that, with the exception of oxalacetic acid the method is suitable for the quantitative determination of organic acids.
of solvents progressively increasing in composition with respect t o n-butyl alcohol. Their solvents were added manually to the column in order of increasing n-butyl alcohol concentration. This procedure, like the first, required constant attention and mas, therefore, more noticeably disadvantageous when mechanical apparatus was employed for the collection of effluent fractions. The above procedures reduced the number of effluent fractions required to collect a given acid by manually but not gradually increasing the polarity of the mobile phase. When the concentration of each fraction n as plotted against its number, the curves on the chromatogram ivere sharper than those obtained Kith a solvent of fixed polarity. The present report, combining the principles underlying the determination of fumaric acid and those of Marvel and Rands, describes a procedure for gradually and automatically increasing the alcohol concentration and thereby the polarity of the solvent entering the column. With this method no false chromatographic peaks or shoulders on the peaks occur, as they sometimes do when the solvent polarity is increased in discrete steps. The applicability of this approach for the separation of certain acids of physiological interest is examined. A typical chromatogram employing the new procedure appears in Figure 1 (left). APPARATUS
A device was arranged so that the stem of the solvent reservoir ( A , Figure 2 ) extended to xithin 3 em. of the bottom of the mixing vessel, B . When 4 was filled Kith 50% (v./v.)n-butyl alcohol-chloroform and B with 175 ml of pure chloroform, the solvent flouing from column C created a fall in pressure within the system which was relieved by solvent delivered by the side arm. The 50y0 mixture from A flowed into B and because of the difference between the density of n-butyl alcohol (0.804 gram per ml.) and chloroform (1.497 gram per ml.) mixing was obtained, as demonstrated below, by the time the ~olvententered C. As a result, the solution entering the column gradual11 increased with respect to alcohol concentration. n-Amyl alcohol (density, 0.810 gram per ml.), where indicated be!ow, was substituted for n-butyl alcohol.
ANALYTICAL CHEMISTRY
186 Table I.
Observed Densities and Calculated Composition of Effluent Fractions
For a blank analysis t o determine rate of mixing of two components of chromatographic solvent) n-Butyl Alcohol-Chloroform n-Amyl Alcohol-Chloroform Density % composition Density % wmposition Fractiona 23' n-butyl alcohol 23 n-amyl alcohol 0.00 1,47
