V O L U M E 2 8 , NO. 6, J U N E 1 9 5 6
1063
LITERATURE CITED
(1) Bowman, R. L., Caulfield, P. A,, Udenfriend, S., Science 122, 32 (1955). ( 2 ) Burdett, R. A., Jones, L. C., J . O p t . SOC.Amer. 37, 554 (1947). (3) Germann, F. E. E., Brown. F. T., Wissell, R., Waugh, T. D., Science 120, 540 (1954). (4) Gornall, A. G., Kalant, H., ANAL.CHEN.27, 474 (1955). (5) Huke, F. B., Heidel, R. H., Fassel, V. A., J . O p t . SOC.Amer. 4 3 , 400 (1953). (6) Lauer, J. L., Rosenbaum, E. J., Ibid.,41,450 (1951). (7) McAnally, J. S., A x . 4 ~CHEM. . 26, 1526 (1954).
Buffer flow rate is controlled by a constant buffer level and a system of wicks. The following modifications of this apparatus allow controlled temperature, regulated current by control of pH and conductivity of buffer in electrode vessels, and easy manipulation of sample tubes.
Modiflcation of Continuous Electrophoresis Apparatus Ernil R. Adamik, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Bethesda, Md.
apparatus described by Saroff [Nature 896 (1955)] T for continuous electrophoresis employs a sheet of filter paper wrapped around a cylindrical jar containing the buffer solution. 175,
HE
Figure 3.
Top view of plate (0)
The temperature of the apparatus is maintained constant by circulating water a t constant temperature through coil A (Figure 1). Cooling the apparatus necessitates the removal of condensate from the bottom of the buffer cylinder, K. This removal is accomplished by the filter paper, L, and wicks, M , as illustrated (Figure 1). Conductivity and p H changes of the buffer in the electrode vessels are controlled by mixing of the buffer in the vessels by means of the two air-lift pumps, B, in a common reservoir, C (Figure 2). The air-lift pump system is arranged to prevent the flow of electricity through the exchange system. The air-lift pump traps a small volume of buffer, pushes it up through tubing, which is relatively perpendicular, and discharges the buffer into the electrode vessels. Overflow tubes then feed the buffer back to reservoir, C . (In long runs, the reservoir may be cooled for control of bacterial growth.) A Lucite plate, D,with funnel-shaped holes, E (Figures 1 and 3), is held a t proper height above a false base, F , by means of a short cylinder, G, of Lucite permitting easy access to wick threads H for changing collecting bottles I and taking test samples during a run. Threads H used to carry sample from paper 0 to collecting bottles I pass through the funnel-shaped holes, E, and are knotted above and below the plate to prevent accidental detachment from the paper. Figure 1. Continuous electrophoresis apparatus
Apparatus for Automatically Increasing Solvent Polarity in Partition Chromatography Earl J. Roberts and Austin Mason, Southern Regional Research Laboratory, New Orleans, La.
chromatography on columns of adsorbents has found E wide application in the separation of small quantities of materials. The development of automatic fraction-collecting LUTION
Figure 2.
Circulating system for buffer in electrode vessels
devices has done much to eliminate the tedium of manual operation. However, certain types of partition chromatography, such as those described by Claborn and Patterson (I), Marvel and Rands (4), and Higuchi, Hill, and Corcoran (S),in which a series of solvents of increasing polarity is employed, still require constant attention, because the solvent must be changed manually. Donaldson, Tulane, and Marshall ( 2 ) have described an apparatus for automatically increasing the solvent polarity by allowing a mixture of a polar and a nonpolar solvent contained in a reservoir to flow into a mixing chamber containing only the nonpolar solvent. The effluent from the mixing chamber increases logarithmically in polarity and approaches the composition of the solvent in the reservoir as a limit. This method is
ANALYTICAL CHEMISTRY
1064 satisfactory for the separation of certain groups of acids, but fails to effect complete separation in some cases. This system is also limited, in that it does not permit the use of nater-saturated solvents, R-hich is advantageous when dilute sulfuric acid is used as the immobile phase a t high elution volumes.
r7
Figure 1. Apparatus
-*
Figure 2. Comparison of techniques to increase solvent polarity A . Solvent channed manuallv B. Apparatus of-Figure 1 uskd C . Method of Donaldson, Tulane, a n d Marshall a i t h reservoir solution of 6 0 R butanol-40R chloroform and 5OO-in1. mixing chamber D. Apparatus of Figure 1 wit11 butanolhexane mixtures
O/o
BUTANOL
the column is provided with a spherical joint, A, and the bottom with a stopcock, B, and a spherical joint, C, to match that of the chromatographic tube. A vent is provided through stopcock D for applying pressure t o the column independent of the solvent apparatus. Spherical stoppers are provided for the side arms and are held in place by clamps when the sections are filled. For operation, stopcock B is closed and the bottom section is filled completely with the nonpolar solvent and sealed by clamping the spherical stopper to the joint on the side arm. The remaining sections are then filled in the same way with polarnonpolar solvent mixtures in which the polar component is increased stepwise. When all the sections have been filled, the apparatus is attached a t joint C to a chromatographic column which has previously been prepared in the customary manner. Stopcock B is then opened, so that the solvent flows into the chromatographic tube. The solvent in each section then flows into the one immediately below it. Piessure may be applied a t the top of the apparatus, if necessary to obtain the desired f l o ~ rate.
The apparatus described (Figure 1) overcomes these difficulties making solvent changing automatic and permitting the use of water-saturated solvents. The solvent composition is changed stepwise R-ith solvent mixtures such as butanol-chloroform, in which the increase in polarity is accompanied bv a decrease in density. With solvent mixtures such as butanol-hexane, in which the increase in polarity is accompanied by an increase in density, diffusion occurs as the solvent mixture in each section slorvly flows into the slightly less dense mixture immediately beloT it. The resulting increase in solvent polarity is logarithmic, but more nearly approaches linearity than that obtained by the method of Donaldson, Tulane, and RIarshall. Therefore the behavior of the apparatus with each new solvent mixture should be determined before use. The apparatus was designed for routine analysis, and a new one must be constructed for each procedure that requires the solvent to be changed in steps of some other volume. The apparatus consists of a glass tube 51 mm. in diameter and 640 mm. long, equipped with nine partitions spaced a t 60-mm. intervals. Each partition has an opening 1 mm. in diameter in the center. Each section is provided with a side arm, terminating in an outer spherical joint, and extending upward so that the bottom of the joint is even with the top of the section to which it is attached. I n this way it is possible to fill each section through its side arm, reducing the air space to a minimum The combined volume of the section and side arm is 100 ml. The top of
This apparatus has been used very satisfactorily in this laboratory with butanol-chloroform mixtures and closely repi oduces the results obtained when the solvent is changed manually. -2 comparison of the composition of the influent to the column produced by three methods of increasing the solvent polarity is shown in Figure 2. The solvent mixture for curves -4) B , and C was butanol-chlor3form, and that for curve D was butanol-hexane. For curves -4, B, and D the solvents n-ere used in 100-nil. portions, in which the butanol content was increased in 5 5 steps from 0 to 457,. The composition of the effluents from the apparatus was determined by refractive index. The composition of the influent produced by manually changing the solvent is shown by A . B shows the composition produced by the apparatus of Figure 1 and C shows the composition produced by the method of Donaldson, Tulane, and Marshall with a reservoir solution of 60% b u t a n o l 4 0 5 chloroform and a mixing chamber of 500-ml. capacity. D s h o w the change in solvent composition produced by the apparatus of Figure 1 with butanol-hexane as the solvent mixture. LITERATURE CITED (1) Claborn, H. V., Patterson, W. I.,J . Assoc. O B c . Agr. Chemists 31, 134 (1948). (2) Donaldson, K. O., Tulane, V. J., llarshall, 31., ASAL.CHEW.24, 185 (1952). (3) Higuchi, T., Hill, iY.C., Corcoran, G. B., Ihid., 24, 491 (1952). (4) hlarvel. C. S., Rands R. D., Jr., J . A m . Chem. Soc. 72, 2642
(1950).
