1210
ANALYTICAL CHEMISTRY
lin G is not preferentially precipitated by N-ethylpiperidine from samples of high benzylpenicillin content. The isotopedilution method for penicillin cannot compete with chemical and biological methods of assay on a cost basis. The advantage of the isotope-dilution assay lies in the fact that i t most nearly approaches an absolute measure of total penicillins. Furthermore, penicilloic acid, the most likely contaminant of penicillin in broth, is not an interfering substance in this analysis. Statistical evaluation of the 14 replicate assays shown in Table I1 assigns a 95% confidence value of f 5 4 units or &2.4% to these data. I n addition, for a single sample assayed in duplicate, the 95% confidence limits are &5.9% on the mean of the duplicate assays. There are no significant differences in results between the made and recrystallized samples. Therefore, the Y-ethylpiperidine precipitation gives isotopically homogeneous material in one crystallization and the only limitations to its precision are those inherent in radioactive-counting procedures. ACKNOWLEDGMENT
The authors are indebted to Mavis Carroll for statistical evaluation, to J. F. Alicino for chemical assays on penicillin samples, to
S. C. Pan for providing a low-sulfur medium for penicillin production, and to Harry Hulit for technical assistance. LITERATURE CITED
(1) Nicino, J. F., IND. ENG.CHEX.,ANAL.ED.,18, 619 (1946). (2) Calvin, M., et al., ‘‘Isotopic Carbon,” pp. 113-20, New York, John Wiley & Sons, Inc., 1949.
(3) Craig, J. T., Tindall, J. B., and Senkus, M . , ANAL.CHEM.,23, 332 (1951). (4) Goodall, R. R., and Levi, .4.A , , .Vatwe, 158, 675 (1946). (5) Howell, S. F., Science, 107, 299 (1948). (6) Levy, G. B., et al., ANAL. CHEM.,21, 664 (1949). (7) Rfaas, E. A., and Johnson, h1. J., J.Bactereol., 57, 415 (1949). (8) Mader, W. J., and Buck, R. R., ANAL.CHEM.,20, 284 (1948). (9) Numerof, P., and Reinhardt, C., Ihid., 25, 364 (1953). (10) Rowley, D., et al., Nature, 161, 1009 (1948); 163, 480 (1949); J . Chem. Soc., 1949, 5-405; Biochem. J., 46, 157 (1950). (11) Sheehan, J. C., et al., J . Am. Chem. Soc., 68, 2407 (1946). (12) Smith, E. L., Brit. Med. Bull., 8 , 203 (1952). (13) Subcommittee, Ministry of Health Conference on Differential Assay of Penicillin, Analyst, 74, 79 (1949). RECBIIVZID for review July 8, 1953. Accepted April 3, 1954.
Elution Chromatography with Thick Filter Paper W. J. FRIERSON’, P. F. THOMASON,
and
HELEN P. RAAEN
O a k Ridge National Laboratory, O a k Ridge, Tenn.
T
ECHNIQUE using thick filter paper was developed for the separation of milligram quantities of uranium from other
elements by elution chromatography. The technique has the advantages of chromatography with thin filter paper, which was first applied to inorganic analysis by Arden et al. ( I ) , and to the separation of uranium by Arden, Burstall, and Linstead ( 2 ) . I n addition, it is suitable for amounts of inorganic substances larger than the approximately 1-mg. maximum that can be handled successfully on thin filter paper ( 3 ) . The technique is more simple, reproducible, and adaptable than the use of cellulose pulp columns, which are often employed for chromatographic separations on the milligram scale. With a thick filter-paper strip, a single elution can effect simultaneous separations for more than one test solution. The apparatus is similar to that of Strain and Sullivan (6) for analysis by electromigration and chromatography.
the strip, cut two V-shaped sections to serve m small cells for holding excess solvent, thus preventing it from running down either side of the glass plates. If a separation is to be effected on a single volume of sample taper the other end of the strip to a point. If two volumes o/ solution are to be chromatographed simultaneously in the same assembly, cut a 0.25 6 inch section out of the center of the strip from the tip to within 2 inches of the top (Figure 2). Taper the ends of the two legs thus formed. For either type of strip, enrlose the outside (longest) edges of the
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EXPERIMENTAL
Apparatus. The assembly for elution chromatography with thick filter paper, (Figure 1) consists of: Eaton-Dikeman No. 320 filter paper, 0.1 inch thick, rapid filtration rate; two platinum foil strips, 2 mils thick; two glass plates approximately 6.5 X 6.5 X 0.25 inches; four stainless steel strips, approximately 1 X 12 X 1/16 inches; two plywood strips approximately 3 X 14 X 0.25 inches, and screws with wing nuts. Sargent polarograph, modified. Precision Scientific Co., automatic titrator, modified. Reagents. All reagents were analytical reagent grade. Diethyl ether, anhydrous. Sitric acid, concentrated. Perchloric acid, 857,. Cranyl nitrate. Standard uranium solutions that were prepared from this reagent were standardized gravimetrically by ignition to uranyl uranate. Cupric nitrate, added directly to the synthetic sample solutions. Aluminum nitrate, added directly to the synthetic sample solutions. Standard ceric sulfate solution, prepared from ceric ammonium sulfate and standardized against arsenious oxide. PROCEDURE
From a sheet of 0.1-inch-thick Eaton-Dikeman No. 320 filter paper, cut a strip 2 X 8 inches. Near each edge of an@end of 1
Preaend address, Agnes Scott College, Decatur, Ga.
I I
Id I
I I I
I
Id
C
1 E
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I Figure 1. Assembly for Elution Chromatography with Thick Paper A-. Snlvmt. back one screwed to plywood - -- . - ..- added strip Front one tightened B . Solution spotted
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C. Plywood
p. Edges of paper covered with
E.
platinum foil Two stainless steel strips,
17
on glass with wing nuts Glass plates with strip snndwiched between
V O L U M E 26, NO. 7, J U L Y 1 9 5 4 strip in platinum foil in order t o prevent the solvent from creeping from the paper onto the glass. Now place the strip between the two glass plates 60 that the top extends about 0.25 inch above the glass and only the tip of the lower end extends below the glass. Support the plates in a vertical position and clamp them tightly on each side, a little more tightly a t the top than a t the bottom so that the top part of the paper will not absorb more solvent than can be absorbed by the bottom portion. Mount a dropping funnel above the strip in such a way that the tip of the funnel just touches the middle of the strip that extends above the glass. In order to remove from the filter paper any organic matter Figure 2. Strip for or ions that may be soluble in Simultaneous Elution of the eluting solvent, prervashthe a Standard and Sample paper by allowing about 100 ml. of the solvent to flow onto the strip a t a rate of about 2 drops per second or a t a rate not to exceed the rate of its absorption by the paper. Remove the strip from between the plates and dry it under an infrared lamp. On a strip cut for a single sample, deposit the test solution in the center of the strip and about 1 inch from the top. On a double-sample strip apply the separate volumes to each of the legs of the strip just below the upper end of the center cut. The spot should be completely covered by glass. Effect the separation by applying the eluting solvent in the same manner used for prewashing the paper. Collect the eluate, which ail1 drip from the tip of the strip, in a suitable container and prepare it for subsequent analysis. The removal of a metal from a thin filter paper .trip, by tapering the strip and allowing the solvent to run off the end, has been described for gold by Kember and Wells ( 5 ) . RESULTS
1211
A second synthetic sample was prepared that contained 3.00 mg. of ura,nium(VI), 35.1 mg. of aluminum(III),and 0.038 mg. of copper(I1) per milliliter. From this solution, three 250-pl. aliquots and two 500-p1. aliquots were chromatographed as described previously. After being treated with nitric and perchloric acids, the residues from the eluates of these aliquots were fumed once with several drops of concentrated sulfuric acid t o ensure complete removal of nitrate and then transferred, with water and 1 drop of the acid, to the cell of an automatic titrator. The uranium was determined by titration with standard ceric sulfate solution. The results are given in Table 11. To determine if other elements were eluted with the uranium, a solution that contained radioisotopes of ruthenium, zirconium, niobium, and the rare earths was chromatographed by this technique. The activity of the radioisotope solution was 5 x 106 counts per minute per ml. The approximate average activity of the eluate was 400 counts per minute per ml., which indicated that about 0.1% of the other elements was being eluted with the uranium.
Table I.
(Cianium taken, 31.5 -,/rnI.) r r a n i u m Recovered, illiquot Nuniher ?/fill. 28 0 33 0 28 4 32 2 28.3 6 30.9 28 3 7 31.0 8
AT. 30.0
Table 11. Yolumetric Determination of Uranium Recovery Aliquot, ill. 250 250 250 500
500
Elution chromatography with thick filter paper was evaluated for the separation of uranium(V1) from two synthetic test solutions that contained uraniumVI), copper(II), and aluminum(111). The eluting solvent used was the diethyl ether-nitric acid (5 volume) mixture described by Burstall and Wells ( 4 ) for the separation and purification of uranium by the use of cellulose columns.
.4 synthetic sample \vas prepared that contained 1.26 mg. of uranium(VI), 35.1 mg. of aluminum(III), and 0.038 mg. of copper(I1) per milliliter. -425O-pl. aliquot of this solution was spotted on a strip of thick filter paper that had been prewashed once with 100 ml. of the eluting solvent and alloived to partially air-dry. The strip was clamped between the glass plates of the apparatus, and a total of 100 ml. of a diethyl ether-nitric acid solution ( 5 volume %) was allowed t o drip slowly onto the top central portion. The solvent addition was regulated so that the eluate was received a t the rate of about 1 drop per second. The temperature during the elution was 25" =k 2" C. The eluate was prepared for the polarographic determination of uranium as follows. About 10 ml. of distilled water was added and the solution slowly evaporated to dryness. The residue was treated four times mith 2-ml. volumes of a 1 to 1 mixture of 85% perchloric and concentrated nitric acids and evaporated to dryness after each treatment. The final residue was taken up with 0.LV nitric acid to a volume of 10 ml. This solution should have contained 31.5 y of uranium per ml. A 5-ml. aliquot of the solution was placed in a cell, and the uranium determined polarographically. The results are given in Table I. Burstall and Wells ( 4 ) indicated that the nitrates of copper and aluminum remained stationary or moved only slightly on a cellulosecolumn elutedwith diethylether-nitric acid (5 volume %) Also, in this work, no copper wave appeared on the polarogram of the eluate from the thick filter paper. Any further evidence for the immobility of copper and aluminum and for their absence from the eluate was unnecessary.
Polarographic Determination of Uranium Recovery
Vranium, h l g . Taken Recovered 0 75 0.72 0.75 0.74 0.75 0.75 1.50 1.43 1.50 1.38
Difference, RIg. -0.03 -0.01 0.00 -0.07 -0.12
DISCUSSION
The type of thick filter paper used is impure, containing both metallic ions and soluble organic matter, thus necessitating prewashing. One prewash of the paper with the diethyl ethernitric acid solvent did not remove all the soluble organic material. An appreciable amount of the yellow organic material was extracted in the first few milliliters by the second wash and even a small amount by the third. The effect of size of the strip on a separation has not been investigated, nor has the quantity range over which the technique is applicable. Possibly a much smaller strip could be used as effectively with a resultant decrease in the required volume of eluting solvent. The approximate maximum volume of test solution that can be handled under the conditions described is believed to be 0.5 ml. ACKNOW LEDG.M ENT
The authors wish to thank L. T . Corbin of the Oak Ridge National Laboratory, dnalytical Chemistry Division, for supplying the solution of radioisotopes. LITERATURE CITED
(1) A4rden,T. V., Burstall, F. H., Davies, G. R., Lewis, J. A , and Linstead, R. P., .jrature,162, 691 (1948).
(2) Arden, T. V., Burstall, F. H., and Linstead, R. P., J. Chem. SOC.,1949 (Suppl. Issue N o . Z ) , S 311. (3) Burstall, F. H., Davies, G. R.. and Wells, R. A., Discussions Faraday SOC.,7, 179 (1949). (4) Burstall, F. H., and Wells, R. 4.,Analyst, 76, 396 (1951). (5) Kember, N. F., and Wells, R. A . , Ibid., 579. (6) Strain, H. H., and Sullivan, J. C., ANAL.CHEM.,23, 816 (1951). RECEIVED €or review January 6 , 1954. Accepted April 7, 1954.
