(I) ; thus, all extractions took place a t room temperature (25' + 1' C.). To prove that the back extraction was complete, the ether layer was evaporated on a planchet and counted. Less than 1 c.p.m. was detected which indicated a good back extraction. The average error was about 20%. Experimental results are shown in Table I and Figure 1. DISCUSSION
TCO4- is an example of the MXaclass of complexed metal ions. The presence of donor atoms in the organic liquid enhances the extraction of the pertechnetate ion. Although the pertechnetate anion is poorly extracted into diethyl ether, there is evidence that the partition is significant. In
the region of 0.0 to 6.ON HKOa thr extraction coefficient is significantly dependent upon hydrogcn ion concentration. Increasing thc acidity of the aqueous layer favors protonation of the ether molecule to yield the diethH yloxonium ion, CzHs-0-CzH5+. This might provide an explanation of the increased extractibility of pertechnetate ion with increasing hydrogen ion concentration by the "oxonium" salt
H formation C:H~-0-CC2H5+TcO4-. In the region of 6.0 to 10.ON HNO,, it appears that maximum protonation has been obtained, and the partition of the pertechnetate ion is a t a relative maximum.
ACKNOWLEDGMENT
The author tliniiks P. K. Kuroda for his tlirec*tionof this research and for his cnc~our:igrnietit. LITERATURE CITED
(1) Boyd, G. E., Larson, Q. V., J . Phys. Chem. 64, 988 (1960). (2) Gerlit, J. B., PTOC.Intern. Cmj. Peaceful Uses At. Energy, Geneva, 1966, Vol. 7 , p. 145 (1956). (3) Morgan, F., Sizeland, M. L., U . K .
At. Energy Authority Report AERE C/M 96 (1950). MOSESA'ITREP, JR Department of Chemistry University of Arkansas
Fayetteville, Ark. THISinvestigation waa performed under the auspices of the U. S. Atomic Energy Commission Contract At-(40-1)-1313.
Water Transpiration as an Aid to Quantitative Paper Chromatography of C144abeled Compounds Paul D. Hoeprich and James N. Whitesides, Department of Internal Medicine, University of Utah College of Medicine, and the Salt Lake County General Hospital, Salt Lake City, Utah
ELIABLE
RADIOCARBON
R compounds chromatography
ASSAY
Of
separated by paper requires quantitative recovery of each compound in a form suitable for measurement of radioactivity. The burden of nonsample carbon must be minimal because high efficiency in carbon-14 measurement generally requires combustion to yield the sample carbon as carbon dioxide. Various elution techniques (4, 6, 8) useful to chemical analysis result in relatively large volumes of aqueous solutions. The additional procedure of drying required before combustion can be carried out is tedious and carries with it the risk of loss of some carbon if heating is used to hasten the process (CY) E~risionof the filter paper locus of a separated rompound, followed by comhistion and recovery of the samplr (*arbon,phis tlw rarbon of the supporting paper, should yield quantitativc recovery of the sample carbon. However, the location of the compound must be precisely defined; if this is done by color development, loss of sample carbon is again a hazard. Color development loss of sample carbon can be avoided by cutting the paper chromatogram so as to leave a wide border around a zone known to 1350
ANALYTICAL CHEMISTRY
contain a compound by comparison with a concomitantly irrigated, colordeveloped guide strip. But after combustion, the sample radiocarbon may be so diluted by the large amount of supporting paper stable carbon that counting of radioactivity may be seriously hampered. Glass fiber sheets for chromatography ought to avoid dilution of sample carbon. However, in our hands, such sheets were friable and difficult to handle, even after treatment with monopotassium phosphate (3) or alumina ( 5 ) . In addition, we did not obtain satisfactory chromatographic separation of mixtures of monosaccharides with glass fiber sheets. Transfer of separatrd sugars from filter paper to small fiber glass sheets has been accomplished with 1 0 0 ~ orecovery by chemicnl analysis (7). Our experience, using C"4-labelrd monosaccharides indicated transfer was not romplete. Quantitative recovery of C14-labeled monosaccharides after chromatographic resolution on filter paper has attended concentration by water transpiration. That other water soluble C14-labeled compounds should be amenable to quantitative recovery from paper chromatograms by this technique is sug-
gested both by our radioassay data with monosaccharides (Figure 1) and by the chemical data accumulated by Davis, Dubbs, and Adams with purines and pyrimidines (2). EXPERIMENTAL
Ten- and 5O-pl. aliquots of an aqueous solution containing D-glucuronic acid (-6-C14) (0.40j0),D-glucose (uniformly Clelabeled) (0.8%), and >rhamnose (-l-C14) (0.870) were applied marginally and centrally, respectively, on two 15 X 57 cm. sheets of Whatman No. 2 filter paper (sites A and B of Figure 1). Irrigation a t room temperature, descending, with ethyl acetatepyridine-water (120: 50: 40) was continued for 18 hours. A 2-cm. wide marginal strip bearing the resolved sugars of the 10-11. aliquot (deposited at A , Figure 1) was cut off and sprayed with aniline-salicylate reagent (aniline, 0.93 gram; salicylic acid, 2.76 grams; acetone q.s. 100 ml.) before heating for 5 minutes a t 106' Cy. In descending order from the origin spots appeared representing: 1)-glucuronic acid, Dglucose, Lrliamnose ( G A , G, and R, Figure 1). Guided by the location of the marginal strip spots, the remainder of each sheet was cut into transverse segments, allowing a t least 2 cm. above and below the predicted locus of a separated monosaccharide. Each segment was trimmed to a blunted point a t one
edge of the paper. After air drying at room temperature, end zones of ahout a 1-sq. em. area (hatched, in Figure 1) were cut off and burned (9) and the resultant CO, was assayed for radioactivity by gas phase proportional counting (1).
____ ____
RESULTS
&.,
70
____. 4.0
____. 6.3 1.0 ____.
5.0
____. 3.0
101.11
Figure 1. Results of radiocarbon assay carried out after water transpiration concentrationof monosaccharidesseparated from a mixture b y paper chromatography. The asterisks refer to C14 radioactivity most likely due to D glucuronolactone spontaneously formed from the D-glucuronic acid (-6-C14) component of the mixture opplied a t B
-
end as indicated in Figure 1 (the shaded areas were discarded). The segments were held in quasicylindrical shape (&xisperpendicular to direction of irrigation) by insertion into a hole cut through an acrylic plastic plate supported ahout 1 em. off the bottom of a 600-ml. beaker containing distilled water to depth of ahout 2 em. (see Figure 2). Left at room temperature overnight, transpired water deposited all solutes in a rime at the upper
Count data for the end zones of each segment are tabulated as part of Figure 1. Recovery was satisfactory as judged by counts predicted by prior assay of aliquok of the solutions of individual monosaccharides which were used in compounding the mixture applied to the paper sheets for chromatography. The asterisks call attention to radioactivity which was probably contributed hy n-glucuronolactone spontaneously formed from 0-glucuronic acid (-64Y4). According to C14 measurements, 3 monosaccharides were recovered quantitatively from a mixture after paper chromatographic resolution by concentration of the individual monosaccharides to a small end zone of paper by water transpiration. It is anticipated that this simple, effective means for sample eoncentration should he widely applicable to quantitation of C"-laheled compounds separahle by paper chromatography. LITERATURE CITED
( 1 ) Bernsteh, W., Ballantine, R., Rev. Sei. In&. 21, 158 (1950). (2) Davis, F., Dubbs, C . A,, Adams, W. S., ANAL.CHEM.34, 175 (1962).
(3) Dieckert, J. W., Morris, N. J., J . Agr. Food Chem. 6,930 (1958). (4) Dimler, R. J., Shaefer, W. C., Wise,
Figure 2. Arrangement for supporting segments of paper chromatograms for tip concentrotion of solutes by water transpiration. The horizontal lines mark 1 -cm. intervals
, Rist, C. E., ANAL. CHEM.24,
j . "
\-""",.
Ionald, E. J., ANAL. CAEM.29, 157).
Ilenberger, -- R. S., Moore, R. G., Ibid., 29, 27 [ 1YSiJ. (9) Vsn Slyke, D. D., Plash, J., Weisiger, J. R.,J. B i d . Chem. 191, 299 (1951). ~~
SUPPORTED in art hv Grants E-2068 and 23-39, Nationd Institute of Allergy and Infectious Diseases, U. S. Public Health Service, Department of Health, Educe tion, and Welfare, Bethesda. 14, Md.
Automatic Attenuator for a Hydrogen Flame Gas Chromatograph id J.
Frt
m e auvamages 01 gaJ chrois the wide concentration range that can he covered. Routinely, measurements are now made from the parts per million level to components that are almost 100% of the sample. To maintain sufficient accuracy over this wide range, i t is customary to use a range changer or attenuator to change the input signal to the recorder. Most chromatographs provide for this selection manually. Electronic stepping switches have been used to switch voltaee divider circuits automatically wit< thermal conductivity detectors [Darling, D. J., Miller, F. D., Bartsch, R. C., Trent, F. M., ANAL.CHEM.32, 144 (1960)l. A similar method employing a motordriven switch has been described NE 01
0 matography
F. Johnson, California
Research Corp., Richmond, Calif.
(Johnson, J. F.,Waver, R. F., Baumann, F., Beach, J. Y., Journtes Intern. Etudes Separation Immediate et de Chromatographie 1961, 136). These devices
are well worth their cost because of the saving of operator time. With automatic attenuators, one operator can handle many chromatographs lorated at diverse points. The Beckmaii hydrogen flame accessory employs two separate sttenuators to enable the amplifier to handle a wide dynamic range. One attenuator switches the output of the amdifier in the normal manner. The other switches feedback resistors in the amplifier. This arrangement is not cornpatihle with the normal automatic attenuators, as they are equipped to switch only one attenuator. These types also expose the output
attenuator to switch transients which are transmitted to the amplifier hy the feedback loop. On the more sensitive ranges, the resulting output of the amplifier is sufficient to drive the attenuator through another step. Thus, a vicious circle is established. It was evident that a different type of attenuator was needed because the feedback resistors, which are part of a high impedance circuit, could not he removed from the electrometer tube box. Therefore, it was decided to rotate the attenuator switch automaticallllv.
A part
Ledex (Ledex, Inc,, Dayton, Ohio, No, 215210-031) bidirectional
~ i ~ i + , , ~ t was ~ ~ geared to the shaft. The pulses required by the Digi-motor are generated by separate relays for the up and down directions. VOL. 34, NO. 10, SEPTEMBER 1962
1351
