Table 111. Distribution Coefficients of IsoSeveral Metal Ions in 90% propanol-1 0% 6 N Hydrochloric Acid Medium (Dowex 50)
(5-mg. load per 1 gram of resin)
Metal ion
Distribution coefficient 250 450 6 20
Cd(I1) Cdi 11)
Ce(II1) Zr(1V) Th(1Y) v1 I v-v I
1 1
very high very high very high 5
(about 0.1 pg.) was found in 50 ml. of the effluent after 500 ml. of the wash solution had already passed through the resin bed. Because of this high breakthrough capacity, uranium in microgram and milligram amounts can readily be separated from bismuth even if this
metal ion is present in very great excess. Results of such separation experiments (carried out according to the recommended procedure) using variable amounts of uranium and bismuth are shown in Table I1 from which it is seen that within the limits of error of the fluorometric procedure of *2%, all separations are quantitative. For the elution of uranium, 4 to 12N hydrochloric acid is preferred over more dilute acid because it allows the rapid removal of uranium. By investigating the adsorption behavior of other metal ions under the conditions used for the separation of uranium from bismuth, the results recorded in Table I11 were obtained. From the values of the distribution coefficients of the various metal ions shown in Table I11 it is seen that all elements which are known to form strong anionic complexes in pure aqueous, and also mixed aqueous, organic solvents containing hydrochloric acid (7) have very low Kd values, except for cobalt which shows the same anomalous behavior as uranium. All these elements can be easily separated from uranium by using the working procedure, except Fe(II1) which is in the medium used, partly present as Fe(II), [formed by the reduction of Fe(II1) by isopropanol and the resin], and which is strongly retained by the resin. The result of an experiment in which 2.5
mg. of uranium plus 250 mg. of cadmium were used showed that quantitative separation is achieved as in the case of bismuth. LITERATURE CITED
( 1 ) Banerjee, G., Heyn, A. H. A., ANAL. CHEM. 30, 1795 (1958). (2) Faris, J. P., Buchanan, R. F., Rept. ANL-6811, July 1964. (3) Fritz, J. S., Abbink, J. E., ANAL.
CHEM.,in press.
(4) Hazan, I., Korkisch, J., Arrhenius, G., 2. Analyt. Chem., in press. ( 5 ) Korkisch, J., Rept. to Z.A.E.A. and U . S . At. Energy Comm. under Contract 67/US (At(30-1)-2623),April 1965. ( 6 ) Korkisch, J., Hazan, I., ASAL.CHEM. 36,2464 (1964). ( 7 ) Korkisch, J., Hazan, I., Tahnta 11, 1157 (1964). (8) Korkisch, J., Hazan, I., unpublished
results.
19) Korkisch. J.. Hazan., I.., ANAL. CHEM.. ' 37, 707 (1965): (10) Korkisch, J., Tera, F., 2. Analyt. Chem. 186, 290 (1962). ( 1 1 ) Kraus, K. A , , hlichelson, I). C., Nelson. F.. J . Am. Chem. SOC.81. 3204 (1959).' ' (12) Nelson, F., Murase, T., Kraus, K . A., J . Chromatog. 13,503 (1964). (13) Strelow, F. W. E., ANAL.CHEM. 32, 1186 (1960). (14) Strelow, F. W. E., Rethemeyer, R., Bothma, C. J. C., Ibid., 37, 106 (1965).
RECEIVEDfor review February 23, 1965. Accepted March 29, 1965. Research sponsored by the International Atomic Energy Agency and the U. S. Atomic Energy Commission under Contract 67/US (AT(30-1)-2623).
Refractometric Column Monitoring in Ion Exchange Chromatography of Carboxylic Acids KAZUKO SHIMOMURA and HAROLD F. WALTON University of Colorado, Boulder, Colo.
A flowing differential refractometer has been used with a potentiometric recorder to monitor effluents in anion exchange elution chromatography. The characteristics and limitations of this monitoring system have been examined. Nitrate-borate solutions were used as eluents; elution volumes for 1 2 carboxylic acids are reported. Elution volumes are greater for dicarboxylic acids than monocarboxylic, and they are increased by the presence of hydroxyl groups and ethylenic double bonds.
A
hindrance to the use of ion exchange chromatography has been the difficulty of analyzing the column effluents. Continuous monitoring can be used if the substances absorb in the ultraviolet or visible region, PRACTICAL
10 1 2
ANALYTICAL CHEMISTRY
and sometimes simple chemical tests can be made on individual fractions. These tests can be automated by the Technicon AutoAnalyzer (4). Nevertheless there has been a great need for a simple, general method of column monitoring that would be comparable in convenience with the methods used in gas chromatography. Refractive index provides such a method. Equipment is available that permits continuous recording of very small differences in refractive index. In column chromatography the difference in refractive index between the solution flowing into the column and that flowing out can be measured and recorded. Refractive index differences of the order and less can easily be detected. We have established the usefulness of this method by applying it to the elution
chromatography of a number of simple aliphatic carboxylic acids, using a strong base anion exchange resin and eluting solutions containing sodium nitrate, sodium borate, and boric acid. We repeated the work of Schenker and Rieman ( 5 ) on the separation of malic, tartaric, and citric acids, then went on to study the effect of eluent composition on elution volume, and measured elution volumes of 12 acids ranging in complexity from acetic to citric. EXPERIMENTAL
Refractometer. The instrument used was a flowing differential refractometer, Model R4, made by Waters Associates, Inc., Framingham, Mass. The construction of this instrument has been described (3). The sample solution and the reference solutions each flow through one half of a
Flow time (one space = 1 5 min.)
Figure 1 .
Recorder tracing of elution curve
2.5 mg. succinic acid, 2.5 mg. maleic acid Peak volumes, 5 3 ml. and 75 ml., respectively Eluant, 0.08M NoNaa, 0 . 0 2 M HaBOs, p H 8.2 Flow rate, 0 . 3 ml./min. Arrow morks romple introduction
rectangular glass cell which is divided by a diagonal plate. The volume of each part of the cell is only 0.07 ml., though the inlet and outlet tubes and the heat exchanger (needed to ensure constant and uniform temperature) add some 2 ml. to the overall volume of the flow system. Light passes through the cell, strikes a mirror, and is reflected back. Any difference in refractive index betwem the two liquids causes the beam to be deflected. The returning beam meets a second mirror which splits it into two parts, each of which strikes a photocell. The currents from these photocells oppose one another. Any imbalance drives a servo motor which turns a parallel-sided glass plate. This displaces the light beam until the photocell currents are equal. At the same time a signal is transmitted to a recorder, which records an electromotive force proportional to the refractive index difference between the sample and reference solutions. The refractometer panel has “span” controls which allow one to vary the sensitivity of the instrument within wide limits. For reasons to be discussed, we have operated a t relatively low sensitivity. A t scale factor 2, span 60, we found that one scale division on our recorder (0.15 mv.) corresponded to a refractive index difference of 1.0 X 10-6 unit. The maximum sensitivity of the instrument, with scale factor 16, span 100, would have been 8 X 100/60 = 13 times 0.15 mv. per R.I. unit. The highest sensitivity we have used is about 3 x 10-7 refractive index unit per recorder scale division, or 3 X 10-6 refractive index unit for full scale deflection on a 15-mv. Brown recorder. Resin Column. The ion exchanger used was Dowex-1, a quaternary ammonium-type anion exchange resin based on polystyrene, with 8% crosslinking and 100- to 200-mesh particle size. It was obtained from the BioRad Corp., Richmond, Calif. The column was 15 X 1 om., with a glass frit a t the bottom and a ground glass cap a t the top, and luer fittings top and bottom which carried three-way Hamil-
ton valves. The glass column was filled with resin nearly to the top, care being taken to minimize dead volume at each end. Samples were introduced through the three-way valve a t the top, using a calibrated syringe; their volume did not exceed 1 ml. and was usually less, Connections to and from the refractometer were made with small-diameter nylon tubing. The eluting solution was fed by gravity. I t flowed first through the “reference” side of the refractometer cell, then into the column, then through the “sample” side of the refractometer and out to waste. The flow rate was measured; it was virtually constant for any given run. Rates of 0.3 to 1.2 ml./min. were used. The sample solutions were prepared by adding the appropriate pure acid or its sodium salt to a portion of the buffer solution to be used as eluent, then adjusting the pH to that of the original buffer by adding sodium hydroxide. Before introducing the sample, the eluting buffer was allowed to flow through the cell, with the refractometer turned on, for 0.5 to 1 hour until the recorder showed a stable base line. RESULTS
General Observations. After introducing a sample, the recorder pen would continue along its base line, except for minor trembling which seemed to be caused by pressure fluctuation, until about 5 ml. (approximately one void column volume) had passed, when it would suddenly move off scale on the low refractive index side. About 0.5 ml. later it would return and travel rapidly to the high refractive index side, again going off scale, as a rule, for some 2 to 3 ml.; then it would drop to the bottom end of the scale for another 2 ml., then return to the base line, where it would stay until the first elution band appeared. The same
behavior, but with much smaller changes, was observed if a few drops of relatively concentrated eluent buffer were introduced. This behavior was puzzling, but seemed to be clarified when we noted that injection of diluted eluent caused the reverse behavior-Le., a brief rapid rise in refractive index, then a drop, then a second brief rise, then a return to the base line. Our tentative explanation is as follows. When a sample solution containing, say, sodium tartrate in addition to the eluent buffer is introduced, the tartrate ions displace nitrate and borate ions from the resin. A pulse of relatively roncentrated sodium nitrate passes down the column a t the same speed as the flowing liquid. As the front of this pulse strikes each resin bead the bead contracts unsymmetrically, becoming temporarily pearshaped as it is “pinched” on the upflow side. The relatively dilute solution inside the bead is squeezed out ahead of the front. This would account for the brief drop in refractive index. The depth of this drop was greater for more concentrated sample solutions. When diluted buffer is injected in place of the sample, the resin beads swell as the front of more dilute solution strikes them; in so doing they suck in some water from the solution just ahead of the low-concentration band. The rise in refractive index that follows the brief initial drop in normal sample introduction presumably results from the displacement of nitrate ions from the resin by the tartrate (or other anion) of the sample. Then, as the band of displaced sodium nitrate passes, we get a zone of relatively dilute solution caused by the relaxation of the beads. We do not pretend that this explanation is correct in every detail, and it is obviously subject to further testing, but the idea that fluctuations in effluent concentration are caused by swelling and contraction of resin beads seems a sound one. I t would explain the random and unpredictable minor displacements of the recorder base line which happened from time to time. These fluctuations prevented us from using the full sensitivity of the refractometer. They were associated with the flow of solution through the resin column and were not due to instability of the electronic circuits, or to optical or mechanical instability. Pressure fluctuations deflect the light beam and cause the recorder pen to move. Elution Curves. The recorder tracings of t u o typical elution curves are shown in Figures 1 and 2. These were obtained with a 50-mv. Leeds and Northrup recorder. The “square” appearance a t the peaks and troughs is typical; it is not because of a lag in the recorder. The VOL. 3 7 ,
NO. 8, JULY 1965
1013
Flow time (one space = 15 min.)
Recorder tracing of elution curve
Figure 2.
0.5 mg. a-methyllactic acid,5 mg. each of succinic, tartorlc, and furmarlc acids Peak volr., 20 ml., 6 2 ml., 83 ml., and 1 2 8 ml., rerpectlvely Eluant, 0.08M N a N 0 3 , 0.02M HaBOa, p H 7.1 Flow rate, 0.67 ml./min.
experiments with one acid a t a time, are summarized in Table I. They are uncorrected for the retention volume of the column; as we have noted, the void volume (volume between the beads, plus volume of connecting tubes between the bottom of the column and the refractometer cell) was about 6 ml.
proportionality between peak height and sample size was tested with tartaric acid; it was linear between 1 and 10 mg. of tartaric acid, and 1 mg. caused a refractive. index increase of 1 X lop5 unit a t the flow rate chosen. Effect of Effluent Composition ; Comparison of Different Acids. The peak elution volumes, obtained by
Table 1.
Eluent No. Eluent corn osition NaN03, H3B0zr hf
h
nH
~ ~ L t i ovolumes n (ml.)for acids Acetic Lactic 2-Methyllactic Oxnlir -. .
Malonic Succinic Maleic, Fumaric hlalic Tartaric Citric
1014
Peak Elution Volumes of Organic Acids
(Resin, Dowex-1 X 8; bulk volume, 14 ml.) 1 2 3 4 5 0.16 0.30 6.1 , . .
.,. ..
,
... , . .
... ... , . .
...
60 77
0.16 0.30 7.1
... 16 16 .
.
I
... 25 ... ... 25 35 62
ANALYTICAL CHEMISTRY
0.16 0.30 8.1
... . I
... ... .
I
I
, . .
,
,
,
, ,
,
, . .
27 48
0.08 0.15 7.1 _21_
20 20 109 71 59 103 122 65
90 ...
0.08 0.02 7.1 20 . _
20 22 124 75 63 86 130 67 86 .
I
6
7
0.08 0.02 8.1
0.04
19 ~~
...
19 118 68 59 95 124 63 80 .
0.01 7.1
I
.
34 34
... , . .
... ... ...
...
... .
.
.
I
The effect of p H was as expected. At the pH values chosen the organic acids are almost completely ionized, and increasing the p H increases the borate ion concentration of the eluent and so increases its displacing power. Schenker and Rieman (5) used pH 6.1; we made most of our tests at pH 7.1, where the borate-boric acid system has better buffering power. A higher pH, with more dilute boric acid, might have been better. Boric acid complexes polyhydroxy compounds and might or might not aid their elution, depending on the distribution of borate between exchanger and solution. Only with tartaric acid was there any sensible effect of boric acid concentration on the elution volume. The only purpose in adding boric acid, therefore, is to buffer the solution. The borate-nitrate eluent was chosen in preference to the acetate eluent used by Goudie and Rieman ( 2 ), primarily because borate and nitrate had less effect on the refractive index of the solution. Table I indicates possibilities for analytical uses. Dicarboxylic acids are bound more strongly than monocarboxylic; hydroxy groups and C :C double bonds strengthen the binding. The sequence oxalic-malonic-succinic suggests that the binding is stronger the closer the two negative charges, which is not surprising; with maleic and fumaric acids, however, the smaller charge separation gives the weaker binding. We find the same order of elution of lactic, malic, and tartaric acids that Courtoisier (1) found with acetic acid eluent, but our elution volumes are much less. LITERATURE CITED
(1) Courtoisier, A. J., Ribereau-Gayon, J., Bull. SOC.Chim. 1963, p. 350. (2) . , Goudie. A. J., Rieman, W. 111, A n d . Chim. Acta 26, '419 (1962). (3) Maley, L. E., I S A Trans. 1, 245
(1962).
( 4 ) Samuelson, O., Swenson, B., A n d . C h i n . Acta 28, 426 (1963).
(5) Schenker. H. H., Rieman, W. 111, ANAL.CHEM.25, 1637 (1953): RECEIVED for review January 11, 1965. Accepted May 3, 1965. Work was supported by the US.Atomic Energy Commission under Contract No. AT( 11-1)-499.
