Fo.am Fractionation of Organic Compounds BARRY
L.
KARGER' and
L. B.
ROGERS
Department o f Chemistry, Massachusetts Institute o f Technology, Cambridge, Mass. Addition of a foaming agent makes possible the separation of surface.active organic compounds which do not form stable foams by themselves. Meihyl orange, crystal violet, and naphthalene derivatives were used in these exploratory studies. When the solute was ionic, it could be concentrated most effectively by using an ionic foaming agent of the opposite charge.
I
THE PAST, organic compounds separated by foam fractionation (1, 2) have been ones capable of producing a stable foam such as methyl cellulose (12), acid phosphatase (8), albumins (4, fa), and urease and catalase (7). Most of the literature on foam fractionation has been summarized
N
(2).
Our goal was to examine the feasibility of extending the applicability of foam fractionation by adding an agent to produce the foam necessary for isolating surface-active organic solutes which had little or no foaming ability. A literature search revealed only one previous attempt, a successful one in which gelatin had been added to produce a foam suitable for the isolation of methyl violet (6). The work of Walling e& al. (14), in which inorganic cations were found to be concentrated by anionic detergents, suggested that enrichments of organic ions might be increased by using a surface-active ion of the opposite charge. In recent studies by Gaden and coworkers (II), radioPresent address, Baker Chemical Laboratory, Cornell University, Ithaca, N. Y.
active strontium has been isolated by adding a foam-producing anion. Enrichment of the methyl orange anion by a cationic foaming agent was used for most of the preliminary work because changes in methyl orange concentration within the column could be followed visually. A few enrichment tests were also made with cationic crystal violet using an anionic wetting agent. Then, 1-naphthylamine and the 1- and Znaphthoic acids were studied because they represented similar molecules Wering only by a single functional group or position of substitution. They also represented a more nearly realistic separation problem. In general, the results indicated that ionpair formation offers a useful basis for foam separations. EXPERIMENTAL
Apparatus. As shown in Figure 1, nitro en used to produce the foam was a r s t passed through a precision flowrator, then a presaturator containing distilled water, and finally an extra-coarse dispersion tube in the sample solution. The foam pawed through a 24-cm. Vigreux column, into an ada ter, and into a graduated cylinder. if portion of the liquid was then diluted and its absorbance measured using a Beckman DU spectrophotometer. The pH of a collected sam le as well aa the pH of the solution in t t e flask, before and after foaming, was determined using a k e d s & Northrup Model 7664 pH indicator and a glaas electrode. Surface tension waa measured with a Cenco Model 70535 tensiometer. Reagents. All solutions, including samples, contained 5% methanol. Solutions were usually made up fresh
in order to minimize changes that might occur with time. Stock solutions of cationic, uncharged, and anionic foaming agents were repared: approximately 8.0 x 1 0 - d ' Armour and Co.'s Ethoquad 18/25 polyethoxy [15] stearyl methyl ammonium chloride; 1.7 X 10-4M Tergitol N P X (nonyl phenyl polyethylene glycol ether) of Carbide and Carbon Chemical Co.; and 2.2 X 10-aM Duponol W. A. Flakes (sodium lorol sulfate) from DuPont. Procedure. A 200-ml. sample was pipeted into a three-necked flask for foaming. .A flow of about 2.0 ml. per second of nitrogen was needed to bring the foam to the top of the column in about 3 minutes. The flow was then reduced to approximately 1.0 ml. per second to keep the foam a t the top of the column without overflowing to the adapter. Reflux was continued for 15 minutes, following which the flow of gas was increased to about 3.0 ml. per second to force the foam into the adapter where most of the bubbles soon broke. A 3-ml. sample could be collected at a nearly steady rate in about 6 minutes. A 2.00-ml. portion of the collected sample was pipeted into a 10-ml. volumetric flask, followed by two drops of either 0.1M hydrochloric acid or sodium hydroxide and distilled water to the mark. Methyl orange and the mphthoic acids were determined in alkaline solution (about p H 9.5) ;all other solutes were usually measured in acid (about 2.5). After allowing a waiting period of 10 minutes so that most of the foam in the column could return to the original flask, a 2-ml. sample was taken and treated in the same way. It was then possible to calculate the enrichment ratio, X / R , in which X is the concentration in the foam and R the concen-
4 Figure 1.
Foam fractionation set-up
1.
Tank of nitrogen 2. Flowmeter (Fixher and Porter, Model 068) 3. Saturator 4. TWO-WOYStopcock 5. Gas dispersion tube 6. 3-Necked distilling flask, 200 ml.; Corning No. 4965 7. Vigreux distilling column 2.5 X 24 cm.; Corning No. 3 5 2 5 8. 3 - W o y connecting tube; Corning No. 8980 9. 2-way connecting tube; Corning No. 8940 10. Funnel, 65-mm. 1 1. 1 0-MI. graduated cylinder 12. Ground gloss stopper
VOL 33, NO. 9, AUGUST 1961
1 165
tration of the residue in the flask. In most of our experiments it was possible to compare enrichments by calculating X / X o , in which X o is the original concentration in the sample before foaming, because a large volume of sample was used and only a relatively small decrease in concentration occurred. Enrichment values for duplicates usually showed a spread of about 5 to 7%.; the worst spread was 15%. RESULTS
Sample Vohme. Variation of A 5 ml. was a practical limit. When the volume was greater than 205 ml., some solution was already in the column before nitrogen flow began. As a result, there was irregular mixing of foam with liquid that bumped higher in the column and receded with the production of giant bubbles that led to eddy currents (6). Large bubbles interfered with reflux by sweeping h e foam ahead of them. At volumes less than 195 ml., coalescence of bubbles occurred in the flask and again produced a great number of large bubbles. Reflux Time. A series of preliminary runs made on portions of a sample containing 4.5 X lO-'M Ethoquad and 3.1 X 10-4M methyl orange showed that enrichment values increased rapidly with reflux time and then nearly leveled off a t 15 minutes. Even though the optimum time might be somewhat influenced by changes in solute concentration in the original sample, a period of 15 minutes was selected as the standard time for future studies. A t the end of the rrflux period, the top 5 cm. of foam were much darker than the rest, but it was necessary to collect a moderate amount of unenriched foam to bring the volume of condensed foam t o 3 ml. If 0.5-ml. portions were collected with a 15-minlite reflux period between each, en-
richment increased to 250% of its regular value. Obviously the longer procedure was advantageous, but the excessive time required for a run did not seem to justify making a semiquantitative evaluation of varibles. These results did emphasize, however, that enrichment was occurring primarily a t the top of the column where most of the bubbles broke. Column Variations. The use of three Vigreux columns connected end-to-end increased enrichment S%, a percentage gain only slightly larger than the limits of reproducibility. In contrast, a 45 mm. diameter column made from plain tubing with suitable glass joints sealed onto both ends tripled the enrichment for a single Vigreux column, thereby showing that enrichment occurred primarily a t the top of the column. In another experiment a simple straight-wall condenser having an inner tube 12 mm. in diameter and 45 cm. long was substituted for the Vigreux column. A 6 mm. diameter glass rod inside the column was connected through a stopper in the adapter to a stirring motor. The resulting enrichment value of 5.8 was identical with that for the wider Vigreux column. However, the net enrichment offered no improvement over a simple Vigreux column, so the use of a rotated rod was discontinued. Bubbler. The size and uniformity of bubbles are known to be variables (8, 9), and the use of a single carefully oriented capillary has been recommended. Using 1.53 X 10-4X Ethoquad and 1.25 X 10-4Mmethyl orange, a coarse fritted-glass bubbler produced an average enrichment of 6.5 as opposed to one of 7.5 from an extra coarse frit. The difference is not great but it is significant. In contrast, a 0.5-mm. capillary gave bubbles eight times the normal 1-mm. size and an enrichment of 18.2, but the column was more dif-
Table I. Agent,
ni x 10-4 Ethnniiarl 1 53- 1 53
1 53 1 49 Duponol 6 68 6 63 5 75 4 21 Tergitol 0 380 0 380 0 380 0.380
1 166
Enrichments of Naphthalene Derivatives Solute Concn. Xo, Charged Type M X lo-' pH X/Xo
Uncharged pH X/Xo
1-Saphthoic acid 2-Xaphthoic arid 1-Naphthylamine 1-(Chloromethy1)naphthalene
0 88 1 24 1 37
9 5 9 5 2 5
4 1 5 4 131
2 5 2 5 9 5
302 254 256
1 06
...
...
9.5
1.85
1-Xaphthoic acid 2-Naphthoic acid 1-Kaphthylamine 1-(Chloromethy1)naphthalene
0.86 0.96 1.37
9.5 9.5 2.5
1.33 1.21 10.5
2.5 2.5 9.5
2.91 2.48 3.80
0.92
...
...
9.5
2.15
1-Kaphthoic acid 2-Naphthoic acid 1-Naphthylamine 1-(Chloromethy1)naphthalene
0.86 0.96 1.37
9.5 9.5 2.5
3.4 4.2 4.4
2.5 2.5 9.5
3.6 3.9 4.8
...
9.5
3.6
ANALYTICAL CHEMISTRY
1.16
e . .
ficult to reproduce because of lower stability of the bubbles. Temperature. Using 1.3 X 10-41M Ethoquad and 1.2 X lO-4M methyl orange a t pH 9.5, enrichment changed from 3.50 a t the temperature of crushed ice to 5.42 a t 25' C., and to 7.5 a t 45' C. Although the increase observed with temperature may have arisen from a more favorable equilibrium value, the lower stability of the bubbles and greater reflux a t 45' C. contrasted sharply with the stable foams a t ice temperature. This temperature variation helped to explain why duplicates run a t the same time showed very good agreement, whereas day-to-day agreement was less satisfactory. The effect of temperature changes in later studies was reduced by making all of the runs for a particular variable within a short period of time. Chemical Variables. CHARGEEFFECTS. There are two general approaches for investigating charge effects in the foaming process. I n one approach the charge of the foaming agent is changed while that of the solute is held constant. By changing from Ethoquad to Tergitol to Duponol, the charge of the wetting agent goes, respectively, from positive to zero to negative. A second approach for studying the charge effect is to change the charge on the solute by changing the pH. Both approaches involve some uncertainties. Preliminary studies using methyl orange and crystal violet in distilled water indicated a significant charge effect. With cationic, uncharged, and anionic agents, enrichments of 4.8, 1.3, and 0.73, respectively, were observed for methyl orange; the corresponding values for crystal violet were 0.71, 1.10, and 4.1. More extensive studies were then made of naphthylamine and the naphthoic acids in solutions containing excess acid or base to control the charge on the solute. The first point to note in Table I is the marked difference in enrichment obtained on going from a cationic t o an anionic agent. Enrichment for the naphthoates decreased sharply whereas that for the protonated naphthylamine increased. The second point to note is that the values obtained for the uncharged agent fell between the values for the other two detergents. I t seems reasonable to conclude that when the charge is opposite to that on the solute, enrichment is improved compared to that obtained using an uncharged agent; when the charges are the same, the enrichment is less. The data for uncharged naphthalene derivatives show significant enrichment values. The striking feature is that the enrichment of 1-(chloromethy1)naphthalene is significantly greater using the uncharged agent than
3.0
? 0
=! 2.0
0
c
0
-
I
X
a
P
2 3 Collecied Sample Number
I 4
5
I.O*
C
u C V
% 1.0 0 a U
B
0 0
5
IO
15
Collected Sample Number
Figure 2. Recovery of methyl orange using approximately 1.53 X 10-'M Ethoquad for each fraction Cdlectlon procedure*
A. 1 -MI.; XO = 3.24 X 1 O-'M 8. 3-MI.; Xo = 3.24 X lO-'M C. l-Ml.;Xo = 1.21 X lO-'M D. MI., xo = 1.21 x 1 0 - 4 ~
with either of the charged agents. The same is true for the other three solutes, but to a less spectacular degree. Spectral evidence points clearly to the formation of very stable ion pairs in the case of Ethoquad and methyl orange (3). It should, therefore, be possible to decrease the enrichment by adding another ion to compete for the charged agent. Table I1 shows that 0.1111 iodide did not compete successfully with methyl orange, whereas it did with the naphthoates. It is true that salting out (IO) might also hqve affected the enrichment. CONCENTRATIONS OF AQENTS AND SOLUTES.Because adsorption isotherms are rarely linear over more than a short range of concentration, it was clear that concentrations of the agent and the solute would be critical factors. Therefore, enrichments were determined for a large number of combinations of Ethoquad and methyl orange using concentrations in the ranges of 2 to 24 X 10-M and 3 to 50 X 10-sM, respectively. At the highest concentration of methyl orange, enrichment was nearly constant a t 3.0. As the concentration of methyl orange decreased, enrichment increased to values near 18 using 24 X 10-6M methyl orange, and near 24 using 2 X lO-6M. Hence, the increased efficiency of the foaming process a t lower concentrations of solute was confirmed (798). Separations. RECOVERY OF A SINGLE COMPONENT.It was found by trial and error t h a t after a 3-ml. portion had been removed, the original foaming characteristics could be re-
stored by adding 50% of the original amount of surface-active agent. In addition, distilled water made alkaline with sodium hydroxide was added t o restore the original volume. Figure 2 shows that virtual completeness of recovery was indeed attainable, and that it was reached more quickly for a more dilute solution of methyl orange. As expected, removal of successive l-ml. portions was much slower in effecting complete recovery of 3-mi. portions, but the former procedure permitted the solute to be concentrated in a smaller total volume. Departure of the curves from exponential behavior reflects changes in enrichment with solute concentration. BINARYMIXTURES. When an attempt was made to prepare a mixture of methyl orange and crystal violet for separation, interaction was severe. Freshly prepared mixtures gave absorbance values that deviated from Beer's law by more than a factor of 10. Within a few hours, a precipitate formed. Hence, further study of this binary system had to be abandoned. The lower portion of Figure 3 shows the separation of two naphthalene species, one charged and the other uncharged. Although no attempt was made to effect complete recovery of the naphthoate, appreciable enrichment was effected with respect to the amine. Even a modest increase in reflux should lead to marked improvement in this separation. The top part of F'igure 3 shows enrichment values for three successive foaming operations on a mixture of 1and Znaphthoic acids. Although the
;0 2
I
E*-
I
o
1
I
I
2
3
4
5
Collected Somple Number
Figure 3. Foam fractionations of naphthalene derivatives using approximately 1.53 X 1 O-4M Ethoquod at pH 9.5 1.53
X lO-'M Ethoquad at pH 9.5
Tap: Two anions. 1. 1-Naphthaate 5.79 X lO-'M 2. 2-Naphthoate 1.92 X lO-'M Bottom: One anion and one neutral species. 1 -Naphthylamine 1.38 X 1 O-4M 1-Naphthoate 8.75 X IO-%
A. 8.
conditions for the three steps differed so that the absolute values for enrichment were markedly different, the ratio of the two enrichment values in each step was nearly constant a t 2.6. This indicated that the extent of separation was not critically dependent on drainage of liquid from the foam. The value of 2.6 is also significant because Table I shows that the quotient for the enrichments of the individual solutes was close to 1.3. The larger value for the binary mixture indicates that competition for the interface resulted in a more favorable quotient for the 2-naphthoic acid. These incomplete separations indicated that the procedure involving addition of a foaming agent was sufficiently promising to warrant further study. If oppositely charged solutes had been involved, separation should have been more favorable.
Table 11. Effect of 0.1M Sodium Iodide on Enrichments Obtained Using 1.53 X 1 O-4M Ethoquad at pH 9.5
Solute Methyl orange 1-Nauhthoicacid .2-Naphthoic acid a b
Solute Concn.,
xoM x
10-4 1.28O 1.24b
0 . 97n 0 . 8Sb 1.42a 1.24*
x/xs 9.1 8.7 1.32 4.2 1.70
5.5
Added salt. No added salt.
VOL. 33, NO. 9, AUGUST 1961
1167
CONCLUSIONS
The present study has demonstrated the feasibility of using added foaming agents to fractionate organic compounds which show little inherent ability to foam (and have relatively low surface activities). The fact that the product is contaminated with an added agent should be relatively unimportant in analytical determinations. However, if charged agents are used in preparative work, removal should be rapid and complete using a short column of ion exchanger. Foam fractionation should receive more attention from analysts. The simplicity of the technique and the fact that separability is usually improved in dilute solutions make foam fractionation attractive for trace analysis. Studies designed to evaluate structural effects on enrichment probably should be carried out using simple
solutes and agents, but there is little question that ultimately the technique will be more useful in dealing with the more complex systems to which it was originally applied-i.e., those involving large polar heat-sensitive molecules of the type encountered in biological samples. LITERATURE CITED
(1) Cassidy, H. G., “Technique of Or-
ganic Chemistry,” Vol. V I Interscience, New York, 1951. (2) Cassidy, H. G., Zbid., Vol. X, 1957. (3) Catoggio, J. A., Olver, J. W., Rogers, L. B., unpublished data, 1961. (4) Gaden E. L. Jr., Schnepf, R. W., J . B i o c h ~idicrobiol. Technol. Eno. 1,1(1959).
( 5 ) Jirgensons, B., Strsumsnis, M. E.,
“A f3,)jm-t Textbook of Colloid Chemistry, Wiley, New York, 1958. (6) Kenrick, F. B., J . Phys. Chem. 16, 513 (1912). (7) London, M., Cohen, M., Hudson, P. B., Arch. Biochem. Biophys. 46, 141 ( 1953).
(8) London, M., Cohen, M., Hudson, P. B., Biochem. et Biophys. Acta 13, 111 (19541. (9) Perri, J. M., Hazel, F., Ind. Eng. Chem. 38, 549 (1946). (10) Sargent, R., Rieman, W., 111, J . Om. Chem. 21, 594 (1956). (11) ’schne f, R. W., Gaden, E. L., Miroczni!, E. Y., Schonfeld, E., Chem. Eng. Prog. 55, 42 (May, 1959). (12) Schutz, F., Trans. Faraday SOC. 38, 85 and 94 (1942). (13) Thurman, W. C., Brown, A. G., McBain, J. W., J . Am. Chem. Soc. 71, 3129 (1949). (14) Walling, C., Ruff, E. E., Thornton, J. L., Jr., J . Phys. Chem. 56, 989 (1952). \ - - - - , -
RECEIVED for review December 12, 1960. Accepted May 5, 1961. Taken in part
from a thesis submitted by Barry L. Karger in partial fulfillment of the requirements for a B.S. degree, Massachusetts Institute of Technology, May 1960. Work was supported in part by the Atomic Energy Commission under Contract AT(30-1)-905.
Particle Size Distribution Measurements below Five Microns R. R. IRAN1 and E. F. KAELBLE Research Department, Inorganic Chemicab Division, Monsanto Chemical Co., St. louis 66, Mo.
b Centrifugal sedimentation and electron microscopy coupled with electronic counting and sizing are intercompared for the measurement of particle-size distributions of calcium phosphate and silica below 5 microns. Weight-size distributions from the two techniques agree within experimental error for materials whose particle shape does not deviate significantly from sphericity. Similar agreement was observed on flour, clay, and glass particles.
P
from this laboratory on particle-size technology have dealt primarily with the size range above 5 microns (3,4, 7, 8). This paper compares centrifugal sedimentation and electron microscopy for determination of particle-size distributions below 5 microns and in the submicron range. REVIOUS PUBLICATIONS
EXPERIMENTAL
Sedimentation. The apparatus was a commercial unit manufactured by Mine Safety Appliance Co., Pittsburgh, and has been described in detail previously (12). It utilizes the layer sedimentation technique (9), so that the amount of particulate matter collected at the Centrifugal
1168
ANALYTICAL CHEMISTRY
bottom of the sedimentation tube is directly proportional to the per cent by weight of particles with a size equal to or greater than that computed from Stokes’ equation. The speeds of the various centrifuges were checked using a tachometer, and agreed well with the manufacturers’ values. However, the time corrections for a centrifuge start-up and stoppage must be determined every 2 to 3 months. Obviously, the minimum t i e for running any centrifuge is the time required to reach full speed. One of the assumptions in this method is that sediment height is proportional to sediment weight. When complete dispersion of particulate matter is achieved, this is a good assumption because monosized particles are essentially settling at any one time, and void space is independent of size for monodisperse systems. However, when strong aggregates of smaller particles exist, the sediments compact more as higher centrifugal fields are used and bulk density correction factors must be used (8). For example, 2-micron silica particles are strong aggregates of many ultimate particles with a size of 0.01 to 0.03 micron. Therefore, in all determinations, the product of sediment volume times bulk density a t the specific revolutions per minute of centrifugation was taken as proportional to sediment weight. Absolute ethyl alcohol was the sedi-
mentation fluid, and the concentration of particulate matter in the suspension was 0.1% by volume. Spatulation (10) was utilized to disperse the powders, followed by vigorous stirring in a highspeed homogenizer. Electron Microscopic Technique. The electron photomicrographs mere made with an RCA EMU-3E microscope. Magnification mas 4080 diameters, as determined with a carbon replica of a ruled 28,800-line-per-inch diffraction grating. Suspensions of calcium phosphate and silica containing 0.1% solids were sprayed from a compressed air atomizer onto thin nitrocellulose films supported on 200-mesh specimen screens. Absolute ethyl alcohol was the dispersion medium for calcium phosphate, and silica was suspended in 2-ethylhexyl alcohol. Dispersion was accomplished by vigorous stirring in a high-speed homogenizer. The photomicrographs were recorded on 3l/, x 4 inch plates, and positive plates were prepared for counting and sizing purposes. The electron photomicrographs were counted and sized (size corrected for magnification) using a Cintel flying spot particle resolver manufactured by Cinema Television, Ltd., London ( I , 11). In all determinations, a total of a t least 2000 particles were counted and sized to give the number-size dis-
