Separation of Phenols by Reversed-Phase Chromatography JAMES S. FRITZ and C. E. HEDRICK' Institute for Atomic Research and Department of Chemistry, Iowa State University, Ames, Iowa
b Mixtures of monohydroxy phenols are separated by reversed-phase chromatography using Teflon4 or porous polyethylene as the solid support. The stationary phase is cyclohexane or cyclohexane containing some 1-hexanol. The mobile phase is aqueous sodium chloride or a watermethanol mixture. Column elution behavior of phenols can b e predicted from batch distribution ratios. Satisfactory results were obtained for separation and quantitative spectrophotometric determination of the separated phenols.
C
reversed-phase column chromatographic separations have been reported, probatdy because really good solid supports have been lacking. Recently, however, some excellent inorganic separations (1, 4-6) and a few organic separations ( 2 , 3, 7) have been obtained by reversed-phase chromatography using porous polyethylene or fluorocarbon polymers as supports. (The references are representative rather than a complete list.) I n the present work monohydroxy phenols are separated by the technique of reversephase chromatography using 1- X 14.5cm. columns packed with 80- to 100mesh microporous polyethylene or 70- to 80-mesh Teflon-6 (a variety of polytetrafluoroethylene) Low molecular weight phenols can be separated using a stationary phase consisting of small percentages of 1-hexanol in cyclohexane, and a mobile phase of aqueous 0.5M sodium chloride. Higher molecular weight phenols are separated using water-methanol mixtures as the mobile phase and cyclohexane as the stationary phase. The separation of closely related monohydroxy phenols has been achieved. Preliminary experiments indicate that more than two phenols can be separated on short columns using gradient elution with water-methanol mixtures. OMPARATIVELY FEW
EXPERIMENTAL
Reagents. T h e solvents used were Eastman White Label or equivalent and were used without further pu1
Present address, Dept. of Chemistry,
1-niversity of Pennsylvania, Philadelphia,
Pa.
rification. Phenolic compounds were obtained from various manufacturers. Teflon-6 (DuPont, 70-80 mesh) was obtained from Analytical Engineering Laboratories, Hamden, Conn. The Teflon-6, a variety of polytetrafluoroethylene, was washed with acetone and diethyl ether and air-dried before using. Microporous polyethylene was obtained from U. S. Industrial Chemicals Co., Chicago, Ill. The substance, called Microthene-710 by the manufacturer, was ground in a mortar with dry ice and diethyl ether. The product was sieved to 80-100 mesh before using. Capillary tubing of 0.1-inch i.d. Teflon was obtained from Bel-A4rt Products, Inc., Pequannock, P;.J. The stock number of the tubing used was T-2 1195. A solution of 0.5M sodium chloride was prepared and pre-equilibrated with the stationary phase before using by shaking equal volumes of the solution with the stationary phase for 1 minute in a separatory funnel. Various concentrations of methyl alcohol were prepared by diluting the proper volume of methyl alcohol with water. The methyl alcohol solutions were equilibrated with the stationary phase in the same way as the sodium chloride solutions. Apparatus. Glass 1- x 15-cm. columns were used. These were equipped with coarse glass frits to support the packing a n d had stopcocks of Teflon. A gradient elution apparatus similar to that used by Schwab, Rieman, and Vaughan was used to obtain separations of more than two phenols (9). Elution curves were recorded using a Uviscan I1 ultraviolet flow monitor. The signal was led to a strip-chart recorder. All connections involving contact with the flowing solutions were made with tubing made of glass or Teflon. A Cary Model 14 recording spectrophotometer was used to analyze compounds collected in batches after passing through the flow monitor. Column Packing. Columns were filled with the liquid t o be used as the stationary phase. T h e dried inert support (Teflon-6 or Microthene-710) was then added to t h e column until t h e desired bed height was obtained. T h e excess stationary liquid in the column was then replaced by the mobile liquid phase using gentle suction. Distribution Ratios. Distribution ratios were measured by shaking 15
rnl. of pre-equilibrated nonpolar solvent with 15 ml. of a solution of the phenol in the equilibrated polar phase. The solutions contained approximately 0.05 mg. of phenolic compound per milliliter of t h e polar solvent. A portion of the nonextracted polar phase containing the phenol was reserved, and the distribution ratio, D , was calculated using the equation, - A, .____ - A. ___ D = concn. (nonpolar) concn. (polar) A, where AO represents the absorbance of
the polar phase before extraction (at the wavelength of maximum absorption of the phenol), and A , represents the absorbance of the polar phase after extraction. Elution Curves. Elution curves obtained using the ultraviolet flow monitor were converted to curves in which the fraction of total solute in t h e effluent fraction is plotted as a function of volume of effluent. This procedure avoids the effect of differences in loading of t h e phenols on the column; a high loading causes the solute to appear earlier in the effluent. Separations and Analysis. Standard phenol solutions were prepared by dissolving a n accurately-weighed 100- to 200-mg. sample in 5 ml. of 2-propanol and diluting to 100 ml. with t h e nonequilibrated mobile phase to be used in the separation. Samples were added to the column in 1.00-ml. aliquots from these standard solutions, A flow rate of approximately 2 ml. per minute was used for all elutions. Standards were prepared in the same way as the samples using the equilibrated mobile phase, and diluting the aliquot to the same volume as the sample which was collected from the column. Any haze that forms in the undiluted eluate is cleared up by adding a few milliliters of 2-propanol to the solution before final dilution in a volumetric flask of appropriate size. The samples and standards were analyzed in 1-em. silica cells by reading the absorbance of the compound a t the peak of the absorption curve taken with the Cary Model 14 recording spectrophotometer. RESULTS
I n the previous work of Fritz and Hedrick, the inert support was Kel-F 300 low density molding powder (4). However, since this material has been VOL. 37, NO. 8, JULY 1965
1015
70
I
I
I
1
I
I
I
PHENOLS: A 0-ISOPROPYL I p-PHENYL
so ’
15
I
1
ao. G 2,6-II-TERT. BUTYL H 2,6-dl-TERT. IUTYL-
e
2a
IO
%” 5E 20 5
E IO
E
0
20 30 40 50 60 70 60 90 VOLUYE PERCENTAGE YETHYL ALCOHOL IN WATER
0
0 0
I
4
3
2
5
7
6
8
9
VOLUME PERCENTAGE I-HEXANOL IN CYCLOHEXANE
Figure 1 . Distribution ratios of phenols between 0.5M sodium chloride and cyclohexane containing increasing concentrations of 1 -hexonol
I
I
I
I
I
I
I
100
Figure 2. Distribution ratios of high molecular weight phenols between cyclohexane and increasing concentrations of methyl alcohol in water
regenerated by washing with acetone and diethyl ether, followed by airdrying. This support was chosen for subsequent work with phenols, although microporous polyethylene can be used. Cyclohexane was chosen as the solvent to be used as the stationary phase for the separation of phenols. Cyclohexane is less dense than Teflon-6 and is transparent in the ultraviolet region of the spectrum, thus permitting the analysis of phenols by ultraviolet absorption spectrometry. Sara et al. (6) studied the partition of phenols between 0.1% aqueous sodium chloride and cyclohexane. They showed that a n increase in the salt concentration would slightly improve the extraction of phenols into cyclohexane. I n the present work the aqueous phase contains 0.5M sodium chloride. We found that the separation factor between phenol and o-cresol can be improved
withdrawn from production by the manufacturer, new inert supports were tested for possible use in organic separations. The new Kel-F 600 is not suitable for use owing to its low absorption of organic liquids. Microporous polyethylene showed promise as an inert support. This substance is obtainable in 40-60 mesh and can be ground to a fine powder after cooling with dry ice and diethyl ether. However, this support tends to dissolve somewhat in nonpolar hydrocarbon solvents. The polyfluorocarbon called Teflon-6 can be purchased in 70 to 80 mesh. Although this support has only 80% of the absorptive capacity for stationary phase as Kel-F 300, Teflon-6 is hard and is useful for column separations. Teflon-6 absorbs approximately 25gib of its own volume of organic solvents. This causes no perceptible swelling of the Teflon. Teflon-6 can be 0.35
IO
IO
and that the extractability of most phenols into cyclohexane can be increased by the addition of a small percentage of a water-insoluble alcohol t o the cyclohexane. At first 1-butanol was used, but the effluent became hazy with droplets of solvent which were removed from the column. A more insoluble alcohol, 1-hexanol, was used with better success. The effect on the distribution ratios for several phenols caused by increasing the percentage of 1-hexanol in cyclohexane is plotted in Figure 1. The polar phase is aqueous 0.5.V sodium chloride. The cyclohexane-1-hexanol and aqueous sodium chloride system is not amenable to the separation of high molecular weight phenols because these substances are insoluble in the aqueous sodium chloride. The distribution ratios of these phenols are very high, and they are completely extracted by the cyclohexane. The distribution ratios for phenols of higher molecular
I
PHENOL 3,4-DIMETHYL PHENOL
OlSO
5: kl
-
0.125-
-
0.20
2,6-DIYETH% PHENOL
4
MILLILITERS OF O.5M NoCl
Figure
3. Separation of phenol and o-cresol
Approximately 1 mg. o f each compound is eluted with 0.5M sodium chloride from o 1 X 12.5-cm. 70/80mesh Teflon-6 column on which i s sorbed 6% 1 -hexanol in cyclohexane
-
1016
ANALYTICAL CHEMISTRY
0
Figure 4. phenol
IO
PO
30 40 50 60 70 MILLILITERS OF 0.SM NoCl
80
90
LOO
Separation of 3,d-dimethylphenol and 2,6-dimethyl-
Conditions similar t o those of Figure 3, except stationary phase is cyclohexane
I a30
I
1
1
50 X ALCOHOL-
-
-METHYL
-METHYL
ALCOHOL-
2,6-di- ISOPROPYL PHENOL
i
4
0.05
0.00
0
IO
20
30
1 .
40 MILLILITERS OF ELUATE
Figure 5. Separation of 2-isopropylphenol and 2,6-diisopropylphenol
-
70/80 mesh Teflon-6 on which Is sorbed cycloColumn is 1 X 12.5-cm. hexone. Approximately 2 mg. of each compound used
Figure 6. Separation of phenols by gradient elution using methyl alcohol-water mixture A, o-cresol; B, 2-isopropyl phenol; C, 2,6-di-isopropyl phenol; D , 2,6-di-tert-butyl phenol Percentage methyl alcohol in water listed on right-hand axis
weight can be reduced by adding methanol to the polar phase. Sodium chloride and 1-hexanol are eliminated from this system to avoid dealing with a ternary system which might change in composition with a change in the temperature or pressure of the environment. The decrease in the distribution ratios of a number of higher molecular weight phenols as a function of increasing concentration of methanol is presented in Figure 2. The batch distribution ratios plotted in Figures 1 and 2 were used to predict which separations of phenols might be feasible and to select conditions for the separations. I n general the column separations work very well using conditions where batch distribution ratios show that there is a favorable separation factor. Elution curves are shown in Figures 3 and 4 for typical column separations using aqueous sodium chloride solution as the mobile phase and cyclohesane or cyclohexane plus 1hexanol as the stationary phase. Higher molecular weight phenols can be separated by using various concentrations of methanol in water as the mobile phase, and cyclohexane as the stationary phase. Preliminary experiments showed that simple elution with one concentration of methanol was not as suitable as the use of successive different concentrations of methanol. An esample of the latter technique is the separation of 1-isopropylphenol and 2,6-diisopropylphenol (Figure 5) More than one phenol can be separated using short columns. Preliminary experiments showed that Ocresol, 2-isopropylpht~no1, 2,Gdiisopropylphenol, and 2,6-di-tert-butylphenol can be separated by gradient elution with water-methanol mixtures. The first three compounds are only partially separated on 1- X 14-cm. columns, but they are completely
ser?arated from the 2.6-di-tert-butvlphenol. The results of a typical elutibn of approximately 15 mg. of each compound is presented in Figure 6. Column separations of a number of mixtures were done quantitatively by collecting each eluted phenol in a single fraction and measuring the phenol content by ultraviolet spectrophotometry. The data for typical quantitative separations are given in Table I. DISCUSSION
A significant advantage of separations by Craig countercurrent distribution over separations by partition chromatography is that the behavior of solutes in countercurrent distribution is accurately predictable from a knowledge of the distribution ratios of the various solutes. Most liquid-liquid partition chromatographic separations are not accurately predictable from distribution ratio d a t a because of absorption by the solid support or other interaction between the support and the sample solutes.
Table I.
However, for pure liquid-liquid partition chromatography where solute-solid support interaction is absent, the relation of the column retention volume (V,) to distribution ratio ( D ) is given by the equation,
V,
=
V,
+ DV,
where V , is the volume of the interstitial mobile phase and V , is the volume of the stationary phase in the column. The advantage of reversed-phase chromatographic separations described above is that the column elution behavior appears to be calculable from batch distribution ratios. For example, consider the separation of 3,4-dimethylphenol and 2,6-dimethylphenol reported in Figure 4. The column, which is 12.5 cm. long and 1.0 em. inside diameter, contains 4.0 ml. of cyclohexane (V, = 4.0) and 5 ml. of the mobile phase ( V , = 5 ) . Thus the predicted retention volume for 3,4-dimethylphenol is 14 ml., compared to a n experimental retention volume of 18 ml. The predicted retention volume of 2,6-dimethylphenol is
Separation of Phenol Mixtures
Col. is 1- X 14.5-cm. Teflon-6. Stationary phase is cyclohexane except for first mixture where cyclohexane containing 6% 1-hexanol was employed. Eluent req'd., Taken, Found, Difference, Compound Mobile phase ml . mg. mg. mg. 1.243 0 5 M Sodium chloride 1.250 Phenol 35 +0 007 0 5M Sodium chloride 100 1.281 1,280 -0 001 o-Cresol 2097, Methanol 4-Nitrophenol 25 0.285 0.287 +o 002 2-Nitrophenol 25 0,420 20% hlethanol 0,421 10 001 dilute BuJOH 1,327 1.332 3,4-Dimethylphenol 0 5 M Sodium chloride 30 -0.005 0 5M Sodium chloride 1.595 1.587 100 -0.008 2,6-I)imethylphenol 1.558 507c hlethanol 1 560 35 2-Isopropylphenol +o. 002 2,6-Diisopropylphena11 75% Methanol 1.957 1.984 50 + 0 . 027 2-Isopropylphenol 50% Methanol 30 1 558 1 570 + O 012 2-Nony lphenol 7 5 7 , Methanol 50 1 957 1 937 -0 020
+
VOL. 37, NO. 8, JULY 1 9 6 5
1017
74 ml., compared with an actual value of 62 ml. While these predictions are not exact, the agreement with the actual values is good enough to be quite useful. The predicted retention volume depends on V,, which is not easy to measure with high precision. I t may be of interest to mention that for the column separation just described, calculations based on plate theory indicate that the column has 20 theoretical plates. Thus the height equivalent of a theoretical plate is approximately 0.6 em. During part of this research, our results for most phenols tended to be high. This difficulty was caused by our use of surgical rubber tubing on the column exit, which introduced significant amounts of ultraviolet-ab-
sorbing immrities into the test solutions. +he use of Teflon capillary tubing to connect the to the flow monitor and from the flow monitor to the receiving - vessel avoids this source of error. A standard o-cresol solution in 0.5M aqueous sodium chloride which stood in contact for 0.5 hour with surgical rubber showed a significant increase in absorbance; no change in absorbance was found when this solution was in contact with silicone stopcock grease or the solid support for similar iime periods. ACKNOWLEDGMENT
The authors thank Jack Horowitz for making available the ultraviolet flow monitor used in part of this work.
LITERATURE CITED
( 1 ) cerrai, E.,Testa, c., J . Chromatog. 9, 216 (1962). ( 2 ) Chen, P. S., Jr., Terepka, A. R., Remaen, Tu'., AXAL.CHEM.35, 2030 (1963). ( 3 ) Frevtacr. W.,Fette, Seifen. Anstrichtmitted 65;'603'(1963); C.A.' 60, 2310a (1964). ( 4 ) Fritz, J. S., Hedrick, C. E., A N ~ L . cHEM. 36, 1324 (19641, ( 5 ) Hamlin, A. G., Roberts, B. J., Loughlin, W., Walker, S. G., Z b d . , 33, 1547 (1961). (6) Hayes, T. J., Hamlin, A. G., Analyst 87, 770 (1962). ( 7 ) Rindi, G., Perri, V,,A n d . Biochem. 5, 179 (1963). (8) Sara, S . C., Bhattacharjee, A., Basak. X . G.. Lahiri., 8.. , J. Chem. E m . Data 8 , 405 (1963). ( 9 ) Schwab, H., Rieman, W., Vaughan, P. A., ANAL.CHEM.29, 1357 (1957).
R~~~~~~~for review J~~~~~~13, 1965. Accepted May 24, 1965.
Empirical Formulae of Some Alcohol Complexes of Quad rivalent Ceriu m from Spectrometric Measurements H. G. OFFNER' and D. A. SKOOG Chemistry Department, Stanford University, Stanford, Calif.
b The
empirical formulae of the quadrivalent cerium complexes of n-, sec-, and tert-butanol and ethylene glycol were determined from spectrometric measurements, and in all cases a 1 : 1 ratio of cerium to alcohol in the complexes was observed. The ethylene glycol complex was the most stable, possibly because of formation of a chelated five-membered ring structure. Experiments were carried out in both nitric and perchloric acid solutions, and the effect of acid and nitrate concentrations on the observed "instability" constants was examined.
Q
cerium ion forms adducts with many anions such as sulfate (Z), chromate ( 8 ) , nitrate (9),oxalate ( 5 ) , and hydroxyl (6) in aqueous solution. Highly colored complexes are also formed with aliphatic alcohols, and analytical procedures have been developed for the determination of methanol, ethanol, and ethylene glycol by absorbtion spectrometry ( 3 , 4). Although the composition of some of the inorganic complexes has been determined, similar work on the organic complexes has not been reported. Consequently, it was considered of interest to further investigate this problem. UADRIVALEST
1 Present address, Rocketdyne, A Division of Xorth American Aviation, Inc., Canoga Park, Calif.
1018
ANALYTICAL CHEMISTRY
The determination of the combining ratios of quadrivalent cerium with n-, sec-, and krt-butanol and ethylene glycol by a spectrometric method is described in this report.
ceric ion or the alcohol concentrations are in large excess in the solution. The following chemical equilibria are considered to exist in a perchloric acid solution containing an alcohol: p Ce+'
THEORY
Experiments were carried out in both perchloric and nitric acid solutions. The mathematical equation used to reduce the data obtained in the perchloric acid series of experiments is derived in detail, while the equation applicable to the nitric acid experiments, being of the same form, is only indicated. The regions of absorption in the spectra of the aquo-ceric ion overlap those of the ceric alcohol complex; therefore, the equations are derived with an appropriate correction term. The following theoretical treatment parallels the method of Bent and French ( I ) . The several equilibria existing in solution are first specified. Then a rigorous expression is derived relating the observed absorbance values to the concentrations of the various species in solution. This expression, containing unknown terms, is not in a usable form; however, approximate relations are developed, based on selected experimental conditions, from which the combining ratio may be calculated from experimentally measurable quantities. The experimental conditions include the cases where the
Ce+'
+
?a
+ ROH = Cep (ROH) HzO = Ce (OH).+('--))
+'p
+ n H+
The molar concentrations are denoted as follows: co = Ce+' c o = Ce (OH).+(4-.)
co = CeP(ROH)+'p Also, let A be the observed absorbance and eo, e., and e, the corresponding molar absorptivities. ch
c t = co
=
H30f
+ c. + p c ,
(total cerium concentration)
cI = ROH
c,
= c,
+ cc
(total alcohol concentration)
The stability constant equations are:
'.Ch" CO
=
K,
