ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978
1911
Separation of Alkylphenols by Normal-Phase and Reversed-Phase High-Performance Liquid Chromatography J. F. Schabron and R. J. Hurtubise” Department of Chemistry, University of Wyoming, Laramie, Wyoming 8207 7
H. F. Silver Mineral Engineering Department, University of Wyoming, Laramie, Wyoming 8207 1
Empirical correlation factors were developed which relate log k’ values for alkylphenols, the naphthols, and two phenylphenols to structural features. Both normal-phase and reversed-phase chromatographic systems were studied. The stationary phases employed in the normal-phase work were p-Bondapak CN, p-Bondapak NHz, and p-Porasil. The structural features which affect retention in the normal-phase chromatographic systems are the number of ortho substituents, the number of aliphatic carbons, and the number of aromatic rings. The stationary phases employed in the reversed-phase work were p-Bondapak C18 and p-Bondapak CN. The structural features which affect retention in the reversed-phase chromatographic systems are the number of allphatlc carbons and the number of aromatic double bonds. On p-Bondapak C,,, the presence or absence of a nonaromatic ring is of added importance.
T h e isolation of phenols is important in the study of coal liquefaction processes ( I ) , in industrial and in environmental pollution work (2),and in other areas (3-5). T h e separation of phenols by high-performance liquid chromatography (HPLC) has been studied by a few workers. Kirkland ( 6 ) separated phenol and six alkylphenols on a polymeric ether bonded-phase packing. Karger et al. ( 7 ) separated phenol, 4-isopropylphenol, and 2,6-di-tert-butyl-4-methylphenol on surface etched glass beads coated with /3,/3’-oxydipropionitrile. T h e separation of phenols on reversed-phase HPLC systems has been reported by Karch e t al. (8) and by Wolkoff and Larose (9). Husain e t al. (10)explored t h e separation of 13 isomeric alkylphenols on microparticulate silica gel columns. They found t h e H P L C method t o be superior to gas-liquid chromatography (GLC). Liquid-liquid chromatographic systems consisting of long-chain aliphatic amines on 4-7 pm solid supports with aqueous acid solution mobile phases for t h e separation of acidic compounds have been studied (11, 12). Several variables affecting the separation process were explored. Callmer et al. (13)studied structural effects on the separation of 33 alkyl-substituted phenols on microparticulate nitrile and octadecylsilane bonded phases. They pointed out t h a t one disadvantage of using GLC for the separation of alkylphenols was t h a t t h e structural information in GLC chromatograms is not straightforward because of the simultaneous influence of vapor pressure and polar interactions. Structure-retention relationships were studied on both nitrile and octadecylsilane HPLC stationary phases (13). The separations on t h e nitrile phases were divided into three groups depending on the number of ortho substituents. For each group, log k ’ decreased linearly with increasing aliphatic carbon number. T h e log k’ values on t h e octadecylsilane stationary phase increased linearly with aliphatic carbon number regardless of the position of substitution (13). Although Bark and Graham (14-1 7 ) had studied extensively structure-retention relationships of alkylphenols in both normal-phase and reversed-phase thin-layer chromatographic 0003-2700/78/0350-191 l$Ol.OO/O
(TLC) systems, Callmer et al. (13)were the first to study the separation of phenols by HPLC in this manner. In the present work, the retention behavior of phenol, several alkylphenols, the naphthols, and two phenylphenols was studied in three normal-phase and two reversed-phase HPLC systems. T h e stationary phases employed in t h e normal-phase work were p-Bondapak CN, pi-Bondapak NH2, and p-Porasil. The stationary phases employed in the reversed-phase work were p-Bondapak CI8 and p-Bondapak CN. Empirical correlation factors were developed which relate log k’values to structural features of the phenols studied in both the normal-phase and reversed-phase systems.
EXPERIMENTAL High-Performance Liquid Chromatograph. The liquid chromatograph used was a Waters model ALC/GPC 244 equipped with a model 6000-A pump, a U6K injector, a free standing ultraviolet detector set at 254 nm, and a 10-mV strip chart recorder. Columns. The columns used were ,all 3.9 mm i.d. X 30 cm columns prepacked and obtained from Waters Associates, Milford, Mass. The columns were p-Porasil packed with 10-pm porous silica, p-Bondapak CIB,p-Bondapak CN, and p-Bondapak NH2. The last three columns consisted of octadecyl groups, propylnitrile groups, and propylamine groups, respectively, chemically bonded to Waters p-Porasil (18). Reagents. HPLC grade methanol and HPLC grade n-heptane were obtained from Fisher Scientific, St. Louis, Mo. These solvents were prefiltered through 0.2-pni filters. Distilled water was filtered through a Millipore type G-S 0.22-pm filter. Chloroform was J. T. Baker AR grade and for the HPLC work was filtered through a Millipore type F-H 0.5-pm filter. Isopropyl alcohol was Mallinckrodt spectro grade and was not filtered. Ethanol was reagent grade absolute ethanol from U S . Industrial Chemicals, New York, N.Y. The phenol standards were obtained from commercially available sources and were purified when necessary. Chromatographic Systems Studied. 1. Normal-phase, p-Bondapak CN with a n-heptane mobile phase at 2 mL/min. 2. Normal-phase, p-Bondapak NH2 with a n-heptane:isopropyl alcohol (99:l) mobile phase at 2 mL/min. 3. Normal-phase. p-Porasil with a n-heptane:chloroform (9010) mobile phase at 2 mL/min. 4. Reversed-phase, p-Bondapak CI8 with a methano1:water (65:35)mobile phase at 1 mL/min. 5 . Reversed-phase, p-Bondapak CN with a methano1:water (50:50) mobile phase at 1 mL/min. Phenol Standards. Solutions of 3-12 mgjmL of the phenol standards were prepared in both ethanol and chloroform. The retention volumes of the phenol standards were determined by injecting 2-4 p L of the standard solutions onto the chromatographic systems listed above. Two sets of solutions were used, one set with chloroform and the other sei; with ethanol. Two sets were used because it was found that distorted or split peaks resulted if some standards were injected onto system 4 in chloroform solution, or onto systems I and 3 in ethanol solution. For example, 2,6-dimethylphenol gave a ‘splitpeak when injected onto system 4 in a chloroform solution, but a single peak when injected in an ethanol solution. Split peaks were observed for
0 1978 American
Chemical Society
1912
ANALYTICAL CHEMISTRY, VOL. 50, NO.
13, NOVEMBER 1978
Table I. Log k ’ Values for the Standards Chromatographed compound non-ortho substituted phenol m-cresol p-cresol 3,4-dimethylphenol 3,5-dimethylphenol 3-ethylphenol 4-ethylphenol 3-isopropy lphenol
4-isopropylphenol 4-n-pentylphenol 3,5-diisopropylphenol 3,5-di-tert-butylphenol 5-indanol 5,6,7,8-tetrahydro- 2-naphthol 2-naphthol 4-phenylphenol mono-ortho substituted o-cresol 2,3-dimethylphenol 2,4-dimethylphenol 2,5-dimethylphenol 2,3,5-trimethylphenol 2-ethylphenol 2-isopropy lphenol
2,4-di-tert-butylphenol 2,5-di-tert-butylphenol 5,6,7,8-tetrah ydro- 1-naphthol 1-naphthol 2-phenylphenol di-ortho substituted 2,6-dimethylphenol 2,3,6-trimeth ylphenol 2,4,6-trimethylphenol 2,3,5,6-tetramethylphenol 2,6-di-sec-butylphenol 2,6-di-tert-butylphenol 2,6-di-tert-butyl-4-methylphenol not retained.
1 (P-CN)
1.10 1.02 1.02 0.98 0.94 1.01 1.01 0.96 1.00
0.98 0.87 0.82 0.98 0.98 1.35 1.37 0.81 0.78 0.73 0.71 0.68 0.79 0.75 0.59 0.49 0.75 1.26 0.43 0.24 0.22 0.19 0.14 0.26 Na
N
3,5-diisopropylphenol, 3,5-di-tert-butylphenol, and 2,4- and 2,5-di-tert-butylphenolwhen they were injected onto system 1 in ethanol solutions, but single peaks were observed when they were injected in chloroform solutions. Peak splitting was observed for phenol when it was injected onto system 3 in ethanol solution, but a single peak was observed when it was injected in chloroform solution. This was not studied further, but similar peak splitting phenomena were observed by Tseng and Rogers (19) and Snyder (20). The chloroform solutions were used for injection onto the normal-phase systems, and the ethanol solutions were used for injection onto the reversed-phase systems. The capacity factor, k’, was calculated by k’ = (VR- V,)/V,, where VR (mL) is the measured retention volume and V , (mL) is the column void volume. The value of V, was determined to be 3.3 mL for the systems studied and was obtained by eluting methanol in the reversed-phase systems and toluene in the normal-phase systems. The method of least squares was employed for the lines in Figures 1-3. RESULTS AND DISCUSSION Normal-Phase Retention. The log k ’ values for the standards chromatographed in systems 1-3 were determined and are listed in Table I. T h e log k’ values for the phenol standards chromatographed by Callmer e t al. (13)on Cyano Sil-X-I with a n-heptane:isopropyl alcohol (99.5:0.5) mobile phase were considered also. This chromatographic system is labeled system 6. An attempt was made to correlate empirically the log k’values from these chromatographic systems with the structural features of the standards chromatographed.
2 (fi-NH,)
1.07 0.99 0.98 0.94 0.91 0.94 0.97 0.91 0.95 0.92 0.73 0.65 0.93 0.93 1.27 1.25 0.80 0.73 0.70 0.68 0.62 0.72 0.68 0.40 0.16 0.72 1.11
0.72 0.23 0.15 0.17 0.050
0.56
N N
3 ( p -Porasil)
1.23 1.19 1.20 1.19 1.17 1.17 1.20 1.15 1.19
1.18 1.08 1.04 1.20
1.19 1.31 1.34 0.92 0.86 0.87 0.85 0.81 0.81 0.75 0.38 0.1 3 0.85 1.07 0.39 0.37 0.31 0.41 0.27 -0.44
N N
4 (fi-C,*)
-0.74 - 0.40 -0.40 - 0.20 -0.16 --0.16 - 0.14 0.050 0.073 0.59 0.57 0.83 - 0.056 0.13 -0.14 0.15 - 0.37 -0.16 -0.12 - 0.10 0.084
-0.10
0.094 0.99 1.02 0.16 - 0.056 0.15 -0.16 0.061 0.11 0.21 0.93 1.08 1.25
5 (fi-CN) -0.56 -0.37 - 0.37 -0.22 - 0.22 - 0.22 -0.22 - 0.087 -0.12
___
0.22 0.38 -0.14 0.050 0.061 0.22 0.31 0.20 - 0.20 - 0.20 -0.056 -0.16 - 0.056 0.54 0.62 0.084 0.14 0.17 -
-
-0.20
-0.056 0.087 0.038 0.49 0.57 0.66
-
Most of the standards chromatographed were alkylphenols. The naphthols and 2- and 4-phenylphenol were also studied. For each chromatographic system, the structural effects on log k’were assumed to be additive. Initially, each aliphatic carbon, regardless of type, was assigned arbitrarily a value of 1, and a plot of log k‘vs. aliphatic carbon number (C,) was made. The log k’vs. C, plot for the alkylphenols chromatographed on the p-Bondapak CN column (Chromatographic system 1) is presented in Figure 1. T h e points grouped around three lines, as in chromatographic systems 2 and 3, depending on the number of alkyl ortho substituents. This type of behavior was also observed by Callmer et al. (13). By observing the relative positions of the three lines in Figure 1, it appeared possible to merge the lines into one continuous line by making several assumptions. T h e presence of one substituent ortho to the hydroxyl group was assumed to exhibit a constant additive effect on retention, and the presence of a second ortho substituent was assumed to exhibit a constant additive effect on retention. The magnitude of these effects was not necessarily the same. Also, these constant effects were considered to be independent of the size of the group or groups ortho to the hydroxyl group. To merge line 2 into line 1, a value of 8 aliphatic carbons was added to C, for each point in line 2 as shown in Figure 1. The new “C,,” value for o-cresol, for example, with a log k’value of 0.81 is 9, which puts the point for o-cresol near the point for 3,sdi-tert-butylphenol, with a log k’value of 0.82 and a C, value
ANALYTICAL CHEMISTRY, VOL. 50, NO. 13, NOVEMBER 1978 1.2 1
Table 11. Values of the Constants in Equation 1 system 1 2
3 6
.
k.,
-0.2 O.O!
-401.-
-0.60
2
4
6
8
10
12
14
16
18
Cn
Flgure I. Log k’vs.C, for the alkylphenol standards in chromatographic system 1. Non ortho-substituted (O),monoortho substituted (0), di-ortho substituted (A) I .4 1.2 1.0
0.8 0.6 r
0.4 1
0.2
0.0
-0.2 A
-0.4 -0.6 -6
1913
-2
2
6
IO
14
.
18
.
22
.
26
\
.
30
FI
Figure 2. Log k’vs. F , for the standards in chromatographic system 1. Non-ortho substituted (O), mono-ortho substituted (0),di-ortho substituted (A)
of 8. Line 3 was shifted to the right in a similar manner by adding 8 13 or 21, to the value of C, for each point in line 3 (Figure 1). The merged lines for chromatographic system 1 are shown in Figure 2. The lines for the other normal-phase chromatographic systems were merged in a similar manner. It was also assumed that the presence of a second aromatic ring exhibits a constant increase in retention for a given chromatographic system. For example, with chromatographic systems 1-3, 2-naphthol and 2-phenylphenol have similar log k’ values which are larger than the log k ’ value for phenol (Table I). With the assumptions above, the data in Table I and Reference 13, and the results from Figure 1,an empirical correlation factor, F,, for the compounds in chromatographic systems 1-3 and 6 was developed and is defined by Equation 1. This equation was obtained by making the several assumptions discussed previously and presently we have no strong theoretical justification for it. F, = A, E , + n-C,- D, (1) where F, = correlation factor for chromatographic system i, A, = effect of one ortho substituent on log k’, B, = effect of a second ortho substituent on log k’, n = the number of aliphatic carbons, C, = l for each aliphatic carbon, and D , =
+
+
A, 8.0 5.0
12 5.0
B, 13 6.0 4.0 4.0
c,
D,
1.0 1.0 1.0 1.0
9.0 3.0 3.0
-
effect of a second aromatic ring. With the correlation factor in this form, F, for phenol is zero for all the chromatographic systems. ‘The values of A,, B,, C,, and D, for chromatographic systems 1, 2, 3, and 6 are listed in Table 11. The values of the terms A , and B, were determined graphically as described above for chromatographic system 1 (Figure 1). The value of the term C, is 1 for each aliphatic carbon for all the Chromatographic systems. The values of the D, terms for chromatographic systems 1-3 were obtained by using the log k ’ values of 2-naphthol and 4phenylphenol and reading the values of F, from the X-axis of the correlation graphs (Figure 2). For these compounds, A,, B,, and n are all zero, so F , = -D,. The calculated values of F1,F,, and F3 for the standards are listed in Table 111. The log K’vs. F , plot for chromatographic system 1 is presented in Figure 2 and is typical of the chromatographic systems investigated. It was apparent that for each chromatographic system instead of one correlation line, two lines appeared. For chromatographic systems 1 and 2, the two lines intersected near the data points corresponding to :!,6-dimethylphenol and 2,4-di-tert-butylphenol, respectively. The points of intersection of the correlation lines for chromatographic systems 3 and 6 occurred near the data points for 2,3,5-trimethylphenol and 2,5-dimethylphenol, respectively. It is at these points of intersection that the assumptions of the presence of one or two ortho substituents results in an additive effect, and that this effect is independent of the size of the ortho group or groups, appear to break down. For example, in chromatographic system 1, the slope of line 2 in Figure 1 is about the same as the slope of line 1,and thus the points for lines 1 and 2 can be combined to give a single line (Figure 2). The slope of line 3 in Figure 1 is steeper and line 3 cannot be merged with the line resulting from combining lines 1 and 2 (Figure 2). Beyond the point a t which the elope changes with increasing values of F,, the presence of one or two ortho groups and the size of the ortho groups cause a decrease in retention greater than that predicted by the merged lines 1 and 2 (Figure 2). For the F-Bondapak CN stationary phase (chromatographic system l),the slope of the correlation line does not change for non-ortho and mono-ortho substituted phenols (Figure 2). For the w-Bondapak NH2 stationary phase (chromatographic system 2), the slope of the correlation line did not change for non-ortho and mono-ortho substituted phenols with the exception of the data point for 2,5-ditert-butylphenol. For the F-Porasil and Cyano Sil-X-I (13) stationary phases, (chromatographic systems 3 and 6), the correlation lines had unchanging slopes only for non-ortho substituted phenols, and mono-ortho substituted phenols with C,
