Anal. Chem. 1996, 68, 437-446
Retention Behavior of Ionizable Isomers in Reversed-Phase Liquid Chromatography: A Comparative Study of Porous Graphitic Carbon and Octadecyl Bonded Silica Qian-Hong Wan, Martyn C. Davies, P. Nicholas Shaw, and David A. Barrett*
Department of Pharmaceutical Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, U.K.
The retention behaviors of 36 positional isomers of ionizable substituted benzene compounds have been compared on two different packing materials: porous graphitic carbon (PGC) and octadecyl bonded silica (ODS) using 35% aqueous acetonitrile as the mobile phase. The effect of the mobile phase pH on the solute retention was studied over a range of pH values from pH 2.0 to 7.0. The retention as a function of pH was modeled using equations based on solute ionization. With PGC, the theoretical equations fitted the observed retention data for each class of solute, indicating that the retention mechanism was uniform over the whole pH range. However, with ODS, only the acidic solutes showed agreement with the theoretical model; for the amine-containing compounds, serious deviations from the theory were observed, suggesting that strongly acidic silanols gave added retention at low mobile phase pH. Overall, PGC demonstrated a higher selectivity toward positional isomers than ODS. This was attributed to the greater steric discriminating ability arising from the flat surface of the PGC compared with the more fluid nature of the ODS bonded phase. The retention behaviors of positional isomers in reversed-phase liquid chromatography (RPLC) have been studied extensively in the last decade.1-10 It has been shown that the retention order of ortho, meta, and para isomers varies widely, depending on the surface properties of the packing material, the composition of the mobile phase, and the nature of the solute substituent groups. The potentially complex interplay between these variables means that the interpretation of the retention behaviors of positional isomers poses formidable difficulties. However, carefully controlled chromatographic studies using selected isomer solutes are capable of revealing the dominant factors in solute retention and (1) Kawaguchi, Y.; Tanaka, M.; Nakae, M.; Funazo, K.; Shono, T. Anal. Chem. 1983, 55, 1852. (2) Tanaka, M.; Kawaguchi, Y.; Shono, T. J. Chromatogr. 1983, 267, 285. (3) Harino, H.; Kimura, K.; Tanaka, M.; Shono, T. J. Chromatogr. 1990, 522, 107. (4) Barman, B. N.; Martire, D. E. Chromatographia 1992, 34, 347. (5) Knox, J. K.; Kaur, B.; Millward, G. R. J. Chromatogr. 1986, 352, 3. (6) Knox, J. H.; Kriz, J.; Adamcova, E. J. Chromatogr. 1988, 447, 13. (7) Weber, T. P.; Carr, P. W. Anal. Chem. 1990, 62, 2630. (8) Kriz, J.; Adamcova, E.; Knox, J. H.; Hora, J. J. Chromatogr. A 1994, 663, 151. (9) Bassler, B. J.; Hartwick, R. A. J. Chromatogr. Sci. 1989, 27, 162. (10) Wan, Q. H.; Shaw, P. N.; Davies, M. C.; Barrett, D. A. J. Chromatogr. 1995, 697, 219. 0003-2700/96/0368-0437$12.00/0
© 1996 American Chemical Society
selectivity, and also the subtle differences in surface features that may influence the chromatographic properties of the packing materials. Chromatographic materials studied in this way include cyclodextrin bonded silica,1,2 crown ether bonded silica,3 octadecyl bonded silica (ODS),4-7 and porous graphitic carbon (PGC).5-9 However, such studies have often used unionizable solutes such as alkylbenzene isomers as probes, which are not typical of the vast majority of compounds analyzed by RPLC. This has led to a lack of comparative experimental data on the chromatographic behaviors of ionizable isomers associated with different reversedphase packing materials. Previously,10 we investigated the effect of eluent pH on the retention of ionizable isomers with electron-donating substituents on PGC, which demonstrated that the retention factor as a function of eluent pH was effectively predicted by a retention model based on solute ionization. The retention order of ortho, meta, and para isomers correlated directly with their pKa values, the isomers with the higher ionization constants being less retained. These observations were interpreted as showing that ionic interactions with ionized solutes were essentially absent on the surface of PGC and that the retention order was probably governed by a solute molecular orientation effect induced by competing interactions of solute between the stationary phase and the mobile phase. In this paper, we directly compare the retention behaviors of ionizable isomers on PGC and ODS packing materials using an extended series of solutes including isomers with electronwithdrawing substituents (see Table 1). The same eluent composition was used throughout the work to ensure that any difference observed in retention behaviors on the two columns was solely ascribed to the stationary phase effects. EXPERIMENTAL SECTION Apparatus. The chromatographic system consisted of a Gilson 305 pump (Villiers le Bel, France), a Gilson 805 manometric module, a Gilson 231 XL sampling injector, a Gilson 401 diluter, and an ABI 759A absorbance detector (Foster City, CA) connected to a Gilson HPLC 715 system controller via a Gilson 506B interface. The HPLC columns used in this study, including Hypersil ODS (150 mm × 4.6 mm i.d., particle diameter 5 µm) and Hypercarb (50 mm × 3.0 mm i.d., particle diameter 5 µm) were supplied by Shandon HPLC (Runcorn, UK). pH measurements were carried out with a Corning Model 7 pH meter (11) Serjeant, E. P., Dempsey, B., Eds. Dissociation Constants of Organic Acids in Aqueous Solution; Pergamon: Oxford, 1979. (12) Perrin, D. D., Ed. Dissociation Constants of Organic Bases in Aqueous Solution; Butterworths: London, 1965; Supplement, 1972.
Analytical Chemistry, Vol. 68, No. 3, February 1, 1996 437
Table 1. Acid Dissociation Constants, pKa, of Ionizable Benzene Isomers, C6H4XY substituents compound
X
Y
pKa(1)a
OCH3 OCH3 OCH3 CH3 CH3 CH3 Br Br Br NO2 NO2 NO2 OH OH OH
4.0 4.09 4.4 3.91 4.27 4.38 2.84 3.86 3.97 2.16 3.47 3.44 10.28 10.09 10.17
OCH3 OCH3 OCH3 OC2H5 OC2H5 OC2H5 CH3 CH3 CH3 C2H5 C2H5 C2H5 Br Br Br NO2 NO2 NO2
4.52 4.23 5.34 4.43 4.18 5.20 4.44 4.73 5.08 4.37 4.70 5.00 2.53 3.58 3.86 -0.26 2.47 1.00
o-anisic acid m-anisic acid p-anisic acid o-toluic acid m-toluic acid p-toluic acid o-bromobenzoic acid m-bromobenzoic acid p-bromobenzoic acid o-nitrobenzoic acid m-nitrobenzoic acid p-nitrobenzoic acid o-cresol m-cresol p-cresol
Acidic COOH COOH COOH COOH COOH COOH COOH COOH COOH COOH COOH COOH CH3 CH3 CH3
o-anisidine m-anisidine p-anisidine o-phenetidine m-phenetidine p-phenetidine o-toluidine m-toluidine p-toluidine o-ethylaniline m-ethylaniline p-ethylaniline o-bromoaniline m-bromoaniline p-bromoaniline o-nitroaniline m-nitroaniline p-nitroaniline
NH3 NH3 NH3 NH3 NH3 NH3 NH3 NH3 NH3 NH3 NH3 NH3 NH3 NH3 NH3 NH3 NH3 NH3
o-aminobenzoic acid m-aminobenzoic acid p-aminobenzoic acid
Zwitterionic COOH NH3 COOH NH3 COOH NH3
pKa(2)a
k ) (tR - tM)/tM where tR is the solute retention time and tM is the column holdup time, which may be taken as the “retention time” of the solvent disturbance peak. The retention factors reported in this study were the averages of at least triplicate determinations. The retention factor as a function of eluent pH for monoprotic ionizable isomers can be expressed by the following equation, based on the work of Horvath et al.:13
Basic
2.11 3.07 2.50
k ) k1/[1 + 10(pH-pKa)] + k2/[1 + 10(pKa-pH)]
k ) ku/[1 + 10((pH-pKa)] 4.95 4.73 4.87
equipped with automatic temperature compensation. The electrode was calibrated with pH 4.0, 7.0, or 10.0 standard solutions, depending on the range investigated. Chemicals. Orthophosphoric acid (analytical grade) was obtained from Sigma Chemical Co. Ltd. (Dorset, UK), triethylamine (HPLC grade) from Romil Chemicals (Loughborough, UK), and acetonitrile (HPLC grade) from Fisons plc (Loughborough, UK). Deionized, purified water was obtained using an Elgastat water purification system (Elga Ltd., High Wycombe, UK). The ortho, meta, and para isomers of substituted anilines, benzoic acids, and cresols used as test compounds were purchased from Aldrich Chemical Co. Ltd. (Dorset, UK). Chromatography. The retention data were recorded at ambient temperature under isocratic conditions with a flow rate of 1 mL/min for the Hypersil ODS column and 0.5 mL/min for the Hypercarb column. UV detection at 254 nm was used for both columns. The mobile phase consisted of aqueous bufferacetonitrile in 65:35 volume ratio. The pH value of the buffer was adjusted by adding an appropriate amount of triethylamine or orthophosphoric acid to 0.01 M orthophosphoric acid solution. The retention factors of substituted benzene isomers were determined over the range of pH 2.0-7.0. Mobile phase pH Analytical Chemistry, Vol. 68, No. 3, February 1, 1996
(1)
where pKa is the negative logarithm of the acid dissociation constant in the mobile phase and k1 and k2 are the limiting retention factors of the solute at pH , pKa and pH . pKa, respectively. When the ionized form of the solute is unretained, as was the case for all the acids and bases examined in this study, this equation can be simplified to
a Uncorrected literature values (in water, 25 °C) taken from refs 11 and 12.
438
higher than 7.0 was not used on ODS because of the likely instability of the silica packing material in alkaline solution. The mobile phases were degassed in an ultrasonic bath before use. When a change in mobile phase pH was made, the passage of 30 column volumes of eluent was usually required to attain equilibration with the new mobile phase before any retention data were taken. The solute solutions were prepared by dissolving the test solutes in the mixture of water and acetonitrile (1:1) to give a concentration of 1-10 µg/mL. Injections of 1-10 µL of these solute solutions were made to produce adequate UV responses. Data Analysis. Retention factors, k, were calculated from retention times with the following equation:
(2)
where “+” applies to acids and “-” to bases, and where ku is the retention factor for the un-ionized form. The retention factor of amphoteric substances, such as aminobenzoic acids, is given by
k ) k1/[1 + 10(pH-pKa(1))+10(2pH-pKa(1)-pKa(2))] + k2/[1 + 10(pKa(1)-pH)+10(pH-pKa(2))] + k3/[1 + 10(pKa(2)-pH)+10(pKa(1)+pKa(2)+2pH)] (3) where k1, k2, and k3 are the retention factors of the cationic, the zwitterionic, and the anionic forms of the amphoteric solute and Ka(1) and Ka(2) are the corresponding dissociation constants. If it is assumed that the cationic and anionic forms of the solute are unretained, then this equation can be simplified to
k ) kz/[1 + 10(pKa(1)-pH)+10(pH-pKa(2))]
(4)
where kz is the retention factor for the zwitterionic form. On truly reversed-phase packings, the retention factor of a weakly acidic/basic solute as a function of pH can be represented by a typical sigmoidal/antisigmoidal curve because the retention of the ionized species is much lower than that of the un-ionized species. Simulated curves using eqs 2 and 4 illustrate the predicted effect of pH on the retention of acidic, basic, and zwitterionic compounds (Figure 1). The best fits of the theoretical curves to the experimental data were found by using a nonlinear least-squares curve-fitting software package, MINIM 2.0.2, developed by R. D. Purves, (13) Horvath, C.; Melander, W.; Molna, I. Anal. Chem. 1977, 49, 142.
Figure 1. Dependence of retention on eluent pH for (1) acidic solute, (2) basic solute, and (3) zwitterionic solute according to eqs 1 and 2. Parameters used: (1) k1 ) 8, k2 ) 0, pKa ) 4.5; (2) k1 ) 0, k2 ) 8, pKa ) 4.5; (3) k1 ) 0, k2 ) 8, k3 ) 0, pKa(1) ) 2, pKa(2) ) 4.5.
Figure 3. Effect of eluent pH on the retention of ortho (b), meta (2), and para (9) isomers of toluic acid. Conditions as for Figure 2.
Figure 2. Effect of eluent pH on the retention of ortho (b), meta (2), and para (9) isomers of anisic acid. Conditions: (PGC) column, Hypercarb, 50 mm × 3.0 mm i.d.; eluent, 35% acetonitrile-aqueous phosphate buffer; flow rate, 0.5 mL/min; UV detection, 254 nm; (ODS) column, Hypersil ODS, 150 mm × 4.6 mm i.d.; eluent, 35% acetonitrile-aqueous phosphate buffer; flow rate, 1 mL/min; UV detection, 254 nm. The solid lines indicate the ideal retention behavior based on the use of the experimentally determined limiting value of retention of the un-ionized solute and the appropriate theoretical ionizationretention equation.
Department of Pharmacology, University of Otago, New Zealand. This fitting procedure enables the estimation of the pKa of the solute in the HPLC mobile phase, assuming that the experimental data are consistent with the proposed theoretical model. RESULTS AND DISCUSSION Comparison of pH-Retention Effects on ODS and PGC Columns. Figures 2-13 show plots of the chromatographically determined retention factors for all of the acidic, basic, and zwitterionic positional isomers between pH 2.0 and 7.0. The superimposed curve for each isomer indicates the ideal retention behavior based on the use of the experimentally determined limiting value of retention of the un-ionized solute and the appropriate theoretical ionization-retention equation.
Figure 4. Effect of eluent pH on the retention of ortho (b), meta (2), and para (9) isomers of bromobenzoic acid. Conditions as for Figure 2.
Figures 2-6 show the dependence of retention factor on mobile phase pH for the acidic series of positional isomers: anisic acid, toluic acid, bromobenzoic acid, nitrobenzoic acid, and cresol. For both the ODS and PGC packing materials, the retention of the acidic solutes decreased as the pH of the mobile phase was increased, with a rapid change in retention close to the pKa of the solute. This behavior correlates well with the predictions from Analytical Chemistry, Vol. 68, No. 3, February 1, 1996
439
Figure 5. Effect of eluent pH on the retention of ortho (b), meta (2), and para (9) isomers of nitrobenzoic acid. Conditions as for Figure 2.
Figure 7. Effect of eluent pH on the retention of ortho (b), meta (2), and para (9) isomers of anisidine. HPLC conditions as for Figure 2. The dotted lines indicate the ideal behavior of basic isomers on the ODS material plotted according to eq 2, with the limiting k values for the ionized forms being set to zero.
Figure 6. Effect of eluent pH on the retention of ortho (b), meta (2), and para (9) isomers of cresol. Conditions as for Figure 2. Figure 8. Effect of eluent pH on the retention of ortho (b), meta (2), and para (9) isomers of phenetidine. Conditions as for Figure 7.
the ionization-retention model, which assumes that the retention is solely controlled by a reversed-phase type interaction with the stationary phase. The limiting retention factors and the pKa values determined from the measured retention factors are given in Table 2. The retention factors for un-ionized solutes were generally greater on PGC than those on ODS, confirming previous observa440 Analytical Chemistry, Vol. 68, No. 3, February 1, 1996
tions that neutral compounds are more strongly retained on the PGC material. The pKa values determined from the curve-fitting were very close for both materials, supporting a predominantly reversed-phase mechanism of isomer separation on both ODS and PGC for these acidic solutes.
Figure 9. Effect of eluent pH on the retention of ortho (b), meta (2), and para (9) isomers of toluidine. Conditions as for Figure 7.
Figure 10. Effect of eluent pH on the retention of ortho (b), meta (2), and para (9) isomers of ethylaniline. Conditions as for Figure 7.
Figures 7-12 show the dependence of retention factor on eluent pH for the basic series of positional isomers: anisidine, phenetidine, toluidine, ethylaniline, bromoaniline, and nitroaniline. In contrast to our observations for the acidic isomers, the retention behaviors of the basic solutes on ODS were distinctly different from those on PGC. In agreement with our previous report,10 the retention factor as a function of eluent pH conformed well
Figure 11. Effect of eluent pH on the retention of ortho (b), meta (2), and para (9) isomers of bromoaniline. Conditions as for Figure 7.
Figure 12. Effect of eluent pH on the retention of ortho (b), meta (2), and para (9) isomers of nitroaniline. Conditions as for Figure 7.
with the predictions from the ionization-retention model for all the basic isomers studied on PGC. The limiting retention factors and the pKa values determined from the curve-fitting of the experimental data are given in Table 2. However, the retention behaviors of the basic solutes on ODS were anomalous compared with those on PGC. Between pH 2 and 4, all the basic solutes Analytical Chemistry, Vol. 68, No. 3, February 1, 1996
441
previous observations that the surface of ODS is not homogeneous. The enhancement of basic solute retention at low pH is most likely associated with the existence of a proportion of silanol groups remaining intact after chemical modification of the silica surface.14,15 As weak acids, these residual silanol groups have a theoretical pKa value of 7.1 ( 0.5,16 and the average values determined by infrared studies of silica have also suggested that the pKa of the silanol groups is around 7.1.17 Secondary interactions may therefore arise from either the SiOH or SiO- groups. If this is the case, then any ionic interaction between the basic solute and the residual silanol groups should be minimal at pH < 3, as a result of the suppression of ionization of the silanols. But the increased retention at pH < 3 for substituted aniline isomers found in the present study suggests that the pKa values of a proportion of the residual silanols in the ODS material used in this study may be considerably lower. This is in agreement with the observations that a small proportion of isolated surface silanols are extremely acidic, and hence remain ionized at low pH values.17 These silanols can act as highly retentive centers for positively charged solutes such as the anilines used in this study. It is likely that an ion exchange mechanism is responsible for the anomalous retention at low pH:
SiOH + A+ S SiOA + H+ Figure 13. Effect of eluent pH on the retention of ortho (b), meta (2), and para (9) isomers of aminobenzoic acid. Conditions as for Figure 2. The dotted lines represent the ideal behavior of the aminobenzoic acid isomers on the ODS material plotted according to eq 4.
are predominantly in their ionized form, and hence should be less retained than the un-ionized forms by a reversed-phase packing material, as has already been observed with the PGC material. But on the ODS packing material, a large increase in retention was observed for all the basic solutes (Figures 7-12), which was in contradicion to the expected result from the ionizationretention model. Hence, pKa values of the solutes could not be derived from the retention factors in this case, and it was not possible to use the ionization-retention model. To provide comparative curves, the pKa values obtained from the PGC data were used to illustrate the substantial deviations from the theoretical predictions of retention, based on the fact that the pKa value is a property of the solute and is not influenced by the packing material. In Figures 7-12, the dotted lines, representing the ideal retention behaviors of basic isomers on the ODS packing material, are plotted according to eq 2, with the limiting k values for the ionized forms being set to zero. Figure 13 shows the dependence of retention factor on eluent pH for the zwitterionic aminobenzoic acid isomers. On PGC, the zwitterionic solutes behaved exactly as expected from theory: as the mobile phase pH increased, the retention factor rose initially to a peak value and then began to fall. The solutes behaved similarly on ODS but with apparent deviations in the low pH region, as was observed for the basic solutes. With PGC, the retention behavior was consistent with the ionization-retention model over the whole pH range, strongly suggesting the existence of a uniform mechanism of solute retention. However, the behavior of ODS was anomalous and indicated a more complex retention mechanism, especially for the amine-containing compounds in the low pH region. This anomalous behavior of the basic solutes on ODS supports the many 442
Analytical Chemistry, Vol. 68, No. 3, February 1, 1996
where A+ is a protonated basic solute. It has been suggested that metal impurities in the silica lattice can influence the acidity of silanols, and it is known that the Hypersil silica used in this study has relatively high levels of trace metals in the bulk silica,18 which might be responsible for this anomalous retention behavior. Knowledge of the exact pKa values of the residual silanols on ODS would make it possible to incorporate the ionic interaction between solutes and the silanol groups into the ionization-retention equation, and hence account for the retention behavior of basic solutes.19 However, it is likely that a wide range of silanol pKa values are present on bonded silica columns, making this approach to modeling of retention a difficult one. Chromatographically Determined pKa Values. The chromatographically determined pKa values of the isomer solutes found by the curve-fitting technique (Table 2) were in reasonable agreement with literature values determined by potentiometric methods in water (Table 1) for the PGC material. This is to be expected, since the pKa value is purely a property of the compound in solution and will not be influenced by the packing material. These results support the validity of the theoretical model of reversed-phase retention on PGC. For ODS, pKa values determined by curve-fitting were obtainable only for the acidic isomers, confirming that the retention of the amine-containing isomers did not fit with the theoretical equations. However, it was noted that there was a small difference between the chromatographically determined value of pKa compared with the literature value. For acids, the pKa determined chromatographically was consistently greater than the literature pKa, and for bases, the pKa determined chromatographically was consistently less than the literature pKa. (14) Dorsey, J. G.; Cooper, W. T. Anal. Chem. 1994, 66, 857A. (15) Barrett, D. A.; Brown, V. A.; Shaw, P. N.; Davies, M. C.; Ritchie, H.; Ross, P. J. Chromatogr. Sci., in press. (16) Nawrocki, J.; Buszewski, B. J. Chromatogr. 1988, 449, 1. (17) Cox, G. B. J. Chromatogr. A 1993, 656, 353. (18) Watson, R. C.; Davies, M. C.; Nankervis, R.; Shaw, P. N.; Ritchie, H. J.; Ross, P.; Barrett, D. A. Unpublished data. (19) Wan, Q. H.; Shaw, P. N.; Davies, M. C.; Barrett, D. A. Unpublished work.
Table 2. Experimental Values of k1, k2, and pKa for Ionizable Benzene Isomers PGCa
ODSb
compound
k1
k2
o-anisic acid m-anisic acid p-anisic acid o-toluic acid m-toluic acid p-toluic acid o-bromobenzoic acid m-bromobenzoic acid p-bromobenzoic acid o-nitrobenzoic acid m-nitrobenzoic acid p-nitrobenzoic acid o-cresol m-cresol p-cresol
9.86 23.04 26.21 15.17 18.41 20.98 20.90 54.51 71.01 12.23 71.91 92.51 4.78 4.23 4.47
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Acidic 4.58 4.38 4.51 4.26 4.39 4.39 3.34 3.81 3.86 2.90 3.52 3.45 10.26 10.24 10.35
o-anisidine m-anisidine p-anisidine o-phenetidine m-phenetidine p-phenetidine o-toluidine m-toluidine p-toluidine o-ethylaniline m-ethylaniline p-ethylaniline o-bromoaniline m-bromoaniline p-bromoaniline o-nitroaniline m-nitroaniline p-nitroaniline
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
4.26 4.30 2.80 12.16 11.03 6.92 4.31 3.90 3.97 8.60 5.84 5.67 17.35 18.02 17.48 45.11 30.77 44.64
Basicc 3.24 3.09 3.80 3.34 3.16 3.85 3.26 3.47 3.69 3.27 3.46 3.69 1.63 2.53 2.84 0.00 1.57 0.00
o-aminobenzoic acid m-aminobenzoic acid p-aminobenzoic acid
0.00 0.00 0.00
27.39 5.02 13.36
k3
0.00 0.00 0.00
pKa(1)
pKa(2)
k1
k2
1.24 2.04 1.88 2.87 3.08 2.98 2.73 4.81 5.31 1.80 2.20 2.42 3.18 2.90 2.97
0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
k3
pKa(1)
pKa(2)
4.54 4.43 4.68 4.66 4.79 4.77 3.44 4.21 4.33 2.86 3.75 3.67 10.26 10.24 10.35
2.07 1.45 0.96 3.90 3.90 1.80 2.50 2.55 2.42 4.60 4.52 4.62 5.60 5.20 4.60 3.38 2.66 2.20
Zwitterionicc 0.93 4.06 2.71 4.16 1.85 4.05
1.28 0.50 0.50
0.00 0.00 0.00
4.87 4.87 4.87
a Chromatographic conditions: column, Hypercarb, 50 mm × 3.0 mm i.d.; eluent, 35:65 acetonitrile-0.01 M H PO , with addition of H PO or 3 4 3 4 triethylamine to adjust pH; flow rate, 0.5 mL/min; detection, 254 nm. b Chromatographic conditions: column, Hypersil ODS, 150 mm × 4.5 mm i.d.; flow rate, 1 mL/min; others same as above. c The chromatographically determined pKa values were unavailable for substituted aniline isomers eluted from the ODS column.
This difference was initially attributed to the presence of acetonitrile in the mobile phase, which is known to suppress solute ionization because of its low dielectric constant. However, pKa determinations performed in the presence of 35% acetonitrile for a selection of the acidic and basic isomers (not reported here) did not show the same magnitude of shift in pKa as those done chromatographically. At present, we have no firm explanation for this effect, but we speculate that the ionization constant of a solute may be altered when the solute is in close proximity to the chromatographic surface, due to electronic interactions with the packing material. Further experimental work is required to determine the correct explanation for the observed pKa shift. Comparison of Isomer Retention Order. Table 3 summarizes the retention orders of the ortho, meta, and para isomers for the 12 sets of ionizable benzene derivatives. The orders were based on the retention data obtained with the eluents of pH 2.0 for acidic solutes, pH 7.0 for the basic solutes, and pH 3.0 for the amphoteric solutes. At these pH values, the acidic and basic isomers are in their un-ionized forms, and the effect of solute ionization on retention is essentially absent. Therefore, the retention order under these conditions is determined predomi-
nantly by the steric interaction between the individual isomer and the PGC or ODS stationary phase. For the acidic isomers, the retention orders on ODS were the same as those on PGC, with the single exception of the anisic acid isomers. The order of retention of the isomers was ortho > meta > para for the majority of the acidic solutes, with the exception of the cresol isomers, which were retained in the order meta > para > ortho. In agreement with our previous report,10 the retention orders correlated inversely with the ionization constants of the isomers (see Table 1). However, for the basic isomers, the retention order on ODS is significantly different from that on PGC, since four out of six of the isomer sets show different retention orders. The aminobenzoic acid isomers (zwitterionic) have identical retention orders on ODS and PGC. The differences in isomer retention order between PGC and ODS suggest that the molecular interactions determining solute retention are different for the two materials. Hydrophobic (or dispersion) interactions are believed to mediate the attractive forces between the solutes and the retentive surface in reversedphase liquid chromatography. In the case of ODS, there is now evidence to suggest that the retention of small solutes is governed Analytical Chemistry, Vol. 68, No. 3, February 1, 1996
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Table 3. Retention Order for the Ionizable Isomers Separated on PGC and ODS retention ordera
a
isomer
PGC
ODS
pH
anisic acid toluic acid bromobenzoic acid nitrobenzoic acid cresol
Acidic o (9.98) < m (23.56) < p (27.33) o (15.07) < m (18.47) < p (20.86) o (18.93) < m (52.43) < p (68.77) o (10.44) < m (66.97) < p (86.74) m (4.37) < p (4.67) < o (4.94)
o (1.27) < p (1.87) < m (2.08) o (2.87) < m (3.08) < p (3.11) o (2.64) < m (4.79) < p (5.29) o (1.61) < m (2.20) < p (2.42) m (2.90) ) p (2.90) < o (3.25)
2.0 2.0 2.0 2.0 2.0
anisidine phenetidine toluidine ethylaniline bromoaniline nitroaniline
Basic p (2.74) < m (4.15) < o (4.2) p (6.67) < m (10.76) < o (11.76) m (3.84) < p (3.96) < o (4.30) p (5.57) < m (5.74) < o (8.46) o (17.28) < p (17.74) < m (17.83) m (30.17) < p (42.83) < o (43.28)
p (1.00) < m (1.45) < o (2.07) p (1.74) < m (2.63) < o (3.93) p (2.42) < o (2.49) < m (2.59) o (4.62) < m (4.68) < p (4.82) p (4.62) < m (5.23) < o (5.64) p (2.05) < m (2.66) < o (3.38)
7.0 7.0 7.0 7.0 7.0 7.0
aminobenzoic acid
Zwitterionic m (0.44) < p (8.73) < o (25.64)
m (0.41) ) p (0.41) < o (1.21)
3.0
The measured retention factors are presented in parentheses; conditions as for Table 2.
by a partitioning process (i.e., the full embedding of the solute in the chains of the stationary phase).14 These primary interactions are augmented to a variable extent by other interactions, which are commonly known as secondary interactions, the nature of which differs between PGC and ODS. It is these secondary interactions which are likely to be responsible for the changes in isomer retention order and any observed differences in solute selectivity between PGC and ODS. In the case of PGC, there is growing evidence to suggest that polar interactions have a significant secondary influence on retention under certain conditions. The mechanism of this effect is unclear, but it is possibly due to π-π stacking interactions between aromatic solutes and the planar graphite surface. With ODS, the secondary interactions are better understood and are known to arise predominantly from the SiOH and SiO- groups, as has been discussed previously. The involvement of additional adsorptive interactions on PGC is borne out by inspecting the retention data in Table 3. The retention of the un-ionized isomers (for example, at pH 2.0 for acidic solutes), in particular those with electron-withdrawing substituents such as bromo and nitro groups, is substantially greater on PGC than that on ODS, despite an identical eluent composition. As previously proposed for neutral isomer solutes,5,9 the retention order of the un-ionized isomers is essentially determined by how well the shape of the solute molecule fits to the retentive surface of the stationary phase. A large contact area between them would be favorable for both π-π stacking and hydrophobic interactions. The solutes adsorbed onto the flat PGC surface would be expected to be closer to the surface than they would be in relation to the fluid alkyl chains of ODS. Since the hydrophobic energy of interaction between atoms is proportional to 1/r6, where r is the distance between atomic nucleii, a small decrease in r will greatly increase the energy of interaction. Based on this explanation, a greater retention of solutes would be expected on PGC. However, the ODS chains can interact with the whole solute molecule by one or more chains “wrapping around” the molecule to constitute a partitioning type of interaction, thus giving an increased adsorption compared with the one-sided contact on graphite. From the chromatographic data, it is evident that the increased energy of solute-surface interaction on PGC more than 444
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Figure 14. Isomer separations for anisic acid. Conditions as for Figure 2, with eluent pH 2.0 for both PGC and ODS columns.
compensates for the alkyl chain interactions on ODS, thus leading to an increased adsorption of a given solute. Comparison of Isomer Resolution. The relative retention of the ortho, meta, and para isomers varied with changes in mobile phase pH. Therefore, it was possible to optimize the separation of the isomers by selecting an appropriate pH value for the mobile phase. For the acidic and zwitterionic isomers, a pH of 2.0 gave the optimal separations on both ODS and PGC columns. The substituted aniline isomers were best separated at a pH of 3.0 with PGC and at a pH of 7.0 with ODS. At the pH 3.0, the basic isomers eluted from ODS showed severe peak tailing, making the practically useful pH range even narrower. Figures 14-25 show the separations of the 12 sets of ionizable isomers on PGC and ODS under these optimized conditions. Eleven out of 12 sets of isomers were baseline resolved on PGC, compared with five out of 12 on ODS. These results for a range of acidic, basic, and zwitterionic benzene isomers support previous observations5,9 that PGC exhibits a superior selectivity toward isomer separations compared with ODS. The discrepancies in the retention order between the two reversed-phase packing materials suggest that the ionic interaction between an ionized solute and the residual silanol groups on ODS packing materials may have marked influence on the differential migration of the isomers. Although
Figure 15. Isomer separations for toluic acid. Conditions as for Figure 2, with eluent pH 2.0 for PGC and ODS columns.
Figure 18. Isomer separations for cresol. Conditions as for Figure 2, with eluent pH 2.0 for PGC and ODS columns.
Figure 16. Isomer separations for bromobenzoic acid. Conditions as for Figure 2, with eluent pH 2.0 for PGC and ODS columns.
Figure 19. Isomer separations for aminobenzoic acid. Conditions as for Figure 2, with eluent pH 2.0 for PGC and ODS columns.
Figure 17. Isomer separations for nitrobenzoic acid. Conditions as for Figure 2, with eluent pH 2.0 for PGC and ODS columns.
the exact mechanism remains to be characterized, the present work suggests that a subtle change in the solute ionization could lead to a substantial change in retention, and therefore, a strict control of eluent pH should be exercised to maximize the selectivity of the isomer separation. To account for the lack of isomer selectivity of ODS compared with that of PGC, we have to consider its specific surface structure. Unlike the surface of PGC, which is composed of randomly twisted and interleaved graphite sheets, the surface of ODS is essentially
Figure 20. Isomer separations for anisidine. Conditions as for Figure 2, with eluent pH 3.0 for PGC and pH 7.0 for ODS.
made up of alkyl chains bonded to the silanol groups of silica. These alkyl chains are highly flexible and mobile, so that they can change configurations to fit the rigid solute molecule, and this reduces the difference in interaction energies between the positional isomers. In addition to this, the flexible linear configuration of the alkyl chains allows only part of the solute molecule to engage in a close contact interaction with a single chain. This interaction may be nonselective with regard to solute shape, thus Analytical Chemistry, Vol. 68, No. 3, February 1, 1996
445
Figure 21. Isomer separations for phenetidine. Conditions as for Figure 2, with eluent pH 3.0 for PGC and pH 7.0 for ODS.
Figure 24. Isomer separations for bromoaniline. Conditions as for Figure 2, with eluent pH 3.0 for PGC and pH 7.0 for ODS.
Figure 22. Isomer separations for toluidine. Conditions as for Figure 2, with eluent pH 3.0 for PGC and pH 7.0 for ODS.
Figure 25. Isomer separations for nitroaniline. Conditions as for Figure 2, with eluent pH 3.0 for PGC and pH 7.0 for ODS.
carbon can provide an enhanced reversed-phase resolution of ionizable aromatic isomers compared to a commonly used ODS packing material. With PGC, the retention mechanism is uniform over the entire pH range and is more simply explained than that on ODS. Hence, the retention behavior of ionizable solutes on PGC can be readily predicted, whereas this is difficult to achieve on ODS, especially for weakly basic solutes. Although this conclusion is based on the results obtained from our experiments, these trends should be generally applicable to a wider range of aromatic isomers and ODS stationary phases. For the practicing analytical chemist, it should be possible to exploit the superior selectivity of PGC to provide improved resolution in the separation of positional isomers. Figure 23. Isomer separations for ethylaniline. Conditions as for Figure 2, with eluent pH 3.0 for PGC and pH 7.0 for ODS.
further diminishing the isomer selectivity of the ODS packing materials. CONCLUSIONS These investigations on the retention behaviors of a series of ionizable isomers of benzene have provided valuable information on how the surface structure of the stationary phase can affect solute retention and selectivity. In particular, it has shown that the homogeneous and flat surface structure of porous graphitic 446 Analytical Chemistry, Vol. 68, No. 3, February 1, 1996
ACKNOWLEDGMENT This work was supported by a grant from the UK Biotechnology and Biological Sciences Research Council via the Separation Processes Initiative. The authors are grateful to Shandon HPLC, Runcorn, UK, for the provision of materials and to Professor J. H. Knox for his helpful comments on the manuscript. Received for review August 8, 1995. Accepted November 9, 1995.X AC950794D X
Abstract published in Advance ACS Abstracts, December 15, 1995.
