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A New Look at the Selectivity of RPC Columns
L. R. Snyder J. W. Dolan LC Resources
P. W. Carr University of Minnesota
The hydrophobic subtraction model evaluates the selectivity of HPLC reversed-phase columns so that researchers can choose a suitable substitute or a sufficiently orthogonal second column.
© 2007 AMERICAN CHEMICAL SOCIETY
B
onded-phase columns for reversed-phase chromatography (RPC), also called reversed-phase HPLC, are widely used by chemists and biochemists for analysis and purification. Since their introduction in the early 1970s, these columns have continued to inspire new research aimed at a better understanding of their nature and use (1–5). The behavior of these columns can be described in terms of their kinetic and equilibrium properties. The contribution of column kinetics to chromatographic separation determines the width of resulting peaks in a chromatogram; column kinetics and widths can be described by the column plate number. The dependence of plate number and width on such experimental conditions as column length, particle size, flow rate, and other variables is now well understood (6). The equilibrium properties of the column can be summarized in terms of equilibrium constants K or retention factors k for the partitioning of sample molecules (solutes) between the column packing and the surrounding mobile phase within the column. Values of k are proportional to values of K. The separation in time of two adjacent peaks within the chromatogram is determined by the ratio of their k values; this ratio is defined as the separation factor a and is commonly referred to as selectivity. Selectivity for a given pair of sample compounds varies with mobilephase composition, temperature, and the nature of the stationary phase (the column). In this article, we will focus on the effect of the column on separation selectivity (i.e., column selectivity) in RPC. Despite an extensive prior literature, a fundamental understanding of RPC column equilibria (as measured by values of K and a) is M A Y 1 , 2 0 0 7 / A N A LY T I C A L C H E M I S T R Y
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(a)
(c)
(f)
H O
O
Hydrophobic
O
O 2N
O 2N
C5 5 N
much less well developed than is the case for column kinetics.
O
O
H-bonding O
In the beginning
NO2
NO2
B
O
(d)
– O2 + C5 N-R + 5N –
In the early 1970s, RPC was p– p (b) carried out using C18 columns O H from different sources; these columns contained silica partiO O X cles with attached octadecylO O O silyl groups. At first, all C18 colH-bonding umns were assumed to be (g) (e) equivalent or interchangeable, but eventually selectivity was found to vary among different BH+ RPC columns (7 ). This someO O O O O– O O times resulted in unacceptable changes in separation. In time, Steric resistance Cation exchange Dipole – dipole column manufacturers came to appreciate the need for column tests that would ensure FIGURE 1. Illustrations of various sample–column interactions that can affect selectivity. repeatable batch-to-batch sep- B, solute hydrogen-bond acceptor group (e.g., NH ); BH+, protonated solute group (e.g., NH+); X, hydrogen-bond acceptor 2 3 arations (8, 9). group in the stationary phase; R, alkyl group; green figures are solute molecules. By the end of the 1990s, manufacturers had greatly improved column reproducibility (10, dictions of separation and column selectivity require an accuracy 11). However, a practical, quantitative understanding of the basis of no worse than ±3% in a; this makes the LSER approach of of column selectivity has been lacking, which has hampered chro- very limited practical value. matographers. For example, a replacement column for a routine Early experimental work suggested an additional solute– col+ assay might be unavailable; until recently, however, proven guide- umn interaction—the ionic interaction of protonated bases BH – lines for selecting an “equivalent” column from another source with ionized silanols SiO (15; Figure 1e). The importance of (i.e., a column with comparable selectivity) were lacking. Alterna- ionic interactions in affecting column selectivity was fully docutively, the development of orthogonal or 2D separations requires mented over the next two decades (16). A C18-type stationary columns of very different selectivity, again necessitating a quanti- phase also differs in important respects from analogous liquids tative assessment of column selectivity. Finally, chemists believe it such as octadecane. Molecules of liquid octadecane are free to is important to have a fundamental understanding of the materi- move, whereas C18 groups in RPC columns are tethered to the als and processes with which they work. This is especially impor- surface of the silica particles. This constraint varies among differtant in HPLC, for which literally hundreds of different RPC ent columns, leading in extreme cases to so-called shape selectivcolumns are commercially available, most of which differ signifi- ity (17 ), similar to Figure 1b. A large number of chromatographic tests that involve differcantly from each other in terms of selectivity and their relative ent solutes and chromatographic conditions have since been deability to separate different samples. Initial attempts at understanding and characterizing RPC col- scribed, which were intended to measure various kinds of umn selectivity assumed that the interaction of a sample mole- solute–column interaction and to characterize column selectivity cule with the column packing could be described by the same in- (3, 18 –20). The design of these column-test procedures has teractions (dispersion, dipole – dipole, hydrogen bonding, etc.) mostly been guided by theory, with limited experimental confirthat apply in solution (12). Figure 1 provides representations of mation that these tests actually measure specific solute–column some sample–column interactions that we believe contribute to interactions. Furthermore, it has not been shown that some differences in a. Hydrophobic interaction (Figure 1a) is the at- combination of such column tests is sufficient to fully describe traction of relatively nonpolar sample molecules (green) to the the selectivity of different columns and allow a reliable choice of surrounding nonpolar C8 stationary phase (12). This classical ap- column for a particular application. A significant exception is a proach to understanding retention was later placed on a sounder column-characterization procedure that has evolved in the past basis by the use of a linear solvation energy relationship (LSER; several years (21). 13), which provided insight into the relative importance of various hydrophobic and hydrogen-bonding interactions between Hydrophobic subtraction applied to alkylsilica the sample and the column. LSER equations have since been ap- columns plied in many dozens of studies of RPC retention and selectivity This approach to characterizing column selectivity (21) began (14), with a predictive accuracy of no better than ±10–15% (1 with the recognition that RPC retention is dominated by hystandard deviation) in k, or ±20% in a. Unfortunately, useful pre- drophobic interactions among sample molecules, the mobile 3256
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phase, and the column, as described by solvophobic theory (22; Figure 1a). However, other, weaker interactions between the column and solute also can contribute to retention and especially to selectivity, as suggested by Figures 1b–1g; that is, the solvophobic model only approximately describes RPC retention and selectivity. Within experimental error, the solute–column interactions in Figures 1a–1e now appear to fully account for differences in selectivity among type-B alkylsilica columns (e.g., C8, C18). Type-B columns are made from high-purity, less acidic silica and are preferred over the older, more acidic type-A columns that are contaminated with trace metals. The separation factor a of a given solute (relative to the reference solute ethylbenzene) can be represented by log a = h9H – s9S* + b9A + a9B + k9C
(1)
the various terms in Equation 1. Figure 2 shows a retention plot of log k versus log k for two different C18 columns and 90 solutes of very diverse structure. If the selectivity characteristics of each column were identical (i.e., equal values of H, S*, and so on), all points would fall on a single line of unit slope (23). The correlation of Figure 2 is quite pronounced (r 2 = 0.995) because of the well-known dominance of the h9H term in reversed-phase retention (22). However, a closer inspection (inset) reveals significant deviations (dlog k) from the correlation line. The overall standard deviation of the plot is ±0.034 in log k (±8% in k), which is a much larger value than the experimental error for these data (±0.5% in k). These significant deviations therefore reflect additional, nonhydrophobic interactions between the solute and the column. Values of dlog k (the result of hydrophobic subtraction) calculated as in Figure 2 for nine different C18 columns and 90 solutes represent the combined contributions to retention in Equation 1 by factors other than h9H. If the values of dlog k for two different solutes and these nine columns are highly correlated, this suggests that a single interaction (any of the last four terms in Equation 1) is largely responsible for these values of dlog k (for two or more solute pairs). From ~4000 pairs of solutes, ~200 pairs were identified (with r 2 > 0.8) and organized into four groups that appear to correspond to the interactions represented by Figures 1b –1e. Average values of dlog k for each
log k (Inertsil ODS-3)
The five terms in this equation correspond to the interactions shown in Figures 1a–1e, respectively. In this equation, a for a given solute is related to the complementary properties of the solute and the column. The solute properties are h9, s9, b9, a9, and k9, where h9 is solute hydrophobicity, s9 is solute bulkiness, b9 is solute hydrogen-bond basicity, a9 is the solute hydrogenbond acidity, and k9 is the effective charge on the solute molecule. The corresponding column properties are H, S*, A, B, and C. These five column-selectivity parameters are of primary practical interest because they determine the selectivity and applicability of most RPC col2.0 umns. Hydrophobicity H can be represented by Figure 1a. Steric interaction S* (Figure 1b) is a measure of the resistance to insertion of 1.5 bulky solute molecules into the stationary phase — conceptually similar to, but not quite the same as, shape selectivity (17 ). The col1.0 umn hydrogen-bond acidity A is predominantly attributable to nonionized silanols (Figure 1c). The column hydrogen-bond ba0.5 sicity B is likely the result (for type-B alkylsilica columns) of water sorbed in the stationary phase (represented by X in Figure 1d). The 0.1 log units 0.0 column cation-exchange activity C is caused by ionized silanols and therefore varies with the pH of the mobile phase (Figure 1e). H, –0.5 S*, A, B, and C are relative to values for a hypothetical average C18 column (for which H dlog k [ 1.00 and S*, A, B, and C [ 0.00). To a first –1.0 approximation, the dependence of H, S*, A, and B on the column temperature or mobilephase composition can be ignored. Values of –1.5 C are determined by silanol ionization and 0.0 0.5 1.0 1.5 –1.0 –0.5 therefore change with mobile-phase pH. log k (StableBond C18) The derivation of Equation 1 is based on experimental measurements of k (proportional to K) for several columns and solutes. Val- FIGURE 2. The importance of hydrophobic interaction in determining sample retention, ues of log k for different solutes and columns and the related measurement of other solute–column interactions. were critically analyzed as follows to obtain dlog k refers to the distance of a data point from the best-fit line. (Adapted with permission from Ref. 21.) M A Y 1 , 2 0 0 7 / A N A LY T I C A L C H E M I S T R Y
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Equation 1 takes into account all significant contributions to column selectivity.
of these groups were equated to S*, A, B, and C, respectively, for each column; values of H are proportional to the slopes of the plots (as in Figure 2). These values were used to derive the solute parameters (h9, s9, and so on) of Equation 1, which was subsequently extended to a total of 90 type-B alkylsilica columns (C3– C18, but mainly C8 and C18) and 150 different solutes. The average standard deviation when Equation 1 is applied to these data is equivalent to only ±1% in k (n = 2733); this suggests that Equation 1 takes into account all significant contributions to column selectivity. No previously reported procedure, combination of procedures, or theoretical model for characterizing the selectivity of type-B, monomeric alkylsilica columns can make this claim. These results suggest that values of the solute and column parameters correspond to individual solute–column interactions of the kind illustrated in Figure 1. If this is the case, then values of h9, s9, and so on should make sense in terms of solute molecular structure and the various solute–column interactions. Similarly, values of H, S*, and so on should be reconcilable with column properties such as ligand length (e.g., C18 and C8) and surface concentration, particle pore diameter, and the presence or absence of column end-capping (which further reduces the number of unreacted silanols). These expectations are generally in line with experimental data, thereby confirming a physicochemical basis for Equation 1 (21).
Correlation of parameter values with solute and column properties Hydrophobic interaction: h9 and H. Because it is well established that this is the primary contribution to RPC retention, h9 correlates well with log k for different solutes and a given column plus specified separation conditions. Likewise, H increases with an increasing ligand length (i.e., C18 vs C3), end-capping of the column, and an increased concentration of the alkyl groups at2 tached to the particle (2.9 vs 0.9 µmol/m ), whereas H decreases for an increase in particle pore diameter from 6 to 30 nm (Table 1, items 1– 4). These changes in H with column properties are expected, hence confirming the identification of this parameter with column hydrophobicity (21). Steric interaction: s9 and S*. Steric interaction leads to smaller values of k as a result of bulky solute molecules being less able to penetrate closely spaced alkyl groups attached to the RPC particle (Figure 1b). Values of s9 increase with molecular length but are less affected by molecular thickness. In this respect, steric interaction differs significantly from shape selectivity and appears to be more related to size exclusion (where retention also decreases with increasing molecular length or hydrodynamic diameter; 17, 24). Note also that S* correlates negatively with a commonly accepted measure of shape selectivity. S* is primarily important for more commonly used monofunctional (monomeric) column packings, whereas shape selectivity is more pronounced for polyfunctional (polymeric) packings. Monomeric packings result from the reaction of monofunctional silanes with silica; polymeric packings arise 3258
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from the use of polyfunctional silanes. As expected, values of S* increase with an increase in ligand length and concentration and decrease with an increase in particle pore diameter (Table 1, items 1– 4). Hydrogen bonding of basic solutes with acidic column groups: b9and A. Unreacted silanols on the silica surface are commonly believed to be responsible for the preferential retention of hydrogen-bond acceptor molecules (Figure 1c). Values of b9 correlate with hydrogen-bond basicity as measured in solution, but only for basic solute groups with similar intramolecular steric hindrance. This pronounced dependence of b9 on steric hindrance near the solute acceptor group arises from the crowded environment created by the attachment of silanol groups to the silica surface, combined with the further crowding of the silanol by adjacent alkyl ligands as in Figure 1c. Values of A should decrease sharply when the surface concentration of silanols is decreased by end-capping; this is observed (Table 1, item 2). Hydrogen bonding of acidic solutes with basic column groups: a9 and B. Values of k9 follow the Brønsted acidity (pKa value) of the solute molecule: neutrals (average a9 = 0.0) < alcohols (0.1) < phenols (0.2) < weak carboxylic acids (0.9) < strong carboxylic acids (≈2.3). The only potential acceptor groups that form a permanent part of the stationary phase are silanol and siloxane groups. End-capping was expected to markedly reduce access to these groups by a solute molecule, with a major reduction in B; instead, end-capping has a slightly positive effect on B (Table 1, item 2). This suggests that silanol and siloxane groups are not responsible for the a9B term in Equation 1. For type-B alkylsilica columns, other evidence suggests that sorbed water in the stationary phase accounts for column hydrogen-bond basicity and values of B (21). Ionic interaction: k9 and C. Values of k9 increase with increasing positive charge on the solute molecule, as expected for an ionic interaction of the solute with an ionized silanol (Figure 1e). Thus, protonated bases have positive values of k9, whereas ionized acids have negative values (k9 = 0.00 for neutral compounds). Presumably, BH+ groups are attracted electrostatically to ionized silanols, whereas ionized carboxyl groups are repelled. As is the case for A, values of C at pH 2.8 decrease markedly for end-capped columns. As expected, C is also observed to increase with an increase in mobile-phase pH, which corresponds to increasing silanol ionization. The practical importance of these five contributions to column selectivity for type-B alkylsilica columns increases as H
