liquid I
t would be diflicult to name an advance more important in the history of modem LC than the development of chemically bonded stationary phases. The rebirth of LC in the late 1960s was driven both by the successes and failures of GC. Theory developed primarily for GC predicted the possibility of high-efficiency separationsby performing LC with small-diameter stationary phases. The need for this separation method was driven by the inability of GC to handle polar and high molecular weight materials. Borrowing from techniques developed for GC, workers first used inert supports coated with viscous liquids insoluble in the mobile phase as stationaryphases. However, the problems of keeping a liquid stationary phase physically coated on a s u p port in the presence of a liquid mobilt phase proved intractable; consequently, liquid-liquid chromatographywas phased out in the early 1970s. In the mid- to late 1970s, LC developed a somewhat unfavorable reputation as manufacturers marketed chemically bonded phases with poor lot-to-lotreproducibility. Often, chemists noted fairly large shifts in retention from one column to another, and in some cases they observed peak elution reversals. This irreproducibility was a result of many factors, including poor control of the physical and chemical properties of the silica support and the bonding reaction. Fortunately,
John G. Dorsey William T. Cooper Florida State University
0003-2700/94/0366 -857A/$04.50/0 0 1994 American Chemical Society
A complete
understanding of retention will allow researchers to use the chromatographic column to measure physical parameters that are otherwise dificult to obtain
these problems were solved relatively quickly, and modern columns typically show variations of capacity factor of < 5% from lot to lot. Differences in retention and selectivity among columns from different manufacturers still may be dramatic, because variations in synthetic methodology and the surface characteristics of the silica affect retention properties. Realizing the importance of the starting silica material, many column manufacturers now make their own starting silica. Since the beginning of bonded-phase chromatographya significant effort has been expended toward understanding the retention mechanism. Retention in normal-phase bonded LC columns was first thought to be similar to that in liquidsolid LC (adsorption chromatography). However, subtle nuances in retention caused by the presence of flexible hydrocarbon chains with the active functional group some distance from the underlying silica surface have required significant modifications in bonded-phasechromatographic theory. In reversed-phaseLC, the solvophobic theory attributed the retention process to the mobile phase, ignoring contributions from the bonded stationary phase. Although this early theory is still widely quoted, it is best viewed in historical perspective as the first rigorous attempt to relate liquid chromatographicretention to physical chemistry principles. Likewise, early attempts to understand the contribution of the stationary phase focused on the carbon content of the bonded phase. This still is one of the most widely quoted, and most misused, figures of merit.
Analytical Chemistry Vol. 66, No. 17, September 7 , 7994 857 A
are dependent on the underlying silica support. It is generally accepted that the surface-active sites on silica are Si-OH groups (silanols). Siloxane groups (Si-0-Si) are also present, but the activity of these essentially hydrophobic sites is so low that they are rarely considered to be of any consequence. Silanols serve as attachment points for the covalent silyl ether bonds that anchor bonded phases to the silica support. Because of steric hindrance, the density of silanols on chromatographic-grade silica ( 8 * 1pmol/m2) is much greater than the maximum possible concentration of alkyl groups in a bonded phase ( 4.5 pmol/m2 for C,, ligands). Therefore, after the silica surface has been modified, numerous unreacted (residual) silanol groups are left within the bonded phase. These residual silanols are weakly acidic, with pK, values typically between 5 and 7; thus, they can interact with polar compounds through strong hydrogen bond and dipole-dipole interactions. The resulting heterogeneous surface leads to mixed retention mechanisms, peak tailing, and loss of chromatographic resolution, particularly when basic solutes are involved. Residual silanols are thus considered by most practicing chromatographers to be deleterious, and many efforts have been made to prepare bonded phases with the fewest possible active silanol sites. However, the heterogeneity induced by the presence of silanols may contribute to stationary-phase selectivity (5,6). Currently, positive contributions of silanols seem more likely in the normal-phase mode. One feature of silanols not generallya p Silica preciated is that they are not all alike (FigAlthough materials such as alumina and ure 1).Indeed, many studies suggest that zirconia continue to receive attention as substrates for chromatographic bonded it is not the absolute number of surface silan01 groups that is importantin determinphases, silica is still by far the most commonly used support material. We will fo- ing the character of a bonded phase, but rather the relative distribution of free, cus on the role of the silica surface in regeminal, and vicinal silanols (7-14).Each tention in both normal- and reverseddifferent surface group has its own unique phase LC. Silica is discussed in detail in numerous reviews, including three arti- properties that affect both bonding reactions and adsorptive character. cles of particular interest to chromatogControversy exists in the literature reraphers (1,3,4).Silica plays a direct role in retention through interactions between garding the properties of each group. Some consider free, isolated silanols to be solutes and its surface-active sites. It also plays an indirect role because the charac- the primary reaction and adsorption teristics of a bonded phase (e.g., bonding sites, with vicinal and geminal groups showing lower reactivity (7-9). Snyder density, specific surface area, porosity)
Understandingthe exact chemistry of the retention process has several motivations. Current attempts to develop expert systems for LC separations will be empirically based until the retention process is clearly understood. Moving these expert systems from an empirical basis to a firm theoretical foundation will result in rapid computer-based method development and will greatly simplifywhat is now often a trial-and-error process. Moreover, a clear understanding of the driving force for retention will result in the predictive development of unique stationary-phase materials for improved separations and will allow chromatographers to solve problems that plague the analyst. A complete understanding of retention will allow researchers to use the chromatography column as a physical chemistry “lab oratory”for measuring physical parameters that are difficult to obtain with other methods. Some of these goals have been achieved, and the future holds promise for attaining the others. In this Report we address all the important factors that control retention in bonded-phaseLC. We will begin with an overview of the silica starting material, briefly address the synthetic methodology, and focus on the use of these materials for both normal- and reversed-phaseLC. For more detail, consult Sander and Wise’s extensive review of bonded phases for LC ( I ) , and each biannual Fundamental Review issue of Analytical Chemistry, which addresses recent advances in both normal- and reversed-phase chromatograPhY (2).
N
N
850 A Analytical Chemistry, Vol. 66, No. 17, September 1, 1994
and Ward (IO),however, argue that internally hydrogen-bonded vicinal groups are more reactive than isolated silanols. Other studies, some based largely on 29Si NMR spectroscopy, point to geminal groups as the most reactive (11-14). These differences in opinion have led to considerablydifferent approaches for preparing silanol-freebonded phases, particularly with regard to pretreating silica before carrying out bonding reactions. One approach assumes that only fully hydroxylated silica surfaces yield dense, homogeneous bonded phases, but it is now clear that fully hydroxylated also means homogeneously hydroxylated. Kohler and Kirkland focused on techniques that selectively rehydroxylate the silica surface and produce a homogeneous distribution of associated silanols (15).They maintain that silica in this form yields bonded phases that have higher pH values, less adsorptivity for basic solutes, and improved hydrolytic and mechanical stability. Ritchie and van den Driest, on the other hand, claim that their rehydroxylation treatment results in a
Figure 1. Active surface groups on silica gel. H, indicates bound hydrogen; H, indicates free hydrogen.
silica having a large population of geminal silanols (14).The resulting bonded phases also show improved chromatographic efficiency. Another approach to silica pretreatment involves reducing the number of surface silanols by some sort of deactivation process so that they all can be reacted during attachment of the bonded phase. This can be done by either selectively deactivating a small fraction of silanols before synthesizing the bonded phase or by heating the silica at temperatures between 200 and 500 “C. Marshall et al. demonstrated improved chromatographic efficiencies using the former a p proach (16);they selectively deactivated the more reactive silanols using chlorotrimethylsilane (17). However, most silica deactivation strategies have focused on thermal pretreatments. Several research groups have attempted to partially dehydroxylate silica by heating before derivatization, followed by either low- or high-temperature silylation (7, 8).High-temperature silylation appears to yield more efficient bonded phases, and few or no residual silanol interactions are apparent, Boudreau and Cooper (7) demonstrated that the surface energies of silica heated at 180 “C and 400 “C were not significantly different, despite the fact that IR spectroscopy of the material subjected to the higher temperature indicated that its surfacewas composed entirely of free, unassociated silanols. Furthermore, all of these free silanols were reacted during the high-temperaturesilylation. The authors of this study concluded that free silanols are the reactive surface species on silica and that thermal removal of less reactive associated silanols reduced steric hindrance to the point whereby almost complete stoichiometric derivatization of all surface silanols could be achieved. Further chromatographicevaluation of an octyl reversed phase prepared with this thermally pretreated silica indicated no silanophilic interactions and showed hydrolytic stability (no rehydroxylation) after exposure to 2500 column void volumes of water (18).The improvements in chromatographic efficiency resulting from these widely varying pretreatment strategies suggest that the controversyregarding the actual active sites on silica surfaces still needs to be resolved.
No discussion of the surface properties of silica can ignore the contributions of trace metals in the silica gel matrix. Chromatographic-grade silica normally contains 0.1-0.3% metallic impurities, and these impuritiesgreatly enhance the adsorptivity of silica gels (3,4). Metals act as additional adsorption sites; they also enhance the activity of adjacent silanols. Acid treatment is generally recommended before silanization to leach metals and produce a more homogeneous bonded phase.
coverage and, in spite of a few anecdotal studies, this remains a largely unexplored area for improving chromatographicmaterials. Applying the experimentaldesign for selection of the leaving group, scavenger base, solvent, and other conditions might prove fruitful for improving existing technology. A second approach involves a polymeric reaction in which either two or three leaving groups are present on the silane. This provides for a more complicated surface chemistry and makes it possible to Bonding chemistry have multiple tie-down points to the silica. More important, the leaving groups can A generalizedview of a typical derivatization reaction is shown above. This repre- hydrolyze before reaction with the surface sents the monomeric reaction, in which to create -OH sites that will react with leaving groups from other silanes, resultonly one leaving group is present on the reactive silane and only one tie-down point ing in a polymeric network extending away from the surface of the silica. This yields to the silica surface is possible. This reaction provides well-defined singlelayer cov- more bonded mass but less well-defined surface coverage, because different erage and is easily reproducible.Singlesilanes may anchor to the surface or to layer coverage does not mean, however, that there is complete coverage of the sur- other silanes at some distance from the surface. Reproducibility is more difficult to face-only that there is no network of bonded material extending away from the control in this reaction; it is very sensisurface. In fact, we will see that the detive to several conditions, especially trace water content, which can cause hydrolygree of surface coverage greatly affects secondary retention, chemical selectivity, sis of the leaving groups. and pH stability of the resulting stationary Sander and Wise (19)showed that phase. these phases can be made reproducibly by Typically the silica and silane are slur- using a single lot of silica and carefully controlling the water content in the reacried in a suitable solvent; a “scavenger base,” or catalyst, is added to the reaction tion mixture. They reported the preparation of a polymeric phase with a relative mixture to remove the acid generated; standard deviation of only 0.96%in surface and the mixture is refluxed for several hours. With careful control of the reaction coverage over four trials. One of the most apparent differences conditions a reproducible phase is produced that typically has a bonding density in the performance of the monomeric and polymeric materials is in shape selectivbetween 2.5 and 3.0 pmol/m2 for CI8 materials and a somewhat higher density for ity. In fact, the National Institute of Standards and Technology has issued a stanshorter chains. Experiment and theory show that as many as 8 pmol/m2 of silanol dard reference material (SRM 869) for the classitication of commercial columns acgroups are available. Although steric hincording to their selectivityfor three polyardrance will never permit all of them to react, there is still room for improvement in omatic hydrocarbons (20).The elution order of the solutes is sensitive to the surface coverage. “chain density”of the bonded alkyl The choice of reactants and reaction groups, and columns derivatized by a moconditions greatly affects the attainable Analytical Chemistry, Vol. 66, No. 17, September l , 1994 859 A
# e B
nomeric process give a different elution order than columns synthesized by the polymeric reaction. SRM 869 is useful both for classification of commercial columns and for studies of retention processes. It would be helpful in choosing stationary phases and reproducing literature separations if column vendors would report the separation of SRM 869 for all commercial columns. The choice of the ligand, or -R group, defines the interactions that occur at the surface and the type of chromatography that will result. Although many different -R groups have been proposed and tried, only a few are popular and commercially available. Clearly the most popular are the reversed-phasematerials, in which the -R group is most commonly a saturated hydrocarbon chain of 8 or 18 carbons in length. However, commercially available chain lengths include 1,2,3,4,6,8,10,12, 18, and 22 carbons. Cyclohexyl, diphenyl, and phenylethyl reversed phases also are available. Other, more polar functionalities may be used in normal-phase chromatography. The most popular are the polyol, propylcyano, and propylamino phases, which provide for more specific interactions at the surface and have operating advantages over traditional silica columns.
in reversed-phasechromatography (21). Yet the solvophobic theory treats the stationary phase as a passive entity that plays no role in the separation process other than providing a sorptive site for retention. Furthermore, hydrophobicity, as strictly defined, plays a small role in the energetics of a typical reversed-phasepartitioning process. These conclusions have come from recent theoretical and experimental studies of the retention process and provide an understanding of retention. More important for the practicing analyst, however, are the improved separation processes that come from these studies. It is now clear that retention is governed by a partitioning process, rather than by adsorption; we define partitioning as full embedding of the solute between the chains of the stationary phase. This partitioning is regulated by the chemical potential difference of the solute between the two phases, and energetics of the solute in both phases are important. Although the word hydrophobic is often used to describe the driving force for r e tention, the strict meaning of this word implies a certain temperature dependence of solubility that is usually not observed with commonly used reversedphase mobile phases. Two pieces of evidence prove that the stationaryphase plays a highly active role in the retention process. First, the shape Reversed=phase chromatography selectivity of polyaromatic hydrocarbons Retention is still often described in terms varies widely among commercial columns of hydrophobic driving forces, and the sol- (19, 20); second, partitioning and selecvophobic theory is still widely quoted. A tivity are both highly dependent on the recent issue of theJouma2 of Chromatogra- chain density of the bonded hydrocarphy was devoted to the Understanding of bon phase (22,23).The failure of the solretention processes (and ongoing debate) vophobic theory to account for these differences arises from its reliance on an incorrect model of the relevant solution processes. It supposes that retention can be modeled in terms of the association of two solute molecules in a single solvent rather than on the transfer of a solute from one solvent to another. A more recent theory of the retention process was described by Dill et al. (2426), who proposed that two driving forces dominate the retention process. One is the difference in the free energy attributFigure 2. Interphase model of able to contact interactions of the solute molecular organization of the with surrounding molecular neighbors stationary-phase chains at high density. in the stationary and mobile phases as Partitioning of solute induces chain ordering. measured by binary interaction constants. 860 A Analytical Chemistry, Vol. 66, No. 17, September 1, 7994
The other is the partial ordering of the grafted stationary-phase chains, which at sufficiently high bonding density leads to an entropic expulsion of solute from the stationary phase relative to what would be expected in a simpler amorphous oil phase/water partitioning process. This theory has been extensively tested by our group and others. The stationary phase is viewed as having both order and an order gradient, with disorder increasing with distance from the silica surface. Figure 2 depicts the organization of the stationary-phase chains and shows that, for a solute to partition to the stationary phase, a cavity must be created in the stationaryphase. This induces further chain ordering, making the partitioning process entropically expensive. Three major lines of research have been used to investigate reversed-phase retention mechanisms. Chromatography experiments have been the most heavily used method for investigating retention processes. As long as careful controls are run and the conditions are described accurately, these experiments can provide highly useful information. More recently, spectroscopicmethods have been used to probe the structure of the stationary phase. These methods include optical experiments, which look at the environment of a sorbed probe, and NMR spectroscopy, which provides information on the conformation of the bonded alkyl chains. Finally, solution thermodynamic studies have elucidated the actual driving forces for the retention process, have provided unambiguous data showing that the predominant means of association of small molecules with the stationary phase is by a partitioning process, and have shown-in direct contradiction of the solvophobic theory-that most of the free energy of retention comes from interactions in the stationaryphase. Chromatographyexperiments. In addition to being useful for chemical analysis, chromatographycan be used to study itself. These investigations run the gamut from simple retention versus mobilephase concentrationexperiments to more sophisticated experiments designed to look at the thermodynamics of the partitioning process. For example, it has been shown that the value of the partition coefficient of a solute from the mobile to the
stationary phase is dependent on the chain density of the bonded alkyl groups, and this chain density affects shape selectivity (22,23). More specifically, this study showed that the partition coefficient goes through a maximum as a function of chain density and that the predominant driving force changes from an enthalpic to an entropic mechanism. These experiments were performed by simple retention measurements made on stationary phases synthesized in house. We should caution against the use of carbon content as a descriptor of the density of the stationary phase. Although this is still the most popular descriptor, in both the commercial and the research literature, when reported alone it is an uninterpretable value. The only useful figure of merit is the chain density on the surface, commonly reported in units of pmol/m2, which takes into consideration both the carbon content and the surface area of the underivatized silica. van't Hoff analysis has been used to probe the thermodynamics of the partitioning process and to investigate possible phase transitions of the bonded, aligned alkyl chains. The van't Hoff expression for chromatography is
Ink'
=
(-AH/RT) + (AS/@ + In
Q,
r * = 0.9952
A H
= -2.19 kcal mol-'
(1)
where k' is the capacity factor, AH and AS represent the enthalpy and entropy of transfer of the solute from the mobile to the stationary phase; R and Tare the gas constant and absolute temperature, respectively; and @ is the volume phase ratio (stationary/mobile). Plotting In k' versus l/Twill give a slope of -M/R, and the entropy and phase ratio are combined in the intercept. Much information has been collected from these experiments. Cole et al. recently used van't Hoff analysis to show that hydrophobicity is not the driving force for retention with most mixed aqueous-organic mobile phases (27, 28).They also showed that the change in entropy during the transfer process increases with increasing chain density of the bonded alkyl groups (27,28).This is further evidence for a partitioning mechanism and has implications for the choice of stationary phases to model other partitioning processes, such as bioavailability estimations.
Figure 3. van't Hoff plots for the solute benzene on a C,. cdumn. (a) Mobile phase of 60:40 acetonitrile-water. (b) Mobile phase of 5:95 mpropanol-water.
van't Hoff plots also have been used to investigate the possibility of phase transitions in the aligned alkyl chain stationary phases. Wheeler et al. recently reviewed this area, including pertinent theory relating to these phenomena (29).A word of caution is necessary about the use and interpretation of van't Hoff analyses. In most reports, a very narrow temperature range-often only 30" or less-was used, and experiments typically are run from 25
"C to 50 or 60 "C. Linearity in van't Hoff plots can easily be found over this range, but it has become increasingly clear that there are significant deviations from linearity if the temperature range is widened. Care should be taken not to draw hasty conclusions based on limited temperature ranges. In fact, maxima in van't Hoff plots are sometimes seen, and this temperature is referred to as TH,the temperature at
Analytical Chemistry, Vol. 66, No. 17, September 1, 1994 861 A
which the enthalpy change is zero. For a largely aqueous mobile phase, THis 20 C (28) and is characteristic of the hydrophobic effect (30).These phenomena are shown in Figure 3, a comparison of van't Hoff plots for benzene on a typical reversed-phase column with mobile phases of acetonitrile-water (60:40) (Figure 3a) and n-propanol-water (5:95) (Figure 3b). The hydrophobic effect may offer a reasonable explanation for the driving forces of retention in a largely aqueous mobile phase, but it is clearly different for more common mobile phases. Spectroscopy exfien'ments. Early spectroscopy experiments were run on stationary phases derivatized with groups designed to be sensitive to spectroscopy. That is, instead of using traditional c8 or cl8 phases, workers derivatized the c18 silica with specially functionalized silanes. Although these experiments showed the power of spectroscopy for interpreting features of the stationary phase, they were not necessarily relevant to traditional reversed-phase materials. More recently, spectroscopic probes have been intercalated into traditional reversed-phase stationary phases and have provided useful information about the organization of the alkyl chains and the environment of sorbed solutes. Marshall et al. (31,32) measured the interfacialpolarity of a C,, material in methanol-water slurries. They also measured the surface fluidity of C,, surfaces and reported a surface viscosity of 4.2 cP, which is 12 times the value for neat acetonitrile. In another series of experiments, Sander, Glinka, and Wise used small-angle neutron scattering to measure the thickness of alkyl-bonded silica surfaces (33). These experiments showed that the average thickness for a monomeric c18 phase is 17A considerably thinner than the fully extended length of octadecane, which is 23 k This is direct evidence that the chains are bent, or disordered, resulting in about a 25%reduction in phase thickness compared with the extended conformation. They also reported that the alkyl chain volume fraction of this same stationary phase was 0.65 5 0.15; the remaining volume fraction would be associated solvent-100% methanol in these experiments. NMR spectroscopy also has been used O
extensively to study the conformation of the alkyl chains. Ilg et al. reported magnetic resonance imaging in a reversedphase LC column (34);they were able to noninvasively observe band profiles in a column. The wall effect, which had been predicted theoretically,was visually confirmed, and thermal effects were directly revealed. Bliesner and Sentell have recently developed a method for wetting the stationary phase under column pressures and then performing NMR experiments (35). This allows investigation of alkyl chain solvation under realistic conditions and should advance our understanding of the interfacialregion. For example, they found that the degree of water associated with the stationary phase was almost constant and was limited, yet the degree of methanol association was considerably greater and was a function of the amount of methanol present in the bulk mobile phase.
spectral shifts induced by solvent effects, and on the study of the forces of the mobile and stationary phases. Carr has reviewed the use of this concept for mechanistic chromatographic studies (36). In an important study, Carr's group (37) used headspace GC to measure gasliquid partition coefficients. They demonstrated that over the entire range of solvent composition, most of the free energy of retention in reversed-phase chromatography arises from attractive dispersive interactions between the solute and the stationary phase, not from net repulsive interactions in the mobile phase. This provides further evidence for a partition mechanism and agrees with other reports. Their comparison of experimental and computed activity coefficientsshowed that regular solution theory is grossly wrong and should not be used for any quantitative predictions involving aqueous systems. Clearly, the thermodynamics of solute transfer from the mobile to the stationary phase are much more complex than has been believed.
Classical LSC theory is usefil as Normal-phasechromatography bare silica was the first common a reference poivtt Although LC stationary phase, the heterogeneity interactions creates a plethfar retentian studies oforaitsofsurface problems in the routine use of this material. For this reason the surface is ifi normal often derivatized to provide for the same type b ~ ~ d e LC d ~ p of interactions ~ ~ but~ with~more home geneous energetics. Most NMR experiments previously had been conducted with wetting of the stationary phase performed at ambient pressure. Because of the pressure dependence of capillary action, it is likely that these phases did not have the same amount or proportion of solvent associated with the stationary phase chains as in the chromatography experiments. Solution thermodynamics. Chromate graphic retention measurements provide information about the combined nature of the mobile and stationary phases. In contrast, solution measurements are made independently of the other phase, allowing the effect of changes in a single-phase composition to be examined independently. The greatest effort has been expended on the use of solvatochromism,
862 A Analytical Chemistry, Vol. 66, No. 17, September I, 1994
If the importance of the stationary phase in reversed-phase LC is just now being recognized, it has always been considered to play the primary role in retention in the normal-phase mode. Adsorbents used as stationary phases in liquidsolid chromatography (LSC) have therefore received considerable attention over the past three decades, and a number of theories have evolved by which the retention of a wide variety of solutes could be predicted with reasonable accuracy. Although there is increasing evidence that bonded phases exhibit unique properties separating them from pure solid adsorbents, classical LSC theory has proven useful as a reference point for studies of retention in what we refer to as normal bonded-phase LC. Before discussing retention in normal
bonded-phase columns, we should point out two important advantages of bonded phases versus solid adsorbents. First, bare silica gel-the most useful adsorbent in high-performance normal-phase LC-is extremely heterogeneous. Deactivation of the strongest active sites on silica by trace amounts of polar impurities in the mobile phase can therefore cause considerable day-to-day variations in retention, particularly for polar solutes. The use of small amounts of water in normal-phase solvents to deactivate strong silanol adsorption sites has become a rather routine, if not entirely satisfactory, practice. Derivatization with alkyl ligands to make bonded phases does not completely eliminate surface heterogeneity; however, it is much more effective and reproducible than using mobile-phase modifiers. A second, less obvious but possibly more significant, advantage of bonded phases in the normal-phase mode is that they provide the opportunity to enhance chromatographic selectivitythrough the adjustment of both mobile and stationary phases. Indeed, it now appears that the fundamental molecular processes controlling retention in different normal bonded-phase columns vary, even in the presence of the same mobile phase (38).It should be possible to serially couple normal bonded-phase columns and do true multidimensional chromatography. Although it is now obvious that reversed bonded phases are not passive participants in retention, it is not possible to serially couple reversed-phase columns and produce truly orthogonal retention mechanisms. The most successful models of retention in LSC (i.e., the models that have the greatest predictive utility) are those of Snyder et al. (39-42) and Soczewinski (43).In actuality,these models are quite similar and can be brought into essential agreement through small modifications. Both models assume that retention is the product of competitive adsorption between solute and solvent (mobile phase) molecules for active sites on the stationary-phase surface. Each assumes a surface monolayer of solute and/or solvent molecules, but the model of Snyder et al. further assumes a homogeneous surface, so that adsorption energies of both solvent and solute molecules are constant.
The stoichiometry of solute-solvent competition can therefore be expressed by
Xm + nSa t)Xa + nS,
(2)
where m and a refer to solute ( X ) and solvent (S) molecules in the mobile and adsorbed phases, respectively. The coefficient n takes into account different adsorption cross sections for solutes and solvents; that is, adsorption of a solute molecule displaces n solvent molecules in the adsorbed monolayer. By assuming a binary mobile phase consisting of a weak, nonpolar solvent (e.g., hexane) and a strong, polar solvent, adsorption of the weak solvent can be ignored
Here, A, is the solute cross-sectional area, n b is the molecular area of the strong solvent, Nb is the mole fraction of the Strong solvent in the mobile phase, k, is the capacity factor of the solute in the binary mobile-phase mixture, and k , is the capacity factor in the strong solvent alone. The Soczewinski model is similar but assumes an energetically heterogeneous surface where adsorption occurs entirely at high-energy active sites, leading to discrete, one-to-one complexes of the form
Xm + n’S-A*
normal bonded-phase LC.Normal bonded phases do contain strongly adsorbing active sites (Soczewinski model), but the solute molecular area, not just polar substituents, is known to play an important role in competitive adsorption (Snyder et al. model). In addition, neither model accounts for so-called secondary solvent effects. These effects, resulting from solutesolvent interactions in both the mobile and adsorbed phases, give rise to some of the most useful changes in retention and often are the source of chromatographic selectivity. These deficiencies were addressed by revising the model of Snyder et al. To account for preferential adsorption of solutes and solvents onto strong sites, empirical A, and n b values larger than those calculated from molecular dimensions are used. This approach was the result of many studies that yielded experimental values of A, and #b that were constant for
n’S, + X - A *
(4) where A* is an active surface site and n’ refers to the number of substituents on a solute molecule that are capable of simultaneously interacting with the active sites. Equation 4 takes into account the possibility of multisite attachment. Capacity factors can be predicted with this model by using an equation similar to that of Snyder et al. t)
Comparison of Equations 3 and 5 reveals that the models of Snyder et al. and Soczewinski both predict that a plot of log k, versus log Nbshould yield a straight line. The model of Snyder et al. predicts that the slope of this line should be the ratio of the molecular areas of solute and solvent, whereas Soczewinski’s model predicts that the slope is the number of strongly adsorbing substituent groups on the solute. Neither model is entirely satisfactory in the forms presented, particularly for
I
I
I
I
I
I
I
I
I
I
2 3 4 5 6 7 8 91( 3inary solvent strength
Figure 4. Logarithm of capacity factors plotted versus binary solvent strength for a cyanopropyl column. (a) Phenol. (b) Aniline. (c) Nitrobenzene. Blue lines represent binary mixtures of hexane and chloroform. Red lines represent mixtures of hexane and dichloromethane. Green lines represent mixtures of hexane and MTBE.
Analytical Chemistry, Vol. 66, No. 17, September 1, 1994 863 A
several systems but were higher than those predicted. The revised model acknowledges the tendency of polar molecules to localize on strongly adsorbing active sites. Localization phenomena appear to be important in almost all normal bonded-phase systems. The revised model of Snyder et al. yields the following equation, which relates the variation of solute retention to changes in solvent strength log k,
=
log k , + dA,(El
- E,)
(6)
where a’ is an adsorbent activity factor, E, and E, are solvent strengths for solvents 1 and 2, and A, is the experimentally determined adsorption cross section. Solvent strengths are determined empirically by using polyaromatic hydrocarbons (e.g., phenanthrene) known to be nonlocalizing; they lie flat on a surface and their adsorption cross sections can be predicted accurately from molecular dimensions. Note that the solvent strengths in Equation 6 are not restricted to pure solvents. Binary solvent strengths of mixtures of weak (a) and strong (b) solvents can be calculated from pure solvent strengths (E, and Ed) by using Equation 7.
E&
=
Ea +
By itself, Equation 10 does not add any insight into retention processes in normal bonded-phase columns. However, Equation 10 and others similar to it have proven extremely useful for comparing experimental data and for understanding how each term varies with solute, solvent, and bonded-phase structure. Solute localiiation can be identified by comparing the slope of a log k, versus E, regression with a calculated molecular cross section. Similarly, comparing intercepts of such a regression for ‘different solvents but the same solute in the same column yields information about secondary solvent effects. Because retention data are plotted against empirically determined solvent strengths in Equation 10, differences in solvent selectivity rather than solvent strength become apparent. As an example of the usefulness of analyzing retention data according to Equation 10, consider Figure 4, in which log k’ for various solutes and a cyanopropyl column are plotted against binary solvent strengths of mobile phases made up of hexane and a strong solvent. Figure 4a is a plot of retention data for phenol in the three binary solvent mixtures. Because the data are plotted as log k’ versus empirical solvent strength (determined by using nonlocalizing polyaromatic hydrocarbons), all three plots should overlap unless there are specific localization and
solvent selectivity effects. Clearly, methyl-tert-butyl ether (MTBE) is a stronger solvent for phenol than predicted from its pure solvent strength. Similar behavior, although not as pronounced, is observed with aniline and nitrobenzene as solutes. Another interesting feature of the data plotted in Figures 4a and 4b is that both slope and intercept of the MTBE regression line for phenol differ from the other two solvents, whereas with aniline it is primarily the slope of the MTBE regression that sets it apart from the other two. Both localization and secondary solvent effects are responsible for the decreased retention of phenol in a hexane-MTBE mobile phase, whereas localization alone contributes to decreased retention of aniline in the same mobile phase. The nature of these localization effects can be identifiedby calculating the pure solvent strengths of the strong, polar modifiers in the binary mixtures through rearrangement of Equation 8. Note that the model assumes constant pure solvent strengths for polar modifiers, regardless of modifier content in the mobile phase, but Figure 5 clearly reveals that the pure solvent strength of MTBE decreases as its concentration in the binary mixture increases. This behavior is a manifestation of restricted-access delocalization, in which polar modifier molecules preferen-
Equation 7 can be simplified if the mole fraction of b is not too low (i.e., N,, < 0.1)
Secondary solute-solvent interactions are incorporated into the revised model of Snyder et al. by adding extra terms A for each solvent to Equation 6. logk,
\
Cyano A
n
I
=
Equation 9 can be further simplified if a nonlocalizing, nonpolar solvent such as hexane is the weak solvent. By assuming that hexane induces no secondary solvent effects and by assigning it a solvent strength of 0, Equation 9 becomes log k,
=
-dA,E2 + logk, +
4
I
A Diol
n
P I
(10)
where k,, is the solute capacity factor in pure hexane.
Figure 5. Plot of pure solvent strength of MTBE in hexane-MTBE binary mixtures versus volume percent for three common normal-phase columns.
864 A Analytical Chemistry, Vol. 66, No. 17, September 1, 1994
tially localize on strong, rigid surfaceactive sites. In contrast, site-competition delocalization involves flexible surface sites of the type found in polar bonded phases. Localization of solvent modifier molecules on surfaceactive sites by either phenomenon disrupts localization of solute molecules, but in subtly different ways and with significantly different consequences. Site-competition delocalization @ i r e 6a) is the most straightforward mechanism that can be used to understand quantitative models of retention. When polar solute and polar solvent modifier molecules, both of which tend to localize at a surface-active site, have essentially unrestricted access to the active site, an increase in the m o d ~ econcentration r will weaken the solute localization. Therefore, retention will decrease to a greater extent than what would be expected from a simple solvent strength determined with nonlocalizing solutes. Correctingfor such an event is rather straightforward because the solvent strength of the modifier can be altered to account for its localizing character. However, this must be done on a specific solvent-solute bonded-phasebasis. Although no comprehensivetabulation of such solvent strength corrections is currently available, Cooper and Smith presented a qualitative description of the necessary site-competition delocalization corrections for the most widely used polar solvent bonded-phase combinations (38). Restricted-access delocalization pigw e 6b), which occurs when polar solvent and solute molecules compete for localization on rigid, fixed surface-active sites where access is highly limited, is more difficult to predict and quantify. When the modifier content is low, modifier molecules will adsorb with localization and effectively disrupt solute localization; the effective solvent strength of the modifier will be relatively large. However, with increasing coverage of the adsorbent surface, a point is reached at which adsorbed modifier molecules interfere with further modifier localization, and further adsorption occurs without localization. The effective solvent strength of the modifier thus decreases with increasing surface coverage. This is the behavior demonstrated in Figure 5.
Clearly, a simple modification to pure solvent strengths is not suitable to account for restricted-accessdelocalization. One promising approach uses localized and delocalized pure solvent strengths along with a delocalization function. The effective pure solvent strength of a localizing modifier (44-46) is given by
Eeff = I Y+
- E?
(11)
Analogous hydrogen bonding with silanols and amino or hydroxy groups would certainly be expected as well. Interestingly, this hydrogen bonding can be disrupted by the presence of small amounts of strongly localizing modifiers such as chloroform (Figure 6c).We refer to this as mixed-site delocalization. Clearly, as we seek to understand the complicated surface characteristics of normal bonded-phasecolumn materials, the underlying silica support surface must never be forgotten.
where E’is the delocalized modifier pure solvent strength, E is the localized modifier pure solvent strength, and %oc is the localization function that measures relative total localization of the modifier on Conclusions the stationaryphase. The ,%, function var- Bonded-phase LC is a mature technique. ies from 1for small values of surface covAdvances in normal- and reversed-phase erage (localized) to 0 for large surface coverages (delocalized). Recently, values for E’, E”,and hocwere determined for the most localizing modifier MTBE and cyano, amino, and diol bonded phases (47). It might appear that all of these localiiation phenomena, being specific to solute solvent stationary-phase combinations, would make method development and routine application of normal bonded-phase HPLC virtually impossible. This is not the case. A substantial amount of literature on these systems is available, and one can predict at least the qualitative behavior of a sample mixture in a particular bondedphase solvent mixture with confidence. The rewards can be elegant, highly selective separations not available in the reversed-phasemode. In addition, as more applications become obvious, twodimensional normal bonded-phase separations should become more routine. As a final word on normal bonded phases, one feature of retention in these columns becomes clearer with each study: the impact of the underlying silica sup port. Not only do residual silanols provide another active site for retention, they also influence the character of the bonded Figure 6. Representation of various phase through their ability to hydrogen delocalization processes In bond with the polar, active functional normal-phase LC. groups of the bonded phase. We have al(a) Site-competitiondelocalization, where ready discussed the importance of repolar modifier molecules (blue circles) have stricted-access delocalization, which preunrestricted access to the polar functional group of the bonded phase and disrupt sumably occurs at exposed silanols. In localization of solute molecules (green circle). addition, several workers have demon(b) Restricted-access delocalization, where strated that hydrogen bonding occurs beaccess to polar functional group of bonded phase is limited. (c) Mixed-site delocalization, tween cyano groups and silanols and inwhere modifier disrupts hydrogen bonding fluences the retention characteristics of between the bonded-phase functional group the cyanopropyl bonded phase (48,49). and the surface silanol group (yellow).
Analytical Chemistry, Vol. 66, No. 1 7, September 1, 1994 865 A
methods are subtle increments to understanding, rather than the large leaps of knowledge associated with the early stages of development. Questions remain and improvements still are needed, however. "he stability of the bonded phases, especially with regard to high- and low-pH conditions, is far from ideal. New approaches to the synthesis of bonded phases may alleviate this problem. For example, Wirth's group has combined selfassembled monolayer technology with the design of chromatographic stationary phases and has developed horizontally polymerized materials that appear to be more resistant,to hydrolysis (5U, 51). As the technique continues to evolve, the chromatographycolumn may become a physical chemistry "laboratory" for the determination of physical and thermodynamic constants. This is already being done to some extent (52).Improvements in our understanding of the bonded-phase structure will certainly help to establish. future applications.
JGD is grateful for support from the Air Force Office of ScientificResearch and the National Institute of EnvironmentalHealth Sciences and also gratefullyacknowledges continued support of our research by Pfizer, Inc., and Merck, Sharp & Dohme Research Laboratories.
References (1) Sander, L. C.; Wise, S. A. CRC Crit. Rev. Anal. Chem. 1987,18,299. (2) Dorsey, J. G.; Cooper, W. T.; Barford, R. A.; Barth, H. G.; Foley, J. P. Anal. Chem. l994,66,500R (3) Nawrocki,J. Chromatographia 1991,3i, 177. (4) Nawrocki,J. Chromatographia 1991,31, 193. (5) Sadek, P. C.; Koester, C. J.; Bowers, L. W. J. Chromatogr. Sci. 1987,25,489. (6) Guillemion,C. L.; Le Page, M.; de Vries, A. I.J. Chromatogr. Sci. 1971,9,470. (7) Boudreau, S. P.; Cooper, W. T. Anal. Chem. 1989,61,41. (8) Welsch, T.; Frank, H. J. J. Chromatogr. 1983,267,235. (9) Mauss, H.; Engelhardt, H. J. Chromatogr. 1986,371,235. (10) Snyder, L. R; Ward, J. W.J. Phys. Chem. 1966,70,3941. (11) Sindorf, D. W.; Maciel, G. E.J. Am. Chem. SOC.1983,105,1487. (12) Sindorf, D. W.; Maciel, G. E.J. Phys. Chem.
1982,86,5208. (13) Weiderer, B.; Bayer, E. J. Chromatogr. 1989,468,67. (14) van den Driest, P. J.; Ritchie, J. Chromatographia 1987,24,324. (15) Kohler, J.; Kirkland,J. J. J. Chromatogr. 1987,385,125. (16) Marshall, D. B.; Stutter, K. A; Lochmuller, C. H.J. Chromatogr. Sci. 1984,22, 217. (17) Marshall, D. B.; Cole, C. L.; Connolly, D. E.J. Chromatogr. 1986,361,71. (18) Wei, M. C. M.S. Thesis, Florida State University, Tallahassee, 1991. (19) Sander, L. C.; Wise, S. A. Anal. Chem. 1984,56,504. (20) Sander, L. C.; Wise, S. A LC-GC 1990,8, 378. (21) J. Chromatogr. 1993,656. (22) Sentell, K. B.; Dorsey, J. G. Anal. Chem. 1989,61,930. (23) Sentell, K. B.; Dorsey, J. G.J. Chromatogr. 1989,461,193. (24) Dill, K. A. J. Phys. Chem. 1987,91,1980. (25) Dill, K. A; Naghizadeh,J.; Marqusee, J. A. Ann. Rev. Phys. Chem. 1988,39,425. (26) Dorsey,J. G.; Dill, K. A. Chem. Rev. 1989, 89,331. (27) Cole, L. A.; Dorsey, J. G. Anal. Chem. 1992,64,1317. (28) Cole, L. A.; Dorsey, J. G.; Dill, K. k Anal. Chem. 1992,64,1324. (29) Wheeler, J. F.; Beck, T. L;Klatte, S. J.;
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Cole, L. A; Dorsey, J. G.J. Chromatogr. 1993,656,317. (30) Blokzijl, W.; Engberts, J.B.F.N.Angew. Chem. Int. Ed. Engl. 1993,32,1545. (31) Men, Y. D.; Marshall, D. B. Anal. Chem. 1990,62,2606. (32) Ellison, E. H.; Marshall, D. E.J. Phys. Chem. 1991,95,808. (33) Sander, L. C.; Glinka, C. J.; Wise, S. A. Anal. Chem. 1990,62,1099. (34) Ilg, M.; Maler-Rosenkranz, J.; Mueller, W.; Bayer, E. J. Chromatogr. 1990,517,263. (35) Bliesner, D. M.; Sentell, K., B. Anal. Chem. 1993,65,1819. (36) Carr, P. W. Microchem. J. 1993,48,4. (37) Carr, P. W.; Li, J.; Dallas, A. J.; Eikens, D. I.; Tan, L. C.J. Chromatogr. 1993,656, 113. (38) Cooper, W. T.; Smith, P. L. J. Chromatogr. 1987,410,249. (39) Snyder, L. R Principles ofAdsorption Chromatography; Marcel Dekker: New York, 1968. (40) Snyder, L. R; Schunk,T.C. Anal. Chem. 1982,54,1764. (41) Snyder, L. R; Glajch, J. L.; Kirkland,J. J. J. Chromatogr. 1981,218,299. (42) Snyder, L. R LCMagazine 1983,1,478. (43) Soczewinski, E. Anal. Chem. 1 9 6 9 , 4 1 , 179. (44) Snyder, L. R; Glajch, J. L.J. Chromatogr. 1982,248,165. (45) Eble, J. E.; Grob, R L.; Antle, P. E.; Sny-
der, L. R J. Chromatogr. 1987,384,25. (46) Eble, J. E.; Grob, R L.; Antle, P. E.; Snyder, L. RJ. Chromatogr. 1987,405,l. (47) Hsu, C. W.; Cooper, W. T.J. Chromatogr., submitted. (48) Suffolk, B. R; Gilpin, R K, Anal. Chem. 1985,57,596. (49) Boudreau, S. P.; Smith, P. L.; Cooper, W. T. Chromatography 1987,2(5), 31. (50) Wirth, M. J.; Fatunmbi, H. 0.Anal. Chem. 1992,64,2783. (51) Wirth, M. J.; Fatunmbi, H. 0.Anal. Chem. 1993,65,822. (52) Pochapsky, T. C.; Gopen, Q. Protein Sci. 1992,1,786.
lytical chemistry in 1979f;vom the University of Cincinnati and for 10 years was a member of the faculty at the University of Florida, where he received four departmental, college, and university teaching awards. He returned to Cincinnati as professor in 1989 and recently moved to Florida State University. His research interests are in the areas offmdamental LC, analytical applications of micelles and organized media, FLA, CE, and old Bordeaux wines.
William T. Cooper is an associateprofessor of chemistry, adjunct professorof oceanography, and director of the Terrestrial Waters Institute at Florida State University. He received a B.S. degree in chemistryfiom the University of Tennessee, Knoxville, and a Ph.D. in chemistryfiom Indiana University. Environmental biogeochemistry is the primary focus of his research, which includes the development of two-dimensional separation methods for analyzing complex John G. Dorsey (left) is professorand chair- environmental and biological samples and man of the Department of Chemistry at inverse gas-liquid chromatographic studies Florida State University (Tallahassee,FL of the sugace chemistry of heterogeneous 32303-3006).He received his Ph.D. in ana- geological materials.
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