Retention Mechanisms of Bonded-Phase Liquid Chromatography

Report

Retention Mechanisms of Bonded-Phase Liquid Chromatography

I

t would be difficult to name an advance more important in the history of modern 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 separations by 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 stationary phases. However, the problems of keeping a liquid stationary phase physically coated on a support in the presence of a liquid mobile phase proved intractable; consequently, liquid-liquid chromatography was 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-lot reproducibility. 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 © 1994 American Chemical Society

these problems were solved relatively A complete quickly, and modern columns typically show variations of capacity factor of < 5% understanding of from lot to lot. Differences in retention and selectivity among columnsfromdifferent retention will allow manufacturers still may be dramatic, because variations in synthetic methodolresearchers to use the ogy and the surface characteristics of the silica affect retention properties. Realizing chromatographic the importance of the starting silica material, many column manufacturers now column to measure make their own starting silica. Since the beginning of bonded-phase physical parameters chromatography a significant effort has been expended toward understanding the that are otherwise retention mechanism. Retention in norbonded LC columns was first difficult to obtain mal-phase 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-phase chromatographic theory. In reversed-phase LC, 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 firstrigorousattempt to relate liquid chromatographic retention 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 1, 1994 857 A

Report Understanding the exact chemistry of the retention process has several motiva­ tions. Current attempts to develop expert systems for LC separations will be empiri­ cally 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 simplify what 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 station­ ary-phase materials for improved separa­ tions and will allow chromatographers to solve problems that plague the analyst. A complete understanding of retention will allow researchers to use the chromatog­ raphy column as a physical chemistry "lab­ oratory" for measuring physical parame­ ters 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 impor­ tant factors that control retention in bond­ ed-phase LC. We will begin with an over­ view of the silica starting material, briefly address the synthetic methodology, and focus on the use of these materials for both normal- and reversed-phase LC. For more detail, consult Sander and Wise's extensive review of bonded phases for LC (1), and each biannual Fundamental Review issue of Analytical Chemistry, which addresses recent advances in both normal- and reversed-phase chromatogra­ phy (2). Silica Although materials such as alumina and zirconia continue to receive attention as substrates for chromatographic bonded phases, silica is still by far the most com­ monly used support material. We will fo­ cus on the role of the silica surface in re­ tention in both normal- and reversedphase LC. Silica is discussed in detail in numerous reviews, including three arti­ cles of particular interest to chromatog­ raphers (1,3,4). Silica plays a direct role in retention through interactions between solutes and its surface-active sites. It also plays an indirect role because the charac­ teristics of a bonded phase (e.g., bonding density, specific surface area, porosity) 858 A

are dependent on the underlying silica support. It is generally accepted that the sur­ face-active sites on silica are Si-OH groups (silanols). Siloxane groups (Si-O-Si) are also present, but the activity of these es­ sentially hydrophobic sites is so low that they are rarely considered to be of any consequence. Silanols serve as attach­ ment 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 ± 1 pmol/m2) is much greater than the maximum possible con­ centration of alkyl groups in a bonded phase (~ 4^mol/m 2 forC lg ligands). Therefore, after the silica surface has been modified, numerous unreacted (re­ sidual) silanol groups are left within the bonded phase. These residual silanols are weakly acidic, with pKa values typi­ cally between 5 and 7; thus, they can inter­ act with polar compounds through strong hydrogen bond and dipole-dipole interac­ tions. The resulting heterogeneous sur­ face leads to mixed retention mecha­ nisms, peak tailing, and loss of chromato­ graphic 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. How­ ever, the heterogeneity induced by the presence of silanols may contribute to sta­ tionary-phase selectivity (5,6). Currently, positive contributions of silanols seem more likely in the normal-phase mode. One feature of silanols not generally ap­ preciated is that they are not all alike (Fig­ ure 1). Indeed, many studies suggest that it is not the absolute number of surface sil­ anol groups that is important in determin­ ing the character of a bonded phase, but rather the relative distribution of free, geminal, and vicinal silanols (7-14). Each different surface group has its own unique properties that affect both bonding reac­ tions and adsorptive character. Controversy exists in the literature re­ garding the properties of each group. Some consider free, isolated silanols to be the primary reaction and adsorption sites, with vicinal and geminal groups showing lower reactivity (7-9). Snyder

Analytical Chemistry, Vol. 66, No. 17, September 1, 1994

and Ward (10), however, argue that inter­ nally 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 considerably different approaches for preparing silanol-free bonded phases, par­ ticularly 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 tech­ niques that selectively rehydroxylate the silica surface and produce a homoge­ neous distribution of associated silanols (15). They maintain that silica in this form yields bonded phases that have higher pH values, less adsorptivity for basic sol­ utes, and improved hydrolytic and me­ chanical stability. Ritchie and van den Driest, on the other hand, claim that their rehydroxylation treatment results in a

Ο /\ 7777s'

Si

7777

Siloxane OH

Ο

OH

77777 Si 77777 Si ^77777^77777 "Free" silanols OH OH \ / 77777 S i 77777 Geminol Ο

Ο

I

I

77777 Si

Si 77777

Vicinol ("bound" silanol) H

W

H

0.

/

Η

\

Ο

Ο

Si

Si

I

77777

Η

I

77777

Bound water Figure 1 . Active surface groups on silica gel. HB indicates bound hydrogen; HF 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 approach (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 surface was composed entirely of free, unassociated silanols. Furthermore, all of these free silanols were reacted during the high-temperature silylation. 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 chromatographic evaluation 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 controversy regarding the actual active sites on silica surfaces still needs to be resolved.

3

CH3

3

Si-O-H + CI-Si-(CH2)17CH3 —• ?

CH3

CH3

Si-0-Si-(CH 2 ) 17 CH3 + HCI 3

CH3

No discussion of the surface properties coverage and, in spite of a few anecdotal of silica can ignore the contributions of studies, this remains a largely unexplored trace metals in the silica gel matrix. Chro- area for improving chromatographic mamatographic-grade silica normally conterials. Applying the experimental design tains 0.1-0.3% metallic impurities, and for selection of the leaving group, scaventhese impurities greatly enhance the adger base, solvent, and other conditions sorptivity of silica gels (3,4). Metals act as might prove fruitful for improving existing additional adsorption sites; they also entechnology. hance the activity of adjacent silanols. Acid A second approach involves a polytreatment is generally recommended bemeric reaction in which either two or three fore silanization to leach metals and pro- leaving groups are present on the silane. duce a more homogeneous bonded phase. This provides for a more complicated surface chemistry and makes it possible to have multiple tie-down points to the silica. Bonding chemistry More important, the leaving groups can A generalized view of a typical derivatization reaction is shown above. This repre- hydrolyze before reaction with the surface to create -OH sites that will react with sents the monomelic reaction, in which 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 to the silica surface is possible. This reac- from the surface of the silica. This yields tion provides well-defined single-layer 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 sensitive to several conditions, especially trace surface. In fact, we will see that the dewater content, which can cause hydrolygree of surface coverage greatly affects sis of the leaving groups. secondary retention, chemical selectivity, and pH stability of the resulting stationary Sander and Wise (19) showed that phase. these phases can be made reproducibly by using a single lot of silica and carefully Typically the silica and silane are slurcontrolling the water content in the reacried in a suitable solvent; a "scavenger tion mixture. They reported the preparabase," or catalyst, is added to the reaction tion 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. conditions a reproducible phase is proOne of the most apparent differences duced that typically has a bonding density in the performance of the monomelic and between 2.5 and 3.0 pmol/m2 for C18 ma- polymeric materials is in shape selectivterials and a somewhat higher density for ity. In fact, the National Institute of Stanshorter chains. Experiment and theory dards and Technology has issued a stanshow that as many as 8 pmol/m2 of silanol dard reference material (SRM 869) for the groups are available. Although steric hinclassification of commercial columns acdrance will never permit all of them to re- cording to their selectivity for three polyaract, there is still room for improvement in omatic hydrocarbons (20). The elution surface coverage. order of the solutes is sensitive to the "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 1, 1994 8 5 9 A

Report in reversed-phase chromatography (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-phase partitioning 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 retention, 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 stationary phase plays a highly active role Reversed -phase in the retention process. First, the shape 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 the Journal 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 Mobile phase two solute molecules in a single solvent Without solute With solute 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 Stationary phase 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.

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-phase materials, 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.

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Analytical Chemistry, Vol. 66, No. 17, September 1, 1994

The other is the partial ordering of the grafted stationary-phase chains, which at sufficiently high bonding density leads to an entropie 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 stationary phase. 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, spectroscopic methods 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 stationary phase. Chromatography experiments. In addition to being useful for chemical analysis, chromatography can be used to study itself. These investigations run the gamut from simple retention versus mobilephase concentration experiments 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 den­ sity 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 fig­ ure of merit is the chain density on the surface, commonly reported in units of pmol/m2, which takes into consider­ ation both the carbon content and the sur­ face area of the underivatized silica. van't Hoff analysis has been used to probe the thermodynamics of the parti­ tioning process and to investigate possible phase transitions of the bonded, aligned alkyl chains. The van't Hoff expression for chromatography is In k' = (-AH/RT) + (AS/R) + In Φ (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 Τ are the gas constant and absolute temperature, re­ spectively; and Φ is the volume phase ra­ tio (stationary/mobile). Plotting In k' ver­ sus 1/Γ will give a slope οί-AH/R, and the entropy and phase ratio are combined in Figure 3. van't Hoff plots for the solute benzene on a C 1 8 column. the intercept. Much information has been (a) Mobile phase of 60:40 acetonitrile-water. (b) Mobile phase of 5:95 n-propanol-water. collected from these experiments. Cole et al. recently used van't Hoff analysis to show that hydrophobicity is not the driv­ ing force for retention with most mixed °C to 50 or 60 °C. Linearity in van't Hoff van't Hoff plots also have been used to aqueous-organic mobile phases (27, plots can easily be found over this range, investigate the possibility of phase transi­ 28). They also showed that the change in tions in the aligned alkyl chain stationary but it has become increasingly clear that entropy during the transfer process in­ phases. Wheeler et al. recently reviewed there are significant deviations from lin­ creases with increasing chain density of this area, including pertinent theory relat­ earity if the temperature range is wid­ the bonded alkyl groups (27,28). This is ened. Care should be taken not to draw ing to these phenomena (29). A word of further evidence for a partitioning mecha­ caution is necessary about the use and in­ hasty conclusions based on limited tem­ nism and has implications for the choice perature ranges. terpretation of van't Hoff analyses. In of stationary phases to model other parti­ In fact, maxima in van't Hoff plots are most reports, a very narrow temperature tioning processes, such as bioavailability range—often only 30° or less—was used, sometimes seen, and this temperature is estimations. referred to as TH, the temperature at and experiments typically are run from 25 Analytical Chemistry, Vol. 66, No. 17, September 1, 1994 861 A

Report L which the enthalpy change is zero. For a extensively to study the conformation of largely aqueous mobile phase, TH is 20 °C the alkyl chains. Ilg et al. reported mag(28) and is characteristic of the hydronetic resonance imaging in a reversedphobic effect (30). These phenomena are phase LC column (34); they were able to shown in Figure 3, a comparison of van't noninvasively observe band profiles in a Hoff plots for benzene on a typical recolumn. The wall effect, which had been versed-phase column with mobile phases predicted theoretically, was visually conof acetonitrile-water (60:40) (Figure 3a) firmed, and thermal effects were directly and «-propanol-water (5:95) (Figure 3b). revealed. The hydrophobic effect may offer a reaBliesner and Sentell have recently desonable explanation for the driving forces veloped a method for wetting the stationof retention in a largely aqueous mobile ary phase under column pressures and phase, but it is clearly different for more then performing NMR experiments (35). common mobile phases. This allows investigation of alkyl chain solSpectroscopy experiments. Early spec- vation under realistic conditions and troscopy experiments were run on station- should advance our understanding of the interfacial region. For example, they found ary phases derivatized with groups dethat the degree of water associated with signed to be sensitive to spectroscopy. the stationary phase was almost constant That is, instead of using traditional C8 or C18 phases, workers derivatized the C18 and was limited, yet the degree of methasilica with specially functionalized silanes. nol association was considerably greater and was a function of the amount of methAlthough these experiments showed the power of spectroscopy for interpreting fea- anol present in the bulk mobile phase. tures 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 interfacial polarity of a C18 material in methanol-water slurries. They also measured the surface fluidity of Clg surfaces and reported a surface viscosity of 4.2 cP, which is 12 times the value for neat acetonitrile. In another series of experiments, Most NMR experiments previously had Sander, Glinka, and Wise used small-angle been conducted with wetting of the staneutron scattering to measure the thicktionary phase performed at ambient presness of alkyl-bonded silica surfaces (33). sure. Because of the pressure dependence These experiments showed that the averof capillary action, it is likely that these age thickness for a monomelic C18 phase phases did not have the same amount or is 17 A, considerably thinner than the fully proportion of solvent associated with the extended length of octadecane, which is stationary phase chains as in the chroma23 A. This is direct evidence that the tography experiments. chains are bent, or disordered, resulting Solution thermodynamics. Chromatoin about a 25% reduction in phase thick- graphic retention measurements provide ness compared with the extended coninformation about the combined nature of formation. They also reported that the althe mobile and stationary phases. In conkyl chain volume fraction of this same trast, solution measurements are made instationary phase was 0.65 ± 0.15; the redependently of the other phase, allowing maining volume fraction would be associ- the effect of changes in a single-phase ated solvent—100% methanol in these composition to be examined indepenexperiments. dently. The greatest effort has been exNMR spectroscopy also has been used pended on the use of solvatochromism,

Classical LSC theory is useful as a reference point for retention studies in normal bonded-phase LC

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Analytical Chemistry, Vol. 66, No. 17, September 1, 1994

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 overtileentire 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 coefficients showed 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. Normal-phase chromatography

Although bare silica was thefirstcommon LC stationary phase, the heterogeneity of its surface interactions creates a plethora of problems in the routine use of this material. For this reason the surface is often derivatized to provide for the same type of interactions but with more homogeneous energetics. 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 consid­ erable 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, prac­ tice. Derivatization with alkyl ligands to make bonded phases does not completely eliminate surface heterogeneity; however, it is much more effective and reproduc­ ible 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 selectivity through the adjustment of both mobile and stationary phases. Indeed, it now appears that the fundamental molecular processes con­ trolling retention in different normal bond­ ed-phase columns vary, even in the pres­ ence of the same mobile phase (38). It should be possible to serially couple nor­ mal bonded-phase columns and do true multidimensional chromatography. Al­ though it is now obvious that reversed bonded phases are not passive partici­ pants in retention, it is not possible to seri­ ally couple reversed-phase columns and produce truly orthogonal retention mecha­ nisms. The most successful models of reten­ tion 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 be­ tween solute and solvent (mobile phase) molecules for active sites on the station­ ary-phase surface. Each assumes a sur­ face 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 sol­ vent and solute molecules are constant.

The stoichiometry of solute-solvent com­ petition can therefore be expressed by Xm + nSa « Xa

+

nSm

(2)

where m and a refer to solute (X) and sol­ vent (S) molecules in the mobile and ad­ sorbed phases, respectively. The coeffi­ cient η takes into account different ad­ sorption cross sections for solutes and solvents; that is, adsorption of a solute molecule displaces η 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 logfe2 = logfcj

log 7^

(3)

Here, As is the solute cross-sectional area, nb is the molecular area of the strong sol­ vent, Nb is the mole fraction of the strong solvent in the mobile phase, k2 is the ca­ pacity factor of the solute in the binary mo­ bile-phase mixture, and kx 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 dis­ crete, one-to-one complexes of the form Xm + n'S-A

^ n'Sm + X-A

normal bonded-phase LC. Normal bonded phases do contain strongly adsorbing ac­ tive sites (Soczewinski model), but the sol­ ute molecular area, not just polar substit­ uents, is known to play an important role in competitive adsorption (Snyder et al. model). In addition, neither model ac­ counts for so-called secondary solvent ef­ fects. 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 ac­ count for preferential adsorption of sol­ utes and solvents onto strong sites, em­ pirical As and nb 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 nb that were constant for

(4)

where A' is an active surface site and »' re­ fers to the number of substituents on a solute molecule that are capable of simul­ taneously interacting with the active sites. Equation 4 takes into account the possibility of multisite attachment. Capac­ ity factors can be predicted with this model by using an equation similar to that of Snyder et al. logfc2 = constant - «'log Nb

(5)

Comparison of Equations 3 and 5 reveals that the models of Snyder et al. and Socze­ winski both predict that a plot of log k2 versus log Nh should 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 sol­ vent, whereas Soczewinski's model pre­ dicts 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

01

2 3 4 5 6 7 8 9

10

Binary 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

Report

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 logfe2 = logÀîj + a'As(E1 - E2)

(6)

where a' is an adsorbent activity factor, Ex and E2 are solvent strengths for solvents 1 and 2, and As 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 (£a and Eb) by using Equation 7. *ab = Κ + log[JVb10a,"b(£b"£-) + 1 - Nh]

(7)

By itself, Equation 10 does not add any insight into retention processes in normal bonded-phase columns. However, Equa­ tion 10 and others similar to it have proven extremely useful for comparing experi­ mental data and for understanding how each term varies with solute, solvent, and bonded-phase structure. Solute localiza­ tion can be identified by comparing the slope of a log k2 versus E2 regression with a calculated molecular cross section. Sim­ ilarly, comparing intercepts of such a re­ gression for different solvents but the same solute in the same column yields information about secondary solvent ef­ fects. 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 ana­ lyzing retention data according to Equa­ tion 10, consider Figure 4, in which log k' for various solutes and a cyanopropyl col­ umn 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 empir­ ical solvent strength (determined by us­ ing nonlocalizing polyaromatic hydrocar­ bons), all three plots should overlap un­ less there are specific localization and

solvent selectivity effects. Clearly, methyl-ierf-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 interest­ ing 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 respon­ sible 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 identified by calculating the pure solvent strengths of the strong, polar mod­ ifiers in the binary mixtures through rear­ rangement 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 sol­ vent strength of MTBE decreases as its concentration in the binary mixture in­ creases. This behavior is a manifestation of restricted-access derealization, in which polar modifier molecules preferen-

Equation 7 can be simplified if the mole fraction of b is not too low (i.e., iVb < 0.1) Kb = Eb +

logWu

(8)

anu

0.160

Secondary solute-solvent interactions are incorporated into the revised model of Snyder et al. by adding extra terms Δ for each solvent to Equation 6.

0.140 0.120

log£2 = log*! + a'A^E, - E2) + (Δ2 - Δχ) (9) 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&2 = -a'AsE2 + log£ h + Δ2

d: 0.0800.060 10

15

20

Volume percent MTBE

(10)

where kh is the solute capacity factor in pure hexane. 864 A

0.100

Figure 5. Plot of pure solvent strength of MTBE in hexane-MTBE binary mixtures versus volume percent for three common normal-phase columns.

Analytical Chemistry, Vol. 66, No. 17, September 1, 1994

daily localize on strong, rigid surfaceClearly, a simple modification to pure active sites. In contrast, site-competition solvent strengths is not suitable to ac­ delocalization involves flexible surface count for restricted-access delocalization. sites of the type found in polar bonded One promising approach uses localized phases. Localization of solvent modifier and delocalized pure solvent strengths molecules on surface-active sites by either along with a delocalization function. The phenomenon disrupts localization of sol­ effective pure solvent strength of a localiz­ ute molecules, but in subtly different ways ing modifier (44-46) is given by and with significantly different conse­ E es - E" + \JF ~ E") (ID quences. Site-competition delocalization (Figure where E" is the delocalized modifier pure 6a) is the most straightforward mecha­ solvent strength, Ε is the localized mod­ nism that can be used to understand quan­ ifier pure solvent strength, and %,oc is the titative models of retention. When polar localization function that measures rela­ solute and polar solvent modifier mole­ tive total localization of the modifier on cules, both of which tend to localize at a the stationary phase. The %,oc function var­ surface-active site, have essentially unre­ ies from 1 for small values of surface cov­ stricted access to the active site, an in­ erage (localized) to 0 for large surface cov­ crease in the modifier concentration will erages (delocalized). Recently, values for weaken the solute localization. Therefore, Ε', E", and %loc were determined for the retention will decrease to a greater ex­ most localizing modifier MTBE and cytent than what would be expected from a ano, amino, and diol bonded phases (47). simple solvent strength determined with It might appear that all of these localiza­ nonlocalizing solutes. tion phenomena, being specific to soluteCorrecting for such an event is rather solvent stationary-phase combinations, straightforward because the solvent would make method development and rou­ strength of the modifier can be altered to tine application of normal bonded-phase account for its localizing character. How­ HPLC virtually impossible. This is not the ever, this must be done on a specific sol­ case. A substantial amount of literature vent-solute bonded-phase basis. Although on these systems is available, and one can no comprehensive tabulation of such sol­ predict at least the qualitative behavior of vent strength corrections is currently a sample mixture in a particular bondedavailable, Cooper and Smith presented a phase solvent mixture with confidence. qualitative description of the necessary The rewards can be elegant, highly selec­ site-competition delocalization correc­ tive separations not available in the retions for the most widely used polar sol­ versed-phase mode. In addition, as more vent bonded-phase combinations (38), applications become obvious, two-dimen­ sional normal bonded-phase separations Restricted-access delocalization (Fig­ ure 6b), which occurs when polar solvent should become more routine. and solute molecules compete for localiza­ As afinalword on normal bonded tion on rigid, fixed surface-active sites phases, one feature of retention in these where access is highly limited, is more dif­ columns becomes clearer with each study: ficult to predict and quantify. When the the impact of the underlying silica sup­ modifier content is low, modifier mole­ port. Not only do residual silanols provide cules will adsorb with localization and ef­ another active site for retention, they fectively disrupt solute localization; the ef­ also influence the character of the bonded fective solvent strength of the modifier phase through their ability to hydrogen will be relatively large. However, with in­ bond with the polar, active functional creasing coverage of the adsorbent sur­ groups of the bonded phase. We have al­ face, a point is reached at which ad­ ready discussed the importance of re­ sorbed modifier molecules interfere with stricted-access delocalization, which pre­ further modifier localization, and further sumably occurs at exposed silanols. In adsorption occurs without localization. addition, several workers have demon­ The effective solvent strength of the modi­ strated that hydrogen bonding occurs be­ fier thus decreases with increasing sur­ tween cyano groups and silanols and in­ face coverage. This is the behavior demon­ fluences the retention characteristics of strated in Figure 5. the cyanopropyl bonded phase (48, 49).

Analogous hydrogen bonding with sil­ anols 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 nor­ mal bonded-phase column materials, the underlying silica support surface must never be forgotten. Conclusions Bonded-phase LC is a mature technique. Advances in normal- and reversed-phase

Figure 6. Representation of various delocalization processes in normal-phase LC. (a) Site-competition delocalization, where polar modifier molecules (blue circles) have unrestricted access to the polar functional group of the bonded phase and disrupt localization of solute molecules (green circle). (b) Restricted-access delocalization, where access to polar functional group of bonded phase is limited, (c) Mixed-site delocalization, where modifier disrupts hydrogen bonding between the bonded-phase functional group and the surface silanol group (yellow).

Analytical Chemistry, Vol. 66, No. 17, September 1, 1994 8 6 5 A

Report

methods are subtle increments to under­ standing, rather than the large leaps of knowledge associated with the early stages of development. Questions remain and improvements still are needed, how­ ever. The stability of the bonded phases, especially with regard to high- and low-pH conditions, is far from ideal. New ap­ proaches to the synthesis of bonded phases may alleviate this problem. For ex­ ample, 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 (50, 51). As the technique continues to evolve, the chromatography column may become a physical chemistry "laboratory" for the determination of physical and thermody­ namic 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 Scientific Research and the National Institute of Environmental Health Sciences and also gratefully acknowledges continued support of our research by Pfizer, Inc., and Merck, Sharp & Dohme Research Laboratories.

1982,86, 5208. (13) Pfleiderer, B.; Bayer, E.J. Chromatogr. 1989,468, 67. (14) van den Driest, P. J.; Ritchie, J. Chro­ matographia 1987,24,324. (15) Kohler, J.; Kirkland, J. J.J. Chromatogr. 1987,385,125. (16) Marshall, D. B.; Stutter, K. A; LochmulReferences ler, C. E.J. Chromatogr. Sci. 1984, 22, 217. (1) Sander, L. C; Wise, S. A. CRC Crit. Rev. (17) Marshall, D. B.; Cole, C. L; Connolly, Anal. Chem. 1987,18, 299. D. E.J. Chromatogr. 1986,361, 71. (2) Dorsey, J. G.; Cooper, W. T.; Barford, (18) Wei, M. C. M.S. Thesis, Florida State Uni­ R. Α.; Barth, H. G.; Foley, J. P. Anal. versity, Tallahassee, 1991. Chem. 1994, 66, 500R. (3) Nawrocki, J. Chromatographia 1991,52, (19) Sander, L. C; Wise, S. A. Anal. Chem. 1984,56,504. 177. (4) Nawrocki, J. Chromatographia 1991,32, (20) Sander, L. C; Wise, S. A LC-GC 1990,8, 378. 193. (5) Sadek, P. C; Koester, C. J.; Bowers, L. W. (21) /. Chromatogr. 1993, 656. (22) Sentell, K. B.; Dorsey, J. G. Anal. Chem. /. Chromatogr. Sci. 1987,25, 489. 1989, 61,930. (6) Guillemion, C. L.; Le Page, M.; de Vries, (23) Sentell, Κ Β.; Dorsey, J. G.J. Chromatogr. A I./. Chromatogr. Sci. 1971, 9,470. 1989,461,193. (7) Boudreau, S. P.; Cooper, W. T. Anal. (24) Dill, K.A.J. Phys. Chem. 1987, 91,1980. Chem. 1989, 61,41. (25) Dill, K. A; Naghizadeh, J.; Marqusee, J. A. (8) Welsch, T.; Frank, H. J. / Chromatogr. Ann. Rev. Phys. Chem. 1988,39,425. 1983,267,235. (9) Mauss, H.; Engelhardt, H./. Chromatogr. (26) Dorsey, J. G.; Dill, Κ A. Chem. Rev. 1989, 89,331. 1986,371,235. (10) Snyder, L. R.; Ward, J. W.J. Phys. Chem. (27) Cole, L. A; Dorsey, J. G. Anal. Chem. 1992, 64,1317. 1966, 70,3941. (11) Sindorf, D. W.; Maciel, G. E.J. Am. Chem. (28) Cole, L. A; Dorsey, J. G.; Dill, K. A Anal. Chem. 1992, 64,1324. Soc. 1983,105,1487. (12) Sindorf, D. W.; Maciel, G. E.J. Phys. Chem.(29) Wheeler, J. F.; Beck, T. L.; Klatte, S. J.;

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Cole, L. Α.; Dorsey, J. G.J. Chromatogr. 1993,656,317. Blokzijl, W.; Engberts, J.B.F.N. Angew. Chem. Int. Ed. Engl. 1993,32,1545. Men, Y. D.; Marshall, D. B. Anal. Chem. 1990, 62, 2606. Ellison, E. H.; Marshall, D. B.J. Phys. Chem. 1991,95,808. Sander, L. C; Glinka, C. J.; Wise, S. A. Anal. Chem. 1990, 62,1099. Ilg, M.; Maler-Rosenkranz, J.; Mueller, W.; Bayer, E.J. Chromatogr. 1990,517, 263. Bliesner, D. M.; Sentell, Κ. Β. Anal. Chem. 1993, 65,1819. Carr,F.W.Microchem.J 1993,45,4. Carr, P. W.; Li, J.; Dallas, A. J.; Eikens, D. I.; Tan, L. C.J. Chromatogr. 1993,656, 113. Cooper, W.T.; Smith, P. L.J. Chromatogr. 1987, 410, 249. Snyder, L. R. Principles ofAdsorption Chro­ matography; Marcel Dekker: New York, 1968. Snyder, L. R.; Schunk, T. C. Anal. Chem. 1982,54,1764. Snyder, L. R.; Glajch, J. L.; Kirkland, J. J. /. Chromatogr. 1981,228,299. Snyder, L. R. LC Magazine 1983,1,478. Soczewinski, E.Anal. Chem. 1969, 41, 179. Snyder, L. R.; Glajch, J. L.J. Chromatogr. 1982,248,165. Eble, J. E.; Grob, R. L.; Antle, P. E.; Sny-

der, L. R./. Chromatogr. 1987,384,25. lytical chemistry in 1979 from the Univer­ (46) Eble, J. E.; Grob, R. L; Antle, P. E.; Sny­ sity of Cincinnati and for 10 years was a der, L R.J. Chromatogr. 1987,405,1. member of the faculty at the University of (47) Hsu, C. W.; Cooper, W. T.J. Chromatogr., Florida, where he received four departmen­ submitted. (48) Suffolk, B. R.; Gilpin, R. K. Anal. Chem. tal, college, and university teaching awards. 1985,57, 596. He returned to Cincinnati as professor in (49) Boudreau, S. P.; Smith, P. L.; Cooper, 1989 and recently moved to Florida State W. T. Chromatography 1987,2(5), 31. (50) Wirth, M. J.; Fatunmbi, H. O. Anal. Chem. University. His research interests are in the areas offundamental LC, analytical ap­ 1992, 64, 2783. (51) Wirth, M. J.; Fatunmbi, H. O. Anal. Chem. plications of micelles and organized media, 1993, 65, 822. FIA, CE, and old Bordeaux wines. (52) Pochapsky, T. C; Gopen, Q. Protein Sci. 1992,1, 786. William T. Cooper is an associate professor of chemistry, adjunct professor of oceanog­ raphy, and director of the Terrestrial Wa­ ters Institute at Florida State University. He received a B.S. degree in chemistry from the University of Tennessee, Knoxville, and a Ph.D. in chemistry from Indiana Uni­ versity. Environmental biogeochemistry is the primary focus of his research, which in­ cludes the development of two-dimensional separation methods for analyzing complex John G. Dorsey (left) is professor and chair­ environmental and biological samples and inverse gas-liquid chromatographic studies man of the Department of Chemistry at of the surface chemistry of heterogeneous Florida State University (Tallahassee, FL 32303-3006). He received his Ph.D. in ana­ geological materials.

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