Column Characterization and Selection Systems in ... - ACS Publications

Review Cite This: Chem. Rev. XXXX, XXX, XXX−XXX

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Column Characterization and Selection Systems in Reversed-Phase High-Performance Liquid Chromatography Petar Ž uvela,† Magdalena Skoczylas,‡ J. Jay Liu,*,§ Tomasz Ba̧czek,∥ Roman Kaliszan,⊥ Ming Wah Wong,† and Bogusław Buszewski‡ †

Department of Chemistry, National University of Singapore, Singapore 117543, Singapore Department of Environmental Chemistry and Bioanalytics, Center for Modern Interdisciplinary Technologies, Nicolaus Copernicus University, Wileńska 4, 87-100 Toruń, Poland § Department of Chemical Engineering, Pukyong National University, 365 Sinseon-ro, Nam-gu, 48-513 Busan, Korea ∥ Department of Pharmaceutical Chemistry and ⊥Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdańsk, Hallera 107, 80-416 Gdańsk, Poland

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ABSTRACT: Reversed-phase high-performance liquid chromatography (RP-HPLC) is the most popular chromatographic mode, accounting for more than 90% of all separations. HPLC itself owes its immense popularity to it being relatively simple and inexpensive, with the equipment being reliable and easy to operate. Due to extensive automation, it can be run virtually unattended with multiple samples at various separation conditions, even by relatively low-skilled personnel. Currently, there are >600 RP-HPLC columns available to end users for purchase, some of which exhibit very large differences in selectivity and production quality. Often, two similar RP-HPLC columns are not equally suitable for the requisite separation, and to date, there is no universal RP-HPLC column covering a variety of analytes. This forces analytical laboratories to keep a multitude of diverse columns. Therefore, column selection is a crucial segment of RP-HPLC method development, especially since sample complexity is constantly increasing. Rationally choosing an appropriate column is complicated. In addition to the differences in the primary intermolecular interactions with analytes of the dispersive (London) type, individual columns can also exhibit a unique character owing to specific polar, hydrogen bond, and electron pair donor−acceptor interactions. They can also vary depending on the type of packing, amount and type of residual silanols, “end-capping”, bonding density of ligands, and pore size, among others. Consequently, the chromatographic performance of RP-HPLC systems is often considerably altered depending on the selected column. Although a wide spectrum of knowledge is available on this important subject, there is still a lack of a comprehensive review for an objective comparison and/or selection of chromatographic columns. We aim for this review to be a comprehensive, authoritative, critical, and easily readable monograph of the most relevant publications regarding column selection and characterization in RP-HPLC covering the past four decades. Future perspectives, which involve the integration of state-of-the-art molecular simulations (molecular dynamics or Monte Carlo) with minimal experiments, aimed at nearly “experiment-free” column selection methodology, are proposed.

CONTENTS 1. Introduction 2. Fundamentals of High-Performance Liquid Chromatography 2.1. Mechanism of High-Performance Liquid Chromatographic Separation 2.2. Chromatographic Columns 2.3. Chromatographic Column Materials 2.3.1. Silica and Other Supports 2.3.2. Chemically Bonded Phases 2.4. Characterization of Chromatographic Columns 2.4.1. Surface Topology Characterization 2.4.2. Microscopic Imaging Techniques for Column Characterization 2.4.3. Thermal Analysis for Column Characterization © XXXX American Chemical Society

2.4.4. Spectroscopic Techniques for Column Characterization 2.4.5. Electromigration Methods for Column Characterization 2.4.6. Chromatographic Methods for Column Characterization 3. Chromatographic Column Selection Systems 3.1. Engelhardt Column Selection System 3.1.1. Experimental Characterization of Chemically Bonded RP-HPLC Columns 3.1.2. Retention Test as an RP-HPLC Column Selection System 3.2. Tanaka Column Selection System 3.2.1. Layne Column Selection System

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L Received: April 15, 2018

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DOI: 10.1021/acs.chemrev.8b00246 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews 3.3. 3.4. 3.5. 3.6. 3.7. 3.8. 3.9.

Euerby Column Selection System Walters Column Selection System Kulikov Column Selection System McCalley Column Selection System Organon Column Selection System Dolan Column Selection System Quantitative Structure−Retention Relationships in Chromatographic Column Selection 3.9.1. Linear Free Energy Relationships 3.9.2. Szepesy Column Selection System 3.9.3. Galushko Column Selection System 3.9.4. Kaliszan Column Selection System 3.9.5. Application of the Kaliszan Column Selection System in Proteomics 3.9.6. Hydrophobic Subtraction Model for Column Selection 3.10. Jandera Column Selection System 3.12. Neue Column Selection System 3.13. Katholieke Universiteit Leuven Column Selection System 3.14. Lesellier Carotenoid Column Selection System 4. Comparative Column Selection Studies 5. Application of Molecular Simulations in RP-HPLC 6. Conclusions and Future Perspectives Author Information Corresponding Author ORCID Notes Biographies Acknowledgments References

Review

Several types of HPLC columns, classified by their mode of separation, are known, including normal-phase (NP-HPLC), reversed-phase (RP-HPLC), size-exclusion (SEC), and ionexchange (IC). RP-HPLC columns are the most popular, accounting for more than 90%9,10 of all HPLC separations, especially in pharmaceutical, environmental, and wastewater applications, the food industry, medical, biomedical, and life sciences fields, and so on. Due to the versatility of HPLC, one can separate nonpolar, polar, ionizable, and ionic compounds, even simultaneously. Moreover, in RP-HPLC, the mobile phase is simply (buffered) water and a water-miscible mobile phase organic modifier, such as methanol or acetonitrile, both of which are relatively safe and easily accessible.11 More than 600 RP-HPLC columns are available on the market,12−14 some with large differences in selectivity as well as manufacturing standards. Most of them differ from each other only subtly, but the difference is often sufficient to achieve a required separation, otherwise not attainable with another column. The differences among columns can present themselves due to several factors. Among them are the purity of the media used for manufacturing the phase and the method of packing the columns. The variability among the RP stationary phases may be beneficial to the analyst, for example, in cases where the contaminants to be separated are closely related to the test substance. On the other hand, this also forces analytical laboratories to maintain a multitude of diverse columns. Such specificity may result in the lack of a requisite column in the market. Suitable column selection is, therefore, a crucial segment in the development of RP-HPLC methods for a particular analysis, especially with regard to the rising sample complexity. The trialand-error method of column selection based on the empirical knowledge of the analyst (and/or on his/her chemical intuition) is often used. However, such an approach is labor-intensive and exorbitant and can lead to conflicting results. Therefore, a good column classification system based on objective (numerical) criteria, which also allows for a ranking of the columns, is currently required. The challenges of column characterization have long been the subject of intensive research. Throughout the past several decades, numerous attempts have been made to evaluate the differences in the quality and separation power of stationary phases in RP-HPLC, as well as to characterize the underlying separation mechanism at the molecular or submolecular level. Modern column selection, classification, characterization tests, and systems have evolved from the early works of Buszewski,15−18 Engelhardt,19−32 Euerby,33−35 Gilpin,36−39 Guiochon,40−51 Horvath,1,3,4,52−54 Jandera,55−61 Kirkland,62−68 Le Mahipan, 69−71 Majors,72−74 Neue,75−83 Sander and Wise,84−90 Snyder,91−94 Tanaka,95−111 and Walters.112,113 On the basis of these, more complex approaches to column selection, utilizing advanced chemometric data processing, have been proposed. They include Héberger’s sum of ranking differences,114−117 Hoogmarten−Haghedooren’s Katholieke Universiteit Leuven (KUL) system,118−123 Kaliszan’s quantitative structure−retention relationships124−127 based on linear free energy relationships (LFERs),128−133 Lesellier’s carotenoid test,134−136 Snyder and Dolan’s hydrophobic subtraction model,137−140 and others.13,141 These works aimed to simplify the end users’ choice of RP-HPLC columns and led to the emergence of new comprehensive column characterization and selection systems (CSSs).14

W X Y Y Z Z Z Z Z AA AA AB AC AG AH AH AJ AJ AL AL AM AM AM AM AM AN AN

1. INTRODUCTION High-performance liquid chromatography (HPLC) is one of the foremost analytical techniques used for scientific purposes and laboratory measurements. It is widely used in chemical, pharmaceutical, and biomedical analysis, as well as in drug treatment monitoring, due to its inherent ability to analyze, separate, and purify a variety of chemical samples, including, but not limited to, acidic, basic, and neutral analytes. What is known today as HPLC is the result of the decade-long dedication of a Hungarian-American chemical engineer, Csaba Horváth.1−4 Building the instrument out of his own laboratory equipment, he laid the groundwork for large-scale commercialization of the revolutionary work of Martin and Synge.5 As a result, over the course of half a century, HPLC has gained immense popularity in most analytical laboratories.6−8 It has become the third most popular instrument after the pH meter and weighing scale. This is because HPLC is a relatively simple and inexpensive technique. The currently available equipment is easy to operate and reliable. Due to the use of automated sample trays and valve changes for the column and mobile phase, chromatography allows for almost unattended analysis of multiple samples at various separation conditions, even by relatively low-skilled personnel. Naturally, like any other popular technique, it is constantly improving. Each year, there are new instrument models, chromatographic columns, and chromatographic system technical solutions available. The development and production of chromatographic columns represent the largest segment of the vast global market of HPLC instrumentation and consumables. B


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and adsorption processes exhibit a significant impact on their retention. For analytes with polar moieties, adsorption constitutes the general retention mechanism on alkyl-bonded phases, regardless of the alkyl chain length.146,157 The incorporation of polar functional groups (so-called embedded polar groups (EPGs)) into the structure of alkyl ligands induced the extension of possible interaction pathways.158−164 The polar solutes exhibit a higher retention on the polar embedded phases, while hydrophobic solutes are less retained. This is owing to more groups capable of hydrogen bond creation (EPGs and sorbed solvent) and the competition between the hydrophobic analyte molecules and solvent molecules for space within the stationary phase, respectively.154 The polar embedded phases also exhibit diminished peak tailing for basic analytes. This is due to (i) the solute interactions with residual silanols (by hydrogen bonds) being diminished because of the presence of EPGs and (ii) the free energy of the transfer of polar analytes from the bulk phase to the surface silanols being reduced.154 The displacement of organic solvent molecules from the stationary phase, caused by solute molecules (the aforementioned competition), occurs in any case. However, its strength depends on the mobile-phase composition, the coverage density of bonded ligands, their length, the presence of EPGs, and the pore curvature.155,157,165 Overall, the bonded phases participate in the retention phenomenon as a heterogeneous phase with multiple sorption sites such as bonded ligands, residual silanols, EPGs, and the interfacial sphere. Thus, the retention mechanism in RP-HPLC is multifaceted with regard to the molecular-level phenomena, and each separation case should be considered discretely.145,157 Normal-phase (adsorption) liquid chromatography (NPHPLC) and hydrophilic interaction liquid chromatography (HILIC) represent two other chromatographic modes dedicated to the analysis of hydrophilic compounds. NP-HPLC historically represents the first chromatography mode, which, over time, lost popularity due to the introduction of RPHPLC.166 In this mode, the stationary phase exhibits a higher degree of polarity than that of the mobile phase. Consequently, the elution order depends on the polarity of the analytes: from the more hydrophobic to the more hydrophilic compounds. According to the Snyder−Soczewiński model,142,143 the retention of analytes and the selectivity of stationary phases are strongly correlated with the size of the analyzed molecules.142,167−169 Strongly hydrophilic compounds, which cannot be studied in an NP-HPLC system due to their irreversible adsorption and insufficient solubility in nonpolar or weakly polar mobile phases, can be analyzed in the HILIC mode.170 This chromatography mode combines the above two systems: NP-HPLC and RP-HPLC. HILIC employs polar stationary phases (similar to NP-HPLC) and aqueous−organic mobile phases (similar to RP-HPLC, but with the organic solvent content higher than 50%). Over the years, the HILIC system has been extensively evaluated and has gained much popularity in chromatographic science.171 It is commonly assumed that the retention occurs through the following phenomena: (i) the partitioning process, (ii) adsorption phenomenon, and (iii) ion-exchange interactions.170−172 The retention in HILIC, under isocratic and gradient conditions, can be predicted and described using the following models: the quadratic model, 173 Snyder−Soczewińs ki model, 174 Lu model,175 Jandera model,176 and Neue and Kuss model.177 Indepth studies and interpretations of retention models of HILIC have been presented by Liang’s and Fisher’s groups.178−180 Overall, the retention of polar analytes increases with a decrease

CSSs may be considered as tools for classification of columns in accordance with specific physicochemical parameters (e.g., their hydrophobicity, silanol activity, and so on) of the RPHPLC separation mechanism. However, since many different CSSs involve the use of complex chemometric methods, an antithetical effect is achieved. End-users and method developers may not only find it difficult to decide on a suitable column for a particular separation, but also ironically end up troubled with a decision choosing a suitable CSS. These issues are addressed critically and objectively, disseminating both the advantages and disadvantages of CSSs, as well as the differences between them. Following the Introduction, this paper consists of a section on the fundamentals of RP-HPLC. Its purpose is to familiarize the reader with chromatography in general, focusing more on the materials for the development of RP-HPLC columns and their characterization. It is followed by a thorough description of several CSSs and a section focused solely on their limitations and the ways of addressing them. Naturally, as the authors, we realize that column testing and selection concerns not only the column, but the whole chromatographic system, i.e., the mobile phase, buffers, any other eluent additives, temperature, other operating conditions, and so on. That being said, this review aims to be a comprehensive, authoritative, critical, and easily readable collection of the most relevant publications related to column selection, classification, and characterization in RP-HPLC. The future perspectives in the field, which seems to be stagnating, are proposed, involving the integration of molecular modeling and simulations aimed at nearly “experiment-free” column selection. Therefore, the review will be of interest not only to end-users and developers of RP-HPLC methods, but also to the (analytical) chemistry community as a whole.

2. FUNDAMENTALS OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY 2.1. Mechanism of High-Performance Liquid Chromatographic Separation

In reversed-phase liquid chromatography (RP-HPLC), a nonpolar stationary phase and a polar hydro-organic mobile phase (optionally spiked with a buffer salt) are employed for analysis. The retention increases with an increase of the (i) hydrophobicity of the solutes, (ii) hydrophobicity of the stationary-phase surface, and (iii) polarity of the mobile phase.142−144 Separation is achieved through two phenomena, i.e., the partitioning process (analyte molecules entirely immerse themselves into the bonded phase) and/or adsorption phenomenon (which occurs at the bonded-phase/solvent interface).145 From a thermodynamic point of view, the partitioning process can be considered according to Horváth et al.146−149 theory or the mechanistic studies of Carr et al.150−153 The solvophobic theory, proposed by Horváth et al., assumed that the retention is governed by the hydrophobic interactions of the solutes with the hydro-organic mobile phase. In contrast, Carr et al. favored the lipophilic interactions (between the nonpolar bonded phase and hydrophobic solutes) as the driving force of retention.146−153 The contribution of the particular phenomenon (partition or adsorption) to the retention mechanism varied and was proven by molecular simulations.146,154−156 The molecular-level comparison of the alkyl phases showed that the nonpolar solute molecules are mainly retained through the adsorption phenomenon on materials with shorter ligands (e.g., the C8 phase), while for longer bonded phases (such as the C18 phase), both partition C


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silanization/hydrosilylation process.172,188 Type A silica is characterized by a higher acidity due to a high metal contaminant concentration in contrast to that of the ultrapure type B silica. The high metal content induces the activation of surface silanol groups, which can lead to chelation with some solutes. This results in strong retention or peak shape deformation. Type B silica has a higher population of surface silanols (at least 7 μmol of SiOH/m2) than that of type A silica (about 5.5−6 μmol of SiOH/m 2 ). 67 The stability at intermediate and higher pH (up to a pH of 9) and the overall better separation properties of type B silica resulted in its common use as a chromatographic support. The details about type C silica are discussed in the following paragraphs.172,189 Silica is also characterized by surface (different types of silanols and siloxanes) and structural (cylindrical, funnelshaped, and ink-bottle-shaped pores) heterogeneity. Generally, three types of silanol groups can be distinguished: isolated (free, single) (total content 6−19% of all surface silanols), vicinal (bridge) (60−65%), and geminal (10−12%) (Figure 1). Vicinal

in the water content in the mobile phase, and the separation depends on the polarities of the compounds as well as the degree of solvation phenomena which occur on the stationary-phase surface. 2.2. Chromatographic Columns

The chromatographic column is the “heart” of each chromatographic system. Its development is subject to the greatest changes among all the chromatographic elements.181 Researchers are constantly focusing their efforts on improving the efficiency and durability of columns to minimize the differences in chromatographic properties between successively produced batches. In recent years, the greatest emphasis has been placed on the possibilities of shortening the time and increasing the robustness of chromatographic analysis. Different manufacturers introduced further modifications of the column packings, which led to the availability of a large selection of columns on the market. Due to differences in stationary-phase production, almost none of the >600 RP-HPLC12−14 columns currently available on the market are identical. This signifies that it is indeed possible to choose the most appropriate system for a particular separation. The question remains how to achieve this. The properties of different chromatographic columns are affected by the differences in their chemical structure and their physicochemical properties such as the type of medium (monolithic, porous, or nonporous), geometry (area of the bed, diameter and pore volume, and particle size and shape), chemical properties of the bed (type of attached ligands and their density), and composition of the stationary-phase carrier (silica, polymers, or carbon). Chromatographic column packing material is usually a carrier of the actual immobilized stationary phase. However, in adsorption or NP-HPLC, the carrier itself may be the final stationary phase. The most common types of packings are comprised of fully porous spherical particles. Since the turn of the century, monolithic columns have gained popularity.182−184 An increase in popularity has also been noted for columns made of particles with a solid core surrounded by a porous layer.8

Figure 1. Scheme of the forms of silica pores (A, cylindrical; B, inkbottle-shaped; C, funnel-shaped) and the types of silanol groups. Adapted with permission from ref 187. Copyright 2012 John Wiley & Sons, Inc.

silanols are formed by hydrogen bonds between neighboring silicon atoms, whereas geminal silanols are bonded to the same silicon atom. Siloxane groups are formed by the condensation of vicinal and geminal silanol groups and comprise 20−35% of the total number of surface functional groups.190 It should be emphasized that the introduction of new silanol groups and the creation of additional siloxane moieties occur during the bonding of organosilanes, while extra silanol groups are created from the functional groups in bi- or trifunctional silanes that are susceptible to hydrolysis. Different types of silanols are responsible for various molecular interactions, i.e., hydrogen bonding, van der Waals forces, and London forces. The isolated and geminal silanols are the moieties with the greatest influence on the retention and form polar active centers. Unlike silanols, siloxanes are hydrophobic entities that cannot form donor−acceptor interactions.187,189 In silica supports, surface heterogeneity is often caused by structural heterogeneity. The macroscale physical properties of silica (i.e., the particle size, shape, and distribution) are well recognized.191−193 The microproperties of silica that include the specific surface area, mean pore diameter, specific pore volume, and pore size distribution play a significant role both in silica derivatization and in the chromatographic features of silicabased columns. Three types of pores are present in silica: macropores (diameter >50 nm), mesopores (2−50 nm), and micropores (diameter R−Cl > R−OH ≈ R−OCH3 > R−OC2H5 ≫ R− O−R. These differences depend on the basicity of the atom bonded directly to the silicon atom and thereby the hydrogen bond strength. When a stronger hydrogen bond is formed by a particular group, a higher reactivity and coverage density are observed. Furthermore, the bond lengths within the organosilane molecule, Si−O (0.174 nm), Si−Cl (0.183 nm), and Si− N (0.189 nm), also have an impact on hydrogen bond creation, which is not negligible. Hence, the observed coverage density for methoxysilanes can be similar to that of chlorosilanes despite the fact that the energy difference for the Si−O and Si−Cl bond formations is 71.2 kJ mol−1 and in favor of Si−O bond

Figure 3. Synthesis schemes of (A) monomeric and (B) polymeric structures of chemically bonded phases and (C) hydride silica, according to the silanization process and then subjected to catalytic hydrosilylation (D). Reaction free energies (ΔG298) were calculated with the M06-2X functional274 at the 6-311+G**275 and 6-31G*276 levels of theory.

formation.272 The formation of byproducts (such as HCl) also has an influence on the reaction yield, which shifts its equilibrium.15,273 In a mechanistic study of the C−H bond activation with cluster oxides, there are different reactivity scenarios for the thermal reactions of protonated [SiO2]+ with methane. The [OSi(OH)]+ cluster reacts with CH4, and two products are generated, i.e., [Si(OH)]+ and [Si(OCH3)]+. According to Shwarz et al., the mechanistic pathways of this reaction involve C−H bond activation in a σ-bond metathesis-like reaction, followed by the intramolecular migration of the methyl group to one of the hydroxide ligands. This intermediate can generate either methanol, with the formation of [Si(OH)]+, or [Si(OCH3)]+ species, accompanied by the loss of a water molecule. Therefore, these activation pathways lead to the formation of different products, which have a visible effect on the reaction yield, in particular, the surface coverage density.277 Silane coupling is a more commonly applied approach in hydride− silica synthesis. In contrast to the organosilanization process described above, the addition of a small moiety (namely, hydrogen) to the organosilane structure, instead of the bulky organic groups (e.g., octyl, octadecyl), results in maximum siloxane formation. Silicon hydride groups are formed during the silanization process (Figure 3C). Furthermore, the hydrosilylation process is aimed at bonding the organic moieties such as alkynes, olefins (nonterminal and terminal), cyano, etc. This process leads to the formation of direct Si−C bonds, which are especially preferred due to their higher hydrolytic stability over that of the other bonds formed during organosilanization.197,206 2.3.2.1. Stationary Phases for Reversed-Phase Liquid Chromatography. Currently, there are many columns on the market that are specialized for reversed-phase applications. Along with the particle type (porous, core−shell, monoliths, and polymeric), particle size, and column dimensions (which were G


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Figure 4. Various sample−column interactions that can affect selectivity (B, solute hydrogen bond acceptor group; BH+, protonated solute group; X, hydrogen bond acceptor group in the stationary phase; R, alkyl group; color figures, solute molecules; green circles, water molecules). Intermolecular interactions considered: electrostatic (hydrogen bond, dipole−dipole, ion−dipole, and ion−ion interactions), π−π interactions, dispersion, and hydrophobic interaction. See ref 285 for information about the nature of these intermolecular interactions.

Table 1. Overview of Select Stationary Phases Used in RP-HPLC Systems

a

Molecular structures are schematic drawings and do not reflect relevant conformations.

the higher the hydrophobicity of the stationary phase. Phenyland aryl-bonded stationary phases represent another essential group of columns for RP-HPLC. The derivatization of the phenyl ring and alkyl ligands via fluorine atom binding resulted in an independent group of chromatographic materials: fluorinated stationary phases. A specific feature of such beds is the ability to form π−π interactions with analytes capable of

described in the previous sections), the type of bonded ligands can be generalized among the manufacturers. The most common stationary phase is the octadecyl-bonded C18 column. Packings with the shorter alkyl chain C8 (octylsilane) are in second place among the mostly used reversed-phase columns.10 Generally, hydrophobic alkyl-bonded phases ranging from C1 to C30 are available.278 Naturally, the longer the aliphatic chain, H


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forming them. That, in turn, provides a different selectivity in comparison with that of the most popular aliphatic columns (Figure 4).10,279−284 Porous graphitic carbon is currently less popular among the HPLC users; however, it is commercially available and exhibits good ruggedness as well as compatibility with both the RP-HPLC and NP-HPLC modes. Cyano-bonded phases, which were initially dedicated solely to NP-HPLC or HILIC separations, are also commonly used for the RP-HPLC mode in aqueous solutions.10 The general structures of the commonly used and commercially available stationary phases as well as the corresponding analytical targets are presented in Table 1. Among the reversed-phase packings, chemically bonded stationary phases based on the unique hydride silica (type C silica) have also been used and commercialized.206 It should be emphasized that, under RP-HPLC conditions, pure silica hydride exhibits low hydrophobic selectivity and retention.286 In addition, silica hydride with chemically bonded cholesterol and bidentate C18 moieties is also available on the market.170 Stationary phases based on hydrides exhibited compatibility for the whole range of mobile-phase compositions, i.e., from pure aqueous to pure nonpolar organic solvents. Rapid equilibration of these separation materials enables a change in the chromatographic system from the RP to the aqueous normal phase (ANP). The separation of hydrophobic and hydrophilic analytes in isocratic conditions is, at times, possible during the simultaneous operation of both mechanisms.197,287 In addition to the diverse types of bonded alkyl ligands, the number of such ligands on the silica support is also essential. This quantity depends on the purity and morphology of the silica, type of organosilanes used, reaction activators, and secondary modification processes (e.g., “end-capping”). If there are typically ca. 8 μmol/m2 free silanols on the surface of the bed, then in a completed C18 phase, less than half the silanols are connected with modifiers. Naturally, the higher the degree of packings, the higher the hydrophobicity of the column, which leads to longer retention times of analytes. Most column manufacturers declare the percentage of carbon in the bed. This can be directly related to the degree of suppression of free silanols and the total hydrophobicity of the column. Highly dense packings (αRP = 4.2−4.3 μmol/m2, i.e., ∼2.6 molecules/nm2) were achieved by Szabó et al., who applied Cab-O-Sil gel and (N,N-dimethylamino)octadecylsilane.308 The influence of the type of activator on the coverage density of chemically bonded phases was investigated by Kinkel and Unger309 and Buszewski et al.310 The studies resulted in the possibility of preparing materials with “controlled coverages”. As this is not the topic of this review, interested readers are referred to a review of the preparation of chemically bonded phases with controlled coverage by Buszewski et al.311 It should also be noted that packings with around 2 times higher coverage density (a maximum coverage of ∼4.5 molecules/nm2) in comparison to Szabó’s highly dense packings were reported.308 These types of materials, the so-called self-assembled monolayers (SAMs), can be prepared by polymerization of trichlorosilanes and hydrated silicas, which occurs via selfassembly at the surface. SAMs however revealed poor masstransfer kinetics in the separation process due to the nearly complete surface modification. This difference in coverage affords the chromatography packings considerable conformational disorder and the ability for the analytes to partition into the bonded phase, whereas only adsorption is possible on SAMs.312−314

Among the available types of commercial columns, endcapped (or double-end-capped) phases are common. The endcapping process is based on the reaction of the residual silanols with a small, reactive hydrophobic or hydrophilic molecule (e.g., trimethylchlorosilane or amino- or hydroxyl-terminated silanes). Consequently, this reduces the number of residual silanols or blocks them and improves the surface coverage. Thus, the strong adsorption of basic compounds, which induces peak tailing, could be eliminated.161,315 The so-called polar embedded stationary phases are another group of materials used in RP-HPLC. This type of bed modification comprises a polar moiety (e.g., amide, urea, ether, sulfonamide, or carbamate) introduced between the support material and a bound aliphatic chain. This polar group is assumed to interact with the silanols on the surface of the solid matrix, thus lowering their activity. Such a phenomenon has a beneficial effect, particularly on the separation of polar and basic compounds. Another advantage of this bed type is the ability to work even when the mobile phase has a very low content of organic modifier.161,315 The combination of hydrophobic groups with ion-exchange moieties (cation-exchange (CEX) or anion-exchange (AEX)) within one column (so-called mixed-mode column) constitutes other alternative packings used in RP-HPLC and HILIC.299−301,316−320 Among this group of materials, RP/ CEX and RP/AEX bimodal materials, as well as RP/AEX/CEX trimodal materials, can be distinguished. The ligand arrangement can also be diversified into four categories of bimodal packings. Type I consists of mixed RP and AEX or CEX stationary phases in a single column. In type II, a modification of the support surface is carried out using a mixture of hydrophobic and ion-exchange silyl groups. However, the difference in hydrolytic stability of particular linkages induces a loss in selectivity. Types III and IV are obtained by applying silyl ligands, which contain both groups, in the synthesis procedure. Type III comprises an embedded ion-exchange group within the alkyl ligands, whereas type IV has this group at the end of the aliphatic chain. This results in different chromatographic properties for these two types of mixed-mode materials, particularly the RP-derived and ion-exchange (IEX)-derived materials. For trimodal columns, the retention mechanism is a combination of the phenomena occurring in RP, CEX, and AEX; therefore, they have a wider applicability than that of bimodal materials.299,320 In addition to the stationary phases commonly used in RPHPLC, which are widely offered by manufacturers, new materials have been developed to obtain more selective separations and tackle the challenges associated with the increase in the number of analytes and sample complexity.160 Biological systems became one of the inspirations. The most popular phase that imitates a biological system is the immobilized artificial membrane (IAM) phase, described and patented by Pidgeon and Venkataram.321 This type of packing is also commercially available. Monolayers of phospholipid analogues covalently bonded to the silica surface are known as Pidgeon’s materials. There were over 20 different IAM phases synthesized using the following phospholipids and their analogues as headgroups: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), and phosphatic acid (PA). Among the different structures of IAM phases, three types of acyl/alkyl− silica and headgroup linkages can be distinguished, i.e., ester and ether linkages, as well as phosphoester bonds, because of the I


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reviews on the preparation, chromatographic behavior, and application of ILs in HPLC.345−349 The separation of enantiomers constitutes a large research field in liquid chromatography using the so-called chiral stationary phases (CSPs). Among them, there are brush CSPs (low molecular mass or donor−receptor) in which stereoisomeric ligands are bound to silica gel, exploiting the phenomenon of charge transfer.350 Hyun and Pirkle350 developed three generations of brush-type CSPs and proposed the principle of “direct target design” for the design of their structure, exploiting the properties of a racemic structure.351,352 Lämmerhofer and Lindner353 worked out chiral stationary phases based on cinchona alkaloids, particularly quinine and quinidine. Such chiral selectors bind to the silica surface via the C9-carbamoyl group and have led to the molecular recognition of enantiomers at the C8/C9 stereogenic centers in the cinchona backbone. Among the polymeric CSPs, we distinguish polysaccharide CSPs,354,355 biopolymer CSPs (i.e., proteinbased materials),307,356 and synthesized polymer CSPs (including molecularly imprinted CSPs).357,358 Protein-based columns exhibit excellent enantioselectivity; however, their stability and column capacity are deficient.359 Molecularly imprinted CSPs show high specificity; furthermore, this feature is also observed when the structures of the analytes and the template are similar. 360 Macrocyclic materials are also used for the preparation of chiral stationary phases. The following groups of macrocyclic materials are commonly used: cyclodextrin,361,362 crown ethers,363,364 and macrocyclic antibiotics.365,366 The selectivity of cylodextrin CSPs is strongly related to the size of the bound macrocyclic molecules. If the molecule is either smaller or larger than the active cavity of the CSP, the selectivity is lost. Crown ether-bound CSPs exhibit a unique cavity which activates their selectivity for polar compounds. However, these materials have several drawbacks, most notably their expense and toxicity. Macrocyclic antibiotic CSPs constitute the most widely used materials due to their high capacity, stability, and versatility.360,367 As these materials comprise a wide group of chromatographic packings, interested readers are referred to reviews on modern chiral stationary phases (including commercially available materials) and their mechanisms.368−375 2.3.2.2. Stationary Phases for Hydrophilic Interaction Chromatography. In general, stationary phases for hydrophilic interaction chromatography (HILIC) can be classified into five groups: (i) pure silica, (ii) neutral hydrophilically bonded ligands, (iii) charged bonded moieties, (iv) zwitterionic materials, and (v) phases with mixed HILIC and RP ligands. Table 2 shows the structures and separation targets of stationary phases that are commonly used in the HILIC mode.376 Columns with unmodified silica provide better performance over time compared with those with chemically bonded stationary phases due to the absence of the “bleeding” process. Depending on the mobile-phase pH, the silanol groups constitute neutral (and polar) moieties or negatively charged groups, which can induce cation-exchange properties. Among the second group, stationary phases with chemically bonded amino, amide, diol, and cyano ligands are included. The charged bonded phases include phases such as charged amino columns, but poly(succinimide) and related materials are also a crucial group of HILIC columns. The stationary phases based on sulfoalkylbetaine and phosphorylcholine modifications comprise another group of widely used zwitterionic columns. Figure 5 depicts the schematic structures of the charge distribution in zwitterionic stationary

glycerol backbone removal. In addition, the IAM phases can also be differentiated according to the end-capping process using decanoyl (C10) and propionic acid (C3) groups. Analyses of these materials have shown that the drug−membrane interactions and permeability of IAM materials can be predicted. Furthermore, the prediction model is valid for passive transport domination in the overall transport process.322 Alongside the development and improvement of commercially available columns, research is being conducted within the scope of scientists. Liu et al.306 presented other types of IAM phases, i.e., materials containing a long-chain alcohol with polar hydroxyl groups or a long-chain fatty acid molecule with a methoxy moiety. These stationary phases allowed the estimation of drug permeability through a biological membrane and a rapid screening of the interactions between a drug and membrane.323 (Aminopropyl)silica modified with cholesterol molecules can also be included in the biologically inspired materials category. It exhibits unique properties, such as a relatively high hydrophobicity, chirality, and the ability to create a “hydrophilic pillow” on the silica surface, causing electrostatic shielding. The selective nature of the immobilized cholesterol molecule makes it applicable in both NP-HPLC and RP-HPLC.324−328 The calixarene-bonded silica gel stationary phases developed by Ding et al. are yet another example of novel packing materials.329 It was shown that the improvement in separation selectivity with use of the calixarene-phase column arises from supramolecular interactions, including π−π interactions, space steric hindrance, and hydrogen bond interactions.329 Liang’s group attached oligo(ethylene glycol) (OEG)330 and α-azido-L-phenylglycine dipeptide198 to a silica support via “click chemistry”. The OEG phase provided satisfactory separation selectivity, comparable to that of C18, for phenyl compounds and natural product samples.330 On the other hand, the “click dipeptide” phase and C18 presented orthogonality and a great separating power in an off-line 2D RP/RP-HPLC system for the separation of a traditional Chinese medicine (TCM), Rheum Palmatum L.198 Bound peptides were also an essential component of the internal surface reversed-phase (ISRP) columns introduced by Hagestam and Pinkerton.331 Peptide-bound ISRP packings were specifically developed for the separation of drugs in blood serum or plasma by direct injection.332 He at al.333 prepared a perhydro 26-membered hexaazamacrocyle-based silica (L1GlySil) stationary phase which exhibited multimode separation behavior. Simultaneous interactions (e.g., dipole−dipole and π−π interactions, acid−base equilibrium, etc.) allowed the application of this material in RP-HPLC and NP-HPLC. Li et al.334 also synthesized a polar embedded stationary phase by incorporating an s-triazine ring into the silica surface. As such, this column resulted in good peak shapes (asymmetry factor in the range of 1.04−1.39) of basic compounds and a high shape selectivity for polyaromatic hydrocarbons (PAHs) and compounds containing benzene rings.334 Ionic liquids (ILs) have also been applied as stationary phases in liquid chromatography.335−338 Initially, ILs were applied as mobile-phase additives to improve the separation of basic compounds in RP-HPLC.339,340 However, the covalent binding of ILs to the silica surface led to much attention on the usefulness of the new surface-confined ILs (SCILs). SCILs such as single-cationic and multicationic ILs, polymeric ILs, and chiral ILs were developed.338,341−344 Overall, IL-based chromatographic columns provide excellent separation of a wide range of analytes due to their multimodal retention mechanism (electrostatic, ionic, π−π, and dipole− dipole interactions). Interested readers are referred to many J


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structure. Depending on the organic modifier content in the mobile phase, the retention on these HILIC columns may be governed by RP-like and NP-like processes. Each of the described column groups has been commercialized and is widely used in the fields of pharmaceutical analysis, proteomics, metabolomics, glycomics, medicine, agriculture, and food chemistry. This is possible due to their properties, which vary depending on the type of chemically bonded ligands. A detailed description of the properties and applications of HILIC columns was presented by Jandera170 and Buszewski.172 HILIC is gaining popularity; therefore, the development and evaluation of novel stationary phases comprises a crucial aspect of scientific research.170,172 To present the developments and research directions in column evaluation, a brief description of novel stationary phases for HILIC applications is provided. Padivitage and Armstrong407 developed stationary phases comprised of silica-bonded sulfonated cyclofructan 6 (SCF6). The resultant columns have been successful in separating hydrophilic compounds, e.g., nucleic acid bases, maltooligosaccharides, water-soluble vitamins, and amino acids.407 In a subsequent study, Qiu et al.408 introduced a zwitterionic stationary phase which involved the bonding of diphenylphosphonium-propylsulfonate to silica. The authors noted that the newly developed material provided greater retention, higher peak efficiency, and better peak symmetry of nucleic bases, nucleosides, and water-soluble vitamins in the HILIC mode, compared with those of the commercial ZIC-HILIC column and bare silica gel. However, poor separation of salicylic acid and its analogues is an application constraint of this material with regard to the baseline separation on the conventional ZIC-HILIC column.408 Ray et al.409 developed a hydrophilic tripeptidebased Boc-Tyr-Ala-Tyr-OH organosilica hybrid stationary phase for the analysis of bioactive polar compounds under HILIC conditions. The same group of researchers prepared another peptide-based material with chemically bonded BocPhe-Aib-Phe-OH tripeptides. This material could be applied in both the RP-HPLC and HILIC modes.410 Li et al.411 demonstrated the differences between linear and cyclic tetrapeptide (Ala-Ala-Ala-Glu)-bound stationary phases. The results of their comparative study showed that cyclopeptidebased stationary phases exhibited size selectivity for PAHs and a weaker hydrophilicity with respect to that of linear peptidebased stationary phases.406Modification of the silica surface with a single layer of amino acids also allowed the preparation of zwitterionic stationary phases. Shen et al.412 prepared a cysteinebound stationary phase that was applied for the separation of oligosaccharides, peptides, nucleosides, basic compounds, and protein digests using HILIC−ESI-MS. Guo et al.413 prepared a “click lysine” zwitterionic stationary phase. Yin et al.414 prepared a tridentate zwitterionic stationary phase by binding (Nbenzylimino)diacetic acid to silica gel. The resultant material exhibited a high efficiency in the separation of both polar (e.g., organic acids and bases) and highly hydrophilic (e.g., cephalosporins and carbapenems) solutes.414 Cheng et al.200 prepared and characterized zwitterionic stationary phases in which the ratio of positively and negatively charged groups can be controlled. The molar ratios of the tertiary amine (trimethylamine) and carboxyl groups (2-((2-(trimethoxysilyl)ethyl)thio)acetic acid) can be chosen according to the assumed ratio of the oppositely charged moieties. The authors noted quite different selectivities and retention behaviors for various polar compounds (e.g., nucleosides and water-soluble vitamins) depending on the ratio of positively and negatively charged

Table 2. Overview of Select Stationary Phases Used in HILIC Systems

a Molecular structures are schematic drawings and do not reflect relevant conformations.

Figure 5. Model of charge distribution in zwitterionic stationary phases. Reprinted with permission from ref 200. Copyright 2015 Elsevier.

phases. Finally, the mixed HILIC/RP retention mechanism can provide the most polar stationary phases (except for bare silica) which have hydrophobic parts (such as hydrocarbons) in their K


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groups within a given stationary phase.200 Most recently, Hou et al.415 prepared and developed a fully pH-stable, positively charged HILIC porous graphitic carbon stationary phase. This material was prepared by creating a thin coating of poly(vinyl alcohol) (PVA) and poly(diallyldimethylammonium chloride) (DDAC) copolymer on porous graphitic carbon. The resultant column exhibited a higher pH stability (pH 2.1−12.7) and lower bleeding compared with those of silica-based packings.415 Interested readers are also referred to an excellent review on the most recent advances in stationary phases and retention mechanism recognition in HILIC systems, published by Jandera and Janás.171

their surface roughness. Nevertheless, there is still a lack of a complete library of NLDFT and QSDFT kernels due to the limited number of pore shapes (planar slit, cylindrical, and spherical), sorbate molecules (nitrogen, argon, and carbon dioxide), and materials (silicas and carbons) and, as a consequence, a limited continuous discovery of novel porous materials.423−425 Inverse size-exclusion chromatography (ISEC) is a method that is complementary to BET measurements. It enables the characterization of the interstitial and pore volumes of porous packings by the injection of a series of monodispersive species of known dimensions.426,427 2.4.2. Microscopic Imaging Techniques for Column Characterization. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) are the primary techniques for imaging microparticle systems. They can provide information about the elemental composition, structure, and charge or force measurements.428 Polymer-based and hybrid silica packings are the main types of stationary phases analyzed using microscopic imaging techniques. SEM images of hybrid silica monoliths are essential for optimizing their synthesis conditions.429 SEM and AFM can also be used for the evaluation of novel polymer adsorbents with a dendrimer structure.430 SEM is a powerful technique to investigate molecularly imprinted monolithic stationary phases for the separation of enantiomers and diastereomers.431 The high depth of field in SEM provides a three-dimensional visual representation. In TEM, staining with a heavy metal cocktail is needed for the visualization of organic matter comprised of only light elements. Among the family of scanning probe microscopies, AFM provides a subnanometer resolution. It is necessary to emphasize that although the height measurements are very accurate, the lateral dimensions are greatly overestimated.428 2.4.3. Thermal Analysis for Column Characterization. Elemental analysis (CHN) allows the accurate determination of the carbon, hydrogen, and nitrogen contents within the packing material. However, elemental contents alone do not provide complete information about the nature of the surface. Better information is provided by calculating the ligand coverage density according to the Berendsen and de Galan equation432 for monofunctional silanes:

2.4. Characterization of Chromatographic Columns

The structure of newly designed stationary phases must be strictly defined and experimentally determined before their implementation for HPLC analysis. There are, however, difficulties in the characterization of commercially available columns, because most of them are trade secrets, and complete information on the chemistry and structure is not shared with end-users. Therefore, of all the available tests, the ones based on chromatography are the most important for day-to-day endusers who do not have access to the instruments, expertise, or time for in-depth analyses of the stationary phases to be used for a particular analysis. Figure 6 depicts the most commonly used techniques for chromatographic column characterization.416

Figure 6. Visual representation of the most commonly used techniques for chromatography column characterization.

αRP (μmol m−2) =

2.4.1. Surface Topology Characterization. The surface and structural properties of chromatographic column packings are commonly determined using the low-temperature nitrogen adsorption (LTNA) method. The measurements can be modeled or complemented with the following theories: the Brunauer−Emmett−Teller (BET) theory,417 Barret−Joyner− Halenda (BJH) theory,418 and density functional theory (DFT),419,420 including the nonlocal (NLDFT)421 and quenched solid (QSDFT)422 density functional theories. However, the conventional BET and BJH models failed to determine the pore sizes and pore structure morphologies (particularly in the case of micropores). Additionally, the obtained results were affected by the type of the applied model and the method of micropore treatment in the data evaluation. DFT calculations overcome these limitations to some extent. Thereby, the NLDFT method has been applied for the characterization of micro- and mesopores of different materials, while the QSDFT method has also allowed for the description of

106PC 1 1200nC − PC(M1 − nx) SBET

(1)

where αRP is the coverage density (μmol m−2), PC is the percentage of carbon content (%), nC is the number of carbon atoms in the ligand, M1 is the molecular mass of the ligand, nx is the number of functional groups in the reactive group of the silane, and SBET is the specific surface area (m2 g−1). Elemental analysis is usually performed along with spectroscopic techniques (described in section 2.4.4). The surface coverage of chemically modified silica can also be determined using thermogravimetric analysis (TGA). Heating the chromatographic packings between 200 and 600 °C induces weight loss, which is attributed to the loss of organic groups attached to the surface. Therefore, TGA is an integral part in the characterization of chromatography column packings.342,433,434 TGA is frequently coupled with differential scanning calorimetry (DSC). This technique, in addition, enables the measurement of the heat adsorbed by the sample, as well as the corresponding specific heat capacity. DSC has been a longstanding part of the L


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Figure 7. Relationships between the natural logarithm of the capacity factor and 1/T (van’t Hoff plot) for (A) benzo[a]anthracene (empty shapes) and naphthacene (filled shapes) on monomeric and polymeric C18 and alkylamide phases, measured in a methanol/water (80/20 (v/v)) mobile phase, and (B) 4-hydroxybiphenyl on the polymeric alkylamide phases, measured in pure aqueous conditions. Adapted and enhanced with permission from refs 447 and 449 Copyright 2010 John Wiley & Sons and 1994 Friedrich Vieweg & Sohn Verlagsgesellschaft mbH, respectively.

temperature, the phase transition which may induce a change from adsorption-dominated to partition-dominated retention can be assumed.88,445−450 Therefore, it should be kept in mind that the stationary phase has the main effect on the thermodynamic processes occurring within a chromatography system, rather than the nature of both the solute and eluent, except for molecules with a tertiary structure, rotamers, or those having a temperature-dependent pKa. 2.4.4. Spectroscopic Techniques for Column Characterization. Spectroscopic techniques provide more detailed information about the structure, conformation, and dynamics of chromatographic column packings. They include cross-polarization magic-angle-spinning nuclear magnetic resonance (13C, 29 Si, 15N, and 31P CP/MAS NMR),451 Fourier-transform infrared (FTIR) spectroscopy,329 spectrofluorometry,452 and Raman spectroscopy.453 The 29Si CP/MAS NMR spectrum provides quantitative data about the distribution of particular silanols and siloxane groups and thus the surface coverage with organic ligands. Other modes of NMR measurements only allow the confirmation of organic ligand immobilization, depending on the type of heteroatoms incorporated into their structure.454 Currently, the use of spectroscopic techniques constitutes a prerequisite step of column characterization, and normally, at least two spectroscopic analyses are carried out.198 Chromatographic column packings can also be investigated using X-ray photoelectron spectroscopy (XPS) and static or time-of-flight secondary ion mass spectrometry (ToF-SIMS). A quantitative elemental analysis of a silica surface may be obtained from XPS measurements. XPS operates under conditions that provide an upper limit of the sampling depth of ∼10 nm. Static SIMS, on the other hand, yields surface information up to a depth of 1−3 nm and provides structural information related to the bound functional groups.455,456 Both variants of SIMS are complementary to XPS. XPS is a much more quantitative technique when compared with ToF-SIMS. Nevertheless, ToF-SIMS provides more chemical information. ToF-SIMS does not give chemical state information about elements other than the hydrogen atom, and XPS is not sensitive to hydrogen.457 In addition, these techniques allow for the characterization of the changes in the contents of various functional groups immobilized onto the silica surface, which are associated with the method of silica fabrication, thermal history, or surface treatment.455 2.4.5. Electromigration Methods for Column Characterization. The ζ potential is a useful parameter in the study of the charge distribution on the surface of chromatography column packings and is defined as the potential of the electric

HPLC-packing characterization process for a variety of chromatographic materials.435−438 Microcalorimetry is yet another method that can provide useful information about ligand conformation and their interactions with solvent molecules.439 Using it, the thermal effect which accompanies the stationary phase “wetting” with an organic solvent can be measured. Specific (hydrogen-bonding and polar interactions) and nonspecific (van der Waals forces and London forces) interactions generate heat, which can be directly measured. While each chromatographic solvent interacts with silica nonspecifically, methanol and acetonitrile can form specific polar interactions. Overall, the changes in the surface accessibility for interactions with solvent molecules cause changes in the heat of immersion.440,441 Microcalorimetric measurements also provide valuable information about the presence of residual silanols on the surface of chromatographic packings. Generally, the heat of solvent adsorption increases with an increase in the number of residual silanols. On the other hand, the number of residual silanols decreases with an increase in the coverage density of the packings. Therefore, microcalorimetric measurements can provide information about the polarity or silanol activity of the chemically bonded phases.442,443 Chromatographic column packings can also be characterized from a thermodynamic point of view. The changes in the retention mechanism or lack thereof over a particular temperature range can be inferred from the van ’t Hoff equation:444 ln ki =

V B ΔS° ΔH ° + ln s − = Ai + i R VM RT T

(2)

This expression describes the relationship among the free energy of transport (ΔG°), retention factor (k), and thermodynamic temperature (T). The parameters Ai and Bi represent the standard partial molar entropy (ΔS°) and standard partial molar enthalpy (−ΔH°) of transfer of the solute i from the mobile phase to the stationary phase, respectively. Parameter Ai is comprised of the ratio between the volumes of the stationary (VS) and mobile (VM) phases in the chromatography system. The enthalpic and entropic contributions to the retention and selectivity can be determined by plotting ln k versus T−1 over a sufficiently broad temperature range (Figure 7A). Moreover, this relationship enables the evaluation of the properties and conformational changes of bonded ligands as a function of the temperature and mobile-phase composition. The deviations from linearity of the ln k and reciprocal of temperature relation (Figure 7B) allow the estimation of the reordering/resolvation temperature of chemically bonded phases. At this onset M


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field formed on the solid−liquid interface. Chemically bonded stationary phases based on silica,458 polymer,459 and hydride silica460 supports exhibit nonzero ζ potential. The silica-based materials always contain residual silanol groups on the surface due to the steric effects of bonded ligands, which prevent their blockage or removal. These residual silanols are partially ionized under conditions where the contacting liquid splits off protons from them. Thus, under chromatographic conditions, a partial ionization of silanol groups always occurs. Silica-based materials possess, in such cases, some charges on the surface. The model of the electric double layer on the surface of chemically bonded stationary phases is presented in Figure 8. If caused by silica gel

allows for the characterization of the surface properties of ion exchangers obtained by chemical modification of organic polymers and silica gel.459 Kulsing et al.460 correlated ζ potential changes with the retention factors of basic compounds and polar peptides on silica hydride stationary phases under different mobile-phase conditions. Despite almost complete silanol group elimination, the silica hydride still exhibited a nonzero ζ potential. The authors highlighted that a knowledge of the ζ potential values of stationary phases allows a better rationalization of both the analyte retention and the impact of the ionic interaction descriptor (C values) in linear solvation energy relationships (LSERs). Therefore, ζ potential measurements can also provide an insight into the retention mechanism of the tested chromatographic packings.460,463,464 2.4.6. Chromatographic Methods for Column Characterization. The separation properties of chromatographic columns can be quantified in terms of equilibrium constants (K) or retention factors (k) for partitioning the analyte molecules between the stationary phase and the surrounding mobile phase. The values of k are proportional to those of K. The separation of two adjacent peaks within a chromatogram is determined by the ratio of the k values. This ratio is known as the separation factor, which is commonly referred to as the selectivity (α). The selectivity for a given pair of sample analytes depends on the mobile-phase composition, temperature, and nature of the stationary phase in the column. Despite the existence of comprehensive literature, a fundamental understanding of RP-HPLC column equilibria (as measured by the values of K and α) has been much less established than that of column kinetics.464 Bonded-phase columns for RP-HPLC have been widely used since their introduction in the early 1970s. As already mentioned, the behavior of these columns can be described in terms of their kinetics and equilibrium properties. The contribution of column kinetics to the chromatographic separation accounts for the width of the analyte peaks in a chromatogram. The column kinetics and peak widths can be characterized using the number of theoretical plates of a column (N). The dependence of the number of plates and peak widths on the experimental conditions such as the column length, particle size, flow rate, and other variables is often discussed.465 In the early 1970s, C18 columns from different sources were applied in RP-HPLC. These columns normally contained silica particles with attached octadecylsilyl groups. To prevent unacceptable differences in separation between different manufacturing procedures, the needs for column tests, ensuring repeatable separations, became obvious during the 1980s and 1990s. Those first tests helped improve column reproducibility. However, there was still a lack of a deeper understanding of the basis of intercolumn selectivity.464,466 The classical approach for the characterization of the RP-HPLC column selectivity assumed that the interactions of a sample molecule with the column packing are similar to the interactions in a solution (dispersion, dipole−dipole, and hydrogen-bonding interactions and so on).467 The concept and importance of various hydrophobic and hydrogen-bonding interactions between the sample components and column, known as LSERs, were often assumed at the time.468 However, to be useful in practice for the prediction of the separation and column selectivity, an accuracy of no less than ±3% for α is required. This makes the LSER approach of limited value in routine analysis.464 Subsequently, additional phenomena, namely, the ionic interactions of protonated bases with ionized silanols, as well as their ion pair

Figure 8. Model of the electric double layer on the surface of chemically bonded stationary phases. Adapted with permission from ref 460. Copyright 2014 Elsevier.

charges, the ζ potential appears at a particular distance from the surface. It is assumed that it is measured at the boundary between the liquid phase that can move either by the action of a pressure gradient or that of an outer electric field and stagnant liquid phase. The ζ potential of the stationary phase in a given solvent (or mixture) is calculated from the electrophoretic mobility μe using the Smoluchowski equation:461 μη ζ= e ε0εr (3) where η is the viscosity of the solution, εr the relative permittivity of the medium, and ε0 the permittivity of a vacuum. ζ potential measurements can be employed to better characterize the possible ionization of the residual silanols localized on specific stationary phases in different mobile phases during chromatographic analysis and their interaction with polar solutes.458 Buszewski et al.458 introduced ζ potential measurements as a new way of characterizing chromatographic packings. The authors studied octadecylsilica-based stationary phases with different coverage densities, with and without “end-capping”. The influence of these parameters on the ζ potential of the tested phases was evaluated. It was also noted that these investigations might be useful for selecting chemically bonded stationary phases for capillary electrochromatography (CEC). Recently, the same group of researchers investigated the effect of polar functional groups incorporated into the structure of chemically bound ligands. Materials with moieties capable of forming proton acceptor interactions (i.e., amine groups) exhibit, in contrast to those of typical hydrophobic adsorbents, positive values of the ζ potential.462 Additionally, this approach N


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second USP procedure, referred therein as the Product Quality Research Institute procedure (the PQRI provided support for the evaluation of the HS model). According to the USP Web site, the user is entitled to a comparison of the selectivity of two columns by two procedures. Two other recommended methods for comparing column selectivity are described herein: Advanced Chemistry Development (ACD, Toronto, Ontario, Canada) Laboratories Column Selector, which emerged out of the Euerby CSS (https://www.acdlabs.com), and the Katholieke Universiteit (Leuven, Belgium) Column Classification System.11,137 Various tests have been reported for chromatographic characterization of stationary phases. It could certainly be confusing for chromatographers to decide which procedure they should use. Nonetheless, the majority of column characterization procedures can be divided into (i) empirical methods, (ii) thermodynamically based methods, and (iii) model-based methods. The first group are empirical methods. The chromatographic information obtained using these methods depends on the arbitrarily chosen test analytes (chromatographic probes), assumed to reflect the specific properties of the columns, e.g., silanol activity. The methods developed by Walters,112 Tanaka et al.,107 Daldrup et al.,475 Eymann,476 and Engelhardt et al.28,31 belong to this group. In methods based on thermodynamics, assays are directed at the determination of the enthalpies and entropies of transfer of the analytes from the mobile phase to the stationary phase. Model-based methods are based on a specific model, such as the attenuation model of silanol activity by Nahum and Horváth,477 the model of solute−solvent complex formation by Jaroniec et al.,478 the model of interaction indicators by Jandera,479 the calculated solvation model by Galushko’s group,480,481 and models employing the quantitative relationships between the parameters of the chemical structure of the analytes and their chromatographic retention quantitative structure−retention relationships (QSRRs) which were proposed by the Abraham,482 Carr,483 Poole,484 and Kaliszan groups.485 Among the chromatographic approaches, an investigation of the solvation processes can also be applied as an effective way to characterize the surface properties of chromatography column packings. A selective distribution of eluent components occurs when a hydro-organic mobile phase is in contact with the stationary-phase surface and depends on its chemical properties. In HILIC, water molecules exhibit stronger adsorption than that of organic solvents. On the other hand, the favored excess adsorption of the hydrophobic component of the mobile phase onto the hydrophobic stationary-phase surface occurs in RP-HPLC.486,487 The following stationary-phase surface properties imply changes in the excess amount of adsorbed solvent: coverage density,486 length and number of organic ligands,488 and presence and type of polar functional groups within the ligand structure.489,490 These measurements can be carried out using various (static and dynamic) chromatographic methods. For these purposes, the following methods are employed: frontal analysis (FA),491,492 excess isotherm measurements,486,493 microcalorimetry,440 and computer simulation.494,495 Excess adsorption measurements are the most effective and popular method for solvent adsorption determination due to the unrestricted mobile-phase composition (0−100%), whereas FA cannot provide measurements across the whole concentration range of the mobile-phase modifier. Nevertheless, it is more commonly used for solute adsorption isotherm determination.496 Finally, molecular simulations constitute a novel approach in column character-

formation with counterions present in the solution, were included.469 The influence of analogous liquids such as octadecane, peak shape, and selectivity were studied as well.90 The use of simple chromatography probes and various mobile phases led to the evolution of chromatographic procedures considering the effect of the pH on the silanol activity. The resultant data appeared to be more relevant than the data from physicochemical measurements alone. Eventually, the chemical/ chromatographic column characterization scheme was combined with different chemometric tools (e.g., principal component analysis (PCA)470 or cluster analysis (CA)471) to visually categorize RP-HPLC columns. In the initial studies,472,473 the number of commercially available RP-HPLC products amounted to 79 packings and 85 columns. These systems have been characterized in terms of efficiency, hydrophobicity, steric selectivity, and hydrogen-bonding capacity, as well as in terms of ion-exchange capacity at both low and high pH. Data processing has been focused on PCA to provide a simple graphical comparison and visualization of the variation in column selectivity among different columns. Revealing the patterns in the retention of individual RP-HPLC columns appeared to be vital for the selection of the most appropriate stationary-phase materials possessing the required selectivity characteristics and for the reliability of the indicated column equivalence. Column differences are particularly important for the separation of geometric isomers. The separation of isomers often poses a considerable challenge because of the close similarities in chemical and physical properties, with the only distinctive difference being at the level of the molecular shape. The ability to separate and quantify individual isomers within a specific class of analytes can be of great significance, because biological processes are often highly dependent upon the molecular shape. Certain chromatographic packings (polymeric phases with chain length from C16 to C30) provide enhanced separations of shape-constrained isomers. These phases are referred to as “shape-selective”. Extensive research has been carried out on the role of the stationary phase, ligand density, chain length, and separation temperature on shape-selective separations. Through chromatographic tests, spectroscopic analyses, and molecular simulations, a comprehensive picture of the factors giving rise to the shape selectivity of stationary phases has started to emerge.474 The design of column-test procedures, which have been intended to account for various types of analyte−column interactions, and to characterize column selectivity, has been supported mostly by theoretical considerations, with a limited experimental confirmation that these tests actually measure specific analyte− column interactions. Furthermore, it has not been proven that some combination of such column tests is sufficient to fully describe the selectivity of different columns and to allow the reliable selection of an optimal column for a particular separation.464 The efficient selection of a matching column, based on the European Pharmacopoeia (EP) or United States Pharmacopeia (USP) monographs, is an important part of the analytical work in a pharmaceutical company, especially that concentrating on the production of generic pharmaceuticals. Obtaining a matching column via a trial-and-error approach can take up to several months, if one of the investigated columns is to perform as well as the one suggested in the pharmacopoeial monographs. Testing the entire column repository available in the company/institution would, therefore, be expensive, tedious, and time-consuming. The hydrophobic-subtraction model (HSM) (described in section 3.9.6) is considered the O


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Table 3. Exhaustive List of Analytes Used as Probes in Column Selection Systems

P


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Table 3. continued

Q


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Table 3. continued

R


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Table 3. continued

a

Abbreviations: D, Dolan; BK, Kaliszan; E, Engelhardt; EP, Euerby−Petersson; G, Galushko; K, Kulikov; KUL, Haghedoren−Hoogmartens; L, Lesselier; Ly, Layne; M, McCalley; MP, Mapihan; N, Neue; O, Organon; S, NIST (SRM 870); Sz, Szepesy; T, Tanaka; W, Walters.

need an elaborate uniform test protocol for an objective, rational, and reproducible column characterization and classification procedure.500

ization and provide useful information about the retention mechanism in liquid chromatography. The application of molecular simulations in RP-HPLC considered aspects is described in section 5. Although RP-HPLC has achieved practical maturity, column selection is still often at the trial-and-error stage. The same holds true for many of the currently used tests to characterize columns. This hampers the objective intercomparison of the still-growing number of columns. There is a need to develop column test protocols that reliably describe the chromatographic properties of columns. These protocols should be comprised of multiple tests of compounds and chromatographic conditions to provide the optimal column selection for specific application fields. In addition, the tests should be based on physicochemical principles that clearly reflect the diverse mutual interactions among analytes and mobile or stationary phases, as well as their respective components affecting the solvation of the bonded stationary phase.497 On the other hand, although it has been widely recognized that for ionizable analytes the manipulation of the pH affects the selectivity of a separation, continuous, programmed eluent pH changes (i.e., pH-gradient HPLC) are still insufficiently exploited in RP-HPLC method development.498 Furthermore, the influence of the temperature on the selectivity has been a popular subject of research.499 Overall, the physical properties of the matrix materials and the structural features of stationary phases for RP-HPLC are the deciding factors in determining the column efficiency and retention. Hence, for the synthesis of well-characterized and reproducible RP-HPLC stationary phases, these properties must be strictly controlled during the production. Unfortunately, until recently, none of the above evaluation methods have been commonly accepted as a uniform method for the assessment of RP-HPLC stationary phases. Furthermore, no actual consensus exists with respect to the type of test substances and experimental conditions, as well as the calculation procedures to be used in a given column test protocol. This lack of uniformity obviously hampers the objective comparison and classification of RPHPLC columns. Furthermore, severe constraints contributing to the CSS unification problem are associated with the given application areas where RP-HPLC columns are to be used and where analytes of highly diverse chemical nature are to be separated. In summary, it must be emphasized that the test methods for RP-HPLC columns using different test compounds, eluents, experimental conditions, and calculation procedures

3. CHROMATOGRAPHIC COLUMN SELECTION SYSTEMS The selection of columns for a particular RP-HPLC separation is not always clear-cut. Although many CSSs have been developed, there is not a single CSS that inherently accounts for the entire set of properties and complex solute−stationary-phase interactions. Therefore, the benefits that such evaluation systems provide are of great interest to chromatographers. Some of the first and most widely used CSSs were the tests named after Engelhardt,31 Tanaka,107 Sander and Wise,85 and Walters.112,113 Two column properties, hydrophobicity and silanol activity, were considered. On average, 20 silica-, alumina-, and polymerbased C8 and C18 columns were initially used in these early studies. The hydrophobicities evaluated using different CSSs were generally consistent and interchangeable. In contrast, the evaluated silanol activities considerably differed.501 Therefore, the selection of an appropriate chromatography column based on the silanol activity strongly depends on the CSS.502 CSSs which determine the silanol activity are usually operated in the isocratic elution mode. Gradient elution is often necessary for the separation of particular analytes. Nevertheless, isocratic conditions allow timely testing, which in turn reduces the time and required resources for column selection.501 Exemplified by widespread use, it can be confidently said that the RP is the most useful and rigorously studied mode of LC separation. Despite major advances and progress in the development of RP-HPLC columns, there is no ideal solution for their preparation. Microparticulate silica has been and is still being used for their synthesis, due to the properties which make this material very suitable for derivatization. Out of the many properties of silica (see section 2.3.1), three make it an attractive stationary-phase material: (i) easily controlled particle size, (ii) porosity, and (iii) mechanical stability. There are various types of surface silanols that have unique properties facilitating both derivatizations and adsorption interactions with analytes. Their distribution considerably influences the characteristics of chromatographic columns based on silica, more than that of the absolute number of silanol groups on the surface. Essentially, any analytical technique (thoroughly discussed in section 2.4; electron microscopy, microcalorimetry, S


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thermogravimetry, spectroscopy, ζ potentiometry, to name a few) can be used for the characterization of prepared stationary phases for RP-HPLC separations. Although these instruments can be found in well-equipped analytical laboratories, in practice, for the selection of a suitable column, most of them are inappropriate. Furthermore, there is no universal agreement on how to measure the number of silanols accessible for interaction with the analyzed compounds. Toward that goal, several chromatography CSSs have been developed. However, they are predominantly focused on the structure of the molecules used as chromatography probes (exhaustively summarized in Table 3). When selecting an appropriate column, one must pay close attention to the results of individual tests, which can often exhibit diverse interactions, even if performed on the same column. That being said, in this section, we critically review and describe chromatographic tests as systems with an aim to ease column selection for day-to-day users of chromatography.

as simple and easily predictable as was initially thought. The affinity of the columns is, in part, governed by the bonded moieties themselves, while the silica carrier plays a large role as well. There are often inconsistencies or even contradictory conclusions in discussions on the selectivity of RP-HPLC bonded phases. To remedy this, pure hydrocarbons and aliphatic alcohols were used to discriminate the hydrophobic selectivities of the investigated RP-HPLC columns, and one of the first parameters for their selection, the relative retention of hexane/nonane (k′hexane/nonane), was introduced. Assuming a constant surface coverage, it increases with an increase in the chain length of the bonded alkylsilanes. This was tested on two C5 and C6 columns (Figure 9), both of which exhibited nearly

3.1. Engelhardt Column Selection System

What is today known as the Engelhardt test or column selection system (CSS) is essentially a set of chromatographic procedures which allow the classification of stationary phases according to their ability to separate neutral and basic analytes.31 Prior to its inception, comprehensive experimental studies directed at the optimization of RP-HPLC systems and characterization of chemically bonded RP-HPLC columns were performed. 3.1.1. Experimental Characterization of Chemically Bonded RP-HPLC Columns. The influence of the eluent and column composition on the retention times of organic analytes separated in the RP mode was described as early as 1976, also accounting for peak tailing.503 Ten mixtures common in routine chromatographic analysis were separated on two silica columns, with chemically bonded C4 and C18 groups. Observations were made on the basis of the polarity of the sample, and one of the first insights into the RP-HPLC mechanism was given. It was shown that the more polar the analyte within a sample, the earlier it is eluted, while dispersive or London forces were thought to be the main driving force of the separation process. The evaluated columns were compared using relative retention times of a variety of analytes with respect to the retention time of methanol. Interestingly, the relative retention times were generally higher for the C18 chemically bonded phases than those of their C4 counterparts. The relative retention factors, on the other hand, were shown to have depended greatly on the eluent composition. Homologous series of analytes exhibited a linear increase in the relative retention time for linear eluent programming. We have noted that RP-HPLC columns with chemically bonded alkyl groups could not have been, at that time, considered the best for chromatographic analysis. However, their largest benefit stems from the fact that the water−solid equilibrium is achieved considerably faster in the RP mode, whereas upon increasing the water content in the eluent, the peak shapes are mostly unaffected, exhibiting tailing for analytes that are not well soluble in the eluent used. On the other hand, the introduction of small polar or nonpolar groups into a large sample considerably influenced the retention times of the analytes. Building upon the results of this optimization study, Engelhardt and Ahr20 thoroughly reviewed the properties of chemically bonded phases, with a focus on C18 columns, in a subsequent work.29 From the point of view of column selection, it has already been considered that the RP-HPLC system is not

Figure 9. Chromatograms depicting analyses on (A) a C5 RP-HPLC column (green color) and (B) a C6 RP-HPLC column (red color). A 7/ 3 (w/w) methanol/H2O mixture was used as the mobile phase. Key: (1) inert analyte, (2) n-hexane, (3) cyclooctane, (4) n-nonane; and (5) cyclohexylcyclohexane. Reprinted and ehanced with permission from ref 20. Copyright 1981 Springer International Publishing AG.

equivalent selectivities for both n-aliphatic and cycloaliphatic alcohols. On the other hand, when compared to that of its cyclohexyl counterpart, C6 columns have shown differences in selectivity, with the former being more selective toward cycloaliphatic rather than n-aliphatic alcohols. With an increase in the number of alkyl groups substituted with cyclohexanol, these differences are less discernible. Apart from the chemistry of the bonded phases, the water content of the eluent has been shown to be another crucial parameter affecting the selectivity of an RP-HPLC column. At high water concentrations, the stationary phase is no longer “wetted” by the eluent, resulting in a decrease in the differences in selectivity across columns. Column selection is, therefore, heavily influenced by properties such as the type and length of the bonded alkyl group only if the water content is below 50% (w/w). Berendsen et al.432 and Krstulović et al.44 showed this in their extensive studies of the changes in the slopes for columns with alkyls of higher lengths (∼12−14 C atoms). Engelhardt and Ahr20 also clearly showed this with an example of neighboring members of a homologous series on RP columns, with increasing length of the bonded alkyl group (i.e., C4 to C18). The linear dependence between log k′ and the number of carbons has been studied. The slopes of the corresponding curves increased with the length of the bonded alkyl group when methanol was used as the eluent. Although the water content in the eluent strongly affects the slopes of log k versus the number of carbon atoms in the solute, when water is used as the sole T


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This essentially blocks the silanol groups from interacting with basic analytes. Another aspect to consider in column selection is the pH value of the silica surface, which generally has a slightly acidic characteristic. When preparing the stationary phases, they are usually washed to neutrality. For separation of basic or acidic compounds, retaining a slightly acidic or basic character is an alternative strategy to optimize the preparation process. With an increase in the surface acidity and basicity, the peak symmetry of the basic and acidic compounds, respectively, decreases. In practice, the choice of an optimal column is strongly dependent on the encountered separation problem. Columns with the highest surface coverage are better when high retentions are needed. For the separation of basic analytes, “end-capping” has an antithetical effect, as seen from a considerable difference in k′N‑methylaniline/benzene. A lower water content in the eluent is recommended for the separation of linear hydrocarbons and polar analytes, whereas for the separation of basic analytes, a medium water content is optimal. 3.1.2. Retention Test as an RP-HPLC Column Selection System. The suitability of a chromatographic column depends not only on the chemical characteristics, but also on the testing conditions (eluent and test analytes). It can, therefore, be proven that an evaluated column is suitable, but only for a particular separation. The initial work of Engelhart et al.29,30,503 resulted in this discouraging conclusion. Further investigations were, therefore, aimed at designing a more general test/system for column selection. As a result, a variety of empirical CSSs were developed. The Engelhardt CSS31 uses two hydrophobic analytes (toluene and ethylbenzene), a weak acid (phenol), and weak bases (aniline and toluidine isomers) (Table 3, compounds 2, 3, 6, 7, 11, 14, and 20; Table 4). A methanol/water ratio of 49:51

eluent, the differences have been indistinguishable. The phenomenon occurs because the analytes interact with the whole length of the column if the bonded alkyl groups are fully solvated by the eluent. Using water as the eluent makes these interactions less favorable than the interaction between the alkyl groups themselves. Consequently, a hydrocarbon film forms on the silica surface, and the total porosity of the column decreases. When selecting an appropriate column, RP phases with shorter alkyl groups can be used to decrease the analysis time; however, if the solubility of the analytes is quite low, as it often is, C18 phases are preferred. The fraction of the organic-phase modifier can be considerably higher for shorter alkyl groups while achieving a virtually identical analysis time. It was challenging to compare RP phases bonded with silanes comprised of variable functional groups. The surface coverage varied across several reaction batches, and there was also considerable hydrophobic selectivity, on top of the desired selectivity that was to be achieved by the introduction of a specific functional group.29 The evaluation and characterization of C18 columns29 was performed through not only the analysis of linear hydrocarbons, but also that of polar and basic hydrocarbons. While the relative retention of linear hydrocarbons is a good indicator of the differences in hydrophobicity between the columns, the retention of polar and basic analytes provides information on the differences in the accessible silanol groups and silanol concentration. The effect of “end-capping”, which does not affect the separation of hydrocarbons such as benzene, toluene, or ethylbenzene, can then be clearly demonstrated, whereby the largest differences were observed, e.g., in the analysis of aniline. The relative retention times of polar (k′toluene/benzene and k′ t o l u e n e / e t h y l b e n z e n e ) and basic (k′ a n i l i n e / b e n z e n e and k′N‑methylaniline/benzene) analytes were therefore promising column selection parameters. The characteristics of the C18 phases and the effect of the silanol concentration and water content were evaluated on three columns, both with (C18,1,tr, C18,2,tr, C18,3,tr) and without (C18,3,untr) “end-capping”. If not subjected to “endcapping”, the third C18 column (C18,3,untr) exhibits a very large deviation in the value of k′aniline/benzene. The values of k′toluene/benzene slightly increase with an increase in the water content in the eluent for all the columns, while the values of k′aniline/benzene and k′N‑methylaniline/benzene are constant for C18,1,tr and C18,2,tr over the whole range of water concentrations. The third C18 column if subjected to “end-capping” (C18,3,tr) shows a virtually identical behavior at lower and higher eluent water contents. For a medium concentration of water in the eluent (∼34.6% (w/w)), the “end-capped” C18 column C18,3,tr exhibits the largest deviations of k′N‑methylaniline/benzene compared with those of the other two columns. Although the first two (C18,1,tr and C18,2,tr) phases have shown to be equivalent, for higher eluent water concentrations, their chemistries are actually distinct. These C18 phases need to be evaluated not only over a range of water content, but also with different eluent mixtures. For the separation of basic solutes, as exemplified by the analysis of N-methylaniline, the lack of ion-exchange interactions between the surface silanols and the amine contributes to peak tailing and a greater asymmetry.29,30 At lower water content (less than 10% (w/w)), silanol groups do not dissociate, making them unable to interact with the amine. Eluents with higher water content (more than 50% (w/w)) cause “dewetting” of the stationary phase. Under these conditions, a hydrocarbon film forms, since dispersive interactions between the alkyl groups of the stationary phase become stronger than those with the eluent.

Table 4. Parameters of the Engelhardt CSS

a

k refers to the retention factor, while As refers to the asymmetry factor. All other abbreviations are explained in the text.

(v/v) or 55:45 (v/v) for the mobile phase and a temperature of 25 °C (±0.3 °C) were found to be the optimal test conditions. Initially, the mobile phase was nonbuffered to prevent the buffer components from interacting with the surface silanols. Subsequent applications of the Engelhardt CSS achieved better reproducibility using a 1−40 mM phosphate buffer at a pH of 7.504,505 The retention factor for toluene (ktoluene) can be related to the amount of carbon in the bed (carbon loading). The ratio U


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of the retention factors of toluene and ethylbenzene (ktoluene/ kethylbenzene) is a measure of the hydrophobic selectivity of the stationary phase. Three isomers of toluidine have, naturally, the same hydrophobicity but differ in pKa. Therefore, these isomers are not separated when the effect of residual silanol is negligible. Because of their carcinogenic properties, it was suggested to replace toluidines with p-ethylaniline.196 The original Engelhardt test was to determine the carbon loading, hydrophobicity, and silanol activity with a mixture of test analytes composed of uracil, phenol, aniline, p-toluidine, m-toluidine, o-toluidine, N,N-dimethylaniline, ethyl benzoate, toluene, and ethylbenzene. For stationary phases with a low silanol activity, the following were observed: a poor separation of the toluidines, phenol eluting after aniline, a ratio of the asymmetry factors of aniline and phenol of less than 1.3, and asymmetry factors of the basic analytes between 0.9 and 1.6. Schmitz et al.32 used the Engelhardt test to classify 26 reversed stationary phases (seven C8, eleven C18, and eight polymercoated phases). A high correlation was observed among the retention factors of both highly retained analytes (N,Ndimethylaniline, ethyl benzoate, toluene, and ethylbenzene) and poorly retained test analytes (aniline, phenol, and the toluidines). On commercial C18 stationary phases, ethyl benzoate eluted before toluene. To the contrary, ethyl benzoate eluted after toluene on commercial C8 phases. On both C18 and C8 phases, ethyl benzoate eluted close to toluene, and N,Ndimethylaniline eluted before both toluene and ethyl benzoate. On the other hand, on the polymer-coated phases, ethyl benzoate eluted before N,N-dimethylaniline. Only small variations were noted in the retention factors of toluene and p-toluidine and in the ratios of the asymmetry factors of aniline and phenol.502 Some of the test analytes in the Engelhardt test have subsequently been changed.28 The substitution of toluidines with p-ethylaniline and the use of the asymmetry factor of p-ethylaniline alone, to assess the silanol activity, were introduced. Stationary phases producing asymmetry factors of less than 2 were classified as those with low silanol activity, and the stationary phases with asymmetry factors of 2−5 were classified as possessing intermediate silanol activity. The stationary phases with asymmetry factors above 5 were classified as phases of high silanol activity.

Table 5. Parameters of the Tanaka CSS

a k refers to the retention factor, while As refers to the asymmetry factor. All other abbreviations are explained in the text.

described properties, four eluent compositions and four analyte mixtures were required.502 More precisely, in the Tanaka CSS, hydrophobic effects were accounted for with the retention factor of pentylbenzene (kpentylbenzene), while methylene selectivity (αCH2) was obtained using the ratio of k values between pentylbenzene and butylbenzene (kpentylbenzene/kbutylbenzene). The shape selectivity was indicated by the ratio of k values between triphenylene and o-terphenyl (ktriphenylene/ko‑terphenyl). All the mentioned analytes have similar hydrophobic properties but differ in molecular shape. Triphenylene possesses a rigid structure and a single conformation, whereas the conformations of o-terphenyl can considerably differ. The silanol activity is quantified through two physicochemical properties of the stationary phase: the ability to form hydrogen bonds and the ion-exchange capacity. Hydrogen bond formation is modeled as the ratio of the retention factors of caffeine (able to form hydrogen bonds with free silanols) and phenol in a nonbuffered 30:70 (v/v) methanol/water mobile phase (kcaffeine/kphenol). The ion-exchange capacity is calculated as the ratio of the retention factors of benzylamine (which is highly basic: pKa < 9) and phenol (weakly acidic: pKa value of 10) (kbenzylamine/kphenol). For its determination, a mobile phase comprised of 30:70 (v/v) methanol/phosphate buffer was used. The concentration of the phosphate buffer was adjusted to set the pH at two values: 7.6 and 2.7. These parameters were visualized using radar plots for the columns with in-house-prepared packing materials as well as commercial stationary phases. Thus, the characteristics of the stationary phases were related to the corresponding methods of synthesis. Figure 10 depicts three distinct cases where the stationary phases were prepared using monochlorosilane, dichlorosilane, and trichlorosilane. With an increase in the values of axes A, B, and C, the hydrocarbon retention, hydrophobicity, and steric selectivity increase. On the other hand, for a decrease in the values of axes D, E, and F, the number of accessible silanols or ion exchange sites also decreases. Commercial columns characterized in the same manner exhibited similar trends.107 3.2.1. Layne Column Selection System. The Layne CSS506 is based on the Tanaka CSS.107 A similar buffered mobile phase for all the tests, comprised of 20 mM potassium phosphate (KH2PO4−K2HPO4) buffer, was used. Different compositions

3.2. Tanaka Column Selection System

Drawing inspiration from the Engelhardt CSS, Tanaka et al. developed one of the first systematic procedures for chromatography column selection (Table 3, compounds 4−6, 12, 19, 20, 24, and 25; Table 5).107 The optimal chromatographic conditions were methanol/water ratios of 80:20 (v/v) and 30:70 (v/v) as the mobile phase, buffered with a 0.02 M phosphate buffer or nonbuffered, with the temperature kept at 30 °C. Beyond the assessment of the stationary-phase surface coverage and hydrophobic properties (ratios of retention and selectivity between pentylbenzene and butylbenzene, k′pentylbenzene/butylbenzene, αpentylbenzene/butylbenzene), the Tanaka CSS also accounted for the shape selectivity (ratio of the retention of triphenylene as “bent” molecules to that of the plane of oterphenyl). In addition, it included an evaluation of the ability to interact through hydrogen bonds (which was represented through the relative retention factor of caffeine and phenol), as well as the ion-exchange capacity of the stationary phase at pH 7.6, when the majority of the residual silanols are dissociated, and at pH 2.7, when only a few very acidic silanols can participate in the retention. Therefore, to determine all the V


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Table 6. Parameters of the Layne CSSa

Figure 10. Visualization of the Tanaka column selection system (CSS) using radar plots. Empty circles represent C18 phases with maximum coverage, full circles represent C18 phases with maximum coverage subjected to “end-capping”, and upward-facing triangles represent C18 phases with medium coverage subjected to “end-capping”. Axes A to F represent parameters of the Tanaka CSS, and their values are denoted in (A). Other plots correspond to columns with packing materials synthesized from (B) monochlorosilane, (C) dichlorosilane, and (D) trichlorosilane. Adapted and enhanced with permission from ref 107. Copyright 1989 Oxford Academic Press.

a

All abbreviations are explained in the text.

from 26 to 8. The other parameters were the relative retention time of pentylbenzene (kPB), representing the number of alkyl chains, the selectivity factor between amylbenzene and butylbenzene (αCH2), representing the hydrophobicity, the selectivity factor between caffeine and phenol (αC/P), accounting for the hydrogen bond capacity, the selectivity factor between triphenylene and o-terphenyl (αT/O), related to the steric selectivity, and the selectivity factors between benzylamine and phenol at pH values of 2.7 and 7.6 (αB/P,pH2.7 and αB/P,pH7.6), accounting for the ion-exchange capacity at pH < 3 and pH > 7, respectively. The procedure was initially tested by characterizing 30 commercial chromatographic columns. The probes 2,7- and 2,3-dihydroxynaphthalene were subsequently removed because they were not sensitive to low concentrations of metal impurities and strongly depended on the column history.118 The number of theoretical plates per meter of pentylbenzene (NPB) was introduced instead.473 The correlation matrix, PCA,470 cluster analysis,471 and radar plots were used to evaluate this sevenparameter CSS. The differences among the columns were clearly revealed using PCA score plots. Euerby et al.473 extended the number of studied columns to 85 and stationary phases to 135 in a subsequent study.34 PCA analysis resulted in seven clusters of columns, and it was shown that the NPB was highly correlated with its retention factor (kPB).473 It has been removed from the CSS as redundant. The final version of the CSS, therefore, comprises six parameters consistent with the parameters of the earlier Tanaka CSS (Table 7).107 These final six parameters were reduced to a single factor, named the column difference factor (CDF),35 which represents the Euclidian distance between column i and an a priori selected reference column:

of acetonitrile/buffer were used to evaluate different physicochemical properties of each investigated column (Table 6). All the columns were maintained at a temperature of 30 °C. Small changes in the organic modifier compositions were introduced. The methylene selectivity was determined by the retention of a series of alkylbenzene homologues: from benzene to pentylbenzene (Table 3, compounds 1−5, 16, 30, 32, 40, 41, 54, 55, and 66−70; Table 6). The conventional, polar embedded, and polar end-capped RP-HPLC stationary phases showed marked differences in their chromatographic behavior. From the point of view of column selection, the Layne CSS has been shown to be advantageous in the selection of polar embedded phases for the separation of basic and acidic analytes. The retention behavior of these analytes exhibits ionic and dipole interactions with the stationary phases in question and is readily captured.506 3.3. Euerby Column Selection System

Euerby and Petersson extensively studied and rationally modified the Tanaka CSS.33−35,473,507−509 With time, their efforts grew into an independent system, for which they proposed 26 compounds as chromatographic probes (Table 3, compounds 2, 4−6, 10, 12, 24, 25, 30−33, and 54−67). Further development33 of the Euerby CSS involved the introduction of two additional chromatographic probes, 2,7-dihydroxynaphthalene, to represent the relative number of theoretical plates, and 2,3-dihydroxynaphthalene, to account for the presence of metal impurities, and a reduction of the total number of parameters W


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Table 7. Parameters of the Euerby CSSa

mechanisms of test analytes (Table 3, compounds 1, 2, 10, 15, 20, and 23; Table 8).112 The eluent was acetonitrile/water, Table 8. Parameters of the Walters CSSa

a


a

All abbreviations are explained in the text. 2

65:35 (v/v), and the temperature of the test was 40 °C. Hydrophobic and silanophilic interactions were taken into account because those interactions depend on the hydrocarbon coverage and amount of unreacted silanol sites, respectively. The hydrophobic interactions were evaluated using the ratio of the retention factor of anthracene to that of benzene (kanthracene/ kbenzene). The amount of unreacted silanols was assumed to be reflected by the ratio of the retention factor of N,N-diethyl-mtoluamide (DETA) to that of anthracene (kN,N‑diethyl‑m‑toluamide/ kanthracene). The column efficiency, which largely depends on the type and particle size of the packing material, was included as a third “classification” criterion in the test. Twelve brands of C18 packings were subjected to the procedure and classified into three major groups on the basis of three criteria: hydrophobicity index, free silanol index, and efficiency. The scheme was introduced to aid the selection of similar (or equivalent) columns and the choice of column type suitable for a given analytical method. The third criterion, the efficiency of columns, was examined by applying three mobile phases: mobile phase A, containing acetonitrile, mobile phase B, which included nheptane, and mobile phase C, which consisted of acetonitrile and water in the ratio 65:35 (v/v). Three solutions of test analytes were comprised of DETA and anthracene (1), nitrobenzene and benzene (2), and uracil, benzene, toluene, and anthracene (3). Two test procedures were developed for the determination of residual silanols. The first (and preferred) method was based on the measurements of the retention factor ratio of DETA and anthracene. This was because the retention of DETA is sensitive to the silanol activity, whereas the retention of anthracene is claimed to be determined only by hydrophobic interactions. However, the retention of anthracene can also be affected by the molecular shape of the analytes. The test then shows a combined effect of the two interactions. A test for residual silanols constituted the measurements of the retention factor of nitrobenzene after 50 mL of acetonitrile, methylene chloride, and each mobile phase was passed in sequence through the column. This second test of silanols was finally discarded because of the timely equilibration and conditioning requirements for the column. However, a

2

CDF = [(kPB, i − kPB,ref ) + (αCH2, i − αCH2,ref )

+ (αC/P, i − αC/P,ref )2 + (αT/O, i − αT/O,ref )2 + (αB/P,pH2.7, i − αB/P,pH2.7,ref )2 + (αB/P,pH7.6, i − αB/P,pH7.6,ref )2 ]1/2

(4)

The results of this comprehensive study have shown which phases are suitable for use in mobile phases with a high water content. It was further shown that perfluorophenyl and perfluoroalkyl stationary phases formed distinct clusters in PCA owing to the basic analytes exhibiting an unusually extended retention on perfluoro phases.34 The results of the hydrophobicity tests appeared to be very sensitive to the nearly negligible changes in methanol content. Therefore, controlling the methanol content was found to be especially important.508 Finally, in 2005, Euerby et al.509 compared their CSS to three others: the Neue CSS, the Layne CSS, and the original Tanaka CSS. Columns with embedded polar groups, and amines which altered the deactivation of the residual surface silanols, were used in the study. The robustness of the Euerby CSS was confirmed using reduced factorial design (RFD), multiple linear regression (MLR),510 and PCA. Eighteen phases, including 17 alkyl phases (some of which contained novel polar end-capping, i.e., amino), were grouped using PCA. The similarities/ dissimilarities among the stationary phases and their corresponding C-alkyl amino end-capped phases of enhanced polar selectivity were revealed.509 Twenty-one commercially available phenyl-type RP-HPLC silica-based packing materials have also been characterized using the Euerby CSS and PCA.35 PCA analysis revealed that columns were clustered according to their selectivities (hydrophobic, shape, and aromatic, i.e., π−π, interactions), as well as hydrogen-bonding and ion-exchange capacities. 3.4. Walters Column Selection System

Walters proposed an approach to classifying C18 columns on the basis of two predominant reversed-phase retention X


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trile/water eluents could only be achieved for low-pKa bases, because of the variable ionization effects. The buffered aqueous acetonitrile mobile phases produced the worst peak shapes for the nine studied higher pKa bases.512 The performance of eight silica-based RP-HPLC columns was once again evaluated with mostly the same solutes (Table 3, compounds 12 and 36−42; Table 10).

correlation has been found between the results of the two methods.23,501 3.5. Kulikov Column Selection System

In the Kulikov CSS, 11 stationary phases were characterized using 15 test compounds511 (Table 3, compounds 2, 3, 6, 7, 11, 21, 23, and 28−35; Table 9). Two micellar mobile phases were Table 9. Parameters of the Kulikov CSS

Table 10. Parameters of the McCalley CSSa

a

nnaphthalene represents the number of theoretical plates calculated using the classical equation based on the retention of naphthalene. All other abbreviations are explained in the text.

used. One was at pH 7.0 (0.075 mol/L sodium dodecyl sulfate and 1.5% (v/v) 1-pentanol) and the other at pH 2.7 (0.075 mol/ L sodium dodecyl sulfate and 1.5% (v/v) 1-pentanol); the pH was adjusted with trifluoroacetic acid. Sodium nitrate was used for determination of the void time for each evaluated column. The operating temperature was 40 ± 0.1 °C. The naphthalene retention factors were correlated to the hydrophobicities of the test analytes. All the other features, i.e., hydrophobic selectivity, methylene selectivity, hydrophilicity, steric selectivity, hydrogen-bonding capacity, ion-exchange capacity, and metal impurity, were taken as ratios of the retention data for the test compounds. Unfortunately, these selectivity factors appeared to exhibit strong mutual correlation and are therefore redundant. Up to three orthogonal abstract factors are sufficient to substitute the nine initial ratios, accounting for 87% of the total data variance. 3.6. McCalley Column Selection System

The performance of eight silica-based RP-HPLC columns was determined using methanol-, acetonitrile-, and tetrahydrofurancontaining mobile phases in combination with aqueous solutions of phosphate buffer at pH 7.0.512 The following basic analytes were chosen for the test: pyridine, nicotine, amphetamine, codeine, diphenhydramine, nortriptyline, procainamide, quinine, and 2-[N-methyl-N-(2-pyridyl)amino]ethanol (PAE) (Table 3, compounds 36−44). The retention coefficient (k′), asymmetry factor (As), and number of theoretical plates (N calculated from the peak widths at half-height, Ndf calculated from the peak widths at 10% of the peak height at the rear and front sides of the peak) were calculated. The practical evaluation of the columns with unbuffered methanol/water and acetoni-

a


The mobile phase was buffered at pH 3.0. The differences in column performance were considerably lower at acidic pH. Tetrahydrofuran showed a significantly better performance than that of acetonitrile or methanol. The ranking of columns according to the average peak asymmetry factor of the set of basic analytes varied to some extent with the pH. All the analytes except pyridine exhibited superior performance at acidic pH.513 Y


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3.7. Organon Column Selection System

columns and to simplify the quality control procedure for RPHPLC−MS analysis.515

In this test, 14 commercially available RP-HPLC stationary phases were compared with respect to the retention of five basic pharmaceutical compounds (Table 3, compounds 45−49; Table 11) synthesized at N.V. Organon (Oss, The Nether-

3.9. Quantitative Structure−Retention Relationships in Chromatographic Column Selection

QSRRs appeared in the early 1970s as an innovative approach for the prediction of chromatographic retention from the molecular structure. Pioneered by Kaliszan,124,516−518 they have since found numerous applications in all types of chromatography for the description of retention mechanisms, optimization of chromatographic methods, prediction of elution order, and column selection, to name a few. QSRRs themselves have been thoroughly described in the literature, and interested readers are referred to two books516,518 and several reviews.124,517,519 Under the scope of this paper, only QSRRs related to column selection are reviewed. First, an overview of linear free energy relationships (LFERs),520 upon which most QSRR-based CSSs were built, is given. It is followed by descriptions of the Szepesy CSS,521 Galushko CSS,480,481,522 Kaliszan CSS,124 and hydrophobic subtraction model (HSM) of Snyder and Dolan.138,140,159,464,523−527 Eventually, the Kaliszan CSS and HSM became standalone and are being widely used for column selection. In QSRR-based column selection systems, the coefficients of the developed models are used to rank the columns, while their parameters are used to evaluate the correlations and causal relationships between the parameters and chromatographic columns. 3.9.1. Linear Free Energy Relationships. One of the earliest QSRR models was introduced by Abraham’s group.520 Solvophobic parameters146,468,528−531 derived from equilibrium measurements were used to predict the chromatographic retention factors (log k) using the following functional relationship:

Table 11. Parameters of the Organon CSS

a

Detailed information about the mobile-phase composition has not been reported by Vervoort et al.514 All abbreviations are explained in the text.

lands).514 The effect of silanol blocking agents on the analyte retention and peak shape was investigated using phosphate buffers at two pH values: 3 and 7. The asymmetry (As), plate height (HETP), and retention coefficients (k′) were determined for each stationary phase. PCA was employed for analysis, and the stationary phases were organized into several groups. The phases clearly differed in their suitability for the analysis of basic compounds. It must be noted here that the addition of N,N-dimethyloctylamine (a known free silanol suppressor) to the mobile phase, buffered at pH 3, considerably improved the peak shapes, biasing the column selection system in the process.340

log k = c + rR 2 + sπ2* + a ∑ α2 H + b ∑ β2 H + vVx (5)

3.8. Dolan Column Selection System

where R2 represents the excess molar refraction, π2* the dipolarity/polarizability, ∑α2H the hydrogen bond donor acidity, ∑β2H the hydrogen bond acceptor basicity, and Vx the characteristic McGowan volume. The resulting model was termed LFERs (Table 12). The regression coefficients in the above equation are determined by multiple regression analyses and are assumed to characterize the stationary-phase/mobilephase system investigated. Hence, r is a measure of the propensity of the stationary-phase π-electron pairs to interact with analyte n, s accounts for the stationary-phase dipolarity/ polarizability, a is a measure of the stationary-phase basicity, b is a measure of the stationary-phase acidity, and v is a measure of the stationary-phase hydrophobicity or the ability to participate in dispersion (London) interactions. 3.9.2. Szepesy Column Selection System. Sándi and Szepesy521 studied 15 different RP-HPLC columns using 34 test analytes (Table 3, compounds 2, 3, 6, 7, 10, 11, 14, 31, 33, 36, 58, 59, 63, and 71−93). They compared the information gained from PCA of the retention data and from the solvation parameters of the Abraham equation (LFERs) (Table 13).133 Although the abstract factors in PCA, called principal components, do not have explicit physical meaning, the absolute and relative values of their so-called loadings provide information on the type and extent of intermolecular interactions cooperating on the various stationary phases. The regression coefficients of eq 5 reflect the differences in the individual intermolecular interactions between the components

Within the Dolan CSS, a kit of four compounds, namely, aspartame, cortisone, reserpine, and dioctyl phthalate, termed the LC−MS performance test mixture (LPTM), are recommended as representative analytes, suitable for RP-HPLC−MS method development and column selection (Table 12). The Table 12. Parameters of the Dolan CSSa

a


procedure is carried out by analyzing the retention times (tR) and order of the analytes. The mobile phase is comprised of solvent A (0.05% formic acid in water (v/v)) and solvent B (0.05% formic acid in acetonitrile (v/v)). Gradient elution was used with different gradient programs. The analyses were carried out at a temperature of 40 °C. It has been claimed that the LPTM suffices for the performance evaluation of RP-HPLC Z


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Table 13. Parameters of the LFERs Modela

Accordingly, the surface layer of the stationary phase was assumed to be a quasi-liquid with its own characteristics, such as surface tension and dielectric constant. The retention can therefore be determined from the difference in analyte molecule solvation energies between the mobile phase and surface layer. To calculate these solvation energies, a molecule is assumed to consist of volume (“bulkiness”) fragments and dipoles, each of which separately interacts with the surrounding medium. In the system, the test analytes are uracil, aniline, phenol, benzene, and toluene. The hydrophobic (methylene) selectivity parameter is calculated from the phenol, toluene, and benzene retention data. The silanol activity is obtained from the relation of aniline and phenol retention, and the size selectivity is calculated from the retention data of benzene, phenol, and toluene.480,481,522 This approach was reported to be successful for the calculation of the retention coefficients of diverse aromatic compounds on C18 columns, eluted with water/organic mobile phases.522 3.9.4. Kaliszan Column Selection System. QSRRs124 are statistically derived relationships between chromatographic retention data and features (descriptors) encoding the structure (or physical properties) of the analytes. Among notable applications, QSRRs are used to describe the mechanisms of the intermolecular interactions of analytes with compounds of chromatographic systems, identify the structural features of analytes that contribute the most to separation, and facilitate chromatographic column selection. Reliable QSRRs can be used for predicting the retention of new analytes in each stationary/ mobile-phase system. The most commonly used retention parameter is the logarithm of the retention factor (log k) or its extrapolation to a pure water mobile phase (log kw), as a kind of normalization allowing a better comparison of the properties of hydrophobic analytes. The simplest approach in QSRR is to regress the retention factors against the theoretically calculated log Koctanol/water (clogP) values.127,534,535

a


of the stationary and mobile phases. If the same mobile phase is used with different columns, the regression coefficients can be applied to characterize the individual stationary phases. LFER theory is believed to provide a rational description of the role and extent of the different molecular interactions operating in a given RP-HPLC system. Within the Szepesy CSS, the authors investigated the effect of different mobile-phase compositions in the range of 20−70% (v/v) organic modifiers (acetonitrile and methanol). Their results showed that the mobile-phase composition has a considerable (relative) effect on the individual molecular interactions.521 Hydrophobic selectivity is not identical to the hydrophobic strength of the column, and its values naturally depend on the structure and polarity of the homologous series used for calculation. The polar selectivity for different types of analytes depends on the propensity of the stationary phase to get involved in the polar (mainly hydrogen bond donor and hydrogen bond acceptor) interactions.532 The difference in hydrogen bond donor acidity between the stationary and mobile phases (coefficient b in eq 5) is highly correlated with the difference in hydrophobicity (v coefficients).533 3.9.3. Galushko Column Selection System. The Galushko CSS is another empirical test based on solvophobic theory (LFERs) designed to evaluate retention in RP-HPLC from the molecular structure of analytes and the characteristics of both the stationary and mobile phases.480,481,522 Five compounds were used as chromatography probes (Table 3, compounds 1, 2, 6, 11, and 20; Table 14). The mobile phase employed for the test was methanol/water in a ratio of 60:40 (v/ v), and the operating temperature was 30 °C. To calculate the retention and selectivity, a two-layer continuum model of a chromatographic system was employed.

log k w = k1 + k 2(clogP)

(6)

Molecular modeling allows for the differentiation of chromatographic stationary phases in terms of the chemical properties of the ligands and matrix. Reliable and physically interpretable results are ensured when using the following structural descriptors of analytes: the total dipole moment (μ), describing the dipole−dipole interactions and dipole−induced dipole interactions between the analyte and the molecules of the mobile and stationary phases; the largest negative excess atomic charge in the molecule (δmin), specifying the local polarity or submolecular polarity of the analyte and hence the ability to participate in polar interactions; and the molecular area of analytes available for contact with the solvent of the mobile phase (AWAS), characterizing the strength of the dispersive interactions (London type) of the analyte with the molecules of the chromatographic phases.126,536 Such a QSRR model describes equations of the following form:

Table 14. Parameters of the Galushko CSSa

log k w = k′1 + k′2 μ + k′3 δmin + k′4 AWAS

(7)

where k′1, k′2, k′3, and k′4 are regression coefficients.124 Since the 1970s, QSRRs have been occasionally used for the comparison of various RP-HPLC columns.126,485,540−544 The QSRR recommended for the objective characterization and comparison of columns is derived for a set of 15 carefully selected analytes (Table 3, compounds 1, 21, 28, 77, 83, and 95−104; Table 15). To determine the retention of these analytes, the following chromatographic conditions have been used. The mobile phase consisted of methanol and 100 mM Tris

a

All abbreviations are explained in the text. AA


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Table 15. Parameters of the General Kaliszan CSSa

HPLC system.126 This allowed the classification of modern chromatographic packings according to the type and strength of the intermolecular interactions that affect the retention of the test analytes. 3.9.5. Application of the Kaliszan Column Selection System in Proteomics. Specific QSRR models have been reported for the rational selection of chromatographic columns in the RP-HPLC analysis of complex proteomic mixtures. Michel et al.125 evaluated 17 physicochemically diverse RPHPLC columns (Figure 11) for the analysis of proteomic mixtures. Twenty-five synthetic peptides were used as chromatography probes, ranging from just a few amino acids (e.g., AF, GM, and WF) to longer peptides (e.g., LPPGPAVVDLTEKLEGQGG-NH2). The chromatographic conditions for the analysis of the peptides were as follows. Gradient elution was carried out with solvents A (water with 0.1% trifluoroacetic acid (TFA)) and B (acetonitrile with 0.1% TFA). In a gradient time of 20 min, the gradient was formed from 0 to 60% solvent B. The measurements were performed at 40 °C, with an eluent flow rate of 1 mL/min and a sample volume of 10 μL. Prior to analysis, the peptide samples were dissolved in water with 0.1% TFA. The Kaliszan CSS described

a


buffer of pH 2.5 and 7.2 to suppress the dissociation of individual analytes. Gradient elution was carried out using 5− 100% methanol with gradient times of 10 and 30 min to compute the log kw values. The measurements were performed at 35 °C with a flow rate of 1 mL/min and sample volume of 20 μL. Using such derived QSRRs, it is possible to discuss the molecular mechanism of separation operating in a given RP-

Figure 11. Molecular structures of some of the stationary phases evaluated for the separation of peptides. Reprinted with permission from ref 125. Copyright 2007 Elsevier. AB


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in the previous subsection (section 3.9.4.) was modified using structural descriptors tailored to the unique properties of peptides such as their amino acid sequence and hydrophobicity, among others (Table 16). Table 16. Parameters of the Kaliszan CSS Applied in Proteomicsa

Figure 12. PCA score plot of 17 RP-HPLC columns for the first two principal components constructed out of the coefficients of the peptide QSRR model obtained through MLR. Analysis was performed using data obtained from ref 125. Numbers represent the following chromatographic columns: (1) XTerra, (2) LiChrospher RP-18, (3) LiChrospher CN, (4) Discovery RP-Amide C16, (5) Discovery HS F54, (6) Chromolith, (7) PRP, (8) Macrosphere 300 C4 5 μm, (9) Macrosphere 300 C8 5 μm, (10) RP-18e Purospher Star, (11) ProntoSIL 200-5-C30, (12) SG-CHOL, (13) SG-Ar, (14) Nucleosil CN, (15) Ascentis RP-Amide, (16) SG-Ph, and (17) HILIC. Dashed and solid red lines represent 95% and 99% confidence ellipses, respectively.

a


three silanol groups are replaced with a methyl group, whereas Chromolith is monolithic). 3.9.6. Hydrophobic Subtraction Model for Column Selection. Another QSRR-based chromatography CSS, the HSM, proposed by Snyder, Dolan, and co-workers has been described in a series of publications.137−140,159,464,524,525,527,548,549 The concept of the HSM137,464 originated from the previously described LFERs, to which it is complementary.137 Because of its fundamental nature, it seems to be suitable for a wide range of applications concerning the evaluation of column selectivity. Some of the applications were reported for the first time in 2004 and 2010, e.g., (i) selection of similar columns as backups for routine assays, (ii) selection of dissimilar columns for a change in separation during the method development, including the development of orthogonal separations to ensure that no “new” peaks appear, and to perform two-dimensional separation, (iii) avoiding columns that are more likely to produce tailing peaks mostly for bases, but occasionally for acids as well, (iv) development of new stationary phases with unique selectivity, (v) prediction of column “dewetting”, (vi) improved control of column manufacturing, (vii) better understanding of stationaryphase degradation in routine use, (viii) “slow” column equilibration for ionized solutes, (ix) deeper insights into the nature of reversed-phase retention and column selectivity, as well as factors determining the selectivity, and (x) quantitative prediction of separations for different columns, with the possibility of choosing the “best” column for a given sample.11,137 On the basis of the retention of the analytes in different test mixtures (Table 17), obtained under identical separation conditions (50% acetonitrile/buffer, pH 2.8 and 7.0, 35 °C), the selectivity of each chromatographic column can be characterized with six parameters:464 the relative retention factor of ethylbenzene (kethylbenzene), hydrophobicity (H), spatial selectivity (S*), hydrogen bond acidity (A) and basicity (B), and the degree of silanol ionization or cation-exchange capacity (C) (Table 18).

These structural descriptors were obtained from optimized peptide geometries and simple gradient RP-HPLC analyses. The following equation was obtained:546 t R = b0 + b1 log sumAA + b2 log VDWvol + b3(clogP) (8)

where log sumAA represents the sum of the gradient retention times of individual amino acids comprising the analyzed peptide, log VDWvol the van der Waals volume, and clogP545 the calculated 1-octanol/water coefficient. The coefficients of the QSRR models for each of the 17 stationary phases were determined using multiple linear eegression (MLR)510 and found to be statistically significant and physically meaningful. Superior QSRR models were obtained for both monolithic and regular octadecylsilica stationary phases.125 PCA470 was used on the obtained coefficients to describe the similarities and dissimilarities of the 17 evaluated RP-HPLC columns. It is worth noting that the authors have also replaced the log kw parameter with the retention time of the peak apex (tR). In a recent study, it was shown that QSRR models built using tR as the dependent variable are considerably more reliable and unbiased.547 For the purposes of this review, the calculations were repeated using OriginPro 2017 (OriginLab Corp., Northampton, MA), and the results were generally similar to those presented in ref 125. Two significant components were extracted, accounting for 97.58% of the variance in the QSRR coefficients. Figure 12 depicts the score plot for the first two principal components of the obtained PCA model. Eight columns are grouped into one cluster (columns with numbers 8−15), which indicates their close similarity. All of them were comprised of octadecyl-bonded stationary phases. Others were localized further away, with a few pairs of columns grouped together. Although these are also comprised of stationary phases prepared using octadecylsilica, they differ in their packing (i.e., in the XTerra column, every AC


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Table 17. Analytes Used as Chromatographic Probes in the Snyder−Dolan Column Selection System

AD


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Table 17. continued

a

Compounds 1−6, 10, 17, 21, 31, 62, 71, 74, 75, 77, and 98 detailed in Table 3 are not repeated here.

the high repeatability, and probably best physicochemical description of the separation-determining interactions, the Snyder and Dolan CSS still exhibits a high degree of redundancy. In contrast to the previously described simpler selection systems, in which only a few chromatographic probes are used, in HSM, for each chromatographic column, a staggering 67 compounds have to be analyzed. This makes the HSM-based CSS complex and inherently time-consuming, which might drive end-users away from its use for column selection. The authors managed to subsequently decrease the number of test compounds to 18, decreasing its redundancy.550 The HSM model has been comprehensively tested with >300 chromatographic columns, and on the basis of the results of multiple tests on a subset of 42 C8 and C18 columns, a column comparison function parameter, Fs, was proposed, which allows the pairwise evaluation of equivalent columns or the ranking of the investigated columns with respect to a reference.140 The Fs metric for two columns (subscripts 1 and 2) is defined in the following manner:137,464

Table 18. Parameters of the Hydrophobic Subtraction Modela

a


Quantitatively, the relationship between the column selectivity and the aforementioned experimental descriptors is as follows: log α ≡ log

k = η′H − σS* + βA + αB + κ′C kEB

(9)

H, S*, A, B, and C were determined in four independent laboratories, and consistent results were reported. Regardless of AE


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Marchand et al.527 correlated the values of the Fs metric with the SD values for a variety of type B C18, cyano, fluoro, and phenyl columns (Figure 13). Except for the fluoro columns, the

Fs = {[wH (H2 − H1)]2 + [wS *(S2* − S1*)]2 +[wA(A 2 − A1*)]2 + [wB(B2 − B1)]2 + [wC2,B(C2 − C1)]2 }1/2

(10)

where H1 and H2 correspond to the H values of columns 1 and 2, respectively, and the parameters S*, A, B, and C correspond similarly. Weighting factors wH, wS*, wA, wB, and wC2,B represent the weighting factor for the difference in hydrophobicity, steric interactions, hydrogen bond acidity, hydrogen bond basicity, and charge interactions at pH 2.8, respectively. Initially, the values of the weighting factors were 12.5, 100, 30, 143, and 83. Presently, the weighting factors can be easily adjusted at the Web site http://www.hplccolumns.org in case one wants to give different relative importance to a certain parameter. For Fs values of 3, but this is not certain. If it is known that the sample does not contain ionized components, in particular, basic analytes, the parameter C2 − C1 can be omitted, with the consequence of lower values of Fs for the two compared columns. If carboxylic acids (either ionized or nonionized) are not present in the sample, the parameter B2 − B1 can also be omitted, similarly reducing the value of Fs. If the eligible columns are definitely of different selectivities, two columns with the greatest value of Fs should be selected. The Web site of the U.S. Pharmacopoeia551 offers instructions to end-users on how to identify the appropriate column according to the HSM. Furthermore, extensive experimental studies resulted in formation of a database which comprises 588 RPHPLC columns with HSM parameter values. Snyder et al. commercialized this database, and it has since become a major segment of the DryLab software (Molnar Institute, Berlin, Germany) for the optimization of chromatographic separations. Eventually, the database was extended by Dwight Stoll in 2017 with parameters for ∼700 chromatographic columns and is freely available online at the aforementioned Web site: http:// www.hplccolumns.org/. The Fs metric originally derived for alkylsilica columns of type B with ligands C1−C30 was extended to columns with built-in polar moieties such as urea, carbamate, and amide, as well as chromatographic columns with a reduced number of residual silanols,139 cyano columns,526 and fluoro-substituted and phenyl columns.527 Apart from Fs, another metric, the standard error (deviation) of the linear fit of the HSM (SD),525 was also introduced to discern the columns. It was defined as follows:

Figure 13. Comparison of two column selection metrics: the standard deviation (SD), correlating log k values for two columns, and the column comparison function Fs, computed from the H, S*, A, B, and C values for each column. Evaluated chromatographic columns include (A) type B alkylsilica columns, (B) cyano columns, (C) fluoro columns, and (D) phenyl columns. Adapted and enhanced with permission from ref 527. Copyright 2005 Elsevier.

other types exhibit a strong correlation between the two metrics. Larger deviations for the fluoro columns suggest either that their retention mechanism is not fully accounted for by the HSM model or that the parameters are simply not accurate enough. Therefore, from the point of view of column selection, the values of H, S*, A, B, and C are not sufficiently reliable for the selection of RP-HPLC fluoro chromatographic columns. Finally, in a recent application of the HSM model, Johnson et al.552 compared >500 RP-HPLC columns using the system selectivity cube (SSC; now can also be calculated at http://www. hplccolumns.org) concept.552,553 The use of the SSC has allowed for the depiction of numerous differences in selectivity, even among columns within the same class (such as alkylsilica and cyano columns or those with embedded polar groups). The suitability of the method has been shown for the selection of similar alternative columns, as well as orthogonal columns for particular separations. Figure 14 depicts an SSC (from two angles, panels A and B) for the selection of columns orthogonal to the observed column Symmetry C18. It is constructed out of the intercept, slope, and R2 values for the correlations of the log k (obtained using HSM) values of an observed column (in this case, Symmetry C18) and the columns in question (in this case, Xterra C8 RP, Thermo CN, Bondoclone C18, Discovery HS-F5, and Zirchrom EZ). The retention factors of the Xterra C8 and Bondoclone C18 are more strongly correlated with that of Symmetry C18 than anticipated from their chromatograms (not shown). The authors note that this showed that the solutes must take advantage of the characteristics distinguishing one phase from the other to maximize the column potentials and thereby offer different selectivities. This does not seem to be the case with the solutes of these two columns. They are indeed not truly

SD = −0.006 − 0.001|H − Hb| + 0.030|S* − S*b | + 0.041|A − Ab| + 0.311|B − B b| + 0.010|C − C b| (11)

where Hb, S*b, Ab, Bb, and Cb represent the average values of H, S*, A, B, and C for type B alkylsilica columns used to derive the HSM solute parameters. The basis of this metric is the reduced accuracy of the HSM model equation due to its approximate nature, and not because additional analyte−stationary-phase interactions are not accounted for. Therefore, the SD of the linear fit and the absolute differences between the values of the HSM parameters of each evaluated column and their averages exhibit a strong correlation. AF


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Figure 14. Two views of the SSC for the Symmetry C18 column compared with other stationary phase types. Adapted and enhanced with permission from ref 552. Copyright 2012 Elsevier.

orthogonal. On the other hand, when compared with the retention factor of the Symmetry C18 column, those of the Zirchrom EZ and Discovery HS-F5 columns were shown to be considerably different. There is a weak correlation between them, indicating their orthogonality. Despite its wide applicability and possibly the best physicochemical description of the retention mechanism in RP-HPLC that it provides, the HSM CSS suffers from a serious limitation. As noted in a recent discussion by Shackman,554 the raw experimental data of HSM typically have limited verification. The HSM parameters and the Fs metric itself may exhibit substantial deviations for an identical column at different periods of time. Besides the typical RP column information (e.g., type of silica, specific surface area, carbon load), it is thereby crucial to also include the column production date. Unfortunately, this problem is not a characteristic of only the HSM CSS. Therefore, for a reliable and objective column selection, unless modeling the so-called “usage drift”, only new (

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