Journal of Chromatographic Science Advance Access published September 18, 2014 Journal of Chromatographic Science 2014;1– 18 doi:10.1093/chromsci/bmu090

Review

Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases for Liquid Chromatography Endler M. Borges* Nu´cleo Biotecnolo´gico, Universidade do Oeste de Santa Catarina, Rua Paese, 198, Bairro Universita´rio—Bloco K. Videira, SC CEP 89560–000, Brazil *Author to whom correspondence should be addressed. Email: [email protected] Received 23 January 2014; revised 7 June 2014

Free silanols on the surface of silica are the “villains”, which are responsible for detrimental interactions of those compounds and the stationary phase (i.e., bad peak shape, low efficiency) as well as low thermal and chemical stability. For these reasons, we began this review describing new silica and hybrid silica stationary phases, which have reduced and/or shielded silanols. At present, in liquid chromatography for the majority of analyses, reversed-phase liquid chromatography is the separation mode of choice. However, the needs for increased selectivity and increased retention of hydrophilic bases have substantially increased the interest in hydrophilic interaction chromatography (HILIC). Therefore, stationary phases and this mode of separation are discussed. Then, non-silica stationary phases (i.e., zirconium oxide, titanium oxide, alumina and porous graphitized carbon), which afford increased thermal and chemical stability and also selectivity different from those obtained with silica and hybrid silica, are discussed. In addition, the use of these materials in HILIC is also reviewed. Introduction The analysis of basic compounds by liquid chromatography (LC) continues to be of interest, as over 70% of pharmaceuticals are bases (1), while peptides and proteins, which have several basic and acid functions, are an emerging class of therapeutic agents currently being developed by many pharmaceutical companies (2 – 4). Thus, all the discussions are devoted to ionizable compounds. Silica is the most used material to prepare stationary phases. However, free silanols on the surface of silica are the “villains”, which are responsible for detrimental interactions of those compounds and the stationary phase (i.e., bad peak shape, low efficiency). For these reasons, several new silica and hybrid silica stationary phases, which have reduced and/or shielded silanols, are introduced in the market. In addition, the most common mode of LC is reversed-phase liquid chromatography (RPLC), which poorly retains polar compounds. Hydrophilic interaction liquid chromatography (HILIC), which uses a polar stationary phase and reversed-phase solvents, has proved to be an effective approach to retain hydrophilic bases (5 –8). In this context, Type C stationary phases are a very promising material for HILIC, which have a negligible amount of free silanols, and non-silica stationary phases afford complementary selectivity to silica and hybrid silica stationary phases. Based on the criteria discussed above, this review is divided into three parts: (i) silanol interaction, in which approaches used to reduce the amount of free silanols on the surface of silica are reviewed; (ii) Type C silica and HILIC, in which some applications of this mode and advantages of it over the reversed-phase # Crown copyright 2014.

mode are discussed and (iii) non-silica stationary phases, in which the uses of these materials are discussed as an alternative to silica and hybrid silica stationary phases and HILIC applications using non-silica stationary phases are also included. This review is focused on commercial stationary phases that most of readers could use to achieve their goals. There are many “interesting” stationary phases developed by research groups over the world, which are not commercially available. Interested readers can find a description of many of them in the Qiu et al. review (9). The Chromatographic Support LC has undergone unprecedented advancement in technology within the last decade. Modern LC as we know it today with dedicated instrumentation and widespread commercial application began in the mid-1960s to 1970s (1). From 1975 to 2000, the industry had exclusively used fully porous 5 –10 mm particles in the high-performance liquid chromatography (HPLC) column for most applications (10). Those stationary phases were spherical silica particles, which had organic groups bonded to its surface. These stationary phases were prepared by the reaction of silanes with silanol groups on the silica surface. The silanes most commonly used are based on the following template: XSi(R1)2R2, wherein X is typically a chloro or alkoxy, the R1 groups are small alkyl chains (i.e., methyl) while the R2 group is a long alkyl chain, typically such as an octyl (C8) or octadecyl (C18) group (11). Following the development of adsorbents in LC over four decades, the major action was to reduce the average particle diameter to sub-2 mm particles to enhance the column efficiency, to gain faster analysis and to achieve higher mass sensitivity (12), and the use of smaller particles to shorten the analytes’ diffusion path is a well-known approach to provide improved separation efficiencies (13). This is evident from the simplified van Deemter equation (equation (1)), which describes the relation between efficiency (expressed as the height equivalent to a theoretical plate, H), linear velocity (m) and particle size (dp), in which l is a packing constant, g is an obstruction factor for diffusion in a packed bed, Dm is the diffusion coefficient of analyte in the mobile phase and f(k) is a function of the retention factor (k). The dependance of C-term, which is considered to mainly represent the resistance to mass transfer in the mobile phase, is directly proportional to the square of the particle size. Thus, a decrease in particle diameter results into a large decrease in the plate height, especially at high linear velocities (14). H ¼ 2l d p þ

dp2 2gDm B þ f ðkÞ m ¼ A þ þ C m: m Dm m

ð1Þ

The position of the minimum on the height equivalent to the theoretical plate curve, and the optimum linear velocity, can be determined by the use of differential calculus. The optimum linear velocity occurs when the slope of the H versus u curve is zero, i.e., when dH/dm ¼ 0. This condition is satisfied in equation (2) (15).

mopt

rffiffiffiffi B ¼ C

ð2Þ

In 2004, the Waters Corp. (Milford, MA, USA) presented the first LC equipment, which is able to work with reduced sub-2 mm particles (15), it was named ultra-performance liquid chromatography (UPLC) and later generally accepted as ultra-highperformance liquid chromatography (UHPLC) and propagated as a “high-throughput” method (12), while LC using particles with diameter 3 mm was named HPLC. The price one had to pay was a high pressure (16). UHPLC afforded a higher efficiency and reduced analyses time compared with HPLC, and method transfer between UHPLC and LC is simple with several tools available in the literature (17– 20) such as method transfer between 5 mm and sub-2 mm stationary phases could be achieved easily using commercial modeling software. For example, Korma´ny et al. (21) had shown the possibility to automatically transfer RPLC methods between different column dimensions and instruments using DryLab 4.1 software. The main drawbacks in UHPLC were the higher inlet pressure, which is needed to pass a mobile phase through columns packed with very fine particles, as it is proportional to d 2p according to the Darcy’s law, and even to d 3p when working under optimal linear velocity conditions (22). According to equation (2), the use of smaller particles implies in a higher mopt, and according to Darcy’s law, the flow rate (or m) are directly proportional to the back pressure. Nova´kova´ et al. (23) had illustrated through several examples how method transfer from HPLC to UHPLC result in decreased analyzes time and limits of quantification. To overcome this drawback, core –shell stationary phases were developed, this technology was originally developed by Kirkland (24 – 27) and became commercially available in 2001 under the trademark Poroshell 300 with from Agilent Technologies, it has particle size of 5 mm. In 2007, 2.7 mm core-shell stationary phases became commercially available under the trademarks Halo from Advanced Materials Technology, Ascentis Express from Sigma – Aldrich, Kinetex and Aeris from Phenomenex and (28), more recently, there are several suppliers such as Macherey-Nagel (Nucleoshell), Agilent (Poroshell), Thermo Scientific (Accucore), Wissenschaftliche Geratebau (BlueShell), Perkin – Elmer (Brownlee), Waters (CORTECS), Shiseido (Capcell core), Nacalai Inc. (CosmoCore and SunShell) and ACE (UltraCore) (11, 16, 29). Core– shell stationary phases were available in 2.7 mm particles with a relatively thick shell of 0.5 mm, overcame the low loading capacity, which was one of the shortcomings of the earliest generation particles. Compared with fully porous particles, this second generation of core – shell particles shows a slightly lower total mass loadability (30, 31). Examples of core –shell stationary phases are given Figure 1 A and C while a totally porous stationary phase is shown in Figure 1B (32). Core– shell stationary phases also have a narrow 2 Borges

particle size distribution (33). For example, Ola´h et al. (33) had measured the particle size of 300 particles of Kinetex C18, Ascentis Express C18, Acquity Ethylene Bridged Hybrid (BEH). and Hypersil Gold, and the particle sizes of these stationary phases claimed by suppliers are 2.5, 2.7, 1.7 and 1.9, respectively. However, the mean particle size and relative standard deviation observed were 2.50 (5.95), 2.72 (5.30), 1.81 (15.79) and 1.95 (15.18), respectively. For large molecules such as intact monoclonal antibodies, due to its core – shell structure and narrow particle distribution, core – shell stationary phases have lower B and C terms in the van Deemter equation (equation (1)). Thus, core-shell stationary phases afford a higher efficiency/pressure value than totally porous stationary phases. For example, Fekete et al. (34) showed that Aeris WP C18 (3.6 mm particle size) afforded a peak capacity comparable with Acquity BEH300 C18 (1.7 mm particle size). For small molecules, core– shell stationary phases had a comparable C term with totally porous particles due to their rough surface (see Figure 1). However, core –shell stationary phases afford better efficiency than totally porous stationary phases due their narrow particle distribution. For example, Cunliffe and Maloney (35) compared sub-3 mm core – shell stationary phases (Halo C18 and Supelco Ascentis Express C18) with sub-2 mm totally porous stationary phases (Waters Acquity BEH C18, Waters Acquity BEH C18 and Zorbax Extended-C18) using micromolecules such as toluene and acetophenone, tests were carried out in the isocratic and gradient mode and the chromatographic efficiency of core –shell and totally porous particles was comparable, while core– shell stationary phases generated a smaller back pressure than totally porous stationary phases. Columns packed with superficially porous particles in the range of 5 –1.3 mm have been commercially available from multiple suppliers since 2012 (36, 37). Columns packed with Sub-2 core-shell and totally porous particles mm have the same drawbacks, which are the need to acquire a dedicated system, optimized in terms of high back pressure pumps and an injector, acquisition rate of the detector, injection cycle time, dwell volume and system dead volume (28, 38). Thus, despite the impressive improvements made in separation efficiency and analysis time by the introduction of small particle columns operated at ultra-high pressures, pharmaceutical analyses are still largely performed on conventional, 250  4.6 mm fully porous 5-mm particle columns (39, 40). This certainly has to do with the fact that numerous official methods (cf. Pharmacopoeia analyses) are described on these large particle columns and that many pharmaceutical laboratories simply are not yet equipped with the most recent, low-dispersion UHPLC (39). This review is focused on different support materials, which are silica, hybrid silica, Type C silica, zirconia, titania and porous graphitic carbon (PGC). Core – shell stationary phases commercially available are silica stationary phases, sub-2 mm are silica or hybrid silica stationary phases and non-silica stationary phases are 5 mm and 2 mm, excepted by ZirChrom polybutadiene (PBD) that is available in sub-2 mm particle size (41). Silica and Hybrid Silica Stationary Phases When basic compounds are analyzed by RPLC, additional interactions besides those that are hydrophobic may occur between basic compounds and residual silanols on the surface of the silica

Figure 1. SEM images of the 3.6 mm core –shell Aeris WP C18 (A), totally porous 1.7 mm stationary phase BEH300 C18 (B) and a cut Aeris WP C18 particle (C). Image (C) was measured and provided by the vendor (Phenomenex). Source: Reproduced with permission from reference (32).

(42– 44). In this case, the compound may be retained by ionic interactions with residual silanols as well as hydrophobic interactions with the bonded moiety in a synergistic manner, forming highly retentive sites (44). Thus, stationary phases that are nominally equal (i.e., C18 or C8), prepared with the same silane on different types of silica, could afford different selectivities for ionizable solutes. For example, Szulfer et al. (45) have shown that two nominally equal stationary phases (Nucleosil 100-5 C18 HD and Hypersil Elite C18) afford different selectivities in the separation of alfuzosin hydrochloride from its main impurities. Borges (46) had shown that the principal phenomena that make nominally equivalent stationary phases (i.e C18) different are ion-exchange and hydrogen-bonding interactions which take place in free silanols of the stationary phase. The reader can find an interesting material about stationary phase in references (47–50). There is the hypothesis that highly acidic silanols—their abundance, variation in reactivity and their effects on bonded ligand heterogeneity—are primarily caused by metal contamination (51 – 56). Thus, before 2000, when the silica had a high metal content, an alternative approach to reduce the interactions between basic compounds and free silanols on the silica surface has been to minimize the level of surface metals (51 – 56), which are known to increase the acidity of the silanol groups and hence result in stronger interactions with basic compounds than less acidic silanols (51–56). Silica with a high metal content is commonly referred to as Type A silica. Type A silica used to prepare RPLC stationary phases afforded asymmetric peak shapes with low efficiencies when employed in the analysis

of basic compounds because of their high acidity and a nonhomogeneous population of the residual silanol groups (51 – 56). The poor peak symmetry and low efficiency were attributed to the existence of high energy retention sites, afforded by the existence of silanols with different degrees of acidity (51 – 56). In order to minimize the amount of metals on the silica and to improve peak shape, many manufacturers employ a mineral acid wash of their silica before bonding; these are commonly referred to as “base deactivated silica (BDS)”. Modern stationary phases are now prepared using extremely pure silica containing low metal concentrations, typically referred to as Type B silica. These silica materials are normally generated via the polymerization of an organosilane such as tetraethoxysilane (TEOS). Several attempts have been made to reduce the amount of residual silanols on the surface of silica (51 –56). The most common approach has involved the subsequent reaction of the silica surface with short alkyl chain silanes after the silica surface was initially reacted with long alkyl chain silanes (this step is called endcapping). However, even after successive endcapping procedures, .50% of silanols are not reacted because of the steric hindrance created by the long alkyl chains linked to the surface of the silica (51 –56). Another approach to avoid undesired interactions of basic compounds with free silanols are stationary phases possessing polar- embedded groups (PEG), where the embedded polar moieties can shield the free silanols from interaction with basic solutes. Engelhardt et al. (57) have shown that the RP C18 stationary phase (Prontosil H) afforded low efficiency and bad peak shape in the separations of basic tricyclic antidepressants, Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases 3

while a stationary phase based on identical silica, but containing an embedded amide group, resulted in good peak shape and high efficiencies. The reader can find more complete descriptions of PEG stationary phases in references (11, 49, 58– 65). The C18 ligands are too bulky to react completely with all silanols; thus, ,50% of the free silanols might be chemically derivatized with C18 and other kinds of organic silanes. Additional reactive silanols can be “endcapped” by reaction with smaller silylating agents such as trimethylchlorosilane, but as many as 50% of the original silanol groups remain unreacted on a typical reversed-phase material. For this reason, other approaches had to be developed to reduce the concentration of free silanols on the stationary phase surface (1). The first approach used to diminish the amount of free silanols on the stationary phase surface was the development of spherical porous hybrid particles for use as reversed-phase packing materials. These phases are made by the co-condensation of methyltriethoxysilane (1 equiv.) with TEOS (2 equiv.) and have an empirical formula of SiO2(CH3SiO1.5)0.5. These methyl hybrid materials displayed pore characteristics similar to those of contemporary silica-based packing materials and could be surface C18 and C8 modified using common chlorosilane-bonding protocols. These stationary phases have been commercially available since 1999 under the trade name XTerra, and materials prepared using this technology are referred to as the first-generation hybrid (66). The XTerra stationary phases have less free silanols than ordinary silica stationary phases since a part of the free silanols in these stationary phases is substituted by methyl groups. The hybrid phases show good peak shape for basic pharmaceuticals. For example, Neue et al. (66) have shown that XTerra stationary phases afford good peak shape for several basic pharmaceuticals at pH 2.5, 7.0 and 9.5. The second approach used to reduce stationary phase residual silanols was the preparation of BEH stationary phases. These materials have an empirical formula of SiO2(O1.5SiCH2CH2 SiO1.5)0.25. They are synthesized by the co-condensation of 1,2-bis(triethoxysilyl)ethane (1 equiv.) with TEOS (4 equiv.) (67– 71). The BEH technology as introduced by Waters in 2004 and stationary phases prepared using the BEH approach are known as second-generation hybrid materials. They have fewer free silanols than the first-generation hybrid stationary phases. The BEH technology was introduced in 2003 (67), stationary phases with particle diameters .1.7 mm are marketed under the trade name XBridge and stationary phases with particle diameters of 1.7 mm have the trade name Acquity BEH. The second-generation hybrid stationary phases have even fewer free silanols than the first-generation hybrid material, which means that they afford even better efficiency and peak shape for ionizable compounds (67 –71). Phenomenex also sells hybrid stationary phases. Its firstgeneration hybrid stationary phase is sold under the trade name Gemini, while its second-generation material is marketed under the trade name Gemini NX. Phenomenex’s phases have a hybrid surface and silica core while Waters’s stationary phases are totally hybrid (72), whereas Kromasil Eternity from AkzoNobel had a structure similar to Gemini. In addition, some stationary phases, such as YMC-Triart C18 from YMC GMBH, and InertSustain C18 from GL Sciences, are prepared with novel bonding and endcapping procedures that are multistep endcapping procedures with 4 Borges

a series of different endcapping reagents, in which suppliers claim that free silanol amounts are negligible (11). Unfortunately, there has not been any study comparing all these stationary phases, but Undin et al. (73) compared the hybrid Phenomenex Gemini-NX C18 and Kromasil Eternity columns with the Kromasil-C18, observing that the amount of free silanols are reduced in the following order: Gemini-NX C18 , Eternity , Kromasil-C18. At present, the coupling between LC with mass spectrometry (LC –MS) is being used with increasing frequency (74 –77). The development of bioanalytical methods has become more and more challenging over the past years because of very demanding requirements in terms of method reliability, sensitivity, speed of analysis and sample throughput (78). LC – MS and LC – MS/MS have become established as methods of choice for routine analysis of biological materials. Very often weak ion strength mobile phases and volatile buffers are required and despite all the improvements in column technology, serious peak deformation can still occur under certain experimental conditions, especially when low ionic-strength mobile phases are employed in LC–MS. For example, Heinisch et al. (79) observed poor peak shape for basic pharmaceuticals in low-ionic-strength mobile phases using Acquity BEH C18 and Kinetex C18 stationary phases. When large biomolecules, which have several acidic and basic functionalities, were analyzed, the problems caused by free silanols are even worse. Fekete et al. (34, 80, 81) have shown that at temperatures ,508C, monoclonal antibodies are irreversibly retained on Phenomenex Aeris Wide pore, which is prepared with ultra-pure silica, and Waters Acquity stationary phases. Relatively new additions to the chromatographic arsenal of stationary phases are charged surface hybrid (CSH) stationary phases. The CSH technology was introduced by Waters in 2010 and stationary phases with particle diameters .1.7 mm are marketed under the trade name, XSelect, and stationary phases with particle diameters of 1.7 mm have the trade name, Acquity CSH. CSH stationary phases are designed to alleviate the problem of low performance in LC – MS at low ionic strength. The concept used is to add a few fixed charges at the surface of the stationary phase. The XSelect stationary phases consist of hybrid silica (the same hybrid silica used in XBridge and Acquity stationary phases), which are tethered with a well-controlled number of amine groups (82 –84). At pH ,3, the bonded amine groups on the silica are quantitatively protonated rendering the surface of the adsorbent its positive charge. Under such conditions of electrostatic repulsion between the charged surface and the analyte, access to the silanol groups is precluded. Thus, a significant reduction of the peak tailing of protonated bases like amitriptyline and metoprolol compared with the shape of their peaks on ethylene hybrid stationary phase under the same conditions was reported in 0.1% formic acid mobile phases (82). Similar observations were made recently with 5 – 50 mM phosphoric acid/potassium hydrogen phosphate buffers for nortriptyline (83, 84). It was demonstrated that the charges on the surface of CSH repel positive ions avoiding the harmful, strong interactions with the active silanol sites. However, as the surface charge density decreases, electrostatic repulsion decreases and the cationic sample regains access to the surface of CSH. The protonated amine groups become neutral at weakly acidic and neutral mobile phases, and the amine groups behave like active sites for charged

compounds. In contrast to what is observed for ethylene hybrid stationary phases in acidic mobile phases, basic pharmaceuticals tail significantly, which demonstrate the existence of new active sites (the amine groups) in addition to the silanol groups (85). Nova´kova´ et al. (86) studied the chromatographic pattern of acidic and basic pharmaceuticals using low-ionic-strength mobile phases at low pH using Acquity CSH and Acquity BEH stationary phases. They observed that in low-ionic-strength acidic mobile phases, the CSH stationary phases afforded better peak shape for basic pharmaceuticals than BEH stationary phases. However, acidic pharmaceuticals had poor peak shape due to detrimental interactions with the amino groups in the CSH stationary phase. In addition, they reported that the Acquity BEH Shield RP 18, which is a PEG stationary phase, had better peak shapes for acidic pharmaceuticals than Acquity CSH stationary phases and the peak asymmetry observed for the basic pharmaceuticals were similar. Type C Silica Silica-hydride materials, which are also known as Type C silica, represent a new type of HPLC stationary phase based on highpurity silica, whereby the surface of this new material is largely populated with the less polar silicon-hydride (Si –H) groups, instead of the polar silanol groups (Si –OH) that cover the surface of all other varieties of modified silica (87 –132) (the differences between Type B silica and Type C silica are illustrated in Figure 2). Figure 3 lists examples of silica-hydride phases (e.g., small alkyl, phenyl, cholesterol, octadecyl and alkyl carboxylic acids) that have resulted in significant applications to date. In this material, referred to as silica hydride, Si –H moieties replace 95% of the Si –OH groups on the surface (128, 133). Type C silica has a negligible amount of free silanols, because the SiOH groups on the surface were converted to SiH groups. Thus, basic pharmaceuticals can be analyzed with good peak shape on silica-hydride stationary phases. For example, the USP method for quinine sulfate requires a resolution of not less than 1.2 from its main impurity, dihydroquinine. In the United States Pharmacopeia (USP) method, the ion pair agents, methanesulfonic acid and diethylamine, are used in the mobile phase. Ion pair agents are often needed to reduce peak tailing of basic analytes such as quinine, when conventional type B silica-based HPLC columns are used. However, when a stationary phase prepared with Type C silica (Phenyl HydrideTM ) was used, quinine is separated from its main impurity with a resolution of 2.6, with excellent peak shape, using only 0.1% trifluoroacetic acid (TFA) in the mobile phase. In addition, the method equilibrates

Figure 2. Surface configuration of ordinary silica (left) and silica hydride (right). Source: Reproduced with permission from reference (92).

rapidly with only a 1 min post time after the gradient and the column shows extended lifetimes, and this example is shown in Figure 4. Type C silica is one of the most promising chromatographic support materials. However, this material has never reached the utility, breadth and importance of Type B silica for HPLC separations. It happens due to its limited number of suppliers and because Type C stationary phases are not available as core –shell and sub-2 mm stationary phases. Hydrophilic Interaction Liquid Chromatography Hydrophilic ionizable compounds may have low retention on reversed-phase stationary phases, which necessitate the use of mobile phases containing low organic modifiers (even 100% aqueous mobile phases). Hence, stationary phases must be compatible under these conditions (i.e., that they do not exhibit phase de-wetting/phase collapse). Phases such as polar embedded and polar endcapped are frequently used for these high aqueous chromatographic conditions. Recently, the technique of HILIC has become an increasingly popular alternative method for the separation of polar compounds when polar stationary phases and mobile phases rich in organic solvents (usually acetonitrile, ACN) are employed. HILIC is a variation of normal phase LC that employs more eco-friendly and less toxic solvents than chlorinated solvents and hexane used in normal phase liquid chromatography. Nowadays, HILIC is a LC mode that get more attention, but its retention mechanism has remained largely unknown (134). HILIC separates polar, hydrophilic analytes between a polar stationary phase, for example bare silica, and an aqueous –organic mobile phase, for which ACN has become the solvent of choice. HILIC closes the application gap for compounds that are insufficiently retained on reversed phases, are insoluble in the non-aqueous mobile phases used in normal phase chromatography and lack the necessary charges for ion-exchange chromatography (5). The high amount of organic modifier used in HILIC provides enhanced LC– MS detection and low back pressure. One of the greatest advantages of HILIC is its totally different selectivity than that obtained in the RP mode. In LC – MS mode, HILIC provides enhanced detection compared with RPLC. Periat et al. (7) have shown that HILIC affords increased sensitivity with electrospray ionization source (ESI/ MS) ( for a complete description of ESI/MS, see reference (135)). They compared peak heights, background noises and signal-to-noise ratios (S/N) obtained with HILIC – MS/MS and RPLC – MS/MS using a dataset of 56 basic drugs, which have diverse physico-chemical properties, in different mobile phase pH levels (3 and 6 in HILIC; 3, 6 and 9 in RPLC) and flow rates (300, 600 and 1,000 mm/min). They observed (7) that the median gain in sensitivity obtained between HILIC and RPLC was 4 and for 90% of the tested compounds and analytical conditions, the best S/N was systematically attained under HILIC mode. In addition, they had shown that the basic compounds with pKa between 6 and 8 generally had the best sensitivity in HILIC at pH 6, while the best sensitivity for basic analytes possessing pKa .8 was usually obtained in HILIC at pH 3. Thus, the sensitivity gain in HILIC versus RPLC was explained by the difference in ACN concentration at elution (in average 29% ACN in RPLC and 82% ACN in HILIC at pH 6) leading to better analytes’ Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases 5

Figure 3. Some examples of commercial hydride phases. Courtesy: Joseph J. Pesek from San Jose State University.

desolvation, and it seems that this high proportion of solvent also favorably influenced the ionization by modifying pH and pKa. For example, weakest bases studied ( pKa between 2 and 5) showed an unexpectedly strong gain in sensitivity, between 20- and 100-fold in comparison with RPLC. These results prove the ionic character of analytes in solution (i.e., pKa and pH) and the ionization mechanism (i.e., proton transfer). In HILIC mode, selectivity is different from the selectivity obtained in the RP mode. In RP mode, retention of neutral, basic and acidic analytes is highly correlated with log D values. However, in HILIC, log D was not correlated with log k. An interesting tool to optimize selectivity in HILIC mode is the use of an experimental design. Rakic´ et al. (136) used this approach to optimize the separations of a model mixture of antidepressants (selegiline, mianserin, sertraline, moclobemide, fluoxetine and maprotiline). The experimental design resulted in the understanding and manipulation of important variables (ACN content in the mobile phase, buffer concentrate ion and pH of the mobile phase) to achieve the best separation. The reader can find in Rakic´ et al. (136) an interesting tool to optimize separations of basic pharmaceuticals in the HILIC mode. The reader can also find some interesting reviews about HILIC in references (8, 137–149). An interesting example of the advantages of the HILIC with respect to the RP mode was given by Ruta et al. (31). They employed RPLC and HILIC methods for the analysis of a drug cocktail

6 Borges

containing two substrates and their numerous desmethylated metabolites, which have different log P, log D and pKa values. They used Acquity BEH C18 and SCH 18 (100  2.1 mm, dp 1.7 mm) stationary phases in the RPLC mode and Acquity BEH silica and BEH amide (100  2.1 mm, dp 1.7 mm) stationary phases in the HILIC mode. In this case, the selectivity of RPLC and HILIC were totally different. In the RPLC mode on both stationary phases, the best separation of solutes was achieved using an alkaline mobile phase [ammonium formate (pH 9; 10 mM)], and the separations were carried out with a gradient profile: 5–95% ACN in 8 min and then 3 min at 95% ACN. In the HILIC mode, the separations were better in a near neutral mobile phase [ammonium acetate (pH 6; 20 mM)]. The separations carried out in the HILIC mode were faster, used a higher amount of organic modifier and generated lower back pressure than the separations done in the RPLC mode. Ruta et al. (31) highlight that lower backpressure was obtained in the HILIC mode than in the RPLC mode due to the high amount of organic modifier used in HILIC, which means that longer columns could be used. In this context, Louw et al. (150) had difficulty separating several acidic and basic pharmaceuticals by HILIC using an Ascentis Si stationary phase (250  4.6 mm, dp 5 mm) with an ACN – ammonium formate buffer ( pH 5; 5 mM) (85:15, v/v) mobile phase, at a 1 mL/min flow rate, at 308C, with a 45 min analysis time. Thus, they increased the temperature to 808C making possible the coupling of five columns at a flow rate of 2 mL/min with a back pressure of 350 bar. At

Figure 4. Separation of quinine from its main impurity. Column: Cogent Phenyl HydrideTM (75  4.6 mm, dp 4 mm). Mobile phase: A, 0.1% trifluoroacetic acid (TFA) and B, 0.1% TFA in acetonitrile. Gradient: Time (min) and % B, 0 and 10%, 6 and 30%, 7 and 10%. Post-time: 1 min. Temperature: 408C. Flow rate: 1.0 mL/min. Injection volume: 10 mL. Sample: Stock solution—1.0 mg quinine (90% by label claim) was dissolved in 1 mL 50% solvent A/50% solvent B mixture. Working solution: A 100 mL aliquot of the stock was diluted with 900 mL of 50% solvent A/50% solvent B mixture. Peaks: 1. Minor impurity; 2. quinine (API) and 3. dihydroquinine (main impurity). Detection: UV 235 nm. t0: 0.9 min. Courtesy: Joseph J. Pesek from San Jose State University.

Figure 5. Analyses of alprazolam in HILIC mode with bare Type C silica. Column: Cogent Diamond HydrideTM (75  4.6 mm, dp 4 mm). Mobile phase: A, 0.1% formic acid and B, acetonitrile 0.1% formic acid in acetonitirle (v/v). Gradient: time (min) and % B, 0 min 95%, 1 min 95%, 6 min 50%, 7 min 95%. Post-time: 3 min. Injection volume: 1 mL. Flow rate: 1.0 mL/min. Peaks: 1. Triazolam (internal standard); 2. alprazolam (API); 3 and 4. Impurities detection: 254 nm. t0: 0.9 min. Courtesy: Joseph J. Pesek from San Jose State University.

808C, they were able to separate the ionizable pharmaceuticals and reduced the analysis time to 30 min. Louw et al. (150) reported that an increase in temperature (30– 808C) reduced the C term of the van Deemter equation. However, one of the advantages of the HILIC mode is its reduced C term. For example, McCalley (1) reported low C terms for HILIC stationary phases (dp 5 mm). Thus, basic pharmaceuticals could be analyzed in high-speed separations at low back pressure. The nature of the adsorbed water layer in HILIC methods is thought to contribute to a lack of robustness in many instances of gradient usage. As such, HILIC columns may require lengthy equilibration that consumes both time and solvents (151). Because the silica hydride surface is slightly hydrophobic, it will adsorb and desorb the mobile phase differently and more quickly. This feature leads to both faster equilibration and higher precision when gradients are used. The HILIC mode is commonly used for the analyses of hydrophilic pharmaceuticals, which are not retained in the RPLC mode. In this context, the reader must keep in mind that Type C silica can afford higher retention than Type B silica. For example, Bawazeer et al. (152) have shown that sugars as well as basic and acidic pharmaceuticals were more strongly retained on bare Type C silica than on bare Type B silica, which means that Type C silica should be considered for the analysis of pharmaceuticals compounds not retained in the RPLC mode or poorly retained in the HILIC mode with Type B silica.

Stationary phases based on Type C silica have demonstrated a number of unique properties that are especially advantageous for bioanalyses. They have excellent retention capabilities for hydrophilic compounds, which have been the most difficult to analyze by standard RPLC methods. In addition, all columns utilizing silica-hydride materials can retain both polar and nonpolar compounds simultaneously. These stationary phases have a high degree of reproducibility and long-term stability and its applications in HILIC and RPLC modes were recently reviewed (91, 92, 108, 115, 129). A good example of Type C silica used in pharmaceutical analysis was given by Pesek et al. (96). They were able to analyze very hydrophilic pharmaceuticals (cycloserine and cycloserine dimer), which are unretained in RPLC mode, using a hydride stationary phase [Cogent Diamond Hydride (DH) (150  2.1 mm, dp 4.0 mm)] with high amounts of organic modifier in the mobile phase under LC–MS friendly conditions (i.e., 0.1% formic acid in ACN /water). The HILIC mode could be a very versatile and powerful tool in pharmaceutical analyses, and the reader should consider changing their normal phase methods to a HILIC format. For example, Figure 5 highlights that the USP assay method for Alprazolam (Xanax), which is a normal phase method, could be simplified when the separation is done in the HILIC mode. The USP assay method for Xanax uses a bare silica column and a complex mobile phase consisting of ACN, chloroform, butyl alcohol and acetic acid. However, this method was simplified when the Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases 7

separation was done by HILIC and a simple LC – MS compatible mobile phase is used. It produces excellent peak shapes for both alprazolam and its USP internal standard. Furthermore, a resolution of 4.3 was obtained between the two peaks, which meets the USP system requirement of Rs . 2.0. Two impurity peaks are also observed, which further illustrates the resolution capabilities of the column. The Non-Silica Stationary Phases The non-silica stationary phases are another option for the analysis of basic pharmaceuticals, since they provide selectivity different from silica and hybrid silica stationary phases.

Zirconia and titania stationary phases Silica is the most commonly used support for the preparation of RPLC stationary phases. However, ZirChrom has commercialized a range of stationary phases prepared by either polymer coating (PBD or polystyrene) or graphitization, which may be followed by subsequent derivatization with alkyl groups onto zirconium oxide particles (153 –167). Stationary phases based on zirconia also are marketed by Sigma – Aldrich under the Discoveryw Zr brand name. The ZirChrom and Discovery Zr stationary phases are the same materials, but they have different brand names. The Sigma – Aldrich brand is found in most countries, which make polymer-coated zirconia stationary phases available over the entire world. At Pittcon 2005, Sachtleben Chemie GmbH (Duisburg, Germany) and ZirChrom Separations introduced two new titania-based stationary phases for HPLC: Sachtoporew-RP—a polymer-coated RPLC titania and Sachtoporew-NP—a titaniabased normal phase (166). The zirconia and titania polymer-coated stationary phases retain basic pharmaceuticals by synergic interactions between ion-exchange sites and reversed-phase sites. Thus, basic pharmaceuticals are highly retained on these stationary phases. On silica surface, free silanols that interact with basic analytes and silica surface act as a Bro¨nsted – Lowry acid, but silica surface does not act as a Lewis acid, because the silica surface has not acid Lewis sites. On the other hand, TiO2 and ZrO2 have sites that can act as Lewis acids and those materials strongly react with basic analytes as Lewis acids [interested readers can find a complete description of the mechanism of retention that takes place on zirconia stationary phases in references (2, 168)]. An example of the high retention of basic pharmaceuticals on zirconia-coated stationary phases is the Dai et al. (155) work. They compare the separation of basic drugs on several octadecyl silane bonded silica stationary phases prepared with silica of different degrees of purity, and a PBD-coated zirconia stationary phase, ZirChrom PBD. The retention characteristics were investigated in detail using a variety of cationic drugs with ammonium phosphate mobile phases at pH 3.0 and 6.0. They observed that the ZirChrom PBD stationary phase has larger pore size and smaller surface area (500 A˚ and 11.2 m2/g, respectively) than the C18 stationary phases studied (80 – 100 A˚ and 179 – 436 m2/g, respectively). However, ZirChrom PBD afforded retention factors higher than those obtained with C18 stationary phases, especially with respect to hydrophobic bases such as amitriptyline and nortriptyline, due to the synergic effects between ion-exchange and hydrophobic interactions. Despite this, the 8 Borges

ZirChrom PBD afforded asymmetry factors and efficiencies as good as those on C18 stationary phases prepared with highpurity silica. In addition, it also provides very significant differences in selectivity toward basic pharmaceuticals compared with C18 stationary phases. Nawrocki et al. (153, 154) have shown that hard buffers used in the mobile phase result in higher retention factors and efficiencies of basic pharmaceuticals. For example, when some antihistamines were analyzed with a ZirChrom-PBD stationary phase using ACN –Lewis base additives (phosphate, fluoride and acetate ammonium salts) ( pH 7; 20 mM) (30 : 70, v/v), the shortest retention times and lowest efficiencies were observed for acetate-containing mobile phases, while the highest retention factors and efficiencies were obtained using phosphate buffer. The explanation given by Nawrocki et al. (153, 154) was that acetate is a weaker Lewis base than fluoride or phosphate that block Lewis sites more effectively than acetate. Thus, these ions introduce a larger negative charge on a ZirChrom PBD surface than does acetate and it becomes a stronger cation-exchanger. According to the manufacturer, Sachtopore-RP (50  4.6 mm, dp 3 mm) shows increased retention factors for basic pharmaceuticals when hard buffers are used in the mobile phase (for example, the retention of basic pharmaceuticals obtained using ammonium acetate buffer is lower than the retention obtained using ammonium phosphate buffer), and discrete small increases in the buffer concentration dramatically reduce the retention factors of basic pharmaceutical as described by Nawrocki et al. (153, 154) for zirconium-coated stationary phases. These properties are illustrated in Figure 6.

Figure 6. Basic pharmaceuticals analysis using a titania stationary phase, Sachtopore-RP (50  4.6 mm). LC conditions: Flow rate: 1.0 mL/min. Temperature: 408C. Detection: UV 254 nm. (A) Effect of Lewis base additive on separation of basic drugs. Mobile phase: acetonitrile – buffer ( pH 7; 20 mM) [(30 : 70), (v/v)] (a) ammonium acetate, (b) ammonium fluoride, (c) ammonium phosphate. Solutes: (1) lidocaine, (2) quinidine, (3) tryptamine, (4) amitriptyline and (5) nortriptyline.(B) Effect of ionic strength on mobile phase: acetonitrile– buffer ( pH 7; 20 mM) [(30:70), (v/v)] (a) 10 mM, (b) 15 mM and (c) 15 mM. Solutes: (1) lidocaine, (2) quinidine, (3) tryptamine, (4) amitriptyline and (5) nortriptyline. Adapted from the supplier web site: http://www.zirchrom.com/, accessed in July 2014.

Figure 7. Basic pharmaceutical analysis in a zirconia stationary phase. (A) Separation of opioids. Column ZirChrom-Diamondbond C18 Mobile Phase: 26.5:73.5 THF – ammonium phosphate (20 mM; pH 11). Flow rate: 1.0 mL/min. Temperature: 408C. Compound identification: 1 ¼ naloxone, 2 ¼ codeine, 3 ¼ ethylmorphine and 4 ¼ oxycodone. (B) Separation of b-blockers. Column: ZirChrom-Diamondbond C18 100  4.6 mm. Mobile phase 20:20:60 acetonitrile – THF – ammonium phosphate buffer (10 mM; pH 11.0). Flow rate: 1.0 mL/min. Temperature: 7588 C. Compound identification: 1 ¼ atenolol, 2 ¼ metoprolol, 3 ¼ oxprenolol and 4 ¼ alprenolol. Adapted from “Technical Bulletin #231. Separation of Opioids ZirChrom Separations Inc. (Anoka, MN)” and “Technical Bulletin #236. Separation of b-blockers. ZirChrom Separations Inc. (Anoka, MN)”.

The zirconia- and titania-coated stationary phases possess sites, which can act as Bro¨nsted acids/bases and Lewis acids and, when basic compounds were chromatographed, poor chromatographic performance was expected unless a hard Lewis base was present in the mobile phase. Thus, the presence of fluoride or phosphate in the mobile phase can greatly improve the chromatography of basic compounds. For example, Figure 7 shows some examples of successful separations of basic pharmaceuticals using mobile phases containing phosphate buffer and Zirchrom-DiamondBond C18, which is a zirconia graphitized followed by subsequent derivatization with C18 groups. Hu et al. (156) have shown that basic solutes were much more retained at near neutral mobile phase pH than at high alkaline mobile phases, basic solutes were poorly retained, because they were unionized and the ion-exchange interactions, which is one of the principal retention mechanisms on zirconia-based stationary phases, were suppressed, for example, when amitriptyline and nortriptyline were analyzed using a ZirChrom-PBD stationary phase (50  4.6 mm, dp 4.1 mm) with an ACN -phosphate buffer ( pH 4 –12; 20 mM) (30 : 70, v/v) mobile phase. The maximum retention was obtained at pH 7, while lower retention was obtained at pH 12. They also reported that the buffer concentration in the mobile phase strongly affects the retention of basic solutes due to the high participation of the ion-exchange mechanism in the retention of basic solutes on zirconia polymercoated stationary phases. For example, the retention factor of nortriptyline was .50, when the mobile phase conditions

cited above were used, while an increase in the buffer concentration to 100 mM resulted in a reduction of amitriptyline retention by .50%. Zˇizˇkovsky´ et al. (159) used the stationary phases Sachtopore-RP (150 mm  4.6 mm, dp 5 mm) and ZirChrom-PBD (150 mm  4.6 mm I.D, dp 5 mm) to separate ondansetron and related compounds. In this study, they reported that both titania and zirconia stationary phases possess similar properties (increased retention at near neutral mobile phase pH, increased retention at low buffer concentration and with harder Lewis buffers), but different selectivity. In addition, the separation of ondansetron and its five impurities was achieved with both stationary phases in 10 min. Zirconia and titania polymer-coated stationary phases are promising choices in pharmaceutical analysis because of their unique properties and selectivity different from silica and hybrid silica stationary phases. The work carried out by Zˇizˇkovsky´ et al. (164) illustrates how a polystyrene-coated zirconium stationary phase (Discovery Zr-PS, 150  4.6 mm, dp 5 mm) could be an alternative in the analysis of a parent drug (doxazosin) and its impurities. The analysis of doxazosin and its five pharmacopeial impurities was done within 16 min and the method was precise, sensitive and robust enough. Due to the unique selectivity of zirconium-coated stationary phases, it was possible to obtain sufficient selectivity at reduced retention times. In addition, they showed that the ZirChrom-PS stationary phase could be a suitable candidate for replacing silica-based C18 stationary phases, which are commonly used for separation of doxazosin impurities. For example, in pharmaceutical analysis, coated zirconia stationary phases could be used instead of silica stationary phases are the quantification of ibuprofen and its impurities (158–162) and the separation of ondansetron and its five pharmacopeial impurities (160). Hard Lewis bases are LC – MS incompatible. Thus, ZirChrom developed the ZirChrom-MS stationary phases (169). This stationary phase is prepared in three steps: first, ethylenediamine N, N, N 0 ,N 0 -tetra(methylenephosphonic) acid (EDTPA) is reacted with the zirconia to deactivate the Lewis sites on the surface; second, amines on the zirconia surface are quaternized with allyl iodide; and third, PBD is coated onto the modified zirconia surface. Finally, PBD is crosslinked with allyl groups using dicumyl peroxide as an initiator. Because of the high coverage and deactivation of the Lewis acid sites, this stationary phase is suitable for basic pharmaceutical analysis using LC –MS compatible buffers (a separation of some basic pharmaceuticals with ZirChrom-MS is shown in Figure 8). Kalafut et al. (166) compared the chromatographic properties ( pH, buffer concentration and buffer type) of ZirChrom-MS and ZirChrom-PBD (both 150  4.6 mm, dp 5 mm) stationary phases using acidic (ibuprofen, diclofenac, flufenamic acid and meclofenamic acid) and basic ( procaine, lidocaine, prilocaine and trimecaine) pharmaceuticals. The pH effect study was carried out using an ACN –phosphate ( pH 2 –9.5; 20 mM) (25 : 75, v/v) mobile phase, where both stationary phases showed increased retention for basic solutes at near neutral pH and increased retention for acid pharmaceuticals at pH values lower than their pKa ( pH , 4). ZirChrom-MS was much more retentive for acidic pharmaceuticals than ZirChrom-PBD in acidic mobile phases ( pH , 4). The buffer effect test was carried out with an ACN –phosphate buffer ( pH 5.5; 20, 25, 30, 35 and 40 mM) (25 : 75, v/v) mobile Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases 9

synergistic ion-exchange and RP interactions. The overall balance of these synergistic interactions are higher in ZirChrom-MS than in ZirChrom-PBD. Thus, ZirChrom-MS is more retentive than ZirChrom PBD. The increased retention available in ZirChrom-MS makes possible an analysis of ondansetron and its five pharmacopeial impurities with an ACN – acetate buffer ( pH 7; 5 mM) (40 : 60, v/v) mobile phase. With this mobile phase, all the analytes co-eluted on ZirChrom-PBD. Thus, the analysis of ondansetron and its impurities were carried out in LC –MS compatible conditions with higher amounts of organic modifiers in mobile phase. The zirconia is the most stable chromatographic support, it is also worldwide available by Sigma –Aldrich under the trade name discovery-Zr and it is available as sub-2 m. Thus, it is surprising that zirconia stationary phases have been rarely used.

Figure 8. Separations of some basic pharmaceuticals using ZirChromw-MS (50 3 4.6 mm). Mobile phase: acetonitrile – ammonium acetate ( pH 5.0; 10 mM) [65 : 35 (v/v)]. Temperature: 3588 C. Flow rate: 1.0 mL/min. Injection volume 5 mL. Pressure drop: 59 bar. Detection: UV at 254 nm. Solutes identification: (A) separation of 1 ¼ lidocaine, 2 ¼ atenolol, 3 ¼ metoprolol, 4 ¼ oxprenolol and 5 ¼ alprenolol. (B) Separation of 1 ¼ lidocaine, 2 ¼ atenolol, 3 ¼ metoprolol, 4 ¼ oxprenolol and 5 ¼ alprenolol. Source: Adapted from LC/MS compatible separation of antidepressants on ZirChromw-MS. ZirChrom [Adapted from Technical Bulletin #298, ZirChrom Separations Inc. (Anoka, MN) and LC/MS compatible separation of b blockers on ZirChromw-MS#296, ZirChrom Separations Inc. (Anoka, MN)].

phase. Both stationary phases showed reduced retention at higher buffer concentrations for basic solutes, while the retention of acid compounds was unchanged by the buffer concentration. However, improved peak shapes were obtained at higher buffer concentrations. This improvement in peak shape with buffer concentration increase is smaller in ZirChrom-MS. The buffer type effect was carried out using ACN –Lewis base ( phosphate, fluoride, and acetate) ( pH 6.5; 20 mM) (25 : 75, v/v). Both stationary phases showed increased retention for basic and acidic pharmaceuticals with harder buffers, while ZirChrom-MS showed higher retention than ZirChrom-PBD. For example, the retention factors obtained with phosphate are higher than those obtained with fluoride on both stationary phases, while the retention factors obtained with ZirChrom-MS are higher than those obtained with ZirChrom-PBD, except for the retention factors of acid pharmaceuticals on ZirChrom-PBD with acetate buffer, which are higher than the retention factors obtained with ZirChrom-MS under the same conditions. ZirChrom-MS appears to have less ion-exchange sites (due EDTPA treatment). It has a higher carbon content and crosslinking than ZirChrom PBD, but it retains ionizable compounds by 10 Borges

HILIC with titania and zirconia stationary phases There are some examples of basic and acidic pharmaceutical analysis in the literature using bare metal oxide stationary phases in the HILIC mode. For example, El Debs et al. (167) studied the retention of b-blockers as a function of water content in the mobile phase using Zirchrom Sachtopore-NP (100  2.1 mm, 5 mm) with an mobile phase consisting of ACN –ammonium acetate ( pH 4.7; 10 mM), at a flow rate of 0.4 mL/min, at 258C. They observed that the main mechanism of retention of b-blockers on bare titania was ion-exchange, because the retention factors of some b-blockers were greatly reduced at increased buffer concentration. This HILIC separation of b-blockers, on bare titania, showed high retention factors and selectivity in mobile phases with ,10% aqueous buffer, while retention factors in the range 2.5 – 4 and poor selectivity were obtained using .20% water in the mobile phase. This analysis is an example of an LC – MS compatible separation of basic pharmaceuticals, where hydrophilic pharmaceuticals were well separated using a 90% organic mobile phase and ammonium acetate buffer. Thus, further studies using bare titania in the HILIC mode deserve attention. Zhou and Lucy (170) studied the separation of a series of carboxylic acids (i.e., 3-aminophthalate, 1,3,5-benzenetricarboxylate, 1,2,4-benzenetricarboxylate) on bare titania [SachtoporeNP column (150  4.6 mm, dp 5 mm)]. The carboxylic acids were poorly retained with 0 – 60% ACN in the mobile phase, while at .70% of ACN in the mobile phase the test solutes were strongly retained. [The test was carried out with an ACN –phosphate buffer ( pH 6; 5 mM) (0 – 80%, (v/v) mobile phases, at 1 mL/min]. They observed that hard buffers gave higher efficiencies and lower retention than soft buffers. For example, carboxylic acids were more retained with acetate buffer than with phosphate buffer. Interestingly, carboxylic acids were more retained on bare titania at higher buffer concentrations than at lower buffer concentrations. (This is also an example of how bare titania could be used with success to analyze acidic compounds in the HILIC mode). At this point, it must be mentioned that in all studies cited in this section successful separations were achieved under HILIC conditions using zirconia and titania stationary phases, but poor peak shapes and low efficiencies were also reported. For example, Kucˇera et al. (163) showed that the separation of carboxylic acids in the HILIC mode is much better with an Atlantis

HILIC silica column than with bare zirconia or a ZirChrom-PBD stationary phase. At present, there are few reports using bare metal oxide stationary phases in the HILIC mode and more studies comparing silica, zirconia and titania stationary phases in the HILIC mode must be done to draw a clear conclusion. (Are metaloxide stationary phases useful in the HILIC mode?)

Alumina stationary phases Alumina has a higher surface area than zirconia and titania (170 m2/g). Alumina stationary phases are commercialized by Merck (Darmstadt, Germany) under the trade name Aluspher RP-Select B. It is an alumina coated with PBD (171 – 173). ES Industries also markets alumina-based stationary phases under the trade name GammaBond. It is available in two stationary phase types: GammaBond RP-1 and RP-8. GammaBond RP-1 is a low load PBD-coated alumina. GammaBond RP-8 is an aluminabased polysiloxane polymer to which n-octyl groups are appended. Both GammaBond stationary phases are prepared with alumina particles of 5 mm in diameter and 80 A˚ pore size. Claessens et al. (174) have reported that Aluspher RP-Select B possesses high ion-exchange properties, and it is highly retentive for aromatic compounds. For example, Jandera et al. (175) were able to separate a series of alkylbenzenes with a bare alumina stationary phase, while Sy´kora et al. (176) demonstrated that Aluspher RP-Select B has increased retention for pyridines in neutral mobile phases (with phosphate buffer). The only example in the literature of the use of alumina stationary phases for basic pharmaceutical analysis was provided by Lambert et al. (177), where several basic pharmaceuticals (i.e., desipramine, amitriptyline and chlorpromazine) were separated using gradient elution (isocratic for 5 min at 10% A, then linear to 90% A over 15 min, hold at 90% A for 5 min. A: 15 mM sodium hydroxide in methanol and B: 15 mM sodium hydroxide) under alkaline conditions using an Aluspher RP-select B stationary phase (125  4.0 mm, dp 5 mm). Thus, the potential of alumina stationary phases for basic pharmaceutical analysis remains unknown. Interested readers can see a description of the relevant properties of alumina as a support material for LC and a number of applications of its use in the Pesek and Matyska review (178).

Porous graphitic carbon Chromatographic stationary phases based on PGC were invented over 30 years ago, while columns have been commercially available for only 20 years. West et al. (179) did a review on this material describing the current knowledge on PGC stationary phases, based on over 400 fundamental studies and applications. However, in this article and other articles (180– 182), the advantage highlighted for using PGC for the analysis of basic compounds was due to its high chemical and thermal stability and high retentivity. These properties permit one to separate basic compounds in their neutral form at high pH values and also afford increased retention for hydrophilic compounds. The PGC stationary phase is commercialized by Thermo-Fisher under the brand name Hypercarb. Its physical properties are described in Table I. The PGC structure is illustrated in Figure 9. This stationary phase is highly retentive because it consists of 100% carbon and hydrophilic drugs, which are poorly retained

Table I Physical Properties of Hypercarb Particles Particle size Surface area Median pore diameter % Carbon Mechanical strength

Spherical, fully porous 3, 5 and 7 mm 30 mm 120 m2/g ˚ 250 A 100 .400 bar

Manufactured from silica template Ideal for LC and LC –MS preparative columns Particle size of choice for SPE applications Ensures retention linearity and good loading capacity Ensures good mass transfer for wide range of analyte shapes and sizes Chemical stability; long lifetime; no silanols

Courtesy: Tony Edge from Thermo Fisher Scientific, Runcorn, UK.

in the RP mode, could be analyzed in reversed-phase using Hypercarb. In this context, Sroka-Markovic et al. (183) used Hypercarb, silica and hybrid silica stationary phases (Zorbax Extend, XTerra RP18, CapCell Pak, Hypurity Advance, XTerra Phenyl, Zorbax SB-CN, Zorbax SB-phenyl, Zorbax SB-CN, all with 150  4.6 mm, dp 5 mm) for the determination of related impurities in prilocaine. Prilocaine and its impurities are hydrophilic bases, which are poorly retained in the RP mode. They achieved the best separation of prilocaine and its main impurities using a Hypercarb stationary phase due to the unique selectivity and high retentivity of this material. In addition, the separation is achieved with .60% organic modifier using Hypercarb, while prilocaine and its main impurities were unretained with .20% organic modifier in mobile phase using silica and hybrid silica stationary phases (183). Small polar analytes have proved difficult to analyze due to lack of retention on conventional RP-LC mode. Typical reversedphase materials are hydrophobic and have been developed for the retention and separation of hydrophobic compounds. On these phases, polar – ionizable compounds are eluted at or near the dead volume of the column, making effective analysis of these compounds challenging. Approaches to induce retention of polar compounds include solute derivatization and ionpairing (184). Derivatization is time consuming, and ion pairing has disadvantages when MS detection is used, namely, ionization suppression and contamination of the ion source with involatile reagents. Recent developments in the manufacturing of stationary phases such as polar-embedded have created phases with some polar character, which have the ability to retain certain polar species. However, retention of very polar species on these columns still may require the use of highly aqueous mobile phases, which leads to reduced MS sensitivity (184) [see reference (7)]. For example, Figure 10 shows that Hypercarb is much more retentive than a polar endcapped stationary phase with the same size and particle diameter. HILIC has become an increasingly popular alternative approach to effectively retain and separate small polar species. A disadvantage of HILIC is that it can retain only polar species, while hydrophobic species are eluted at or just after the column void volume and the advantages of HILIC are lost with compounds that have high water solubility and do not easily transfer to the organic phase in extraction. However, PGC offers a further alternative, because it can retain hydrophobic analytes as well as very polar and ionic species through dispersive interactions and polar species through dipole – dipole interactions in Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases 11

Figure 9. Illustration of the structure of PGC. Courtesy: Tony Edge from Thermo Fisher Scientific.

PGC stationary phase as shown in Figure 11A, the separation of these herbicides is difficult in the RP mode due the poor retention of these hydrophilic compounds. In Figure 11B, the separation of a glucosamine sulfate using 50% ACN in the mobile phase is shown. Unfortunately, Hypercarb afford bad peak shape for basic pharmaceuticals. For example, Albert et al. (187) observed poor peak shapes for basic pharmaceuticals (codeine, quinine, chloroprocaine, propranolol and diphenhydramine) using Hypercarb. Conclusion

Figure 10. Columns: 100  0.32 mm, 5 mm, mobile phase: A, H2O þ 0.1% formic acid and B, ACN þ 0.1% formic acid. Gradient: 0 – 25% B in 15 min. Flow rate: 8 mL/min. Temperature: 258C. Detection: UV 254 nm. Analytes: 1. cytosine, 2. uracil, 3. guanine, 4. adenine, 5. xanthine and 6. thymine. Courtesy: Tony Edge from Thermo Fisher Scientific.

both reversed phase-type mobile phases or HILIC-type mobile phases (184 – 186). For example, diquat and paraquat, which have high water solubility, are separated in RPLC mode using a 12 Borges

The development of new second-generation hybrid silica, Type C silica, PGC and zirconia polymer-coated stationary phases now allows the chromatographer to exploit a much wider design space (i.e., temperature and pH), including alkaline mobile phases that were previously prohibited, and hence the probability of satisfying the desired chromatographic selectivity/resolution is increased. These new types of stationary phases have also been shown to provide high efficiencies and excellent peak shape for the chromatography of basic compounds especially when they are analyzed in their ion-suppressed mode. Thin and symmetrical peaks under high and low ionic-strength mobile phases have been demonstrated. This property is of major importance in rapid generic screening using LC–MS, which often employs solvent switching to evaluate compound purity in low and high pH mobile phases. HILIC is a good choice to analyze ionizable pharmaceuticals, because it affords good peak shape often under LC –MS/MS compatible conditions with high organic content mobile phases. In this context, Type C silica is a valuable choice because it affords higher retention than bare Type B silica and it is much more stable than bare Type B silica. Stationary phases that possess either polar-embedded functionality, polar endcapping group (i.e., CSH, aqua) have been

Figure 11. Separation of some hydrophilic compounds in Hypercarb column. Columns: (A) High hydrophilic herbicides, column: Hypercarb (50  4.0 mm, dp 5 mm). Mobile phase: A, 0.05% TFA and B, 0.05% TFA in acetonitrile. Gradient: 5 –35% B in 10 min. Flow rate: 0.8 mL/min. Detection: UV 295 nm, 0 –3 min; 245 nm, 3– 10 min. Analytes: 1 ¼ diquat and 2 ¼ paraquat. (B) Glucosamine sulfate analysis under LC–MS friendly conditions. Column: Hypercarb 3 mm 100 3 2.1 mm. Mobile phase: A, 0.1% ammonia and B, ACN. Isocratic (A : B) 50 : 50. Flow rate: 0.2 mL/min. Temperature: 608C. Detection: Negative ESI-MS (4508C, 3.5 kV, 22 V). Analyte: Glucosamine sulfate. Courtesy: Tony Edge from Thermo Fisher Scientific.

demonstrating to be valuable choices/alternatives for the analysis of basic pharmaceuticals. However, these phases are normally less stable to alkaline conditions than simple C18 functionalized hybrid silica and zirconia polymer-coated stationary phases.

4. 5.

Acknowledgments The author acknowledge financial support and the fellowship (Processo: 11/07466-0) from FAPESP (Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜ o Paulo), Conselho Nacional de Desenvolvimento Cientı´ fico e Tecnolo´ gico (CNPq 150098/ 2014-6) and Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´ vel Superior (CAPES). The author also thank Prof. Melvin R. Euerby (University of Strathclyde), Joseph J. Pesek (San Jose State University), Szabolcs Fekete (University of Geneva), Tony Edge (Thermo Fisher Scientific) and Radim Kucˇera (Charles University in Prague) for their help, friendship and endless support.

References 1. McCalley, D.V.; The challenges of the analysis of basic compounds by high performance liquid chromatography: some possible approaches for improved separations; Journal of Chromatography A, (2010); 1217(6): 858– 880. 2. Fekete, S., Guillarme, D.; Reversed-phase liquid chromatography for the analysis of therapeutic proteins and recombinant monoclonal antibodies; LC-GC Europe, (2012); 25(10): 540–550. 3. Fekete, S., Gassner, A.L., Rudaz, S., Schappler, J., Guillarme, D.; Analytical strategies for the characterization of therapeutic

6.

7.

8.

9.

10.

11.

monoclonal antibodies; TrAC—Trends in Analytical Chemistry, (2013); 42: 74 –83. Sano, A., Nakamura, H.; Recent advances in materials for separation; Bunseki Kagaku, (2007); 56(5): 279–297. Periat, A., Grand-Guillaume Perrenoud, A., Guillarme, D.; Evaluation of various chromatographic approaches for the retention of hydrophilic compounds and MS compatibility; Journal of Separation Science, (2013); 36(19): 3141–3151. Periat, A., Debrus, B., Rudaz, S., Guillarme, D.; Screening of the most relevant parameters for method development in ultra-high performance hydrophilic interaction chromatography; Journal of Chromatography A, (2013); 1282: 72 –83. Periat, A., Boccard, J., Veuthey, J.L., Rudaz, S., Guillarme, D.; Systematic comparison of sensitivity between hydrophilic interaction liquid chromatography and reversed phase liquid chromatography coupled with mass spectrometry; Journal of Chromatography A, (2013); 1312: 49 –57. Jandera, P.; Stationary and mobile phases in hydrophilic interaction chromatography: a review; Analytica Chimica Acta, (2011); 692(1–2): 1– 25. Qiu, H., Liang, X., Sun, M., Jiang, S.; Development of silica-based stationary phases for high-performance liquid chromatography; Analytical and Bioanalytical Chemistry, (2011); 399(10): 3307– 3322. Zoffoli, H.J.O., do Amaral-Sobrinho, N.M.B., Zonta, E., Luisi, M.V., Marcon, G., Tolo´n-Becerra, A.; Inputs of heavy metals due to agrochemical use in tobacco fields in Brazil’s Southern Region; Environmental Monitoring and Assessment, (2013); 185(3): 2423– 2437. Borges, E.M., Euerby, M.R.; An appraisal of the chemical and thermal stability of silica based reversed-phase liquid chromatographic stationary phases employed within the pharmaceutical environment; Journal of Pharmaceutical and Biomedical Analysis, (2013); 77: 100–115.

Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases 13

12. Unger, K.K., Liapis, A.I.; Adsorbents and columns in analytical highperformance liquid chromatography: a perspective with regard to development and understanding; Journal of Separation Science, (2012); 35(10–11): 1201–1212. 13. de Villiers, A., Lestremau, F., Szucs, R., Ge´le´bart, S., David, F., Sandra, P.; Evaluation of ultra performance liquid chromatography: part I. Possibilities and limitations; Journal of Chromatography A, (2006); 1127(1):60– 69. 14. Fekete, S., Ganzler, K., Fekete, J.; Facts and myths about columns packed with sub-3mm and sub-2 mm particles; Journal of Pharmaceutical and Biomedical Analysis, (2010); 51(1): 56 –64. 15. Swartz, M.E.; UPLCTM : an introduction and review; Journal of Liquid Chromatography & Related Technologies, (2005); 28(7-8): 1253–1263. 16. Borges, E.M., Rostagno, M.A., Meireles, M.A.A.; Sub-2 mm fully porous and partially porous (core – shell) stationary phases for reversed phase liquid chromatography; RSC Advances, (2014); 4(44): 22875– 22887. 17. Guillarme, D., Nguyen, D.T., Rudaz, S., Veuthey, J.-L.; Method transfer for fast liquid chromatography in pharmaceutical analysis: application to short columns packed with small particle. Part II: gradient experiments; European Journal of Pharmaceutics and Biopharmaceutics, (2008); 68(2): 430–440. 18. Guillarme, D., Nguyen, D.T.-T., Rudaz, S., Veuthey, J.-L.; Method transfer for fast liquid chromatography in pharmaceutical analysis: application to short columns packed with small particle. Part I: isocratic separation; European Journal of Pharmaceutics and Biopharmaceutics, (2007); 66(3):475–482. 19. Guillarme, D., Veuthey, J.-L. UHPLC in life sciences; Royal Society of Chemistry, Cambridge, UK (2012). 20. Nova´kova´, L., Veuthey, J.L., Guillarme, D.; Practical method transfer from high performance liquid chromatography to ultra-high performance liquid chromatography: the importance of frictional heating; Journal of Chromatography A, (2011); 1218(44): 7971 – 7981. 21. Korma´ny, R., Fekete, J., Guillarme, D., Fekete, S.; Reliability of computer-assisted method transfer between several column dimensions packed with 1.3–5 mm core–shell particles and between various instruments; Journal of Pharmaceutical and Biomedical Analysis, (2014); 94: 188–195. 22. Fekete, S., Kohler, I., Rudaz, S., Guillarme, D.; Importance of instrumentation for fast liquid chromatography in pharmaceutical analysis; Journal of Pharmaceutical and Biomedical Analysis, (2014); 87: 105–119. 23. Nova´kova´, L., Matysova´, L., Solich, P.; Advantages of application of UPLC in pharmaceutical analysis; Talanta, (2006); 68(3): 908–918. 24. Kirkland, J.J.; Superficially porous silica microspheres for the fast high-performance liquid chromatography of macromolecules; Analytical Chemistry, (1992); 64(11): 1239– 1245. 25. Kirkland, J.J.; Ultrafast reversed-phase high-performance liquid chromatographic separations: an overview; Journal of Chromatographic Science, (2000); 38(12): 535–544. 26. Kirkland, J.J., Langlois, T.J., DeStefano, J.J.; Fused core particles for HPLC columns; American Laboratory, (2007); 39(8): 18– 21. 27. Kirkland, J.J., Truszkowski, F.A., Dilks, C.H., Jr, Engel, G.S.; Superficially porous silica microspheres for fast high-performance liquid chromatography of macromolecules; Journal of Chromatography A, (2000); 890(1): 3–13. 28. Guillarme, D., Ruta, J., Rudaz, S., Veuthey, J.-L.; New trends in fast and high-resolution liquid chromatography: a critical comparison of existing approaches; Analytical and Bioanalytical Chemistry, (2010); 397(3): 1069–1082. 29. Schuster, S.A., Wagner, B.M., Boyes, B.E., Kirkland, J.J.; Optimized superficially porous particles for protein separations; Journal of Chromatography A, (2013); 1315: 118–126. 30. Vanderheyden, Y., Cabooter, D., Desmet, G., Broeckhoven, K.; Isocratic and gradient impedance plot analysis and comparison of some recently introduced large size core – shell and fully porous particles; Journal of Chromatography A, (2013); 1312: 80 –86.

14 Borges

31. Ruta, J., Zurlino, D., Grivel, C., Heinisch, S., Veuthey, J.-L., Guillarme, D.; Evaluation of columns packed with shell particles with compounds of pharmaceutical interest; Journal of Chromatography A, (2012); 1228: 221–231. 32. Fekete, S., Berky, R., Fekete, J., Veuthey, J.-L., Guillarme, D.; Evaluation of recent very efficient wide-pore stationary phases for the reversed-phase separation of proteins; Journal of Chromatography A, (2012); 1252: 90– 103. 33. Ola´h, E., Fekete, S., Fekete, J., Ganzler, K.; Comparative study of new shell-type, sub-2 mm fully porous and monolith stationary phases, focusing on mass-transfer resistance; Journal of Chromatography A, (2010); 1217(23): 3642–3653. 34. Fekete, S., Veuthey, J.-L., Eeltink, S., Guillarme, D.; Comparative study of recent wide-pore materials of different stationary phase morphology, applied for the reversed-phase analysis of recombinant monoclonal antibodies; Analytical and Bioanalytical Chemistry, (2013); 405(10): 3137–3151. 35. Cunliffe, J.M., Maloney, T.D.; Fused-core particle technology as an alternative to sub-2-mm particles to achieve high separation efficiency with low backpressure; Journal of Separation Science, (2007); 30(18): 3104–3109. 36. Fekete, S., Guillarme, D.; Possibilities of new generation columns packed with 1.3 mm core – shell particles in gradient elution mode; Journal of Chromatography A, (2013); 1320: 86 –95. 37. Broeckhoven, K., Cabooter, D., Eeltink, S., Desmet, G.; Kinetic plot based comparison of the efficiency and peak capacity of highperformance liquid chromatography columns: theoretical background and selected examples; Journal of Chromatography A, (2012); 1228: 20– 30. 38. Fountain, K.J., Neue, U.D., Grumbach, E.S., Diehl, D.M.; Effects of extra-column band spreading, liquid chromatography system operating pressure, and column temperature on the performance of sub-2-mm porous particles; Journal of Chromatography A, (2009); 1216(32): 5979–5988. 39. Kahsay, G., Broeckhoven, K., Adams, E., Desmet, G., Cabooter, D.; Kinetic performance comparison of fully and superficially porous particles with a particle size of 5mm: intrinsic evaluation and application to the impurity analysis of griseofulvin; Talanta, (2014); 122: 122– 129. 40. Gritti, F., Guiochon, G.; Speed-resolution properties of columns packed with new 4.6 mm Kinetex-C18 core – shell particles; Journal of Chromatography A, (2013); 1280: 35 –50. 41. Yang, X., Dai, J., Carr, P.W.; Analysis and critical comparison of the reversed-phase and ion-exchange contributions to retention on polybutadiene coated zirconia and octadecyl silane bonded silica phases; Journal of Chromatography A, (2003); 996(1– 2): 13 –31. 42. Yang, X., Dai, J., Carr, P.W.; Analysis and critical comparison of the reversed-phase and ion-exchange contributions to retention on polybutadiene coated zirconia and octadecyl silane bonded silica phases; Journal of Chromatography A, (2003); 996(1 – 2): 13– 31. 43. Marchand, D.H., Carr, P.W., McCalley, D.V., Neue, U.D., Dolan, J.W., Snyder, L.R.; Contributions to reversed-phase column selectivity. II. Cation exchange; Journal of Chromatography A, (2011); 1218(40): 7110– 7129. 44. Neue, U.D., Tran, K., Me´ndez, A., Carr, P.W.; The combined effect of silanols and the reversed-phase ligand on the retention of positively charged analytes; Journal of Chromatography A, (2005); 1063(1– 2): 35– 45. 45. Szulfer, J., Plenis, A., Baczek, T.; Evaluation of a column classification method using the separation of alfuzosin from its related substances; Journal of Chromatography A, (2012); 1229: 198– 207. 46. Borges, E.M.; How to select equivalent and complimentary reversed phase liquid chromatography columns from column characterization databases; Analytica Chimica Acta, (2014); 807: 143–152. 47. Snyder, L.R., Dolan, J.W.; Optimizing selectivity during reversedphase high performance liquid chromatography method development: prioritizing experimental conditions; Journal of Chromatography A, (2013); 1302: 45 –54.

48. Neue, U.D., Van Tran, K., Iraneta, P.C., Alden, B.A.; Characterization of HPLC packings; Journal of Separation science, (2003); 26(3 –4): 174– 186. 49. Euerby, M.R., Petersson, P.; Chromatographic classification and comparison of commercially available reversed-phase liquid chromatographic columns using principal component analysis; Journal of Chromatography A, (2003); 994(1): 13– 36. 50. Chamseddin, C., Molna´r, I., Jira, T.; Intergroup cross-comparison for the evaluation of data-interchangeability from various chromatographic tests; Journal of Chromatography A, (2013); 1297: 146– 156. 51. Nawrocki, J., Buszewski, B.; Influence of silica surface chemistry and structure on the properties, structure and coverage of alkyl-bonded phases for high-performance liquid chromatography; Journal of Chromatography A, (1988); 449(C): 1 –24. 52. Nawrocki, J.; The strength of interaction of highly retentive silanols with hydrocarbons on porasil C; Chromatographia, (1989); 28(3 – 4): 139–142. 53. Nawrocki, J., Moir, D.L., Szczepaniak, W.; Investigation of small populations of reactive silanols on silica surfaces; Journal of Chromatography A, (1989); 467(C): 31 –40. 54. Nawrocki, J., Moir, D.L., Szczepaniak, W.; Trace metal impurities in silica as a cause of strongly interacting silanols; Chromatographia, (1989); 28(3 –4): 143– 147. 55. Nawrocki, J.; Silica surface controversies, strong adsorption sites, their blockage in removal. Part II; Chromatographia, (1991); 31(3– 4): 193–205. 56. Kirkland, J.J., Van Straten, M.A., Claessens, H.A.; Reversed-phase high-performance liquid chromatography of basic compounds at pH 11 with silica-based column packings; Journal of Chromatography A, (1998); 797(1– 2): 111–120. 57. Engelhardt, H., Gru¨ner, R., Scherer, M.; The polar selectivities of non-polar reversed phases; Chromatographia, (2001); 53(1): S154–S161. 58. O’Sullivan, G.P., Scully, N.M., Glennon, J.D.; Polar-embedded and polar-endcapped stationary phases for LC; Analytical Letters, (2010); 43(10–11): 1609–1629. 59. Layne, J.; Characterization and comparison of the chromatographic performance of conventional, polar-embedded, and polarendcapped reversed-phase liquid chromatography stationary phases; Journal of Chromatography A, (2002); 957(2): 149–164. 60. Rimmer, C.A., Sander, L.C.; Shape selectivity in embedded polar group stationary phases for liquid chromatography; Analytical and Bioanalytical Chemistry, (2009); 394(1): 285– 291. 61. Qiu, H., Mallik, A.K., Takafuji, M., Liu, X., Jiang, S., Ihara, H.; A new imidazolium-embedded C18 stationary phase with enhanced performance in reversed-phase liquid chromatography; Analytica Chimica Acta, (2012); 738: 95 –101. 62. Maldaner, L., Collins, C.H., Jardim, I.C.; Modern stationary phases for reversed phase high performance liquid chromatography; Quı´mica Nova, (2010); 33(7): 1559–1568. 63. Przybyciel, M., Majors, R.E.; Phase collapse in reversed-phase liquid chromatography; LC GC North America, (2002); 20(6): 516– 523. 64. Majors, R.E., Przybyciel, M.; Columns for reversed-phase LC separations in highly aqueous mobile phases; LC GC North America, (2002); 20(7): 584– 593. 65. Rafferty, J.L., Siepmann, J.I., Schure, M.R.; Molecular-level comparison of alkylsilane and polar-embedded reversed-phase liquid chromatography systems; Analytical Chemistry, (2008); 80(16): 6214–6221. 66. Neue, U.D., Walter, T.H., Alden, B.A., Jiang, Z., Fisk, R.P., Cook, J.T., et al..; Use of high-performance LC packings from pH 1 to pH 12; American Laboratory, (1999); 31(22): 36– 39. 67. Wyndham, K.D., O’Gara, J.E., Walter, T.H., Glose, K.H., Lawrence, N.L., Alden, B.A., et al.; Characterization and evaluation of C18 HPLC stationary phases based on ethyl-bridged hybrid organic/inorganic particles; Analytical Chemistry, (2003); 75(24): 6781–6788. 68. O’Gara, J.E., Wyndham, K.D.; Porous hybrid organic– inorganic particles in reversed-phase liquid chromatography; Journal of Liquid

69.

70.

71.

72.

73.

74.

75. 76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

Chromatography and Related Technologies, (2006); 29(7 – 8): 1025–1045. Neue, U.D., Kele, M., Mazzeo, J.R., Grumbach, E.S., Wyndham, K.D., Morawski, J., et al.; Ultra-performance LC: from high to ultra; Ultra-performance LC: Von “high” zu “ultra”. Nachrichten aus der Chemie, (2004); 52(11): 1217–1218. Liu, Y., Grinberg, N., Thompson, K.C., Wenslow, R.M., Neue, U.D., Morrison, D., et al..; Evaluation of a C18 hybrid stationary phase using high-temperature chromatography; Analytica Chimica Acta, (2005); 554(1–2): 144– 151. Wang, Y., Ai, F., Ng, S.-C., Tan, T.T.Y.; Sub-2 mm porous silica materials for enhanced separation performance in liquid chromatography; Journal of Chromatography A, (2012); 1228: 99 –109. Peng, L., Farkas, T.; Analysis of basic compounds by reversed-phase liquid chromatography– electrospray mass spectrometry in high-pH mobile phases; Journal of Chromatography A, (2008); 1179(2): 131–144. Undin, T., Samuelsson, J., To¨rncrona, A., Fornstedt, T.; Evaluation of a combined linear – nonlinear approach for column characterization using modern alkaline-stable columns as model; Journal of Separation Science, (2013); 36(11): 1753– 1761. Rodriguez-Aller, M., Gurny, R., Veuthey, J.L., Guillarme, D.; Coupling ultra high-pressure liquid chromatography with mass spectrometry: constraints and possible applications; Journal of Chromatography A, (2013); 1292: 2– 18. Holcˇapek, M., Jandera, P.; Coupling of liquid chromatography and mass spectrometry; Chemicke Listy, (1998); 92(4): 283– 286. Holcˇapek, M., Jira´sko, R., Lı´ sa, M.; Recent developments in liquid chromatography-mass spectrometry and related techniques; Journal of Chromatography A, (2012); 1259: 3–15. Guillarme, D., Schappler, J., Rudaz, S., Veuthey, J.L.; Coupling ultra-high-pressure liquid chromatography with mass spectrometry; TrAC—Trends in Analytical Chemistry, (2010); 29(1): 15– 27. Nova´kova´, L.; Challenges in the development of bioanalytical liquid chromatography mass spectrometry method with emphasis on fast analysis; Journal of Chromatography A, (2013); 1292: 25– 37. Heinisch, S., D’Attoma, A., Grivel, C.; Effect of pH additive and column temperature on kinetic performance of two different sub-2 mm stationary phases for ultrafast separation of charged analytes; Journal of Chromatography A, (2012); 1228: 135–147. Fekete, S., Rudaz, S., Veuthey, J.L., Guillarme, D.; Impact of mobile phase temperature on recovery and stability of monoclonal antibodies using recent reversed-phase stationary phases; Journal of Separation Science, (2012); 35(22): 3113–3123. Fekete, S., Rudaz, S., Fekete, J., Guillarme, D.; Analysis of recombinant monoclonal antibodies by RPLC: toward a generic method development approach; Journal of Pharmaceutical and Biomedical Analysis, (2012); 70: 158– 168. Wyndham, K., Walter, T., Iraneta, P.C., Alden, B., Bouvier, E., Hudalla, C.J., et al..; Synthesis and applications of BEH particles in liquid chromatography; LC-GC North America, (2012); 30(S4): 20– 29. Gritti, F., Guiochon, G.; Effect of the pH and the ionic strength on overloaded band profiles of weak bases onto neutral and charged surface hybrid stationary phases in reversed-phase liquid chromatography; Journal of Chromatography A, (2013); 1282: 113–126. Gritti, F., Guiochon, G.; Adsorption behaviors of neutral and ionizable compounds on hybrid stationary phases in the absence (BEH-C18) and the presence (CSH-C18) of immobile surface charges; Journal of Chromatography A, (2013); 1282: 58 –71. Gritti, F., Guiochon, G.; Effect of the ionic strength on the adsorption process of an ionic surfactant onto a C18-bonded charged surface hybrid stationary phase at low pH; Journal of Chromatography A, (2013); 1282: 46 –57. Nova´kova´, L., Vlcˇkova´, H., Petr, S.; Evaluation of new mixed-mode UHPLC stationary phases and the importance of stationary phase choice when using low ionic-strength mobile phase additives; Talanta, (2012); 93: 99– 105. Bocian, S., Rychlicki, G., Matyska, M., Pesek, J., Buszewski, B.; Study of hydration process on silica hydride surfaces by microcalorimetry

Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases 15

88.

89.

90.

91.

92.

93.

94.

95.

96.

97.

98.

99.

100.

101.

102.

103.

and water adsorption; Journal of Colloid and Interface Science, (2014); 416: 161– 166. Young, J.E., Matyska, M.T., Azad, A.K., Yoc, S.E., Pesek, J.J.; Separation differences among phenyl hydride, UDC cholesterol, and bidentate C8 stationary phases for stability indicating methods of tetracyclines; Journal of Liquid Chromatography and Related Technologies, (2013); 36(7): 926–942. Yang, Y., Matyska, M.T., Boysen, R.I., Pesek, J.J., Hearn, M.T.W.; Simultaneous separation of hydrophobic and polar bases using a silica hydride stationary phase; Journal of Separation Science, (2013); 36(7): 1209– 1216. Yang, Y., Boysen, R.I., Kulsing, C., Matyska, M.T., Pesek, J.J., Hearn, M.T.W.; Analysis of polar peptides using a silica hydride column and high aqueous content mobile phases; Journal of Separation Science, (2013); 36(18): 3019– 3025. Pesek, J.J., Matyska, M.T., Kim, A.M.; Evaluation of stationary phases based on silica hydride for the analysis of drugs of abuse; Journal of Separation Science, (2013); 36(17): 2760–2766. Pesek, J.J., Matyska, M.T., Boysen, R.I., Yang, Y., Hearn, M.T.W.; Aqueous normal-phase chromatography using silica-hydride-based stationary phases; TrAC—Trends in Analytical Chemistry, (2013); 42: 64– 73. Le, R., Young, J.E., Pesek, J.J., Matyska, M.T.; Separation of 1,3-dimethylamylamine and other polar compounds in a dietary supplement formulation using aqueous normal phase chromatography with MS; Journal of Separation Science, (2013); 36(16): 2578–2583. Dang, A., Pesek, J.J., Matyska, M.T.; The use of aqueous normal phase chromatography as an analytical tool for food analysis: determination of histamine as a model system; Food Chemistry, (2013); 141(4): 4226–4230. Yeman, H., Nicholson, T.M., Friebolin, V., Steinhauser, L., Matyska, M.T., Pesek, J.J., et al.; Time-dependent column performance of cholesterol-based stationary phases for HPLC by LC characterization and solid-state NMR spectroscopy; Journal of Separation Science, (2012); 35(13): 1582– 1588. Pesek, J.J., Matyska, M.T., Dang, A.; Analysis of cycloserine and related compounds using aqueous normal phase chromatography/mass spectrometry; Journal of Pharmaceutical and Biomedical Analysis, (2012); 64– 65: 72– 76. Pesek, J.J., Matyska, M.T.; A new approach to bioanalysis: aqueous normal-phase chromatography with silica hydride stationary phases; Bioanalysis, (2012); 4(7): 845–853. MacNeill, R., Stromeyer, R., Urbanowicz, B., Acharya, V., Moussallie, M., Pesek, J.J.; Silica hydride-based chromatography of LC – MS response-altering compounds native to human plasma; Bioanalysis, (2012); 4(24): 2877–2886. Buszewski, B., Bocian, S., Rychlicki, G., Matyska, M., Pesek, J.; Determination of accessible silanols groups on silica gel surfaces using microcalorimetric measurements; Journal of Chromatography A, (2012); 1232: 43–46. Bocian, S., Soukup, J., Matyska, M., Pesek, J., Jandera, P., Buszewski, B.; The influence of the organic modifier in hydro-organic mobile phase on separation selectivity of steroid hormones separation using cholesterol-bonded stationary phases; Journal of Chromatography A, (2012); 1245: 90–97. Young, J.E., Matyska, M.T., Pesek, J.J.; Liquid chromatography/mass spectrometry compatible approaches for the quantitation of folic acid in fortified juices and cereals using aqueous normal phase conditions; Journal of Chromatography A, (2011); 1218(15): 2121–2126. Pesek, J.J., Matyska, M.T., Nshanian, M.; Open-tubular capillary electrochromatography of small polar molecules using etched, chemically modified capillaries; Electrophoresis, (2011); 32(13): 1728–1734. Pesek, J.J., Matyska, M.T., Lee, P.; Synthesis of a preparative C30 stationary phase on a silica hydride surface and its application to carotenoid separation; Journal of Liquid Chromatography and Related Technologies, (2011); 34(3): 231–240.

16 Borges

104. Pesek, J.J., Matyska, M.T., Fischer, S.M.; Improvement of peak shape in aqueous normal phase analysis of anionic metabolites; Journal of Separation Science, (2011); 34(24): 3509– 3516. 105. Buszewski, B., Bocian, S., Matyska, M., Pesek, J.; Study of solvation processes on cholesterol bonded phases; Journal of Chromatography A, (2011); 1218(3): 441–448. 106. Boysen, R.I., Yang, Y., Chowdhury, J., Matyska, M.T., Pesek, J.J., Hearn, M.T.W.; Simultaneous separation of hydrophobic and hydrophilic peptides with a silica hydride stationary phase using aqueous normal phase conditions; Journal of Chromatography A, (2011); 1218(44): 8021–8026. 107. Pesek, J.J., Matyska, M.T., Prajapati, K.V.; Synthesis and evaluation of silica hydride-based fluorinated stationary phases; Journal of Separation Science, (2010); 33(19): 2908– 2916. 108. Pesek, J.J., Matyska, M.T.; Silica hydride—chemistry and applications; Advances in Chromatography, (2010); 48: 255–288. 109. Matyska, M.T., Pesek, J.J., Shety, G.; Type C amino columns for affinity and aqueous normal phase chromatography: synthesis and HPLC evaluation; Journal of Liquid Chromatography and Related Technologies, (2010); 33(1): 1–26. 110. Matyska, M.T., Pesek, J.J., Duley, J., Zamzami, M., Fischer, S.M.; Aqueous normal phase retention of nucleotides on silica hydridebased columns: method development strategies for analytes relevant in clinical analysis; Journal of Separation Science, (2010); 33(6– 7): 930– 938. 111. Bocian, S., Matyska, M., Pesek, J., Buszewski, B.; Study of the retention and selectivity of cholesterol bonded phases with different linkage spacers; Journal of Chromatography A, (2010); 1217(44): 6891–6897. 112. Plumere´, N., Speiser, B., Dietrich, B., Albert, K., Pesek, J.J., Matyska, M.T.; Thermally induced radical hydrosilylation for synthesis of C18 HPLC phases from highly condensed SiH terminated silica surfaces; Langmuir, (2009); 25(23): 13481– 13487. 113. Pesek, J.J., Matyska, M.T., Loo, J.A., Fischer, S.M., Sana, T.R.; Analysis of hydrophilic metabolites in physiological fluids by HPLC-MS using a silica hydride-based stationary phase; Journal of Separation Science, (2009); 32(13): 2200–2208. 114. Pesek, J.J., Matyska, M.T., Hearn, M.T.W., Boysen, R.I.; Aqueous ormal-phase retention of nucleotides on silica hydride columns; Journal of Chromatography A, (2009); 1216(7): 1140–1146. 115. Pesek, J.J., Matyska, M.T.; Our favorite materials: silica hydride stationary phases; Journal of Separation Science, (2009); 32(23 – 24): 3999– 4011. 116. Friebolin, V., Bayer, M.P., Matyska, M.T., Pesek, J.J., Albert, K.; 1H HR/ MAS NMR in the suspended state: molecular recognition processes in liquid chromatography between steroids and a silica hydridebased cholesterol phase; Journal of Separation Science, (2009); 32(10): 1722–1728. 117. Pesek, J.J., Matyska, M.T., Venkat, J.P.; Evaluation of protein, peptide, and amino acid retention on C5 hydride-based stationary phases; Journal of Separation Science, (2008); 31(14): 2560– 2566. 118. Pesek, J.J., Matyska, M.T., Sukul, D.; Capillary liquid chromatography and capillary electrochromatography using silica hydride stationary phases; Journal of Chromatography A, (2008); 1191(1 – 2): 136–140. 119. Pesek, J.J., Matyska, M.T., Salgotra, V.; Retention of proteins and metalloproteins in open tubular capillary electrochromatography with etched chemically modified columns; Electrophoresis, (2008); 29(18): 3842–3849. 120. Pesek, J.J., Matyska, M.T., Fischer, S.M., Sana, T.R.; Analysis of hydrophilic metabolites by high-performance liquid chromatography-mass spectrometry using a silica hydride-based stationary phase; Journal of Chromatography A, (2008); 1204(1): 48–55. 121. Pesek, J.J., Matyksa, M.T., Sharma, A.; Use of hydride-based separation materials for organic normal phase chromatography; Journal of Liquid Chromatography and Related Technologies, (2008); 31(1): 134– 147.

122. Santali, E.Y., Edwards, D., Sutcliffe, O.B., Bailes, S., Euerby, M.R., Watson, D.G.; A Comparison of Silica C and Silica Gel in HILIC Mode: The Effect of Stationary Phase Surface Area; Chromatographia, (2014); 77(13–14): 873–881. 123. Yang, Y., Boysen, R.I., Matyska, M.T., Pesek, J.J., Hearn, M.T.W.; Open-tubular capillary electrochromatography coupled with electrospray ionization mass spectrometry for peptide analysis; Analytical Chemistry, (2007); 79(13): 4942–4949. 124. Pesek, J.J., Matyska, M.T., Larrabee, S.; HPLC retention behavior on hydride-based stationary phases; Journal of Separation Science, (2007); 30(5): 637– 647. 125. Pesek, J.J., Matyska, M.T., Hearn, M.T., Boysen, R.; Temperature effects on solute retention for hydride-based stationary phases; Journal of Separation Science, (2007); 30(8): 1150–1157. 126. Pesek, J., Matyska, M.T.; A comparison of two separation modes: HILIC and aqueous normal phase chromatography; LC-GC North America, (2007); 25(5): 480–490. 127. Pesek, J.J., Matyska, M.T., Velpula, S.; Open tubular capillary electrochromatography migration behavior of enkephalins in etched chemically modified fused silica capillaries; Journal of Chromatography A, (2006); 1126(1–2): 298– 303. 128. Pesek, J.J., Matyska, M.T., Gangakhedkar, S., Siddiq, R.; Synthesis and HPLC evaluation of carboxylic acid phases on a hydride surface; Journal of Separation Science, (2006); 29(6): 872– 880. 129. Pesek, J.J., Matyska, M.T.; How to retain polar and nonpolar compounds on the same HPLC stationary phase with an isocratic mobile phase; LC-GC North America, (2006); 24(3): 296–303. 130. Pesek, J.J., Matyska, M.T.; Silica hydride surfaces: versatile separation media for chromatographic and electrophoretic analyses; Journal of Liquid Chromatography and Related Technologies, (2006); 29(7 –8): 1105– 1124. 131. Pesek, J.J., Matyska, M.T.; How to retain polar and nonpolar compounds on the same HPLC stationary phase with an isocratic mobile phase; LC-GC North America, (2006); 24(6 Suppl.): 90 –95. 132. Pesek, J.J., Matyska, M.T., Young, J.E.; Analysis of thiopurines using aqueous normal phase chromatography; Journal of Pharmaceutical and Biomedical Analysis, (2014); 95: 102– 106. 133. Pesek, J.J., Matyska, M.T., Bloomquist, T., Carlon, G.; Analysis of antibiotics in milk using open tubular capillary electrochromatography; Journal of Liquid Chromatography and Related Technologies, (2005); 28(19): 3015–3024. 134. Melnikov, S.M., Ho¨ltzel, A., Seidel-Morgenstern, A., Tallarek, U.; A molecular dynamics study on the partitioning mechanism in hydrophilic interaction chromatography; Angewandte Chemie International Edition, (2012); 51(25): 6251– 6254. 135. Banerjee, S., Mazumdar, S.; Electrospray ionization mass spectrometry: a technique to access the information beyond the molecular weight of the analyte; International Journal of Analytical Chemistry, (2012); 2012:doi:10.1155/2012/282574. 136. Rakic´, T., Jancic Stojanovic, B., Malenovic´, A., Ivanovic´, D., Medenica, M.; Improved chromatographic response function in HILIC analysis: application to mixture of antidepressants; Talanta, (2012); 98: 54 –61. 137. Buszewski, B., Noga, S.; Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique; Analytical and Bioanalytical Chemistry, (2012); 402(1): 231–247. 138. Hemstro¨m, P., Irgum, K.; Hydrophilic interaction chromatography; Journal of Separation Science, (2006); 29(12): 1784– 1821. 139. Bernal, J., Ares, A.M., Po´l, J., Wiedmer, S.K.; Hydrophilic interaction liquid chromatography in food analysis; Journal of Chromatography A, (2011); 1218(42): 7438–7452. 140. Dejaegher, B., Vander Heyden, Y.; HILIC methods in pharmaceutical analysis; Journal of Separation Science, (2010); 33(6– 7): 698–715. 141. Gama, M.R., da Costa Silva, R.G., Collins, C.H., Bottoli, C.B.G.; Hydrophilic interaction chromatography; TrAC—Trends in Analytical Chemistry, (2012); 37: 48 –60. 142. Garcı´ a-Go´mez, D., Rodrı´ guez-Gonzalo, E., Carabias-Martı´ nez, R.; Stationary phases for separation of nucleosides and nucleotides

143.

144.

145.

146.

147.

148.

149.

150.

151.

152.

153.

154.

155.

156.

157.

158.

159.

160.

161.

by hydrophilic interaction liquid chromatography; TrAC—Trends in Analytical Chemistry, (2013); 47: 111– 128. Guo, Y., Gaiki, S.; Retention and selectivity of stationary phases for hydrophilic interaction chromatography; Journal of Chromatography A, (2011); 1218(35): 5920– 5938. Kahsay, G., Song, H., Van Schepdael, A., Cabooter, D., Adams, E.; Hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics; Journal of Pharmaceutical and Biomedical Analysis, (2014); 87: 142– 154. Noga, S., Felinger, A., Buszewski, B.; Hydrophilic interaction liquid chromatography and per aqueous liquid chromatography in fungicides analysis; Journal of AOAC International, (2012); 95(5): 1362–1370. Nova´kova´, L.; Challenges in the development of bioanalytical liquid chromatography– mass spectrometry method with emphasis on fast analysis; Journal of Chromatography A, (2013); 1292: 25 –37. Zauner, G., Deelder, A.M., Wuhrer, M.; Recent advances in hydrophilic interaction liquid chromatography (HILIC) for structural glycomics; Electrophoresis, (2011); 32(24): 3456–3466. Greco, G., Letzel, T.; Main interactions and influences of the chromatographic parameters in HILIC separations; Journal of Chromatographic Science, (2013); 51(7): 684–693. Jovanovic´, M., Rakic´, T., Jancˇic´ –Stojanovic´, B.; Theoretical and empirical models in hydrophilic interaction liquid chromatography; Instrumentation Science & Technology, (2014); 42(3): 230–266. Louw, S., Lynen, F., Hanna-Brown, M., Sandra, P.; High-efficiency hydrophilic interaction chromatography by coupling 25 cm  4.6 mm ID  5 mm silica columns and operation at 808C; Journal of Chromatography A, (2010); 1217(4): 514– 521. Ikegami, T., Tomomatsu, K., Takubo, H., Horie, K., Tanaka, N.; Separation efficiencies in hydrophilic interaction chromatography; Journal of Chromatography A, (2008); 1184(1): 474–503. Bawazeer, S., Sutcliffe, O.B., Euerby, M.R., Bawazeer, S., Watson, D.G.; A comparison of the chromatographic properties of silica gel and silicon hydride modified silica gels; Journal of Chromatography A, (2012); 1263: 61– 67. Nawrocki, J., Dunlap, C., McCormick, A., Carr, P.W.; Part I. Chromatography using ultra-stable metal oxide-based stationary phases for HPLC; Journal of Chromatography A, (2004); 1028(1): 1–30. Nawrocki, J., Dunlap, C., Li, J., Zhao, J., McNeff, C.V., McCormick, A., et al.; Part II. Chromatography using ultra-stable metal oxide-based stationary phases for HPLC; Journal of Chromatography A, (2004); 1028(1): 31– 62. Dai, J., Yang, X., Carr, P.W.; Comparison of the chromatography of octadecyl silane bonded silica and polybutadiene-coated zirconia phases based on a diverse set of cationic drugs; Journal of Chromatography A, (2003); 1005(1–2): 63– 82. Hu, Y., Yang, X., Carr, P.W.; Mixed-mode reversed-phase and ionexchange separations of cationic analytes on polybutadiene-coated zirconia; Journal of Chromatography A, (2002); 968(1– 2): 17 –29. Hu, Y., Carr, P.W.; Synthesis and characterization of new zirconiabased polymeric cation-exchange stationary phases for highperformance liquid chromatography of proteins; Analytical Chemistry, (1998); 70(9): 1934–1942. Zˇizˇkovsky´, V., Kucˇera, R., Klimesˇ , J., Hola´skova´, P., Dohnal, J.; RP-ZrO2 stationary phase as an alternative to separate of doxazosin impurities; Chromatographia, (2009); 70(1–2): 185–189. Zˇizˇkovsky´, V., Kucˇera, R., Klimesˇ, J., Dohnal, J.; Titania-based stationary phase in separation of ondansetron and its related compounds; Journal of Chromatography A, (2008); 1189(1–2): 83–91. Zˇizˇkovsky´, V., Kucˇera, R., Klimesˇ , J.; Potential employment of non-silica-based stationary phases in pharmaceutical analysis; Journal of Pharmaceutical and Biomedical Analysis, (2007); 44(5): 1048–1055. Kucˇera, R., Zˇizˇkovsky´, V., Sochor, J., Klimesˇ, J., Dohnal, J.; Utilization of zirconia stationary phase as a tool in drug control; Journal of Separation Science, (2005); 28(12): 1307– 1314.

Silica, Hybrid Silica, Hydride Silica and Non-Silica Stationary Phases 17

162. Kucˇera, R., Sochor, J., Klimesˇ , J., Dohnal, J.; Use of the zirconia-based stationary phase for separation of ibuprofen and its impurities; Journal of Pharmaceutical and Biomedical Analysis, (2005); 38(4): 609–618. 163. Kucˇera, R., Kovarˇ ı´ kova´, P., Klivicky´, M., Klimesˇ, J.; The retention behaviour of polar compounds on zirconia based stationary phases under hydrophilic interaction liquid chromatography conditions; Journal of Chromatography A, (2011); 1218(39): 6981– 6986. 164. Kalafut, P., Kucˇera, R., Klimesˇ, J., Sochor, J.; An innovative approach to the analysis of 3-[4-(2-methylpropyl)phenyl]propanoic acid as an impurity of ibuprofen on a carbon-coated zirconia stationary phase; Journal of Pharmaceutical and Biomedical Analysis, (2009); 49(5): 1150–1156. 165. Kalafut, P., Kucˇera, R., Klimesˇ , J.; The influence of a carbon layer deposited on a zirconia surface on the retention of polar analytes in an organic rich mobile phase; Journal of Chromatography A, (2012); 1232: 242– 247. 166. Kalafut, P., Kucˇera, R., Klimesˇ , J.; The retention behavior of acidic, basic and neutral pharmaceuticals on the deactivated polybutadiene zirconia phase; Current Analytical Chemistry, (2012); 8(4): 574–582. 167. El Debs, R., Abi Jaoude´, M., Morin, N., Miege, C., Randon, J.; Retention of b blockers on native titania stationary phase; Journal of Separation Science, (2011); 34(15): 1805– 1810. 168. Yang, X., Dai, J., Carr, P.W.; Effect of amine counterion type on the retention of basic compounds on octadecyl silane bonded silicabased and polybutadiene-coated zirconia phases; Analytical Chemistry, (2003); 75(13): 3153– 3160. 169. Kalafut, P., Kue`era, R., Klimesˇ , J.; The retention behavior of acidic, basic and neutral pharmaceuticals on the deactivated polybutadiene zirconia phase; Current Analytical Chemistry, (2012); 8(4): 574–582. 170. Zhou, T., Lucy, C.A.; Separation of carboxylates by hydrophilic interaction liquid chromatography on titania; Journal of Chromatography A, (2010); 1217(1): 82 –88. 171. Kaliszan, R., Haber, P., Baczek, T., Siluk, D.; Gradient HPLC in the determination of drug lipophilicity and acidity; Pure and Applied Chemistry, (2001); 73(9): 1465– 1475. 172. Ulrich, N., Mu¨hlenberg, J., Schu¨u¨rmann, G., Brack, W.; Linear solvation energy relationships as classifier in non-target analysis—an approach for isocratic liquid chromatography; Journal of Chromatography A, (2014); 1324: 96 –103. 173. Koba, M., Stasiak, J., Bober, L., Baczek, T.; Evaluation of molecular descriptors and HPLC retention data of analgesic and anti-inflammatory drugs by factor analysis in relation to their pharmacological activity; Journal of molecular modeling, (2010); 16(8): 1319–1331. 174. Claessens, H.A., Van Straten, M.A., Cramers, C.A., Jezierska, M., Buszewski, B.; Comparative study of test methods for reversed-

18 Borges

175.

176.

177.

178.

179.

180.

181.

182.

183.

184. 185.

186.

187.

phase columns for high-performance liquid chromatography; Journal of Chromatography A, (1998); 826: 135–156. Jandera, P., Novotna´, K., Beldean-Galea, M.S., Jı´ sˇ a, K.; Retention and selectivity tests of silica-based and metal-oxide bonded stationary phases for RP-HPLC; Journal of Separation Science, (2006); 29(6): 856– 871. Sy´kora, D., Tesarˇ ova´, E., Popl, M.; Interactions of basic compounds in reversed-phase high-performance liquid chromatography influence of sorbent character, mobile phase composition, and pH on retention of basic compounds; Journal of Chromatography A, (1997); 758(1): 37– 51. Lambert, W.E., Meyer, E., De Leenheer, A.P.; Systematic toxicological analysis of basic drugs by gradient elution of an alumina-based HPLC packing material under alkaline conditions; Journal of Analytical Toxicology, (1995); 19(2): 73 –78. Pesek, J.J., Matyska, M.T.; Modified aluminas as chromatographic supports for high-performance liquid chromatography; Journal of Chromatography A, (2002); 952(1): 1 –11. West, C., Elfakir, C., Lafosse, M.; Porous graphitic carbon: a versatile stationary phase for liquid chromatography; Journal of Chromatography A, (2010); 1217(19): 3201–3216. Michel, M., Buszewski, B.; Porous graphitic carbon sorbents in biomedical and environmental applications; Adsorption, (2009); 15(2): 193–202. Monser, L.; Liquid chromatographic determination of four purine bases using porous graphitic carbon column; Chromatographia, (2004); 59(7– 8): 455–459. Barrett, D., Pawula, M., Knaggs, R., Shaw, P.; Retention behavior of morphine and its metabolites on a porous graphitic carbon column; Chromatographia, (1998); 47(11– 12): 667–672. Sroka-Markovic, J., Johansson, L., Andersson, M.B., Granelli, I.; Development of an HPLC method for determination of related impurities in prilocaine substance; Chromatographia, (2012); 75(7 – 8): 329– 336. Pereira, L.; HILIC-MS sensitivity without silica; LC GC North America, (2011); 29(3): 262–269. Robb, C.S., Eitzer, B.D.; The direct analysis of diquat and paraquat in lake water samples by per aqueous liquid chromatography; LC GC North America, (2011); 29(1): 54– 59. dos Santos Pereira, A., David, F., Vanhoenacker, G., Sandra, P.; The acetonitrile shortage: is reversed HILIC with water an alternative for the analysis of highly polar ionizable solutes? Journal of Separation Science, (2009); 32(12): 2001– 2007. Albert, M., Cretier, G., Guillarme, D., Heinisch, S., Rocca, J.L.; Some advantages of high temperature for the separation of pharmaceutical compounds with mass spectrometry detection; Journal of Separation Science, (2005); 28(14): 1803–1811.