ADVANCES IN HYDROPHILIC INTERACTION LIQUID
2. COLUMNS FOR HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY SEPARATIONSCHROMATOGRAPHY SEPARATIONS
2.4 MONOLITHIC COLUMNS FOR HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY SEPARATIONSCHROMATOGRAPHY SEPARATIONS
2.4.1 Silica Gel and Hybrid Monoliths
Bare silica monoliths generally show low HILIC retention. The domain size (i.e., the sum of the average sizes of the through-pores and skeleton) controls the efficiency of a silica gel monolithic
column (Vervoort et al., 2004). Coating the monolithic silica surface with cationic latex nanoparticles by electrostatic attachment provides separation media that retain the high efficiency and permeability of the native silica monolith, but show a significantly larger amount of adsorbed water, leading to stronger HILIC/ion-exchange retention (Ibrahim and Lucy, 2012). Fast separations of benzoates, nucleotides, and amino acids at high acetonitrile concentrations in the mobile phase were reported on latex-coated monoliths, based on mixed-mode electrostatic repulsionehydrophilic liquid interactions (ERLIC) (Ibrahim et al., 2010).
Hybrid monolithic silica capillary columns for HILIC were prepared by on-column polymerization of acrylic acid on monolithic silica in a fused silica capillary modified with anchor groups. The products maintained their high permeability and provided a theoretical plate height (H) of 10e20mm for polar solutes, including nucleosides and carbohydrates (Horie et al., 2007).
Zwitterionic silica-based monolithic capillary columns were prepared by grafting a layer of zwitterionic monomer ([2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide or 2-methacryloyloxyethyl phosphorylcholine) on the silica monolithic surface (Moravcova´ et al., 2014). Two types of efficient capillary zwitterionic organic-silica hybrid monolithic columns were synthesized in a single-step thermally induced polymerization, by using [2-(methacryloyloxy)-ethyl]
dimethyl-(3-sulfopropyl)ammonium hydroxide or [2-(methacryloyloxy)-ethyl] phosphorylcholine as the organic monomers and [3-(methacryloxy)-propyl]trimethoxysilane. The columns were suitable for HILIC separations of various low-molecular-weight neutral, basic, and acidic analytes, as well as small peptides in tryptic digests (Lin et al., 2012).
2.4.2 Organic Polymer Hydrophilic Interaction Liquid Chromatography Columns
Monolithic (poly)methacrylate diol or (poly)hydroxymethacrylate columns were successfully employed for HILIC separations of oligonucleotides. Unfortunately, organic polymer monolithic columns traditionally have shown rather low separation efficiency for low-molecular-weight compounds (Nischang and Bruggemann, 2010). Organic polymer-based monoliths have a hetero- geneous structure, which rather resembles a net of interconnected nonporous cauliflower-like microglobules with low surface area. This morphology is suitable for separation of polymers requiring large pores (15e100 nm) with relatively low specific surface area (10e150 m2/g) (Svec, 2012). Large molecules of biopolymers cannot diffuse into the small pores in the microglobules of organic monoliths and can access only the large pores by convection; hence they provide narrow peaks. On the contrary, small molecules can penetrate into the narrow pores by slow diffusion, generally resulting in a poor separation efficiency (Svec and Lv, 2015). Essential improvement in the performance of monoliths was achieved by controlling the inner pore morphology of the monolithic bed structure. The proportion of mesopores should be increased while preserving a sufficient amount of through-pores (Nischang et al., 2010; Nischang and Clauson, 2016). Several strategies were followed to this end:
1. Chemistry and proportions of the functional monomers, cross-linkers, and porogen solvents have major impacts on the morphology of the final monolith (Arrua et al., 2012).
2. The initiation conditions and the temperature of polymerization should be carefully adjusted (Urban and Jandera, 2013).
3. Termination of the polymerization reaction at an early stage, in less than 1 h, provided incomplete polymerization, and consequently, less cross-links and larger porosity of the final monoliths
(Greiderer et al., 2009). However, nanostructural heterogeneity in the polymer network and a void volume can be formed during polymerization because of different local compositions of monomer and cross-linkers. This may cause poor reproducibility of monoliths prepared using this approach.
4. The chromatographic performance of the organic polymer monoliths could also be improved via adjustment of the pore morphology using postpolymerization modifications or two-step monolith fabrication (Currivan and Jandera, 2014), hypercrosslinking (Urban et al., 2010), or incorporation of additional structural elements into the monolithic skeleton, such as carbon nanotubes (Svec and Lv, 2015).
Organic polymer monoliths with zwitterionic groups incorporated into (poly)methacrylate monolithic structures are suitable for HILIC separations of neutral, basic, and acidic polar compounds in aqueouseorganic mobile phases with 60% or more acetonitrile. Polymer monolithic columns containing sulfoalkylbetaine moieties were prepared by photo-induced (Viklund et al., 2001) or thermal (Jiang et al., 2007) copolymerization of ethylene dimethacrylate (EDMA) andN,N-dimethyl- N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine (MEDSA) inside fused silica capillaries (Fig. 2.5). Capillary monolithic columns prepared by copolymerization of methacryloxyethyl-N-(3- sulfopropyl) ammonium betaine (MEDSA) with EDMA or with 1,2-bis(p-vinylphenyl) ethane (BVPE) were employed for separations of nucleic bases and other neutral, basic, and acidic polar analytes in aqueouseorganic mobile phases with 60% or more acetonitrile (Jiang et al., 2007; Foo et al., 2012). Their performance compared favorably to particle-packed silica-based zwitterionic columns.
Porosity, permeability, selectivity, and retention characteristics of monolithic sulfobetaine columns improve when using water-containing porogen solvents in the polymerization process (Viklund et al., 2001; Jiang et al., 2007; Foo et al., 2012; Urban et al., 2009). Long-chain alkyl monomers and cross- linkers increased the proportion of the mesopores in polymethacrylate monolithic columns, resulting in improved separation efficiency up to 70,000e80,000 theoretical plates/m for small molecules.
Monolithic stationary phases prepared by copolymerization of 3-chloro-2-hydroxypropyl methacry- late and ethylene dimethacrylate in a fused silica capillary could be further functionalized by reaction with triethanolamine ligands and used for separations of nucleobases and substituted benzoic acids (Qiao and Shi, 2015).
Dimethacrylate cross-linkers with relatively long polar (poly)oxyethylene chains generally improve the performance of polar monolithic columns for HILIC applications. By copolymerizing MEDSA withN,N0-methylenebisacrylamide (MBA) (Yuan et al., 2013), 1,4-bis(acryloyl)piperazine (PDA) (Liu et al., 2014), or bisphenol A glycerolate dimethacrylate (BIGDMA) (Stankova´ et al., 2013) cross-linkers, columns with significantly improved efficiency for small molecules were prepared. For the last type of monolithic column, excellent long-term stability, reproducibility of preparation within 1%e3% (relative), and efficiency up to 60,000 theoretical plates/m were reported at the optimum flow rate of 1e3mL/min. After correction for extra-column contributions to band broadening, the efficiency increased to 120,000 theoretical plates/m (Jandera and Stankova´, 2015).
Recently,Li et al. (2016)investigated the effect of three charged hydrophilic groups introduced into hydrophilic sulfobetaine stationary phases, namely N,N-dimethyl-N-acryloyloxyethyl-N-(3- sulfopropyl)ammonium betaine (SPDA), [2-(acryloyloxy)ethyl] trimethylammonium chloride (AETA), and 3-sulfopropyl acrylate potassium salt (SPA), on the chromatographic properties of
zwitterionic monolithic capillary columns. Depending on the combination of stationary phaseemobile phaseesolute, hydrophilic interactions and electrostatic and hydrogen-bonding interactions, together with molecular shape, contribute to the retention and separation selectivity of analytes. Because of the weak anion exchange properties, the zwitterionic poly(SPDA-co-MBA) hydrophilic monoliths exhibit the best separations for phenols,b-blockers, and small peptides, in terms of selectivity, peak shape, and analysis time. On the other hand, the cationic poly(AETA-co-MBA) monolith provided the best separation for nucleobases and nucleosides.
A two-step surface modification of a poly(styrene-co-vinylbenzyl chloride-co-divinylbenzene) nonpolar monolithic layer, including hypercross-linking and thermally initiated surface grafting of [2- (methacryloyloxy)ethyl]dimethyl(3-sulfopropyl)ammonium hydroxide, has been used to form a sec- ond zwitterionic layer for efficient separation of small polar compounds (Skerı´kova´ and Urban, 2013).
A primary monolithic poly(lauryl methacrylate-co-tetraethyleneglycol dimethacrylate) layer could be used as a stable scaffold for a second, polar monolithic layer with zwitterionic functionality by in situ copolymerization of poly(N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine- co-bisphenol A glycerolate dimethacrylate) on the primary monolith; FriedeleCrafts grafting onto the scaffold layer was not necessary. The column was suitable for HILIC separations of phenolic acids, flavones, nucleosides, and bases of nucleic acids, with similar efficiencies, but with selectivities differing from zwitterionic methacrylate monolithic columns prepared by a single step polymerization (Currivan et al., 2015).