Introduction to Modern Liquid Chromatography, Third Edition part 27 doc

216 THE COLUMN 0.2 0.4 0.6 0.8 Time ( min ) 1 2 3 4 5 Figure 5.12 High-temperature separation of a pharmaceutical mixture. Sample: 1, doxy- lamine; 2, methapyrilene; 3, chloropheniramine; 4, meclizine; 5, triprolidine. Conditions: 100 × 4.6-mm ZirChrom-PBD  column (zirconia); 20% acetonitrile/water with added tetramethylammonium hydroxide to control pH-13; 4.2 mL/min; 140 ◦ C; 2850 psi. Courtesy of ZirChrom Separations, Inc. low- and high-pH mobile phases (1 ≤ pH ≤ 13), and they can be used at very high temperatures (≤ 160 ◦ C) [34]. Figure 5.12 shows a separation on a zirconia column at pH-13 and 140 ◦ C. Early columns packed with polymer-coated zirconia displayed rather poor efficiency, apparently because of poor stationary-phase mass transfer. While their performance continues to be improved, zirconia columns are still waiting (as of the time this book was written) for a critical application where they perform demonstrably better than silica-based RPC columns. However, this may reflect the present limited impact of high-temperature separation ( > 60 ◦ C). For ionizable solutes and zirconia-based columns, poor peak shapes often result, regardless of how the packing is prepared. Consequently, when zirconia-based packings are used with ionizable solutes, special mobile-phase additives (e.g., phos- phate or fluoride) are required for good peak shape and reasonable values of N [34, 35]. For a sufficiently large concentration of the additive, values of N and peak shape for basic solutes are similar to those found for alkylsilica columns (for pH ≤ 10). At the time this book was published, somewhat poorer results were obtained for the separation of peptides and proteins, compared to separations with alkylsilica columns. Zirconia-based ion-exchangers are also available commercially. A weak anion-exchanger can be formed by coating zirconia particles with polyethyleneimine, followed by cross-linking with 1,4-butanedioldiglycidylether. This approach pro- duces columns that are stable from pH range 3 to 9 and can be used to separate organic acids, inorganic anions, and highly polar compounds such as sugars. Chemically and thermally stable, strong anion-exchange columns are also available for separations at ≤ 100 ◦ C, over the pH range of 1 to 13. These packings are formed by cross-linking zirconia-coated polyethyleneimine with 1,10-diiododecane or a similar compound [34]. Carbon-clad zirconia is a uniquely selective packing, compared to other RPC columns; it is prepared by passing a reduced pressure of organic vapor over porous zirconia at a temperature of ∼700 ◦ C [34]. Carbon-clad zirconia differs from alkyksilica packings in being more hydrophobic, is better able to separate polar and nonpolar geometrical isomers, and is also capable of π –π interactions 5.3 STATIONARY PHASES 217 (Section 5.4.1). This packing is stable from pH 0.3 to 14 at 40 ◦ C, and is thermally stable to ≥ 200 ◦ C at neutral pH. Peak shape and column efficiency tend to be poor at lower temperatures (e.g., 35 ◦ C), but these columns have been under development for a much shorter time than silica-based columns—so future improvements seem likely. 5.2.5.2 Alumina and Titania RPC columns based on an alumina support were reported in the early 1980s [36], and have since been reviewed [37]. The chemistry of the alumina surface is more like that of zirconia than silica, and alumina columns are also stable at higher pH. As covalently bonded alumina is not stable, coating or polymerizing a polymer onto the surface is used in place of silane derivatization. Because of its strongly adsorptive properties, few reversed-phase applications of coated alumina have been reported. Unbonded alumina is not used at present for HPLC separation, but it has found a role for sample preparation (Section 16.6.5.2). Commercial columns packed with titania particles are also available, but these are used mainly for normal-phase separations. Reversed-phase materials based on titania generally show no advantages over more traditional silica- and zirconia-based particles, so they have not achieved widespread use. On balance, silica remains by far the most popular support, because of a better compromise among important column properties (Table 5.5). 5.2.5.3 Graphitized Carbon Porous, graphitic carbon (PGC) is a very different column-packing, consisting of fully porous, spherical carbon particles that are formed from flat sheets of hexagonally arranged carbon atoms [38, 39]. The carbon atoms have a fully satisfied valence that results in very different retention and selectivity, compared to other columns. PGC can be used for both reversed- and normal-phase separation, and is stable at 1 ≤ pH ≤ 14 and ≤ 200 ◦ C. However, its reduced particle strength limits the maximum pressure that can be used with these columns. PGC retains polar compounds by a combination of strong hydrophobic, electronic, and dipolar interactions [38], so that polar solutes can be preferentially retained even under RPC conditions. The selectivity of graphitized-carbon columns is difficult to predict, compared to conventional bonded-phase columns, and this can make method development more difficult. Also column efficiency and peak shape can be somewhat poorer than for conventional RPC columns. However, porous carbon shows a special ability to separate stereo- and diastereoisomers, as well as positional isomers for which poor or no separation occurs with conventional packings. Figure 5.13 shows a separation of hippuric acid and its methyl-substituted isomers on a PGC column, using a low-pH mobile phase. 5.3 STATIONARY PHASES The column stationary phase determines retention and selectivity (Sections 2.3, 5.4), and it must meet certain practical requirements, for example, acceptable stability, 218 THE COLUMN 1 05 Time ( min ) 10 2 3 4 Figure 5.13 Separation on a graphitized-carbon column of hippuric acid and its methyl-substituted isomers. Sample: 1, 2-methylhippuric acid; 2, hippuric acid; 3,3-methyl- hippuric acid; 4, 4- methylhippuric acid. Conditions: 100 × 4.6-mm Hypercarb column; mobile phase is 30% acetonitrile, 30% isopropanol, and 40% water with 0.1% TFA; 1.0 mL/min; 25 ◦ C. Courtesy of Thermo Scientific. reproducibility, peak shape, and column efficiency N. In this section we review the preparation, nature, and properties of different stationary phases—apart from their selectivity, which is discussed in the following Section 5.4. Most stationary phases are organic in nature, either covalently bound to or (rarely) mechanically deposited on the particle. In some cases the surface of the unmodified particle is the stationary phase, for example, unmodified silica for use in normal-phase chromatography (including hydrophilic interaction chromatography, HILIC). We will assume that we start with a silica particle, prior to adding the stationary phase. Procedures used for other supports were referred to in the previous Sections 5.2.3–5.2.5. 5.3.1 ‘‘Bonded’’ Stationary Phases RPC packings usually are made by covalently reacting (‘‘bonding’’) an organosilane with the silanols on the surface of a silica particle to form the stationary phase or ligand R: X 3 −Si−R + ≡ Si−OH → ≡ Si−O−Si(X 2 )−R + HX (5.1) (silane) (silanol) (final phase) The functional group X is often –Cl or –OEt, and/or –CH 3 (Fig. 5.14), in which case the reaction by-product HX is HCl or ethanol. Silanes substituted with other groups X are also used, as will be discussed. Some bonded-phase packings are made via the monofunctional reaction of Figure 5.14a. Here a single silane reagent reacts with a single, surface-silanol group, for example, chlorodimethyl-octadecylsilane (where the ligand R = C 18 ) reacts to form a monomeric C 18 column. Other commercial packings are formed from a surface reaction with a trifunctional (or difunctional) silane, as illustrated in Figure 5.14b, c (although two silane-silica bonds are shown here, three such bonds are also possible). Depending on the reaction conditions, polymeri- zation of the stationary phase can result in the latter case (use of a difunctional 5.3 STATIONARY PHASES 219 Si−OH + Cl−Si(CH 3 ) 2 R Si−O−Si(CH 3 ) 2 −R + HCl (a) (b) Si OH Si OH + Cl 3 Si-R Si O Si O Si(Cl)−R + 2 HCl (c) + (EtO) 3 Si−R Si O Si O Si(OEt)−R + 2 EtOH Si OH Si OH (d) Si-OH + (EtO)Si(CH 3 ) 2 −R Si−O−Si(CH 3 ) 2 −R + EtOH Figure 5.14 Synthesis of various bonded-phase column packings by the reaction of a silane with silica. (a, d), Monomeric packings; (b, c), potentially polymeric packings. or trifunctional silane), yielding a polymeric stationary phase or column (not to be confused with a ‘‘polymer column’’; Section 5.2.3). As we will see, the properties of monomeric and polymeric columns are significantly different in important respects. Several different kinds of silane–silica reactions have been used to prepare HPLC columns, as illustrated in Figure 5.15. Figure 5.15a illustrates a ‘‘vertical’’ polymerized phase that results from the reaction of a di- or trifunctional silane (as in Fig. 5.14b or Fig. 5.14c). In the example of Figure 5.15a, the silane that initially reacts with the surface further reacts with one or more additional silane molecules to give a polymeric phase (in the presence of water; see below). These phases tend to be more stable than monomeric phases at both low and high pH, as the ‘‘heavier’’ surface coverage of these packings slows down the attack of the mobile phase on both the silica and the ligand–silica bond. However, packings of this type tend to be less reproducible in terms of retention and selectivity because of variable (inadequately controlled) silane polymerization. ‘‘Horizontal’’ polymerization with self-assembled silanes (C 3 plus C 18 ) yields the general structure shown in Figure 5.15b. Here Si atoms of adjacent silanes are connected to each other through oxygen atoms (siloxane linkages, Si–O–Si), while each silane is connected to the silica via another siloxane bond. Columns prepared in this way have been reported to exhibit superior stability in both low- and high-pH applications [40], but no commercial columns of this type had been announced at the time this book went to press. The monomeric phase of Figure 5.15c is most widely used for RPC columns; packings with several different functional groups (ligands) are commercially available (Section 5.3.3, Table 5.7); the silane side-groups are usually methyl groups, as in Figure 5.15c. These packings are commonly prepared by reacting dimethylchloro- or dimethylethoxy-silanes with the silica support (Fig. 5.14a, d): one silane molecule reacts with one silanol group. The advantage of this one-to-one reaction is that a 220 THE COLUMN Si Si Si O OH O OH O Si Si Si Si Si Monomeric (dimethyl-substituted) (c) Si Si OOHOHOOH Si Si Si Si Si Monomeric (steric protected) (d) methyl i-butyl Si Si-OH Si-OH OOOH OO Si Si Si Si Si O OH Polymeric Vertical polymerization (a) Si Si Si Si Si OO O O O Si Si Si Si Si OO OOOO Polymeric Horizontal polymerization (b) Figure 5.15 Some alternative bonded phases based on different reaction conditions. Adapted from [40]. reproducible, well-defined bonded phase results. Packings made in this way often exhibit the highest column efficiency because of rapid diffusion of the solute into and out of the less-crowded stationary-phase layer. Conversely, packings with multifunctional, highly polymerized stationary phases can exhibit slower solute diffusion and lower values of N, especially at higher flow rates. The silane reactions of Figure 5.14c, d are typically carried out with alkoxysilanes that have reactive R-groups (ligands) such as –C 3 –NH 2 or –C 3 –O–CH(OH)–CH 2 OH (to give an amino or diol column, respectively). Stationary phases with certain ligands (e.g., those containing reactive amino or hydroxyl groups) cannot be prepared from chlorosilanes because of undesirable secondary reactions of the ligand. These reactive stationary phases are instead made from alkoxysilanes, as in Figure 5.14c or d. Alkoxysilane reactions are somewhat slower than those with chloro- and dimethylamino-silane, generally requiring longer reaction times and higher silane concentrations for equivalent reaction yields. The sterically protected silane stationary phase of Figure 5.15d [41–43] is a variation of the monomeric phase of Figure 5.15c, where the methyl groups of the silane in Figure 5.15c are replaced by i-propyl or i-butyl in Figure 5.15d.The latter large, bulky side-groups interfere with the hydrolysis of the bonded silane, as illustrated in Figure 5.16b andcomparedwithFigure5.16a. Each Si–O–Si bond is 5.3 STATIONARY PHASES 221 (b)(a) (c)(d) Si Si Si Si Si Si OOHOHOH + (CH 3 ) 3 Si−Cl Si Si Si Si Si Si O O OH OH TMS TMS = trimethylsilyl = (CH 3 ) 3 Si− Si Si Si O OH O OH O Si Si Si Si Si H 3 O + Si Si OOHOHOOH Si Si Si Si Si H 3 O + Figure 5.16 Options for increasing the stability of alkylsilica columns. (a, b), protection of the—Si–O–bond by a steric-protected bonded phase (for low-pH conditions only); (c, d)pro- tection of the bonded phase by end-capping. individually protected by the size of the two bulky side-groups in Figure 5.16b.Steric protection is useful for separations at low pH (but not at high pH) because low pH catalyzes the breaking of the O–Si bond [41]. Sterically protected stationary phases are available with a variety of ligands (e.g., C 8 ,C 18 , cyano, phenyl), each of which show exceptional stability for use with low-pH mobile phases. Because low-pH mobile phases are preferred for the separation of ionic samples (Section 7.3.4.2), sterically protected columns are especially useful for the separation of biological samples such as peptides and proteins. Because of the steric bulk of the protecting silane groups, these packings have a lower surface concentration of the ligand, and exhibit lower retention than comparable dimethyl-substituted phases—as shown by the data of Table 5.6 for monomeric columns. Bonded bidentate-silane stationary phases (as in Fig. 5.14b) are more stable for high-pH applications [44]. Prior to the silane reaction, a fully hydroxylated silica will have a surface-silanol concentration of ≈ 8 μmol/m 2 . However, the size of an attached silane results in some overlap of adjacent silanols, which inhibits their further reaction with the silane reagent. The ligand concentration for a fully reacted packing will therefore seldom exceed 4 μmol/m 2 (leaving half or more of the original silanols unreacted). As shown in Table 5.6, as the chain length or cross section of the silane increases, the percentage of reacted silanol groups (and ligand concentration) decreases. Almost 50% of the silanol groups remain unreacted for the smallest silane (trimethyl). Although unreacted silanols may be inaccessible to a reacting silane, they may still be able to interact with the solute molecule (Section 5.4.1). Polymeric phases are used (to a limited extent) because of their greater stability and unique selectivity (Section 5.4.1.2). Whereas the preparation of monomeric phases (as in Fig. 5.14a) must be carried out in a water-free reaction medium, 222 THE COLUMN Table 5.6 Effect of Silane Bonded-Phase Chain Length and Bulk on Silica Support Coverage Bonded Phase Ligand Surface Coverage (μmol/m 2 ) Reacted Silanols (%) Trimethyl 4.1 51 Dimethyl-3-cyanopropyl 3.8 48 Dimethyl-n-butyl 3.8 48 Dimethyl-n-octyl 3.5 44 Dimethyl-n-octadecyl 3.2 40 Sterically protected columns Triisopropyl 2.2 28 Diisopropyl-3-cyanopropyl 2.1 26 Diisopropyl-n-octyl 2.0 25 Diisobutyl-n-octadecyl 1.9 25 polymeric phases require the presence of water during part of their synthesis. The extent of reaction or polymerization is controlled by varying the amount of water added to the reaction [45]. The reaction of an alkyltrichlorosilane with silica particles is carried out in the presence of water [45]. To minimize unwanted interactions with residual silanol groups (Section 7.3.4.2), column packings for RPC are usually endcapped,byafurther reaction of the bonded phase with a small silane such as trimethylchlorosilane or dimethyldichlorosilane (Fig. 5.16d). This procedure decreases the concentration of unreacted silanols, as well as their interaction with retained solute molecules—but does not totally eliminate silanol-solute interaction (end-capping increases the percentage of reacted silanols by only 20–30% [46], corresponding to a somewhat smaller decrease of unreacted silanols). Small ligands (e.g., end-capping trimethylsilyl groups) are more susceptible to hydrolysis and loss at low pH, which can lead to changes in retention and selectivity. On the other hand, end-capped columns are more stable at intermediate and higher pH. Still another kind of bonded phase is based on ‘‘type-C’’ silica. The polar silanols of a typical type-B silica are first reacted to form a nonpolar, silicon-hydride surface (Fig. 5.17a). The latter type-C silica can be used without further change for either RPC or normal-phase chromatography. Type-C silica can be modified (for RPC) by the addition of alkyl groups (Fig. 5.17b). Good reproducibility and stability are claimed for these packings, even when used in 100% water mobile phases over a pH range of 1.5 to 10 [47]. These packings were relatively new at the time this book was published. Information on their properties and use was quite limited, but they were commercially available (MicroSolve Technology; Eatontown, NJ). Alkyl and aromatic ligands present in a packing (including end-capping) can be identified following their removal from the silica particle [48]. The packing is first treated with aqueous hydrofluoric acid, which cleaves the ligand–silica bond. The ligand reaction-product can then be characterized by GC, NMR, and/or mass spectrometry. 5.3 STATIONARY PHASES 223 HHHH Si O O OSi Si O Si OO O O Si H Si O Si Si Si Si (b)(a) CH CH Si Si Si Si OOOO Si Si Si Si OO O Figure 5.17 Type-C silica (a) and the resulting bonded phase (b) (bidentate C 18 ). 5.3.2 Other Organic-Based Stationary Phases 5.3.2.1 Mechanically Held Polymers As in the case of metal-oxide supports other than silica (Section 5.2.5), mechanically held polymers such as cross-linked polystyrene and polybutadiene have been used as stationary phases for silica-based particles [49]. Because of their poorer efficiency and reproducibility, as well as the lack of phases with different functional groups, little use has so far been made of these columns. 5.3.2.2 Hybrid Particles Hybrid particles are formed by polymerizing two monomers (e.g., tetramethoxysilane and tetraethoxysilane), to form an organic/inorganic structure as in Figure 5.18. The chemistry for these materials was first introduced by Unger et al. [50] in R SiEt O O Si Et O O Et O Si Si O O R O O O n Polyethoxyoligosiloxane Polymer OEt Si EtO OEt OEt R Si EtO OE t OEt + Figure 5.18 Synthesis of organic/inorganic hybrid particle. Courtesy of Waters Corporation. 224 THE COLUMN 1977, and later developed more fully by Waters Corporation. Hybrid particles are formed using a silane that contains hydrolytically stable Si–C bonds. These stable bonds are part of the matrix, which improves the pH stability of such packings relative to silica-based packings. The first such packing was prepared from a 2 to 1 ratio of tetraethoxysilane and methyltriethoxysilane (XTerra). Later a hybrid packing with further improved pH stability was created from tetra-ethoxysilane and ethyl-bis-triethoxysilane (XBridge). These particles possess excellent stability when used with both low- and high-pH mobile phases, as well as a mechanical strength that allows their use at pressures to 15,000 psi. As with silica particles, hybrid particles can be derivatized with various ligands (C 8 ,C 18 , etc.) [51]. Hybrid packings are especially useful for the high-pH separation of non-ionized basic solutes—allowing improved peak shapes and larger sample weights (Section 15.3.2.1). 5.3.2.3 Columns for Highly Aqueous Mobile Phases RPC separations of very polar samples may require small values of %B in order to achieve values of k ≥ 1, although other means exist for increasing the retention of such samples (Sections 6.6.1, 7.3.4.3, and Chapter 8). When RPC is used with low values of %B, several problems may be encountered: a decrease in sample retention with time, decrease in values of N, and long equilibration times when changing from one mobile phase to another [52, 53]. This behavior is the result of stationary-phase de-wetting (sometimes incorrectly called phase collapse), with the consequent expulsion of mobile phase from the pores of the particle [54, 55]. Thus the pressure P required to force the mobile phase into a particle pore of diameter d pore is P = −4γ cos θ d pore (5.2) where γ is the surface tension of the mobile phase, and θ is the contact angle between the stationary and mobile phases. The value of θ is > 90 ◦ for a C 18 stationary phase and water as mobile phase, meaning that pressure is required to force water into the pores. If the pressure P is insufficient to force a highly aqueous mobile phase into all the pores of the particle, solute molecules will be excluded from these pores. The required pressure increases for more hydrophobic columns (C 30 , more pressure; C 1 less pressure), and for particles with smaller pores. P also increases for smaller values of %B. Dewetling and the loss in retention can be reversed by flushing the column with methanol or another organic solvent [56]. Problems from column de-wetting arise mainly when the column is de-pressured. If the column is initially filled with mobile phase of > 50% B, all pores will be filled with mobile phase at pressures normally used in HPLC. This will commonly be the case when beginning a series of RPC separations, as it is recommended to store the column with 100% acetonitrile as fill solvent. If the mobile phase is then changed to a lower value of %B while maintaining the column pressure, de-wetting is less likely to occur. However, when planning to carry out RPC separations with <5% B, it is advisable to select a column that is less likely to undergo de-wetting (shorter ligand lengths, more polar ligands, lower ligand concentration; see also Section 5.4.4.2). 5.3 STATIONARY PHASES 225 In an attempt to solve the problem of de-wetting, special ‘‘aqueous reversed-phase’’ packings have been developed that allow the use of mobile phases that contain > 95% water. Examples of this kind include columns with embedded polar groups, polar end-capping (indicated by such terms as ‘‘polar,’’ ‘‘AQ,’’ ‘‘hydrosphere,’’ ‘‘aqua,’’ ‘‘aquasil’’), or a lower concentration of the alkyl ligand. Wide-pore columns or columns with a shorter ligand are also less susceptible to de-wetting, but such columns are also less retentive and therefore less useful for the RPC separation of very polar samples. 5.3.3 Column Functionality (Ligand Type) Apart from the differences in RPC stationary phases described in Sections 5.3.1 and 5.3.2, the chemical composition of the ligand can vary. Ligands for several, commercially available column types are described in Table 5.7 and illustrated in the Table 5.7 Functional Groups Found in HPLC Stationary Phases Functional Group Mode a Comment C 3 RPC Used primarily for separations of proteins C 4 RPC C 5 RPC C 8 RPC Most commonly used columns; similar retention and selectivity C 18 RPC C 30 RPC Used mainly for carotene separation Phenyl RPC Commonly used column, mainly for a change in selectivity Embedded-polar-group (amide, carbamate, urea) RPC Commonly used column, mainly for or use with water-rich mobile phases (<5% B), to improve peak shape for basic solutes, or for a change in selectivity Perfluorophenyl (PFP) RPC Less commonly used column, mainly for a change in selectivity Cyano RPC, NPC Less commonly used column NH 2 (amino) RPC, NPC, IEC Less commonly used column Diol RP, NP, SEC Mainly used for SEC WAX IEC Used mainly for separating inorganic ions or large biomolecules (Section 13.4.2) WCX IEC SAX IEC SCX IEC a RPC, used for reversed-phase chromatography; NPC, used for normal-phase chromatography; IEC, used for ion-exchange chromatography; SEC, used for size-exclusion chromatography. . hydrophilic interaction chromatography, HILIC). We will assume that we start with a silica particle, prior to adding the stationary phase. Procedures used for other supports were referred to in the previous. meaning that pressure is required to force water into the pores. If the pressure P is insufficient to force a highly aqueous mobile phase into all the pores of the particle, solute molecules will. is recommended to store the column with 100% acetonitrile as fill solvent. If the mobile phase is then changed to a lower value of %B while maintaining the column pressure, de-wetting is less likely to occur.