ADVANCES IN HYDROPHILIC INTERACTION LIQUID
3. SEPARATION MECHANISM AND EFFECTS OF THE ADSORBED WATER AND MOBILE PHASEAND MOBILE PHASE
3.3 DUAL HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY/REVERSED- PHASE RETENTION MECHANISMPHASE RETENTION MECHANISM
concluded that the transition from a partitioning to a surface adsorption mechanism for neutral compounds occurs at more than 75%e80% acetonitrile on diol, silica, and amino columns, depending on the different degree of hydration of the stationary phases. For electrostatically attracted compounds, surface adsorption remains the dominant retention mechanism even at lower acetonitrile concentrations.
In spite of differences in the conclusions of some studies of HILIC mechanisms, the adsorption and the partition retention mechanisms most probably actually coexist in many HILIC systems, depending on the solute, the stationary phase polar functional groups, and the eluting conditions (Dinh et al., 2013; Soukup and Jandera, 2014). For example, less hydrophilic nortriptyline was reported to be retained by a partition-like mechanism and cytosine by a more hydrophilic mechanism, rather than by an adsorption-like mechanism, which was attributed to slower diffusion of the more retained polar species in the viscous diffuse water layer, even though diffusion is not a thermodynamic phenomenon (Karatapanis et al., 2011; Heaton and McCalley, 2014).
Many ionized compounds can be separated in HILIC systems. Very often, either attractive (ion exchange) or repulsive (ion exclusion or ion repulsion) electrostatic interactions participate in the retention mechanism, especially on strong (SAX) or weak (WAX) anion-exchange columns. The addition of salts, weak acids, bases, or ion-pairing reagents as mobile phase additives usually significantly improves the separation in the mixed HILIC/ion-exchange mode (Mant and Hodges, 2008). Adjusting the pH and salt (buffer) concentrations may significantly improve the retention selectivity, peak profile, and separation, however, with very different selectivity effects for acids and bases (Heaton et al., 2014). On bare silica columns, acids show much stronger retention in mobile phases containing trifluoroacetic acid than that in ammonium formate buffers, wheredon the contrarydbases are better retained (McCalley, 2015).
Some mixed-mode silica-based HILIC/IEX stationary phases can be used for separations of polar and ionic solutes under HILIC conditions in organic solvent-rich mobile phases and for separations of less-polar compounds under RP conditions in more aqueous mobile phases. The RP/WAX phases differ from the typical HILIC stationary phases, TSKGel Amide-80, ZIC-HILIC, or polysulfoethyl A, to which they provide a certain degree of complementary application possibilities (Lammerhofer et al., 2008).
At increased concentration of acetonitrile, adequate retention and satisfactory resolution of both basic and acidic peptides can be achieved in a single run, on either SAX or WAX columns at low pH, where HILIC and electrostatic repulsion retention mechanisms superimpose to produce the ERLIC mode, which offers possibilities for independently adjusting the HILIC and the ion-exchange selec- tivities in highly organic mobile phases (Alpert, 2008;Alpert et al., 2015).
3.3 DUAL HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY/REVERSED-
Hence, many polar columns show a dual HILIC-RP retention mechanism: nonpolar (RP) at high water concentrations and polar (HILIC) in mobile phases with high concentrations of organic solvents. In this case, the graphs of the sample retention factors,k, versus the volume fraction of water,4(H2O), show characteristic U-profiles over a broad range of binary aqueouseorganic mobile phases (see examples inFig. 2.8). The dual HILIC-RP behavior depends both on the column and sample, and it probably increases for polar stationary phases containing significant nonpolar moieties in the structure.
In the presence of a dual HILIC-RP mechanism, the effects of the volume fraction of the more polar solvent, water,4ðH2OÞ, on the retention factor,k, can often be described byEq. (2.6)over a broad range of compositions of aqueouseorganic mobile phases at4ðH2OÞ>0:02
: logk¼aþmRP$4H2OmHILIC$log 1þb$4H2O
(2.6)
The parametermRPcharacterizes the effect of the increasing concentration of water in the mobile phase on the retention because of the RP mechanism in water-rich mobile phases, whereas the parametermHILICis a measure of the water contribution to the decrease of retention in highly organic mobile phases (i.e., the HILIC range). The system constant,a, depends on the solute and type of organic solvent. The empirical term,b, improves the description of the retention at low water con- centrations (Jandera and Ha´jek, 2009).
Eq. (2.6) describes the U-shaped experimental graphs, where the “U-turn” transition from the HILIC to the RP mode can be localized at the minimum retention corresponding to the4mincon- centration of water in the mobile phase:
4min¼0:434mHILIC=mRP (2.7)
FIGURE 2.8
Effect of the volume fraction of aqueous buffer,4H2O, in aqueous-acetonitrile mobile phase on the retention factor, k, of sulfonamides on monolithic column BIGDMA-MEDSA (A) and flavonoids on carbamoyl bonded silica column TSKgel Amide-80 (B). Points are the experimental data and lines the best fit plots ofEq. (2.5).
Based on unpublished results from P. Jandera, M. Stankova´, P. Jana´s.
The “U-turn” mobile phase composition generally depends on the polarity of sample and on the type of stationary phase. The transition between the HILIC and the RP behavior is observed at a lower volume fraction of the aqueous buffer on less PEG columns (such as bonded polyethylene glycol in 70%e90% acetonitrile), in comparison to more polar columns, e.g., diol, zwitterionic, etc., in 40%e70% acetonitrile (Jandera and Ha´jek, 2009).
Zwitterionic polymethacrylate monolithic columns show dual HILIC and RP mechanisms.
Orthogonal selectivity in the HILIC and the RP ranges may provide excellent separations of flavonoids and phenolic acids (as well as others) on a single column in both retention modes. Silica hydride columns with bidentate or cholesterol nonpolar ligands bonded on the surface also show dual retention mechanisms, despite very low affinity to water (Soukup and Jandera, 2012a,b).
Some columns may provide practically useful separations only either at high organic solvent concentrations (HILIC) or in highly aqueous mobile phases (RP), ruling out from practical use the major part of the medium mobile phase composition range, where the retention is too low. The useful mobile phase range depends both on the stationary phase and on the analyte. For example, organic polymer zwitterionic columns provide relatively broad mobile phase ranges both for HILIC and RP separation modes.Fig. 2.8A shows examples of U-turn retention plots of sulfonamides on a home- made capillary zwitterionic column (Stankova´ and Jandera, 2016) and Fig. 2.8B the U-turn reten- tion plots of flavonoids on a commercial bonded amide column.
In a recent study, the retention of 35 small polar compounds was compared among aminopropyl, amide, diol, and cyanopropyl columns with silica-based, hybrid, and fused-core particles (Vlckova´, et al., 2014). Most acidic and neutral compounds experienced greater retention under RP conditions than that under HILIC conditions. Only with the beta-blockers atenolol and propranolol were typical U-profile retentionemobile phase graphs observed. The aminopropyl stationary phase strongly retains
Inject
waste
Short C18 core-shell or monolithic RP 2Dcolumn, 30 or 50 mm
Loop 1, 2 μL 20 cm 0.5 mm i.d. capillary BIGDMA-MEDSA 1D colum,
Loop 2, 2 μL
10-port valve fraction transfer interface
Detecto
FIGURE 2.9
Instrumental two-dimensional comprehensive HILICRP HPLC setup with a zwitterionic polymethacrylate capillary column in the first dimension and a short efficient C18 column in the second dimension.
acids because of electrostatic interactions, even under RP conditions. Bare silica exhibits a preference for basic compounds by interactions with the acidic surface of silanols.
Because the separation selectivities on a dual-mechanism column are highly complementary (orthogonal), a single column can be used to obtain useful information on the sample injected sub- sequently in acetonitrile-rich (HILIC) and highly aqueous (RP) mobile phases (Jandera et al., 2010a,b). The dual mechanism is best used with alternating increasing (HILIC) and decreasing (RP) concentration gradients of water in buffered mobile phase at pH¼3.1, both in unidimensional and 2D HPLC separations (Jandera et al., 2013).