ADVANCES IN HYDROPHILIC INTERACTION LIQUID
3. SEPARATION MECHANISM AND EFFECTS OF THE ADSORBED WATER AND MOBILE PHASEAND MOBILE PHASE
3.2 MOBILE PHASE IN HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY SEPARATIONSSEPARATIONS
35.4% (v/v) water, was observed for the Ascentis Express OH column, whereas the Xbridge HILIC, Atlantis HILIC, Ascentis Express ES-CN, and Ascentis Express F5 columns have lower saturation capacities, less than 9% v/v. At full column saturation, the excess adsorbed water,Vex, fills up to 45.3%
of the pore volume of normal silica-based columns but only 2.6%e5.5% of the pore volume of hydrosilated silica columns. Because of the low affinity of the hydrosilated silica material to water, saturation capacities are as low as 0.2%e0.4% water in the inner pore volume, which are achieved in mobile phases containing 3%e6% v/v water (for the hydrosilated bare silica or bonded C18 Bidentate columns), in agreement with the Pesek ANP model (Pesek and Matyska, 2009). This low water saturation capacity is similar to the long nonpolar alkyl chain (C30) columns. On the other hand, polar columns used frequently in HILIC show much less steep water isotherms and relatively high saturation capacities, which are not approached even in mobile phases containing 20% v/v water in acetonitrile (Soukup and Jandera, 2014).
Fig. 2.7(B)shows the water uptake in terms of the number of “hypothetical” monomolecular water layer equivalents,Nw, at full saturation capacity of the 19 columns tested (Soukup and Jan- dera, 2014). The number of adsorbed water layer equivalents generally agrees with the order of column sorption capacities inFig. 2.7(A)but with some exceptions from the rule. Less than one monomolecular water layer equivalent (full horizontal line) was adsorbed on the silica hydride- based stationary phases and on moderately polar coreeshell columns (Ascentis Express F5 and Ascentis Express CN) at the column saturation capacity. On strongly polar stationary phases, several water layer equivalents are captured from the mobile phase. The sample partition between the bulk mobile phase and a submonomolecular layer of adsorbed water lacks a physical meaning. Rather, competition between the adsorbed water and polar solutes based on a NP adsorption mechanism (Pesek and Matyska, 2009) is more realistic. Hence, a low number of the adsorbed monomolecular equivalents,Nw<1, may be used to distinguish between aqueous normal phase (ANP) and tradi- tional HILIC (Soukup and Jandera, 2014). The strongest affinity to water was observed on the ZIC- cHILIC stationary phase, where more than nine water layer equivalents were adsorbed at the saturation capacity. Columns with bonded hydroxyl and diol ligands show stronger water adsorption in comparison to bare silica.
3.2 MOBILE PHASE IN HYDROPHILIC INTERACTION LIQUID CHROMATOGRAPHY
retention and separation selectivity among all water-miscible organic solvents in HILIC (or in ANP) chromatography. Methanol and other lower alcohols are protic solvents that provide significant hydrogen bonding interactions and compete with water for solvation of the polar adsorbent surface, decreasing the amount of adsorbed water layer on polar stationary phases (Quiming et al., 2007b).
Theoretically, it is possible to substitute water by an organic solvent and to employ, e.g., a methanoleacetonitrile mobile phase in “nonaqueous HILIC chromatography,” but usually the reten- tion is weaker than that in conventional HILIC separations (Bicker et al., 2008; Soukup and Jandera, 2013).
Mobile phases containing methanol (and to lesser extent ethanol, 2-propanol, or other protic sol- vents) often provide insufficient sample retention and broad or nonsymmetric peak shapes. The poor performance of alcohols as organic components of HILIC mobile phases is attributed to their similarity to water in providing strong hydrogen-bonding interactions. The retention is obviously promoted by increasing differences in selective polar interactions between the highly aqueous liquid layer occluded on the surface of polar adsorbents and the bulk organic-rich mobile phase. These differences are higher with acetonitrile-rich mobile phases in comparison to the mobile phases containing protic organic solvents.
The attempts to replace acetonitrile with a less toxic solvent have not been very successful so far.
Acetone has similar polarity as acetonitrile but shows lower sample retention under HILIC conditions.
Moreover, it cannot be used with UV detection and provides lower intensity MS signals (Fountain et al., 2010). At high concentrations of carbon dioxide added to ethanol-water mobile phases, HILIC separations of nucleic bases and other simple polar compounds on bare silica were reported to resemble those in acetonitrile-water mobile phases (Pereira et al., 2010). However, the routine use of such mobile phases can be subject to serious practical problems.
HILIC mobile phases usually contain a buffer, whose pH and ionic strength are usually selected to enhance the sample ionization, retention, and separation selectivity, i.e., pH>7 for acids and pH<7 for bases (Jandera, 2008; Hao et al., 2008). The retention in HILIC systems with uncharged stationary phases usually increases with increased salt (buffer) concentrations, probably because of enhanced hydrogen-bonding interactions between the analyte and the stationary phase. On the other hand, the retention of ionic samples on a stationary phase with ionic or ionizable functionalities may decrease with increasing ionic strength because the salt counterions displace the retained ionized sample molecules by ion-exchange interactions. That is why the retention of acidic compounds on bonded amino phases decreases at higher salt concentrations, in contrast to other simple polar bonded silica- based stationary phases (Nguyen et al., 2010).
High ionic strengths often reduce peak tailing in HILIC systems. Some samples may require a buffer concentration of 100 mmol/L or higher for acceptable peak shapes. The addition of trifluoro- acetic acid (TFA) to the mobile phase may also improve peak shapes of basic compounds by formation of ion associates. However, high ionic strengths or TFA additives are not recommended in HILIC/MS applications because they may suppress sample ionization in the mass spectrometer.
In some applications, water was partly replaced by a weaker polar protic solvent. For example, the retention of methacrylic acid, cytosine, nortriptyline, and nicotinic acid on a BEH-HILIC column increased considerably and the separation improved when substituting 5% of water with a second organic solvent (methanol, ethanol, or 2-propanol) in the original buffered mobile phase containing 90% acetontrile (Grumbach et al., 2008). Water can even be fully replaced by a polar organic solvent in so-called “nonaqueous HILIC chromatography” (NA-HILIC), employing mixed
polar organic mobile phases. There is no water in the mobile phase; instead, the liquid diffuse layer adsorbed on the stationary phase contains an increased concentration of an organic “protic modi- fier.” NA-HILIC may fill the gap between traditional nonaqueous NP (employing a nonpolar and one or more polar organic solvents) and aqueouseorganic HILIC systems. It can potentially be useful in the analysis of some poorly soluble oligomers or weakly polar compounds, which may precipitate in water-containing HILIC mobile phases. Selecting the type of protic solvent added to acetonitriledethylene diol, methanol, or ethanold(with elution strength decreasing in that order), the retention and separation selectivity of nucleobases, nucleosides, and deoxynucleosides could be adjusted in HILIC on bare silica, diol (Luna HILIC), bonded thioglycerol, and oxidized thioglycerol polar bonded phases.
In HILIC mode, the retention on polar columns increases with decreasing concentration of water in a water/organic mobile phase. The effect of water in the mobile phase on the sample retention factor, k¼tR/tM, can sometimes be described byEq. (2.3), or byEq. (2.4)(Jandera and Chura´cek, 1974), in the limited range of high water concentrations:
logk¼a1mHILIC$log4H2O (2.3)
logk¼a0mHILIC$4H2O (2.4)
where4H2Ois the volume fraction of water, “a1” is the (extrapolated) logarithm of the retention factor in pure water, and “a0” is the logarithm of the retention factor in pure organic solvent. (tRis the sample retention time andtMis the elution time of a nonretained standard, i.e., the column hold-up time.) The parameter “mHILIC” characterizes the rate of decreasing retention with increasing content of water in the highly aqueous mobile phases. Traditionally, it has been believed thatEq. (2.3)characterizes the retention in adsorption systems andEq. (2.4)in partition systems (Snyder and Poppe, 1980). In fact, various experimental HILIC systems show better data fit for either of the two equations (Jandera, 2011). However, a good fit of any model equation to the experimental data does not guarantee the correctness of the model. Furthermore, the original idea of the validity ofEq. (2.4) was applied to immiscible stationary and mobile phases, such as RP-LC with long-chain chemically bonded alkyl- silica materials, or organic NP-LC (Snyder, 1974). The correct application of theoretical models relies on knowledge of the exact volumes of both the stationary and mobile phases in the column, which can hardly be guaranteed in HILIC systems, where the amount of adsorbed water changes with the composition of the bulk aqueouseorganic mobile phase.
In many cases, neitherEq. (2.3)norEq. (2.4)fits the HILIC experimental data satisfactorily. For example, this was the case with 35 acidic, basic, and neutral polar compounds on bare silica and bonded aminopropyl, amide, diol and cyanopropyl columns, including fully porous, hybrid, and coreeshell types of particles (Vlckova´ et al., 2014). CombiningEq. (2.3)andEq. (2.4), we obtainEq.
(2.5), which takes into account a dual HILIC-RP retention mechanism and provides improved fit to the retention data over a broader range of aqueouseorganic mobile phase compositions, in comparison to polynomial empirical equations (Jin et al., 2008; Jandera and Ha´jek, 2009):
lnk¼aþbln4H2Oc4H2O (2.5)
The ratio of termsbandcofEq. (2.5)was tentatively employed to estimate the importance of the relative contributions of partitioning and the surface adsorption mechanism (Karatapanis et al., 2011).
Based on the results of application ofEq. (2.5) to the retention of water-soluble vitamins, it was
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).