Practical examination of flow rate effects and influence of the stationary phase water layer on peak shape and retention in hydrophilic interaction liquid chromatography.

The effect of flow velocity on retention and peak shape of neutral, acidic and basic probe compounds was studied using seven different UHPLC hydrophilic interaction chromatography (HILIC) columns. Surprisingly on some columns, the retention factor k was found to vary somewhat with flow velocity, due to the combined effects of pressure and of frictional heating on retention. The selectivity of different HILIC columns was much greater than typically found with RP columns. The volume of the water layer on the HILIC columns was measured using the toluene exclusion procedure. For the neutral solute uridine, a good correlation was found between the volume of the water layer and retention, indicating the likely domination of a partition mechanism. For the ionogenic solutes, the correlation was generally poor, due to the presence of strong additional mechanisms such as ionic retention and repulsion. Reduced Van Deemter plots for uridine showed a negative correlation between the reduced b coefficient and the volume of the water layer, which can be attributed to reduced surface diffusion in this viscous layer. Once again, the behaviour of ionic solutes was complex on some columns making detailed interpretation difficult.

Managing sample introduction problems in hydrophilic interaction liquid chromatography.

Sample injection can cause serious problems in hydrophilic interaction liquid chromatography (HILIC) when the injection solvent has higher elution strength than the mobile phase (mp). It can lead to asymmetric peak shapes and poor efficiency. The problem can occur when the mp contains a high proportion of organic e.g. 95% acetonitrile (a weak solvent) whereas the injection solvent contains a higher proportion of water (a strong solvent) that is necessary to dissolve polar samples. We investigated different strategies to overcome this problem. A simple method is pre-column dilution where the injector is programmed to deliver a plug of weak solvent (e.g. pure acetonitrile) along with the sample dissolved in a solvent with higher water content than the mp. Another option is to use alternative organic solvents to acetonitrile in the injection solvent, e.g. isopropanol, acetone or tetrahydrofuran, that may give enhanced sample solubility. The role of the volume of injection solvents was investigated as well as the possible effects of mass overload on the results. The use of small sample volumes is always recommended to reduce mismatch effects.

Influence of metals in the column or instrument on performance in hydrophilic interaction liquid chromatography.

A method is proposed for measuring the relative contribution of extracolumn and column effects to the detrimental interactions which occur between metal-sensitive solutes and the complete HPLC system. The method involves the substitution of a length of narrow bore silica tubing for the column and measuring the extracolumn contribution, which is subtracted from the total bandspreading measured with a column in place to yield the column contribution. The investigation focussed on HILIC separations, which have been relatively little studied compared with similar effects in RPLC. Metal-solute interactions can lead to tailing peaks and reduced sensitivity or even irreversible adsorption of particularly challenging solutes such as mono-, di- and triphosphorylated nucleotides, which show strong interactions between their phosphate groups and metals. A deactivated HILIC column, treated by a vapour deposition procedure gave generally good results when using high pH (pH 9.0) mobile phases, which suppress the effects of metals. The addition of metal complexing agents such as citrate at low millimolar concentration gave further improvements in peak shape at high pH, and even micromolar concentrations of citrate or medronic acid showed good results. These lower concentrations are more favourable for LC-MS. Addition of the higher concentration of citrate gave acceptable results for the nucleotides even at low pH (pH 3.0). With the standard UHPLC instrument used, loss of efficiency due to metal solute interactions was 25% or less, with most losses due to interactions with the column, although this result will depend on the condition and design of the instrument, which is easily assessed by the proposed procedure.

Evaluation of a linear free energy relationship for the determination of the column void volume in hydrophilic interaction chromatography.

The application of a linear free energy relationship (LFER) to a variety of hydrophilic interaction chromatography columns with different bonded ligands and pore sizes was studied in order to determine their void volume Vm. The method was based on the determination of the elution volume of a series of alkylbenzene standards from C1 (toluene) to C17 (heptadecylbenzene). Results were compared with those obtained by injection of toluene alone, which has traditionally been used as a simple Vm marker. Vm was smaller when derived from the LFER plot than when measured with toluene with differences between the two methods ranging from 2.7 to 12.7 % for the columns studied. This result could be due to the small but appreciable retention of toluene due to its solubility in the water rich layer, which partially constitutes the stationary phase in HILIC. Larger pore size columns showed less difference in Vm between LFER and toluene procedures. This result may be due to size sieving effects of non-excluded solutes in the pores of the stationary phase, or to differences in phase ratio between columns of different pore size.

Alternative mobile phase additives for the characterization of protein biopharmaceuticals in liquid chromatography - Mass spectrometry.

When analyzing large complex protein biopharmaceuticals, ion-pairing agents imparting low pH are widely used as mobile phase additives to improve the chromatographic performance. However, one of the most effective additives in RPLC and HILIC, trifluoroacetic acid (TFA), is known as a strong suppressor of the MS signal and limits its use in hyphenated techniques. In this study, we evaluated a wide range of acidic additives to find alternatives to TFA that provided comparable chromatographic performance and improved MS sensitivity. It was observed that stronger acidic additives were required for intact level analysis compared to subunit level analysis and that the additive nature had a larger impact on the chromatographic performance in HILIC mode compared to RPLC. Therefore, four additives were identified as valuable alternatives to TFA in RPLC mode, namely, difluoroacetic acid (DFA), dichloroacetic acid (DClAA), trichloroacetic acid (TClAA), and methanesulfonic acid (MSA). Only one of these additives provided acceptable performance in HILIC mode, namely, TClAA. After evaluation of the MS performance, TClAA was discarded due to the apparent loss of intensity in both RPLC-MS and HILIC-MS mode. Together, these results demonstrate that for HILIC-MS analysis TFA remains the gold standard additive. However, DFA was found as promising alternative to TFA for RPLC-MS analysis and could play an important role in the development of methods for the characterization of the increasingly complex protein biopharmaceuticals.

Managing the column equilibration time in hydrophilic interaction chromatography.

For a wide variety of hydrophilic interaction chromatography stationary phases, a repeatable partial equilibration was demonstrated in gradient elution after purging with as little as 12 column volumes of mobile phase. Relative standard deviations of retention time of on average ~0.15% could be obtained after 1 or 2 conditioning (blank) runs. The equilibration period must be kept strictly constant, otherwise selectivity changes occur, but this is not problematic on modern instruments. Partial equilibration was largely independent of stationary phase or gradient slope. Alternatively, full column equilibration is favoured for stationary phases that do not trap extensive water layers, and for materials with a wider pore size that have a lower surface area. Temperatures somewhat above ambient also shorten the equilibration time. Some stationary phases under optimum conditions can achieve full column equilibration using purging with ~12 column volumes, which is useful for rapid set-up of isocratic separations or for conventional gradient analysis.

Evaluation of additives on reversed-phase chromatography of monoclonal antibodies using a 1000 Šstationary phase.

A wide pore (1000 Å) diphenyl stationary phase was evaluated for the analysis of monoclonal antibodies (mAbs), comparing a conventional mobile phase of acetonitrile-water containing overall 0.1% trifluoracetic acid (TFA) with a similar mobile phase incorporating in addition 5% butanol. Alternatively, TFA was replaced by ammonium formate (AF) buffer (pH 3.0) and by methane sulfonic acid. Addition of 5% butanol to the mobile phase reduces the minimum temperature at which suitable UV analysis of the mAbs can be obtained from about 70 °C with TFA alone to about 60 °C thus potentially improving column lifetime and reducing the possibility of sample degradation. AF buffers produce satisfactory UV sensitivity at 70 °C and have the advantage of reducing signal suppression in mass spectrometry (MS). Some peak tailing was noted in comparison with TFA separations. Methane sulfonic acid at the same molar concentration as TFA produced the best chromatographic peaks, maintaining reasonable UV sensitivity down to 50 °C, also giving acceptable results even at only 3 mM concentration of the additive. The good results with this additive were attributed to its stronger acidity and consequent suppression of the ionisation of column silanols. Surprisingly, peak response (as measured by the size of the peaks) was rather poorly correlated with the peak capacity of the gradient analysis. A possible explanation is self-deactivation of active column sites by a portion of the sample.

Retention characteristics of some antibiotic and anti-retroviral compounds in hydrophilic interaction chromatography using isocratic elution, and gradient elution with repeatable partial equilibration.

The separation of some zwitterionic, basic and neutral antibiotic and antiretroviral compounds was studied using hydrophilic interaction chromatography (HILIC) on bare silica, bonded amide and urea superficially porous phases. The differences in the selectivity and retentivity of these stationary phases were evaluated for compounds with widely different physicochemical properties (logD -3.43 to 2.41 at wwpH 3.0). The mobile phase was acetonitrile-ammonium formate buffered at low wwpH. Compounds containing quinolone and serine groups showed poor peak shapes on all columns, attributed to metal-oxide interactions with system metals. Peak shapes were improved by addition of citrate buffers. Gradient elution, particularly with regard to column equilibration, was also studied due to the large differences in retention factors observed under isocratic conditions. Full equilibration in HILIC was slow for both ionogenic and neutral solutes, requiring as much as ∼40 column volumes. However, highly repeatable partial equilibration, suitable for gradient elution, was achieved in only a few minutes. Pronounced selectivity differences in the separations were shown dependent on the partial equilibration time.

Evaluation of the properties of a superficially porous silica stationary phase in hydrophilic interaction chromatography.

The sample capacity, column efficiency (and its variation with flow) of a superficially porous unbonded silica phase (Halo) was investigated using hydrophilic interaction chromatography (HILIC), particularly for separation of basic compounds. Sample capacity compared with totally porous silica phases was somewhat reduced, broadly in line with the decreased surface area, but still favourable compared with reversed-phase separations of these solutes. Efficiencies in excess of 100,000 plates were obtained at room temperature in reasonable analysis times by using a 45 cm coupled column, while generating back pressures compatible with conventional HPLC. Shorter columns offered the possibility of fast analysis of bases, and the unfavourable mass transfer properties reported by others at high flow rate for similar reversed-phase columns, were not apparent. While excellent peak shapes were obtained for many bases on silica HILIC phases, problems may still occur for some solutes.

Estimation of the extent of the water-rich layer associated with the silica surface in hydrophilic interaction chromatography.

The possible presence of a mobile phase layer rich in water on the surface of silica columns used under conditions typical in hydrophilic interaction chromatography was investigated by the injection of a small hydrophobic solute (benzene) using acetonitrile-water mobile phases of high organic content. Benzene does not partition into this layer and is thus partially excluded from the pores of the phase up to a water content of about 30%, after which hydrophobic retention of the solute on siloxane bonds is observed. In 100% acetonitrile, the retention volume of benzene was smaller than that estimated either by pycnometry or by calculation from the basic physical parameters of the column. This result might be attributable to the larger size of the benzene molecule: the elution volume of a molecule is the pore volume minus a surface layer half the diameter of the analyte molecule. However, some influence of strongly adsorbed water that remains on the surface of the phase even after extensive purging with dry acetonitrile cannot be entirely discounted. The results suggest that about 4-13% of the pore volume of a silica phase is occupied by a water-rich layer when using acetonitrile-water containing 95-70% (v/v) acetonitrile.

Analysis of basic compounds by reversed-phase high-performance liquid chromatography using hybrid inorganic/organic phases at high pH.

The retention and mass overload of five bases and a quaternary ammonium compound were studied on a bridged ethyl hybrid inorganic/organic phase (XBridge C18) over a pH range 2.7-12.0 using acetonitrile-phosphate and carbonate buffers. Some comparisons were drawn with results on a methyl hybrid (XTerra) phase. At low pH, rapid mass overload was observed with severe decreases in efficiency as sample mass was increased over the range 0.04-5 microg of solute. At intermediate pH (swpH 7.0), generally good peak shapes for small sample mass were still obtained on the ethyl hybrid, but with somewhat increased tailing of bases compared with swpH 2.7. At higher pH (swpH 10), good peak shape and improved loadability were obtained for moderately strong bases, due to their occurrence mostly as neutral species. However, stronger bases gave poor efficiency, attributed to interaction of the charged basic solute with increasingly ionised column silanol groups. Results were broadly similar on the methyl hybrid at swpH 10. At swpH 11, unusual profiles of increasing followed by decreasing efficiency were obtained on the ethyl hybrid for some bases as sample mass was increased; improved results were obtained at swpH 12. While the column loadability increased substantially at the highest pH studied, tailing for small sample mass was still more severe than at low pH, even though all compounds were <1% ionised in the mobile phase.

Investigation of the effect of pressure on retention of small molecules using reversed-phase ultra-high-pressure liquid chromatography.

The effect of inlet pressure on the retention of a series of low molecular weight acids, bases and neutrals, was investigated at constant temperature in reversed-phase liquid chromatography using a commercial ultra-high-pressure system (Waters UPLC instrument). For neutral compounds, relatively small increases in retention factor of up to approximately 12% for a pressure increase of 500bar were noted; the largest values were obtained for polar solutes, or solutes of higher molecular weight. Ionisable acids and bases gave much larger increases in retention with pressure, in some cases as high as 50% for a pressure increase of 500bar. Thus, such compounds could show increases in retention factor approaching 100% over the pressure range available in the commercial UPLC instrument. Due to these differential increases, significant selectivity effects can be obtained for mixtures of different types of solute merely by changing the pressure.

A study of the analysis of acidic solutes by hydrophilic interaction chromatography.

The analysis of acidic solutes was compared on a cross-linked bonded amino phase and a neutral hybrid inorganic-organic amide phase, previously shown to give reasonable retention of acidic solutes. The amino column gave strong selective retention of acids, which was governed by ionic interactions that mostly increased as the solute became more negatively charged at higher pH. While the relative selectivity of the amide column towards acids, bases and neutrals was completely different to that of the amino column, the selectivity of both columns towards acidic solutes alone was surprisingly similar. It is possible that solute charge also controls retention on the amide column, through increased solute hydrophilicity and increased hydrogen bonding between the ionised form of the acid and neutral polar column groups. On both these silica-based columns there appeared to be a competitive effect between repulsion of acidic solutes from silanols, which become increasingly ionised as the pH is raised. This effect was absent when using a polymer-based amino phase which has no silanols.

A study of column equilibration time in hydrophilic interaction chromatography.

The time taken to achieve full column equilibration for isocratic analysis of acidic, basic, and neutral solutes in hydrophilic interaction liquid chromatography (HILIC) was compared using the mobile phase disturbance method, column efficiency measurements and retention time stability. Full equilibration, which could take up to an hour, was best measured by the last procedure and was found to depend on the nature of the stationary phase, the pre-equilibrium (e.g. storage) solvent and the flow rate. While longer equilibration times are a relatively minor inconvenience in isocratic analysis, they are surprisingly not a barrier to the use of gradient elution in HILIC. A repeatable partial equilibration giving retention time stability equivalent to that in isocratic analysis was demonstrated for an equilibration time of only ∼5 min, using as few as 2 preliminary conditioning runs on a column that had taken the longest time to achieve full equilibration. Due to selectivity changes that occur dependent on the equilibration time, it is necessary to use identical gradient conditions in a series of analyses, which however appears to be facile on a modern HPLC instrument.

Is hydrophilic interaction chromatography with silica columns a viable alternative to reversed-phase liquid chromatography for the analysis of ionisable compounds?

The separation of acidic, neutral and particularly basic solutes was investigated using a bare silica column, mostly under hydrophilic interaction chromatography (HILIC) conditions with water concentrations >2.5% and with >70% acetonitrile (ACN). Profound changes in selectivity could be obtained by judicious selection of the buffer and its pH. Acidic solutes had low retention or showed exclusion in ammonium formate buffers, but were strongly retained when using trifluoroacetic acid (TFA) buffers, possibly due to suppression of repulsion of the solute anions from ionised silanol groups at the low (s)(s)pH of TFA solutions of aqueous ACN. At high buffer pH, the ionisation of weak bases was suppressed, reducing ionic (and possibly hydrophilic retention) leading to further opportunities for manipulation of selectivity. Peak shapes of basic solutes were excellent in ammonium formate buffers, and overloading effects, which are a major problem for charged bases in RPLC, were relatively insignificant in analytical separations using this buffer. HILIC separations were ideal for fast analysis of ionised bases, due to the low viscosity of mobile phases with high ACN content, and the favourable Van Deemter curves which resulted from higher solute diffusivities.

A study of retention and overloading of basic compounds with mixed-mode reversed-phase/cation-exchange columns in high performance liquid chromatography.

The retention and overload of bases were studied on two new mixed-mode, silica based phases possessing ionic carboxylate functionalities of different acidity embedded within a hydrophobic ligand (SiELC Primesep). At low pH, good peak shapes were obtained for small solute mass, suggesting that the mere presence of a mixed-mode hydrophobic/ionic retention mechanism is not responsible for the poor peak shape that can occur on conventional reversed-phases with ionised silanols. Somewhat inferior, but still acceptable peak shape for bases was obtained on a column containing a mixture of discrete ion exchange and reversed-phase particles (Hypersil Duet). In both types of column, the ionic sites favourably increased the capacity for ionised bases, reducing considerably the deterioration of peak shape with load observed with conventional RP columns. The combined ionic and reversed-phase interaction can give strong retention of bases under certain conditions, necessitating careful choice of stationary and mobile phase.

Effect of mobile phase additives on solute retention at low aqueous pH in hydrophilic interaction liquid chromatography.

Trifluoracetic acid (TFA) added to the aqueous acetonitrile mobile phase induces some unexpected changes in the ionic component of retention in hydrophilic interaction separations when using Type B silica and amide-bonded silica columns. TFA use results in anion exchange properties which contrast with the cation exchange typically found with ammonium salt buffers. The significant cation exchange properties of silica hydride columns are also moderated by TFA. Similar behaviour was shown in a metal- free amide column operated on a system washed with a metal complexing agent, suggesting that adsorbed metal cations were not responsible for this anion exchange behaviour. Both suppression of silanol ionisation at low pH and ion pairing of bases with TFA could contribute to this effect. It is also possible that the column surface acquires some positive charges at the low pH of TFA. A surprising reversal of the properties of the columns back to predominately cation exchange behaviour was shown using methanesulfonic acid (MSA), which appears to be a stronger acid than TFA in high concentrations of acetonitrile. MSA maintains sufficient ionic strength in the mobile phase even at low concentrations, giving good peak shape, which could be useful for mass spectrometry detection. Besides giving different selectivity to TFA, MSA also gives different selectivity to that of ammonium salt buffers, suggesting it may be useful in manipulating the selectivity of a separation. Similar changes to the selectivity with TFA could be achieved by adding neutral methylsulfonate salts to the TFA mobile phase. While it is possible that methylsulfonate ions are retained on the stationary phase surface, experiments using ion pair reagents of opposite charge yielded the same results as MSA salts. It therefore seems more likely that the higher ionic strength of these solutions negates the influence of charges that may be formed in TFA solutions. Ion pairing effects with MSA are expected to be limited.

Understanding and manipulating the separation in hydrophilic interaction liquid chromatography.

Hydrophilic interaction liquid chromatography (HILIC) has emerged as a valuable complimentary technique to reversed-phase (RP), being especially suited for the analysis of polar and ionised solutes, which are difficult to retain in RP. For solutes amenable to both separation mechanisms, HILIC provides a different selectivity to RP, and also offers possibilities as an orthogonal mechanism for 2-dimensional LC when combined with RP. HILIC has further advantages of lower column back pressures, and increased sensitivity with mobile phase evaporative detectors such as electrospray mass spectrometry. This review covers progress in our understanding of the HILIC technique, principally over the last ten years, including the classification of columns, the factors that control retention and selectivity, and attempts to model the separation process and its kinetics.

Study of overload for basic compounds in reversed-phase high performance liquid chromatography as a function of mobile phase pH.

The retention and overloading properties for eight basic solutes and two quaternary ammonium compounds were studied over the pH range 2.7-10.0 using phosphate and carbonate buffers. At low pH, a hybrid inorganic-organic silica-ODS phase (XTerra RP-18, 15 cm x 0.46 cm) showed substantial loss in efficiency when sample masses exceeded about 0.5 microg; these results were similar to those obtained previously on pure silica ODS and wholly polymeric phases, suggesting a common overloading mechanism. At pH 7-8.5, substantial improvements in loading capacity were obtained on XTerra due apparently to the unexpectedly strong influence of small decreases in solute ionisation. Data from the quaternary compounds suggested that silanol ionisation on this phase was still small even at intermediate pH. For many bases, loading capacity continued to improve as the pH was raised to 10, in line with the decrease in the proportion of ionised solute. However, for the highest pK(a) solutes, peak shape worsened at high pH, possibly due to the negative influence of increasing column silanol ionisation.