Influence of pressure on the retention of sugars in hydrophilic interaction chromatography.

Pressure can influence the retention of analytes in hydrophilic interaction chromatography as well as in RP chromatography. We demonstrate that the retention of sugars in hydrophilic interaction chromatography decreases with pressure, and interpret the observation as a gain in solvation, or more specifically hydration, as the sugar molecules enter the water-rich stationary phase.

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.

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.

The application of novel 1.7 microm ethylene bridged hybrid particles for hydrophilic interaction chromatography.

An un-derivatized 1.7 microm ethylene bridged hybrid (BEH) particle was evaluated for its utility in retaining polar species in hydrophilic interaction chromatography (HILIC), and was compared to a 3 microm un-derivatized silica material. Retentivity as a function of mobile phase pH, polar modifier and ACN content was examined. Also, the efficiency of the two particle substrates was compared by plotting HETP vs. linear velocity. Improved chemical resistance of the un-derivatized BEH particle was compared to un-derivatized silica at pH 5, demonstrating no performance deterioration over the course of 2000 injections for the BEH particle, while the silica particle deteriorated rapidly after 800 injections. Lastly, ESI-MS sensitivity as a function of particle size and separation mode was demonstrated. A 2.2 to 4.7-times higher ESI-MS response was observed on the 1.7 microm particle compared to the 3 microm particle, whereas a 5.6 to 8.8-times higher ESI-MS response was observed using HILIC as when compared to traditional RP chromatography.

Peak capacity in gradient reversed-phase liquid chromatography of biopolymers. Theoretical and practical implications for the separation of oligonucleotides.

Reversed-phase ultra-performance liquid chromatography was used for biopolymer separations in isocratic and gradient mode. The gradient elution mode was employed to estimate the optimal mobile phase flow rate to obtain the best column efficiency and the peak capacity for three classes of analytes: peptides, oligonucleotides and proteins. The results indicate that the flow rate of the Van Deemter optimum for 2.1 mm I.D. columns packed with a porous 1.7 microm C18 sorbent is below 0.2 mL/min for our analytes. However, the maximum peak capacity is achieved at flow rates between 0.15 and 1.0 mL/min, depending on the molecular weight of the analyte. The isocratic separation mode was utilized to measure the dependence of the retention factor on the mobile phase composition. Constants derived from isocratic experiments were utilized in a mathematical model based on gradient theory. Column peak capacity was predicted as a function of flow rate, gradient slope and column length. Predicted peak capacity trends were compared to experimental results.

Performance of idealized column structures under high pressure.

We have examined the influence of the longitudinal temperature and pressure gradients in columns operated under very high pressures on the coefficients of the van Deemter equation under the idealized condition of complete radial uniformity. These gradients change the diffusion coefficients over the length of the column, and the equation takes a new form, where the classical linear C-term is replaced by more complex forms that capture the effects of these axial gradients. The details of the derivations are shown and the implications are discussed.

Peak compression in reversed-phase gradient elution.

Previous reports suggest that peak widths in linear gradient elution are consistently larger than predicted by theory; however, if gradient compression is ignored, experiment and theory are in reasonable agreement. This suggests that gradient compression might represent an incorrect or poorly understood concept. In the present study, an experimental program was carried out to better understand the role of gradient compression and the reason for past differences between experiment and theory. It is concluded that the concept of gradient compression is correct.

Selectivity in reversed-phase separations Influence of the stationary phase.

The selectivity difference between 15 different stationary phases was measured using a large number of analytes at 2 or 3 different pH values (3, 7 and 10) with acetonitrile and methanol as the mobile phase modifiers. The packings discussed include standard C(8) and C(18) packings, packings with embedded polar groups, a phenyl packing, a pentafluoro-phenyl packing, an adamantylethyl packing and others. The major selectivity differences observed are discussed in detail. Specific effects such as pi-pi interactions on phenyl packings or hydrogen-bond interactions on phases with embedded polar groups are confirmed.

Theory of peak capacity in gradient elution.

Peak capacity is the best measure of the performance of a gradient separation. In this paper, the theory of peak capacity for the standard operating conditions of reversed-phase and ion-exchange chromatography is outlined. The influence of the operating conditions on the peak capacity of a separation are discussed. Finally, bandspreading phenomena in gradient chromatography are analyzed.

The combined effect of silanols and the reversed-phase ligand on the retention of positively charged analytes.

The nature of the interaction of positively charged analytes with the surface of reversed-phase bonded phases has been investigated as a function of both pH and volume fraction of organic modifier. Studies of the combined effect of both the parameters have been previously reported by us, and the data presented here further demonstrate a multiplicative interaction between pH and the concentration of organic modifier in the mobile phase. Fitting of the data as functions of pH and eluent composition clearly shows that the hydrophobically assisted ion-exchange process dominates over a purely reversed-phase or a pure ion-exchange retention mechanism. The underlying theory is developed in detail, and the mechanism is elucidated using several reversed-phase packings of substantially different character.

Comparison of the acidity of residual silanol groups in several liquid chromatography columns.

The silanol acidity of Waters Resolve C18, Waters Resolve silica, Waters Symmetry C18, Waters Symmetry silica, Waters XTerra MS C18 and underivatized XTerra columns has been measured from the retention of LiNO3 with a methanol/water (60:40) mobile phase buffered to different pH values. The Li+ cation is retained by cationic exchange with the background cation of the mobile phase (Na+) through the ionized silanols. The number of active silanols increases in the order: XTerra MS C18 << Symmetry C18 < underivatized XTerra << Resolve C18 < Resolve silica approximately equal to Symmetry silica. XTerra MS C18 does not present any residual silanol acidity up to s(s)pH 10.0 (pH in 60% methanol) as measured by LiNO3. The underivatized XTerra packing and Symmetry C18 present active silanols only at s(s)pH values higher than 7.0. For the other three columns, two different types of silanols with different acidity (s(s)pKa values about 3.5-4.6 and 6.2-6.8, respectively) have been observed. Symmetry C18 shows evidence of the presence of active basic sites that retain NO3- by anionic exchange.

Implications of column peak capacity on the separation of complex peptide mixtures in single- and two-dimensional high-performance liquid chromatography.

Column peak capacity was utilized as a measure of column efficiency for gradient elution conditions. Peak capacity was evaluated experimentally for reversed-phase (RP) and cation-exchange high-performance liquid chromatography (HPLC) columns, and compared to the values predicted from RP-HPLC gradient theory. The model was found to be useful for the prediction of peak capacity and productivity in single- and two-dimensional (2D) chromatography. Both theoretical prediction and experimental data suggest that the number of peaks separated in HPLC reaches an upper limit, despite using highly efficient columns or very shallow gradients. The practical peak capacity value is about several hundred for state-of-the-art RP-HPLC columns. Doubling the column length (efficiency) improves the peak capacity by only 40%, and proportionally increases both the separation time and the backpressure. Similarly, extremely shallow gradients have a positive effect on the peak capacity, but analysis becomes unacceptably long. The model predicts that a 2D-HPLC peak capacity of 15,000 can be achieved in 8 h using multiple fraction collection in the first dimension followed by fast RP-HPLC gradients employing short, but efficient columns in the second dimension.

Development of an accelerated low-pH reversed-phase liquid chromatography column stability test.

The important experimental design criteria for an accelerated low-pH RPLC column stability test are discussed. The influence of method variables on the amount and rate of retention-loss and the final optimized parameters for the accelerated low-pH RPLC stability test are presented. The retention-loss curves for selected C8 and C18 stationary phases are compared. These studies indicate that ligand chain length, functionality and bonding density play an important role in determining the low-pH stability of a stationary phase. Additionally, elemental analysis data are used to infer the mechanism responsible for the observed retention-loss under low-pH conditions.

Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction.

An ion-pair reversed-phase HPLC method was evaluated for the separation of synthetic oligonucleotides. Mass transfer in the stationary phase was found to be a major factor contributing to peak broadening on porous C18 stationary phases. A small sorbent particle size (2.5 microm), elevated temperature and a relatively slow flow-rate were utilized to enhance mass transfer. A short 50 mm column allows for an efficient separation up to 30mer oligonucleotides. The separation strategy consists of a shallow linear gradient of organic modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases have been traditionally used for oligonucleotide separations with good result. However, the oligonucleotide retention is affected by its nucleotide composition. We developed a mathematical model for the prediction of oligonucleotide retention from sequence and length. We used the model successfully to select the optimal initial gradient strength for fast HPLC purification of synthetic oligonucleotides. We also utilized ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA-HFIP aqueous buffers are useful for a highly efficient and less sequence-dependent separation of heterooligonucleotides.

Role of mass spectrometry in the purification of peptides and proteins.

Experiments were carried out to evaluate the fractionation of proteins and peptides according to mass. Model mixtures were separated by either reversed-phase or ion-exchange chromatography with mass spectrometry-compatible mobile phase additives. Fraction collection was triggered by the mass/charge ratio of each one of the components of the mixture. Chromatography was additionally monitored with a UV-Vis detector in order to compare the new technique with generally accepted in separations. The results indicated that adequate purification is achieved by this new technique. Fraction collection triggered by changes in the mass/charge ratio reduces sample handling and analysis time. This study demonstrates the utility of mass-directed fractionation of peptides and proteins when mass spectrometry-compatible mobile phase additives are used.

The determination of the plate count for large molecules under reversed-phase gradient conditions.

The true performance of HPLC columns in the gradient separation of macromolecules can be assessed by measuring the true plate count, if we know the retention factor of the analyte at the point of elution. We are demonstrating in this short communication how this can accomplished in a straightforward fashion. The procedure used here is a significant simplification over previous approaches, and enables the chromatographer to do so without complex algebra or additional experiments.

Further investigations of the effect of pressure on retention in ultra-high-pressure liquid chromatography.

In this study, we investigated further the large increases in retention with pressure that we observed previously in RP-LC especially for ionised solutes. These findings were initially confirmed on a conventional silica C(18) column, which gave extremely similar results to the hybrid C(18) phase originally used. Large increases in retention factor of approximately 50% for a pressure increase of 500 bar were also shown for high MW polar but neutral solutes. However, experiments with the same bases in ionised and non-ionised forms suggest that somewhat greater pressure-induced retention increases are found for ionised solutes. Retention increases with pressure were found to be considerably smaller for a C(1) column compared with a C(18) column; decreases in retention with increasing pressure were noted for ionised bases when using a bare silica column in the hydrophilic interaction chromatography (HILIC) mode. These observations are consistent with the partial loss of the solvation layer in RP-LC as the solute is forced into the hydrophobic environment of the stationary phase, and consequent reduction in the solute molar volume, while the water layer on the surface of a HILIC packing increases the hydration of a basic analyte. Finally, retention changes with pressure in RP-LC can also be observed at a mobile phase pH close to the solute pK(a), due to changes in pK(a) with pressure. However, this effect has no influence on the results of most of our studies.

Effects of extra-column band spreading, liquid chromatography system operating pressure, and column temperature on the performance of sub-2-microm porous particles.

The effects of extra-column band spreading, LC system operating pressure, and separation temperature were investigated for sub-2-microm particle columns using both a conventional HPLC system as well as a UPLC system. The contributions of both volume- and time-based extra-column effects were analyzed in detail. In addition, the performance difference between columns containing 2.5 and 1.7-microm particles (same stationary phase) was studied. The performance of these columns was compared using a conventional HPLC system and a low dead volume UPLC system capable of routine operation up to 1000 bar. The system contribution to band spreading and the pressure limitations of the conventional HPLC system were found to be the main difficulties that prevented acceptable performance of the sub-2-microm particle columns. Finally, an increase in operating temperature needs to be accompanied by an increase in flow rate to prevent a loss of separation performance. Thus, at a fixed column length, an increase in temperature is not a substitute for the need for very high operating pressures.