Uwe D. Neue

Dependence of reversed-phase retention of ionizable analytes on pH, concentration of organic solvent and silanol activity

In reversed-phase chromatography, the retention of ionizable analytes is influenced by the ionic properties of the packing caused by surface silanol groups. We have measured the ion-exchange properties of both reversed-phase bonded phases and their underlying base materials. The probe used in this part of the study was bretylium tosylate. The acquired knowledge is then used for a complete and quantitative understanding of the retention behavior of ionizable compounds as a function of the pH of the mobile phase and the solvent composition. We have studied the retention pattern of a broad range of acids, bases, and polyfunctional analytes over the pH range from 2 to 11 and from water to 80% acetonitrile. A few application examples demonstrate the relevant findings.

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.

Universal procedure for the assessment of the reproducibility and the classification of silica-based reversed-phase packings: II. Classification of reversed-phase packings

Abstract The tools developed for the testing of the reproducibility of several commercial packings have been used to study the differences and similarities of over 50 different commercial reversed-phase packings. The tests employed allow a characterization of the hydrophobicity of packings, the silanol activity at neutral and acidic pH, a differentiation between classical packings and packings with an embedded polar functional group, and a differentiation between C18 and C8 packings.

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.

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

Universal procedure for the assessment of the reproducibility and the classification of silica-based reversed-phase packings : I. Assessment of the reproducibility of reversed-phase packings

A method is described that has been used since 1985 to assess the quality and reproducibility of several popular reversed-phase packings. Both the precision of the method and the reproducibility of the packing materials are described. The reproducibility of the newer packings surpasses that of the older packings. In addition, improved results are achieved today for the packings that existed in 1985.

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.

Peak capacity in unidimensional chromatography

The currently existing knowledge about peak capacity in unidimensional separations is reviewed. The majority of the paper is dedicated to reversed-phase gradient chromatography, covering specific techniques as well as the subject of peak compression. Other sections deal with peak capacity in isocratic chromatography, size-exclusion chromatography and ion-exchange chromatography. An important topic is the limitation of the separation power and the meaning of the concept of peak capacity for real applications.

A theoretical study of the optimization of gradients at elevated temperature

In order to achieve fast chromatographic separations of unknown entities, very rapid gradients need to be performed. We have studied the influence of temperature on such rapid gradient separations carried out under reversed phase conditions. The tool by which the quality of a gradient separation is measured is the peak capacity. Elevated temperature has two major influences. The first one is a reduction in viscosity, which reduces the backpressure for a given set of operating conditions. The second effect is an improvement in the diffusivity of the analytes, which speeds up mass transfer. We have assessed the influence of flow rate, gradient duration, and temperature on peak capacity using columns with different particle sizes and column lengths. The beneficial effect of temperature on both the viscosity and the diffusivity of the sample results not only in an improvement of the speed of the separations but also in a reduction of the back pressure under optimal operating conditions. At reasonable pressures, good separation power can be achieved in as little as one minute or less.

The application of novel 1.7 μm 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.