Harald Ritchie

Enhanced separation performance using a new column technology: parallel segmented outlet flow.

A new column technology - termed parallel segmented outlet flow was employed here to illustrate gains in separation performance that are achievable by the active management of flow as it exits from the outlet of the chromatography column. Parallel segmented outlet flow requires a column be fitted with an outlet fitting that separates flow from the central region of the column from that of wall region. Each region of flow is able to be processed independently, such that post column detection emulates end column localised detection. As a result of this flow segmentation and the subsequent more efficient means of detection, column efficiency was observed to increase by more than 20%, with gains in sensitivity by as much as 22%, and a decrease in peak volume by up to 85%.

Transition from enantioselective high performance to ultra-high performance liquid chromatography: A case study of a brush-type chiral stationary phase based on sub-5-micron to sub-2-micron silica particles

Three brush-type chiral stationary phases (CSPs) differing in the particle size of the starting silica particles have been prepared by covalent grafting of the pi-acidic bis-(3,5-dinitrobenzoyl)-derivative of trans-1,2-diaminocyclohexane (DACH-DNB). Starting silica particles of 4.3, 2.6 and 1.9micron were used to generate the final CSPs using an improved, highly reproducible synthetic methodology, that allowed to assemble and surface-graft the whole chiral selector in only two steps. The different CSPs have been packed in columns of various length and diameters, and fully characterized in terms of flow permeability, kinetic performances and enantioselectivity using a set of test solutes. Very high speed and high resolution applications together with stereodynamic HPLC examples are demonstrated on the columns with reduced particle diameters, on which separations of several enantiomeric pairs are routinely obtained with analysis times in the 15-40s range.

Introducing enantioselective ultrahigh-pressure liquid chromatography (eUHPLC): theoretical inspections and ultrafast separations on a new sub-2-μm Whelk-O1 stationary phase.

A new chiral stationary phase for ultrahigh-pressure liquid chromatography (UHPLC) applications was prepared by covalent attachment of the Whelk-O1 selector to spherical, high-surface-area 1.7-μm porous silica particles. Columns of varying dimensions (lengths of 50, 75, 100, and 150 mm and internal diameters of 3.0 or 4.6 mm) were packed and characterized in terms of permeability, efficiency, retention, and enantioselectivity, using both organic and water-rich mobile phases. A conventional HPLC Whelk-O1 column based on 5.0-μm porous silica particles and packed in a 250 mm × 4.6 mm column was used as a reference. Van Deemter curves, generated with low-molecular-weight solutes on a 100 mm × 4.6 mm column packed with the 1.7-μm particles, showed H(min) (μm) and μ(opt) (mm/s) values of 4.10 and 5.22 under normal-phase and 3.74 and 4.34 under reversed-phase elution conditions. The flat C term of the van Deemter curves observed with the 1.7-μm particles allowed the use of higher-than-optimal flow rates without significant efficiency loss. Kinetic plots constructed from van Deemter data confirmed the ability of the column packed with the 1.7-μm particles to afford subminute separations with good efficiency and its superior performances in the high-speed regime, compared to the column packed with 5.0-μm particles. Resolutions in the time scale of seconds were obtained using a 50-mm-long column in the normal phase or polar organic mode. The intrinsic kinetic performances of 1.7-μm silica particles are retained in the Whelk-O1 chiral stationary phase, clearly demonstrating the potentials of enantioselective UHPLC in terms of high speed, throughput, and resolution.

Parallel segmented flow chromatography columns: Conventional analytical scale column formats presenting as a ‘virtual’ narrow bore column☆

Narrow bore columns find advantage in HPLC applications when volumetric flow is important, For example, for detection processes that are volume limited. Yet there are significant drawbacks to narrow bore columns. Due to their small column volume relative to analytical scale columns, narrow bore columns are more affected by system dead volume. In addition the wall effect and the variation in packing density from the centre to the wall are more significant in these columns relative to larger scale analytical columns. In this study we operate a 4.6mm i.d. parallel segmented flow column in such a manner that it emulates 2.1mm i.d. and 3.0mm i.d. columns. By using a parallel segmented flow column in this way, it was possible to combine the benefits of narrow bore and analytical scale columns.

Retention studies on mixed-mode columns in high-performance liquid chromatography

The retention properties of a column prepared by mixing together strong cation exchange (SCX) and reversed-phase (C18) packing materials were investigated using a range of test solutes. The column was found to exhibit chromatographic properties characteristic of both phases. The effects of changes in eluent composition, buffer ion, ionic strength and pH on the capacity factors of different compounds were determined. The dual nature of the retention mechanism allowed the retention of ionisable molecules to be adjusted by altering the composition of the aqueous component of the mobile phase while those of compounds uncharged over the pH range investigated remained unaffected. Results were compared those obtained on a C18 column and it was found that the acidic and weakly basic compounds had higher capacity factors on this column whereas strongly basic compounds had higher capacity factors on the mixed-mode column.

One-Pot Synthesis of Spheres-on-Sphere Silica Particles from a Single Precursor for Fast HPLC with Low Back Pressure

Spheres-on-sphere (SOS) silica particles are prepared in a one-pot scalable synthesis from mercaptopropyltrimethoxysilane with hydrophilic polymer and cationic surfactant under alkaline conditions. The SOS particles exhibit solid-core porous-shell properties. The fast separation of small molecules and proteins with low back pressure are demonstrated by high-performance liquid chromatography (HPLC) for the columns packed with SOS-particles.

Enantioselective ultra-high and high performance liquid chromatography: a comparative study of columns based on the Whelk-O1 selector.

This paper reports on the thermodynamic and kinetic evaluation of a new ultra-high performance liquid chromatography broad-spectrum Pirkle-type chiral stationary phase (CSP) for enantioselective applications (eUHPLC). The well-known Whelk-O1 selector was covalently immobilized onto 1.7-μm high-surface-area, porous spherical silica particles to produce a totally synthetic, covalently bonded CSP that was packed into 150 mm, 100mm, 75 mm and 50mm columns, either 4.6 or 3.0mm ID. A 100 mm × 4.6 mm ID column was fully characterized from a kinetic and thermodynamic point of view, using as reference a conventional HPLC Whelk-O1 column, 250 mm×4.6mm ID, based on 5-μm porous silica particles. On the eUHPLC column, van Deemter plots generated H(min) values of 3.53 μm for 1,3-dinitrobenzene, at an interstitial mobile phase linear velocity (μ(inter)) of 5.07 mm/s, and H(min) of 4.26 and 4.17 μm for the two enantiomers of acenaphthenol, at μ(inter) of 4.85 mm/s and 4.24 mm/s, respectively. Resolution of 21 enantiomeric pairs including alcohols, epoxides, sulfoxides, phosphine oxides, benzodiazepines and 2-aryloxyproprionic esters used as herbicides, were obtained with significant advantages in terms of efficiency and analysis time. Speed gain factors were calculated for the different column geometries (150 mm, 100mm, 75 mm and 50mm, either 4.6 or 3.0mm ID), with respect to the standard HPLC column (250 mm ×4.6 mm ID), and were as high as 13, in the case of the 50-mm-long column, affording sub-minute separations of enantiomers with excellent resolution factors. In particular, trans-stilbene oxide was resolved in only 10s, while a 50 mm×3.0 mm ID column was used as a compromise between reduced mobile phase consumption (less than 1 mL per analysis) and smaller extra-column band-broadening effect. Given the relatively low viscosity in NP mode, and the excellent permeability of these eUHPLC columns, with backpressure values under 600 bar for a wide range of flow rates, the use of standard HPLC hardware is possible. In this case, however, a significant loss in resolution is observed, compared to the UHPLC instrumentation, if no modifications are introduced in the HPLC apparatus to reduce extra-column variance. The excellent efficiency and selectivity, conjugated with the very high-throughput and the ultra-fast analysis time, prove the potentials of the eUHPLC Whelk-O1 columns in the development of enantioselective UHPLC methods.

The use of silica for liquid chromatographic/mass spectrometric analysis of basic analytes

Silica stationary phases of varying purity have been compared and evaluated for the LC separation of pharmaceutically relevant basic compounds of differing molecular weight, log P, and pK a using aqueous/organic mobile phases. The least pure silica gave the best separations. All phases showed a linear relationship between In k and volume fraction of ACN (30-70%) or MeOH (15-35%). The investigations confirmed that all the unbonded phases examined possessed a hydrophobic/adsorption and ion-exchange character. The use of silica with MS friendly mobile phases, and high volume fractions of ACN, has been shown to be applicable to LC/MS analysis of basic analytes.

Gradient elution chromatography with segmented parallel flow column technology: A study on 4.6 mm analytical scale columns☆

A new column format known as parallel segmented flow has recently been introduced, whereby improvements in column performance are observed. These improvements are achieved via the separation of eluent from the column core from that of the column wall region. The segmentation of flow is accomplished immediately as the eluent exits the column through the use of a multi-channel end fitting. The ratio of flow exiting through the column central port relative to the peripheral ports, known as the segmentation ratio, can be tuned to optimise chromatographic performance. Investigations into the use of parallel segmented flow chromatography columns have demonstrated increased sensitivity and theoretical plates in analytical scale isocratic separations, but so far no studies have detailed the performance of these columns in gradient elution. The current study addresses the performance of parallel segmented flow columns in gradient elution, detailing the reproducibility of the gradient at various segmentation ratios and compares the performance to conventional columns. The study found that there was no observable difference in the gradient shape, or reproducibility of the gradient profiles generated at any segmentation ratio, tested on three different types of stationary phases. A separation of an 11-component test mixture verified that the primary advantage of parallel segmented flow columns was that the peak volume was reduced in proportion to the segmentation ratio.

Investigation on synthesis of spheres-on-sphere silica particles and their assessment for high performance liquid chromatography applications.

There is a continuing challenge in improving the separation speed while keeping both the resolution high and the back pressure low in high performance liquid chromatography (HPLC). The recent renaissance of core-shell (or fused-core) silica particles has shown great promise in this respect. However, the fused-core silica particles are typically synthesized by the time-consuming multiple-step layer-by-layer technique. An one-pot synthesis of spheres-on-sphere (SOS) silica microspheres is presented here. The preparation parameters including Si precursors, mixing methods (magnetic stirring, mechanical stirring and homogenization), heating (microwave heating and conventional heating), and reaction temperature are investigated in order to control the morphology and improve the size distribution of the SOS particles. The improvement and influence on SOS morphology, particle size and particle size distribution are discussed. Furthermore, the calcined and modified SOS particles are packed into stainless steel columns, which are then assessed for the separation of various test mixtures containing small molecules and proteins under normal phase, reversed phase, and HILIC conditions. The HPLC tests demonstrate fast and efficient separation with very low back pressure, suggesting that SOS particles are a type of new and highly promising packing materials for liquid chromatography.