Relationship between the particle size distribution of commercial fully porous and superficially porous high-performance liquid chromatography column packings and their chromatographic performance

The separation efficiency and kinetics of several commercial HPLC particle types (both fully porous and superficially porous) have been investigated using a pharmaceutical weakly basic N-containing compound as a test molecule. A strong trend between the particle size distribution (PSD) of the particles and the typically employed “goodness of packing”-parameters was observed. The relative standard deviation of the PSD of the tested particles ranged between 0.05 and 0.2, and in this range, a near linear relationship between the A-term constant, the h_min-value and the minimal separation impedance was found. The experimental findings hence confirm the recent observations regarding the relationship between the narrow PSD of the recently commercialized porous-shell particles and their superior efficiency and kinetic performance. The outcome also suggests that the performance of the current generation of fully porous particle columns could be significantly improved if the PSD of these particles could be reduced.     

Evaluation of a new polymeric stationary phase with reversed-phase properties for high temperature liquid chromatography

The performance of a polymeric stationary phase with reversed-phase properties (ET-RP1) was evaluated for LC separations at elevated temperature. The most significant observation was that the reduced plate height (h) decreased from 3.4 at 25 °C (optimal flow 0.5 mL/min) to 2.4 at 150 °C (optimal flow 2.5 mL/min) which is comparable to the efficiency obtained with silica-based reversed-phase columns of 4.6 mm ID operated at 0.8 mL/min. The phase showed no deterioration after long use at 150 °C within the pH range 1–9. Catalytic activity originating from the stationary phase material, e.g. as experienced on zirconium columns operated at elevated temperature, was absent. The performance of ET-RP1 is illustrated with the analysis of some pharmaceutical samples by LC and LC–MS. Operation at elevated temperature also allows to reduce the amount of organic modifier or to replace acetonitrile and methanol by the biodegradable ethanol.     

Total pore blocking as an alternative method for the on-column determination of the external porosity of packed and monolithic reversed-phase columns

An alternative method to determine the interstitial void volume and the external porosity inside a packed or a monolithic column was developed. The method is based on the total blocking of the mesopores of a porous support by filling them with a hydrophobic solvent. The strong interaction of the latter with the hydrophobic coating inside the pores keeps the solvent in position during the subsequent measurements. With the pores of the stationary phase material completely inaccessible for any type of polar molecules, the method allows to perform interstitial void measurements using small molecular weight (MW) molecules instead of the large MW molecules that need to be used in inverse size exclusion chromatography (ISEC). These small MW molecules are able to penetrate every corner of the interstitial volume and therefore lead to a very accurate determination of the external porosity. Since only one type of molecules needs to be injected, the often troublesome regression analysis needed in ISEC is omitted as well. In the present contribution, the method has been applied to a packed bed and a monolithic column to investigate the optimal conditions of flow velocity, liquid compositions, and unretained marker selection. The robustness and the repeatability of the method are discussed as well.     

High performance liquid chromatography analysis of wine anthocyanins revisited: Effect of particle size and temperature

The complex anthocyanin fraction of red wines poses a demanding analytical challenge. We have found that anthocyanins are characterised by extremely low optimal chromatographic velocities, and as a consequence generic HPLC methods suffer from limited resolving power. Slow on-column inter-conversion reactions, particularly between carbinol and flavylium species, are shown to occur on the same time scale as chromatographic separation, leading to increased plate heights at normal chromatographic velocities. In order to improve current routine HPLC separations, the use of small (1.7 μm) particles and high temperature liquid chromatography (HTLC) were investigated. 1.7 μm particles provide better efficiency and higher optimal linear velocities, although column lengths of ∼20 cm should be used to avoid the detrimental effects of conversion reactions. More importantly, operation at temperatures up to 50 °C increases the kinetics of inter-conversion reactions, and implies significantly improved efficiency under relatively mild analysis conditions. It is further demonstrated using relevant kinetic data that no on-column thermal degradation of these thermally labile compounds is observed at 50 °C and analysis times of <2 h.     

Ultra-rapid separation of an angiotensin mixture in nanochannels using shear-driven chromatography

The present paper reports on the separation of a mixture of fluorescein isothiocyanate-labeled angiotensin I and II peptides in a shear-driven nanochannel with a C18-coating and using an eluent consisting of 5% acetonitrile in 0.02 M aqueous phosphate buffer at pH 6.5. The flat-rectangular nanochannel in fused silica consisted of an etched structure in combination with a flat moving wall. The very fast separation kinetics that can be achieved in a nanochannel allowed to separate the angiotensin peptides in less then 0.2 s in a distance of only 1.8 mm. Plate heights as small as 0.4 microm were calculated after substraction of the injection effect.     

The kinetic plot method applied to gradient chromatography: Theoretical framework and experimental validation

The kinetic plot method, originally developed for isocratic separations, was extended to the practically much more relevant case of gradient elution separations. A set of explicit as well as implicit data transformation expressions has been established. These expressions can readily be implemented in any calculation spread-sheet program, and allow to directly turn any experimental data set representing the relation between the separation efficiency and the flow rate measured on a single column into the kinetic performance limit curve of the tested separation medium. Since the kinetic performance limit curve is based on an extrapolation to columns with a different length, it should be realized that the curve is only valid under the assumption that the gradient time and the delay time (if any) are adapted such that the analytes are subjected to the same relative mobile phase history when the column length is changed. Both experimental and numerical data are presented to corroborate the fact that the kinetic performance limit curves that are obtained using the proposed expressions are indeed independent of the column length the experimental data were collected in. Deviations might arise if excessive viscous heating occurs in columns with a pronounced non-adiabatic thermal behaviour.     

Future of high pressure liquid chromatography: do we need porosity or do we need pressure?

Making a theoretical study supported by experiments of the kinetic advantages of increased inlet pressures versus increased external porosity using impedance plots of analysis time versus required plate number, it is found that both approaches more or less have the same effect on the kinetic performance. The need to change a given system to one with an increased inlet pressure or with an increased external porosity can best be assessed from the optimal plate number (N(opt)) of the system. When the pursued application requires a plate number that is larger than N(opt), any increase in inlet pressure and external porosity is beneficial. When the required plate number is smaller than N(opt), any increase in inlet pressure and external porosity should preferentially be accompanied by an overall reduction of the feature sizes of the support. The degree to which this feature size reduction can be realized in practice will to a large extent determine which of the two approaches will be the dominant system of the future.     

Atmospheric Pressure Ionization Using a High Voltage Target Compared to Electrospray Ionization

A new atmospheric pressure ionization (API) source, viz. UniSpray, was evaluated for mass spectrometry (MS) analysis of pharmaceutical compounds by head-to-head comparison with electrospray ionization (ESI) on the same high-resolution MS system. The atmospheric pressure ionization source is composed of a grounded nebulizer spraying onto a high voltage, cylindrical stainless steel target. Molecules are ionized in a similar fashion to electrospray ionization, predominantly producing protonated or deprotonated species. Adduct formation (e.g., proton and sodium adducts) and in-source fragmentation is shown to be almost identical between the two sources. The performance of the new API source was compared with electrospray by infusion of a mix of 22 pharmaceutical compounds with a wide variety of functional groups and physico-chemical properties (molecular weight, logP, and pKa) in more than 100 different conditions (mobile phase strength, solvents, pH, and flow rate). The new API source shows an intensity gain of a factor 2.2 compared with ESI considering all conditions on all compounds tested. Finally, some hypotheses on the ionization mechanism, similarities, and differences with ESI, are discussed.

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One drop chemical derivatization – DESI‐MS analysis for metabolite structure identification

Structural elucidation of metabolites is an important part during the discovery and development process of new pharmaceutical drugs. Liquid Chromatography (LC) in combination with Mass Spectrometry (MS) is usually the technique of choice for structural identification but cannot always provide precise structural identification of the studied metabolite (e.g. site of hydroxylation and site of glucuronidation). In order to identify those metabolites, different approaches are used combined with MS data including nuclear magnetic resonance, hydrogen/deuterium exchange and chemical derivatization followed by LC‐MS. Those techniques are often time‐consuming and/or require extra sample pre‐treatment. In this paper, a fast and easy to set up tool using desorption electrospray ionization–MS for metabolite identification is presented. In the developed method, analytes in solution are simply dried on a glass plate with printed Teflon spots and then a single drop of derivatization mixture is added. Once the spot is dried, the derivatized compound is analyzed. Six classic chemical derivatizations were adjusted to work as a one drop reaction and applied on a list of compounds with relevant functional groups. Subsequently, two successive reactions on a single spot of amoxicillin were tested and the methodology described was successfully applied on an in vitro incubated alprazolam metabolite. All reactions and analyses were performed within an hour and gave useful structural information by derivatizing functional groups, making the method a time‐saving and efficient tool for metabolite identification if used in addition or in some cases as an alternative to common methods. Copyright © 2015 John Wiley & Sons, Ltd.     

High-efficiency high performance liquid chromatographic analysis of red wine anthocyanins

The analysis of anthocyanins in natural products is of significant relevance in recent times due to the recognised health benefits associated with their consumption. In red grapes and wines in particular, anthocyanins are known to contribute important properties to the sensory (colour and taste), anti-oxidant- and ageing characteristics. However, the detailed investigation of the alteration of these compounds during wine ageing is hampered by the challenges associated with the separation of grape-derived anthocyanins and their derived products. High performance liquid chromatography (HPLC) is primarily used for this purpose, often in combination with mass spectrometric (MS) detection, although conventional HPLC methods provide incomplete resolution. We have previously demonstrated how on-column inter-conversion reactions are responsible for poor chromatographic efficiency in the HPLC analysis of anthocyanins, and how an increase in temperature and decrease in particle size may improve the chromatographic performance. In the current contribution an experimental configuration for the high efficiency analysis of anthocyanins is derived using the kinetic plot method (KPM). Further, it is shown how analysis under optimal conditions, in combination with MS detection, delivers much improved separation and identification of red wine anthocyanins and their derived products. This improved analytical performance holds promise for the in-depth investigation of these influential compounds in wine during ageing.