Deirdre Cabooter

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.

Hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics

This paper presents a general overview of the application of hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics in different sample matrices including pharmaceutical, plasma, serum, fermentation broths, environmental water, animal origin, plant origin, etc. Specific applications of HILIC for analysis of aminoglycosides, β-lactams, tetracyclines and other antibiotics are reviewed. HILIC can be used as a valuable alternative LC mode for separating small polar compounds. Polar samples usually show good solubility in the mobile phase containing some water used in HILIC, which overcomes the drawbacks of the poor solubility often encountered in normal phase LC. HILIC is suitable for analyzing compounds in complex systems that elute near the void in reversed-phase chromatography. Ion-pair reagents are not required in HILIC which makes it convenient to couple with MS hence its increased popularity in recent years. In this review, the retention mechanism in HILIC is briefly discussed and a list of important applications is provided including main experimental conditions and a brief summary of the results. The references provide a comprehensive overview and insight into the application of HILIC in antibiotics analysis.

Tryptic digest analysis by comprehensive reversed phase x two reversed phase liquid chromatography (RP-LC x 2RP-LC) at different pH's

As an alternative to the classical approach of combining strong cation exchange SCX-LC and RP-LC for the separation of complex proteomic samples, this essay describes the online comprehensive RP-LCxRP-LC separation of BSA and human blood serum. High orthogonality and peak capacity are achieved through the application of a significantly different pH in the two dimensions. The coupling of fused-core columns in series ensures high efficiency in the first dimension, while a previously designed interface with parallel second dimension columns further enhances the separation capability of the comprehensive system.

Kinetic plot based comparison of the efficiency and peak capacity of high-performance liquid chromatography columns: theoretical background and selected examples.

The present contribution reviews the foundations of the kinetic-plot method for the direct comparison of the kinetic performance of different chromatographic support and operating modes. The method directly uses experimental data collected for a specific sample and operating condition of ones interest, and is applicable both under isocratic- and gradient-elution conditions. Experimental proof is provided for the strong relation between the kinetic performance of a given support under isocratic and gradient conditions: a material offering superior kinetic performances under isocratic conditions will remain superior under gradient conditions and vice versa provided the comparison occurs under unbiased conditions. In addition, a review is made of the recent literature using the kinetic-plot method to compare and assess the kinetic performance of high performance HPLC columns and their operation mode.

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 microm) particles and high temperature liquid chromatography (HTLC) were investigated. 1.7 microm particles provide better efficiency and higher optimal linear velocities, although column lengths of approximately 20 cm should be used to avoid the detrimental effects of conversion reactions. More importantly, operation at temperatures up to 50 degrees 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 degrees C and analysis times of <2h.

Comparison of performance of high-performance liquid chromatography columns packed with superficially and fully porous 2.5 μm particles using kinetic plots.

A recently introduced 2.5 μm fully porous support (Kromasil Eternity) is compared with three different brands of superficially porous material (Kinetex, Halo and Poroshell 120) by means of the kinetic plot method using pharmaceutical compounds from GlaxoSmithKline as probe molecules. The kinetic plot method immediately shows the range of plate numbers wherein a support performs better than another. Results from experiments carried out at pH 4.5 and 8.0 are presented in order to assess the pH stability of the tested phases. Moreover, since all supports are able to withstand pressures higher than 400 bar, they have been evaluated both on HPLC and UHPLC instrumentation. True average particle sizes were determined by SEM images taken from loose stationary phases. Kinetex outperforms the other columns in HPLC conditions for practically relevant efficiencies, but shows poor packing quality in the 100×2.1-mm format. Kromasil is advantageous for simple and fast separations on short columns both in HPLC and in UHPLC conditions. Halo achieves the highest efficiencies of all columns at the lowest pressure cost and shows a noticeable lower axial diffusion. Poroshell 120 has the best packing quality reproducibility across the tested formats. All columns preserve their performance at high pH.

Isocratic and gradient impedance plot analysis and comparison of some recently introduced large size core-shell and fully porous particles.

The intrinsic kinetic performance of three recently commercialized large size (≥4μm) core-shell particles packed in columns with different lengths has been measured and compared with that of standard fully porous particles of similar and smaller size (5 and 3.5μm, respectively). The kinetic performance is compared in both absolute (plot of t0 versus the plate count N or the peak capacity np for isocratic and gradient elution, respectively) and dimensionless units. The latter is realized by switching to so-called impedance plots, a format which has been previously introduced (as a plot of t0/N(2) or E0 versus Nopt/N) and has in the present study been extended from isocratic to gradient elution (where the impedance plot corresponds to a plot of t0/np(4) versus np,opt(2)/np(2)). Both the isocratic and gradient impedance plot yielded a very similar picture: the clustered impedance plot curves divide into two distinct groups, one for the core-shell particles (lowest values, i.e. best performance) and one for the fully porous particles (highest values), confirming the clear intrinsic kinetic advantage of core-shell particles. If used around their optimal flow rate, the core-shell particles displayed a minimal separation impedance that is about 40% lower than the fully porous particles. Even larger gains in separation speed can be achieved in the C-term regime.

Method development for pharmaceutics: some solutions for tuning selectivity in reversed phase and hydrophilic interaction liquid chromatography.

In LC method development, the choice of suitable experimental conditions is often challenging for the analyst because of the huge diversity of stationary phases, mobile phase pH and organic modifiers, that could significantly alter the selectivity. The influence of these parameters on selectivity was experimentally tested in both RPLC and HILIC conditions for the analysis of 45 pharmaceutical compounds covering a wide range of physico-chemical properties. Principal component analysis (PCA) models were built to assess the resulting multivariate dataset. The complementarity between RPLC and HILIC was clearly demonstrated. The importance of mobile phase pH as one of the main experimental factors to be considered was confirmed. The RPLC and HILIC methods were thus employed for the analysis of a drug cocktail containing two substrates and their numerous desmethylated metabolites. All the compounds were finally resolved in both modes, with a very distinct elution order. In addition, the possibility to combine columns of different selectivity was highlighted using a column coupler set-up and found to be extremely promising. The same type of experiments was also carried out for the impurity profiling of an antihistaminic drug. In this example, compounds of very distinct polarity were satisfactorily eluted in both RPLC and HILIC modes, using suitable conditions of pH and stationary phase. In conclusion, this study demonstrates the complementary and interest of RPLC and HILIC in the case of pharmaceutical method development.

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.

Errors involved in the existing B-term expressions for the longitudinal diffusion in fully porous chromatographic media Part II: experimental data in packed columns and surface diffusion measurements.

Peak parking experiments have been performed on three RP-HPLC different columns, using two different components and a variable mobile phase composition. The aim of the study was to investigate whether the B-term diffusion expressions currently used in the literature (which are all Knox-type models) should be replaced by the effective diffusion expressions that have been developed in the frame of the effective medium theory (EMT). Although the EMT-expressions are not fully accurate either (the mathematics of the complex interactions between different diffusion zones that are in close contact are too demanding to catch them in an exact analytical expression), they at least are physically sound and do not violate Maxwells basic law of diffusion. Further they also provide a much better approximation of the numerically calculated effective diffusivity in the theoretical test situation considered in part I. The present study shows that the values of the surface or stationary phase diffusion coefficient that are derived from peak parking models can depend heavily on the employed B-term model. The EMT-based B-term expressions lead to values of the surface diffusion coefficient that vary much less strongly with the phase retention factor than if one of the Knox-type models is used to analyze the data. This implies that, since all peak parking experiments that have been performed in the past have all been interpreted with a Knox-type model, the conclusions that have been drawn from these studies should all be moderated or at least revisited.