Patrik Petersson

Critical comparison of performances of superficially porous particles and sub-2 mu m particles under optimized ultra-high pressure conditions

The performance of 2.7 microm superficially porous particles at 600 bar and sub-2 microm fully porous particles at 1000 bar were compared by the Poppe plot method. Theoretical Poppe plots were first constructed for each stationary phase to compare their kinetic performance at different analysis times. The theory was then verified by experiments under the optimized conditions identified from the Poppe plot calculation. We found that the 2.7 microm superficially porous particles at 600 bar can provide similar performance compared to the sub-2 microm fully porous particles at ultra-high pressure (1000 bar) when analysis times are very short (e.g. sub-minute). As analysis time increases, the superficially porous particles start to outperform the sub-2 microm particles and can give much higher efficiencies (e.g. > 2 times higher plate count) at very long analysis times (>3h). The comparison was extended to gradient elution of a mixture of pharmaceutical interest by constructing gradient peak capacity Poppe plots and similar behavior was observed.

Maximizing peak capacity and separation speed in liquid chromatography.

The practical effects of gradient time and flow rate on the peak capacities of a range of analytes of differing molecular weights (MWs) and physico-chemical properties have been evaluated using ultra high pressure LC instrumentation with sub-2 mum and superficially porous particle phases. Optimum peak capacity, in RP gradient LC, for small molecules, including typical pharmaceutical drugs and peptides with MWs up to 1300, was demonstrated at a maximum flow rate for a given gradient time (i.e. up to 40 min). Flow rates significantly higher than the optimum in the van Deemter plots and also higher than those typically employed by the majority of the chromatographers today are recommended for gradient LC (i.e. up to 1.0 mL/min on 50-150x2.1 mm 1.7 mum columns). This recommendation is applicable for temperatures above 40 degrees C, i.e. temperatures typically utilized for separations employing sub-2 mum particles to reduce column back pressure. Van Deemter and pseudo van Deemter plots were determined and combined with chromatographic gradient elution theory to explain our unexpected observations. The derived models exhibited good agreement between experimental and predicted peak capacities (absolute average error 4%, max. error 12%).

An automatic peak finding method for LC‐MS data using Gaussian second derivative filtering

A highly automated procedure for localising and characterising peaks in the chromatographic time domain of LC-MS data has been developed. The work was initiated by an identified need to facilitate the detection and tracking of chromatographic peaks during method development for the analysis of impurities in pharmaceutical products. The algorithm is mainly based on a digital filter for which the settings are automatically adapted to the data set under study. The procedure was evaluated for synthetic data sets with various S/N levels, peak widths and baseline properties. It was found that even for the worst case tested with S/N=10 and a high variability in the baseline, 94% of the simulated analytical peaks could be detected without producing any false-positive identifications. Furthermore, the number of correctly estimated peak heights and peak widths falling within a 10% error of the true values were 94 and 91%, respectively. For experimental data sets, peak height, and width estimations were more difficult, but the processed reconstructions showed an excellent agreement with the analytical signals of the raw data, and also a clearly improved visualisation in total ion- and base-peak chromatograms.

Why ultra high performance liquid chromatography produces more tailing peaks than high performance liquid chromatography, why it does not matter and how it can be addressed

The purpose of this study is to demonstrate, with experiments and with computer simulations based on a firm chromatographic theory, that the wide spread perception of that the United States Pharmacopeia tailing factor must be lower than 2 (T(f)<2) is questionable when using the latest generation of LC equipment. It is shown that highly efficient LC separations like those obtained with sub-2 μm porous and 2.7 μm superficially porous particles (UHPLC) produce significantly higher T(f)-values than the corresponding separation based on 3 μm porous particles (HPLC) when the same amount of sample is injected. Still UHPLC separations provide a better resolution to adjacent peaks. Expressions have been derived that describe how the T(f)-value changes with particle size or number of theoretical plates. Expressions have also been derived that can be used to scale the injection volume based on particle size or number of theoretical plates to maintain the T(f)-value when translating a HPLC separation to the corresponding UHPLC separation. An aspect that has been ignored in previous publications. Finally, data obtained from columns with different age/condition indicate that T(f)-values should be complemented by a peak width measure to provide a more objective quality measure.

Efforts to improve detection sensitivity for capillary electrophoresis coupled to atmospheric pressure photoionization mass spectrometry.

Electrospray ionization performs best with volatile buffers. However, generally the best separation performance for capillary electrophoresis (CE) is achieved with non-volatile buffers. Hyphenation of CE with mass spectrometry (MS) utilizing atmospheric pressure photoionization (APPI) enables use of a wider range of separation buffers without compromising detection sensitivity. As APPI is considered to be mass flow sensitive, the use of a larger inner diameter separation capillary (75 microm) allows larger volumes to be injected, without decreased separation performance, thus providing improved sensitivity (approx. a factor of 10), compared to the use of a 25 microm capillary. However, nebulizing gas flow and position of capillary tip in the sprayer have to be carefully optimized to prevent excessive band broadening. Further improvement in sensitivity (approx. a factor of 2) was obtained by decreasing the distance between the sprayer and ionization region, indicating that a specially designed CE/APPI-MS interface for low flow rates will be favourable.

Combined use of algorithms for peak picking, peak tracking and retention modelling to optimize the chromatographic conditions for liquid chromatography-mass spectrometry analysis of fluocinolone acetonide and its degradation products.

A strategy for rapid optimization of liquid chromatography column temperature and gradient shape is presented. The optimization as such is based on the well established retention and peak width models implemented in software like e.g. DryLab and LC simulator. The novel part of the strategy is a highly automated processing algorithm for detection and tracking of chromatographic peaks in noisy liquid chromatography-mass spectrometry (LC-MS) data. The strategy is presented and visualized by the optimization of the separation of two degradants present in ultraviolet (UV) exposed fluocinolone acetonide. It should be stressed, however, that it can be utilized for LC-MS analysis of any sample and application where several runs are conducted on the same sample. In the application presented, 30 components that were difficult or impossible to detect in the UV data could be automatically detected and tracked in the MS data by using the proposed strategy. The number of correctly tracked components was above 95%. Using the parameters from the reconstructed data sets to the model gave good agreement between predicted and observed retention times at optimal conditions. The area of the smallest tracked component was estimated to 0.08% compared to the main component, a level relevant for the characterization of impurities in the pharmaceutical industry.

A component tracking algorithm for accelerated and improved liquid chromatography-mass spectrometry method development

A method for tracking of sample components during liquid chromatography-mass spectrometry (LC-MS) method development has been proposed. The method manages to, fully automatically and without user intervention, find the chromatographic peaks in the data sets, discriminate them to sample components and track them when the separation conditions have been changed. The algorithm utilises the resolution obtained from all considered data sets and has the ability to discriminate the non informative parts. The technique has a great sensitivity even in cases where a majority of the tracked components cannot easily be spotted by means of traditional total ion chromatogram (TIC) or base peak chromatogram (BPC) representations. The method was tested on an experimental sample using six different columns and an average of 79% of the suggested sample components could be successfully tracked at a minimum area of 0.05% of the main component in the sample. 66 components with 79-92% of the total suggested component area were able to be tracked between all data sets. The method could be used to rapidly investigate selectivity during different types of separation conditions.