Development of two-dimensional liquid chromatographic systems for the optimised separation and targeted isolation of complex samples

Abstract

Chromatography is a powerful separation technique that was initially developed for the isolation of natural components in a highly purified form from complex mixtures. However, early applications of chromatography that were preparative in nature were quickly surpassed by analytical separations as the need for qualitative and quantitative information about components present in simple and complex mixtures became the primary objective. HPLC is the commonly used analytical separation technique for the determination of components in complex mixtures as it offers high sensitivity and also high selectivity. In general, HPLC is carried out in a single dimension using one primary retention mechanism dictated largely by the stationary phase. Although modern HPLC column technology has made available stationary phases with improved efficiency, particularly for the separations of complex mixtures, there are limits to its use. In reality there is only so much space available for components in a given sample to be separated into individual peaks, which is limited by the peak width and determined by the efficiency of the column. This is highly dependent upon the number of theoretical plates (N) available for the separation and therefore the peak capacity. Multidimensional HPLC is a technique that is gaining appreciable support at the analytical level due to the vastly expanded separation space that allows for increased resolution of components in complex samples. The introduction of a second dimension, which offers a change in selectivity to that of the first dimension, is a means of increasing the total peak capacity of the separation process and therefore expanding the separation space. Two-dimensional HPLC is an effective separation technique for the analysis of complex mixtures if the sample's complexity can be reduced as the separation mechanism of the first dimension may be tailored towards the sample's multidimensionality and/or its physical characteristics such as size, polarity, charge and shape. Reducing the complexity can be as simple as ensuring co-elution of the key components in the first dimension, but then utilising the second dimension to resolve those components co-eluting in the first dimension. Here the first dimension can be considered essentially as a 'clean-up' step in order to isolate the required targets in the second dimension. This essentially reduces the required peak capacity of the first dimension and the separation space in the second dimension more than compensates for this reduction as the second dimension is required to separate a lower number of components. Preparative HPLC has only seen resurgence in the last few decades with traditional methods such as distillation, centrifugal extraction and crystallisation unsuitable for the problems encountered by the various industries. The stringent regulations of governing bodies for the approval of highly purified products to be released into a highly competitive market dictate largely the advancement of chromatographic methods for preparative separations. Chapter 1 is a general introduction with a brief history of chromatography presented. The theory and practice of two-dimensional chromatographic separations and preparative chromatographic separations are also discussed. Sample dimensionality and its importance to the determination of orthogonality of separation steps are also considered. Chapter 2 details the chemicals and equipment used to perform the experiments outlined throughout the thesis. Specific general methods are also included here for reference. Chapter 3 describes a model framework for the development of a two-dimensional liquid chromatographic system and was reliant on low molecular weight oligostyrenes as they are complex, are indefinitely stable and easily characterised. The ultra high resolution separation of diastereomers of low molecular weight oligostyrenes on carbon adsorption stationary phases are also discussed. Chapter 4 examines the practical aspects in the optimisation of targeted isolations in two-dimensional HPLC with emphasis on analytical scale analysis. The emphasis in this chapter was on the isolation of "a" target analyte from "a" complex mixture, where effectively 'a' represents a generic sample, complex in nature. Chapter 4 also examines the practical aspects in the optimisation of targeted isolations in two-dimensional HPLC with emphasis on analytical scale analysis. However this chapter focuses on maximising the recovery of the target component at analytical scale analysis. Chapter 5 investigates the practical aspects in the optimisation of preparative scale two-dimensional isolations by the establishment of a continuous batch-wise 2D purification process, with the intent to preparative scale-up. In this chapter the experimental variables that effect the production of a target component are introduced and the influences they have on the separation are discussed. Chapter 5 also investigates the use of the system introduced at the preparative level however high sample loads are now used to determine the effect of the recovery in both the first and second dimensions; the purity of the collected product; and finally the product recovery yield, effective production rate and the practical production rate. Chapter 6 focuses on improving the quality of the isolation process by increasing the peak capacity of the second dimension firstly, followed by an increase in the peak capacity in both the first and second dimensions. This improved the resolution and therefore reduced the number of components transferred to the second dimension. This had the benefit of decreasing the performance demand of the second dimension column. Chapter 7 summarises all of the findings contained throughout this thesis.

Date of Award	2010
Original language	English