The complex nature of biological samples demands pre-separation prior to mass spectrometric analysis. Chromatographic techniques essentially form the foundation of the separation sciences and hence are at the core of analytical chemistry. In recent years, high-performance liquid chromatography (HPLC) has become the method of choice for separations and analysis of complex mixtures in various fields such as general chemistry, pharmaceuticals, natural products, food safety, forensic analysis and ‘-omic’ sciences (McMaster 2007). The need for separation of complex mixtures with varying physical and chemical properties along with technological advancements has led to the origins of a number of variants of liquid chromatographic techniques such as high-performance, ultra-pressure, supercritical fluid, hydrophilic interaction (HILIC) and ion chromatography. The versatility offered by liquid chromatographic techniques for the analysis of chemically diverse compounds is due to the customisation of LC through a choice of stationary phases along with additives such as buffers. Consequently, it offers greater control over separations. Selectivity for analytes is achieved in liquid chromatography by choice of columns in conjunction with modification of composition of mobile phases (Ardrey 2003). The core fundamental concepts of liquid chromatography have been extensively reviewed elsewhere (Ardrey 2003; McMaster 2007). Liquid chromatography is commonly combined with MS for high-throughput analysis of complex samples due to its high resolving power, robustness and easy hyphenation using electrospray ionisation (ESI).
Reversed phase liquid chromatography is the most commonly used form of LC separations and is based on the principle of hydrophobic interactions between analytes and a stationary phase. A non-polar stationary phase is employed which is typically a straight chain alkyl group (such as C18H37, known as C18 or ODS) linked covalently to a silica particle through a covalent siloxane bond. An aqueous, moderately polar mobile phase such as water combined with an organic solvent (methanol, acetonitrile etc.) is employed to separate analytes based on their hydrophobicity (Kellner et al. 2004). This results in the fast elution of polar molecules whilst less polar molecules are retained. A gradient elution can be applied to this system wherein the percentage of organic solvent in the eluent is gradually increased; the affinity of hydrophobic analytes towards the stationary phase is thus reduced resulting in faster elution times for such analytes. The
20
optimisation of the percentage of the organic solvent and the gradient profile is critical for ensuring maximum separation of analytes from any complex mixture prior to mass spectral analysis.
Recent years have seen evolution of traditional HPLC through technical advances giving rise to ultra-high performance liquid chromatography (UHPLC). UHPLC utilises smaller particle sizes (typically sub ~2 µm) or superficially porous particles for increased efficiency of chromatographic separation. The back-pressure generated by UHPLC columns is inversely related to the particle size of the media used to pack the column. Over the years the trend in liquid chromatography has been the development of efficient media (i.e. stationary phases) to improve chromatographic performance and reduce analysis times. This is achieved by building high performance pumps and instrumentation which can withstand high back-pressures generated by LC columns with smaller particle sizes or by using high efficiency particles such as core-shell stationary phases. These two technologies have been used in the research presented in this thesis and are outlined in the following sections:
1.1.3.1 UPLC
In HPLC columns, as the particle size decreases to less than 2.5 µm there is a significant increase in chromatographic efficiency which does not diminish even at increased mobile phase flow rates (Swartz 2005). As a result of using smaller particle size, speed and peak capacity can be increased. Ultra performance liquid chromatography employs sub 2 µm particles of materials such as bridged ethylene hybrid (BEH) C18 as a packing material (Waters Corporation, Manchester, UK). This has been termed UHPLC or UPLC™ (Waters Corporation, Manchester, UK). UPLC takes advantage of advancements in particle chemistry combined with instrumentation which is capable of operating at higher pressures as compared to that used in HPLC systems.
1.1.3.2 Poroshell
Porous silica is widely used as a column packing material in analytical and preparative LC systems. Porous silica particles have proved useful for the analysis of macromolecules such as proteins, polypeptides and nucleic acids (Kirkland et al. 2000). The concept of pellicular particles was first proposed by Horvath et al. in late 1960s and
21
were initially intended for analysis of macromolecules (Horvath & Lipsky 1969). The evolution of porous silica particles over the years has led to the development of superficially porous silica microspheres as a packaging material for HPLC (Cohen et al. 2011). These have been termed core-shell or poroshell particles (Kirkland et al. 2000).
Figure 1.10 Schematic depicting the structure of core-shell particles (Agilent
Technologies 2013).
Poroshell particles consist of an impermeable solid inner core surrounded by a uniform porous silica layer and thus are significantly different to conventional porous silica particles (Kirkland et al. 2000). These particles typically have a solid core of ~1.7 µm coated by a porous layer of 0.5 ~µm thickness. These stationary phases have been claimed to offer 80-90% of the efficiency at reduced system back-pressures of the order of ~40-50% compared to that offered by sub 2µm particles (Eddinger-Anderson et al. 2010). Poroshell particles provide increased column performance comparable with UHPLC systems without generating excessive back-pressure; as a result these can be used using conventional HPLC setup without the loss of chromatographic efficiency. However, the nature and design of these particles render them difficult for manufacturing. The preparation of thin porous shells (of the order of 0.25 to 0.5 µm) around spherical non-porous silica cores (of 1.2 to 1.7 µm diameters) is an expensive process. Along with the difficulties in manufacturing of core-shell particles, the efficient packing of the narrow bore columns with these particles presents further challenges in production of poroshell columns (Cohen et al. 2011).
22
The use of poroshell columns has not been limited to macromolecules and with technological advancement in the manufacturing of stationary phases, poroshell particles with smaller pore sizes which are well suited for small molecule analysis (of the order of 120 Å as opposed to 300 Å for macromolecule analysis) have been made possible, extending the analytical range of this technique (Agilent Technologies 2013).
In the research described in this thesis, both UPLC systems and Poroshell C18 particle columns have been used in separate metabolite profiling studies providing comparable chromatographic performance.