High performance liquid chromatography (HPLC)

The development of GLC reduced interest in liquid chromatography until recently, when HPLC almost began superseding GLC for many analytical purposes. High Performance Liquid Chromatography, while eliminating the need for vapourising the sample, rivals GLC with high resolution, speed, sensitivity, automatic operation and the unsurpassed range of applications. High performance liquid chromatography is now recognized as a powerful analytical and preparative technique. The principle of operation of HPLC is that is common to liquid chromatography; it exploits different affinities of the components for a stationary phase packed into a column and a mobile liquid phase that percolates through. While the use of a single solvent (isocratic separation) may often be adequate to achieve a desired separation, more complex mixtures may require a gradient elution. In gradient elutions the eluting power of a mobile phase is increased with the gradual addition of a more polar solvent. Separation is determined by monitoring the column efflluent using a sample detector. A chromatogram relates the concentration of the components in the mobile phase with time of retention by the column. Efficient separations require minute and regular-shaped support media, a constant flow rate of the mobile phase and an efficient detection system. This method is often referred to as high pressure liquid chromatography, a name descriptive of the comparatively high pressures it employs (40 MPa). However, the

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nomenclature high performance liquid chromatography better describes all aspects of this method relating to speed, capacity and sensitivity.

6.3.1. HPLC equipment and operation

An HPLC system consists of five separate components: the mobile phase delivery system (pump), the injector, the column, the detector and the recorder. While the column is the component that effectuates the separation, the other components of the system provide support necessary for efficient and reproducibile separations. Major manufacturers usually build individual, compatible modules that are linked together.

Pumps: Chromatographic separation requires the delivery of the mobile phase through the stationary phase at a predetermined constant rate. In modem HPLC systems this is mechanically achieved with the use of pumps. The pump is usually the major component of the solvent delivery system, which incorporates facilities for degassing the solvents and mixing them in solvent gradients. Efficient and reproducible separations require a constant flow rate of solvent through the column. Pulsation within the system can result in a noisy detector baseline and thus raise the detection limits in sensitive assays. Mechanical reciprocating pumps (single, dual or triple-headed) with electrically driven pistons are usually employed. A pump that achieves virtually pulse free flow rates with the use of electronic pulse compensation circuitry has recently been introduced by a major supplier of HPLC equipment.

Injectors: For accurate separations, the sample needs to be injected at the top of the column without any dilution. Syringes and valves are the two main injection devices available for this purpose at present. Syringes inject the samples in-line at the top of the columns. Valves inject samples into loops which subsequently are brought in-line with the HPLC system. Injection valves provide major advantages of precision, reliability, and ease of operation and are therefore, the widely used system in modem HPLC apparatus.

Columns'. The column is the single most important component of the HPLC assembly which determines, together with the mobile phase, the quality of a separation achieved. The narrow metal tube containing the tightly packed stationary phase constitutes the column. Microfilters are usually placed at column ends to prevent particulate matter accumulating on top of the stationary phase material. At present the majority of the packing materials for HPLC columns are based on silica gels due to its versatility as an adsorbent. These gels consist of fused aggregates of colloidal silica which have surface

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areas up to 800 irr and pore sizes of 5 x 10'6 mm Adsorbents with high surface area, although producing high resolution, have a restricted polarity range and can separate only solutes that have polarities close to that of the mobile phase (Scott, 1976). However, microparticulate supports of 5-10 Jim diameter, which render separations extremely efficient, are being widely used. A large number of different types of modifier bonded silica packed columns are presently used in different modes of chromatography. Octadecanoylsilyl (ODS), amino (NH2), and cyano (CN) groups are widely used in reversed phase HPLC columns. Normally, a small precolumn (40 mm long, 3 mm internal diameter) is used in order to filter particles in the sample and solvents that may accumulate on the top of the column. The precolumn is packed with microparticulate material similar to those in the analytical column. The relatively high pressures (41.3 MPa) employed in HPLC systems have led to the replacement of glass tubes with stainless steel column housings. A short column together with a narrow diameter column packing is necessary to achieve rapid, high efficiency resolutions. Most analytical columns are 10-20 cm in lengths and 4-6 mm in internal diameter. The length to diameter ratio should be at least 10 and preferably larger. An important recent advancement in column technology is the emergence of radially packed columns.

Characterization of a new column in terms of a number of theoretical criteria prior to use is essential to determine its efficiency, and to be able to compare its performance over time. Two of the most important of these criteria are the capacity factor (k’) and the number of theoretical plates (N). The measurement of the capacity factor ensures that the sample has been sufficiently retained on the column for separation to occur. The capacity factor and the number of theoretical plates are calculated as shown in figure 6-1.

Detectors: The concentrations of individual components of a mixture separated by the HPLC column and being eluted are monitored by a detector. Efficient use of a HPLC column depends on the use of a detector of high sensitivity. Detection can be based on the measurement of a bulk property of the eluting solvent (refractive index) or a physical property of the solute. The most widely used solute property is ultraviolet light (uv) absorption. The variable wavelength uv detectors are being increasingly used.

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O

Capacity factor k’ = v i - y 2

Separation factor a =

V i - V Q

Number of theoretical plates N = 16

VQ, and V2 are ml equialents of the retention times of the peaks 1, 2 and 3 respectively

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