High-Performance Liquid Chromatography

High-performance liquid chromatography has become an indispensable analytical tool. HPLC has applications not only in forensics but also in biochemistry, environmental science, food science, pharmaceutical chemistry, and toxicology.

High-performance liquid chromatography (HPLC) is the most versatile and widely used type of elution chromatography. The technique is used by scientists for separating and determining species in a variety of organic, inorganic, and biological materials. In liquid chromatography, the mobile phase is a liquid solvent containing the sample as a mixture of solutes.

The types of high-performance liquid chromatography classified by separation mechanism or type of stationary phase. These include partition, or liquid-liquid chromatography; adsorption, or liquid-solid chromatography; ion-exchange, or ion chromatography; size-exclusion chromatography; affinity chromatography; and chiral chromatography.

Early liquid chromatography was performed in glass columns having inside diameters of perhaps 10 to 50 mm. The columns were packed with 50- to 500-cm lengths of solid particles coated with an adsorbed liquid that formed the stationary phase. To ensure reasonable flow rates through this type of stationary phase, the particle size of the solid was kept larger than 150 to 200 µm.

Even with these particles, flow rates were a few tenths of a milliliter per minute at best. Application of vacuum or pressure to speed were not effective because increases in flow rates were accompanied by increases in plate heights decreases in column efficiency. The theory of liquid chromatography, it was recognized that large decreases in plate heights would be realized if the particle size of packings were reduced.

Figure 33-1 Effect of particles size of packing and flow rate on plate height in liquid chromatography. (From R. E. Majors, J. Chromatrogr. Sci, 1973, Vol. 11, (2), 1973: 88–95, Fig 5)

The reason for this difference is that diffusion in liquids is much slower than in gases, Therefore, its effect on plate heights is observed only at extremely low flow rates.

In late 1960s, technology was developed for producing and using packings with particle diameters as small as 3 to 10 µm. This technology required instruments capable of much higher pumping pressures than the simple devices that preceded them. Simultaneously, detectors were developed for continuous monitoring of column effluents.

The name high-performance liquid chromatography (HPLC) is often used to distinguish this technology from the simple column chromatographic procedures that preceded them. Simple column chromatography, however, still finds considerable use for preparative purposes.

Fig. 33-2 Applications of liquid chromatography Fig. 33-2 Applications of liquid chromatography. Methods can be chosen based on solubility and molecular mass. In many cases, for small molecules, reversed-phase methods are appropriate

Various types of liquid chromatography tend to be complementary. For example: For analytes having molecular masses greater than 10,000, one of the two size-exclusion methods is often used: 1- gel permeation for nonpolar species and 2- gel filtration for polar or ionic compounds. For ionic species, ion-exchange chromatography is often the method of choice. In most cases for nonionic small molecules, reversed-phase methods are suitable.

33A Instrumentation Pumping pressures of several hundred atmospheres are required to achieve reasonable flow rates with packings in the 3- to 10-µm size range, Because of high pressures, equipment for high-performance liquid chromatography tends to be more elaborate and expensive. Fig. 33-3 is a diagram showing the important components of a typical HPLC instrument.

Fig. 33-3 Block diagram showing components of a typical apparatus for HPLC.

33A-1 Mobile-Phase Reservoirs and Solvent Treatment Systems A modern HPLC instrument is equipped with one or more glass reservoirs, each of which contains 500 mL or more of a solvent. Provisions are often included to remove dissolved gases and dust from the liquids. Dissolved gases can lead to irreproducible flow rates and band spreading. In addition, both bubbles and dust interfere with the performance of most detectors.

Degassers may consist of vacuum pumping system distillation system device for heating and stirring Or a system for sparging by swept out the dissolved gases of solution by fine bubbles of an inert gas that is not soluble in mobile phase (Fig. 33-3) . An elution with a single solvent or solvent mixture of constant composition is termed an isocratic elution. In gradient elution, two (and sometimes more) solvent systems that differ significantly in polarity are used and varied in composition during the separation.

The ratio of the two solvents is varied in a preprogrammed way, sometimes continuously and sometimes in a series of steps. Gradient elution frequently improves separation efficiency, just as temperature programming helps in gas chromatography (Fig. 33-4) . Modern HPLC instruments are often equipped with proportioning valves that introduce liquids from two or more reservoirs at ratios that can be varied continuously (Fig. 33-3).

Fig. 33-4 Improvement in separation effectiveness by using gradient elution.

Memo Sparging is a process in which dissolved gases are swept out of a solvent by bubbles of an inert, insoluble gas. An isocratic elution in HPLC is one in which the solvent composition remains constant. A gradient elution in HPLC is one in which the composition of the solvent is changed continuously or in a series of steps.