Chapter 32 HIGH-PERRORMANCE LIQUID CHROMATOGRAPHY High-performance liquid chromatography (HPLC) is the most versatile and widely used type of elution chromatography. In liquid chromatography, the mobile phase is a liquid solvent containing the sample as a mixture of solutes. There are several types of high- performance liquid chromatography, depending on the nature of the stationary phase. They include (1) partition, or liquid-liquid, chromatography; (2) adsorption, or liquid-solid, chromatography; (3) ion-exchange, or ion, chromatography; (4) size-exclusion chromatography; and (5) affinity chromatography.

Instruments for High-Performance Liquid Chromatography Pumping pressure of several hundred atmospheres are required to achieve reasonable flow rates with packing in the 3- to 10-  m size range, which are common in modern liquid chromatography. As a consequence of these high pressures, the equipment for high-performance liquid chromatography tends to be considerably more elaborate and expensive than that encountered in other types of chromatography.

Mobile-Phase Reservoirs A modern HPLC apparatus is equipped with one or more glass or stainless steel 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. An elution with a single solvent to constant composition is called isocratic. In gradient elution, two (and sometimes more) solvent systems that differ significantly in polarity are employed. 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.

Pumping Systems The requirements for liquid-chromatographic pumps include (1) the generation of pressures of up to 6000 psi, (2) pulse-free output, (3) flow rates ranging from 0.1 to 10 mL/min, (4) flow reproducibilities of 0.5% relative or better, and (5) resistance of corrosion by a variety of solvents. Three types of pumps are employed: (i) a screw- driven syringe type, (ii) a reciprocating pump, and (iii) a pneumatic or constant-pressure pump.

Columns for High-Performance Liquid Chromatography Liquid-chromatographic columns are usually constructed from stainless steel tubing, although glass or Tygon tubing is sometimes employed for lower pressure application (<600 psi). Most columns range in length from 10 to 30 cm and have inside diameters of 4 to 10 mm. Column packing typically have particle sizes of 5 or 10  m. Columns of this type often contain 40,000 to 60,000 plates/m.

Column Thermostats For many application, close control of column temperature is not necessary and columns are operated at room temperature. Often, however, better chromatograms are obtained by maintaining column temperatures constant to a few tenths of a degree Celsius. Most modern commercial instruments are now equipped with heaters that control column temperatures to a few tenths of a degree from near ambient to 150 o C.

Detectors No highly sensitive, universal detector system, such as those for gas chromatography, is available of HPLC. Thus, the detector used will depend on the nature of the sample. The most widely used detectors for liquid chromatography are based on absorption of ultraviolet or visible radiation. Modern instruments use diode-array instruments that can display an entire spectrum as an analyte exits the column. The combination of HPLC with a mass spectrometry detector is currently receiving a great deal of attention. Such HPLC/MS systems can identify the analytes exiting from the, HPLC column.

Column Packing The most common packing for partition chromatography is prepared from silica particles. The particles are coated with thin organic films, which are chemically or physically bonded to the surface. Two types of partition chromatography are distinguishable based on the relative polarities of the mobile and stationary phases. Early work in liquid chromatography was based on highly polar stationary phases; a relatively nonpolar solvent such as hexane served as the mobile phase. This type of chromatography is now called normal- phase chromatography.

…continued… In reversed-phase chromatography, the stationary phase is nonpolar, often a hydrocarbon, and the mobile phase is a relatively polar solvent (such as water, methanol, or acetonitrile). In normal-phase chromatography, the least polar component is eluted first; increasing the polarity of the mobile phase then decreases the elution time. In contrast, in the reversed-phase method, the most polar component elutes first, and increasing the mobile phase polarity increases the elution time.