1. Chapter 33 High-Performance Liquid Chromatography

2. 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.

3. The types of high-performance liquid chromatography are often classified by the separation mechanism or by the type of stationary phase. These include (1)partition, or liquid-liquid, chromatography; (2)adsorption, or liquid-solid, chromatography; (3)ion-exchange, or ion, chromatography; (4)size-exclusion chromatography; (5)affinity chromatography; and (6)chiral chromatography.

4. High-performance liquid chromatography combines a liquid mobile phase and a very finely divided stationary phase. In order to obtain satisfactory flow rates, the liquid must be pressurized to several hundred or more pounds per square inch.

5. Instrumentation Pumping pressures of several hundred atmospheres are required to achieve reasonable flow rates with packings in the 3- to 10-mm size range. Because of the high pressures required, the equipment for HPLC tends to be considerably more elaborate and expensive.

7. 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 such as degassers are often included to remove dissolved gases and dust from the liquids. Dissolved gases can lead to irreproducible flow rates and band spreading. Sparging is a process in which dissolved gases are swept out of a solvent by bubbles of an inert, insoluble gas. An elution with a single solvent or solvent mixture of constant composition is termed an isocratic elution. In gradient elution, two (or more) solvent systems that differ significantly in polarity are used and varied in composition during the separation.

9. Pumping Systems The requirements for liquid chromatographic pumps include (1)the generation of pressures of up to 6000 psi (lb/in2), (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 to corrosion by a variety of solvents. Two major types of pumps are used in HPLC instruments: the screw driven syringe type and the reciprocating pump. Reciprocating types are used in almost all commercial instruments. Syringe-type pumps produce a pulse-free delivery whose flow rate is easily controlled.

11. The most widely used method of sample introduction in liquid chromatography is based on a sampling loop. Many HPLC instruments incorporate an auto sampler with an automatic injector. These injectors can introduce continuously variable volumes from containers on the auto sampler.

12. Columns for HPLC Liquid chromatographic columns are usually constructed from stainless steel tubing, although glass and polymer tubing, such as polyetheretherketone (PEEK), are sometimes used. Analytical Columns Most columns range in length from 5 to 25 cm and have inside diameters of 3 to 5 mm. Micro columns, which are packed with 3- or 5-mm particles, contain as many as 100,000 plates/m and have the advantage of speed and minimal solvent consumption.

13. Precolumns Two types of precolumns are used. A precolumn between the mobile phase reservoir and the injector is used for mobile-phase conditioning and is termed a scavenger column. A second type of precolumn is a guard column. It is positioned between the injector and the analytical column and prevents impurities, such as highly retained compounds and particulate matter, from reaching and contaminating the analytical column.

14. Column temperature control Better, more reproducible chromatograms are obtained by maintaining constant column temperature. Most modern commercial instruments are equipped with heaters that control column temperatures to a few tenths of a degree from near room temperature to 150°C. Column Packings Two types of packings are used in HPLC: pellicular particles - spherical, nonporous, glass or polymer beads with typical diameters of 30 to 40  m. porous particles - microparticles having diameters ranging from 3 to 10  m.

15. HPLC Detectors The most widely used detectors for liquid chromatography are based on absorption of ultraviolet or visible radiation.

18. 33 B Partition chromatography In this type of chromatography, the stationary phase is a second liquid that is immiscible with the liquid mobile phase. Partition chromatography can be subdivided into 1.liquid-liquid partition chroma-tography where the stationary phase is a solvent held in place by adsorption of the surface of the packing particles. 2. liquid-bonded-phase chromatography where the stationary phase is an organic species that is attached to the surface of the packing particles by chemical bonds. The difference between the two lies in the way that the stationary phase is held on the support particles of the packing.

19. Bonded-Phase Packings Most bonded-phase packings are prepared by reaction of an organochlorosilane with the --OH groups formed on the surface of silica particles by hydrolysis in hot dilute hydrochloric acid. The organic group is often a straight chain octyl- or octyldecyl-group. Other organic functional groups that have been bonded to silica surfaces include aliphatic amines, ethers, and nitriles as well as aromatic hydrocarbons.

20. Normal-and Reversed-Phase Packings Two types of partition chromatography are distinguishable based on the relative polarities of the mobile and stationary phases. 1.In normal-phase partition chromatography, the stationary phase is polar and the mobile phase nonpolar. The least polar analyte is eluted first. 2.In reversed-phase partition chromatography, the stationary phase is non- polar, and the mobile phase is a relatively polar solvent. The least polar analyte is eluted last. Ion-pair chromatography is a subset of reversed-phase chromatography in which easily ionizable species are separated on reversed-phase columns.

21. Choice of Mobile and Stationary Phases Successful partition chromatography requires a proper balance of intermolecular forces among the three participants in the separation process: the analyte, the mobile phase, and the stationary phase. The polarity of the stationary phase is matched roughly with that of the analytes; a mobile phase of considerably different polarity is then used for elution.

25. 33 C Adsorption chromatography Adsorption, or liquid-solid, chromatography was first introduced by Tswett. Finely divided silica and alumina are the only stationary phases that find use for adsorption chromatography. It follows many of the principles and techniques used for the normal-phase partition chromatography. 33 D Ion chromatography Two types of ion chromatography are currently in use: suppressor-based and single-column. They differ in the method used to prevent the conductivity of the eluting electrolyte from interfering with the measurement of analyte conductivities.

26. Ion Chromatography Based on Suppressors In suppressor-based ion chromatography, the ion-exchange column is followed by a suppressor column, or a suppressor membrane, that converts an ionic eluent into a nonionic species that does not interfere with the conductometric detection of analyte ions. The suppressor column is packed with a second ion-exchange resin. The resin effectively converts the ions of the eluting solvent to a molecular species of limited ionization without affecting the conductivity due to analyte ions. The disadvantage of the suppressor columns is the need to regenerate them periodically (typically every 8 to 10 hr) to convert the packing back to the original acid or base form.

28. Single-Column Ion Chromatography In single-column ion-exchange chromatography, analyte ions are separated on a low-capacity ion exchanger by means of a low-ionic strength eluent that does not interfere with the conductometric detection of analyte ions. The advantage offered is that no special equipment is required for suppression. However, it is a somewhat less sensitive method for determining anions than suppressor-column methods.

29. 33 E Size-exclusion chromatography Size-exclusion, or gel chromatography, is a powerful technique that is particularly applicable to high-molecular-mass species. Fractionation is based on molecular size and to some extent molecular shape. The average residence time of analyte molecules depends on their effective size. Molecules that are significantly larger than the average pore size of the packing travel through the column at the rate of the mobile phase. Molecules that are appreciably smaller than the pores can penetrate throughout the pore maze and are the last to elute. Intermediate-size molecules penetrate into the pores of the packing depending on their diameters.

30. Two types of packing for size-exclusion chromatography are encountered: polymer beads and silica-based particles, both of which have diameters of 5 to 10 mm. Gel filtration is a type of size-exclusion chromatography in which the packing is hydrophilic. It is used to separate polar species. Gel permeation is a type of size- exclusion chromatography in which the packing is hydrophobic. It is used to separate nonpolar species.

32. 33 F Affinity chromatography In affinity chromatography, a reagent called an affinity ligand is covalently bonded to a solid support. Typical affinity ligands are antibodies, enzyme inhibitors, or other molecules that reversibly and selectively bind to analyte molecules in the sample. Only the molecules that selectively bind to the affinity ligand are retained in the column. The stationary phase is a solid, such as agarose, or a porous glass bead to which the affinity ligand is immobilized.

33. 33 G Chiral chromatography Compounds that are nonsuper-imposable mirror images of each other are called chiral compounds. Such mirror images are called enantiomers. Chiral mobile-phase additives or chiral stationary phases are required for these separations. A chiral resolving agent is a chiral mobile-phase additive or a chiral stationary phase that preferentially complexes one of the enantiomers.

35. 33 H Comparison of high-performance liquid chromatography and gas chromatography