Chapter 33

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 • partition, or liquid-liquid, chromatography • adsorption, or liquid-solid, chromatography • ion-exchange, or ion, chromatography • size-exclusion chromatography • affinity chromatography • chiral chromatography

4. Early in the development of the theory of LC, it was recognized that large decreases in plate heights would be realized if the particle size of packings were reduced. • The minimum shown in Figure is not reached. It is because that diffusion in liquids is much slower than in gases and its effect on plate heights is observed only at extremely low flow rates.

5. In the late 1960s, particle diameters are developed as small as 3 to 10 mm. • The instruments was required of much higher pumping pressures than the simple devices that preceded them. • Detectors were developed for continuous monitoring of column effluents. • High-performance liquid chromatography (HPLC) 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.

6. Applications of LC. • For analytes having molecular masses greater than 10,000, one of the two size-exclusion methods is often used: • Gel permeation for nonpolar compound • Gel filtration for polar or ionic compounds.

7. 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.

8. Mobile-Phase Reservoirs and Solvent Treatment System • 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 degassersare often included to remove dissolved gases and dust from the liquids. • Dissolved gases can lead to irreproducible flow rates and band spreading. • Both bubbles and dust interfere with the performance of most detectors. • Spargingis a process in which dissolved gases are swept out of a solvent by bubbles of an inert, insoluble gas.

9. Mobile-Phase Reservoirs and Solvent Treatment System • 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. • Modern HPLC are equipped with proportioning valves that introduce liquids from two or more reservoirs at ratios that can be varied continuously.

10. Pumping System • The requirements for liquid chromatographic pumps include: • the generation of pressures of up to 6,000 psi (lb/in2) • pulse-free output • flow rates ranging from 0.1 to 10 mL/min • flow reproducibilities of 0.5% relative or better • resistance to corrosion by a variety of solvents • Two major types of pumps are used in HPLC instruments: 1) the screw driven syringe type and 2) 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. Pumping System • The screw driven syringe pump: • Advantage: flow rate is easily controlled • Disadvantage: relatively low capacity, inconvenient when solvents must be changed.

12. Pumping System • The reciprocating pump consists of a small cylindrical chamber that is filled and then emptied by the back-and-forth motion of a piston. • Modern HPLC uses dual pump heads or elliptical cams to minimize pulsations. • A tandem-piston pump provides a continuous output flow.

13. Pumping System • The advantages of reciprocating pumps: • small internal volume (35 ~ 400 mL), • high output pressure (up to 10,000 psi), • adaptability to gradient elution, • constant flow rate. • Computer-controlled device measures the flow rate by determining the pressure drop across a restrictor located at the pump outlet.

14. Sample Injection Systems • The most widely used method of sample introduction in liquid chromatography is based on a sampling loop. • An interchangeable loops are capable of providing a choice of sample sizes ranging from 1 to 100 mL or more. • Many HPLC instruments incorporate an auto sampler with an automatic injector. • These injectors can introduce continuously variable volumes from containers on the auto sampler.

15. 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. • PEEK • Analytical Columns • Most columns range in length from 5 to 25 cm and have inside diameters of 3 to 5 mm. • The most common particle size of packings is 3 ~ 5 mm. • The common columns are 10 ~ 15cm long, 4.6 mm in inside diameter, and packed with 5-mm particles. Columns of this type provide 40,000 ~ 70,000 plates/m.

16. Columns for HPLC • In 1980s, microcolumns became available with inside diameters of 1 to 4.6 mm and length of 3 to 7.5 cm. • Microcolumn, 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. • Example: a separation based on a microbore column. MS/MS was used to monitor the separation of rosuvastatin from human plasma.

17. Columns for HPLC • 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, positioned between the injector and the analytical column. • A guard column is a short column packed with a similar stationary phase as the analytical column. • The purpose of the guard column is to prevent impurities, such as highly retained compounds and particulate matter, from reaching and contaminating the analytical column. • The guard column is replaced regularly and serves to increase the lifetime of the analytical column.

18. Columns for HPLC • Column Temperature Control • For some applications, close control of column temperature is not necessary and columns are operated at room temperature. • Better and 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. • Columns can also be fitted with water jackets fed from a constant-temperature bath to give precise temperature control.

19. Columns for HPLC • Column Packings • Two types of packings are used in HPLC: 1) Pellicular Particles and 2) Porous Particles. • Pellicular Particles - spherical, nonporous, glass or polymer beads with typical diameters of 30 to 40 m. • A thin, porous layer of silica, alumina, a polystyrene-divinyl benznene synthetic resin, or an ion-exchange resin was deposited on the surface of these beads. • In recent year, only small pellicular packings (~5 mm) was developed for separation of proteins and large biomolecules.

20. Columns for HPLC • Porous Particles - microparticles having diameters ranging from 3 to 10 m. • The particles are composed of silica, alumina, the synthetic resin polystyrene-divinyl benzene, or an ion-exchange resin. • Silica is the most common packing in LC. • Silica particles are often coated with thin organic films , which are chemically or physically bonded to the surface.

21. HPLC Detectors • The ideal detector for HPLC should have all the characteristics of the ideal GC detector expect that it has no need to have great temperature range. • HPLC detector must have low internal volume (dead volume) to minimize extra-column band broadening. • The detector should be small and compatible with liquid flow.

22. HPLC Detectors • The most widely used detectors for liquid chromatography are based on absorption of ultraviolet or visible radiation. • Both photometers and spectrophotometers are available for HPLC. • Photometers often make use of the 254- and 280- nm lines from a mercury source because many organic functional groups absorb in the region. • Deuterium sources or tungsten-filament sources with interference filters also provide a simple means of detecting absorbing species. • Spectrophotometeric detectors are widely used in high-performance instruments. • Diode-array detector (DAD) can display an entire spectrum as an analyte exits the column.

23. HPLC Detectors • The combination of HPLC with a mass spectrometry detector produces a very powerful analytical tool. • LC/MS systems can identify the analytes exiting from the HPLC column. • The refractive index detector (RI) is general rather than selective and responds to the presence of all solutes. • RI has limited sensitivity. • Potentiometric, conductometric, and voltametric measurements can also be used in HPLC.

24. Five Mechanical LC Techniques

25. Partition Chromatography • The most widely used type of HPLC is partition chromatography in which the stationary phase is a second liquid that is immiscible with the liquid mobile phase. • Partition chromatography can be subdivided into liquid-liquid and liquid-bonded-phase chromatography. • The difference between the two lies in the way that the stationary phase is held on the support particles of the packing. • In liquid-liquid partition chromatography, the stationary phase is a solvent held in place by adsorption of the surface of the packing particles. • In liquid-bonded-phase chromatography, the stationary phase is an organic species that is attached to the surface of the packing particles by chemical bonds. • The bonded-phase method predominate because of their greater stability and compatibility with gradient elution.

26. 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 product is an organosiloxane. • The organic group R is often a straight chain octyl- (C8) or octyldecyl- (C18) group. • Other organic functional groups that have been bonded to silica surfaces include aliphatic amines, ethers, and nitriles as well as aromatic hydrocarbons. • Many different polarities for the bonded stationary phase are available.

27. Normal- and Reversed-Phase Packings • Two types of partition chromatography are distinguishable based on the relative polarities of the mobile and stationary phases. • Early work in LC was based on highly polar stationary phases (triethylene glycol or water) and a relatively nonpolar solvent (hexane or i-propyl ether) served as the mobile phase. • For historic reasons, this type of chromatography is called normal-phase chromatography (NPLC). • In normal-phase partition chromatography, the stationary phase is polar and the mobile phase is nonpolar. • The least polar analyte is eluted first. • Increasing the polarity of the mobile phase then decreases the elution time.

28. Normal- and Reversed-Phase Packings • In reversed-phase partition chromatography (RPLC), the stationary phase is non-polar, and the mobile phase is a relatively polar solvent. • The least polar analyte is eluted last. • The most polar component elutes first. • Increasing the mobile phase polarity increases the elution time. • More than ¾ of all HPLC separations are performed with reversed-phase, bonded, octyl- or octyldecyl- siloxane packings. • The long-chain hydrocarbon groups are aligned parallel to one another and perpendicular to the surface of the particle giving a nonpolar hydrocarbon surface. • The mobile phase used is often an aqueous solution containing various concentrations of MeOH, Acetonitrile, or Tetrahydrofuran.

29. Normal- and Reversed-Phase Packings • Ion-pair chromatography is a subset of reversed-phase chromatography in which easily ionizable species are separated on reversed-phase columns. • In this type of chromatography, an organic salt containing a large organic counterion, such as a quarternary ammonium ion or alkyl sulfonate, is added to the mobile phase as an ion-pairing reagent. • The separation mechanism is: • The counterion forms an uncharged ion pair with a solute ion of opposite charge in the mobile phase. • The ion pair then partitions into the nonpolar stationary phase giving differential retention of solutes based on the affinity of the ion pair for the two phases. • Separation of organic solute ions of the opposite charge then occurs by formation of reversible ion-pair complexes with the more strongly retained solutes forming the strongest complexes with the stationary phase.

30. Normal- and Reversed-Phase Packings • An example illustrates the separation of ionic and nonionic compounds using alkyl sulfonates of various chain lengths as ion-pairing agents. • A mixture of C5- and C7-alkyl sulfonates gives the best separation results.

31. Choice of Mobile and Stationary Phases • Successful partition chromatography requires a proper balance of intermolecular forces among the three participants in the separation process: 1) the analyte, 2) the mobile phase, and 3) the stationary phase. • These intermolecular forces are described qualitatively in terms of the relative polarity possessed by each of the 3 components. • The polarities of common organic functional groups in increasing order are: • hydrocarbons < ethers < ketones < aldehydes < amides < alcohols • Water is more polar than compounds containing any of these functional groups.

32. Choice of Mobile and Stationary Phases • 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. • If the polarities of the analyte and the mobile phase are matched but are different from that of the stationary phase, the stationary phase cannot compete successfully for the sample components. • Retention times will be too short for practical application. • The order of polarities of common mobile phase solvents are: • Water > acetonitrile > methanol > ethanol > tetrahydrofuran > propanol > cyclohexane > hexane

33. Applications Gradient elution C8 bonded-phase column • Figure shows typical applications of bonded-phase partition chromatography for separating soft drink additives and organophophate insecticides. Isocratic elution Nitile bonded-phase column

34. Applications • Table illustrates the variety of samples to which the technique is applicable.

35. Adsorption Chromatography • Adsorption, or liquid-solid, chromatography is the classic form of liquid chromatography first introduced by Tswett at the beginning of the 20th century. • In many normal-phase separations, adsorption/displacement processes govern retention. • Finely divided silica and alumina are the only stationary phases that find use for adsorption chromatography. • Silica is preferred because of its higher sample capacity. • For both silica and alumina, retention times become longer as the polarity of the analyte increases. • Because of the versatility and ready availability of bonded stationary phase, traditional adsorption chromatography with solid stationary phases has seen decreasing use in recent years in favor of normal-phase chromatography.

36. Ion Chromatography • Ion chromatography was first developed in the mid-1970s when it was shown that anion or cation mixtures can be resolved on HPLC columns packed with anion-exchange or cation-exchange resins. • The detection was generally performed with conductivity measurements, but it is notideal because of high electrolyte concentrations in the mobile phase. • The development of low-exchange-capacity columns allowed the use of low-ionic-strength mobile phase that could be deionized to allow high sensitivity conductivity detection. • Currently, spectrophometric and eletrochemical detectors are available for ion chromatography.

37. 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.

38. Ion Chromatography Based on Suppressors • Conductivity detectors are highly sensitive, universal for charged species, and respond in a predictable way to concentration changes. • Conductivity detectors are simple to operate, inexpensive to construct and maintain, easy to miniaturize and usually give prolonged, trouble-free service. • The only limitation to the use of conductivity detectors was due to the high electrolyte concentrations required to elute most analyte ions in a reasonable time. • The conductivity from the mobile-phase components tends to swamp that from the analyte ions greatly reducing the detector sensitivity.

39. Ion Chromatography Based on Suppressors • The problem created by the high conductance of eluents was solved by the introduction of an eluent suppressor column immediately following the ion-exchange column. • 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. • For example: when cations are being separated and determined, hydrochloric acid is chosen as the eluting reagent, and the suppressor column is an anion-exchange resin in the hydroxide form. The product of the reaction in the suppressor is water, • H+(aq) + Cl-(aq) + resin+OH-(s) resin+Cl-(s) + H2O • the analyte cations are not retained in the 2nd anion-exchange column.

40. Ion Chromatography Based on Suppressors • For anion separations, the suppressor packing is the acid form of a cation-exchange resin and sodium bicarbonate or carbonate is the eluting agent, • Na+(aq) + HCO3-(aq) + resin-H+(s) resin-Na+(s) + H2CO3(aq) • 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. • Micromembrane suppressors operates continuously and they are capable of removing all Na+ from a 0.1M NaOH solution with an eluent flow rate of 2 mL/min.

41. Ion Chromatography Based on Suppressors • Figure shows two applications of IC based on a suppressor column and conductometric detection. • Ions are present in ppm range, the sample size is 50 mL in one case and 100 mL in the 2nd one.

42. Single-Column Ion Chromatography • Single-Column IC depends on the small differences in conductivity between sample ions and the prevailing eluent ions. • 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.

43. Size-Exclusion Chromatography • Size-exclusion, or gel chromatography, is a powerful technique that is particularly applicable to high-molecular-mass species. • Packings for size-exclusion chromatography consist of small (~10mm) silica or polymer particles containing a network of uniform pores into which solute and solvent molecules can diffuse. • While in the pores, molecules are trapped and removed from the flow of the mobile phase. • The average residence time of analyte molecules depends on their effective size.

44. Size-Exclusion Chromatography • Molecules that are larger than the average pore size of the packing are excluded and thus suffer no retention, that is, they 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 entrapped for the greatest time; so they are last to elute. • Intermediate-size molecules penetrate into the pores of the packing depending on their diameters. • Fractionation is based on molecular size and to some extent molecular shape.

45. Size-Exclusion Chromatography • The size-exclusion separation differ from the other chromatographic methods in the respect that there are no chemical or physical interactions between analyte and the stationary phase. • Unlike other chromatographic methods, size-exclusion separation has an upper limit to retention time because no analyte species is retained longer than those small molecules that totally permeate the stationary phase.

46. Column Packings for Size-Exclusion • 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. • Silica particles are more rigid, which leads to easier packing and permits higher pressures to be used. • Silica particles are more stable, allowing a great range of solvents to be used and exhibiting more rapid equilibration with new solvent.

47. Column Packings for Size-Exclusion • Packings for size-exclusion: some are hydrophilic for use with aqueous mobile phase; hydrophobic are used with nonpolar organic solvents. • 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. • Generally, size-exclusion packings accommodate a 2- to 2.5-decade range of molecular mass.

48. Applications of Size-Exclusion • Figure illustrates typical applications of size-exclusion chromatography. Fatty acids separation Epoxy resin separation

49. Applications of Size-Exclusion • Another important application of size-exclusion chromatography is the rapid determination of the molecular mass or the molecular mass distribution of large polymers or natural products. • Calibrations can be accomplished by means of standards of known molecular mass. • Molecules separated in size-exclusion are according to hydrodynamic volume. • A universal calibration curve can be obtained by plotting logM vs. the retention volume VR. • Absolute calibration can be achieved by using a molar mass-sensitive detector such as a low-angle light-scattering detector (LSD).

50. 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. • When the sample passes through the column, only the molecules that selectively bind to the affinity ligand are retained. • Molecules that do not bind pass through the column with the mobile phase. • After the undesired molecules are removed, the retained analytes can be eluted by changing the mobile phase conditions.