1. High-Performance Liquid Chromatography

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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)

8. 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.

9. 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.

10. 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.

11. Fig. 33-2 Applications of liquid chromatography
Fig 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

12. 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.

13. 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 is a diagram showing the important components of a typical HPLC instrument.

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

15. 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.

16. 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.

17. 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).

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

19. 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.

20. 33A-2 Pumping Systems
The requirements for liquid chromatographic pumps include
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.

21. The high pressures generated by liquid chromatographic pumps are not an explosion hazard because liquids are not very compressible.
Thus, rupture of a component results only in solvent leakage.
Such leakage may constitute a fire or environmental hazard with some solvents, however.
Two major types of pumps are used in HPLC instruments:
the screw-driven syringe type and
he reciprocating pump.

22. 1. Syringe-type pumps produce a pulse-free delivery whose flow
1. Syringe-type pumps produce a pulse-free delivery whose flow rate is easily controlled.
They suffer, however, from relatively low capacity (250 mL) and are inconvenient when solvents must be changed.
2. Reciprocating types are used in almost all commercial instruments.
Fig illustrates the operating principles of the reciprocating pump.
This device consists of a small cylindrical chamber that is filled and then emptied by the back-and-forth motion of a piston.

23. Figure 33-5 A reciprocating pump for HPLC.

24. The pumping motion produces a pulsed flow that must be subsequently damped because the pulses appear as baseline noise on the chromatogram.
Modern HPLC instruments use dual pump heads to minimize such pulsations.
Advantages of reciprocating pumps include
small internal volume (35 to 400 mL)
high output pressure (up to 10,000 psi),
ready adaptability to gradient elution, and
constant flow rates, which are largely independent of column back-pressure and solvent viscosity.

25. As part of their pumping systems, many commercial instruments are equipped with computer-controlled devices for measuring the flow rate by determining the pressure drop across a restrictor located at the pump outlet.
Any difference in signal from a preset value is then used to increase or decrease the speed of the pump motor.

26. Most instruments also have a means for varying the composition of the solvent either continuously or in a stepwise fashion.
For example, the instrument shown in Figure 33-3 contains a proportioning valve that permits mixing of up to four solvents in a preprogrammed and continuously variable way.

27. 33A-3 Sample Injection systems
The most widely used method of sample introduction in liquid chromatography is based on a sampling loop, such as that shown in Fig
These devices are often an integral part of liquid chromatography equipment and have interchangeable loops capable of providing a choice of sample sizes ranging from 1 to 100 mL or more.

28. Figure 33-6 A sampling loop for liquid chromatography.

29. The reproducibility of injections with a typical sampling loop is a few tenths of a percent relative.
Many HPLC instruments incorporate an autosampler with an automatic injector.
These injectors can introduce continuously variable volumes from containers on the autosampler.

30. 33A-4 Columns for HPLC
Liquid chromatographic columns are usually constructed from stainless steel tubing, and less common from glass and polymer tubing, such as polyetheretherketone (PEEK)
In addition, stainless steel columns lined with glass or PEEK are also available.
Hundreds of packed columns differing in size and packing can be purchased from HPLC suppliers.
The cost of standard-sized, nonspecialty columns ranges from $200 to $500. Chiral columns can cost more than $1000.

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

32. In the 1980s, microcolumns became available with inside diameters of 1 to 4.6 mm and lengths of 3 to 7.5 cm.
These columns, which are packed with 3- or 5-µm particles, contain as many as 100,000 plates/m and have the advantage of speed and minimal solvent consumption.
This latter property is of considerable importance because the high-purity solvents required for liquid chromatography are expensive to purchase and to dispose of after use.

33. Fig. 33-7 illustrates the speed with which a separation can be performed on a microbore column.
In this example, MS/MS was used to monitor the separation of rosuvastatin from human plasma components on a column that was 5 cm in length with an inside diameter of 1.0 mm.
The column was packed with 3-µm particles. Less than 3 minutes were required for the separation.

34. Figure 33-7 High-speed gradient elution separation of rosuvastatin from human plasma-related components.
Column: 5 cm x 1.0 mm i.d. Luna C18 and 3 μm. Monitored by MS/MS at m/z = and

35. A scavenger column between the mobile-phase container and the injector is used to condition the mobile phase.
Usually called Guard column

36. Pre-columns
Two types of pre-columns are used.
A pre-column between the mobile phase reservoir and the injector is used for mobile-phase conditioning and is termed a scavenger column.
The solvent partially dissolves the silica packing and ensures that the mobile phase is saturated with silicic acid prior to entering the analytical column.
This saturation minimizes losses of the stationary phase from the analytical column.

37. A second type of pre-column 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.

38. Column Temperature Control
For some applications, close control of column temperature is not necessary, and columns are operated at room temperature.
Often, however, better, more reproducible chromatograms are obtained by maintaining constant column temperature.

39. 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.
Many chromatographers consider temperature control to be essential for reproducible separations.

40. Two types of packings are used in HPLC,
Column Packings
Two types of packings are used in HPLC,
pellicular and
porous particle.
The original pellicular particles were spherical, nonporous, glass or polymer beads with typical diameters of 30 to 40 µm.
A thin, porous layer of silica, alumina, a polystyrene-divinylbenzene synthetic resin, or an ion-exchange resin was deposited on the surface of these beads.
Small porous microparticles have completely replaced these large pellicular particles.

41. In recent years, small (<5 mm) pellicular packings have been reintroduced for separation of proteins and large biomolecules.
The typical porous particle packing for liquid chromatography consists of porous microparticles having diameters ranging from 3 to 10 µm;
for a given size particle, a very narrow particle size distribution is desirable.

42. The particles are composed of silica, alumina, the synthetic resin polystyrene-divinyl benzene, or an ion-exchange resin.
Silica is by far the most common packing in liquid chromatography.
Silica particles are often coated with thin organic films, which are chemically or physically bonded to the surface.

43. 33A-5 HPLC Detectors
an 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.

44. Unfortunately, no highly sensitive, universal detector system is available for high-performance liquid chromatography.
Thus, the detector used will depend on the nature of the sample.
Table 33-1 lists some of the common detectors and their properties.
The most widely used detectors for liquid chromatography are based on absorption of ultraviolet or visible radiation (Fig. 33-8).