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 highperformance 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 screwdriven 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
150oC.
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 normalphase 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.