Page 207 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

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Encyclopedia of Physical Science and Technology EN008B-382 June 30, 2001 18:58

674 Liquid Chromatography

a particle-supported stationary phase is the technique
termed liquid chromatography (LC). There are two major
types of LC, classical and high-performance liquid chro-
matography (HPLC). Classical LC uses large columns
approximately 2 × 50 cm, packed with large porous par-
ticles 50–250 µm in diameter. Sample volumes in the
milliliter range are often required. The mobile phase is
generally gravity-fed at slow flow rates because the deep
pores of the packing limit mass transfer and separation
times can be on the order of hours. Fraction collection
of the components for later spectroscopic identification is
common. Although inefficient and not sensitive, classical
LC requires no special equipment and is often adequate
for some organic synthesis and biochemical research. The
evolution of HPLC occurred in the late 1960s as the tech-
nology became available to manufacture pellicular (see
Glossaryfordefinition)and10-µmsizeporoussilicaparti-
cles. These tiny particles when packed in smaller columns
of 0.4 × 25 cm require pumping of the mobile phase to
overcome the pressure drop of 1000 to 3000 psi. Because FIGURE 1 Block diagram of a high-performance liquid chromato-
of the shallower pores in these packing, mass transfer of graph; —–, Mobile phase flow; ---, computer control of device;
—·—· data acquisition and readout.
the analyte from the bulk of the mobile phase to the sur-
face of the stationary phase is faster, permitting separation
units are commercially available. Two micrometer inlet
times on the order of 10 to 30 min with good resolution.
filters are attached to the liquid-carrying Teflon tubing
Because smaller sample volumes of usually 20 µl are nec-
to prevent any insoluble particles from being drawn to
essary to not exceed the capacity of these columns, detec-
the pump where they could cause check valve malfunc-
tors equipped with flow cells have been developed for
tion. As shown in Fig. 1, blending of the mobile phase
more sensitive and convenient detection of the separated
constituents in the proper ratio is accomplished by the
components. This description of HPLC is considered to
solenoid proportioning valves A, B, and C. By control-
be analytical in nature and will be the major emphasis of ling the fraction of time each valve is opened using the
this article. However, preparative and microbore HPLC computer system through a digital-to-analog (DAC) in-
which bracket analytical HPLC with respect to flow rates, terface, a reproducible mobile phase made up of two or
sample size, and column dimensions will be briefly dis- three components can be formed in the mixing chamber.
cussed. Capillary HPLC and the relatively new technique, The volume of the mixing chamber should be kept small
capillary electrochromatography, will also be compared. to minimize gradient lag time at the column. The mixing
chamber, often magnetically stirred, contains a 2 µm frit
for final mobile phase filtration before it reaches the pump.
I. ANALYTICAL HPLC High-pressure mixing is an alternative method for gen-
eration of the desired mobile phase. The outlets of two
A block diagram for a typical instrument or high- or more HPLC pumps are connected into a mixing cham-
performance liquid chromatograph is shown in Fig. 1. ber. Basically, the solvents are mixed on the high-pressure
Each part of the chromatograph will be described in some side of the pumps. The composition of the desired mo-
detail with respect to analytical HPLC first. bile phase is dependent on the selected pumping speeds
of each pump, which are often computer controlled. The
A. Solvent Mixing resultant flow rate represents the combined output of the
individual pump flow rates. Although precise control of
The mobile phase reservoirs are generally screw-top
the gradient composition is possible using this system, the
plastic-coated glass bottles with inlet lines to allow for
expense of multiple pumps is a disadvantage.
degassing of the solvents for 10–15 min either by vacuum
or sparging with helium or both. A positive pressure of
B. Pump
helium should be maintained in the solvent bottles. De-
gassing is important to prevent gas bubbles from lodging The HPLC pump must meet several criteria. These are
in the pump, column, or the detector. On-line degassing (1) pumping capability up to 6000 psi, (2) pulseless