Chemistry Reference
In-Depth Information
a detector, and some type of data recording device. However, during the past decade,
and certainly to a greater extent within the past five years or so, the basic compo-
nents have become much more sophisticated, and specialized systems have emerged
for specific applications, including those for method development. There are several
key components of any HPLC system, and systems used specifically for method
development are really no different. HPLC systems can be modular or integrated,
and use either isocratic or gradient solvent delivery. Modular systems consist of
separate modules connected in such a way as to function as a single unit, and can
provide a degree of flexibility to exchange different components in and out of the
system, sometimes necessary for maintenance purposes or experimental require-
ments. However, in regulated laboratories, this flexibility may not be viewed as an
advantage due to compliance issues with analytical instrument qualification.
In integrated systems, the individual components can share electrical, commu-
nication, and fluid connections and control, and can operate in ways that provide
better solvent and sample management than modular systems. Modern integrated
systems are holistically designed to take advantage of managing both the sample
and the solvent in ways that can significantly decrease injection cycle time and pro-
vide increased precision and accuracy while still providing flexibility in detection
choices. HPLC system architecture can be further classified by how the solvents in
the mobile phase are blended, as illustrated in Figure 3.1. Traditional high-pressure
systems use two or more pumps to blend solvents under high pressures. A separate
controller is used to alter the flow rate to blend different proportions of solvent or to
generate gradients, using external mixers. While the system has the disadvantage of
the cost and maintenance of multiple pumps, high-pressure systems typically have
lower system (gradient delay) volumes, important when using smaller diameter, sub-
2-µm particle columns and fast LC techniques. High-pressure systems are usually
binary systems, although optional solvent-select valves can grant access to more than
one solvent at a time per pump. In low-pressure designs, a single pump draws the sol-
vent through a multiport proportioning valve. Software algorithms control and time
the opening of the ports with the pump stroke under microprocessor control to blend
the solvent or generate a gradient in the pump head under low pressure. Degassing,
either by helium sparge or membrane modules, is required to prevent outgassing
during solvent blending. The simplicity of a single pump is certainly an advantage,
but the pump head and other downstream components contribute to a typically larger
system or gradient delay volume than that found in high-pressure systems. However,
low-pressure systems offer more flexibility in solvent selection than high-pressure
systems, and can be used to generate different mobile phase compositions on-line
(as opposed to premixing), adding more flexibility during method development. In
addition, in low-pressure systems, any solvent volume change that might occur dur-
ing mixing is accomplished before the solvent is pressurized; therefore, flow rate
changes that might result from this effect in high-pressure systems that do not pre-
compress the solvent are not a problem with low-pressure systems.
In general, gradient systems are preferred over isocratic systems for method
development because of their multisolvent capability. Gradient multisolvent systems
can be used to prepare mobile phases on-the-fly, often referred to as “dial-a-mix”
or “auto blend,” providing maximum flexibility for method development (especially
