High Performance Liquid Chromatography (HPLC)
is an analytical process utilizing special instruments designed to
separate, quantify and analyze components of a chemical mixture.
Samples of interest are introduced to a solvent flow path; carried
through a column packed with specialized materials for component
separation; and component data is obtained through the combination of
a detection mechanism coupled with a data recording system. All this
occurs under pressures which may reach or exceed 6,000 psi!
The basic components of an HPLC system include
- solvent resevoir
- sample injector
- pump(s)
- analytical column
- detector(s)
- data recorder
- waste container
(or fraction collector)
Other important elements are an inlet
solvent filter, post-pump inline filter, sample filter, precolumn
filter, guard column, back-pressure regulator and/or solvent sparging
system. The function of each of these components is briefly described
below.
An HPLC system begins with the solvent
reservoir, which contains the solvent used to carry the sample
through the system. The solvent should be filtered with an inlet
solvent filter to remove any particles that could potentially
damage the system's sensitive components.
Solvent is propelled through the system by the
pump. This often includes internal pump seals, which slowly
break down over time. As these seals break down and release particles
into the flow path, an inline solvent filter prevents any post-pump
component damage.
The next component in the system is the
sample injector, also known as the injection valve. This valve,
equipped with a sample loop of the appropriate size for the analysis
being performed, allows for the reproducible introduction of sample
into the flow path. Because the sample often contains particulate
matter, it is important to utilize either a sample filter or a
precolumn filter to prevent valve and column damage. Some
systems utilize an automatic injector or autosampler to accomplish
this task.
Following the injector, an analytical column
allows the primary sample separation to occur. This is based on the
differential attraction of the sample components for the solvent and
the packing material within the column. However, a sacrificial
guard column is often included just prior to the analytical column
to chemically remove components of the sample that would otherwise
foul the main column.
Following the analytical column, the separated
components pass through a detector flow cell before they pass
into the waste reservoir. The sample components' presence in
the flow cell prompts an electrical response from the detector, which
is digitized and sent to a recorder. The recorder helps analyze
and interpret the data. Recorders can be as simple
as a pen recording what happens in the detector onto moving chart
paper or as sophisticated as a computer with powerful software for
hardware control and data interpretation.
As a final system enhancement, a back
pressure regulator is often installed immediately after the
detector. This device solvent bubble formation until the solvent is
completely through the detector. This is important because bubbles in
a flow cell can interfere with the detection of sample components.
Alternatively, an inert gas sparging system may be installed to
force dissolved gasses out of the solvent being stored in the solvent
reservoir.
Each of the components described above requires
fittings to couple it into a system.
It is important to note that improper selection
or installation of these fittings can lead to leaks or the formation
of dead volume, both of which can result in poor HPLC performance.
GMI can assist in component
choice or complete system configuration to accomplish most lab and
production requirements. We spend extra time in our
reconditioning 'up front' so that you, our customer, receive a
COMPLETE working system upon arrival to your laboratory.
HPLC LINGO:
ELUTION:
This term describes the transport of a species through the stationary
phase by the continuous flow (addition) of mobile phase.
ELUANT:
Mobile phase that carries the sample through the column.
ELUATE
or EFFLUENT: Mobile phase with separated components after
they emerge from the column.
ISOCRATIC
ELUTION:
A separation in which the mobile phase composition remains unaltered.
The mobile phase may comprise of a single solvent or a pre-mixed
mixture of solvents.
GRADIENT
ELUTION:
HPLC is frequently used for the separation of mixtures that contain
compounds with a wide range of polarities. In such situations,
isocratic conditions may not provide an acceptable separation (i.e.,
it is not possible to obtain sufficient resolution or the separation
takes an unacceptably long period of time). To solve these problems,
the composition of the mobile phase is changed during the separation.
Two or three solvents that differ in polarity are employed. After
sample introduction, the ratio of these solvents is programmed to vary
either continuously or in steps, resulting in enhanced separation
efficiency.
The terms ‘binary
gradient’, ternary gradient’, and quaternary gradient’ refer to the
use of 2, 3, and 4 solvents, respectively, to make up the mobile phase
composition in a gradient elution method.
CHROMATOGRAM:
When a detector that responds to solute concentration is placed at the
column outlet, a plot of the generated signal versus time (or volume
of mobile phase) is called a chromatogram. Such a plot, which usually
comprises of one or more peaks, may be used for qualitative and
quantitative purposes – the location of a peak on the time (or volume)
axis serves to identify the component, and the area under the peak
provides a quantitative measure of that component.
SENSITIVITY:
Minimum limit of detection of a given species. Determined by the
smallest ratio of ‘peak height-to-baseline noise’ (signal-to-noise
ratio) that allows accurate and reproducible determination of peak
height or area. This varies with detection method, instrument used and
species being detected.
UNRETAINED
COMPOUND:
Component of a mixture that moves through a column at the same rate as
the mobile phase, i.e., migration is not retarded by physical or
chemical interaction with the stationary phase.
VOID
VOLUME:
Total volume of mobile phase in a fully wet packed column - the space
between the particles of the stationary phase (interstitial volume)
plus the volume within the particles (pore volume). It is also defined
as the volume of mobile phase required to carry an unretained
component through a column.
RESOLUTION:
Measure of the degree of separation between two successively eluting
components in a chromatographic run (two adjacent peaks in a
chromatogram).
The resolution
between species A and B may be expressed as:
R = 2 [tB
– tA]/ (WA + WB), where t and W
correspond to the retention time and peak width at baseline,
respectively. A resolution value of 1.5 implies a complete separation
of the two species
