Chromatography is a technique to separate mixtures of substances into their

components on the basis of their molecular structure and molecular composition. This
involves a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase
(a liquid or a gas). The mobile phase flows through the stationary phase and carries the
components of the mixture with it. Sample components that display stronger
interactions with the stationary phase will move more slowly through the column than
components with weaker interactions. This difference in rates cause the separation of
various components. Chromatographic separations can be carried out using a variety of
stationary phases, including immobilized silica on glass plates (thin-layer
chromatography), volatile gases (gas chromatography), paper (paper chromatography)
and liquids (liquid chromatography).

High perfomance Liquid Chromatography

High performance liquid chromatography (HPLC) is basically a highly improved form

of column liquid chromatography. Instead of a solvent being allowed to drip through a
column under gravity, it is forced through under high pressures of up to 400
atmospheres. That makes it much faster. All chromatographic separations, including
HPLC operate under the same basic principle; separation of a sample into its
constituent parts because of the difference in the relative affinities of different
molecules for the mobile phase and the stationary phase used in the separation.

Types of HPLC

There are following variants of HPLC, depending upon the phase system (stationary) in
the process :

1. Normal Phase HPLC:

This method separates analytes on the basis of polarity. NP-HPLC uses polar
stationary phase and non-polar mobile phase. Therefore, the stationary phase
is usually silica and typical mobile phases are hexane, methylene chloride,
chloroform, diethyl ether, and mixtures of these. Polar samples are thus
retained on the polar surface of the column packing longer than less polar
materials.
2. Reverse Phase HPLC:
The stationary phase is nonpolar (hydrophobic) in nature, while the mobile
phase is a polar liquid, such as mixtures of water and methanol or acetonitrile.
It works on the principle of hydrophobic interactions hence the more nonpolar
the material is, the longer it will be retained.
3. Size-exclusion HPLC:
The column is filled with material having precisely controlled pore sizes, and
the particles are separated according to its their molecular size. Larger
molecules are rapidly washed through the column; smaller molecules
penetrate inside the porous of the packing particles and elute later.
 4. Ion-Exchange HPLC:
The stationary phase has an ionically charged surface of opposite charge to the
sample ions. This technique is used almost exclusively with ionic or ionizable
samples. The stronger the charge on the sample, the stronger it will be
attracted to the ionic surface and thus, the longer it will take to elute. The
mobile phase is an aqueous buffer, where both pH and ionic strength are used
to control elution time.

Instrumentation of HPLC

As shown in the schematic diagram in Figure above, HPLC instrumentation includes

a pump, injector, column, detector and integrator or acquisition and display system.
The heart of the system is the column where separation occurs.

1. Solvent Resorvoir : Mobile phase contents are contained in a glass resorvoir.

The mobile phase, or solvent, in HPLC is usually a mixture of polar and
non-polar liquid components whose respective concentrations are varied
depending on the composition of the sample.
2. Pump : A pump aspirates the mobile phase from the solvent resorvoir and
forces it through the systems column and detecter. Depending on a number of
factors including column dimensions, particle size of the stationary phase, the
flow rate and composition of the mobile phase, operating pressures of up to
42000 kPa (about 6000 psi) can be generated.
3. Sample Injector : The injector can be a single injection or an automated
injection system. An injector for an HPLC system should provide injection of
the liquid sample within the range of 0.1-100 mL of volume with high
reproducibility and under high pressure (up to 4000 psi).
4. Columns : Columns are usually made of polished stainless steel, are between
50 and 300 mm long and have an internal diameter of between 2 and 5 mm.
They are commonly filled with a stationary phase with a particle size of 310
m. Columns with internal diameters of less than 2 mm are often referred to as
microbore columns. Ideally the temperature of the mobile phase and the
column should be kept constant during an analysis.
 5. Detector : The HPLC detector, located at the end of the column detect the
analytes as they elute from the chromatographic column. Commonly used
detectors are UV-spectroscopy, fluorescence, mass-spectrometric and
electrochemical detectors.
6. Data Collection Devices : Signals from the detector may be collected on chart
recorders or electronic integrators that vary in complexity and in their ability
to process, store and reprocess chromatographic data. The computer integrates
the response of the detector to each component and places it into a
chromatograph that is easy to read and interpret.

Applications of HPLC

The information that can be obtained by HPLC includes resolution, identification and
quantification of a compound. It also aids in chemical separation and purification. The
other applications of HPLC include :

Pharmaceutical Applications
1. To control drug stability.
2. Tablet dissolution study of pharmaceutical dosages form.
3. Pharmaceutical quality control.
Environmental Applications
1. Detection of phenolic compounds in drinking water.
2. Bio-monitoring of pollutants.
Applications in Forensics
1. Quantification of drugs in biological samples.
2. Identification of steroids in blood, urine etc.
3. Forensic analysis of textile dyes.
4. Determination of cocaine and other drugs of abuse in blood, urine etc.
Food and Flavour
1. Measurement of Quality of soft drinks and water.
2. Sugar analysis in fruit juices.
3. Analysis of polycyclic compounds in vegetables.
4. Preservative analysis.
Applications in Clinical Tests
1. Urine analysis, antibiotics analysis in blood.
2. Analysis of bilirubin, biliverdin in hepatic disorders.
3. Detection of endogenous Neuropeptides in extracellular fluid of brain etc.

How Does High Performance Liquid Chromatography Work?

The components of a basic high-performance liquid chromatography [HPLC] system
are shown in the simple diagram in Figure E.
A reservoir holds the solvent [called the mobile phase, because it moves]. A
high-pressure pump [solvent delivery system or solvent manager] is used to generate
and meter a specified flow rate of mobile phase, typically milliliters per minute. An
injector [sample manager or autosampler] is able to introduce [inject] the sample into
the continuously flowing mobile phase stream that carries the sample into the HPLC
column. The column contains the chromatographic packing material needed to effect
the separation. This packing material is called the stationary phase because it is held
in place by the column hardware. A detector is needed to see the separated compound
bands as they elute from the HPLC column [most compounds have no color, so we
cannot see them with our eyes]. The mobile phase exits the detector and can be sent to
waste, or collected, as desired. When the mobile phase contains a separated
compound band, HPLC provides the ability to collect this fraction of the eluate
containing that purified compound for further study. This is called preparative
chromatography [discussed in the section on HPLC Scale].
Note that high-pressure tubing and fittings are used to interconnect the pump, injector,
column, and detector components to form the conduit for the mobile phase, sample,
and separated compound bands.

Figure E: High-Performance Liquid Chromatography [HPLC] System

The detector is wired to the computer data station, the HPLC system component that
records the electrical signal needed to generate the chromatogram on its display and to
identify and quantitate the concentration of the sample constituents (see Figure F).
Since sample compound characteristics can be very different, several types of
detectors have been developed. For example, if a compound can absorb ultraviolet
light, a UV-absorbance detector is used. If the compound fluoresces, a fluorescence
detector is used. If the compound does not have either of these characteristics, a more
universal type of detector is used, such as an evaporative-light-scattering detector
[ELSD]. The most powerful approach is the use multiple detectors in series. For
example, a UV and/or ELSD detector may be used in combination with a mass
spectrometer [MS] to analyze the results of the chromatographic separation. This
provides, from a single injection, more comprehensive information about an analyte.
The practice of coupling a mass spectrometer to an HPLC system is called LC/MS.
 Figure F: A Typical HPLC [Waters Alliance] System

HPLC Operation
A simple way to understand how we achieve the separation of the compounds
contained in a sample is to view the diagram in Figure G.
Mobile phase enters the column from the left, passes through the particle bed, and
exits at the right. Flow direction is represented by green arrows. First, consider the top
image; it represents the column at time zero [the moment of injection], when the
sample enters the column and begins to form a band. The sample shown here, a
mixture of yellow, red, and blue dyes, appears at the inlet of the column as a single
black band. [In reality, this sample could be anything that can be dissolved in a
solvent; typically the compounds would be colorless and the column wall opaque, so
we would need a detector to see the separated compounds as they elute.]
After a few minutes [lower image], during which mobile phase flows continuously
and steadily past the packing material particles, we can see that the individual dyes
have moved in separate bands at different speeds. This is because there is a
competition between the mobile phase and the stationary phase for attracting each of
the dyes or analytes. Notice that the yellow dye band moves the fastest and is about to
exit the column. The yellow dye likes [is attracted to] the mobile phase more than the
other dyes. Therefore, it moves at a faster speed, closer to that of the mobile phase.
The blue dye band likes the packing material more than the mobile phase. Its stronger
attraction to the particles causes it to move significantly slower. In other words, it is
the most retained compound in this sample mixture. The red dye band has an
intermediate attraction for the mobile phase and therefore moves at
an intermediate speed through the column. Since each dye band moves at different
speed, we are able to separate it chromatographically.
 Figure G: Understanding How a Chromatographic Column Works Bands

What Is a Detector?
As the separated dye bands leave the column, they pass immediately into the detector.
The detector contains a flow cell that sees [detects] each separated compound band
against a background of mobile phase [see Figure H]. [In reality, solutions of many
compounds at typical HPLC analytical concentrations are colorless.] An appropriate
detector has the ability to sense the presence of a compound and send its
corresponding electrical signal to a computer data station. A choice is made among
many different types of detectors, depending upon the characteristics and
concentrations of the compounds that need to be separated and analyzed, as discussed
earlier.
What Is a Chromatogram?
A chromatogram is a representation of the separation that has chemically
[chromatographically] occurred in the HPLC system. A series of peaks rising from a
baseline is drawn on a time axis. Each peak represents the detector response for a
different compound. The chromatogram is plotted by the computer data station [see
Figure H].

Figure H: How Peaks Are Created

In Figure H, the yellow band has completely passed through the detector flow cell; the
electrical signal generated has been sent to the computer data station. The resulting
chromatogram has begun to appear on screen. Note that the chromatogram begins
when the sample was first injected and starts as a straight line set near the bottom of
the screen. This is called the baseline; it represents pure mobile phase passing through
the flow cell over time. As the yellow analyte band passes through the flow cell, a
stronger signal is sent to the computer. The line curves, first upward, and then
downward, in proportion to the concentration of the yellow dye in the sample band.
This creates a peak in the chromatogram. After the yellow band passes completely out
of the detector cell, the signal level returns to the baseline; the flow cell now has, once
again, only pure mobile phase in it. Since the yellow band moves fastest, eluting first
from the column, it is the first peak drawn.
A little while later, the red band reaches the flow cell. The signal rises up from the
baseline as the red band first enters the cell, and the peak representing the red band
begins to be drawn. In this diagram, the red band has not fully passed through the
flow cell. The diagram shows what the red band and red peak would look like if we
stopped the process at this moment. Since most of the red band has passed through the
cell, most of the peak has been drawn, as shown by the solid line. If we could restart,
the red band would completely pass through the flow cell and the red peak would be
completed [dotted line]. The blue band, the most strongly retained, travels at the
slowest rate and elutes after the red band. The dotted line shows you how the
completed chromatogram would appear if we had let the run continue to its
conclusion. It is interesting to note that the width of the blue peak will be the broadest
because the width of the blue analyte band, while narrowest on the column, becomes
the widest as it elutes from the column. This is because it moves more slowly through
the chromatographic packing material bed and requires more time [and mobile phase
volume] to be eluted completely. Since mobile phase is continuously flowing at a
fixed rate, this means that the blue band widens and is more dilute. Since the detector
responds in proportion to the concentration of the band, the blue peak is lower in
height, but larger in width.