High-performance liquid chromatography or commonly known as

HPLC, is an analytical technique used to separate, identify or

quantify each component in a mixture

The mixture is separated using the basic principle of

column chromatography and then identified and quantified by
spectroscopy.
In the 1960s, the column chromatography LC with its low-pressure suitable
glass columns was further developed to the HPLC with its high-pressure
adapted metal columns.
HPLC is thus 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.

HPLC Principle

 The purification takes place in a separation column between a stationary and

a mobile phase.
 The stationary phase is a granular material with very small porous particles in
a separation column.
 The mobile phase, on the other hand, is a solvent or solvent mixture which is
forced at high pressure through the separation column.
 Via a valve with a connected sample loop, i.e. a small tube or a capillary made
of stainless steel, the sample is injected into the mobile phase flow from the
pump to the separation column using a syringe.
 Subsequently, the individual components of the sample migrate through the
column at different rates because they are retained to a varying degree by
interactions with the stationary phase.
 After leaving the column, the individual substances are detected by a suitable
detector and passed on as a signal to the HPLC software on the computer.
 At the end of this operation/run, a chromatogram in the HPLC software on
the computer is obtained.
 The chromatogram allows the identification and quantification of the
different substances.

Instrumentation of HPLC
 The Pump

 The development of HPLC led to the development of the pump system.

 The pump is positioned in the most upper stream of the liquid
chromatography system and generates a flow of eluent from the solvent
reservoir into the system.
 High-pressure generation is a “standard” requirement of pumps besides
which, it should also to be able to provide a consistent pressure at any
condition and a controllable and reproducible flow rate.
 Most pumps used in current LC systems generate the flow by back-and-forth
motion of a motor-driven piston (reciprocating pumps). Because of this piston
motion, it produces “pulses”.

Injector

 An injector is placed next to the pump.

 The simplest method is to use a syringe, and the sample is introduced to the
flow of eluent.
 The most widely used injection method is based on sampling loops.
 The use of the autosampler (auto-injector) system is also widely used that
allows repeated injections in a set scheduled-timing.

Column

 The separation is performed inside the column.

 The recent columns are often prepared in a stainless steel housing, instead of
glass columns.
 The packing material generally used is silica or polymer gels compared to
calcium carbonate.
The eluent used for LC varies from acidic to basic solvents.
 Most column housing is made of stainless steel since stainless is tolerant
towards a large variety of solvents.

Detector

 Separation of analytes is performed inside the column, whereas a detector is

used to observe the obtained separation.
 The composition of the eluent is consistent when no analyte is present. While
the presence of analyte changes the composition of the eluent. What detector
does is to measure these differences.
 This difference is monitored as a form of an electronic signal. There are
different types of detectors available.
 Recorder

 The change in eluent detected by a detector is in the form of an electronic

signal, and thus it is still not visible to our eyes.
 In older days, the pen (paper)-chart recorder was popularly used. Nowadays,
a computer-based data processor (integrator) is more common.
 There are various types of data processors; from a simple system consisting of
the in-built printer and word processor while those with software that are
specifically designed for an LC system which not only data acquisition but
features like peak-fitting, baseline correction, automatic concentration
calculation, molecular weight determination, etc.

Degasser

The eluent used for LC analysis may contain gases such as oxygen that are non-
visible to our eyes.
 When gas is present in the eluent, this is detected as noise and causes an
unstable baseline.
 Degasser uses special polymer membrane tubing to remove gases.
 The numerous very small pores on the surface of the polymer tube allow the
air to go through while preventing any liquid to go through the pore.

Column Heater

The LC separation is often largely influenced by the column temperature.

 In order to obtain repeatable results, it is important to keep consistent
temperature conditions.
 Also for some analysis, such as sugar and organic acid, better resolutions can
be obtained at elevated temperatures (50 to 80°C).
 Thus columns are generally kept inside the column oven (column heater).

Advantages over low-pressure column liquid chromatography

There are many advantages of High-performance liquid chromatography
(HPLC) over traditional low-pressure column liquid chromatography.

 Greater sensitivity (various detectors can be employed)

 Improved resolution
 Speed
 Easy sample recovery (less eluent volume to remove)
 A wide variety of stationary phases
Types of HPLC
The following variants of HPLC depend upon the phase system
(stationary) in the process.

1. Normal Phase HPLC

 They are also known as normal-phase or absorption
chromatography. This method separates analytes based on polarity.
 It has a polar stationary phase and a non-polar mobile phase.
 Therefore, the stationary phase is usually silica, and typical mobile
phases are hexane, methylene chloride, chloroform, diethyl ether,
and mixtures.
 The technique is used for water-sensitive compounds, geometric
isomers, cis-trans isomers, class separations, and chiral
compounds.

2. Reverse Phase HPLC

 The stationary phase is nonpolar (hydrophobic), while the mobile
phase is an aqueous, moderate polar.
 It works on the principle of hydrophobic interactions; hence the
more nonpolar the material is, the longer it will be retained.
 This technique is used for non-polar, polar, ionizable, and ionic
molecules.

3. Size-exclusion HPLC
 It is also known as gel permeation chromatography or gel filtration
chromatography.
 The column is filled with a material having precisely controlled pore
sizes, and the particles are separated according to their molecular
size.
 Larger molecules are rapidly washed through the column; smaller
molecules penetrate the porous packing particles and elute later.
 Size-exclusion chromatography is also helpful in determining the
tertiary and quaternary structure of proteins and amino acids.
 It is also used for the determination of the molecular weight of
polysaccharides.
4. Ion-Exchange HPLC
 In this type of chromatography, retention is based on the attraction
between solute ions and charged sites bound to the stationary phase.
 Same charged ions are excluded.
 This technique is used in purifying water, Ligand and Ion-exchange
chromatography of proteins, high-pH anion-exchange
chromatography of carbohydrates and oligosaccharides, etc.

5. Bio-affinity HPLC
 In this type of chromatography, separation is based on the reversible
interaction of proteins with ligands.

Applications of HPLC
The information that HPLC can obtain 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 the extracellular fluid of
the brain etc.

Limitations
The limitation of using high-performance liquid chromatography (HPLC)
is the following.

 HPLC is much more costly requires a large number of expensive

organics.
 HPLC may have low sensitivity for certain compounds, and some
cannot even be detected as they are irreversibly adsorbed.
 Complexity
 Volatile substances are much better to be separated by gas
chromatography.