The document discusses high-performance liquid chromatography (HPLC), including its basic principles, types, instrumentation, and applications. HPLC forces solvents through columns under high pressure to separate sample components faster than traditional chromatography. There are four main types depending on the stationary phase used: normal phase uses polar stationary and nonpolar mobile phases; reverse phase uses nonpolar stationary and polar mobile; size-exclusion separates by molecular size; ion-exchange uses charged stationary phases. HPLC instrumentation includes pumps, injectors, columns, detectors, and data collection systems. Key applications are in manufacturing quality control, environmental monitoring, forensics, food analysis, research, and medical analysis of substances in blood, urine, and tissues.

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High-Pressure Liquid Chromatography
(HPLC)
High performance/pressure liquid chromatography (HPLC) is 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, this 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 the following variants of HPLC, depending upon the phase system
(stationary) in the process:
 Normal Phase HPLC: It uses 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 of these.
Polar samples are thus retained on the polar surface of the column packing longer
than less polar materials.
 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.
 Size-exclusion HPLC: The column is filled with material having precisely controlled
pore sizes, and particles are separated according to their molecular size. Larger
molecules are rapidly washed through the column; smaller molecules penetrate
inside the pores of the packing particles and are removed later.

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 Ion-Exchange HPLC: The stationary phase has a charged surface of the 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 be removed. The
mobile phase is an aqueous buffer, where both pH and ionic strength are used to
control removal time.
INSTRUMENTATION / WORKING OF HPLC
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.
Figure: Components of the HPLC apparatus.
The details of all of these components (and their functions) are as follows:
Solvent Reservoir: Mobile phase contents are contained in a glass reservoir. 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.
Pump: A pump sucks the mobile phase from the solvent reservoir and forces it through
the system’s column and detector. Depending on several factors including column
dimensions, particle size of the stationary phase, the flow rate and composition of the

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mobile phase, operating pressures of up to 42000 kPa (about 6000 psi) can be
generated.
Sample Injector: The injector can be a single injection or an automated injection
system. An injector for an HPLC system should provide an injection of the liquid
sample within the range of 0.1-100 mL of volume under high pressure (up to 4000 psi).
Columns: Columns are usually made of polished stainless steel, 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. Columns with internal diameters of less than 2 mm are
often referred to as micro-bore columns. Ideally, the temperature of the mobile phase
and the column should be kept constant during an analysis.
Detector: The HPLC detector, located at the end of the column, detects the analytes
as they are removed from the chromatographic column. Commonly used detectors are
UV-spectroscopy, fluorescence, mass-spectrometric and electrochemical detectors.
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 following are the applications of high performance/pressure liquid
chromatography (HPLC):
Manufacturing: HPLC has many applications in both laboratory and clinical science. It
is a common technique used in pharmaceutical development, as it is a dependable
way to obtain and ensure product purity. While HPLC can produce extremely high
quality (pure) products, it is not always the primary method used in the production of
bulk drug materials. An increase in specificity, precision, and accuracy that occurs with

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HPLC unfortunately corresponds to an increase in cost. In the pharmaceutical
industry, it is used for the following reasons:
 To control drug stability.
 Tablet dissolution study of pharmaceutical dosages form.
 Pharmaceutical quality control.
Environmental Applications: It is used in various environmental monitoring tests such
as:
 Detection of phenolic compounds in drinking water.
 Bio-monitoring of pollutants.
Legal / Forensics: This technique is also used for chemical analysis of various samples
such as urine, blood, etc. The most common method of drug detection is an
immunoassay. This method is much more convenient. However, convenience comes
at the cost of specificity and coverage of a wide range of drugs. As HPLC is a method
of determining (and possibly increasing) purity, using HPLC alone in evaluating
concentrations of drugs is somewhat insufficient. With this, HPLC in this context is
often performed in conjunction with mass spectrometry. It is used in this regard for the
following:
 Quantification of drugs in biological samples.
 Identification of steroids in blood, urine, etc.
 Forensic analysis of textile dyes.
 Determination of cocaine and other drugs of abuse in blood, urine, etc.
Food Industry: It is used in the food industry for the following reasons:
 Measurement of Quality of soft drinks and water.
 Sugar analysis in fruit juices.
 Analysis of polycyclic compounds in vegetables.
 Preservative analysis.

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Research: Similar assays can be performed for research purposes, detecting
concentrations of potential clinical candidates like anti-fungal and asthma drugs. This
technique is useful in observing multiple species in collected samples, as well, but
requires the use of standard solutions when information about species identity is
sought out. It is used as a method to confirm the results of synthesis reactions, as
purity is essential in this type of research. However, mass spectrometry is still a more
reliable way to identify species.
Medical: Medical use of HPLC can include drug analysis, but falls more closely under
the category of nutrient analysis. While urine is the most common medium for
analyzing drug concentrations, blood serum is the sample collected for most medical
analyses with HPLC. While an expensive tool, the accuracy of HPLC is nearly
unparalleled. Some examples are as follows:
 Urine analysis, antibiotics analysis in blood.
 Analysis of bilirubin, biliverdin in hepatic disorders.
 Detection of endogenous Neuropeptides in extracellular fluid of brain, etc.