The document provides an overview of high performance liquid chromatography (HPLC). It describes key components of HPLC systems including pumps to deliver solvent at stable flow rates, columns for molecular separation, and detectors for recognizing analytes. The separation principle is based on the distribution of analytes between a mobile liquid phase and stationary column packing material. Different constituents are eluted at different times, achieving separation. HPLC is widely used in pharmaceutical applications such as drug development, production quality control, and stability testing.

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HIGH PERFORMANCE
LIQUID
CHROMATOGRAPHY
(HPLC)
UNNATI GARG
M. Pharm (Pharmaceutics)
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INTRODUCTION
• Chromatography" is a technique for separation,
"chromatogram" is the result of chromatography, and
"chromatograph" is the instrument used to conduct
chromatography.
• Devices dedicated for molecular separation called columns and
high-performance pumps for delivering solvent at a stable flow
rate are some of the key components of chromatographs.
• HPLC separates compounds dissolved in a liquid sample and
allows qualitative and quantitative analysis of what
components and how much of each component are contained
in the sample.
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• The separation principle of HPLC is based on the distribution
of the analyte (sample) between a mobile phase (eluent) and a
stationary phase (packing material of the column).
• The specific intermolecular interactions between the molecules
of a sample and the packing material define their time “on-
column”. Hence, different constituents of a sample are eluted
at different times. Thereby, the separation of the sample
ingredients is achieved.
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PRINCIPLE

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• A detection unit (e.g. UV detector) recognizes the analytes
after leaving the column.
• The signals are converted and recorded by a data management
system (computer software) and then shown in a
chromatogram.
• After passing the detector unit, the mobile phase can be
subjected to additional detector units, a fraction collection unit
or to the waste.
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TYPES OF HPLC
Normal Phase HPLC
• Mobile phase in NP HPLC are based on non polar solvents
with addition of small quantity of polar solvent.
• The stronger the mobile phase interactions with the stationary
phase, the lower the difference between the stationary phase
interaction and the analyte interactions, thus lower analyte
retention.
• Packing materials used in NP HPLC are generally Silica or
Alumina
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TYPES OF HPLC
Reverse Phase HPLC
• Mobile phase in RP HPLC is Polar and the stationary phase is
Hydrophobic
• Employs mainly dispersive forces (hydrophobic or van der
wall interactions.
• 90 % of analysis are done using this technique.
• It has the advantage of low energy generation due to the
presence of weak forces of interaction.
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Instrumental Setup
of HPLC
Solvent Reservoirs
• Clean and inert container
• Storage of sufficient amount of solvent
• Cap is placed with a tube that feeds mobile phase to the
solvent delivery system
• In-line degassers are also present, which are primarily used to
remove small gas bubbles and reduce dissolved air.
• An additional filter is often placed at the end of the mobile
phase inlet line to remove any particulates or precipitates that
may form in the mobile phase during its use.
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Pump:
• Continuous flow of mobile phase through the system
• Pressure from 500 to 20,000 psi.
• The purpose of the pump is to deliver a precise, accurate,
reproducible, constant, and pulse-free flow of mobile phase to
the column.
• The majority of commercial high pressure pumps available
today are designed around a simple reciprocating piston pump.
Injector:
• Introduce analyte mixture into the stream of mobile phase
without depressurizing the HPLC system
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PUMP PRINCIPLE APPLICATIONS PUMPING
CAPACITY
Constant Pressure
Pump
Mobile phase is driven
through the column with
the use of pressure from
the gas cylinder
Suitable for isocratic
elution
70 ml
Reciprocating Piston
Pump
It has a small motor-driven
piston which pushed the
mobile phase due to the
forward and backward
action of the piston
present.
The reciprocating pump
can work as an isocratic
and gradient mode.
35 – 400 µL per
cycle
Syringe Type Pump This type of pump
consists of a big syringe
that is motorized by an
electronic motor used to
drive a steady flow rate.
suitable for isocratic
elution but inconvenient
for the gradient elution
250 – 500 ml
TYPES OF PUMPS

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Column:
• Produces separation of the analytes in the mixture
• Currently columns are made of heavy-walled glass, titanium, and
plastic (e.g., PEEK) to offer the analyst better performance for
particular analytes.
• Common dimensions for analytical scale columns are in the range of
10–30 cm long and 4–10mm i.d.
• The common particle sizes of packing are 3, 5, and 10μm.
• Columns are easily degraded by the irreversible adsorption of
impurities from samples and solvents. Hence, a guard column is
often used to protect the integrity of the analytical column, which is
much more expensive.
• Also, for analytes that may contain particulates, an in-line filter can
be placed between the injector and guard column.
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COLUMN Mobile Phase Stationary Phase
Normal Phase
Columns
More polar stationary
phase than mobile
phase; packing
material should be
more polar than the
mobile phase (eg
silica)
Methyl chloride,
hexane, chloroform
Amino bonded with
siloxane
Reverse Phase
Columns
It has non-polar or
less polar stationary
phase than the more
polar mobile phase
Aqueous organic
solution like water-
methanol is used
Bonded hydrocarbons
like C8, C18, C4
Ion Exchange
Columns
Stationary phase is
acidic or basic while
mobile phase is polar
liquid
Aqueous buffer Charged surface like
Sulfonic acid or
primary or tertiary
amine
TYPES OF COLUMS

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Detector:
• Continuous registration of specific physical properties of the
column effluent.
• HPLC detectors are classified as either bulk property detectors,
which respond to a bulk property of the eluent (e.g., refractive
index (RI) or conductivity), or solute property detectors, which
respond to some property of the analyte (e.g., UV absorbance).
• In either case, the response of the detector is modulated by the
presence and amount of the analyte.
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Data acquisition and control system:
• Computer based system that controls all parameters of HPLC
instrument
• The data collection device takes the electronic signal produced
by the detector and outputs a plot of response versus time.
• This resulting chromatogram can then be evaluated for both
qualitative and quantitative information.
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APPLICATIONS
• HPLC is an integral analytical tool applied in all stages of drug
discovery, development, and production.
• It can help (bio-) pharmaceutical researchers and
manufacturing facilities fully characterize potential drug or
treatment candidates, and ensure the medicines are
manufactured in a safe and consistent way.
• HPLC can be useful in ensuring critical quality attributes such
as strength/concentration, content uniformity, the detection
and quantification of impurities, and the quality and identity of
raw material
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• Impurities can pose a serious safety risk to patients, and their
detection and identification is often facilitated by the use of
HPLC.
• HPLC can also be useful in determining shelf life; “for
example, some biotherapeutics are sensitive to aggregation
over time, or if not stored properly, and HPLC can be used to
monitor this aggregation.”
• It can be used in evaluating the pharmaceutical stability of a
product.
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REFERNCES
• https://www.shimadzu.com/an/service-support/technical-
support/analysis-basics/basic/what_is_hplc.html#1
• LaCourse, Margaret E. (2017). Liquid Chromatography ||
General instrumentation in HPLC *. , (), 417–
429. doi:10.1016/B978-0-12-805393-5.00017-8
• https://books.google.co.in/books?id=csAk-
MpeTocC&lpg=PR5&ots=Do30Tn1ANm&dq=principle%20a
nd%20instrumentation%20of%20hplc&lr&pg=PA10#v=onepa
ge&q&f=false
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