HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY
(HPLC)

Presenter: Nandit P B
Overview
Chromatography and its principle
Liquid chromatography
High Performance Liquid Chromatography
Components - HPLC.
Separation process.
Chromatogram.
Applications
Advantages
References
 Introduction
Chromatography is a separation technique in which separation of
components takes place between two phases – a stationary phase and a
mobile phase.

Used in biochemistry and analytical chemistry to identify, quantify and

purify the individual component of a mixture.

Can separate a mixture of compounds based on polarity, charge,

molecular weight, presence of functional group.

Stationary Phase : Substance on which adsorption of the analyte

(substance to be separated during chromatography) takes place. It can be
solid, gel or solid liquid combination.

Mobile Phase: Solvent which carries the analyte. (liquid or gas)

 Introduction contd…
Chromatographic techniques are divided into different types
based on :

The type of chromatographic bed used i.e

- Column chromatography (Gas chromatography) and
- Planar chromatography (Paper and thin)

 The physical state of mobile phase:

- Gas chromatography and liquid chromatography.

 The separation mechanism:

- Ion-exchange and size exclusion.
HPLC
 HPLC is a type of liquid chromatography where the
sample is forced through a column that is packed with a
stationary phase composed of irregularly or spherically
shaped particles, a porous monolithic layer or a porous
membrane by a liquid (mobile phase) at high pressure.

 Yields high performance at high speed compared to

traditional column chromatography because of forcibly
pumped mobile phase.
 PRINCILPE

Liquid chromatography is a separation technique that

involves:
placement (injection) of a small volume of liquid sample
into a tube packed with porous particles (stationary phase)
where individual components of the sample are
transported along the packed tube (column) by a liquid
moved by gravity.

The main principle of separation is adsorption .

When a mixture of components are introduced into the
column, various chemical and or physical interactions take
place between the sample molecules and the particles of the
column packing.
They travel according to their relative affinities towards the
stationary phase:
 The component which has more affinity towards the
adsorbent, travels slower.
 The component which has less affinity towards the stationary
phase travels faster.
 Since no two components have the same affinity towards the
stationary phase, hence the components are separated
 Separated components are collected at the exit of the column

 Identified by an external measurement technique such as a

spectrophotometer that measures the intensity of the color or
by device that can measure their amount/quantity.
 HPLC is a separation technique that involves:

 Injection of a small volume of liquid sample into a tube

packed with tiny particles (3 to 5 micron in diameter
called the stationary phase)

 Individual components of the sample are moved down the

packed tube (column) with a liquid (mobile phase) forced
through the column by high pressure delivered by a
pump.
 These components are separated by the column packing
that involves various chemical and/or physical interactions
between their molecules and the packing particles.

 Separated components are detected at the exit of this tube

(column) by a flow-through device (detector) that
measures their amount .

 The output from the detector is called a liquid

chromatogram

 In principle, LC and HPLC work the same way except the

speed, efficiency, sensitivity and ease of operation of
HPLC is vastly superior
 TYPES OF HPLC
I.BASED ON MODE OF SEPERATION
1.Normal phase chromatography - stationary phase is polar
(hydrophilic) and mobile phase is non-polar (hydrophobic).

2.Reverse phase chromatography - stationary phase is non-

polar (hydrophobic) and mobile phase is polar (hydrophilic).

Polar-Polar bonds and Non Polar-Non Polar bonds have

more affinity than Polar-Non Polar bonds.

Reverse phase chromatography is more commonly used

when drugs are usually hydrophilic.
II. Based on Principle of Separation:

 Absorption Chromatography:
o Solute molecules bound directly to the surface of
stationary phase, the component which has more
affinity towards mobile phase elutes first & the
component which has less affinity towards stationary
phase elutes later.

o No two components have same affinity towards

mobile phase and stationary phase.
 Ion-exchange Chromatography:
o Process that allows the separation of ions and polar
molecules based on their charge.

o Can be used for almost any kind of charged molecule

including large proteins, small nucleotides and amino
acids.

o Retention is based on the attraction between solute ions

and charged sites bound to the stationary phase.

o Ions of same charge are excluded.

 Ion-pair Chromatography:
o Form of chromatography in which ions in solution can be
“paired” or neutralized and separated as an ion pair on a
reversed phase column.
o Ion pairing agents are usually ionic compounds that
contain a hydrocarbon chain that imparts a certain
hydrophobacity so that the ion can be retained on a
reversed phase column.
 Gel permeation Chromatography :
o This type of chromatography lacks an attractive interaction
between the stationary phase and solute.
o The liquid or gaseous phase passes through a porous gel
which separates the molecules according to its size.
 Affinity Chromatography :
o The stationary phase consists of a support medium (eg:
cellulose beads) on which the substrate (or sometimes a
coenzyme) has been bound covalently, so that the reactive
groups that are essential for enzyme binding are exposed.
The technique is based on the high affinity of specific
proteins for specific chemical groups. Thus co-enzymes
can be used to purify enzymes.
For example, NAD is used to purify dehydrogenases. By
using antibodies, antigens could be easily separated.
Conversely, antibodies can be purified by passing through
a column containing the antigen.
III. BASED ON ELUTION TECHNIQUE
1.Isocratic elution
A separation in which the mobile phase
composition remains constant throughout the
procedure is termed as isocratic elution

In isocratic elution, peak width increases with

retention time linearly with the number of theoretical
plates. This leads to the disadvantage that late-eluting
peaks get very flat and broad.

Often used in quality control applications.

2. Gradient elution
A separation in which the mobile phase composition is
changed during the separation process is described as a
gradient elution

Gradient elution decreases the retention of the later-

eluting components so that they elute faster, giving
narrower peaks . This also improves the peak shape and
the peak height

Best for the analysis of complex samples

Often used in method development for unknown mixtures

 IV. BASED ON TYPE OF ANALYSIS
1.Qualitative analysis
 Analysis of a substance in order to ascertain the nature of
its chemical constituents
 We can separate individual components but cannot assess
the quantity in this analysis

2.Quantitaive analysis
 Determining the amounts and proportions of its chemical
constituents .
 Quantity of the impurity and individual components can
be assessed
Solvent Reservoirs

Pump

Injector

Column

Detector
 A . Solvent delivery system(Mobile phase)
 Mobile phase in HPLC refers to the solvent being
continuously applied to the column or stationary phase.
 Acts as a carrier to the sample solution

 Sample solution is injected into the mobile phase of an

assay through the injector port

 Solution flows through a column with the mobile phase,

the components of that solution migrate according to the
non-covalent interaction of the compound with the
column
 B. Pumps
 Role of the pump is to force a liquid (called the mobile
phase) through the liquid chromatograph at a specific flow
rate, expressed in (mL/min).
 Normal flow rates in HPLC are 1-2 mL/min range.

 Typical pumps can reach pressures in the range of 6000-

9000 psi.
 During the chromatographic experiment, a pump can
deliver a constant mobile phase composition (isocratic) or
an increasing mobile phase composition (gradient).

 There are several types of pumps used for HPLC analysis,

most common are reciprocating piston pump, syringe
pump and constant pressure pump.
 C. Injector:
 Injector serves to introduce the liquid sample into the flow
stream of the mobile phase.
 Typical sample volumes are 5-20 μL.
 Should be able to withstand the high pressures of the
liquid system.
 An auto sampler is the automatic version, when the user
has many samples to analyze or when manual injection is
not practical .
 D. Column
 Considered the “heart of the chromatography”.

 Stationary phase separates the sample components of

interest using various physical and chemical parameters.
 It is usually made of stainless steel to withstand high
pressure caused by the pump to move the mobile phase
through the column packing.

 Small particles inside the column are called the “packing”

which cause the high back pressure at normal flow rates.
Column packing is usually silica gel because of its particle
shape, surface properties and pore structure to give us a good
separation.

Porous plug of stainless steel or Teflon are used in the end of

the columns to retain the packing material.

According to the mode of HPLC , they are available in

different size, diameters, pore size or they can have special
materials attached (such as antigen or antibody) for immuno
affinity chromatography.
Guard Column: These are placed anterior to the
separating column. This serves as protective factor.

They are dependable columns designed to filter or remove

:
Particles that clog the separation column, compounds and
ions that could ultimately cause “ Baseline drift ”,
decreased resolution, decreased sensitivity and create false
peaks.

These columns must be changed on a regular basis in

order to optimize their protective function
Fast Column: One of the primary reasons for using these column is
to obtain improved sample output ( amount of compound per unit
time).
 Fast column are designed to decrease the time of chromatographic
analysis
 Here internal diameter is same but length is short and packed with
smaller particles, that are 3 μm diameter.
 Advantages- Increased sensitivity, Decreased analysis time,
Decreased mobile phase usage, Increase reproducibility

Capillary Column:
 Also known as micro columns
 Has a diameter much less than a millimeter and are of 3 types:
 Open tubular
 Partially packed
 Tightly packed
 They allow the user to work with nanoliter of sample volume with
decreased flow rate and decreased solvent usage volume, leads to
 Modes of HPLC

 Citric acid, Acetic acid.

 Ammonia, Methylamine

 Lithium Fluoride, NaCl

- Hexane, Heptane, Octane

- Phosphate ions, Chloride

ions

- Myosin, Macroglobulin,
Transferrin
 E . Detector:
 Detector can detect the individual molecules that elute from
the column and convert the data into an electrical signal.

 Provides an output to a recorder or computer that results in the

liquid chromatogram.

 Selected based on the analyte or the sample under detection.

 Types of detectors are Ultraviolet detectors, Fluorescence

detector, Mass Spectrometry, Refractive index detectors, etc.
 F. Data processing unit (Computer)
 Frequently called the data system.

 Computer controls all the modules of the HPLC instrument

 It takes the signal from the detector and uses it to determine

the time of elution (retention time) of the sample
components(qualitative analysis) and the amount of sample
(quantitative analysis).

 The concentration of each detected component is

calculated from the area or height of the corresponding
peak and reported.
 In a chromatogram, different peaks correspond to different
components of the separated mixture.

 Retention Time: Time elapsed between sample introduction and

maximum of response is the characteristic time which a analyte
takes to pass through the system.

 Negative peaks occur if mobile phase absorbance is larger than

sample absorbance.

 Peak doubling occurs due to the co- elution of interfering

compound, column over load, channeling in column.

 Base line spikes occur due to the air bubbles in the mobile
phase or detector or column deterioration.
i) Retention time t’R = tR – tM

ii) Retention factor k=tR / tM

iii) Resolution of analyte:

R = (√N/4)[(a-1)/a)](k/(1+t’R )
 Applications of HPLC
This technique is used for -
 Analyzing complex mixtures like Macroglobulins, Transferrin
etc.

 Purifying chemical compounds like Hexane, Octane, Sodium

sulfate.

 Developing processes for synthesizing chemical compounds,

isolating natural products, or predicting physical properties.

 Quality control to ensure the purity of raw materials, control

and improve process yields, to quantify assays of final products
or to evaluate product stability and monitor degradation.
 Advantages of HPLC
1. Separation is fast and efficient (high resolution power)

2. Continuous monitoring of the column effluent

3. Separation and analysis of very complex mixtures

4. Accurate quantitative measurements.

5. Repetitive and reproducible analysis using the same column.

6. Adsorption, partition, ion exchange and exclusion column

separations are excellently made.
Advantages contd…,
7. HPLC is more versatile than GLC in some respects,
because it has the advantage of not being restricted to
volatile and thermally stable solute and the choice of
mobile and stationary phases is much wider in HPLC.

8. Both aqueous and non aqueous samples can be analyzed

with little or no sample pre treatment

9. A variety of solvents and column packing are available,

providing a high degree of selectivity for specific analyses.

10. It provides a means for determination of multiple

components in a single analysis.
References
 HPLC instrumentation – Agilent Technologies
 Introduction to HPLC – Agilent Technologies
 Wilson Keith and Walker John – Text book of
Principles and Technique of Biochemistry and
Molecular Biology
 HPLC THEORY INTRODUCTION AND
INSTRUMENTATION HARDWARE - Dr Cristina
Legido-Quigley, Lecturer in Pharmaceutical Chemistry
(Separation Science) at KCL
THANK YOU