HPLC

Presented by- Taniska Sharma and Aditi Khali

M.Pharm 1st Sem [Pharmaceutical Chemistry]
 Contents
• Introduction
• Principle
• Instrumentation
• Chromatographic parameters
• Factors affecting resolution
• Applications
 What is High-Performance Liquid
Chromatography [HPLC]?
• HPLC is an analytical techniques used to separate, identify, or quantify each
components in the mixture.
• The mixture is separated using the basic principle of column
chromatography and then identified and quantified by the spectroscopy.
• In the 1960s, the column chromatography LC [liquid chromatography] with its
low pressure suitable glass column 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, solvent is forced through under high pressure of upto 400
atmosphere.
 Principle
• 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].
• Depending upon the chemical structures of the analyte, the
molecules are retarded while passing the stationary phase.
• 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 particle 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 the 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 passes on as a signal to the HPLC
software on the computer.
• At the end of this operation, a chromatogram in the HPLC
software on the computer is obtained.
• The chromatogram allows the identification and quantification
of the different substances.
Normal phase Types of HPLC
Column packing is polar (e.g. silica) and the mobile phase is non-
polar. It is used for water-sensitive compounds, geometric isomers, and
chiral compounds.

Reverse phase
Column packing is non-polar (e.g. C18), the mobile phase is polar
(e.g. methanol). It can be used for polar, non-polar, ionizable, and ionic
samples.

Ion exchange
Column packing contains ionic groups and the mobile phase is buffer. It is
used to separate anions and cations.

Size exclusion
Molecular diffuse into pores of a porous medium and are separated
according to their relative size to the pore size. Large molecules elute
first and small molecules elute later.
Instrumentation of HPLC
Components of HPLC :
1.Solvent Reservoir / Mobile Phase Reservoir
 A modern LC apparatus is equipped with one or more glass reservoirs , each of
which contains 500ml or more of solvent.
 The mobile phase ,or solvent in HPLC is usually a mixture of polar and non-
polar liquid components whose respective concentration are varied depending
on the composition of the sample.
2. SOLVENT DEGASSING: Several gaseous are soluble in organic solvent. When solvent
are pumped under high pressure, gas bubbles are formed which will interfare with the separation
process, steady base line and shape of the peak.
This can be done by using following technique:
1)Vaccumm filtration: which can remove air bubbles , but it is not always reliable and complete
2) Helium purging : by passing helium through the solvent . This is very effective but
expensive
3) Ultrasonification : by using ultrasonicator , which converts ultra high frequency to
mechanical vibrations . This causes the removal of air bubbles.
3. MIXING UNIT:- Mixing unit is used to mix solvents in different proportions and
pass through the column. There are two types of mixing units :
1) Low pressure mixing chamber which uses helium for degassing solvents
2) High pressure mixing chamber does not require helium for degassing solvents .
4) INJECTOR:- The injector serves to introduce the liquid sample into the slow
stear the mobile phase.
▪ Typical sample volume are 5-20microlitres.
▪ The injector must also be able to withstand the high pressure of the liquid system.
▪ An auto sampler is an automatic version for when the user has many samples to
analyze or when or manual injection is not practical.
Types of injector:
1) Septum injectors: injecting sample through a rubber septum .
2) Stop flow: flow of Mobile phase is stopped for a while and the sample is
injected through a valve device .
3) Rheodyne injector: most popular injector .Injector has 2 modes ,i.e., load
position when the sample is loaded and inject mode when the sample is injected .
5.Pump
• The role of pump is to force a liquid( called the mobile phase )through the liquid
chromatography at a specific flow rate expressed in millimetres per minute(ml/min)
• Normal flow rates in HPLC are in the 1- 2 mL/min range
• During the chromatographic experiment a pumpkin deliver a constant mobile phase
composition( isocratic) or an increasing mobile phase composition( gradient )best for the
analysis of complex samples .
Types of pumps
• Constant flow reciprocating pump
• Syringe type pump
• Pneumatic pump
Constant flow reciprocating pump
Syringe type pump
Pneumatic pump
6.Column
1.Guard column
 It is placed before the analytical column , so also called as pre columns .
 They are used to protect the analytical column from the impurities and other
contaminations from solvent .
 Guard columns are compulsorily used during the bioanalytical studies to protect
the analytical column from biological matrix.
2. Analytical column
 Analytical column is considered as the heart of an HPLC system because this is
the part where separation of mixture takes place.
 The efficiency of the separation is purely depends on the column .
Packing material :
• Packing material is prepared
from silica particle aluminium
particle and ion exchange resins.
• Porous plug of stainless steel or
Teflon are used in the end of
the column to retain the packing
materials.
 Types of Detectors

Bulk property Solute property

Others Detectors
Detectors Detectors

UV-visible
Refractive Infrared Absorption
Absorption
index Detectors Detectors
Detectors

Evaporative Light
Fluorescene
Scattering
Detectors
Detectors (ELSD)

Amperometric Mass Spectrometric

Detectors Detectors
UV Visible detector
• UV visible detector is widely used as it detects large number of compounds because most drugs
have appropriate structural characteristics for light absorption .
• These are useful for aromatic compounds and other type of unsaturated system
• These are classified as fixed or variable wavelength detector
• Fix wavelength detector employee filter as a source to provide appropriate wavelength most
common fixed wavelength detectors are based on 254nm .
• variable wavelength detectors are employ a spectrophotometer to provide dispersion of light and
selection of any wavelength in UV visible region.
Fluorescence detectors :
• It is based on the fluorescent radiation emitted by some compounds.
• The excitation source passes through the flow cell to a photo detector while a
monochromator measures the emission wavelengths .
• More sensitive and specific.
• The disadvantage is that most compounds are not fluorescent in nature .
Mass Spectrometric Detectors
• Nowadays the highly sophisticated mass spectrometric detectors are widely used due to their
sensitivity and reliability.
• When mass spectrometer is used as a detector for LC (LC -MS), it can greatly aid in identifying
species as they elute from a chromatographic column.

8.Data collection device or recorder

• Recorders are used to record the responses obtained from the detectors after amplification
• They record the baseline and all the peaks obtained with respect to time(Rt)
• But that area of the individual peaks cannot be known
Advantages of Using HPLC
1.High Sensitivity and Specificity: Detects even trace levels of
compounds.
2.Scalability: Adaptable from research labs to large-scale
manufacturing.
3.Robust and Reliable: Delivers consistent and repeatable results
across batches.
4.Integration with Advanced Tools: Combines with mass
spectrometry and fluorescence for enhanced analysis .
 HPLC PARAMETERS
• Retention time
• Adjusted retention time
• Retention volume
• Retention factor
• Selectivity factor
• Theoretical plate
• Column efficiency
• Asymmetric peaks
• Resolution
Retention time[tR]
Difference in time between the sample injection and appearance of
the peak maxima.
Different compounds have different retention time.
It may depend upon-
I. Pressure used
II. Nature of the Statinary phase
III. Temperature of the column
IV. Composition of the mobile phase

Adjusted retention time[t’R]

It is the measurement between retention time and unretained
time.

Retention volume [VR]

The volume of mobile phase that passed through the column from
the point of injection to the detector.
Retention factor [K]
It is defined as the ratio of adjusted retention time and unretained
time.
Higher the K value, greater the resolution.
K value
1-10 Good seperation
<1 Poorly retained
>10 Too long to separate and
broaden the separation

Selectivity factor [separation factor]

It is defined as the ratio of the partition coefficient of two
components to be separated or ratio of adjusted retention time.
Higher the selectivity factor, greater the resolution.
Theoretical plates
It is an imaginary units of column where distribution of analytes
between stationary phase and mobile phase has attained
equilibrium.
It is otherwise called as functional units of the column.
Ideal column Theoretical plate more than 2000.
Higher the Theoretical plates, higher the efficiency of column.

Column efficiency
It is also known as plate count, or number of theoretical plates
Narrow peaks take up less space in the chromatogram and thus
allow more peaks to be separated.
Resolution
It is defined as the difference in retention time between the two
components divided by the combined widths of the elution peaks.
OR
It is measure of the extent of separation of two components and
the baseline separation achieved.
Ideal resolution value should be greater than 2.

Factors affecting resolution

Resolution is affected by three important parameters, they are-
1. Selectivity [separation factor]
2. Efficiency
3. Retention [capacity factor]
 Application of HPLC
1. Pharmaceutical industry
• Quantity of drug determination from pharmaceutical dosage form
• To control the drug stability
• Quantity of drug determination from biological fluids , ex : blood glucose level.
2. Analysis of natural contamination
• Phenol and mercury from sea water
3.Forensic test
 Determination of steroid in blood, urine and sweat
 Detection of psychotropic drug in plasma
4.Food and essence manufacture
 Sweetener analysis in the fruit juice
 Preservative analysis in sausage
MCQs based on HPLC
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