TOPIC: HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY (HPLC)
Presented by : Muttu B Mugali
Department of pharmaceutical chemistry
M Pharm 1st SEM

Submitted to: Dr. B.S. Kittur

Head and Professor
Department of Pharmaceutical Chemistry
HSK College of Pharmacy Bagalkot.
CONTENTS

Introduction
Principle
Instrumentation & Working
Chromatographic parameters
Factors affecting resolution
Applications
 INTRODUCTION

• HPLC (high performance liquid chromatography) is an analytical

Instrument which is used for separation of compounds in a mixture.
• Some time referred as high pressure liquid chromatography.
• It is an analytical technique for separate, identify and quantifying a
components in mixtures.
• It gives high performance due to the small particle size of stationary phase
(3.5-10µm).
• High pressure is applied for rapid separation of compound that is 1000-
4000psi.
Principle
 HPLC is a form of liquid chromatography, where separation occurs
between a mobile phase and a stationary phase.
 It is the ability with which the sample constituents will distribute
themselves between the two phases that will effect the separation.
 Depending on the nature of the stationary phase the separation process
can be of four different modes.

Adsorption chromatography
Partition chromatography
Ion-exchange chromatography
Size exclusion chromatography
 Adsorption chromatography
• It is a process of separation of liquid mixtures based on principle of
adsorption.
• It is used for solid-gas chromatography and solid-liquid chromatography.

Partition chromatography
• It is a process of separation of liquid mixtures based on theory of Partition
coefficient.
• The separation will occurred by the polarity.
• partition chromatography is used for the liquid–gas and liquid-liquid
mixtures.
Ion-exchange chromatography
• It is a process of separation of liquid mixtures based on principle of
exchange of resins.
• This chromatography is used mainly for the proteins.

Size exclusion chromatography

• It is a chromatographic method in which molecules are separated by their

size and in some cases by their molecular weight.
• This technique is widely used for the separation and purification of
proteins.
INSTRUMENTATION

1. Mobile phase
2. Solvent Pump
3. Liquid Sample
4. Column
5. Detector
 MOBILE PHASE

• In HPLC ,the reservoir are usually made up of stainless steel or borosilicate glass
with the capacity of 500 ml to 1 L of solvent ( HPLC grade ).
• Beside they are duly equipped with adequate mean for the removal of dissolved
gaseous product viz O2 and N2 that may produce undesired small bubbles in the
column as well as detector system engaged.
• Used to store the mobile phase.
• The Gradient solvents are used here.
• Two types of solvent reservoirs are
i. Binary system
ii. Quaternary system.
DISPLACEMENT PUMP

Provide a small internal volume

(10– 100ul ) .
Accomplish high output pressure
(up to 10,000 psi).
Constant flow rate attained is
invariably independent of viscosity
of solvent.
RECEPROCATING PUMPS

•The reciprocating pump it consist of a small

chamber where in the solvent is moved in
and by an eccentric system or gear.
•The forward stroke shut down the inlet check
valve, where as the outlet check valve open
the flood gate and allow the respective
intended mobile phase to pumped in to the
Colum efficiency.
PNEUMATIC PUMPS

In these pumps the mobile phase is

driven through the Colum with use of
pressure produced from a gas cylinder.

It has a limited capacity of solvent.

Solvent is pumped back and forth by a

motor driven piston.
 FLOW CONTROL SYSTEM
 Modern HPLC system is duly provided with a custom designed pumping system to
manage control the compound aided devices that are capable of determine.
 The pressure meticulously importantly, one may either increase or decrease the
flow by critically adjusting the speed of the motor of the pump.
Working of HPLC
Separation Process
Graph reading
 GUARD COLUMN
 Hplc system are dully provided with a short guard column which is strategically
positioned just before the analytical column to remove particulate matter as well
as inadvertently
admitted contaminated from solvents.
 It also serve to cause and effect the saturation of the mobile phase with the ensuring
stationary phase so as the check prevent and minimize the possibly incurred looses
of this specific HPLC solvent from column .
 Packing guard column must be same as used for the analytical column however the
particle size of the packing material may a little bigger in size than the analytical
column (10-15 um) in order to reduce the change of any pressure drop in the HPLC
system.
 ANALYTICAL COLUMN
 Stainless steel tubing- HPLC columns are made from smooth borosilicate tubing.
 Heavy walled glass tubing – it also finds its enamorous utility
in HPLC system, however the application is restricted only to lower pressure of
about 600 psi.

In additional to the HPLC column ,there are 2 physical feature.

• Column length – the columns , length in a HPLC system may be increased by
coupling together two or more column in a series as and when required and the
usual length is 25 cm.
• Column inside diameter – the inside diameter of the columns ranges between 4 to
10mm whereas the particle size commonly found to be form 3 to 10 um.
 DETECTOR

• The detector can detect the individual molecule that elute from the
Colum and convert the data into electrical signal.
• A detector service to measure the amount of these molecules.
• The detector provide an out put to a recorder or computer that result in
liquid chromatogram.
• Detector is selected based on the analyte or the sample under detection.
 COMMONLY USED DETECTOR ARE

• Ultra violet detector

• Fluorescence detector
• Mass spectroscopy detector
• Infrared detector
• Diode array detector
• Conductivity
• Refractive index.
 UV – Visible absorption detector
• UV visible absorption detectors are regarded to be the most commonly employed
variants based upon the following three extremely important factual evidence such
as:
1. High degree of sensitivity
2. Based spectrum linear response range.
3. Excellent capability to organize and monitor.
• several analyte variants in the sequential manner they get eluted from a HPLC
column
• In UV visible detector in a HPLC system essentially light source along with 1 or 2
photodiode plus an efficient flow cell
to enable the smooth passage of the eluents.
 FLUORESENCESE DETECTOR

• They are only useful for assay and to monitor fluorescent analyte and
may even afford support sensitive almost 10 folder greater in
comparison to valves obtainable by uv visible detector.
• Usually simple Hg excitation source along with one or more filter is
just enough to isolate band emitted radiation.
 INFRARED DETECTOR

• There are 2 type of IR spectrophotometer that find their usage in a HPLC analytical
system .
• Dispersive IR Spectrophotometer shows a ranges of wave length from 2.5 um or a
frequency from 4000- 690cm .
• Fourier transform , IR Spectrophotometer provide a fast and sensitive mode of
complete spectrum scan that may be accomplished in a few minute duration only.
• Possible to accomplished multiple spectra.
• Sensitivity of analysis may be altered with S/N ratio to obtain both improved
and reproducible scans.
 MASS SPECTROMETER DETECTOR

• Mass spectrometer may be intelligently and skillfully exploited and employed as

highly efficient powerful and selective mode of detection in a HPLC system
because the specific mass of each and every molecular entities may be identified
critically as it elutes from the HPLC.
Chromatographic Parameters

1. Efficiency
2. Retention factor
3. Selectivity
4. Pressure
 Efficiency (N)

• Column efficiency is used to compare the performance of different columns. It

is probably the most frequently cited parameter of column performance and is
expressed as the theoretical plate number, N.

where,
N = 16 (tr/Wt)2 N - Efficiency of column
tr - Retention time of sample
Wt - Peak width at base

• Higher the column efficiency lesser selectivity required to completely resolve narrow
peaks.
Retention factor (k)
• Also known as a capacity factor
• It is a measure of retention period of sample which resides in a stationary phase
relative to the time it resides in a mobile phase.
Where,
K = Retention factor.
K =(tr-t0 )/ t0 t0 = Retention time for un-retained peak
tr = Retention time for the sample peak
Selectivity or separation factor (α)
• The separation factor is a measure of the time or distance between the maxima of two
peaks. If α = 1, the two peaks have the same retention time and co-elute.

Where,
α=k2/k1 k1 = Retention factor for 1st peak.
k2 = Retention factor for 2nd peak.
Pressure

• It mainly acts on 5 things. Those are,

Viscosity of solvent ηFL

Flow rate ΔP=
K0πr2dp2
Column length
Column radius
Particle diameter
Resolution (Rs)
• Resolution describes the ability of a column to separate the peaks of
interest. So the higher the resolution, easier it is to achieve baseline
separation between two peaks.

Tr2 – Tr1
Rs=
0.5(Wb1 + Wb2)

Where,
• Tr2 & Tr1 = Retention time of 2nd and 1st peak.
• W & W = Baseline width of 2nd and 1st peak.
Applications of HPLC

Water purification.
To analyze finished drug products.
Drug discovery.
Applied in pharmaceuticals to control drug
stability.
Applies in forensic test to determine steroid in
the blood.
Used for separation of anti-biotic form.
ADVANTAGES

• High accuracy
• High Resolution
• Speed of analysis
• Quantification of results
• Good Sensitivity
DISADVANTAGES

• High cost
• Required skilled person
• Low sensitive for some compounds
• only applicable for volatile and thermally stable compounds
• Co-elution is difficult to detect
• Only gradient solvents are used
REFERENCES
• LaCourse ME, LaCourse WR. General instrumentation in HPLC. InLiquid
Chromatography 2017 Elsevier.
• Belanger JM, Paré JJ, Sigouin M. High performance liquid chromatography (HPLC):
principles and applications. In Techniques and Instrumentation in Analytical
Chemistry 1997 (Vol. 18, pp. 37-59). Elsevier.
• Int. J. Pharm. Sci. Rev. Res., 55(2), March - April 2019; Article No. 09, Pages: 46 – 50
• HPLC Method development for pharmaceticals, ediated by Satinder Ahuja, Henrik
Rasmussen. Vol-8.
• Instrumentation method of chemical analysis by Gurudeep .R. Chatawal , Sham .K.
Anand
• Swartz M. Seeing is believing: detectors for HPLC. LCGC North America. 2010 Oct
1;28(10):880-9.
Thank you.