HPLC- Introduction, Theory, Instrumentation, Advantage, Applications High-performance liquid chromatography or commonly known as HPLC, is an analytical technique used to separate, identify or quantify each component in a mixture. The mixture is separated using the basic principle of column chromatography and then identified and quantified by spectroscopy. In the 1960s, the column chromatography LC with its low-pressure suitable glass columns 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 pressures of up to 400 atmospheres. 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 on the chemical structure of the analyte, the molecules are retarded while passing the stationary phase. Instrumentation 1.Solvent reservoir and degassing system 2. Pumping System (Screw- driven syringe pump, Reciprocating pump, Pneumatic or constant- pressure pump) 3. Sample injection system(Septum injectors, Stop flow septum- less injection, Micro- volume sampling valves) 4. Columns- (1. Guard columns 2.separating column) 5. Detectors( The commonly used detectors in HPLC are Bulk property detectors- examples 1. Refractive-index detectors 2. Conductivity detectors Solute property detectors- Examples 1. UV detectors 2. Fluorescence detectors Multipurpose detectors- Example- 1. Perkin-Elmer 3D system (UV absorption+ fluorescence + conductometric detection altogethers) Electrochemical Detectors- Examples 1.Amperometric, 2. Coulometric detectors) 6. Recorder( There are various types of data processors; from a simple system consisting of the in-built printer and word processor while those with software that are specifically designed for an LC system which not only data acquisition but features, like peak-fitting baseline correction, automatic concentration calculation, molecular weight determination, etc.) Type of HPLC- Normal phase, Reverse Phase, ion exchange, size exclusion, Applications-Stability study- eg Atropin Bioassays- HPLC is commonly used for the bioassay and analysis of peptide harmones and some antibiotics- cotrimoxazole, penicillins, sulphates and chloramphenicol In cosmetic industries- used for analyzing the quality of various cosmetic products such as lipsticks, gels, creams etc Isolation of Natural pharmaceutically Active Compounds– use in the isolation of different type of Alkaloids and Glycosides ( analysis of cinchona, liquorice, ergot extracts and digitalis.) Control of microbiological processes- HPLC is used in analyse antibiotics (eg. Tetracyclines, chloramphenicol, strptomycin and penicillins ) Assay of cephalosporins Advantage, Limitation

1. Mrs Vandana Sharma
Assistant Professor
HPLC instrument

2.  Introduction
 Theory
 Instrumentation
 Advantage
 Applications

3. HPLC- different parts’ Name

4.  Explain the various type of pumps used in HPLC-8
 Discuss the basic principle of TLC. Explain how TLC differs from
HPTLC. Also mention the distinguishing points of HPTLC and
HPLC. 4+6+6
 Detectors used in HPLC. 8
 Columns used in HPLC. 4
 b) Write principle, instrumentation and applications of HPLC. 8
 Discuss the principle, instrumentation and various applications of
HPLC. 16
 . Explain partition chromatography with reference to the
principle, procedure, different types and applications in pharmacy
field. 16
 Write short notes on
HPLC. 4
 Discuss the methodology and applications partition column
chromatography 5

5.  High-performance liquid chromatography or commonly known as
HPLC, is an analytical technique used to separate, identify or
quantify each component in a mixture.
 The mixture is separated using the basic principle of
column chromatography and then identified and quantified by
spectroscopy.
 In the 1960s, the column chromatography LC with its low-
pressure suitable glass columns 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 pressures of up to 400 atmospheres.

6. 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
on the chemical structure of the analyte, the molecules are
retarded while passing the stationary phase.
may use any diagram of HPLC- to answer the
questions regarding HPLC
1.

7. Or 2.

8. 1. The purification takes place in a separation column between a
stationary and a mobile phase.
2. The stationary phase is a granular material with very small
porous particles in a separation column.
3. The mobile phase, on the other hand, is a solvent or solvent
mixture which is forced at high pressure through the separation
column.
4. Via a valve with a 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.
5. 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.

9. 6. After leaving the column, the individual substances are
detected by a suitable detector and passed on as a signal to
the HPLC software on the computer. At the end of this
operation/run, a chromatogram in the HPLC software on the
computer is obtained.
7. The chromatogram allows the identification and
quantification of the different substances.

11. Instrumentation of HPLC

12. High Performance Liquid Chromatography

13. Instrumentation
1.Solvent reservoir and degassing system
2. Pumping System
3. Sample injection system
4. Columns
5. Detectors
6. Recorder
 Mobile phase used in the HPLC may be a mixture of organic
solvents or an aqueous-organic mixture or a buffer solution.
 Selection of mobile phase depends on the chromatographic
method and the detector to be used.
 Solvents- completely free from UV absorbing impurities and
any particulate matter

14. Solvent reservoir
 Solvent reservoir consists of a glass bottles with a lid and PTFE
tube to transfer the mobile phase from reservoir to the
degassers and pump.
Degasser
 The eluent used for LC analysis may contain gases such as
oxygen that are non-visible to our eyes.
 When gas is present in the eluent, specially affecting the
working of pump and the detectors (this is detected as noise
and causes an unstable baseline).
 Degasser uses special polymer membrane tubing to remove
gases.
 The numerous very small pores on the surface of the polymer
tube allow the air to go through while preventing any liquid to
go through the pore.

15.  The development of HPLC led to the development of the pump
system.
 The pump is positioned in the most upper stream of the liquid
chromatography system and generates a flow of eluent from the
solvent reservoir into the system.
 High-pressure generation is a “standard” requirement of pumps
besides which, it should also to be able to provide a consistent
pressure at any condition and a controllable and reproducible
flow rate.
 The type of pumps that are used in HPLC
1. Screw- driven syringe pump
2. Reciprocating pump
3. Pneumatic or constant- pressure pump
 Most pumps used in current LC systems generate the flow by
back-and-forth motion of a motor-driven piston (reciprocating
pumps). Because of this piston motion, it produces “pulses”.

16.  An injector is placed next to the pump.
 The simplest method is to use a syringe, and the sample is
introduced to the flow of eluent.
 The most widely used injection method is based on sampling
loops.
 The use of the autosampler (auto-injector) system is also widely
used that allows repeated injections in a set scheduled-timing.
 The following three modes of sample injection system are used in
HPLC-
1. Septum injectors-
2. Stop flow septum- less injection-
The first two methods are inexpensive.
3. Micro- volume sampling valves

17. Additional information
1. Septum injectors- In this system sample is introduced through a
high pressure syringe via self-sealing septum.
Limitation- Mobile phase is immediate contact with the
septum, gives rise to a leaching effect that results in ghost or
psedo peaks.
2. Stop flow septum- less injection- the flow of mobile
phase through the column is stopped for a few
movements, and when column attains ambient pressure,
the column top is opened and the sample is introduced
at the top of the packing
3. Micro- volume sampling valves-
having good precision and
adaptable for automatic injection
Sampling to be done reproducibly

18.  The recent columns are often prepared in a stainless steel
housing, instead of glass columns. At the present time, micro-
columns with 1-4.6mm diameter and 3-7.5cm length are
available- require les solvent and have a greater speed.
 The packing material generally used is silica or polymer gels
compared to calcium carbonate. Thin layers of organic solvents
are coated on these silica particles, and the solvent bound to the
particle surface physically and chemically. Alumina particles, ion
exchange resins, and porous polymer particles can also be used
as column packing material.
The eluent used for Liquid Chromatography (LC) varies from
acidic to basic solvents.
 Most column housing is made of stainless steel since stainless is
tolerant towards a large variety of solvents.

19. 1. Guard column-
Short column present between injector and analytical column
Packing composition of guard columns and analytical is similar,
but particle size is larger in guard columns to aid reduction of
pressure drop.
Benefits-
They eliminate foreign particles and contaminants from the
solvents, thereby improving the life of analytical column
2. Column thermostat/ Column Heater
 The LC separation is often largely influenced by the column
temperature.
 In order to obtain repeatable results, it is important to keep
consistent temperature conditions.
 To achieve a constant and precise temperature control, water
jackets are fitted in the columns.
Two types of columns are used in HPLC
1. Guard columns
2. Column thermostat

20.  Separation of analytes is performed inside the column,
whereas a detector is used to observe the obtained
separation.
 The composition of the eluent (Mobile phase) is consistent
when no analyte is present. While the presence of analyte
changes the composition of the eluent (mobile phase).
Detector - measure these differences.
 This difference is monitored as a form of an electronic signal.
There are different types of detectors available.
 The Modern commercial instruments contain heaters for
controlling the column temperature to few tenths of degree from
near ambient to 150o C.
 Some analysis, such as sugar and organic acid, better resolutions
can be obtained at elevated temperatures (50 to 80°C).
 Thus columns are generally kept inside the column oven (column
heater).

21. In HPLC, the detector monitors the mobile phase passing out of the
column----which further releases electrical signals directly
proportional to = the characteristic of the solute or mobile
phase.
The commonly used detectors in HPLC are
 Bulk property detectors- examples
1. Refractive-index detectors
2. Conductivity detectors
 Solute property detectors- Examples
1. UV detectors
2. Fluorescence detectors
 Multipurpose detectors- Example-
1. Perkin-Elmer 3D system (UV absorption+ fluorescence +
conductometric detection altogethers)
 Electrochemical Detectors- Examples
1.Amperometric
2. Coulometric detectors

22.  The change in eluent (mobile phase) detected by a detector is in
the form of an electronic signal, and thus it is still not visible to
our eyes.
 In older days, the pen (paper)-chart recorder was popularly
used. Nowadays, a computer-based data processor (integrator)
is more common.
 There are various types of data processors; from a simple
system consisting of the in-built printer and word processor
while those with software that are specifically designed for an LC
system which not only data acquisition but features, like peak-
fitting baseline correction, automatic concentration calculation,
molecular weight determination, etc.

23.  Normal phase:
 Column packing is polar (e.g silica) and the mobile phase is
non-polar. It is used for water-sensitive compounds, geometric
isomers, cis-trans isomers, and chiral compounds.
 Reverse phase:
 The column packing is non-polar (e.g C18), the mobile phase is
water+ miscible solvent (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:
 Molecules diffuse into pores of a porous medium and are
separated according to their relative size to the pore size. Large
molecules elute first and smaller molecules elute later.

24. Normal phase chromatography
Normal phase chromatography is a type of HPLC which
uses a polar stationary phase eg Silica and
a non-polar mobile phase (hexane, methylene chloride, chloroform,
diethyl ether and mixture of these selected mobile phase.
Method used for --- separation of mixture components on the basis of
polarity
Adsorption strengths (adsorptive mechanism) can be increased by by
increasing polarity of components
Polar component interact with Polar stationary phase and retained-----
elution time is increased
Method used for the analysis of solutes readily soluble in organic
solvents, based on their polar differences such as amines, acids, metal
complexes, etc

25. Reversed-phase HPLC
•Stationary Phase – Hydrophobic /Non polar stationary phase--
-Non polar hydrocarbon
•Mobile phase- Polar- An aqueous, moderately polar mobile
phase
RPC is based on the principle of Hydrophobic interaction,
resulting from repulsive forces between a polar eluent (Mobile
Phase), relatively non-polar component, and Non-polar
stationary phase.
The binding of component to the stationary phase occurs
according to the contact surface area around the non-polar
segment of the component molecule upon association with the
ligand in the aqueous eluent.

26.  A C18 column is an example of a "reverse phase" column.
 Reversed-phase chromatography,
 a partition mechanism, is typically used for separations by non-
polar differences.
Size – Exclusion HPLC (SEC) or gel permeation or Filtration
Chromatography
 Size exclusion chromatography works by trapping smaller
molecules in the pores of the chromatographic column.
Molecules that are larger than the pores are unable to defuse on
the beads. Therefore, they are eluate first. Smaller molecules
penetrate deep inside the pores and they are eluate last.
 Used to determine the tertiary and quaternary structures of
proteins and amino acids
 Useful for determination of molecular weight of polysaccharides

29.  Ion Exchange HPLC
 In this method retention occurs according to the attraction between
the solute ions and charges sites bound to the stationary phase,
excluding the similarly charged ions.
 The ion exchange-high-performance liquid chromatography is a
high-throughput analytical method that allows to determine the
charge profile of purified antibodies.

30. Widely used in
Purifying water
Ion exchange chromatography of protein
High – pH anion-exchange chromatography of carbohydrates and
oligosaccharides

31. This method involves separation ----according to the specific
reversible interaction of proteins with legands that are attached to
the solid support on a bio-affinity matrix through covalent bonds.
• Proteins interact with these column-bound ligands, are
retained and can be eluted by following 2 ways:
1. Biospecific Elution:- Inclusion of free ligands in elution buffer
which competes with column bound ligand
2. Aspecific elution – Change in pH, salt etc. weakens the intraction
of protein with a column – bound substrate.
Examples include antibody/antigen, enzyme/substrate, and
enzyme/inhibitor interactions.
The steps involve in affinity chromatography
1: The two phases of an affinity chromatography: The mobile and the
stationary phase.
2: First step - Add cell lysate to the column.
3: Protein of interest bind to the the affinity beads
Bio- affinity chromatography

32. 4: Add wash buffer and remove remaining unspecific protein and
other substances.
5: Elute your protein of interest from the affinity beads through an
elution buffer.

34. 1. Stability study- eg Atropin
1. Bioassays- HPLC is commonly used for the bioassay and
analysis of peptide harmones and some antibiotics-
cotrimoxazole, penicillins, sulphates and chloramphenicol
2. In cosmetic industries- used for analyzing the quality of various
cosmetic products such as lipsticks, gels, creams etc
3. Isolation of Natural pharmaceutically Active Compounds– use in
the isolation of different type of Alkaloids and Glycosides (
analysis of cinchona, liquorice, ergot extracts and digitalis.)
4. Control of microbiological processes- HPLC is used in analyse
antibiotics (eg. Tetracyclines, chloramphenicol, strptomycin
and penicillins )
5. Assay of cephalosporins

35. 7. Assay of Furosemide
8. Assay of theophylline
9. Assay of corticosteroids
10. Assay of Dichlorphenamide
1. It is highly sensitive
2. Is shows better performance
3. Rapid process and less time consuming
4. Resolution and separation capacity is high
5. Accurate and precise
6. Utilizes a chemically inert mobile phase for developing chamber
7. It needs a small amount of mobile phase for developing
chamber

36. 8. It involves early recovery of separated components.
9. It enables easy visualization of separated components.
10. Good reproducibility and repeatability
11. Useful in qualitative and quantitative analysis
12. Used for analytical and preparative purposes
13. Qualitative control studies of product
 Cost: Despite its advantages, HPLC can be costly, requiring large
quantities of expensive organics.
 Complexity
 HPLC does have low sensitivity for certain compounds, and some
cannot be detected as they are irreversibly adsorbed.
 Volatile substances are better separated by gas chromatography.

37. The commonly used detectors in HPLC are
A. Bulk property detectors- examples
1. Refractive-index detectors
2. Conductivity detectors
B. Solute property detectors- Examples
1. UV detectors
2. Fluorescence detectors
C. Multipurpose detectors- Example-
1. Perkin-Elmer 3D system (UV absorption+ fluorescence +
conductometric detection altogethers)
D. Electrochemical Detectors- Examples
1.Amperometric
2. Coulometric detectors

38. A. Bulk Property Detectors: Bulk property detectors are those that
measure the changes in solute and mobile phase in
combination. Such detectors show fluctuation in readings even
with slight change in mobile phase combination.
Examples are:
 1.Refractive index (refractive index detector (RI or RID) is a
detector that measures the refractive index of an analyte
(Sample) relative to the mobile phase only. They are often used
as detectors for high-performance liquid chromatography and
size exclusion chromatography.)
Principle: The RI detectors measure a bulk property of the
mobile phase leaving the column: its ability to refract to bend
light (i.e., its refractive index). This property changes as the
composition of the mobile phase changes, such as when solutes
from the column.

39.  In refractive index deterctor light emitted from the source(s) is
concentrated into cell containing the sample or reference sample
and reference sample.
 Bothe the chambers are of cells are separated by diagonal glass
sheet.
 The light passes through the cell and reaches the beam splitter
(B) that diverts the light towards two photocells (P1 and P2 ).
 A change in the observed refractive index of the sample results
in difference in their relative output, and this difference in
amplified and recorded.
 2. conductivity detectors

40. B. Solute property detectors are also called as selective detectors
because they give response for a particular physical or
chemical property of the analyte, being ideally independent of
the mobile phase.
Examples- UV- Detectors
Fluorescence Detectors
1. UV detector or UV-Vis detector is an ultraviolet/visible light
detector. UV detectors are nondestructive chromatography
detectors that measure the amount of ultraviolet or visible
light that is absorbed by components of a mixture being
eluted off the chromatography column.
Originally, dual – wavelength instruments with 254 and 280nm
were used but now more sophisticated and updated variable
wavelengths ranging between 210 -800 nm are used for
performing more selective detector

41. UV detector or UV-Vis detector

42. 2. Fluorescence Detectors
 Many compounds (solute) are present in mobile phase.
 When they allowed to pass as column effluent through a cell
irradiate with xenon or deuterium source , first UV radiation is
absorbed and subsequently radiation of a longer wavelength is
emitted in the following two ways:
1. If instantly, named as “Fluorescence” and
2. If after a time gap, named as “ Phosphorescence”
Fluorescent Compounds: The number of inorganic and organic
compounds exhibiting natural fluoresce property is very less,
while most of the pharmaceutical substance and environmental
contaminants ( eg. Polycyclic Aromatic Hydrocarbons (PAH)) with
a conjugated – cyclic system are fluorescent in nature.
Energy absorbed by these substances is re-emitted and can be
detected by a fluorescent detector
For the detection of non- fluorescent compounds, they firstly
converted to fluorescent derivatives by treating with appropriate
solvents

43. Radiation emitted from a xenon or deuterium source is
concentrated on the flow cell using a filter. Usually at 90o to the
incident beam,
the fluorescent radiation emitted by the sample is measured. A
second filter is used to filter only a suitable wavelength and reject
all scattered light to reach the photo multiplier tube

44.  A multipurpose detector include three detectors that are
combined and kept together in single unit.
 Example- Perkin-Elmer 3D system
 The three different detectors perform the following functions-
 Fluorescence function
 UV- Function
 Conductance function
Perkin- Elmer

45. It is based on the measurement of current resulting from the
oxidation reduction reaction of the analyte at the suitable electrode
The level of current is directly proportional to the analyte
concentration.
Electrochemical (EC) detection (ECD) coupled with HPLC is a
powerful tool for the detection of neurotransmitters, environmental
assessment, and the detection of phenol compounds from food
samples. Various neurotransmitters are detectable.
Electrochemical detectordetector