HPLC

Contents
• Introduction
• Theory
• Instrumentation
• Applications
• Limitations
 Importance
• Chromatography has application in every branch of
the physical and biological sciences
– 12 Nobel prizes were awarded between 1937 and 1972
alone for work in which chromatography played a vital role

3
 Chromatography Definition
• Chromatography is defined as the physical
method of separation, in which the mixture
of analytes is separated using two phases,
one is stationary phase and other a mobile
phase which percolates through the
stationary phase. The separation occurs
because of difference in affinity between
analytes and stationary phase.
4
 Introduction
➢High-performance liquid chromatography
(HPLC) is a form of liquid chromatography to
separate compounds that are dissolved in
solution

• HPLC is characterized by the use of

high pressure to push a mobile phase solution
through a column of stationary phase allowing
separation of complex mixtures with high
performance /resolution
5
• The different components in the mixture pass
through the column at different rates due to five
different reasons:
• 1: Polarity of solutes 2: ionic nature 3:Mol.Wt
4: partitioning ability 5: Ability to form affinity
complexes

• This requires sophisticated instrument.

• Provides specific, sensitive and precise method for

analysis of different complicated samples.
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CHROMATOGRAPHY

7
 Advantages of HPLC
➢ Sensitive method for analysis of different
complicated samples

➢ There is ease of sample preparation and introduction

➢ There is speed of analysis

➢ Accuracy and precision

➢ Operating at low temperature so can be used for

separation of thermolabile, high polarity substances
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 Basic Components
• Pump to deliver mobile phase at high
pressure
• Injection system to introduce sample
• Column for separating sample components
• Detector to detect the separated components
• Data System to operate, control instrument
and process the signal given by the detector

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10
 HP/LC
LC- Liquid Chromatography (Liquid- mobile phase)

•on nature of stationary phase

•LSC •LLC
•Adsorption •Partition
Chromatography Chromatography
•on polarity of stationary phase &
mobile phase

•Normal •Reversed
Phase Phase
 Chromatography Stationary Phases

•Silica Gel •Derivatized Silica Gel

O O O O O O
| | | | | |
−O−Si−O−Si−O−Si−O−H −O−Si−O−Si−O−Si−O−R
| | | | | | •Where R = C18H37
O O O O O O •hydrocarbon chain
| | | | | | •(octadecylsilyl deriv.
−O−Si−O−Si−O−Si−O−H −O−Si−O−Si−O−Si−O−R
| | | | | | •silica or “C18”)
O O O O O O

•bulk (SiO2)x •surface •bulk (SiO2)x •surface

•relatively polar surface • relatively nonpolar surface

•“normal phase” •“reversed phase”
 Normal vs. Reversed Phase Chromatography
Normal Phase Reversed Phase
Stationary phase Polar (silica gel) Non-polar (C18)
Non-polar Polar
Mobile phase
(organic solvents) (aqueous/organic)
Sample movement Non-polar fastest Polar fastest
Different polarities Different
Separation based on
(functionality) hydrocarbon content
 HPLC

• HPLC as compared with the classical technique is

characterized by:
• Small diameter (2-5 mm), reusable stainless steel columns;
• Column packings with 3, 5 and 10 µ particles.
• High inlet pressures and controlled flow of the mobile phase;
• Precise sample introduction
• Special continuous flow detectors capable of handling small
flow rates and detecting very small amounts.
• Automated standardized instruments;
• Rapid analysis; and
• High resolution.

15
 HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
• Very small particles of narrow distribution range.

• Uniform pore size and distribution.

• High pressure column slurry packing technique.

•
• Accurate low volume sample injectors.

• Sensitive low volume detector.

•
• Good pumping systems.
•

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 Instrumentation in HPLC

➢Mobile phase reservoir

➢Pump and Gradient Controller
➢Mixing Chamber
➢Sample Conditioning Column
➢Sample injector unit
➢Precolumn
➢Analytical column
➢Detectors
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 HPLC INSTRUMENTATION
• PUMP
• INJECTOR
• COLUMN
• DETECTOR
• RECORDER OR DATA SYSTEM

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•Reservoir •Reservoir
•Recorder

•Pump •Gradient •Pump

•controller
•Fraction •Detector
•collector

•Mixing •Analytical
•chamber •column

•Solvent
•Injector •Precolumn
•Conditioning
•column

• Fig. Block diagram of HPLC

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 Classification of HPLC on the internal
diameter of column
• Semi Micro (0.3-1mm ID)
• micro (1-3mm ID)
• Conventional (4-8mm ID)
• Semi preparative (10-20mm ID)
• Preparative (20-50mm ID)
• Process (less than 50mm ID)

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 Solvent/ mobile phase reservoirs

• Glass or stainless-steel containers capable of

holding up to 1 liter mobile phase (pure
organic solvents or aqueous solutions of salts
and buffers)
• Inert to a variety of aqueous and non aqueous
mobile phases.
• Stainless steel should be avoided for use with
solvents containing halide ions.
 Degassing & filtration of mobile phase

• In many cases, aqueous solvents & some organic

solvents are degassed prior to use
• Degassing is done to prevent formation of gas
bubbles in the pump or detector ( Mobile phases are
degassed by stirring of the mobile phase under
vacuum, sonication or sparging with helium gas)
• The mobile phase are filtered to remove particulate
matter that may clog the system
 Tubing
• Should be inert,
• have ability to withstand pressure
• able to carry sufficient volume
 PUMPS
– FUNCTION
– Deliver constant , reproducible flow of eluent to the column under
high pressure.

– What is a good HPLC pump?

– Capable of delivering a range of flow rates suitable for different modes of
HPLC.
– Reproducibility of flow rates.
– Ability to operate under high pressure - 6000 psi
– Stable flow without pulsations to minimise detector noise.
– Compatibility of components with the wide range of eluents
– Easy access for regular maintenance.

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 Solvent delivery system (Pump)
• Must be constructed from material that are
inert to all mobile phases
• Materials commonly used are glass, stainless
steel, Teflon.
• The solvent flow rate produced by pump
should be pulseless or should be dampened in
order to remove pulses (pulses may cause
spurious results with some detectors)
 •HPLC
Pump
•Manner in which they
operate

•Constant flow rate (Mechanical) Pump •Constant Pressure Pump

(Pneumatic)

•syringe •Amplifier
•Reciprocating •Direct pressure
pump pump
pump pump
(screw
driven)

•Single
•Reciprocati
piston •Double ng
reciprocatin piston
diaphragm
g pump reciprocatin pump
g pump
 Constant Pressure Pumps
• Advantages of constant pressure pumps
Simple
Free from pulsation's resulting in smooth baselines.
Inexpensive, easy to operate, and easy to maintain.
• Disadvantages
Flow rate must be monitored carefully and constantly,
Factors affecting flow rate -
Solvent viscosity due to a temperature or composition change.
• Changes in flow rate can affect-
• Qualitative - dependent on retention time.
• Quantitative analysis - detectors are concentration dependent, affects the peak
area to be taken for calculation.

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 Pumps
• Pneumatic pump : which produce a constant pressure
• Gas displacement type: which use direct pressure from
a highly compressed gas to force solvent out of a tube
• Pneumatic amplifier type: in which compressed gas at a
lower pressure impinges on the large end of the piston
to force the smaller end to deliver the liquid.
• The pneumatic pumps have the advantage of pulseless
operation.
 Constant Flow Pumps
• Advantages -
Ability to repeat elution volume and peak area, regardless of viscosity
changes.

• Two types
- Reciprocating piston -
1. Can maintain a liquid flow for indefinitely long time.
2. Causes flow and pressure pulsation.
Used - Most of the HPLC applications.

- Positive displacement (syringe) pumps -

1. Has to be refilled after it displaces the whole syringe volume.
2. Does not have any flow and pressure pulsation.
Used - Micro-HPLC applications where a constant flow at the
micro-liter per minute flow rate range is essential.
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 Reciprocating Piston Pumps

The piston expels liquid through a one-way valve (check valve).

The pumping rate is usually adjusted by controlling the distance the piston retracts.

•Disadvantage -

•Pressure pulsations.

•Pulse dampers are used to reduce pulsation. 30

 Reciprocating dual piston pump
• Dual Piston Pumps - provides a constant and almost pulse free flow.
• Both pump chambers are driven by the same motor through a common
eccentric cam; this common drive allows one piston to pump while the
other is refilling.
• As a result, the two flow-profiles overlap each other significantly
reducing the pulsation downstream of the pump.

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 ]

Advantages of dual piston pumps

• A computer-designed camshaft is used to achieve maximum overlap of

pump strokes, eliminating pulsation or ripple.

• Use of staggered inlet/outlet lines to allow complete flushing when

liquids are changed or if air is drawn through the pump.

• Small-volume check valves are used to allow the pumps to function

reliably at flow rates as low as 0.001 mL/min.

• Fewer moving parts, with maintenance-requiring components pump

seals, check valves) readily accessible from the front of the instrument.

• A wide flow rate range (0.01 to 10 ml/min) is provided without gear

change.
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 MODES OF ELUTION
1. Isocratic elution.
– Solvent composition remains constant.
2. Gradient elution.
– Steady changes in the mobile phase composition.
– Used when solutes of different retention times need to be analysed.

– Reduce retention times of the later eluting peaks by increasing the solvent
strength of the eluent.
– Stepwise switching from one eluent to another after a certain interval of time.
– Continuos gradient of solvent strength. 33
• If gradient analysis is necessary for separation, most
common way of forming gradient is to include
second reservoir and pump and a gradient controller.

• Gradient controller is an electronic device combines

the operation of two pumps, to provide mobile phase
mixture of desired conc. E.g. if 50:50 mixture of
solvent in two reservoirs is desired at an flow rate of
1mL/min., controller adjust the rate of delivery of
each pump to 0.5 mL/min. The solvents then are
combined in the mixing chamber.

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 INJECTORS
Injection device should deliver
1. Sample within the range of 0.1 to 100 ml of volume.
2. High reproducible volumes under high pressure (up to the 4000 psi).
3. Produce minimum band broadening.
4. Minimize possible flow disturbances.

• Rheodyne injector
• Valves are commonly used.
• Samples introduced reproducibly into pressurized columns without significant
interruption of flow, even at elevated temperatures

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 Injection device
• The solute mixture is introduced into the
chromatograph by means of suitable injection device.
•Type of injector

•syringe •Stopped flow •Solvent flowing,

•Injection device •(Loop injectors)

•Septum injectors : are available, in which the sample

solution is injected through a self sealing rubber or Teflon disk
using a micro liter syringe.
•-This may be done while the mobile phase is flowing or while
it has been stopped temporarily.
•-Inexpensive and easy to use
•-Problem : lack of reproducibility
 Rotary-valve and loop injector

• This consist of a stainless-steel and Teflon

block that has been drilled to provide two
alternative paths for solvent flow each
selectable by a rotating valve.
Rheo-dyne injector

•load

•inje
ct
 INJECTORS

•Advantages of valve injection - rapid, reproducible, and operator independent

delivery of a wide range of sample volumes from 60 nl up to several milliliters at
pressures up to 7000 psi .
•Disadvantage - sample loop must be changed to obtain various sample volumes.
40
Precolumn:
It contains a packing chemically identical to that in
analytical column.
Mainly used to remove the impurities from the
solvent and thus prevents contamination of the
analytical column.

It is also called as guard column.

it is having large particle size.

It is having short length of 2 to 10 cm, so does not

affect separation.
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➢ Analytical column:
➢ The success or failure of analysis depends upon choice of
column.
➢ Actual separation is carried out here.
➢ Stainless –steel tube
➢ size – length -25 to 100 cm
Internal diameter – 2 to 4.6 mm
Column is filled with small particles 5 – 10 micron. The
solid support can be silica gel, alumina.
The separation is result of different components adhering to
or diffusion into the packing particles when the mobile
phase is forced through column.

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➢ Analytical column:
• Both C8 and C18 columns are considered as examples of
reversed phase liquid chromatography (RP).

• The stationary phase here is seen as a thin film of non-polar

liquid phase that has been designed to be chemically similar
to an inert material (Silica gel particles).

• The non-polar layer is chemically linked to the silica

particles surface by reaction with the polar silanol groups on
the stationary phase surface and so rendering them less
polar or non-polar.

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• The difference between the two columns will be in the
length of the carbon-chain attached to the silica surface.

• Acoordingly C8 hplc columns have packing material

composed of silica particles attached to C8 carbon units

• C18 will, of course, have packing materials coated with C18

hydrophobic units.

• Categorically both are reversed phase but C18 columns will

definitely be more "hydrophobic rather than the C8
columns.

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COLUMN

Must operate in high pressure

Usually constructed of metals
Typical dimensions
10-30 cm long
1-3 cm ID
Contains packing material which
holds the stationary phase
Many types exist
Typical packing materials are 5-
10 µm in diameter
Guard column used to extend life of
main column

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 DETECTORS
• Function of a detector -
– High Precision, High sensitivity & High stability.
• Basic detector should -
1) be capable of detecting 1 part or less of solute in 106 parts of eluent.
2) cause no re-mixing of solute bands.
3) have a wide linear dynamic range to ensure good quantitative analysis.
4) have low noise level and drift.
5) have fast response time to record rapidly eluting peaks.
6) be insensitive to flow rate and temperature changes.
7) be insensitive to eluent composition changes to allow gradient elution.
8) be reliable and reproducible.
9) be easy to operate and maintain.
10) be non-destructive.

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 DETECTORS
• What is Noise ?
• Any disturbance of the detector baseline which is not related to eluted solute
is termed as ‘Noise’.
• Short term noise is the short variation of the baseline from a straight line
caused by
– electric signal fluctuations, lamp instability,temperature fluctuations and
other factors.
– Noise usually has much higher frequency than actual chromatographic
peak.

Drift is continuos increase or decrease of the detector signal as a

result of temperature or mobile phase composition changes.
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Detectors:
Features of good detectors are

1. should respond to all components in

the mixture

2. should not respond to mobile phase

3. should be unaffected by the changes

in temp. and flow rate.

4. should have high sensitivity, low

noise 49
 •Major Types of HPLC Detectors
•Fixed l
•Variable l
•UV-Vis
•Photodiode Array
•Amperometry
•Solute Property •Electrochemical

•Pulse Amperometry
•Fluorescence •Voltammetry
•Coulometry

•Deflectance Type
•Refractive Index
•Reflectance Type
•Bulk Property •Suppressed
•Conductivity
•Non-suppressed
 OPTICAL DETECTORS

• Ultraviolet (UV) - Most popular and widely used.

– The solutes that contain a chromophore at the monitoring wavelength, absorb the
incident light as they pass through the flow cell.
– Amount of light absorbed produces a signal proportional to the concentration of solute.
–
» Absorbance (A) = ECL

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 FIXED WAVELENGTH DETECTOR
• HPLC detectors which does not allow to change the wavelength of the
radiation called fixed-wavelength detectors.
• Cost effective.

• Low-pressure mercury vapor lamp emit very intense light at 253.7 nm.
By filtering out all other emitted wavelengths, the 254 nm line is utilised
to provide stable, highly sensitive detector.
• The 254 nm was chosen since the most intense line of mercury lamp is
254 nm, and most of UV absorbing compounds have some absorbance at
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254 nm.
 VARIABLE WAVELENGTH DETECTOR
• Detectors which allow the selection of the operating wavelength
called variable wavelength.

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54
Characteristics:

•These detectors are nondestructive.

•These are insensitive to changes in solvent flow rate

and temperature.

•Drawback:

•Nonuniformity of response for different compounds.

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 •Photodiode Array Detectors (PDA) or DAD
•--Even much more rapid scanning of the absorption spectra of the eluted peak is
•possible using a photodiode array detector
•--The optical arrangement of the photodiode array detection is shown below:
•--Optical arrangement is referred to as
•“reverse optics”. This is because the
•dispersion device (holographic gratings) is
•placed after the flow cell (opposite to UV-Vis)
• Working of DAD
a) Light from a continuum source (e.g., D2
• Lamp) passes through a lens system which
• focusses polychromatic light onto the
• flow cell (containing the sample)
•b) The transmitted light then falls on a
•holographic gratings where it is dispersed
•into a photodiode array (PDA).
•c)PDA is a several hundreds of photodiodes
•arranged in a linear fashion. A typical
•photodiode array has 512 diodes to cover a
•range of wavelength (190-800 nm), each photodiode has a bandwidth of 2 nm.
•d) A range of wavelengths of light falls on a photodiode array and each diode picks
•up a different wavelength of light.
 PHOTO DIODE ARRAY DETECTOR-PDA

• Special feature is the ability to perform spectroscopic scanning.

•Precise absorbance readings at a variety of wavelengths while the peak is passing

though the flow cell.

•Allows for the best wavelength(s) to be selected for actual analysis.

•Allows for the determination of peak purity when the peak shape in itself does not
57
reveal that it actually corresponds to two (or even more) components.
 REFRACTIVE INDEX DETECTOR
• Principle- Measuring of the change in refractive index of the column
effluent passing through the flow-cell.

• The deflection of a light beam is changed when the composition in the

sample flow-cell changes in relation to the reference side.
• When no sample is present in the cell, the light passing through both
sides is focused on the photo-detector.
• As sample elutes through one side, the changing angle of refraction
moves the beam. 58
 R I DETECTOR
Advantages :
– (1) universal response
– (2) low sensitivity to dirt and air bubbles in the cells
– (3) the ability to cover the entire refractive index range from 1.000
to 1.750 RI with a single, easily balanced cell.

Disadvantages:
– Low Sensitivity
– Complex mixtures, may cover a wide range of refractive index
values and some may closely match that of the mobile phase,
becoming invisible to the detector.
– Changes in the eluent composition require the re-balancing of the
detector.
– Cannot be used in the analyses requiring the gradient elution.
– Disability to easily remove and clean or replace the cell when
filming or clogging occurs.
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 FLUORESCENCE DETECTORS

• Fluorescence detectors are very specific and selective

• Most sensitive detector.

• Fluorescence intensity depends on both the excitation and emission
wavelength, allowing selectively detect some components while suppressing
the emission of others.

60
• Fluorescence detector:
• These are very sensitive and selective
• Certain compounds emit light when excited by
UV light.

• In this detector the photodetector is placed at

right angles to the excitation light in order to
detect only fluorescence light.
• Wavelength selection is for the excitation and
emission can be accomplished by either filters or
monochromators.
61
• The sensitivity of this detector is increased by
increasing power of excitation source.

• This is widely used in pharmaceutical analysis

for analysis of biological fluids and this is 100
times more sensitive than UV detection

• Drawback:
• Its Relatively narrow linear dynamic range.

62
63
– Based on the measurements of the current resulting from
oxidation/reduction reaction of the analyte at a suitable electrode.
– Used for analyzing phenols and organic acids.

64
•The conductivity of the column effluent is continuously
measured and the appearance of the analyte in the cell is
indicated by a change in conductivity.
•Used most successfully in ion-exchange chromatography of
anions and cations.

65
 Principle

• A liquid mobile phase is pumped under pressure

through a stainless steel column containing particles of
stationary phase with a different diameter of 3-10 μm.
• The analyte is loaded onto the head of the column via
a loop valve and separation of a mixture occurs
according to the relative lengths of time spent by its
components in the stationary phase.
• It should be noted that all components in a mixture
spend more or less the same time in mobile phase in
order to exit the column.
• Monitoring of the column effluent can be carried out
with a variety of detectors.
 Applications
• The combination of HPLC with monitoring by UV/Visible
detection provides an accurate, precise and robust method
for quantitative analysis of pharmaceutical products. It is
the industry standard method for this purpose.
• Monitoring of the stability of pure drug substances and of
drugs in formulations, with quantitation of any degradation
products.
• Measurement of drugs and their metabolites in biological
fluids.
• Determination of partition coefficients and pKa values of
drugs and drug protein binding.
 Application
➢Qualitative analysis

➢Natural Products
➢Stability Studies

➢Quantitative analysis

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70
 Strengths
• Easily controlled and precise sample introduction ensures
quantitative precision.
• HPLC is the chromatographic technique which has seen
the most intensive development in recent years, leading to
improved columns, detectors and software control.
• The variety of columns and detectors means that the
selectivity of the method can be readily adjusted.
• Compared to GC there is less risk of sample degradation
because heating is not required in the chromatographic
process.
• Readily automated.
 Limitations
• There is still a requirement for a reliable and
inexpensive detectors which can monitor
compounds that lack a chromophore.
• Drugs have to be extracted from their formulations
prior to analysis.
• Large amounts of organic solvent waste are
generated, which are expensive to dispose of.
73