DEPARTMENT OF PHARMACEUTICAL

CHEMISTRY

HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY [HPLC]ERFORMANCE
LIQUID
CHROMATOGRAPHY [HPLC]

Mr. TUSHAR BHATKAR

[Department of Pharmacy (CHEMISTRY)]
Dr. Rajendra Gode Institute of Pharmacy, Amravati
[email protected]

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 CHROMATOGRAPHY
 It is define as, it is analytical method in which separation of active components in
complex mixture, and the mixture was distributed in two phases i.e. stationary
phase and mobile phase is known as chromatography.
 It is technique is used for separation, purification,
Identification and extraction of compound.
 It is method it can consist of two phases stationary phase and
mobile
phase.
 Stationary phase is constant phase or column packaging material.
 Mobile phase is moveable phase.
 The basic principle of chromatography is based on
Adsorption and partition chromatography.
 Adsorption chromatography - The affinity of molecules towards
stationary
phase is known as Adsorption chromatography.
 Partition chromatography - The molecule can moves in two phases of
liquid is known as partition chromatography.
 It is important for qualitative and quantitative analysis.
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 TYPES OF CHROMATOGRAPHY
 Based on modes of chromatography
1. Normal phase mode
2.Reverse phase mode
 Based on principle of separation
1. Adsorption chromatography
2. Ion exchange chromatography
3. Partition chromatography
4. Size exclusion
 Based on elution technique
1. Isocratic separation
2. Gradient separation
 Based on the scale of operation
1. Analytical HPLC
2. Preparative HPLC
 Based on the type of analysis
1. Qualitative analysis
2. Quantitative analysis

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 HPLC
 HPLC is a High Performance liquid Chromatography.
 High Pressure Liquid Chromatography.
 High Priced Liquid Chromatography.
 It is column chromatography.
 It is Liquid Chromatography.
 It is modified from of gas chromatography, it is applicable for both Volatile as
well as Non volatile compound.
 It can mainly divided by two types 1. Normal phase HPLC 2. Reversed Phase
HPLC.
 It is having a high resolution and separation capacity.
 It is used as qualitative as well as quantitative analysis.
 High performance liquid chromatography (HPLC) is a chromatographic
technique used to separate a mixture of compounds in analytical chemistry and
biochemistry with the purpose of identifying, quantifying or purifying the
individual components of the mixture. 4

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 PRINCIPLE
 High Performance Liquid Chromatography [HPLC] is principle is based on
adsorption as well as partition chromatography is depending on the
nature of stationary phase, if stationary phase is solid principle is based on
adsorption chromatography and if stationary phase is liquid principle is based on
partition chromatography.

 It is important for determination of volatile and non volatile compounds.

 It is important for determination qualitative and quantitative analysis.

 It is important for determination of Retention Time (the time is required , after

sample injection maximum angle peak reaches to detector)

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 ADVANTAGES
 It is simple, rapid , reproducible.
 High sensitivity.
 High performance.
 Rapid process and hence time saving.
 It is having a high resolution and separation capacity.
 Accuracy and Precision.
 Stationary phase was chemically innert.
 Wide varities of stationary phase.
 Mobile phase was chemically innert.
 Less requirement of mobile phase in developing chamber.
 Early recovery of separated component.
 Easy visualization of separated components.
 It is having Good reproducibility and repeatability.
 It is analytical technique is important for validation of product, quality
control studies of product.
 It is important for qualitative and quantitative analysis.
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 It is used for both analytical and preparative purpose.

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 TYPES OF HPLC SEPARATIONS
 Normal Phase: Separation of polar analytes by partitioning onto a polar, bonded
stationary phase.

 Reversed Phase: Separation of non-polar analytes by partitioning onto a

non-polar, bonded stationary phase.

 Adsorption: In Between Normal and Reversed. Separation of moderately polar

analytes using adsorption onto a pure stationary phase (e.g. alumina or silica)

 Ion Chromatography: Separation of organic and inorganic ions by their

partitioning onto ionic stationary phases bonded to a solid support.

 Size Exclusion Chromatography: Separation of large molecules based in the

paths they take through a “maze” of tunnels in the stationary phase.

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 BLOCK DIAGRAM OF HPLC
Reservoir
Reservoir Recorder

Pump Gradient Pump

controller
Fraction Detector

collector
Mixing Analytical
chamber column

Solvent Injector
Conditioning Precolumn
column

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 WHY USE HPLC
 Simultaneous analysis
 Identification

 Seperation

 Purification

 High resolution
 High sensitivity

 Good repeatability

 Moderate analysis condition

 Easy to fractionate and purify

 Not destructive

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 INSTRUMENTATION OF HPLC
 Mobile phase storage bottle
 Gradient controller and mixing unit

 De-gassing of solvents

 Pump

 Pressure gauge

 Pre-column

 Sample introduction system

 Column

 Detector

 Recorder

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 OUTLINE OF LC-2010

Reservior Tray

System
Auto sampler Controller

Column Oven
UV detector
Degassing Unit
Pump Unit

Low pressure 23
gradient device
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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.

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 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 sparing with helium gas)
 The mobile phase are filtered to remove particulate matter that may
clog the
System

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 PUMP
 The solvents or mobile phase must be passed through a column
at high pressures at up to 400-600 bar.

 As the particle size of stationary phase is smaller (5 to 10µ) the resistance

to the flow of solvent will be high.
 That is, smaller the particle size of the stationary phase the greater
is the resistance to the flow of solvents.
 Hence high pressure is recommended.

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 REQUIREMENTS FOR PUMPS

 Generation of pressure of about 6000 psi.

 Pulse free output & all materials in the pump should be chemically
resistant to solvents.
 Flow rates ranging from 0.1 to 10 mL/min
 Pumps should be capable of taking the solvent from a single
reservoir or more than one reservoir containing different solvents
simultaneously.

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 TYPES OF
PUMPS USED
IN HPLC

DISPLACEMENT RECIPROCATING PNEUMATIC

PUMPS PUMPS PUMPS

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 HPLC Pump

Manner in which they operate

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

syringe Direct pressure pump Amplifier pump

(Displace Reciprocating pump
ment
pump)

Single
Reciprocating
piston Double piston diaphragm
reciprocati reciprocating pump
ng pump pump

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 DISPLACEMENT PUMPS

⚫ It consists of large, syringe like chambers equipped with a

plunger activated by a screw driven mechanism powered by
a stepping motor.
⚫ So it is also called as Screw Driven Syringe Type Pump.
⚫ Advantages:- It produces a flow that tends to be independent
of viscosity & back pressure.
⚫ Disadvantages:- It has a limited solvent
capacity(~250) &
considerably inconvenient when solvents must be changed.

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 RECIPROCATING PUMPS

 This pump transmits alternative pressure to the solvent via a

flexible diaphragm ,which in turn is hydraulically pumped
by a reciprocating pump.
 Disadvantages

Produces a pulsed flow which is damped because pulses

appear as baseline noise on the chromatograph.

This can be overcome by use of dual pump heads or

elliptical
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cams to minimize such pulsations.
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 out

pump head check valve

motor and cam

5 - 50µL

plunger
check valve
plunger seal

in
Mobile phase

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 Solvent is pumped back and forth by a motor driven piston
 Two ball check valves which open & close which controls the
flow
 The piston is in direct contact with the solvent
 Small internal volume 35-400μL
 High output pressure up to 10,000 psi
 Ready adaptability to gradient elution and constant flow rate

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 Advantages
 Have small internal volume of 35-400µL
 Higher output pressures up to 10,000 psi.
 Adaptability to gradient elution.
 Large solvent capacities & constant flow rates.
 Largely independent of column back pressure & solvent
viscosity.

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 PNEUMATIC PUMPS

 In this pumps, the mobile phase is driven through the column with the use
of pressure produced from a gas cylinder.
 It has limited capacity of solvent
 Due to solvent viscosity back pressure may develop.

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 SAMPLE INJECTOR SYSTEM

 Several injector devices are available either for manual or

auto injection of the sample.

(i) Septum Injector

(ii) Stop Flow Injector

(iii) Rheodyne Injector

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 I. SEPTUM INJECTOR
⚫ These are used for injecting the sample through a rubber
septum.
⚫ This kind of injectors cannot be commonly used , since the
septum has to withstand high pressures.

ii. Stop
Flow

 In this type the flow of mobile phase is stopped for a while &
the
sample is injected through a valve.

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 III. RHEODYNE INJECTOR

 It is the most popular injector and is widely used.

 This has a fixed volume of loop, for holding sample until its injected into
the column, like 20µL, 50µL or more.
 Through an injector the sample is introduced into the column.
 The injector is positioned just before the inlet of the column.

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SELECTION VALVE
BY USING THE SELECTION VALVE
WE CAN SELECT WHETHER THE
PURPOSE IS FOR ANALYTICAL
PURPOSE OR PREPARATIVE
PURPOSE.

LOAD POSITION
In this position the sample is
loaded into the sample
loop .

INJECT POSITION
In this position the loaded
sample is injected into the
column by the forceful flow
of the solvent into the sample
loop by which the sample is
introduced into the column.
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HPLC AUTO INJECTORS

Inside of SIL-20AC
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 COLUMN
1. Precolumn

1. It contains a packing chemically identical to that in analytical column.

2.Mainly used to remove the impurities from the solvent and thus prevents contamination of the analytical
column, it can protect analytical column.
3. It is also called as guard column or protective column.
4. it is having large particle size.
5. It is having short length of 2 to 10 cm, so does not affect separation.

2. Analytical column
1. The success or failure of analysis depends upon choice of column.
2. Actual separation is carried out here.
3. Stainless –steel tube
4. size – length -25 to 100 cm
5. Internal diameter – 2 to 4.6 mm
6. Column is filled with small particles 5 – 10 micron. The solid support can be silica gel, alumina.
7. 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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8.C8 and C18 columns are considered as examples of reversed phase
liquid chromatography (RP).
9.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).
10.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.
11. The difference between the two columns will be in the length of
the carbon-
chain attached to the silica surface.
12.Accordingly C8 hplc columns have packing material composed of
silica particles attached to C8 carbon units.
13. C18 will, of course, have packing materials coated with C18
hydrophobic
units.
14.Categorically both are reversed phase but C18 columns will
definitely be more "hydrophobic rather than the C8 columns.
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 DETECTORS
 Absorbance (UV/Vis and PDA)
 Refractive index (detects the change in turbidity)
 Fluorescence (if the analyte is fluorescent)
 Electrochemical (measures current flowing through a pair of
electrodes, on which a potential difference is imposed, due to
oxidation or reduction of solute)
 Conductivity (for ions)
 Light scattering
 Mass spectrometry (LC-MS)

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TYPES OF HPLC DETECTORS
Name Advantage Disadvantage
UV-Vis Works w/all molecules Non-specific; complex
samples; absorption
wavelength

DAD Works for all wavelengths High LOD

Fluorescence Very specific; low LOD Not everything fluoresces

IR Works w/all molecules Many solvents IR active

Refractive Index Works w/nearly all Temperature sensitive;

molecules high LOD

Scattering Uniform response; Non-specific; interference

5ng/25mL LOD from solvent

Electrochemical Commercially available Non-specific; high LOD

Mass Spec Low LOD; analyte Ability to ionize analyte

identification 49

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 IDEAL DETECTOR PROPERTIES
 High Sensitivity
 Universality or predictable specificity
 Large linear response range
 Low dead volume
 Non-Destructive
 Insensitive to temperature & mobile phase
 Continuous operation
 Reliable and easy to use
 No single detector fits all these criteria

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 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
systems.
 These are classified as fixed or variable wavelength detectors.
 Fixed wavelength detectors employ filter as a source to provide
appropriate wavelength.
 Most common fixed wavelength detectors are based on 254 nm.
 Variable wavelength detectors are employ a spectrophotometer to
provide
dispersion of light and selection of any wavelength in UV visible regions.
 Diffraction gratings are frequently used for wavelength dispersion.

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 PHOTODIODE
ARRAY (PDA)
 A photodiode array (PDA) is a linear array of discrete photodiodes on an
integrated circuit (IC) chip.
 Allows a range of wavelengths to be detected simultaneously. In this regard it can
be thought of as an electronic version of photographic film. Array detectors are
especially useful for recording the full Uv- vis is a absorption spectra of samples
that are rapidly passing through a sample flow cell, such as in an HPLC detector.
 PDAs work on the same principle as simple. Photovoltaic detector
 similar to UV detector, non destructive 190-600 nm for quantization &
identification Spectra is 3D, Response vs time vs WL.

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 RECORDERS AND INTEGRATORS
 Recorders are used to record responses obtained from the detectors after
amplification, if necessary.
 They record the baseline & all the peaks obtained with respect tot time.
 Retention time can be found out from this recordings, but area under curve
cannot be
determined.
 The Integrators are improved versions of recorders with some data
processing capabilities.
 They can record the individual peaks with retention time, height, width of
peaks, peak area, percentage area, etc.
 Integrators provides more information on peaks than recorders.
 In recent days computers and printers are used for recording and processing the
obtained data & for controlling several operations.

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 APPLICATION
 Drug Discovery
 Seperation
 Identification
 Purification
 Clinical Analysis
 Proteomics
 Forensic Chemistry
 Drug Metabolism study
 Environmental chemistry
 Diagnostic studies
 Cosmetic analysis
 Determination of Green Florescent Protein
 Structural Determination
 Pharmaceutical Applications
 Identification of Bile Acid Metabolite
 Clinical Applications
 qualitative and quantitative analysis
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 Therapeutic Drug Monitoring
 Biochemical Genetics
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 REFERENCE
 Knox JH, Done JN, Fell AF et al. High-Performance Liquid
Chromatography. Edinburgh: Edinburgh University Press; 1978.
 Simpson CF. Practical High-Performance Liquid Chromatography. London: Heyden
and Son; 1976.
 Pungor E. A Practical Guide to Instrumental Analysis. Boca Raton: CRC Press;
1995.
 Moffat AC, Osselton MD, Widdop B. Clarke’s Analysis of Drugs and Poisons.
London: Pharmace utical Press; 2004.
 Y. Shen and R.D. Smith, Electrophoresis 23 (2002) pp. 3106-3124.
 D.A. Skoog and D.M. West, Fundamentals of Analytical Chemistry, 3rd edit., pp.
643-649.
 D. Sievers, M.E. Swartz and B.J. Murphy, G.I.T. Laboratory Journal (2004)
pp.43-45.(2004) pp.
 503-504
 L.A. Colon, J.M. Cintron, J.A. Anspach, A.M. Fermier and K.A. Swinney, The
Analyst 129
 L.R. Snyder, J.J. Kirkland and J.L. Glajch, Practical HPLC Method development,
2nd edit., pp. 41- 43.
 J.C. Giddings, Dynamics of Chromatography: Part I Principles and Theory 64
(Marcel Dekker, Inc., New York: 1965) pp. 25-26, 229-230.
 J.M. Cintron and L.A. Colon, The Analyst 127 (2002) pp. 701-704. A.D.
Jerkovich, J.S. Mellors 47
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