HPLC type, principal, instrumentation,application

1. Seminar on
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY
Presented By
Mr. ROYAL DEB NATH
M pharmacy 1st year.
H.Tno.636218885006
Under guidance of
Dr.S.Y.Manjunath.Ph.D
DEPARTMENT OF PHARMACEUTICALANALYSIS
SRIKRUPA INISTITUTE OF PHARMACEUTICAL
SCIENCES
(Approved by AICTE;PCI)
(Affiliated to osmania university)
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2. CONTENT
1. INTRODUCTION.
2. PRINCIPLE.
3. TYPE OF HPLC.
4. INSTRUMENTATION.
5. APPLICATION.
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3.  HPLC stands for “High performance liquid
chromatography”(sometimes referred to as High pressure liquid
chromatography).
 HPLC is a chromatographic technique that can separate a mixture
of compounds
 It is used in biochemistry and analytical chemistry to identify,
quantify and purify the individual components of a mixture.
 Chromatography:
 physical method in which separation of components takes place
between two phases
 1.stationary phase 2. mobile phase
stationary phase : The substance on which adsorption of analyte
take place. It can be a solid, a gel, or a solid liquid combination.
Mobile phase: solvent which carries the analyte (a liquid or gas)
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4. • Liquid chromatography:
 It is a chromatographic technique in which mobile phase is a
liquid.
 The main principle involved in liquid chromatography is
adsorption
PRINCIPLE:
 When a mixture of components are introduced into the column
various chemical and/or physical interactions take place between
the sample molecules and the particles of the column packing .
 They travel according to their relative affinities towards the
stationary phase. The component which has more affinity towards
the adsorbent, travels slower.
 The component which has less affinity towards the stationary phase
travels faster.
 Since no two components have the same affinity towards the4

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6. TYPES OF HPLC
 Based on mode of separation
 Based on principle of separation
 Based on elution technique
 Based on scale of operation
 Based on type of analysis
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7. BASED ON MODE OF SEPARATION
1.Normal phase chromatography - stationary phase is
polar (hydrophilic) and mobile face is non-polar
(hydrophobic).
2.Reverse phase chromatography- stationary face is non-
polar (hydrophobic) and mobile face is Polar
(hydrophilic).
•Polar-Polar bonds and Non Polar-Non Polar bonds have
more affinity than Polar-Non Polar bonds.
•Reverse phase chromatography is more commonly used
as drugs are usually hydrophilic
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9. Based on principle of
separation
Adsorption chromatography:
 In which stationary phase is an adsorbent
 The compounds separated based on their affinity towards
stationary phase
 More affinity-slow elution
 less affinity-fast elution
Partition chromatography:
 A process of separation of solutes utilizing the
partition of the solutes between two liquid phases
 The original solvent and the film of solvent on the adsorption colu
mn.
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10. Size-Exclusion HPLC
In which the stationary phase is a gel having a closely
controlled pore size.
Molecules are separated based on molecular size and shape, and
mainly Agarose , Dextran used as mobile phase.
Ion-exchange chromatography :-
•Ion exchange chromatography is a process that allows the separation
of ions and polar molecules based on their charge.
•It can be used for almost any kind of charged molecule including large
proteins, small nucleotides and amino acids.
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11. BASED ON ELUTION
TECHNIQUE
During the chromatographic experiment, a pump can deliver a
constant mobile phase composition(isocratic) or an increasing
mobile phase composition (gradient).
Isocratic elution - Delivers constant mobile phase composition;
 solvent must be pre-mixed;
 lowest cost pump
 Best for simple preparation
Gradient elution - Deliveres variable mobile phase composition;
 In this mobile phase is programmed to change in composition
during elution time
 Best for complex preparations
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12. BASED ON SCALE OF OPERATION
Analytical HPLC:
 No recovery of individual components of substance
Preparative HPLC:
 Individual components of substance can be recovered
BASED ON TYPE OF ANALYSIS
Qualitative analysis:
 Determine the quality of sample
Quantitative analysis:
 Determine the quantity(concentration) of sample
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13. Solvent reservoir & Mixing system
High pressure pump
Sample injector
Column
Detectors
Data recording system
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14. HPLC
INSTRUMENTATION
OVER-VIEW
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15. 1.Solvent reservoir & Mixing unit
& degassing system :
 The appropriate solvents [mobile phase] from the reservoirs
are allowed to enter the mixing chamber where a homogenous
mixture is obtained.
 Several gases are soluble in organic solvents.
 When solvents are pumped under high pressure, gas
bubbles are formed which will interfere with the separation
process, steady base line and the shape of the peak.
 Hence degassing of solvent is important. This can be done by
 Vacuum filtration,
 Ultrasonication
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16. 2.High pressure pump:
 The role of the pump is to force a liquid (called the mobile
phase)through the liquid chromatograph at a specific flow
rate, expressed in milliliters per min (mL /min).
 Normal flow rates in HPLC are in the 1-to 2-mL/min range.
 Typical pumps can reach pressures in the range of 6000-
9000psi (400-to 600bar).
 There are several types of pumps used for HPLC most
commonly used are
1.Reciprocating piston pump,
2.syringe pump,
3.constant pressure pump.
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17. 3. Injector:
 The injector serves to introduce the liquid sample into the flow stream of
the mobile phase.
 Typical sample volumes are 5-to 20microliters(μL).
 The injector must also be able to withstand the high pressures of the
liquid system.
Types of injectors :
Manual injectors:
 User manually loads sample into the injector using a syringe. and then
turns the handle to inject sample into the flowing mobile
Auto sampler injector:
a. measures the appropriate sample volume,
b. injects the sample,
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18. MANUAL INJECTOR
AUTO SAMPLE INJECTORS
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19. 4. Column:
 The columns used for HPLC are generally made up of stainless
steel . so they can withstand up to high pressure [8000psi]
 Straight columns of 20-50 cm in length & 1-4mm in diameter are
used
 Particle size should be for porous particles 20-40μm. For porous
micro particles it should be 3-10 μm
• Stationary phases used in column:
 Alumina
 Silica
 Polyvinyl acetate beads
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20. TYPES OF COLUMNS IN HPLC:
 Guard Column
 Fast Column
 Preparative Column (lengths50-250mm)
 Capillary Column (various lengths)
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21. Guard column:
Guard columns, set between the injector and an analytical
column, are used to protect analytical columns from chemical
impurities in samples. We have two types of guard columns :
1. cartridge type,
2. packed type,
• Fast column:
 One of the primary reasons for using these column is to obtain
improved
• sample output ( amount of compound per unit time).
 Fast column are designed to decrease the time of chromatographic
analysis
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22. • Capillary column:
 They allow the user to work with nano liter sample volume ,
decreased flow rate and decreased solvent usage volume , led to
cost effectiveness
• Preparative column:
 It Used when objective is to prepare bulk ( milligrams) of sample
for
• laboratory preparatory application.
 It has usually a large column diameter , which is designed to
facilitate
• large volume injections into the HPLC system
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23. 5.DETECTORS:
 Uv-visible detectors
 Photo diode array detectors
 Refractive index detector
 Fluorescence detectors
 Conductivity detectors
 Mass spectrometer
 Evaporative light scattering detector
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24. 1. Uv-visible Detector:-
 This detector is based upon the light absorption characteristic of the
sample.
 Two types of this detector are available, one is the fixed wavelength
detector which operates at 254nm where most drug compounds absorb.
 The other is the variable wavelength detector which can be operated
from 190nm to 600nm.
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25. 2. Refractive index Detector:-
 This is a non specific or universal detector.
 This is not much used for analytical applications because of low
sensitivity and specificity.
3. Conductivity Detector:-
 Based upon electrical conductivity, the
response is recorded.
 This detector is used when the sample has
conducting ions like anions and cations.
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26. 4. Flourimetric Detector:-
 This detector is based on the fluorescent radiation emitted by some
class of compounds.
 The exitation wavelength and emission wavelength can be selected for
each compound.
 This detector has more specificity and sensitivity.
 The disadvantage is that some compounds are not fluorescent.
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27. HPLC ADVANTAGES:
 Speed(minutes)
 High resolution
 Sensitivity
 Accuracy
 Automation
HPLC DISADVANTAGES
 co-elution
 Cost
 Complexity
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28. APPLICATIONS :
 Purification of samples
 Identification of compounds
 Determination of impurities
 Separation of mixture of samples
ex: carbohydrates
 Determine the concentration of drug
 Biopharmaceutical &pharmacokinetic studies
 Drug stability studies
 Qualitative & quantitative analysis
 Environmental applications[analysing air & water pollutants]
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29. QUANTITATIVE
ANAYSIS:
 To measure the concentration of each compound in a sample
 There are 2 main ways to interpret a chromatogram.
 Determination of the peak height of a chromatographic peak as
measured from the baseline
 Detection of the peak area
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30. QUALITATIVE ANALYSIS:
 The identification of individual compounds in the sample;
 The most common parameter for compound identification is its
retention time [is the amount of time a compound spends on the
column after injection]
 Depending on the detector used, compound identification is also
based on the chemical structure, molecular weight or some other
molecular parameter.
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31. REFERENCES:
 Principles of instrumental analysis- Doglas A skoog
 Instrumental methods of chemical analysis-
Gurudeep R.chatwal, sham K.anand
 Textbook of chemical analysis-
Francis Rouessac and Annick Rouessac 2nd edition john
wiley & sons ltd
 Text book of modern analytical chemistry-
David harvey
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