high performance liquid chromatography

1. HIGH PERFORMANCE LIQUIDHIGH PERFORMANCE LIQUID
CHROMATOGRAPHYCHROMATOGRAPHY
BYBY
Dr. SHANTHA ARCOTDr. SHANTHA ARCOT

2. CHROMATOGRAPHYCHROMATOGRAPHY
ChromatographyChromatography is the term used to describe ais the term used to describe a
separation techniqueseparation technique in which a mobile phasein which a mobile phase
carrying a mixture is caused to move in contactcarrying a mixture is caused to move in contact
with awith a selectively absorbent stationary phase.selectively absorbent stationary phase.

3. TYPES OF CHROMATOGRAPHYTYPES OF CHROMATOGRAPHY
 HPLC - Normal PhaseHPLC - Normal Phase
-Reverse Phase-Reverse Phase
 Size exclusion chromatographySize exclusion chromatography
 Super critical fluid chromatographySuper critical fluid chromatography
 Ion chromatographyIon chromatography
 Counter current chromatographyCounter current chromatography
 Capillary electrophoresisCapillary electrophoresis
 Gas chromatographyGas chromatography

4. WHAT DOES HPLC MEAN ?WHAT DOES HPLC MEAN ?
 High Pressure Liquid ChromatographyHigh Pressure Liquid Chromatography
 High Price Liquid ChromatographyHigh Price Liquid Chromatography
 High Performance Liquid ChromatographyHigh Performance Liquid Chromatography
 High Patience Liquid ChromatographyHigh Patience Liquid Chromatography
 Hewlett – Packard Liquid ChromatographyHewlett – Packard Liquid Chromatography

5. CHROMATOGRAPHIC PROCESSCHROMATOGRAPHIC PROCESS
 Separation of theSeparation of the
components A and Bcomponents A and B
depends on their relativedepends on their relative
affinity towards stationaryaffinity towards stationary
phase.phase.
 Component A – lessComponent A – less
affinity towards SP, elutedaffinity towards SP, eluted
first.first.
 Component B – moreComponent B – more
affinity towards SP , elutedaffinity towards SP , eluted
later.later.

6. MODES OF CHROMATOGRAPHYMODES OF CHROMATOGRAPHY
1.1. Normal Phase ModeNormal Phase Mode ::
 More polar analytes retainedMore polar analytes retained
longer.longer.
 Structural isomers are oftenStructural isomers are often
separated.separated.
 Analytes with larger number ofAnalytes with larger number of
polar functional groups arepolar functional groups are
retained longer.retained longer.

7. MODES OF CHROMATOGRAPHYMODES OF CHROMATOGRAPHY
Reverse Phase mode :Reverse Phase mode :
 More polar analytesMore polar analytes
retained less.retained less.
 Analytes with largeAnalytes with large
hydrophobic part arehydrophobic part are
retained longer.retained longer.
 Almost no separation ofAlmost no separation of
structural isomers.structural isomers.

8. MOBILE PHASEMOBILE PHASE
 Acetonitrile, dioxane ,ethanol, methanol,Acetonitrile, dioxane ,ethanol, methanol,
isopropanol, THF, water, etc are some of theisopropanol, THF, water, etc are some of the
mobile phases used in RP-HPLC.mobile phases used in RP-HPLC.
 The liquid mobile phases are used to adjust theThe liquid mobile phases are used to adjust the
chromatographic separation and retention inchromatographic separation and retention in
liquid chromatography .liquid chromatography .
 The selection of mobile phase for a particularThe selection of mobile phase for a particular
drug is based on its retention timedrug is based on its retention time

9. INSTRUMENTATIONINSTRUMENTATION
Pump
Injector
Column
Detector
Mobile Phases
Gradient
Controller
•

10. PUMPPUMP
 To provide a continuous constant flow of theTo provide a continuous constant flow of the
eluent through the HPLC injector, column, andeluent through the HPLC injector, column, and
detectordetector
 It should generate a pressure up to 6000psiIt should generate a pressure up to 6000psi
 Flow rate ranging from 0.1 to 10 ml/ minFlow rate ranging from 0.1 to 10 ml/ min
 It should be Corrosion resistant.It should be Corrosion resistant.

11. TYPES OF PUMPSTYPES OF PUMPS
 Reciprocating PistonReciprocating Piston
PumpsPumps
 Dual Piston PumpsDual Piston Pumps
 Syringe-Type PumpsSyringe-Type Pumps

12. INJECTORSINJECTORS
 Injectors for liquidInjectors for liquid
chromatographic systemschromatographic systems
should provide theshould provide the
possibility of injecting thepossibility of injecting the
liquid sample within theliquid sample within the
range of 0.1 to 100 ml ofrange of 0.1 to 100 ml of
volume with highvolume with high
reproducibility and underreproducibility and under
high pressure (up to thehigh pressure (up to the
4000 psi)4000 psi)

13. RHEODYNE INJECTORRHEODYNE INJECTOR
 A syringe is used to injectA syringe is used to inject
sample in to loopsample in to loop
connected between twoconnected between two
ports of the multiport valve.ports of the multiport valve.
 When the valve is switchedWhen the valve is switched
fromfrom load to injectload to inject
positionposition, mobile phase is, mobile phase is
directed through loopdirected through loop
carrying sample in to thecarrying sample in to the
head of the column.head of the column.

14. COLUMNCOLUMN
 Most commonly used 4.6mm x 260 mm.Most commonly used 4.6mm x 260 mm.
 many variations possible:many variations possible:
- packing material – silicon dioxide , RP-8,RP- 18, CN,- packing material – silicon dioxide , RP-8,RP- 18, CN,
Phenyl..Phenyl..
- different particle size. The smaller the better- different particle size. The smaller the better
however the larger the pressure. Typical 5µmhowever the larger the pressure. Typical 5µm
particle size.particle size.

15. NORMAL PHASE MATERIALNORMAL PHASE MATERIAL
 30% silica gel30% silica gel
 47% silica based bonded phases.47% silica based bonded phases.
- diols- diols
-amine-amine
- cyano- cyano
 3% alumina3% alumina
 20% chiral bonded phase20% chiral bonded phase

16. REVERSE PHASE MATERIALREVERSE PHASE MATERIAL
 80% Octadecylsilica (ODS C18)80% Octadecylsilica (ODS C18)
 10% octylsilica ( C8 )10% octylsilica ( C8 )
 5% butylsilica (C4 )5% butylsilica (C4 )
 3% phenyl3% phenyl
 2% cyano (CN)2% cyano (CN)

18. GUARD COLUMNGUARD COLUMN
 Before the sample enter into the column theyBefore the sample enter into the column they
reaches thereaches the guard columnguard column in order to preventin order to prevent
the contamination of the main column.the contamination of the main column.
 Guard column has the same packing material asGuard column has the same packing material as
in main column .But differs in the particlein main column .But differs in the particle
size(10size(10µm).µm).

19. DETECTORDETECTOR
 A detector translates concentration changes in theA detector translates concentration changes in the
HPLC column effluent in to electrical signals.HPLC column effluent in to electrical signals.
 A detector must be applicable to wide range ofA detector must be applicable to wide range of
samples.samples.
 Its response should be unaffected by temperature,Its response should be unaffected by temperature,
flow rate or characteristics of mobile phase.flow rate or characteristics of mobile phase.
 It should be highly sensitive to small concentration.It should be highly sensitive to small concentration.

20. TYPES OF DETECTORSTYPES OF DETECTORS
 The Refractive Index DetectorThe Refractive Index Detector
 Deflection detectorsDeflection detectors
 Ultraviolet/Visible Spectroscopic DetectorsUltraviolet/Visible Spectroscopic Detectors
 Diode-array detectorsDiode-array detectors
 Fluorescence detectorsFluorescence detectors
 Electrochemical DetectorsElectrochemical Detectors
 Electrolytic Conductivity DetectorElectrolytic Conductivity Detector

21. PARAMETERS USED IN HPLCPARAMETERS USED IN HPLC
 Retention timeRetention time
 Retention volumeRetention volume
 Separation factorSeparation factor
 ResolutionResolution
 Theoretical plateTheoretical plate
-HETP (height equivalent to theoretical plate)-HETP (height equivalent to theoretical plate)
-Efficiency-Efficiency
 Asymmetry factorAsymmetry factor

22. RETENTION TIMERETENTION TIME
 It is the difference in timeIt is the difference in time
between the point ofbetween the point of
injection and appearanceinjection and appearance
of peak maxima.of peak maxima.
 Retention time,Retention time, tRtR isis
inversely proportional toinversely proportional to
the eluent flow rate.the eluent flow rate.

23. RETENTION VOLUMERETENTION VOLUME
 Retention volume,Retention volume, VRVR represent the volume of the eluentrepresent the volume of the eluent
passed through the column while eluting a particularpassed through the column while eluting a particular
component.component.
 Component retention volumeComponent retention volume VRVR could be split into twocould be split into two
parts:parts:
 Reduced retention volumeReduced retention volume is the volume of the eluent thatis the volume of the eluent that
passed through the column while the component was sittingpassed through the column while the component was sitting
on the surface.on the surface.
 Dead volumeDead volume is the volume of the eluent that passedis the volume of the eluent that passed
through the column while the component was moving withthrough the column while the component was moving with
the liquid phase.the liquid phase.

24. SEPARATION FACTORSEPARATION FACTOR
 It’s a ratio of adsorption co-It’s a ratio of adsorption co-
efficient of the two componentsefficient of the two components
to be separatedto be separated
Kb (tb - to)Kb (tb - to)
S = -------- =S = -------- =
------------------------
Ka (ta - to)Ka (ta - to)
Where,Where,
to = retention time ofto = retention time of
unretained substanceunretained substance
Kb, Ka = adsorption co-efficientKb, Ka = adsorption co-efficient
of b and aof b and a
ta, tb = retention time ofta, tb = retention time of
substance of b and a.substance of b and a.

25. RESOLUTIONRESOLUTION
 Resolution is the parameterResolution is the parameter
describing the separationdescribing the separation
power of the completepower of the complete
chromatographic systemchromatographic system
relative to the particularrelative to the particular
components of the mixture.components of the mixture.
 By convention, resolution (By convention, resolution (RR) is) is
expressed as the ratio of theexpressed as the ratio of the
distance between two peakdistance between two peak
maxima to the mean value ofmaxima to the mean value of
the peak width at the base line:the peak width at the base line:
2
(Rt1 – Rt2 )
Rs =
-----------------------
W1 + W2

26. THEORETICAL PLATESTHEORETICAL PLATES
 A theoretical plate is anA theoretical plate is an
imaginary or hypothetical unitimaginary or hypothetical unit
of a column where distributionof a column where distribution
of solute between a stationaryof solute between a stationary
phase and a mobile phase hasphase and a mobile phase has
attained equilibrium.attained equilibrium.
 Efficiency ( no. of theoreticalEfficiency ( no. of theoretical
plates)plates)
Rt2Rt2
n = 16 -------n = 16 -------
W2W2

27. HEIGHT EQUIVALENT TOHEIGHT EQUIVALENT TO
THEORETIACL PLATES (THEORETIACL PLATES (HETPHETP))
 HETP is given by Van DeemterHETP is given by Van Deemter
equationequation
A BA B
H = ---------------+ -------------+CsU + CmUH = ---------------+ -------------+CsU + CmU1/21/2
1+Cm / U1+Cm / U1/21/2
UU
H = column efficiency, smaller the term theH = column efficiency, smaller the term the
more efficient the column.more efficient the column.
A = eddy diffusionA = eddy diffusion
B = rate of diffusion of moleculeB = rate of diffusion of molecule
C= mass transferC= mass transfer

28. ASYMMETRY FACTORASYMMETRY FACTOR
 Chromatographic peakChromatographic peak
should be symmetricalshould be symmetrical
about its centre andabout its centre and
said to followsaid to follow
GAUSSIANGAUSSIAN
distributiondistribution..

29. ASYMMETRY FACTORASYMMETRY FACTOR
 FrontingFronting
Fronting is due to theFronting is due to the
saturation of stationarysaturation of stationary
phase and can be avoidedphase and can be avoided
by using less quantity ofby using less quantity of
sample.sample.
TailingTailing
It is due to the moreIt is due to the more
active adsorption sites.active adsorption sites.

30. WHAT CAUSES TAILING ?WHAT CAUSES TAILING ?
 HydrophobicHydrophobic
interactions withinteractions with
bonded phase.bonded phase.
 Ion-exchangeIon-exchange
interaction withinteraction with
charged sites.charged sites.

31. APPLICATIONSAPPLICATIONS
 Qualitative analysisQualitative analysis
 Checking the purity of the compound.Checking the purity of the compound.
 Presence of impurityPresence of impurity
 Quantative analysis:Quantative analysis:
1. Direct comparison method1. Direct comparison method
2. Calibration curve method.2. Calibration curve method.
3. Internal standard method.3. Internal standard method.

33. THANK YOUTHANK YOU