History of ChromatographyHistory of Chromatography Early LC carried out in glass columnsEarly LC carried out in glass columns

diameters: 1-5 cmdiameters: 1-5 cm lengths: 50-500 cmlengths: 50-500 cm

Size of solid stationary phaseSize of solid stationary phase diameters: 150-200 diameters: 150-200 mm

Due to this, the flow rates were low and the separation times Due to this, the flow rates were low and the separation times long.long.

Decrease particle size of packing causes increase in column Decrease particle size of packing causes increase in column efficiency.efficiency. diameters 3-10 diameters 3-10 mm

This technology required sophisticated instrumentsThis technology required sophisticated instruments new method called HPLCnew method called HPLC

Chromatography by M. TswettChromatography by M. Tswett

ether

CaCO3

chlorophyll chlorophyll

ChromatoChromatography

colourscolours

HPLC

originally referred to:High Pressure Liquid

Chromatography high pressure to be able to use small high pressure to be able to use small

particle size to allow proper separation at particle size to allow proper separation at reasonable flow ratesreasonable flow rates

HPLC

Laterly referred to:High Performance Liquid

Chromatography high performance due to its reproducibilityhigh performance due to its reproducibility

currently refers to:High Precision Liquid

Chromatography

IntroductionIntroduction

Compounds are separated by injecting Compounds are separated by injecting a sample mixture into the column. The a sample mixture into the column. The different component in the mixture pass different component in the mixture pass through the column at different rates through the column at different rates due to differences in their partition due to differences in their partition behavior between the mobile phase and behavior between the mobile phase and the stationary phase. the stationary phase.

What is HPLC?What is HPLC? The most widely used analytical separations The most widely used analytical separations

techniquetechnique Utilizes a liquid mobile phase to separate Utilizes a liquid mobile phase to separate

components of mixturecomponents of mixture Uses high pressure to push solvent through Uses high pressure to push solvent through

the columnthe column PopularityPopularity: :

sensitivitysensitivity ready adaptability to accurate quantitative ready adaptability to accurate quantitative

determinationdetermination suitability for separating nonvolatile species or suitability for separating nonvolatile species or

thermally fragile onesthermally fragile ones

Principle of HPLC:Principle of HPLC:

The principle of HPLC are based on Van Deemter equation The principle of HPLC are based on Van Deemter equation which relates the efficiency of the chromatographic which relates the efficiency of the chromatographic column to the particle size of the column, molecular column to the particle size of the column, molecular diffusion and thickness of stationary phase.diffusion and thickness of stationary phase.The Van Deemter Equation is given asThe Van Deemter Equation is given as

H or HETP = A + H or HETP = A + B B + C + C υυ υυ

where, A represents eddy diffusionwhere, A represents eddy diffusion

B represents molecular diffusionB represents molecular diffusion C represents rate of mass transferC represents rate of mass transfer υυ represents flow rate represents flow rate

Advantages to HPLCAdvantages to HPLC Higher resolution and speed of analysisHigher resolution and speed of analysis HPLC columns can be reused without repacking HPLC columns can be reused without repacking

or regenerationor regeneration Greater reproducibility due to close control of the Greater reproducibility due to close control of the

parameters affecting the efficiency of separationparameters affecting the efficiency of separation Easy automation of instrument operation and Easy automation of instrument operation and

data analysisdata analysis Adaptability to large-scale, preparative Adaptability to large-scale, preparative

proceduresprocedures

Components Of A Liquid Chromatograph System

Mobile Phase / Solvent ReservoirMobile Phase / Solvent Reservoir DegasserDegasser Solvent Delivery System (Pump)Solvent Delivery System (Pump) InjectorInjector PrecolumnPrecolumn ColumnColumn Temperature ControlTemperature Control Detectors Detectors Recorder (Data Collection)Recorder (Data Collection)

HPLC Basic Instrumentation

Mobile phase

Pump

Solvent Delivery

Injector

Sample Injection

Column

Separation

Detector

Data Processor

1. HPLC

HPLC systemHPLC system

HPLC SolventReservoirs

SolventDelivery System

VariableUV/Vis Detector

RheodyneInjector

HPLCColumn

ComputerWorkstation

HPLC system

Solvent ReservoirSolvent Reservoir

DegasserDegasser

Solvent Delivery System Solvent Delivery System (Pump)(Pump)

InjectorInjector

Column &ovenColumn &oven

Detectors Detectors Recorder (Data Collection)Recorder (Data Collection)

Components

Solvent Reservoir Usually one or more glass or stainless steel Usually one or more glass or stainless steel

reservoirs each of which contains 200-1000 ml of reservoirs each of which contains 200-1000 ml of

solventsolvent Isocratic elution - single solvent separation Isocratic elution - single solvent separation

techniquetechnique Gradient elution - 2 or more solvents, varied Gradient elution - 2 or more solvents, varied

during separationduring separation

Degasser Problems caused by dissolved air(OProblems caused by dissolved air(O22, N, N22)in mobile phase)in mobile phase

Unstable delivery in pumpUnstable delivery in pump Bigger noise and large baseline-drift in detector cellBigger noise and large baseline-drift in detector cell

In order to avoid causing the problems,In order to avoid causing the problems, mobile phase should be degassedmobile phase should be degassed..

vacuum pumping systemsvacuum pumping systems distillation systemdistillation system a system for heating and stirring the solventsa system for heating and stirring the solvents sparging system - bubbles an inert gas of low solubility sparging system - bubbles an inert gas of low solubility

through the solvent through the solvent

Solvent Delivery SystemThree basic types of LC Pumps

used are: pneumatic pumpspneumatic pumps

motor driven syringe type pumpsmotor driven syringe type pumps

reciprocating pumpsreciprocating pumps

Injectors For injecting the solvent through the columnFor injecting the solvent through the column Minimize possible flow disturbances Minimize possible flow disturbances Limiting factor in precision of liquid chromatographic Limiting factor in precision of liquid chromatographic

measurementmeasurement Volumes must be smallVolumes must be small .1-500 .1-500 LL Sampling loopsSampling loops

interchangeable loops (5-500 interchangeable loops (5-500 LL at pressures up to 7000 psi) at pressures up to 7000 psi)

ColumnColumn

straight, 15 to 150 straight, 15 to 150 cm in length; 2 to 3 cm in length; 2 to 3 mm i.d.mm i.d.

packing - silica gel, packing - silica gel, alumina, Celitealumina, Celite

Detector

Mostly opticalMostly optical Equipped with a flow cellEquipped with a flow cell Focus light beam at the center Focus light beam at the center

for maximum energy for maximum energy transmissiontransmission

Cell ensures that the separated Cell ensures that the separated bands do not widenbands do not widen

Types of Detector

UV/Visible UV/Visible - Fixed wavelengthFixed wavelength- variable wavelength- variable wavelength

Photo Diode ArrayPhoto Diode Array Refractive indexRefractive index FluorescenceFluorescence Evaporative light scatteringEvaporative light scattering ConductivityConductivity ElectrochemicalElectrochemical