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Basic Principles of HPLC : 

Basic Principles of HPLC Mr. Pravin M. Botre. QMS Dept.


HPLC Introduction & history lesson Classification of chromatography Types of liquid chromatography Components of HPLC Types of Detector Evaluation parameters

History lesson : 

History lesson Early LC carried out in glass columns diameters: 1-5 cm lengths: 50-500 cm Size of solid stationary phase diameters: 150-200 m Flow rates still low! Separation times long! Eureka! Decrease particle size of packing causes increase in column efficiency! diameters 3-10 m This technology required sophisticated instruments new method called HPLC

Chromatography by M. Tswett : 

Chromatography by M. Tswett ether CaCO3 chlorophyll Chromatography colours


HPLC originally referred to:High Pressure Liquid Chromatography high pressure to be able to use small particle size to allow proper separation at reasonable flow rates


HPLC originally referred to: High Pressure Liquid Chromatography high pressure to be able to use small particle size to allow proper separation at reasonable flow rates


HPLC Laterly referred to: High Performance Liquid Chromatography high performance due to its reproducibility currently refers to: High Precision Liquid Chromatography

Introduction : 

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

What is HPLC? : 

What is HPLC? The most widely used analytical separations technique Utilizes a liquid mobile phase to separate components of mixture uses high pressure to push solvent through the column Popularity: sensitivity ready adaptability to accurate quantitative determination suitability for separating nonvolatile species or thermally fragile ones

Principle of HPLC: : 

Principle of HPLC: The principle of HPLC are based on Van Deemter equation which relates the efficiency of the chromatographic column to the particle size of the column, molecular diffusion and thickness of stationary phase. The Van Deemter Equation is given as H or HETP = A + B + C υ υ where, A represents eddy diffusion B represents molecular diffusion C represents rate of mass transfer υ represents flow rate

Advantages to HPLC : 

Advantages to HPLC Higher resolution and speed of analysis HPLC columns can be reused without repacking or regeneration Greater reproducibility due to close control of the parameters affecting the efficiency of separation Easy automation of instrument operation and data analysis Adaptability to large-scale, preparative procedures

Slide 12: 

Chromatography Mobile Phase Liquid Mobile Phase gas Stationary Phase Stationary Phase Solid Liquid Solid Liquid Solid Liquid Liquid Liquid Solid Gas Gas Liquid Chromatography Chromatography Chromatography Chromatography (Adsorption Chromatography) (Partition Chromatography) (G.S.C) (G.L.C) Examples : Examples : (ii) Ion – Exchange Chromatography (i) Reverse phase & Normal Chromatography (iii) Thin Layer Chromatography (ii) Paper Chromatography (iv) Size Exclusion Chromatography -

Types of Liquid Chromatography : 

Types of Liquid Chromatography Partition (liquid-liquid) reverse-phase nonpolar stationary phase normal-phase polar stationary phase Adsorption (liquid-solid) used to separate relatively nonpolar species with MW < 5000 a particular strength of this method is that it resolve isomeric mixtures such as meta and para benzene derivatives much easier than other techniques Ion-exchange Gel-permeation packing is hydrophobic-used to separate nonpolar species Gel-filtration packing is hydrophilic -used to separate polar species

Components Of A Liquid Chromatograph System : 

Components Of A Liquid Chromatograph System Mobile Phase / Solvent Reservoir Degasser Solvent Delivery System (Pump) Injector Precolumn Column Temperature Control Detectors Recorder (Data Collection)

Slide 15: 

HPLC Basic Instrumentation Mobile phase Pump Solvent Delivery Injector Sample Injection Column Separation Detector Data Processor 1. HPLC

HPLC system : 

HPLC system HPLC Solvent Reservoirs Solvent Delivery System Variable UV/Vis Detector Rheodyne Injector HPLC Column Computer Workstation

HPLC system : 

HPLC system Solvent Reservoir Degasser Solvent Delivery System (Pump) Injector Column &oven Detectors Recorder (Data Collection)

Slide 18: 

Solvent Delivery System Rheodyne Injector %A %B %C Flow Rate Pressure {H2O} {MeOH} (mL/min) (atmos.) Ready Ternary Pump A C B from solvent reservoir Column to detector to column through pulse dampener to injector through pump load inject

Components : 

Components Solvent Reservoir Usually one or more glass or stainless steel reservoirs each of which contains 200-1000 ml of solvent Isocratic elution - single solvent separation technique Gradient elution - 2 or more solvents, varied during separation

Gradient system : 

Gradient system Isocratic system Fixed (un-changeable) mixing ratio during analysis Gradient system Changeable mixing ratio during analysis HPGE (High Pressure Gradient) LPGE (Low Pressure Gradient)

Aim of gradient - problems in isocratic mode - : 

Aim of gradient - problems in isocratic mode - in isocratic mode Long analysis time Poor separations Methanol / water = 6 / 4 Methanol / water = 8 / 2 (Column : ODS type)

Aim of gradient - solution - : 

Aim of gradient - solution - Gradual change of the mixing ratio during analysis Methanol concentration in mobile phase Short analysis time & Excellent separation

Degasser : 

Degasser Problems caused by dissolved air(O2, N2)in mobile phase Unstable delivery in pump Bigger noise and large baseline-drift in detector cell In order to avoid causing the problems, mobile phase should be degassed. vacuum pumping systems distillation system a system for heating and stirring the solvents sparging system - bubbles an inert gas of low solubility through the solvent

Solvent Delivery System : 

Solvent Delivery System Requirements ability to mix solvents and vary polarity of mobile phase during run “unlimited” solvent reservoir generation of pressures up to 6000 psi flow rates ranging from 0.1 to 10 mL/min flow reproducibility’s of 0.5 % or better resistance to corrosion by a variety of solvents pulse-free output

Components : 

Components Three basic types of LC Pumps are: pneumatic pumps motor driven syringe type pumps reciprocating pumps

Motor driven syringe type pumps : 

Motor driven syringe type pumps Advantages: simple inexpensive pulse free Disadvantages limited capacity not amenable to gradient elution

Components : 

Components Three basic types of LC Pumps are: pneumatic pumps motor driven syringe type pumps reciprocating pumps

Reciprocating Pumps : 

Reciprocating Pumps Advantages small internal volume high output pressures (up to 10,000 psi) readily adaptable to gradient elution “unlimited” solvent reservoir Disadvantages produces a pulsed flow expensive

A reciprocating pump for HPLC : 

A reciprocating pump for HPLC The piston expels liquid through a one-way valve (check valve). The pumping rate is usually adjusted by controlling the distance the piston retracts, thus limiting the amount of liquid pushed out by each stroke, or by the cam rotating speed.

Reciprocating Pump : 

Reciprocating Pump Motor & Cam Plunger Plunger seal Check valves Pump head

A dual piston reciprocating pump for HPLC : 

A dual piston reciprocating pump for HPLC 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.

A dual piston reciprocating pump for HPLC : 

A dual piston reciprocating pump for HPLC Another type of dual piston pump uses only two check valves, but piston volumes are different. While the smaller piston dispenses an eluent in the HPLC system, the bigger piston is sucking an eluent. When pistons change their direction, the bigger piston simultaneously refill the smaller chamber and dispenses eluent into the system.

Gradient Controller : 

Gradient Controller The gradient controller is the device that allows you to create a gradient program (vary the composition of the solvent during the run) Gradients are produced differently for different types of pumping systems

Injectors : 

Injectors Sample Injection System sample valve syringe

Sample Injection Systems : 

Sample Injection Systems For injecting the solvent through the column Minimize possible flow disturbances Limiting factor in precision of liquid chromatographic measurement Volumes must be small .1-500 L Sampling loops interchangeable loops (5-500 L at pressures up to 7000 psi)

Slide 40: 

Manual injector Valve injectors Isolated from the pump eluent Stream (LOAD position) Positioned in it (INJECT position)

Direct injection auto sampler : 

Direct injection auto sampler from Pump from Pump to Column Vial Needle Measuring Pump to Column LOAD INJECT

Precolumn : 

Precolumn remove impurities from solvent saturates mobile phase with liquid of stationary phase before the analytical column

Column : 

Column straight, 15 to 150 cm in length; 2 to 3 mm i.d. packing - silica gel, alumina, Celite

Columns : 

Columns Solid Support - Backbone for bonded phases. Usually 10µ, 5µ or 3µ silica or polymeric particles. Bonded Phases - Functional groups firmly linked (chemically bound) to the solid support. Extremely stable Reproducible Guard - Protects the analytical column. Particles Interferences Prolongs the life of the analytical column Analytical - Performs the separation.

Common Reverse Phase Solvents : 

Common Reverse Phase Solvents Methanol Acetonitrile Tetrahydrofuran Water CH3OH CH3CN H2O

Columns : 

Columns straight, 15 to 150 cm in length; 2 to 3 mm i.d. packing - silica gel, alumina, Celite

Partitioning : 

Partitioning Separation is based on the analyte’s relative solubility between two liquid phases

Separation Mechanism of sample : 

Separation Mechanism of sample

Detector : 

Detector Mostly optical Equipped with a flow cell Focus light beam at the center for maximum energy transmission Cell ensures that the separated bands do not widen

Some Properties of Detector : 

Some Properties of Detector Adequate sensitivity Stability and reproducibility Wide linear dynamic range Short response time Minimum volume for reducing zone broadening

More Properties of Detector : 

More Properties of Detector High reliability and ease of use Similarity in response toward all analytes Selective response toward one or more classes of analytes Non-destructive

Types of Detector : 

Types of Detector UV/Visible - Fixed wavelength - variable wavelength Photo Diode Array Refractive index Fluorescence Evaporative light scattering Conductivity Electrochemical

Ultraviolet detector cell for HPLC : 

Ultraviolet detector cell for HPLC

UV/Visible : 

UV/Visible Advantages high sensitivity &small sample volume required can be used with gradient elution Is relatively cheap and not sensitive to temperature Can be used in gradient elution Sensitive to large number of organic compounds. Disadvantage does not work with compounds that do not absorb light at this wavelength region Cannot be used with the solvents having large absorption in the UV region Cannot be used for the sample components which cannot be absorbed in the UV region.

Diode Array Detectors : 

Diode Array Detectors Diode Array Detector (DAD) The more common tool for research-grade HPLC instruments quite versatile... Non-destructive, non-universal DAD scans a range of wavelengths every second or few seconds. At each point in the chromatogram one gets a complete UV-VIS spectrum! Huge volumes of data Detailed spectra for each peak and each region of each peak

Diode Array Detectors : 

Diode Array Detectors Diode array alternative puts grating, array of photosens. Semiconductors after the light goes through the sample. Advantage, speed, sensitivity, The Multiplex advantage Disadvantage, resolution is 1 nm, vs 0.1 nm for normal UV

HPLC – Diode Array Detector Output : 

HPLC – Diode Array Detector Output

Refractive Index Detector : 

Refractive Index Detector One of a very few Universal HPLC detectors. Non-destructive Responds to analytes changing the RI of the mobile phase requires a separate reference flow of mobile phase Extremely temperature sensitive sensitive to temp changes of +/- 0.001 °C No longer really widely used Absorbance detectors are relatively cheap. Useful for process work, on-line monitoring, etc.

Refractive Index Detector : 

Refractive Index Detector

Light Scattering Detector : 

Light Scattering Detector

Slide 64: 

Parameters used in HPLC 2. Parameters used in HPLC EVALUATION CAPACITY FACTOR RESOLUTION ASYMMETRY FACTOR ( TAILING FACTOR ) EFFICIENCY Retention : When a component in a sample interacts with the stationary phase in the column and a delay in elution occurs. Column efficiency : Goodness of a column

Slide 65: 

2. Parameters used in HPLC Retention parameters tR : retention time (the time between the injection point and the maximum detector response for correspondent compound) vR : retention volume (tR x eluent flow rate) k’ : capacity factor t0 : the time required for the component not retained by the column to pass through the column

Slide 66: 

2. Parameters used in HPLC Resolution tR1 tR2 k’1 k’2 W1 W2 Resolution : Separation factor : = k’1 k’2 The resolution of two bands is a function of both their relative Retentions and peak width.

Slide 67: 

2. Parameters used in HPLC Peak symmetry S : Symmetry factor ( T : Tailing factor ) f W0.05 h x 0.05 h S = 1 : The peak is completely symmetric. S > 1 : Tailing S < 1 : Leading

Slide 68: 

2. Parameters used in HPLC Efficiency: Keeps the bands from spreading and gives narrow peaks.

Slide 69: 

Thank You

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