Basic Principles of HPLC

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Basic Principles of HPLC : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 1 Basic Principles of HPLC Miss.Anagha Bhangale ARDL-I FDC LIMITED

HPLC : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 2 HPLC Introduction & history lesson Classification of chromatography Types of liquid chromatography Components of HPLC Types of Detector Evaluation parameters

History lesson : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 3 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 : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 4 Chromatography by M. Tswett ether CaCO3 chlorophyll Chromatography colours

HPLC : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 5 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 : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 6 HPLC Laterly referred to: High Performance Liquid Chromatography high performance due to its reproducibility currently refers to: High Precision Liquid Chromatography

Introduction : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 7 Introduction Compounds are separated by injecting a sample mixture onto the column. The different component in the mixture pass through the column at different rates due to differences in their partition behavior between the mobile phase and the stationary phase.

Principle of HPLC: : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 8 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 : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 9 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

Types of Liquid Chromatography : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 10 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 : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 11 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 12: 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 12 HPLC Basic Instrumentation Mobile phase Pump Solvent Delivery Injector Sample Injection Column Separation Detector Data Processor

HPLC system : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 13 HPLC system Solvent Reservoir Degasser Solvent Delivery System (Pump) Injector Column &oven Detectors Recorder (Data Collection)

Components : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 14 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 : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 15 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 - : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 16 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 - : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 17 Aim of gradient - solution - Gradual change of the mixing ratio during analysis Methanol concentration in mobile phase Short analysis time & Excellent separation

Degasser : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 18 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 : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 19 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 : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 20 Components Three basic types of LC Pumps are: Pneumatic pumps Motor driven syringe type pumps Reciprocating pumps

Reciprocating Pumps : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 21 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

Slide 22: 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 22

A reciprocating pump for HPLC : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 23 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 : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 24 Reciprocating Pump Motor & Cam Plunger Plunger seal Check valves Pump head

A dual piston reciprocating pump for HPLC : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 25 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.

Injectors : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 26 Injectors Sample Injection System sample valve syringe

Sample Injection Systems : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 27 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)

Direct injection auto sampler : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 28 Direct injection auto sampler from Pump from Pump to Column Vial Needle Measuring Pump to Column LOAD INJECT

Precolumn : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 29 Precolumn remove impurities from solvent saturates mobile phase with liquid of stationary phase before the analytical column

Column : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 30 Column straight, 15 to 150 cm in length; 2 to 3 mm i.d. packing - silica gel, alumina, Celite

Slide 31: 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 31

Columns : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 32 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.

Slide 33: 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 33

Slide 34: 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 34

Common Reverse Phase Solvents : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 35 Common Reverse Phase Solvents Methanol Acetonitrile Tetrahydrofuran Water CH3OH CH3CN H2O

Columns : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 36 Columns straight, 15 to 150 cm in length; 2 to 3 mm i.d. packing - silica gel, alumina, Celite

Partitioning : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 37 Partitioning Separation is based on the analyte’s relative solubility between two liquid phases

Detector : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 38 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 : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 39 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 : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 40 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 : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 41 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 : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 42 Ultraviolet detector cell for HPLC

UV/Visible : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 43 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 : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 44 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 : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 45 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 : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 46 HPLC – Diode Array Detector Output

Refractive Index Detector : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 47 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 : 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 48 Refractive Index Detector

Slide 49: 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 49 System Suitabilty 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 50: 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 50 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 51: 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 51 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 52: 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 52 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 53: 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 53 Efficiency: Keeps the bands from spreading and gives narrow peaks.

Slide 54: 

Analytical Research and Developement Laboratory - I,FDC Limited,Mumbai 54 Thank You