High Performance Liquid Chromatography (HPLC)

PowerPoint Presentation: 

Narmdeshwar Dev Mishra M.Pharm (Pharmaceutics) narmdeshwar25@rediffmail.com Mob. No.9039822909 Monika Bhairam M.Pharm (Pharmaceutics) monikab430@gmail.com Columbia Institute Of Pharmacy Raipur (C.G) High Performance Liquid Chromatography

Introduction : 

Introduction Chromatography may be defined as a method of separating a mixture of component distribution component though equilibrium distribution between tow phase. Chromatographs separation techniques was introduced by j. sweet in 1903.by adsorption chromatography. Firstly plant pigments are separated. HPLC was introduced by Kirkland and haler.

PowerPoint Presentation: 

HPLC is an analytical process utilizing special instrument designed designed to separate, quailing and analyze components of Introduction a cervical armature. Sample of interest are introduced to a solvent flow path, carried through a column paced with specialized materials for component separation & component dada is obtained though the combination of a detection mechanism in cooled with a dada rewording system. All this occurs under pressure which may reach or exceed 6000 psi. Introduction

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

HPLC is….: 

HPLC is…. Popularity: widespread applicability to substances that are of prime interest to industry, to many fields of science, and to the public Ideally suited for separation and identification of amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, pharmaceuticals, pesticides, pigments, antibiotics, steroids, and a variety of other inorganic substances

PowerPoint Presentation: 

HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector . 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. The mobile phase must be degassed to eliminate the formation of air bubbles. Definition

Types of HPLC: 

Types of HPLC Based on modes of chromatography :- POLAR-POLAR = MORE AFFINITY NONPOLAR-NONPOLAR = MORE AFFINITY POLAR-NONPOLAR = LESS AFFINITY Normal phase In this column type, the retention is governed by the interaction of the polar parts of the stationary phase and solute. For retention to occur in normal phase, the packing must be more polar than the mobile phase with respect to the sample .(SP-MN=NP travel faster deu to less affinity)

Reverse phase : 

Reverse phase In this column the packing material is relatively nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile -water.(SN-MP=P travel faster deu to less affinity)

Based on principle of separation :-: 

Liquid-solid (adsorption) chromatography Liquid-liquid (partition) chromatography Ion-exchange chromatography Size exclusion chromatography Based on principle of separation :-

Adsorption Chromatography: 

Adsorption Chromatography The most common & simple method of adsorption chromatography consist in the separation of substance in solution by filtration through a column of a finely powdered adsorbent placed in a glass table the two widely wed techniques for the separation by column adscription chromatography are :- Displacement Elution

Partition Chromatography : 

Partition Chromatography Reverse Phase Chromatography Nonpolar Stationary Phase Polar Mobile Phase Normal Phase Chromatography Polar Stationary Phase Nonpolar Mobile Phase Column Selection Mobile-Phase Selection

Partition Chromatography: 

Partition Chromatography Most widely used Bonded-phase Chromatography Silica Stationary Phase: OH OH OH OH O O O Si Si Si Si Siloxanes : O CH 3 Si O Si R R = C 8 , C 18 O CH 3

What is Ion Chromatography?: 

Modern methods of separating and determining ions based on ion-exchange resins Mid 1970s Anion or cation mixtures readily resolved on HPLC column Applied to a variety of organic & biochemical systems including drugs, their metabolites, serums, food preservatives, vitamin mixtures, sugars, pharmaceutical preparations.(Resins are…. What is Ion Chromatography?

Ion-Exchange Packings: 

Types of packings pellicular bead packing large (30-40 µm) nonporous, spherical, glass, polymer bead coated with synthetic ion-exchange resin sample capacity of these particles is less coating porous microparticles of silica with a thin film of the exchanger faster diffusion leads to enhanced efficiency Ion-Exchange Packings

Ion-Exchange Equilibria: 

Exchange equilibria between ions in solution and ions on the surface of an insoluble, high molecular-weight solid Cation exchange resins sulfonic acid group, carboxylic acid group Anion exchange resins quaternary amine group, primary amine group Ion-Exchange Equilibria CM Cellulose Cation Exchanger DEAE Cellulose Anion Exchanger

Size Exclusion Chromatography(SEC): 

Gel permeation(GPC), gel filtration(GFC) chromatography Technique applicable to separation of high-molecular weight species Rapid determination of the molecular weight or molecular-weight distribution of larger polymers or natural products Solute and solvent molecules can diffuse into pores -- trapped and removed from the flow of the mobile phase Size Exclusion Chromatography(SEC)

SEC(continued): 

Specific pore sizes.average residence time in the pores depends on the effective size of the analyte molecules larger molecules smaller molecules intermediate size molecules SEC(continued)

SEC Column Packing: 

Small (~10 µm) silica or polymer particles containing a network of uniform pores Two types (diameters of 5 ~ 10 µm) Polymer beads silica-based particles SEC Column Packing

Advantages of Size Exclusion Chromatography: 

Short & well-defined separation times Narrow bands--> good sensitivity Freedom from sample loss, solutes do not interact with the stationary phase Absence of column deactivation brought about by interaction of solute with the packing Advantages of Size Exclusion Chromatography

Disadvantages: 

Only limited number of bands can be accommodated because the time scale of the chromatogram is short Inapplicability to samples of similar size, such as isomers. At least 10% difference in molecular weight is required for reasonable resolution Disadvantages

Based on elution technique :-: 

Isocratic elution single solvent of constant composition Gradient elution 2 or more solvents of differing polarity used Based on elution technique :-

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

Advantages to HPLC: 

Advantages of HPLC are result of 2 major advances: stationary supports with very small particle sizes and large surface areas appliance of high pressure to solvent flow Advantages to HPLC

Instrumentation: 

Instruments required: Mobile phase reservoir Pump Injector Column Detector Data system Instrumentation

Mobile phase reservoir: 

Glass/stainless steel reservoir Removal of dissolved gases by degassers vacuum pumping system heating/stirring of solvents sparging vacuum filtration Mobile phase reservoir

The Mobile Phases are...: 

Aqueous solutions containing methanol, water-miscible organic solvents also contain ionic species, in the form of a buffer solvent strength & selectivity are determined by kind and concentration of added ingredients ions in this phase compete with analyte ions for the active site in the packing The Mobile Phases are...

Properties of the Mobile Phase : 

Must dissolve the sample have a strong solvent strength leads to reasonable retention times interact with solutes in such a way as to lead to selectivity Properties of the Mobile Phase

Schematic of liquid chromatograph: 

Schematic of liquid chromatograph

Pumping System I: 

Provide a continuous constant flow of the solvent through the injector Requirements pressure outputs up to 6000 psi pulse-free output flow rates ranging from .1-10 mL /min flow control and flow reproducibility of .5% or better corrosion-resistant components Pumping System I

Pumping System II: 

Two types: constant-pressure constant-flow Reciprocating pumps motor-driven piston disadvantage: pulsed flow creates noise advantages: small internal volume (35-400 L) , high output pressures (up to 10,000 psi), ready adaptability to gradient elution, constant flow rates Pumping System II

Pumping System III: 

Displacement pumps syringe-like chambers activated by screw-driven mechanism powered by a stepper motor advantages: output is pulse free disadvantage: limited solvent capacity (~20 mL ) and inconvenience when solvents need to be changed Flow control and programming system computer-controlled devices measure flow rate increase/decrease speed of pump motor Pumping System III

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) Sample Injection Systems

Liquid Chromatographic Column: 

Smooth-bore stainless steel or heavy-walled glass tubing Hundreds of packed columns differing in size and packing are available from manufacturers ($200-$500) Add columns together to increase length Liquid Chromatographic Column

Liquid Chromatographic Columns II: 

Column thermostats maintaining column temperatures constant to a few tenths degree centigrade column heaters control column temperatures (from ambient to 150 o C) columns fitted with water jackets fed from a constant temperature bath Liquid Chromatographic Columns II

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 Detector

Some Properties of Detector: 

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

Types of Detector: 

Refractive index UV/Visible Fluorescence Conductivity Evaporative light scattering Electrochemical Types of Detector

Refractive Index I: 

Measure displacement of beam with respect to photosensitive surface of dectector Refractive Index I

Refractive Index II: 

Advantages universal respond to nearly all solutes reliable unaffected by flow rate low sensitive to dirt and air bubbles in the flow cell Refractive Index II

Refractive Index III: 

Disadvantages expensive highly temperature sensitive moderate sensitivity cannot be used with gradient elution Refractive Index III

UV/Visible I: 

Mercury lamp  = 254nm  = 250, 313, 334 and 365nm with filters Photocell measures absorbance Modern UV detector has filter wheels for rapidly switching filters; used for repetitive and quantitative analysis UV/Visible I

UV/Visible II: 

UV/Visible II

UV/Visible III: 

Advantages high sensitivity small sample volume required linearity over wide concentration ranges can be used with gradient elution UV/Visible III

UV/Visible IV: 

Disadvantage does not work with compounds that do not absorb light at this wavelength region UV/Visible IV

Fluorescence I: 

For compounds having natural fluorescing capability Fluorescence observed by photoelectric detector Mercury or Xenon source with grating monochromator to isolate fluorescent radiation Fluorescence I

Fluorescence II: 

Advantages extremely high sensitivity high selectivity Disadvantage may not yield linear response over wide range of concentrations Fluorescence II

Conductivity: 

Measure conductivity of column effluent Sample indicated by change in conductivity Best in ion-exchange chromatography Cell instability Conductivity

Evaporative Light Scattering I: 

Nebulizer converts eluent into mist Evaporation of mobile phase leads to formation of fine analyte particles Particles passed through laser beam; scattered radiation detected at right angles by silicon photodiode Similar response for all nonvolatile solutes Good sensitivity Evaporative Light Scattering I

Evaporative Light Scattering II: 

Evaporative Light Scattering II

Electrochemical I: 

Based on reduction or oxidation of the eluting compound at a suitable electrode and measurement of resulting current Electrochemical I

Electrochemical II: 

Advantages high sensitivity ease of use Disadvantages mobile phase must be made conductive mobile phase must be purified from oxygen, metal contamination, halides Electrochemical II

Data System: 

For better accuracy and precision Routine analysis pre-programmed computing integrator Data station/computer needed for higher control levels add automation options complex data becomes more feasible software safeguard prevents misuse of data system Data System

Electrophoresis…charged species migrate in electric field Separation based on charge or mobility: 

Electrophoresis…charged species migrate in electric field Separation based on charge or mobility

Capillary electrophoresis higher voltages can be used as the heat can be dissipated: 

Capillary electrophoresis higher voltages can be used as the heat can be dissipated

THANK YOU: 

THANK YOU