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CH ROM ATO GRA PHY. Jigarkumar B. Patel K.B.I.P.E.R. INTRODUCTION. History of chromatography. Chromatography was first developed and defined by the russian botanist Mikhail S. Tswett in 1903.
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CHROMATOGRAPHY Jigarkumar B. Patel K.B.I.P.E.R.
History of chromatography Chromatography was first developed and defined by the russian botanist Mikhail S. Tswett in 1903. He produced a colorful separation of plant pigments using a column of calcium carbonate (chalk). The word chromatography comes from the Greek words for color "chroma" and write "graphein". So chromatography means 'to write with color' 1938 Paper and TLC 1952 Gas-liquid chromatography GLC 1968 High pressure liquid chromatography HPLC 1980s Super critical fluid chromatography SFC
How it works? In all chromatography there is a mobile phase and a stationary phase. The stationary phase is the phase that doesn't move and the mobile phase is the phase that does move. The mobile phase moves through the stationary phase picking up the compounds to be tested. As the mobile phase continues to travel through the stationary phase it takes the compounds with it. At different points in the stationary phase the different components of the compound are going to be absorbed and are going move with the mobile phase. This is how the separate from the other components is achieved.
Is a technique used to separate and identify the components of a mixture. • Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium. • Molecules that spend most of their time in the mobile phase are carried along faster.
USP 2000: “Chromatography is procedure by which solutes are separated by dynamic differential migration process in a system consisting of two or more phases, one of which moves continuously in given direction and in which the individual substance exhibits different mobilities by reasons of differences in adsorption, partition, solubility, vapor pressure, molecular size or ionic charge density. The individual substance thus separated can be identified or quantitatively measured by suitable analytical method.”
USP 2009 • Chromatography is defined as a procedure by which solute are separated by a dynamic differential migration process in a system consisting of two or more phases, one of which moves continuously in a given direction and in which the individual substances exhibit different mobilities by reason of differences in adsorption, partition, solubility, vapor pressure, molecular size, or ionic charge density. The individual substances thus separated can be identified or determined by analytical procedures.
IUPAC 1993: • “Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction.”
mobile phase: solvent • may be a liquid or a gas. • stationary phase : column packing material • may be a solid, liquid supported on a solid or a gel. • This definition suggests that chromatographic separations have three distinct features: • They are physical methods of separation; • Two distinct phases are involved, one of which is stationary while the other is mobile; and • Separation results from differences in the distribution constants of the individual sample components between the two phases.
Purpose of Chromatography • Analytical - determine chemical composition of a sample • Preparative - purify and collect one or more components of a sample
Uses for Chromatography Chromatography is used by scientists to: Analyze – examine a mixture, its components, and their relations to one another Identify – determine the identity of a mixture or components based on known components Purify – separate components in order to isolate one of interest for further study Quantify – determine the amount of the a mixture and/or the components present in the sample
Uses for Chromatography Real-life examples of uses for chromatography: Pharmaceutical Company – determine amount of each chemical found in new product Hospital – detect blood or alcohol levels in a patient’s blood stream Law Enforcement – to compare a sample found at a crime scene to samples from suspects Environmental Agency – determine the level of pollutants in the water supply Manufacturing Plant – to purify a chemical needed to make a product
Types of Chromatography Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase) Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase) Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase) Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)
Basically, molecules can be separated from one another if there are differences in one or more of the following: • 1. Volatility • 2. Polarity • 3. Molecular size • 4. Charge
Separation of closely related components of a complex mixture. Mobile phase forced through a stationary Phase (column or a planar surface).Components of a mixture distribute between mobile and stationery phase to varying degree. More solubility in Mobile Phase – fast progress- low retention Time (Rt). More solubility in stationery phase – slow progress – long retention time(Rt)
In the animation below the red molecules have more affinity towards the stationary phase than the green molecules.
For example, suppose we have a mixture of two solutes, A and B, dissolved in a suitable buffer solution. • A column is filled with beads of an insoluble substance that is expected to bind differentially and reversibly to A and B. • The column is initially equilibrated by running a certain volume of the buffer through it, and the mixture applied to the top of the column as a narrow layer. • If the stopcock of the column is carefully opened, the layer of solutes will pass into the column, and the solutes can be washed out slowly by pouring more buffer into the column. • This process is called elution, and the solution emerging at the bottom of the column is called the eluate.
Chromatogram – Detector signal vs. retention time or volume
Let us assume, for the purpose of this illustration, that solute B binds more strongly to the matrix than solute A. • Then, as the mixture travels down the column, the molecules of B will be retarded with respect to the molecules of A. • In time, they will separate into bands, and be eluted at different times.
Classification of chromatography • Depending on depth of separation(Geometry): • 1) Column Chromatography: • The stationary phase is held in a narrow tube through which the mobile phase is forced under pressure or by gravity. • 2) Planar Chromatography: • The stationary phase is supported on a flat plate or the interstices of a paper and the mobile phase moves through the stationary phase by capillary action or by gravity.
Classification of chromatography Nature of mobile phase : On the basis of types of Mobile Phase and Stationary Phase and the kinds of equilibrium involved in the transfer of solutes between the two phases. Liquid Chromatography Gas Chromatography Supercritical Chromatography
CLASSIFICATION OF CHROMATOGRAPHY Based on instrument/ apparatus Based on working principle/ Mode of chromatography Nature of Stationary Phase Column Chromatography Paper Chromatography TLC Gas Chromatography HPLC SCF Chromatography Electrophoretic Chromatography 1-3: Gives separation basically 4-7: give separation and separated compound is analyzed quantitatively and qualitatively. Adsorption Chromatography Partition Chromatography Ion exchange Chromatography Size exclusion Chromatography Miscellaneous
BASED ON APPARATUS/ INSTRUMENT USED • Column Chromatography: • used for the separation of large quantity of drug. • a) Adsorption Column Chromatography • b) Partition Column Chromatography • 2) Paper Chromatography: • Separation and identification of very small quantity of drug (µg) e.g. amino acids. • a) Ascending Paper Chromatography • b) Descending Paper Chromatography • c) Circular Paper Chromatography • d) Two Dimensional Paper Chromatography
BASED ON APPARATUS/ INSTRUMENT USED 3) Thin Layer Chromatography: Separation and identification of very small quantity of drug (µg), limit test, assay. Advantage: fast as compared to PC. a) Two Dimensional Thin Layer Chromatography b) Continuous development TLC c) High Performance Thin Layer Chromatography (HPTLC) 4) Gas Chromatography: For gaseous and volatile substance. a) Gas-Solid Chromatography (GSC) b) Gas-liquid Chromatography (GLC)
BASED ON APPARATUS/ INSTRUMENT USED 5) High Performance/Pressure Liquid Chromatography: a) Normal Phase HPLC b) Reverse Phase HPLC 6) Super Critical Fluid Chromatography 7) Electro Chromatography/ Capillary Electrophoretic Chromatography
BASED ON WORKING/SEPARATION PRINCIPLE • Adsorption (liquid-solid) Chromatography: • Substance of different chemical type. • S.P. : Adsorbent • Adsorb different solute to different extent.
Adsorption Chromatography Adsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibriation between the mobile and stationary phase accounts for the separation of different solutes.
BASED ON WORKING/SEPARATION PRINCIPLE 2) Partition (liquid-liquid) Chromatography: Substance having similar chemical type. Both the phases must be liquid. one having greater partition co-efficient in m.p. than s.p., it will move fast and vice versa. Chromatography in which separation is based mainly on differences between the solubility of the sample components in the stationary phase (gas chromatography), or on differences between the solubilities of the components in the mobile and stationary phases (liquid chromatography)
Partition Chromatography This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibriates between the mobile phase and the stationary liquid
BASED ON WORKING/SEPARATION PRINCIPLE 3) Ion-exchange Chromatography: Ionic and inorganic Substance. Separation is based upon ionic charge density. S.P.: ion exchangers which has capacity to exchange ions. Support chemically bonded to anionic or cationic group Separation on the basis of charge on the solute on the principle of opposite charges attracting each other. Resolution influenced by pH & ionic strength of buffer. Cationic Sulfonate exchanger strongly acidic Carboxylate exchanger weak Anionic Quaternary ammonium strongly basic Tertiary amine weak
Ion Exchange Chromatography In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces.
BASED ON WORKING/SEPARATION PRINCIPLE 4) Size exclusion(MolecularExclusion/gel permeation) chromatography: Size exclusion chromatography is a type of chromatography in which the stationary phase is a molecular sieve (Different gel material). Therefore, the molecules are separated according to differences in their molecular size. Non interactive mode of separation. The particles of column packing have various size pores and pore networks Solute molecules are retained or excluded on the basis of their hydrodynamic volume. Column packing: - semi rigid cross linked - polymer gels - rigid controlled pore size glasses or silicas
Molecular Exclusion Chromatography Also known as gel permeation or gel filtration, This type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. The pores are normally small and exclude the larger solute molecules, but allows smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones.
BASED ON WORKING/SEPARATION PRINCIPLE • 3) Miscellaneous Chromatography: • a) Ion pair Chromatography: • Control of hydrophobicity and hydrophilicity counter ion is added. • Production of ion pair comprising a sample ion and oppositely charged ion component of mobile phase. • Non polar interaction of ion-pair with stationary phase. • Ion pair agent first absorbs onto stationary phase via non polar interaction. • Sample ion interacts with the agent.
BASED ON WORKING/SEPARATION PRINCIPLE • 3) Miscellaneous Chromatography: • b) Chiral Chromatography: • S.P.: Optically active • Thus, entiomer can be separated. • c) Affinity Chromatography: • Specific interactions between stationary phase and solute molecule. • Antigen - antibody • enzyme - substrate or inhibitor • Hormone - binding protein • S.P.: Enzyme (highly selective in interaction with substrate) • OR • Substrate
Affinity Chromatography This is the most selective type of chromatography employed. It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. For example, the immobilized molecule may be an antibody to some specific protein. When solute containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later extracted by changing the ionic strength or pH.
