Introduction of chromatography.pptx

Introduction, types, mechanism and application of chromatography

1. Introduction of Chromatography By Shubhrat Maheshwari Subject - Instrumental methods of analysis Subject code- BP701T

2. Introduction Chromatography is a physical process where the components (solutes) of a sample mixture are separated as a result of their differential distribution between stationary and mobile phases. Greek chroma meaning ‘color’ and graphein meaning ‘writing’ ADVANTAGES OF CHROMATOGRAPHY OVER OTHER METHODS Separation of mixtures of chemicals having closer physical or chemical properties. Gentle method. Separation in even micrograms is possible. Simple, rapid and efficient technique.

3. Introduction Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components.

4. Stationary Phase

5. Introduction 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

6. Definition ‘A technique by which a mixture is separated into its components on the basis of relative ability of each component to be moved along/through a stationary phase by mobile phase’ The technique of chromatography is based on the differences in the rate at which the components of a mixture move through a porous medium (called stationary phase) under the influence of some solvent or gas (called moving/mobile phase). Chromatography is a nondestructive procedure. Applied for both qualitative and quantitative studies

7. History The subject of Chromatography was introduced into scientific world in a very modest way by M. Tswett in 1906. He employed a technique to separate various pigments such as chlorophylls and xanthophylls by passing the solution of these compounds into the glass column which was packed with finely divided calcium carbonate. After the later, Thompson and Way had realized the Ion- Exchange properties of soils. Almost after three decades, in 1935 Adams and Holmes observed the Ion Exchange characteristics in crushed phonograph. This observation opened the field for preparation of Ion Exchanged resins. The concept of Gas-Liquid Chromatography was first introduced by Martin and Synge in 1941.

8. History They were also responsible for the development in Liquid- Liquid chromatography. In 1944, from Martin laboratory, the separation of amino acid by paper chromatography was reported. In 1952, the importance of the chromatography was observed when both Synge and Martin were awarded with Nobel Prize. In 1959, a technique known as Gel Filtration chromatography was observed which is used to separate low molecular weight substances from high molecular substances. In 1960, further improvement in liquid chromatography led to the development of High Performance Liquid Chromatography. The following decade of 1970 improvement in the field of adsorption chromatography in the form of Affinity chromatography which was mainly based on biological interactions.

9. History A new field was originated which was supercritical fluid chromatography. Supercritical fluid chromatography is a hybrid of gas and liquid chromatography and combine advantageous feature of the both gas and liquid chromatography. It will not be wrong to say that the entire twentieth century can be named as the century of chromatography.

10. Principle Chromatography is usually based on principle of partition of solute between two phases. It usually consists of a Mobile Phase and a Stationary Phase. The Mobile Phase usually refers to the mixture of the substances to be separated dissolved in a liquid or a gas . The Stationary Phase is a porous solid matrix through which the sample contained in the mobile phase percolates.

11. Principle Like-dissolve-like or like-prefer-like. The basis-partition or distribution coefficient ‘K’ which describes the way in which a compound distributes itself between two immiscible phases. Defined as the molar concentration of analyte in the stationary phase divided by the molar concentration of the analyte in the mobile phase 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑖𝑛 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑖𝑛 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒 K =

12. Commonly used terms in chromatography The analyte is the substance to be separated during chromatography. Analytical chromatography is used to determine the existence and the concentration of analyte(s) in a sample. A chromatogram is the visual output of the chromatograph. In the case of an ideal separation, different peaks or patterns on the chromatogram represent different components of the separated mixture.

13. Commonly used terms in chromatography A chromatograph is an equipment that enables the separation e.g. gas chromatographic or liquid chromatographic separation. The eluate is the mobile phase leaving the column. The eluent is the solvent that carries the analyte. An eluotropic series is a list of solvents ranked according to their eluting power. Elution is the process of extracting a substance that is adsorbed to another by washing it with a solvent. An immobilized phase is a stationary phase that is immobilized on the support particles, or on the inner wall of the column tubing. The mobile phase is the phase that moves over the stationary phase. It may be a liquid (LC) or a gas (GC). The mobile phase moves through the stationary phase where the sample interacts with the stationary phase and is separated.

14. Commonly used terms in chromatography The retention time is the time required for the mobile phase to sweep a component from the stationary phase. The retention volume is the volume of the mobile phase required to sweep a component through stationary phase. The sample is the matter analyzed in chromatography. It may consist of a single component or it may be a mixture of components. When the sample is treated, the phase or the phases containing the analytes of interest is/are referred to as the sample whereas everything else separated from the sample before or during analysis is referred to as waste. The solute refers to the sample components in partition chromatography. The solvent refers to any substance capable of solubilizing another substance, and especially the liquid mobile phase in liquid chromatography.

15. Commonly used terms in chromatography The stationary phase is the substance fixed in place for the chromatography procedure. It may be solid, gel or a liquid. e.g ; silica, alumina, cellulose The detector refers to the instrument used for qualitative and quantitative detection of analytes after separation. Rf value or Retention factor ( R f ) is defined as the ratio of the distance traveled by the center of a spot (solute) to the distance traveled by the solvent front (solvent) 𝑅𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟(𝑅𝑓) = 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡

16. Chromatography Mechanisms Adsorption Partition Ion exchange Size-exclusion Affinity Chromatography

17. Adsorption Based on relative polarities . Compounds having high affinity towards the stationary phase travel slower Compounds having lesser affinity towards the stationary phase travel faster No two components have same affinity for a combination of stationary phase, mobile phase and other conditions

18. Partition Based on relative solubility. Solutes will be distributed according to their partition coefficients. Components which are more soluble in the stationary phase – Travel slower Components which are less soluble in the stationary phase –Travel faster No two components have same partition coefficient for a particular combination of stationary phase, mobile phase and other conditions.

19. Ion exchange Stationary phase contains fixed charged groups and mobile counter ions Counter ion exchange with ions of solute Reversible exchange of ions takes place between similar charged ions of solute in the mobile phase and that of an ion exchange resin.

20. Size exclusion Retention depends on the extend to which the solute molecules are trapped in the pores of inert stationary phase. This depends on size of molecules. Smaller molecules diffuses into the pores of the stationary phase – Travel slower. Larger molecules do not enter the pores of stationary phase – Travel faster.

21. Affinity Affinity chromatography  is a method of separating a biomolecule from a mixture, based on a highly specific  macromolecular binding  interaction between the biomolecule and another substance. The specific type of binding interaction depends on the biomolecule of interest;  antigen  and  antibody ,  enzyme  and  substrate ,  receptor  and  ligand , or  protein  and  nucleic acid  binding interactions are frequently exploited for isolation of various biomolecules. Affinity chromatography is useful for its high  selectivity  and  resolution  of separation, compared to other chromatographic methods.

22. Classification of Chromatography

23. Classification of Chromatography

24. Classification of Chromatography Adsorption Chromatography 1. Thin Layer Chromatography (TLC) 2. Column Chromatography 3. Ion Exchange Chromatography 4. High Performance (pressure) Liquid Chromatography (HPLC) 5. Gel Permeation Chromatography 6. Gas Solid Chromatography (GSC) Partition chromatography 1. Paper Chromatography 2. Gas Liquid Chromatography (GLC)

25. HOW TO CHOOSE A METHOD Polarity of the sample. Solubility and volatility of sample. Resolution required. Concentration of analyte. Detection limit. Physical and chemical properties of sample.

26. Uses of 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

27. Applications Applications of Chromatography in the Pharmaceutical Industry Applications of Chromatography in the Food Industry Applications of Chromatography in the Chemical Industry Applications of Chromatography in the Field of Molecular Biology