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Introduction to chromatography

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An introduction to chromatography and its pplication in research

An introduction to chromatography and its pplication in research

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  • 1. INTRODUCTION AND APPLICTIONS OF CHROMATOGRAPHY 1 Table of Contents 1. Introduction…………………………………………………………………………………………………………………….1 1.1. Definition………………………………………………………………………………………………………………….1 2. Principle Of Chromatography ………………………………………………………………………………………….2 3. Commonly Used Terms In Chromatography……………………………………………………………………..2 4. Classification Of Chromatography…………………………………………………………………………………….3 5. Types Of Chromatography……………………………………………………………………………………………….4 5.1. Adsorption Chromatography…………………………………………………………………………………....4 5.2. Partition Chromatography…………………………………………………………………………………………6 6. Other Types Of Chromatography…………………………………………………………………………………..…7 7. Quantification Of Chromatogram…………………………………………………………………………………….7 8. Visualization Of The Spots Of Chromatogram………………………………………………………………..…8 9. Applications Of Chromatography……………………………………………………………………………………..8 10. References……………………………………………………………………………………………………………………….9
  • 2. INTRODUCTION AND APPLICTIONS OF CHROMATOGRAPHY 2 1. Introduction: Chromatography, literally "color writing", was first employed by Russian-Italian scientist Mikhail Tsvet in 1900, primarily for the separation of plant pigments such as chlorophyll, carotenes, and xanthophylls. The word chromatography is derived from two Greek words Chroma ….……..color Graphos ………..writing 1.1. Definition: Chromatography may be defined as 'A method of separating a mixture of components into individual components through equilibrium distribution between two phases’. OR ‘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’ One of these phases is a mobile phase and the other is a stationary phase. Porous medium through which the mobile phase migrates is called the support. 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 for resolving a multi-component mixture of minor or major constituents into its individual fractions. It can be applied both for both qualitative and quantitative studies as it is a separation technique. 2. Principle of Chromatography: The principle of chromatography is like-dissolve-like or like-prefer-like. The basis of all forms of chromatography is the partition or distribution coefficient ‘K’ which describes the way in which a compound distributes itself between two immiscible phases. The Partition coefficient; is defined as the molar concentration of analyte in the stationary phase divided by the molar concentration of the analyte in the mobile phase 𝐾 = 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑖𝑛 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑛𝑒𝑛𝑡 𝑖𝑛 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒 The distribution of analytes between phases can be described simply. An analyte is in equilibrium between the two phases; Amobile Astationary
  • 3. INTRODUCTION AND APPLICTIONS OF CHROMATOGRAPHY 3 3. 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.
  • 4. INTRODUCTION AND APPLICTIONS OF CHROMATOGRAPHY 4  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.  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.  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 (Rf) 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 = 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑢𝑡𝑒 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 = 𝑅𝑓 4. Classification of chromatography: The chromatography is either preparative or analytical o Analytical ……….. to determine the chemical composition of a sample o Preparative ………. used to purify and collect one or more components of a sample
  • 5. INTRODUCTION AND APPLICTIONS OF CHROMATOGRAPHY 5 5. Types of chromatography: 5.1. Adsorption chromatography 5.2. Partition chromatography 5.1. Adsorption chromatography: It uses a mobile phase or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes. Following are the chromatographic techniques that are included in this category: 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) 1. Thin layer chromatography (TLC): Thin layer chromatography is similar to paper chromatography, but the stationary phase is a thin layer of a solid such as alumina or silica supported on an inert base such as glass, aluminum foil or insoluble plastic. The mixture is ‘spotted’ at the bottom of the TLC plate and allowed to dry. The plate is placed in a closed vessel containing solvent (the mobile phase) so that the liquid level is below the spot. TLC has advantages over paper chromatography in that its results are more reproducible, and that separations are very efficient because of the much smaller particle size of the stationary phase. TLC is also called as drop, strip, spread layer, surface chromatography and open column chromatography.
  • 6. INTRODUCTION AND APPLICTIONS OF CHROMATOGRAPHY 6 2. Column chromatography: A solvent acts as the mobile phase while a finely divided solid surface acts as the stationary phase. The stationary phase will adsorb the components of the mixture to varying degrees. As the solution containing the mixture passes over the adsorbent, the components are distributed between the solvent and adsorbent surface. This process may be described by three-way equilibrium between the sample, the solvent and the adsorbent. The solvent and sample compete for positions on the solid adsorbent, the solvent displacing the sample reversibly and continuously in the direction of the solvent flow. Consequently, a weakly adsorbed compound will spend more time in the solvent, and will therefore be eluted first. 3. Ion exchange chromatography: Ion exchange chromatography is used to remove ions of one type from a mixture and replace them by ions of another type. The column is packed with porous beads of a resin that will exchange either cations or anions. There is one type of ion on the surface of the resin and these are released when other ions are bound in their place – e.g. a basic anion exchange resin might remove nitrate ions (NO3–) from a solution and replace them with hydroxide ions (OH–). 4. High Performance (pressure) Liquid Chromatography (HPLC): HPLC involves a liquid sample being passed over a solid adsorbent material packed into a column using a flow of liquid solvent under pressure. o Normal Phase HPLC: NP-HPLC uses a non-polar, non-aqueous mobile phase (e.g. Chloroform), the analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity. o Reversed Phase HPLC: RP-HPLC has a non-polar stationary phase and an aqueous, moderately polar mobile phase. With such stationary phases, retention time is longer for molecules
  • 7. INTRODUCTION AND APPLICTIONS OF CHROMATOGRAPHY 7 which are less polar, while polar molecules elute more readily (early in the analysis). 5. Gel Filtration or Gel Permeation Chromatography: The separation of large molecules, often in biochemical situations, can be achieved in a column which works on the basis of molecular exclusion. The mixture of solutes is carried through the column by a solvent. The stationary phase (the gel) typically consists of particles of a cross-linked polyamide which contains pores. Separation occurs according to molecular size – the larger molecules passing through the column fastest. Different gels are available that allow the separation of proteins with relative masses. The greatest resolution is achieved by using very small gel particles, but the flow rate through the column then becomes much slower. 5.2. 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 equilibrates between the mobile phase and the stationary liquid. a. Paper Chromatography b. Gas Liquid Chromatography (GLC) a. Paper chromatography: Cellulose filter paper is often used as stationary phase in paper chromatography. Since it is hydrophilic, it is usually covered with a thin film of water. That’s why it is often called as liquid-liquid chromatography. Principle: The substances are distributed between liquid phases. One phase is the water which present in pores of filter paper and other phase is mobile phase which moves on the paper. The separation of mixture is due to different attraction force towards stationary phase (water) and mobile phase (solvents). Types of paper chromatography: The classification is based on the procedure of the development of chromatogram on the paper. So there are five types of paper chromatography.  Ascending chromatography
  • 8. INTRODUCTION AND APPLICTIONS OF CHROMATOGRAPHY 8  Descending chromatography  Ascending- descending mode  Radial mode  Two dimensional chromatography Mostly ascending type or radial type chromatography is used because they are easy to handle and not time consuming. They give chromatogram with fast speed. b. Gas chromatography (GC): o Gas-liquid chromatography (GLC): In GLC the mobile phase is a gas and stationary phase is a thin layer of a non-volatile liquid bound to a solid support thus a partition process occurs. In such case small inert particles such as Diatomaceous earth is coated with the liquid so that a large surface area exists for the solute to equilibrate with. o Gas-solid chromatography (GSC): GSC utilizes a solid adsorbent as the stationary phase while gas as a mobile phase and an adsorption process takes place. The separation method can be affected by the polarity of stationary phase, temperature, carrier gas flow, length of column, material amount etc. 6. Other types of Chromatography: i. Affinity Chromatography: This is the most specific type of chromatography. It is based on the specific interaction between two molecules. The one is solute molecule and a second molecule is immobilized on a stationary phase. For example, the immobilized molecule can be an antibody which interacts on the particular area of protein. When a solute with mixture of proteins is passed by immobilized molecule, only the particular protein is reacted to this antibody. Thus the antibody binds to the stationary phase. After this interaction, the extraction of protein is completed by altering its ionic strength or pH. The packing material is called the affinity matrix. It should be inert. ‘Agarose’ is used for this purpose. ii. Countercurrent Chromatography (CCC): Countercurrent chromatography (CCC), the liquid-liquid chromatography, technique, is much less widely known and used than solid liquid chromatography. This technique uses two immiscible liquids to form the stationary and mobile phase of a chromatographic system. In all CCC systems, the stationary phase is held and maintained by gravitational
  • 9. INTRODUCTION AND APPLICTIONS OF CHROMATOGRAPHY 9 force. All modern, commercially manufactured CCC instruments use high gravitational force fields and are essentially centrifuges. 7. Quantification of Chromatogram: The components elute from the column can be quantified by a detector and/or collected for further analysis. An analytical instrument can be combined with a separation method for on- line analysis. Examples of such "hyphenated techniques" include gas and liquid chromatography with mass spectrometry (GC-MS and LC-MS), Fourier-transform infrared spectroscopy (GC-FTIR), and diode-array UV-VIS absorption spectroscopy (HPLC-UV-VIS). 8. Visualization of the Spots of Chromatogram: It can be done by following method, o Use of fluorescent material like manganese with activated zinc silicate o Spray of Iodine vapors o UV exposure o Ninhydrin for identification of amino acids 9. Applications of chromatography: o It is commonly used technique for the separation of molecule. For example, it is used to remove pesticides and insecticides like DDT in the water and poly chlorinated biphenyls. o It is widely used to determine the purity of a substance. o In pharmaceutical companies, it is used for producing pure materials for medicines and also for checking the contamination presence in medicines. o It is used in pharmacy for detecting the chiral compounds (Enantiomers and optical isomers). o In the food industry, this technique is very useful for analyzing and the separation of additives, proteins and amino acids etc. o It used in forensic science for detecting the presence of drugs. o Chromatography has been extensively used in the isolation, characterization and determination of the 60 or so carotenoids at present known. 10. References 1. M. Younus, Ilmi Kitab Khana, 2011. Organic Spectroscopy And Chromatography, Chromatograph, pg. 269, ed. 3rd . 2. Scott, R. P. W. Introduction to Analytical Gas Chromatography; 2nd ed.; Marcel Dekker, 1998. 3. Jennings, W. G.; Mittlefehldt, E.; Stremple, P. Analytical Gas Chromatography; 2nd ed.; Academic Press, 1997. 4. McNair, H. M.; Miller, J. M. Basic Gas Chromatography; Wiley, 1997. 5. Grant, D. W. Capillary Gas Chromatography; Wiley, 1996. 6. Fowlis, I. Gas Chromatography; 2nd ed.; Wiley, 1995.
  • 10. INTRODUCTION AND APPLICTIONS OF CHROMATOGRAPHY 10 7. Scott, R. P. W. Techniques and Practices of Chromatography; 2nd ed.; Marcel Dekker, 1995. 8. Grob, R. L. Modern Practice of Gas Chromatography; 3rd ed.; Wiley, 1995. 9. Baugh, P. E. Gas Chromatography: A Practical Approach; Oxford, 1994. 10. Hinshaw, J. V.; Ettre, L. S. Introduction to Open Tubular Column Gas Chromatography; Advanstar, 1994. 11. Grob, K. Split and Splitless Injection in Capillary Gas Chromatography; 3rd ed.; Hüthig, 1993. 12. Hill, H. H.; McMinn, D. G. Detectors for Capillary Chromatography; Wiley, 1992. 13. Grob, K. On-Column Injection in Capillary Gas Chromatography; 2nd ed.; Hüthig, 1991. 14. Poole, C. F.; Poole, S. K. Chromatography Today; Elsevier, 1991. 15. Baars, B.; Schaller, H. Fehlersuche in der Gaschromatographie; VCH, 1994. 16. Kolb, B. Gaschromatographie in Bildern; Wiley-VCH, New York, 1999. 17. Kenndler, E.; Huber, J. F. K. In Analytiker Taschenbuch; Springer, 1989