Dr Debalina Basu Department of Microbiology Surendranath College Paper Chromatography Principle What is Descending Paper Chromatography Descending paper chromatography is an analytical technique in which the mobile phase moves downward through the stationary phase In other words i ID: 1022520
Download Presentation The PPT/PDF document "CHROMATOGRAPHY Presented by" is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
1. CHROMATOGRAPHYPresented by Dr Debalina BasuDepartment of MicrobiologySurendranath College
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14. Paper Chromatography
15.
16. Principle
17.
18.
19.
20.
21.
22.
23.
24.
25. What is Descending Paper Chromatography?Descending paper chromatography is an analytical technique in which the mobile phase moves downward through the stationary phase. In other words, in this method, the development of the paper occurs due to the movement of the solvent downwards on the paper. Therefore, the solvent reservoir should be at the top of the paper. In this process, the movement of the solvent is governed by gravity as well as capillary action.
26. Two-dimensional chromatography is a type of chromatographic technique in which the injected sample is separated by passing through two different separation stages. Two different chromatographic columns are connected in sequence, and the effluent from the first system is transferred onto the second column.What is 2-D paper chromatography?The purpose of employing this technique is to separate mixtures that one dimensional liquid chromatography otherwise cannot separate effectively. Two dimensional liquid chromatography is better suited to analyzing complex mixtures samples such as urine, environmental substances and forensic evidence such as blood.
27.
28.
29.
30. What is column chromatography?Column chromatography is described as the useful technique in which the substances to be isolated are presented onto the highest point of a column loaded with an adsorbent (stationary phase), go through the column at various rates that rely upon the affinity of every substance for the adsorbent and the solvent or solvent mixture, and are typically gathered in solution as they pass from the column at various time. The two most common examples of stationary phases for column chromatography are silica gel and alumina while organic solvents are regarded as the most common mobile phases.
31. Column Chromatography PrincipleThe main principle involved in column chromatography is the adsorption of the solutes of the solution with the help of a stationary phase and afterward separates the mixture into independent components.At the point when the mobile phase together with the mixture that requires to be isolated is brought in from the top of the column, the movement of the individual components of the mixture is at various rates.The components with lower adsorption and affinity to the stationary phase head out quicker when contrasted with the greater adsorption and affinity with the stationary phase. The components that move rapidly are taken out first through the components that move slowly are eluted out last. The adsorption of solute molecules to the column happens reversibly. The pace of the movement of the components is communicated as:Rf = the distance traveled by solute/ the distance traveled by the solvent Where Rf is called retardation factor
32.
33. Column chromatography ProcedureThe steps included in the column chromatography are:Preparation of the columnMostly the column is comprised of a glass tube with an appropriate stationary phaseThe bottom end of the column is packed with a glass wool/cotton wool or an asbestos pad after which the stationary phase is packed.After packing the column, a paper disc is placed on the top to avoid the disturbance of the stationary phase during the introduction of the sample or mobile phase.The disturbance in the stationary phase (adsorbent layer) leads to the irregular bands of separation. Two types of preparing the column, known as packing techniques namely:Dry packing technique – The amount of absorbent needed is added as a fine dry powder in the column and the solvent flows freely through the column until equilibrium is achieved.Wet packing technique – The slurry of adsorbent is prepared along with the mobile phase and is poured into the column.It is regarded as the ideal technique for packaging.The column should be properly washed and completely dried before in-use.
34. Elution techniqueThrough this technique, the individual components are separated completely from the column.The process of elution can be carried out by employing two techniques:Isocratic elution technique – Throughout the procedure, a solvent of the same polarity or same solvent composition is utilized.Example: Use of chloroform aloneGradient elution technique – Throughout the separation procedure, solvents of gradually increased polarity or increased elution strength are utilized.Example: Benzene → Chloroform → Ethyl acetate → ChloroformDetection of ComponentsIn case the mixture separated in a column chromatography procedure are colored compounds, then monitoring the separation progress is simple.In case the compounds undergoing separation are colorless, then small fractions of the eluent are sequentially collected in tubes that are labeled. Thorugh TLC, the composition of each fraction is determined.Introduction of the sampleThe sample (a mixture of components) is dissolved in the minimum amount of the mobile phase.At one instant, the sample is introduced into the column and on the top portion of the column, it is absorbed.Through the elution process, the individual sample can be isolated from this zone.
35. Types of Column chromatographyAdsorption column chromatography – Technique of separation in which compounds to be separated (solute) is retained or adsorbed on the surface of the adsorbent (stationary phase).Partition column chromatography – It is based on the variance in partition coefficient of the individual components of the mixture, where the stationary phase and the mobile phase both are in the liquid state.Gel column chromatography – Here, the separation is carried out through a column packed with gel and possesses a porous stationary phase. It is also referred to as size exclusion chromatographyIon exchange column chromatography – The basis relies on the charge of the molecules. The separation is done when molecules get attracted to the oppositely charged stationary phase.
36. Column chromatography usesColumn chromatography is one of the versatile methods for purifying and separating both solids and liquids. Major applications:To isolate active constituentsTo separate compound mixturesTo remove impurities or carry purification processTo isolate metabolites from biological fluidsTo estimate drugs in drug formulations or crude extracts
37.
38. Affinity chromatography 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.
39. The technique involves use of ligands covalently attached to inert and porous matrix in a column. It is mainly based on the biological affinity (or) biological specificity. The materials to be isolated are capable of binding reversibly to a specific ligand i.e., attached to an insoluble matrix.The matrix should be inert to other molecules to minimize non specific adsorption . The immobilized ligands acts as molecular hooks to selectively pickup the desired protein while the remaining proteins pass through the column. Alternatively some reagents that can break protein ligand interaction can also be employed for the separation .
40.
41.
42. The Advantages of Affinity Chromatography are: High sensitivity . Affinity chromatography is used in the production of vaccines. Affinity chromatography is used in the purification of protein and enzyme. Affinity chromatography gives High specificity. Enzymes and other proteins are studied by affinity chromatography. To maintain the quality of the product, this chromatography is used in the pharmaceutical manufacturer in the production of vaccines. Affinity chromatography doesn’t rely on ionic strength, pH, temperature, and composition of the buffer. The high degree of purity can be obtained by Affinity Chromatography. This is a very reproducible process. This is the simplification method. Used to increase the solubility. In genetic engineering, affinity chromatography is used.
43.
44. The Disadvantages of Affinity Chromatography are:It takes a lot of skill to handle it.It interferes with the structure.Transfer and the leakage of metal ion lead to protein loss.Sometimes ligands leakage is observed.The volume of the sample is limited.The carrier gas used must be pure such as pure nitrogen.The ligands used in affinity chromatography are costly.Relatively low productivity.It has a non-specific adsorption.Degradation of the solid support.Metal-ion transfer and metal ion leakage lead to loss of protein.Affinity chromatography is non-specific for adsorption than other chromatography methods.
45. Gel filtration is a technique of partition chromatography in which the partitioning is based on the molecular size of the substances to be separated.Size-exclusion chromatography (SEC) is also known as gel permeation chromatography (GPC) or gel filtration chromatography and separates molecules according to their size, shape & molecular weight. It is also referred to as molecular sieving or molecular exclusion chromatography. Smaller molecules are able to enter the pores of the media and, therefore, molecules are trapped and removed from the flow of the mobile phase. However, molecules that are larger than the average pore size of the packing are excluded and thus suffer essentially no retention; such species are the first to be eluted. This is how the molecules are separated. It is generally a low-resolution chromatography technique and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining the tertiary structure and quaternary structure of purified proteins, especially since it can be carried out under native solution conditions.GEL FILTRATION CHROMATOGRAPHY
46.
47. Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is a separation technique in which the mobile phase is a gas.It is the method of choice for the separation of volatile substances or the volatile derivatives of certain non-volatile substances. Stationary phase is an inert solid material impregnated with a non-volatile liquid. In gas chromatography, a sample is rapidly heated and vaporized at the injection port. The sample is transported through the column by a mobile phase consisiting of an inert gas. Sample components are separated based on their boiling points and relative affinity for the stationary phase, which is most often a viscous liquid (wax) within the column. The higher a component's affinity for the stationary phase, the slower it comes off the column. The components are then detected and represented as peaks on a chromatogram.GAS-LIQUID CHROMATOGRAPHY
48. The mixture of volatile material is injected into the column along with the mobile phase. The separation of the volatile mixture is based on the partition of the components between the mobile phase(gas) and stationary phase (liq.), hence the name GAS-LIQUID CHROMATOGRAPHY. It is well suited for use in the petrochemical, environmental monitoring and industrial chemical fields. Sensitive, rapid and reliable.
49. Ion exchange chromatography (usually referred to as ion chromatography) uses an ion exchange mechanism to separate molecules on the basis of their electrical charges.Ion exchange chromatography uses a charged stationary phase to separate charged compounds including anions, cations, amino acids, peptides, and proteins.Cation exchangers & anion exchangers are used as ion exchange resins. In conventional methods the stationary phase is an ion exchange resin that carries charged functional groups that interact with oppositely charged groups of the compound to retain.ION EXCHANGE CHROMATOGRAPHY
50.
51.
52.
53.
54. Application
55.
56. Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or a plane. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as high performance liquid chromatography (HPLC).The chromatographic techniques are slow & time consuming, hence the separation can be greatly improved by using high pressure in the range of 5000-10000 psi(pounds per square inch),hence this technique is also referred to as high pressure liquid chromatography. In HPLC the sample is forced by a liquid at high pressure (the mobile phase) through a column that is packed with a stationary phase composed of irregularly or spherically shaped particles. The interaction between the mobile and the stationary phase leads to the separation of the mixture.HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC )
57.
58.
59. Advantages of HPLC • Seapartion of voltile and non- voltile components.• Thermally unstable compounds isolated.• Quick analysis• High resolution• Less cumbersome• More reproducibilityDisadvantages of HPLC are• Tedious to detect co-elution.• High cost.• Complex to operate.
60.