chromatography and its types

chromatography,history,major types of chromatography,principle,working,advantages and disadvantages

1. Presentation of “Biochemistry 2” Presented by :- Asifa Bibi Department :- “ Bs.Biotechnology ” Topic :- “Chromatography”

2. TOPIC OF PRESENTATIOn CHROMATOGRAPHY AND ITS TYPES

3. CHROMATOGRAPHY Chromatography is an analytical technique commonly used for separating a mixture of chemical substances into its individual components, so that the individual components can be thoroughly analyzed. I t is also defined as the process of separation of the individual components of a mixture based on their relative affinities towards stationary and mobile phases.

4. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase.

5. Its name is derived from two words: "chromo" meaning colour , and " graphy " meaning writing. in other words, colour bands are formed in the procedure, which are then measured or analysed . these bands are due to separation of individual compounds at different lengths on the column, as seen in column chromatography and on paper in paper chromatography. Origin of name:-

6. T he word “chromatography” was firstly coined by the italian -born scientist M ikhail tsvet in 1903 in russia . H e continued to work with chromatography in the first decade of the 20th century, primarily for the separation of plant pigments C hromatography technique developed substantially as a result of the work of archer john porter martin and richard laurence millington synge during the 1940s and 1950s. Histroy :-

7. C hromatography is based on the principle of separation of compounds into different bands (color graphs) and then identification of those bands. B asically, the samples are subjected to flow by mobile liquid onto or through the stable stationary phase. the sample components are separated into fractions based on their relative affinity towards the two phases during their travel. Thefraction with a greater affinity to stationary layer travels slower and at a shorter distance, while that with a lesser affinity travels faster and longer Principle:-

8. Types Of chromatography Types of chromatography to be discussed:- Paper Chromatography Column Chromatography Gel Filtration Chromatography Ion Exchange Chrmoatography Affinity Chromatography

9. P aper chromatography is an analytical chemistry technique for separating and identifying color mixtures. S ubstances are distributed between stationary phase and a mobile phase. Stationary phase is usually a piece of filter paper and mobile phase is colors that travels up stationary phase. C omponents of the samples will separate readily according to how strongly they absorb on the stationary phase vs. how readily they dissolve in the mobile phase. Paper Chromatography:

10. 1.Paper Chromatography

11. T he discovery of paper chromatography in 1943 by M artin and S ynge provided, for the first time, the means of surveying constituents of plants and for their separation and identification. there was an explosion of activity in this field after 1945. History:-

12. T he principle involved is partition chromatography wherein the substances are distributed or partitioned between liquid phases. one phase is the water, which is held in the pores of the filter paper used; and other is the mobile phase which moves over the paper. the compounds in the mixture get separated due to differences in their affinity towards water (in stationary phase) and mobile phase solvents during the movement of mobile phase under the capillary action of pores in the paper. Principle:-

13. T he principle can also be adsorption chromatography between solid and liquid phases, wherein the stationary phase is the solid surface of paper and the liquid phase is of mobile phase. but most of the applications of paper chromatography work on the principle of partition chromatography, i.e. partitioned between to liquid phases. Principle:-

14. Ink is a solution containing a number of different molecules. These different molecules have different characteristics such as size and solubility. Solubility is a molecule's ability to dissolve in a particular solvent such as alcohol, water or nail polish remover. Because of their different characteristics, each molecule travels at a different speed when pulled along the piece of paper toweling by the solvent. The lightest particles, which are not necessarily the lightest coloured particles, move more quickly and a greater distance than the heavier particles. Thus, all of the pigments that make up an ink sample are separated out. What Happen In paper chromatography:-

15. P aper chromatography is specially used for the separation of a mixture having polar and non-polar compounds. separation of amino acids. Determine organic compounds, biochemical in urine I n the pharma sector it is used for the determination of hormones, drugs, etc. S ometimes it is used for evaluation of inorganic compounds like salts and complexes Uses And applications:-

16. 2 .“Column Chromatography”

17. Column chromatography is a preparative technique used to purify compounds depending on their polarity or hydrophobicity. In column chromatography, a mixture of molecules is separated based on their differentials partitioning between a mobile phase and a stationary phase. Stationary phase:- is confined to a glass or plastic tube and is mostly silica gel Mobile phase:- (a solvent or buffer) is allowed to flow through the solid adsorbent. Sample:- to be analyzed is layered on top of the column. “2.Column chromatography”

18. Russian- italian botanist M ikhail T svet . Tsvet applied his observations with filter paper extraction to the new methods of column fractionation for separating the components of petroleum. “ HIStory ”

19. The rate at which the components of a mixture are separated depends on the activity of the adsorbent polarity of the solvent. If the activity of the adsorbent is very high and polarity of the solvent is very low, then the separation is very slow but gives a good separation. On the other hand, if the activity of adsorbent is low and polarity of the solvent is high the separation is rapid but gives only a poor separation, “principle”

20. “procedure” Packing the column Loading the column Eluting The column Collecting the Eluent Detection of Eluting Components

21. ♦ Column chromatography is best suited to separate active principle from plant materials. ♦ In separation of compounds after organic synthesis to obtain desired molecule. ♦ To separate or purify natural compound mixtures like alkaloids, glycosides. “applications”:-

22. 3.“Gel Filtration Chromatography”

23. The chromatographic methods discussed up to this point allow the separation of molecules according to size.The method of gel-exclusion chro - matography (also called gel filtration, molecular-sieve chromatography, or gel- permeation chromatography) Stationary phase consists of beads of porous polymeric materials(gel beads). Mobile Phase will be a solvent “Gel filtration”

24. The technique was invented by grant H enry lathe and C olin R ruthven , working at queen charlotte’s hospital, london.They later received the john scott award for this invention. “History”

25. Analytes larger than the pores cannot enter the interior of the gel beads, so they are limited to the space between the beads. As a result, they are not slowed in their progress through the column and elute rapidly in a single zone. Small molecules capable of diffusing in and out of the beads have a much larger volume available to them. Therefore, they are delayed in their journey through the column bed. Molecules of intermediate size migrate through the column at a rate somewhere between those for large and small molecules. “Principle”

26. 1. Select columns not greater than 100 cm in length. 2. Selection the proper gel, which act as stationary phase 3.Layer sample on the column. 4.Add buffer to wash proteins through the column. 5. Eluting the sample 6. At th end collect the fraction. “How Does It works?”

27. Fractionation of molecules and complexes within a predetermined size range Size analysis and determination Removal of large proteins and complexes Desalting Removal of small molecules Assessment of sample purity purification. molecular weight determination and quantitative analysis of molecular interactions. “applications”:-

28. Ion exchange chromatography

29. 4 .“Ion Exchange Chromatography”

30. Allows the separation of ions and polar molecules based on the charge properties of the molecules. A type of column chromatography  Useful in the separation of charge compounds like proteins differing by only one charged amino acid. Stationary phase (ion exchanger components) Mobile phase (buffered sollution ) Sample ( positive/negative) “Introduction”:-

31. Components of Ion exchanger :- 1-Resin Polymers that are capable of exchanging particular ions within the polymer with ions in a solution that is passed through them A-polystyrene: polymerisation reaction Of styrene and divinylbenzene , useful For separating small molecular weight Compounds B-cellulose: readily obtained in a high Pure state,greater permeability to Macromolecules “Component of ion exchange”:-

32. 2-Exchange Medium The choice of ion exchangers depends upon the stability, molecular weight, and ionic strength of the sample components Volume of exchanger 2.5 fold greater than to exchange with the ion in the sample Anion exchangers Retains positively charged cations Because the stationary phase displays A negatively charged group Cation exchangers Retains anions using positively charged group “Component of ion exchange”:-

33. Principle:- Ion exchange chromatography relies on the attraction between oppositely charged stationary phase, known as an ion exchanger, and analyze. To these covalently bound functional groups the oppositely charged ions are bounded (mobile counter ion), which will be exchanged with like charge ions in the sample having charge magnitude more than the ions bounded to the matrix. Thus if anion exchange chromatography is performed, negatively charged sample components will interact more with the stationary phase and will be exchanged for like charged ions already bounded to the matrix. “principle”:-

34. Types of ion exchange Cation exchange chromatography Positively charged molecules are attracted to a negatively charged solid support. Commonly used cation exchange resins are s-resin, sulfate derivatives; and CM resins, carboxylate derived ions “Types of ion exchange chromatography”:-

35. Anion exchange chromatography Negatively charged molecules is attracted to a positively charged solid support. Commonly used anion exchange resins are q-resin and DEAE resin “Types of ion exchange chromatography”:-

36. Procedure Step1 A sample introduced into a sample Loop of known volume Step 2 “Set the column ( equlibrium phase)” The mobile phase carries the sample From the loop onto a column that contains Some form of stationary phase material Stationary phase material is a resin or gel matrix consisting of cellulose beads with covalently bonded charged functional groups. “ procedue ”:-

37. Step3 “ sample application into the column” The target analytes (anions or cations ) are retained on the stationary phase Step4 “elution” When we increased the concentration of a similarly charged species it will displace the analyte ions from the stationary phase. The analytes of interest must then be detected by some means, typically by conductivity or uv /visible light absorbance. Step5 “regeneration” Cation exchanger = by acid then washed with water Anion exchanger =by alkali then washed with water

38. Complete overview

39. Applications of IEC 1-Separating proteins 1. Separating the proteins Separates proteins according to their net charge, which is dependent on the composition of the mobile phase(by adjusting the ph or the ionic concentration) Proteins are charged molecules. At specific ph , it can exist in anionic (-), cationic (+) or zwitterion (no net charge) stage. cationic pH = pI anionic pH increase “applications”:-

40. 2. Softening of hard water Hardness due to the presence of ca 2+ .Mg 2+ Removed by passing hard water to cation exchanger changed with na + nar+nca 2+ car + nna + Resin hard water resin solution Canr 2 + 2nna + nca 2+ +2nanr Resin solution resin regenerated “applications”:-

41. Separation of similar ions from one another antibody purification monoclonal antibodies Hemoglobins seperation Separation of vitamins Separation of inorganic cations and anions “applications”:-

42. Also known as bioselective adsorption It is a protein purification technique developed thirty years ago. It is the isolation of a particular protein on a bioligand that is attached to an inert matrix by a spacer arm. It relies on the specificity of a protein binding site for a particular ligand “introduction”:-

43. 1930s,first developed by A.W tiselius -a swedish biochemist won the noble price in 1948 Used to study enzymes and other proteins. “history”:-

44. Specificity is based on three aspects: Matix : for ligand attachment Spacer arm : bind ligand to matrix Ligand : bind with protein to purify it “specificity”:-

45. Principle of affinity chromatography is that the matrix combines with the specific ligand due to specific functional groups present on matrix . as the mixture of proteins is passed through the chromatography column, those proteins that have a binding site for the immobilised ligand will bind to the matrix, while all other proteins will be eluted from the column. “principle”:-

46. Step 1 Attachment of ligand to matrix Step 2 Attachment of proteins to complex Step 3 After washing proteins that are unable to bind separated Step 4 Finally elution occurs and protein is purified “procedure”:-

47. “Examples”:-

48. Advantages Purify and concentrate protein Used in genetic engineering Production of vaccines The binding sites of biological molecules can be investigated “advantages”:-

49. Expensive ligands Non-specific adsorption Relatively low productivity “disadvantages”:-