Chromatography

Chromatography and its types

1. Paper Chromatography

2. Introduction First introduced by the German scientist Christian Friedrich Schonbien in 1865. It is a type of simplest Chromatography. It is the most simplest and widely used type of chromatography procedures which runs on specialized paper.

3. Definition Analytical method used to separate colored chemicals and substances. Applicability to isolation, identification, and Quantitative determination of organic and inorganic compounds.

4. Two Principles of Paper Chromatography 1) . Paper Partition Chromatography . Paper impregnated with alumina and silica act as absorbent ( stationary phase ) and solvent as mobile phase. 2).Paper Adsorption Chromatography . The moisture or water present in pores of cellulose fibers present in filter paper act as stationary phase and another solvent as mobile phase.

5. Principle of separation Principle of separation is mainly partition rather than adsorption. Types of paper Chromatography 1). Ascending 2). Descending 3). Ascending-Descending mode. 4). Radial mode 5). Two-Dimensional chromatography

6. Ascending Chromatography As the name indicates ,the chromatogram ascends .Here the development of paper occurs due to the solvent movement or travel in upward direction on the paper. Descending Chromatography Development of papers occurs due to solvent travels downward on the papers.

7. Ascending Descending mode In it the solvent first travels upward and then downward on the paper. Radial Mode Here the solvent travels from center (midpoint) towards the periphery of Circular chromatographic paper.

8. Two Dimensional Chromatography Here the chromatogram development occurs in two directions at right angles.

9. Applications Used in forensic studies. In analytical chemistry for identifying and separating colored mixtures like pigments. Sugars, amino acids, lipids and nucleic acids and other biomolecules can be easily identified by spraying with appropriate reagents to detect these specific compounds. Paper chromatography can be reproduced easily as long as the conditions are controlled and maintained.

10. Retention Factor Indicated by Rf = Distance travelled by applicant from application point Distance travelled by solvent from application point

11. APPLICATioNS: Among all the chromatography methods paper chromatography is an inexpensive and rapid method that provides graphic and clear results . Used as a qualitative method for identifying the components in a mixture . Used in several scientific studies in identification of unknown organic and inorganic compounds from a mixture.

12. Column Chromatography Is a method used to purify chemical compounds from mixture of compounds on the basis of their polarity. Large scale application. This is a solid liquid technique in which Stationary phase is a solid Mobile phase is liquid Vertical glass columns is used support the stationary phase

13. P rinciple The principle of column chromatography is adsoption. Mixture of components dissolved in M.P is introduced in column. C omponents move according to their relative affinity. C omponents adsorbed with the statonary phase Weakly adsorbed components move faster separated first

14. Apparatus Column Stopcock Cotton prevent solid materials to pass Sand provide level surface. Stationary phase ( silica , alumina ) Mobile phase ( solvent )

15. Gels Silica and alumina are both polar adsorbents. Silica gel is less polar than alumina. Silicais preferentially retains basic compounds. Alumina preferentially retains acidic compounds. Non Polar compounds  elute first

16. Steps Packing column  Bottom portion of the column is packed with cotton wool. Then sand is added Packing can be either Dry A dd dry solid phase to the column and pass buffer to saturate the solid Wet M ix both liquid and solid outside of the column and pour it into the column.

17. Loading column is loaded using pipette to make sure even layer on top of silica gel. Then mobile phase is poured. Stop cock is opened and solvent start moving along sample which will separat different components according to their relative affinity with stationary phase.   components that strongly interacts with the stationary phase retained in the column thus moves slowly But when the interactions are weak the components elutes easily so, exits first

18. 3. Analyzing   Depend on the type of molecules separated  protein or DNA gel   Plant extracts run a TLC plate

19. Factors Affecting Column Efficiency Dimension of the column (length/width ratio ) Particle size of column packing. Activity of the adsorbent. Temperature of the column. Packing of the column. Quality of solvents (low viscosity )

20. Advantages Any type of mixture can be separated Any quantity of mixture can be separated Wider choice of Mobile Phase Automation is possible

21. Disadvantages Time consuming more amount of Mobile Phase are required Automation makes the techniques more complicated E xpensive

22. High-Performance Liquid Chromatography (HPLC)

23. HPLC HPLC stands for High performance liquid chromatography sometimes referred as High Pressure Liquid Chromatography. HPLC is a powerful tool in analysis because it yields high performance as compared to traditional column chromatography. It can separate mixture of compounds.

24. Conti... High performance liquid chromatography is improved form of column chromatography .   It pumps a sample mixture in a solvent (known as the mobile phase) at high pressure through a column with chromatographic packing material (stationary phase ).  HPLC has the ability to separate, and identify compounds that are present in any sample that can be dissolved in a liquid in trace concentrations.

25. Instrumentation The components use in HPLC system include: the solvent reservoir or multiple reservoirs a high-pressure pump a column injector system the detector.

26. HPLC work A reservoir holds the solvent.  A pump is used to generate a specified flow of the mobile phase.  An injector is able to inject the sample into the continuously flowing mobile phase stream that carries the sample into the HPLC column . The column contains the chromatographic packing material needed to effect the separation . This packing material is known as stationary phase. Then a detector is needed to see the separated compound bands as they elute from the high pressure column.

27. Conti... The information is sent to the computer by the detector, which generates the chromatogram.

28. Mobile phase enters the column from the left and exits at the right. when the sample enters the column and begins to form a band . The sample shown here, a mixture of yellow, red, and blue dyes, appears at the inlet of the column as a single black band .  individual dyes have moved in separate bands at different speeds.

29. As the separated dye bands leave the column, they pass immediately into the detector.  The detector contains a flow cell that  detects each separated compound band.

30. AFFINITY CHROMATOGRAPHY

31. INTRO ... Affinity chromatography separates proteins on the basis of a reversible interaction between a protein and a specific ligand coupled to a chromatography matrix. AC is designed to purify a particular molecule from a mixed sample.

32. The technique can be used to separate active biomolecules from denatured or functionally different forms, to isolate pure substances present at low concentration in large volumes of crude sample and also to remove specific contaminants.

33. Principle ... A ffinity chromatography is principally based on the molecular recognition of a target molecule by a molecule bound to a column. It r elies on the reversible interactions between the protein to be purified and the affinity ligand coupled to chromatographic matrix. Binding is; ** Reversible, & ** Specific.

34. Components ... Wh ile affinity chromatography is used for the purification and separation of large biomolecules from complex mixtures, these components is to be considered; T he support (matrix), S pacer arms, and L igand .

35. Matrix ... chemical inertness ; does not react (or bind) with sample. chemical stability ; r esistance toward microbial and enzymatic attack . Chemical reactivity allowing ligands and spacers to be introduced . Good flow properties for rapid separation Such as; Agarose, Polyacrylamide, Cellulose, and silica.

36. Ligands ... Ligands are ions or neutral molecules attach to the matrix that bond to a biomolecule (protein). The selection of the ligand for affinity chromatography is influenced by two factors: the ligand must exhibit specific and reversible binding affinity for the target substance and it must have chemically modifiable groups that allow it to be attached to the matrix without destroying binding activity.

37. Spacer Arms ... The binding sites of the target molecule are sometimes deeply located and difficult to access due to steric hindrance, a spacer arm is often incorporated between the matrix and ligand to facilitate efficient binding and create a more effective and better binding environment.

38. Steps: Affinity purification involves 3 main steps: Incubation of a crude sample with the affinity support to allow the target molecule in the sample to bind to the immobilized ligand. Washing away non-bound sample components from the support. Elution (dissociation and recovery) of the target molecule from the immobilized ligand by altering the buffer conditions so that the binding interaction no longer occurs.

39. Step 1. Loading affinity column

40. Step 2. Proteins sieve through matrix of affinity beads.

41. Step 3. Proteins interact with affinity ligand with some binding loosely and others tightly.

42. Step 4. Wash off proteins that do not bind and that are loosely bond .

43. Step 5 . Elute proteins that bind tightly to ligand and collect purified protein of interest.

44. Kinds of ELUTION ... Elution by change in buffer composition that elutes the bound substance without harming it . pH Elution; high pH applied that elute the substance but damage it either temporary or permentaly. Competitive elution; by addition of substance that competes for binding to protein with ligand.

45. Ion Exchange Chromatography T he separation of molecules based on their total charge . This technique enables the separation of similar types of molecules that would be difficult to separate by other techniques because the charge carried by the molecule of interest can be readily manipulated by changing buffer pH . Used in separation of proteins.

46. Protein Made of 20 type of Amino Acids Amino acid carry different charges based on their side chains Protein carry net – ive or + ive charge depending on types of AA present Protein differ by charge from one another separated by ion exchange

47. Stationary phase Solid phase Gel beads – 0.30 to 0.85nm in diameter Carbohydrate polymers Cellulose Agarose Fixed and inert Side chains added to make them + ive or – ive charge

48. Stationary phase

49. Anion exchange Matrix X - A + ………… B - Mixture: D- E + Ion Exchange: X - A + ……. D- B- E+ Cation exchange Matrix X – A - ………… C + Mixture: D- E + Ion Exchange: X – A - ……. E+ C+ D- Elute Elute

50. Assembly Funnel Colum filled with gel beads Eluent collected at bottom

51. Process( eg cationic exchange) Mixture of 3 types of protein Coloumn filled with – ively charged resins Pour mixture in funnel Initially they will collect at top After sometime move downward due to gravity

52. The negative protein repels the resin and move fastest, collected at bottom Slightly positive will bind to resin at middle and move downward after sometime Strongly positive will bind at top To seprate bound proteins elution is carried out

53. Elusion Two types Salt gradient PH gradient

54. Salt gradient Principle: increase net + ive ions Salt solution poured in column e.g. NaCl Na+ Ions produced, compete with protein ions to bind with resin Less strongly bound protein elute first Concentration of salt increased and more strongly bound proteins separate Proteins collected at bottom

55. PH Gradient Principle:

56. Mixture of buffer added Binding Buffer, low pH Elusion Buffer, high pH pH gradient produced cationic exchange: low pH to High pH buffer Anionic exchange: high pH to low pH buffer

57. Example of cationic exchange Low pH buffer added first, causes formation of more + ive charge on protein Then high pH buffer added, it generates pH gradient protein moves to region that is equal to their isoelectric point More elusion buffer added that cause increase in pH and protein now carry negative charge Proteins desorb and elute from column