Chromatography by narayan sarkar and simi baruah

chromatography basic principle, types and procedure ,application of paper , column and thin layer chromatography
NARAYAN SARKAR 2021, MSC BOTANY

1. CHROMATOGRAPHY Presented by – Simi Baruah Roll No-18 Narayan Sarkar Roll No-6 M.Sc. 3 rd semester Guided by- Dr. Nabanita Bhattacharyya Assistant Professor Dept. of Botany

2. CONTENTS INTRODUCTION HISTORY PRINCIPLE TYPES OF CHROMATOGRAPHY COLUMN CHROMATOGRAPHY PAPER CHROMATOGRAPHY THIN LAYER CHROMATOGRAPHY CONCLUSION

3. INTRODUCTION Chromatography is a Greek word ( colour writing), chroma =“ colour ” & Graphein = “to write ” It is the collective term for a set laboratory techniques for the separation of mixtures. Definition: Chromatography is a technique for the separation of a mixture by passing it in solution or suspension through a medium ,in which the components move at different rates.

4. HISTORY OF CHROMATOGRAPHY To write with colors -- literally translated from its Greek roots   chroma   and   graphein Chromatography: Russian botanist Mikhail Tswett in 1903 , first developed this technique. It has since developed into an invaluable laboratory tool for the separation and identification of compounds.

5. PRINCIPLE OF CHROMATOGRAPHY

6. Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is separating from each other while moving with the aid of a mobile phase. The factors: Molecular characteristics related to Adsorption (liquid-solid), Partition (liquid-solid) , and Affinity or differences among their molecular weights.

7. Because of these differences, some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into the mobile phase, and leave the system faster.

8. Basis of the chromatography technique: Stationary phase :  This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on the surface solid support”. Mobile phase :  This phase is always composed of “liquid” or a “gaseous component.” Separated molecules: The type of interaction between the stationary phase, mobile phase, and substances contained in the mixture is the basic component effective on the separation of molecules from each other.

9. Eluent : An eluent is a solvent used to carry the components of a mixture through a stationary phase. It is alternative term used for the mobile phase. Eluate : The mobile phase that exits the column is termed as eluate . Elution : The process in which solutes are washed through a stationary phase by the movement of a mobile phase .

10. TYPES OF CHROMATOGRAPHY

11. 1)Adsorption chromatography Separation is based on differences between the adsorption affinities of the sample components for the surface of an active solid stationary phase. 2)Partition chromatography It is a type of chromatography in which the components of the mixture get distributed into the two phases due to differences in partition coefficients( Kd ),which is the ratio of the concentration of solutes in two phases . Kd = concentration of solute in phase A concentration of solute in phase B The distribution of solutes between two phases is based on solubility differences .

12. 3)Ion exchange chromatography : It is applicable for the separation of charged molecules.

13. 4) Size exclusion chromatography : It separates molecules on the basis of size and shape.A column matrix filled with porous gel beads made of insoluble and hydrated polymer such as polyacrylamide or agarose acts as stationary phase.

14. PAPER CHROMATOGRAPHY What Is Paper Chromatography? It was discovered by Synge and Martin in the year 1943. It is the technique that uses paper sheets or strips as the adsorbent being the stationary phase through which a solution is made to pass. Inexpensive method of separating dissolved chemical substances by their different migration rates. Powerful analytical tool that uses very small quantities of material.

15. PAPER CHROMATOGRAPHY PRINCIPLE It is a partition chromatography technique Cellulose paper is a supporting medium over which the solvents flow. Water bound to the polar cellulose is the stationary phase and organic solvent which flows over it is a mobile phase. Organic solvent moves over the hydrated cellulose fibres As the solvent passes through an area of paper containing a solute (mixture of components), the solute begins to partition itself between the aqueous and organic phases in proportion to its relative solubility in the two phases

16. The components of the solute more soluble in organic phase will be carried faster along the organic phase. Conversely, greater the affinity for water, slower the solute will move with respect to the solvent front. Thus if several compounds possess different solubility rates, each will move across the paper at specific rate which is generally different from that of any other compound

17. The distance the solute moves, in relation to the distance the solvent moves, serves as a means of identifying the solute and is called R f .

18. PROCEDURE OF PAPER CHROMATOGRAPHY Selecting a suitable type of development:   It is decided based on the complexity of the solvent, paper, mixture, etc. Usually ascending type or radial paper chromatography is used as they are easy to perform Selecting a suitable filter paper : Selection of filter paper is done based on the size of the pores and the sample quality. Prepare the sample :   Sample preparation includes the dissolution of the sample in a suitable solvent (inert with the sample under analysis) used in making the mobile phase

19. Spot the sample on the paper :   Samples should be spotted at a proper position on the paper by using a capillary tube. Chromatogram development :   Chromatogram development is spotted by immersing the paper in the mobile phase. Due to the capillary action of paper, the mobile phase moves over the sample on the paper. Paper drying and compound detection : Once the chromatogram is developed, the paper is dried using an air drier. Also, detecting  solution  can be sprayed on the chromatogram developed paper and dried to identify the sample chromatogram spots.

20. Paper Chromatography Applications Some of the uses of Paper Chromatography in different fields are discussed below: 1)To study the process of fermentation and ripening. 2 )To check the purity of pharmaceuticals. 3)To inspect cosmetics. 4)To detect the adulterants. 5)To detect the contaminants in drinks and foods. 6)To examine the reaction mixtures in biochemical laboratories. 7)To determine dopes and drugs in humans and animals.

21. COLUMN CHROMATOGRAPHY What Is Column Chromatography? This method is a type of  adsorption chromatography  technique. It is a technique which is used to separate a single chemical compound from a mixture dissolved in a fluid. It separates substances based on differential adsorption of compounds to the adsorbent as the compounds move through the column at different rates which allow them to get separated in fractions. This technique can be used on a small scale as well as large scale to purify materials that can be used in future experiments.

22. Principle of column chromatography When the mobile phase along with the mixture that needs to be separated is introduced from the top of the column, the movement of the individual components of the mixture is at different rates The components with lower adsorption and affinity to stationary phase travel faster when compared to the greater adsorption and affinity with the stationary phase. The components that move fast are removed first whereas the components that move slowly are eluted out last.

23. The adsorption of solute molecules to the column occurs in a reversible manner. The rate of the movement of the components is expressed as: R f   = the distance travelled by solute the distance travelled by the solvent R f   is the retardation factor .

24. Column Chromatography Procedure Mobile phase – This phase is made up of solvents and it performs the following functions: It acts as a solvent – sample mixture can be introduced in the column. It acts as a developing agent – helps in the separation of components in the sample to form bands Some examples of solvents used as mobile phase based on their polarity are – ethanol, acetone, water,  acetic acid , pyridine, etc. Stationary phase – It is a solid material which should have good adsorption property.

25. The stationary phase is made wet with the help of solvent as the upper level of the mobile phase and the stationary phase should match. In the first step the compound mixture that needs to be separated, is added from the top of the column without disturbing the top level . Without disturbing the stationary phase solvent mixture is added slowly by touching the sides of the glass column. 

26. The tap is turned on to initiate the movement of compounds in the mixture. The movement is based on the polarity of molecules in the sample. The non-polar components move at a greater speed when compared to the polar components.

27. For example, a compound mixture consists of three different compounds viz red , blue, green then their order based on polarity will be as follows blue > red > green As the  polarity  of the green compound is less, it will move first. When it arrives at the end of the column it is collected in a clean test tube. After this, the red compound is collected and at last blue compound is collected. All these are collected in separate test tubes .

28. Column Chromatography Applications Column Chromatography is used to isolate active ingredients. It is very helpful in Separating compound mixtures. It is used to determine drug estimation from drug formulations It is used to remove impurities. Used to isolation metabolites from biological fluids.

29. THIN LAYER CHROMATOGRAPHY What Is Thin Layer Chromatography? It is a technique used to isolate non-volatile mixtures. The experiment is conducted on a sheet of aluminium foil, plastic, or glass which is coated with a thin layer of adsorbent material. The material usually used is   aluminium oxide , cellulose , or silica gel . On completion of the separation, each component appears as spots separated vertically.

30. Each spot has a retention factor ( R f ) expressed as: R f   = dist. travelled by sample dist. travelled by solvent The factors affecting retardation factor are the- solvent system, amount of material spotted, absorbent and temperature.

31. Thin Layer Chromatography Principle Like other chromatographic techniques, thin-layer chromatography (TLC) depends on the separation principle. The separation relies on the relative affinity of compounds towards both the phases. The compounds in the mobile phase move over the surface of the stationary phase.

32. The movement occurs in such a way that the compounds which have a higher affinity to the stationary phase move slowly while the other compounds travel fast. On completion of the separation process, the individual components from the mixture appear as spots at respective levels on the plates. Their character and nature are identified by suitable detection techniques.

33. Thin Layer ChromatographyExperiment The stationary phase that is applied to the plate is made to dry and stabilize. To apply sample spots, thin marks are made at the bottom of the plate with the help of a pencil. Apply sample solutions to the marked spots. Pour the mobile phase into the TLC chamber and to maintain equal humidity, place a moistened filter paper in the mobile phase. Place the plate in the TLC chamber and close it with a lid. It is kept in such a way that the sample faces the mobile phase.

34. Immerse the plate for development. Remember to keep the sample spots well above the level of the mobile phase. Do not immerse it in the solvent. Wait till the development of spots. Once the spots are developed, take out the plates and dry them. The sample spots can be observed under a UV light chamber .

35. Thin Layer Chromatography Applications 1) The qualitative testing of Various medicines such as sedatives, local anaesthetics , anticonvulsant tranquilisers , analgesics, antihistamines, steroids, hypnotics is done by TLC . 2) TLC is extremely useful in Biochemical analysis such as separation or isolation of biochemical metabolites from its blood plasma, urine, body fluids, serum, etc. 3) Thin layer chromatography can be used to identify natural products like essential oils or volatile oil, fixed oil, glycosides, waxes, alkaloids, etc.

36. 4) It is widely used in separating multicomponent pharmaceutical formulations. 5) It is used to purify of any sample and direct comparison is done between the sample and the authentic sample. 6) It is used in the food industry, to separate and identify colours ,  sweetening agent , and preservatives 7) It is used in the cosmetic industry. 8) It is used to study if a reaction is complete .

37. Thin–layer chromatography offers several benefits over the paper chromatographic separations. Some of the benefits are: Time Saving The biggest advantage offered to the chromatographer is time- saving. Paper chromatography can take several hours to develop the plate whereas development in thin layer chromatography can be completed in much shorter time (about half an hour or so) Automation Paper chromatography has not seen much automation over the years but thin layer chromatography instruments available have automation capabilities which include autosampler , constant volume sample dispenser, documentation and camera for retaining pictorial record of separations

38. Rigid Support The cellulose paper support in paper chromatography is flexible whereas the adsorbent in TLC is coated onto a rigid metal, glass or plastic plate. This contributes to reproducibility of spots and faster development. Due to support rigidity there is less diffusion and as a result well-defined spots are formed. Choice of Support TLC presents a vast choice of support adsorbent phases including liquid coated adsorbents which can include fluorescence inducers as well. On the other hand choice of papers in paper chromatography is very much limited . Development Chamber Design It is not necessary to suspend the plate as required in paper chromatography from a rod on top of the development chamber. It can be simply placed in a slanting position with its bottom edge resting on the chamber base.

39. Sample Volume The quantity of sample applied is small( in microliters ) and can be reproducibly applied with the help of automated sample dispensers Choice of Spray Reagents Corrosive spray reagents can char the filter paper and can even deteriorate the sample spots. Coated plates used in thin layer chromatography can withstand corrosive spray reagents to a greater extent. Heating Thin-layer chromatography plates can be heated if required for spot development. Paper chromatography sheets cannot withstand heating beyond a point.

40. CONCLUSION Now a days, chromatography is a very popular biophysical technique for the separation of bimolecules from both plants and animals. It is used for the separation of plant pigments such as chlorophyll , xanthophyll etc . Separation of amino acids is done by this method.

41. REFERENCES A text book of plant physiology , biochemistry and biotechnology by Verma and Verma Biophysics and molecular biology by Pranav Kumar https://Byjus.com www.biochemden.com

42. THANK YOU