Thin Layer Chromatography by Nitik Kalra.pptx

The powerpoint presentation is about the Thin layer chromatography it's uses and techniques with special reference to amino acids, carbohydrates and organic compounds.

1. ST. ANDREW’S DEGREE COLLEGE NAME: NITIK KALRA CLASS: M.Sc. IV SEMESTER (CHEMISTRY) ROLL NO: 2313097530006

2. Seminar M.Sc. Chemistry (IV SEMESTER) UNDER THE GUIDANCE OF PROF. AMIT MASIH

3. Seminar M.Sc. Chemistry (IV SEMESTER) UNDER THE GUIDANCE OF PROF. M.H. KHAN AND PROF. AMIT MASIH

4. Seminar M.Sc. Chemistry (IV SEMESTER) UNDER THE GUIDANCE OF PROF. AMIT MASIH ARE YOU READY?

5. Table of contents 01 03 02 04 Introduction to Chromatography Thin-Layer Chromatograhy Chemistry behind TLC Application and Uses SO LET’S BEGIN

6. Table of contents 01 02 03 Introduction to Chromatography Thin-Layer Chromatograhy Application and Uses Of TLC

7. Introduction to Chromatography 01

8. What is Chromatography? The term chromatography is derived from Greek, chroma meaning, “color,” and graphene meaning, “to write.” Chromatography is a process for separating components of a mixture. To get the process started, the mixture is dissolved in a substance called the mobile phase, which carries it through a second substance called the stationary phase.

9. What is Chromatography? The term chromatography is derived from Greek, chroma meaning, “color,” and graphene meaning, “to write.” Chromatography is a process for separating components of a mixture. To get the process started, the mixture is dissolved in a substance called the mobile phase, which carries it through a second substance called the stationary phase. Mikhail S. Tsvet

10. Terms related to Chromatography Stationary Phase : substance that stays fixed inside the column Mobile phase : Solvent moving through column Eluent: fluid entering the column. Eluate: fluid exiting the column Analyte: mixture whose individual components have to be separated and analyzed.

11. Terms related to Chromatography Stationary Phase : substance that stays fixed inside the column Mobile phase : Solvent moving through column Eluent: fluid entering the column. Eluate: fluid exiting the column Analyte: mixture whose individual components have to be separated and analyzed.

12. Applications of chromatography The chromatographic techniques is used for separation of amino acids, proteins and carbohydrates. Used for analysis of drugs, hormones, vitamins. Helpful for qualitative and quantitative analysis of complex mixtures. Useful for determination of molecular weight of proteins.

13. Different types of chromatography Paper Chromatography Thin Layer Chromatography Gel Chromatography Column Chromatography Ion exchange Chromatography Gel filtration Chromatography Gas Liquid Chromatography Affinity Chromatography

14. Different types of chromatography Paper Chromatography Thin Layer Chromatography Gel Chromatography Column Chromatography Ion exchange Chromatography Gel filtration Chromatography Gas Liquid Chromatography Affinity Chromatography

15. Thin Layer Chromatography

16. INTRODUCTION TLC is one of the simplest, fastest, easiest and least expensive of several chromatographic techniques. Separation or identification of mixtures of components by using finely divided adsorbent liquid/solid spread over a glass plate and liquid as mobile phase. TLC is a form of liquid chromatography consisting of : A mobile phase (developing solvent). A stationary phase (a glass slide coated with a layer of silica) Separation of adsorbed substances by mobile phase

17. Principle The separation principle of the TLC procedure is based on the given compound’s relative affinity towards the mobile and stationary phase. The mobile phase moves over the stationary phase surface. During this the compounds that are more attracted towards stationary phase secure their positions at lower levels while others move towards higher level resulting in separation.

18. MATERIALS REQUIRED TO PERFORM TLC:- Glass Slide/Plate Watch glass / Jar cover Stationary Phase (Silica Gel SiO2) Mobile Phase Oven Developing Chamber Storage facility for prepared plates.

19. EXPERIMENTAL PROCEDURE TLC plate preparation Activation of TLC plate Spotting the TLC plate Development of plate Visualize the spots.

20. TLC PLATE PREPARATION TLC plates are prepared by mixing the adsorbent (stationary phase) such as silica gel with water to from a slurry. This slurry is spread uniformly on the glass slide creating a layer of uniform thickness. The thickness of the adsorbent layer is typically around 0.5 – 2.0 mm.

21. Activation of TLC Plate Plate is kept for drying in oven at 100 deg Celsius for 30 mins. This step is called activation of plate. By doing this surface area of adsorbent increases. TLC PLATE

22. SPOTTING THE TLC PLATE Take the prepared TLC slide and draw a line with pencil near the bottom of plate. (2cm above) Spot the plate on the marked line using capillary tube In a jar containing the solvent insert the plate vertically, the spot should not be immersed in the solvent. Cover the jar and the solvent will begin to rise up. Remove the plate before the solvent hits the top of plate. APPLY SAMPLE HERE

23. Solvent front Separation of components VISUALIZATION (On application of visualizing reagent)

24. Rf value (Retention factor) It is defined as the ratio of distance travelled by the compound mixtures to the distance travelled by the solvent front. Rf= distance travelled by compound distance travelled by solvent front

25. Rf value (Retention factor) It is defined as the ratio of distance travelled by the compound mixtures to the distance travelled by the solvent front. The Rf value is always less than one. Rf= distance travelled by compound distance travelled by solvent front

26. Advantages of TLC Separation is sharper in TLC. TLC is much more rapid. Non volatile compounds can be separated. It is more accurate. The components of complex mixtures easily separate.

27. Fields of application of TLC Biomedical use Pharmacy Food industry 30% 18% 25% 12% Cosmetics

28. Mark two straight lines on the activated TLC plate. Spot the amino acids samples. Place the TLC plate in the jar with appropriate solvent. Allow the solvent to reach the top line. Dry the TLC plate and apply the visualizing reagent. Look for different colored spots and calculate their Rf value. SEPARATION OF AMINO ACIDS BY TLC

29. The ninhydrin reaction is used to detect the presence of amino acids. Amino acids contain a free amino and carboxyl group which reacts together with ninhydrin to produce a characteristic blue color (or occasionally pale yellow). In this reaction first an amino group is attached to the first or alpha carbon of the amino acid’s carbon chain and then the nitrogen atom of the amino group reacts with ninhydrin to give a blue-purple product known as Ruhemann’s purple. Some amino acids (e.g. proline, secondary amine) give yellow-orange colour . SEPARATION OF AMINO ACIDS BY TLC

30. Amino acids Rf value Developing S olvent Locating reagent Spot colour Alanine 0.34 Butanol:Acetic acid:water (4:1:1) Ninhydrin Purple Glycine 0.45 Butanol: Acetic acid: water (4:1:1) Ninhydrin Purple Serine 0.56 Butanol: Acetic acid: water (4:1:1) Ninhydrin Purple Valine 0.71 Butanol: Acetic acid: water (4:1:1) N inhydrin Purple Threonine 0.82 Butanol: Acetic acid: water (4:1:1) N inhydrin Purple

31. Developing S olvent Locating reagent Spot colour Chloroform:methanol:water P-Anisidine hydrochloride Green brown Chloroform:methanol:water P-Anisidine hydrochloride Green brown Chloroform:methanol:water P-Anisidine hydrochloride Green brown SEPARATION OF CARBOHYDRATES

32. Organic compounds Rf value Developing S olvent Locating reagent Spot colour Acetic acid 0.34 Butanol:water Iodine vapours Tan brown Citric acid 0.45 Ethyl acetate: acetic acid: water Iodine vapours Tan brown Oxalic acid 0.56 Ethyl acetate : acetic acid: water Iodine vapours Tan brown SEPARATION OF ORGANIC COMPOUNDS

33. Thanks!