1. THIN LAYER CHROMATOGRAPHY

2. CONTENT Definition Principle Components Apparatus Procedure Steps Retention factor Factors affect retardation factor Advantages & Disadvantages of TLC Applications Experiment Analysis for colourless substance References

3. THIN LAYER CHROMATOGRAPHY What is thin layer chromatography? Thin Layer Chromatography is a technique used to isolate non-volatile mixtures. It is a technique used to separate the components of a mixture using a thin stationary phase. What Is Thin Layer Chromatography?Thin Layer Chromatography is a technique used to isolate non-volatile mixtures.What Is Thin Layer Chromatography?Thin Layer Chromatography is a technique used to isolate non-volatile mixtures.

5. PRINCIPLE. All forms of chromatography work on the same principle.They all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates.

6. COMPONENTS IN CHROMATOGRAPHY There are three components in thin layer chromatography. 1.Stationary phase 2.Mobile phase 3.Solute

7. APPARATUS OF TLC Chamber Watch glass Capillary plate Solvent Pencil UV light

9. PROCEDURE A few components involved in the TLC procedure are as follows. TLC Plates: These are used for applying the thin layer of stationary phase. They are stable in nature. The layer of stationary phase is kept even throughout these plates for better analysis. Mobile Phase: This comprises a solvent (or solvent mixture). The taken solvent needs to be chemically inert, of the highest possible purity, and particulate-free. Only then can the TLC spots be able to develop.

10. CONTINUE… TLC Chamber: This is where the thin layer chromatography procedure takes place. It keeps the dust particles away from the process and does not let the solvent evaporate. Filter Paper: This gets placed inside the chamber after being moistened with the mobile phase solution. It ensures that the mobile phase rises uniformly throughout the TLC plate’s length. After collecting all these components, the process begins.

11. STEPS 1.Here are the steps followed in it: 2.1.The process starts by making a thin mark on the TLC plate’s bottom with a pencil. These spots are kept at equal distances. 3.2.The sample is then applied to these spots made on the line. 4.3.Then the TLC chamber is filled with the mobile phase up to a few centimetres of its bottom. 5.4.After pouring the mobile phase, the moistened filter paper is placed along with the inside of the chamber wall. This helps to avoid the edge effect by maintaining equal humidity.

12. CONTINUE…. 5.Finally, the prepared stationary phase plate is put inside the chamber. 1. 6.At this point, the sample spots are kept on the mobile phase’s side. The chamber is then closed after placing the plate into it. 2.7.Once enough time has elapsed for the process, the plate is taken out and allowed to dry. 3.8.At last, the sample spots get analyzed through a suitable method for sample, such as UV light, KMnO4 stain, and iodine staining. This way, the TLC procedure gets completed.

13. RETENTION FACTOR Each component appears as spots separated vertically. Each spot has a retention factor.. (Rf) expressed as: Rf = dist. travelled by sample / dist. travelled by solvent.

14. FACTORS AFFECTING RETENTION FACTOR The factors affecting retention factor are:: 1.the solvent system, 2.amount of material spotted, 3.adsorbent 4.temperature.

15. ADVANTAGES TLC Advantages of TLCThese include: 1.The separated spots of TLC can be further visualized without any trouble. 2.This chromatography process is cost-effective as compared to other methods. 3.It can be used for a number of compounds, and it does not take much time. 4.TLC makes it simple to analyze any given compound’s purity standards. 5.Several compounds can easily get isolated through TLC.

16. DISADVANTAGES The drawbacks of the process are: 1. The TLC procedure can not be used for lower detection limit experiments because it has a high detection limit. 2. The plates used in TLC do not possess a more extended stationary phase. Result reproduction is challenging in TLC. 3. TLC is limited to qualitative analysis, and it can not be used for quantitative analysis. 4. The separation length is also restricted as compared to other chromatography methods. 5. The process here does not take place in a closed system. Therefore, aspects like temperature and humidity can affect the results.

17. APPLICATIONS OF TLC Thin Layer Chromatography Applications The qualitative testing of Various medicines such as sedatives, local anaesthetics, analgesics, antihistamines, steroids,, is done by TLC. TLC is extremely useful in Biochemical analysis such as isolation of biochemical metabolites from its blood plasma, urine, body fluids, etc. It is also use in cosmetics.

18. CONTINUE… It is widely used in separating multicomponent pharmaceutical formulations. It is used for the purification of samples and direct comparison is done between the sample and the authentic sample. It is used in the food industry, to separate and identify colors, sweetening agent, and preservatives.

19. TLC EXPERIMENT Thin Layer Chromatography Experiment: 1.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. 2.Apply sample solutions to the marked spots. 3Pour the mobile phase into the TLC chamber and to maintain equal humidity, place a moistened filter paper in the mobile phase.

20. CONTINUE…. 4.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.Immerse the plate for development. 5.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. 6.Once the spots are developed, take out the plates and dry them. The sample spots can be observed under a UV light chamber.,

22. WHAT IF THE SUBSTANCES YOU ARE INTERESTED IN ARE COLOURLESS? There are two simple ways of getting around this problem.Using fluorescence,the stationary phase on a thin layer plate often has a substance added to it which will fluoresce when exposed to UV light. That means that if you shine UV light on it, it will glow.That glow is masked at the position where the spots are on the final chromatogram - even if those spots are invisible to the eye. That means that if you shine UV light on the plate, it will all glow apart from where the spots are. The spots show up as darker patches.While the UV is still shining on the plate, you obviously have to mark the positions of the spots by drawing a pencil circle around them. As soon as you switch off the UV source, the spots will disappear again.

23. REFERENCES https://byjus.com/chemistry/thin-layer-chromatography/ https://en.m.wikipedia.org/wiki/Thin-layer_chromatography