Thin Layer Chromatography (TLC) Lecture Notes PDF

THIN LAYER CHROMATOGRAPHY PRINCIPLES AND APPLICATIONS OF Dr. Ume Kulsoom Lecture 04 VARIOUS TYPES OF CHROMATOGRAPHY 16th sept, 2024 THIN LAYER CHROMATOGRAPHY (TLC) Principle: TLC is based on the principle of adsorption. When the sample mixture is appli...

THIN LAYER CHROMATOGRAPHY PRINCIPLES AND APPLICATIONS OF Dr. Ume Kulsoom Lecture 04 VARIOUS TYPES OF CHROMATOGRAPHY 16th sept, 2024 THIN LAYER CHROMATOGRAPHY (TLC) Principle: TLC is based on the principle of adsorption. When the sample mixture is applied to the TLC plate, its components interact with the stationary phase (silica or alumina) by adsorbing onto it. As the mobile phase (solvent) moves up the plate, the components that have less affinity for the stationary phase will move faster, while those with stronger interactions will move more slowly, leading to separation. The separation is based on the relative affinities of each compound for the stationary phase and the solvent. PROCEDURE 1. Preparation of the TLC Plate: o Take a TLC plate coated with a thin layer of adsorbent material (usually silica gel or alumina). 2. Application of Sample: o Using a capillary tube, apply a small amount of the sample mixture onto the plate, about 1-2 cm from the bottom edge (this is the baseline). Allow the sample spots to dry. PROCEDURE 3. Choosing the Solvent: o Select an appropriate solvent (or mixture of solvents) that will serve as the mobile phase. The solvent should move the components of the mixture at different speeds. 4. Developing the Plate: o Place the TLC plate in a development chamber (a glass jar) containing a shallow layer of solvent. Make sure the solvent level is below the baseline so the sample doesn't dissolve into the solvent directly.  PROCEDURE 5. Movement of the Solvent: o The solvent travels up the plate by capillary action, carrying the components of the mixture with it. Different components move at different rates based on their interactions with the stationary phase and the solvent. 6. Visualizing the Results: o After the solvent front has moved up the plate, remove the plate from the chamber and mark the solvent front. o Depending on the compounds, you may use a UV lamp or chemical stains to visualize the separated spots. PROCEDURE 7. Calculating Rf Value: The Retention Factor (Rf) is a numerical value that represents how far a compound travels on the TLC plate relative to the solvent front. It is calculated using the formula: The Rf value (Retention factor) for each spot is calculated as: APPLICATIONS Identification of Compounds TLC is commonly used to identify compounds in a mixture by comparing their Rf values with known substances. Purity Testing It helps to check the purity of a substance by observing whether there are any additional spots indicating impurities. Drug Analysis Widely used in pharmaceutical industries to analyze drug compositions. Food Industry Used to detect additives, food colorants, and contaminants.  ADVANTAGES Simple and Quick is easy to perform and provides fast results, making it ideal for routine analysis. TLC Cost-effective is inexpensive compared to other chromatographic techniques. It Multiple Samples Several samples can be analyzed simultaneously on one plate Visualization Allows for easy visualization of compounds, especially with UV light or chemical stains.  LIMITATIONS Low Sensitivity TLC is less sensitive compared to other techniques like HPLC (High-Performance Liquid Chromatography), meaning it may not detect very low concentrations of substances. Limited Quantification is not ideal for quantitative analysis as it gives approximate results. It Not Suitable for Large Samples TLC is typically used for small-scale separations and cannot handle large quantities of samples. Manual Process The results may vary due to inconsistencies in handling or environmental conditions like temperature and humidity. QUESTION # 1 What is the stationary phase in Thin Layer Chromatography (TLC)? A) Glass plate B) Silica or alumina on a glass or plastic plate C) Solvent D) Sample mixture Correct Answer: B) Silica or alumina on a glass or plastic plate In TLC, the stationary phase is a thin layer of silica or alumina coated on a glass, plastic, or metal plate, which helps in the separation of substances. QUESTION # 3 Which principle is Thin Layer Chromatography (TLC) based on? A) Partitioning B) Adsorption C) Crystallization D) Precipitation Correct Answer: B) Adsorption TLC works on the principle of adsorption, where the components of the mixture adhere to the surface of the stationary phase (silica or alumina), and their movement is determined by how strongly they are adsorbed. QUESTION # 2 What is the mobile phase in TLC? A) The plate B) The mixture being separated C) The solvent that moves up the plate D) The silica gel Correct Answer: C) The solvent that moves up the plate The mobile phase in TLC is the solvent, which moves up the plate carrying the components of the mixture for separation. QUESTION # 4 Why is it important to cover the TLC chamber when performing the experiment? A) To prevent evaporation of the solvent B) To speed up the process C) To keep the stationary phase clean D) To allow air circulation Correct Answer: A) To prevent evaporation of the solvent Covering the TLC chamber helps prevent the solvent from evaporating and ensures that the atmosphere inside remains saturated, allowing the solvent to move up the plate evenly. QUESTION # 5 How are spots visualized in TLC after the solvent has run? A) By looking at the plate in normal light B) By using a UV lamp or chemical stains C) By heating the plate D) By dissolving the spots in water Correct Answer: B) By using a UV lamp or chemical stains The separated spots may not be visible under normal light, so they are often visualized using UV light or chemical reagents that react with the substances. QUESTION # 6 The Rf value in TLC is calculated by A) Dividing the distance traveled by the solvent by the distance traveled by the sample B) Dividing the distance traveled by the sample by the distance traveled by the solvent C) Multiplying the distances traveled by the solvent and sample D) Adding the distances traveled by the solvent and sample Correct Answer: B) Dividing the distance traveled by the sample by the distance traveled by the solvent The Rf value is the ratio of the distance traveled by the substance (solute) to the distance traveled by the solvent front. QUESTION # 7 What could cause the spots to streak or appear as smudges in TLC? A) Using too little solvent B) Applying too much sample C) Running the solvent too slowly D) Using a UV lamp Correct Answer: B) Applying too much sample If too much sample is applied, the spots can become overloaded, leading to streaks or smudges instead of clear spots, which can affect the separation quality. QUESTION # 8 TLC is commonly used for A) Separating gases B) Separating liquid mixtures C) Purifying large quantities of a substance D) Identifying the components of a mixture Correct Answer: D) Identifying the components of a mixture TLC is a useful tool for identifying different components of a mixture by separating them and comparing their Rf values with known substances. QUESTION # 9 What is an advantage of Thin Layer Chromatography (TLC) over paper chromatography? A) TLC is faster and can separate more complex mixtures B) TLC uses less solvent C) TLC provides more accurate Rf values D) TLC can separate only colored compounds Correct Answer: A) TLC is faster and can separate more complex mixtures TLC can separate more complex mixtures more efficiently and quickly compared to paper chromatography, and it allows for a wider range of solvents and detection methods. QUESTION # 10 Which of the following is an application of Thin Layer Chromatography? A) Identifying components in food additives B) Separating gases in air samples C) Measuring the molecular weight of proteins D) Determining the density of liquids Correct Answer: A) Identifying components in food additives TLC is commonly used for analyzing mixtures like food additives, separating the components, and comparing them to known standards. THANK YOU