Paper Chromatography Lecture PDF

PAPER CHROMATOGRAPHY PRINCIPLES AND APPLICATIONS OF Dr. Ume Kulsoom Lecture 03 VARIOUS TYPES OF CHROMATOGRAPHY 16th sept, 2024 PAPER CHROMATOGRAPHY Principle: The principle of paper chromatography is based on the partitioning of substances between two p...

PAPER CHROMATOGRAPHY PRINCIPLES AND APPLICATIONS OF Dr. Ume Kulsoom Lecture 03 VARIOUS TYPES OF CHROMATOGRAPHY 16th sept, 2024 PAPER CHROMATOGRAPHY Principle: The principle of paper chromatography is based on the partitioning of substances between two phases: the stationary phase (the paper) and the mobile phase (the solvent). The different components in a mixture interact differently with these phases based on their solubility and affinity. As the solvent moves up the paper by capillary action, substances with higher solubility in the solvent move further, while those with higher affinity for the paper move less, leading to the separation of the mixture.  KEY CONCEPTS Mobile Phase: Stationary Phase: The mobile phase is the liquid that moves through the The stationary phase is the paper that stays still during the paper, carrying the different parts of the mixture with process. Some parts of the mixture stick to the paper while it. It helps separate the substances as it travels up the others move with the mobile phase, causing them to paper. separate. Partitioning Principle The different components of the mixture partition Differential Movement between the water molecules in the paper (stationary The varying solubilities of the components in the solvent and phase) and the solvent (mobile phase). This their interaction with the paper cause them to travel at partitioning is governed by the component’s affinity different rates, resulting in their separation. for each phase. Capillary Action The solvent moves up the paper by capillary action, carrying the components of the mixture along with it.  PROCEDURE 1. Preparation of Stationary Phase (Paper)  The filter paper acts as the stationary phase. The molecules of the sample interact with this surface.  The paper is often made of cellulose, which contains water molecules. As a result, paper chromatography works on the principle of partition chromatography (compounds dissolve in both the water retained in the paper and the solvent).  PROCEDURE 2. Application of Sample  A small drop of the sample mixture (such as ink or plant extract) is placed on the baseline (a line drawn in pencil about 2 cm from the bottom of the paper).  This is where the separation process begins. 3. Introduction of Mobile Phase (Solvent) A suitable solvent or solvent mixture (e.g., water, alcohol, acetone) is used as the mobile phase. The bottom of the paper (not touching the sample) is dipped into the solvent, which begins to rise through the paper via capillary action.  PROCEDURE 4. Movement of the Solvent  As the solvent moves upward, it carries the components of the sample with it.  The different components of the sample mixture (pigments, amino acids, etc.) have varying degrees of solubility in the solvent and affinity for the paper. 5. Differential Partitioning Each component in the mixture partitions itself between the water in the paper and the solvent. More soluble components in the solvent will move further up the paper, as they travel with the mobile phase easily. Less soluble components in the solvent, or those with a higher affinity for the paper, will move more slowly and travel shorter distances.  PROCEDURE 6. Separation of Components  As a result, the different components of the sample separate from each other based on their solubility and interaction with the stationary phase.  Components with a greater affinity for the solvent (mobile phase) will travel further, while those with greater affinity for the paper (stationary phase) will remain closer to the origin. 7. Formation of Spots The separated components form distinct spots on the paper, each representing a different substance in the mixture.  PROCEDURE 8. Retention Factor (Rf) Calculation  After the experiment, the distance traveled by each component is measured.  The retention factor (Rf) is calculated as: Formula: ○ Dsolute = Distance traveled by the solute (each spot) ○ Dsolvent = Distance traveled by the solvent front  APPLICATIONS Identifying Compounds in Inks or Dyes Used in forensic science or food industries to identify different ink or dye components.  APPLICATIONS Separation of Pigments Paper chromatography use to separate plant pigments (chlorophyll, carotenoids, etc.)  APPLICATIONS Biochemical Analysis Helpful in separating amino acids, nucleotides, and sugars in biological samples.  ADVANTAGES Simple and Inexpensive Requires minimal equipment and materials. Fast and Easy results with straightforward procedures. Quick Minimal Sample Requirement a small amount of sample is needed for analysis. Only Good for Soluble Compounds Effective for separating soluble substances like dyes, pigments, and inks. Portable Can be performed outside of a traditional laboratory environment. LIMITATIONS Limited Resolution Paper chromatography cannot separate very similar compounds efficiently. Qualitative, Not Quantitative is usually used for identification, not for precise quantification. It Not Suitable for Volatile Substances volatile substances may evaporate before separation occurs. The Limited to Small Sample Sizes Paper chromatography cannot efficiently separate large amounts of a sample. Time-Consuming for Complex Mixtures It may take longer to separate more complex mixtures compared to simpler compounds.  EXAMPLE USING CHROMATOGRAPHY TO ANALYZE FOOD DYES Imagine you're working in a lab that tests food safety. You need to check if the dyes used in a candy are safe and legal. How: You could use Paper Chromatography to separate the different dyes in the candy. After separating them, you could compare the colors that appear on your TLC plate to a standard set of dyes to see if anything unexpected or illegal is present. If further analysis is needed, you might use High-Performance Liquid Chromatography (HPLC) to get a more detailed look at the dyes and quantify exactly how much of each is in the candy. QUESTION # 1 What is the stationary phase in paper chromatography? A) Solvent B) Filter paper C) Mixture D) Glass plate Correct Answer: B) Filter paper In paper chromatography, the filter paper acts as the stationary phase, where the substances are absorbed and separated. QUESTION # 2 The mobile phase in paper chromatography refers to? A) The solvent that moves through the paper B) The paper itself C) The substance being analyzed D) The ink spot applied Correct Answer: A) The solvent that moves through the paper The mobile phase is the solvent that moves through the paper by capillary action, carrying the components of the mixture with it. QUESTION # 3 What principle does paper chromatography mainly rely on? A) Adsorption B) Partitioning C) Sublimation D) Precipitation Correct Answer: B) Partitioning Paper chromatography works on the partitioning principle, where substances separate based on their affinity for the stationary phase (water in the paper) and the mobile phase (solvent). QUESTION # 4 What does the Rf value in chromatography represent? A) The speed of the solvent B) The ratio of the distance traveled by the solute to the distance traveled by the solvent C) The thickness of the paper D) The amount of mixture used Correct Answer: B) The ratio of the distance traveled by the solute to the distance traveled by the solvent The Rf value (Retention Factor) is calculated by dividing the distance traveled by the solute (substance) by the distance traveled by the solvent front. It helps in identifying substances. QUESTION # 5 In paper chromatography, substances that have a higher affinity for the mobile phase will: A) Stay near the baseline B) Move farther up the paper C) Form a single spot D) Evaporate from the paper Correct Answer: B) Move farther up the paper Substances that are more soluble in the solvent (mobile phase) will travel farther up the paper, while those with more affinity for the stationary phase will stay closer to the baseline. QUESTION # 6 Which of the following is an example of a solvent that can be used as the mobile phase in paper chromatography? A) Water B) Petroleum ether C) Ethanol D) All of the above Correct Answer: D) All of the above Water, petroleum ether, and ethanol are examples of solvents that can be used as mobile phases depending on the type of substances being separated. QUESTION # 7 Why is it important to mark the solvent front in paper chromatography? A) To identify the paper type B) To calculate the Rf value C) To stop the solvent from evaporating D) To know where to apply the sample Correct Answer: B) To calculate the Rf value The solvent front is marked so that after the experiment, you can measure how far the solvent traveled and use this to calculate the Rf values of the separated substances. QUESTION # 8 What would happen if a substance is highly soluble in the mobile phase? A) It will stay at the origin B) It will move slowly up the paper C) It will move quickly and further up the paper D) It will not appear on the paper Correct Answer: C) It will move quickly and further up the paper A substance that is highly soluble in the mobile phase will travel further because it has a stronger interaction with the solvent compared to the stationary phase. QUESTION # 9 What is the purpose of paper chromatography in ink analysis? A) To change the color of ink B) To identify the different pigments in the ink C) To remove pigments from ink D) To test the paper quality Correct Answer: B) To identify the different pigments in the ink Paper chromatography can separate the different pigments present in the ink, showing which colors are mixed to form the ink. QUESTION # 10 Which of the following factors does NOT affect the separation of components in paper chromatography? A) The solubility of the component in the solvent B) The thickness of the paper C) The affinity of the component for the stationary phase D) The temperature of the solvent Correct Answer: B) The thickness of the paper The separation of components is mainly affected by their solubility in the solvent and their affinity for the stationary phase, not by the thickness of the paper itself. QUESTION # 11 Why is it important to use a pencil for drawing the baseline in paper chromatography? A) Pencil marks help the solvent move faster B) Pencil does not dissolve in the solvent C) Pencil marks absorb the solute D) Pencil reacts with the mixture Correct Answer: B) Pencil does not dissolve in the solvent A pencil is used to draw the baseline because it is made of graphite, which will not dissolve in the solvent and interfere with the experiment. QUESTION # 12 In which of the following would paper chromatography be least effective? A) Separating plant pigments B) Identifying different amino acids C) Separating gases D) Identifying food colorings Correct Answer: C) Separating gases Paper chromatography is effective for liquids and soluble substances but not ideal for separating gases, which require other techniques like gas chromatography. QUESTION # 13 What happens if the solvent level is above the baseline where the sample is spotted? A) The sample will not move B) The sample will dissolve into the solvent and wash away C) The solvent will not rise D) The solvent will evaporate Correct Answer: B) The sample will dissolve into the solvent and wash away If the solvent level is above the baseline, the sample will mix directly with the solvent and wash away, preventing separation. QUESTION # 14 What can cause poor separation of components in paper chromatography? A) Using a solvent that is too strong B) Incorrect solvent selection C) Too much sample applied on the paper D) All of the above Correct Answer: D) All of the above Poor separation can result from several factors, such as using the wrong solvent, applying too much sample, or using a solvent that dissolves all the components too quickly. QUESTION # 15 What is the main use of the Rf value in paper chromatography? A) To measure the distance traveled by the solvent B) To identify unknown substances by comparing their Rf values with known ones C) To determine the quality of the paper D) To calculate the speed of the solvent Correct Answer: B) To identify unknown substances by comparing their Rf values with known ones The Rf value is used to identify substances by comparing the distance they traveled relative to the solvent front. Matching the Rf values to known standards helps in the identification of unknown compounds THANK YOU