Lecture 05-High Performance TLC-23 Sep 2024 PDF

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY PRINCIPLES AND APPLICATIONS OF Dr. Ume Kulsoom Lecture 05 VARIOUS TYPES OF CHROMATOGRAPHY 23rd sept, 2024 HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY (HPTLC) High-Performance Thin Layer Chromatography (HPTLC) is...

HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY PRINCIPLES AND APPLICATIONS OF Dr. Ume Kulsoom Lecture 05 VARIOUS TYPES OF CHROMATOGRAPHY 23rd sept, 2024 HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY (HPTLC) High-Performance Thin Layer Chromatography (HPTLC) is an advanced version of traditional TLC that offers higher resolution, sensitivity, and reproducibility. It uses a thin layer of adsorbent material applied uniformly on glass or plastic plates and allows for more precise and automated analysis of complex mixtures. HPTLC is widely used in research, pharmaceuticals, and quality control to separate and identify compounds. HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY (HPTLC) Principle: HPTLC works on the same basic principle as TLC: Adsorption: A sample is applied to the plate, and as the mobile phase (solvent) moves up the plate, the components of the sample are separated based on their affinity for the stationary phase (adsorbent like silica gel) and the mobile phase. The main improvement in HPTLC is the high precision and automation of the sample application, development, and analysis processes, which lead to more consistent and reproducible results. PROCEDURE Adsorbent Sheets 1. Preparation of the HPTLC Plate: Use a high-performance TLC plate coated with a fine and uniform layer of silica gel or another adsorbent. 2. Selection of Mobile Phase: A solvent or a mixture of solvents is chosen as the mobile phase. The choice of solvent depends on the nature of the sample being analyzed. PROCEDURE 3. Sample Application: The sample is applied using an automatic sample applicator that ensures precise and consistent spot sizes. Multiple samples can be applied on a single plate. 4. Development of the Plate: The plate is placed in an automatic development chamber, which controls temperature, humidity, and solvent vapor. The mobile phase travels up the plate, separating the sample components based on their interactions with the stationary phase.  PROCEDURE 5. Visualization of Separated Components: After development, the separated spots are visualized using UV light or chemical stains. Automated visualization systems can also be used to detect and quantify the separated compounds. 6. Quantification: Automated densitometers or scanners are used to measure the intensity of the spots and quantify the amount of each compound. The Retention Factor (Rf) value is calculated for each component: PROCEDURE 7. Interpretation & Documentation: After the separation of compounds, spots will be analyze for qualitative or quantitative data, often using software for more complex analysis. The results are documented using specialized software that generates clear reports with images of the developed plate and data on the separated components.  ADVANTAGES High Resolution performance thin layer chromatography (HPTLC ), provides better separation and clarity of High components compared to conventional TLC. Versatile & Increased Sensitivity It can detect very small amounts of compounds. HPTLC can handle a wide variety of samples, from simple to highly complex mixtures. Fast and Efficient Multiple samples can be processed simultaneously, saving time. Automation Automated sample application, plate development, and analysis lead to greater reproducibility and accuracy. APPLICATIONS Pharmaceutical Analysis High performance thin layer chromatography (HPTLC ), is used for the identification and quality control of active ingredients in drugs. Food Industry It is used to detect contaminants, additives, and food colors Environmental Testing Analyzing pollutants and toxic substances in water, air, and soil samples can be achieved by high performance thin layer chromatography. Herbal Medicine It is use for the standardizing and ensuring the quality of herbal products by analyzing active compounds.  LIMITATIONS High Initial Setup Cost The equipment used for HPTLC, such as automated sample applicators and densitometers, can be expensive. Specialized Training Required HPLTC requires trained personnel to operate the automated systems. Not Suitable for Large Scale Purification Similar to TLC, HPTLC is not suitable for purifying large quantities of substances. Requires Careful Solvent Selection The choice of solvent is critical for achieving good separation, and finding the right solvent mixture can be time-consuming QUESTION # 1 1. What is the main principle behind HPTLC? A) Crystallization B) Adsorption C) Filtration D) Diffusion Correct Answer: B) Adsorption HPTLC is based on the adsorption principle, where compounds in a mixture adhere to the stationary phase and are separated based on their relative affinities for the stationary and mobile phases QUESTION # 2 2. What is the stationary phase in HPTLC typically made of? A) Paper B) Silica gel or alumina C) Metal D) Plastic Correct Answer: B) Silica gel or alumina The stationary phase in HPTLC is usually a thin layer of adsorbent material like silica gel or alumina coated on a plate QUESTION # 3 Which of the following is an advantage of HPTLC over traditional TLC? A) More expensive B) Higher resolution and sensitivity C) Slower results D) Less automation Correct Answer: B) Higher resolution and sensitivity HPTLC provides higher resolution, sensitivity, and automated processes compared to traditional TLC, making it more accurate and efficient. QUESTION # 4 What does the term "Retention Factor (Rf)" represent in HPTLC? A) The speed of the solvent B) The ratio of the distance traveled by the solvent and the compound C) The amount of sample applied D) The density of the plate coating Correct Answer: B) The ratio of the distance traveled by the solvent and the compound Rf value is the ratio of the distance traveled by the compound to the distance traveled by the solvent front QUESTION # 5 What is used to visualize the separated components in HPTLC? A) Chemical Stains B) Heat C) Capillary Action D) Light Microscope Correct Answer: A) Chemical Stains Separated components in HPTLC can be visualized using UV light or chemical stains that react with the components to make them visible. QUESTION # 6 6. Which of the following is a typical application of HPTLC? A) Separation of gases B) Quality control in pharmaceuticals C) Filtration of solids D) DNA sequencing Correct Answer: B) Quality control in pharmaceuticals HPTLC is widely used in pharmaceutical industries for analyzing drug purity and quality control. QUESTION # 7 7. Which of the following is NOT an advantage of HPTLC? A) Increased sensitivity B) Automation C) Low setup cost D) High resolution Correct Answer: C) Low setup cost HPTLC has many advantages, one limitation is the high initial setup cost of the specialized equipment QUESTION # 8 8. What role does the mobile phase play in HPTLC? A) It holds the sample in place B) It separates the components by dissolving them C) It stops the separation process D) It prevents the plate from moving Correct Answer: B) It separates the components by dissolving them The mobile phase carries the sample components up the plate by capillary action, separating them based on their solubility in the solvent and interaction with the stationary phase. QUESTION # 9 9. How is the sample applied to the HPTLC plate? A) By pouring it on the plate B) By capillary tube manually C) Using an automatic sample applicator D) Spraying it on the surface Correct Answer: C) Using an automatic sample applicator HPTLC uses automated sample applicators to ensure consistent and precise spot application. QUESTION # 10 10. What is a limitation of HPTLC? A) High sensitivity B) Cannot handle large-scale purification C) Can only analyze organic compounds D) Does not require a solvent system Correct Answer: B) Cannot handle large-scale purification HPTLC is typically used for analytical purposes, and it is not suitable for large-scale purification of substances. THANK YOU