CHROMATOGRAPHY - TLC - HPLC (YTLINKS).pptx

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1. _____ _____ CHROMATOGHRAPHY

2. ____ ____ INTRODUCTION Chromatography is a technique used for separating and analyzing mixtures of compounds based on their differential distribution between a stationary phase and a mobile phase . Chromatography is widely used in scientific research, quality control, and pharmaceutical industries to analyze and separate complex mixtures. Chromatography is a versatile and powerful tool due to its ability to separate and quantify individual components in a mixture.

3. ____ ____ KEY ELEMENTS OF CHROMATOGRAPHY Stationary Phase: This is a solid or liquid phase that does not move during the chromatographic process. It can be a packed column or a thin layer on a solid support. The choice of stationary phase depends on the specific separation requirements. Mobile Phase: This is a fluid that moves over or through the stationary phase, carrying the sample with it. The interaction between the components of the sample and the mobile phase is crucial for the separation process. Sample: The mixture of compounds to be separated and analyzed .

4. ____ ____ TYPES OF CHROMATOGRAPHY According to the chromatographic bed shape: Column Chromatography. Paper Chromatography. Thin-Layer Chromatography (TLC). According to state of phases: Gas Chromatography. High Performance Liquid Chromatography (HPLC). Supercritical Liquid Chromatography. According to mechanism of separation: Affinity Chromatography. Ion Exchange Chromatography. Gel Filtration Chromatography.

5. _____ _____ THIN-LAYER CHROMATOGRAPHY (TLC) https://www.youtube.com/watch?v=rMGQavOMAmc

6. ____ ____ WHAT IS THE TLC ? TLC is a chromatographic technique that involves the separation of compounds in a mixture based on their differential migration rates through a thin layer of adsorbent material.

7. ____ ____ BASIC PRINCIPLE OF TLC 1- Stationary Phase: T hin layer of adsorbent material, commonly silica gel or alumina, coated on a glass or plastic plate. The choice of the adsorbent depends on the type of compounds being separated. 2- Mobile Phase: S olvent or a mixture of solvents that moves up the plate via capillary action. The interaction between the sample and the mobile phase causes the components to move at different rates. 3- Sample Application: A small spot of the sample mixture is applied near the bottom of the TLC plate. This can be done using a capillary tube or micropipette.

8. ____ ____ … BASIC PRINCIPLE OF TLC 4- Development: the plate is then placed in a developing chamber, where the solvent rises up the plate. As the solvent travels, it carries the sample with it, and the different components in the sample move at varying rates. 5- Differential Migration: the different components of the mixture have varying degrees of affinity for the stationary phase and the mobile phase. As a result, they move at different rates up the plate. Components with a higher affinity for the stationary phase move more slowly, while those with a higher affinity for the mobile phase move faster. 6- Separation : the interaction between each component and the stationary phase determines the extent to which it is retained on the plate. This leads to the separation of the components along the length of the plate.

9. ____ ____ … BASIC PRINCIPLE OF TLC 7- Visualization: After development, the plate is taken out and dried. The separated components are often invisible, so various visualization techniques can be used, such as UV light, staining with specific reagents, or by exposing the plate to iodine vapours. 8- Retention Factor Value (RF): is a ratio that represents the distance travelled by a compound relative to the distance travelled by the solvent. It is a characteristic parameter used for identification and comparison of compounds. 9- Analysis & Interpretation: the positions and characteristics of the spots on the TLC plate are analyzed to identify and characterize the components of the mixture.

10. ____ ____ ADVANTAGES OF TLC Quick and cost-effective. Minimal sample requirements. Compatibility with various detectors. Versatility in stationary phase. ____ ____ LIMITATIONS OF TLC Difficulty in scale-up. Overlapping spots. Low resolution. Difficulty in automation .

11. _____ _____ HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) https://www.youtube.com/watch?v=eCj0cRtJvJg

12. ____ ____ HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) HPLC is a powerful analytical technique used for separating, identifying, and quantifying components in a mixture. It is a form of liquid chromatography that employs a high-pressure pump to force a liquid solvent (the mobile phase) through a column packed with a stationary phase.

13. ____ ____ BASIC PRINCIPLE OF HPLC 1- Stationary Phase: finely divided solid adsorbent (such as silica or a polymer) packed into a stainless steel column. Its choice depends on properties of analytes and the separation goal. 2- Mobile Phase: liquid (usually a solvent or a mixture of solvents) that is pumped through the chromatographic column under high pressure. The mobile phase carries the sample through the column and interacts with the stationary phase, leading to the separation of components. 3- High Pressure: to force the mobile phase through the column. This high pressure is essential for achieving fast and efficient separations. 4- Sample Injection: into the mobile phase flow, typically through an autosampler, which ensures precise and reproducible injections.

14. ____ ____ … BASIC PRINCIPLE OF HPLC 5- Separation mechanisms: various separation mechanisms, as adsorption, partition, ion exchange, and affinity interactions. The choice depends on the nature of sample and analytes. 6- Detection: common detectors as UV-visible spectrophotometers, fluorescence detectors, refractive index detectors, and mass spectrometers. The choice depends on the nature of the analytes and the sensitivity required. 7- Retention Time: time it takes for a particular component to travel from the injection point to the detector. Retention time is used for qualitative and quantitative analysis. 8- Chromatogram: analysis output is a chromatogram, which is a plot of detector response (intensity) versus time or elution volume.

15. ____ ____ TYPES OF HPLC Normal-Phase HPLC: Non-polar solvent. Polar stationary phase. Polar compounds move slower. Non-polar compounds move faster. Reverse-Phase HPLC: Polar solvent. Non-polar stationary phase. Non-polar compounds move slower. Polar compounds move faster.

16. ____ ____ ADVANTAGES OF HPLC High sensitivity , high resolution & high speed of analysis. Wide applicability . Automation and high throughput . Adaptability to large scale Sample stability, so suits thermally sensitive compounds. ____ ____ LIMITATIONS OF HPLC Complexity of operation. High cost. Co-elution difficult to detect.

17. ____ ____ THANK YOU BY: HOSSAM KHAYYAL