Size_Exclusion_Chromatography_Presentation.pptx

HPLC

1. Size-Exclusion Chromatography (SEC) Overview, Principles, Components, and Applications

2. What is Size-Exclusion Chromatography (SEC)? Size-Exclusion Chromatography (SEC) is a chromatographic method that separates molecules based on their size. It is also known as Gel Filtration Chromatography when applied to aqueous solutions. SEC is widely used in the purification and analysis of proteins, polymers, and other biomolecules.

3. Principles of Size-Exclusion Chromatography SEC separates molecules based on their size as they pass through a porous gel matrix: - Large molecules elute first as they cannot enter the pores. - Small molecules elute later as they enter and exit the pores. The size of the pores in the gel determines the range of molecules that can be separated.

4. Key Components of SEC - **Column Gel:** Filled with porous particles (Sephadex, Sepharose, or synthetic polymers). - **Mobile Phase (Eluent):** A buffer or solvent that carries the molecules through the column. - **Detector:** Detects eluted molecules, such as UV-Vis or Refractive Index detectors.

5. Procedure of Size-Exclusion Chromatography 1. Prepare the column with gel matrix. 2. Inject the sample containing molecules of various sizes. 3. Elute the molecules using a buffer (mobile phase). 4. Large molecules elute first, followed by smaller molecules. 5. Collect fractions and analyze using detectors.

6. Advantages of SEC - No chemical interactions between the sample and the stationary phase. - Ideal for sensitive molecules like proteins and enzymes. - Allows for separation of molecules based solely on size.

7. Limitations of SEC - Limited resolution for molecules with similar sizes. - Only small sample volumes can be used. - Limited separation range based on pore size of the gel.

8. Applications of SEC 1. Purification of proteins and enzymes. 2. Determining molecular weights of proteins and polymers. 3. Desalting or removing small molecules from biomolecules. 4. Analyzing the quaternary structure of proteins (e.g., oligomers). 5. Separating viruses or large particles.

9. Example of SEC In a typical protein purification procedure using SEC: 1. Protein samples are injected into the column. 2. Large protein aggregates or oligomers elute first, followed by the target protein. 3. Small molecules or impurities elute last. 4. Fractions are collected and analyzed for protein purity.

10. Optimizing Size-Exclusion Chromatography - Choose the correct pore size for the gel based on target molecule size. - Ensure proper flow rate to maintain resolution. - Use the appropriate sample volume to avoid dilution or loss of resolution.