Chromatographic techniques: Size Exclusion Chromatography (SEC)

A topic from the subject of Chromatography in Chemistry.

11 months ago
9 min read

Chromatographic Techniques: Size Exclusion Chromatography (SEC)

Introduction

Size exclusion chromatography (SEC), also known as gel filtration chromatography, is a powerful separation technique used in chemistry, biochemistry, and other fields to separate molecules based on their size. This technique is widely employed in various applications, including protein purification, polymer analysis, and particle characterization.

Basic Concepts

Separation Mechanism: SEC operates on the principle of molecular size discrimination. A porous gel or matrix is used as the stationary phase, and the sample molecules are eluted based on their ability to enter and diffuse through the pores of the gel. Smaller molecules can access the pores and elute later, while larger molecules are excluded from the pores and elute earlier.
Stationary Phase: The stationary phase in SEC is typically a porous gel or matrix, often composed of agarose, dextran, or synthetic polymers. These gels have a defined pore size distribution that allows for size-based separation of molecules.
Mobile Phase: The mobile phase in SEC is a liquid solvent or buffer that carries the sample molecules through the chromatographic column. The choice of mobile phase depends on the nature of the sample and the gel used.

Equipment and Techniques

Chromatographic Column: SEC is typically performed using a chromatographic column packed with the stationary phase gel. The column is designed to allow the mobile phase to flow through the gel bed.
Sample Injection: The sample is injected into the mobile phase, which carries it through the chromatographic column. The sample molecules interact with the stationary phase gel, and the separation process occurs.
Elution: The mobile phase elutes the sample molecules from the column based on their size. Smaller molecules elute later, while larger molecules elute earlier.
Detection: As the eluted molecules exit the column, they are detected using various methods, such as UV absorbance, fluorescence, or refractive index detectors. The detector generates a chromatogram that shows the elution profile of the sample components.

Types of Experiments

Analytical SEC: Analytical SEC is used to determine the molecular weight distribution and other physical properties of a sample. It is commonly employed in protein analysis, polymer characterization, and particle size determination.
Preparative SEC: Preparative SEC is used to isolate and purify specific molecules from a sample mixture. This technique is widely employed in protein purification, drug isolation, and other preparative applications.

Data Analysis

Elution Profile: The chromatogram obtained from SEC provides information about the molecular weight distribution of the sample. The elution profile shows the concentration of eluted molecules as a function of elution time or elution volume.
Molecular Weight Determination: The molecular weight of the sample molecules can be estimated using calibration curves or other methods based on the elution behavior of standard samples with known molecular weights.
Polydispersity Index: The polydispersity index (PDI) is a measure of the heterogeneity of a sample. It is calculated from the elution profile and provides information about the distribution of molecular weights in the sample.

Applications

Protein Purification: SEC is extensively used to purify proteins from complex mixtures, such as cell extracts or fermentation broths. It allows for the separation of proteins based on their molecular size and is often used in conjunction with other purification techniques.
Polymer Analysis: SEC is employed to characterize polymers, such as molecular weight distribution, branching, and composition. This information is crucial for understanding the properties and behavior of polymers.
Particle Size Determination: SEC can be used to determine the size distribution of particles in suspensions or emulsions. This technique is widely applied in fields such as colloid chemistry and pharmaceutical sciences.

Conclusion

Size exclusion chromatography (SEC) is a versatile and powerful technique used in various fields to separate molecules based on their size. With its ability to provide information about molecular weight distribution, purity, and other physical properties, SEC plays a crucial role in protein purification, polymer analysis, particle characterization, and other applications.

Size Exclusion Chromatography (SEC): A Versatile Separation Technique

Size exclusion chromatography (SEC), also known as gel permeation chromatography (GPC), is a widely used separation technique in chemistry for the analysis of macromolecules and particles based on their size.

Key Points:

Principle: SEC separates molecules based on their hydrodynamic size. A porous gel or matrix is employed as the stationary phase, and molecules are eluted from the column in order of decreasing molecular size.
Stationary Phase: SEC utilizes porous materials like cross-linked dextran, agarose, or silica gels as the stationary phase. The pore size of the matrix determines the size range of molecules that can be separated.
Mobile Phase: SEC uses a liquid mobile phase, typically organic solvents or buffers, to elute molecules through the column.
Elution: Larger molecules are eluted first as they cannot enter the pores, while smaller molecules penetrate the pores and are retained for a longer time. This results in the separation of molecules based on their size.
Detection: Detectors used in SEC include ultraviolet (UV) absorbance, refractive index (RI), or evaporative light scattering (ELS) detectors, which provide information about the concentration or mass of the eluted molecules.
Applications:
- Characterization of Polymers: SEC is used to determine the molar mass distribution, molecular weight averages, and polydispersity index of polymers.
- Protein Analysis: SEC is employed to separate proteins based on their molecular size, enabling the determination of molecular weight and purity.
- Separation of Nanoparticles: SEC can be used to separate and characterize nanoparticles based on their size and aggregation state.
- Food and Beverage Analysis: SEC finds applications in the analysis of carbohydrates, proteins, and lipids in food and beverage products.

Main Concepts:

Molecular Size Separation: SEC separates molecules primarily based on their size, making it useful for analyzing polymers, proteins, and particles.
Calibration: SEC requires calibration using standards of known molecular weight to establish a relationship between elution volume and molecular size.
Choice of Stationary Phase: The selection of the stationary phase is crucial, as the pore size distribution determines the size range of molecules that can be separated.
Elution Conditions: The choice of mobile phase and flow rate affects the separation efficiency and resolution of SEC.
Quantitative Analysis: SEC can be used for quantitative analysis by integrating the detector signal and comparing it with standards.

Conclusion:
SEC is a powerful chromatographic technique that separates molecules based on their size. It has a wide range of applications in chemistry, biochemistry, and other fields for the analysis of polymers, proteins, nanoparticles, and other macromolecules.

Chromatographic Techniques: Size Exclusion Chromatography (SEC) Experiment

Introduction:

Size Exclusion Chromatography (SEC), also known as Gel Filtration Chromatography, is a separation technique that separates molecules based on their size. Larger molecules are eluted first, followed by smaller molecules. SEC is widely used in biochemistry and chemistry to analyze and purify proteins, polymers, and other macromolecules.

Experiment Overview:

This experiment demonstrates the separation of a mixture of proteins using SEC. The experiment involves the following steps:

Materials:

SEC column (specify type and pore size if known, e.g., Sephadex G-75 column)
Mobile phase (buffer solution): (Specify buffer, e.g., Phosphate Buffered Saline (PBS), pH 7.4)
Sample containing a mixture of proteins: (Specify proteins, e.g., BSA, Myoglobin, Cytochrome C)
Chromatographic system (HPLC or FPLC): (Specify make and model if possible)
UV detector: (Specify wavelength, e.g., 280 nm)
Fraction collector (optional, but helpful)
Pipettes and other standard lab equipment

Procedure:

Sample Preparation: Prepare a solution of the protein mixture at a known concentration (e.g., 1 mg/mL) in the mobile phase. Filter the sample to remove any particulate matter.
Column Equilibration: Equilibrate the SEC column with several column volumes of the mobile phase. Monitor the baseline absorbance to ensure stable conditions before proceeding.
Sample Injection: Inject a known volume (e.g., 100 µL) of the prepared protein sample onto the equilibrated column using a syringe or autosampler.
Elution: Elute the proteins from the column using the mobile phase at a constant flow rate (e.g., 0.5 mL/min). Collect the eluate in fractions (e.g., 1 mL fractions) using a fraction collector.
Detection: Monitor the eluate continuously using a UV detector at a suitable wavelength (e.g., 280 nm for proteins). Record the absorbance as a function of elution volume or time.
Data Acquisition: The chromatographic software or data acquisition system should record and process the data for analysis.

Data Analysis:

The data from the experiment will be a chromatogram showing UV absorbance versus elution volume. Each peak represents a different protein. The order of elution reflects the relative sizes of the proteins; larger proteins elute first. Determine the retention volume (Ve) for each peak. By comparing these to the void volume (Vo) and total volume (Vt), you can calculate the partition coefficient (Kav) and estimate molecular weights using a calibration curve with proteins of known molecular weight.

Significance:

This experiment demonstrates the principle and application of Size Exclusion Chromatography (SEC) for the separation and analysis of proteins. SEC is a powerful technique that can be used to determine the molecular weight and size distribution of proteins. It is also used for the purification of proteins and other macromolecules. The results allow for qualitative and quantitative analysis of the protein mixture.

Related Topics