Size Exclusion Chromatography

Size Exclusion Chromatography

Size exclusion chromatography is a technique used to separate molecules based on their size, allowing for the analysis of samples in a liquid solution.

Size Exclusion Chromatography




Principles Of Size Exclusion Chromatography

Size Exclusion Chromatography (SEC), also known as gel filtration chromatography, is a widely used technique in the field of analytical chemistry. SEC is based on the principle of separating molecules based on their size, allowing researchers to determine the molecular weight or size distribution of a sample. This technique is particularly useful for analyzing polymers, proteins, and other large biomolecules, where size plays a crucial role in their function.

Molecular Size Exclusion

In SEC, the separation is achieved using a stationary phase consisting of porous particles. These particles have specific pore sizes that allow molecules of different sizes to interact with the stationary phase to varying degrees. Smaller molecules can enter the pores, leading to slower elution from the column, while larger molecules are excluded and elute faster. The result is a separation of molecules based on their size, with smaller molecules spending more time in the column and larger molecules passing through more rapidly. This separation process is highly valuable for determining the molecular weight of a sample or analyzing size variations within a mixture.

Pore Size Of The Stationary Phase

The successful separation of molecules in SEC depends on the careful selection of the stationary phase, particularly its pore size. The pore size dictates the range of molecule sizes that can be separated. Columns with larger pores are suited for separating larger molecules, while columns with smaller pores can resolve smaller molecules with higher resolution. Pore size is typically expressed in terms of the “exclusion limit,” which is the molecular weight at which a molecule can no longer enter the pores and is effectively excluded from the stationary phase. The choice of a stationary phase with the appropriate pore size is crucial for obtaining accurate and reliable separation results.

Instruments And Components

Size Exclusion Chromatography (SEC) is a powerful technique used to separate and analyze molecules based on their size and shape. To perform SEC effectively, a set of instruments and components are essential. Let’s take a closer look at the key components required for successful SEC analysis.


The most critical component of SEC is the column. Columns used in SEC are packed with stationary phases designed to create a size-based separation of molecules. The stationary phase typically consists of porous particles, referred to as the packing material, that allow smaller molecules to enter more freely, while larger molecules are excluded.

SEC columns are available in different lengths, internal diameters, and pore sizes to suit the specific needs of the analysis. When selecting a column, it’s important to consider the desired separation range and the nature of the molecules being analyzed. Different columns offer various resolving powers and can handle different sample volumes, so choosing the right column is crucial.

Mobile Phase

The mobile phase, also known as the eluent, is a crucial component of SEC. It is responsible for carrying the sample through the column and facilitating the separation process. The choice of mobile phase depends on the characteristics of the molecules being analyzed.

In SEC, the mobile phase is typically a non-interacting solvent such as water, an aqueous buffer, or an organic solvent. The mobile phase should be selected carefully to ensure compatibility with both the sample and the column. For instance, the mobile phase should have a viscosity suitable for efficient flow through the packed particles.

In some cases, the addition of additives or modifiers to the mobile phase can enhance the separation performance. These additives may include salts, surfactants, or organic modifiers. The optimization of the mobile phase composition is crucial for achieving the desired separation and resolution.

In conclusion, the SEC requires specific instruments and components to achieve accurate and reliable results. The choice of columns and mobile phases plays a significant role in successful SEC analysis. By understanding these key components, researchers can optimize their SEC method and obtain valuable insights into the size and shape of molecules.

Sample Preparation


Size exclusion chromatography is a sample preparation technique that helps separate molecules based on their size. It is a powerful tool used in various industries, including pharmaceuticals and biochemistry, to achieve accurate results and analyze complex samples efficiently.

Sample Preparation Sample preparation is a critical step in the process of Size Exclusion Chromatography (SEC), ensuring that the sample is in an optimal condition for analysis. This step involves the removal of soluble aggregates and buffer exchange. Let’s delve into each of these aspects in detail. Removal of Soluble Aggregates Soluble aggregates, if present in the sample, can interfere with accurate chromatographic analysis. These aggregates can potentially lead to broadened peaks, incorrect molecular weight determination, and poor resolution. To prevent such issues, it is crucial to effectively remove soluble aggregates prior to SEC. There are several methods available to remove soluble aggregates, including centrifugation, filtration, and ultrafiltration. Centrifugation can be particularly effective for samples with large aggregates, whereby high-speed centrifuges separate the aggregates from the desired sample. Filtration and ultrafiltration, on the other hand, utilize porous membranes to retain aggregates while allowing the desired sample to pass through. Buffer Exchange Buffer exchange is another crucial step in sample preparation for SEC. This process involves replacing the original buffer of the sample with a new buffer solution that is compatible with the SEC column and detection system. Buffer exchange is essential to maintain the stability and homogeneity of the sample throughout the chromatographic process. To achieve buffer exchange, an efficient method is dialysis, where the sample is placed in a semipermeable membrane and immersed in the desired buffer solution. Over time, small molecules, including the original buffer components, diffuse out of the sample while the desired buffer molecules diffuse in. This gradual exchange ensures that the sample is free from any carryover of incompatible buffer components. In Summary, Effective sample preparation is crucial for successful Size Exclusion Chromatography analysis. By removing soluble aggregates and performing buffer exchange, the sample condition is optimized for accurate and reliable results. Whether through centrifugation, filtration, ultrafiltration, or dialysis, these sample preparation methods play a significant role in ensuring the quality of the sample before it enters the SEC column. Remember, sample preparation is the foundation upon which the success of SEC analysis is built. By implementing these steps meticulously, researchers can enhance the accuracy and reliability of their results, ultimately advancing their understanding of biomolecules and their interactions.

Sample Injection And Separation

Sample injection and separation are critical steps in Size Exclusion Chromatography (SEC) that play a vital role in separating and analyzing molecules based on their size and shape. These steps are crucial for obtaining accurate and reliable results in various research and analytical applications. Let’s take a closer look at each step:

Sample Loading

Sample loading, also known as sample injection, is the initial step in SEC where the sample is introduced into the chromatographic system. The goal here is to ensure that the sample is loaded uniformly and without any disruption to the column packing material. This step requires precision and careful handling to avoid any loss of sample or misrepresentation of the molecular size distribution.

During sample loading, the sample is injected into the SEC column using a syringe or an automated injection system. It is essential to choose an appropriate injection volume to ensure that the sample is within the capacity of the column and does not cause any overloading. Typically, a smaller injection volume is preferred to minimize the risk of overloading and to enhance the resolution of the separation.


Once the sample is successfully loaded onto the column, the elution process begins. Elution refers to the movement of the sample through the column matrix and the separation of molecules based on their size. The elution process relies on the principle of size exclusion, where smaller molecules penetrate deeper into the column pores and take a longer path, while larger molecules elute faster, experiencing less interaction with the column material.

The elution process in SEC typically involves the use of a mobile phase, which is a solvent or buffer that flows through the column, carrying the sample along. Throughout the elution, the column separates the molecules based on their size, allowing them to elute at different times. This step is crucial for the accurate determination of molecular weight distributions and the identification of different molecular species present in the sample.

It is important to note that the elution conditions, such as the flow rate of the mobile phase and the column temperature, can significantly impact the separation efficiency and resolution of the SEC analysis. Careful optimization of these parameters is necessary to achieve optimal results and obtain reliable data.

Detection Methods

When it comes to analyzing the size and shape of molecules, Size Exclusion Chromatography (SEC) is an invaluable technique. However, in order to obtain accurate and precise results, it is essential to have reliable detection methods in place. Detection methods play a crucial role in quantifying and characterizing macromolecules, allowing scientists to gain deeper insights into their structure and behavior. In this section, we will explore two commonly used detection methods in Size Exclusion Chromatography: Ultraviolet (UV) Detection and Multi-Angle Light Scattering (MALS).

Ultraviolet (UV) Detection/u

The Ultraviolet (UV) detection method utilizes the absorbance of molecules in the ultraviolet spectrum. This method is particularly useful for detecting molecules with chromophores, such as aromatic amino acids or nucleic acids, which can absorb UV light. The absorbance of these molecules is directly proportional to their concentration, allowing for accurate quantification.

During the SEC process, as the sample containing the molecules passes through the UV detector, the molecules absorb UV light at specific wavelengths. The detector measures the absorbance of the sample at these wavelengths, and the data is then used to generate a chromatogram, showing the concentration of the molecules as a function of elution time.

Umulti-angle Light Scattering (mals)/u

Multi-Angle Light Scattering (MALS) is an advanced technique used for measuring the absolute molar masses and sizes of macromolecules in solution. Unlike other detection methods, MALS does not rely on the sample’s ability to absorb or emit light. Instead, it utilizes the scattering of laser light by the molecules.

As the molecules pass through the MALS detector, they scatter the laser light at different angles. The scattered light is then detected, and the intensity of the scattering is measured at multiple angles. By analyzing the scattering pattern, the absolute molar mass and size of the molecules can be determined, regardless of their UV absorption properties.

MALS provides valuable information about the size and structure of macromolecules, including their oligomeric state, aggregation behavior, and conformational changes in solution. It is particularly useful for studying complex biological molecules, such as proteins and polymers, providing insights into their interactions and stability.

Size Exclusion Chromatography






Size Exclusion Chromatography offers significant advantages, allowing for precise separation and analysis of biomolecules based on size, leading to accurate protein characterization and purification. This technique facilitates efficient sample preparation and provides insights into protein structure and function.

Size Exclusion Chromatography (SEC) offers several advantages that make it a popular technique in the field of analytical chemistry. With its non-destructive nature and high resolution, SEC provides valuable insights into the molecular weight and size distribution of a sample.


One of the key advantages of the SEC is its non-destructive nature. Unlike other chromatographic techniques that may alter the integrity of the sample, SEC allows for the analysis of delicate and sensitive molecules without damage. This means that after separation, the sample can be collected and further analyzed or utilized for other experiments.

High Resolution

SEC offers high resolution, enabling the separation and detection of molecules based on their size. The stationary phase of SEC consists of porous beads, with different pore sizes available for different applications. The larger molecules are excluded and eluted first, followed by smaller molecules. This separation process allows for the detection and quantification of individual components within the sample, providing valuable information about the molecular weight distribution.


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Size Exclusion Chromatography (SEC), also known as Gel Filtration Chromatography, is a versatile technique widely used for separating and analyzing molecules based on their size. It is commonly applied in various scientific fields due to its ability to provide important insights into the molecular weight, size, and distribution of a wide range of substances. Let’s explore some of the key applications of SEC:

Protein Analysis

Size Exclusion Chromatography plays a crucial role in protein analysis, enabling scientists to determine the molecular weight and oligomeric state of proteins. By separating proteins based on their size, SEC helps in identifying protein aggregates or impurities that may affect the quality, stability, and functionality of a protein sample. Furthermore, SEC can be utilized for studying protein-protein interactions, ligand binding, and structural characterization of proteins. Its accuracy and sensitivity make it an invaluable tool in various biological research fields such as biochemistry, biotechnology, and pharmaceutical sciences.

Polymers And Nanoparticles

SEC also finds extensive applications in the analysis of polymers and nanoparticles. The technique allows for the determination of molecular weight distribution, branching, and aggregation of polymers. By separating polymers based on their size, SEC aids in characterizing their physical properties, such as viscosity, rheology, and elasticity. Additionally, SEC is widely used in the analysis of nanoparticles, enabling researchers to evaluate their size distribution and purity. By providing precise and reliable data, SEC facilitates the development and quality control of polymeric materials and nanoparticle-based formulations used in industries such as plastics, cosmetics, and nanotechnology.

Overall, Size Exclusion Chromatography is a powerful analytical technique with broad applications in various scientific domains. It allows for the accurate determination of molecule size and distribution, making it an essential tool for protein analysis, polymer characterization, and nanoparticle research.

Size Exclusion Chromatography





While Size Exclusion Chromatography (SEC) is a powerful technique for separating and characterizing macromolecules, it does have certain limitations that need to be taken into account. Some of these limitations include:

Sample Retention Time Variability

One of the limitations of SEC is the variability in sample retention time. The retention time of a sample can vary depending on various factors such as the composition of the sample, the size and shape of the macromolecules, and the hydrodynamic volume. This variability in retention time can sometimes make it challenging to accurately compare and analyze different samples. It is important to consider this limitation when interpreting SEC results and to take into account other characterization techniques for a more holistic understanding of the sample.

No Information On Chemical Composition

Another limitation of SEC is that it does not provide information on the chemical composition of the sample. SEC primarily separates molecules based on their size and hydrodynamic volume, but it does not provide information about the specific chemical groups present in the sample. This means that SEC alone may not be sufficient for complete characterization of a sample, especially when detailed information about the chemical structure is required. In such cases, complementary techniques such as mass spectrometry or Fourier-transform infrared spectroscopy (FTIR) can be used to gain more insights into the chemical composition of the sample.

Future Developments

As science continues to advance, size exclusion chromatography is not exempt from the constant drive to improve and fine-tune existing methods. Researchers and scientists are exploring new avenues and pushing the boundaries of this separation technique. These future developments hold great promise in terms of improving column technology and enhancing detection methods.

Improved Column Technology

New improvements in column technology are set to revolutionize size exclusion chromatography, enabling even better separation and analysis of complex mixtures. Advanced materials and designs are being developed to enhance the efficiency, resolution, and selectivity of the columns used in the process. These innovative columns will allow for more accurate and precise determination of molecular weight distributions, helping researchers unravel the complexities of various biomolecules and polymers.

Enhanced Detection Methods

To further augment the capabilities of size exclusion chromatography, scientists are investing efforts into developing enhanced detection methods. By leveraging cutting-edge technologies, such as high-performance detectors and advanced data acquisition systems, researchers aim to achieve even greater sensitivity, specificity, and accuracy in detecting and quantifying target molecules in complex samples. These improved detection methods will facilitate deeper insights into the chemical composition, interactions, and structural characteristics of the analytes under study.

Frequently Asked Questions For Size Exclusion Chromatography


What Is Size Exclusion Chromatography Used For?


Size exclusion chromatography is a technique used to separate and analyze molecules based on their size. It is commonly used in biochemistry, analytical chemistry, and pharmaceutical research to determine the molecular weight, purity, and stability of a sample. This method is particularly effective for separating large biomolecules such as proteins, DNA, and polysaccharides.


How Does Size Exclusion Chromatography Work?


Size exclusion chromatography works by using a porous stationary phase called a gel matrix. When a sample is injected into the column, smaller molecules are able to enter the pores of the gel and take longer to elute from the column, while larger molecules pass more freely and elute faster.


As a result, the sample is separated based on size, with larger molecules eluting first and smaller molecules eluting later.


What Are The Advantages Of Size Exclusion Chromatography?


Size exclusion chromatography has several advantages over other separation techniques. It requires no sample pre-treatment, allows for non-destructive analysis, provides high resolution and reproducibility, and is suitable for a wide range of sample types. Additionally, this technique is relatively quick, easy to use, and provides valuable information about the size distribution and molecular weight of the sample.


Can Size Exclusion Chromatography Be Used To Determine Protein Size?


Yes, size exclusion chromatography is commonly used to determine the size of proteins. By comparing the elution times of protein standards with the elution times of the protein of interest, researchers can estimate the molecular weight and size of the protein.


This information is crucial for understanding protein function, interactions, and structure.




Size exclusion chromatography is a valuable technique for separating and analyzing biomolecules based on their size. By utilizing porous beads, this method allows smaller molecules to enter the pores and take longer to elute, while larger molecules pass through more quickly.


This process offers researchers a powerful tool for characterizing and purifying biomolecules in a variety of industries, from pharmaceuticals to biotechnology. With its numerous advantages and widespread applications, size exclusion chromatography continues to be an essential method in the field of analytical chemistry.

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