Type Of Chromatography That Separates Molecules Based On Size.

In the realm of analytical chemistry, size exclusion chromatography (SEC) stands out as a powerful technique for separating molecules based on their size. This method, also known as gel permeation chromatography (GPC) or gel filtration chromatography, plays a crucial role in various fields, from polymer science and biochemistry to pharmaceuticals and food chemistry. By exploiting the differences in hydrodynamic volume, SEC allows researchers to analyze complex mixtures, determine molecular weights, and study the aggregation behavior of macromolecules.

Introduction to Size Exclusion Chromatography

Size exclusion chromatography is a chromatographic technique in which molecules are separated based on their size, or more accurately, their hydrodynamic volume. Unlike other chromatographic methods that rely on chemical interactions between the analyte and the stationary phase, SEC uses a porous matrix to separate molecules based on their ability to enter the pores. Smaller molecules can access a larger fraction of the pore volume, thus spending more time inside the pores and eluting later. Conversely, larger molecules are excluded from most of the pores and elute earlier. This separation mechanism makes SEC particularly useful for analyzing polymers, proteins, and other macromolecules.

The term "size exclusion" is somewhat of a misnomer because it implies that the separation is purely based on size. In reality, the separation is based on the hydrodynamic volume, which is related to the size and shape of the molecule in solution. This distinction is important because molecules of the same molecular weight can have different hydrodynamic volumes depending on their conformation and degree of solvation.

Principles of Size Exclusion Chromatography

The fundamental principle of SEC revolves around the use of a porous stationary phase. This stationary phase consists of beads or particles with a well-defined pore size distribution. The mobile phase, typically a liquid, carries the sample through the column. As the sample passes through the column, molecules partition themselves between the mobile phase and the pores of the stationary phase.

Large molecules, too big to enter the pores, flow through the interstitial spaces between the beads and elute first. These molecules experience a smaller effective column volume and thus have a shorter retention time.
Small molecules, on the other hand, can enter the pores and explore a larger effective column volume. This leads to a longer retention time as they spend more time inside the pores, effectively delaying their elution.
Molecules of intermediate size can partially enter the pores, resulting in intermediate retention times.

The separation process is governed by the equilibrium between the mobile phase and the stationary phase, specifically by the accessibility of the pores to molecules of different sizes. The distribution coefficient, Kav, describes this equilibrium and is defined as:

Kav = (Ve - V0) / Vt

Where:

Ve is the elution volume of the molecule.
V0 is the void volume of the column (the volume accessible to molecules that are completely excluded).
Vt is the total volume of the column.

Kav ranges from 0 to 1. A Kav of 0 indicates that the molecule is completely excluded from the pores, while a Kav of 1 indicates that the molecule can freely enter all the pores.

Components of a Size Exclusion Chromatography System

A typical SEC system consists of several key components:

Mobile Phase Reservoir: This holds the solvent that carries the sample through the column. The mobile phase must be compatible with the sample and the stationary phase, and it should not interact with the sample molecules.
Pump: The pump delivers the mobile phase at a constant flow rate through the system. Precise flow control is essential for reproducible separations.
Injector: The injector introduces the sample into the mobile phase stream. It should be able to deliver a precise and reproducible volume of sample.
Column: The column is the heart of the SEC system. It contains the porous stationary phase that separates the molecules based on size.
Detector: The detector measures the concentration of the eluting molecules. Common detectors include UV-Vis spectrophotometers, refractive index detectors, and light scattering detectors.
Data Acquisition System: This system collects and processes the detector signal, generating a chromatogram that shows the elution profile of the sample.

Stationary Phases in Size Exclusion Chromatography

The stationary phase is a critical component of the SEC system. It consists of porous particles that provide the size-based separation. The choice of stationary phase depends on the size range of the molecules being separated and the properties of the mobile phase. Common stationary phase materials include:

Silica-based materials: These are widely used for separating proteins and other biomolecules in aqueous mobile phases. Silica particles can be modified with different functional groups to control their surface properties.
Polymer-based materials: These are used for separating polymers in organic solvents. Common polymer materials include polystyrene, polymethacrylate, and polyacrylamide.
Cross-linked agarose or dextran gels: These are used for separating large biomolecules, such as proteins and polysaccharides. These gels have a high water content and are biocompatible.

The pore size of the stationary phase is a critical parameter. It determines the range of molecular sizes that can be separated. Stationary phases with small pores are suitable for separating small molecules, while stationary phases with large pores are suitable for separating large molecules. The pore size distribution should be narrow to provide good resolution.

Mobile Phases in Size Exclusion Chromatography

The mobile phase plays a crucial role in SEC. It carries the sample through the column and interacts with the stationary phase and the sample molecules. The choice of mobile phase depends on the nature of the sample and the stationary phase. Important considerations include:

Solubility: The mobile phase must dissolve the sample completely.
Compatibility: The mobile phase must be compatible with the stationary phase and the detector.
Inertness: The mobile phase should not interact with the sample molecules or the stationary phase.
Viscosity: The viscosity of the mobile phase affects the pressure drop across the column and the separation efficiency.

Common mobile phases for SEC include:

Aqueous buffers: These are used for separating proteins and other biomolecules. The buffer should maintain a constant pH and ionic strength.
Organic solvents: These are used for separating polymers. Common organic solvents include tetrahydrofuran (THF), dimethylformamide (DMF), and toluene.
Mixed solvents: These are used to optimize the solubility of the sample and the separation efficiency.

Detection Methods in Size Exclusion Chromatography

Several detection methods can be used in SEC to measure the concentration of the eluting molecules. The choice of detector depends on the properties of the sample and the sensitivity required. Common detectors include:

UV-Vis Spectrophotometers: These detectors measure the absorbance of the eluting molecules at a specific wavelength. They are widely used for detecting proteins, nucleic acids, and other molecules that absorb UV or visible light.
Refractive Index (RI) Detectors: These detectors measure the difference in refractive index between the mobile phase and the eluting molecules. They are universal detectors that can be used for detecting any molecule that has a different refractive index than the mobile phase. However, they are less sensitive than UV-Vis detectors and are sensitive to changes in temperature and flow rate.
Light Scattering Detectors: These detectors measure the amount of light scattered by the eluting molecules. They are particularly useful for determining the molecular weight and size of polymers and other macromolecules. There are two main types of light scattering detectors: static light scattering (SLS) and dynamic light scattering (DLS).
Viscosity Detectors: These detectors measure the viscosity of the eluting solution. When combined with light scattering data, viscosity data can be used to determine the branching and conformation of polymers.
Mass Spectrometers (MS): These detectors measure the mass-to-charge ratio of the eluting molecules. They provide detailed information about the identity and structure of the molecules.

Applications of Size Exclusion Chromatography

SEC has a wide range of applications in various fields, including:

Polymer Science: SEC is widely used to determine the molecular weight distribution of polymers. This information is crucial for understanding the properties of polymers and for controlling their synthesis.
Biochemistry: SEC is used to separate and analyze proteins, nucleic acids, and other biomolecules. It can be used to determine the molecular weight of proteins, to study protein aggregation, and to purify proteins.
Pharmaceuticals: SEC is used to analyze the purity and stability of pharmaceutical products. It can be used to detect aggregates and other impurities in protein therapeutics.
Food Chemistry: SEC is used to analyze the composition of foods and beverages. It can be used to determine the molecular weight distribution of polysaccharides in food products.
Environmental Science: SEC is used to analyze the size distribution of particles in environmental samples. It can be used to study the transport and fate of pollutants in the environment.

Advantages and Limitations of Size Exclusion Chromatography

SEC offers several advantages over other chromatographic techniques:

Non-destructive: SEC does not involve strong interactions between the sample and the stationary phase, so it does not alter the sample molecules.
Predictable separation: The separation mechanism is well understood, making it easier to predict the elution behavior of molecules.
Versatile: SEC can be used to separate a wide range of molecules, from small organic molecules to large biomolecules.

However, SEC also has some limitations:

Limited resolution: SEC has lower resolution than other chromatographic techniques, such as reversed-phase chromatography. This is because the separation is based on size, which is not always a very discriminating property.
Peak broadening: SEC peaks can be broad due to diffusion and other factors. This can make it difficult to accurately determine the molecular weight of molecules.
Column limitations: The choice of stationary phase and mobile phase is limited by the need to maintain the integrity of the porous structure.

Gel Permeation Chromatography (GPC) vs. Gel Filtration Chromatography (GFC)

While SEC is the overarching term, two specific types are commonly used: Gel Permeation Chromatography (GPC) and Gel Filtration Chromatography (GFC). The distinction primarily lies in the mobile phase used:

Gel Permeation Chromatography (GPC): This term is typically used when the mobile phase is an organic solvent. GPC is primarily employed for the analysis of synthetic polymers, as they are often soluble in organic solvents like tetrahydrofuran (THF), dimethylformamide (DMF), or toluene. The stationary phases used in GPC are also designed to be compatible with organic solvents, often being composed of cross-linked polystyrene or other organic polymers.
Gel Filtration Chromatography (GFC): This term is used when the mobile phase is an aqueous solution. GFC is mainly used for the separation of biomolecules such as proteins, peptides, and polysaccharides, which are typically soluble in aqueous buffers. The stationary phases used in GFC are hydrophilic and biocompatible, often consisting of cross-linked agarose, dextran, or polyacrylamide gels.

Factors Affecting Separation in SEC

Several factors can affect the separation in SEC, and understanding these factors is crucial for optimizing the method.

Pore Size of the Stationary Phase: The pore size distribution of the stationary phase determines the range of molecular sizes that can be separated. Choosing a stationary phase with appropriate pore sizes for the sample is essential for achieving good separation.
Column Length and Diameter: Longer columns provide better resolution but also increase the analysis time and pressure drop. The column diameter affects the sample capacity and the sensitivity of the detection.
Flow Rate: The flow rate of the mobile phase affects the separation efficiency and the peak broadening. Higher flow rates can reduce the analysis time but also decrease the resolution.
Temperature: The temperature can affect the viscosity of the mobile phase and the interactions between the sample and the stationary phase. Controlling the temperature is important for reproducible separations.
Mobile Phase Composition: The mobile phase composition affects the solubility of the sample and the interactions between the sample and the stationary phase. Optimizing the mobile phase composition can improve the separation efficiency.
Sample Concentration and Injection Volume: High sample concentrations can lead to overloading of the column and peak broadening. The injection volume should be optimized to avoid band broadening.
Column Packing Quality: The uniformity and quality of the column packing can significantly affect the separation efficiency. Poorly packed columns can lead to peak broadening and reduced resolution.

Calibration and Molecular Weight Determination

SEC is often used to determine the molecular weight distribution of polymers and other macromolecules. However, SEC is a relative technique, meaning that it requires calibration with standards of known molecular weight. The calibration process involves running a series of standards through the column and plotting the elution volume versus the logarithm of the molecular weight. The resulting calibration curve can then be used to determine the molecular weight of unknown samples.

Several types of standards can be used for SEC calibration:

Narrow Polydispersity Standards: These are polymers with a narrow molecular weight distribution. They provide the most accurate calibration curves.
Broad Polydispersity Standards: These are polymers with a broad molecular weight distribution. They are less accurate than narrow polydispersity standards, but they are often easier to obtain.
Protein Standards: These are proteins with known molecular weights. They are used for calibrating SEC columns for protein analysis.

The accuracy of the molecular weight determination depends on the quality of the calibration curve and the similarity between the standards and the samples. It is important to use standards that are chemically similar to the samples and to run the standards under the same conditions as the samples.

Troubleshooting in Size Exclusion Chromatography

Like any analytical technique, SEC can be subject to various problems that can affect the quality of the results. Common troubleshooting issues include:

Peak Broadening: This can be caused by various factors, such as high sample concentration, high flow rate, poor column packing, or extra-column band broadening.
Peak Tailing: This can be caused by interactions between the sample and the stationary phase or by column overloading.
Poor Resolution: This can be caused by inappropriate pore size of the stationary phase, high flow rate, or poor column packing.
Pressure Increase: This can be caused by blocked frits, contaminated mobile phase, or column degradation.
Ghost Peaks: These can be caused by contamination of the mobile phase or the column.
Baseline Drift: This can be caused by temperature fluctuations, changes in mobile phase composition, or detector instability.

Conclusion

Size exclusion chromatography is a versatile and powerful technique for separating molecules based on their size. Its applications span across diverse fields, making it an indispensable tool for researchers and scientists worldwide. By understanding the principles, components, and factors affecting separation in SEC, one can optimize the method to achieve accurate and reliable results. Whether it's determining the molecular weight distribution of polymers, analyzing the purity of pharmaceutical products, or studying the aggregation behavior of proteins, SEC provides valuable insights into the properties and behavior of macromolecules.