Gel Permeation Chromatography- Definition, Principle, Parts, Steps, Uses

Gel permeation chromatography (GPC) is a type of size-exclusion chromatography (SEC) that separates molecules based on their size, typically in organic solvents. The technique is often used for the analysis of polymers, such as plastics, rubbers, proteins and polysaccharides.

GPC can provide information about the molecular weight distribution, average molecular weight and molecular structure of polymers. These properties are important for determining the physical, chemical and mechanical properties of polymer materials.

The principle of GPC is that molecules of different sizes pass through a column packed with porous beads at different rates. Larger molecules cannot enter the pores and elute faster than smaller molecules that can penetrate the pores partially or completely. The elution time of each molecule is related to its size and can be calibrated using standards of known molecular weight.

GPC can be performed in a wide range of solvents, depending on the solubility and stability of the polymer sample. The most common solvents are tetrahydrofuran (THF), chloroform, dimethylformamide (DMF) and water. The choice of solvent affects the swelling and shrinking of the porous beads and the interaction between the polymer and the stationary phase.

GPC requires specialized instrumentation and components, such as columns, pumps, detectors and software. The columns are filled with gel-like materials that have well-defined pore sizes and are chemically inert and mechanically stable. The pumps deliver a constant flow rate of solvent and sample through the column. The detectors measure the concentration or other properties of the eluting molecules, such as refractive index, ultraviolet absorbance, light scattering or viscosity. The software controls the operation of the system and analyzes the data to calculate the molecular weight parameters.

GPC is a simple, fast and reliable technique that can be used for various applications, such as:

• Protein fractionation and purification
• Molecular weight determination of natural and synthetic polymers
• Characterization of polymer branching, cross-linking and end-groups
• Quality control of polymer products
• Investigation of polymer degradation and aging
• Separation of polymer additives, such as antioxidants, stabilizers and plasticizers

In this article, we will discuss the principle, components, steps, uses, advantages and limitations of GPC in more detail.

Gel permeation chromatography (GPC) is a type of size exclusion chromatography (SEC), which separates molecules based on their size or molecular weight. The principle of GPC is that larger molecules pass through a column faster than smaller molecules, because they have less interaction with the porous stationary phase.

The stationary phase in GPC is a gel composed of spherical beads with pores of different sizes. The pores are filled with the mobile phase, which is a solvent that carries the sample through the column. The sample molecules can enter the pores depending on their size and shape. Larger molecules are excluded from entering the smaller pores, while smaller molecules can access more pores. Therefore, larger molecules have a shorter path and elute faster than smaller molecules.

The separation of molecules in GPC depends on two factors: the molecular weight and the hydrodynamic volume. The molecular weight is the mass of a molecule, while the hydrodynamic volume is the space occupied by a molecule in solution. The hydrodynamic volume is affected by the shape and conformation of the molecule. For example, a linear molecule has a larger hydrodynamic volume than a branched or coiled molecule of the same molecular weight.

The elution order of molecules in GPC is determined by their hydrodynamic volume, not by their molecular weight. Molecules with smaller hydrodynamic volume elute later than molecules with larger hydrodynamic volume. However, if the molecules have similar shapes and conformations, then their elution order will correspond to their molecular weight. Therefore, GPC can be used to estimate the molecular weight of molecules by comparing their elution times with those of known standards.

GPC can also be used to measure the molecular weight distribution (MWD) of a sample, which is the range of molecular weights present in a sample. The MWD can be calculated from the area under the peaks in the chromatogram, which represents the relative amount of each molecular weight fraction. The MWD can provide information about the polymerization process, degradation, branching, and cross-linking of polymers.

Gel permeation chromatography (GPC) is a technique that requires specialized equipment and components to perform the separation of molecules based on their size. The main components of a GPC system are:

• Stationary phase: This is the porous polymer matrix that fills the column and acts as the sieving medium for the sample molecules. The stationary phase has a range of pore sizes that can accommodate different sizes of molecules. The stationary phase should be chemically inert, mechanically stable, and have a uniform and homogeneous porous structure. Some examples of stationary phases are dextran, agarose, and acrylamide gels.

• Mobile phase: This is the liquid solvent that carries the sample molecules through the column. The mobile phase should dissolve the sample molecules, wet the packing surface, and permit high detection response. The mobile phase should also be compatible with the stationary phase and the detectors. Some examples of mobile phases are water, buffer solutions, organic solvents, and mixtures of solvents.

• Columns: These are cylindrical tubes that contain the stationary phase and allow the passage of the mobile phase. The columns can vary in diameter, length, and material depending on the type and scale of analysis. For analytical purposes, columns with diameters of 7.5–8 mm and lengths of 25–60 cm are commonly used. For preparative purposes, columns with diameters of 22–25 mm are used. Narrow-bore columns with diameters of 2–3 mm have also been introduced to reduce solvent consumption and increase sensitivity.

• Pumps: These are devices that deliver a constant and precise flow rate of the mobile phase through the column. The pumps can be either syringe pumps or reciprocating pumps. The flow rate can be adjusted according to the desired resolution and speed of analysis.

• Detectors: These are devices that measure the properties of the eluted sample molecules and generate signals that can be recorded and analyzed. The detectors can be either concentration-sensitive detectors or bulk property detectors. Concentration-sensitive detectors measure the amount of sample molecules in the mobile phase, such as UV-visible absorbance, fluorescence, or mass spectrometry detectors. Bulk property detectors measure the changes in physical properties of the mobile phase caused by the presence of sample molecules, such as refractive index, viscosity, or light scattering detectors.

The components of a GPC system work together to achieve the separation and characterization of sample molecules based on their size. By choosing the appropriate stationary phase, mobile phase, column, pump, and detector, different types of molecules can be analyzed by GPC with high efficiency and accuracy.

The stationary phase in gel permeation chromatography (GPC) is composed of semi-permeable, porous polymer gel beads with a well-defined range of pore sizes. The pore size determines the molecular weight range that can be separated by the gel. The gel beads are packed into a column and immersed in the mobile phase, which is a solvent that dissolves the sample molecules.

The stationary phase has the following properties:

• Chemically inert: The gel should not react with the sample molecules or the mobile phase, and should not leach any impurities that may interfere with the detection or analysis.
• Mechanically stable: The gel should withstand the pressure and flow rate of the mobile phase, and should not deform or break during operation.
• With ideal and homogeneous porous structure: The gel should have uniform and spherical pores that allow different sizes of molecules to enter and exit according to their molecular weight. The pore size distribution should be narrow to achieve high resolution and reproducibility.
• A uniform particle and pore size: The gel should have consistent and well-defined particle and pore sizes to ensure even packing and flow distribution in the column.

Some examples of gels used as stationary phases in GPC are:

• Dextran (Sephadex) gel: An α 1-6-polymer of glucose natural gel that can separate molecules with molecular weights ranging from 100 to 150,000 Da. It is widely used for protein purification and fractionation.
• Agarose gel: A 1,3 linked β-D-galactose and 1,4 linked 3,6-anhydro-α, L-galactose natural gel that can separate molecules with molecular weights ranging from 1,000 to 40,000,000 Da. It is commonly used for DNA and RNA analysis.
• Acrylamide gel: A polymerized acrylamide synthetic gel that can separate molecules with molecular weights ranging from 100 to 2,000,000 Da. It is often used for synthetic polymers and oligonucleotides analysis.

The choice of the stationary phase depends on the type, size, and solubility of the sample molecules, as well as the desired resolution and speed of separation. Different gels can be mixed or stacked in a column to achieve optimal separation conditions.

The mobile phase is the liquid that carries the sample molecules through the column. It should be able to dissolve the sample molecules and wet the surface of the gel beads. The choice of the mobile phase depends on several factors, such as:

• The solubility and stability of the sample molecules
• The compatibility with the gel material and the detector
• The viscosity and polarity of the liquid
• The buffer capacity and pH of the solution

Some examples of mobile phases used in gel permeation chromatography are:

• Water or aqueous buffers for polar samples and hydrophilic gels
• Organic solvents such as tetrahydrofuran, chloroform, or hexane for non-polar samples and hydrophobic gels
• Mixed solvents such as methanol-water or acetonitrile-water for samples with intermediate polarity

The mobile phase should be filtered and degassed before use to remove any particulate matter and dissolved air that could interfere with the separation or detection. The flow rate of the mobile phase should be constant and optimal for the column and the sample. A higher flow rate can reduce the analysis time but also decrease the resolution. A lower flow rate can increase the resolution but also prolong the analysis time and increase band broadening. The optimal flow rate depends on factors such as:

• The column dimensions and packing material
• The molecular weight and size of the sample molecules
• The viscosity and pressure of the mobile phase

The mobile phase plays an important role in gel permeation chromatography as it determines the elution time and order of the sample molecules. Larger molecules that are excluded from the pores of the gel beads will elute faster than smaller molecules that can enter the pores. Therefore, the mobile phase should have a suitable solvent strength and polarity to ensure that the sample molecules do not interact with the gel beads or each other. This way, the separation is based solely on the size exclusion principle.

Columns are the essential components of gel permeation chromatography, as they contain the stationary phase that separates the sample molecules based on their size. Columns can vary in their dimensions, materials, and types of gel used. Some of the factors that affect the choice of columns are:

• The molecular weight range of the sample: Different gels have different pore sizes and exclusion limits, which determine the range of molecular weights that can be separated by them. For example, Sephadex G-25 has an exclusion limit of 5000 Da, which means that molecules larger than 5000 Da will not enter the pores and will elute first. On the other hand, Sephadex G-200 has an exclusion limit of 200000 Da, which means that molecules smaller than 200000 Da will enter the pores and will elute later. Therefore, the column should be selected according to the molecular weight range of the sample to achieve optimal separation.
• The resolution required: Resolution is the ability to distinguish between two adjacent peaks in a chromatogram. It depends on several factors, such as the column length, the particle size, and the pore size of the gel. Generally, longer columns, smaller particles, and smaller pores provide higher resolution, but they also increase the back pressure and the analysis time. Therefore, a balance between resolution and efficiency should be considered when choosing a column.
• The sample volume and concentration: The sample volume and concentration affect the loading capacity and the sensitivity of the column. The loading capacity is the maximum amount of sample that can be applied to a column without compromising the separation. It depends on the column volume and the gel type. Generally, larger columns and gels with larger pores have higher loading capacities. The sensitivity is the minimum amount of sample that can be detected by a detector. It depends on the column diameter and the detector type. Generally, smaller columns and more sensitive detectors have higher sensitivity. Therefore, the column should be selected according to the sample volume and concentration to achieve optimal loading and detection.
• The compatibility with the mobile phase: The column material and the gel type should be compatible with the mobile phase used for elution. The mobile phase should not damage or dissolve the column material or the gel beads. It should also not interfere with the detection or cause unwanted interactions with the sample molecules. Therefore, the column should be selected according to the mobile phase to ensure stability and reproducibility.

Some examples of columns used for gel permeation chromatography are:

• Sephadex columns: These are made of cross-linked dextran (a polysaccharide) gel beads with different pore sizes and exclusion limits. They are suitable for separating proteins, peptides, nucleic acids, and other biomolecules in aqueous buffers.
• Sepharose columns: These are made of cross-linked agarose (a polysaccharide) gel beads with different pore sizes and exclusion limits. They are suitable for separating proteins, antibodies, enzymes, and other biomolecules in aqueous buffers or organic solvents.
• Bio-Gel columns: These are made of cross-linked polyacrylamide (a synthetic polymer) gel beads with different pore sizes and exclusion limits. They are suitable for separating proteins, peptides, nucleic acids, carbohydrates, and other biomolecules in aqueous buffers or organic solvents.
• Styragel columns: These are made of cross-linked polystyrene (a synthetic polymer) gel beads with different pore sizes and exclusion limits. They are suitable for separating synthetic polymers, rubbers, plastics, and other organic molecules in organic solvents.

Pumps are devices that deliver a constant and precise flow of the mobile phase through the column and the detector. They are essential for maintaining the separation efficiency and reproducibility of gel permeation chromatography.

There are two main types of pumps used in gel permeation chromatography: syringe pumps and reciprocating pumps.

Syringe pumps

Syringe pumps are simple and inexpensive devices that use a motor-driven plunger to push the mobile phase through a syringe. They have a low pulsation and can generate high pressures. However, they have some disadvantages, such as:

• Limited volume capacity: The syringe size determines the maximum volume of mobile phase that can be delivered per run. Larger syringes may be cumbersome and difficult to handle.
• Frequent refilling: The syringe needs to be refilled manually after each run or when the volume is low. This may interrupt the analysis and introduce errors or contamination.
• Flow rate variation: The flow rate may vary depending on the viscosity of the mobile phase, the temperature, and the plunger speed. This may affect the retention time and peak shape of the analytes.

Reciprocating pumps

Reciprocating pumps are more complex and expensive devices that use a piston or a diaphragm to move the mobile phase through a check valve system. They have a high pulsation and can generate very high pressures. However, they have some advantages, such as:

• Large volume capacity: The volume of mobile phase that can be delivered per run is only limited by the reservoir size. Larger reservoirs can accommodate longer runs or multiple columns.
• Continuous operation: The reservoir can be refilled automatically or manually without interrupting the analysis. This reduces the downtime and improves the productivity.
• Flow rate control: The flow rate can be controlled by adjusting the piston or diaphragm stroke, frequency, and speed. This allows for more precise and consistent separation conditions.

Both types of pumps require regular maintenance and calibration to ensure their optimal performance and accuracy. Some factors that may affect the pump performance include:

• Leaks: Leaks may occur in the seals, valves, tubing, or fittings of the pump system. They may cause pressure drops, flow rate fluctuations, air bubbles, or contamination of the mobile phase.
• Wear: Wear may occur in the moving parts of the pump system due to friction, corrosion, or abrasion. They may cause noise, vibration, or reduced efficiency of the pump.
• Degassing: Degassing is the removal of dissolved gases from the mobile phase to prevent bubble formation in the pump system. Bubbles may cause pressure spikes, flow rate instability, or detector noise. Degassing can be achieved by using vacuum, ultrasonic, or membrane methods.

Pumps are critical components of gel permeation chromatography that affect the quality and reliability of the analysis. Therefore, they should be selected carefully based on their specifications, compatibility, and suitability for the intended application.

Detectors are devices that measure some property of the eluted components and generate a signal that can be recorded and analyzed. There are different types of detectors that can be used in gel permeation chromatography, depending on the information required and the nature of the sample.

The most common detector is the refractive index (RI) detector, which measures the change in refractive index of the eluent as different components pass through it. This detector is concentration-sensitive and can be used for any sample that has a different refractive index than the solvent. However, it has low sensitivity and cannot distinguish between components with similar refractive indices.

Another widely used detector is the ultraviolet-visible (UV-vis) detector, which measures the absorption of light at a specific wavelength by the eluted components. This detector is also concentration-sensitive and can be used for samples that have chromophores or absorb light in the UV-vis range. It has higher sensitivity than RI detector and can provide some information on the chemical structure of the components.

A more advanced type of detector is the light scattering detector, which measures the intensity and angle of light scattered by the eluted components. This detector is not concentration-sensitive but mass-sensitive, meaning that it can measure the absolute molecular weight of the components without calibration. It can also provide information on the molecular size and shape of the components, such as radius of gyration and branching. There are different types of light scattering detectors, such as low-angle light scattering (LALS), right-angle light scattering (RALS), and multi-angle light scattering (MALS).

Another sophisticated type of detector is the viscometer detector, which measures the change in viscosity of the eluent as different components pass through it. This detector is also mass-sensitive and can measure the intrinsic viscosity of the components, which is related to their molecular weight and conformation. It can also provide information on the hydrodynamic radius and branching of the components.

Some gel permeation chromatography systems use a combination of detectors, such as triple detection, which consists of RI, light scattering, and viscometer detectors. This allows for a more comprehensive characterization of the sample, as each detector provides complementary information on different aspects of the components.

Gel permeation chromatography (GPC) involves three major steps: preparation of the column, loading the sample, and eluting the sample.

Preparation of the column

The first step is to prepare the column for gel filtration. The column is a tube filled with a porous polymer gel that acts as the stationary phase. The gel has a well-defined range of pore sizes that determine the separation of molecules based on their size.

The preparation of the column involves the following steps:

• Swelling of the gel: The gel beads are soaked in a solvent that matches the mobile phase to be used. This allows the gel to swell and fill the pores with the solvent. The swelling time depends on the type and size of the gel.
• Packing the column: The swollen gel beads are packed into the column using a slurry method. The slurry is prepared by mixing the gel beads with some solvent in a flask. The slurry is then transferred to the column using a pump or a syringe. The packing should be done carefully to avoid air bubbles and channeling in the column.
• Washing: After packing, several column volumes of buffer solution are passed through the column to remove any air bubbles and to test the column homogeneity. The buffer solution should have the same composition and pH as the mobile phase. The washing also equilibrates the column with the mobile phase.

Loading the sample

The second step is to load the sample onto the column using a syringe. The sample should be dissolved or suspended in a solvent that is compatible with the mobile phase. The sample should also be filtered or centrifuged before loading to remove any particulate matter that could clog or damage the column.

The sample volume should be small enough to avoid overloading the column and broadening the peaks. Typically, 0.1-1% of the column volume is used as the sample volume. The sample is injected into the column through an injection port using a syringe.

Eluting the sample and detection of components

The third step is to elute the sample and detect its components. The elution is done by passing a continuous flow of mobile phase through the column using a pump. The mobile phase carries the sample molecules through the column and separates them based on their size.

The larger molecules are excluded from entering the pores of the gel and travel faster through the column. The smaller molecules can enter some or all of the pores of the gel and travel slower through the column. Thus, the molecules are eluted in order of decreasing size.

The eluted molecules are detected by a detector at the end of the column. The detector measures some property of the molecules, such as their concentration, refractive index, absorbance, fluorescence, etc. The detector generates a signal that is proportional to the amount of molecules passing through it.

The signal is recorded as a function of time or volume and plotted as a chromatogram. The chromatogram shows peaks corresponding to different components of the sample. The peak area or height can be used to estimate the relative amount of each component. The retention time or volume can be used to estimate the molecular weight or size of each component using a calibration curve.

Before loading the sample onto the column, the column must be prepared for gel filtration. This involves the following steps:

• Swelling of the gel: The gel beads that form the stationary phase must be swollen in a suitable buffer solution to achieve the desired pore size and volume. The swelling time depends on the type and size of the gel beads, and can range from a few hours to overnight. The swollen gel beads should be stored in a sealed container to prevent drying and shrinking.
• Packing the column: The column is filled with the buffer solution and then the swollen gel beads are added slowly and evenly to avoid air bubbles and channeling. The column should be packed tightly enough to prevent settling of the gel beads, but not too tightly to cause excessive back pressure. The packing can be done manually or using a packing device.
• Washing: After packing, several column volumes of buffer solution are passed through the column to remove any air bubbles and to test the column homogeneity. The flow rate and pressure should be monitored and adjusted as needed. The washing also equilibrates the column with the buffer solution and removes any impurities or contaminants from the gel beads.

After preparing the column for gel filtration, the next step is to load the sample onto the column using a syringe. The sample should be dissolved or diluted in the same buffer as the mobile phase to avoid any changes in pH, ionic strength, or solvent composition that might affect the separation. The sample volume should be small enough to minimize band broadening and peak overlap, but large enough to ensure adequate detection and recovery. Typically, the sample volume should be less than 5% of the column volume.

To load the sample onto the column, a syringe with a needle or a blunt tip is used. The syringe is connected to the injection port of the column and the sample is injected slowly and gently to avoid disturbing the gel bed. The injection port is then closed and the mobile phase is pumped through the column at a constant flow rate. The flow rate should be optimized according to the column dimensions, gel type, and sample characteristics. A higher flow rate can reduce the analysis time, but it can also decrease the resolution and increase the back pressure. A lower flow rate can improve the resolution, but it can also increase the analysis time and cause diffusion of the sample bands.

As the mobile phase flows through the column, it carries the sample molecules along with it. The sample molecules interact differently with the gel matrix depending on their size and shape. Larger molecules are excluded from entering the pores of the gel and elute faster than smaller molecules that can penetrate deeper into the pores. This results in a separation of the sample components based on their molecular weight or size. The elution order is from largest to smallest molecule.

The eluted sample components can be detected by various detectors such as refractive index, ultraviolet-visible, fluorescence, or light scattering detectors. The detectors generate signals that are recorded as chromatograms showing peaks corresponding to different sample components. The peak area or height can be used to quantify the amount of each component in the sample. The retention time or volume of each peak can be used to estimate the molecular weight or size of each component by comparing it with a calibration curve obtained from known standards. Alternatively, online coupling with mass spectrometry can provide more accurate and detailed information about the molecular structure and identity of each component.

Loading the sample onto the column using a syringe is an important step in gel permeation chromatography that requires careful optimization of several parameters such as sample concentration, volume, buffer composition, injection speed, flow rate, and detection method. By following these guidelines, one can achieve a successful separation of complex mixtures based on their molecular weight or size using gel permeation chromatography.

After loading the sample onto the column using a syringe, the mobile phase is pumped through the column at a constant flow rate. The mobile phase carries the sample molecules through the gel matrix, where they are separated based on their size or molecular weight. The larger molecules, which cannot enter the pores of the gel, will elute faster than the smaller molecules, which can penetrate deeper into the gel. This results in a size-based separation of the sample components.

The eluted components are detected by a detector at the end of the column. The detector measures a property of the mobile phase that changes as different components pass through it. For example, a refractive index (RI) detector measures the change in refractive index of the mobile phase caused by different solutes. A concentration-sensitive detector measures the change in concentration of the solutes in the mobile phase.

The detector generates a signal that is proportional to the amount of component present in the mobile phase at any given time. The signal is recorded as a function of time or volume of mobile phase eluted. This produces a chromatogram, which is a graphical representation of the separation process. The chromatogram shows peaks corresponding to different components in the sample. The retention time or volume of each peak indicates how long it took for that component to elute from the column. The peak area or height indicates how much of that component was present in the sample.

The molecular weight or size of each component can be estimated by comparing its retention time or volume with those of known standards that have been run under the same conditions. Alternatively, a calibration curve can be constructed by plotting the logarithm of molecular weight versus retention time or volume for a series of standards with known molecular weights. The molecular weight of an unknown component can then be determined by interpolating its retention time or volume on the calibration curve.

Gel permeation chromatography (GPC) is a versatile technique that can be used for various purposes in different fields of science and industry. Some of the common applications of GPC are:

• Separation of proteins, polysaccharides, enzymes, and synthetic polymers based on their size and molecular weight. GPC can resolve molecules with a wide range of molecular weights, from a few hundred to several million Daltons . GPC can also separate molecules with different shapes, such as linear, branched, or cyclic.
• Purification of biomolecules and polymers from contaminants, such as salts, solvents, monomers, oligomers, or additives. GPC can remove these impurities by eluting them either before or after the desired molecules . GPC can also be coupled with other techniques, such as ion exchange chromatography or affinity chromatography, to achieve higher purity levels.
• Determination of molecular weight distributions and averages of polymers and biomolecules. GPC can provide information on the number average molecular weight (Mn), weight average molecular weight (Mw), size average molecular weight (Mz), and polydispersity index (PDI) of a sample by using appropriate calibration methods and detectors . GPC can also measure the intrinsic viscosity and hydrodynamic radius of the molecules.
• Determination of quaternary structure and conformation of purified proteins. GPC can reveal the presence of aggregates, subunits, or complexes in a protein sample by comparing the elution profiles with those of known standards . GPC can also indicate the degree of folding or unfolding of a protein by measuring its hydrodynamic volume.
• Analysis of natural and synthetic macromolecules in various fields, such as biochemistry, biotechnology, pharmaceuticals, food, cosmetics, plastics, rubber, coatings, paints, adhesives, and petroleum. GPC can help to characterize the molecular properties and quality of these materials by providing information on their molecular weight distributions, composition, branching, cross-linking, end-groups, functionality, degradation, and stability .

These are some of the main applications of gel permeation chromatography. However, there are many more possibilities and variations that can be explored by using different types of columns, solvents, detectors, and operating conditions. GPC is a powerful and flexible technique that can meet various analytical needs and challenges.

Gel permeation chromatography (GPC) is a powerful technique for separating and analyzing molecules based on their size. It has several advantages over other methods of chromatography, such as:

• Short analysis time: GPC can separate molecules in a matter of minutes, depending on the column length and flow rate. This makes it suitable for rapid screening and quality control of samples.
• Well defined separation: GPC can resolve molecules with different molecular weights or sizes within a narrow range, depending on the pore size distribution of the stationary phase. This allows for accurate determination of molecular weight averages and distributions, as well as identification of molecular species.
• Narrow bands and good sensitivity: GPC produces narrow and symmetrical peaks for each component, which improves the resolution and detection of the analytes. The detectors used in GPC, such as refractive index or light scattering, are sensitive to small changes in concentration or molecular weight, respectively.
• No sample loss: GPC does not involve any chemical interaction between the sample and the stationary phase, which means that there is no sample loss or degradation during the separation. The sample can be recovered from the column after elution, if desired.
• Small amount of mobile phase required: GPC uses a liquid mobile phase that is compatible with the sample and the stationary phase. The amount of mobile phase required is relatively small compared to other chromatographic techniques, which reduces the cost and waste generation.
• The flow rate can be set: GPC allows for adjusting the flow rate of the mobile phase according to the desired separation speed and resolution. A higher flow rate can reduce the analysis time but may compromise the resolution, while a lower flow rate can improve the resolution but may increase the analysis time.

Gel permeation chromatography (GPC) is a powerful technique for separating and analyzing molecules based on their size, but it also has some limitations that need to be considered. Some of the limitations are:

• GPC cannot provide absolute molecular weight information. It can only give relative molecular weight values based on a calibration curve obtained from known standards. Therefore, the accuracy and precision of GPC results depend on the quality and suitability of the standards used. Moreover, different types of molecules may have different shapes and interactions with the gel matrix, which can affect their elution behavior and cause errors in molecular weight estimation.
• GPC requires high purity and homogeneity of the sample and the mobile phase. Any impurities or contaminants in the sample or the mobile phase can interfere with the separation and detection of the analytes. For example, dust or particulates can clog the pores of the gel matrix, reduce the column efficiency, and damage the detectors. Therefore, filtration and degassing of the sample and the mobile phase are essential steps before performing GPC analysis.
• GPC has a limited resolution and separation range. The resolution of GPC depends on several factors, such as the column length, the particle size and pore size distribution of the gel matrix, the flow rate and composition of the mobile phase, and the molecular weight distribution of the sample. However, even under optimal conditions, GPC can only resolve a limited number of peaks within a short time scale. Moreover, GPC can only separate molecules within a certain molecular weight range that matches the pore size range of the gel matrix. Molecules that are too large or too small for the gel matrix will be excluded or permeated completely, respectively, and will not be separated by GPC.
• GPC is not suitable for analyzing volatile or thermally unstable molecules. Since GPC operates at ambient or elevated temperatures and uses organic solvents as mobile phases, it can cause degradation or evaporation of some molecules that are sensitive to heat or solvents. Therefore, GPC is not recommended for analyzing volatile or thermally unstable molecules, such as some polymers or proteins. Alternatively, other techniques such as size-exclusion chromatography (SEC) or field-flow fractionation (FFF) can be used for such molecules.

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