Gel Permeation Chromatography – Definition, Principle, Parts, Steps, Applications

What is Gel Permeation Chromatography?

  • Gel permeation chromatography (GPC), also known as gel filtration or size exclusion chromatography (SEC), is a separation and analysis technique for molecules based on their size. GPC’s stationary phase is a porous matrix composed of substances such as cross-linked polystyrene, cross-linked dextrans, polyacrylamide gels, or agarose gels.
  • The porous gel matrix functions as a molecular filter, allowing smaller molecules to enter the pores and elute slowly, while bigger molecules are excluded from the pores and elute quickly. As a result, the molecules are classified according to their molecular sizes. This method is especially effective for separating proteins, polysaccharides, enzymes, and synthetic polymers.
  • Size exclusion chromatography may be traced back to 1955, when it was invented by Lathe and Ruthven. However, J.C. Moore of the Dow Chemical Company created the phrase “gel permeation chromatography” in 1964. Moore’s research on the approach resulted in the creation of unique column technology, which was ultimately licenced to Waters Corporation. This method was commercialised by Waters Corporation in 1964, making GPC equipment and consumables readily available. GPC systems and consumables are now available from a variety of manufacturers.
  • When characterising polymers with GPC, it is critical to consider not only the molecular weight but also the dispersity (), which provides information on molecular weight distribution. Molecular weight can be defined in several ways, including number average molecular weight (Mn), weight average molecular weight (Mw), size average molecular weight (Mz), and viscosity molecular weight (Mv). GPC can calculate dispersity () and viscosity molecular weight (Mv), as well as number average molecular weight (Mn), weight average molecular weight (Mw), and size average molecular weight (Mz) based on supplementary data. This extensive characterisation is critical for efficiently analysing and purifying polymers.

Gel Permeation Chromatography Principle

The separation of components based on their molecular weight or size is the basis of gel permeation chromatography (GPC), also known as gel filtration or size exclusion chromatography. The method employs a stationary phase composed of a porous polymer matrix. The solvent that serves as the mobile phase entirely fills the pores of this matrix.

The sample molecules are pumped through specialised columns containing the microporous packing material, also known as the gel, in GPC. The separation is accomplished by utilising the gel matrix’s size-based exclusion features. Molecules bigger than a particular size are completely barred from entering the pores of the gel, whilst smaller molecules can reach the inside of the pores partially or completely.

Larger molecules can move through the column unhindered since they do not penetrate the gel matrix as the mobile phase passes through it. Smaller molecules, on the other hand, experience mobility slowed as a result of partial or complete gel penetration. As a result, the capacity of the molecules to access and infiltrate the gel matrix determines their elution or retention period.

It is possible to separate molecules depending on their size or molecular weight by monitoring their elution times. Larger molecules will elute faster because they are not influenced by the gel matrix, but smaller molecules will elute later due to their interaction with the gel. This differential elution behaviour enables the separation and analysis of sample components dependent on their molecular size.

Overall, the GPC principle is based on the exclusion of larger molecules from the gel pores while smaller molecules penetrate the gel to varied degrees, resulting in differential elution periods and the separation of components based on size or molecular weight.

Important Note

  • Gel permeation chromatography/size-exclusion chromatography is a kind of high-performance Liquid Chromatography (LC).
  • GPC is a process that can be carried out using a range of solvents. From organics with non-polar polarity to aqueous formulations.
  • GPC/SEC utilizes columns that are packed with tiny round, porous, round particles that separate molecules in the solvent being pumped through them.
  • GPC/SEC is a method of separating molecules on the base of the size they are, hence the term’size exclusion’.
  • The initial GPC/SEC columns were packed with the materials known as gels. This is why they were called ‘gel permeation’..
  • GPC/SEC is used to measure the molecular weight distributions of polymers.
  • The particles of the columns are made of polymers which have been cross-linked to create insoluble or inorganic substances like the spherical silica.

Instrumentation of Gel Permeation Chromatography

The gel permeation process is carried out mostly in columns for chromatography. The design of the experiment isn’t any different from other methods for liquid chromatography. Samples are mixed with an appropriate solvent. In the instance of GPC they are typically organic solvents. After filtering, the solution is then injected into the column. Separation of the multi-component mix is carried out inside the column. The constant flow of fresh eluent into the column is made possible by using a pump. Because most analytes cannot be visible to the naked eye, the need for a detector is essential. In most cases, multiple detectors are employed to gather additional information about this polymer. The presence of detectors makes fractionation easy and precise.

Components/ Instrumentation of Gel Permeation Chromatography
Components/ Instrumentation of Gel Permeation Chromatography

1. Stationary phase

Gel permeation chromatography (GPC), also known as gel filtration or size exclusion chromatography, uses semi-permeable, porous polymer gel beads with a well-defined range of pore sizes as the stationary phase. These gel beads have unique features that make them appropriate for use as the stationary phase in GPC.

For starters, the gel beads are chemically inert, which means they don’t interact much with the molecules being separated. This guarantees that the separation process is mostly dependent on the analytes’ size or molecular weight rather than any chemical interactions.

Second, the stationary phase is mechanically stable, allowing for effective gel packing into columns and guaranteeing the gel’s integrity during the chromatographic process.

The ideal stationary phase has a consistent particle and pore size and a homogeneous porous structure. This consistency is critical for achieving consistent and reliable separations. Pore size changes might result in decreased resolution and reduced separation efficiency.

Gel materials of various sorts are utilised as the stationary phase in GPC. Here are several examples:

  1. Dextran (Sephadex) gel: Dextran (Sephadex) gel is a natural gel made of 1-6-polymerized glucose. Because of its well-defined pore size range and stability, it is commonly utilised in GPC.
  2. Agarose gel: Agarose gel is a natural gel that is composed of 1,3 linked -D-galactose and 1,4 linked 3,6-anhydro-, L-galactose. It offers a flexible stationary phase for GPC applications.
  3. Acrylamide gel: Acrylamide gel is a synthetic gel generated through the polymerization of acrylamide monomers. It allows for the customization of pore size and characteristics to meet individual separation needs.

These examples demonstrate the variety of gel materials used as the stationary phase in GPC, with each gel type offering unique benefits and characteristics suitable to certain applications. The stationary phase used is determined by criteria such as analyte type, required separation resolution, and overall chromatographic performance requirements.

2. The Mobile Phase

  • In gel permeation chromatography (GPC), also known as gel filtration or size exclusion chromatography, the mobile phase is a liquid that performs several critical tasks in the chromatographic process.
  • The mobile phase’s principal function is to dissolve the biomolecules or analytes being analysed. This ensures that the analytes are soluble, allowing them to pass through the chromatographic system and interact efficiently with the stationary phase.
  • Furthermore, the mobile phase is designed to allow for a rapid detection reaction. This means that it should be compatible with the GPC detecting method, such as UV absorption, refractive index (RI) detection, or light scattering techniques. The mobile phase should improve analyte detection by delivering accurate and sensitive concentration measurements.
  • Furthermore, the mobile phase acts to moisten the surface of the stationary phase’s packing material or gel beads. This wetting is essential for mass transfer between the stationary and mobile phases. The mobile phase enhances the interaction and separation of analytes based on their size or molecular weight as they travel through the gel matrix by wetting the packing surface.
  • The specific liquid used as the mobile phase is determined by a number of criteria, including the type of the analytes and the detection method used. Organic solvents, aqueous solutions, or solvent combinations are commonly employed mobile phases in GPC to optimise solubility and detection response.
  • To summarise, the mobile phase in GPC is a liquid that dissolves bio-molecules or analytes, allows for a high detection response, and wets the packing surface of the stationary phase. The mobile phase, by performing these activities, enables effective separation and analysis of analytes according on their size or molecular weight.

3. Column

The column is an integral component of the chromatographic system in gel permeation chromatography (GPC), also known as gel filtration or size exclusion chromatography. The stationary phase is housed in the column, allowing analytes to be separated according on their size or molecular weight.

Depending on the unique application and required analytical scale, multiple types of columns can be employed in GPC. These are some examples:

  • Analytical columns: Analytical columns are widely used in everyday analysis and research. Their sizes normally range from 7.5 to 8 mm. These columns are intended for smaller sample quantities and produce high-resolution separations, allowing for exact analyte characterisation.
  • Preparative columns: These are used when higher sample amounts are required for purification or isolation. The sizes of these columns are normally between 22 and 25 mm. They are intended to handle bigger sample volumes and to separate and collect larger amounts of purified analytes.
  • Column lengths: Columns in GPC come in a variety of lengths, including 25, 30, 50, and 60 cm. The length of the column is determined by the specific separation needs and the analyte resolution sought. Longer columns improve separation efficiency but may necessitate longer analytical durations.
  • Narrow-bore columns: GPC now includes narrow-bore columns with diameters ranging from 2 to 3 mm. These columns are utilised in applications that require lower sample quantities and higher sensitivity. They provide benefits such as lower sample and solvent usage while still providing reliable separations.

The optimum column dimensions are determined by the sample size, desired separation resolution, and detection technologies available. Analytical columns are typically used for routine analysis, whereas preparative columns are typically employed for larger-scale purification. The length and diameter of the column can be modified to match specific analytical needs, and narrow-bore columns provide enhanced sensitivity and efficiency for lower sample amounts.

Overall, the column in GPC is an important component that houses the stationary phase and is used to separate and analyse analytes based on their size or molecular weight. The column type and dimensions are determined by the application, sample size, and required resolution.

3. Eluent

In gel permeation chromatography (GPC), the eluent, also known as the mobile phase, is a crucial component that carries the sample through the chromatographic system. The eluent should possess certain characteristics to ensure optimal separation and detection of the polymer sample.

The eluent in GPC should fulfill the following requirements:

  1. Good solvent for the polymer: The eluent should be capable of dissolving the polymer sample effectively. It should have good solvating properties for the polymer under analysis to ensure proper interaction and separation during the chromatographic process.
  2. High detector response: The eluent should permit a high detector response from the polymer. This means that the eluent should enable effective detection and measurement of the polymer analyte by the chosen detectors, such as UV absorption or refractive index detectors. The eluent should enhance the sensitivity and accuracy of the detection process.
  3. Wetting of the packing surface: The eluent should wet the packing surface of the column. This ensures uniform and efficient flow of the eluent through the column, enabling proper interaction and separation of the polymer sample.

Commonly used eluents in GPC for polymers that dissolve at room temperature include:

  • Tetrahydrofuran (THF): THF is a widely used eluent in GPC for a variety of polymer samples. It is an effective solvent for many polymers and offers good solvating properties.
  • O-dichlorobenzene and trichlorobenzene at 130-150 °C: These eluents are specifically used for crystalline polyalkynes, which require higher temperatures for dissolution and separation.
  • M-cresol and o-chlorophenol at 90 °C: These eluents are suitable for crystalline condensation polymers such as polyamides and polyesters. The elevated temperature helps in effective dissolution and separation of these polymers.

The selection of the eluent depends on the specific polymer being analyzed and its solubility characteristics. Different polymers may require different eluents to achieve optimal separation and detection. The choice of eluent can impact the resolution and accuracy of the GPC analysis.

In summary, the eluent in GPC should be a good solvent for the polymer, permit high detector response, and wet the packing surface of the column. Common eluents used in GPC for polymers include THF, o-dichlorobenzene, trichlorobenzene, m-cresol, and o-chlorophenol, depending on the specific polymer being analyzed and its solubility properties.

4. Pumps

Pumps are an essential component of the chromatographic system in gel permeation chromatography (GPC). They are in charge of supplying the mobile phase, which transports the sample through the column for separation and analysis.

In GPC, two types of pumps are commonly used:

  1. Syringe pumps: In GPC systems, syringe pumps are frequently used. These pumps distribute the mobile phase at a steady flow rate using a syringe. They enable precise flow rate control, enabling for accurate and repeatable separations. Syringe pumps are ideal for analytical-scale GPC applications that require smaller sample quantities.
  2. Reciprocating pumps: Another form of pump utilised in GPC is the reciprocating pump. To deliver the mobile phase, these pumps reciprocate back and forth. Throughout the chromatographic process, they maintain a high constant flow rate. Where greater sample quantities and higher flow rates are required, reciprocating pumps are often utilised in both analytical and preparative-scale GPC systems.

The choice between syringe pumps and reciprocating pumps is determined by the GPC application’s specific requirements. Syringe pumps provide great precision and flow rate control, making them ideal for precise and accurate analytical separations. Conversely, reciprocating pumps can offer higher flow rates, making them more suitable for preparative-scale separations or applications involving bigger sample volumes.

The pumps guarantee a steady flow rate of the mobile phase in both circumstances, which is critical for achieving dependable and consistent separations. The flow rate is meticulously managed to ensure that the analytes move through the column at the appropriate pace, allowing for effective separation depending on size or molecular weight.

Pumps, in general, play an important part in GPC by supplying the mobile phase at a high constant flow rate. Pumps offer efficient and accurate separations in gel permeation chromatography, whether through syringe pumps for precise analytical separations or reciprocating pumps for greater flow rates in preparative applications.

5. Detector

Detectors play a crucial role in gel permeation chromatography (GPC) by monitoring and measuring various properties of the analytes as they elute from the column. There are different types of detectors used in GPC, categorized into concentration-sensitive detectors and molecular weight-sensitive detectors.

  1. Concentration-sensitive detectors: These detectors monitor the concentration of the polymer in the eluting solvent as it passes through the column. Some commonly used concentration-sensitive detectors include:
  • UV absorption detectors: UV detectors measure the absorption of UV light by the analyte. They are particularly useful for analyzing polymers with UV-absorbing functional groups.
  • Differential refractometer (DRI) or refractive index (RI) detectors: These detectors measure changes in refractive index caused by the analyte as it elutes from the column. RI detectors are commonly used in GPC due to their high sensitivity and wide applicability.
  • Infrared (IR) absorption detectors: IR detectors measure the absorption of infrared light by the analyte. They are useful for analyzing polymers with specific functional groups that exhibit characteristic IR absorption bands.
  • Density detectors: Density detectors measure changes in density caused by the presence of the analyte in the eluent. These detectors can provide valuable information about the composition and concentration of the analyte.
  1. Molecular weight-sensitive detectors: These detectors are used to determine the molecular weight distribution of polymers. They include:
  • Low angle light scattering detectors (LALLS): LALLS detectors measure the scattering of light at low angles by the analyte. This scattered light provides information about the size and molecular weight of the polymer.
  • Multi-angle light scattering detectors (MALLS): MALLS detectors measure the scattering of light at multiple angles, providing more comprehensive information about the size and molecular weight distribution of the polymer.

The choice of detectors depends on the specific requirements of the analysis. The most sensitive detector is the differential UV photometer, while the most commonly used detector is the differential refractometer (DRI). When characterizing copolymers, it is often necessary to use multiple detectors in series. For accurate determination of copolymer composition, at least two of the detectors should be concentration-sensitive detectors. Common combinations for copolymer analysis include UV and RI detectors.

In summary, detectors in GPC are used to monitor and measure the concentration and molecular weight distribution of the analytes. Concentration-sensitive detectors monitor the concentration of the polymer, while molecular weight-sensitive detectors provide information about the molecular weight distribution. The choice of detectors depends on the analyte characteristics and the specific analysis requirements.

6. Injector

Incorporates the polymer solutions in its mobile phase.

7. Automated data processing equipment

  • By handling and analysing the data generated during the chromatographic process, automated data processing equipment plays a critical role in gel permeation chromatography (GPC). These systems automate the calculation, storing, and reporting of numerous factors, resulting in efficient and precise results analysis.
  • The computation, storing, and reporting of critical parameters like as Mz (size average molecular weight) and Mw (weight average molecular weight) is one of the fundamental responsibilities of automated data processing devices in GPC. These numbers are crucial markers of the polymer sample’s molecular weight dispersion. Other important characteristics recorded by the equipment include Mv (viscosity molecular weight), Mn (number average molecular weight), and MWD (molecular weight distribution).
  • Furthermore, GPC data systems provide complete control over the entire chromatographic system. They enable the automated execution of a large number of tests, allowing for the efficient examination of several samples in a single run. The raw data produced by the chromatographic process can be processed automatically, saving time and lowering the possibility of human mistake in data processing.
  • Modern GPC software programmes are built with advanced features and computations in mind to improve data processing and analysis. These software programmes incorporate multi-detection processing computations that take into consideration data from numerous detectors used in GPC, such as UV, RI, or light scattering detectors. This allows for more precise characterization of the polymer sample.
  • Another useful feature provided by GPC software is band broadening adjustment. It accounts for the dispersion or spreading of analyte bands as they move through the chromatographic system, ensuring that the molecular weight distribution is accurately determined.
  • GPC software also includes calibration methods to calibrate the system and ensure precise and reliable measurements. These techniques involve the use of known molecular weight standard reference materials to construct a calibration curve and properly determine the molecular weights of the sample components.
  • Furthermore, modern GPC software can aid in the determination of polymer branching, a property that provides vital information about the structure and properties of the polymer.
  • In general, automated data processing equipment in GPC is critical for handling and analysing data collected during the chromatographic process. It automates essential parameter computations, storage, and reporting, providing control over the GPC system and enabling efficient analysis of many samples. The software programmes include advanced features and computations for reliable data processing, band broadening correction, calibration algorithms, and polymer branching characterization, among other things.

Steps in Gel Permeation Chromatography

Gel permeation chromatography (GPC), also known as gel filtration or size exclusion chromatography, involves three major steps in the process:

A. Preparation of the column for gel filtration:

  1. Swelling of the gel: The first step is to prepare the gel by swelling it in an appropriate solvent. The gel used in GPC is a semi-permeable, porous polymer with a well-defined range of pore sizes. Swelling the gel allows it to reach its maximum volume and ensures optimal separation during the chromatographic process.
  2. Packing the column: Once the gel is swollen, it is packed into the column. The gel beads, with their defined range of pore sizes, are carefully placed in the column to create a porous matrix for separation. Proper packing ensures uniform flow of the eluent and efficient separation of the sample components based on their molecular sizes.
  3. Washing: After packing the column, it is essential to remove any air bubbles and test the column’s homogeneity. Several column volumes of buffer solution are passed through the column to wash and stabilize it. This step ensures that the column is ready for sample loading and provides consistent results.

B. Loading the sample onto the column using a syringe:

Once the column is prepared, the next step is to load the sample onto the column. The sample, typically a solution containing the target analytes (e.g., proteins, polymers, or other biomolecules), is carefully injected onto the column using a syringe. The sample is introduced at the top of the column and allowed to flow into the gel matrix.

C. Eluting the sample and detection of components:

After the sample is loaded onto the column, the elution process begins. The eluent, or mobile phase, is continuously passed through the column, causing the components of the sample to move through the gel matrix. The separation is based on the molecular sizes of the analytes, as the gel behaves like a molecular sieve. Larger molecules are excluded from the pores, while smaller molecules partially or wholly access the interior of the pores. As a result, the larger molecules pass through the column more quickly, while smaller molecules are retarded based on their penetration of the gel.

During elution, the components of the sample are detected using appropriate detectors. These detectors may include concentration-sensitive detectors (e.g., UV absorption or refractive index detectors) or molecular weight-sensitive detectors (e.g., light scattering detectors). The detectors measure specific properties of the eluting components, providing information about their concentration, molecular weight, or other relevant parameters. The resulting chromatogram represents the weight distribution of the polymer or analyte as a function of retention volume.

In summary, the steps in gel permeation chromatography involve the preparation of the column by swelling and packing the gel, loading the sample onto the column using a syringe, and then eluting the sample through the column while detecting and analyzing its components.

GPC Sample Preparation

The most crucial aspect to consider when the preparation for the GPC analysis is locating the right solvent to dissolve the polymer. This may sound trivial however, be aware this: GPC is actually a sorting method dependent by the dimension of the polymer that is present. Polymer chains expand to a specific, relaxed state in solution and the solvent selected will determine what the dimensions will be. Most polymers are soluble at room temperature in a variety of solvents, however in certain instances (especially for crystallized polymers) the use of high temperature is necessary to dissolve. Another aspect that is crucial that is crucial to GPC preparation of the sample is the concentration that is selected. If the weight of the sample on the columns is excessive it could cause impacts of viscosity or concentration which can lead to improper volume of elution. Another thing to consider is whether or not to remove the solution of polymer. We will examine the factors to consider when preparing the sample.

It is essential to clean the eluent in vacuum prior to use in the chromatographic process. For organic solvents, the fluorocarbon filter is typically employed. The size of the membrane typically 0.45m (micron).

A. Concentration

After we have selected the correct solvent for analysis then the following step will be to make the standard and samples. It is important to select a concentration that is sufficient to achieve an acceptable signal-to noise ratio, however without risk of overloading the column or causing the effects of concentration. The following table is an approximate “rule of thumb” to be followed in determining the concentration to be made. The concentrations are expressed in percentages, where 1.0 mg/ml corresponds to 0.10 percent. The corrections are not made for temperature, therefore it is assumed that everything was made at ambient temperature. The concentrations listed are to be taken as assuming that the maximum is 100ul.

GPC Sample Preparation
GPC Sample Preparation

B. Preparing the Sample

After we have successfully dissolving the samples and standards in the solvent we have chosen, and put in our GPC columns We are now ready to begin injecting. The next decision we need to determine if we should remove the solution sample. In most instances, we need to filter the sample before injecting it.

In general, just like for the solvent filtration mentioned earlier, we’d recommend the 0.45 millimeter membrane fluorocarbon filtration. In certain instances, where there is extremely tiny particles (such like carbon black, titanium dioxide silica, or any other fillers) it is possible to use it is possible to use a 0.45 millimeter filter can be utilized. Naturally, once we begin using very small filter sizes, polymer shear could be a problem. Filtering a very high molecular weight polymer with an 0.20m filter will certainly result in some degradation in shear. It is possible to decide whether or not the polymer is filtered and hope for no increase in pressure because of the plugging of the system’s in-line filter, or frit in the column.

We can now begin making injections of the samples and standards. As we mentioned earlier, we can inject up to 100ul of each column, according to the concentrations on the chart. The run time is roughly 15 minutes per column with an average speed of 1.0 milliliters per minute, so the time to analyze for a set of three columns is approximately 45 minutes.

After the sample set is completed, it’s necessary for data management systems to analyze the results in accordance with the integration technique we chose to use and provide a complete report. This can be accomplished automatically by using the “Run and Report” mode within Empower Software, or we could choose to go to every raw data file and manually integrate every sample.

How GPC is working?

GPC is a method of separating the molecules present in a solution according to its “effective size in solution.” To prepare a sample to be used for GPC analysis, the resin is first dissolved in a suitable solvent.

In the gel permeation chromatograph, the dissolving resin is injected into an continuously moving liquid (mobile phase). The mobile phase moves through thousands of extremely porous hard pieces (stationary particle) tightly packed into columns. The sizes of the pores that these particle have are controlled. They are accessible in a variety of sizes.

The size of the individual peak peaks is indicative on the dimensions of molecules in any resin as well as its constituents. It is called the molecular weight distribution (MWD) curve. When taken together, the peaks represent the MWD of a specimen. The larger the MWD the greater the size of the peaks get and the reverse is true. The greater that the molecular mass average the farther along the molecular-weight line the curve changes and in reverse

Applications of Gel Permeation Chromatography

Gel permeation chromatography (GPC), also known as gel filtration or size exclusion chromatography, has various applications in the field of analytical chemistry and biochemistry. Some of the common applications of GPC are:

  1. Proteins fractionation: GPC is widely used for fractionating proteins based on their size. The porous gel matrix acts as a sieve, allowing smaller proteins to penetrate the pores while larger proteins are excluded. This technique enables the separation and isolation of different protein fractions, which is useful for studying their individual properties and functions.
  2. Purification: GPC can be employed as a purification technique for biomolecules. By selecting an appropriate gel matrix with specific pore sizes, impurities or unwanted components can be separated from the target molecule based on their size. The larger impurities are excluded from the pores, while the target molecule passes through, resulting in its purification.
  3. Molecular weight determination: GPC is commonly used for determining the molecular weight of polymers, proteins, and other macromolecules. The elution behavior of the analyte is correlated with its molecular weight. By calibrating the column with known molecular weight standards, the molecular weight of the sample can be estimated by comparing its elution volume with those of the standards.
  4. Separation of sugar, proteins, peptides, rubbers, and others based on size: GPC is applicable for separating various biomolecules and polymers based on their size. It can be used to separate sugars, proteins, peptides, rubbers, and other macromolecules according to their molecular dimensions. This size-based separation provides valuable information about the sample composition and can aid in further analysis and characterization.
  5. Determination of the quaternary structure of purified proteins: GPC can be utilized to study the quaternary structure of purified proteins. By analyzing the elution behavior of protein samples under native conditions, GPC can provide insights into the oligomeric state or assembly of proteins. This information is crucial for understanding protein function, interactions, and stability.

In summary, Gel permeation chromatography (GPC) finds diverse applications in the field of biochemistry and analytical chemistry. It is commonly used for proteins fractionation, purification, molecular weight determination, separation of various biomolecules based on size, and studying the quaternary structure of purified proteins. GPC provides valuable insights into the composition, characteristics, and behavior of biomolecules and polymers, contributing to research, development, and quality control in various industries.

Advantages of Gel Permeation Chromatography

Gel permeation chromatography (GPC), also known as gel filtration or size exclusion chromatography, offers several advantages as a separation technique. Some of the key advantages of GPC are:

  1. Short analysis time: GPC provides a relatively quick method of analysis. Most samples can be thoroughly analyzed in an hour or less, making it a time-efficient technique for determining molecular weights and distributions of polymers.
  2. Well-defined separation: GPC offers a well-defined separation based on the size or molecular weight of the analytes. There is a final elution volume for all unretained analytes, ensuring consistent and reproducible separation.
  3. Narrow bands and good sensitivity: GPC can provide narrow bands during separation, which contributes to high resolution and sensitivity. This allows for accurate detection and quantification of the analytes in the sample.
  4. No sample loss: One of the advantages of GPC is that the analytes do not interact chemically or physically with the column. This reduces the chance of sample loss during the separation process, ensuring that the analytes are retained and detected efficiently.
  5. Small amount of mobile phase required: GPC requires a relatively small amount of mobile phase compared to other chromatographic techniques. This reduces the consumption of solvents and makes GPC more cost-effective.
  6. Adjustable flow rate: GPC allows for the setting of the flow rate, providing flexibility in the separation process. The flow rate can be optimized to achieve optimal separation and analysis conditions for specific samples.

Limitations of Gel Permeation Chromatography

Gel permeation chromatography (GPC), despite its advantages, also has some limitations. These limitations include:

  1. Limited resolution: Due to the short time scale of GPC runs, there is a limited number of peaks that can be resolved. This can be a constraint when analyzing complex samples with multiple components or closely spaced molecular weights.
  2. Requirement for filtration: Prior to GPC analysis, filtration of the sample is necessary to prevent dust and particulate matter from interfering with the columns and detectors. While this protects the instrument, there is a possibility that the pre-filtration process may remove higher molecular weight samples, affecting the representation of the sample on the column.
  3. Broad peaks for similar molecular weights: In the case of polymers, where the molecular masses of most chains are closely spaced, GPC separation may result in broad peaks that do not provide detailed resolution. This limitation can hinder the accurate determination of molecular weight distribution and subtle differences in polymer samples.

To overcome these limitations, alternative techniques such as field-flow fractionation (FFF) can be explored. FFF offers the advantage of separating particles based on their size and other physical properties using a flowing carrier fluid, without the limitations of column-based chromatography. FFF can provide better resolution and separation for samples with closely spaced molecular weights or complex compositions.

While GPC has its limitations, it remains a valuable technique for molecular weight determination and size-based separation in various applications. Understanding these limitations helps researchers and analysts choose the appropriate analytical approach for their specific samples and objectives.

FAQ

What is gel permeation chromatography (GPC)?

Gel permeation chromatography, also known as gel filtration or size exclusion chromatography, is a technique used for the separation and analysis of polymers and other macromolecules based on their size or molecular weight.

How does gel permeation chromatography work?

GPC utilizes a porous stationary phase, typically composed of gel beads, which acts as a molecular sieve. Sample molecules are separated based on their ability to access the pores of the stationary phase. Larger molecules pass through the column more quickly, while smaller molecules are retained longer.

What are the applications of gel permeation chromatography?

GPC is widely used in various fields, including polymer chemistry, biochemistry, pharmaceuticals, and environmental science. It is used for molecular weight determination, polymer characterization, quality control, protein fractionation, and the separation of various macromolecules.

What are the advantages of gel permeation chromatography?

Some advantages of GPC include short analysis time, well-defined separation, narrow bands, good sensitivity, no sample loss, and the ability to control the flow rate. It provides a convenient and efficient method for determining molecular weights and distributions of polymers.

What are the limitations of gel permeation chromatography?

GPC has limitations in terms of the number of resolved peaks within a short time scale, broad peaks for similar molecular weights, and the need for sample filtration to prevent interference. Some samples with closely spaced molecular weights may not show detailed resolution using GPC.

What types of detectors are used in gel permeation chromatography?

Detectors commonly used in GPC include concentration-sensitive detectors (such as UV absorption, refractive index, and infrared detectors) and molecular weight-sensitive detectors (such as light scattering detectors). These detectors provide information about the composition and molecular weight of the separated components.

How is the gel permeation chromatography column prepared?

The column is prepared by swelling the gel beads, packing them into the column to form a porous matrix, and then washing the column with a buffer solution to remove air bubbles and test column homogeneity.

What is the role of the eluent in gel permeation chromatography?

The eluent, or mobile phase, is a solvent that dissolves the bio-molecules and allows them to elute through the column. It should be a good solvent for the analyte, permit high detector response, and wet the packing surface for efficient separation.

Can gel permeation chromatography be automated?

Yes, gel permeation chromatography can be automated using data processing equipment. Automated systems calculate, store, and report values for molecular weights and distributions. They also offer control over GPC systems, allowing multiple tests to be run simultaneously and enabling automatic processing of raw data.

How is gel permeation chromatography different from other chromatographic techniques?

Gel permeation chromatography is specifically designed for the separation of macromolecules based on their size or molecular weight. Unlike other chromatographic techniques, such as gas chromatography or liquid chromatography, GPC focuses on the analysis of polymers and other large molecules rather than small organic compounds.

References

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  • GPC – Gel Permeation Chromatography aka Size Exclusion Chromatography- SEC, Wendy Gavin, Biomolecular Characterization Laboratory
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  • https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206469/
  • https://www.malvernpanalytical.com/en/products/technology/liquid-chromatography/gel-permeation-chromatography
  • https://www.agilent.com/cs/library/primers/Public/5990-6969EN%20GPC%20SEC%20Chrom%20Guide.pdf
  • http://library.umac.mo/ebooks/b28050630.pdf
  • https://www.slideshare.net/asabuwangwa/gel-permeation-chromatography-gpc
  • https://en.wikipedia.org/wiki/Gel_permeation_chromatography
  • http://www.materials-talks.com/blog/2016/08/30/an-introduction-to-gel-permeation-chromatography-in-30-minutes/

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5 Best Microbiology Books For B.Sc 1st Year Students What is a digital colony counter? Why do Laboratory incubators need CO2? What is Karyotyping? What are the scope of Microbiology? What is DNA Library? What is Simple Staining? What is Negative Staining? What is Western Blot? What are Transgenic Plants?
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