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Gel Permeation Chromatography

Gel permeation chromatography may also be known as gel size exclusion or filtration. For size-exclusion chromatography it is the case that stationary...

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This article writter by MN Editors on February 01, 2022

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Gel Permeation Chromatography
Gel Permeation Chromatography

Gel Permeation Chromatography Introduction

Gel permeation Chromatography (GPC) can be described as a kind of size-exclusion chromatography (SEC) which is a method of separating analytes according to size, usually with organic solvents. The technique is commonly employed to analyze polymers. It is a technique SEC was first invented in the year 1955 through Lathe as well as Ruthven. Gel permeation chromatography could be traced all the way back in the late 1950s by J.C. Moore of the Dow Chemical Company who investigated the method in 1964. The technology for the column has been licensed to Waters Corporation, who subsequently commercialized the technology in the year 1964. GPC equipment and consumables is available from several manufacturers. It is usually required to separate polymers for both analysis and to make the product you want to use.

When analyzing polymers, it’s important to take into account the dispersion (D) and molecular weight. Polymers can be identified with a wide range of definitions of molecular weight such as the molecular weight of the average number (Mn) and the molecular weight of the weight average (Mw) and the size molecular weight (Mz) and Mv, which stands for viscosity. (Mv). GPC allows the calculation of D and Mv, and, using other information, Mn as well as Mw and Mz are able to be calculated.

Gel Permeation Chromatography Principle

It is a method where the separation of the components is based on the molecular size or weight. The stationary phase employed is porous polymer matrix, that has pores completely lined with the solvent in order that is used for the mobility phase. The molecules within the sample are then pumped through specially designed columns that contain this microporous packing materials (gel).

The reason for this separation lies in the fact that molecules that are larger than the size of a certain amount are completely exempt from pores, smaller molecules can access the inside of the pores partially or completely. The mobile phase will therefore cause larger molecules to move through the column without restriction and without piercing the gel matrix. On the other hand, smaller molecules will be impeded in proportion to their penetration into the gel.

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.

Components 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. Gel

Gels serve as a stationary phases for GPC. The size of the pore of the gel has to be precisely managed to allow the application of the gel for a particular separation. Other desirable characteristics of the gel-forming agent include lack of any ionizing elements and, when used in a solvent, low affinity to the components that are to be separated. Commercial gels like PLgel & Styragel (cross-linked polystyrene-divinylbenzene), LH-20 (hydroxypropylated Sephadex), Bio-Gel (cross-linked polyacrylamide), HW-20 & HW-40 (hydroxylated methacrylic polymer), agarose gel and are often used based on different separation requirements.

2. Column

The column used to make GPC has been filled up with porous material for packing. This column will be filled gel. Commercially available columns comprise

  • Analytical column- 7.5–8mm diameters.  
  • Preparative columns-22–25mm  
  • Usual column lengths-25, 30, 50, and 60 cm.  
  • Narrow bore columns- 2–3mm diameter have been introduced

3. Eluent

The liquid (mobile phase) must be a great liquid for polymers. It must allow a high response to detectors from the polymer, and be able to wet the surface of the packing. The most commonly used eluents to dissolve polymers at ambient temperature include tetrahydrofuran (THF) as well as o-dichlorobenzene, Trichlorobenzene, at 130-150 degrees Celsius for crystallized polyalkynes, and O-chlorophenol and m -cresol at 90 degC for crystal polymers that are condensation like polyesters and polyamides.

4. Pump

There are two kinds of pumps that are able to provide consistent delivery of small volumes of liquids for GPC pumps: piston and the peristaltic pumps.

5. Detector

In GPC the concentration measured by weight of polymer within the solvent that is eluting can be continuously monitored by an instrument. There are numerous types of detectors that are available, and they can be classified into two major types. The first is the concentration sensitive detectors, which include UV absorption and differential refractiveometer (DRI) also known as refractive index (RI) detectors as well as infrared (IR) absorption as well as density detectors. The second type is the molecular weight sensitive detectors that include Low angle Light Scattering detectors (LALLS) and multi-angle light scattering (MALLS). The resulting chromatogram therefore represents an indication of the weight distribution of the polymer in relation to of the volume of retention.

Most sensitive is the differential UV photometer. The most popular detector is called the differential refractometer (DRI). For the purpose of determining copolymer composition it is essential to include two detectors in series. To ensure the accuracy of the copolymer composition, at least two detectors need to contain concentration sensors. The analysis of the majority of copolymer compositions is made by using UV as well as RI detectors, but other combinations may be employed.

Examples of Detector

Concentration sensitive detectors 

  • Bulk Property Detectors- Refractive Index (RI) Detector
  • Solute Property Detectors- Ultraviolet (UV) Absorption Detector 
  • Evaporative Detectors- Evaporative Light Scattering Detector (ELSD) 

Molar mass sensitive detectors 

  1. Light Scattering Detectors 
    1. Low Angle Light Scattering (LALS) Detectors 
    2. Multiangle Light Scattering (MALS) detectors 
  2. Viscosity Detectors- Differential Viscometers

6. Injector

Incorporates the polymer solutions in its mobile phase.

7. Automated data processing equipment

Automatically calculates, stores and reports numbers for the values Mz and Mw. It also records Mv, Mw, Mn, and MWD.

Data systems also offer total control over GPC systems, allowing huge numbers of tests can be run at a time and the raw data can be processed automatically. The current GPC software products must offer special calculations for multi-detection processing band broadening correction, specific calibration routines , and the determination of polymer branching to mention just a few.

Steps in Gel Permeation Chromatography

It involves three major steps:

A. Preparation of column for gel filtration

It includes:

  1. Gel swelling
  2. The column is packed with semi-permeable, pores of polymer gel, with the most well-defined range of pores sizes.
  3. Washing: Following packing, various column volumes of buffer solution are passed through the column to eliminate any air bubbles, and to check the homogeneity of the column.

B. Loading the sample onto the column using a syringe

C. Eluting the sample and detection of 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

  • Proteins fractionation
  • Purification
  • Molecular weight determination.
  • Separation of proteins, sugars and peptides, rubbers and other substances by their dimensions.
  • It can serve to identify the quternary structure of purified proteins.

Advantages of Gel Permeation Chromatography

  • It is separated by a clearly defined time because there is a definitive elution volume for all non-retained analytes.
  • GPC provides narrow bands, but this feature is a limitation of GPC is more challenging for polymer samples with broad spectrums of molecular weights.
  • Since the analytes cannot interact physically or chemically with the column it is less likely that there will be a likelihood that loss of analytes to occur.
  • To study specific properties in polymer sample specifically, GPC can be very beneficial.
  • GPC offers a faster and more efficient method to determine the molecular weights of polymers.
  • The majority of tests can be completely analysed within a half one hour, or even less.
  • These were very labor-intensive processes mass distributions and molecular weights generally were not examined.
  • GPC has enabled the simple and quick estimation of molecular weights as well as distribution of polymer samples

Limitations of Gel Permeation Chromatography

  • There are an extremely limited number of peak that can be resolvable within the time frame that is GPC. GPC run.
  • As a technique, GPC requires at minimum at least 10 percent difference in molecular weight to allow an acceptable resolution of the peak events to occur.
  • Concerning polymers, molecular mass of most chains are too close to the GPC separation to reveal anything other than broad peak.
  • Another drawback for GPC in polymers is filtrations have to be done prior to the use of the instrument in order to stop dust and other particles from damaging the columns and causing interference to the detectors.

References

  • Ugbolue, Samuel C.O. (2017). Polyolefin Fibres || Testing, product evaluation and quality control of polyolefins. , (), 313–338. doi:10.1016/B978-0-08-101132-4.00010-2 
  • GPC – Gel Permeation Chromatography aka Size Exclusion Chromatography- SEC, Wendy Gavin, Biomolecular Characterization Laboratory
  • https://chromatography.conferenceseries.com/events-list/applications-of-chromatography
  • 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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Microbiology Notes is an educational niche blog related to microbiology (bacteriology, virology, parasitology, mycology, immunology, molecular biology, biochemistry, etc.) and different branches of biology.

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