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Gel Permeation Chromatography

Gel permeation chromatography used in laboratory

What is gel permeation chromatography?

In modern research, the examination of molecular weight and molecular weight distribution of polymeric materials is one of the important tasks to measure the material properties. Molecular weight determination has a wide range of applications in material development and preparation, quality control, and failure analysis. The various properties of many polymer materials are directly related to molecular weight, such as impact strength, heat, oxygen aging, corrosion resistance, tensile and bending, etc. One of the most common methods used to study polymers is gel permeation chromatography (hereinafter referred to as GPC). Gel Permeation Chromatography system is used to determine the fractionation and distribution of highly fractionated materials and to analyze the content of each component of the material, and the mobile phases available for GPC testing include water, tetrahydrofuran (THF), dichloromethane (DCM), dimethylformamide (DMF), trichlorobenzene (TCB), and chloroform.

Gel permeation chromatography (GPC), also known as volume exclusion chromatography (SEC), is a liquid phase chromatography method that uses a solvent as the mobile phase and porous packing material (such as porous silica gel or porous resin) as the separation medium. The GPC system consists of a pump system, an (auto) injection system, a gel chromatography column, a detection system, and a data acquisition and processing system.

Composition of the GPC instrument

a. The pump system consists of a solvent reservoir, a degassing unit, and a high-pressure pump. Its job is to make the mobile phase (solvent) flow into the column at a constant flow rate. The good or bad working condition of the pump directly affects the accuracy of the final data. The more precise the instrument, the more stable the working condition of the pump is required. The error of the required flow rate should be less than 0.01mL/min.

b. The chromatographic column is the core component of the gel permeation chromatography for separation. A chromatographic column is a stainless steel hollow thin tube with particles of different pore sizes as packing. Each column has a certain relative molecular mass separation range and permeation limit, and the column has an upper and lower limit of use. When the size of the smallest molecule of the polymer is larger than the size of the largest gel in the column, the polymer does not enter the pore size of the gel particles and flows through the outside of the gel particles, which does not achieve the purpose of separating polymers of different relative molecular masses. This result also means that the upper limit of the column has been reached. And there is a possibility of blocking the gel pore, which affects the separation effect of the column and reduces its service life. The lower limit of the use of the chromatographic column is when the largest size of the molecular chain in the polymer is smaller than the minimum pore size of the gel pore, which also does not achieve the purpose of separating different relative molecular masses. Therefore, when using gel chromatography to determine relative molecular masses, a column that matches the relative molecular mass range of the polymer must first be selected.

c. The filler needs to be selected according to the solvent to be used. The most basic requirement for the filler is that the filler cannot be dissolved by the solvent.

d. The material of the column is usually glass or stainless steel.

e. The detection systems are available as universal detector, differential refractometer detector, UV absorption detector and selective detector. Universal detectors are suitable for the detection of all polymers and organic compounds. The refractive index of the solvent for the differential refractometer detector is as different as possible from the refractive index of the sample to be measured. UV absorption detectors are characterized by the absence of strong absorption of the solvent near the characteristic absorption wavelength of the solute. Selective detectors are suitable for polymers and organic compounds that have a specific response to this detector.

Gel permeation chromatography principle

Basic working principle of gel permeation chromatography 

A. Separation principle

The gel is chemically inert and it does not have adsorption, partitioning, or ion exchange. The polymer solution being measured needs to pass through a column with different pore sizes inside. The paths available for molecules to pass through the column are the inter-particle gaps (larger) and the through-pores within the particles (smaller). As the polymer solution flows through the column (gel particles), larger molecules (larger in size than the gel pores) are excluded from the small pores of the particles and can only pass through the gaps between the particles at a faster rate, while smaller molecules can enter the small pores in the particles and pass at a much slower rate. In contrast, medium-sized molecules can penetrate into larger pores but are excluded by smaller pores, between the two cases mentioned above. After a certain length of the column, the molecules are separated according to their relative molecular masses, with those with larger relative molecular masses in front and those with smaller relative molecular masses at the back. The total volume of leachate received from the time the sample is fed into the column to the time it is eluted is called the leachate volume of the sample. When the apparatus and experimental conditions are determined, the leachate volume of the solute is related to its molecular weight. The larger the molecular weight, the smaller the leachate volume.
The separation range of gel permeation chromatography columns is very wide, from a few hundred to several million, even tens of millions. Compounds have a short residence time in the gel permeation column and are eluted before the solvent molecules elute. In general, strongly retained molecules do not accumulate in the column, thereby increasing the lifetime of the gel chromatography column. However, for some compounds with close relative molecular weights but different chemical properties or structures, the gel permeation chromatography column is unable to distinguish them and achieve the purpose of separation and purification.

B. Calibration principle

A line corresponding to the volume or time of drenching and the relative molecular mass of a monodisperse standard polymer with a known relative molecular mass is preformed and is called a "calibration curve". Monodisperse standards are hardly found in polymers and are usually replaced by narrowly distributed specimens. Under the same test conditions, a series of GPC standard spectra were made. The retention times of the samples with different relative molecular masses are plotted as lg M against t. The resulting curve is the "calibration curve". The calibration curve allows the calculation of the required relative molecular mass and relative molecular mass distribution information from the GPC spectra. There are not many types of polymers for which standards can be made, and it is not possible to have calibration curves for polymers without standards, nor is it possible to obtain the relative molecular masses and relative molecular mass distributions of polymers using the GPC method. In this case, the universal calibration principle can be used.

C. Universal calibration principle

Since the separation of polymers by GPC is based on molecular hydrodynamic volumes, this means that for the same molecular hydrodynamic volume, the hydrodynamic volumes are the same if the outflow is at the same retention time.

Application of gel permeation chromatography

Gel permeation chromatography application

Gel permeation chromatography can be used not only for the separation and determination of relative molecular masses and relative molecular mass distributions of polymers but also for the separation of fat-soluble and water-soluble substances depending on the gel packing used. Its separation relative molecular masses range from a few million to less than 100.

In recent years, gel permeation chromatography has also been widely used for small molecule compounds. It is impossible to achieve complete separation and purification of substances with similar relative molecular masses but different chemical structures by gel permeation chromatography. Gel chromatography cannot distinguish compounds with similar molecular sizes, and the difference in relative molecular masses needs to be above 10% to obtain a separation.

A. Application of gel permeation chromatography in quality inspection of drugs & chemical raw materials

Drugs are usually composed of components of different sizes. Theoretically, the GPC spectrum of a branded drug is as characteristic as a human fingerprint under certain chromatographic conditions. By comparing the similarities and differences between the GPC spectra of two samples, it can be determined whether they belong to the same brand. By the same token, GPC can be used for the quality inspection of chemical raw materials.

B. Application of gel permeation chromatography in the pre-treatment of pesticide residues analysis

Pesticide residue analysis is the analysis of trace components in complex matrices. The process mainly includes two parts: sample pretreatment and determination. The difficulty of the sample pretreatment process is to extract multiple pesticide trace components simultaneously and remove impurities effectively while maintaining high recovery, which is directly related to the accuracy and sensitivity of pesticide residue analysis. GPC plays an important role in the pre-treatment of pesticide residue analysis due to its own characteristics and can separate pesticide residues from a variety of complex matrices. GPC can also be used for pre-treatment of pesticide residue analysis, quality testing of drugs and chemical materials, etc. Users should use GPC reasonably according to the actual situation.

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