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Gel permeation chromatography

physical science Maturity 11-13 Next article →

Gel permeation chromatography, or GPC, is a specialized method of size-exclusion chromatography (SEC).

GPC instrument
GPC instrument

It is used to separate high molecular weight or colloidal analytes based on their size or diameter. This process typically occurs within organic solvents. Scientists primarily use GPC to analyze and purify polymers. Characterizing these polymers requires understanding their molecular weight and their size distribution. This distribution is often expressed as dispersity (Đ). By using GPC, researchers can determine various types of molecular weight. These include the number average molecular weight (Mn), the weight average molecular weight (Mw), and the size average molecular weight (Mz). It can also determine the viscosity molecular weight (Mv).

GPC Chromatogram
GPC Chromatogram

The mechanism of GPC relies on the physical size of molecules rather than chemical interactions. Unlike other chromatography types, GPC does not depend on physical or chemical attractions between the mobile and stationary phases. Instead, separation happens through the use of porous gel beads packed inside a column.

Pore size schematic
Pore size schematic

These beads act as the stationary phase. The principle of separation depends on whether macromolecules are included in or excluded from the pores. Larger molecules are too big to enter the pores. Consequently, they elute, or come out of the column, much earlier. Smaller molecules can enter the pores. This causes them to stay inside the column longer, increasing their retention time. The entire process occurs without the analytes interacting with the surface of the stationary phase.

Different types of molecules interact with the pores in distinct ways. If an analyte is too large for the pores, it is totally excluded. These excluded molecules elute with the free volume outside the particles, known as Vo. Conversely, if an analyte is very small, it may be totally permeating. These small molecules elute with the solvent, which represents the total permeation volume (Vi). Most molecules fall somewhere in between. They are partially retained based on their hydrodynamic volume, which is their radius of gyration. Because each column has specific pore sizes, each column has a specific range of molecular weights it can separate.

Size Limits for GPC
Size Limits for GPC

If a sample has a very broad molecular weight range, scientists might use several columns with different pore volumes in tandem to resolve the sample fully.

The history of GPC is tied to several key developments in the mid-20th century. The broader technique of size-exclusion chromatography was first developed in 1955 by Lathe and Ruthven. The specific term "gel permeation chromatography" is traced back to J.C. Moore. Moore worked for the Dow Chemical Company and investigated the technique in 1964. Following this, the proprietary column technology was licensed to the Waters Corporation. Waters Corporation subsequently commercialized the technology in 1964. Today, GPC systems and consumables are manufactured by many different companies.

To perform GPC, a specific set of instrumentation is required.

GPC instrument
GPC instrument

The process begins with an autosampler, which can run several samples without user interaction. A pump, such as a piston or peristaltic pump, provides a constant supply of eluent. This constant flow is vital for precision because the flow rate is used for calibration. The eluent is the mobile phase, which must be a solvent that dissolves the polymer. Common eluents include tetrahydrofuran (THF) or o-dichlorobenzene. Finally, a detector is needed to monitor the concentration of the polymer. Concentration-sensitive detectors include UV-VIS absorption and differential refractometers (DRI). Molecular weight-sensitive detectors include low angle light scattering (LALLS) and multi-angle light scattering (MALS).

Calibration is a critical step to ensure the accuracy of the results. GPC measures relative molecular weight, which can be determined with ± 5% accuracy if comparable standards are used.

Size exclusion standardisation
Size exclusion standardisation

Polystyrene standards are typically used for calibration. These standards usually have dispersities of less than 1.2. However, polystyrene is a very linear polymer. This means it is most useful when comparing it to other linear polymers of similar size. Scientists also use the hydrodynamic volume to create a universal calibration curve. This involves plotting the logarithm of the molecular weight against the retention time or volume.

GPC offers several significant advantages for laboratory work. It provides a well-defined separation time because there is a final elution volume for all unretained analytes. This makes the process efficient and predictable. Additionally, GPC can provide narrow bands, though this is harder with broad molecular weight samples. Because analytes do not interact chemically with the column, there is a lower chance of analyte loss. Most importantly, GPC is a very fast method. Most polymer samples can be thoroughly analyzed in one hour or less. This speed makes it much more efficient than older methods like fractional extraction or fractional precipitation.

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🖼️ Images & Media (8)
File:Pore size schematic.svg
Pore size schematic.svg
File:Size Limits for GPC.jpg
Size Limits for GPC.jpg
File:GPC instrument.jpg
GPC instrument.jpg
File:sample holder.jpg
sample holder.jpg
File:GPC Chromatogram.jpg
GPC Chromatogram.jpg
File:Anionic Polystyrene GPC Spectrum.jpg
Anionic Polystyrene GPC Spectrum.jpg
File:Free Radical Polystyrene GPC.jpg
Free Radical Polystyrene GPC.jpg
File:Size exclusion standardisation.png
Size exclusion standardisation.png
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