Gel Permeation Chromatography (GPC) is a specialized variation of size-exclusion chromatography (SEC), gathering the most attention in this field. This method is most often attributed to the profiling of macromolecules, polymers that are both natural (sand, cellulose) and synthesized (nylon, polystyrene), or cellular agents such as antibodies, membrane proteins, and oligopeptides.
First implemented in 1963 by Jim Water, GPC is akin to other analytical techniques, expounding upon a given analyte's molecular weight and composition. However, GPC provides further detail into the intrinsic viscosity and the hydrodynamic radius of the matrix it's analyzing. It is often associated with polymer analysis and can efficiently resolve individual monomer units.
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The Hull of the GPC
The GPC Apparatus has distinct components that one should be familiar with. First is the solvent supply/mobile phase, which is fixed to a solvent delivery system. Perpendicular to this section is the sample injector. The pump pushes the target sample and the solvent to form a homogenous matrix that will flow through the column. The Column is where the chemistry of interest takes place, encompassing a large tube with solution passing through it.
Here, the entire solution flows through a stationary phase, allowing different species to pass depending on their molecular weight. This stationary phase often forms polystyrene or glass, which will act as microdialyzer units. These units will cause larger polymer molecules within the column to flow quicker, while smaller molecules trace these beads and flow through more slowly. These polystyrene/glass beads are mechanically stable, chemically inert, and have an ideal homogenous porous structure.
The detector fit to this apparatus is variable and dependent on the analyte one wishes to assay. A differential refractometer (DRI) for polymer samples should be implemented- an example of a detector that will measure the analyte's refractive index relative to the mobile phase/solvent. This analytical approach trumps that of other polymer assaying methodologies such as ultracentrifugation in areas such as speed and extreme dissolution conditions (such as high temperatures or low pH). Often these detectors are fit to high-performance liquid chromatography (HPLC) hulls, but it is also used in size exclusion chromatography.
An alternate detector variant that can be employed is the evaporative light-scattering detector (ELSDs), which can expound upon the absolute molecular weight of the analyte. In addition, ELSDs provide data on monomers' radius of gyration, their intrinsic viscosity, and the viscosity of their average molecular weight.
Gel Permeation Chromatography's (GPCs) Operational Design
By design, the contents of the matrix will diffuse through the pores within the gels matrix and will not be halted by the stationary phase. In contrast, the smaller analytes within this medium will penetrate the pores of the gel at variable depths, all based on their size. To grasp a broader view of this methodology and to solve for unknowns, the following equation is often implemented in textbooks:
Vt = V0 +Vi + VM
Where…
Vt = the total volume of the column
V0 = the volume of liquid outside the gel matrix (dead volume)
Vi = the volume of liqid inside the matrix
VM = the volume of gel matrix
When we speak of "size" in the context of this scale, we mean that access to these pores is limited by steric hindrance, whereby the physical presence of surrounding ligands will hinder certain atoms within a molecule. This steric hindrance is attributed to the "charge" of any given moiety or element and can lead to the deterring or complete seizing of reactions between that element and others.
This principle of size exclusion especially pertains to polymers. When performing GPC for polymer assays, the polymers segregate based on their hydrodynamic size once the sample begins to pass through the porous packing material. Larger polymers may only pass through larger pores within the system; hence they can travel through the stationary phase more quickly.
The GPC system is unique in the sense that it can resolve many interesting characteristics of a polymer sample. Using a DRI, one can quantify the average molecular weight per polymer subunit, the dispersity Đ (the distribution of uniformity of the polymers subunits), and the refractive index value.
Limitations of GPC
When implementing an experimental design that utilizes GPC, it is important to consider the column's nature and the analyte's steric effects. Akin to steric hindrance, the structural characteristics of any polymer may also inhibit the target sample from passing through the glass beads that make up the stationary phase.
Finally, the interaction of the sample with the solvent itself may interfere with the permeability of the stationary phase, depending on the type of detector in use. A limited number of peaks can be resolved within the narrow time frame that the GPC runs. In addition, the molecular masses of some analytes may be too alike to resolve with GPC, resulting in broad and indistinguishable peaks.
Sources:
- Striegel A. M. (2017). Specific refractive index increment (∂n/∂c) of polymers at 660 nm and 690 nm. Chromatographia, 80(6), 989–996. https://doi.org/10.1007/s10337-017-3294-2
- Milana MR, Denaro M, Arrivabene L, Maggio A, Gramiccioni L. Gel permeation chromatography (GPC) of repeatedly extruded polyethylene terephthalate (PET). Food Addit Contam. 1998 Apr;15(3):355-61. doi: 10.1080/02652039809374651. PMID: 9666895.
- Striegel, A. M., & Sinha, P. (2019). Absolute molar mass determination in mixed solvents. 1. Solving for the SEC/MALS/DRI "trivial" case. Analytica chimica acta, 1053, 186–195. https://doi.org/10.1016/j.aca.2018.11.051
- le Maire M, Arnou B, Olesen C, Georgin D, Ebel C, Møller JV. Gel chromatography and analytical ultracentrifugation to determine the extent of detergent binding and aggregation, and Stokes radius of membrane proteins using sarcoplasmic reticulum Ca2+-ATPase as an example. Nat Protoc. 2008;3(11):1782-95. doi: 10.1038/nprot.2008.177. PMID: 18974737.
- Webster GK, Jensen JS, Diaz AR. An investigation into detector limitations using evaporative light-scattering detectors for pharmaceutical applications. J Chromatogr Sci. 2004 Oct;42(9):484-90. doi: 10.1093/chromsci/42.9.484. PMID: 15693189.
- Engel, P., Hein, L., & Spiess, A. C. (2012). Derivatization-free gel permeation chromatography elucidates enzymatic cellulose hydrolysis. Biotechnology for biofuels, 5(1), 77. https://doi.org/10.1186/1754-6834-5-77
- Whitfield, R., Truong, N. P., & Anastasaki, A. (2021). Precise Control of Both Dispersity and Molecular Weight Distribution Shape by Polymer Blending. Angewandte Chemie (International ed. in English), 60(35), 19383–19388. https://doi.org/10.1002/anie.202106729
Further Reading