Size-Exclusion Chromatography (SEC)

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Characterizing lipid nanoparticles (LNPs) is a developing delivery modality in biotherapeutic analysis. We address some of the current challenges and opportunities in this field, including an examination of the most common tools used to characterize LNPs.

Determination of molar mass distributions and the molar mass averages derived therefrom are the main objectives of gel permeation chromatography/size-exclusion chromatography (GPC/SEC) analysis. But what is the meaning of these averages and how are they influenced by the setting of baselines and integration limits? This instalment of Tips and Tricks in GPC/SEC will try to provide a better understanding.

In 2015, the American Chemical Society’s Committee on Professional Training added a requirement to the ACS degree certification program that undergraduates learn about macromolecules, supramolecular aggregates, and nanomaterials (MSN). This requirement can be met by a specialized course in these topics, but many programs are also choosing the distribute these topics across the curriculum.

Light scattering detectors are ideally suited for size-exclusion chromatography (SEC) because they provide the molecular weight and radius of gyration information of polymeric samples without column calibration. In this article, the fundamentals of light scattering as applied to SEC are introduced, with emphasis on the origin of the Rayleigh equation. This tutorial is geared to those new to the field or who already utilize light scattering and seek clarification regarding the multitude of equations associated with light scattering. Part X of this series will present a brief summary of commercially available light scattering instrumentation with emphasis on new detector technology. Owing to the complexity of data analysis and the many equations involved with light scattering measurements, part X will also include a glossary of principal symbols and a summary of relevant equations.

The macroscopic properties of material based on poly(D,L-lactic-co-glycolic acid) (PLGA) polymers are tunable by molar mass distribution and degree of branching, enabling optimization for applications in the pharmaceutical and medical industries. Size-exclusion chromatography followed by online multi-angle light scattering with intrinsic viscosity detection (SEC–MALS–IV) is an advanced analytical method for determining absolute molar mass distributions, identifying polymer conformation and quantifying branching. SEC–MALS–IV overcomes the errors that can be encountered in molar mass determined by conventional SEC, which arise from chemical composition and molecular structure, and provides comprehensive characterization of PLGA to facilitate the targeted development of optimized polymer.

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Water-soluble macromolecules require water as a mobile phase, and method development therefore seems to be straightforward. At second glance, however, aqueous gel permeation chromatography/size-exclusion chromatography (GPC/SEC) is complex and the choice of the stationary phase and pH are crucial. In addition, mobile phase additives are often required to allow for interaction-free separations.

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The chemistry of samples analyzed using gel permeation chromatography/size-exclusion chromatography/gel filtration chromatography (GPC/SEC/GFC) is very diverse. Different chemistries of stationary phases are required to allow for true size separation. Several types of materials are available, all of which have their advantages and limitations. While silica‑based stationary phases are most common in high performance liquid chromatography (HPLC), for macromolecules polymer-based phases are popular.

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The “greenest solution” is certainly using no solvent but gel permeation chromatography/size-exclusion chromatography (GPC/SEC) as a liquid chromatography (LC) technique requires the use of a mobile phase. The growing awareness of the need for more sustainable (greener) solutions has focused attention on environmentally- and health-friendly solvents and solutions.