Size-Exclusion Chromatography (SEC)

This exploration into commercially available light scattering detection systems for size-exclusion chromatography (SEC) emphasizes new technologies and will help users understand the options.

This instalment describes the interplay between column length, pore size distribution, and particle size to optimize GPC/SEC separations.

Hydrolyzed collagens (collagen peptides) are water-soluble products obtained by hydrolysis of natural proteins and used for dietary supplements. A simple GPC/SEC method is described for molar mass determination of collagen peptides, allowing reliable molar mass determination using ultraviolet (UV) detection.

Analytical size-exclusion chromatography (SEC) often suffers from several limitations. This article shows how some of these limitations can be partly (or even completely) resolved by a multi-angle light scattering (MALS) detector for the determination of molar mass distributions of synthetic and natural polymers.

How to create a calibration curve when no chemically-matching reference materials with narrow molar mass distribution are available.

A review of alternative approaches to narrow standard calibration.

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.

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.

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.

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.

The accuracy of a measurement describes how close the measured result is to the true value. Accuracy is influenced by systematic errors, which are difficult to detect even for experienced scientists.

One of the major problems with plastics is recycling. Only a few materials can be recycled and the acceptance of recyclates is sometimes low. GPC/SEC can be applied to investigate the quality of materials containing recycled portions.

The addition of a light scattering detector dramatically increases the capabilities of gel permeation chromatography/size-exclusion chromatography (GPC/SEC) analysis. However, the complexity of the system also increases. This instalment of Tips & Tricks discusses column issues when working with light scattering detectors.

Although modern GPC/SEC instruments are generally very reliable, scientists sometimes encounter problems. This instalment of GPC/SEC Tips & Tricks offers advice on how to efficiently identify the root cause of problems, such as dealing with a too high pressure, loss of resolution, or drifting baselines.

The most commonly applied detector in gel permeation chromatography/size-exclusion chromatography (GPC/SEC) is the differential refractive index detector, RI. How UV–vis detection, if applicable, adds true value to GPC/SEC applications is discussed in this instalment of Tips & Tricks.

The potential of TD SEC for in situ analyses of thermoreversibly bonded polymers is discussed. TD SEC allows the evolution of the polymer’s molar mass distribution to be monitored during temperature-sensitive bonding and debonding reactions. Through quantitative evaluation of the chromatograms, the reaction-influencing parameters can be studied, which is crucial for the effective development of novel functional materials. By using TD SEC, the effect of polymer size and flexibility on the debonding temperatures of DA polymers was confirmed, their debonding and bonding ability studied, and the de-crosslinking of thermoreversibly cross-linked DA polymers assessed. TD SEC offers a versatile platform for a broad variety of different polymer materials and to assess a variety of different analytical questions.

While gel permeation chromatography/size-exclusion chromatography (GPC/SEC) of uncharged molecules in organic eluents in most cases is a straightforward task, aqueous GPC/SEC of polyelectrolytes usually requires more parameters to be considered and optimized. This instalment of Tips & Tricks explains more.

Gel permeation chromatography/size-exclusion chromatography (GPC/SEC) is a relative method and that requires a calibration to obtain molar mass distribution information. The way that the type of calibrants and the quality of the calibration curve influences the results is discussed in this instalment of Tips & Tricks.

The chemical composition of a copolymer material has a substantial impact on its properties and performance. Defining a composition profile is therefore an essential aspect of product development for these materials. The copolymer’s composition often varies considerably with molecular weight (MW), and in the R&D process, therefore, it requires a detailed analysis which maps out composition across the entire MW distribution.

The refractive index (RI) detector is the most common detector in gel permeation chromatography/size-exclusion chromatography (GPC/SEC). The advantage of this universal detector is that it detects everything; the disadvantage is that it detects everything. This instalment of “Tips & Tricks” offers some advice when working with RI detectors.

Ultrahigh-pressure size-exclusion chromatography (UHP-SEC) offers multiple benefits for synthetic polymer characterization. However, despite the many advantages of UHP-SEC, its rapid, low-volume separation is more sensitive to column calibration errors and drift than traditional high performance (HP)-SEC. Additionally, only a small selection of column chemistries are available. It is therefore essential to combine UHP-SEC with online, low-volume multi-angle light scattering instrumentation (UHP-SEC-MALS) to overcome these challenges.

Monitoring lipid oxidation during the shelf life of lipid-containing food emulsions, such as mayonnaise, is challenging. It is, however, essential for the development of improved, consumer-preferred products. Determining the nonvolatile lipid oxidation products (NONVOLLOPS), the precursor compounds for rancidity, is required to determine the effectiveness of product stabilization technologies. A method based on normal-phase liquid chromatography with atmospheric pressure photo ionization-mass spectrometry (LC–APPI-MS) was developed for this purpose. The inclusion of a size-exclusion chromatography (SEC) step was needed to remove interfering diacylglycerides and free fatty acids from the samples. The combined SEC and normal-phase LC–APPI-MS method allowed the identification of a wide range of oxidized species including hydroperoxides, oxo-2½ glycerides, epoxides, and other oxidized species. The method was found to be more suitable for the analysis of large sample sets.

Fluorescence polarization (FP) is a highly regarded technique for studying drug–protein interactions, but has limited value regarding protein mixtures. As a novel approach to drug target discovery, the possibility of combining FP with liquid chromatography (LC) was explored. Nondenaturing protein LC principles such as size-exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), and ion exchange chromatography (IEX) were found to be orthogonal and compatible with FP because the mobile phases used do not negatively affect detection. For simple protein mixtures, the SEC/HIC/IEX–FP approach was able to identify tankyrase as the target of a triazole-based inhibitor of the Wnt signaling pathway, which is heavily associated with colon cancer. However, the total peak capacity of the three LC dimensions was not sufficient to resolve at cell-proteome level, calling for higher resolution of intact proteins to enable stand-alone drug target discovery with LC and FP.

With many new biopharmaceuticals now being developed, robust analytical methods are needed to ensure that these protein-based drugs are of high purity and safe with a minimum amount of side effects. Size-exclusion chromatography is an important technique in investigating purity and is useful to identify and monitor protein aggregation, which can have economic and immunogenicity effects. This article discusses those column parameters that are most important in the selection of the optimum phase for SEC separations.

Hamish Small discusses the secrets to his success in separation science and the value of vague thoughts in scientific progress.

Size-exclusion chromatography (SEC) and pyrolysis-gas chromatography (Py-GC) are commonly used to characterize copolymers. SEC is a powerful method to determine the molecular-weight distribution of polymers whereas Py-GC provides valuable information on their chemical composition. The combination of these two techniques could yield combined size and composition information for copolymers or polymer mixtures. A fully automated system was constructed to perform these two-dimensional (2D) characterizations. Several examples of the use of this new technique to comprehensively characterize polymers are described.

The authors describe a new laser-induced flourescence detector for application to both capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC).