#WYATT ASTRA SOFTWARE FULL#

The most common use of IEX chromatography is as an intermediate purification step for separating target proteins from aggregates, host cell proteins and other contaminants. Anion exchange (AIEX) and cation exchange (CIEX) matrixes bind negatively and positively charged variants respectively. The principle of IEX is based on protein separation according to their surface charge, leading to different ionic interactions with the support matrix 14, 15. Ion exchange chromatography (IEX) is another chromatographic method commonly used for protein separation and characterization 13. Therefore, relatively high protein concentrations may be required (mainly for small macromolecules) in order to obtain enough LS signal for MALS analysis. Separation in analytical SEC is highly influenced by the injection volume and this volume limitation does not exist in other chromatographic procedures. SEC-MALS usually requires extensive equilibration for achieving a clean baseline signal, since SEC columns often release particles from the stationary phase that interfere with the light scattering measurements (вЂњcolumn sheddingвЂќ) 4 5. Aggregates have a very intense light scattering signal, thus the presence of even low amounts of aggregates in the protein peak can introduce a significant error into the calculated molar mass.

#WYATT ASTRA SOFTWARE FULL#

However, most of the analytical SEC columns have limited separation ability due to their short length and thus full separation of oligomeric forms can be difficult 3. For a suitable oligomeric analysis, SEC-MALS should be used only for well-resolved peaks 4. The technique separates proteins only by size, so molecules with the same size cannot be separated and properly analyzed 2. The process of protein aggregation is an essential topic of substantial research and can results from different factors such as protein misfolding, chemical environment and concentration 11, 12.Дespite its advantages and potential, SEC-MALS has several limitations: 1.

SEC-MALS is also useful for studying and characterizing aggregations occurring in protein samples due to the high sensitivity of light scattering to high molecular weights species 4, 10. Coupling an analytical SEC column in-line with multi-angle light scattering, UV detector and refractometric detector (SEC-MALS) provides a widespread approach and a useful tool for accurate analysis of molar mass, oligomeric states and hydrodynamic radius of proteins in native solution, independent of the protein retention time analyzed by SEC 3, 8, 9. In addition, the interactions that could occur between the macromolecule and the stationary phase may affect the retention time relative to the theoretical expected value for the studied sample 4, 7. However, mass estimation by SEC is often inaccurate, since the retention time of the macromolecule depends on its hydrodynamic radius and not only on its molecular mass. SEC is a liquid chromatography method that separates macromolecules of different sizes based on the partial exclusion of these molecules from the pores of the stationary phase 6, and is widely used as an analytical tool for protein characterization and for molecular mass estimation 7. Size exclusion chromatography (SEC) and field flow fractionation (FFF), which separate macromolecules by size, are the frequent separation techniques coupled in-line with MALS for biological macromolecules characterization 3, 4, 5. Combining MALS with analytical separation techniques allows molar mass calculation at any point in the elution chromatogram and characterization of each population in a mixed sample. Characterization of a mixed sample using MALS can be performed for several types of macromolecules such as polymers, though for protein characterization MALS should be utilized with a pure sample or coupled to a separation technique 4. In particular, multi-angle light scattering (MALS) is utilized for molar mass determination of macromolecules such as proteins according to Rayleigh theory, and dynamic light scattering (DLS) can be used for measuring the hydrodynamic radius of the protein 3. Light scattering (LS) based techniques are commonly used in the research and characterization of different molecules and particles 1, 2.

Wyatt astra software