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Liquid Chromatography
Published in Ernő Pungor, A Practical Guide to Instrumental Analysis, 2020
One of the main parameters which influences the analysis time is the temperature. If the temperature increases the viscosity will decrease, and the pressure drop on the column will decrease. The increased temperature will increase the plate number by a factor of two or three, decreasing the mass transfer resistance. The retention is a temperature-independent process in SEC. If a temperature-dependent retention can be obtained, the solute can adsorb onto the surface of the support used in SEC. This mainly happens when biopolymers are separated with an inorganic support such as silica gel. To reduce this unfavorable effect surface modification or adding masking agent to the eluent are widely used. It is also found that separation is independent of flow rate. The SEC can be used for separation of both synthetic polymers and biopolymers. One class of the rigid and porous stationary phase is based on silica gel.99 Unger stated the separation of polymers on silica gel columns can be done if the two solutes differ in their molecular weight by a factor of 2. There are two possibilities to separate a polydisperse solute. The first is a solution coupling of the columns of decreasing exclusion limit or using a packing which has two distinct average pore sizes with narrow distribution and nearly equal pore volume. This can be reached by mixing two different stationary phases or using a special stationary phase which meets the requirement stated above, a bimodal pore size which differs in diameter by of a factor 10.
Downstream Processing
Published in Maik W. Jornitz, Filtration and Purification in the Biopharmaceutical Industry, 2019
Resins are available which separate molecules within particular size ranges (Table 15.3). SEC is therefore used for the fine fractionation of molecules by size in the same way that gel electrophoresis exploits the sieving potential of agarose and polyacrylamide gels. Indeed, many of the SEC media available commercially are based on agarose, polyacrylamide, and other polymers. An important concept in SEC is that the separation medium is the pores on the beads and not the beads themselves. Therefore, 95%–99% of the column volume remains unused in any operation, and feed volumes must be adjusted accordingly, representing a significant bottleneck. For this reason, SEC is often the very final stage in biopharmaceutical purification, and is used to separate the target protein from very similar molecules such as degradation products and multimers. The most important variables in SEC are the column length and linear flow rate. Slow mass transfer of macromolecules can cause peak broadening and loss of resolution, which can be addressed by reducing the flow rate.
Dynamics of Dilute Polymer Solutions
Published in Timothy P. Lodge, Paul C. Hiemenz, Polymer Chemistry, 2020
Timothy P. Lodge, Paul C. Hiemenz
SEC is one of several modes of liquid chromatography in which a mixture of solutes is separated by passing a solution through an appropriate column. As the solution (the mobile phase) passes through the column, different solutes are retained to various degrees according to their interaction with the column packing (the stationary phase). Surface adsorption and ion exchange are examples of interactions that serve as the basis for other types of liquid chromatography. In SEC, the columns are filled with porous particles and the separation occurs because molecules of different sizes penetrate the pores of the stationary phase to varying degrees. The method is akin to a “reverse sieving” at the molecular level. The largest molecules are excluded from the pores to the greatest extent and, hence, are the first to emerge or elute from the column. Progressively smaller molecules permeate the porous stationary phase to increasing extents and elute sequentially. The emerging liquid, or eluent, is monitored for the presence of solute by a suitable detector and an instrumental trace of the detector output, a chromatogram, provides distinct peaks for well resolved mixtures and broad peaks for a continuous distribution of molecular sizes. With suitable calibration or multiple detection schemes, this information can be translated into a quantitative characterization of the sample in terms of molecular weight, molecular weight distribution, chemical composition, and even architecture. Owing to this wealth of potential information, the relatively rapid sample throughput (typically ∼ 30 min or less per solution), and the ease of automation, SEC is currently the single most important characterization tool in polymer science.
Colloidal lead in drinking water: Formation, occurrence, and characterization
Published in Critical Reviews in Environmental Science and Technology, 2023
Javier A. Locsin, Kalli M. Hood, Evelyne Doré, Benjamin F. Trueman, Graham A. Gagnon
More specialized methods provide continuous size discrimination via the mobility of the particles when exposed to a medium (i.e., column) or external field (Fig. 1). In SEC and HDC, smaller, lower molecular mass, colloids diffuse more slowly through the column (Fig. 1). By contrast, larger colloids will elute from the column more quickly. The mechanism of retention of the two techniques is different, however: in SEC, retention is due to preferential sampling of the pore volume (Prazeres, 1997), while in HDC it is due to preferential sampling of the streamlines of flow (Striegel & Brewer, 2012). SEC has a limited size separation range and surface adsorption may result in unintended intermolecular interactions of analyte components with the stationary phase (i.e. column packing material) (Stulík et al., 2003). Unlike SEC, the use of non-porous beads as the stationary phase in HDC considerably reduces interactions with particles but may be less efficient compared to other techniques (Striegel & Brewer, 2012).