Porous polymer packings, HPLC

More recently, columns have been developed where the stationary phase is formed of a porous polymer network inside the capillary. These are called monolithic phases, and have emerged as an alternative to traditional packed bed columns for use in micro-HPLC. They hold many advantages over traditional packed bed columns, being easy to manufacture since the monolith is formed in situ, often via a one-step reaction process, and its properties such as porosity, surface area, and functionality can be tailored. Another major advantage is that they eliminate the need for retaining frits. These columns can be manufactured from a variety of materials, but the most common include sol-gel, methacrylate-based, acrylamide-based, and styrene-based polymeric structures. 

Depending on the particular method of polymer HPLC, is defined in different ways. It is the total volume of pores, Vp, in a porous packing but it can be also related to the total surface of packing (mostly to the surface situated within the pores) or to the effective volume of bonded phase. The volume of pores is relatively well defined in the case of many packings applied in polymer HPLC and plays an especially important role in the exclusion-based separations (Sections 16.3.3, 16.3.4, and 16.4.1). The exclusion processes, however, play an important role in the coupled techniques of polymer HPLC (Section 16.5). In the latter cases, the surface of packings and the effective volume of bonded phase are to be taken into account. In some theoretical approaches also, surface exclusion is considered. 

FIGURE 16.3 Dependences of the polymer retention volume on the logarithm of its molar mass M or hydrodynamic volume log M [T ] (Section 16.2.2). (a) Idealized dependence with a long linear part in absence of enthalpic interactions. Vq is the interstitial volume in the column packed with porous particles, is the total volume of liquid in the column and is the excluded molar mass, (b) log M vs. dependences for the polymer HPLC systems, in which the enthalpic interaction between macromolecules and column packing exceed entropic (exclusion) effects (1-3). Fully retained polymer molar masses are marked with an empty circle. For comparison, the ideal SEC dependence (Figure 16.3a) is shown (4). (c) log M vs. dependences for the polymer HPLC systems, in which the enthalpic interactions are present but the exclusion effects dominate (1), or in which the full (2) or partial (3,4) compensation of enthalpy and entropy appears. For comparison, the ideal SEC dependence (Figure 16.3a) is shown (5). (d) log M vs. dependences for the polymer HPLC systems, in which the enthalpic interactions affect the exclusion based courses. This leads to the enthalpy assisted SEC behavior especially in the vicinity of For comparison, the ideal SEC dependence (Eigure 16.3a) is shown (4). 

In certain cases, affinity columns can be used to fractionate within classes of bound materials for instance, protein A antibody columns have been used to separate the various subtypes of IgG. In this case, the packings are micro-porous, heavily cross-linked polymers and benefit from HPLC operating conditions. Eluting conditions are usually step gradients of buffers with different pHs. The last step of a protein G column is at a very acidic pH and the sample is eluted into a buffer solution that quickly raises the pH to prevent protein denaturation. 

In GC and LC the adsorbent is fixed into a cylinder that is usually made of glass, polymer or stainless steel (column). In this column the adsorbent is present as a porous or non-porous randomly arranged packing or as a monolithic block. Because of the high separation efficiency of packed columns made of small particles this type of chromatography is called high-performance liquid chromatography (HPLC). 

HPLC packings are generally porous, to maximize the surface area available for interaction with the analyte. The surface inside the pores must be accessible to the analyte without excessive restriction of diffusion. The technology to manipulate the pore size, surface area, and pore volume of silica was developed quickly and is well understood today. Thus the pore size may be tailored to the size of the analyte, and padcings can be created and optimized for small molecules as well as for polymers. 

Packed column SFC stationary phases are very similar or identical to those used for HPLC. With neat CO2 mobile phases, polymer or polymer-coated silica stationary phases have typically been used. With modified-C02 mobile phases, bonded-phase silica columns are typically used. For structural separations, diol, amino, or cyano stationary phases are most often used. For stereochemical separations, derivatized polysaccharide-bonded sihca columns are most often the stationary phases of choice. A powerful feamre of modified-C02 pSFC is the ability to serially connect different stationary phases to obtain enhanced or multiple mechanism separations. With subcritical (super heated) water mobile phases, the use of polymer, porous graphitic carbon, and polymer-coated zirconia stationary phases has been described. 

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