Nonporous polymer column

Figure 7.33. HPLC analysis of nuclei acids (pBR322-DNA HAE-III digest) using gradient weak-anion exchange chromatography on a column packed with 2.5-pm nonporous polymer support (weak anion exchange) with UV detection at 260nm. Reprinted with permission from reference 40.

The nonporous spherical gels for PCHdC are often specially prepared for research purposes. However, nonporous polystyrene/divinylbenzene beads. Solid Bead, can be obtained in various particle sizes from Jordi Associates, Inc. (Bellingham, MA). Columns packed with these gels can be used for HdC of the polymers that are currently analyzed using polystyrene/divinylbenzene SEC columns. Fumed silica nanospheres are offered by Cabot (Tuscola, IL) (17), and nonporous silica (NPS) microspheres are offered by Micra Scientific, Inc. (Northbrook, IL). These nonporous silica gels may also be used for HdC. 

FIG U RE 1.13 Gradient separation of polypeptides on silica rod column and particle-packed columns. Mobile phase velocity 4mm/s, gradient 5%-60% ACN in the presence of TFA, gradient time 5min, columns (a) silica rod column, (b) Capcellpak SG (5 pm), (c) LiChrospher WP 300 RP-18e (5 pm), (d) nonporous NPS-ODS-1 HPLC column (1.5pm) (e) polymer-based TSKgel Octadecyl-NPR (2.5pm). (Reprinted from Minakuchi, H. et al., J. Chromatogr. A, 828, 83, 1998. Copyright 1998, with permission from Elsevier.)... 

The column length varies from 3 to 30 cm, the inner diameter from 1 to 10 mm. Columns are made of very resistant materials to withstand high pressures (<40 MPa) Most commonly, a stainless steel or heavy-walled glass tube is inserted into a metal tube. Columns are packed with a solid material of particle size <10 pm (spherical and nonporous microglass beads or polymer particles). A guard column is usually placed before the column to remove suspended particles from solvents, as well as certain sample constituents that could irreversibly bind to the stationary phase. 

Because HIC supports are designed for macromolecules, they either possess pore diameters of at least 300 A to allow inclusion or are nonporous. Both silica and polymer matrices are used because the hydrophilic polymeric coating minimizes or ehminates most matrix-based effects. The absolute retention and selectivity of an HIC support may be affected by the specific composition of the bonded phase, as well as the ligand. For example, protein mixtures have shown distinct selectivity on different HIC columns which have propyl functional groups [5]. 

The HPLC of large biomolecules such as proteins and DNA often requires specialized columns packed with wide-pore polymer or silica-based bonded phase with extra-low silanol activity.1215 Alternate approaches are pellicular materials or very small nonporous particles. Some of these columns are packed in PEEK or titanium hardware to allow the use of high-salt mobile phase and to prevent possible protein denaturing by metallic leachates. Further details on bio-separations and application examples are discussed in Chapter 7. 

In GC and LC the adsorbent is fixed into a cylinder (column) that is usually made of glass, polymer, or stainless steel (column). In this column the adsorbent is present as a porous or nonporous randomly arranged packing or as a monolithic block. 

The occurence of the complete adsorption of macromolecules forms a base of the full adsorption-desorption liquid chromatography-like separation method, FAD (Berek and Nguyen, 1998). A very weak mobile phase is employed, which acts as an adsorb for n-1 sample constituents. They are completely retained within the appropriate full adsorption-desorption column packed with nonporous particles. The unretained sample constituent is directly forwarded to an online SEC column for its molecular characterization. In the next stage, eluent strength is stepwise increased and sample constituents are successively one-by-one desorbed and forwarded into the SEC column. Successful separation of up to six distinct polymers with help of FAD method was reported. In the FAD method, solvents are chosen so that they always act as a desorb just for one sample constituent. 

Since there is no back pressure resisting the pump s ability to deliver the solvent at a given flow rate, as in HPLC, capillaries can be made longer. Thus, it is practical to utilize packings as small as 1 /u.m. These can be porous or nonporous, spherical or irregular, coated or uncoated, pure silica or mixed mode (silica/polymer, sil-ica/alumina, etc.). The combination of longer capillaries packed with smaller-diameter particles makes it possible to obtain and use columns with 100,000-500,000 and perhaps even 1,000,000 plates per meter. 

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