Stationary phase nonporous

The two techniques differ in that HDC employs a nonporous stationary phase. Separation is affected as a result of particles of different size sampling different velocities in the interstitial spaces. Size exclusion chromatography is accomplished by superimposing a steric selection mechanism which results from the use of a porous bed. The pore sizes may vary over a wide range and the separation occurs as a result of essentially the same processes present in the gel permeation chromatography of macromolecules. 

The simplest way to eliminate the problems of porous particles is to eliminate the pores. Nonporous particles of 10 pm diameter with a thin layer of stationary phase on their surfaces were in fact introduced early in the evolution of HPLC packings as a solution to the pore problem, but achieved little popularity because of their low capacity. Recently, nonporous materials have returned to the marketplace in the form of very small particles with diameters of 1.5 to 2.5 pm. The use of small particles compensates to some extent for the loss in capacity.11 However, because of the high flow resistance of microparticulate nonporous packings, they are generally packed in short lengths and often operated at elevated temperatures. 

The second stationary phase architecture (electrostatic agglomerated films on nonporous substrates, EANPS) was one of the first types of... 

Figure 25-10 shows a rapid separation of proteins on superficially porous particles, which consist of a 0.25-p.m-thick porous silica layer on a 5-pm-diamctcr nonporous silica core. A stationary phase such as Clg is bonded to the thin, porous outer layer. Mass transfer of solute into a 0.25-p.m-thick layer is 10 times faster than mass transfer into fully porous particles with a radius of 2.5 pm. enabling high efficiency at high flow rate. Superficially porous particles are especially suitable for separation of macromolecules such as proteins, which diffuse more slowly than small molecules. 

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. 

An important advance in the RP-HPLC impurity analysis of proteins has been the advent of short columns containing micropellicular stationary phases.42 These short columns contain nonporous particles of the order of 2 pm that are capable of attaining high flow rates ( 4 mL/min) at elevated temperatures ( 80° C). Such a configuration leads to very rapid and highly... 

Figure 28 Capillary electrochromatography of four t-RNAs. Capillary column, packed with 2-pm nonporous ODSS stationary phase, 20.5/27 cm x 100 pm i.d. running voltage, 20 kV, electrokinetic injection, 1 kV for 2 s mobile phase in (a), hydroorganic eluent containing 1 5 mM phosphate and composed of 40% (v/v) methanol. Solutes 1, t-RNAGIU 2 t-RNAVal 3, t-RNALys 4, t-RNAphe. (Reprinted from Ref. 119, with permission.)...

The elevated mechanical and thermal stabilities of pellicular stationary phases having a solid, fluid-impervious core have favored the development of nonporous particles tailored for the rapid HPLC analysis of peptides, proteins, and other biopolymers. Most of these applications are in RPC. 

HP-RPC separates compounds according to their relative nonpolarity or hydrophobicity. In RPC, the polarity of the stationary and mobile phase is to the reverse of that used in NPC. HP-RPC is performed on porous or nonporous stationary phases with immobilized nonpolar polymers (i.e., -alkylsilicas) or nonpolymer polymers (i.e., microparticulate polystyrenes). The most commonly accepted retention mechanism in RPC is based on the solvophobic theory, which describes the hydrophobic interaction between the nonpolar surface regions of the analytes and the nonpolar ligands/surfaces of the stationary phase.15 16... 

Almost all stationary phases used in chromatography have a bimodal pore size distribution. The first mode corresponds to the macropores or throughpores that allow the percolation of the column by the stream of mobile phase. The second distribution corresponds to the mesopores that combine to give the conventional internal porosity distribution described in the previous section. The mesopores are responsible for most of the specific surface area necessary to provide the retention and the saturation capacity that are needed to permit the retention of the mixture components in a good solvent, a condition for chromatographic separation. Nonporous particles have been used with only moderate success because very weak solvents must be used to achieve sufficient retention, which often causes solubility problems, and the saturation capacity of these particles is small. The terms of macro- and meso-pores apply as well to columns made of packed particles and to monolithic columns. 

Figure 1.1 HPLC separation of benzodiazepines (T. Welsch, G. Mayr and N. Lammers, Chromatography, InCom Sonderband, Dusseldorf, 1997, p. 357). Conditions samples 40 ng each column 3 cm x 4.6 mm i.d. stationary phase ChromSphere UOP CIS, 1.5 im (nonporous) mobile phase 3.5mlmin water-acetonitrile (85 15) temperature 35 C UV detector 254nm. Peaks 1 =bromazepam 2 = nitrazepam 3 = clonazepam 4 —oxazepam 5 = flunitra-zepam 6 = hydroxydiazepam (temazepam) 7 = desmethyldiazepam (nordaze-pam) 8 = diazepam (valium).

Various types of stationary phases are in use Porous particles, nonporous particles of small diameter, porous layer beads, perfusive particles, and monolithic materials. 

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