Sorbents, micropellicular

K. Kalghatgi and C. Horvath, Micropellicular sorbents for rapid reversed-phase chromatography of proteins and peptides, in Analytical Biotechnology, Capillary Electrophoresis, and Chromatography, C. Horvath and J.G. Nikelly (Eds.), American Chemical Society, Washington, D.C., 1990, p. 162. 

Micropellicular Sorbents for Rapid Reversed-Phase Chromatography of Proteins and Peptides... 

Due to recent advances in column technology, novel stationary phases have become available for such applications. This communication deals with the use of micropellicular sorbents which consist of a fluid-impervious microspherical support with a thin retentive layer at the surface. For biopolymer analysis by HPLC, such stationary... 

KALGHATGI HORVATH Micropellicular Sorbents for Rapid HPLC... 

Due to small particle size, the columns packed with micropellicular stationary phases have low permeability (27) and therefore, can not be operated at very high flow rates due to pressure limitations of commercial HPLC instruments. In comparison to porous particles, the surface area of stationary phases per unit column volume is low, and hence, their loading capacity is correspondingly smaller. This is particularly evident in the isocratic analysis of small molecules where the column can be easily overloaded. Therefore, micropellicular sorbents do not appear to offer advantages in the HPLC of small molecules. 

On the other hand, the lack of internal pore structure with micropellicular sorbents is of distinct advantage in the analytical HPLC of biological macromolecules because undesirable steric effects can significantly reduce the efficiency of columns packed with porous sorbents and also result in poor recovery. Furthermore, the micropellicular stationary phases which have a solid, fluid-impervious core, are generally more stable at elevated temperature than conventional porous supports. At elevated column temperature the viscosity of the mobile phase decreases with concomitant increase in solute diffusivity and improvement of sorption kinetics. From these considerations, it follows that columns packed with micropellicular stationary phases offer the possibility of significant improvements in the speed and column efficiency in the analysis of proteins, peptides and other biopolymers over those obtained with conventional porous stationary phases. In this paper, we describe selected examples for the use of micropellicular reversed phase... 

COLUMN STABILITY. The absence of a porous support structure results in enhanced column stability at elevated temperature and pH even with micropellicular sorbents prepared from siliceous supports (14). This is illustrated by the chromatogram in Figure 5 which shows the separation of minor conformers of human growth hormone by using a moderately alkaline mobile phase (pH 8.5). Prior to obtaining the above chromatogram, the column was perfused with 4000 column volumes of the mobile phase at 80°C, yet no noticeable changes in retention behavior, separation efficiency and sample recovery had been observed with respect to initial column performance. 

Maa Y F, Ftorvath C (1988). Rapid analysis of proteins and peptides by reversed-phase chromatography with polymeric micropellicular sorbents. J. Chromatogr. 445(l) 71-86. 

The need for enhanced detection sensitivity and automation has steadily increased for the separation and analysis of peptides from natural sources or proteolytic digestion of low abundance proteins this is also partly a consequence of the greater usage of combinatorial solid-phase synthetic approaches. Narrow bore (1-2 mm i.d.), microbore (0.5-1.0 mm i.d.), and capillary (100-500 pm i.d.) columns have provided attractive solutions to these problems. 1221 An important attribute of very small particle diameter micropellicular, porous, or nonporous RPC sorbents is that they are ideally suited to such microbore or capillary... 

Melittin, the main component of bee venom, was analyzed by gradient elution (Figure 12.22a) and purified by displacement chromatography (Figure 12.22b) on micropellicular CIS silica [50]. An isotachic train at two different temperatures is shown. Note that the flow rate on the 30x4.6 mm analytical column was 3 mL/min, while on the 105x4.6 mm displacement column best results were obtained at only 0.6 mL/min, in spite of the fast mass transfer kinetics on pellicular sorbents [50]. 

A variety of micropellicular packing materials has been developed for the analysis of both small and large molecules by various HPLC modes, including ion exchange (lEC), metal interaction (MIC), reversed-phase (RPC), and affinity chromatography (AC). Besides analytical applications, other possible utilization of micro-pelhcular stationary phases includes fundamental kinetic and thermodynamic studies of the retention mechanisms on a well-defined surface. Nevertheless, a relatively limited variety of micropellicular columns are commercially available. They are mainly restricted to ion-exchange and reversed-phase stationary phases. This may reflect certain practical disadvantages of micropelhcular sorbents. 

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