Micropellicular stationary phases

Huber, C.G., Micropellicular stationary phases for high-performance liquid chromatography of double-stranded DNA,. Chromatogr, A, 806, 3, 1998. 

Table I. Micropellicular Stationary Phases for HPLC Analysis of Biopolymers...

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... 

Figure 1. Schematic representation of the micropellicular stationary phase.

RAPID ANALYSIS OF PEPTIDES AND PROTEINS. Columns packed with micropellicular stationary phases can be utilized in many areas of protein chemistry and biotechnology where high analytical speed and resolution are required. Peptide mapping by HPLC is routinely employed for the determination of structure of proteins (2, 3,32). 

MICROPELLICULAR AND POROUS STATIONARY PHASES. In order to compare the features of micropellicular and porous stationary phases in rapid protein HPLC, experiments were conducted with two columns of similar size, each one of which was packed with different stationary phase and operated under comparable conditions. In this experiment the results of which are shown in Figure 10, the operational conditions were optimized for the micropellicular stationary phase (conditions A) and used subsequendy for separation of the same mixture with the porous stationary phase under identical conditions. Thereafter, the elution conditions were optimized for the column packed with the porous stationary phase (condition B) and the experiment was repeated with the column packed with micropellicular stationary phase. The chromatograms are depicted in Figures 10 and 11 and the results of the two approaches are summarized in Table III. 

The performance of micropellicular stationary phase in either case is superior when compared by the peak width of sample components or the resolution of the critical eluite pair (Cyt.C / Lys) in the sample. 

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... 

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) [4], and affinity chromatography (AC) [5]. Besides analytical applications, other possible utilization of micropellicular stationary phases includes fundamental kinetic and thermodynamic studies of the retention mechanisms on a well-defined surface. Nevertheless, a relatively limited variety of micropellicular... 

---