Perfusive stationary phases

The most important feature of monolithic media is that the mobile phase flows exclusively through the separation unit. In contrast, there is no flow inside the conventional porous chromatographic particles and only a partial flow through the perfusion beads. Just as with the membrane adsorbers, monolith stationary phases may be operated with a minimum in mass transfer resistance with the concomitant advantages in terms of speed and throughput. 

A high electroosmotic flow through the stationary-phase particles may be created when the appropriate conditions are provided. This pore flow has important consequences for the chromatographic efficiency that may be obtained in CEC. From plate height theories on (pressure-driven) techniques such as perfusion and membrane chromatography, it is known that perfusive transport may strongly enhance the stationary-phase mass transfer kinetics [30-34], It is emphasised... 

One effect of pore flow is that it enhances the mass transfer rate between the pore and interstitial volumes. Instead of by molecular diffusion only, which is by nature slow in solution, mass exchange occurs also by perfusive EOF. This effect can be treated as a form of stimulated diffusion. Following the original treatment for pressure-driven LC according to Rodrigues et al. [31], the plate height contribution from stationary-phase mass transfer resistance HCs in the presence of pore flow can be written as... 

In order to improve the separation efficiency and speed in biopolymer analysis a variety of new packing materials have been developed. These developments aim at reducing the effect of slow diffusion between mobile and stationary phase, which is important in the analysis of macromolecules due to their slow diffusion properties. Perfusion phases [13] are produced from highly cross-linked styrene-divinylbenzene copolymers with two types of pores through-pores with a diameter of 600-800 mu and diffusion pores of 80-150 nm. Both the internal and the external surface is covered with the chemically bonded stationary phase. The improved efficiency and separation speed result from the fact that the biopolymers do not have to enter the particles by diffusion only, but are transported into the through-pores by mobile-phase flow. 

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

The chemostat vessel was a modified 500-mL Virtis fermentor with a teflon impeller. The medium consisted of 350 mL MSM containing alachlor (100 mg/L), glucose (100 mg/L), and yeast extract (50 mg/L). The chemostat was inoculated with 20 mL of soil perfusate, the inoculum was allowed to grow to stationary phase as a batch culture,... 

Monolithic columns are another approach to provide lower pressure drops and higher rates of mass transfer. These are continuous solid columns of porous silica stationary phase instead of packed particles. Like perfusion packings, they have a bimodal pore structure (Figure 21.7). Macropores, which act as flowthrough pores, are about 2 fim in diameter. The silica skeleton contains mesopores with diameters of about 13 nm (130 A). It can be surface modified with stationary phases like Cig. The rod is shrink-wrapped in a polyetheretherketone (PEEK) plastic holder to prevent waU effects of solution flowing along the walls. The surface area of the mesopores is about 300 mVg, and the total porosity is 80%, compared with 65% for packed particles. The colunm exhibits a van Deemter curve approximating... 

Comprehensive multidimensional liquid chromatography is a relatively new development and has yet to develop a diverse application base. For the time being applications are dominated by the separation of proteins and synthetic polymers. For proteins the first dimension separations are usually based on ion exchange and the second dimension separations on reversed-phase liquid chromatography. Gradient elution was often used for both separation modes with a separation time less than 2 minutes for the second dimension separation and from 30 minutes to several hours for the first dimension separation. Current trends include the use of non-porous particles and perfusive stationary phases for the second dimension separation to reduce the total separation time and wider internal diameter columns in place of packed capillary columns to simplify interface construction and instrument operation and to allow the loading of larger sample sizes. 

Dearie H S, Smith N W, Moffatt F, et al. (2002). Effect of ionic strength on perfusive flow in capillary electrochromatography columns packed with wide-pore stationary phases. J. Chromatog. A. 945 231-238. 

The methods which are used for the preparation of HPLEC stationary phases are similar to those which have been developed for soft gel matrices by treating controlled pore glass with 3-(2-aminoethy-lamino)propyl-trimethoxysilane and subsequently perfusing the column with copper sulphate, a copper-chelate support is prepared (Masters and Leyden, 1978). Direct treatment of silica with copper sulphate can also be used (Caude and Foucault, 1979) (Fig. 9.6). Methods used in the preparation of these stationary phases can be found in the literature (Sugden et al., 1980 Caude et al., 1984). 

Fig. 7.4 Perfusion chromatography. Left particle of the stationary phase right fast separation of immunoglobulin G from cell culture. (Reproduced with permission from N. B. Afeyan, S. P. Fulton and F. E. Regnier, J. Chromatogr., 544, 267 (1991).) Conditions column, 10cmx4.6mm i.d. stationary phase, Poros M for hydrophobic interaction, 20 pm mobile phase, gradient from 2 to OM ammonium sulphate in water in 5 min, 10 ml minUV detector, 280 nm.

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