Wide-pore packings

Tweeten, K.A. and Tweeten, T.N., Reversed-phase chromatography of proteins on resin-based wide-pore packings, J. Chromatogr., 359, 111, 1986. 

The use of wide-pore packings in CEC was examined [46] and it was found that for normal pore size packings (120 A), there is a gradual decrease in... 

A material. Note that wide-pore packings typically have lower surface area and therefore lower sample capacities. Other approaches for bioseparations are the use of very-wide-pore materials,34 45 nonporous,35 or pellicular materials (Figure 7.26). Nonporous and pellicular materials are particularly useful for fast separations. 

Table 1 lists a number of commercially available wide-pore packings, mostly intended for protein use. In addition to RP supports several packings intended for hydrophobic interaction chromatography have been included as they may sometimes be useful used in a RP mode for very hydrophobic proteins. It is not possible to recommend a single brand of packing which is ideal for all circumstanees. Some small proteins can be... 

Tanaka, N., Kimata, K., Mikawa, Y., Hosoya, K., Araki, T., Ohtsu, Y., Shiojima, Y., Tsuboi, R., Tsuchiya, H. (1990). Performance of wide-pore silica- and polymer-based packing materials in polypeptide separation the effect of pore size and alkyl chain length. J. Chromatogr. 535, 13-31. 

Mercury poroslmetry data of these packings are given In Table IV. It Is of Interest to note that the pore-size distribution of CPG Is significantly more narrow than that of Syn-Chropak, a surface-modified porous silica (LlChrospher). These different physical characteristics may help to explain the existence of micropores In SynChropak. Because of the wide pore-size distribution of this packing. It seems reasonable that this material also contains a population of micropores which are only accessible to D2O. In mercury poroslmetry measurements, the lower pore size limit Is about 30A. 

From these studies with SynChropak SEC packings and controlled porosity glass, it is concluded that the silica packing contains a population of micropores which are differentially accessible to low molecular weight probes of total permeation volume. It is not known, however, if the microporosity in the 100 and 300A SynChropak SEC packings is the result of the rather wide pore-size distribution and whether all silicas contain micropores. 

In addition to the packed bed acting as an ultrafilter, the porous frits used at both ends of the column may act as very effective filtering devices. Thus a 2-vim porosity frit would have an average pore radius of 1 lun. Because of the tortuosity and relatively wide pore-size distribution present in frits, it would be safe to assume that it contains much smaller crevices which can entrap macromolecules. 

Assay of proteins using wide-pore HPLC packings... 

Capillaries packed with poly-A-acryloyl-L-phenylalanine ethyl ester (Chiraspher) modified silica were used for electrochromatographic enantiomer separation of ben-droflumethiazide. To suppress bubble formation, the inlet buffer vial was pressurized to 12 bar and the outlet buffer vial to 4 bar [42], Acetonitrile or methanol were used as organic modifier whereby acetonitrile was superior to methanol. Non-aqueous p-CEC was performed on helical poly(diphenyl-2-pyridylmethylmethacrylate) coated on wide-pore aminopropyl silica [56]. With this chiral stationary phase, the enantiomer separation of Trogers base, benzoin acetate, methylbenzoin and trans-stilbene oxide was achieved by pressure-supported CEC, applying a higher pressure to the inlet than to the outlet buffer vial. 

For those samples that are not compatible with GC, the first question to ask involves the size (molecular weight) of the solute molecules. Their size should be compared to the pores of the packing materials that can be used in LC. If the size of the molecules is not negligible relative to the (average) pore size, then part of the pores and hence part of the stationary phase present in the column will not be accessible to the solute molecules. Hence, the simple relationship between chromatographic retention and thermodynamic distribution (eqn.l. 6) loses its significance. To avoid that, wide pore materials can be used for the separation of large molecules (e.g., proteins) based on their distribution over the two phases [202]. 

Since electroosmotic flow can exist in both the interparticle and intraparticle spaces, numerous studies have focused on the existence of intraparticle flow in CEC. Several groups have investigated the existence of electroosmotic flow in wide-pore materials [41-44], A model was developed to estimate the extent of perfusive flow in CEC packed with macroporous particles [41] by employing the Rice and Whitehead relationship. Results showed the presence of intraparticle EOF in large-pore packings (> 1000 A) at buffer concentrations as low as 1.0 mM. Additional parameters had been investigated [43,44] to control intraparticle flow by the application of pressure to electro-driven flow. Enhancement in mass transfer processes was obtained at low pore flow velocities under the application of pressure. The authors pointed out that macroporous particles could be used as an alternative to very small particles, as smaller particles were difficult to pack uniformly into capillary columns. 

Figure 6 The effect of increasing the ionic strength on the EOF mobility of Nucleosil C-18 wide-pore octadecylated silica stationary phases. Conditions column 55 cm (40 cm packed length) x 100 pm i.d. Eluent actonitrile-Tris (pH 9.0) (60 40, v/v) ionic strength variable. Temperature 25°C. Dectection 214 nm. Voltage 30 kV. (o) 7-pm Nucleosil 4000 A, ( ) 7-pm Nucleosil 500 A, (A) 5-pm Nucleosil 120 A, and ( ) 3-pm Spherisorb ODS-1, 80 A. (Reprinted with permission from Ref. 46, copyright 2001, with permission from Elsevier Science.)...

Clearly, these studies demonstrated that it is desirable to chromatograph a high-MW protein sample on a wide-pore (200-500 A) silica coated with a medium-polarity reversed phase. Unfortunately, manufectured columns generally do not meet these criteria, and thus the researcher must either exercise caution in the selection of a commercial column or prepare his own packing material. 

We now have a fairly adequate understanding of the different properties, including the particle diameter i/p, the pore size, the degree of permeability, and the chemical composition of the surface of the support matrix, to know which type of stationary phase can be successfully used with a particular class of peptides. Most of the HPLC packing materials now in use for peptide separations are based on the wide pore microparticulate silica gels with polar or nonpolar carbonaceous phases chemically bonded to the surface of the matrix. Methods for the preparation of these chemically bonded stationary phases, their available sources of supply. 

The porosity of particles suitable for packing HPLC columns depends on the size of molecules to be separated. Totally porous particles with a pore size of 7-12 nm and specific surface area of 150-400 m"/g are suitable for the separation of small molecules, but wide-pore particles with a pore size of 15-100 nm and relatively low specific surface area (10-150 nr/g) are required for the separation of macromolecules to allow easy access to the interactive surface within the pores. Packings with perfusion particles contain very broad pores (400-800 nm) throughout the whole particle interconnected by smaller pores. The mobile phase flows through the pores in the particle, which minimises both band broadening and column backpressure (111. Perfusion materials have been designed especially for the separation and isolation of biopolymers. 

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