Wide-pore analytical column

The separation of basic and metal-sensitive compounds is difficult on silica-based stationary phase materials, but these separations can be performed on vinyl alcohol copolymer gels. Examples are the separation of methallothionein from dolphin kidney, a-, j8-, and y-endorphin, and nucleotide and nucleoside mixtures.8 However, an analytical-scale separation may also be performed on surface-modified wide-pore silica gels (pore size 300 A or more), using columns which showed a negative response in the heavy metal test described above. 

Perhaps the biggest limitation to RP-LC is the difficulty with adequate retention for very polar analytes. Three undesirable issues are associated with poor retention (1) poor chromatographic separation, (2) greater ionization suppression, and (3) reduced sensitivity due to poor analyte desolvation in highly aqueous mobile phases. Column manufacturers have used several formats to improve retention of polar solutes that include extended alkyl phases, polar-endcapped alkyl phases, polar-embedded alkyl phases, nonendcapped short alkyl phases, and wide pore-diameter phases [94]. Interested readers are referred to a recent two-part review on this important subject [94,95]. 

A bicyclic 13-amino-aeid a-conotoxin chimera was synthesized and purified on a wide-pore C]g column (A == 215 nm) using a 30-min 95/5 -> 75/25 water (0.1% TFA)/(90/10 acetonitrile/water with 0.09% TFA) gradient. The monocyclic peptide and its oxidation product were well resolved from the analyte of interest. Elution was complete in 22 min. A 20 pg injection was readily detected [1298]. 

Metal Oxides. These include (a) silicagel, which is a specific type of adsorbent because there are free hydroxyl groups on the surface (polar molecules are easily separated on these materials, and wide-pore silicas with homogeneous surfaces are used for analytical gas-solid chromatography ) and (b) alumina. These columns can be easily baked out to remove contaminants and reused with good reproducibility. 

Figure 2. Analytical anion-exchange HPLC of an oligofrA) preparation on a PEI column. Sample, 20 pg commercial poly(rA) digested with 1 N NaOH at 56°C for 3 min and then neutralized with 1 N HCI column. Baker PEI wide pore (4.6 x 250 mm) apparatus. Bruiser LC 21 B with a Shimadzu SPD-6A spectrojAiotometer set at 270 nm with 0.04 AUFS elution, linear gradient from 1 mM potassium phosphate, pH 6.3, 60% (v/v) formamide to 0.225 M potassium phosphate, pH 6.3, 60% (v/v) formamide inlSO min flow-rate, 1 ml/min.

Figure 5. Anlytical HPLC separation of 300 pg venom peptides (A) and 10 ml conditioned water (B) from Conus textile. Details on preparation of the substances are given in the legend to Fig. 4. Separations were performed on an analytical CIS reverse phase column (Vydac wide pore, 4.6 x 250 mm, 5 pm particle size) at a flow rate of 0.5 ml/min. Substances were loaded on the column in aqueous 0.1% tri-fluoroacetic acid (TFA) and eluted with a linear gradient of 0-60% acetonitrile in 0.1% aqueous TFA in 0-60 minutes. On-line detection and spectral analysis was performed with a Hewlett-Packard diode array detector. The spectrum of the main peak obtained from the CW (B) is not identical to those of any of the venom derived peptides (A) that are eluted at similar times from the column (not shown). Attempts to isolate the active component(s) of Conus textile CW on reverse phase cartridge columns and Amicon filters were not successful, due to loss of the biological activity.

The analytical capability of a SEC column is sometimes judged by the peak capacity, which is the number of unique species that can be resolved on any given SEC column. This number will increase with decreased particle size, increased column length, and increased pore volume. Because small particlesized medium generally has a lower pore volume and a shorter column length, peak capacities of ca. 13 for fully resolved peaks can be expected for high-resolution modern media as well as traditional media, (see Eig. 2.5). It was found that SEC columns differ widely in pore volume, which affects the effective peak capacity (Hagel, 1992). 

The column packings used for SEC must be compatible with aqueous mobile phases and therefore must be hydrophilic in nature. The support surface must be inert to minimize interactions with protein analytes. The packing must be available in pore sizes suitable for permeation of a wide range of proteins, and the pores should be uniform in diameter. Because the separation only takes place within the pore system, the porosity of the packing should be as large as possible. The support material should be chemically compatible with SEC mobile phases and mechanically stable under high flow rates and pressures. 

---