Bonded phase chromatography column preparation

Development of new HPLC stationary phases is ongoing. A multimodal phenylpropanolamine-coated silica column was recently prepared and applied to the quantitation of ascorbic acid in orange juice and the separation of ascorbic acid from its epimer, isoascorbic acid (Table 28) (199). Other examples include new bonded phases and columns made of continuous porous rods that permit fast chromatography with low backpressure and high resolution (200-202). 

The suspension of influenza virus, strain A/X-53, was separated from the constituents of alantoic fluid by preparative size exclusion chromatography on a 10 x 120 cm column. The hemo-agglutination method revealed an elevated level (93%) of viral activity. The leakage of the bonded phases can be more efficiently minimized, however, with the use of positively charged polymers. 

The experiments below use reverse phase chromatography with bonded silica columns and uv absorbance detection. If more extensive experimental facilities are available, some additional experiments are suggested. These are concerned with the preparation and evaluation of columns, and with the use of other detectors and modes of hplc. It should be possible to complete each experiment within a three hour practical period. 

Bonded-phase silica and ion-exchange resins in plastic cartridges and mini-columns are very useful for off-line prepurification of samples, especially those for preparative chromatography, when appropriate pre- or guard columns may not be available for on-line clean-up of a sample. 

More than 90% of the column packings are based on silica gel and its bonded phases [1], The primary reasons for its widespread use is because of high surface area and porosity, easy preparation, adjustable polarity, and good mechanical strength. Silica gel optimized for chromatography should have the characteristics listed in Table 5.1 [2]. 

The most popular reversed-phase columns are manufactured from silica-based bonded phases, and, among those, the Cig-type bonded phase is most frequently used. These bonded phases are prepared by reacting silica with a reactive silane that carries the hydrophobic ligand after which the phase is named. However, the poor stability of silica at extreme pH values, along with the presence of underivatized silanol groups that most frequently cause secondary interactions, limits the application of silica-based supports in liquid chromatography. 

Normal-phase chromatography used an amino bonded-phase column (Du Pont Zorbax, 25- X 0.48-cm i.d., 5-p,m spherical particle size) n-hexane and dichloromethane mixtures were used as the mobile phases. Reverse-phase separations used an octadecyl bonded-phase column (Vydac 201TP5, 25- X 0.46-cm i.d. for analytical scale, and 25- X 0.94-cm i.d. for preparative scale, both with 5-p,m spherical particles, Separations Group) methanol and dichloromethane were the mobile phases. As in previous work (5-7, 9), the concentration of strong solvent in the mobile phase was varied from 0% to 100% for reverse-phase studies and from 0% to 30% for normal-phase studies. The peaks isolated from preparative-scale runs were further purified by separation on the analytical-scale octadecyl bonded-phase column. 

Porous silica (or silica gel) is by far the most important adsorbent for liquid-solid chromatography and is also the substrate used to prepare most chemically bonded phases, giving it a preeminent position in modem column technology [3-15]. Silica particles are mechanically strong and easily prepared in a wide range of particle size ranges and pore diameters suitable for chromatography. As a substrate for the... 

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