Bonded phases from silica

The method most commonly used to prepare bonded phases from silica involves reaction of the silica with a substituted dimethylchlorosilane. Fig. 3.2a shows the reaction, in which HC1 is eliminated between a surface silanol group and the silylating agent. 

Since selectivity in HPLC involves both the stationary and mobile phases [5-9,58-60], it is important to note that the solvent strength of the mobile phase, as compared to the stationary phase, (composed of mobile-phase components reversibly retained by the bonded phase and silica support) determines the elution order or k of the retained components. Unfortunately, the columns with the same stationary phase can exhibit significant variabilities from one manufacturer to another and even from the same manufacturer [5-8]. Based on discussions heard at various scientific meetings, this situation has not changed much. Variabilities can occur in the packing process even where all other conditions are supposedly constant. These factors have to be considered prior to developing an understanding as to how separations occur in HPLC. 

Pesek, I.I., Matyska, M.T., WiUiamsen, E.J., Evanchic, M., Hazari, V., Konjuh, K., Takhar, S., and Tranchina, R., Synthesis and characterization of aUcyl bonded phases from a silica hydride via hydrosUation with free radical initiation, J. Chromatogr. A, 786, 219, 1997. 

In conclusion, the enthalpic partition processes in the columns for polymer HPLC substantially differ from the adsorption processes. Enthalpic partition can be employed for the separation of polymers of the low-to-medium polarity in combination with the alkyl bonded phases on silica gels. The extent of the enthalpic partition and consequently also of the polymer retention is controlled primarily by the thermodynamic quality of eluent toward separated species and by the extent of the bonded phase solvation. 

Hydrophilic size separation columns for use with aqueous samples are very popular choices for purifying proteins and carbohydrates. Protein separation columns are available on both silica and polymeric supports. It is surprising that the best of these protein purification columns in terms of resolution and in recovery of native protein are silica-based columns. One would expect that protein release from silica would be a real problem. It certainly is in many other silica columns. These columns, however, especially the TSK family of columns, give excellent recovery of enzymatic activity. I have talked to other column manufacturers who have investigated the problem. They say that when you remove the bonded phases from these columns they appear to be identical to bonded phases from a number of other, less successful, columns designed for protein purification. All of these bonded phases are primarily diol ether polymers, very hydrophilic, but of intermediate polarity. Some modification of... 

Select columns packed with 3- or 5-pm high-purity silica-bonded phases from a reputable manufacturer. 

Three columns packed by bonded phases from three different silica lots Mobile Phase... 

Gas chromatography (GC) was performed on a Varian Associates Inc. (Palo Alto, CA) model 3700 gas chromatograph equipped with a flame ionization detector (FID) and modified to accommodate a laboratory-made capillary on-column injector (ref. 12). All gas chromatographic separations were carried out on a J W Scientific (Folsom, CA) bonded phase fused silica capillary column (60 m X 0.32 mm ID, DB-1 stationary phase, 0.25 fim film thickness) unless otherwise noted. The GC oven temperature was programmed from 50°C to 300°C at 20°C/min. 

NPLC stationary phases include metal oxides and moderately or strongly polar chemically bonded phases. Unmodified silica gel and silica-based bonded phases are most frequently used nowadays. Considerable effort in the development of new HPLC column packing materials in the past years has resulted in significant improvement of the column efficiency, reproducibility, and increased stability at elevated temperatures and at higher pH, enabling better compatibility with HPLC/mass spectrometry techniques and rapid analyses. Even though the new column technologies were primarily focused on RPLC separations, normal-phase HPLC also benefits from the improved properties of the support materials with uniform small particles and well-defined pore size. 

Loss of stationary phase from silica-based columns is accelerated at higher temperatures. Temperatures above 40°C should be used with caution when operating at intermediate or high pH with phosphate buffers. Operation at pH < 3 and elevated temperatures can degrade the bonded stationary phase more rapidly and cause retention reproducibility problems. However, there are specially end-capped stationary phases available from several of column manufacturers that can withstand higher temperature with less hydrolysis. 

A Hewlett-Packard (HP) Model 5890 gas chromatograph equipped with a flame ionization detector (FID) and a 30 m x 0.25 mm i.d. DB-Wax bonded-phase fused silica capillary column (J W Scientific, Folsom, CA) was used to quantitate heptanoic acid formed from heptanal oxidation. The GC peak areas of heptanoic acid obtained at various time intervals were divided by the GC peak area of the internal standard (nonadecane, 400 mg) to calculate a relative peak area (RPA). 

Sedimentary rocks (like sandstone) have a microstructure rather like that of a vitreous ceramic. Sandstone is made of particles of silica, bonded together either by more silica or by calcium carbonate (CaCOj). Like pottery, it is porous. The difference lies in the way the bonding phase formed it is precipitated from solution in ground water, rather than formed by melting. 

Alhedai et al also examined the exclusion properties of a reversed phase material The stationary phase chosen was a Cg hydrocarbon bonded to the silica, and the mobile phase chosen was 2-octane. As the solutes, solvent and stationary phase were all dispersive (hydrophobic in character) and both the stationary phase and the mobile phase contained Cg interacting moieties, the solute would experience the same interactions in both phases. Thus, any differential retention would be solely due to exclusion and not due to molecular interactions. This could be confirmed by carrying out the experiments at two different temperatures. If any interactive mechanism was present that caused retention, then different retention volumes would be obtained for the same solute at different temperatures. Solutes ranging from n-hexane to n hexatriacontane were chromatographed at 30°C and 50°C respectively. The results obtained are shown in Figure 8. 

The above data were obtained on a polymeric bonded phase and not a brush phase. The so-called brush phases are made from monochloro-sxlants, (or other active group) and, thus, the derivative takes the form of chains attached to the silica surface [2]. The bulk phases are synthesized from polyfunctional silanes in the presence of water and, thus, are cross linked and form a rigid polymeric structure covering the silica surface. These two types of phases behave very differently at low concentrations of moderator. 

The other restriction of SynChropak size exclusion columns is a general one for silica-based supports, that of pH. The most harmful pH is that above 7.5 due to silica dissolution. The bonded phase of SynChropak GPC has some polymeric properties therefore, it is not removed rapidly from the silica at pH 2-3. The bonded phase of SynChropak CATSEC is polymeric and stable at pH 2-7.5. 

Scientific (Northbrook, IL) contain a silica support with a -y-glycidoxypropylsi-lane-bonded phase to minimize interaction with anionic and neutral polymers. The columns come in five different pore sizes ranging from 100 to 4000 A. The packing material has a diameter from 5 to 10 /cm and yields in excess of 10,000 plate counts. With a rigid silica packing material, the columns can withstand high pressure (maximum of 3000 psi) and can be used under a variety of salt and/or buffered conditions. A mobile phase above pH 8, however, will dissolve the silica support of the column (21). A summary of the experimental conditions used for Synchropak columns is described in Table 20.8. 

The use of bonded, silica column supports has also become a useful way to characterize cationic, water-soluble polymers. CATSEC SEC columns from Micra Scientific contain a silica support with a polymerized polyamine-bonded phase. This imparts a cationic surface charge on the packing that can be... 

The polymeric resin beads fill a need that arises from the instability of silica gel and its products to mobile phases of extreme pH (outside a pH range of about 4.0-7.0) and, consequently, are employed in most ion exchange separations. Organic moieties containing ionic groups can be bonded to silica and produce an effective ion exchange media, but the restrictions of pH on phase stability still apply. It follows that ion exchange bonded phases are less popular than the polymer bead alternatives. 

The hydrogel is allowed to stand for a few days during which time a process called sinerisis takes place. During sinerisis the condensation of the primary particles, one with another, continues and the gel shrinks further, accompanied by the elimination of more saline solution that exudes from the gel. After three or four days, sinerisis is complete and the gel becomes firm and can now be washed free of residual electrolytes with water. The washed product is finally heated to 120°C to complete the condensation of the surface silanol groups between the particles, and a hard xerogel is formed. It is this xerogel that is used as the LC stationary phase and for bonded phase synthesis. It is not intended to discuss the production of silica gel in detail and those interested are referred to "Silica Gel and Bonded Phases", published by Wiley (1). 

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