Silica-based columns

The separation of the same charged compounds were also accomplished on an ethyl-pyridine bonded silica surface and 30 0% methanol/C02 mobile phases without the need of added sulfonate modifier. Anionic compounds did not elute from the ethyl-pyridinium surface that lead the authors to hypothesize that the surface was positively charged. To further test this hypothesis, the separation of the same compounds on a strong anion exchange column, silica-based propyltri-methylammonium cationic surface, which exhibits are permanent positive charge was attempted. The same retention order was observed on the strong cation exchange surface. 

Figure 2.8 Similar influence of pH and temperature change on the selectivity of a separation of acids and bases. Column silica based CIS, mobile phase phosphate buffer/ACN 50/50 v/v, peaks (1) 2-phenylpyridine, (2) ketoprofen, (3)...

The efficiency of sample clean-up of lAC columns in determination of MC has been compared by several authors to conventional SPE sorbents.For cleanup of tap water samples, the use of C18 sorbent did not remove numerous coconcentrated impurities that make identification of MC signals more difficult. The functioning of two different I AC columns, silica-based and Sepharose-based ones, has been compared to that of... 

The first work concerning the generic capture of the nucleic acids was completed by using the affinity chromatography s columns (silica-based columns) and recently polymer particles such as stimuli-responsive latexes (sensitive to the pH, salinity, and the temperature). 

A liquid mobile phase is far denser than a gas and, therefore, carries more momentum. Thus, in its progress through the interstices of the packing, violent eddies are formed in the inter-particular spaces which provides rapid solute transfer and, in effect, greatly increases the effective diffusivity. Thus, the resistance to mass transfer in that mobile phase which is situated in the interstices of the column is virtually zero. However, assuming the particles of packing are porous (i.e., silica based) the particles of packing will be filled with the mobile phase and so there will... 

Select mobile phases for HPSEC based on their ability to dissolve the sample and their compatibility with the column. Zorbax PSM columns are compatible with a wide variety of organic and aqueous mobile phases (Table 3.4), but analysts should avoid aqueous mobile phases with a pH greater than 8.5. As mentioned earlier, select mobile phases that minimize adsorption between samples and silica-based packings. Sample elution from the column after the permeation volume indicates that adsorption has occurred. If adsorption is observed or suspected, select a mobile phase that will be more strongly adsorbed onto the silica surface than the sample. For example, N,N-dimethyl-formamide (DMF) is often used for polyurethanes and polyacrylonitrile because it eliminates adsorption and dissolves the polymers. When aqueous mobile phases are required, highly polar macromolecules such as Carbowax can be used to coat the silica surface and eliminate adsorption. Table 3.5 provides a list of recommended mobile-phase conditions for some common polymers. 

I. SILICA-BASED TSK-GEL SW/SWxl COLUMNS FOR GEL-FILTRATION CHROMATOGRAPHY (GFC)... 

Resin-based TSK-GEL PW columns are durable to alkaline conditions, which cannot be used with silica-based columns. Such durability facilitates separations with basic eluents and allows the use of alkaline cleaning reagents. 

The packed columns of Shodex PROTEIN KW-800 series are packed with hydrophilic silica-based gels and are best suited for analyses of proteins and water-soluble polymers (Table 6.14, page 213). 

SynChropak GPC supports were introduced in 1978 as the first commercial columns for high-performance liquid chromatography of proteins. SynChropak GPC columns were based on research developed by Fred Regnier and coworkers in 1976 (1,2). The first columns were only available in 10-yu,m particles with a 100-A pore diameter, but as silica technology advanced, the range of available pore diameters increased and 5-yu,m particle diameters became available. SynChropak GPC and CATSEC occasionally were prepared on larger particles on a custom basis, but generally these products have been intended for analytical applications. 

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. 

An important difference between Protein-Pak columns and other size exclusion columns is the silica backbone of the Protein-Pak columns. Because the silica structure is unaffected by the solvent, these columns do not swell or shrink as a function of the solvent. This is a general advantage compared to other size exclusion columns. However, silica-based columns can only be used up to pH 8, which limits their applicability. Also, surface silanols are accessible for interaction with the analytes, but this phenomenon has been minimized by proper derivatization techniques. Generally, a small amount of salt in the mobile phase eliminates interaction with silanols. 

The pressure capability of silica-based columns extends beyond the range of organic polymers. Protein-Pak columns are stable to above 25 MPa (4000 psi), the smaller pore sizes to above 40 MPa (6000 psi). 

The Jordi polyamine column is a polar column for simple sugar and polysaccharide applications. The amine groups are bonded to the DVB backbone and are stable in aqueous mobile phases. This material does not self-hydrolyze as do many silica-based amino packings (Fig. 13.14). 

Problems with adsorption onto the packing material are more common in aqueous GPC than in organic solvents. Adsorption onto the stationary phase can occur even for materials that are well soluble in water if there are specific interactions between the analyte and the surface. A common example of such an interaction is the analysis of pEG on a silica-based column. Because of residual silanols on the silica surface, hydrogen bonding can occur and pEG cannot be chromatographed reliably on silica-based columns. Eikewise, difficulties are often encountered with polystyrenesulfonate on methacrylate-based columns. 

A CSP based on the adsorption of a chiral anthrylamine on porous graphitic carbon successfully resolved the enantiomers of tropic acid derivatives and anti-inflammatory agents in SFC [65]. The carbon-based CSP produced superior results when compared to an analogous silica-based CSP. Occasional washing of the column was necessary to remove highly retained substances. 

An example of the efficacy of the resin phases used as an alternative to a conventional silica based reverse phase is shown in figure 12 where the separation of the three tocopherols are shown separated on the Cl 8 Polymer Column and The ODA-A 120A silica gel based columns. The columns were 15 cm long, 4.6 mm i.d., operated at a flow rate of 0.5 ml/min at 30°C with a mobile phase of 98% methanol/2% water. 

Comparison of the Separation of Some Tocopherols on a Polymer C18 Column and a Conventional Silica Based ODS... 

Polymer C18 Column ODS-A 120A Silica Based Reverse Phase Column... 

It is seen that the polymer resin does not have the same retentive capacity as the conventional reverse phase column and thus, will not exhibit the same resolution or the equivalent loading capacity. Nevertheless, the polymer column will function over a wide range of pH whereas the silica based columns will be restricted to operating within a pH of 4.0 to 8.0 at the most. 

The analysis demonstrates the elegant use of a very specific type of column packing. As a result, there is no sample preparation, so after the serum has been filtered or centrifuged, which is a precautionary measure to protect the apparatus, 10 p.1 of serum is injected directly on to the column. The separation obtained is shown in figure 13. The stationary phase, as described by Supelco, was a silica based material with a polymeric surface containing dispersive areas surrounded by a polar network. Small molecules can penetrate the polar network and interact with the dispersive areas and be retained, whereas the larger molecules, such as proteins, cannot reach the interactive surface and are thus rapidly eluted from the column. The chemical nature of the material is not clear, but it can be assumed that the dispersive surface where interaction with the small molecules can take place probably contains hydrocarbon chains like a reversed phase. 

The more useful types of chirally active bonded phases are those based on the cyclodextrins. There are a number of different types available, some of which have both dispersive or polar groups bonded close to the chirally active sites to permit mixed interactions to occur. This emphasizes the basic entropic differences between the two isomers being separated. A range of such products is available from ASTEC Inc. and a separation of the d and / isomers of scopolamine and phenylephrine are shown in figure 4. The separations were carried out on a cyclodextrin bonded phase (CYCLOBOND 1 Ac) that had been acetylated to provide semi-polar interacting groups in close proximity to the chiral centers of the cyclodextrin. The column was 25 cm long, 4.6 mm in diameter and packed with silica based spherical bonded phase particles 5pm in diameter. Most of the columns supplied by ASTEC Inc. have these dimensions and, consequently, provide a... 

An example of the separation of a mixture of explosives on a C8 column is shown in figure 7. The column was 3.3 cm long, 4.6 mm in diameter and packed with 3 pm C8 silica based reverse phase. This short column has a potential efficiency of 5,500 theoretical plates. 

Another example of the use of a C8 column for the separation of some benzodiazepines is shown in figure 8. The column used was 25 cm long, 4.6 mm in diameter packed with silica based, C8 reverse phase packing particle size 5 p. The mobile phase consisted of 26.5% v/v of methanol, 16.5%v/v acetonitrile and 57.05v/v of 0.1M ammonium acetate adjusted to a pH of 6.0 with glacial acetic acid and the flow-rate was 2 ml/min. The approximate column efficiency available at the optimum velocity would be about 15,000 theoretical plates. The retention time of the last peak is about 12 minutes giving a retention volume of 24 ml. 

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