Silica-based reversed phase

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. 

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

The newcomer to chromatography, faced with a hitherto unknown sample, would do well to start with a C8 silica based reverse phase and an acetonitrile water mixture as a mobile phase and carry out a gradient elution from 100% water to 100% acetonitrile. From the results, the nature and the complexity of the sample can be evaluated and a more optimum phase system can be inferred. 

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. 

De Jong, G. J. Optimization and characterization of silica-based reversed-phase liquid chromatographic systems for the analysis of basic pharmaceuticals./. Chromatogr. A 2000, 897,1-22. 

Feibush, B., A new chemically deactivated silica-based reversed phase/ion exchange support, /. Liq. Chromatogr. Relat. Technol, 19(14), 2315, 1996. 

Recent advances in column stationary phases are remarkable. High performance silica-based reversed-phase 3 to 5 jxm packing materials have been developed for biological sample separations... 

Neue, U. D., Serowik, E., Iraneta, P., Alden, B. A. and Walter, T. H., A Universal Procedure for the Assessment of the Reproducibility and the Classification of Silica-Based Reversed-Phase Packings 1. Assessment of the Reproducibility of Reversed-Phase Packings. /. Chromatogr. A, 849 87—100, 1999. 

The CEC phases must be capable of carrying a charge to generate an EOE and appropriate moieties to facilitate the chromatographic processes. Silica-based reversed-phase packing materials have been most widely used in CEC. The use of polymeric and mixed-mode bonded particles has also been reported. Eor the silica-based phases, the carbon chains bonded on the silica surface provide the retention and selectivity for analytes, and the residual silanol groups on the surface of the silica are ionizable and generate the EOF. 

The use of nonpolar chemically bonded stationary phases with a polar mobile phase is referred to as reverse-phase HPLC. This technique separates sample components according to hydrophobicity. It is widely used for the separation of all types of biomolecules, including peptides, nucleotides, carbohydrates, and derivatives of amino acids. Typical solvent systems are water-methanol, water-acetonitrile, and water-tetrahydrofiiran mixtures. Figure 3.15 shows the results of protein separation on a silica-based reverse-phase column. 

To avoid basic silica dissolution that leads to poor reproducibility, reduced efficiencies, poor peak shapes and high back pressure carbon-based stationary phases were also tested. Unlike silica-based reversed phases, they can be exposed to both highly acidic and alkaline environments (pH 1 to 14) and very high temperatures without degradation [15] they proved very useful in the analysis of organic acids [16]. 

In order to optimize the LC/MS/MS system, the authors investigated the effects of methanol content, ammonium acetate concentration, and the percentage of acetic/formic acids in the mobile phase on the ESI response (m/z 744 to 495 transition) (Figure 7-19). They found that the best ESI response was obtained at -80% (v/v) of methanol (Figure 7-20A).They also discovered that the ammonium acetate (5 mM) could be applied as a buffer in the mobile phase to achieve better reproducible separation between ET-743 and the internal standard. It is well known that the addition of acetic acid or formic acid in the mobile phase can suppresse the ionization of residual silanols on silica-based reversed-phase columns for LC/MS analysis (Figure 7-19C). In positive ion mode, however, the acids can form an ion pair with the MH+... 

With regard to the silica-based reversed-phase stationary phase, unreacted residual silanol groups may play a significant role in IIC because it was shown that... 

The reproducibility of results obtained with silica-based reversed phases, of the same declared characteristics but from different manufacturers, was sometimes poor, probably because of the properties of the silica used and the different reaction conditions in the alkylation of the support. 

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