Silanol activity mobile phase

The most widely used particulate support is diatomaceous earth, which is composed of the silica skeletons of diatoms. These particles are quite porous, with surface areas of 0.5-7.5 m /g, which provides ample contact between the mobile phase and stationary phase. When hydrolyzed, the surface of a diatomaceous earth contains silanol groups (-SiOH), providing active sites that absorb solute molecules in gas-solid chromatography. 

The use of TFA as a mobile-phase additive in LC-MS can be problematical when using electrospray ionization. In negative ion detection, the high concentration of TFA anion can suppress analyte ionization. In positive ion detection, TFA forms such strong ion pairs with peptides that ejection of peptide pseudo-molecular ions into the gas phase is suppressed. This problem can be alleviated by postcolumn addition of a weaker, less volatile acid such as propionic acid.14 This TFA fix allows TFA to be used with electrospray sources interfaced with quadrupole MS systems. A more convenient solution to the TFA problem in LC-MS is to simply replace TFA with acetic or formic acid. Several reversed-phase columns are commercially available that have sufficient phase coverage and reduced levels of active silanols such that they provide satisfactory peptide peak shapes using the weaker organic acid additives.15... 

Retention in RP chromatography is based on the interaction of the hydrophobic part of the analyte with the hydrophobic section of the stationary phase. This interaction can be modulated with the type and the concentration of the organic modiher in the mobile phase. The selectivity is mainly inflnenced by the interaction of the polar fnnctional gronps of the analyte with constituents of the mobile phase (bnffer, salts, etc. in the aqneons part) and with the amonnt and activity of residual surface silanols, which are, of course, also modihed by mobile phase constituents. 

The silica gel used in TLC has the same properties as that used in LSC in columns, and the discussion about silica in Chapter 9 is relevant. In brief, the silica has a heterogeneous energy surface and many very active silanol groups. It picks up water from the atmosphere very readily and will preferentially adsorb the most polar component in a mobile phase mixture, as just described. Most of the discussion about LSC is also applicable to TLC. 

An area of intense investigation is choosing the column with the best selectivity. The selectivity is dependent on the bonded phase (i.e., bonded ligand, silanol activity), the probe analytes, the pH of the mobile phase, the type and... 

Temperature, pressure, and density may also influence SFC selectivity in other ways. For example, water solubility in superaitical fluids generally increases with temperature, causing a shift the equihbrium of the number of water-deactivated silanol groups to carbon-dioxide-deactivated groups [1]. Therefore, the solubihty of analytes in the mobile phases inaeases but so does retention for polar analytes due to increased stationary-phase activity. 

George and Patel investigated some reversed-phase column packing materials for the presence of active silanol groups, by using the columns in the adsorption mode with -heptane as solvent. Four materials were tested for the analysis of theophylline and caffeine. These two compounds were well separated on all materials with the mobile phase acetonitrile - water - acetic acid (95 5 0.2). However, the sequence of caffeine and the internal standard 8-hydro-xypropyltheophylline varied for the columns. 

One of the key advances in column technologies is the development of high-purity silica.1,9 In recent years, it has become a de facto industry standard for almost all new column offerings. This development stems from the realization that batch-to-batch reproducibility and peak tailing of basic solutes are mostly caused by acidic residual silanols. Figure 3.9 shows different types of silanols and their relative acidity. The worst culprits turned out to be the very acidic silanols adjacent to and activated by metallic oxides. Many older silica materials have high metallic contents (e.g., Spherisorb) and are extremely acidic. They often require the use of amine additives in the mobile phase (e.g., tri-ethylamine) to prevent adsorptive interaction with basic analytes. The inherent variations of these active (acidic) silanols are also responsible for the lack of batch-to-batch consistency of these acidic silica materials. 

The HPLC of large biomolecules such as proteins and DNA often requires specialized columns packed with wide-pore polymer or silica-based bonded phase with extra-low silanol activity.1215 Alternate approaches are pellicular materials or very small nonporous particles. Some of these columns are packed in PEEK or titanium hardware to allow the use of high-salt mobile phase and to prevent possible protein denaturing by metallic leachates. Further details on bio-separations and application examples are discussed in Chapter 7. 

These unreacted silanol sites lie closer to the mobile phase than in GC, and can easily act as active sites degrading the chromatography as described in Section 11.4. 

Very often it is accepted that the higher the loading, the higher the acetonitrile content should be in the mobile phase to obtain elution. It is observed that the percentages needed in order to elute the cephalosporins within 40 min are very different and no clear correlation can be seen with the carbon content. However, not only the amount of organic material plays a role, but also the residual silanol activity. Therefore we modified the methyl red adsorption test of Shapiro and Kolthoff ( ) in order to use it for the quantitative measurement of residual silanol groups. A stock solution of 400 mg/100 ml of methyl red was used and the results are given in Table III. For Zorbax, a very low methyl red adsorption... 

The amount and activity of surface silanols on silica>based packings or their suppression by modification of the bonded phase or the mobile phase are significant. 

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