Silanol, content, reversed-phase .column

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+... 

The columns used for the separation of phenolic acids are mainly reversed phase (RP), other sihca-based chemically bound phases, and non-silica polymers or mixed inorganic-organic phases. Special silica sorbents with reduced metallic residue contents and minimum residual silanol groups on the surface could positively influence peak symmetry without the strict demands for the successful separation of acidic analytes. Almost exclusively, RP C18 phases ranging from 100 to 250 mm in length and usually with an internal diameter of 3.9 to 4.6 mm are recommended. Particle sizes are in the range of 3-10 pm. Short 50- 100-mm columns with 3-pm particles have also been tested for fast phenolic acid separations. Narrow bore columns (internal diameter 2 mm) are recommended especially for HPLC-MS applications.Some problems could arise with the applications of narrow or microbore columns in the direct injections of plant extracts there is the possibility of plugging the column after repeated injections. In these cases, an additional clean-up step has to be applied instead of just the simple extraction... 

The interaction of analytes with the residual silanols can be a nuisance to the user of reversed-phase packings. They make the retention behavior of basic analytes more difficult to interpret and less predictable. In some extreme cases, especially for basic peptides, plots of the retention factor versus the percent organic modifier exhibit a minimum while for pure hydrophobic interaction, a monotonous decline with increasing organic content of the mobile phase is expected. The increased tailing caused by the interaction of silanols with polar, but especially basic, compounds makes peak integration more difficult and reduces column performance. 

In fact, every silica-based reversed phase represents a mixed-mode column, because besides the alkyl chains it has silanol groups, which are groups capable of weak cation exchange. Some C18 materials of the first generation, such as Partisil ODS from Whatman or Spherisorb ODSl from Waters, have only a very low carbon content (<5%) and a high number of free silanol groups. With these... 

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