The number and nature of silanol groups could be estimated in mesoporous silica prepared with cetyltrimethylammonium bromide (CTMBr) in acidic medium to obtain a material appropriate as a stationary chromatographic phase, using thermogravimetric analysis (TGA) and diffuse reflectance infrared spectroscopy (DRIFT). The temperature-programmed desorption of pyridine in the DRIFT cell was quantitatively correlated to the change in intensity of the two IR bands, 1595 and 1446 cm-1, assigned to pyridine interacting respectively with hydrogen-bonded and free silanol groups, after the determination of the absorptivities for the two bands. For calcined or solvent extracted samples, the silanol density (3−4 groups/nm2), is represented mainly by hydrogen-bonded silanol groups. A significant increase is obtained by rehydroxylation. Activation by heating above 200 °C promotes the condensation of the vicinal silanol groups.