100 mg of 3-mercaptopropyl silica gels with different
sulfhydryl coverage and samples of 2-mercaptoethanol stock
solution, all equivalent to thiol amounts in the range of
5–125 mol, were transferred to 25 ml volumetric flasks. Unmodified LiChrospher® Si100 10 m and acetonitrile were
also tested as blank samples. 1.3 ml of the 0.1 M DPDS solution was added and a yellow-greenish color started to develop in the case of thiol-containing samples. All flasks were
covered with aluminum foil to provide light protection and
the reaction was allowed to proceed for 3 h at room temperature under vigorous shaking. The volumetric flasks were then
filled up to 25 ml with acetonitrile. In the case of silica gel
samples, the suspensions were centrifuged and the upper clear
solution was used for quantification. The concentration of all
solutions at this step was in the range of 0.2–5 mol SH ml−1.
An aliquot of each solution was diluted with acetonitrile
(1:10), reaching concentrations in the range of 0.02–0.5 mol
SH ml−1. The extinction of all samples was determined spectrophotometrically in the spectral range between 300 and
450 nm, using a 1 cm cuvette and acetonitrile as blank.
Another aliquot of each solution, without further dilution,
was analyzed by HPLC, using a Hypersil BDS-C18 column
(5 m, 125 mm × 4 mm i.d.) from Agilent Technologies (Vienna, Austria). The mobile phase was double distilled water
(A) and acetonitrile (B), and a linear gradient was run from 5
to 90% B in 20 min. The temperature was 25 ◦C and the flowrate was adjusted to 1 ml min−1. The detection wavelengths
were 369 and 254 nm and the injected volumes 20 l.