Waters 486 chromatograph

A total of 50 mg of immobilized lipase was added to 3 mL of 50 mM R- or S-methyl mandelate in 50 mM sodium phosphate solution at pH 7.0. The reaction was carried out at room temperature under gentle stirring. The quantification of hydrolysis was determined by the release of mandelic acid in the reaction medium. A Waters 486 chromatograph (Waters, Millford, USA) presenting a Kromasil C18 column (15 cm × 0.46 cm) and a UV/VIS detector (set to 230 nm) was employed in the analyses to determine the degree of conversion (two points over 5% and under 25%, to ensure linearity and minimize experimental error caused by the initial acid content of the samples) and enzymatic activity [112 (link)]. The mobile phase was 10 mM ammonium acetate and acetonitrile (65–35% (v/v)) at pH 2.8 with a flow rate of 1 mL/min. The retention times were 2.5 min for mandelic acid and 4.2 min for the R- or S-methyl mandelate [113 (link)]. The activities ratio was defined as the activity versus the R-isomer/activity versus the S-isomer.

A volume of 0.3 mL of immobilized enzyme suspension (166 mg/mL) was added to 3 mL of 50 mM of triacetin prepared in 50 mM of sodium phosphate buffer at pH 7.0. Hydrolysis was carried out at 25 °C under magnetic stirring (100 rpm). The hydrolytic activity in triacetin was quantified by detection of 1,2 and 1,3 diacetin (under these conditions, 1,2 diacetin suffers acyl migration, giving 1,3 diacetin) [118 (link)]. The degree of conversion was calculated by HPLC in a Waters 486 chromatograph (Waters, Millford, UK.) equipped with a UV/VIS detector (set to 230 nm) [118 (link)] using a Kromasil C18 column (15 cm × 0.46 cm) with a mobile phase composed of 15% (v/v) of water and 85% (v/v) of acetonitrile with a flow rate of 1 mL/min. The retention times were 4 min for 1,2 and 1,3 diacetins (under these conditions eluted at the same time) and 18 min for triacetin. Conversions of 15–20% were used to calculate enzyme activity [119 (link)].

A mass of 0.05 g of commercial immobilized lipase were added to 3 mL of 50 mM R- or S-methyl mandelate in 50 mM sodium phosphate buffer solution at pH 7.0. Hydrolysis was carried out at 25 °C under magnetic stirring (100 rpm). The substrate and product concentrations were determined by HPLC using a Waters 486 chromatograph (Waters, Millford, UK) equipped with a UV/VIS detector (set to 230 nm) [114 (link)] using a Kromasil C18 column (15 cm × 0.46 cm) with a mobile phase composed of 10 mM ammonium acetate and acetonitrile (35–65% (v/v)) at pH 2.8 with a flow rate of 1 mL/min. The retention times were 2.5 min for mandelic acid and 4.2 min for the R- or S-methyl mandelate. Conversions of 15–25% were used to calculate enzyme activity [120 (link)].