Spd 20a prominence uv vis detector

The synthesis of mandelonitrile from benzaldehyde was analyzed by HPLC with a chiral column as described in a previous report^{18 (link)} with slight modifications. The enzyme samples were mixed with 200 µl 300 mM citrate buffer (pH 4.2) containing 50 mM benzaldehyde and 100 mM potassium cyanide. The mixture was incubated at 22 °C for 5 min. Then, 50 µl reactant was added to 450 µl of n-hexane:2-propanol = 85:15, mixed vigorously, and the mixture was centrifuged at 20000 g for 3 min. A 5-µl aliquot of the organic phase was analyzed as described previously^{40 (link)} using a UFLC Prominense Liquid Chromatograph LC-20AD equipped with a CHIRALCEL OJ-H column (particle size: 5 µm; 4.6 mm i.d. × 250 mm; Daicel Corporation, Tokyo, Japan) connected to a Prominence UV-Vis detector SPD-20A (Shimadzu, Kyoto, Japan). The separation conditions were as follows: mobile phase, n-hexane:2-propanol (85:15); flow rate, 1 mL/min. Eluted products were detected by monitoring absorbance at 254 nm.

A 250 mg of Lyophilized whole venom of Naja naja was dissolved in 5 ml of de-ionized water and given a heat treatment for 30 min at 60^oc in a water bath followed by centrifugation at 2500 rpm for 20 min. 50 mg of the supernatant was loaded onto a CM-cellulose column (100 × 20 mm) which was equilibrated with 0.02 M phosphate buffer (pH 7.2). A total of 42 fractions (each of 5 ml volume) were collected using the stepwise gradient of sodium chloride (0.02 M – 1 M in phosphate buffer, pH 7.2) with a constant elution rate of 30 ml/min at room temperature. Protein content in the fractions was estimated by Lawry’s method [18 ]. All the fractions were checked for their cytotoxic activity against MCF-7 cells. The fraction which was showing cytotoxic activity was further purified by Reverse phase HPLC (Shimadzu LC-2010HT, Japan) using C18 column (4.6 × 250 mm) (Waters, USA) equilibrated with 0.1% Trifluoroacetic acid (TFA) in water and eluted with a linear gradient of 100% acetonitrile in 0.1% TFA at a flow rate of 1 ml/min. The HPLC profile of the fraction was monitored at 280 nm for 60 min using Shimadzu Prominence UV/Vis detector (SPD-20A).