Waters 2489 dual lambda absorbance detector

The chromatographic determinations were carried out on a Waters Alliance Separation Module (Waters, USA) quarternary gradient system and Waters 2489 dual lambda absorbance detector. The system control and data acquisition was carried out using Empower 3 software (Waters, USA). The separation was carried out in reverse phase Waters C18 column (250 mm × 4.0 mm, 5 μ; Waters, USA). The mobile phase was a mixture of 40% (v/v), 5.4 mM phosphate buffer (pH = 2.8) and 60% (v/v) acetonitrile which was filtered through 0.45 μm Millipore filter paper. The flow rate was 0.6 mL/minute and the column was maintained at ambient temperature and the injection volume was 20 μL. The detection wavelength was 280 nm. Prior to chromatographic separation both the standard and the sample were filtered through 0.2 μm membrane filter (Pall Life Sciences, India).

A Waters Alliance Separation Module (Waters, USA) quaternary gradient system and Waters 2489 dual lambda absorbance detector were used for the quantification purpose. The analysis was carried out using Empower 3 software (Waters, USA). A reverse phase Waters C18 column (Waters, USA) of 100 mm length, 4.0 mm internal diameter, 5 μm particle size, and a gradient elution flow rate of 0.6 mL/min was used for analysis. The mobile phase was a combination of filtered and degassed 5.4 mM phosphate buffer (pH = 2.8) and acetonitrile (ACN) in proportions as presented in Table 1.
The analysis was carried out at ambient temperature and the injection volume was 20 μL. The detection wavelength was 210 nm for DP and 280 nm for RC. Prior to chromatographic separation both the standard and the sample solutions were filtered through 0.2 μm membrane filter (Pall Life science, India).