67C-30

In recent years derivative ratio spectrophotometric method has been successfully applied to determine combined chemicals in synthetic mixtures and in pharmaceutical preparations. However, to our knowledge there is no report on using this method to simultaneously determine the β-carotene and astaxanthin in the Phaffia rhodozyma culture. Therefore, the objective of this research was to test whether this method could also be applied for a simultaneous determination of β-carotene and astaxanthin whose spectrums were overlapped in a great degree within the range from 360nm to 520nm. This technique depends on first derivative of the ratio spectra by division of the absorption spectrum of the binary mixture by a standard spectrum of one of the components and then calculating the first derivative of the ratio spectrum. In our tests suitable wavelength interval (Δλ) of 2nm was used to obtain the first derivative values. The first derivatives of the ratio amplitudes at 461nm for β-carotene and 466nm for astaxanthin were selected for the determination. Under this condition, obtained calibration graphs complied with Beer’ law and displayed a good linearity in the range of 0–6 μg/ml for β-carotene and 0–5μg/ml for astaxanthin, whose corresponding regressive equations were: Y=0.0014X+0.0048 and Y=0.0146X-0.00002, respectively. Their R square values exceeded 0.999.  Analysis of the β-carotene and astaxanthin in the Phaffia rhodozyma culture was performed using this model. The result showed there was marginal larger quantity of β-carotene in the early stage than in the later stage of culturing, while astaxanthin showed opposite tendency. In addition, sample recovery rates were from 99% to 101%, which confirmed the validity of the method for the determination of binary combinations in synthetic mixtures. In general, this method is simple, accurate, non-destructive and suitable for quality control applications.