UV-Vis Spectrophotometer

In the experiment, UV-Vis Spectrophotometer is used to determine the reducing sugar content of commercial natural honey by 3,5-Disnitrosalysilic acid (DNS) method. By scanning the standard solution (0.2, 0.4, and 0.6mg/ml) from 200nm to 800nm, the spectra with different concentration of standard solution is obtained. From the data, it clearly showed that the value of maximum absorbance increase with the increase of concentration of standard solution where we can see that the maximum absorbance for standard solution at 0.2, 0.4, and 0.6mg/ml are 0.406, 0.667, and 1.030 respectively. Then, three wavelength are chosen which are 490nm (up slope), 500nm (apex), and 510nm (down slope) and the graph of absorbance against concentration of glucose anhydrase

This is usually corresponds to the apex of the highest absorption peak because the measurement obtained will have maximum sensitivity which defined as the absorbance change per unit change in analyte concentration. Besides, the measurement obtained will also have greater adherence to Lambert-Beer’s law since the spectral region making up the radiation beam is composed of wavelengths with relatively small differences in their molar absorptivity for the analyte being measured (Nielsen, 2009). Furthermore, taking the readings at the peak maximum wavelength is the best at low absorbances because it gives the best signal-to-noise ratio that improves the precision of measurement. When comparing the three standard curves, we can see that the curve with wavelength at 500nm which is the λmax is steepest compared to the curve with wavelength 490nm and 510nm. This indicated that the standard curve at 500nm is the most sensitive because the steeper the slope of curve, the greater the sensitivity. Besides, the R2 value obtained from the standard curve at 500nm is the closest toward 1.0 which is 0.9892. As we know, R-squared is a statistical measure of how close the data are to the fitted regression line. In another word, the closest the value of R-squared toward 1.0, the better the model fits the data (Glantz & Slinker, 1990). Therefore, the wavelength at 500nm which is also the λmax of

This is because absorbance is directly proportional to the concentration of the absorbing species in the solution under appropriate conditions. As we can see, the equation obtained from the standard curve at wavelength 500 nm is y= 1.7148x. From the calculation, the percentage of reducing sugar content in sample is range from 70.98% to 73.66%. According to Food Act 1983 and Food Regulation, Part VIII Standards and Particular Labelling Requirements for Food (130.2.a), honey shall contain not less than 60 per cent of reducing sugars calculated as dextrose anhydrous (Food Act 1983 & Regulation, 2011). The result obtained from our experiment is slightly higher than the standard. It can be due to some experimental errors which affect the accuracy of our experimental result. For example, the sample may not be weighted properly and mixed well during the experiment. Besides, the cuvette may not be washed properly after used which will result in sample contamination. Other than that, it is important to make sure that there is no fingerprints deposited any on the sides of the cuvette because fingerprints will scatter laser light and create scatter signals that are not related to the sample (Clark, 1993). Furthermore, air bubbles that trapped in the cuvette will also alter the measurement obtained. Therefore, carefully