P-Nitrophenol Spectrophotometry

1. The standard curve is a graph that shows a relationship between concentration and absorbance of a solution. A standard curve was experimentally created in this experiment using 10mL solutions of phenol red with concentrations 10µM, 7.5 µM, 5.0 µM and 2.5 µM then the absorbance of each sample was measured using a spectrophotometer. This generated curve with resulting average absorbances of 1.273nm, 1.0275nm, 0.585nm, 0.285nm and 0.124nm provided a means to determine the phenol red concentrations of two unknown samples after experimentally identifying the absorbance of the two samples using a spectrophotometer.

2. Based on the results in the standard curve exercise, the two unknown samples (unknown 1 and unknown 2) had measured absorbance’s of 0.533 and 1.581. As 1.581 was off the standard curve graph, a diluted solution was created for unknown 2. This resulted in absorbances of 0.533 and 0.929 and concentrations of 4.33µm and 7.66µm and mass of 4.88µ and 17.31µg for unknown 1 and unknown 2, respectively.

3.
The pKa of a weak acid (p-nitrophenol) and its conjugated base (p-nitrophenolate ion) was determined by plotting pH as a function of [A-]/[HA] in order to represent the ionized and unionized regions across pH. The optimal wavelength (λm), where the maximum absorbance occurs was determined by making twelve solutions where p-nitrophenol/pH is completely ionized (pH 9.0). The importance of determining the proper λm is this is where wavelength signal is the greatest; therefore minimizing risk of interfering with the instrumentation, which was 400nm at the peak absorbance of