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Spectrophotometric Determination of the Dissociation Constant of an Indicator Ramiya Sureshkumar 1004391302 08/11/17 22/11/17 TA: Christopher DiPietrantonio PRA 0127
Experiment 4: Spectrophotometric Determination of the Dissociation Constant of an Indicator Purpose The purpose of this experiment is to use spectrophotometric analysis to determine the dissociation constant, K IN, and the pK IN of the bromophenol blue indicator. The pH indicator under study, bromophenol blue, is yellow in its acid form (HIn) and is blue in its basic form (In - ). The equation dictating the change from HIn to In - from when a proton from a hydroxyl group is lost in the reaction is listed in the table below as equation 1. Seeing that the dissociation constant was determined spectrophotometrically, the concentration of solute in solution was measured by measuring the amount of light absorbed by the solute. The Beer-Lambert law states that the absorbance of light at a specific wavelength is directly proportional to the concentration of the indicator absorbing light at said specific wavelength. 1 Subsequently, the Beer-Lambert law is assumed to follow during the experiment, the relative concentration of the two forms of the indicator varies as the pH varies. The concentration of the indicator in its acidic and basic form were used to calculate the dissociation constant, as indicated in equation 2 in the table below. Furthermore, the data was used in determination of the molar absorptivity constant, a measure of the strength of the absorption of light at a specific wavelength by a chemical species. 2 Said constant can be calculated using equation 4 of the table below. The relevant theories applied to this lab include spectrophotometry, applications of the Beer-Lambert law, dissociation constants, and applications of indicators. Said theories can be applied to real life situations, as, for example, spectrophotometry is used in forensic examination, measurement of enzyme activity, determination of protein concentrations, and a variety of other industries. This is due to the ability of a spectrophotometer to determine, through calibration, the present substances and quantity of said substances in a target through calculations of observed wavelengths. Equation 1 HIn + H 2 O ⇌ In - + H 3 O + Equation 2 K IN = ([H 3 O + ][In - ])/[HIn] Equation 3 pK IN = pH + log [HIn]/[In - ] Equation 4 A = ɛlc 1 Clark, J. The Beer-Lambert Law (accessed Nov 19, 2017). 2 Robinson, A. How to Calculate Molar Absorptivity - 5161812.html (accessed Nov 19, 2017). 1
Experiment 4: Spectrophotometric Determination of the Dissociation Constant of an Indicator Experimental method The pH and absorbance of the solution was measured in the first part of the experiment. The solution had been a mixture of 5.00 mL of 0.02% bromophenol blue and 95.00 mL of 0.05M KHP. From the absorbance values collected, the two absorbance peaks were identified (λ 1 and λ 2 ). In the second part of the lab, the solution from part 1 had been transferred into two individual 50.00 mL beakers and