Absorbance and spectrophotometry

Absorbance and Spectrophotometry





Abstract

Spectrophotometry is used to calculate the absorbance spectrum of a specific compound in a solution. This is a characteristic of thecompound. Therefore measuring the absorbance of light of a diluted substance through a spectrophotometer, allows the concentration of the solute to be determined. Methylene blue (0.0005%), carmine red(0.005) and an unknown concentration were used as samples. After determining at which wavelength (nm) does maximum absorption occur using the spectrophotometer for both solutions and building a graph,Beer’s law was used to calculate the molar extinction coefficient ( ). Therefore, through the equation and the spectrophotometer, the concentration of the unknown solution of methylene blue wascalculated: 5.78 . Then it was placed into a ‘standard graph’ relating the concentration to the absorbance of five different solutions for the direct estimation of the concentration when absorbanceknown.

Introduction

The ability of certain diluted substances to absorb visible light, or light at any other particular wavelength depending on the substance, is essential. The reason is thatthe maximum absorption spectra can be used as an identifying feature of a compound or in calculating its concentration. The concentration of a given solute in a fixed thickness holder solute isdirectly proportional to the absorbance. That, can be seen in Beer’s law (Absorbance=k Concentration t), used to calculate any of the above mentioned. In biochemical processes, when using aspectrophotometer, quantitative assays of diluted biomolecules require simply a measurement of their absorbance of light at their specific wavelength. The aim of the experiment was to understand the procedure of usinga spectrophotometer and Beer’s law to calculate the concentration of different dilutions.

Materials and Methods

* Methylene blue (0.0005%) and carmine red (0.005%) solutions were used as...