Molar extinction coefficient of methylene blue

Experiment #6: Molar absorptivity

Introduction

In this experiment, students will learn how to use the UV-visible spectrophotometer to determine the molar extinction coefficient of organic dyes. In the same time they will learn how to use UV-vis spectroscopy to construct a calibration plot for light absorbing compound. Then, they will be able to identify unknown concentrations of this compound, in this case the organic dye, based on their calibration plot.

Theory

When monochromatic light passes through a sample, the ratio of power transmitted to the original power is called the transmittance T. The Absorbance is equal to the logarithm of the reciprocal of the transmittance:

A = -log T = log (1/T)

In this case, the light absorbance of the sample depends on the molar concentration (c), light path length in centimeters (L), and molar extinction coefficient (ε) for the dissolved substance. The molar extinction coefficient (ε) is a term that determines how strongly a substance absorbs light at a given wavelength per molar concentration. It is also called molar absorptivity or attenuation coefficient.

Therefore, the concentration of the substance dissolved in the solvent can be determined experimentally using a spectrophotometer and based on Beer’s law: A= εLC

Plotting the absorbance against the concentration for a series of dilutions will result in a linear line with the slope = ε, where the light path length (through the sample solution) is 1 cm.

Procedure

1- Carefully perform every step in this procedure, as the grade for this lab will be based on precision and accuracy.

2- In a set of 20 mL glass vials, prepare a series of aqueous solutions of the methylene blue solution, 10, 30, 50, 70, and 100 M. You may prepare a sample of the highest concentration (100 M) and then perform serial dilutions.

3- In a set of 20 mL glass vials, prepare a series of aqueous solutions of the rhodamine B solution, 10, 30, 50, 70, and 100 M. You may prepare a sample of highest concentration (100 M) and then perform serial dilutions.

4- Set the UV-vis spectrophotometer for scan absorbance over the range of 300 − 700 nm.

5- Use DI water to obtain baseline readings of the spectrophotometer.

6- Measure the UV-vis spectra for your series of methylene blue solutions using disposable cuvettes over the range on 300 − 700 nm.

7- Measure the UV-vis spectra for your series of rhodamine B solutions using disposable cuvettes over the range on 300 − 700 nm.

8- Perform the analysis of each concentration in triplicate.

9- Measure the absorbance of the unknown concentration of each dye.

Data Manipulation and Calculations

1- Export data to Excel and calculate average absorbance of each sample.

2- Plot average absorbance vs wavelength and identify the peak maximum.

3- Plot the absorbance at peak maximum against concentration of the dye.

4- Find the slope and regression of the linear fit line. Slope equals extinction coefficient (ε).

5- Calculate the concentration of the unknown sample using extinction coefficient and absorbance.

Equipment

20 mL Glass Scintillation vials

UV/VIS Spectrophotometer

Disposable cells (cuvettes) for UV-VIS