Absorbance and Spectrophotometry

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  • Topic: Light, Color, Red
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  • Published : November 26, 2012
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Experiment 2: Absorbance and Spectrophotometry


This was an investigation into the effects of different wavelengths of light on methylene blue and carmine red on the absorbance value on a spectrophotometer. A spectrophotometer is used to measure light intensity by emitting a single light source through a cuvette of coloured solution. The particles in the solution, which are coloured, absorb the light depending on how concentrated it is and this produces an electronic reading from the photometer which is the absorbance value. The maximum absorption was found for both solutions and was used to calculate the molar extinction coefficient of methylene blue. An unknown concentration of methylene blue was calculated by using graphs produced in the dilution experiments prior. The results produced supported Beer’s Law because the absorbance was directly proportional to the concentration, and so, we can be assured that the concentration of the unknown methylene blue solution calculated is relatively accurate.


A spectrophotometer is used to measure the absorbance of light by coloured solutions. The absorbance value is produced by a photometer that compares the light detected with a blank cuvette (a cuvette containing just water/clear colourless solvent, which should be 0), with the amount of light detected with a test solution – in this case, methylene blue or carmine red. Using Beer’s Law, we know that the absorbance is directly proportional to the concentration, therefore, knowing the absorbance of a solution can be very useful as the concentration of the solution can be find by substituting known values into the equation:

Absorbance = k c t

Where: k = constant
c = concentration of absorbing molecules
t = thickness of absorbing layer

The aims of this experiment were to use solutions methylene blue and carmine red to confirm that Beer’s Law is true by finding the maximum absorption value for each solution, and then using this, find the absorption of methylene blue solution at various dilutions. By plotting these results on a calibration curve (concentration against absorbance), this allows the experimenter to read the concentration at a particular absorbance directly, such as the unknown concentration of methylene blue.


A spectrophotometer was used throughout this experiment.


After finding the absorption for 0.0005% methylene blue solution and 0.0005% carmine red solution at different wavelengths of light, we plotted a graph to show our findings to make it easier to see what region of wavelength the maximum absorption would occur at. Please refer to figure 1.

From this graph, we can see that the maximum absorption for methylene blue is around 650nm-675nm as the peak on the line for methylene is around these values; for carmine red, we can see that the maximum absorption for carmine red is 500-550nm. To obtain a more accurate wavelength value, I placed more cuvettes of methylene blue and carmine red around their regions of maximum absorption.

After finding the absorption values around each region, I plotted the findings of each solution on separate graphs to show the maximum absorption value. Figure 2 shows that the maximum absorption of methylene blue is 665nm because this has the peak absorption of 0.965. However, this is not as accurate a value as it could be because the spectrophotometer did not go to more accuracy than 5nm.

From Figure 3, we can see that the maximum absorption of carmine red is 0.207 at wavelength 520nm as this is the peak on the graph.

|Maximum Absorption at 0.0005% |
|Methylene Blue |Carmine Red |
|0.965 |0.207 |

After we found the maximum absorption for methylene blue, 0.965, at 665nm, we made up various dilutions of methylene...
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