Absorbance and Spectrophotometry
Abstract
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.
Introduction
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 found by substituting known values into the equation:
Absorbance = k c t
Where: k = constant
c = concentration of absorbing molecules
t = thickness of the 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.
Method
A spectrophotometer was used throughout this experiment.
Results
After finding the absorption for 0. 005% 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 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 blue and put each solution through the spectrophotometer at wavelength 665nm to find the molar extinction coefficient. I plotted these results on a graph (figure 6) and did the line of best fit through the points to find the gradient, which is the molar extinction coefficient. Figure 4. Graph showing dilutions of methylene blue and the absorptions each solution gives
The black line in figure 4 represents the regression line. We can use this to find the concentration of the unknown concentration of methylene blue solution by drawing a tangent to the regression line at absorbance 0. 262 (where the unknown absorbed) and reading down from that point on the graph to the concentration. The concentration of the unknown methylene blue is 4. 4 x 10-6 mol dm-3. We can find the molar extinction coefficient by substituting values of absorbance and the concentration of the unknown concentration of methylene blue into Beer’s laws equation.
Absorbance = k c t k = absorbance / c t k = 0. 262 / 4. 4 x 10-6 x 1 k = 59545 mol dm-3 cm-3
Therefore, k, the molar extinction coefficient is 59545 mol dm-3 cm-3.
Discussion
The main objectives of this experiment were to find the unknown concentration of methylene blue by using a spectrophotometer. I found the maximum absorption for methylene blue and carmine red (please refer to figure 1) and using this I determined a more accurate maximum absorption value for each solution by taking further readings around the peak of each line to determine the maximum.
However, the findings of maximum absorption for methylene blue and carmine red may not be as accurate as we think because there are extraneous variables that we can not necessarily control. One is that the outside of the cuvette may have been dirty (however, this was controlled to an extent as I wiped each side down of the cuvette with a paper towel before placing it in the spectrophotometer); another variable is that the dial on the spectrophotometer only measured in wavelength intervals of 5nm, and so, we could not get more accurate readings than the ones we concluded with.
From figure 1, we can also see that high (maximum) absorptions for carmine red occur at around 475nm-550nm. This is because the light absorbs most light at this wavelength, and therefore, reflects light at approximately 675nm-725nm which are the wavelengths of the colour red, so we see a red solution. The same can be applied to methylene blue solution because we can see from figure 1 that high absorptions for methylene blue occur around 600nm-675nm – the light absorbs most colours at this wavelength and reflects light at approximately 400nm-450nm which are the wavelengths of the colour blue, so we see a blue solution.
We could use the maximum absorption of methylene blue found to make dilutions of methylene blue with water to plot a graph proving that Beers Law is true – that the absorbance is directly proportional to the concentration. This is confirmed by the graph produced as the line of best fit is accurate and goes through the origin.
Appendix
Finding the maximum absorbance:
| Wavelength/nm | Absorption | Absorption |
| – | Methylene Blue | Carmine Red |
| 350 | 0. 33 | 0. 156 |
| 375 | 0. 015 | 0. 018 |
| 400 | 0. 015 | 0. 046 |
| 425 | 0. 018 | 0. 048 |
| 450 | 0. 006 | 0. 127 |
| 475 | 0. 029 | 0. 093 |
| 500 | 0. 041 | 0. 65 |
| 525 | 0. 040 | 0. 186 |
| 550 | 0. 077 | 0. 144 |
| 575 | 0. 186 | 0. 068 |
| 600 | 0. 476 | 0. 039 |
| 625 | 0. 622 | 0. 028 |
| 650 | 0. 800 | 0. 005 |
| 675 | 0. 95 | 0. 013 |
| 700 | 0. 102 | 0. 004 |
More accurate values of methylene blue: More accurate values of carmine red:
| Methylene Blue | |
| Wavelength/nm | Absorption |
| 630 | 0. 623 |
| 640 | 0. 679 |
| 655 | 0. 885 |
| 660 | 0. 929 |
| 665 | 0. 965 |
| 670 | 0. 913 |
| Carmine Red | |
| Wavelength/nm | Absorption |
| 510 | 0. 205 |
| 515 | 0. 204 |
| 520 | 0. 207 |
| 530 | 0. 191 |
| 540 | 0. 169 |
The table below shows the dilutions and the absorbance values of methylene blue at 665nm:
| Tube | Water: Methylene Blue (ml) | Absorption | Concentration of methylene blue in water/mol dm-3 |
| 1 | 4:1 | 0. 171 | 3. 13 x 10-6 |
| 2 | 3:2 | 0. 376 | 6. 26 x 10-6 |
| 3 | 2:3 | 0. 595 | 9. 9 x 10-6 |
| 4 | 1:4 | 0. 762 | 12. 51 x 10-6 |
| 5 | 0:5 | 0. 963 | 15. 64 x 10-6 |
| Blank | 5:0 | 0. 000 | 0 |
Unknown solution absorbance: 0. 262
The formula mass of methylene blue: 319. 6
Working out the concentration of methylene blue from %:
- 0. 0001% methylene blue so, 100/0. 001 = 1000000 so, 1/1000000 = 1 x 10-6 g cm-3 so, conc. = 1 x 10-6 g cm-3 / 319. 6 g mol-1 = 3. 13 x 10-6 mol dm-3
- (3. 13 x 10-6) x 2 = 6. 26 x 10-6 mol dm-3
- (3. 13 x 10-6) x 3 = 9. 39 x 10-6 mol dm-3
- (3. 13 x 10-6) x 4 = 12. 51 x 10-6 mol dm-3
- (3. 13 x 10-6) x 5 = 15. 64 x 10-6 mol dm-3
- Figure 1. Methylene blue and carmine red’s absorption at regular intervals of wavelengths
- Figure 2. More accurate wavelengths to find the max. absorption for methylene blue
- Figure 3. More accurate wavelengths to find the max. absorption for carmine red
