Absorption Spectra and the Beer-Lambert Law

LS1120 Practical 3
ABSORPTION SPECTRA AND THE BEER-LAMBERT LAW

The purpose of this practical was to measure the amount of a chemical substance present in a sample. Primarily, the aim of Experiment 1 was to measure the absorption spectrum of a particular coloured substance (in this case Bromophenol Blue and Methyl Orange) at varying wavelengths of light. For Experiment 2, the process of the experiment focuses on the substance and records its absorption levels at different concentrations.
The absorption levels (A) that are obtained when samples of any given substance exposed to a source of visible light are measured by: The length (l) of the light path (i.e. the distance over which the light had to travel), and the concentration (c) of that substance. This, essentially, is the Beer-Lambert law. Therefore, a molecule can only absorb a specific amount of visible light because of the size of its container (the length from one side to the other) and the concentration.
Absorption levels vary across compounds. To measure the absorbance, a reference sample has to be used along with the actual dye sample being tested. The intensity of the light passing through the reference cell is measured for each wavelength of light used. (Chemguide, 2007)
Method
Experiments 1 and 2 were carried out as specified in the LS1120 Practical Schedules booklet.
In doing so, a problem was encountered. While measuring the absorbance using Bromophenol Blue, it was discovered that the liquids leaked out into the compartment. After this was cleaned up, it became evident that the problem arose due to a zeroing of the colorimeter that was too forceful. This error was corrected and the experiment then continued smoothly.
Discussion
A very important step in the course of both experiments was to zero the spectrophotometer using distilled water. This was done in order to improve the accuracy of the results, because water has been found to be an optimum standard for comparison due to the fact that it has zero absorbance.
The results are accurate to a good extent, but are unlikely to be perfectly accurate because of the existence of human error. It is possible that measuring for a specific concentration of the tested solution, the container mat have been inserted the wrong way round, thus blocking out some of the light meant to go through the solution. Otherwise, the container again could have been held erratically resulting in fingerprints on the side of the container. This will again adversely affect the results obtained, because it will render the
Another source of error (specifically in Experiment 2) could be that the wrong measurements were made, and so the concentrations of the substance in the test tubes that were produced were not correct. Consequentially, the results obtained will be incorrect.

Experiment 1: Bromophenol Blue Wavelength (nm) | Absorbance | 400 | 0.089 | 420 | 0.049 | 440 | 0.044 | 460 | 0.057 | 480 | 0.080 | 500 | 0.124 | 520 | 0.215 | 540 | 0.330 | 560 | 0.505 | 580 | 0.796 | 600 | 0.855 | 620 | 0.287 | 640 | 0.096 | 660 | 0.041 | 680 | 0.046 | 700 | 0.049 | Further readings to determine ‘λ max’ | Wavelength (nm) | Absorbance | 590 | 0.795 | 595 | 0.605 | 600 | 0.696 | 605 | 0.523 | 610 | 0.316 | 615 | 0.186 |

Highest Absorbance Value (λ max) for Bromophenol Blue dye obtained at wavelength 590nm

Experiment 1: Methyl Orange Wavelength (nm) | Absorbance | 400 | 0.376 | 420 | 0.473 | 440 | 0.629 | 460 | 0.681 | 480 | 0.588 | 500 | 0.461 | 520 | 0.236 | 540 | 0.085 | 560 | 0.021 | 580 | 0.019 | 600 | 0.029 | 620 | 0.033 | 640 | 0.063 | 660 | 0.059 | 680 | 0.050 | 700 | 0.069 | Further readings to determine ‘λ max’ | Wavelength (nm) | Absorbance | 450 | 0.643 | 455 | 0.627 | 460 | 0.642 | 465 | 0.630 | 470 | 0.650 | 475 | 0.600 |

Highest Absorbance Value (λ max) for Bromophenol Blue dye obtained at wavelength 590nm

Experiment 2: Absorbance values at ‘λ max’ for Bromophenol Blue TubeNumber | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | Stock Solution (10mg/l) (ml) | 0.3 | 0.6 | 0.9 | 1.2 | 1.5 | 1.8 | 2.1 | 2.4 | 2.7 | 3.0 | Distilled Water (ml) | 2.7 | 2.4 | 2.1 | 1.8 | 1.5 | 1.2 | 0.9 | 0.6 | 0.3 | 0.0 | Concentration (mg/l) | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | Concentration (1%) | 0.0001 | 0.0002 | 0.0003 | 0.0004 | 0.0005 | 0.0006 | 0.0007 | 0.0008 | 0.0009 | 0.0010 | Absorbance | 0.080 | 0.152 | 0.221 | 0.300 | 0.371 | 0.449 | 0.526 | 0.605 | 0.665 | 0.743 | ε (1%) Epsilon | 800 | 760 | 736.67 | 750 | 742 | 748 | 751.43 | 756.25 | 738.89 | 743 |

Experiment 2: Absorbance values at ‘λ max’ for Methyl Orange TubeNumber | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | Stock Solution (10mg/l) (ml) | 0.3 | 0.6 | 0.9 | 1.2 | 1.5 | 1.8 | 2.1 | 2.4 | 2.7 | 3.0 | Distilled Water (ml) | 2.7 | 2.4 | 2.1 | 1.8 | 1.5 | 1.2 | 0.9 | 0.6 | 0.3 | 0.0 | Concentration (mg/l) | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | Concentration (1%) | 0.0001 | 0.0002 | 0.0003 | 0.0004 | 0.0005 | 0.0006 | 0.0007 | 0.0008 | 0.0009 | 0.0010 | Absorbance | 0.367 | 0.131 | 0.145 | 0.326 | 0.322 | 0.230 | 0.222 | 0.208 | 0.286 | 0.304 | ε (1%) Epsilon | 3670 | 655 | 483.32 | 815 | 644 | 383.3 | 317.14 | 260 | 317.78 | 304 |

References
Chemguide (2007) The Beer-Lambert Law. Available at: http://www.chemguide.co.uk/analysis/uvvisible/beerlambert.html [Acccessed 04 December 2012]

References: Chemguide (2007) The Beer-Lambert Law. Available at: http://www.chemguide.co.uk/analysis/uvvisible/beerlambert.html [Acccessed 04 December 2012]