Abstract This report investigates the absorbance of methylene blue and carmine red using a spectrophotometer to determine the absorption spectra of both solutions. The concentration of the unknown solution of methylene blue was found to be 1. 07 x 10-5 M by using the molar extinction coefficient, with absorption of 0. 547. It was also found that the results concluded confirmed beer’s law with an R2 value of 0. 9989. Introduction Spectrophotometry is the quantitative measurement of the absorbance or transmittance properties of an analyte as a function of wavelength.
Beer’s law states that the absorbance is directly proportional to the concentration of the analyte. A straight line should be observed on an absorbance vs. concentration graph if the experiment is to confirm to Beer’s law. [The range of where the maximum peak occurred was identified and analysed in detail using smaller increments to fully establish the wavelength at maximum absorption (methods section)] The aims of this experiment were to confirm Beer’s law as well as apply the molar extinction coefficient.
The molar extinction coefficient was used to predict the concentration of the unknown solution of methylene blue from the collected data that was utilized to plot an absorbance against concentration graph. Spectrophotometry spans a wide range of scientific fields, such as biochemistry, molecular biology, materials science and physics, therefore it is important to familiarise the techniques and uses of the spectrophotometer. Materials and Methods The absorbance of methylene blue and carmine red were measured using an Ultrospec 1100 Pro spectrometer.
Absorbance of the several diluted methylene blue solutions were recorded at the wavelength at which maximum absorbance occurred using the spectrometer, this data was then used to calculate the concentration of the unknown solution of methylene blue. Results Figure 1 shows the absorption spectrum of black filled dots; carmine red and white filled dots; methylene blue Figure 2 (top left) shows the graph of methylene blue at a smaller range of wavelengths at which the maximum absorbance occurs as well as analysing the shoulder in detail shown in figure 1.
Figure 3 (top right) shows the graph of carmine red at a range of wavelengths at which the maximum absorbance occurs Both solutions absorb at different wavelengths which can be seen in figure 1. It can also be seen that methylene blue has a low absorbance between the wavelengths of 350-550nm corresponding to the colours of violet, green and blue. However in carmine red, the wavelength at which low absorbance occurs ranges from 600-700nm, corresponding to the colours of orange and red. These are the colours that are mostly reflected resulting in the colour of these solutions.
Methylene blue has a maximum absorbance at a wavelength (? max) of 665nm seen in figure 2 which corresponds to the colour red. In comparison to carmine red, it’s ? max is at 575nm seen in figure 3 corresponds to the colour of green-yellow. A shoulder can be seen for methylene blue between the wavelengths at 625-650nm shown in figure 1. This is also further analysed in the discussions section. Figure 4 calibration curve for methylene blue concentration There is a linear proportion between concentration and absorbance, therefore conforming to Beer’s law.
This shows that the absorbance of light increases with increased concentration. [talk about the solutes? ] Although the value of R2 is very close to 1, there were a few limitations that arose in the experiment which is further discussed in the discussions section. See Appendix A, calculation 2 for the concentration of the unknown solution of methylene blue Discussions and Conclusion Although the graph is observed to conform to Beer’s law which states that the absorbance is directly proportional to the concentration of the analyte, there were a few limitations to this experiment that could result to the kewness of the graphs and the R2 value. Seen in appendix B, tables 2 and 3, the average absorbance was taken as the repeats were not the same as those shown in appendix B, table 1. A reason for this was the handling of the cuvettes. Dust particles entering the cuvettes will reflect light so less light is absorbed. In avoiding this error, the cuvettes should be cleaned with distilled water, blotted and placed upside down in a designated plastic rack to further drain the excess water.
The shoulder of the graph for methylene blue explained in the results section was analysed in smaller intervals of wavelength between 625-675nm shown in figure 2. This concluded that there were no measurement errors [how? ] Another limitation is due to parallax error when diluting methylene blue which contributed to the relatively lower R2 value. Parallax error was avoided through looking at the level parallel to the surface of the water when measuring with a pipette Appendices Appendix A – Calculations Methylene blue (0. 005%) in water was used A 0. 0005% wt/vol solution contains 0. 0005g solute per 100ml 0. 005g solute per 1L Using the moles calculation of mass , whereby the Mr of methylene blue is 319. 5g mol-1, Mr A concentration of 1. 56 x 10-5 M was obtained. Calculation 1 The absorbance of the unknown solution of methylene blue was 0. 547Au. Using beer’s law A= kct, concentration was able to be calculated. As A= 0. 547k=51009andt=1 Rearranging the equation gives C=0. 547/51009 C=1. 07 x 10-5 M Calculation 2
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