Laboratory 2 Dilutions and Introduction to Spectrophotometry

September 15, 2012

I. Introduction Dilutions are an essential procedure in the microbiology laboratory, whether one is working with bacterial samples, blood samples, or chemicals, they are used to ensure that an optimal concentration is used for safety and for the accuracy of the measurements to be carried out. Dilutions are carried out by taking a small volume of the stock solution and mixing it with a larger quantity of solvent. The new concentration can then be calculated by dividing the mass or volume of the solute added by the new volume of the solution. Beers law provides a useful way to measure the accuracy of a dilution. Using an instrument known as a photospectrometer that measures the intensity of a particular wavelength of light passing through a sample, one can calculate the absorbance, which according to Beers law is linearly related to the concentration of a sample. The equation for Beers law is: (1)

A = lc

Where A is absorbance, is an absorptivity constant particular to the material, l is the length of the path that the light travels through the sample, and c is the concentration of the sample. (Clark, 2007) The purpose of this experiment is to practice making dilutions, and to experimentally confirm Beers law. The hypothesis is that the absorbance of a solution increases in a linear trend with increasing concentration, as stated by Beers law. II. Method Materials used for this experimentation are: concentrated dye, two 10mL pipettes, distilled water, and a total of 11 test tubes. The procedure consisted of preparing 11 different dilutions of dye in distilled water and measuring their absorbance using a spectrophotometer, then comparing the obtained absorbance with the theoretical value as predicted by Beers law. The dilutions were prepared so that the total volume in each test tube was always 5mL. Thus the first sample contained 5mL of concentrated dye, the second contained 4mL of dye and 1mL of water, and so on until the last test tube contained 5mL of water. This test tube containing pure water was used to zero the spectrophotometer (which was set at a wavelength of 640nm), then each test tube was inserted in turn and the absorbance was recorded. The theoretical absorbance was calculated by taking the measured absorbance of the sample of pure concentrated dye and multiplying it times the percent of concentration.

III. Results
Concentration Theoretical Actual (% of solute) Absorbance Absorbance % Error 100 1.460 1.460 0.00 90 1.314 1.315 0.08 80 1.168 1.205 3.17 70 1.022 1.054 3.13 60 0.876 0.936 6.85 50 0.730 0.806 10.41 40 0.584 0.638 9.25 30 0.438 0.520 18.72 20 0.292 0.362 23.97 10 0.146 0.221 51.37 0 0 0.051 Table 1: Theoretical and measured absorbances for each concentration. Note the increasing percentage error as concentration decreases.
Beer's Law
1.600 1.400 1.200 Absorbance 1.000 0.800 0.600 0.400 0.200 0.000 0 10 20 30 40 50 60 70 80 90 100 Concentration (%) y = 0.0139x + 0.0827 R2 = 0.9985

Graph 1: Plot of the experimental values for absorbance versus the concentration of the samples, showing a strong linear correlation, as shown by the equation and R2 value.

IV. Summary and Conclusions The linear trend obtained from plotting absorbance versus concentration coincides with that predicted by Beers law, supporting the hypothesis. However, the measured results did show an increasingly significant deviation from the predicted values as the concentration of solute became smaller, probably due to experimental or systematic error. V. References: 1. Clark, J. (2007). The Beer-Lambert law. Retrieved from Chemguide website: http://www.chemguide.co.uk/analysis/uvvisible/beerlambert.html