dilutions and introduction to spectrophotometry

7/29/2019 Dilutions and Introduction to Spectrophotometry

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Laboratory 2

Dilutions and Introduction to Spectrophotometry

September 15, 2012


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I. Introduction

Dilutions are an essential procedure in the microbiology laboratory, whether oneis 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 solutionand 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 equationfor Beers law is:

(1) A = lc

Where A is absorbance, is an absorptivity constant particular to the material, l is thelength of the path that the light travels through the sample, and c is the concentration ofthe 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 solutionincreases 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 11different dilutions of dye in distilled water and measuring their absorbance using aspectrophotometer, 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 always5mL. 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 wasset 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.


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III. Results

Concentration(% of solute)

TheoreticalAbsorbance

ActualAbsorbance % 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

y = 0.0139x + 0.0827

R2 = 0.9985

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

0 10 20 30 40 50 60 70 80 90 100

Concentration (%)

Absorbance

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 coincideswith 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

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