Introduction
Serial dilution is an important experimental technique used in the field of science to obtain specific concentrations of a solution. As the name states, it is a series of dilutions used to obtain smaller concentrations of the particular solution so that it is more usable. Dilution factor at each dilution is usually constant and a ten-fold dilution is called a logarithmic dilution. Common uses of this technique are such as obtaining very small amounts of drugs or reagents, which are usually difficult to measure, from highly concentrated solutions to reduce measurement errors as well as diluting bacterial or virus cultures to estimate the concentration of an unknown sample by counting the colonies cultured in diluted samples and then back track the counts to the unknown concentration.
In this experiment, we were provided with stock solutions of Methylene Blue dye with known concentration as well as one sample with an unknown concentration. A similar method to the estimation of bacterial colonies by serial diluting will be used to find the unknown concentration of the sample. The only difference is instead of counting the colonies, we will be using spectrophotometry to measure the light absorbance.
Methods
Concentration of Stock Solution (M) Volume of Stock Used (ml) Volume of Water Used (ml) Total Volume of Solution (ml) Concentration of Final Solution (M)
1.00 1 9 10 0.100
0.10 1 9 10 0.010
0.01 1 9 10 0.001
1.00 3 7 10 0.300
0.30 1 9 10 0.030
0.03 2 18 20 0.003
Serial Dilutions
Table 1: Volume of stock solution and water (diluent) used for each stock concentration during serial dilution to produce solutions of final concentration 0.100M, 0.010M, 0.001M, 0.300M, 0.030M and 0.003M.
A Methylene Blue dye stock solution of 1M had been provided and solutions ranging from 1M to 0.001M as well as 0.3M to 0.003M were prepared using serial dilutions. First, to prepare the 0.1M solution from the 1M stock solution, since the dilution factor is by ten-fold, 1ml of stock solution was added, followed by 9ml of diluent which is distilled water to the 10ml volumetric flask. Then, similar procedure was used to prepare the 0.01M and 0.001M solutions but just with the 0.1M and 0.01M solutions as stock solutions respectively as shown in Table 1. The 0.3M solution, on the other hand, was prepared by adding 3ml of 1M stock solution to 7ml of water since the dilution factor is 3/10. The 0.03M and 0.003M solutions were prepared using a stock volume to water volume used ratio of 1:9 from stock concentrations of 0.3M and 0.03M respectively as seen in Table 1. All the volumetric flasks were gently shook after the dilution to ensure that solutions were evenly mixed.
* Dilution Factor = Volume of Stock Used / (Volume of Stock Used + Volume of Water Used)
* Final Concentration of Solution = Stock Concentration x Dilution Factor
Spectrophotometry
Each solution was transferred to individual cuvettes with separate pastettes to about three quarter full. The cuvettes were then inserted into a spectrophotometer with wavelength set to 663nm, optimum absorbance of Methylene Blue, to measure the absorbance for each concentration. However, since the water is a diluent, there was no need to measure the amount of light absorbed by water in the solution. Hence, before measurement of each sample, we calibrated the spectrophotometer with a “blank” vial containing water also known as ‘zeroing’.
Data Analysis
Graphs were plotted with absorbance against concentrations on both arithmetic and semi-logarithmic graph papers as well as another between absorbance and logs of the concentrations. This was to cross check between graphs and find a more accurate estimation for the unknown concentration. The unknown concentration was then estimated by plotting its absorbance on the graphs and retrace the corresponding concentration from the line or curve.
Results
Figure 1: Line graph on absorbance for different concentration of Methyl Blue solutions in Molarity (M). Red numbers on both axes show the absorbance of unknown solution and its corresponding concentration.
Figure 2: Line graph on a semi-log paper on absorbance for different concentration of Methyl Blue solutions in Molarity (M). Red numbers on both axes show the absorbance of unknown solution and its corresponding concentration.
Figure 3: Figure 2: Line graph on absorbance for different log (concentration of Methyl Blue solutions) in Molarity (M). Red numbers on both axes show the absorbance of unknown solution and its corresponding log (concentration).
A positive relationship was observed between absorbance and concentration of Methyl Blue solution where an increase in the concentration results in an increase in absorbance from all of the graphs. The absorbance of unknown sample (in red) is 1.348 therefore the estimated concentration is 0.642M for the first graph (Figure 1), 0.64M for the second graph (Figure 2) and log (concentration) for the third graph is -0.19M (Figure 3). Further calculation of result from Figure 3 estimated the concentration to be 0.646M. Therefore, an average of the estimations from the three graphs resulted in unknown concentration of 0.643M.
Discussion
Beer-Lambert Law states that there is a linear relationship between absorbance and concentration of solution. This can be seen from Figure 1, where when concentration is 0.3M, absorbance is 0.632 and 0.6M concentration corresponds to absorbance of 1.264 hence an increase in concentration of solution results in an equal increase in absorbance. However, there is a skewness of large value with 1M concentration resulting in 2.076 absorbance while the other values had absorbance of less than 0.690. That is why Figure 2 and 3 were plotted so that the data values were spread out better with the logarithmic scales. Both Figure 2 and 3 showed similar positive relationship between absorbance and concentration however, unlike the linear relationship in Figure 1, there was a gradual increase in absorbance as concentration increases. Various factors may cause this deviation from the Beer-Lambert’s Law such as alteration of refractive index in solutions of high concentrations, instrumental factors including stray light, noise and polychromatic radiation but a prominent factor may be the dimerisation of Methyl Blue in higher concentrations causing a shift in maximum absorbance (Heger et al. 2005) resulting in a rapid increase in absorbance when concentration is greater than 0.1M as shown in Figure 2 which is equivalent to log(concentration) of -1M in Figure 3.
These experimental techniques still serve as a basis to many experiments even in the industrial level however, more specific and accurate procedures have been improvised over the years. An example includes using Sulforhodamine 101 (S101) dye as an indicator for molecules that solubilized in organic solvent dimethylsulfoxide (DMSO) as this allows dilution range up to 10^7 with minimal cumulative errors enabling higher quality dose with reduced compound consumption (Walling et al. 2011). On the other hand, derivative spectrophotometry is an analytical technique for increased sensitivity in analysis of various compounds (Achariya et al. 2010) and Bayesian analysis outperforms the currently standard approach based on inverting an estimated curve (Gelman et al. 2004).
Conclusion
The methods used to find an estimation of the unknown concentration of Methylene Blue dye solution is quite significant since all three graphs estimated about similar estimations with an average of 0.643M however, further improvements can be made using more specific analytical methods to get a more accurate estimation.