Introduction
Concentration refers to the quantity of a given solute or analyte that is present in a given quantity of solution. Measuring the quantity of a solute in a solution requires the use of different techniques. One of these methods employs use of a standard whose concentration is known and an instrument to detect the solute. Standards refer to set of mixtures whose solute and solvent have been accurately weighed or measured to give the correct concentration. The absorbance of the standards can then be measured using a spectrophotometer. A graph indicating the relationship existing between the solute concentration of the standards and their absorbance can then be plotted. The graph that indicates how the absorbance relates to the standard concentrations is referred to as a standard curve (Sutherland, 2014).
The standard curves are, usually, employed in the determination of the concentration values of samples whose concentrations are not unknown using the easily measured ones (Kenyon College, 2004). The spectrophotometer is a simple machine and consists of a sample compartment, source of light, and a detector. By the use of the desired light wavelength, the absorbance of the sample is determined. The beer-lambert relationship is then used to determine the concentration of the analyte. The relationship may be expressed using the equation A = elc, with A representing the absorbance, c is the analyte concentration in mol/liter, e extinction coefficient in liters/(mol.cm), l path length in cm, and (Burns, 2012).
This experiment aimed to determine the unknown concentration of reagents through the use of the standard curve.
Materials and Methods
Materials
The materials used in the experiment were methylene blue stock solution, Reagent A, test tubes and test tube rack, Gilson pipettes and tips, spectrophotometer, standard cuvettes and distilled water.
Method
The standard solutions were prepared using distilled water and the working solution of methylene blue as shown in Table 1 below.
The spectrophotometer was set to measure at a wavelength of 600 nm and distilled water used to zero the spectrophotometer. The absorbance of all the standard solutions was determined to start with the most dilute solution and to rinse the cuvette between measurements. The standard solutions were poured back into the test tubes. Three milliliters of Reagent A were added into the cuvette, and the absorbance measured.
Results
The measured absorbance of the standard solutions and Reagent A was recorded in Table 2 below. The absorbance of the standards increased as the concentration of the standards increased.
Using the above data, a graph of absorbance against concentration was plotted as shown in Figure 1 below.
Figure 1: Relationship between absorbance and concentration of the standards
Using the equation of the linear curve, the concentration of the Reagent A with an absorbance of 0.337 was determined as follows
y=0.0805x - 0.0728
x=y+0.07280.0805
Concentration=0.337+0.07280.0805
=5.09μg/mL
Discussion
The use of the standard curve in the determination of unknown concentration of samples proves to be a very useful tool of analysis. The tool uses a series of standards whose concentration is known to plot a graph of absorbance against concentration. In this experiment, the standards’ absorbance was measured using a spectrophotometer and the measured absorbance used to plot a standard curve. The absorbance of the standards increased as the concentration of the standards increased. The increased absorbance resulted from an increased absorption of light by the methylene blue added to the solution.
The standard curve gave a linear relationship between the absorbance and the concentrations providing an equation for easier determination of the concentration of other samples. The equation successfully determined the concentration of Reagent A to be 5.09µg/mL. The calculated concentration was greater than the known concentration of Reagent A which is 4.680µg/mL. The difference in concentration may have resulted from errors in the preparation of the standards leading to the wrong equation of the curve or in the preparation of the Reagent A.
In conclusion, the experiment aimed to determine the unknown concentration of reagents through the use of the standard curve. The experiment achieved this through the use of dilutions of a concentrated methylene blue to make standard solutions and using the standards to plot a standard curve. The concentration of Reagent A was determined to be 5.09µg/mL using the equation of the line obtained using the standard solutions.
Reference List
Burns, S. (2012). Bio 120 Lab 5: Quantitative Analysis. Retrieved October 12, 2014, from Napa Valley College: http://www.napavalley.edu/people/sburns/Documents/Biol%20120%20Labs/NVC_Bio120_Lab5_quan_analysis.pdf
Kenyon College. (2004). Standard Curves. Retrieved October 12, 2014, from http://biology.kenyon.edu/courses/biol09/standard%20curve/intro.htm
Sutherland, M. (2014). Graphing. Retrieved October 12, 2014, from http://www2.hendrix.edu/biology/CellWeb/Techniques/GRAPhstdcrv.html
