Introduction
Spectrophotometry is one of the most common and efficient means of chemical analysis currently used in estimating the concentration of some chemicals. The technique exploits the radiation absorption property of various chemical substances in determining their concentrations. Spectrophotometry is based on Beer-Lambert Law. Beer-Lambert Law is the linear relationship between the absorbance and concentration of an absorbing species (Nair, 2010; Burrows et al., 2013). The mathematical expression of Beer-Lambert Law is shown in equation1 below:
A=aλ*b*c Equation 1
In the equation 1 shown above, A represents the measured absorbance, a is the absorptivity coefficient which depends on the wavelength, b denotes the path length, and c represents the concentration of the analyte. The Beer-Lambert Law can also be expressed as shown in equation 2 below (Burrows et al, 2013; Pradeep and Ashokreddy, 2012):
A=ɛ*b*c Equation 2
In the equation 2 shown above, ɛ denotes the wavelength-dependent molar absorptivity coefficient whose unit is L mol-1 cm-1. In experiments, measurements are made on the basis of transmittance (T). Transmittance is defined as shown in the formula below (Atkins, De Paula, and Friedman, 2013; Ramakrishnan, Prasannan, and Rajan, 2001):
T=I/Io Equation 3
Where I refers to the light intensity that has passed through the sample while Io is the light intensity before it passes through the sample. When light enters the sample, its intensity reduces because the analyte in the sample absorbs some energy of specific wavelength.
Spectrophotometer is the apparatus used in conducting spectrophotometric analysis of chemical substances. The apparatus is fitted with a lamp that provides the source of light, a diffraction grating which splits light into its constituent wavelengths and the detector that measures the transmittance and absorbance of the sample. When the lamp is lit, the beam of light it emits strikes the diffraction grating, which then splits the light into its constituent wavelengths and reflect it. The preferred wavelength is selected by rotating the grating to allow only specific wavelength to reach the exit slit. From the exit slit, the beam passes through the sample where it interacts with the components of the sample before exiting. The exiting light strikes the detector which then senses the transmittance of the analyte in the sample and displays this information digitally.
This experiment was intended to estimate the concentration of copper sulphate in two unknown solutions.
Results
A set of five calibration solutions were prepared. The concentrations of the calibration solutions are shown in table 1 below:
The concentrations of copper sulphate in the undiluted solution 1 and 2 were found to be 104 and 54 respectively. The concentrations of copper sulphate in the unknown solutions are shown in table 2 below:
The calibration plot showed a linear relationship between absorbance and the concentration of copper sulphate. The slope of the graph is 0.0124 as shown in the figure. The graph is shown in figure 1 below:
Figure 1: Calibration graph
Sample calculations
The concentration of copper sulphate in measured sample 1 was determined as shown below:
Copper sulphate=Measured Absorbance+regression inteceptregression slope
Copper sulphate=0.953+0.0050.0124=0.6730.0124=77.26=77.26 mmolL-1
The standard deviation of sample 1 was estimated as shown in the calculation below:
so=syxregression slope1m+1n+yo-ȳregression slope2ixi- x212
so=0.01170.01212+17+1.2598-0.51170.00532* 4083.333312
so=0.01170.0127.165112
so=0.975*7.165112
so=0.975*2.6768=2.6099mmol L-1
The standard deviation of sample 2 was estimated as shown in the calculation below:
so=syxregression slope1m+1n+yo-ȳregression slope2ixi- x212
so=0.01170.01212+17+0.9994-0.51170.00532* 4083.333312
so=0.01170.0124.894812
so=0.975*4.894812
so=0.975*2.2124=2.1571mmol L-1
Discussion
In the experiment, the concentration of copper sulphate in the unknown samples was determined as follows. First, calibration solutions were made and a calibration graph of absorbance against concentration was plotted. The concentrations of the two unknown solutions, 1 and 2, were then determined by measuring the absorbance of the two solutions and using the equation of the line of best fit of the calibration graph to calculate the concentrations. Further calculations were done to estimate the concentration of the two unknown undiluted solutions.
Beer-Lambert law is the linear relationship between the concentration of an absorbing species and absorbance. The law is instrumental in determining the concentration of substances that absorb light energy. Indeed, it is the basis on which spectrophotometer operates. Beer-Lambert law is expressed mathematically as shown below:
A=aλ*b*c
Where A represents absorbance, a represents the absorptivity coefficient, b represents the path length, and c represents the concentration of the analyte. Based on the Beer-Lambert law shown above, the absorbance is directly proportional to the concentration of the analyte in the sample. Consequently, a graph of absorbance against concentration should be a straight line that passes through the origin. The gradient of the graph, on the other hand, should be equivalent to the product of the path length, b, and the absorptivity coefficient, a. In the experiment, the calibration graph (graph of absorbance against concentration) produced had a y-intercept of -0.0053 and a gradient of 0.0124. Based on the equation of the line of the calibration graph, the concentration of sample 1 before dilution was found to be 100mmolL-1. On the other hand, the concentration of sample 2 before dilution was found to be an average of 81mmolL-1. After dilution, the concentrations of sample 1 and sample 2 were found to be 76 and 41respectively.
Even though many efforts were made to ensure that accurate results are achieved, certain errors were detected in the results. The errors could have originated from various sources. Some of the possible sources of errors could be the following: contamination of the samples as a result of being exposed during the experiment, the possible presence of fluorescent substances in the sample, and changes in the temperature and humidity conditions. The errors possibly partly accounted for the y-intercept noted in the experiment. Dilution was also found to propagate the errors in the experiment.
Regardless of the errors noted in the experiment, the experiment achieved all its objectives. The concentrations of the original samples were successfully determined even though they were not accurate due to errors. Besides, the students gained many insights into the various techniques of conducting a laboratory experiment. For example, the students learned skills such as pipetting. The students also had the opportunity to test Beer-Lambert law. In conclusion, the experiment was successfully conducted.
References
Atkins, P. W., De Paula, J., & Friedman, R. (2013). Physical chemistry: quanta, matter and change.
Burrows, A., Holman, J., Parsons, A., Pilling, G. and Price, G., (2013). Chemistry³: Introducing Inorganic, Organic and Physical Chemistry. Oxford University Press.Top of Form
Ramakrishnan, S., Prasannan, K. G., & Rajan, R. (2001). Textbook of medical biochemistry. Hyderabad, Orient LongmanBottom of Form
Nair, A.J., (2010). Comprehensive Biotechnology XI. Firewall Media.Top of Form
Pradeep, T., & Ashokreddy, A. (2012). A textbook of nanoscience and nanotechnology. New Delhi, Tata McGraw Hill Education.
