1. Introduction Absorption spectrophotometry is an effective technique in analytical chemistry to determine the concentration of coloured materials in a solution. Beer-Lambert Law (also known as Beer's Law) states that there is a linear relationship between the absorbance and the co ncentration of a sample. Beer's Law is written as:
A= A=ϵlc Where, A is the measure of absorbance (no units), ϵ is is the molar absorptivity (or absorption coefficient), l is the path length, and c is the concentration. The T he molar absorptivity is given as a constant and var ies for each molecule. Since absorbance has no units, the units for ϵ must must cancel out the units u nits of length and concentration. As a result, ϵ has has the units: L·mol-1·cm-1. The path length is measured in centimetres. Because a standard spectrometer uses a cuvett e that is 1 cm in width, l is is always assumed to equal 1 cm. Since absorption, abso rption, ϵ , and path length are known, the concentration c of the sample can thus be calculated. For each wavelength o off light passing through the spectrometer, the intensity of the light passing through the reference cell is measured. This is usually referred to as I o- that’s I for intensity. intensity. The intensity of the light passing through the sample cell is also measured for that wavelength- g iven the symbol, I. If I is less than I o, then the sample has absorbed some of the light. Considering the Beer-Lambert Law, the relationship between A (t he absorbance) and the two intensities, is given by:
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1.1 Aim and Objective
To learn about spectrophotometry and the factors involved with beer’s Law. To determine λmax (maximum wavelength) for Copper (II) Sulphate pentahydrate, Copper Chloride dehydrate and a mixture of the two solution. To find the concentration of the unknown solution.
2. Materials and Apparatus used
2.1 Apparatus used
100ml volumetric flask
Fig2.1.1- 100mL volumetric flask (photo taken on 21st November 2014)
25ml/10ml pipette
50ml beaker
Glass rod
10ml vials
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Fig.2.1.2- vials (photo taken on 21st November 2014)
Electronic Weighing balance
Fig.2.1.3- Electronic balance (photo taken on 21st November 2014)
Hach DR 2500 Spectrophotometer (Wavelength range 365nm-800nm)
Fig.2.1.4-Hach spectrophotometer (photo taken on 21st November 2014)
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2.2 Chemical Reagents used
Copper Chloride Dihydrate
Copper Sulphate Pentahydrate
Distilled water
2.2 Procedures 2.2.1 Copper (II) Sulphate pentahydrate Preparation of Copper sulphate Pentahydrate solution (stock solution)
A 50ml beaker is placed on the electronic balance. The tare is then set to zero. About 2.513g of copper sulphate pentahydrate crystals is then weighed in a dry beaker. Distilled water is then added in the beaker and stirred using a glass rod to dissolve all the crystals. The dissolved solution is then transferred through a filter funnel into a 100ml volumetric flask. The beaker is then rinsed with distilled water and poured in the flask to prevent the lost of any residue in it. More distilled water is added to the volumetric flask up to the mark. The stopper is put in place and the flask is shaken until a homogeneous solution is obtained which have a concentration of 25g/L.
Dilution process
5 other dilutions are done in 100ml volumetric flasks by using the stock solution. The table 2.2.1 shows the different volumes of solution used to prepare the solutions with different concentration in order to perform the experiment. The different volumes of the solution are pipetted using a 25 ml pipette in 6 different 100ml volumetric flasks and distilled water is added up to t he mark. The stopper is put in place and the flask is shaken until a homogeneous solution is obtained.
Table 2.2.1-Volumes re uired to re are different solutions
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Concentration of solution prepared (g/L) Volume of solution pipetted (ml)
6.25
3.125
2.08
1.56
1.25
25
12.5
8.3
6.25
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Obtaining an absorbance spectrum to determine the maximum wavelength
10mL of each diluted solution is pipetted into separate 10mL vials which are labeled carefully to avoid confusion. 10mL of the given sample is also pipetted in a labeled vial. 10ml of distilled water is pipetted and filled in a vial. This is the blank and it is used to zero the spectrophotometer each time the wavelength is changed. 8 vials are obtained with the following concentrations: 25g/L, 6.25g/L, 3.125g/L, 2.083g/L, 1.56g/L, 1.25g/L, 0g/L and sample. The absorbance of each solution in the vials is measured between 400 and 880 nm in increment of 50 nm using the spectrophotometer. Care should be taken to re-zero the spectrophotometer at each wavelength using the blank solution. Once the region from 400 to 880 nm has been measured, the wavelength with the highest absorbance is identified. In increments of 10 nm, two wavelengths below and two wavelengths above the highest absorbance wavelength is chosen. The absorbance at these new wavelengths is recorded. The wavelength with the greatest absorbance values is λ max and is used in for the Beer's law plot.
2.3.2 Copper Chloride Dihydrate Preparation of copper chloride dihydrate solution
A 50ml beaker is placed on the electronic balance. The tare is then set to zero. About 1.5g of copper chloride dihydrate crystals is then measured in a dry beaker. Distilled water is then added in the beaker and stirred using a glass rod to dissolve all the copper chloride dihydrate. The dissolved solution is then transferred through a filter funnel into a 100ml volumetric flask. The beaker is then rinsed with distilled water and poured in the flask to prevent the lost of any left residue in it. More distilled water is added to the volumetric flask up to the mark. The stopper is put in place and the flask is shaken until a homogeneous solution is obtained which have a concentration of 15g/L.
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Dilution process
5 other dilutions are done in a100ml volumetric flask by using the prepared copper chloride dihydrate solution. Table 2.3.1 shows the different volumes of solution used to prepare the so lutions with different concentration in order to perform the experiment. The different volumes of the solution are pipetted using a 25 ml pipette in 6 different 100ml volumetric flasks and distilled water is added up to the mark. The stopper is put in place and the flask is shaken until a homogeneous solution is obtained.
Table 2.3.1- Volumes required to prepare different solutions
Concentration of solution prepared (g/L) Volume of solution pipetted (ml)
3.75
1.875
1.25
0.9375
0.75
25
12.5
8.3
6.25
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Obtaining an absorbance spectrum to determine the maximum wavelength
10mL of each diluted solution is pipetted into separate 10mL vials which are labeled carefully to avoid confusion. 10mL of the given sample is also pipetted in a labeled vial. 10ml of distilled water is pipetted and filled in a vial. This is the blank and it is used to zero the spectrophotometer each time the wavelength is changed. 8 vials are obtained with the following concentrations: 15g/L, 3.75g/L, 1.875g/L, 1.25g/L, 0.9375g/L, 0.75g/L, 0g/L and sample. The absorbance of each so lution in the vials is measured between 400 and 880 nm in increment of 50 nm using the spectrophotometer. Care should be taken to re-zero the spectrophotometer at each wavelength using the blank solution. Once the region from 400 to 880 nm has been measured, the wavelength with the highest absorbance is identified. In increments of 10 nm, two wavelengths below and two wavelengths above the highest absorbance wavelength is chosen. The absorbance at these new wavelengths is recorded. The wavelength with the greatest absorbance values is λ max and is used in for the Beer's law plot.
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2.4 Precautions taken
Before using the vials, care should be taken that they are clean and dry. Care should be taken that each time a solution is pipetted from one vo lumetric flask; the pipette should be washed before pipetting another solution of different concentration The transparent sides of the vials are wiped c lean of any fingerprints before inserting them in the spectrophotometer.
3. Results 3.1 Copper (II) Sulphate pentahydrate 3.1.1 Selecting the best wavelength on the spectrophotometer The absorbance values for the different solutions of different concentration at different wavelengths are recorded as follows: Table 3.1.1-Readings from the spectrophotometer
Concentration g/L 0 ( Blank) avelength(λ) nm 400 0.000 450 0.000 500 0.000 550 0.000 600 0.000 650 0.000 700 0.000 750 0.000 800 0.000 880 0.000
25 0.021 0.015 0.012 0.023 0.112 0.349 0.827 1.315 1.555 1.443
6.25
3.125
0.036 0.018 0.015 0.012 0.038 0.083 0.198 0.301 0.347 0.307
-0.005 0.005 -0.001 0.001 0.011 0.027 0.085 0.129 0.155 0.142
A graph of absorbance against wavelength is plotted
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2.08 0.027 0.010 0.008 0.007 0.012 0.017 0.068 0.108 0.115 0.106
1.56 0.011 0.002 0.004 0.003 0.006 0.010 0.046 0.068 0.075 0.067
1.25 0.054 0.052 0.048 0.040 0.049 0.023 0.066 0.062 0.070 0.060
ample 0.010 0.008 0.006 0.009 0.020 0.043 0.126 0.184 0.208 0.182
e c n a b r o s b A
1.55 1.45 1.35 1.25 1.15 1.05 0.95 0.85 0.75 0.65 0.55 0.45 0.35 0.25 0.15 0.05 -0.05 -0.15 350
0 g/L 25 g/L 6.25 g/L 3.125 g/L 2.08 g/L 1.56 g/L 1.25 g/L
450
550
650
750
850
Wavelength nm Fig.3.1.1- Graph of absorbance against wavelength.
From Figure 3.1.1 it can be seen that the absorbance is at its maximum in the range of 800nm-850nm. Hence another set of readings is recorded for this range of wavelength and another graph is plotted. Table 3.1.2- Absorbance values in the range of 800nm-850nm
Concentration g/L Wavelength nm 800 810 820 830
0( Blank) 0.000 0.000 0.000 0.000
25
6.25 1.555 1.560 1.564 1.536
3.125
0.347 0.364 0.360
0.155 0.166 0.165
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2.08 0.115 0.128 0.132
1.56 0.075 0.087 0.086
1.25 0.070 0.070 0.089
Sample 0.208 0.213 0.216
1.8 1.6 1.4 0 g/L
1.2 e c n a b r o s b A
25 g/L
1
6.25 /L
0.8
3.125 g/L 0.6
2.08 g/L
0.4
1.56 g/L
0.2
1.25 g/L
0 790
795
800
805
810
815
820
825
830
835
840
Wavelength nm Fig.3.1.2- Graph of absorbance v/s wavelength
From the second graph, it can be seen that the maximum absorbance is recorded at 820 nm.
3.1.2 Preparing the calibration curve Using the optimum wavelength which is 820nm, a graph of absorbance against concentration is p lotted to determine the concentration of Copper (II) Sulphate pentahydrate in the sample. Table 3.1.3- Absorbance at 820 nm
Concentration g/L 0( Blank) 25 6.25 3.125 2.08 1.56 1.25 Sample
Absorbance 0.000 1.564 0.360 0.165 0.132 0.086 0.089 0.216
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1.8 y = 0.062x - 0.009
1.6 1.4 1.2 e c n a b r o s b A
1 0.8 0.6 0.4 0.2 0 -0.2
0
5
10
15
20
25
30
Concentration g/L Fig.3.1.3Linear graph of absorbance against concentration
Calculations: Replacing the value of the absorbance of the sample in the equation of the line: 0.216=0.062x-0.009 x= 3.629 g/L
Therefore, the concentration of Copper (II) Sulphate Pentahydrate in the sample is 3.629 g/L.
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3.2 Copper Chloride Dihydrate 3.2.1 Selecting the best wavelength on the spectrophotometer The absorbance values for the different solutions of different concentration at different wavelengths are recorded as follows: Table 3.2.1-Values of absorbance at different wavelengths centration g/L 0( Blank) 15 3.75
1.875
1.25
0.013 0.004 0.004 0.008 0.014 0.050 0.132 0.209 0.246 0.221
0.030 0.023 0.012 0.031 0.029 0.039 0.098 0.157 0.185 0.163
375
0.75
velength(λ) nm 400 450 500 550 600 650 700 750 800 880
0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
0.018 0.001 0.009 0.034 0.129 0.395 0.900 1.394 1.615 1.463
0.100 0.002 0.006 0.011 0.041 0.108 0.254 0.389 0.459 0.404
-0.011 -0.003 -0.005 0.004 0.012 0.026 0.075 0.120 0.137 0.122
-0.011 -0.005 -0.003 0.007 0.008 0.021 0.063 0.107 0.114 0.105
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1.5 0 g/L 15 g/l
1
3.75 g/L e c n a b r o s b A
1.875 g/L 0.5
1.25 g/L 0.9375 g/L 0.75 g/L
0 350 -0.5
450
550
650
750
Wavelength nm
Fig.3.2.1- Graph of absorbance against wavelength.
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850
From Fig.3.2.1 it can be seen that the optimum wavelength lies in the range of 800-850nm. A second set of readings is recorded for this range of wavelength. Concentration g/L Wavelength nm 800 810 820 830
0( Blank)
15
0.000 0.000 0.000 0.000
3.75
1.875
1.25
0.9375
0.75
1.615
0.459
0.246
0.185
0.137
0.114
1.617 1.622 1.594
0.460 0.466
0.238 0.248
0.191 0.193
0.140 0.140
0.118 0.123
1.8 1.6 1.4 0 g/L
1.2
15 g/L 1
3.75 g/L 1.875 g/L
0.8
1.25 g/L
0.6
0.9375 g/L 0.4
0.75 g/L
0.2 0 800
810
820
830
Fig.3.2.2- Graph of absorbance against wavelength in the range of 800-850 nm
From this second graph the optimum wavelength is found to be 820nm. 3.2.2 Preparing the calibration curve A graph of absorbance v/s concentration is plotted at the optimum wavelength and from this graph the concentration for the copper chloride dihydrate in the sample is determined.
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Table3.2.3- Values of absorbance for the different concentrations at optimum wavelength
Concentration g/L 0 15 3.75 1.875 1.25 0.9375 0.75 Sample
Absorbance 0.000 1.622 0.466 0.248 0.193 0.140 0.123 0.182
1.8 y = 0.105x + 0.042
1.6 1.4 1.2 e c n a b r o s b A
1 0.8 0.6 0.4 0.2 0 0
2
4
6
8
10
12
14
Concentration g/L Fig.3.2.3- Graph of absorbance against concentration
Calculations: Replacing the value of absorbance for the sample in the equation of the line: 0.182=0.105x+0.042 0.105x=0.140 x=1.333g/L
Hence the concentration of copper chloride dihydrate is 1.333g/L .
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4. Discussion
The purpose of this experiment is to determine the maximum wavelength and to use that information to calculate the concentration of an unknown solution.
Since a straight line graph is obtained, we ca n observe that the absorbance varies linearly with the concentration. The data supports this because when the concentration is doubled, t he absorbance is doubled and when the concentration is halved, the absorbance is also halved. These two relationships can be combined to yield a general equation called Beer's Law.
Beer’s law can be represented as A= εcI Where: c is
the concentration of the absorbing substance in the solution
is I
the optical path length
ε
is the molar absorptivity.
The molar absorptivity is a constant that depends on t he nature of the absorbing solution system and the wavelength of the light passing through it. The best straight-line fit obtained from the plot has the form y = mx + c Thus, rewriting the line equation in terms of Beer’s Law, y = m x +c is equivalent to A = ε l c
This shows the slope, m, is equal to the product of ε×l and can be used to calculate the concentration of a solution given its absorbance. The straight line graph obtained seems to be almost good since most of the points lie on the line of best fit except for some points.
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From fig.3.1.3, points (3.125, 0.165) and (6 .25, 0.360) are found to be below the line of best fit which may be due to the addition of more distilled water during dilution.
From fig.3.2.3, point (3.75, 0.466) is found to be above the line of best fit which may be due to the addition of less distilled water.
Conclusion: In view of the above results, the purpose of this experiment was to determine the concentration of a colourful solution. The process involved finding the transmittance as well as the absorbance of various concentrations of a sulphate solution. Multiple trials and multiple solutions are done to get a better sense of which solutions absorb certain types of light. Solutions of CuSO4 absorb more light at higher wavelengths than at lower wavelengths, corresponding well with the blue colour of the solution.
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6. References
Absorption Spectrophotometry. 2014. Absorption Spectrophotometry. [ONLINE] Available at: http://www.nfstc.org/pdi/Subject03/pdi_s03_m05_03.htm. [Accessed 05 December 2014].
Spectrophotometry - Chemwiki. 2014. Spectrophotometry - Chemwiki. [ONLINE] Available at: http://chemwiki.ucdavis.edu/Physical_Chemistry/Kinetics/Reaction_Rates/Experimental_Determin ation_of_Kinetcs/Spectrophotometry. [Accessed 02 December 2014].
absorption spectra - the Beer-Lambert Law. 2014. absorption spectra - the Beer-Lambert Law. [ONLINE] Available at: http://www.chemguide.co.uk/analysis/uvvisible/beerlambert.html. [Accessed 05 December 2014].
chemistry. 2014. General chemistry (Michael Mascari). [ONLINE] Available at: https://salve.digication.com/MasteringChemistryMichaelMascari/Conclusion_Discussion7. [Accessed 05 December 2014].
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