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 concentration of a sample. Beer's Law is written as:

A=lc
Where, A is the measure of absorbance (no units), is the molar absorptivity (or absorption coefficient), l
is the path length, and c is the concentration. The molar absorptivity is given as a constant and varies for
each molecule. Since absorbance has no units, the units for must cancel out the units of length and
concentration. As a result, has the units: Lmol-1cm-1. The path length is measured in
centimetres. Because a standard spectrometer uses a cuvette that is 1 cm in width, l is always assumed to
equal 1 cm. Since absorption, , and path length are known, the concentration c of the sample can thus be
calculated.
For each wavelength of 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- thats I for intensity.
The intensity of the light passing through the sample cell is also measured for that wavelength- given the
symbol, I. If I is less than Io, then the sample has absorbed some of the light.
Considering the Beer-Lambert Law, the relationship between A (the absorbance) and the two intensities, is
given by:

1.1 Aim and Objective

To learn about spectrophotometry and the factors involved with beers Law.
To determine max (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

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)

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 25ml 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 2.2.1-Volumes required to prepare different solutions

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

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 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.

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 solutions with different
concentration in order to perform the experiment.
The different volumes of the solution are pipetted using a 25ml 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

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 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 max and is used in for the Beer's law plot.

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 volumetric flask; the pipette
should be washed before pipetting another solution of different concentration
The transparent sides of the vials are wiped clean 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)
Wavelength() 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

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

Sample
0.010
0.008
0.006
0.009
0.020
0.043
0.126
0.184
0.208
0.182

Absorbance

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

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

Absorbance

1.2

25 g/L

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 plotted 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

1.8
y = 0.062x - 0.009

1.6
1.4

Absorbance

1.2
1
0.8
0.6
0.4

0.2
0
-0.2

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
Concentration 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

0.9375

0.75

Wavelength() 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

1.5
0 g/L
15 g/l

1
Absorbance

3.75 g/L
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.

11

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

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
Absorbance

1.2
1
0.8
0.6
0.4
0.2
0
0

10

12

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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14

16

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 can observe that the absorbance varies linearly with
the concentration. The data supports this because when the concentration is doubled, the 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.
Beers law can be represented as A= cI
Where:
c is the concentration of the absorbing substance in the solution
I is the optical path length
is the molar absorptivity.

The molar absorptivity is a constant that depends on the 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 Beers Law,
y = m x +c
is equivalent to
A=lc
This shows the slope, m, is equal to the product of 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.

14

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 CuSO 4 absorb more light at higher
wavelengths than at lower wavelengths, corresponding well with the blue colour of the solution.

15

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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