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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 Io- 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:

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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

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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 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

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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 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.

Concentration

of solution

prepared (g/L)

6.25 3.125 2.08 1.56 1.25

Volume of

solution

pipetted (ml)

25 12.5 8.3 6.25 5

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Table 2.3.1- Volumes required to prepare different solutions

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.

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.

Concentration

of solution

prepared (g/L)

3.75 1.875 1.25 0.9375 0.75

Volume of

solution

pipetted (ml)

25 12.5 8.3 6.25 5

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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 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) 25 6.25 3.125 2.08 1.56 1.25 Sample

Wavelength() nm

400 0.000 0.021 0.036 -0.005 0.027 0.011 0.054 0.010

450 0.000 0.015 0.018 0.005 0.010 0.002 0.052 0.008

500 0.000 0.012 0.015 -0.001 0.008 0.004 0.048 0.006

550 0.000 0.023 0.012 0.001 0.007 0.003 0.040 0.009

600 0.000 0.112 0.038 0.011 0.012 0.006 0.049 0.020

650 0.000 0.349 0.083 0.027 0.017 0.010 0.023 0.043

700 0.000 0.827 0.198 0.085 0.068 0.046 0.066 0.126

750 0.000 1.315 0.301 0.129 0.108 0.068 0.062 0.184

800 0.000 1.555 0.347 0.155 0.115 0.075 0.070 0.208

880 0.000 1.443 0.307 0.142 0.106 0.067 0.060 0.182

A graph of absorbance against wavelength is plotted

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Table 3.1.2- Absorbance values in the range of 800nm-850nm

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.

Concentration g/L 0( Blank) 25 6.25 3.125 2.08 1.56 1.25 Sample

Wavelength nm

800 0.000 1.555 0.347 0.155 0.115 0.075 0.070 0.208

810 0.000 1.560 0.364 0.166 0.128 0.087 0.070 0.213

820 0.000 1.564 0.360 0.165 0.132 0.086 0.089 0.216

830 0.000 1.536

-0.15-0.050.050.150.250.350.450.550.650.750.850.951.051.151.251.351.451.55

350 450 550 650 750 850

Ab

sorb

ance

Wavelength nm

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

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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 Absorbance

0( Blank) 0.000

25 1.564

6.25 0.360

3.125 0.165

2.08 0.132

1.56 0.086

1.25 0.089

Sample 0.216

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

790 795 800 805 810 815 820 825 830 835 840

Ab

sorb

ance

Wavelength nm

0 g/L

25 g/L

6.25 /L

3.125 g/L

2.08 g/L

1.56 g/L

1.25 g/L

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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.

y = 0.062x - 0.009

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 5 10 15 20 25 30

Ab

sorb

ance

Concentration 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 0.9375 0.75

Wavelength() nm

400 0.000 0.018 0.100 -0.013 0.030 -0.011 -0.011

450 0.000 0.001 0.002 -0.004 0.023 -0.003 -0.005

500 0.000 0.009 0.006 -0.004 0.012 -0.005 -0.003

550 0.000 0.034 0.011 0.008 0.031 0.004 0.007

600 0.000 0.129 0.041 0.014 0.029 0.012 0.008

650 0.000 0.395 0.108 0.050 0.039 0.026 0.021

700 0.000 0.900 0.254 0.132 0.098 0.075 0.063

750 0.000 1.394 0.389 0.209 0.157 0.120 0.107

800 0.000 1.615 0.459 0.246 0.185 0.137 0.114

880 0.000 1.463 0.404 0.221 0.163 0.122 0.105

Fig.3.2.1- Graph of absorbance against wavelength.

-0.5

0

0.5

1

1.5

2

350 450 550 650 750 850

Ab

sorb

ance

Wavelength nm

0 g/L

15 g/l

3.75 g/L

1.875 g/L

1.25 g/L

0.9375 g/L

0.75 g/L

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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 0( Blank) 15 3.75 1.875 1.25 0.9375 0.75

Wavelength nm

800 0.000 1.615 0.459 0.246 0.185 0.137 0.114

810 0.000 1.617 0.460 0.238 0.191 0.140 0.118 820 0.000 1.622 0.466 0.248 0.193 0.140 0.123 830 0.000 1.594

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.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

800 810 820 830

0 g/L

15 g/L

3.75 g/L

1.875 g/L

1.25 g/L

0.9375 g/L

0.75 g/L

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Table3.2.3- Values of absorbance for the different concentrations at optimum wavelength

Concentration g/L Absorbance

0 0.000

15 1.622

3.75 0.466

1.875 0.248

1.25 0.193

0.9375 0.140

0.75 0.123

Sample 0.182

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.

y = 0.105x + 0.042

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 2 4 6 8 10 12 14 16

Ab

sorb

ance

Concentration g/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 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 = l c

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.

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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].