PRACTICAL 1 : SPECTROPHOTOMETRY TECHNIQUES

GROUP: C1

GROUP MEMBERS:

1. DHANENDRAN A/L RAJASEGARAN A196260

2. MUHAMMAD TAUFIQ ONN BIN MUHAMAD FIRDAUS ONN A194891

3. PAULE KEONG QI YAN A195653

4. ‘AINAA BINTI ALI A196922

Objective
1. To measure the absorbance spectrum of copper (II) sulphate pentahydrate (CuSO4.5H2O )
2. To determine the unknown concentration of CuSO4.5H2O in the test sample solution.
3. To determine the unknown amount of CuSO4.5H2O in the test sample solution

Principles
Spectrophotometry is a technique used in clinical biochemistry to analyze compounds in
small quantities. This technique is used to measure the absorbance of a chemical solution by
measuring the intensity of the beam light passing through the sample solution. Absorbance of the
sample solution is then related to the concentration of the chemical solution. This is because
based on the basic light principle, each particle absorbs or transmits light of specific wavelength.
The instrument used to undergo this technique is the spectrophotometer. This instrument works
by passing through light with specific wavelength to a sample solution. Some of the light energy
will then be absorbed by the sample solution while some will not be absorbed and pass through
the sample solution. That particular light energy is measured to identify the absorbance and thus
the concentration of the solution. The spectrophotometer is a combination of two instruments
which are the spectrometer and a photometer. Spectrophotometer works for producing light with
different wavelengths while the photometer is for measuring the intensity of light. The cuvette
containing the sample solution is placed in between these two instruments in order to undergo
this process. The spectrometry technique used the Beer’s - Lambert Law which relates the
absorbance and the concentration of solution linearly. The mathematical expression of the law
used is;

I0
Absorbance = log 10 = εcƖ
I

where,

Io = Intensity of the incident light

I or I t = Intensity of the transmitted light

ε = Molar extinction coefficient
c = Molar concentration
Ɩ = Length of the light path
Materials

1. Distilled water
2. CuSO4.5H2O solution (20 g/100 mL)

Apparatus

1. 2 Cuvettes
2. 7 Test tubes
3. Test tube holder
4. Micropipette
5. Pipette
6. Pipette Filler
7. 1 Small beaker
8. Spectrophotometer

Method for using spectrophotometry

1.The cuvette is wiped and dried before use.

2. A series of copper (II) sulphate solutions that cover the concentration range sample is made.
3. The wavelength is set to 600 nm using the wavelength control knob.
4. With the cuvette empty and the lid closed, the display is set to 0% transmittance by using the
zero-control knob.
5. The blank cuvette that contains distilled water is wiped with a Kimwipe to remove any
fingerprints and the blank cuvette is placed into the cuvette holder by aligning the guide marks.
The sample compartment lid is closed.
6. The display is adjusted to 100% transmittance by using the transmittance/absorbance control
knob.
7. The cuvette that contains distilled water is removed from the cuvette holder.
8. The display mode is set to absorbance by pressing the mode control key.
9. The copper (II) sulphate solution is put into a cuvette. The cuvette that contains copper (II)
sulphate solution is wiped and placed into the cuvette holder by aligning the guide marks. The
sample compartment lid is closed and the absorbance is recorded.
10. The cuvette that contains copper (II) sulphate solution is removed from the cuvette holder.
11. Steps 9 and 10 are repeated for the different concentration range of copper (II) sulphate
solutions.
12. The concentration of copper (II) sulphate solution in each standard solution is calculated in
mol/L
unit. A calibration curve for absorbance, OD as the y-axis versus concentration of copper (II)
sulphate solution in mol/L as the x-axis is plotted and the unknown concentration of the test
sample of 5 mL copper (II) sulphate solution in mol/L is determined from the graph.
13. The amount of copper (II) sulphate solution in each standard solution is calculated in grams.
A calibration curve for absorbance, OD as the y-axis versus the amount of copper (II) sulphate
solution in grams as the x-axis is plotted and the unknown amount of the test sample of 5 mL
copper (II) sulphate solution in grams is determined from the graph.
Methods

Preparations of CuSO4.5H2O solution

1. A series of standard CuSO4.5H2O solution is made as follows:

Test tubes Blank 1 2 3 4 5 6 Test
(Unknown)

CuSO4,5H2O 0.5 1.0 2.0 3.0 4.0 5.0 -

(mL)
(20 g/100 mL)

Distilled water 5.0 4.5 4.0 3.0 2.0 1.0 - -

(mL)

Test sample X - - - - - - - 5.0

(mL)

Total volume 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
(mL)

OD (600 nm) 0.000 0.066 0.133 0.270 0.396 0.525 0.655 0.268

Concentration of 0.08 0. 16 0.32 0.48 0.64 0.80 0.33

CuSO4,5H2O
(mol/L)

Amount of 0.10 0.20 0.40 0.60 0.80 1.00 0.41

CuSO4,5H2O
(grams)

2. The solution is mixed well and the absorbance is read at 600 nm wavelength.
Calculations

Calculations for the amount of CuSO4.5H2O (grams) and concentration of CuSO4.5H2O in mol/L

Molar(M)= Weight of compound (gram) X 1 = mol/L

Relative molecular weight Volume(L)
of compound
Figure 1.1: Formula for Calculating Concentration of CuSO4.5H2O in mol/L

Note: Molecular weight for CuSO4 .5H2O = 250, 5 ml = 0.005 L

Weight of compound (grams) = Weight of solvent per unit volume (g/mL) x Volume (mL)

Figure 1.2: Formula for Calculating the Amount of CuSO4.5H2O in mol/L

Table 1.1: Concentration of CuSO4.5H2O (mol/L) and Amount of CuSO4.5H2O (grams)

CuSO4 ∙ 5H20 (mL) Amount of CuSO4.5H2O Concentration of CuSO4.5H2O
(20g/100 mL) (grams) (mol/L)

0.5 20 g 0.10 g 1
× 0.5 mL=0.10 g × =0.08 mol/ L
100 mL 250 0.005 L

1.0 20 g 0.20 g 1
× 1.0 mL=0.20 g × =0.16 mol/ L
100 mL 250 0.005 L

2.0 20 g 0.40 g 1
× 2.0 mL=0.40 g × =0.32 mol/ L
100 mL 250 0.005 L

3.0 20 g 0.60 g 1
× 3.0 mL=0.60 g × =0.48 mol/ L
100 mL 250 0.005 L

4.0 20 g 0.80 g 1
× 4.0 mL=0.80 g × =0.64 mol /L
100 mL 250 0.005 L

5.0 20 g 1.00 g 1
× 5.0 mL=1.00 g × =0.80 mol/ L
100 mL 250 0.005 L
Table 1.2: Concentration of CuSO4.5H2O (mol/L) and Absorbance (OD) Value
Concentration of CuSO4.5H2O (mol/L) Absorbance (OD)

0.00 0.000

0.08 0.066

0.16 0.133

0.32 0.270

0.48 0.396

0.64 0.535

0.80 0.655

Unknown 0.262

Figure 1.3: Calibration Curve - Absorbance (OD) Versus Concentration of CuSO 4.5H2O
(mol/L)
Calculation for co ncentration of CuSO4.5H2O (mol/L) in 5mL of test sample

Based on the Beer-Lambert Law, A = εcƖ

Where, A= Absorbance
ε = Molar absorption coefficient (slope)
c = Molar concentration
Ɩ = Optical path length

y 2− y 1
By using the slope equation,
x 2−x 1
y 2− y 1 0.655−0.00
=
x 2−x 1 0.80−0.00
= 0.81875

Therefore, ε = 0.81875

Since the optical path length which is the y-intercept is 0, the unknown concentration of
CuSO4 .5H2O ,
A
c=
ε
0.268
Hence, c =
0.81875
= 0.3273 mol/L
≈ 0.33 mol/L

Table 1.3 Amount of CuSO4.5H2O (g) and Absorbance (OD)

Amount of CuSO4.5H2O (g) Absorbance (OD)

0.00 0.000

0.10 0.066

0.20 0.133

0.40 0.270

0.60 0.396

0.80 0.535
 1.00 0.655

Figure 1.4: Calibration Curve - Absorbance (OD) Versus Amount of CuSO4.5H2O (grams)

Calculation for amount of CuSO4.5H2O (g) in 5mL of test sample

y 2− y 1
By using the slope equation,
x 2−x 1
y 2− y 1 0.655−0.00
=
x 2−x 1 1.00−0.00
= 0.655

Therefore, m = 0.655

By using the equation, y = mx + c , the amount of CuSO4 .5H2O , c = 0

y = mx
y
x=
m
0.268
x=
0.655
x = 0.4092 g
Discussions:

Spectrophotometry is a quantitative measurement that uses photometers to determine the

intensity of a light beam in order to determine the reflection or transmission qualities of materials
as a function of wavelength (Morris, R. 2015). Spectrophotometers monitor modest variations in
optical density that occur at two wavelengths in the visible area with a narrow spectral interval.
Spectrophotometry is frequently used for analysis in a broad range of domains, particularly
biochemistry, to determine enzyme-catalyzed processes, for example. Spectrophotometry is
divided into two types: atomic absorption spectrophotometry and atomic emission
spectrophotometry.

A spectrophotometer is a device that measures the amount of light absorbed (amount of photons)
after passing through a sample solution. Spectrophotometers are divided into UV-visible
spectrophotometers and infrared spectrophotometers based on the wavelength range of the light
source. UV-visible spectrophotometers utilise light in the ultraviolet (185-400 nm) and visible
(400-700 nm) ranges of the electromagnetic radiation spectrum, whereas IR spectrophotometers
use light in the infrared (700-15000 nm) range of the electromagnetic radiation spectrum.

We used the spectrophotometry technique throughout the experiment to compute the unknown
quantity of CuSO4.5H2O, which is 0.3273 mol/L. With the data collected, the quantity of
absorbance is plotted against the concentration of CuSO4.5H2O (mol/L), it is observed that the
amount of light absorbed by solution rises as the concentration of CuSO4.5H2O (mol/L)
increases. Statistically, the absorbance is directly proportional to the concentration of the
solution. It is disciplined with what we learnt about the Beer-Lamberts' Law, which states that
absorbance (A) is proportional to the duration of light traveling through the sample (I) and the
molar concentration of the chemical that absorbs light (C). The straight line in the graph was
estimated via the data points in this experiment using linear regression.

Due to the element of atmospheric differential refraction, the findings in this experiment diverge
somewhat from the theoretical result. If the slit is not aligned with the direction of air refraction,
the result is influenced by relative light losses in low or moderate light dispersion narrow-
aperture spectrophotometry (Hardesty, J. H., & Attili, B.2010). Moreover, a dirty cuvette also
may affect absorbance. This is because less light will reach the sample in a dirty cuvette.
Therefore, the machine will interpret this as more light being absorbed. Thus, we must ensure
that we clean the cuvette properly and avoid to touch the clear part of the cuvette.
Conclusion

From this experiment, we can measure the absorbance of copper (II) sulphate pentahydrate and
determine the unknown concentration of copper (II) sulphate pentahydrate in each standard
solution using the spectrophotometry technique. The graph for absorbance, OD versus
concentration of copper (II) sulphate pentahydrate, shows a linear relationship. We can see that
the optical density increases as the concentration in the solution increases. Therefore based on
the data collected, we can conclude that this experiment follows the Beer-Lambert Law.

Questions

1. How can this technique be applied in the analyses?

Nowadays, spectrophotometry is an important technique which can analyze organic compounds

in pharmaceuticals. UV spectrophotometers are being used to measure the visible regions of UV
light to provide crucial information about the levels of the active substances present in
pharmaceutical compounds, and also to find any impurities. The qualitative analysis using
spectrophotometry methods can be obtained in a short time with accurate results while using
only a small amount of sample quantities. This quick and effective instrumentation has become a
crucial tool within the pharmaceutical industries thanks to its capabilities and economic value. It
has been proven that this application is highly useful in many major forms of organic compounds
to ensure the patient acquires a safe pharmaceutical compound.

Spectrophotometry is a standard technique to measure light absorption or the amount of

chemicals in a solution. It uses a light beam which passes through the sample, and each
compound in the solution absorbs or transmits light over a certain wavelength. The Beer-
Lambert law can be used to analyse the quality of water. This ensures the water that we drink to
be safe because it can recognize toxic levels of additives that will cause harm to our body.
How does spectrophotometers work in the analysing of water quality? Spectrophotometers are
able to measure APHA colour and specific wavelengths of light. This helps to classify the exact
properties of chemical components found in various sources, and provide a precise water quality
analysis. If a water sample contains more contaminant, the spectrophotometer will measure
higher optical density (OD) value, meaning more light is absorbed while passing through the
water sample.
2. What are the limitations of using this technique in analysis?

According to Beer-Lambert Law, limitations of this technique can be caused by scattering of

light due to turbidity of the sample. For spectrophotometry, it is required to prepare
homogeneous solutions with optical density not too low and not too high. Some compounds can
dissolve only in nonpolar solvents thus you cannot use plastic cuvettes. For ultraviolet, glass
cuvettes are not suitable to be used. If there are a lot of compounds with close characteristic
wavelengths in a specimen, they can obstruct each other. The accurate value of molecular weight
of a substance is merely difficult to be identified using infrared spectroscopy, due to the fact that
it does not adhere with Beer-Lambert Law of complexity spectra.

Besides, one other limitation is fluorescence of the sample phosphorescence of the sample
changes in refractive index at high sample concentration, owing to the fact that not all atoms or
molecules have an equal chance to interact with the light beam; the availability of a reactive
component limits the assay.

Next, if the lights applied are not monochromatic, the outcome will deviate from the Beer-
Lambert Law. Furthermore, changes in temperature will have an impact on the outcome. The
breadth of the instrument must also be correctly set up to ensure that no deviation occurs and that
more accurate readings are achieved. Besides, the spectra measurement accuracy of absorption
will be influenced due to decrease in linearity range and the reduction in absorbency of
substance.
References:

Spectrophotometry (2017, Nov 17) Biocompare.com

https://www.biocompare.com/Editorial-Articles/343518-Specs-on-Spectrophotometry/

Spectrophotometry (2022, Jan 4) LibreTexts.com

https://chem.libretexts.org/Bookshelves/
Physical_and_Theoretical_Chemistry_Textbook_Maps/
Supplemental_Modules_(Physical_and_Theoretical_Chemistry)/Kinetics/
02%3A_Reaction_Rates/2.01%3A_Experimental_Determination_of_Kinetics/
2.1.05%3A_Spectrophotometry

Phillips, K. (2015, March 13). UV spectrophotometry identifies compounds in pharmaceuticals.

● https://blog.hunterlab.com/blog/color-pharmaceuticals/qualitative-analysis-how-uv-
spectrophotometry-help-identify-organic-compounds-in-pharmaceuticals/