CHAPTER ONE

INTRODUCTION

1.1 Introduction

Adsorption is classified as a separation technique. In adsorption, molecules of a substance

tend to distribute themselves in between two phases, one solid and the other liquid. Its
importance in industries cannot be overemphasized (Coulson and Richardson, 2002).

The Adsorbent, which is the solid that is highly porous in nature is key in any adsorption
process. It is introduced to selectively carryout the distribution and separation of molecules in
a bulk fluid mixture. Adsorbents are substances with an amorphous or microcrystalline
structure that might be natural or artificial. In terms of sales volume, the most often utilized
ones are activated carbon, molecular sieves, silica gel, and active alumina (Keller et al.,
1987).

Adsorption takes place when forces coming from a neighbouring surface hold molecules that
are diffusing in the fluid phase for a while. Atoms at the surface of a solid contain residual
molecular forces that are not met by surrounding atoms, such as those in the structure's body.
This surface reflects a significant discontinuity in the solid's structure (Perry and Green,
1999).

The only reason some materials are referred to as "adsorbents" is that they can be made in a
very porous form, giving rise to a large internal surface, because these residuals or van der
Waals forces are universal to all surfaces and responsible for withholding molecules (Coulson
and Richardson, 2002).

Material analysis using a UV-spectrophotometer is a technique in chemical analysis for the

characterization and possible identification of pure and mixed substances. While a UV-
spectrophotometer, the phenomenon is UV-spectroscopy. UV-spectroscopy that is
ultraviolent spectroscopy is based on the absorption of light by a sample. Depending on the
amount of light and its wavelength absorbed by the sample, valuable information can be
obtained, such as the purity of the sample. Moreover, the amount of absorbed light is related
to the amount of sample, and thus, quantitative analysis is possible by optical spectroscopy.

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1.2 Aims and Objectives

The aims of this experiment are:

1. To determine the absorbance, transmittance and concentration of liquid solution at

different wavelengths.
2. To determine the effects of impurities on the transmittance, absorbance and
concentration of distilled water at different wavelengths.
3. To determine the absorbance of different concentration of aqueous solution at
constant temperature, the wavelength of light and the path length of light.

1.3 Significance of Study

The study of the principle of UV-spectroscopy is significant in so many ways, some of which
include:

1. To help in identification and classification as a quantitative analytical technique.

2. It aids in analysis of DNA and RNA, Pharmaceutical analysis and some many other
industrial applications.
3. To determine the impurities in a sample solution.
4. To aid in qualitative and quantitative analysis.

CHAPTER TWO

2
 LITERATURE REVIEW

2.1 Uv-spectrophometer

The term "spectrophotometry" refers to any analytical methods used to gather

physicochemical data from samples by absorbing, transmitting, or reflecting the incident
radiant radiation (Porcu, 2018). One of the most popular analytical methods for
characterizing and determining a variety of organic and inorganic chemicals is light
absorption spectroscopy in the ultraviolet and visible region (UV-Vis) (200-800nm) (Porcu,
2018).

Ultraviolet-visible (UV-Vis) spectroscopy is a commonly used technique in many scientific

fields, including chemical research, bacterial culturing, medication identification, and nucleic
acid purity tests, impurity detection and quantitation (Tom, 2021).

Due to its accessibility, ease of use, versatility, and wide range of applications in a number of
fields, including analytical chemistry and biochemistry, the UV-Vis analytical method has
grown significantly in importance and acceptance in several scientific fields throughout the
world (Porcu, 2018).

The amount of discrete wavelengths of UV or visible light that are absorbed by or transmitted
through a sample in contrast to a reference or blank sample is measured by the analytical
technique known as UV-spectroscopy. The sample composition has an impact on this
attribute, which could potentially reveal what is in the sample and at what concentration
(Tom, 2021)

2.2 Measurement Principle

Considering the characteristics of light which is of great importance as this spectroscopy

method depends on its utilization. The energy of light has a fixed value that is inversely
proportional to its wavelength. As a result, shorter light wavelengths carry more energy while
longer ones carry less. To boost electrons in a substance to a higher energy state, which we
can observe as absorption, a precise quantity of energy is required (Tom, 2021).

3
When light passes through a sample solution in a cuvette, a UV spectrophotometer detects the
intensity of the light and compares it to the intensity of the light before the sample as shown
below

Figure 2.1: The main components in a UV-spectrophotometer

2.2.1 The Light source

A stable source that can emit light over a variety of wavelengths is necessary for this light-
based method. It is typical and most common to employ a single xenon lamp as a high
intensity light source for both UV and visible wavelengths. However, compared to tungsten
and halogen lamps, xenon lamps are more expensive and less stable. A tungsten or halogen
lamp is frequently used for visible light in devices with two lamps, while a deuterium lamp is
typically the source of UV light. The light source in the device must swap during the
measurement since it takes two different light sources to scan both the UV and visible
wavelengths. (Tom, 2021).

2.2.2 The Wavelength

The next step is to choose from among the variety of light wavelengths that the light source
emits those that are suitable for the sample type and analyte for detection for sample
inspection. There are several options for doing this which includes: monochromators,
absorption filters, interference filters, cut off filters and band pass filters. (Tom, 2021) of all
these monochromators are mostly used because it separates light into a narrow band of
wavelength and can be used with a filter for the purpose of being more precise.

4
2.2.3 Sample Analysis

A blank sample, a solvent, such as water or alcohol, is poured into a cuvette, a suitable,
transparent, and impermeable container. The light source's light beam travels through the
solvent-filled cuvette and is used as a reference for the sample whose absorbance and
transmittance are to be detected. A detector placed behind the cuvette containing the solvent
then measures and records the intensity of the transmitted light at a certain wavelength
(Cosimo and Haller, 2015)

A light beam generated by the light source passes through the cuvette with the sample after
the sample has been dissolved in the solvent and added. The sample molecules in the solution
partially absorb the light as it travels through the cuvette. The detector then measures the
transmitted light and the transmitted intensity of the sample solution is divided by the
equivalent values of the blank to determine the light intensity at a given wavelength. Finally,
a recorder stores this ratio. The detector is based on semiconductors and photoelectric coating
and then recorded by a computerized system (Tom, 2021).

2.3 Absorbance and Transmittance

A UV-spectrophotometer's detector monitors the light's intensity after it has passed through
the sample solution. The transmitted intensity, I, represents the percentage of light that the
detector has detected.  For example, light at particular wavelength being absorbed, the sample
solution reduces the intensity of the transmitted light. Its value is consequently less than the
initial intensity I0 at the light source (Cosimo and Haller, 2015)

Figure 2.2: Light Intensity, Absorbance and Transmittance

Transmittance T, which has the unit %, is the ratio of the two intensities I to I0

T=I/I0 Equation 2.1

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The primary, but not the only value that UV spectroscopy can determine is the transmittance.
Also, a second value can be determined while recording UV spectra, the absorbance A.
Absorbance represents an additional outcome that is frequently used. It is known as the
transmittance's negative logarithm and has a significant advantage. Absorbance is a
dimensionless value, that is a unitless value (Cosimo and Haller, 2015).

A = −log(T) Equation 2.2

2.4 Lambert-Beer law

Light is dimmed proportionately to sample concentration when it passes through a transparent

cuvette filled with sample solution. In other words, a sample solution that is more
concentrated will absorb more light. A longer cuvette will result in more light being absorbed
because attenuation is related to cuvette length. The attenuation of the light intensity is
proportional to the concentration of the sample solution as well as the length of the cuvette
(Cosimo and Haller, 2015).

By expressing the absorbance, A as a function of concentration and cuvette length, both

components can be summed together. In specifically, the extinction coefficient, concentration
c, and route length, d, are multiplied to create the absorbance, A.

A= e.c.d Equation 2.3

This relationship is called the Lambert-Beer law where the sample concentration c is
expressed in mol/L, the path length d of the cuvette is expressed in cm and the extinction
coefficient e (epsilon) is a sample specific constant describing how much the sample is
absorbing at a given wavelength in L/(cm*mol) or mL/(cm*g).

The sample concentration can be calculated using the obtained absorbance value and the
Lambert-Beer law. Concentration c can be determined from absorbance A as shown below if
the extinction coefficient and the route length d are known (Cosimo and Haller, 2015).

c = A/e.d Equation 2.4

6
 CHAPTER THREE

METHODOLOGY

3.1 Materials

The materials used in carrying out this experiment include:

1. Distilled water
2. Methylene blue

3.2 Apparatus

The apparatus used in this experiment are:

1. Weigh balance: this is an equipment used to determine the quantity (mass) of

methylene blue to be used. Figure 3.1 shows a diagram of a weigh balance.
2. Beaker: a cylindrical laboratory glass ware. It is used to hold the quantity of
methylene blue mixed with distilled water. A typical example of a beaker is Figure
3.2.
3. Spatula: a laboratory equipment used to mix as well as spread the methylene blue.
Figure 3.3 shows a diagram of a spatula.
4. Measuring cylinder: a graduated cylinder used to know the volume (ppm) of
methylene blue and water from stock solution to be used. Figure 3.4 shows a diagram
of a measuring cylinder.
5. Test tube: a laboratory glassware used that contains samples of the various volume of
the stock mixture. Figure 3.5 shows a diagram of a test tube.
6. Volumetric flask: is a piece of laboratory apparatus, a type of laboratory flask that is
calibrated to contain a precise volume and is used to prepare the stock solution. Figure
3.6 below is a diagram of a volumetric flask.
7. UV- spectrophotometer: an analytical equipment that measures the amount of light
absorbed or transmitted through a sample in comparison to a refence sample. Figure
3.7 below is a diagram of a uv-spectrophotometer.

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Table 3.1: Apparatus Used during the Experimentation

S/No Apparatus Model

1 Weigh balance ABUAD, chemical engineering department

laboratory equipment.

2 Beaker ABUAD, chemical engineering department

laboratory equipment.

3 Spatula ABUAD, chemical engineering department

laboratory equipment.

4 Measuring cylinder ABUAD, chemical engineering department

laboratory equipment.

5 Test tube ABUAD, chemical engineering department

laboratory equipment.

6 Volumetric flask ABUAD, chemical engineering department

laboratory equipment.

7 Uv spectrophotometer ABUAD, chemical engineering department

laboratory equipment.

3.3 Diagrams

Figure 3.1: Weigh balance

8
Figure 3.2: Beaker Figure 3.3: Spatula

Figure 3.4: Measuring cylinder Figure 3.5: Test tube

Figure 3.6: Volumetric flask Figure 3.7: Uv-spectrophotometer

9
3.4 Procedures

1. A clean and dry beaker was placed on a weigh balance and 0.1 gram of methylene
blue was weighed.
2. The 0.1 gram of methylene blue was mixed with distilled water in a beaker and
poured into a volumetric flask which was then topped with water up to the 1000ml
mark which would serve as the stock solution.
3. Then 10ppm of a solution was prepared in a 100ml cylinder by mixing 1.0ml of the
stock solution with 99ml of distilled water.
4. The uv-spectrophotometer was turned on and allowed to normalize for about
5minutes.
5. Then the sample chamber of the uv-spectrophotometer was opened and the rack was
pulled out and the cuvette was filled with a reference sample (water).
6. Then the sample chamber was closed and the reference sample was aligned to the rays
of UV light.
7. The transmittance button was pressed and the reading from the indicator was recoded
as 100% and using the mode button to navigate to the absorbance, the reading of 0%
was recorded.
8. Then the 10ppm solution sample prepared was placed in the sample chamber and
aligned with the position of the rays of light and its transmittance and absorbance
value were indicated and recorded.
9. The above step was repeated for the prepared 15ppm, 20ppm, 25ppm and 30ppm
solutions and their corresponding transmittance and absorbance readings indicated
and recorded.

10
 CHAPTER FOUR

RESULTS AND DISCUSSION OF RESULTS

4.1 Results

Transmittance of reference sample = 100%

Absorbance of reference sample = 0

Wavelength of light = 320nm

Table 4.1: Table of Results obtained

Concentration Transmittance (%) Absorbance

(ppm/mg/L)

10.00 64.40 0.192

15.00 58.30 0.234

20.00 57.20 0.242

25.00 53.50 0.272

30.00 46.30 0.334

Aborbance vs concentrati on
0.4
0.35
0.3 f(x) = 0.00644 x + 0.126
0.25
Absorbance

Absorbance
0.2
Linear (Absorbance)
0.15
0.1
0.05
0
5 10 15 20 25 30 35
Concentration

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Figure 4.1: A graph of Absorbance Vs Concentration

4.2 Discussion of Result

After successfully carrying out the experiment by accurately following the methodology, the
results above were obtained. From the data above, it is observed that there exists a directly
proportional relationship between the concentration of a solution (sample) and absorbance.
that is as the concentration increased the absorbance increases. This relationship is supported
by the Absorbance Vs Concentration graph. Also, there exist an inversely proportional
relationship between the concentration of a solution (sample) and transmittance. That is as the
concentration increased the transmittance decreases.

12
 CHAPTER FIVE

CONCLUSION AND RECOMMENDATION

5.1 Conclusion

Following the successful completion of this study and careful examination of the results
obtained it is concluded that the concentration, transmittance and absorbance of a solution
were determined and there exists a relationship between these values. The relationship
between concentration and transmittance and concentration and absorbance is an inversely
proportional relationship and a directly proportional relationship. Hence, the aims of the
experiment were achieved.

5.2 Recommendation

In view of the success of this experiment, a couple of recommendations are made to further
enhance a more accurate result. Firstly, the liquid solution can be treated to remove impurities
so as to prevent the scattering of light caused by the presence of impurities in the absorbing
liquid. Secondly, the sample chamber should be clean and dry and the cuvette free from
finger prints. Thirdly, samples should be prepared according to specifications by avoid
random and parallax errors during measurements and the sample should be homogenous.
Fourthly, the procedure can be repeated and an average of the values used. Lastly, other
samples such as chromium salt can yield better and more accurate results hence
recommended for use.

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 REFERENCES

1. Coulson, J and Richardson, J. (2002): Chemical Engineering Particle Technology and

Separation Process, Fifth Edition, Volume II, Butterworth-Heinemann, Oxford,
1209p.
2. Cosimo, A and Haller, C. (2015): UV/VIS Spectrophotometry -Fundamentals and
Applications. First Edition, Mettler Toledo, Switzerland, 53p.
3. Keller, G. A., Anderson, R. A. and Yon, C. M (1987): Adsorption, Handbook of
Separation Process Technology, R.W. Rousseau ed., Wiley, New York, 664p.
4. Porcu, O. M. (2018): The Importance of UV-Vis Spectroscopy: Application in Food
Products Characterization, 1, 1-4.
5. Perry, R. H. and Green, D. W. (1999): Perry’s Chemical Engineers’ Handbook,
McGraw Hill, New York, 2580p
6. Tom, J. (2021): UV-Vis Spectroscopy: Principle, Strengths and Limitations and
Applications, 1. 3-15.
7. https://www.pharmatutor.org/pharma-analysis/analytical-aspects-of-uv-visible-
spectroscopy/applications.html

14
 APPENDIX

Transmittance of reference sample = 100%

Absorbance of reference sample = 0

Wavelength of light = 320nm

Preparation of 10ppm concentration of sample solution:

x
x 1000 ppm=10 ppm
100 ml

x = 1.0ml

Therefore, 10ppm concentration of sample solution = 1.0ml of stock solution + 99ml of

distilled water.

Preparation of 15ppm concentration of sample solution:

x
x 1000=1 5 ppm
100 ml

x = 1.5ml

Therefore, 15ppm concentration of sample solution = 1.5ml of stock solution + 98.5ml of

distilled water.

Preparation of 20ppm concentration of sample solution:

x
x 1000 ppm=20 ppm
100 ml

x = 2.0ml

Therefore, 20ppm concentration of sample solution = 2.0ml of stock solution + 98ml of

distilled water.

Preparation of 25ppm concentration of sample solution:

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 x
x 1000 ppm=2 5 ppm
100 ml

x = 2.5ml

Therefore, 25ppm concentration of sample solution = 2.5ml of stock solution + 97.5ml of

distilled water.

Preparation of 30ppm concentration of sample solution:

x
x 1000 ppm=30 ppm
100 ml

x = 3.0ml

Therefore, 30ppm concentration of sample solution = 3.0ml of stock solution + 97ml of

distilled water.

Determination of the concentration for 0.354, 0.760 and 0.910 absorbance

Form Lambert beer equation:

A= e.c.d

The concentration can be evaluated as:

c = A/e.d

where c = concentration

A = absorbance

e = extinction coefficient/molar absorptivity

d = l = path length

The product e.L = slope of the calibration curve of the graph of Absorbance Vs Concentration
= 0.0064.

Therefore, concentration c = A/0.0064.

Therefore, for an absorbance of 0.354, the concentration =

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c = 0.354/0.0064 = 55.3125ppm

Therefore, for an absorbance of 0.760, the concentration =

c = 0.760/0.0064 = 118.75ppm

Therefore, for an absorbance of 0.910, the concentration =

c = 0.910/0.0064 = 142.1875ppm

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