KULLIYYAH OF SCIENCE

SPECTROSCOPY
(BSCH 2321)

PRACTICAL 1:
Determination of Caffeine and Acetylsalicylic Acid in an Analgesic
Tablet by Ultraviolet Spectrophotometry

NAME 1. SITI NUR AFIAH BINTI SHEIKH MOKHTAR (2117412)

2. MUHAMMAD AIMAN BIN MOHD RUSHDI (2113049)

COORDINATOR DR. NUR SALIYANA BINTI JUMALI

EXPERIMENT DATE 21 MARCH 2023

INTRODUCTION

Objectives:
1. To measure the absorbance of the tablet solution at maximum absorption wavelength
for both of the components.
2. To calculate and figure out the amount of each component in the tablet.

Adsorption spectra is a branch of spectroscopy which measures the loss of

electromagnetic energy, which is shown as dark lines, of a sample under study after it was
illuminated by a light source (Chu, S. et al, 2023). Adsorption spectroscopy is an analytical
method which is used as means for measuring the adsorption spectra as it interacts with the
sample. The intensity of the adsorption differs depending on the frequency, meaning that the
more energy the particles of a sample adsorb, the larger the intensity of the adsorption.

To calculate the absorptivity of light, a chemical formula called Beer-Lambert Law

was formulated, which states that the adsorptive capacity of a dissolved substance is
relatively proportional to the concentration of the solution (Rafferty, J. P., 2022). The
chemical formula was expressed below,
𝐴 = ε𝑙𝑐
A is the absorbance, ε is the molar extinction coefficient, l is the length of the path that the
light must pass through, while the c is the concentration of a given solution. However, as the
intensity of light changes due to adsorption, interference, and scattering, another equation
was formulated, which is
Δ𝐼 = 𝐼0 − 𝐼𝑇

From the above equation, derivative equations were created. As absorbance is defined
as the amount of light being adsorbed, it was usually calculated as the negative of
transmittance. Transmittance is identified as the part of the light that moves through the other
side of the surface. It can also be defined as a ratio of the intensity of incident light (I0) to the
amount of intensity that passes through the object (I). Thus, it can be denoted as the
following.
𝐼0 1
𝐴 = 𝑙𝑜𝑔10 𝐼𝑇
= 𝑙𝑜𝑔10( 𝑇 ) =− 𝑙𝑜𝑔10𝑇

In this experiment, two components, caffeine and acetylsalicylic acid in the analgesic tablet,
were studied using a double beam spectrophotometer.
MATERIALS AND APPARATUS

Table 1: The materials and apparatuses used.

Materials Apparatuses

1. Methanol 1. Double beam spectrophotometer

2. Caffeine 2. 50 mL Volumetric flask
3. Acetylsalicylic acid 3. 100 mL Volumetric flask
4. Analgesic tablet 4. 5 mL pipette

METHODOLOGY

Preparation of solutions:

1. Caffeine stock solution — 0.024 g of caffeine was dissolved in a 50 mL volumetric

flask using methanol and diluted to the mark.
2. Acetylsalicylic acid (ASA) stock solution — 0.024 g of ASA was dissolved in a 50
mL volumetric flask using methanol and was diluted to the mark.
3. Analgesic sample solution — 0.06~0.09 g of an analgesic tablet was dissolved using
20 mL of methanol in a 50 mL volumetric flask and diluted using methanol to the
mark. Three sample solutions were made using three different tablets.

Procedure:

1. Working standards and samples from the stock solutions were first prepared as
follows;

a. ASA 1 — 0.5 mL ASA stock added and diluted with methanol in a 50 mL

volumetric flask to the mark.
b. ASA 2 — 1.0 mL ASA stock was added and diluted with methanol in a 50
mL volumetric flask to the mark.
c. ASA 3 — 1.5 mL ASA stock was added and diluted with methanol in a 50
mL volumetric flask to the mark.
d. ASA 4 — 2.0 mL ASA stock was added and diluted with methanol in a 50
mL volumetric flask to the mark.
e. ASA 5 — 2.5 mL ASA stock was added and diluted with methanol in a 50
mL volumetric flask to the mark.
f. Caffeine 1 — 0.5 mL Caffeine stock was added and diluted with methanol in
a 50 mL volumetric flask to the mark.
g. Caffeine 2 — 1.0 mL Caffeine stock was added and diluted with methanol in
a 50 mL volumetric flask to the mark.
h. Caffeine 3 — 1.5 mL Caffeine stock was added and diluted with methanol in
a 50 mL volumetric flask to the mark.
 i. Caffeine 4 — 2.0 mL Caffeine stock was added and diluted with methanol in
a 50 mL volumetric flask to the mark.
j. Caffeine 5 — 2.5 mL Caffeine stock was added and diluted with methanol in
a 50 mL volumetric flask to the mark.
k. Sample 1 — 5.0 mL of sample solution 1 was added and diluted with
methanol in a 100 mL volumetric flask to the mark.
l. Sample 2 — 5.0 mL of sample solution 1 was added and diluted with
methanol in a 100 mL volumetric flask to the mark.
m. Sample 3 — 5.0 mL of sample solution 1 was added and diluted with
methanol in a 100 mL volumetric flask to the mark.

2. The spectra for ASA 5 and Caffeine 5 were run, after which the wavelength of the
maximum absorbance (λmax) for each working standard was determined by scanning
from 200 – 400 nm using methanol as the blank.
3. Using the λmax obtained for both ASA 5 and Caffeine 5, the absorbance of standard
solutions ASA 1 – ASA 4 and standard solutions Caffeine 1 – Caffeine 4 was
determined.
4. Then using the two λmax wavelengths obtained for ASA 5 and Caffeine 5, the
absorbance for all samples 1 – 3 solution was measured and recorded.
 DATA AND RESULTS

Table 2: The concentration and absorbance for all three samples with the working standard
solutions.
Working Caffein stock ASA stock Total volume Concentration Absorbance
Standard added (mL) added (mL) diluted with (M)
Solution methanol
(mL) At 𝜆max = At 𝜆max =
272 nm 298 nm

ASA 1 - 0.5 50 2.66×10-5 0.105 0.156

ASA 2 - 1.0 50 5.32×10-5 0.079 0.244

ASA 3 - 1.5 50 7.98×10-5 0.084 0.332

ASA 4 - 2.0 50 1.06×10-4 0.090 0.448

ASA 5 - 2.5 50 1.33×10-4 0.107 0.568

Caffein 1 0.5 - 50 2.47×10-5 0.266 0.032

Caffein 2 1.0 - 50 4.94×10-5 0.427 -0.002

Caffein 3 1.5 - 50 7.41×10-5 0.679 0.031

Caffein 4 2.0 - 50 9.88×10-5 0.924 0.056

Caffein 5 2.5 - 50 1.24×10-4 1.088 0.029

Sample 1 5 mL of sample solution 1 100 2.23×10-4 1.080 0.444

Sample 2 5 mL of sample solution 2 100 1.58×10-4 1.476 0.094

Sample 3 5 mL of sample solution 3 100 5.95×10-4 1.494 0.491

 Figure 1: Graph of absorbance versus concentration (M) of ASA at 𝜆max of ASA and
Caffeine.

Figure 2: Graph of absorbance versus concentration (M) of Caffeine at 𝜆max of ASA and
Caffeine.

Table 3: The molar absorptivity for each component.

Component Molar absorptivity

At 𝜆max = 272 nm At 𝜆max = 298 nm

ASA 55.556 3807.4

Caffeine 8631.5 208.81

 Table 4: The weight percent of ASA in the original analgesic tablet for each sample.
Sample ASA

Concentration Concentration Mass in Mass in original Weight percent of

in standard before dilution samples (g) analgesic tablet (g) ASA in original
solution (M) (M) analgesic tablet

1 1.0979×10-4 2.1958×10-3 19.828×10-3 0.160 32.99%

(0.06g of
Anarex)

2 1.5326×10-5 3.0652×10-4 2.7679×10-3 0.009 4.50%

(0.06g of
Celebrex)

3 1.1951×10-4 2.3902×10-3 21.5835×10-3 0.120 24.00%

(0.09g
PCM)

Table 5: The weight percent of Caffeine in original analgesic tablet for each sample.
Sample Caffeine

Concentration Concentration Mass in Mass in original Weight percent of

in standard before dilution samples (g) analgesic tablet (g) Caffeine in original
solution (M) (M) analgesic tablet

1 1.2442×10-4 2.4884×10-3 24.1611×10-3 0.195 40.21%

(0.06g of
Anarex)

2 1.7090×10-4 3.4180×10-3 3.3187×10-2 0.111 55.50%

(0.06g of
Celebrex)

3 1.7232×10-4 3.4464×10-3 3.3463×10-2 0.186 37.20%

(0.09g
PCM)
DISCUSSION

From this experiment, it is observed that the concentration of ASA And Caffeine in an
Analgesic Tablet could be determined by using ultraviolet spectrophotometry. Besides, this
experiment was able in analysing the spectrophotometric of a two-component system.
Moreover, the molar absorptivity for both ASA and Caffeine at their’s maximum
wavelengths could also be identified based on the calibration curve. Furthermore, the amount
of each component in the original analgesic tablet could be calculated based on the molar
absorptivity obtained. There are three Analgesic tablets have been investigated in this
experiment which is Annarex, Celebrex and PCM.

Initially, the standard solution of ASA and Caffeine has been prepared from its’s stock
solutions. There are five concentrations used for each standard solution and the concentration
used can be referred to in Table 2. Besides, the calculation to determine the concentration of
each standard solution can be referred to in Table 6. This standard solution was useful in
determining the molar absorptivity at their maximum wavelength. The maximum wavelength
for ASA and Caffeien were identified to be 298nm and 272nm respectively. Afterwards, the
absorbance of all samples was determined followed by all the standard solutions. All the
results of absorbance can be referred to in Table 2. Besides, it is observed that the absorbance
increase as the concentration increase. All of the absorbance and concentration obtained was
then proceeded with the calibration curve by plotting the graph of absorbance versus
concentration as in Figure 1 and 2.

Moreover, the graph in Figure 1 interpreted that the linearity is good for the
absorbance at 𝜆max = 298nm, meanwhile, at 𝜆 = 272nm, the linearity is poor. The same things
occurred on the graph in Figure 2, but it happened vice versa, where the linearity is good for
the absorbance at 𝜆max = 272nm, meanwhile, it is poor at 𝜆max = 298nm. The linearity of both
graphs shown can be referred to based on the value of the coefficient of determination, R2. As
the R2 value is close to 1, the linearity was good, meanwhile, as the R2 value is further from 1,
the linearity is poor. This is due to the component itself. The absorbance of ASA was good at
𝜆max = 298nm since it can absorb the light at those wavelengths. The same goes for Caffeine
but at 𝜆max = 272nm.

From these graphs also the molar absorptivity for both ASA and Caffeine at both
maximum wavelengths has been distinguished. The molar absorptivity for ASA at 272nm
and 298nm was observed to be 55.556 and 3807.400 respectively. Meanwhile, the molar
absorptivity for Caffeine at 272nm and 298nm were identified to be 8631.5 and 208.81
respectively. The molar absorptivity of each component was obtained from the gradient of the
plotted graph. Afterwards, the molar absorptivity for each component at both maximum
wavelengths was used in determining the concentration of each component in each sample.
For sample 1 of 0.06 g of Annarex, it is observed that the concentration of ASA and Caffeine
was found to be 1.0979×10-4 M and 1.2442×10-4 M, respectively. In sample 2 of 0.06 g of
Celebrex, it is identified that the concentration of ASA and Caffeine was found to be 1.5326×
10-5 M and 1.7090×10-4 M, respectively, meanwhile, for sample 3 for PCM, it is observed
that the concentration of ASA and Caffeine was found to be 1.1951×10-4 M and 1.7232×10-4
M. From the concentration of ASA and Caffeine of these samples, the weight percent of each
component could be determined in the original analgesic tablet. The weight percent of ASA
and Caffeine in the 485mg Annarex was identified to be 32.99% and 40.21% respectively.
Besides, the weight percent of ASA and Caffeine in the 500mg Celebrex was identified as
4.50% and 55.50% respectively. Meanwhile, the weight percent of ASA and Caffeine in the
200mg PCM was observed to be 24.00% and 37.20%, respectively. All the calculations can
be referred to in Table 6 to Table 8.

CONCLUSION

In conclusion, this experiment has achieved its objectives. The measurement of

absorbance for each component has been determined at the maximum absorption wavelength.
The amount of each component in the sample's standard solution and the original analgesic
tablet has been calculated and determined perfectly based on the calculation. Besides, the
molar absorptivity of both components has been determined from the calibration curve. Last
but not least, the weight percent of each component also has been determined.

QUESTION

1. Instead of methanol, would benzene be a suitable solvent for the analysis?

➢ Benzene would not be a suitable solvent for replacing methanol, for the analysis.
This is because benzene is a non-polar solvent while both caffeine and ASA are
polar molecules. As ‘like dissolves like’, only polar solvent can properly dissolve
both caffeine and ASA.

2. If a pharmaceutical mixture has three components with different maximum

absorbance, comment on the possibility of determining all three components
simultaneously.

➢ The possibility of determining all three components with different maximum

absorbance simultaneously depends on several factors, such as the absorbance
maximum of each component, the concentration range of the components and the
availability of suitable analytical methods. It is deemed possible to determine all
three components simultaneously if each of their absorbance maxima is separated at
its own unique wavelength at which it absorbs light. However, if one or more
components are present in very low concentrations, it may be very difficult to detect
them accurately in the presence of other components that absorb strongly at similar
wavelengths. Thus, it would sometimes be necessary to use a pre-concentration step
or a more sensitive analytical technique, such as liquid chromatography or mass
spectrometry.
 3. Would it be more desirable to use spectra-grade methanol for the blank solution?
Justify your answer.

➢ Yes, it would be more desirable to use spectra-grade methanol for the blank solution.
This is due to the fact that spectra-grade methanol is a polar solvent that has a higher
purity level than other grades of methanol and is specifically designated to be used
in analytical applications which require a very high degree of purity and consistency
in the chemical properties of the solvent. Spectra-grade methanol can help to
improve the accuracy and precision of the UV-Vis spectrophotometer analysis,
besides providing a better baseline for the calibration curves to improve the
sensibility and detection limit of the method used.

CALCULATION

Table 6: The calculation for the concentration of each working standard solution.
Working No. Number of moles Concentration (M)
Standard Solution (mol)

1 −4
1.332×10 𝑚𝑜𝑙
= 2.66×10-3 M
0.05 𝐿

Diluted 0.5 mL of ASA 1 in 50 mL methanol,

−3
2.66×10 𝑀 × 0.0005 𝐿
0.024 𝑔 = 2.66×10-5 M
ASA = 1.332×10-4 0.05 𝐿
180.16 𝑔/𝑚𝑜𝑙
2 −4
1.332×10 𝑚𝑜𝑙
= 2.66×10-3 M
0.05 𝐿

Diluted 1.0 mL of ASA 2 in 50 mL methanol,

−3
2.66×10 𝑀 × 0.0010 𝐿
= 5.32×10-5 M
0.05 𝐿

3 −4
1.332×10 𝑚𝑜𝑙
= 2.66×10-3 M
0.05 𝐿

Diluted 1.5 mL of ASA 3 in 50 mL methanol,

−3
2.66×10 𝑀 × 0.0015 𝐿
= 7.98×10-5 M
0.05 𝐿

4 −4
1.332×10 𝑚𝑜𝑙
= 2.66×10-3 M
0.05 𝐿

Diluted 2.0 mL of ASA 4 in 50 mL methanol,

−3
2.66×10 𝑀 × 0.0020 𝐿
= 1.06×10-4 M
0.05 𝐿
 5 −4
1.332×10 𝑚𝑜𝑙
= 2.66×10-3 M
0.05 𝐿

Diluted 2.5 mL of ASA 5 in 50 mL methanol,

−3
2.66×10 𝑀 × 0.0025 𝐿
= 1.33×10-4 M
0.05 𝐿

1 −4
1.236×10 𝑚𝑜𝑙
= 2.47×10-3 M
0.05 𝐿

Diluted 0.5 mL of Caffeine 1 in 50 mL methanol,

−3
2.47×10 𝑀 × 0.0005 𝐿
0.024 𝑔 = 2.47×10-5 M
Caffeine = 1.236×10-4 0.05 𝐿
194.19 𝑔/𝑚𝑜𝑙
2 −4
1.236×10 𝑚𝑜𝑙
= 2.47×10-3 M
0.05 𝐿

Diluted 1.0 mL of Caffeine 2 in 50 mL methanol,

−3
2.47×10 𝑀 × 0.0010 𝐿
= 4.94×10-5 M
0.05 𝐿

3 −4
1.236×10 𝑚𝑜𝑙
= 2.47×10-3 M
0.05 𝐿

Diluted 1.5 mL of Caffeine 3 in 50 mL methanol,

−3
2.47×10 𝑀 × 0.0015 𝐿
= 7.41×10-5 M
0.05 𝐿

4 −4
1.236×10 𝑚𝑜𝑙
= 2.47×10-3 M
0.05 𝐿

Diluted 2.0 mL of Caffeine 4 in 50 mL methanol,

−3
2.47×10 𝑀 × 0.0020 𝐿
= 9.88×10-5 M
0.05 𝐿

5 −4
1.236×10 𝑚𝑜𝑙
= 2.47×10-3 M
0.05 𝐿

Diluted 2.5mL of Caffeine 5 in 50 mL methanol,

−3
2.47×10 𝑀 × 0.0025 𝐿
= 1.24×10-4 M
0.05 𝐿
 1 0.060 𝑔 −4
2.227×10 𝑚𝑜𝑙
= 2.227× = 4.45×10-3 M
269.388 𝑔/𝑚𝑜𝑙 0.05 𝐿
10-4
Diluted 5.0 mL of Sample 1 in 100 mL methanol,
Samples −3
4.45×10 𝑀 × 0.005 𝐿
= 2.23×10-4 M
0.10 𝐿

2 0.060 𝑔 −4
1.573×10 𝑚𝑜𝑙
= 1.573× = 3.15×10-3 M
381.373 𝑔/𝑚𝑜𝑙 0.05 𝐿
10-4
Diluted 5.0 mL of Sample 2 in 100 mL methanol,
−3
3.15×10 𝑀 × 0.005 𝐿
= 1.58×10-4 M
0.10 𝐿

3 0.090 𝑔 −4
5.954×10 𝑚𝑜𝑙
= 5.954× = 1.19×10-2 M
151.163 𝑔/𝑚𝑜𝑙 0.05 𝐿
10-4
Diluted 5.0 mL of Sample 3 in 100 mL methanol,
−2
1.19×10 𝑀 × 0.005 𝐿
= 5.95×10-4 M
0.10 𝐿

k = ε, molar absorptivity
kASA: kCaffeine:

At 272 nm = 55.556 At 272 nm = 8631.5

At 298 nm = 3807.4 At 298 nm = 208.81

To determine the concentration of ASA and caffeine in the samples,

A(𝜆) = kASA(𝜆) CASA + kCaffeine (𝜆) CCaffeine

Let, kASA = k1, and kCaffeine = k2

Table 7: The calculation to determine the concentration of ASA and Caffeine in all samples.
Samples Calculation

1 - (0.06 g of Annarex) At 𝜆 = 272 nm,

A(272) = k1(272) CASA + k2(272) CCaffeine

1.080 = 55.556 CASA + 8631.5 CCaffeine Eq. (1)

 At 𝜆 = 298 nm,

A(298) = k1(298) CASA + k2(298) CCaffeine

0.444 = 3807.4 CASA + 208.81 CCaffeine Eq. (2)

Solved the mathematical expression of Eq. (1) and Eq. (2),

[ASA] = 1.0979×10-4 M
[Caffeine] = 1.2442×10-4 M

2 - (0.06 g of Celebrex) At 𝜆 = 272nm,

A(272) = k1(272) CASA + k2(272) CCaffeine

1.476 = 55.556 CASA + 8631.5 CCaffeine Eq. (1)

At 𝜆 = 298nm,

A(298) = k1(298) CASA + k2(298) CCaffeine

0.094 = 3807.4 CASA + 208.81 CCaffeine Eq. (2)

Solved the mathematical expression of Eq. (1) and Eq. (2),

[ASA] = 1.5326 ×10-5 M

[Caffeine] = 1.7090 ×10-4 M

3 - (0.09 g of ppm) At 𝜆 = 272nm,

A(272) = k1(272) CASA + k2(272) CCaffeine

1.494 = 55.556 CASA + 8631.5 CCaffeine Eq. (1)

At 𝜆 = 298nm,

A(298) = k1(298) CASA + k2(298) CCaffeine

0.491 = 3807.4 CASA + 208.81 CCaffeine Eq. (2)

Solved the mathematical expression of Eq. (1) and Eq. (2),

[ASA] = 1.1951×10-4 M
[Caffeine] = 1.7232×10-4 M
 Table 8: The calculation for the weight percent of ASA and Caffeine for all three samples.
Samples Calculation

ASA Caffeine

1 Concentration in standard solution Concentration in standard solution

= 1.0979×10-4 M = 1.2442×10-4 M

Concentration before dilution Concentration before dilution

0.100 𝐿 0.100 𝐿
= (1.0979×10-4 M) × ( ) = (1.2442×10-4 M) × ( )
0.005 𝐿 0.005 𝐿
= 2.1958×10-3 M = 2.4884×10-3 M

No of moles No of moles
= (2.1958×10-3 M) × (0.05 L) = (2.4884×10-3 M) × (0.05 L)
= 1.0979×10-4 mol = 1.2442×10-4 mol

Mass in samples Mass in samples

= 1.0979×10-4 mol)(180.16 g/mol) = (1.2442×10-4 mol)(194.19 g/mol)
= 19.828×10-3 g = 24.1611×10-3 g

Mass in original analgesic tablet Mass in original analgesic tablet

0.485 𝑔 0.485 𝑔
=( )×(19.828×10-3 g) =( )×(24.1611×10-3 g)
0.06 𝑔 0.06 𝑔
= 0.160 g = 0.195 g

Weight percent of ASA in original analgesic Weight percent of ASA in original analgesic
tablet tablet
0.160 𝑔 0.195 𝑔
=( )×(100%) =( )×(100%)
0.485 𝑔 0.485 𝑔
= 32.99% = 40.21%

2 Concentration in standard solution Concentration in standard solution

= 1.5326×10-5 = 1.7090×10-4 M

Concentration before dilution Concentration before dilution

0.100 𝐿 0.100 𝐿
= (1.5326×10-5 M) × ( ) = (1.7090×10-4 M) × ( )
0.005 𝐿 0.005 𝐿
= 3.0652×10-4 M = 3.4180×10-3 M

No of moles No of moles
= (3.0652×10-4 M) × (0.05 L) = (3.4180×10-3 M) × (0.05 L)
= 1.5326×10-5 mol = 1.709×10-4 mol

Mass in samples Mass in samples

= (1.5326×10-5 mol)(180.16 g/mol) = 1.709×10-4 mol)(194.19 g/mol)
= 2.7679×10-3 g = 3.3187×10-2 g
 Mass in original analgesic tablet Mass in original analgesic tablet
0.200 𝑔 0.200 𝑔
=( )×(2.7679×10-3 g) =( )×(3.3187×10-2 g)
0.06 𝑔 0.06 𝑔
= 0.009 g = 0.111 g

Weight percent of ASA in original analgesic Weight percent of ASA in original analgesic
tablet tablet
0.009 𝑔 0.111 𝑔
=( )×(100%) =( )×(100%)
0.200 𝑔 0.200 𝑔
= 4.50% = 55.50%

3 Concentration in standard solution Concentration in standard solution

= 1.1951×10-4 = 1.7232×10-4

Concentration before dilution Concentration before dilution

0.100 𝐿 0.100 𝐿
= (1.1951×10-4 M) × ( ) = (1.7232×10-4 M) × ( )
0.005 𝐿 0.005 𝐿
= 2.3902×10-3 M = 3.4464×10-3 M

No of moles No of moles
= (2.3902×10-3 M) × (0.05 L) = (3.4464×10-3 M) × (0.05 L)
= 1.1951×10-4 mol = 1.7232×10-4 mol

Mass in samples Mass in samples

= (1.1951×10-4 mol)(180.16 g/mol) = 1.7232×10-4 mol)(194.19 g/mol)
= 21.5835×10-3 g = 3.3463×10-2 g

Mass in original analgesic tablet Mass in original analgesic tablet

0.500 𝑔 0.500 𝑔
=( )×(21.5835×10-3 g) =( )×(3.3463×10-2 g)
0.09 𝑔 0.09 𝑔
= 0.120 g = 0.186 g

Weight percent of ASA in original analgesic Weight percent of ASA in original analgesic
tablet tablet
0.120 𝑔 0.186 𝑔
=( )×(100%) =( )×(100%)
0.500 𝑔 0.500 𝑔
= 24.00% = 37.20%
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