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Exploring UV-Vis Spectrophotometry_ Un

derstanding the basic principles and anal
yzing different aspec

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Summary
Exploring UV-Vis Spectroscopy: Understanding the basic principles,
concepts, and utilization of UV-Vis spectrophotometry through
determination of lambda max of Bromocresol Green

Author: Ryan Jazer F. Dino

Location: Adventist University of the Philippines, 4118 Silang, Cavite, Philippines
Class: STCH 223L Analytical Chemistry 2 Lab
Date performed: Feb. 7, 2024

Abstract
UV-Vis spectrophotometry is a type of spectroscopy called optical spectroscopy. UV-Vis spectrophotometry
8
specifically deals with the interaction of light of the ultraviolet and visible region of the electromagnetic
spectrum. In this laboratory, the primary objective was to ascertain the lambda max (𝝀max) (wavelength of
maximum absorption) of bromocresol green (BCG) solution within the visible spectrum (400-700 nm). This
was accomplished using a Hitachi U5100 spectrophotometer in the initial experiment. The subsequent
13
experiment aimed to deepen understanding of the relationship between concentration and absorbance, along
with associated principles and concepts. This was achieved by preparing diluted concentrations extracted from
the standard BCG solution. The 𝝀max of the standardized bromocresol green (BCG) solution was found out to
be 620 nm with the absorbance of 0.277. In the second experiment, the absorbances of the four diluted
samples were 0.278, 0.245, 0.178, and 0.136 with respective calculated decreasing concentrations. The
laboratory experiments not only successfully determined the 𝝀max of the BCG standard solution, but also
provided valuable insights into the fundamental principles and concepts governing the interaction between
concentration and absorbance in UV-Vis spectrophotometry.

Keywords: UV-Vis spectrophotometry, absorbance, lambda max, concentration, bromocresol green.

15
INTRODUCTION represents only a small part of the extensive
1
electromagnetic spectrum, which encompasses
The term spectroscopy is defined as the study of gamma rays, infrared, X-rays, microwaves, and
how light interacts with matter. Spectrophotometry several others (Skoog, 2013, p. 650). There are
is the technique under1
spectroscopy. A longer, but several processes and factors of how a light is being
clearer definition of spectroscopy is defined as the absorbed and transmitted in spectrophotometry. A
“measurement and interpretation of electromagnetic rudimentary diagram for the parts of a UV-Vis
radiation emitted or absorbed, when ions, atoms or spectrophotometry is shown below:
molecules of a sample move from one energy state
to another energy state” (SMACgig World, 2023). In Light source → wavelength selector → sample →
layman terms, the elevation in energy of its Detector
molecules by absorbing light (Agilent Technologies,
2021, p. 3). Electromagnetic radiation, in a strict To simplify in a concise way, in modern equipment
sense, is also called light, an exclusive term in the of spectroscopy, the sample absorbs specific
visible region (Harris, 2011, p. 651). The ultraviolet wavelengths of light, and the transmitted light
and visible light portion exhibits the complementary color visible to the
 14
human eye. For example, if the sample absorbs red graph is met when the Beer-Lambert’s Law is obeyed,
light, it transmits the complementary color, green, as shown below (Royal Society of Chemistry, 2009, p.
and vice versa (Spectroscopy, n.d.). The color of the 6).
bromocresol cresol green standard solution prepared,
appeared to be a darker shade of blue. Color blue has Figure 2
a wavelength spectrum of 580-595 nm. However, a
more diluted BCG stock solution was prepared Calibration graph
beforehand, and the solution has a pale tint of green.
Since the color blue predominates the green color
with less dilution, the color is greenish-blue.
Greenish-blue is the complementary color of orange
(absorbed) and has the wavelength band of 595-650
nm as shown in figure 1. Thus, the possible
wavelengths of the standard BCG are 595-650 nm. A
comparison in color of the solution is made in figures
3 and 4.

Figure 1

Color Measurement

Note. This graph shows the plot of absorbance versus

concentration with the absorbance of ≤ 1.3. As a result,
the concentration was determined to be 13 mg/l. From
3
Royal Society of Chemistry, 2009.
(https://edu.rsc.org/download?ac=12904).

Limitations of Beer-Lambert’s Law. According to

Note. A visual representation of colors of visible light
wavelengths with different absorbed colors and its
respective complementary colors. At a specific
wavelength band, a color is absorbed, which then
transmits the complementary color seen by the naked
human eye. From The basics of UV-Vis Spectroscopy, n.d.
(https://www.agilent.com/cs/library/primers/public/primer
-uv-vis-basics-5980-1397en-agilent.pdf).
18
Beer-Lambert’s
6
law, also known as absorption law,
states that absorbance is directly proportional to11the
concentration of the analyte. It is expressed in the
following equation: A = 𝜀bc, where A =5 absorbance;
𝜀 (epsilon) = molar absorptivity (M-1 cm-1); b = The transmitted light can be expressed in the following
pathlength of the dimensions of a cuvette (cm); and c ratio and its percentage. Eq. (1) (2)
= concentration
10
of sample (mol/L) (Harris, 2011, p.
436). Beer-Lambert’s law can be used to plot a graph
called a calibration graph to determine the
concentration by its absorbance. A linear calibration
Harris (2011), sample solutions must be diluted at
concentrations of ≲0.01 M in monochromatic radiation.
If the conditions are not met, it causes deviations to
the linear calibration graph (Skoog, 2013, p. 669).
Concentration level higher than ≲0.01 M, the solute
molecules influence each other due to their close
distance, leading to the solute becoming the solvent
(Harris, 2011, p. 437). Ultimately, causing inaccurate
data.
Absorbance, transmittance, and concentration.
Mathematical applications of equations can also be
used to understand the principle/concept and
quantitative expression of light and the sample.
T (Transmittance) = P/P0 (1)
%T (Percent transmittance) = 100T (2)

P is the emerging light from the sample (light that
enters the detector), and P0 is the incident light (light
that enters the sample). The value of T ranges from 0
to 1, and the value of percent transmittance ranges
from 0 to 100% (Harris, 2011, p. 435).
If the value of T is 0.6, the percent transmittance
would be 60% = 100(T) = 100(0.6). If the incident
light intensity is 100%, then the light absorbed would
be 40%. Therefore, the relationship between 9
the
absorbance of a sample and its transmittance can be
expressed in the following Equation (3)
7
volumetric pipet, a 3.00 x 10-4 M Bromocresol green
was prepared in a 100 mL volumetric flask by diluting
0.001 M Bromocresol green stock solution through
calculated amount (mL).
II. Determination of lambda max (𝝀max) of BCG
standard solution
For every 10 nm within the set visible range spectrum
(400 - 700 nm) from the wavelength scan list option,
the absorbance was recorded and inserted into a chart
in excel ( Figure 3).

A(Absorbance) = log(P0/P) = -log T (3) III. Influence of dissimilar concentrations on

Setting the numerical value of T to 1 results in an
absorbance of 0, as calculated by A = -log(T) =
-log(1) = 0. Conversely, if the numerical value of T is
0, the absorbance becomes 1. This infers that
transmittance is inversely proportional to
absorbance, while absorbance and concentration is
directly proportional with each other as shown in
figure 2.
absorbance
Four test tubes, each with a length of 150 mm, were
utilized. From the 0.001 M BCG stock solution,
quantities of ten, eight, six, and four mL were precisely
16
transferred into the respective test tubes employing a
10 mL Mohr pipet and a 3 mL volumetric pipet. 3
Subsequently, distilled water was transferred into each
test tube to attain a total volume of 10 mL in each.

METHODOLOGY RESULTS AND DISCUSSION

Equipment/Materials: A spectrophotometer has a light source that may be

absorbed, then reflected, scattered, or transmitted by
the sample. Before the absorption process, a
Latex gloves test tubes 150 mm (4)
wavelength selector called monochromator, which was
100 mL volumetric 10 mL Mohr pipet also the wavelength selector of the spectrophotometer
flasks (4) used in this experiment, separates light into the
selected narrow wavelength band, projecting it through
3 mL volumetric pipet Droppers the length of the sample, which then emerges from the
sample as a lesser beam of light (Harris, 2011, p. 435).
Aspirator bulb Cuvettes The detector then converts the incoming light as a
17
current and translates it into a value known as
50 mL beaker Spectrophotometer absorbance (Royal Society of Chemistry, 2009, p. 5).
(Hitachi U5100)
Determination of lambda max (𝝀max). The 𝝀max of the
25 mL volumetric pipet 5 mL volumetric pipet 3.00 x 10-4 M BCG standard solution shown in figure 5
by utilizing spectrophotometer (Hitachi U5100) was
Reagents: 620 nm with the highest absorption of 0.277. Distilled
0.001 M Bromocresol green solution (BCG) water was used as a blank cell. A blank sample zeroes
Distilled water out the instrument before any measurements happen
(Blanks in Method Validation, 2019).
I. Preparation of Standard Solution
Utilizing a 25 mL serological pipet and a 5 mL
Figure 5: Lambda max (𝝀max) of BCG standard CONCLUSION
solution
The experiment aimed to determine the 𝝀max of a 3.00 x
10-4 M BCG standard solution using a Hitachi U5100
spectrophotometer, resulting in a peak absorption at
620 nm with an absorbance of 0.277. Subsequently, the
impact of concentration on absorbance was
investigated, revealing absorbance values of 0.278,2
0.245, 0.178, and 0.136 for concentrations of 3.00 x
10-4 M, 2.4 x 10-4 M, 1.8 x 10-4 12M, and 1.2 x 10-4 M,
respectively. Ideally, according to Beer-Lambert's Law,
a linear correlation between absorbance and
concentration is to be expected. However, the observed
absorbance values exhibited deviations from this
Effect on concentration on absorbance. The expected linearity, particularly noticeable in the graph
observed absorbances of the four samples were where the absorbance for 10 mL exceeded that of the
0.278, 0.245, 0.178, and 0.136 with concentrations of standard solution for the second experiment. While the
3.00 x 10-4 M, 2.4 x 10-4 M, 1.8 x 10-4 M , and 1.2 x 𝝀max determination was successful, the observed
2

10-4 M respectively. Typically, according to deviations as shown in figure 6 from linearity in the
Beer-Lambert's Law, absorbance should exhibit a concentration-absorbance relationship signifies the
linear correlation with concentration. However, the need for careful attention to experimental procedures,
observed absorbance values deviate from this particularly in the dilution process.
expected linearity as shown in the graph below. One
significant factor could be the accuracy of the
dilution process. Dilution is a critical step in
preparing standard solutions, and any inaccuracies in
this process can lead to deviations from the expected
linear trend. In this case, it seems that the dilution
may not have been achieved accurately for each
concentration. This is much more noticeable in the
fact that the absorbance for 10 mL was 0.278, a
higher absorbance than the standard solution.

Figure 6: Relationship between absorbance and

concentration
 References

Agilent Technologies. (2021). The Basics of UV-Vis Spectroscopy. Agilent.

https://www.agilent.com/cs/library/primers/public/primer-uv-vis-basics-5980-1397en-agilent.pdf
4
EuraChem. (2019). Blanks in Method Validation.

https://www.eurachem.org/images/stories/Guides/pdf/MV_Guide_Blanks_supplement_EN.pdf

Harris, D. C. (2011). Quantitative Chemical Analysis Ninth Edition (9th). New York: W.H.Freeman and
Company.

Royal Society of Chemistry. (2009). Ultraviolet - Visible Spectroscopy (UV).

https://edu.rsc.org/download?ac=12904

SMACgig World. (2023, June 1). UV-VIS Spectroscopy: principle.

https://www.smacgigworld.com/blog/principle-uv-vis-spectroscopy-.php#

Skoog, D. A., West, D. M., Holler, F. J., & Crouch S. R. (2013). Fundamentals of Analytical Chemistry

(9th ed.). Mary Finch.

Spectrophotometry. (n.d.). Technique Primer. Retrieved February 16, 2024 from

https://sitesmedia.s3.amazonaws.com/chem/files/2012/08/Spectrophotometry_Primer.pdf
 Appendix

𝐶𝑎𝑙𝑐𝑢𝑙𝑎𝑡𝑖𝑜𝑛𝑠:

𝐴. 𝑃𝑟𝑒𝑝𝑎𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝐵𝑟𝑜𝑚𝑜𝑐𝑟𝑒𝑠𝑜𝑙 𝐺𝑟𝑒𝑒𝑛 (𝐵𝐶𝐺) 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛

𝑀1𝑉1 = 𝑀2𝑉2
−4
(0. 001 𝑀)(𝑉1) = (3. 00 × 10 𝑀)(100 𝑚𝐿)
−4
(3.00×10 𝑀)(100 𝑚𝐿)
𝑉𝑓𝑖𝑛𝑎𝑙 = (0.001 𝑀)
= 30 𝑚𝐿

−4
𝐵. 𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛𝑠 𝑜𝑓 𝐵𝐶𝐺 𝑠𝑎𝑚𝑝𝑙𝑒𝑠 𝑓𝑟𝑜𝑚 3. 00 × 10 𝑀 𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑆𝑜𝑙𝑢𝑡𝑖𝑜𝑛

10 mL:
−4
(3.00 × 10 𝑀)(10 𝑚𝐿) −4
𝑀𝑓𝑖𝑛𝑎𝑙 = (10 𝑚𝐿)
= 3. 00 × 10 𝑀

8 mL:
−4
(3.00 × 10 𝑀)(8 𝑚𝐿) −4
𝑀𝑓𝑖𝑛𝑎𝑙 = (10 𝑚𝐿)
= 2. 4 × 10 𝑀

6 mL:
−4
(3.00 × 10 𝑀)(6 𝑚𝐿) −4
𝑀𝑓𝑖𝑛𝑎𝑙 = (10 𝑚𝐿)
= 1. 8 × 10 𝑀

4 mL:
−4
(3.00 × 10 𝑀)(4 𝑚𝐿) −4
𝑀𝑓𝑖𝑛𝑎𝑙 = (10 𝑚𝐿)
= 1. 2 × 10 𝑀
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UV-Vis spectrophotometry is a type of spectroscopy

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The basics of UV-Vis

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