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Abstract

This experiment aims to figure out the concentration of unidentified liquids using colorimetric
analysis utilising a spectrophotometer. Standard solutions containing known concentrations of blue
and yellow dyes were prepared, and their absorbance was recorded at specific wavelengths (630.0
nm for blue and 424.0 nm for yellow). A calibration curve was established for each colour, and the
concentration of unknown substances was ascertained by linear regression. The blue unknown
(Sample 38) exhibited a concentration of around 0.0184 mM, while the yellow unknown (Sample
18) displayed a concentration of roughly 0.0307 mM. The findings indicate that the method was
successful in acquiring dependable and precise concentration measurements for both unidentified
substances.

109 Words.

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Introduction

Colorimetry is a standardised scientific method for quantifying a substance's concentration in a

solution through the measurement of light absorption at a certain wavelength. This method relies
on the Beer-Lambert Law, which asserts that the colour intensity of a solution is directly
proportionate to its concentration. This law asserts that absorbance is directly proportional to the
concentration of the solute and the length of the light path. A spectrophotometer is an instrument
that quantifies the amount of light absorbed by a solution at a designated wavelength. This
experiment employed coloured solutions to ascertain the quantity of unknown compounds by a
colorimetric method.

Key chemical terminology utilised in this experiment include "absorbance," denoting the quantity
of light absorbed by a solution, and "standard curve," which is a graph constructed using known
concentrations to demonstrate a correlation between absorbance and concentration. By utilising
the absorbance values of established standards, we may construct a linear regression line to predict
the concentration of an unknown sample based on its absorbance measurement.

The objective of this experiment is to accurately ascertain the concentration of two unidentified
samples: one yellow solution and one blue solution. A reference curve for each colour was
established by measuring the absorbance of known quantities at the designated wavelengths (424.0
nm for yellow and 630.0 nm for blue). The curves were utilised to determine the concentration of
each unknown sample according to the position of their absorbance values on the graph. This
experiment demonstrates the integration of perceived colour change and spectrophotometric data
to produce valuable quantitative results.

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Materials and Methods

Materials Used

This experiment included normal blue and yellow dye solutions with known concentrations of 0.01
mM, 0.02 mM, and 0.05 mM, as well as two unknown samples: one blue (Sample 38) and one
yellow (Sample 18). Both blank measurements and solution dilution were carried out using
distilled water. A spectrophotometer was used to measure absorbance, with the wavelength set to
630.0 nm for blue solutions and 424.0 nm for yellow. Each sample was held in clean cuvettes, and
the cuvettes were wiped off with tissues before measurement to ensure accuracy. For preparation
and handling, the experiment required two 30 mL or 50 mL beakers, two 5 mL glass pipettes, a
pipette bulb, and three 100 mL volumetric flasks with stoppers. Additional tools included droppers
when needed, a plastic bag for waste disposal, and personal protection equipment such as lab
gloves and safety goggles to ensure safe handling during the procedure.

Experimental Procedure

To start the experiment, standard solutions of blue and yellow dyes were made by diluting known
concentrations with distilled water in volumetric flasks. Each dye contained three standard
solutions and one unknown sample. Each solution's absorbance was measured using a
spectrophotometer at its specific wavelength (630.0 nm for blue and 424.0 nm for yellow). Prior
to conducting measurements, the spectrophotometer was calibrated with a blank sample of distilled
water. Cuvettes were properly cleaned and filled with pipettes and droppers to prevent
contamination. Each sample had its absorbance readings recorded. After collecting data, standard
curves for both dyes were created by graphing absorbance versus concentration. A linear equation
was created for each curve, which was then utilised to compute the concentration of the unknown
samples based on their absorbance values. All processes were carried out using approved
laboratory techniques to ensure accuracy and uniformity.

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Figure 1: Flowchart of experimental steps for determining unknown solution concentrations using
colorimetry.

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Results and Calculations

The absorbance of known and unknown dye solutions was measured with a spectrophotometer.
Two sets of data were collected: blue dye at 630.0 nm and yellow dye at 424.0 nm. Blank samples
of distilled water were used to zero the spectrophotometer. The collected data was then being used
to make two standard calibration curves, allowing the accumulation of unknown samples to be
computed using best-fit line equations. The data are summarised in Tables 1 and 2 below.

• Blue Dye Data (Wavelength: 630.0 nm)

Table 1. Absorbance values of standard and unknown blue dye solutions measured at 630.0 nm,
including concentrations and sample numbers.

Sample No. Concentration (mM) Wavelength (nm) Absorbance (A)

Blank 0.00 630.0 0.0003
35 0.01 630.0 0.1680
36 0.02 630.0 0.3009
37 0.05 630.0 0.7628
38 (Unknown) 0.0186 630.0 0.2872

• Yellow Dye Data (Wavelength: 424.0 nm)

Table 2. Absorbance values of standard and unknown yellow dye solutions measured at 424.0 nm,
including concentrations and sample numbers.

Sample No. Concentration (mM) Wavelength (nm) Absorbance (A)

Blank 0.00 424.0 0.0004
15 0.01 424.0 0.0480
16 0.02 424.0 0.1066
17 0.05 424.0 0.2886
18 (Unknown) 0.0304 424.0 0.1718

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Figure 2. The solutions of Blue dye in three 100 mL volumetric flasks with cuvettes prepared for
concentration analysis.

Figure 3. The solutions Yellow dye in three 100 mL volumetric flasks used for concentration
analysis.

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 Figure 4: Standard Curve for Blue Dye

The standard curve of the blue dye connection between absorbance and concentration at 630.0 nm
is displayed in Figure 4 above. The concentration of the unknown sample was ascertained using
the equation of the line.

Explanation for the Blue Dye Graph for Figure 4:

A clear and linear relationship between concentration and absorbance can be seen in the blue dye
standard curve at 630.0 nm. As the concentration of the blue solution rises, so does the absorbance
value. The Beer-Lambert Law, which states that absorbance in a coloured solution is proportionate
to its concentration, is in line with this. To create a straight-line equation, the absorbance
measurements of the known amounts were plotted. This formula was used to map the absorbance
of Sample 38, the unidentified blue sample, to a location on the graph. The concentration was then
estimated to be around 0.0186 mM. This indicates that the concentration of the unknown was
precisely ascertained using the spectrophotometric method and graph.

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 Figure 5: Standard Curve for Yellow Dye

The standard curve of the yellow dye connection between absorbance and concentration at 424.0
nm is displayed in Figure 5. The concentration of the unidentified sample was determined using
the linear trendline.

Explanation for the Yellow Dye Graph for Figure 5:

The yellow dye's standard curve, which is measured at 424.0 nm, shows a consistent linear
relationship between absorbance and concentration. More absorption occurs when the
concentration of yellow dye rises, intensifying the hue. The data's adherence to Beer-Lambert's
Law is demonstrated by this linear trend. A line of best fit was created on the graph using the
absorbance values of the known concentrations. Sample 18, a yellow solution that is unknown, has
an absorbance of 0.1718. The concentration of the unknown was determined to be around 0.0304
mM using this value in the equation of the line. This outcome demonstrates the accuracy with
which the graph may be used to infer unknown concentrations from experimental absorbance data.

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Calculation

1. Calculating the Concentration of the Unknown Samples

We used the standard form of a linear equation:

𝒚 = 𝒎𝒙 + 𝒄

• Blue Standard Curve Equation:

𝑦 = 15.141𝑥 + 0.0052

To find the unknown concentration (Sample 38, absorbance = 0.2872):

(0.2872−0.0052)
𝑥=
15.141
0.282
𝑥= = 0.0186 𝑚𝑀
15.141

• Yellow Standard Curve Equation:

𝑦 = 5.8357𝑥 − 0.0058

To find the unknown concentration (Sample 18, absorbance = 0.1718):

(0.1718+0.0058)
𝑥=
5.8357
0.1776
𝑥 = 5.8357 = 0.0304 𝑚𝑀

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 2. Calculate the energy of the photons that are absorbed at the maximum wavelength. What
coloured light is being absorbed?

Equation: 𝐸 = ℎ𝑐 / 𝜆

• Blue Dye Wavelength (𝜆) = 630.0 𝑛𝑚 = 630.0 × 10⁻⁹ 𝑚

(6.626 × 10⁻³⁴ Js) × (3.0 × 10⁸ m/s)

𝐸 =
(630.0 × 10⁻⁹ m)

(1.9878 × 10⁻²⁵)
𝐸 =
(6.3 × 10⁻⁷)

𝐸 = 3.154 × 10⁻¹⁹ 𝐽

Therefore, blue dye appears blue because it absorbs reddish-orange light.

• Yellow Dye Wavelength (𝜆) = 424.0 𝑛𝑚 = 424.0 × 10⁻⁹ 𝑚

(6.626 × 10⁻³⁴ 𝐽𝑠) × (3.0 × 10⁸ 𝑚/𝑠)
𝐸 =
(424.0 × 10⁻⁹ m)

(1.9878 × 10⁻²⁵)
𝐸 =
(4.24 × 10⁻⁷)

𝐸 = 4.689 × 10⁻¹⁹ 𝐽

Therefore, yellow dye appears yellow because it absorbs violet/blue light.

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Discussion

The findings of this experiment show that the spectrophotometer may be used to accurately
calculate the concentration of unknown dye solutions by comparing their absorbance readings to
a standard calibration curve. Both the blue and yellow dye samples yielded straight-line graphs
with good consistency between measured and predicted values. The predicted concentrations of
the unknowns (0.0186 mM for blue and 0.0304 mM for yellow) are well within the expected range
of accuracy. This implies that the experiment successfully achieved its goal of identifying unknown
concentrations using absorbance data and standard curves.

However, there are aspects that could be improved. One potential improvement would be to use a
more precise and sophisticated spectrophotometer with automated cuvette handling, which could
reduce human error during measurement. Furthermore, employing a higher concentration to
construct the standard curve (five or six instead of three) might improve the calibration line's
accuracy and repeatability. Implementing software that directly combines data and automatically
shows curves may help eliminate manual errors and increase efficiency.

This experiment's methodology and principles have a wide range of applications in biological and
chemical research. For example, the same procedure can be used to measure protein concentration
(e.g., Bradford test), DNA purity, and even contaminant levels in water samples. Understanding
how substances absorb specific wavelengths enables researchers to identify chemicals in complex
combinations, making this technique an important tool in analytical chemistry.

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 1. Why are the calibration curves different even though dye concentrations are the same?

Due to variations in how each pigment absorbs light at particular wavelengths, the calibration
curves for the blue and yellow dyes differed even though their concentration levels were the same.
The absorbance of the yellow dye rose more slowly, but the calibration curve for the blue dye was
steeper (higher sensitivity). This is due to the fact that every hue has a unique chemical makeup
that affects how it responds to electromagnetic radiation. Yellow and blue dyes absorb light in
different parts of the visible spectrum: yellow dye absorbs in the violet-blue region (424 nm),
whereas blue dye absorbs in the red-orange region (about 630 nm). Their chemical makeup and
the size of their conjugated systems affect how strongly they absorb particular wavelengths,
resulting in these differences in absorption (Lakowicz, 2006; Skoog et al., 2013).

References:

Lakowicz, J. R. (2006). Principles of Fluorescence Spectroscopy. Springer.

Libretexts. (2023a, January 30). The Beer-Lambert Law. Chemistry LibreTexts.

https://chem.libretexts.org/Bookshelves/Physical_and_Theoretical_Chemistry_Textbook
_Maps/Supplemental_Modules_%28Physical_and_Theoretical_Chemistry%29/Spectrosc
opy/Electronic_Spectroscopy/Electronic_Spectroscopy_Basics/The_Beer-Lambert_Law
(Libretexts, 2023e)

Skoog, D. A., Holler, F. J., & Crouch, S. R. (2018). Principles of Instrumental Analysis
(7th ed.). Cengage Learning.

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 2. What is it about the chemical structure of these dyes that allows them to absorb radiation
in the visible region of the spectrum?

Dyes absorb visible light due to their unique structures. They have extensive chains of double
bonds (known as conjugated systems), which allow electrons to jump to higher energy levels when
struck by light (Libretexts, 2023f; Ashenhurst, 2022b). This is how colour is seen. Additional
groups, such as –OH or –NH₂, enhance their ability to absorb (Umairkhan.A6a, n.d.-b). These
properties enable the dyes to absorb certain wavelengths of light while reflecting others, giving
them a blue or yellow appearance.

References:

• Libretexts. (2023c, July 30). 21.9: Conjugated systems. Chemistry LibreTexts.

https://chem.libretexts.org/Bookshelves/General_Chemistry/ChemPRIME_%28Moore_et
_al.%29/21%3A_Spectra_and_Structure_of_Atoms_and_Molecules/21.09%3A_Conjuga
ted_Systems (Libretexts, 2023f)
• Ashenhurst, J. (2022, October 31). Conjugation and color (+ How bleach works). Master
Organic Chemistry.
https://www.masterorganicchemistry.com/2016/09/08/conjugation_and_color/
(Ashenhurst, 2022b)
• Umairkhan.A6a. (n.d.). Dyes & Pigments Lecture 1 (22!03!2023). Scribd.

https://www.scribd.com/document/727488908/Dyes-Pigments-Lecture-1-22-03-2023

(Umairkhan.A6a, n.d.-b)

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Conclusion

The purpose of this experiment was to use standard calibration curves created by
spectrophotometry to ascertain the concentration of two dye solutions that the investigator was
unaware of. The fact that the unknown concentrations were accurately anticipated to be
approximately 0.0184 mM for the blue dye and 0.0307 mM for the yellow dye shows that the
experiment was successful. The reliability of this approach for quantitative analysis is
demonstrated by this confirmation.

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 References

Libretexts. (2023b, January 17). 1.8: Serial dilutions and standard curve. Biology LibreTexts.
https://bio.libretexts.org/Bookshelves/Biotechnology/Lab_Manual%3A_Introduction_to_Biotech
nology/01%3A_Techniques/1.08%3A_Serial_Dilutions_and_Standard_Curve

Spectrophotometry & dilutions. (n.d.).

https://websites.nku.edu/~whitsonma/Bio150LSite/Lab%202%20Water/SpectrophotometryII.ht
ml

Uzman, A., Johnson, J., Widger, W., Eichberg, J., Voet, D., Voet, J. G., & Pratt, C. W. (2016).
Fundamentals of Biochemistry, student companion: Life at the Molecular Level. Wiley.

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Pavia, D. L., Lampman, G. M., Kriz, G. S., & Vyvyan, J. R. (2015). Introduction to spectroscopy.

Denniston, K. J., Topping, J. J., Caret, R. L., Caret, R. L., Katherine, D., & Joseph, T. (2006).
General, organic, and biochemistry. McGraw-Hill Science, Engineering & Mathematics.

Atkins, P. W., & De Paula, J. (2014). Atkins’ physical chemistry. Oxford University Press.

Christian, G. D., Dasgupta, P. K., & Schug, K. A. (2013). Analytical chemistry. Wiley Global
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