Laboratory: Biophysics

Exercise number: 1
Exercise title: Preparation of standard (calibration) curves
Name of the teacher: Aleksandra Kalitnik

Authors:
Name Paula Gawron, Sara Niedzielska
Index no. 276242, 276485

Department of Biomedical
Department Engineering, Wroclaw University
of Science and Technology
Date of the classes Monday, 11:15-14:00
Date of the exercise 27.05.2024
Report submission date 3.05.2025
Grade

Lab’s report structure:

1. Introduction (purpose of the exercise, research theses)
2. Methodology (description of the experiment, calculations)
3. Measurements & Results
4. Discussion and conclusions
5. Literature/bibliography
6. Measurement Protocol

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 1. Introduction
Main purpose of the experiment
The purpose of this exercise is to create standard curves for
spectrophotometric measurements of solution concentrations.
These will be utilized in Dialysis experiments and in exercises
focused on dye diffusion through membranes. Additionally, the
exercise aims to evaluate the error associated with different
dilution methods.

Research theses
1. Accurate standard curves can be established using
spectrophotometric measurements.
2. Serial dilution and simple dilution methods can be compared in
terms of accuracy and reliability for preparing calibration curves.

2. Methodology
The experiment involves preparing standard (calibration) curves for
spectrophotometric measurements of methylene blue and erythrosine
solutions. This includes performing serial and simple dilutions of the
dye solutions, measuring their absorbance, and plotting absorbance
versus concentration to create the standard curves. The following
steps outline the procedures for methylene blue and erythrosine
solutions using both dilution methods.

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List of materials used:
• 0.5 mM methylene blue solution

• 4.5 mM erythrosine solution

• Adjustable pipettes

• Volumetric flasks: 10 cm3, 20 cm3

• Test tubes

• Spectrophotometer

Description of the experiment:

Preparation of the Starting Solution

Methylene Blue Solution:

First, the 0.5 mM methylene blue solution is diluted 20 times to obtain

a starting solution of concentration c0/20. To achieve this, 1 cm³ of the
0.5 mM solution is measured using a volumetric flask and then distilled
water is added to make up the final volume of 20 cm³. The solution is
mixed thoroughly to ensure uniform concentration. The absorbance of
the methylene blue solution is then measured three times at a
wavelength of 668 nm to obtain an average absorbance value.

Erythrosine Solution:

Similarly, the 4.5 mM erythrosine solution is diluted 160 times to obtain

a starting solution of concentration c0/160. This is done by measuring
0.125 cm³ of the 4.5 mM solution with a volumetric flask and adding
distilled water to make up the final volume of 20 cm³. The solution is
mixed thoroughly to ensure uniform concentration. The absorbance of
the erythrosine solution is then measured three times at a wavelength
of 532 nm to obtain an average absorbance value.

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Serial Dilution Method

Methylene Blue Solution:

For the serial dilution method, 2 cm³ of the 0.025 mM methylene blue
solution is pipetted into a clean, labeled test tube. Then, 2 cm³ of distilled
water is added to the test tube to achieve a 1:2 dilution (0.0125 mM). The
solution is mixed thoroughly, and the absorbance is measured at 668 nm.
This dilution process is repeated to obtain the following concentrations:
c0/2, c0/4, c0/8, c0/16, c0/32, and c0/64. For each dilution, 2 cm³ of the previous
concentration is pipetted, 2 cm³ of distilled water is added, the solution is
mixed thoroughly, and the absorbance is measured.

Erythrosine Solution:

Similarly, for the erythrosine solution, 2 cm³ of the 0.028125 mM

erythrosine solution is pipetted into a clean, labeled test tube. Then, 2 cm³
of distilled water is added to the test tube to achieve a 1:2 dilution
(0.0140625 mM). The solution is mixed thoroughly, and the absorbance is
measured at 532 nm. This dilution process is repeated to obtain the
following concentrations: c0/2, c0/4, c0/8, c0/16, c0/32, and c0/64. For each
dilution, 2 cm³ of the previous concentration is pipetted, 2 cm³ of distilled
water is added, the solution is mixed thoroughly, and the absorbance is
measured.

Simple Dilution Method

Methylene Blue Solution:

For the simple dilution method, the volume of the 0.025 mM methylene
blue solution needed to achieve specific concentrations (c0/2, c0/4, c0/8,
c0/16, c0/32, c0/64) is calculated. For each target concentration, the
calculated volume of the 0.025 mM solution is pipetted into a volumetric
flask and distilled water is added to make up the final volume of 4 cm³.
The solution is then transferred to a clean, labeled test tube. The
absorbance of each concentration is measured three times at 668 nm.

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Erythrosine Solution:

Similarly, for the erythrosine solution, the volume of the 0.028125 mM

solution needed to achieve specific concentrations (c0/2, c0/4, c0/8, c0/16,
c0/32, c0/64) is calculated. For each target concentration, the calculated
volume of the 0.028125 mM solution is pipetted into a volumetric flask
and distilled water is added to make up the final volume of 4 cm³. The
solution is then transferred to a clean, labeled test tube. The absorbance
of each concentration is measured three times at 532 nm.

By following these detailed steps, standard curves for methylene blue

and erythrosine solutions can be prepared and used for subsequent
experiments involving dialysis and dye diffusion through membranes.

Calculations:

▪ Methylene Blue

• Initial concentration c0 of methylene blue:

0.5
𝑐0 = = 0.025[𝑚𝑀]
20

• Dilution error for c0:

∆𝐶0 ∆𝐶𝑠 ∆𝑣1 𝑠 ∆𝑣2 𝑓𝑖𝑛𝑎𝑙 0 0.030 0.5

𝐶0
= 𝐶𝑠
+ 𝑣1 𝑠
+ 𝑣2 𝑓𝑖𝑛𝑎𝑙
= 0.5 + 1
+ 50 = 0.04

Where:
∆𝑐𝑠𝑡𝑜𝑐𝑘 = 0
𝑐𝑠𝑡𝑜𝑐𝑘 = 0.5 [mM]
∆𝑣1 𝑠𝑡𝑜𝑐𝑘 = ± 0.030 [ml]
𝑣1 𝑠𝑡𝑜𝑐𝑘 = 1 [ml]
∆𝑣2 𝑓𝑖𝑛𝑎𝑙 = ± 0.5 [ml]
𝑣2 𝑓𝑖𝑛𝑎𝑙 = 50 [ml]

∆𝑐0 = 0.025 ∙ 0.04 = 0.001 [ml]

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 • Concentrations c0/2, c0/4, c0/8, c0/16, c0/32, c0/64:
c0 = 0.025 [mM]
c0/2 = 0.0125 [mM]
c0/4 = 0.00625 [mM] ≈ 0.0063 [mM]
c0/8 = 0.003125 [mM] ≈ 0.0031 [mM]
c0/16 = 0.0015625 [mM] ≈ 0.0016 [mM]
c0/32 = 0.00078125 [mM] ≈ 0.0008 [mM]
c0/64 = 0.000390625 [mM] ≈ 0.0004 [mM]

• The error for series dilution:

𝑉0 = 0.030 [ml]

∆𝐶0/2 ∆𝐶0 ∆𝑣0 0.03

=𝐶 +2∙ = 0.04 + 2 ∙ = 0.055
𝐶0/2 0 𝑣0 4
∆𝐶0/4 ∆𝐶0/2 ∆𝑣0 0.03
= +2∙ = 0.055 + 2 ∙ = 0.07
𝐶0/4 𝐶0/2 𝑣0 4
∆𝐶0/8 ∆𝐶0/4 ∆𝑣0 0.03
= +2∙ = 0.07 + 2 ∙ = 0.085
𝐶0/8 𝐶0/4 𝑣0 4
∆𝐶0/16 ∆𝐶0/8 ∆𝑣0 0.03
= +2∙ = 0.085 + 2 ∙ = 0.10
𝐶0/16 𝐶0/8 𝑣0 4
∆𝐶0/32 ∆𝐶0/16 ∆𝑣0 0.03
= +2∙ = 0.1 + 2 ∙ = 0.155
𝐶0/32 𝐶0/16 𝑣0 4
∆𝐶0/64 ∆𝐶0/32 ∆𝑣0 0.03
=𝐶 +2∙ = 0.155 + 2 ∙ = 0.17
𝐶0/64 0/32 𝑣0 4

• The error for simple method dilution:

C = methylene blue volume + distilled water

c0 = 4 [ml]
c0/2 = 2 [ml] + 2 [ml]
c0/4 = 1 [ml] + 3 [ml]
c0/8 = 0.5 [ml] + 3.5 [ml]
c0/16 = 0.25 [ml] + 3.75 [ml]
c0/32 = 0.125 [ml] + 3.875 [ml]
c0/64 = 0.0625 [ml] + 3.9375 [ml]

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∆𝑣1 = 0.030 [ml]
∆𝑣2 = 0.05 [ml]
𝑣𝑠 = (amount of c0 solution)
𝑣2 = 5 [ml] (capacity of cylinder)

• Error of concentration of diluted c0:

∆𝐶0 ∆𝐶𝑠𝑡𝑜𝑐𝑘 ∆𝑣1 ∆𝑣2 0.006 0.05
=𝐶 ++ + =0+ + = 0.018
𝐶0 𝑠𝑡𝑜𝑐𝑘 𝑣1 𝑣2 1 50

∆v1 - 𝑝𝑒𝑟𝑚𝑖𝑠𝑠𝑖𝑏𝑙𝑒 𝑙𝑖𝑚𝑖𝑡 𝑒𝑟𝑟𝑜𝑟 𝑜𝑓 𝑚𝑢𝑙𝑡𝑖 − 𝑚𝑒𝑎𝑠𝑢𝑟𝑖𝑛𝑔 𝑝𝑖𝑝𝑒𝑡𝑡𝑒 [𝑚l]

v1 - 𝑛𝑜𝑟𝑚𝑎𝑙 𝑐𝑎𝑝𝑎𝑐𝑖𝑡𝑦 𝑜𝑓 𝑚𝑢𝑙𝑡𝑖 − 𝑚𝑒𝑎𝑠𝑢𝑟𝑖𝑛𝑔 𝑝𝑖𝑝𝑒𝑡𝑡𝑒 [𝑚l]
∆v2 - 𝑝𝑒𝑟𝑚𝑖𝑠𝑠𝑖𝑏𝑙𝑒 𝑙𝑖𝑚𝑖𝑡 𝑒𝑟𝑟𝑜𝑟 𝑜𝑓 𝑐𝑦𝑙𝑖𝑛𝑑𝑒𝑟 [𝑚l]
v2 - 𝑛𝑜𝑟𝑚𝑎𝑙 𝑐𝑎𝑝𝑎𝑐𝑖𝑡𝑦 𝑜𝑓 𝑐𝑦𝑙𝑖𝑛𝑑𝑒𝑟 [𝑚l]

∆𝐶0/2 ∆𝐶0 ∆𝑣1 ∆𝑣2 0.03 0.05

=𝐶 + + = 0.018 + + = 0.043
𝐶0/2 0 𝑣𝑠 𝑣2 2 5
∆𝐶0/4 ∆𝐶0 ∆𝑣1 ∆𝑣2 0.03 0.05
= + + = 0.018 + + = 0.058
𝐶0/4 𝐶0 𝑣𝑠 𝑣2 1 5
∆𝐶0/8 ∆𝐶0 ∆𝑣1 ∆𝑣2 0.03 0.05
= + + = 0.018 + + = 0.088
𝐶0/8 𝐶0 𝑣𝑠 𝑣2 0.5 5
∆𝐶0/16 ∆𝐶0 ∆𝑣1 ∆𝑣2 0.03 0.05
= + + = 0.018 + 0.25 + = 0.148
𝐶0/16 𝐶0 𝑣𝑠 𝑣2 5
∆𝐶0/32 ∆𝐶0 ∆𝑣1 ∆𝑣2 0.03 0.05
= + + = 0.018 + + = 0.268
𝐶0/32 𝐶0 𝑣𝑠 𝑣2 0.125 5
∆𝐶0/64 ∆𝐶0 ∆𝑣1 ∆𝑣2 0.03 0.05
=𝐶 + + = 0.018 + 0.0625 + = 0.508
𝐶0/64 0 𝑣𝑠 𝑣2 5

▪ Erythrosine

• Initial concentration c0 of erythrosine:

4.5
𝑐0 = = 0.028125 ≈ 0.028[𝑚𝑀]
160

• Dilution error for c0:

∆𝐶0 ∆𝐶𝑠 ∆𝑣1 𝑠 ∆𝑣2 𝑓𝑖𝑛𝑎𝑙 0 0.006 0.100

= + + = 4.5 + 0.125 + = 0.05
𝐶0 𝐶𝑠 𝑣1 𝑠 𝑣2 𝑓𝑖𝑛𝑎𝑙 50

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 Where:
∆𝐶𝑠𝑡𝑜𝑐𝑘 = 0
𝐶𝑠𝑡𝑜𝑐𝑘 = 4.5 [mM]
∆𝑣1 𝑠𝑡𝑜𝑐𝑘 = ± 0.006 [ml]
𝑣1 𝑠𝑡𝑜𝑐𝑘 = 0.125 [ml]
∆𝑣2 𝑓𝑖𝑛𝑎𝑙 = ± 0.100 [ml]
𝑣2 𝑓𝑖𝑛𝑎𝑙 = 50 [ml]

∆𝐶0 = 0.028 ∙ 0.05 = 0.0014 [ml]

• Concentrations c0/2, c0/4, c0/8, c0/16, c0/32, c0/64:

c0 = 0.028125 [mM] ≈ 0.028 [mM]
c0/2 = 0.0140625 [mM] ≈ 0.014 [mM]
c0/4 = 0.00703125 [mM] ≈ 0007 [mM]
c0/8 = 0.003515625 [mM] ≈ 0.004[mM]
c0/16 = 0.0017578125 [mM] ≈ 0.0018[mM]
c0/32 = 0.00087890625 [mM] ≈ 0.0009 [mM]
c0/64 = 0.000439453 [mM] ≈ 0.0004 [mM]

• The error for series dilution:

𝑉0 = 0.006 [ml]

∆𝐶0/2 ∆𝐶0 ∆𝑣0 0.006

=𝐶 +2∙ = 0.05 + 2 ∙ = 0.053
𝐶0/2 0 𝑣0 4
∆𝐶0/4 ∆𝐶0/2 ∆𝑣0 0.006
= +2∙ = 0.053 + 2 ∙ = 0.056
𝐶0/4 𝐶0/2 𝑣0 4
∆𝐶0/8 ∆𝐶0/4 ∆𝑣0 0.006
= +2∙ = 0.056 + 2 ∙ = 0.059
𝐶0/8 𝐶0/4 𝑣0 4
∆𝐶0/16 ∆𝐶0/8 ∆𝑣0 0.006
= +2∙ = 0.059 + 2 ∙ = 0.062
𝐶0/16 𝐶0/8 𝑣0 4
∆𝐶0/32 ∆𝐶0/16 ∆𝑣0 0.006
= +2∙ = 0.062 + 2 ∙ = 0.065
𝐶0/32 𝐶0/16 𝑣0 4
∆𝐶0/64 ∆𝐶0/32 ∆𝑣0 0.006
=𝐶 +2∙ = 0.065 + 2 ∙ = 0.068
𝐶0/64 0/32 𝑣0 4

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3. Measurements & Results
Wavelength λ:

λ for Methylene Blue -> 668 nm

λ for Erythrosine -> 532 nm

Series dilution

ABSORPTION
DILUTION
Methylene Blue [A668nm] Erythrosine [A532nm]
c0 1.542 1.491
c0/2 0.786 0.768
c0/4 0.583 0.389
c0/8 0.398 0.196
c0/16 0.317 0.105
c0/32 0.284 0.054
c0/64 0.264 0.045

Simple dilution

ABSORPTION
DILUTION
Methylene Blue [A668nm] Erythrosine [A532nm]
c0 1.525
c0/2 1.144
c0/4 0.727
c0/8 0.524 no data
c0/16 0.310
c0/32 0.290
c0/64 0.263

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 4. Discussion and Conclusions
In conclusion, our laboratory experiment aimed at establishing
standard curves for spectrophotometric measurements of solution
concentration has provided valuable insights into the principles of
Beer-Lambert’s law and the practicalities of dilution techniques.
Despite encountering challenges with the spectrophotometer used, we
successfully demonstrated the correlation between solution
concentration, absorbance, and transmittance.

While our measurements for erythrosine were obtained from another

lab group due to time constraints, the standard curves generated for
methylene blue and the borrowed erythrosine data offer reliable means
for determining solution concentrations. Our comparison of dilution

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methods revealed that the series dilution method exhibited lower error
rates, likely due to more precise control over volumes.

Through this experiment, we gained practical knowledge about

pipetting uncertainties and the functionalities of spectrophotometers
in taking accurate measurements. Although both double dilution and
series dilution techniques yield similar results, the latter proves more
efficient and less error prone.

Overall, this experiment not only reinforces our understanding of the

physical phenomenon under study but also equips us with
fundamental skills for conducting future experiments in
spectrophotometry and solution concentration analysis.

5. Literature / Bibliography
We based our theoretical understanding on the "Libre Texts
Spectrophotometry 2.1.5" PDF provided by the lecturer.
(You can find it here:
https://eportal.pwr.edu.pl/pluginfile.php/432691/mod_resource/content/1
/2.1.05__Spectrophotometry.pdf ).

Additionally, we were given the necessary formulas during our lab

sessions.

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6. Measurement Protocol

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