Laboratory: Biophysics

Exercise number: 2
Exercise title: Dialysis
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 10.06.2024
Report submission date 17.06.2024
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 investigate the principles and
practical applications of dialysis using a dialyzer. Dialysis plays a
crucial role in medical treatments by selectively removing solutes
from solutions based on their diffusion rates across semi-permeable
membranes. By conducting this experiment, we seek to enhance our
understanding of the physical mechanisms governing dialysis and
the operational parameters influencing its efficiency.

Research theses
1. The efficiency of dialysis, as indicated by dye removal rates, will vary
depending on the direction of dialysate flow through the dialyzer.
2. The application of biophysical principles, such as Lambert-Beer law
and fluid dynamics equations, will enable accurate measurement
and analysis of dialysis parameters.

2. Methodology
The experiment aimed to assess the efficiency of dialysis using a
dialyzer under controlled conditions. This section outlines the
experimental setup, materials used, procedures undertaken, and
discusses the challenges encountered during the experiment.

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List of materials used:
• Methylene blue solution
• Dialyzer
• Peristaltic pump
• Silicone tubing
• Distilled water tank
• Separating funnel
• Measuring cylinders
• Spectrophotometer
• Cuvettes

Description of the experiment:

Preparation of Dialysis Solution
The experiment began with the preparation of the dialysis solution. A total of
400 ml of distilled water was measured using a graduated cylinder and
transferred into a clean beaker. Subsequently, 4 ml of methylene blue
solution was added to the water and thoroughly mixed to achieve a uniform
concentration suitable for dialysis.

Setup and Priming of the Peristaltic Pump

Prior to starting the experiment, the peristaltic pump system was set up and
primed for operation. The distilled water tank was filled to ensure a
continuous supply throughout the procedure. Initially, the separating funnel
was closed, and 100 ml of distilled water was carefully poured into it. Special
attention was given to positioning the silicone tubing outside the peristaltic
pump head to prevent operational issues.

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It was crucial to completely fill the two primary lines: the dialysis solution
line extending from the separating funnel to the outflow, and the dialysis
fluid line stretching from the distilled water tank to the outflow. Both lines
were thoroughly filled with distilled water to remove any air bubbles and
ensure smooth operational flow. After filling, the pump head clamps were
securely tightened to stabilize the lines and prevent leakage.

Initiation and Execution of Dialysis Process

Following meticulous setup, the dialysis of the methylene blue solution
began promptly. The system's configuration (forward or reverse dialysis) was
confirmed, and 200 ml of the prepared dye solution was introduced into the
separating funnel. The valve connecting the dialysis fluid line was then
gradually opened to initiate controlled fluid flow through the system.

Periodic measurements of the dialysate's absorbance were carried out

using a spectrophotometer set at a wavelength of 668 nm, optimal for
detecting methylene blue's absorbance. The whole process was repeated
for reversed flow. Despite facing technical challenges with the
spectrophotometer and time constraints, absorbance readings were
diligently recorded at intervals. However, due to unforeseen circumstances,
the experiment was prematurely concluded after completing 6 cycles
instead of the planned 8, resulting in irregular measurement intervals.

Challenges and Limitations

Throughout the experiment, significant challenges arose, including
equipment malfunctions, time limitations, and an overflow of the waste
collection flask, which threatened experimental integrity and data accuracy.

Despite the encountered challenges, collected data were meticulously

analyzed to assess the dialyzer's efficacy in removing methylene blue from
the solution. Results were interpreted to discern the impact of flow direction
(forward vs. reverse) on dialysis efficiency.

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Calculations:

In a place of A0 we substituted values for A1 because our initial

measurements were made incorrectly and none of the
calculations would make sense, so we took values from
measurements of the first cycle (A1).

• Flow of dialyzed solution

𝑉𝑇
𝐹𝑙𝑜𝑤 =
𝑡𝑇
Where:
𝑉𝑇 − Total volume of used dialysate [mL]
𝑡𝑇 − Total time [min]

Forward Reversed
212 192
𝐹1 = 1.04 = 203.85 [mL/min] 𝐹1 = 0.98 = 195.92 [mL/min]
191
197
𝐹2 = 1.02 = 193.14 [mL/min] 𝐹2 = 0.95 = 201.05 [mL/min]
192
195
𝐹3 = 1.01 = 193.07 [mL/min] 𝐹3 = 0.94 = 204.26 [mL/min]
190
177
𝐹4 = 0.96 = 184.38 [mL/min] 𝐹4 = 0.93 = 204.30 [mL/min]
185
176
𝐹5 = 0.91 = 193.41 [mL/min] 𝐹5 = 0.92 = 201.09 [mL/min]
148
175
𝐹6 = 0.89 = 196.63 [mL/min] 𝐹6 = 0.86 = 172.09 [mL/min]

• Student’s t-test

- average flow velocity

∑𝑛
𝑖 𝐹𝑖
𝑥̅𝑓𝑜𝑟𝑤𝑎𝑟𝑑 = => 194.08 [mL/min]
𝑛

∑𝑛
𝑖 𝐹𝑖
𝑥̅𝑟𝑒𝑣𝑒𝑟𝑠𝑒𝑑 = => 196.45 [mL/min]
𝑛

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 - standard deviation (calculated in Excel =STDEV)
∑𝑛
𝑖 (𝐹𝑖−𝑥̅ 𝑓𝑜𝑟𝑤𝑎𝑟𝑑 )
𝑠𝑓𝑜𝑟𝑤𝑎𝑟𝑑 = √ = 6.3 [mL/min]
𝑛−1

∑𝑛
𝑖 (𝐹𝑖 −𝑥̅ 𝑟𝑒𝑣𝑒𝑟𝑠𝑒𝑑 )
𝑠𝑟𝑒𝑣𝑒𝑟𝑠𝑒𝑑 = √ = 12.32 [mL/min]
𝑛−1

- t-value
𝑥̅𝑓𝑜𝑟𝑤𝑎𝑟𝑑 −𝑥̅𝑟𝑒𝑣𝑒𝑟𝑠𝑒𝑑 194.08 −196.45
𝑡= = 2 2
= -0.4211
𝑠2 2 √(6.3) +(12.32)
√ 𝑓𝑜𝑟𝑤𝑎𝑟𝑑 +𝑠𝑟𝑒𝑣𝑒𝑟𝑠𝑒𝑑 6 6
𝑛 𝑛

• Degrees of freedom
df = nforward + nreverse – 2 = 6 + 6 – 2 = 10
p = 0.05
𝑑𝑓 = 10

• Critical t-value (calculated in Excel =T.INV.2T(p;df)

tcrit = 2.23

• Dye reduction coefficient

- Absorbance mean average

0.314+0.307+0.293+0.292+0.274+0.273
𝐴6 𝑓𝑜𝑟𝑤𝑎𝑟𝑑 = = 0.2921
6

0.289+0.305+0.294+0.277+0.284+0.263
𝐴6 𝑟𝑒𝑣𝑒𝑟𝑠𝑒𝑑 = = 0.2853
6

𝐴1 −𝐴6
CRR = ∙ 100%
𝐴1

0.314−0.2921
CRRforward = ∙ 100% = 0.06847
0.314
0.289−0.2853
CRRreversed = ∙ 100% = 0.01280
0.289

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• Dialyzer clearance
𝐾∙𝑡 𝐴0
= ln( )
𝑉 𝐴𝑡
Where:
K – dialyzer clearance
t – time
V – volume of substance excreted
𝐴0 – initial absorption
𝐴𝑡 – absorption at instant t

[formula transformation]:
𝑉 𝐴
𝐾= 𝑙𝑛( 0
)
𝑡 𝐴 𝑡

Vforward = 212 – 175 = 37

Vreversed = 192 –148 = 44

37 0.314
Kforward = 5.827 𝑙𝑛(0.2925) = 0.451 mL/min

44 0.289
Kreversed = 5.58 𝑙𝑛(0.2853) = 0.114mL/min

With assumption that ct = 0.3 c0:

37 0.314
Kforward = 5.827 𝑙𝑛( 0.314 ∙ 0.3) = 7.645 mL/min

44 0.289
Kreversed = 5.58 𝑙𝑛(0.289 ) = 9.495 mL/min
∙ 0.3

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 3. Measurements & Results
Wavelength λ for Methylene Blue = 668 nm

FORWARD FLOW

Cycle Time Volume Flow rate

Absorbance
number [s] [mL] [mL / min]

1 62.57 212 0.314 203.85

2 61.17 197 0.307 193.14
3 60.48 195 0.293 193.07
4 57.38 177 0.292 184.38
5 54.59 176 0.274 193.41
6 53.43 175 0.273 196.63

REVERSED FLOW

Cycle Time Volume Flow rate

Absorbance
number [s] [mL] [mL / min]

1 58.65 192 0.289 195.92

2 56.80 191 0.305 201.05
3 56.55 192 0.294 204.26
4 55.84 190 0.277 204.30
5 55.38 185 0.284 201.09
6 51.56 148 0.263 172.09

VALUES SUMMARY

𝐱̅ S CRR K
FLOW TYPE t df tcrit
[mL/min] [mL/min] [mL/min]

FORWARD 194.08 6.30 0.06847 7.645

- 0.4211 10 2.23
REVERSED 196.45 12.32 0.01280 9.495

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 4. Discussion and Conclusions
The dialysis experiments aimed to evaluate the efficiency of removing
methylene blue from a solution under forward and reverse flow
conditions. Measurements at a wavelength of 668 nm revealed distinct
patterns for both flow types, highlighting variations in absorbance and
flow rate over time.

In the forward flow setup, data indicated a steady decrease in

absorbance from 0.314 to 0.273 over six cycles, reflecting a continuous
reduction in the concentration of methylene blue in the solution.
Despite minor fluctuations, the flow rate remained relatively stable,
averaging 194.08 mL/min with a standard deviation of 6.30 mL/min. The
absorbance versus time curve displayed a logarithmic decrease with a
high correlation coefficient (R² = 0.8971).

Conversely, under reverse flow conditions, the absorbance values

decreased from 0.289 to 0.263 across six cycles. However, these
changes were less consistent compared to forward flow. The average
flow rate was slightly higher at 196.45 mL/min but with a greater
standard deviation of 12.32 mL/min, indicating more variability. The
absorbance versus time curve for reverse flow had a lower correlation
coefficient (R² = 0.4033).

The comparative analysis of forward and reverse flow conditions

highlights several critical points. Forward flow demonstrated a more
consistent and predictable decrease in absorbance, indicating more
efficient methylene blue removal. The stability of the flow rate was also
better in forward flow, as shown by the lower standard deviation.
Despite the slightly higher average flow rate in reverse flow, forward
flow proved to be more effective, potentially due to more favorable flow
dynamics and mass transfer conditions.

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Several challenges impacted the experiment's execution and data
quality. The spectrophotometer did not function properly throughout
the entire experiment, causing difficulties in obtaining accurate
measurements. Time constraints led to the premature conclusion of
the experiment after six cycles instead of the planned eight, introducing
variability and inconsistency. Additionally, an overflow of the waste
collection flask risked compromising the experimental setup's integrity
and the results' accuracy.

An essential adjustment was made due to initial measurement errors.

Instead of using the actual initial absorbance values (A0), we
substituted them with values from the first cycle (A1). This substitution
was necessary because the initial measurements were incorrect and
using them would have rendered all subsequent calculations
meaningless. By using A1 values, we ensured that the calculations
remained valid and interpretable.

In conclusion, the experiment successfully demonstrated the dialysis

process for methylene blue removal under both forward and reverse
flow conditions. Forward flow exhibited more consistent and efficient
removal, evidenced by a stable decrease in absorbance and a higher
correlation coefficient for the absorbance versus time curve. Despite
the challenges and limitations encountered, including
spectrophotometer malfunctions, measurement difficulties, and the
need to substitute initial absorbance values, the collected data
provided valuable insights into the dialysis performance and the factors
influencing its efficiency.

Future experiments should address these limitations by ensuring

sufficient time for all planned cycles, resolving spectrophotometer
issues, and preventing overflow in the waste collection system. Further
studies could also explore the impact of different flow rates, membrane
materials, and solution concentrations on dialysis efficiency.

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 5. Literature / Bibliography
We based our theoretical understanding on the PDF provided by the
lecturer.

(You can find it here:

https://eportal.pwr.edu.pl/pluginfile.php/435263/mod_resource/cont
ent/1/2.%20Dialysis.pdf ).

Additionally, we were given the necessary formulas during our lab

sessions.

6. Measurement Protocol

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