Name of the Author: Muhammet Hasan Başar

Date of the Experiment: 10.10.2024

Submission Date: 17.10.2024

Name of the Experiment: Preparation of Buffer with Tris Edta and Distilled Water, 70
Percent Ethanol Preparation, 1 M NaCl 25 ml Preparation in Falcon, Serial Dilution in
Eppendorf and Glass Tubes

Buffer Ethanol NaCl solution Preparation and Dilution Procedure

Aysu Özyürek, Samed Muhammed Cılız, Ayşenur Dursun, Ceyda Emgin, Muhammet Hasan
Başar, Feyyaz Tarık Yardımcı

Abstract
Buffer solution was prepared using tris and edta. The aim of the buffer is to keep the pH stable during chemical
reactions which is important in many biological processes. Ph was adjustedby adding small amounts of HCl and
NaOH until correct pH was reached. The experiment showed that how careful pH control is needed to make a
buffer solution. The buffer was successfully made. pH was measured.

70 percent ethanol solution was prepared. Ethanol and distilled water was mixed. Ehtanol is essential for
disinfectant and a solvent. 70 ml of ethanol and 30 ml of water was measured in graduated cylinder. The result
was a clear and homogenous solution.

1M NaCl was prepared with distilled water. Sodium chloride is commonly uses inn biology and chemistry. 1.46
grams of NaCl and dissolved it in 25 ml of water. This experiment showed the process of preparing a salt
solution.

Serial dilution was used as reducing the concentrating of a subsance, in this case, lugol solution. Serial dilution is
a technique was used to gradually lower the concentration a solution which was mixed it with distilled water.
High concentration of lugol was diluated three times, in one it was reduced by half and in the other by a tenth.
After each dilution, the solution became lighter in color.

Introduction
Buffer is a solution that can resist pH change upon the addition of an acidic or basic components. It is able to
neutralize small amounts of added acid or base, thus maintaining the pH of the solution relatively stable. This is
important for processes and rections which require specific and stable pH ranges. These are some examples:
enzyme activities, electrophoretic mobilities, antibody aggregation. Additionally they not only set ph, they can
address specific ion effects in terms of Hofmeister series.(1) Some components are suitable for preparing buffers

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such as tris and edta. Tris (tris(hydroxyethyl)aminomethane) is an organic compound used as a pH buffer(2),
while Edta (ethylenediaminetetraacetic acid) is a chelating agent that forms complexes with metal ions.(3) This
experiment aim is preparation buffer solution with tris, edta and distilled water.

Alcohol is the general name given to organic samples in which a -OH (hydroxyl) group is directly attached to the
carbon atom. Ethanol or ethyl alcohol is a colorless, pleasant-smelling and slightly flammable liquid. It is the
only type of beverages. It is also uses as a solvent. Its chemical formula is C2H5OH. Alcohol’s actions in our
body and environemnt very complex.(4) It is hard for prediction. This experiment aim is prepartion 70 percent
ethanol solution.

Salt is a mineral composed primarily of sodium chloride (NaCl). When is used in food, it is called table salt. Salt
has been used in food preparation in human history. Excess salt intake is strongly linked to increases in blood
pressure (hypertension) and pre-disposes individuals to the onset of cardiovascular disease.(5) It has a huge
effect our environmet and our production. For istance, soil salinity is one of the major abiotic stresses that affects
crop product and quality.(6) This experiment aim is 1M 25 ml NaCl preparation in falcon.

Serial Dilution is the metod of diluting sampple with a asterile diluent, which can be either distilled water or 0,9
percent saline, in sequence of standard volumes. In biology and medicine, besides the more traditional
applications mentioned earlier, serial dilution can also be utilized to decrease the concentration of microscopic
organisms or cells sample. Dilution can be useful for getting more manageable results.(7) Serial dilution is also a
cheaper and simpler method for preparing cultures from a single cell.(8) These experiments aim are that
compairing the initial and final conditions in the glass tube and eppendorf by reducing the concentration of
cherry juice with serial dilution.

Materials and Methods

Tris, edta and distilled water were used to make buffer. After each measurement, the pH meter tip was washed
with distilled water. 2 molar 25 ml tris was made in the bottle , the grammage was calculated for this and the
required amount was measured in precision balance, this amount was 1.21 grams, then It was placed next to the
pH meter to bring the pH to 8 and the pH was measured and it came out as 11.3, then HCl was added to make the
pH 8. 100 microliters of HCl was added 3 times with micropipette. Meanwhile, reduce the pH to 10.1, 9.6 and
9.4. Then 150 microliters of HCl was added 3 times and the pH decreased to 9, 8.7 and 8.3 respectively, then 100
microliters of hcl was added again and the pH decreased to 7.8, then 50 microliters of NaOH was added and the
pH was increased to 7.95. Then distilled water was added 25 ml to make it 2 molar. Then 2.5 ml Tris was poured
into the falcon tube labeled with tris buffer. Edta was prepaired simplier. Edta was used 1.86 grams in 10 ml
water in falcon tube. It was 0,5 molar. PH of edta was measured. It was 5.2. 2M 500 microliter NaOH was added
4 times with micropipette and the pH decreased to 6.3, 6.4, 6.8, 7.2. Then 2M 300 microliter NaOH was added
300 microliter. Thus Edta pH reached 8. Then 100 microliter Edta was poured into the buffer with micropipette.
The 47.5 ml distilled water was poured with graduated pipette. Thus 50 ml buffer was made in falcon. Then
Buffer pH was measured. The pH of the buffer turned out to be 7.7.

70 ml ethanol was poured into graduated cylinder then 30 ml distilled water was poured into the graduated
cylinder. Finally 70 percent ethanol was prepared. The volume measurements of the liquid were made according
to the values in the graduated cylinder.

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1M 25 ml Sodyum Chlorite should be prepared so 1.46 grams NaCl was measured Precision Balance. Then 1.46
grams NaCl was poured into the falcon. Then 25 ml water was poured with graduated pipette. Finally, 1 M
Sodyum Chloroite was prepared.

Lugol was used for serial dilution in both experiment. In the first experiment, micropipette and glass tubes were
used. 1 ml of 10 ml of Lugol was taken and then poured into 9 ml of distilled water with a micropipet. Another
pour of the previous solution was poured into 9 ml of distilled water. Then this process was applied once again
and the difference between the first and last solution was photographed. Lugol, distilled water and Eppendorf
were used in the second experiment. 500 microliters of 1 ml pure Lugol was taken with a micropipette. 500
microliters of Lugol was added to 500 microliters of distilled water. This process was repeated 2 more times.
After each procedure, the liquid was mixed with a micropipette. A photo of the difference between the first and
last one was taken.

Results
In this part of experiment, The pH change of Tris and Edta is as shown in the graph in Figure 1.

Figure 1 pH change graph of tris and edta

The experiment successfully produced tris and edta at appropriate pH for buffer. Tris and edta, with the target pH
values through stepwise addition of acid (HCl) and base (NaOH), respectively.(Figure 2)

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 Figure 2 On the left is buffer, in the middle is EDTA, on the right is Nacl solution.

As a result of the experiment, when 70 ml of ethanol and 30 ml of pure water were combined in a measuring
cylinder, The volume of the mixture was directly determined by the measurements made on the measuring
cylinder and the 70% ethanol solution was successfully prepared.(Figure 3)

Figure 3 There is water on the left, graduated cylinder in the middle and ethanol on the right.

The solution appeared clear and homogenous, indicating that the NaCl dissolved completely. The final
concentration of the solution was calculated to be 1 M, as expected.(Figure 2)

After three dilutions, the difference in color between the initial solution and the last dilution was important. After
the first dilution, the color became lighter. The photograph clearly captured the tranitions.(Figure 4)

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 Figure 4 Lugol's solution is 1 in 10 from left to right

The serial dilution of lugol’s solution was perfomed using eppendorf tubes. After each dilution (1:2 ratio), the
color of the solution was monitored. Similar to the first experiment, the solution appeared much lighter. The
change in color was documented in the photographed in figure 5.

Figure 5 The dilution process is seen from left to right

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 Figure 6 Graph of dilution process

Discussion
The preparation of a buffer solution tris and edta was successful. Ph of both components was carefully adjusted
using HCl and NaOH. The each step ph modificaction shows the importance of precision in buffer. The final pH
was reached, making the buffer able to keep the pH stable for experiments.

The preparation of 70 percent ethanol involved a simple mixing of ethanol and distilled water. The measured
volumes were accurate. The final solution was homogenous. The clear solution indicated proper mixing. Ethanol
is often used in labs as a disinfectant. The clear solution shows that the mixture was done well.

The preparation of a 1M NaCl solution was completed by dissolving the sodium chloride in distilled water. The
solution was clear and homogenous. It is essential for various biological experiments.

The serial dilution experiment showed that how dilutions decrease the concentration of a substance step by step.
Evident is from the changes in the color intensity of the lugol solution. Serial dilution is an essential technique in
microbiology and biochemistry. The difference between the initial and final dilutions was clear.

References
1-Salis, A., & Monduzzi, M. (2016). Not only pH. Specific buffer effects in biological systems. Current
Opinion in Colloid & Interface Science, 23, 1-9.

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2-National Center for Biotechnology Information (2024). PubChem Compound Summary for CID 6503,
Tromethamine. Retrieved October 13, 2024
from https://pubchem.ncbi.nlm.nih.gov/compound/Tromethamine.

3-Guo, Q. M., & ZakhResults-National Center for Biotechnology Information (2024). PubChem
Compound Summary for CID 6049, Edetic Acid. Retrieved October 13, 2024
from https://pubchem.ncbi.nlm.nih.gov/compound/Edetic-Acid. (2008).

4-Commentary: systems biology and its relevance to alcohol research. Alcohol Research &
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5-Durack, E., Alonso-Gomez, M., & Wilkinson, M. G. (2008). Salt: a review of its role in food science
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6-Hussain, T. M., Ch, T., Hazara, M., Sultan, Z., Saleh, B. K., & Gopal, G. R. (2008). Recent advances
in salt stress biology a review. Biotechnology and Molecular Biology Reviews, 3(1), 8-13.

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Appendices