MBG 311- R.

Gül Aydın- 250104007

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Buffer and Solution Preparation

Introduction
Buffer is a type of solution that stabilizes pH for large changes1. They are composed of
weak acid and conjugate base, vice versa. Henderson-Hasselbalch equation is one way of preparing
buffer solutions and estimating the pH of them2.

Acid Conjugate base

pKA is relative acidity.

One way of preparing solutions is dilution which makes the solution’s concentration less
dense by enlarging solution volume, preferably by using water3. Simple dilution is one step and
equation is “C1*V1=C2*V2” (c=concentration, v=volume). For instance, if you are diluting from
5x to 1x for Y solution, you should take 4x volume from initial Y solution and add 1x volume
water. Serial dilution is when a solution is diluted consecutively generally from a stock solution, it
contains multiple steps. Other method is “percentage” (%w/v). “Solute’s mass/solution’s volume”
(g/mL) is one the type of that. It is simply achieved by dissolving specific amount of solute in a
solution. Last one is “molarity” (M) which is number of moles of solute per litre of solution (n/V,
mole/litre). Aim of these experiments was to prepare buffers (0.1 M PBS, Z) and adjust their ph
(7,8) accordingly, also execute simple dilutions for glucose (100mM -> 50 mM) and SDS (10% -
> 0.1%).

Method & Results

1. 5 mL 10% and 0.01% SDS
Firstly 5 mL 10% SDS was prepared. %10 is g/mL. 10%=x gram of SDS/5 mL. So, 0.5 gram of
SDS was mixed which was measured by precision scale with 5 mL distilled water.
For 0.01% SDS equation was =(10% SDS)*(x mL of 10% SDS)=(0.01% SDS)*(5mL). So, 0.05
mL of 10% SDS was mixed with 4.95 mL of distilled water to obtain 0.01% SDS.
2. Glucose dilution
In order to dilute 100 mM glucose to 10 mL final volume of 50 mM, following equation was used=
(100 mM glucose) *(x mL of 100 mM glucose) = (50mM glucose) *(10 mL). So, 5 mL of 100 mM
was mixed with 5 mL distilled water in a falcon to obtain 10 mL 50 mM glucose.
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3. 0.1 M PBS, 250 mL pH=7

n
For 0.06 M Na2 HPO4 -> 0.06M = 0.25l -> 0.015 mol=m/177.99g/mol -> 2.67 g Na2 HPO4
n
For 0.04 M NaH2 PO4 ->0.04M = 0.25l -> 0.01 mol=m/119.98g/mol -> 1.19 g NaH2 PO4

These components were measured by precision scale, combined in 250 mL distilled water, mixed.
Final pH for this buffer was measured as 6.97 (with pH meter) which was adjusted by adding NaOH
since the initial pH was around 6.4.

4. Phosphate, pH=8, Z buffer

n
For 0.06 M Na2 HPO4 -> 0.06M = 0.05l -> 0.003 mol=m/177.99 g/mol -> 0.53 g Na2 HPO4
n
For 0.04 M NaH2 PO4 -> 0.04M = 0.05l -> 0.002 mol=m/119.98 g/mol -> 0.24 g NaH2 PO4
n
For 0.001 M MgSO4 -> 0.001M = 0.05l -> 0.00005 mol = m/246.48 g/mol -> 0.0123 g MgSO4
n
For 0.01 M KCl -> 0.01M = 0.05l -> 0.0005 mol = m/74.65 g/mol -> 0.037 g KCl

These components were measured by precision scale, combined in 50 mL distilled water, mixed.
Final pH for this buffer was measured as 7.83 (with pH meter) which was adjusted by adding NaOH
and HCl respectively since the initial pH was lower than 8

Discussion
0.04 M NaH2 PO4 , 0.06 M Na2 HPO4 were calculated from Henderson-Hasselbalch
equation that was mentioned above. For the molarity of NaH2 PO4 which will ionize into NaHPO4
[0.1−𝑥]
ion, [x] was used. 7 = 6.8 + log [𝑥]
. pH=7 was desired and pKa value for NaH2 PO4 is 6.8. X
was found as 0.04 M for NaH2 PO4 , and since PBS was 0.1 M, 0.04 M was subtracted to obtain
molarity of Na2 HPO4 which is 0.06 M.

In order to obtain different pH from the same solutions, Le Chatelier’s principle can be
used. It specifies that equilibrium shift can take place when conditions are changed, thus
equilibrium is disturbed4,5. As an example, in an equation like HA + H2 O ↔ H3 O+ + A− , if more
acid is added to the environment, the H3 O+ ions will be present. Thus, reaction will shift in reverse
direction. So, if the quantity of acidic compound in solution is increased, the pH will be changed
accordingly. Thus, different pHs can be obtained even though using the same chemicals6,7. While
preparing Z and PBS buffers, this principle was used to shift the buffer’s pH to what we want by
adding either acid or base for decreasing or increasing the pH respectively. But since buffers
stabilize large pH changes1, it is relatively challenging to do so.
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References
1. Holten, D., Gilbertson, M., Proctor, J., & Herman, C. (1999). Blood, Sweat, and Buffers:
pH Regulation During Exercise. Retrieved 11 October 2019, from
http://www.chemistry.wustl.edu/~edudev/LabTutorials/Buffer/Buffer.html
2. Po, H., & Senozan, N. (2001). The Henderson-Hasselbalch Equation: Its History and
Limitations. Journal Of Chemical Education, 78(11), 1499. doi: 10.1021/ed078p1499
3. Yu, C. (2004). V1C1 = V2C2. Retrieved 11 October 2019, from
http://academics.wellesley.edu/Biology/Concepts/Html/V1C1.html
4. Campbell, J. (1985). Le Châtelier's principle, temperature effects, and entropy. Journal Of
Chemical Education, 62(3), 231. doi: 10.1021/ed062p231
5. Fernandez-Prini, R. (1982). Le Châtelier's principle and the prediction of the effect of
temperature on solubilities. Journal Of Chemical Education, 59(7), 550. doi:
10.1021/ed059p550
6. Bender, H., Arter, D., & Francis, E. (2011). Equilibria & Le Chatelier's Principle.
Retrieved 13 October 2019, from
http://dl.clackamas.edu/ch105/lesson7equilbria_lechatelier.htmlMercier, P. (2018).
7. Applying Le Châtelier’s Principle To Model Strong Acid–Strong Base Titration. Journal
Of Chemical Education, 95(4), 521-527. doi: 10.1021/acs.jchemed.7b00575