Ph and Buffer Solution

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Title: pH and buffer solutions
Aim
This experiment was carried out to determine the role of buffer solution and the factor which affect the buffer capacity. Besides, this experiment was carried out to investigate the solubility of protein casein over a range of pH concentration. This experiment also was carried out to determine the isoelectric point of the casein and the effect of the isoelectric point toward the casein solution.

Methods

Verification of the Henderson-Hasselbalch equation

Figure 1: Method for Verification of the Henderson-Hasselbalch equation

The tris (hydroxymethyl) aminomethane buffer system

Figure 2: Method The tris (hydroxymethyl) aminomethane buffer system Effect of temperature and/or dilution on buffering capacity: Temperature

Figure 3: Method for effect of temperature on buffering capacity Dilution

Figure 4: Methods for effect of dilution on buffering capacity Table 1: Table of dilution
Dilution ratiosVolume of Phosphate solution (mL)Volume of tris buffer (mL)Volume of distilled water (mL) 1:102.02.018.0
1:500.40.419.6
1:1000.20.219.8

Dilution
1:10 dilution
Volume of buffer needed = 1/10 × 20 = 2.0 mL
Volume of distilled water = 20-2.0 =18.0 mL
Concentration of buffer = 0.1/1000 × 2.0 = 2 ×10-4 M
1:50 dilution
Volume of buffer needed = 1/50 × 20 = 0.4 mL
Volume of distilled water = 20-0.4 =19.6 mL
Concentration of buffer = 0.1/1000 × 0.4 = 4 ×10-5 M
1:100 dilution
Volume of buffer needed = 1/100 × 20 = 0.2 mL
Volume of distilled water = 20- 0.2 =19.8 mL
Concentration of buffer = 0.1/1000 × 0.2 = 2 ×10-5 M

Isoelectric precipitation of Casein

Figure 5:Methods for Isoelectric precipitation of Casein
Table 2: The amount of casein solution , distilled water and acetic acid added in finding the isoelectric point of casein.
1234567
Casein solution (mL)1.01.01.01.01.01.01.0
Distilled water (mL)8.98.88.58.07.05.00
0.1M acetic acid (mL)0.10.20.51.02.04.09.0

Results
Verification of the Henderson- Hasselbalch equation
Table 3: The initial pH, final pH and change in the pH (Δ pH) of 0.1M phosphate buffer after adding 1mL of 0.2M NaOH pH of 0.1M phosphate bufferInitial pHpH after addition of NaOHΔ pH 6.05.966.300.34

7.06.987.190.21
8.07.988.240.26

0.1M of phosphate buffer at pH 6 shows the largest difference in pH, 0.34 when it is added with 1.0mL of 0.2M NaOH. The smallest change will be the phosphate buffer at pH 7where the change is pH, 0.21. The tris (hydroxymethyl) aminomethane buffer system

Table 4: The initial pH, final pH and change in the pH (Δ pH) of 0.1M tris buffer after adding 1mL of 0.2M NaOH pH of 0.1M tris bufferInitial pHpH after addition of NaOHΔ pH 8.07.867.950.09

Effect of temperature and/or dilution on buffering capacity
Temperature
Table 5: The initial pH, final pH and change in the pH (Δ pH) of 0.1M phosphate buffer and tris buffer at a different temperature. Temperature (0C)0.1M phosphate buffer at pH 70.1 M tris buffer at pH 8

Initial pHFinal pHΔ pHInitial pHFinal pHΔ pH
47.017.140.138.208.300.10
506.927.090.177.597.950.36
706.877.080.217.377.850.48

Both buffers showed the least difference in pH at 40C (phosphate buffer pH, 0.13 and tris buffer pH, 0.10) and the highest differences in pH for both buffer at 700C (phosphate buffer pH, 0.21 and the tris buffer pH, 0.48) Dilutions

Table 6: The initial pH, final pH and change in the pH (Δ pH) of 0.1M phosphate buffer and tris buffer at different dilutions. Dilution ratio0.1M phosphate buffer at pH 70.1 M tris buffer at pH 8

Initial pHFinal pHΔ pHInitial pHFinal pHΔ pH
1:107.1711.724.557.8511.423.57
1:507.1811.694.517.8411.693.85
1:1007.1311.454.327.8711.703.83

For the phosphate buffer at pH 7, the highest difference in pH was for the dilution ratio 1:10 (Δ pH 4.55)...
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