Biochemistry
Essay by j l33 • March 21, 2017 • Lab Report • 1,470 Words (6 Pages) • 1,033 Views
Experiment 3
Materials:
0.1 M phosphate buffer (pH 6.0, 7.0, 8.0), 0.2 M NaOH, 0.1 M tris buffer, Casein solution (0.6% casein in 0.1 M sodium acetate), distilled water, 0.1M acetic acid
Methods:
Verification of the Henderson-Hasselbalch equation:
[pic 1]
The tris (hydroxymethyl) aminomethane buffer system:
[pic 2]
Effect of temperature on buffering capacity:
[pic 3]
Effect of dilution on buffering capacity:
Table 1: Dilution factor of phosphate buffer (pH7.0) and tris buffer (pH8.0)
Buffer | Dilution factor | Volume of buffer (mL) | Volume of ditilled water (mL) |
Phosphate buffer (pH 7.0) | 1/10 | 2.0 | 18.0 |
1/50 | 0.4 | 19.6 | |
1/100 | 0.2 | 19.8 | |
Tris buffer (pH 8.0) | 1/10 | 2.0 | 18.0 |
1/50 | 0.4 | 19.6 | |
1/100 | 0.2 | 19.8 |
[pic 4]
Isoelectric precipitation of casein:
Table 2. Volume of casein solution, distilled water and acetic acid in 7 test tubes.
Tube | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
Casein (mL) | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 |
Distilled water (mL) | 8.9 | 8.8 | 8.5 | 8.0 | 7.0 | 5.0 | 0 |
0.1 M acetic acid (mL) | 0.1 | 0.2 | 0.5 | 1.0 | 2.0 | 4.0 | 9.0 |
[pic 5]
Results:
Table 3. pH determination of 0.1 M phosphate buffers at pH 6.0, 7.0 and 8.0.
0.1 M phosphate buffer | Initial pH | Final pH | ΔpH |
pH 6.0 | 6.24 | 6.56 | 0.32 |
pH 7.0 | 7.23 | 7.46 | 0.23 |
pH 8.0 | 8.15 | 10.34 | 2.19 |
Table 4. pH determination of 0.1 M tris buffer at pH 8.0.
0.1 M tris buffer | Initial pH | Final pH | ΔpH |
pH 8.0 | 8.42 | 10.20 | 1.78 |
Table 5. pH determination of phosphate buffer at pH 7.0 and tris buffer at pH 8.0 at different temperatures.
Buffer | Temperature (°C) | Initial pH | Final pH | ΔpH |
Phosphate buffer (pH 7.0) | Ice | 7.4 | 7.43 | 0.03 |
27 | 7.23 | 7.46 | 0.23 | |
50 | 7.25 | 7.40 | 0.15 | |
70 | 7.26 | 7.45 | 0.19 | |
Tris buffer (pH 8.0) | Ice | 8.25 | 8.28 | 0.03 |
27 | 8.42 | 10.20 | 1.78 | |
50 | 7.55 | 7.93 | 0.38 | |
70 | 7.18 | 7.91 | 0.73 |
Table 6. pH determination of different dilution factor of phosphate buffer at pH 7.0 and tris buffer at pH 8.0.
Buffer | Dilution factor | Initial pH | Final pH | ΔpH |
Phosphate buffer (pH 7.0) | 1/10 | 7.21 | 11.55 | 4.34 |
1/50 | 7.72 | 11.82 | 4.10 | |
1/100 | 8.29 | 11.83 | 3.54 | |
Tris buffer (pH 8.0) | 1/10 | 8.46 | 11.48 | 3.02 |
1/50 | 8.94 | 11.95 | 3.01 | |
1/100 | 8.41 | 11.88 | 3.47 |
Table 9: pH values and amount of precipitation of casein solution with different contents
Tube | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
Experimental pH | 6.00 | 5.00 | 5.00 | 5.00 | 4.00 | 3.00 | 3.00 |
Expected pH | 5.70 | 5.40 | 5.00 | 4.70 | 4.40 | 4.10 | 3.75 |
Amount of precipitation | - | + | + | ++ | +++ | + | - |
Type of precipitation | Clear | Cloudy | Flakes | Granular | Flakes | Cloudy | Clear |
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