Victoire Ndong & ELina Shrestha
Biochemistry 311 Tuesday lab
10/5/07
Instructor: Laurie Lentz-Marino
LAB REPORT #2: pH AND BUFFERS
Many natural reactions that take place in our organism require an environment of
optimum pH. Reactions depending on where they happen require more acidic or basic pH. For
that reason, there are solutions called buffer which are important in keeping the pH required
for those reactions. A buffer is a solution that can keep a narrow range of pH even when strong
acids or bases are added. An example of a buffer is blood which is very good at counter acting
different substance’s pH. In our experiment, we try to see the difference between non pH
solution and pH solutions. For that, we made buffers like Acetic acid/Acetate and
pyrophosphate and we looked for changes that occurred when strong acids or strong bases
were added. The change in the pH will determine whether it was really a buffer. We also
titrated Glycine in order to get it’s pKas from the titration curve.
RESULTS & CALCULATIONS:
Table1: Measured pH and calculated pH
Measured
Substance
pH
Calculated pH
H2O
6.1
7
H2O + HCl
2.7
2.6
11.2
11.3
H2O
Victoire Ndong & ELina Shrestha
Biochemistry 311 Tuesday lab
10/5/07
Instructor: Laurie Lentz-Marino
+NaOH
HCl
NaOH
1.8
1.3
12.4
12.7
Table 2: Titration of glycine with HCl pH and volume
pH
Volume (ml)
7
0
5.6
1.3
4.6
4.5
4
6.3
1.6
8.7
Table 3: Titration of Glycine with NaOH pH and volume
pH
Volume (ml)
6.5
0
8.5
0.3
8.8
0.4
9.1
0.8
10.1
0.9
10.6
1
Victoire Ndong & ELina Shrestha
Biochemistry 311 Tuesday lab
10/5/07
Instructor: Laurie Lentz-Marino
10.9
2
Table 4: Data sheet for the dilution read on the spectrophotometer
Absorbance
Tube Letter
mmoles of HOAc
Initial amount
530nm
pH
calculated
Final amount (x)
A
3.2
1.6
0.0076
3.56
B
1.6
0.8
0.163
3.83
C
0.8
0.4
0.3666
4.14
D
0.4
0.2
0.4231
4.44
E
0.2
0.1
0.4985
4.74
F
0.1
0.05
0.3221
5.04
G
0.05
0.025
0.0798
5.34
H
0.025
0.0125
0.0208
5.64
I
0.0125
0.00625
0.095
5.94
Graph1: Plot of the pH versus the volume of HCl during the titration of Glycine
Victoire Ndong & ELina Shrestha
Biochemistry 311 Tuesday lab
10/5/07
Instructor: Laurie Lentz-Marino
pH of the solution
pH versus volume of HCl
10
9
8
7
6
5
4
3
2
1
0
Series1
0
2
4
6
8
Volume of HCl in ml
Graph 2: Plot of the whole titration of Glycine
Titration of Glycine
12
10
pH value
8
6
Series1
4
2
0
-10
-8
-6
-4
-2
Volume of NaOH and HCl
Graph3: Absorbance versus pH of Casein
0
2
Victoire Ndong & ELina Shrestha
Biochemistry 311 Tuesday lab
10/5/07
Instructor: Laurie Lentz-Marino
Absorbance versus pH
Absorbance 530 nm
0.6
0.5
0.4
0.3
Series2
0.2
Series1
0.1
0
0
2
4
6
8
pH
Calculation
pH of water is 7. When 0.05 N of HCl is added it increases the volume to 21ml and the new
concentration is (.05x0.001)/(.021)=0.00238 M So the new pH is –log(0.00238)= 2.6
pH+pOH=14 The new concentration of OH when NaOH is added is (.05x0.001)/(.021)=0.0023 so
pOH is –log(0.00238)=2.6 and pH=14 – 2.6=11.3pH of HCl=-log(0.05x0.001)= 1.3
pOH of NaOH is -log(0.05x0.001)= 1.3 so the pH= 14 – 1.3= 12.7
pH = pKa +log([base]/[acid]) pKa is 4.74 ratio= 10^.06=1.148. [Base] + [acid]=0.15 so [acid]=
0.15/2.148=0.069 and [base]= 0.15-0.069=0.080 M. We only want 50ml so we will need
6.9/2=3.5ml of acetic acid and 8.0/2= 4ml of acetate
Victoire Ndong & ELina Shrestha
Biochemistry 311 Tuesday lab
10/5/07
Instructor: Laurie Lentz-Marino
Buffer+ 1ml of HCl changes ratio to 0.07995/0.06905=1.22 and the new pH is 4.74 +log (0.063)
= 4.8 and when NaOH is added we have the ratio 0.08005/0.06805=1.17 and the new pH is 4.81
For the pyrophosphate we chose the closest pKa which is 8.44 so the log of the ratio is 8.58.44= 0.06 and the ratio is 10^ (0.06) =1.148. We have molarity is 0.01 so [acid] =0.047 and
[base] = 0.053. This time we will start with 10ml of NaOH and we will add 4.7 ml of the HCl.
When HCl is added the ratio changes: 0.05295 for the base and 0.04705 for the acid. The new
pH = 8.44 + log(0.05295/0.0465)= 8.49
When NaOH is added the ratio shifts the other way the new ratio is 0.05305 and 0.04605 so
pH= 8.44 +log(0.0535/0.04595)=8.51
For the diluted buffer: the concentration changed and the new pH=pka3 + log
([0.00053]/[0.00047])=6.81 when HCl was added the calculated pH is 6.74 + log
(0.00058/0.00465) =
Calculation of the pH in each tube:
pH= pKa+log([base]/[acid]) the pKa of acetic acid is 4.74 so only the ratios change: the
concentration of acid is half for every dilution so we plug it in the equation pH= 4.76 + log (0.1/x)
with X= the concentration of the acid. We will have first x=3.2 next is 1.6, then 0.8 and 0.4 and
0.2, then 0.1, 0.05, 0.025, 0.0125, 0.00625.
The pI of glycine is pKa1+pKa2/2 it is: 6.6
Victoire Ndong & ELina Shrestha
Biochemistry 311 Tuesday lab
10/5/07
Instructor: Laurie Lentz-Marino
DISCUSSION:
A buffer is a solution that maintains a narrow range of pH even when strong acid are added. In
our experiment, we can see how a substance that is not a buffer like water can undergo very
big changes when small amounts of strong acid or strong bases are added. The pH when only
iml of HCl was added went from 7 to 2.6 and when iml of NaOH was added it went to 11.3.
When a buffer is present, it keeps the pH in a more narrow range. The buffer we made of acetic
acid didn’t undergo a lot of changes even when we added HCl and NaOH. With 1ml of HCl the
pH change was almost nothing (<0.1) same for the NaOH. The pyrophosphate buffer also had
the same results. A buffer however can change if diluted. When we diluted our pyrophosphate
we noticed that even with a small addition of HCl the pH went very low and it went high when
NaOH was added. That means that we didn’t have buffer anymore. The dilution changed our
solution into a non-buffer substance.
In our titration of glycine, we noticed that as we added the HCl, there was not a lot of change.
We kept titrating for a very long time without a lot of change but at some point the pH sudently
changed drastically. The same happened for the titration with NaOH. The period where there is
no change is the buffering range. The solution is able to absorb lots of acid or base without
changing a lot. However, big changes can happen as soon as we are out of that buffering range.
In our experiment we might have an error caused by a too fast HCl titration. It may have given
us an inaccurate titration curve causing the pI to be also wrong. The normal pKa1 of Glycine is
Victoire Ndong & ELina Shrestha
Biochemistry 311 Tuesday lab
10/5/07
Instructor: Laurie Lentz-Marino
around 2.1 and in our curve we got 4.1. The other possible source of error is the pH meter
which was not very accurate during our titration.
The pI is the point at which the net charge of the solution is ) so the ion aggregate and they are
able to be detected by the spectrophotometer. The more the aggregation, the highest the
absorbance is. The pI of casein corresponds to pH associated with the highest absorbance; in
our case the pI is ~4.74.