Acids, Bases, Buffers, and Titrations
Kathleen Nolan and Dylan Catterton
September 11, 2014
Purpose: The purpose of this lab is to be able to perform the titration of an unknown amino acid
and to rationalize some of the important conclusions that can be made, practice using a pH meter
and practice how to calibrate it accordingly, understand the Henderson-Hasselbalch equation and
its importance in titrations, and to correctly quantify amino acids (Sparace, 2014).
Data:
Table 1: pH Measurements of Various Common Solutions/Drinks.
Acidic Solution
Lemon Juice
Orange Juice
Vinegar
Coca Cola
Pure Water
Skimmed Milk
0.01M N HCl
pH
2.584
3.928
2.64
2.76
7
6.21
2.30
Basic Solution
Tums Solution
Milk of magnesia
Household bleach
Window cleaner
PBS
Baking Soda
0.01M NaOH
pH
9.49
9.57
10.30
10.53
7.13
8.88
11.94
Table 2: Ninhydrin Detection and Quantification of Amino Acids. This is shared class
data due to ninhydrin problems or human error mistakes that made data collection not
possible.
Sample
AA Standard, 0.0
µmoles (µLAA/H2O)
“. 05”
“0.1”
“0.15”
“0.2”
“0.3”
“0.5”
Seed Extract
Kiwi tissue extract
Skimmed milk
Apple juice
White cranberry juice
Uncolored Gatorade
Aspartic Acid, 10mM
Histidine, 10mM
Proline, 10mM
Tryptophan, 10mM
BSA protein standard
(1µg/mL)
Standard (μL)
0
Water (μL)
100
ABS570
0
5
10
15
20
30
50
10 (of 20 fold
dil.)
50
50 (of 10 fold
dil.)
100
100
100
100 (of 10 fold
dil.)
100
100
100
100
95
90
85
80
70
50
90
.109
.234
.404
.566
.867
1.655
.044
50
50
.316
.382
0
0
0
0
2.128
.566
.005
.543
0
0
0
0
2.418
.060
1.947
-0.05
ABS at 570 nm
Glycine Standard Curve
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
y = 3.3302x - 0.0706
R² = 0.9923
0
0.1
0.2
0.3
0.4
µmoles Glycine
0.5
0.6
Figure 1: The relationship between absorption at 570 nm and the µmoles glycine from
the standard amino acid data in Table 2.
Table 3: Titration of 50 mL of unknown B amino acid with a 1.6 mEq per addition (400
µL).
mEq NaOH
0
1.6
3.2
4.8
6.4
8
9.6
11.2
12.8
14.4
16
17.6
19.2
20.8
22.4
24
pH
1.19
1.36
1.4
1.5
1.7
1.96
2.28
2.61
3
3.73
9.06
9.61
10.01
10.46
11.64
12.5
Relationship Between pH
and mEq for Unknown B
14
12
pH
10
8
6
4
2
0
0
5
10
15
mEq NaOH
20
25
Figure 2: Titration curve of 50 mL unknown B, showing the relationship between pH
and mEq NaOH.
mEq of NaOH: .100 mL of 1N= 0.1 mEq .4mEq for 400 µL
1 N to 4N= 1Nx4=4N
0.4 mEq x 4= 1.6 mEq per addition
Questions:
3. pH= 4.52 when using the Henderson-Hasselbalch equation.
Formula: pH= pKa + log10 [A]/[HA]
pKa of Acetic Acid=4.76
[A]= 100mL/1000= .1 L  .1L x 1 M = .1 mol
[HA]= 10 mL/ 1000= .01 L  .01 L x 17.4 M= .174 mol
pH= 4.76 + log10 ([.1]/[.174])
pH=4.52
8. Skimmed milk was probably the example that contained the most amino acids
considering we had to dilute it 10 fold to have it be under 3 A. This could be related to
the nutritional value of skimmed milk because milk contains 3.3% total protein. Milk
proteins also contain all 9 essential amino acids required by humans, therefore, something
with a lot of protein contains a lot of amino acids.
9. See Figure 2. To calculate the equivalence point, one needs to realize that the pH is
one half of the equivalence point and that point is the same as the pKa. So one would
simply calculate the pKa by finding the original amino acid concentration and the volume
of titrant added to reach equivalence multiplied by the molarity or concentration of the
titrant will give you the number of moles of titrant added. The number of moles of titrant
added, multiplied by the ratio is equal to the number of moles of analyte originally
present. The one divides the number of moles of analyte by original volume of analyte to
find the concentration. pKa couldn’t accurately be found because of it was hard to
distinguish where the buffering range was. The pI (isoelectric point) could not be
calculated.
Conclusion:
In conclusion from this lab we were able to understand how acids, bases, buffers and
titrations are all interrelated. We were able to make conclusions about the pH’s of
various solutions and rank them based on their alkalinity or acidity. We were also able to
calculate amino acids original concentration from their absorbance using a
spectrophotometer. At the end of this lab we were also able to understand how ninhydrin
reactions detect the presence of free amino acids and use/construct a standard curve.
Literature Cited
Sparace, Salvatore, and Brandon Moore. "Acids, Bases, Buffers and Titrations." Biology
4341 Laboratory Manual: Biological Chemistry Laboratory Techniques.
Clemson: Clemson U, 2014. 25-38. Print.