Lab #3 Acids, Bases, Buffers, and Titrations

Kamran Warsi
Lab partner: Kelsey Haymond
BCHM 4341
02/06/14




Introduction
The purpose behind this lab was to learn how to measure the pH of various
acid/base solutions as well as quantifying and detecting free amino acids in
biochemical solutions. The detection of free amino acids is hard to quantify, so it is
useful to perform a colorimetric reaction. Ninhydrin is a very useful reaction that
upon reacting with the amine groups of an amino acid will generate a solution of a
particular color. Based on the color change, the name of the amino acids can then be
quantified by measuring the absorbance of sample.
The mechanism behind the Ninhydrin is that when it reacts with a given
amino acid, it decarboxylates or removes a carboxyl group from it. The products
that result are water, carbon dioxide, and aldehydes. Another important part of the
lab is to analyze titration curves and knowledge of buffers to help determine the
unknown concentration of amino acids. It helps to give the experimenter an idea of
the composition of the amino acid such as the number of titratable groups as well as
the pKas of each one. Each amino acid has a certain number of titratable groups are
function as either weak acids or bases.
Performing a titration allows the experimenter to see when the conjugate
acid is equal in concentration to its conjugate acid. Depending on the
concentrations of the titratable groups conjugate acid/base or vice versa, the pKa of
each group is determined. Lastly, the titration curves of polyprotic amino acids can
used to estimate the PI of an amino acid or the pH when it functions as a Zwitterion.

Results
Table 1. pH meter measurements on drinks or common household solutions.
Acidic Solution pH Basic Solution pH
Lemon juice 3.018 Tums solution 9.15
Orange juice 3.86 Milk of magnesia 10.63
Vinegar 2.84 Household bleach 11.19
Coca Cola 2.776 Window cleaner 10.938
Pure water 6.94 PBS 7.41
Skimmed milk 3.99 Baking soda 7.95
0.01 N HCL 4.02 0.01 N NaOH 10.12
Others Others













Table 2. Absorbances of various prepared samples in analysis for quantification of amino acids.
Sample Sample Vol. (L) Water (L) ABS570 [M]
AA stand., 0.0 moles
(L AA/H2O)
0 100 0.220 0
0.05 5 95 0.450 0.0005
0.1 10 90 0.590 0.001
0.05 15 85 0.726 0.0015
0.2 20 80 0.896 0.002
0.3 30 70 0.906 0.003
0.5 50 50 1.526 0.005
Root extract 10 90 1.254 0.0039
Shoot extract 10 90 0.788 0.002
Seed extract 10 (of 10-fold
dil.)
90 >3 0.011
White Cranberry
extract
50 50 0.028
Grape 50 50 >3 0.011
Milk 100 0 >3 0.011
Apple juice 100 0 >3 0.011
Uncolored gatorade 100 0 0.001
Aspartic acid, 10 mM 100 0 2.156 0.0077
Proline, 10 mM 100 0 0.26
Histidine, 10 mM 100 0 0.551 0.00000
Tryptophan, 10 mM 100 0 0.006 0.002
BSA protein standard
(1 g/mL)
100 0 0.781


Figure 1. Measured absorbances at 570 nm with various types of solutions











y = 244.54x + 0.303
R = 0.9974
0
0.5
1
1.5
2
2.5
3
3.5
0 0.002 0.004 0.006 0.008 0.01 0.012
A
b
s
o
r
b
a
n
c
e


[mM]
Sample absorbances reacted wtih
Ninhydrin at 570 nm
" Sample absorbances
reacted wtih Ninhydrin"
Linear (" Sample
absorbances reacted wtih
Ninhydrin")
Linear (" Sample
absorbances reacted wtih
Ninhydrin")
Figure 2. Titration of a unknown amino acid with 4 N NaOH














0
2
4
6
8
10
12
14
0 5 10 15 20
p
H

o
f

u
n
k
n
o
w
n

a
m
i
n
o

a
c
i
d

mEq of added NaOH
Unknown amino acid Titration Curve
Unknown Amino Acid Curve
pKa
1

pKa
2

Discussion
The pH determination activity involved measuring the pH of everyday
household solutions and drinks. The most acidic solutions started with coca cola
(pH=2.776), vinegar (2.84), Lemon juice (3.018), Orange juice (3.86), pure skimmed
milk (3.99), 0.01 N HCL (4.02), and pure water (6.94). On the other hand, the most
basic solution started with household bleach (11.19), window cleaner (10.938), milk
of magnesia (10.63), 0.01 N NaOH (10.12), tums solution (9.15), baking soda (7.95),
and PBS (7.41). Orthophosphoric acid is responsible for the acidity in Coca Cola,
Citric acid gives orange juice its acidity, and L-ascorbic acid gives lemon juice its
acidity. Sodium hypochlorite makes bleach very basic, Sodium Bicarbonate gives
baking soda its basicity, and calcium carbonate makes Tums solution basic.
pH is vital to measure in Biochemistry because living organisms properly
function during homeostasis. For instance, the enzymes and bacteria in the human
body cant thrive in acidic or basic solutions otherwise there would be denaturation.
pH is very crucial because it affects organic and inorganic chemical processes. The
changes in the acidity in the atmosphere can bring severe consequences to our
environment. In general pH allows scientists to quantify acidity in order to develop
strategies to help improve the ecosystem.
The way acidity is determined is by measuring an acids strength of giving up
a proton. The strength of a weak acid is determined by the pKa or the Ka values
which are constants that give a value to a weak acid to see how well it dissociates. A
weak acid like Carbonic acid with a pKa of 6.637 will dissociate very little compared
to Hydrochloric acid. Hydrochloric acid dissociates a 100% and therefore doesnt
need a pKa value to describe how well it dissociates in solution. Phosphoric acid
could be described as a strong weak acid compared to hydrochloric acid because it
doesnt give up protons as well as hydrochloric acid. The rank of the following acids
from strong to weak would be hydrochloric acid, phosphoric acid, lactic acid, acetic
acid, propionic acid, and carbonic acid.
One of the most important aspects of the lab was the ability to quantify free
amino acids in solution. According to Figure 2, the R^2 value is 0.99 which signifies
precise concentrations going from one sample to the next. The conflict in the data
comes in when the absorbance values are plugged into the linear regression
equation to solve for the unknown concentration of the living tissue, amino acid
solution, etc. The known absorbance values turn out to be less than the y-intercept
for some of the solutions. The solutions of white cranberry sauce, proline, Gatorade,
and BSA turn into negative concentration values. Although the R^2 value is stating
0.99, the conflicting values if known could help make the data more precise.
However, the conflicting data could also signify that the spectrophotometer
could detect very little concentrations of the stated solutions above because they are
less than the given y intercept. The causes of the error could be the solutions
werent tightly sealed when place in the spectrophotometer, debris on the cuvette
caused by the homogenate, or making incorrect dilution calculations.
The relative abundance of the amino acids in plant tissue, seed, and shoot
extracts provides between 0.788 to more than 3 M. The function of the tissues in
pea plants is that there roots allow them to fix nitrogen into ammonia which can be
supplied to rhizobia (Kimball, 2011). The drink that provides the most amino acids
is in milk, which has two major proteins like Casein and Whey, which provide
essential amino acids as well as providing Leucine for muscle protein synthesis
(Sexton, 2013). The calculations for the abundance of amino acids in the tissues are
the following in moles: seed = 0.55, shoot=0.2, and root = 0.39.
The absorbances for this activity were useful for comparing the colorimetric
reactions of the amino acid standards to the plant tissues or drinks. At 570 nm, it
was very interesting to see no traces of Proline because around this region yellow
light is absorbed, but then is reflected or transmitted as red and green light. Plant
tissue and drinks absorbed light strongly because their proteins reacted very well
with the Ninhydrin giving them a dark blue color. According to figure 1, the
standards amino acids and amino acids found at the bottom of the table are
clustered around the bottom of the chart. On the other end of the chart at the very
upper right, the presence of the drinks has high amounts of amino acids.
The data confirms that drinks strongly absorbs light at 570 nm and has much
more protein than the low end of the standard amino acid solutions. The titration
curve preparation was the last activity to be performed in lab. Figure 2. shows a
titration curve of adding a series of 0.8 mEq of NaOH to the titrand. The Figure
depicts a possible diprotic acid because the curve is leveled at the bottom and at the
top because the leveled parts represent when the pH = pKa. It is expected that the
lower leveled piece to have a lower pKa because low amounts of mEq of NaOH are
present.
The significance of two pKa values possibly signify the presence of an amino
and carboxyl group with the presence of a non-titrable side group. The amino acid is
acting as a weak acid for the majority of the curve because the pKa regions depict
the resistance in the increase of pH, the buffer regions. Around 8.8 mEq, the end
point of the amino acid is reached at a pH of 4 because there is a vertical jump to a
pH of 9. The original concentration of the titrand is 8.8 mEq because 8.8 mEq H
+
have been neutralized by the NaOH.
The pKa 1 of the amino acid is around 5.6 and the pKa 2 is about 12. The
buffering capacity of pKa 1 is between 4.6 to 6.6 and pKa 2 is between 11 to 13. The
buffer capacity of pKa 1 is found to be 4.08 mEq and the buffer capacity of pKa 2 is
2.77 mEq. The isoelectric point is (pKa1 + pKa2)/2 or 4.4 is the PI. In conclusion, the
amino acid content in solutions vary and can be found by reacting it with nynhydrin
or a similar colorimetric reaction. Performing a titration can help predict the
structure and original starting concentration of amino acids.









Questions

3. pH = 3.5
5. The reaction of alanine or phenylalanine with ninhydrin results in the formation
of a double nitrogen bond between the carbon that had the two hydroxyl groups on
the ninhydrin and the amino group of the amino acid.














References
Kimball, J. (2011, February 23). Symbiotic nitrogen fixation.
Sexton, C. (2013, October 31). Does milk contain protein?.