CHEM 132.

2 – BIOCHEMISTRY (LABORATORY)
LABORATORY REPORT

Name __John Allan T. Pasana_____ Date Submitted: __Jan 4, 2020______

Lab Schedule: __MTH at 10:00-1:00_ Date Performed: _Jan 4, 2020______

Exercise No. 2
pH and Buffer System
I. OBJECTIVES:
 To calibrate the pH meter
 To choose and prepare appropriate buffer systems
 To titrate an amino acid

II. RESULTS AND DISCUSSION:

A. Calibration of the pH meter

In using a pH meter, an individual could either use a handheld one or a larger and
stationary one. The one utilized in the video was the stationary pH meter. Parts of it
include the meter, arm and the electrode. The arm itself allows the electrode to be
easily moved up and down into a beaker with the sample to be tested. Prior to use a pH
meter should be calibrated using a color-coded buffer of fixed pH. PH of 4, 7 and 10 are
considered a standard. The first step in using a pH meter is to remove the protective cap
from the electrode. Additionally, the electrode of the pH meter needs to be stored
properly, capped and immersed in a certain solution. Next, the electrode must be rinsed
using distilled water. Various pH meters have different procedures for calibration. To
calibrate your pH meter, you will need to review the owner’s manual for its specific
instructions and procedures. Begin the process of calibration using the standard of the
pH of 7. Once the standard has been calibrated, you will need to remove the sample and
rinse the electrode with distilled water. Repeat the process again, this time using the
standard with the pH of 4. Once the standard has been calibrated, you will need to
remove the sample and once again, rinse the electrode with distilled water. Then, you
will calibrate the last standard with the pH of 10. When the pH 10 standard has been
calibrated, rinse off the electrode with distilled water. PH meters must be calibrated
 each time to ensure the accuracy of measurements using the 3 different pH standards
for calibration since it provides a wide range of accuracy. After this, you are now ready
to measure the pH of an unknown solution. To begin, place the unknown solution
beneath the electrode and slowly lower the arms so that the electrode is fully
submerged in the solution. Based on the video, the pH of the unknown solution is 5.57
at 23.9℃. Once the reading is complete, remove your sample. Once again, rinse the
electrode with distilled water. If your final sample is done with the process, replace the
protective electrode cap for storage.

B. Factors Affecting Buffer Capacity

Effect of Concentration of Buffer
Phosphate Buffer

Concentration pH before NaOH was added pH after NaOH was added

DiscDiscussion: 0.005 M 6.9 8.7
0.05 M 7.0 7.2
0.10 M 7.1 7.2

In this part, phosphate buffer solutions were first prepared. For the 0.005 M
phosphate buffer, measure 1.3 mL phosphate buffer using a pipet and read
meniscus at an eye level. Afterwards, it was placed in 25 mL volumetric flask. For
the 0.05 M phosphate buffer, measure 12.5 mL phosphate buffer using graduated
cylinder and place it in 25 mL volumetric flask. And lastly for the 0.1 M
phosphate buffer, measure 25 mL buffer solution using a graduated cylinder or
just directly add solution until 25 mL mark of volumetric flask. Then, it was
diluted to desired volume, adding water gradually until it reaches 25 mL mark.
The same thing was done to the 0.005 M buffer solution and the other solution. A
stopper was then put and the flask was inverted to allow mixing of solutions.
Make sure to label each beaker before transferring the solutions in it. After
transferring, calibrate the pH meter first before you measure the pH of each
solutions and rinse it after with distilled water. Next, measure the pH of each
solutions before NaOH was added (which is shown above). A 2 mL NaOH was
added to each of these solutions. The pH meter was once again rinsed with
distilled water and the pH of the solutions after the addition of NaOH was also
measured (also shown above). As what I’ve observed and based solely on my
 opinion, the higher the concentration of phosphate buffer, the lower its pH will
be after the addition of NaOH. I can say that because 0.10 M is a higher
concentration compared to 0.005 M and 0.05 M and judging on their pH before
and after the addition of NaOH, I’ve noticed that 0.005 M had a much higher pH
level than that of the 0.05 M and 0.10 M, in which both had a pH of 7.2 after the
addition of NaOH.
Acetate Buffer

Concentration pH before NaOH was added pH after NaOH was added

DiscDiscussion: 0.005 M 4.9 11.0
0.05 M 4.8 4.9
0.10 M 4.8 4.9

For the acetate buffer, the same procedure like that of the phosphate buffer was
followed. Based solely on my opinion and mere observation on the results
gathered, I have noticed that the exact opposite thing happened here in
comparison to the phosphate buffer. It is because in this, the lower
concentration of acetate buffer, the higher its pH will be after the addition of
NaOH. I guess I can say that because of the 3, 0.005 M is the one which had the
least concentration compared to 0.05 M and 0.10 M. The 0.05 M and 0.10 M
solutions both had a pH of 4.8 before the addition of NaOH and 4.9 after the
addition of the same thing. However, in 0.005 M, it had a pH of 4.9 before the
addition of NaOH and had a major increase in pH after the addition of NaOH as
seen by its 11.0 pH level.
Effect of the ratio of the conjugate base to weak acid
Phosphate Buffer

[Conjugate Base]
pH pH after NaOH was added
[Weak Acid]
6.2 0.1:1 -----
7.2 1:1 -----

8.2 10:1 -----

Discussion:
The ratio of the conjugate base to weak acid is very crucial in a buffer solution
since improper ratio of base to acid could render the solution ineffective. That is
 why to get the ratio, I used the Henderson-Hasselbach equation and substituted
the values in there (pka=7.20, pH= 6.2,7.2,8.2) and solved it .

Here’s my solution:

Acetate Buffer

[Conjugate Base]
DiscuDiscussion: pH pH after NaOH was added
[Weak Acid]
3.7 0.09:1 -----
4.7 0.93:1 -----

5.7 9.33:1 -----

As what I’ve said, it is vital for one in the laboratory to know the proper ratio of
conjugate base to weak acid in order for our buffer solution to be effective. That
is why I’ve followed the same step as before and that’s by using the Henderson-
Hasselbach equation, substitute the values (pka=4.73 , pH= 3.7,4.7,5.7) and solve
it.
Here’s my solution:
C. Choosing the Proper Buffer Solution
1. Choosing the Proper Buffer Solution
In Protein Precipitation, two liters of 5mM buffer solution with pH 5.2 is needed in the
isolation of albumin. Which among the following buffer solution is best fitted for said
purpose? Justify your answer.

Buffer solutions pKa

Acetate buffer 4.73

Tris- (hydroxymethy) aminomethane 8.08

Phosphate buffer 7.20

Discussion:

To get what we need, we want to use a substance with a pKa= 5.2 +/- 1 so from 4.2
to 6.2 and the closer we get to 5.2 the better it will be. Judging from this, the best
fitted buffer solution to use would be the acetate buffer with the pKa of 4.73.
2. Preparation of the Chosen Buffer System
Calculate and measure the amounts (in grams if solid and in mL if liquid) of weak acid
and conjugate base needed to be able to prepare the chosen buffer system in part A
above. Express your answer in useful units (that is, prepare it from practical amounts or
concentrations of starting materials).

Here’s my solution:
D. Titration of an Amino acid
Graph:

Discussion:
In the titration of the amino acid Glycine, a 10 mL glycine was first taken and was put in a
beaker. An HCl solution was then added. The amount of HCl solution should be sufficient
enough to protonate the glycine. The HCl solution was then added by drops with 20 drops
equivalent to 1 mL. As seen in the graph, with every addition of drops (in mL), the pH level
itself also increases.
 E. pH of the Blood

The normal blood pH needs to sit between the range of 7.35 and 7.45. If it ever goes
below this particular range, acidosis occurs. On the other hand, if it goes above the said
range, alkalosis occurs. When we measure pH, we’re basically just measuring the
quantity or concentration of Hydrogen ions. When we get our blood checked by a
medical professional to have a look whether we have acidosis or alkalosis, they don’t
just look at our blood pH level but they also consider other factors as well. When a
person has acidosis, the concentration of Hydrogen ions is going up. However, if a
person has alkalosis, the concentration of the Hydrogen ions is going down. If we have
an increase of CO2, the more acidic our blood pH will be. When we are talking about
respiratory alkalosis and acidosis, it means that it mostly talks about our breathing
(mostly increase and decrease in CO2). Metabolic acidosis and alkalosis, on the other
side of the story, talks about whether there is an increase or decrease in our hydrogen
and bicarbonate ions. That is exactly why we should be able to take good care of
ourselves to avoid having severe complications whether it be blood-related or not.

III. CONCLUSION:

According to David Stewart (2018), a buffer is a chemical substance that helps maintain a
relatively constant pH in a solution, even in the face of addition of acids or bases. Buffering is
very vital among living systems as a means of maintaining a fairly constant internal
environment or homeostasis, in other words. Even humans’ blood pH level is maintained by
the carbonic acid- bicarbonate ion buffering system. Believe it or not, if ever blood is not on the
right pH level, there is a huge possibility that the cells of our bodies can stop functioning and
we may even die, in worst case scenarios. It is due to the fact that enzymes are very specific in
nature, and function optimally at the right temperature and the right pH; if the pH of our blood
goes out of ranger, the enzymes stop working and sometimes enzymes can even get
permanently denatured, thus disabling their catalytic activity. This in turn affects a lot of
biological process in the human body, leading to various diseases. To recapitulate it up, every
chemical process in our world is very vital to life for without these processes, life itself will
cease to exist. That is exactly the reason why we should learn to appreciate its essence and give
respect to everything in this world.