PH AND BUFFER LAB

BIO CHEMISTRY
LABORATORY EXPERIMENT NO. 1

BY:

MIA CLAIRE ANDERSON

SUBMITTED TO:

DR. GAUTAM DAR

LOS ANGELES TRADE TECHNICAL COLLEGE

March 10, 2019

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ABSTRACT
This experiment will achieve the following objectives:
 Prepare acetic acid and sodium acetate
 Demonstrated the calibration of a pH meter
 Measure the PH of acetic acid and sodium acetate
 Measure the PH on solution prepared by reacting acetic acid with sodium hydroxide
 Calculate the Ph for a weak acid or a base
 Compute for the buffer capacity and buffering range
 Determine the effect of the addition of the acid or the base to the buffer

INTRODUCTION

It is known that the human body operates on a delicate balance of pH. A human body

can only sustain homeostasis between a pH of 7.35-7.45. Once these conditions are not met,

enzymes do not function as expected.

An acid by definition is a proton donor. A base, on the other hand, accepts protons.

Biochemical reactions are affected by changes in the Hydrogen ion concentration. These

changes are measured by its pH. The pH scale is measured from 1-14 where a pH of 7 indicates

that the solution is neutral. A pH of more than 7 is a basic solution while a pH that is less than 7

is an acidic solution.

In this laboratory, acetic acid and sodium acetate will be prepared. Its corresponding pH

will be measured, The pH of the sample will be computed and the effects of the addition of the

acid or base to a buffer will be explored.

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PROCEDURE

PART A: PREPARATION OF ACETIC ACID AND SODIUM ACETATE

1. Obtain 2 250 mL bottles. Prepare 250 mL of 0.25 M acetic acid through dilution.
2. Prepare another 250 L of 0.25 sodium acetate solution. By dissolving the solid salt.

PART B: pH MEASUREMENTS ON SOLUTIONS OF ACETIC ACID AND SODIUM ACETATE

1. Mix the acetic and sodium acetate in a clean 50 mL beaker.
2. Record its ph in the appropriate a table.
3. Plot the pH vs. the log (salt/acid).
4. Report the equation of the line that best fits to the data.

PART C: pH MEASUREMENTS ON SOLUTIONS PREPARED BY REACTION OF ACETIC

ACID WITH SODIUM HYDROXIDE
1. Prepare in a clean 50 mL beaker the indirect buffers that are indicated.

BEAKER mL of 0.250 M HC2H3O2(aq) mL of 0.250 M NaOH (aq)

1 18.0 2.0

2 16.0 4.0

3 14.0 6.0

4 12.0 8.0

5 10.0 10.0

2. Measure the pH and record the results .

3. Compare these with the pKa.
4. Plot pH vs. log (base/acid).
5. Report the best fit equation of the line.

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PART D: EFFECT OF ADDITION OF ACID OR BASE TO A BUFFER
1. Add the following to 3mL of Buffer B3.
a. 1 drop 0.25 M HCl
b. 5 drops 0.25 M HCl
c. 1 drop 0.25 M NaoH
d. 5 drop 0.25 M Naoh
2. Repeat the procedure with 3mL of water.

DATA
PART A
Amount (in g or mL) pH Measured PH calculated

0.25 M. Acetic Acid 3.6 mL 2.6 2.17

O.25 Sodium Acetate 5.1 g 7.5 9.06

Show pH calculation below:

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5
5. PART B: Plot the pH vs. the log (salt/acid).

BEAKER HC2H302 (mL) NAC2H3O2 (mL) pH Measured pH Calculated

B1 18.0 2.0 3.91 4.148

B2 16.0 4.0 4.33 4.57

B3 14.0 6.0 4.53 4.75

B4 12.0 8.0 4.93 5.12

Equation of the line

R2 : 0.999
pKa : 4.76
Ka: 4.533

Show pH Calculation of pH

B1:
pH= pka = log ( salt/acid)
Moles of Acetic Acid= (0.25m ) (0.016L) = 0.0 2L X = 0.2 M
L 0.02 L
________________
0.02 L

Moles of Sodium Acetate= (0.02L) X=0.250 m (0.004L) = 0.05 M

0.012L L
____________
0.012 L
Ph = 4.75 + log ( 0.05/0.2)= 4.15
B2

MOLES OF ACETIC ACID: (0.250M) (0.012L) = 0.020L X = 0.15 M

0.020L 0.020L

MOLES OF SODIUM ACETATE : (0.020L)(X) = (0.250M)( 0.08l) = 0.1 M

(0.020L) (0.020L)

Ph = 4.75 + log ( 0.1/1.5) = 4.57

B3:

MOLES OF ACETIC ACID : (0.250M) (0.01L) = 0.020L X = 0.125 M

0.020L 0.020L

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MOLES OF SODIUM ACETATE : (0.020L)(X) = (0.250M)( 0.01L) = 0.125 M
(0.020L) (0.020L)

Ph = 4.75 + log ( 0.125/0.125) = 4.75

B4

MOLES OF ACETIC ACID : (0.250M) (0.06L) = 0.020L X = 0.075 M

0.060L 0.020L

MOLES OF SODIUM ACETATE : (0.020L)(X) = (0.250M)( 0.014L) = 0.175 M

(0.020L) (0.020L)

Ph = 4.75 + log ( 0.175/0.075) = 5.118

PART C : BUFFERS INDIRECT METHOD

BEAKER HC2H3O2 NaOH pH Calculations pH calculated

(mL) (mL) measured

C1 18.0 2.0 3.55 4.75 + log (0.5/4.5-0.5) =3.84 3.84

C2 16.0 4.0 4.00 4.75 + log( ¼) =4.27 4.27

C3 14.0 6.0 4.44 4.75 + log (1.5/3.5-1.5) =4.62 4.62

C4 12.0 8.0 4.68 4.75 + log (2/3-2) =5.05 5.05

C5 10.0 10.0 5.32 4.75 + log (2.5/0) UNDEFINED

Equation of the line:Y=0.383X =2.827
R2: 0.9828
pKa : 4.76
Ka : 2.827

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PART D: Ph CHANGES

INITIAL PH FINAL PH CHANGE OF PH

a 1 drop 0.25 M HCl IN BUFFER 3 4.53 4.51 0.02

b 5 drop 0.25 M HCl 4.53 4.32 0.2

c 1 drop 0.25 M NaOH 4.53 4.58 -0.05

d 5 drops 0.25 M NaOH 4.53 4.71 -0.28

e 1 drop 0.25 M HCl IN WATER 7.00 2.68 4.32

f 5 drop 0.25 M HCl 7.00 2.37 4.63

g 1 drop 0.25 M NaOH 7.00 10.38 3.38

h 5 drop 0.25 M NaOH 7.00 11.35 -4.35

-
Using Buffer 3, after the addition of 0.25 M HCl there was only a 0.02-0.2 change. With the
addition of drops of the NaOH, that change was -0.05—0.28. Without the buffer solution, in water,
with the addition of 1-5 drops of HCl, there was a marked increase in the pH. A range of 4.32-
4.63 was observed. With the additions of drops of NaOH, a similar marked change was observed
with a range of -4.35 to 3.38.

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INTERPRETATION

PART B

pH vs. Log (salt/acid)

5 4.93

4.53
4.33
4
3.91
pH

0
-0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6
Log (salt/ acid

y = 1.053x + 4.533
R² = 0.999

For this table the equation of the line is y= 1.053x + 4.533. The R2 is less than 1. The R²
represents how much variation in the data can be explained by the model. This means that we can safely
assume the pH is indeed directly caused by changes in the concentration of the salt and the acid.

PART C

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 pH vs. log (base/acid=base)
5
4.68
4.44
4.5
4
4
3.55
3.5

3
ph

2.5

1.5

0.5

0
0 1 2 3 4 5 6
y = 0.383x + 2.827
log (base/ acid-base) R² = 0.9828

In this table, the equation of the line is y= 0.383x + 2.827. The R2 is 0.9828. This indicates that
the graph can account for 98.28% of the relationship between the pH and the concentrations of
the buffers in the given solution. This indicates that we can safely assume that the pH is affected
by the concentration of the base and remaining acid after the solutions were mixed.

PART D.

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Using Buffer 3, after the addition of 0.25 M HCl there was only a 0.02-0.2 change. With the
addition of drops of the NaOH, that change was -0.05—0.28. Without the buffer solution, in water,
with the addition of 1-5 drops of HCl, there was a marked increase in the pH. A range of 4.32-
4.63 was observed. With the additions of drops of NaOH, a similar marked change was observed
with a range of -4.35 to 3.38.

Using Buffer 3, there was only a small change in pH due to the effect of the Buffer. (Please see
answer in Post lab question D). With water, the absence of a buffer was observed and so
changes in the pH were marked as compared to that with only a buffer.

CONCLUSION
With this lab and the data obtained, it can be observed that pH is a function of the concentration
of the acid and its base. It can be concluded that buffers indeed control or limit the changes in
the pH upon the addition of small amounts of acids and bases.

POST LAB QUESTIONS

1. Why can weak acid be used to make buffer but not strong acids?
Buffers cannot be made from a strong acid (or strong base) and its conjugate. This is because they
ionize completely! Weak acids and its conjugate base however can be made into a buffer

2. What would you expect to be the slope of the lines in Part B and C?
The slope of the lines are the pka of the acids and the salts involved.

3. Explain the observations in Part D

A mixture containing acetic acid and sodium acetate is acidic because the Ka of
acetic acid is greater than the Kb of its conjugate base which is acetate. A buffer
because it contains both the weak acid and its salt. Therefore, it will acts to keep
the hydronium ion concentration (and the pH) constant through the addition of
either of a strong acid or a strong base. Once a base is added such as sodium
hydroxide, the hydroxide ions react with the few hydronium ions present. Then
more of the acetic acid reacts with water. This restores the hydronium ion
concentration almost to its original value:

CH3CO2H(aq)+H2O(l)⟶H3O+(aq)+CH3CO−2(aq)(14.6.1)(14.6.1)CH3CO2H(aq)+H
2O(l)⟶H3O+(aq)+CH3CO2−(aq)

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 The pH demonstrates very little change. Once an acid is added such as
hydrochloric acid, most of the hydronium ions from the hydrochloric acid combine
with acetate ions, forming acetic acid molecules:
H3O+(aq)+CH3CO−2(aq)⟶CH3CO2H(aq)+H2O(l)(14.6.2)(14.6.2)H3O+(aq)+CH3C
O2−(aq)⟶CH3CO2H(aq)+H2O(l)

Still, there is very little increase in the concentration of the hydronium ion, and the
pH remains practically unchanged. This demonstrates buffers in action.

4. If 1 drop of acid is equal to 50 microliter. Calculate the concentrations of H+ ions and the
pH of the solution when 1 drop of 0.25 M HCl is added to 3 mL of water. Does that
conform to your observation in part D . if not, why?

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REFERENCES

 https://chem.libretexts.org/Bookshelves/General_Chemistry/Book%3A_Chemistry_(OpenSTAX)
/14%3A_Acid-Base_Equilibria/14.6%3A_Buffers
 https://search.yahoo.com/yhs/search?hspart=iba&hsimp=yhs-
syn&type=asbw_8063_CHW_US_tid1104&p=if%201%20drop%20of%20acid%20is%20eq1ual%2
0to%2050%20microliter.%20Calculate%20the%20concetrationof%20H%2B%20ions%20and%20t
he%20pH%20of%20the%20solution%20when%201%20drop%20of%200.25%20M%20HCl%20is
%20added%20to%203mL%20water.%20Does%20that%20conform%20to%20your%20observatio
n%20in%20part%20D.%20if%20not%2C%20why

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