MARIANO MARCOS STATE UNIVERSITY

City of Batac, Ilocos Norte, Philippines

College of Medicine

Experiment 3

Preparation of Buffer Solutions

Experiment 4

Experimental Determination of pH
Experiment 5

Buffer Action
Biochemistry Laboratory

Submitted by:

Agustin, Aileen Concepcion M.

Doloroso, Gianfranco Emmanuel M.

MD-1

Dr. Gracia Urubio Urmeneta

Date of Experiment:

August 10, 17, 2015

Date of Submission:

August 2015
 MARIANO MARCOS STATE UNIVERSITY
City of Batac, Ilocos Norte, Philippines
College of Medicine
ABSTRACT

The purpose of this laboratory activity was to understand

INTRODUCTION

The experiment was conducted

METHODOLOGY

Experiment 3

Prepare a 0.10 M buffer solution using 0.20 M salt solutions whose pH and volume will be
assigned to you. The pH range will be between 2.0 to 8.2. Show your calculations for the
volumes of the acid (base) and the salt to be used. Measure the calculated volumes using
burettes. Mix and check the pH using a pH meter. Adjust the pH if necessary.

Prepare the following solutions and keep them for experiment 4:

1. 200 ml of 0.10 M NaOAc/HOAc buffer, pH 3.8

2. 200 ml of 0.10 M NaOAc/HOAc buffer, pH 4.7
3. 200 ml of 0.10 M Na2HPO4/NaH2PO4 buffer, pH 6.8
4. 200 ml of 0.01 M Na2HPO4/NaH2PO4 buffer, pH 6.8

RESULTS AND DISCUSSION

Questions

1. Review the following: a) concepts of acids and bases, b) methods of expressing

concentrations of solutions, c) Ka, pKa, pH, Kb, pOH, Kw, pKw.

2. Derive the Henderson-Hasselbach equations for a solution of a weak acid, HA, and its
salt, NaA. Do the same for a solution of a weak base, MOH, and its salt, MA.

3. What is the biological importance of pH?

4. How do you determine the concentration of a buffer solution?

 MARIANO MARCOS STATE UNIVERSITY
City of Batac, Ilocos Norte, Philippines
College of Medicine
5. Explain the possible discrepancy between the calculated pH and the experimental pH of
the prepared buffer.

6. A buffer solution is prepared by mixing 200 ml of 0.20 M salt solution and 400 ml of 0.2 M
acid solution. What is the concentration of the resulting buffer solution?

7. The pKa of HOAc is 4.7. Calculate the concentration of NaOAc that must be present in 0.1
M solution of acetic acid so that the solution will have a pH of a) 5.7 and b) 3.7.

8. How would you prepare 400ml of a 0.10 M buffer solution with a pH of 6.6 from 0.2 M
solutions of acid and its salt?

9. Calculate the pH of a solution prepared by mixing 80.0 ml of 0.20 M NaH 2PO4 and 120.0
ml of 0.20 M NaH2PO4 solution.

METHODOLOGY

Experiment 4

A. Preparation of Colorimetric Buffer Standard Solutions

Prepare the colorimetric buffer standard solution assigned to you by pipetting 4.0 ml of the
buffer into a vial and adding 2 drops (small vial dropper) of the indicator for this pH. (Refer to
the preceding table). Label the vial with the pH and the initials of the indicator and submit to the
instructor.

B. Colorimetric Determination of pH

Determine colorimetrically the pH of 0.01 N HCl, 0.01 N HOAc and 0.2% gelatin solution.

Determine the approximate pH range of the samples by using either a universal indicator
(example: Bogen indicator) or a set of trial indicators. If Bogen indicator is being used, touch its
dropper to a cavity of your spot plate and add a drop of the solution. The colors given below will
indicate the approximate pH.

Color pH
Red 2.0
Orange 4.0
Yellow 6.0
Green 8.0
Blue 10.0
 MARIANO MARCOS STATE UNIVERSITY
City of Batac, Ilocos Norte, Philippines
College of Medicine
A set of three trial indicators:

Trial Indicators pH range Color Change

Methyl Red-Methylene Blue (MR-MB) 4.5-6.0 Purple-Green
Methyl Orange (MO) 3.1-4.4 Red-Yellow
Phenolpthalein (H2Ph) 8.3-10.0 Colorless-Red

The pH range of the sample is first determined by the use of methyl red-methylene blue
(MR-MB). Place 2 drops of the sample in a cavity of a spot plate and add a drop of MR-MB. If
the color obtained is purple or pink, the pH of the sample is 4.5 or below. Place another 2 drops
of the sample in a cavity of a spot plate and add a drop of methyl orange (MO). If the color
obtained with MO is red, then the approximate pH range of the sample is 3.1 or below; if orange,
the pH range is between 3.1 to 4.4. If the color obtained with MR-MB is green, the pH of the
sample is 6.0 or above. Place another 5 drops of the sample in the spot plate and add a drop of
phenolphthalein (H2Ph)). If the color with H2Ph is colorless, the pH range is between 6.0 to 8.3,
if it is red or red, the pH is 8.3 or above.

Sample

MR-MB

Purple (pH ≤ 4.5) Brown (pH = 4.6-5.9) Green (pH ≥ 6.0)

MO H2Ph

Red Orange Yellow Colorless Pink Red

(pH ≤ 3.1) (pH = 3.2-4.3) (pH ≥ 4.4) (pH ≤ 8.2) (pH = 8.3-9.9) (pH ≥ 10.0)

Knowing the approximate pH range, make the final pH determination by pipetting 4.0 ml of
the solution into a vial and adding 2 drops (small vial dropper) of the indicator used in the
preparation of the colorimetric buffer standards covering this pH range. Compare with the buffer
 MARIANO MARCOS STATE UNIVERSITY
City of Batac, Ilocos Norte, Philippines
College of Medicine
standards containing this same indicator. Use a clean piece of bond paper as background. The pH
of the solution is the pH of the buffer standard that matches closely in color intensity.

For solutions that are slightly colored or turbid like the gelatin solution, place a vial of
distilled water behind the gelatin with the indicator and a vial with gelatin behind the buffer
standard during the color comparison.

Calculate the hydrogen ion concentration of the sample tested.

C. Electrometric Determination of pH

Determine the pH of the following samples using the pH meter: soft drinks, vinegar, filtered
fresh pineapple juice, calamansi juice, urine, filtrate from soil suspension, sea water, wine.

RESULTS AND DISCUSSION

Questions

1. Compare the true or actual acidities of 0.01 N HCl and 0.01 N HOAc and account for the
differences, if any. How would their total or titratable acidities compare?

2. Can the colorimetric method be used to determine the pH of water? Milk? Blood? Why?

3. What physical qualities of vinegar, fruit juices, soft drinks, and wine are affected by pH?

METHODOLOGY

Experiment 5

A. Prepare the following samples:

1. Distilled H2O (freshly boiled in an Erlenmeyer flask and cooled under tap H2O with a
cover)
2. 0.10 M NaOAc
3. 0.10 M HOAc
4. 0.1 M acetate buffer (pH 4.74)
5. 0.1 M acetate buffer (pH 3.8)
6. 0.1 M phosphate buffer (pH 6.8)
7. 0.01 M phosphate buffer (pH 6.8)
8. Fruit homogenate or juice
9. Animal tissue homogenate, blood, albumin, or egg white
 MARIANO MARCOS STATE UNIVERSITY
City of Batac, Ilocos Norte, Philippines
College of Medicine
B. Pipet 20.o ml each of samples 1, 2, 3, 4, 8, and 9 into separate beakers. Determine the pH
electrometrically. Add 0.1 ml of 0.01 M NaOH to solutions 1-4. For solutions 8 and 9, add
0.01 ml of 0.01 M NaOH. Mix well. Determine the pH up to the second decimal place.
C. Repeat B adding 0.1 ml of 0.01 M HCl instead of NaOH to solutions 1-4 and 0.01 ml of 0.01
M HCl to solutions 8 and 9.
D. Buffer Capacity
*Pipet 20.0 ml each of samples 4-7 and determine the pH electrometrically. To each of the
solutions, add 2.5 ml of standard 0.1 N NaOH. Mix well and determine the pH
electrometrically. Repeat from * but use 0.1 N HCl instead of NaOH. Calculate the buffer
capacity of the four solutions.

RESULTS AND DISCUSSION

Questions

1. Which of the samples exhibit buffer action? Give the chemical equations involved.

2. Represent with ionic equations the buffering action of a HCO3-/H2CO3 system.

3. Why should the distilled water be boiled and cooled before determining its pH?

4. What factors affect buffer capacity?

5. At what pH does a buffer solution exhibit maximum buffer efficiency?

6. What are universal buffer solutions?

7. What is the pH of a buffer solution containing equal volumes of 0.10 M HOAc and 0.10 M
NaOAc? Calculate the pH when a) 0.01 mole of HCl has been added to 1 liter of the
buffer; b) 0.01 mole of NaOH has been added to 1 liter of the buffer.

CONCLUSION

REFERENCES

1.