Name _______________________________ Date _______________ Pts ______/10

EXPERIMENT 2: PREPARATION OF BUFFERS OF DIFFERENT pH

Adapted from "Experiments in biochemistry: A hands on approach" by Ferrell and Taylor, 2nd edition

PRE-LAB QUESTIONS:

The following preparatory questions should be answered before coming to lab. They are
intended to introduce you to several ideas that are important to aspects of the experiment. You
must turn in your work to your instructor before you will be allowed to begin the
experiment.

1. What is a buffer solution? How you make an acetate buffer solution having pH of 5.2?

2. Calculate the weight of buffer you would use to make a buffer for part A.

3. If you are given HEPES in the basic form and ask you to make a buffer of pH 8.0, will you add
HCl or NaOH to adjust the pH? Why? (With commercial buffers, there is always an acid form and
a basic form that can be bought. It is not obvious from the name of the compound, so look to see if
it is acidic or basic.

1
Name _______________________________ Date _______________ Pts ______/10

EXPERIMENT 2: PREPARATION OF BUFFERS OF DIFFERENT pH

Adapted from "Experiments in biochemistry: A hands on approach" by Ferrell and Taylor, 2nd edition

MATERIALS REQUIRED:
Acetate
CAPS
HEPES
Citrate
Phosphate
Tricine
Tris
pH meter
Distilled water
1M HCl
1M NaOH

Use this space to take notes

2
Name _______________________________ Date _______________ Pts ______/10

EXPERIMENT 2: PREPARATION OF BUFFERS OF DIFFERENT pH

OBJECTIVES:
Upon successful completion of this lab, you will be able to
 Calculate the pH of solutions of strong acids or bases, weak acids or bases, buffers, and/ or
combinations of these.
 Explain how a buffer resists change in pH
 Prepare an appropriate buffer for a given pH.
 Calculate the theoretical pH of the buffer after adding a known quantity of acid or base
 Predict the effect of changing temperature and concentration on buffer pH.
 Standardize and operate a pH meter.
Waste Disposal
All unknowns can be dumped down the sink at the week’s end. Save the HCl and NaOH for future labs.
Discard or save dry buffers depending on quality and quantity.

INTRODUCTION:
Buffers:
A buffer is a solution of a weak acid and its conjugate base. It resists pH change when reasonable amounts
of both forms are present. A buffer is best when used close to its pKa. Buffers are made by taking a weak
acid and titrating with a strong base until the pH is correct or taking a weak base and adding a strong acid
until the pH is correct. A buffer can protect against pH changes from added hydrogen ion or hydroxide ion
as long as there is sufficient basic form and acid form, respectively. As soon as you run out of one of the
forms, you no longer have a buffer. To be a good buffer, the pH of the solution must be within 1 pH unit of
the pKa. The pKa gives an idea of the strength of the acid and tells us what the pH will be when there are
equal amounts of acid form and basic form present.

The original buffers used in the lab were made from simple weak acids and bases, such as acetic acid,
phosphoric acid, and citric acid. There are some limitations of using these buffers. (a) They often change
pH when diluted or temperature changed, and (b) they permeated cells in solution, thereby changing the
chemistry of interiors of the cells. It has been found that buffers such as HEPES, TRIS, TIS, MOPS,
PIPES, etc. does not change their pH with temperature and dilution as well as they do not permeate the
cell. Even though the name of the buffer compounds look different and the structures of these compounds
might be complicated, you do not really need to know the structure to use a buffer correctly. The important
consideration is the pKa of the buffer and the concentration you want. The Handerson - Hasselbalch
equation works just fine whether or not you know the structure of the compound in question.

3
Handerson – Hasselbalch equation:

pH = pKa + log [salt]/[Acid]

Table 1: Acid and base form of some useful biochemical buffers.

METHOD:
Part A: preparation of buffers
Make one buffer starting with solid material, which is the most common way to make buffers. You will be
given a desired pH, and your task is to prepare 100mL of the buffer at a concentration of 0.10 M.
 Using the following table, choose the most appropriate buffer compounds for your pH. Proceed with
steps 2-7 for both buffers.
Buffers pKa1 pKa2 pKa3 formula weight (g/mol)
Acetate 4.76 136.1
CAPS 10.4 221.3
Citrate 3.06 4.74 5.40 294.1
HEPES 7.55 238.3
Phosphate 2.12 7.21 12.32 142.0
Tricine 8.15 179.2
TRIS 8.3 121.1

4
 Calculate the weight of the buffer you would need to make 100 mL of a 0.100 M solution. Weigh out
the correct amount and dissolve in 50 mL water.
 Standardize the pH meter at pH 4, 7, and 10. Set up the beaker with your buffer solution on a stir plate
such that you can stir the solution and read the pH continuously.

CAUTION!!!! Never stir the solution with pH sensor. pH sensor is very fragile and expensive to
replace.

 If you have no stir plate, just swirl the beaker often or use a glass rod while adding acid or base.
 Use 1 M NaOH or 1 M HCl to titrate to the desired pH. Add the acid or base a drop at a time. By doing
this, you effectively change some of the acid form to the basic form or vice versa until the ratio is the
correct one to give you the pH you want.
 Add water until the volume is about 99 mL.
 Recheck the pH to make sure it has not changed. If it has, correct it with NaOH or HCl. Warning! You
might want to use a lower concentration of NaOH or HCl because the pH change with concentrated
HCl or NaOH will be big.
 Bring the volume to 100 mL and save this solution for later.

Part B: Effect of concentration on pH:

For this part, you must have a digital pH meter.
 Take 5 mL of your buffer and dilute with deionized water to 50 mL to give a final concentration of
0.01 M. Save these solutions.
 Take 5 mL of your diluted buffers from step 1 and dilute to 50 mL to a concentration of 0.001 M.
Save these solutions.
 Measure the pH of the two diluted solutions.

Part C: Why a buffer is a buffer

 Put 50 mL of one of your original 0.1 M buffers in a beaker. If your buffer has a pH higher than its
pKa, add 0.5 mL of 1 M HCl to it. Record the new pH. If your buffer has a pH lower than its pKa, add
0.5 mL of 1M NaOH to it. Record the new pH.
 Repeat step 1, but use 50 mL of water instead of your buffer. Add either acid or base, depending on
what you did in step 1.

5
RESULTS:

Data
Part A
Buffer #1: _____________ grams: _________ Original pH: ___________

Part B
Buffer #1 pH of 0.1 M (original pH) _______ pH of 0.01 M _______ pH of 0.001 M _______

Part C
Buffer chosen: _____________ pH: ___________ pKa:___________

Acid or base added:_____________

pH after adding acid or base: ______________

pH of 50 mL of water:__________

pH after adding acid or base: ____________

POSTLAB QUESTIONS:
1. Answer the following questions
(a) What is the ratio of A-/HA in your buffer after you adjusted its pH to the required value?

(b) How many milimoles of A- and HA are present in the solution?

6
(c) If you now add 3 mL of 1M NaOH, will you still have a valid buffer?

2. What would be the most efficient way to make up a HEPES buffer at pH 8.5? What starting
compounds and reagents will you use?

3. If you make up a solution of 50 ml 0.1M TRIS in the acid form, what will be the pH?
Use pH = pKa - log[HA] equation to calculate the pH of the solution.
2

4. If you add 2 ml of 1 M NaOH to the solution in 3, what will be the pH?