Unit 1

Preparing Stock and Buffer Solution

1.1. Course Objectives

This unit aims to provide training and practice for students to prepare and
dilute stock solution. This unit also covers training and practice for the preparation
of some buffer solutions, and measure its capacity to be used for chemical and
biochemical analyses.

1.2. Course Learning Outcomes

After completing this unit, students are expected able to:
1. Prepare solution with various concentration diluted from stock solution.
2. Calculate acid and conjugates used to prepare buffer solution.
3. Prepare buffer solutions and determine its buffer capacity.

1.3. Basic Principles

1.3.1. Preparing Stock Solution
Most experiments and research in food chemistry and biochemistry use liquid
solution. The solution should be accurately prepared to give precise concentration,
even low concentration up to micro-molar (M). A solution with low concentration
is difficult to prepare from powdered materials or solids, because they have to
weighed at a very small amount. To overcome this problem, a stock solution of
higher concentration is prepared and then diluted to obtain a solution with the
desired concentration.
In making the solution, it should be noted that the volume of solution should
meet the experimental treatment or analysis to be performed. The volume of solution
should be calculated carefully, especially for solutions made from expensive
chemicals, difficult to obtain or must always be freshly prepared. In the preparation
of the solution, stirring, heating or addition of acid/alkaline aqueous treatment is
often necessary.

6
Food Chemistry and Biochemistry Laboratory Manual 7

Dilution should be done properly, because it will determine the final calculation
of experimental data. The volume of solvent for dilution is calculated using the
following equation:

V1 x C1  V2 x C2 (1.1)

where: Cn = concentration of solution (n = 1,2)

Vn = volume of solution at each concentration (n = 1, 2) usually in mL

Example 1:
To prepare 25 mL of protein solution (5 mg/mL) from 25 mL of stock solution
(60 mg/mL protein), how much stock solution should be used?
Answer:
By using equation 1.1, the volume of stock solution required is:

V1 x C1  V2 x C2
V1 x 60 mg/mL = 25 mL x 5 mg/mL
25 ml x 5 mg/mL
V1  = 2.08 mL
60 mg/mL

A total of 2.08 mL stock solution is taken and added about 22.92 mL of diluent
solution (usually water) to a volume of 25 mL.
1.3.2. Buffer Solution
A buffer solution serves to maintain the stability of pH in foods. The buffer
solution can also be used to neutralize acidic foods as well. Table 1.1 shows the pH
of some foods. Data shows that most foods have a pH value of <7.
All biochemical reactions occur in aqueous solution at approximately neutral
pH that should be maintained. The little change in pH may alter the enzyme activity
involved in cell metabolism. A buffer system is necessary for the cell physiological
pH to remain stable. Fluids in every living cell have a certain buffer capacity.
Several buffers are naturally available in foodstuffs. In animal products, buffer
systems are usually in the form of amino acids, protein and phosphate salts, whereas
in vegetable products in the form of organic acids (citric acid, malic, oxalic, and
tartaric) that binds with phosphate salts.
8 Preparing Stock and Buffer Solution

Table 1.1. Approximate pH of some foods materials

Food sources pH Food sources pH
Lime 2.0 Pumkin, carrot 5.0
Lemon 2.2 Cucumber 5.1
Vinegar, plum 2.9 Turnip, cabbage, squash 5.2
Gooseberry 3.0 Parsnip, beet 5.3
Prune, apple 3.1 Sweet potato, bread 5.4
Rhubarb, dill pickle 3.2 Spinach 5.5
Apricots, blackberry 3.3 Cauliflower, asparagus 5.6
Strawberry 3.4 Meat (ripened) 5.8
Peach 3.5 Tuna 6.0
Rasberry, sauerkraut 3.6 Potato 6.1
Orange, blueberry 3.7 Pea 6.2
Sweet cherry 3.8 Corn, oyster, date 6.3
Pear 3.9 Egg yolk 6.4
Acidophilus milk 4.0 Milk 6.6
Tomato 4.2 Shrimp 6.9
Buttermilk 4.5 Meat (unripened) 7.0
Banana, pimento 4.6 Egg white 8.0

1.3.2.1. Henderson-Hasselbach Equation

Protonation or deprotonation of groups (carbonyl, carboxyl, amino, hydroxyl,
sulfhydryl, phosphate and methyl) in food components play an important role to
changes in pH. This phenomenon can be determined by the Henderson-Hasselbach
equation (1.2) to calculate (a) the value that produces ionic environment with certain
conditions, or (b) the degree of protonation or deprotonation of a group at a parti-
cular pH. This equation is derived from the equilibrium state for the dissociation of a
weak acid.
[A  ]
pH  pKa  log (1.2)
[HA]
where: [A-] deprotonated form
[HA] protonated form.
In the state in which pH = pKa, then log ([A-]/[HA]) = 0 and [A-]/[HA] = 1. This
means that at pH equal to pKa, deprotonated and protonated form are the same.
This situation also applies to the base by replacing the notation [A -] with [B] as the
deprotonated form and [HB+] replace [HA] as the protonated form. This equation
can be used to create a titration curve or ionization curve as shown in Figure 1.1.
From these curves, it can be seen that:
 At pH = pKa, then half the acid is deprotonated. In other words, the amount of
protonated form is equal to the deprotonated form.
 If the pH is 1 unit above (or below) pKa, then 90% in the group deprotonated
form (or protonated).
Food Chemistry and Biochemistry Laboratory Manual 9

 If the pH of 2 units above (or below) pKa, then> 90% of the group in the form of
deprotonated (or protonated).
 If the pH of 3 units above (or below) pKa, then 99% of the group in the form of
deprotonated (or protonated).
For example, amino acids having -carboxylate group with a pKa=2.4 is
deprotonated at pH 7, while its -amino groups with a pKa of 9.6 will be fully
protonated at pH 7. If pKa and concentration are known, then the pH can be
calculated. Conversely, if the pH and pKa is known, then the value of the concen-
tration ratio can be calculated.
Almost all biochemical systems consist of a mixture of various components, for
example the various substances in the cells. In the experiment, buffer solution to
maintain pH and the reactants are usually added. Buffers are usually used in
sufficient concentration, which is about 0.1 M. It is important to note that that a
buffer solution should give pKa values approaching the desired pH. Two buffer
solutions are commonly used are dihydrogen phosphate with a pKa of 6.8, and a
weak base of tris-hydromethylaminomethane which has pKa of 8.08. The desired pH
can be achieved by mixing the protonated and deprotonated forms appropriately. It
can also be obtained by adding NaOH or HCl solution.

Figure 1.1. Titration curve of acetic acid with the addition of alkali solution

Example 2:
Calculate the pH of a buffer consisting of [tris] = 0.025 M and [TrisH +] = 0.075 M,
with pK a = 8.08 to TrisH+
Answer:
In order to calculate this pH, concentration (M) of protonated form (TrisH+) and
a deprotonated form (tris) buffer component must be known and then inserting
them in the following equation:
pH = pKa + log [deprotonated]/[protonated]
pH = 8.08 + log 0.025 = 7.6
0.075
10 Preparing Stock and Buffer Solution

This buffer should be made by weighing a certain amount of tris base (molar
mass 121.1 g/mole) and trisHCl (molecular weight of 157.6 g/mole) and dissolving
it in a certain volume.
The common method is to weigh a certain amount of tris base, and HCl gra-
dually in order for partial neutralization against tris base. For example, if the 0.075
mol tris base is added to 50 mL of 1.00 M HCl in order to have volume of up to 0.5 L,
the calculation should take into account the pH of the reaction that occurs before the
concentration of protonated and deprotonated form are calculated. Thus, it will be
obtained that:
The number of initial mole Tris = 0.075 mol
The number of initial mole HCl = 0.050 mol
The number of mole of H+ formed Tris = 0.050 mol
The number of mole Tris base remaining = 0.025 mol, so
pH = 8.08 + log 0.025 = 7.78
0.050

For the above calculation, molar concentration should be used rather than
number of mole. However, if the volume is the same, it will neglect each other.
Moreover, error often occurs because of the use of the initial number of moles of tris
base, and not the number of moles of tris remaining bases.
Reactants in the biochemical systems are in low concentrations, ie. in the range
of 10-2 to 10-5 M, or even smaller. Therefore reactants do not affect significantly
change the buffer pH. Instead, pH of buffer solution will dominantly affect the state
of ionization of functional groups in the reactants.
Example 3:
Calculate the pH buffer solution composed of 0.100 mol of NaH2PO4 and 0.040
mol of NaOH with a total volume of 1:00 L solution
Answer:
To answer this question, we must first calculate the result of the reaction
between H2PO4- with a strong base OH-, so that we obtain buffer mixture. Because
H2PO4- is in excess, the amount of HPO42- is equivalent with OH-, and the remaining
portion will be H2PO4-. Changes that occur until the achievement of buffer condi-
tions can be summarized in Table 1.2. If the concentration in the final state is
inserted into the equation Henderson-Hasselbach, then
0.040 mol
pH = 6.76 + log = 6.76 - 0.18 = 6.58
0.060 mol

Table 1.2. Changes of buffer solution

State H2PO4- OH- HPO42-
Initial 0.100 mole 0.040 mole 0
Endpoint 0.060 mole 0 0.040 mole
Food Chemistry and Biochemistry Laboratory Manual 11

Henderson-Hasselbach equation can be used to (1) show that the addition of

small amounts of acid or base in a buffer solution only result to a change in pH than
when added to water or saline solution of strong acids or strong bases, such as NaCl;
(2) calculate the amount of acid or base added to the solution to achieve a particular
pH (or for preparation of buffer); and calculate the pK that is based on data obtained
from experiments.
Example 4:
To determine the stability of the pH buffer above, compare the effect of adding
100 mL of HCl 0:01 M into 1 L of water at pH 7 and in 1 L of buffer 1 M pH 7
(HA/A-). Assuming tat pKa of HA is equal to 7.
Answer:
A 100 mL of HCl 0.01 M contains 0.001 mole of HCl. In acid-water mixture, the
concentration of hydrogen ions changes into 0.001 mole/1.1 L = 0.000909 M, so that
the pH can be calculated as follows:
pH = -log 9.09x10-4 = 0.96 + 4 = 3.04
When the acid is added to the buffer, then most of the proton from HCl will join
the A-:
HCl  A  HA  Cl 
To calculate the concentration of H+ in buffer solution after the addition of HCl,
the proton joins with A- should be calculated. It is important to note that at pH 7,
[HA] = [A-] = 0.5 M, when the HA pKa equal to 7. Buffer concentration is similar to
the concentration of acid plus conjugate base concentration. Addition of 0.001 mol
HCl into the system produces [HA] = 0501 mole/1.1 L and [A -] = 0499 mol/1.1 L.
Based on the equation of Henderson-HasselBach, then it can be calculated:
0.499/1.1
pH  7.00  log  7.00  0.02  6.98
0.501/1.1

It can be seen that the buffer prevent pH changes that occur when the acid
solution is added to water.
1.3.2.2. Buffer capacity
Henderson-Hasselbach equation can be used in the preparation of buffer solu-
tion with a specific pH and in the determination of buffer capacity. Buffer capacity
can be defined as follows: (a) the number of moles of H + or OH- required to indicate
a change in pH in 1 liter of buffer solution; or (b) changes in pH that occur when the
amount of acid or base is added to 1 liter buffer. Buffer capacity is a function of
buffer concentration, where the solution with a higher concentration will have a high
buffer capacity.
Example 5 :
Calculate the amount of acetic acid (pKa = 4.75, Ka = 1.6x10 -5) and sodium
acetate needed to make 1 L solution of 0.2 M acetate buffer with pH of 4.5?
Answer:
12 Preparing Stock and Buffer Solution

To answer this question, we must use Henderson-Hasselbach equation. HA=A-

= acetic acid and sodium acetate. In the buffer 0.2 M, then [HA] + [A -] = 0.2 M. When
[A-]=x, then [HA]=0.2x. Subsequently, incorporate it into the Henderson-Hasselbach
equation:
[A  ]
pH  pKa  log
[HA]
x
4.5  4.75  log
0.2  x
0.2  x
0.25  log
x
0.2  x
1.78   x  0.07
x

To make 0.2 M buffer pH 4.5, 0.13 mole of acetic acid and 0.07 mole sodium
acetate is required, then dissolved into 1 L.
Example 6:
Determine the amount of 0.1 M HCl needed to change 1 L 0.2 M acetate buffer
with pH 4.5 to pH 3.5?
Answer:
Calculate the concentration of OH at pH 3.5 and pH 4.5. Enter in the Henderson-
Hasselbach equation as follows:
[A  ]
pH  pKa  log
[HA]
x
3.5  4.75  log
0.2  x
0.2  x
log  1.25 , then x = 0.01
x
At pH 3.5, the concentration of HA = 0:19 M and the concentration of A- = 0:01
M. [HA] at pH 4.5 is 0.13 M. Thus, in an acid buffer capacity is 0.19 to 0.13 = 0.06
mole/L. In alkaline buffer capacity can be calculated as follows:
x
5.5  4.75  log
0.2  x
x = 0.17
so that A- = 0.17 and buffer capacity is 0.17 – 0.07 = 0.1 mole/L

1.4. Procedures
1.4.1. Experiment 1. Preparing Buffer Solution and Dilution
Equipment
 Analytical balance
 Volumetric flask
 Erlenmeyer flask
Food Chemistry and Biochemistry Laboratory Manual 13

 Porcelain cup
 Volumetric pipette
Reagents
 Standardized solution
 NaOH 2 N
 KOH 1 M
 HCl 8 M
 CH3COOH 1 M
 NaCl 1 M
Procedure
1. Calculate the amount of reagents to make a stock solution. Calculate also the
amount of stock solution needed for dilution to prepare a solution of various
concentrations.
2. Weigh reagents in accordance with the results of calculations to make a stock
solution with a concentration of 1 M. Take into account purity of reagents as
stated by manufacturer.
3. Make a serial dilution to obtain the solution with lower concentrations (0.1, 0.25,
0.4).
4. Use glassware according to its function and purpose. Remember, in preparing
diluted solution, do not allowed to use a beaker glass. Use a volumetric flask
instead.
1.4.2. Experiment 2. Preparing Buffer Solution
Equipment
 Analytical balance
 pH meter
 Volumetric flask
 Erlenmeyer flask
 Porcelain cup
 Volumetric pipette
Reagents
 Glycine-HCl pH 3 buffer solution
 Sodium Citrate - NaOH buffer pH 6
 Phosphate buffer pH 6
 Acetic acid pH 4
 Tris-HCl buffer pH 8
 Citrate - Phosphate buffer pH 3
Procedure
1. To set up a buffer solution, prepare the acid and conjugated base, then calculate
the amount to be mixed, either with acid or conjugated base, and then adjust the
pH with acids or strong bases, at the precise volume. The pH value is checked
and adjusted before it is used to prepare a buffer at desired concentration.
14 Preparing Stock and Buffer Solution

2. Buffer solutions may have slight different concentration compared with the
calculation. It is better to use a standard formula to create a buffer with the
desired pH.
3. Create a buffer solution. Prepare the buffer solution according to the instructions
of making the buffer solution directly in Table 1.3-1.5.
4. Measure pH buffer solution is by using a pH meter and compare with the results
of calculations.

Table 1.3. Standardized buffer, effective pH and temperature

# Buffer pH Range Temp (oC) pH/K
1 Glycine -HCl 1.2 – 3.4 Room 0
2 Na citrate-HCl 1.2 – 5.0 Room 0
3 Phosphate 5.0 – 8.0 20 - 0.003
4 Citric acid-phosphate 2.2 – 7.8 21
5 Acetate 3.5 – 5.6 25
6 Tris-HCl 7.2 – 9.0 23
Table 1.4. Stock solution and formulation to obtain bufer solution
Stock Solution
Buffera Formulac
A B
1 0.1 M Glycine 0.1 M HCl x mL A + (100 – x) mL B
2 0.1 M disodium citrate (21.01 citrate 0.1 M HCl x mL A + (100 – x) mL B
monohydrate salt + 200 mL 1M HCl
diluted up to 1.0 L)
3 1/15 M potassium dihydrogen 1/15 M disodium x mL A + (100-x) mL B
phosphate phosphate
4 0.1 M citric acid 0.2 M disodium x mL A + (100-x) mL B
phosphate
5 0.1 M sodium acetate 0.1 M acetic acid x mL A + (100-x) mL B
6 0.2 M tris (24.23 g tris diluted up to 1.0 L) 0.1 M HCl 25 mL A + x mL B,
diluted up to 100 mL
a See Table 1.3 for buffer
b Stock solution is prepared using free-CO2 destiled water and and chemicals for analysis (p.a)

Table 1.5. The x value for calculation used in Table 1.4

Buffer a
pH
1 2 3 4 5 6
1.2 11.1 9.0
1.4 26.4 17.9
1.6 36.2 23.6
1.8 43.9 27.6
2.0 50.7 30.2
2.2 56.5 32.2 98.8
2.4 62.3 34.1 94.5
2.6 74.4 36.0 90.0
2.8 68.4 37.9 85.1
3.0 81.0 39.9 80.3
3.2 42.1 76.0
3.4 44.8 72.0
3.6 47.8 68.4
3.8 51.2 65.1 10.9
4.0 55.1 62.0 16.6
4.2 60.0 59.1 23.9
Food Chemistry and Biochemistry Laboratory Manual 15

Buffer a
pH
1 2 3 4 5 6
4.4 66.4 56.4 33.5
4.6 74.9 53.7 44.9
4.8 85.6 51.2 56.6
5.0 100 99.2 49.0 67.8
5.2 98.4 46.9 76.8
5.4 97.3 44.7 84.0
5.6 95.5 42.4 89.3
5.8 92.8 40.0
6.0 88.9 37.4
6.2 83.0 34.5
6.4 75.4 31.4
6.6 65.3 27.9
6.8 53.4 23.5
7.0 41.3 19.0
7.2 29.6 13.8 44.7
7.4 19.7 9.8 42.0
7.6 12.8 6.8 39.3
7.8 7.4 4.6 33.7
8.0 3.7 27.9
1.5. References
Miller DD. 1998. Food Chemistry. A Laboratory Manual. John Wiley & Sons, Inc.
Weaver CM and Daniel JR. 2003. The Food Chemistry Laboratory: A Manual for Experi-
mental Foods, Dietetics, and Food Scientists. 2nd edition. CRC Press.
Kenkel JV. 2003. Analytical Chemistry for Technicians. 3 rd edition. CRC Press.