Exercise 1

pH and Buffer Systems

Results and Discussion

In biological systems, maintenance of the optimum pH is needed for biochemical processes to

proceed spontaneously. Cells and tissues depend on the strict regulation of the hydronium ion
concentration - thus, a buffer is needed to resist the drastic change in pH upon addition of an acid or
a base (Boyer, 2012). Proteins, nucleic acids, and other cellular molecules cannot function properly if
the pH is not maintained since these biomolecules interact via hydrogen bonding and ionic interactions
(Lehninger, Nelson, and Cox, 2013). Metabolic processes are dependent on enzymatic activity, which
is also dependent on pH. Buffer systems are the first line of defense of the body against drastic pH
changes. A buffer must be compatible with the body aside from having a pK a value near the pH 7.
Bicarbonate and phosphate buffer systems are two buffer systems found in the body that maintains
the intracellular pH constantly. Bicarbonate buffer systems are found in the blood plasma while
phosphate buffer systems are found in intracellular fluid of cells (Garrett and Grisham, 2013).

Buffers can be prepared in the laboratory using a weak acid or base and its conjugate pair.
Suppose a buffer solution is made from a weak acid, HA, and its conjugate base, A-, being ionized upon
addition of water to form hydronium ion, H3O+.

HA + H2O ⇌ H3O+ + A- (Equation 1.1)

The rate of the forward reaction can be represented as k 1, with the rate of the backward reaction as
k2. The ratio of the forward and backward reaction is equal to the equilibrium constant k a.
k1 [H+] [A-]
ka = = (Equation 1.2)
k2 [HA]

The concentration of the hydronium ion can then be determined by rearranging Equation 1.2 such that
ka [HA]
[H+] = (Equation 1.3)
[A-]

The Henderson-Hasselbalch equation (Equation 1.4) shows how the pH (reported as -log[H+]), pKa
(reported as -log Ka), HA and A- are related (Skoog, West, Holler, and Crouch, 2014; Boyer, 2012).
[A-]
pH = pKa + log (Equation 1.4)
[HA]

For the buffer to be effective, the concentrations of the weak acid HA and the conjugate base A - must
be equal; therefore, pH = pKa. The effective buffering range for a buffer is pKa ± 1.

Correct buffer selection is essential in biochemical studies outside biological systems since
biomolecules and cell organelles are most stable at the normal pH range of 6 to 8 inside the body.
Several natural buffer systems are available depending on the needed pH range needed; however,
several factors must be considered when selecting buffer systems aside from effective pH range. Among
all buffers available, phosphate buffers are widely used due to their high buffering capacity and are
natural components of cells and biological fluids despite having some disadvantages. Binding of Ca2+
and Mg2+, inhibition of biological processes, and limited useful pH range are the major disadvantages
of using phosphate buffers (Boyer, 2012). Dilution and addition of acids and bases can affect the
buffering capacity of a buffer. This laboratory exercise investigated the effects of these factors to buffer
preparation and pH measurement.

Buffer capacity, as defined by Harris (2010), is the measure of how a solution can resist the
change in pH given that a strong acid or base is added. Concentrations of the acid or base and the pH
determines the buffering capacity of a given buffer. A buffer has a greater buffer capacity if it has great
resistance to pH change. Therefore, a buffer reaches its buffer capacity if the concentrations of the
weak acid and its conjugate base is equal (pH = pKa). As the concentrations of the buffer components
increase, so as the buffering capacity of the buffer along with the decrease in pH.
Table 1.1. Data on the effect of buffer concentration on the buffering capacity.
pH after addition of
Buffer concentration Original pH Change in pH
base
Stock solution 6.9 7.0 0.1
1:10 7.1 7.0 -0.1
1:50 7.0 10.9 3.9
1:100 7.0 11.1 4.1

The effect of buffer concentration on the buffering capacity was investigated by linear dilution
of the stock solution to different final volumes (Table 1.1). pH was then measured before and after the
addition of 0.1M NaOH using a pH pen. The pH pen was calibrated before use using solutions of pH 4
and 7. The change in pH was then computed using the equation

∆pH = final pH – initial pH (Equation 1.5)

0.1M phosphate buffer was prepared by measuring 24.71mL of 0.1M Na2HPO4 and 25.29mL of 0.1M
NaH2PO4 to obtain 50mL of the buffer solution. The pH of the stock solution was measured before and
after the addition of 0.1M NaOH. The stock solution was then diluted to 10mL, 50mL, and 100mL with
their respective pH before and after the addition of the base was measured as well. It was observed
that as the buffer concentration decreases, the pH increases. The highest change in pH was observed
at 1:100 dilution with ∆pH = 4.1. Therefore, keeping the pKa at the range of pH ± 1 would give the
chosen buffer its optimum buffer capacity (Skoog, West, Holler, and Crouch, 2014). Buffer range does
not necessarily mean that the concentrations of the acid and the base are effective in resisting pH
change outside the range, whereas buffer capacity ensures that the concentrations of the acid and the
base are sufficient enough to resist pH change outside the range. Phosphate buffers are said to be
effective at pH range close to 6.86, which is 5.86 to 7.86.

Addition of acids and bases also affect the buffer capacity of a given buffer. The chemical
equation for the phosphate buffer system is

NaH2PO4 + H2O ⇌ Na2HPO4 + H3O+ (Equation 1.6)

Upon addition of a small amount of acid,

Na2HPO4 + H3O+ → NaH2PO4 + H2O (Equation 1.7)

Upon addition of a small amount of base,

Na2HPO4 + OH- → NaH2PO4 + H2O (Equation 1.8)

Table 1.2a. Preparation of phosphate buffer.

Volume of 0.1 M Volume of 0.1 M [𝐻𝑃𝑂42− ]
Test tube pH
Na2HPO4 (mL) NaH2PO4 (mL) 𝐻2 𝑃𝑂4−
1 0.60 19.40 5.70 0.0309
2 1.80 18.20 6.21 0.0989
3 10.0 10.0 7.21 1
4 18.20 1.80 8.21 10.111
5 19.40 0.60 8.72 32.330
6 20.0 mL distilled water

Five test tubes of 20mL of 0.1M phosphate buffer solution and a test tube containing 20mL
water was prepared by mixing volumes of 0.1M Na2HPO4 and 0.1M NaH2PO4 listed in Table 1.2a. The
theoretical pH of the mixture was calculated using the Henderson-Hasselbalch equation (Equation 1.4)
along with the ratio of the conjugate base to the acid. The actual pH of the mixture was then measured
using a calibrated pH pen. 2mL of 0.1M NaOH was then added to the buffer solutions and the resulting
pH was measured on all test tubes. Five test tubes of 20mL 0.1M phosphate buffer and a test tube
containing distilled water of the same volume was prepared. After measuring the actual pH of the
mixture, 2mL of 0.1M HCl was added to the buffer solutions. The resulting pH after the addition of both
base and acid were recorded in Tables 1.2b and 1.2c, respectively.

Table 1.2b. Data on the effect of the ration of conjugate base to weak acid (addition of NaOH).
Measured pH
Test tube pH after adding Change in pH
Calculated Actual
1 5.70 5.5 6.1 0.6
2 6.21 6.2 6.5 0.3
3 7.21 7.2 7.4 0.2
4 8.21 8.2 10 1.8
5 8.72 8.2 10.3 2.1
6 (dH2O) - 6.4 11.4 5

Table 1.2c. Data on the effect of the ratio of conjugate base to weak acid (addition of HCl).
Measured pH
Test tube pH after adding Change in pH
Calculated Actual
1 5.70 5.5 2.7 -2.8
2 6.21 6.2 3.1 -3.1
3 7.21 7.2 6.5 -0.7
4 8.21 8.2 7.2 -1
5 8.72 8.2 7.2 -1
6 (dH2O) - 7 2.4 -4.6

It was observed that test tubes 1 and 2 had an increase in pH upon addition of the acid and
base while test tubes 4 and 5 had a decrease in pH upon addition of the acid and the base. The data
gathered illustrated that the extent of the buffer capacity of a given buffer not only lies with the
concentration of the acid and the base but also with their ratio. Buffer capacity is said to decrease as
the concentration ratio of the acid to the conjugate base becomes less than or greater than 1 (Skoog,
West, Holler, and Crouch, 2014). Therefore, having a pH range of pKa ± 1 would give the buffer system
its effective buffering capacity.

Amino acids can act either as an acid or as a base - called zwitterion - when dissolved in water.

Figure 1.1 A zwitterion acting as an acid (left) and as a base (right) (Lehninger, Nelson, and Cox,
2013)

Being weak polyprotic acids, amino acids can dissociate depending on the pH of the medium (Lehninger,
Nelson, and Cox, 2013; Garrett and Grisham, 2013). Substances that have an acid-base nature is said
to be amphoteric. When protonated, the carboxylic group (-COOH) and the nitro group (-NH3+) yields
protons shown in Figure 1.1. Based on this, amino acids can have characteristic titration curves unique
to their own. The last part of the exercise dealt with the titration of an unknown amino acid and the
determination of pKa values of its ionizing groups.
Table 1.3. Data on the titration of an unknown amino acid.
Volume of 0.1 M KOH pH Volume of 0.1 M KOH pH
0 1.5 17.5 6.9
0.5 1.5 18 7
1 1.5 18.5 7
1.5 1.5 19 7.1
2 1.6 19.5 7.2
2.5 1.6 20 7.3
3 1.7 20.5 7.3
3.5 1.7 21 7.4
4 1.8 21.5 7.5
4.5 1.8 22 7.6
5 1.9 22.5 7.7
5.5 1.9 23 7.9
6 2 23.5 8.1
6.5 2 24 8.3
7 2.1 24.5 8.9
7.5 2.2 25 10.3
8 2.3 25.5 10.8
8.5 2.3 26 11
9 2.4 26.5 11.2
9.5 2.5 27 11.3
10 2.6 27.5 11.4
10.5 2.8 28 11.5
11 3 28.5 11.6
11.5 3.2 29 11.7
12 3.8 29.5 11.7
12.5 5.3 30 11.8
13 5.8 30.5 11.8
13.5 6 31 11.9
14 6.2 31.5 11.9
14.5 6.3 32 11.9
15 6.4 32.5 12
15.5 6.5 33 12
16 6.6 33.5 12
16.5 6.7 34 12
17 6.8

Figure 1.2. Titration curve of the unknown amino acid.

 Based on the titration curve of the unknown amino acid, the pKa1= 2 and pKa2 = 7.1. The
probable identity of the amino acid is asparagine since the obtained pKa values are near the theoretical
pKa values of asparagine, pKa1 = 2.02 and pKa2 = 8.08 (Lehninger, Nelson, and Cox, 2013). The
experimental IpH of the amino acid is 4.55 while the theoretical IpH is 5.05. The difference in the
obtained and theoretical pKa2 can be attributed to the experimental errors during the titration, which
affected the computed IpH of asparagine. Since amino acids are zwitterions, they can exist in their
cationic or anionic form, depending on the pH of the solution. Figure 1.3 are the probable identities of
asparagine at different parts of the titration curve.

(A) (B)

(C) (D)
Figure 1.3. Probable identities of the amino acid asparagine in the titration curve.

At pH less than pKa1, asparagine may exist as (A). Asparagine may exist as (B) at pH greater than pK a1
but less than the IpH, wherein the dominant form of asparagine is (C). At pH near pKa2, asparagine
exists as (D). The net charge of asparagine in the IpH is zero, therefore it can resist drastic changes
via addition of an acid or a base since most of the zwitterionic form of asparagine can be found in this
region.

Sample Calculations

1. Preparation of 50 mL of 0.1 M phosphate buffer at pH 7.2. (MM NaH 2PO4 = 141.958 g/mol and
MM of NaH2PO4 = 119.76 g/mol).
[𝐴− ]
pH= pKa log +
[𝐻𝐴]

[𝐻𝑃𝑂42−]
7.2 = pKa + log
[𝐻𝐴]

[𝐻𝑃𝑂42− ]
log = antilog (7.2-7.21) = 0.977237221
[𝐻𝐴]

a. Amount of 0.1 M Na2HPO4 (mL)

mol 1L 0.977237221
(0.1 l )(50 mL X 1000 mL)(1+0.977237221)
V0.1 M Na2HPO4 = mol 1L = 24.71218958 mL
(0.1 )( )
L 1000 mL

b. Amount of 0.1 M NaH2PO4 (mL)

mol 1L 1
(0.1 l )(50 mL X 1000 mL)(1+0.977237221)
V0.1 M NaH2PO4 = mol 1L = 25.28781042 mL
(0.1 )( )
L 1000 mL

2. For dilutions in determining the effect of buffer concentration to buffer capacity.

For 1:10 dilution,
Let X be the vol. of buffer needed
1 𝑋
= → X = 5 mL buffer
10 50
Volume of diluent = 50-5 = 45 mL dH2O
3. Calculation of the pH of the resulting mixture and ratio of the conjugate base to weak
acid.
 a. Test tube 1, 0.60 mL 0.1 M Na2HPO4+ 19.40 mL 0.1 M NaH2PO4
[𝐻𝑃𝑂42− ]
pH = pKa + log
[𝐻𝐴]
0.60
pH = 7.21 + log
19.40
pH = 5.70034952
4. Computation on the change of pH
ΔpH = final pH – initial pH
ΔpH = 6.1 – 5.5
ΔpH = 0.6
5. Computation of IpH
(pKa1 +pKa2 )
IpH =
2
7.1 + 2
IpH =
2
IpH = 4.55

References/ Literature Cited

Boyer, R. F. (2012). pH, Buffers, Electrodes, and Biosensors. Chapter 3: General Laboratory
Procedures. Biochemistry Laboratory: Modern Theory and Techniques, Second Edition. Upper
Saddle River, New Jersey: Pearson Education, Inc. Pp. 53-64

Garrett, R. H. and Grisham, C. M. What Are Buffers, and What Do They Do? Chapter 2: Water: The
Medium of Life. Biochemistry, Fifth Edition. Belmont, CA: Brooks/Cole, Cengage Learning. Pp.
43-45

Harris, D. C. (2010). Buffer Capacity. Chapter 8: Monoprotic Acid-Base Equilibria Quantitative

Chemical Analysis, Eight Edition. New York, NY: W. H. Freeman and Company. Pp. 177
Lehninger, A. L., Nelson, D. L., and Cox, M. M. (2013). Lehninger Principles of Biochemistry, Sixth
Edition. New York, NY: W. H. Freeman and Company. Pp 64-65
Skoog, D. A., West, D. M., Holler, F. J., and Crouch, S. R. (2014). Buffer Solutions. Chapter 9: Aqueous
Solutions and Chemical Equilibria. Fundamentals of Analytical Chemistry, Ninth Edition.
Belmont, CA: Brooks/Cole, Cengage Learning. Pp. 219-221.