Chapter 2

Water: The Solvent for

Biochemical Reactions

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Chapter Outline
(2-1) Water and polarity
(2-2) Hydrogen bonds
(2-3) Acids, bases, and pH
(2-4) Titration curves
(2-5) Buffers

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Electronegativity
• Measure of the force of an
atom’s attraction for
electrons it shares in a
chemical bond with
another atom
• Oxygen and nitrogen are
more electronegative
than carbon and
hydrogen

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Polar Bonds
• Electrons are unequally shared
• More negative charge is found closer to one of the
atoms
• Formed because of the difference in electronegativity
of atoms involved in the bond

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Polar Bonds and Molecules
• Bonds in a molecule may be polar, but the molecule
itself can be nonpolar because of its geometry
• Molecules such as CO2 have polar bonds but, given
their geometry, are nonpolar
• Nonpolar: Bond in which two atoms share electrons
evenly

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Solvent Properties of Water
• Ionic bonds
• Held together by positive and negative ions
• Salt bridge
• Interaction that depends on the attraction that occurs
when oppositely charged molecules are in close
proximity
• Ion–dipole interactions
• Occur when ions in solution interact with molecules
that have dipoles
• Example - NaCl dissolved in H2O

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Figure 2.2 - Ionic Bonds Become Replaced by Ion–
Dipole Interactions

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Figure 2.3 - Ion–Dipole and Dipole–Dipole
Interactions

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Solvent Properties of Water (continued 1)
• van der Waals forces: Noncovalent associations
based on weak attractions of transient dipoles for one
another
• Called van der Waals interactions or van der Waals
bonds
• Include noncovalent bonds that do not involve
electrostatic interactions of fully charged ions
• Dipole–dipole interactions
• Forces that occur between molecules that are dipoles
• Partial positive side of a molecule attracts the partial
negative side of another molecule

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Solvent Properties of Water (continued 2)
• Dipole–induced dipole interactions
• Permanent dipole in one molecule can induce a
transient dipole in another molecule through
momentary distortion of electron clouds
• Weak and generally do not lead to solubility in water
• Induced dipole–induced dipole interactions
• London dispersion force: Attraction between
transient induced dipoles

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Figure 2.4 - Dipole-Induced Dipole Bonds

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Figure 2.5 - Induced Dipole-Induced Dipole
Interactions

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Table 2.2 - Strengths of Bonds Found in
Biochemistry

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Solvent Properties of Water (continued 3)
• Hydrophilic
• Tending to dissolve in water (water-loving)
• Examples - Ionic and polar substances
• Hydrophobic
• Tending not to dissolve in water (water-hating)
• Hydrophobic interactions: Attractions between
nonpolar molecules
• Amphipathic
• Molecule that contains both hydrophobic and
hydrophilic regions
• Example - Sodium palmitate

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Table 2.3 - Examples of Hydrophobic and
Hydrophilic Substances

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Figure 2.6 - Sodium Palmitate, an Amphipathic
Molecule

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Micelle Formation by Amphipathic Molecules
• Spherical arrangement of organic molecules in water
solution clustered so that their:
• Hydrophobic parts are buried inside the sphere
• Hydrophilic parts are on the surface of the sphere and
in contact with the water environment
• Formation depends on the attraction between
temporary induced dipoles

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Hydrogen Bonds
• Noncovalent attractive interactions between dipoles
when the:
• Positive end of one dipole is a hydrogen atom bonded
to a highly electronegative atom (hydrogen-bond
donor)
• Negative end of the other dipole is an atom with a lone
pair of electrons (hydrogen-bond acceptor)

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Figure 2.9 - Hydrogen-Bonding Sites

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Interesting and Unique Properties of Water
• Each water molecule can be involved in 4 hydrogen
bonds
• Acts as a donor in 2 and as an acceptor in the other 2
• Enabled by the tetrahedral arrangement of the water
molecule with bond angles of 104.3°

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Hydrogen Bonding
• Even though hydrogen bonds are weaker than
covalent bonds, they have a significant effect on the
physical properties of hydrogen-bonded compounds

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Hydrogen Bonding between Polar Groups and
Water
• If a polar solute can serve as a donor or an acceptor
of hydrogen bonds, it can:
• Form hydrogen bonds with water
• Participate in nonspecific dipole–dipole interactions

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Other Biologically Important Hydrogen Bonds
• Hydrogen bonding is important in the stabilization of
3-D structures of biological molecules, such as DNA,
RNA, and proteins

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Acids and Bases
• Acid
• Molecule that behaves as a proton donor
• Commonly known as a Brønsted acid
• Base
• Molecule that behaves as a proton acceptor

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Acid Strength
• Tendency of an acid to dissociate to a hydrogen ion
and its conjugate base
• Characterized by acid dissociation constant (Ka)

• Greater the Ka, the stronger the acid

• Correct form of the equation:

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Ionization of Water
• Let us quantitatively examine the dissociation of
water:  H +   OH - 
Ka 
 H 2O
• Molar concentration of pure water, [H2O], is 55.5 M
• Thus,
 H +  OH - 
Ka 
55.5

K a  55.5   H +  OH -   K w

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Ion Product Constant for Water (Kw)
• Measure of the tendency of water to dissociate to
give H+ and OH– ions
• Numerical value can be determined by measuring
[H+] of pure water
• In monoprotIc acids, [H+] = [OH–]
• At 25°C in pure water,

 H +  = 107 M = OH  

• At 25°C, the numerical value of Kw is given by:

K w =  H +  OH   =  107   10 7  = 10 14

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pH and pKa
• Quantity for expressing [H+] and [OH–] in aqueous
solutions
pH  -log10  H 
+

• Difference of one pH unit implies a tenfold difference in

[H+]
• Neutral solutions have pH = 7, acidic solutions have
pH < 7, and basic solutions have pH > 7
• pKa
• Convenient numerical measure of acid strength
pK a =  log10 K a

• Smaller the value, the stronger the acid

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Example 2.1 - pH Calculations
• In pure water, [H+] = 1×10–7 M and pH = 7.0
• Calculate the pH of the following aqueous solutions:
a. 1×10–3 M HCl
b. 1×10–4 M NaOH
• Assume that the self-ionization of water makes a
negligible contribution to the concentrations of
hydronium ions and of hydroxide ions, which will
typically be true unless the solutions are extremely dilute

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Example 2.1 - Solution
• The key points in the approach to this problem are
the definition of pH, which needs to be used in both
parts, and the self-dissociation of water, needed in
the second part
a. For 1×10–3 M HCl, [H3O+] = 1×10–3 M
• Therefore, pH = 3
b. For 1×10–4 M NaOH, [OH–] = 1×10–4 M
• Because [OH–] [H3O+] = 1×10–14, [H3O+] = 1×10–10 M
• Therefore, pH = 10.0

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Henderson–Hasselbalch Equation
• Mathematical relationship between the pKa of an acid
and the pH of a solution containing the acid and its
conjugate base
• Can be determined as follows:
 H    A  
Ka 
 HA 

 A 

 log K a  log  H   log


 HA 
 A  
 log  H     log K a  log
 HA 
 A  
pH  pK a  log
 HA 
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Henderson–Hasselbalch Equation (continued)
• When concentrations of a weak acid and its
conjugate base are equal, the pH of the solution
equals the pKa of the weak acid
• pH versus enzymatic activity

• Note how the activity of a protein can rapidly change

as pH leaves optimum range
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Titration Curves
• Titration: Experiment in which
measured amounts of base
are added to an acid
• Equivalence point: Point in
an acid–base titration at which
enough base has been added
to neutralize the acid

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Example 2.2 - Calculating pH Values for Weak
Acids and Bases
• Calculate the relative amounts of acetic acid and
acetate ion present at the following points when 1
mol of acetic acid is titrated with sodium hydroxide
• Also use the Henderson–Hasselbalch equation to
calculate the values of the pH at these points:
a. 0.1 mol of NaOH is added
b. 0.3 mol of NaOH is added
c. 0.5 mol of NaOH is added

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Example 2.2 - Solution
• Approach this problem as an exercise in
stoichiometry
• There is a 1:1 ratio of moles of acid reacted to moles
of base added
• Difference between the original number of moles of
acid and the number reacted is the number of moles of
acid remaining
• These are the values to be used in the numerator and
denominator, respectively, of the Henderson–
Hasselbalch equation

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Example 2.2 - Solution (continued 1)
a. When 0.1 mol of NaOH is added, 0.1 mol of acetic
acid reacts with it to form 0.1 mol of acetate ion,
leaving 0.9 mol of acetic acid
• Composition is 90% acetic acid and 10% acetate ion
0.1
pH = pK a + log
0.9
0.1
pH = 4.76 + log
0.9
pH = 4.76  0.95

pH = 3.81

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Example 2.2 - Solution (continued 2)
b. When 0.3 mol of NaOH is added, 0.3 mol of acetic
acid reacts with it to form 0.3 mol of acetate ion,
leaving 0.7 mol of acetic acid
• Composition is 70% acetic acid and 30% acetate ion

0.3
pH = pK a + log pH = 4.39
0.7

c. When 0.5 mol of NaOH is added, 0.5 mol of acetic

acid reacts with it to form 0.5 mol of acetate ion,
leaving 0.5 mol of acetic acid
• Composition is 50% acetic acid and 50% acetate ion
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Example 2.2 - Solution (continued 3)

0.5
pH = pK a + log pH = 4.76
0.5
• Note that this one is possible without doing much math
• We know that when the [HA] = [A–], the pH = pKa
• Therefore, the minute we saw that we added 0.5 mol of
NaOH to 1 mol of acetic acid, we knew that we had
added enough NaOH to convert half the acid to the
conjugate base form
• Thus, the pH has to be equal to the pKa

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Titration Curves (continued)
• Dissociation constants for three groups of acids
• Monoprotic acids release one H+ ion and have a single
Ka and pKa
• Diprotic acids release two H+ ions and have two Ka
values and two pKa values
• Polyprotic acids release more than two H+ ions
• When pH < pKa, the weak acid predominates
• H+ on, substance protonated
• When pH > pKa, the conjugate base predominates
• H+ off, substance deprotonated

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Buffer Solutions
• Tend to resist change in pH when small to moderate
amounts of a strong acid or strong base are added
• Consist of a weak acid and its conjugate base
• Examples of acid–base buffers are solutions
containing:
• CH3COOH and CH3COONa
• H2CO3 and NaHCO3
• NaH2PO4 and Na2HPO4

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Figure 2.17 - Buffering

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Mechanism of Buffers - Example
• Consider a solution in which the concentrations are
[HPO42–] = 0.063 M and [H2PO4–] = 0.10 M
• Thus, the conjugate base/weak acid ratio is 0.63
• If 1.0 mL of 0.10 M HCl is added to 99.0 mL of the
buffer, the following reaction takes place and almost all
the added H+ is used up

HPO 24 + H +  H 2 PO 4
• Concentrations of [HPO42–] and [H2PO4–] would change,
and new concentrations can be calculated

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Mechanism of Buffers - Example (continued)

• New pH can be calculated using the Henderson–

Hasselbalch equation and phosphate ion concentrations
• Appropriate pKa is 7.20
 HPO 24  0.062
pH = pK a + log pH = 7.20 + log = 6.99
 H 2 PO 4  0.101

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Mechanism of Buffers
• Buffers follow Le Chatelier’s principle
• If stress is applied to a system in equilibrium, the
equilibrium will shift in the direction that relieves the
stress
• When H+ ion is added to a buffer system, stress is
added to the reaction

HA  H+ + A

• To relieve the stress, H+ reacts with A– to maintain

equilibrium

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Buffer Selection
• Determined by understanding the relationship
between pH and buffering capacity
• pH changes little in the vicinity of the inflection point of
a titration curve
• At the inflection point, half the amount of acid originally
present has been converted to the conjugate base
• When the pH is one unit higher than pKa, the ratio of
conjugate base form to the conjugate acid form is 10
• When pH is two units higher than pKa, the ratio is 100
• Buffer solutions can maintain a relatively constant pH
value, which is met at values at or near the pKa of the
acid
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Favorable Criteria for Selecting a Buffer

Suitable pKa (pKa closest to the pH)

No interference with the reaction or with the assay

Suitable ionic strength of the buffer

No precipitation of reactants or products due to the

presence of the buffer

Nonbiological nature of the buffer

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Table 2.7 - pH Values and Base/Acid Ratios for
Buffers

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Figure 2.18 - Relationship between the Titration Curve
and Buffering Action in H2PO4–

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Buffer Capacity
• Measure of the amount of acid or base that can be
absorbed by a given buffer solution
• Related to the concentrations of the weak acid and its
conjugate base
• Greater the concentration of the weak acid and its
conjugate base, the greater the buffer capacity

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Making Buffers in the Laboratory
• Start with the HA form and add NaOH until the pH is
correct or start with A– and add HCl until the pH is
correct

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Naturally Occurring Buffers
• H2PO4–/HPO42– is the principal buffer in cells
• Buffering system in blood is based on the
dissociation of carbonic acid (H2CO3)
• pKa = 6.37
• CO2 can dissolve in water and water-based fluids
• Dissolved CO2 forms H2CO3, which reacts to produce
bicarbonate ion
• Conversion is catalyzed by carbonic anhydrase, an
enzyme CO 2 ( g )  CO 2 (aq)
CO 2 ( g )  H 2O(l )  H 2CO3 ( aq)
H 2 CO3 (aq)  H + (aq )  HCO3- (aq)
Net equation: CO 2 ( g )  H 2 O(l )  H  ( aq)  HCO3 ( aq)
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Table 2.8 - Acid and Base Form of Some
Useful Biochemical Buffers

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Naturally Occurring Buffers (continued)
• Buffer systems can be:
• In vitro (outside the living body)
• Example - Buffer system based on tris(hydroxymethyl)
aminomethane (TRIS)
• In vivo (inside the living body)
• Example - Phosphate buffer system
• Zwitterions: Compounds that have both a positive
and negative charge
• Considered less likely to interfere in biochemical
reactions than other buffers

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