Module 2: FOUNDATIONS IN BIOLOGY

OCR A Level Biology A (H420)

MPW Cambridge

Module 2:
Foundations in biology
Layla’s summary notes (part 2 of 6)

TOPICS:
 2.1.1 Cell structure
 2.1.2 Biological molecules
 2.1.3 Nucleotides and nucleic acids
 2.1.4 Enzymes
 2.1.5 Biological membranes
 2.1.6 Cell division, cell diversity and
cellular organisation
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2.1.2 Biological molecules

The cells of all living organisms are composed of biological molecules. Proteins,
carbohydrates and lipids are three of the key groups of biological macromolecules that are
essential for life. A study of the structure of these macromolecules allows a better
understanding of their functions in living organisms.

Water
A water molecule (H2O) is comprised of a single oxygen atom, covalently bonded to two
hydrogen atoms. There is an uneven distribution of charge across the molecule: the oxygen
atom has a small negative charge, whilst the hydrogen atoms each have a small positive
charge. These tiny charges are referred to as δ‐ (pronounced delta minus) and δ+ (delta
plus).

This uneven distribution of charge occurs because the pair of electrons in each oxygen‐
hydrogen covalent bond is not shared equally; the electrons are more strongly attracted to
the oxygen atom’s nucleus than to the hydrogen atom’s nucleus. Molecules (or groups of
atoms within a molecule) that have an uneven distribution of charge are referred to as
polar.

Draw a diagram of a water molecule, showing the uneven distribution of charge:

Water molecules are attracted to each other: the δ‐ oxygen atom of one molecule will be
attracted to a δ+ hydrogen atom of another water molecule. This attraction is called a
hydrogen bond. It is represented on diagrams by a dashed line.

Draw a diagram of several water molecules, showing the hydrogen bonds between them:

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Water is by no means the only type of polar molecule: hydroxyl (OH), carbonyl (CO) and
amine (NH2) groups also have polarity, hence any molecule containing such groups can be
described as a polar.

Polar molecules (e.g. amino acids, which have a carbonyl group and an amine group) are
usually soluble in water, since water molecules will be attracted to, and will form hydrogen
bonds with, their polar groups.

A hydrogen bond can form between two water molecules, as shown above (referred to as
cohesion), or between a water molecule and a different type of polar molecule (referred to
as adhesion).

However, hydrogen bonds need not always involve water molecules at all. They can occur
between any two polar molecules of the same type (e.g. two cellulose molecules), or
between two polar molecules that are different types (e.g. adenine with thymine in DNA), or
even between polar groups that are part of the same molecule (e.g. the intramolecular
hydrogen bonding that can occur within a polypeptide).

A polar, and hence water‐soluble, molecule (e.g. a monosaccharide) can also be described
as hydrophilic (water‐loving).

However, molecules which have no polar groups (i.e. they are non‐polar, meaning there is
an even distribution of charge across the molecule) cannot form hydrogen bonds with
water molecules; water molecules will instead be repelled. Such molecules (e.g. a steroid
hormone such as testosterone) can be described as hydrophobic (water‐hating) and will be
lipid‐soluble but not water‐soluble.

Hydrogen bonds within a body of liquid water are not permanent; they are temporary and
so constantly break and reform as the water molecules move around. This allows liquid
water to flow, since the individual water molecules are not in fixed positions.

The hydrogen bonds between water molecules are considered to be individually weak
(certainly far weaker than the covalent bonds that hold the oxygen and hydrogen atoms
together within each water molecule); however, the hydrogen bonds in a body of water are
so very numerous that they have very significant effects on its properties.

Properties of water and importance in living organisms

Excellent solvent
As explained above, water molecules are polar (i.e. there is an uneven distribution of
charge, specifically the oxygen atom has a small negative charge, δ‐, whilst the hydrogen
atoms have small positive charge, δ+). Water molecules therefore form hydrogen bonds
with other polar molecules (e.g. glucose) and also can interact with ions (e.g. Na+ Cl‐). The

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water molecules are strongly attracted to and completely surround the molecules/ions of a
soluble substance so they become dispersed, i.e. they dissolve in the water; the solute, e.g.
glucose, has dissolved in the solvent, water, to form a solution. Most chemical reactions
(metabolism) in living organisms take place in solution, e.g. within the cytosol of a
(prokaryotic or eukaryotic) cell.

Diagram showing how water, with its polar molecules, acts as a solvent for sodium chloride
and for glucose:

Diagram from Biology 1 for OCR (Mary Jones, OUP, ISBN 978‐0‐521‐71763‐2)

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Effective thermal buffer

Water has a high specific heat capacity (due to the numerous hydrogen bonds), i.e. a large
amount of energy is required in order to increase its temperature. This means that the
temperature of a body of water is very stable. This is important within cells, since enzymes
have a narrow optimum temperature range. Also this property means that a pond/lake
provides a stable habitat for aquatic organisms.

High latent heat of vaporisation

Water is a liquid (not a gas) at room temperature due to the large number of intermolecular
hydrogen bonds which hold water molecules relatively close together. High latent heat of
vaporisation means that a large energy input is needed in order to cause liquid water to
change state into a gas (i.e. to vaporise or evaporate). This is due to the numerous
hydrogen bonds which need to be broken, requiring a great deal of energy. Evaporation of
water from the surface of an organism is therefore an effective cooling mechanism since it
takes a lot of heat from the body, e.g. sweating or panting by mammals, transpiration from
plants. Hence water is acting as a coolant.

Low density of ice (compared to liquid water)

Ice is less dense than liquid water. This is because in ice, the solid, the water molecules are
held apart in a regular (semi‐crystalline) arrangement (called an ‘open‐lattice’) by stable
intermolecular hydrogen bonds. A layer of ice on a pond/lake will therefore float on the
surface. This ice will insulate (i.e. reduce heat loss from) the water below, reducing the
chance that these lower regions will themselves freeze. Most aquatic organisms will
therefore survive even if the upper layer of their habitat is frozen, hence this property of
water is significant in providing a stable habitat for many species, from prokaryotic
microorganisms to large eukaryotes such as fish.

Cohesion between water molecules

Water molecules cohere (stick) to each other via hydrogen bonding. This allows water to
form strong, continuous columns within a vessel/tube; these columns have high tensile
strength due to the high number of hydrogen bonds between the water molecules. Liquid
water within a vessel will flow from one region to another (due to gravity or a hydrostatic
pressure difference) by a process called ‘mass flow’. Water therefore makes a very effective
transport medium e.g. the blood of animals and, in plants, the sap of the xylem or phloem.
Cohesion (along with adhesion) also causes water to show capillarity (or capillary action)
i.e. a column of water will tend to spontaneously rise up a narrow tube, against gravity, e.g.
this contributes to upward movement of water in xylem vessels.

High surface tension

Hydrogen bonding by water molecules on the surface to those below (only) causes a
property called high surface tension: water behaves as if it has a strong ‘skin’ at its surface.
Small animals (e.g. pond striders) can move on the surface without breaking through.

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Incompressible
Water, as a liquid, is incompressible (i.e. it cannot be forced to decrease in volume). This
property is especially important in insects, which have a hydrostatic skeleton to support the
body (instead of bones). In plants, cells (which have cell walls) become turgid (stiffened)
when the volume of water inside the cells is great enough to exert of pressure on the cell
wall; this cell turgor is vital in enabling the leaves of a plant to be held up to intercept
sunlight.

Transparent
Light can pass through water, though its ability to do so varies according to wavelength. This
is important for aquatic plants and other photosynthetic organisms, which rely on light
energy to drive glucose production via photosynthesis.

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Biological molecules
Monomers and polymers
Most large biological molecules (macromolecules) consist of long chains of smaller
molecules bonded together, e.g. a polypeptide (protein) is a long chain of amino acids
bonded together. A large molecule of this type is called a polymer; polypeptides (proteins),
polysaccharides and polynucleotides (nucleic acids) are examples.

The smaller ‘building block’ molecules that are bonded together to make the polymer are
called monomers (e.g. amino acids, monosaccharides or nucleotides).

Some types of biological macromolecule are not in fact polymers, notably lipids (see below).

Condensation and hydrolysis

Polymers are formed when their constituent monomers are joined together via
condensation reactions; they are broken down back into individual monomer molecules via
hydrolysis reactions. In biology, both condensation and hydrolysis reactions are enzyme‐
catalysed.

DEFINITONS:

Condensation: a reaction in which two monomers are joined together, forming a new
bond; a molecule of water is released during the reaction.

Hydrolysis: a reaction in which a molecule of water is used in order to break a bond.

Condensation and hydrolysis reactions are opposites of one another. For example,
condensation reactions enable amino acids to be joined via peptide bonds to form a
polypeptide; hydrolysis of the polypeptide breaks the peptide bonds, releasing individual
amino acids.

Whereas a molecule of water is produced during a condensation reaction, a molecule of

water is used during hydrolysis.

An enzyme which catalyses a hydrolysis reaction can (in general) be referred to as a

hydrolase, or a hydrolytic enzyme. For example, a protease is a hydrolytic enzyme which
catalyses the hydrolysis of a protein into amino acids.

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There is not a general name for enzymes that catalyse condensation reactions, but one
example is DNA polymerase which catalyses the condensation reactions that join free DNA
nucleotides together to form a new DNA strand (a DNA polynucleotide) during DNA
replication.

Elemental composition of biological molecules

Each class of biological molecule contains a specific combination of chemical elements,
which always includes carbon (C), hydrogen (H) and oxygen (O) and may also include
sulphur (S), nitrogen (N) and/or phosphorus (P).

Learn the specific elements present in different classes of biological molecules as follows:

Carbohydrates: C, H and O (with H:O in a 2:1 ratio and the number of C approximately equal
to the number of O)

Lipids: C, H and O (the proportion of O relative to C and H is very low; phospholipids also
contain P)

Proteins: C, H, O, N and S

Nucleic acids (DNA, RNA): C, H, O, N and P.

Carbohydrates
Carbohydrates (also known as saccharides or sugars) are biological molecules which contain
only the elements carbon, hydrogen and oxygen; there are always twice as many hydrogen
atoms as oxygen atoms in a carbohydrate. The general formula of a carbohydrate is:

Cx(H2O)y
The simplest form of carbohydrate is a monosaccharide (also called a single sugar or simple
sugar); glucose, galactose, fructose, ribose and deoxyribose are examples.

Two monosaccharides can be bonded together in a condensation reaction to form a

disaccharide (double sugar); examples include maltose (glucose + glucose), lactose (glucose
+ galactose) and sucrose (glucose + fructose).

The bond between the two monosaccharide residues in the disaccharide is called a
glycosidic bond.

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A large number of monosaccharides can be bonded together via multiple condensation

reactions to form a polysaccharide (complex sugar). Polysaccharides are a type of polymer
molecule, with the monosaccharides as the repeating monomer subunit; the
monosaccharides have been joined together in a long chain via glycosidic bonds.

Examples of polysaccharides include starch (which is actually a mixture of two

polysaccharides, amylose and amylopectin, both of which use α–glucose as their
monomer), glycogen (also an α–glucose polymer) and cellulose (a β–glucose polymer).

Hexose monosaccharides: Glucose

Glucose is an example of a monosaccharide (simple sugar). Specifically, it is a hexose
monosaccharide, meaning that it contains six carbon atoms.

Other examples of hexose sugars include fructose (found in combination with glucose in the
disaccharide sucrose, e.g. in fruits) and galactose (found in combination with glucose in the
disaccharide lactose, e.g. in milk).

The molecular formula of glucose is:

C6H12O6
However, this formula disguises the fact that glucose occurs in nature in two different
structural variants (isomers), called α–glucose (alpha‐glucose) and β–glucose (beta‐
glucose). These differ only in the position of the hydroxyl (OH) group on carbon 1.

You need to learn the difference as shown in the diagrams below, which show the ring
structures of the two structural isomers of glucose:

α–glucose β–glucose

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In terms of the properties of glucose, the large number of polar hydroxyl (OH) groups in the
structure of the molecule means that glucose (like all monosaccharides) can be described as
polar, hydrophilic and water‐soluble.

Water molecules can form hydrogen bonds with the hydroxyl groups in the glucose
molecule. Glucose will therefore dissolve in blood plasma and be transported around the
body (e.g. from small intestine or liver, to brain and muscles).

Glucose will also dissolve in the cytosol of a cell and readily diffuse to areas where it is
being used up.

The main role of glucose is as an energy source in respiration (i.e. it is a respiratory

substrate, respired to release energy for ATP production). The covalent bonds within the
molecule, for example carbon‐carbon bonds, contain energy that is released when the
glucose is broken down and oxidised in the series of enzyme‐catalysed reactions that make
up respiration.

Glucose is also a monomer used in the building of a number of important polysaccharides

(starch, glycogen, cellulose – see below).

Pentose monosaccharides: Ribose and deoxyribose

Monosaccharides which contain five carbon atoms are known as pentose sugars. There are
two pentose sugars which have important roles in cells: ribose is part of the structure of an
RNA nucleotide and also part of the structure of an ATP molecule; deoxyribose is part of the
structure of a DNA nucleotide.

The structures of ribose and deoxyribose differ in only one respect: deoxyribose has one
less oxygen atom than ribose (because in deoxyribose, the hydroxyl group on carbon 2 has
been replaced by a hydrogen atom):

Ribose Deoxyribose

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Glycosidic bonds
A glycosidic bond is a covalent bond between two monosaccharide residues; it is formed
via a condensation reaction (i.e. a new bond is formed, such that two smaller molecules
become joined together, and a molecule of water is produced); the reaction is usually
enzyme‐catalysed.

The formation of a glycosidic bond involves a reaction between a hydroxyl (OH) group on
one monosaccharide and a hydroxyl group on the other monosaccharide.

During the reaction, two of the hydrogen atoms and one oxygen atom from this pair of
hydroxyl groups are removed from the monosaccharides and join together to form a water
molecule, leaving the monosaccharides bonded together via the remaining oxygen atom.

Disaccharides and polysaccharides are formed by the joining together of two or more
monosaccharides via glycosidic bonds.

Formation of maltose (a disaccharide consisting of α–glucose + α–glucose):

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Formation of sucrose (a disaccharide consisting of α–glucose + β‐fructose):

Formation of lactose (a disaccharide consisting of β‐galactose + β(or α)–glucose, with the

second sugar staggered or flipped over compared to the first):

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Formation of cellobiose (a disaccharide consisting of β–glucose + β–glucose, with the

second sugar flipped over by 180° compared to the first):

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The breaking of glycosidic bonds (thereby releasing the individual monosaccharides) occurs
via hydrolysis reactions.

A molecule of water is required in order break each glycosidic bond; specifically, the atoms
from one molecule of water (two hydrogens and one oxygen) are required to reform/repair
the two hydroxyl groups which had previously undergone a condensation reaction to form
the bond.

Hydrolysis of maltose:

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Polysaccharides
Starch
Starch is an energy storage polysaccharide found in plant cells (usually as starch grains
within chloroplasts). Starch is actually a mixture of two different α–glucose polymers:
amylose (which is unbranched and adopts a coiled structure) and amylopectin (which has a
branched structure).

Starch stores tend to build up during the daytime (when photosynthesis rate is higher than
respiration rate, leading to excess glucose production); at night (when there is no
photosynthesis), starch can be readily hydrolysed by enzymes, releasing α–glucose. This is
used by the cell as a respiratory substrate or as a building block for other types of biological
molecule (e.g. amino acid synthesis).

Starch is a compact molecule, so that many α–glucose residues, and hence a great deal of
energy, can be stored in a small space. The fact that starch has very low solubility means
that in does not significantly decrease the water potential of the cell (in the way that the
presence of a large number of individual, highly soluble glucose molecules would); hence
starch does not cause osmotic effects.

Glycogen
Glycogen is an energy storage polysaccharide found in animal and fungal cells (usually as
glycogen granules in the cytoplasm). It is an α–glucose polymer with a branched structure.
Glycogen’s structure is in fact almost identical to amylopectin, only with a higher frequency
of branching; this makes glycogen even more compact than starch (i.e. a greater number of
α–glucose residues being stored in a given volume).

The large number of branches also means that glycogen can be very rapidly hydrolysed to
release glucose for respiration (e.g. during exercise); enzymes can simultaneously attach to
the end of each branch, releasing glucose monosaccharides from multiple parts of the
molecules at once. Like starch, glycogen is insoluble and so does not disturb the osmotic
balance of cells/tissues.

Cellulose
Cellulose is a polymer of β–glucose, used to build plant cell walls. The polysaccharide chains
of cellulose are described as linear, i.e. unbranched. Whereas α–glucose polymers consist of
sugar residues that are all in the same orientation, each β–glucose residue in a cellulose
molecule is flipped by 180° relative to the previous one. This has the consequence that
each cellulose molecule is a perfectly straight chain, with no coiling (unlike α–glucose
polymers, where the polysaccharide chains coil quite tightly due to the angle of the
glycosidic bonds).

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Large numbers of these straight cellulose molecules can bundle together in parallel, held to
each other by extensive intermolecular hydrogen bonding; the structure formed is called a
microfibril and it has very high tensile strength, whilst retaining flexibility.

Cellulose microfibrils are so large that they are insoluble. This means a plant cell wall will
not be weakened by parts of it dissolving in water. Cellulose is also relatively inert
(unreactive), meaning that the cell wall won’t be weakened due to parts of it undergoing
inappropriate metabolic reactions.

[See textbook p47‐50 for diagrams of the structures of these polysaccharides.]

The following table summarises the structures and properties of starch, glycogen and
cellulose, and how these relate to their functions in living organisms:

Starch
Feature Glycogen Cellulose
Amylose Amylopectin
Monomer α‐glucose α‐glucose α‐glucose β‐glucose
Type(s) of
1,4 only 1,4 and 1,6 1,4 and 1,6 1,4 only
glycosidic bonds
Each sugar
Orientation of residue is flipped
All the same All the same All the same
sugar residues 180° relative to
the previous one
Linear or
Linear Branched Branched Linear
branched chains
Some coiling Some coiling
Straight or coiled
Coiled (though limited (though limited Straight
chains?
by the branching) by the branching)

Solubility Low Low Low Very low

Name given to
Cellulose
large associations Starch grain Glycogen granule
microfibril
of molecules
Animal and fungal
cells, in the
cytoplasm; in
Plant and algal cells, often inside Plant and algal
Location animals, liver and
chloroplasts cell walls
muscle cells can
store particularly
large quantities
Energy storage Strengthens the
(glycogen is cell wall, which
Energy storage (starch is hydrolysed hydrolysed when prevents the cell
when photosynthesis rate is low, blood glucose bursting due to
Function
releasing glucose to be used in levels are low, entry of water,
respiration) releasing glucose enabling the cell
to be used in to instead
respiration) become turgid

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Feature Starch Glycogen Cellulose

Compact, so
much can be Very high tensile
stored in a small strength, so able
space; branched, to prevent the
Compact so much can be stored in a
so enzymes can cell expanding or
small space; amylopectin is branched
hydrolyse it at rupturing, instead
so enzymes can hydrolyse it at
How properties multiple points to allowing turgor to
multiple points to release much
relate to function release much be established;
glucose quickly; insoluble so does not
glucose quickly; insoluble so does
affect the cell’s water potential
insoluble, so does not dissolve in
(avoiding osmotic effects).
not affect cell’s water (which
water potential would lead to a
(avoiding osmotic weakened wall).
effects).

Lipids
Lipids (fats and oils) are non‐polar, hydrophobic compounds, made up mostly of carbon
and hydrogen atoms, with a small proportion of oxygen.

Lipids do not dissolve in water; this is because the distribution of charge across the lipid is
very even (no/few polar groups, so no δ+ or δ‐ on any of the atoms) meaning that the lipid
cannot form hydrogen bonds with water.

However, lipids are soluble in organic solvents such as benzene, since these are themselves
also non‐polar and hydrophobic.

The term fat refers to a lipid that is solid at room temperature, e.g. the subcutaneous fat
stored under the skin of many animals.

Oils are liquid at room temperature, e.g. the oil stored in a sunflower seed.

Lipids are not polymers, as they do not consist of a chain of specific repeating monomer
subunits. However, they are usually very large and complex molecules and hence can be
referred to as biological macromolecules.

Fatty acids
Fatty acids consist of a carboxyl (‐COOH) group attached to a hydrocarbon chain (i.e. a long
chain of carbon atoms bonded to hydrogen atoms); they are a type of carboxylic acid.

Saturated fatty acids contain no carbon‐to‐carbon double bonds; all carbon atoms in the
chain form the maximum number of bonds with hydrogen atoms. The hydrocarbon chain of

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a saturated fatty acid is straight in shape (no kinks). Animal lipids often contain saturated
fatty acids; because their hydrocarbon chains are straight, saturated fatty acids can pack
closely together, resulting in a lipid which is solid at room temperature, i.e. a fat.

Drawing of a saturated fatty acid:

A monounsaturated fatty acid contains one carbon‐to‐carbon double bond in its

hydrocarbon chain. The presence of the double bond causes a kink (bend) in the chain.

Drawing of a monounsaturated fatty acid:

A polyunsaturated fatty acid contains more than one carbon‐to‐carbon double bond in its
hydrocarbon chain. The chain therefore has more than one kink. Mono‐ and
polyunsaturated fatty acids are typically found in lipids from plants; the kinks in the fatty
acid chains prevent them packing closely together, resulting in a lipid that is liquid at room
temperature, i.e. an oil.

Drawing of a polyunsaturated fatty acid:

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The (un)saturation of fatty acids can be recognised from their molecular formulae. The
principles behind this are that, excluding the carboxyl group (COOH):

 any saturated fatty acid will have double the number of H compared to C atoms,
plus one;
 For each C=C double bond present, 2 H atoms are lost.

Example: state whether each of the following is a saturated, monounsaturated or

polyunsaturated fatty acid, giving a reason for each answer:

C15H31COOH (palmitic acid)

C17H33COOH (oleic acid)

C17H31COOH (linoleic acid)

Fatty acids are significant in being constituents of more complex lipids, including
triglycerides and phospholipids (see below).

Triglycerides
A triglyceride is a type of lipid produced when one glycerol molecule is bonded to three
fatty acid molecules via ester bonds.

Drawing of a triglyceride:

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Glycerol is an alcohol, i.e. it contains hydroxyl (‐OH) groups (in this case, three). Each
hydroxyl group from glycerol is able to react in a condensation reaction with the hydroxyl
group at the end of a fatty acid. A molecule of water is produced in this reaction; the new
bond is formed is called an ester bond, hence this specific type of condensation reaction is
also known as esterification.

The ester bonds in a triglyceride can be broken via a hydrolysis reaction. The atoms from
one molecule of water are needed to break each ester bond (hence three molecules of
water in total would be needed to completely hydrolyse a triglyceride). This hydrolysis
reaction occurs during digestion of triglyceride fats in our small intestines, catalysed by
lipase enzymes.

Diagram summarising the synthesis and breakdown of a triglyceride via the formation
(esterification) and breakage (hydrolysis) of ester bonds between fatty acids and glycerol:

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In many animals, triglycerides are stored under the skin (as subcutaneous fat) and around
internal organs (as visceral fat). Their functions include:

o long‐term energy storage – stable (i.e. metabolically unreactive) enough to be

stored for long periods; compact in structure and containing a high proportion of
energy‐rich C‐C and C‐H bonds so that much energy is stored in a small volume;
insoluble so no effect on cells’ water potential; can be respired (via a process called
beta‐oxidation) when needed to release energy;

o thermal insulation – reducing heat loss from the body, especially important in
animals adapted to cold climates;

o cushioning of internal organs such as the kidneys – reducing the extent of

mechanical damage by acting as a shock absorber if there is a heavy impact to the
abdomen;

o buoyancy of aquatic animals, e.g. whales – lipids are less dense than water so their
presence in the bodies of aquatic animals keeps them buoyant.

Phospholipids
Phospholipids are similar in structure to triglycerides, however the glycerol molecule has
bonded to two (not three) fatty acids, plus one phosphate group. Whilst most lipids contain
only the chemical elements carbon, hydrogen and (a low proportion of) oxygen,
phospholipids additionally contain the element phosphorus.

Drawing of a phospholipid:

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The phosphate group of a phospholipid is significant as it gives the molecule a unique

property: one end of the molecule is water soluble, whilst the other end is not.

Specifically, the phosphate group is negatively charged and hence water soluble, giving the
phospholipid molecule a hydrophilic head; however the two fatty acid chains are not
soluble in water and hence are referred to as hydrophobic tails. These hydrophobic fatty
acid chains are able to interact with one another (and are soluble in organic solvents);
however, due to the hydrocarbon chains being non‐polar, they repel water molecules as
they cannot form hydrogen bonds with them.

Due to these properties, phospholipids spontaneously form stable structures called bilayers
when placed in water. In a bilayer, two layers of phospholipids are arranged such that the
hydrophobic fatty acid tails point towards one another in the interior of the structure
(excluding water from this region), whilst the hydrophilic phosphate heads lie on the outer
faces of the structure, where they interact with water molecules.

Diagram of a phospholipid bilayer:

Phospholipid bilayers form the basis of all biological membranes, including the plasma (cell
surface) membrane of all prokaryotic and eukaryotic cells, as well as the membranes around
eukaryotic cell organelles.

A phospholipid bilayer provides partial permeability, meaning that some molecules can
pass freely through (i.e. those that are small and/or lipid‐soluble) whilst other molecules
and ions cannot pass through (i.e. those that are water‐soluble).

Multiple layers of a membrane comprised of a phospholipid bilayer can also have the
function of providing electrical insulation, e.g. the myelin sheath around the axon of a
neurone.

[For much more on membranes, see section 2.1.5 Biological membranes.]

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Sterols
Sterols (steroid alcohols) have little structural similarity with the other types of lipid: they
have a far more complex structure, based on four fused carbon rings. Most of this structure
is hydrophobic, however there is a hydrophilic hydroxyl (‐OH) group at one end of the
steroid, meaning that the properties of sterols are somewhat similar to those of
phospholipids.

Diagram showing the structure of cholesterol:

Cholesterol is the principal type of sterol produced within our bodies (mainly in the liver). It
has a number of functions including:

o Cholesterol is a constituent of plasma membranes, where it slots in between the

phospholipids, helping to stabilise the membrane structure; cholesterol also
regulates the membrane’s fluidity, by preventing the phospholipid bilayer becoming
too fluid at high temperatures and by keeping it fluid enough at low temperatures;

o Cholesterol is a precursor in the synthesis of Vitamin D, needed for the formation of

strong bones and hence prevention of rickets in children;

o It is also a precursor in the synthesis of steroid hormones, including testosterone

and oestrogen.

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Amino acids and proteins

Amino acids are the monomers from which polypeptides (proteins) are synthesised. There
are 20 different types of amino acid found in naturally‐occurring proteins. However, they all
have the same general features.

Each amino acid has a central carbon atom (sometimes called the alpha carbon). Carbon
atoms are capable of forming four covalent bonds.

In all amino acids, the central carbon makes bonds to the following four components:

1. A carboxyl (also called carboxylic acid) group, ‐COOH

2. An amine group, ‐NH2

3. An individual hydrogen atom, ‐H

4. A variable group known as the R group; this is different in each of the 20 types of
amino acids. The simplest R group consists of a single hydrogen atom (as in the
amino acid called glycine). R groups vary greatly in size and chemical properties (see
further comments overleaf) and may contain some or all of the elements C, H, O, N
and S.

Draw a diagram showing the general structure of an amino acid:

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As noted above, each of the 20 types of amino acid has the same general structure, but has
a unique R group.

Some R groups are water‐soluble, i.e. hydrophilic, in that they contain polar groups or
groups which ionise to leave a positive or negative charge, either of which will attract water
molecules. Others are lipid‐soluble, i.e. hydrophobic, in that they contain mainly or only
non‐polar groups which repel water molecules.

Diagram showing amino acids with R groups that are water‐soluble, capable of hydrogen
bonding and ionic bonding within protein structures:

Diagram showing amino acids with R groups that are lipid‐soluble, capable of hydrophobic
interactions within protein structures:

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Diagram showing the amino acid with R group that contains sulphur, capable of forming
disulphide bonds within protein structures:

Diagrams from Biology 1 for OCR (Mary Jones, OUP, ISBN 978‐0‐521‐71763‐2)

In terms of our diet, some amino acids are referred to as ‘essential’ – these cannot be made
in the body so must be obtained in the diet. Other amino acids are ‘non‐essential’ – these
can be made in the body from the essential amino acids, by a process called
transamination.

Formation and hydrolysis of peptide bonds

Two amino acids joined together form a dipeptide molecule; the new bond formed
between the amino acid residues is called a peptide bond. The reaction occurs between the
amine group of one amino acid and the carboxyl group of the other amino acid. As well as
resulting in the formation of the new peptide bond, this reaction produces a molecule of
water and hence is an example of a condensation reaction.

To separate a dipeptide molecule back into two amino acid molecules, the peptide bond
must be broken via a hydrolysis reaction. This will require the atoms from a molecule of
water.

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Diagram showing the formation and hydrolysis of a peptide bond between two amino acids:

In living cells, amino acids are joined together by ribosomes, in the specific order
determined by an mRNA molecule, during the process of translation. Formation of the
peptide bonds between the amino acids is catalysed by an enzyme called peptidyl
transferase, which is part of the ribosome structure.

If many amino acids are joined by peptide bonds to form a long (unbranched) chain, the
resulting molecule is called a polypeptide. The term protein is used to refer to one or more
polypeptides which have a specific shape and biological function.

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The number of different possible polypeptides that can be made is enormous, given that
any length of chain is possible and there are 20 types of amino acid (each of which can be
included once, more than once or not at all).

For a polypeptide of length n amino acids (given 20 types of amino acid):

number of different polypeptides possible = 20n

Example: How many different pentapeptides are possible?

Following consumption of protein in food, polypeptide molecules are hydrolysed back into
their constituent amino acids during digestion, in the stomach and small intestine; these
reactions are catalysed by protease enzymes such as pepsin (in the stomach) and trypsin (in
the small intestine).

The levels of protein structure

The primary structure of a protein is its specific sequence of amino acids, i.e. the order in
which amino acids are bonded together. The only type of bonding involved in primary
structure is peptide bonding, formed via condensation reactions.

The primary structure of a protein is determined by the order of DNA bases in the gene
which encodes that protein (and the order of bases in the corresponding mRNA).

The secondary structure of a protein is the way in which some parts of the polypeptide
chain coil into regions of α‐helix, or fold back on themselves into regions of β‐pleated
sheet.

The only type of bonding involved in holding secondary structure in place is hydrogen
bonding; this hydrogen bonding is intramolecular, i.e. it occurs between pairs of atoms (one
of which has δ‐, the other δ+) from different parts of the same polypeptide molecule.

Some regions of a polypeptide chain may not adopt either of these specific secondary
structures but instead form a random coil.

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The tertiary structure of a protein is the further folding of the polypeptide into its final 3D
shape. This involves various types of bonding between atoms/groups in the R groups of the
amino acid residues in the polypeptide chain:

o Hydrophobic interactions – these involve hydrophobic (lipid‐soluble), non‐polar R

groups interacting only with one another and excluding water and polar/ionised R
groups from that region of the protein. Globular proteins (see below) often have a
hydrophobic core, held in place by hydrophobic interactions between non‐polar R
groups.

o Hydrophilic interactions (hydrogen bonding) – water‐soluble (hydrophilic), polar R

groups will be attracted to water and to each other; this involves the formation of
(intramolecular) hydrogen bonds between pairs of atoms (one of which has δ‐, the
other δ+) from R groups in different parts of the polypeptide chain, and also
formation of hydrogen bonds between atoms in the R groups and water molecules in
the surrounding solution (e.g. the cytoplasm). Both these interactions help to
determine and stabilise the tertiary structure. In a globular protein, hydrophilic R
groups are generally found on the surface of the folded protein; this causes the
protein to have good solubility in water (even though it may be large and have
hydrophobic groups ‘hidden’ inside).

o Disulphide bonds – these are covalent bonds between two sulphur atoms; each
sulphur atom is part of the R group of a cysteine amino acid from a different region
of the polypeptide chain. In general, disulphide bonds are only seen in proteins that
are secreted from a cell, e.g. antibodies.

o Ionic bonds – these are strong attractions between an atom with a negative charge
(from the R group of one amino acid residue) and an atom with a positive charge
(from the R group of a different amino acid residue, from further along the
polypeptide chain). The two R groups involved have undergone ionisation (usually,
one R group has lost an H+ and so become negatively charged, whilst the other R
group has gained an H+ and so become positively charged). Ionisation of R groups,
and hence how many ionic bonds can form (and where) in the protein structure is
affected by the pH of the surrounding solution.

PRACTICAL: Computer modelling (using specialist software) can be used to investigate the
levels of protein structure within a protein molecule.

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Diagram illustrating that types of bonding between R groups in protein tertiary (or
quaternary) structure:

Diagrams from Biology 1 for OCR (Mary Jones, OUP, ISBN 978‐0‐521‐71763‐2)

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Globular proteins
Globular proteins are those in which the polypeptide chain(s) are folded into a compact,
roughly spherical shape. The specific way that the polypeptide chain folds (i.e. the tertiary
structure) is such that most hydrophobic amino acid R groups are ‘buried’ in the centre of
the structure, forming a hydrophobic core. Meanwhile, hydrophilic R groups are mostly
exposed on the surface of the protein, causing the globular protein as a whole to have good
solubility in water (providing it has not been denatured).

Many globular proteins are conjugated, meaning that they contain a permanently attached
prosthetic group that is essential to the function of the protein, e.g. haemoglobin contains
haem groups.

Globular proteins have a wide range of important biological roles: hormones for cell
signalling; enzymes for catalysing metabolic reactions; oxygen transport (using
haemoglobin) in the blood, channel proteins and carrier proteins for transport across
membranes; antibodies for fighting pathogens; compact packaging of DNA (using histone
proteins) into chromosomes.

You need to be familiar with the structures and functions of the following globular proteins
in particular:

Insulin
Insulin is a hormone which controls blood glucose levels via cell signalling; it is produced by
the beta cells (found in the islets of Langerhans) in the pancreas and released into the blood
stream in response to high blood glucose levels.

Insulin binds to specific complementary receptors on the plasma membranes of liver and
muscle cells, triggering them to absorb more glucose from the blood and hence causing a
decrease in blood sugar level.

Insulin’s tertiary structure must be a very specific 3D shape, i.e. the polypeptide chain must
fold in exactly the same way each time the protein is produced. This is important because
the mechanism of action of the hormone involves it binding to its membrane‐bound
receptor, to which it must be complementary.

Since insulin travels in the blood plasma, it must have good solubility: this is achieved by
having hydrophobic R groups in the core of the protein and hydrophilic R groups on the
surface.

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Catalase
Catalase is an enzyme, meaning that it acts as a biological catalyst, i.e. a protein which
speeds up a metabolic reaction. Specifically, catalase catalyses the breakdown of hydrogen
peroxide (a highly toxic waste product of cellular metabolism) into oxygen and water.

In terms of structure, a catalase is a globular protein containing more than one polypeptide
chain, i.e. it has quaternary structure.

Catalase is also an example of a conjugated protein: this means that it has prosthetic
groups attached. Prosthetic groups are a permanent part of the protein structure, essential
for the protein’s function, however they are not composed of amino acids. In the case of
catalase, the prosthetic groups are haem groups, which contain iron ions (Fe2+). A hydrogen
peroxide molecule binds to a haem group as the first stage of the reaction mechanism.

Haemoglobin
Haemoglobin is found in the cytoplasm of erythrocytes (red blood cells), where its role is to
reversibly combine with oxygen and hence carry oxygen in the blood from the lungs to
respiring body tissues.

Haemoglobin has high solubility, so that it can readily dissolve in the cytoplasm of
erythrocytes in huge quantities; this is achieved via the presence of hydrophilic amino acid R
groups on the surface of the protein, with hydrophobic R groups in the core only.

Like catalase, haemoglobin is globular in terms of tertiary structure and also has quaternary
structure: specifically, it has four polypeptide chains, two each of two different types: 2 α‐
globin and 2 β‐globin polypeptides.

Haemoglobin also contains prosthetic groups (specifically, four haem groups in total, one
per polypeptide), i.e. it is a conjugated protein.

One molecule of oxygen (O2) binds to the Fe2+ ion in each of the four haem groups. This
means that, since there are four haem groups, one haemoglobin protein can carry up to
four oxygen molecules at one time.

At the lungs, oxygen binds to haemoglobin, forming oxyhaemoglobin; this is carried (within
erythrocytes) in the blood to respiring tissues, where the oxygen is released from the
haemoglobin (i.e. dissociation of oxygen occurs) for immediate use in aerobic respiration.

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Fibrous proteins
Fibrous proteins are those that adopt an elongated shape in their tertiary structure (i.e. the
polypeptide chains do not fold up into a spherical shape). Fibrous proteins tend to be very
long and are insoluble in water. Their insolubility is due to their massive size plus the
presence of many amino acids with hydrophobic R groups exposed on the surfaces of the
protein.

In general, fibrous proteins also have high tensile strength (often due to forming rope‐like
structures) and are metabolically unreactive; most are also quite flexible.

Compared to globular proteins, fibrous proteins often have far less variety of different
amino acid types in their primary structure; indeed their primary structures often are quite
repetitive, e.g. a particular amino acid might appear at every third position in the chain.

You need to be familiar with the properties and functions of the following examples of
fibrous proteins (but note that the specification does not require you to learn specific
structural details):

Collagen
This is a protein with very high tensile strength, insoluble but flexible, found in the
connective tissue of skin, ligaments, tendons and surrounding nerves.

The high tensile strength is due to the structure of each collagen molecule, which consists
of three polypeptides wound round one another in a rope‐like structure; numerous such
molecules are then cross‐linked via many hydrogen bonds to form thick fibres, providing
further strength to the overall structure.

Keratin
This is an insoluble fibrous protein with high tensile strength that is relatively inflexible. It is
the main constituent of hair and nails (where its role is to protect softer, more easily
damaged tissues) and is also found in skin.

Keratin proteins contain a very high proportion of the sulphur‐containing amino acid
cysteine in their primary structure; this enables numerous disulphide bonds to form
between polypeptide chains, giving increased strength to the protein. The more of these
bonds form, the stronger the keratin will be.

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Elastin
This is a stretchy (elastic) fibrous protein found in elastic fibres, e.g. in the walls of the
alveoli in the lungs and in artery walls.

The elasticity of elastin proteins enables the alveolar walls and artery walls to expand by
stretching and then (passively) recoil to return to their original size/shape. Elastin is also
found in the skin, giving it elasticity alongside its strength.

Elastin has quaternary structure made up of an association of many individual tropoelastin

polypeptides (which act like miniature springs) cross‐linked to one another.

Inorganic ions involved in biological processes

DEFINTION: An ion is an atom, or a group of atoms, that carries a positive or negative
charge. Ions with a positive charge are called cations; ions with a negative charge are called
anions. Opposite charges attract one another: the strong attraction between a cation and
an anion is called an ionic bond.

Learn the names of the following ions, their chemical symbols, and an example of the
biological significance of each:

Type of Chemical
Name Significance
ion symbol
Binds to vesicles, triggering their
Calcium ion Ca2+ movement towards the plasma membrane
for exocytosis of their contents (e.g.
neurotransmitter release at a synapse).

Sodium ion Na+ Generation and transmission of an action

potential (electrical impulse) in a neurone.

Potassium ion K+ Generation and transmission of an action

potential (electrical impulse) in a neurone.

Cation A high concentration of H+ means the pH

(positive of the solution is low (acidic conditions).
charge) Hydrogen ion (also
called a proton) H+ A proton gradient across the inner
mitochondrial membrane is a key part of
the mechanism whereby ATP is
synthesised via aerobic respiration.
Released into the soil when decomposer
microorganisms break down organic waste
Ammonium ion NH4+ (dead bodies, faeces, urine) via the
process of ammonification; the NH4+ can
be converted to NO2‐ and then NO3‐ by
nitrifying bacteria.

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Type of Chemical
Name Significance
ion symbol
Nitrosomonas bacteria convert NH4+ to
Nitrite ion NO2‐ NO2‐ as the first stage in nitrification.
Nitrobacter bacteria convert NO2‐ to NO3‐
as the second stage in nitrification; the
Nitrate ion NO3‐ NO3‐ is readily absorbed by plants’ root
hair cells and is then used by plant cells as
a source of N for amino acid synthesis.
Formed in red blood cells following the
Anion
(negative
charge)
Hydrogencarbonate
ion HCO3‐ reaction of carbon dioxide with water;
large quantities are carried in the blood
plasma.

Chloride ion Cl‐ A cofactor ion for amylase, without which

the enzyme is inactive.

Phosphate ion PO43‐ Part of the structure of nucleotides, ATP

and phospholipids.
An excess of OH‐ compared to H+ signifies
Hydroxide ion OH‐ a high pH (alkaline conditions).

Tests for biological molecules

PRACTICAL: You need to know how to carry out and interpret the results of several common
chemical tests.

The biuret test for polypeptides/proteins

Add ‘biuret reagent’ (i.e. sodium hydroxide solution mixed with copper sulphate solution)
to the sample to be tested. If the colour changes from pale blue to lilac/purple, this
indicates that proteins are present (since copper ions form a purple complex with peptide
bonds in an alkaline solution). Notice that no heating is required as part of this test.

Benedict’s test for reducing sugars

Reducing sugars are mono‐ or disaccharides which are capable of donating electrons to (i.e.
‘reducing’) other molecules/ions. All monosaccharides (e.g. glucose, galactose, fructose)
are reducing sugars; certain disaccharides are also reducing sugars (e.g. maltose, lactose).
However some disaccharides, notably sucrose, are non‐reducing sugars (meaning that they
are not capable of donating electrons to reduce another molecule/ion).
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To test for reducing sugars, add an equal volume of Benedict’s reagent (alkaline copper (II)
sulphate). Heat the mixture at 80°C for 10min. If (high concentration of) reducing sugars are
present, a change from pale blue solution to brick red precipitate will be seen.

In fact, the quantity of precipitate formed is proportional to the quantity of reducing sugar
which was present; if a low concentration of reducing sugar was present, the precipitate will
appear greenish; medium concentration will appear orange, whilst the classic brick red will
be seen only if there was a high reducing sugar concentration.

Comparing colours of different test solutions gives some indication of relative reducing
sugar content; however, to gain a quantitative (numerical) result, the precipitate could be
filtered out and weighed.

Benedict’s test for non‐reducing sugars

Non‐reducing sugars (e.g. sucrose) give a negative result when tested with Benedict’s
reagent as above; the solution remains pale blue.

However, the non‐reducing sugar could then be hydrolysed into its constituent
monosaccharides (e.g. hydrolysis of sucrose into glucose and fructose). The monosaccharide
products of hydrolysis should then test positive for reducing sugar when the Benedict’s test
is repeated.

To hydrolyse the non‐reducing sugar, treat it in solution with a suitable enzyme (e.g.
invertase/sucrase to hydrolyse sucrose), or boil it in dilute hydrochloric acid. If the test
solution gives a negative (i.e. pale blue) result for presence of reducing sugars before
hydrolysis, but then tests positive (brick red precipitate) after hydrolysis, the conclusion is
that a non‐reducing sugar (usually sucrose) was present in the original sample.

Reagent test strips for reducing sugars

Commercially available test strips (e.g. for the reducing sugar glucose) can be dipped in the
sample (e.g. urine, blood). The end of the test strip may change colour; the colour is then
compared with the manufacturer’s colour chart, which tells you the approximate sugar
concentration corresponding to the colour change obtained.

The iodine test for starch

Add a few drops of ‘iodine solution’ (iodine dissolved in potassium iodide solution) to the
test sample. A colour change from yellow to blue‐black indicates starch (specifically, the
amylose component of starch) is present.

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The emulsion test for lipids

The sample to be tested is mixed with ethanol. The resulting solution is then itself added to
water and shaken. The tube is then left to settle for a short time; if a milky white emulsion
is seen, this indicates the presence of lipid. However, if the solution remains clear and
colourless, this is a negative result (no lipid present).

Quantitative methods to determine the concentration

of a chemical substance in a solution
PRACTICAL: Quantitative methods enable the collection of numerical data, e.g. the
concentration of a specific molecule in a solution. You need to be familiar with colorimetry
and biosensors as examples of quantitative methods.

Colorimetry
A colorimeter is a device with passes light through a sample solution and measures the
transmission of light (i.e. the percentage of light which is able to pass through the solution)
or the absorbance of light (i.e. the percentage of light which is absorbed by components of
the solution and so not able to pass through).

One common use of a colorimeter is to enable Benedict’s test to become a quantitative

method for determining reducing sugar (e.g. glucose) concentration.

First, Benedict’s test is carried out in the usual way on the test solution (which has an
unknown concentration of reducing sugars), and also repeated on a number of ‘standard’
solutions (e.g. prepared by serial dilution), whose reducing sugar concentrations are
already known. All solutions are then filtered to remove any precipitate. (At this stage the
solutions will all look pale blue to colourless.)

A red filter is fitted to the colorimeter (so that only the correct, specific wavelength of light
is used) and a small tube (cuvette) of water (called ‘the blank’) is placed in the device for
calibration: the colorimeter is calibrated such that water gives a transmission reading of
100% (or absorbance reading of 0%).

Then each test and standard solution is placed in the colorimeter in turn and the
transmission (or absorbance) of light is recorded for each one.

A graph called the calibration curve is plotted using the readings from the standard
solutions (of known glucose concentrations); this enables the concentration of glucose in

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the test solution to be determined, by reading off the glucose concentration on the graph
which corresponds to that solution’s transmission (or absorbance).

Note that in this test, a solution which contained very low glucose concentration would
appear ‘very blue’ after Benedict’s test and filtration (as few of the blue copper (II) ions will
have been reduced), so this will have low transmission of light and high absorbance of light
when tested in the colorimeter.

A solution which had very high glucose concentration will be almost colourless (having lost
its blueness when the copper ions were reduced); this will have high transmission of light
and low absorbance of light.

Biosensors
A biosensor is a device that uses biological components (e.g. enzymes and/or antibodies)
to determine the presence and/or concentration of a specific molecule (e.g. a hormone, or
a sugar).

First, the molecule being tested for must be recognised, e.g. by binding to an enzyme or
antibody with a complementary binding site.

Next, there is transduction, which is when the molecular recognition triggers a change,
which is then detected (e.g. a pH change).

This in turn leads to the production of signal which is then displayed, e.g. a numerical
reading is produced on a display. In this way, data can be collected relating to the
concentration of a specific molecule in the samples being tested.

A well‐known example of a biosensor that uses antibodies is the pregnancy test, which
detects the presence of hCG (human chorionic gonadotrophin) hormone in urine (see
Module 5: Excretion topic).

Chromatography
Chromatography is the separation of molecules according to their different properties
(notably solubility in specific solvents and molecular size). It can be used in biology to
separate mixtures of molecules such as proteins, carbohydrates, pigments or vitamins.

Note that in general chromatography is used to separate components that are not
charged; a technique called electrophoresis is used instead if the components do have
charge, e.g. DNA molecules (see Module 6: Manipulating Genomes topic).

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There are a number of forms of chromatography, with the choice of technique depending
on the types of molecule that you wish to separate.

For the separation of amino acids or photosynthetic pigments, thin layer chromatography
(TLC) is suitable. A thin layer of silica gel forms the stationary (i.e. non‐moving) phase,
applied onto a rigid metal/plastic sheet called the chromatography plate. The mixture of
amino acids is applied near to one end of this plate. The base of the sheet (only) is
submerged in an organic solvent (the mobile phase). The solvent is drawn upwards through
the stationary phase, carrying the amino acids with it, up the plate.

Different types of amino acid move upwards at different rates due to differences in the
extent of interactions (e.g. hydrogen bonds) between the amino acid’s R group and the
stationary phase, and also due to differences in the solubility of the amino acids in the
mobile phase. Hence after a fixed period of time, the different types of amino acid have
reached different distances up the sheet and are separated from one another.

If the molecules separated do not already have colour, a stain will be needed to visualise
them on the chromatogram, e.g. ninhydrin can be used to stain colourless amino acids in
purple.

A typical protocol for TLC to separate amino acids is as follows:

1. Draw pencil line (called the start line or origin) across the TLC plate, about 2cm from
its base, high enough that this line will not be submerged when the base of the plate
is placed into the solvent;

2. The sample of amino acids is ‘spotted’ onto the start line (e.g. using a fine pipette
tip), with repeated spots being applied to the same place to build up a high
concentration (allowing each spot to dry before the next is applied);

3. If multiple different samples are to be compared on the same TLC plate, place their
spots far enough apart from each other that they will not merge;

4. Add the chromatography solvent (mobile phase) to a suitable beaker (or

chromatography tank), to a height of around 1cm – it is important that this solvent
level will be significantly lower than the start line on the TLC plate, so the spots of
amino acids will not be lost by dissolving away into the solvent;

5. Very carefully place the plate into the beaker, taking care only to touch the edges of
the plate – ideally wear gloves or use forceps to prevent damage to the stationary
phase (silica gel) or transfer of substances from the hands;

6. Place a lid/cover over beaker to prevent evaporation of the solvent;

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7. The plate should be supported in the beaker (e.g. using a small clamp at the top of
the plate only), to keep plate vertical (not touching the sides of the beaker) and
immobilised so that the solvent soaks up the plate evenly;

8. Take the plate out of the solvent once the solvent reaches around 1cm from the top
and immediately mark the position of the solvent front with a pencil line (before
the solvent evaporates) – this is essential for the calculation of Rf values;

9. Stain the chromatogram with ninhydrin in order to visualise the (colourless) amino
acids;

10. Use a pencil to mark the position of each component as the coloured spots may
fade quickly in bright light, giving each component a letter or number to avoid
confusion;

11. Calculate Rf values (see below) for each component;

12. Ideally, repeat the procedure three times and find mean Rf values in order to achieve
more accuracy in identification, to check repeatability is good and to identify any
anomalies.

The components separated by chromatography can be identified using published retention

(Rf) values. The Rf value for a given chemical compound will always be the same, providing
the identical conditions have been used (e.g. same stationary and mobile phases, same
temperature).

Rf = distance travelled by component*

distance travelled by solvent
*if a component forms a thick line or spot, measure to the middle of this.

Note that Rf values will always be between 0 and 1. For example, the amino acid glutamine
has an Rf of 0.13, whereas phenylalanine has an Rf of 0.59.

Example of Rf value calculation:

The diagram (next page) shows the positions of pigments from a leaf extract following TLC.
Calculate the Rf value for each of the 5 types of pigment.

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Rf for carotenes =

Rf for chlorophyll a =

Rf for chlorophyll b =

Rf for lutein =

Rf for xanthophylls =

PRACTICAL: You need to be familiar with the techniques used in practical investigations to
analyse biological solutions using paper chromatography and/or thin layer
chromatography.

***END OF BIOLOGICAL MOLECULES TOPIC***

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