1.

Introduction 3
Questions 7

2. Physics tools for food technology 8

Questions 14

3A. Interactions 24
Exercises 32

3B. Physics Tools: Steric Interaction 34

3C. Physics Tools: Depletion Interaction 37

4. Physics Tools: Proteins 40

4.1 The role of proteins in food 40
4.2 Protein structures 40
4.3 Transitions between protein structures 50
Exercises 52

5. Bulk Rheology 53
5.1 Fluids 54
5.2 Solids 58
5.3 Viscoelastic materials 61
Exercises 66

6. Physics Tools: Macromolecules 70

6.1 Introduction 70
6.2 Configuration of Macromolecules in Solution 71
6.3 Effect of configuration on physical properties 75
6.4 Limited flexibility 78
6.5 Non-Ideal chains in solution 81
6.6 Polyelectrolytes 84
Exercises 87

7. Physics approach to Sauces 88

Questions 95

8. Physics Tools: Interfaces 96

Questions 119

9. Physics approach to milk and dairy products 124

9.1 Natural milk 124
9.2 Homogenized milk 127
9.3 Cream 129
9.4 Whipped cream 130
9.5 Butter 133
9.6 Margarine 134
9.7 Ice cream 134
9.8 Yoghurt 135
9.9 Buttermilk 136
9.10 Sour cream 136
9.11 Cheese 136
Questions 138
2
 Chapter 1: Introduction

A food technologist faces the following general question:

Which ingredient do I have to put in where, when and how, during the preparation of
a product (new or already existing), and what do I have to assure during processing,
storage and consumption in order to meet the criteria being put forward regarding
safety, taste, stability (physical and chemical), health and convenience?

The disciplines that play a role in answering these questions are chemistry, physics/physical
chemistry, process engineering, microbiology, product design and queality management.

In order to answer the physics/physical chemistry questions for a specific case (i.e. for a
specific product, during the proces of making, storage or consumption) one would like to
have a systematic approach in answering these questions (a sort of structure which can be
followed while addressing any question) and a practical toolbox.

In answering a particular question we have to define which product it is about. Each product
has its specific types of ingredients and for each product we have different expectations form
the consumer point of view. For instance regarding the question how to store something. Ice
cream is cold and a frozen product, and therefore has to be stored in the freezer, while a
mayonaise should not be frozen and therefore should not be stored in the freezer. Both
products need to be stable, but under different storage conditions.

In order to systematically approach any food technology question we need to have a

systematic way of ordering the types of products. Such a systematic categorisation allows to
specify the question in an efficient manner. Many different categorisations have been put
forward in the literature, but many boil down to more or less the same idea. Here we adopt
one given by Mc Gee (1984). See below.

The categorisation in types of products helps to specify the questions posed to the
technologist, but the answer to the question has still to be sought. To answer any questions,
one needs to specify which ingredients are in the product, and one has to have knowledge on
relationships between ingredient properties (down to a molecular scale) and consumer
relevant product properties (up to a macroscopic scale). In answering food technological
questions, answering in terms of such specific relationships is important. To this end, the
herefore mentioned disciplines are all important. In this course we focuss on the physical and
physico-chemical aspects that are relevant to formulating relationships between ingredient
properties and consumer relevant product properties.

Examples of consumer relevant product properties are firmness, pourability, spreadibility,

colour, thickness, crunchiness etc. Examples of physical and physico-chemical properties of
ingredients deal with the shape of the molecule, chemical fine structure etc. The type of
molecules which we focus on in foods are proteins, carbohydrates and fat molecules, water
molecules and molecules in air, as these are the main components of foods. Besides these
ingredients one also has diffferent ingredients in foods, but these are present in relatively low
amounts.

3
There exist two important aspects in formulating physical and physico-chemical relationships
between properties of molecules and consumer relevant properties.
1. There is a factor billion difference in length scale betwen olecular scale and
macroscopic scale (nanometers to meters).
2. Foods are usually not homogeneous on a length scale of microns. Examples are bread,
beer foam, margarine. See figure below.

Droplets in mayonaise Fat crystals in a margarine myosin protein network

Whipped cream Bread dough myosin protein network

Taken from: I. Heertje, Taken from: A. M. Hermanson,

Food Structure 12 (1993) 343 J. Food Sci. and Agriculture,
37 (1986) 69

In order to formulate the desired relationships in a usefull way, one needs to know about the
intermediate, inhomogeneous domain. This domain is referred to as mesoscopic domain. Of
particular relevance are domain sizes between 10 nanometers and millimeters, which is
referred to as the colloidal domain. The phyics that is relevant to this intermediate domain
size is called mesoscopic physics and science relevant to this length scale is classically
referred to as colloid science. The physics and physical chemistry regarding the mesoscopic
domain acts as a bridge for formulating relationships between properties on a molecular scale
and a macroscopic scale.

Mesoscopic Physics

Food Macroscopic
Ingredients Food properties

4
In order to arrive at a systematic approach in solving physics and physical chemistry related
food technological questions it is thus important to

1. Know and be able to distinguish between different types of products

2. Know which type of colloidal structures are present in foods, and which properties
such colloidal structures exhibit
3. Know which ingredients and concomittant functions are present in food products
4. Know different possible functions of ingredients in relation to the relevant
macroscopic properties of a food.

and in addition to

5. Know and apply general rules/laws on which ingredient forms what types of colloidal
structures
6. Know and apply general rules/laws that relate molecular properties of ingredients
with the properties and mutual interactions of the colloidal structures that are formed
by these ingredients
7. Know and apply general rules/laws on how properties and mutual interactions of
colloidal structuresdetermine teh macroscopic properties of the systems that contain
these colloidal structures

In the above list of 7 points the first 4 refer to sheer knowledge related to products (“handy to
know” type of things). This type of knowledge has been summarised in the figure below.

Relevant Food know ledge

Milk and dairy products
E ggs/egg- based products
Meat Ingredients?
F ruit/vegetables and herbs F unction(s) of ingredients?
G rains and nuts Colloidal structures?
Bread and dough Macroscopic properties?
Sauces
Chocolate, sweets, sugars
A lcoholic beverages (wine, beer, liquors, …)
Drinks, juices

Product categorisation and related specific type of food knowledge

Points 5 t/m/ 7 relate to more generic knowledge, which is generally applicable to more than
one problem. This is summarised in the figure below. Ingredients can form a multitude of
different structures on a colloidal scale. One can have platelets, spheres, long thin threads,
and threads that are forming a coil like structure. This is schematically summarised as
“structures”. The macroscopic properties are dependent on how the structures exhibit
interaction, and on the properties of the colloidal structures themselves. During the course
points 1 to 7 will be treated. The combination of product knowledge and generic knowledge
can be used as a toolbox to solve aspects of the general question which was formulated in the
beginning of this chapter.

5
 Generic know ledge
Ingredients Structures Macro-properties
P rotein Mechanical
F low
P olysaccharide
Stability
W ater
…
F ats/oils
P roperties and interactions
Mixtures of on different length scales

nm Colloidal scale micron

Generic physical knowledge relevant to food technology

During the course, examples of the different food categories will be treated, with their
according colloidal structures, including their main ingredients. The function of the
ingredients usually follow from their influence on properties of the colloidal structures and
from their specific nutritional value. For each category one can give a description for relevant
colloidal structures, the relevant ingredients and their functions, and subsequently the main
macroscopic properties in relation to colloidal and ingredient properties.

During the course also generic knowledge aspects wil be treated. These include an overview
of different interactions (including their origin) and phenomena at interfaces (which separate
colloidal structures with their surrounding medium). The interfacial phenomena are in
particular relevant since inhomogeneities on a colloidal scale in a medium imply the
existence of lots of interface in that medium. Apart form these two main aspects, response of
systems upon deformation will be treated (rheology).

In dealing with colloidal structures it is convenient to use a generally accepted nomenclature

on different types of systems. One phase is usually embedded in another phase. Either phase
may be gas, liquid or solid. The various possibilities of e.g. solid in liquid, liquid in liquid etc.
are denoted by different terms. The nomenclature is explained in the figure below.

Nomenclature of some meso-structure containing systems

Dispersed Continuous or
phase Dispersing phase

Solid <1micron Coloidal dispersion or sol Liquid

Solid< 1 micron Hydrosol Water
Polymer Latex Liquid
Solid > 1 micron Suspension Liquid
Liquid Emulsion Liquid
Gas Foam Liquid
Solid/liquid Aerosol (smoke/fog) Gas

Nomenclature of various colloidal (meso-structure) containing systems

6
 Questions Chapter 1

Introduction

1. Name five aspects of foods that are important to consumers

2. Name two examples of a food ingredient

3. Name five disciplines that are important o a food technologist

4. Name three kinds of food that are inhomogeneous on a micron scale

5. Name two issues which makes that formulating relations between physical properties on
a molecular scale and properties on a consumer relevant macroscopic scale becomes a
difficult task.

6. How does one call structures that have a size between 1 nanometer and 1 millimeter?

7. If one faces a question on a relationship between ingredient property and product

property, what would be two essential pieces of knowledge that are essential to answer
the question?

8. Name four most relevant ingredients of foods.

9. Name two most relevant physical characteristics on a meso-scopic scale that are relevant
to formulating relationships between molecular and macroscopic properties.

10. Consider mixtures of two bulk phases. Make a table of type of dispersed phase with
according dispersing phase (including mentioning the size of the dispersed phase where
relevant) which applies to respectively a sol, suspension, hydrosol, foam, emulsion,
suspension, latex, fog, smoke.

7
 Chapter 2: Physics tools for food technology

In treating physics and physical chemistry that is relevant to food technology, one may
distinguish between physical phenomena that play a role in food technology, and physical
laws that can be applied to quantify these phenomena. In this part we will address aspects of
both.

Creaming/sedimentation
During creaming and sedimentation of particles in a liquid (for instance air bubbles in beer,
or the moving downwards of raisins in dough) one has three main factors that are relevant to
the speed, v, of the particles
a. Density difference between particle phase and liquid phase, 
b. Diameter, d, of the particles
c. Viscosity of the continuous phase, 

The influence of , d and  on speed, v, is summarised in the following equation (Stokes)

 . g . d2
v= ----------------------
18 . 

where g denotes the acceleration due to gravitation (g=9.81 m/s2) . This equation does not
take into account interaction between neighbouring particles, and is only strictly applicable to
solid and spherical particles in a fluid. See figure below for streamlines around a particle.

Flow of fluid around particle

8
The background of the equation of Stokes is a balance between the force on the particle due
to gravity, which tends to move the particle, and an opposing force by the surrounding fluid.
More specifically, the movement of the particle forces the fluid in its immediate surroundings
to move along. Far from the particle, the influence of the movement of the particle on the
movement of the fluid ceases to exist. So there is a region where the fluid molecules are
changing their speed from “fast” to zero, i.e. the fluid molecules exhibit a relative motion
towards one another in a certain region of the fluid. This relative motion results in a friction
between the molecules, and an according loss of energy (transformed into heat). The total
amount of friction results in a force that opposes the movement of the particle. In summary,
the opposing force exerted by the surrounding fluid on the moving particle is originating from
the friction between the liquid molecules as they exhibit a relative motion between one
another.
In fact, the opposing force by the liquid is a frictional force. The parameter which expresses
the magnitude of this frictional force is called viscosity.

Brownian movement
Observing micron-sized particles in a fluid, under the light microscope, reveals a random
movement of the particles. This movement is named after its first observer, Brown. The
origin of this movement lies in the fact that the particle for each of the translational
movements in the x,y, and z-direction as well as for each of the rotational movements around
its three principal axes, it has on average an energy of ½ kT available. Each direction has
equal preference for the particle to be taken. The randomness is related to the equal
preference for each direction.

Brownian movement versus creaming/sedimentation

If there is no gravitational force acting on particles, particles will exhibit Brownian
movement, i.e. there will be no preferred direction for the particles to move in, and thus there
will be no creaming nor sedimentation. If a gravitational force is existing, there is one
direction, i.e. the direction of the gravitational force, which yields a lower potential energy
for the particle when it follows that direction, i.e. there exists a preferred direction (since
moving in that direction lowers the potential energy of the particle).
The question arises under which circumstances the gravitational component is negligible, and
the motion of the particle remains still random-like, i.e. exhibits Brownian motion, and under
which circumstances one starts to observe the influence of the gravitational force on the
movement, i.e. when one starts to observe creaming/sedimentation. This can be analysed as
follows. If the energy gain for the particle, when travelling along the direction of the
gravitational force over a distance equal to its diameter, D, is much larger than ½ kT, the
most preferred direction will lie along the force of gravitation. When such gain in kinetic
energy (travelling over a distance of its diameter) is of the order of ½ kT (see Brownian
movement), deviations from a random movement will only just start to become observable.
The gain in kinetic energy due to following the path along the direction of the gravitational
force, F, over a distance D, is equal to F.D. The force F is given by
F=(particle - fluid ).g.(D3/6) where particle and fluid denote the density of particle and fluid,
respectively, and where g denotes the acceleration due to gravity. Using F.D~½kT as the
criterium when random motion starts to be influenced by gravitational force one finds

(D4/6) (oarticle - fluid ).g ~1/2 kT

or

9
D4 ~3 kT/{(oarticle - fluid )g}

Creaming or sedimentation of a spherical object with diameter D can be

estimated to occur when gravitational energy and thermal energy become
comparable, leading to

D~{3kT/.(oarticle - fluid ).g}1/4

Break up of fluid and fat like particles in flow

This is in detail lectured in the Advanced Food Physics course, part on emulsion science.

Heating and time effects

Pasteurisation and sterilisation are two effects that relate to heat diffusion and transport
phenomena. These are treated in the process engineering courses.

Clustering of particles
Leading to increased creaming in milk, and e.g. to gelling phenomena. This phenomenon and
its application to gelling is covered in detail in the Advanced Food Physics course, part on
gels. The specific clustering morphology depends in part on the interaction between the
particles (see interactions).

Whipping, coalescence, and drainage

During whipping, air is being incorporated forcefully into a liquid. Bubbles are being formed
as a result. If one whips water, the bubbles quickly disappear. The amount of bubble surface
is diminished quickly. If one whips water containing a solution of dishwashing liquid the
bubbles remain stable for a much longer time. This is due to the fact that the molecules in the
dishwashing liquid are surface active, i.e. they want to stay at the interface between the air
and the water. This is due to their amphiphilicity. Analogously, protein molecules also are
amphiphilic thus able to stabilise air/water interfaces. In addition, protein molecules may
change their conformation while being at the interface (sometimes becoming more attached
to the surface) and they may intertwine with neighbouring protein molecules, thus forming a
layer which is viscous and elastic, making it hard to be disentangled into smaller pieces
again. So, one prerequisite for whipping is making bubbles that are stabilised by surface
active molecules. An example for comparing the effects of two proteins is in whipping egg
white versus whipping milk. Egg white proteins are more sensitive to conformational changes
and entanglement between neighbouring molecules upon whipping compared to milk
proteins, and thus are more efficient as a whipping agent. Once the bubbles are being formed
they also may encounter one another, and the bubbles may fuse (typically occurring when
bubbles come close together). This fusing should also be prevented. One way is to have a
layer around the bubble which is difficult to be disentangled. This can be realised by suitable
proteins, as indicated above. Another way is to have fat particles around the bubbles
(whipping of cream). So, another prerequisite for efficient whipping is to prevent coalescence
of the bubbles being formed.
In preventing coalescence, the bubbles should be kept as wide apart as possible. One way to
accomplish this is to make sure that the fluid in between the droplets does not flow (drain)

10
away rapidly, in other words to prevent drainage. This drainage occurs due to the fact that
gravity acts on the fluid in between the droplets and thus leads to flow of the fluid. The larger
the viscosity, the less the fluid will flow at equal force. Hence, increasing viscosity will
counteract drainage and thus make the whipping more efficient. Viscosity can be increased
by using filler particles (fat particles in cream act that way) or having more protein in solution
(egg white has a higher viscosity than milk fluid). The elasticity of the whipped cream has its
origin in the elasticity of the network of the fat particles. The reason why cream can be
whipped and not milk is that milk has a lower concentration of fat particles able to form a
network and increase the viscosity of the fluid, and that in the case of milk it has been
homogenised and thus has a different membrane around the fat-globule.

Amphilicity
Amphilicity of a molecule refers to the fact that part of the molecule is relatively oil-soluble,
while another part of the molecule is more water-soluble. This leads to activity at interfaces
where part of the interface is hydrophobic (oil loving, oil soluble) while the other part is
hydrophilic (water soluble, water loving). The word amphiphilic stems from the Greek word
“amphi” which refers to “both kinds” and from the Greek word “philos” which refers to
“affinity to”. Whether a molecule is amphiphilic or not depends on the existing interactions
between parts of the molecule and the fluids in which it is immersed. See interfacial
phenomena.

Interactions
The reason why certain molecular ingredients in solution form certain colloidal structures is a
direct consequence of the existing interactions between the ingredient molecules and the
surrounding molecules of the solute. The reason why, by way of another example, certain
molecules are amphiphilic with respect to, for instance, water and oil is directly related to
how much the specific molecular groups interact with water and with oil. Part of the
molecules interact more preferably with water and another part interacts preferably more with
oil. For amphilicity, mutual interactions between molecules and parts of molecules are
therefore of importance. Such molecular interactions also determine whether molecules mix
with one another (up to an extent with water and alcohol) or whether they phase separate (like
oil and water). Interactions are also important on a larger scale, e.g. on the scale of emulsion
droplets or foam bubbles, as such interactions determine, among other things, the stability of
food products. In a separate chapter one finds a summary of the typical types of interactions
that play a role on various length scales, relevant to Foods.

Interface related phenomena

Many foods are inhomogeneous on a colloidal scale. As a result, such foods contain a
considerable amount of “interfaces”, separating colloidal structures from their surrounding
medium. As the colloidal structures are relatively small, the amount of interface is relatively
large. The properties of such interfaces therefore have a considerable impact on the properties
on a macroscopic level of the entire systems in which they are present. Thus, interface related
phenomena are important to the overall understanding of macroscopic food properties. There
is a separate chapter on interfacial phenomena.

11
Viscosity
The viscosity of a fluid is a measure for the amount of energy which is lost during flow of
that fluid. This energy is lost due to friction between the liquid molecules as they move past
one another in that flow, i.e. as they exhibit a relative motion between one another. The larger
the viscosity, the larger the energy loss is during movement of the fluid molecules.
We may quantitatively define viscosity in the following way. Consider two parallel plates, of
which one is moving with a velocity v with respect to the other. Surface area of both plates is
A. Distance between the plates is D. The liquid at the moving plate will move along with the
plate. The fluid at the resting plate will not move at all. If there is no friction within the fluid,
there does not need to be a constant force exerted on the upper plate to have it move. The
motion will continue to take place. Experience learns that one needs a force to move the
plate. Calling this force F, it is proven experimentally that

F~v*A/D

The proportionality constant is different for each liquid and one defines this constant as the
viscosity. So, we have

F= *v*A/D

One could reword the above by saying that the total frictional force exerted by the fluid, and
which opposes the movement of the upper plate, equals F as given by the above equation.
The above applies to the force for a parallel plate geometry. Note that in case one has another
geometry in which the fluid flows, the total frictional force is more difficult to calculate. For
instance, in the case of fluid around a moving spherical particle, one has a specific flow
pattern of the fluid, where the fluid moves along with the surface of the particle, and where
far away from the particle, the fluid does not flow at all. The factor 18 in the Stokes equation
for creaming/sedimentation of spherical particles in a fluid is a direct consequence of the
particular flow field around the particle.

Kinetic energy
The total kinetic energy of a particle with mass m and velocity vx in the x-direction (and vy =
vz =0) is given by

Ekin=1/2 mvx2

Elasticity
Apart from viscosity as defined above, a food may exhibit elastic properties. For instance,
think of gelatine that gets back to its original shape after being deformed. Or bread that gets
back in its original shape when deformed. The energy which is introduced into the food in
order to have it deformed is in this case released as the food gets back its original shape. One
says that the food has stored energy. This energy resembles a “potential” energy, i.e. it is a
type of energy that can be used again, i.e. can be put back into “work”. The energy which is
stored in this way is also referred to as elastic energy, like the energy which is stored in an
elastic spring as it is deformed, and, which makes the spring jump back into its original shape
as the tension to develop the deformation is released.

Rheology
In general, a food exhibits both viscous as well as elastic properties. It is the combination of
both of these properties that makes the response of a food upon deformation what it is. The

12
science of response of a material as a function of deformation is referred to as rheology.
Deformation refers to small deformations (force linear with extension in the case of a spring
for instance) and large deformations (force not linear with extension), but also situations of
constant flow (for example like flow of chocolate milk through a pipe during its processing).
The perception of a food, like for instance textural perception, is related partially to
rheological response of the food during in mouth processing so to say. This makes rheology
an important topic during the preparation of foods, as well as during its consumption. In
addition, the sedimentation of for instance chocolate particles in a chocolate milk upon time
due to gravity is also rheology related. This is why one separate chapter is devoted to
rheology.

Macromolecules
Many ingredient molecules within foods are huge molecules, i.e. exhibiting a large molecular
weight. These are called macromolecules. Often, they have a linear chemical fine structure,
but the molecules are forming a coil, i.e. parts of the chain almost feel one another.
Depending on the level to which these chains want to be in contact with the solvent, these
coils have a different size. Depending on the way the chemical building blocks are bound
together and how these blocks interact with the neighbouring building blocks, the chains are
having a certain stiffness. The stiffness also determines (besides the level to which the chains
want to be in contact with the solvent) how large and how extended (i.e. how swollen the coil
versus how linear) the molecule appears. The way these molecules interact with the solvents,
with the other colloidal elements, and with themselves is rather diverse and leads to subtle
differences regarding specific phenomena, but with far reaching consequences for the overall
macroscopic behaviour of a food. Therefore, a separate chapter is devoted in this course to
macromolecules.

13
 Questions Chapter 2

Physics Tools General Small items

1. Give the relation for the velocity, v, of a droplet in a viscous medium, with the following
three factors that are relevant to the speed, v, of the particles
d. Density difference between particle phase and liquid phase, 
e. Diameter, d, of the particles
f. Viscosity of the continuous phase, 
which resembles the law of Stokes
2. Draw streamlines along a moving droplet within a fluid
3. How much energy is available per degree of freedom for a particle due to thermal motion?
4. What is the energy related to the displacement over a distance d due to a constant force F?
5. What is the net gravitational force on a particle with diameter D, with density difference
between fluid and particle given by , and gravitational constant g=10, taking into account
the fact that the volume of that particle is given by 1/6D3
6. Derive the diameter for a particle with diameter D for which creaming will be as likely to
occur as Brownian motion, using questions 3 to 5.
7. What would have to be roughly the density difference between fat in fat particles of
homogenised milk and the milk plasma, in order for fat particles to not show significant
sedimentation nor creaming? Make use of the answer to question 6 and of the average
diameter of fat particles in milk.
8. Which ingredient, egg white proteins or milk proteins will be more efficient in yielding
lots of foam (for a short time at least) and which protein characterisitc is responsible for this?
9. Which is better whippable, egg white solution or milk, and why?
10. Which is more efficient in preventing coalescence, proteins or regular surfactants?
11. Which is more eficient in preventing coalsecence, regular surfactants or fat particles
originating from cream?
12. Which fluid shows faster drainage from a foam, egg white or milk?
13. Which fluid shows faster drainage, milk or whipped cream?
14. Name two reasons why milk cannot be whipped that efficient as cream can
15. What is an amphiphilic molecule?
16. What is a surfactant molecule?
17. What is a macromolecule?
18. Name three phenomena that are important for foam stability

14
 Chapter 3A: Interactions
Introduction
In the previous chapter we have seen that many food products consist of structural
elements, like macromolecules, solid particles, liquid droplets, gas bubbles, crystals, and
membranes. The size and shape of these elements, their physical properties, and the
interaction forces between the elements determine the macroscopic properties of a food
product. In this section we will discuss the interaction forces between the structural elements
in more detail. We will discuss the origin of all interaction forces, present an overview of the
various types of interaction forces, discuss their properties (strength, range, temperature
dependence), and end with a discussion of their effect on macroscopic properties like
solubility and stability (DLVO-theory).

Interaction forces affect a system on a wide range of scales, starting at the atomic or
molecular scale, and extending as far as the mesoscopic or colloidal scale. On the molecular
scale the balance between repulsive and attractive interactions governs the formation of
condensed phases (Figure 1a). Gas-liquid coexistence cannot occur in systems without
attractive interactions. Upon isothermal compression a system with only repulsive
interactions between molecules would become denser, but would not show a gas-liquid
transition.

For a macromolecule, the interaction between the building blocks of the

macromolecular chain determines among other things how swollen the macromolecule is.

On the colloidal scale the interaction forces affect the stability of emulsions, foams,
and dispersions (Figure 2). When repulsive interactions between emulsion droplets are
strong, and attractive forces are weak, the emulsion droplets will not aggregate, and the
emulsion will be stable. Margarine contains small fat crystals, and the hardness of margarine
is determined by the strength of the network formed by these crystals. The network strength
is determined by the balance of repulsive and attractive interactions between the fat crystals.

15
 Molecular Scale Macromolecular Scale

Without attractive With attractive

interaction interaction Strong repulsion Weak Repulsion

Figure 1. Left: Interaction forces on the molecular scale. A system without attractive
interaction cannot undergo a gas-liquid phase transition when being compressed. Upon
isothermal compression a system with attractive interactions will form a liquid phase (L) in
equilibrium with a gas phase (G). The dotted line denotes the interface between the phases.
Right: Interaction forces on the macromolecular scale. In a macromolecule with strong
repulsive interactions between chain segments the spherical conformation formed by the
chain “swells” and has a large volume. In a macromolecule with weak repulsive and strong
attractive interactions between chain segments the conformation “collapses” and has a small
volume.

Foams consist of small gas bubbles, surrounded by thin liquid films. When the interaction
forces between the interfaces bounding these liquid films are strongly repulsive, drainage and
rupture of the film can be retarded, and the stability of the foam is increased. There are
numerous other examples of how interaction forces affect colloidal systems. We will discuss
several of these in the remainder of this text.

Figure 2. Interaction forces on the mesoscopic scale. Left: The stability of an oil in water
emulsion is determined by the balance between attractive and repulsive interactions between
the oil droplets. Right: Margarine contains a network of fat crystals, and the strength of the
interaction forces between these crystals determines the strength of this network, and the
hardness of the margarine.

The origin of all interactions on the molecular and colloidal scale is the electromagnetic force
and the gravitational force. The electromagnetic force is the force between positively and
negatively charged particles, like electrons, protons, dipoles, and ions. Its range is between
10-10 and 10-6 m. The gravitational force is the attractive interaction between masses. The
electromagnetic force will be more important than the gravitational force, since masses are
usually small on these scales. The fact that the interactions are predominantly
electromagnetic in origin gives us an easy way to classify the various forms of interaction
forces. We can classify the interactions by the types of charges that are interacting. Here we
will distinguish three types of charges (Figure 3):

16
1. permanent charges,
2. permanent dipoles,
3. induced dipoles

Permanent charges are particles with a net positive or negative charge, like electrons, protons,
and ions. Permanent dipoles are particles with a net neutral charge, but a center of mass of the
positive charges that does not coincide with the center of mass of the negative charges. A
permanent dipole is formed when the constituent atoms in a molecule vary significantly in
electronegativity. Electronegativity quantifies the tendency of an atom to attract electrons.
When a molecule consists of strongly and weakly electronegative atoms, the strongly
electronegative atoms will attract the electrons in a covalent bond stronger than the weakly
electronegative atoms. The strongly electronegative atom will be slightly negative (), and
the weakly electronegative atom will be slightly positive () (Figure 3b). The net charge of
the molecule equals  Strongly electronegative atoms are for example Oxygen (O),
Nitrogen (N), and Fluoride (F). Weakly electronegative atoms are for example Hydrogen (H),
and Carbon (C). So dipoles are commonly found in molecules containing OH groups
(polysaccharides), NH or CO groups (proteins), or HF groups. A well-known example of a
permanent dipole is the water molecule.

a) b) +
+

  
+
+
+ 

c)
+
+ 

Figure 3. Three types of charges: a) permanent charges, b) permanent dipoles, and c) induced dipoles

An induced dipole is a dipole caused by the presence of another charge (Figure 3c). The
presence of the permanent charge in Figure 3c disturbs the charge distribution of the
uncharged molecule, and induces a dipole in the molecule. The inducing charge need not be a
permanent charge, but can also be a permanent or another induced dipole. In fact, two
uncharged molecules can induce a dipole in each other. To understand this we need to realize
that the electron charge distribution around a molecule fluctuates very rapidly. On the time
scale of most common experiments the molecule will appear to be uncharged. But on
molecular time scales (~ 10–18 s) the charges will appear to be localized instead of smeared,
and can repel each other, thereby inducing a dipole in both molecules.
Based on the different forms of charges described above we can construct a convenient
scheme to classify the various types of electromagnetic interactions. This scheme is presented
in Figure 4.

17
 PC PD ID

Permanent Charge (PC) PC-PC PC-PD PC-ID

Permanent Dipole (PD) PD-PD PD-ID

Induced Dipole (ID) ID-ID

Figure 4. Classification scheme for electro-magnetic interactions between Permanent Charges (PC),
Permanent Dipoles (PD), and Induced Dipoles (ID).

Almost all electromagnetic interactions can be reduced to one or more of the interactions
presented in Figure 4. In this text we will focus mainly on the shaded areas in Figure 4.
Among the interactions between permanent charges (electrons, dipoles, and ions) we can
distinguish

1. covalent interactions (short-range attraction),

2. short-range repulsive interactions,
3. the longer-range Coulomb forces.

Covalent interactions arise when two atoms form a chemical bond in which electrons are
being shared between the atoms. Short-range repulsive interactions arise when two atoms or
molecules approach each other to very short distances, such that the electron clouds start to
overlap and repel each other. In case the molecules are ions, this interaction is referred to as
Born Repulsion. In case of uncharged molecules it is referred to as hard core repulsion or
steric repulsion.
The interactions between permanent charges and permanent dipoles, and the interactions
between two permanent dipoles are Coulomb forces. The latter are often called orientation
interactions. The interaction between a permanent and an induced dipole is termed induction
or Debye interaction, whereas the interaction between two induced dipoles is referred to as
dispersion interactions. The sum of the orientation (PD-PD), induction (PD-ID) and
dispersion interactions (ID-ID) is called the van der Waals interaction.

Strength of an interaction
To describe interactions quantitatively we first need to introduce a means to characterize their
strength. From experimental observation we know that the strength of interactions between
two particles depends on the distance between them. Think for instance about two permanent
magnetic particles that repel one another. The further away from one another the more easy it
is to bring them closer together by for instance 1 cm. As they have approached one another
almost, bringing them closer for another 1 cm is harder than when they still are very remote.

The interactions can be either attractive or repulsive, but in both cases the strength increases
for decreasing separation distance. We can characterize this strength either by the
force F (r ) or the energy (potential) V (r ) needed to hold two molecules at a certain separation
distance r. The force F (r ) is defined to be positive when the interactions are repulsive, and
negative when they are attractive (Figure 5a). The energy or potential V (r ) is defined as the
negative of the work performed in displacing two interacting molecules from an infinite
separation distance to a finite distance r .

18
 r

Repulsion: F>0

Attraction: F<0

Figure 5. Quantitative characterization of the strength of interactions: a) The force F(r) or b)

the energy V(r).

These two methods of characterization are of course related. For a particle in a constant force
field, the work associated with a displacement L of the particle in that field is simply equal to
the magnitude of the force multiplied by the displacement distance L , i.e. V  FL . For
example in the case of a particle moving in a gravitational field, the force is mass*g and the
work in moving it over a distance L is m*g*L. The problem which arises in interactions
between small particles, and molecules, is that the magnitude of the force F varies with r , so
we have to replace the simple multiplication by an integral expression

r
V (r )  V    F (r )dr  (1)


where V is the interaction potential at infinite separation, which we will set to zero, and
r  the integration variable. Equation (1) implies

V (r )
F (r )   (2)
r
i.e. the force is equal to the negative of the gradient of the interaction potential.

Interactions most relevant to Foods

Molecular interactions: Coulomb force between two permanent charges

Let us consider two permanent charges, for example two ions, separated by a distance r .
The ions are interacting through the electric fields that emanate from both charges (see Figure
6).

+
X
X+ r

Figure 6. Two positively charged particles at a distance r , interacting through their respective
electric fields.

Let us first consider the strength of the electric field emanating from charge 1. The strength
of this field, E1 , must of course be proportional to the charge of the ion, q1 . The higher the

19
charge of the ion, the stronger the field it emanates. The strength is inversely proportional to
 , the dielectric constant or dielectric permittivity of the medium in between the two
charges. The dielectric constant is a measure for how easy an electric field can penetrate a
medium. In vacuum, the dielectric constant is minimal, an electric field can penetrate
optimally. Each medium has a dielectric constant and it is often expressed in terms of the
dielectric constant of vacuum. A high dielectric constant means that the field has difficulty
penetrating the medium, and therefore E1 (r ) will be smaller at a given distance r from the
ion. Finally, the strength of the field is a function of the distance r from the ion. The further
we are removed from the ion, the weaker the field will be. This is due to energy conservation.
The field cannot be constant throughout entire space, since then the total energy would be
infinite. One can prove from the consideration on finiteness of the total energy, and symmetry
considerations (the field must be spherically symmetric) that E1 arising from particle 1 is
given by
q1
E1 (r )  (3)
4 r 2
The force exerted by this field on particle 2 is proportional the strength of the field at the
location of particle 2, and proportional to the charge of particle 2, q 2 . This can be proven
experimentally. So we find that

q1 q 2
F2 (r )  q 2 E1 (r )  (4)
4 r 2
Conversely, by symmetry, one also has for the force exerted on particle 1 by particle 2 is
given by
qq
F1 (r )  q1 E 2 (r )  1 22 (5)
4 r
and we see that F1 (r )  F2 (r )  F (r ) . We can now use Eq. (1) to calculate the interaction
potential V (r ) between two charged particles as

r
 1
r
qq r 1 qq
V (r )    F (r )dr    1 2  2 dr    1 2  r  

4  r  4  (6)
qq
 1 2
4 r

The dielectric constant is usually expresses as  r 0 , where  0 is the dielectric constant of

vacuum (8.854 x 10-12 C2J-1m-1), and  r the relative dielectric constant of the medium. The
charge qi in case of ions equals zi e , where zi is the valency of the ion and e the elementary
charge of an electron, which equals 1.6 x 10-19 C. Combining this with Eq. (6) we find

z1 z2e 2
V (r )  (7)
4 r 0 r
For an interaction to be effective it must be strong enough to overcome the effects of thermal
motion, i.e. we must have V (r )  kT , where k is the Boltzmann constant and T is the
absolute temperature. So the effective range of this interaction re can be calculated by setting
the expression in Eq. (7) equal to kT and solving for r . The result is

20
 z1 z2e 2
re  (8)
4 r 0 kT
For two monovalent ions ( z1 = z2 =1) in a vacuum (  r =1), at a temperature of 300 K, the
effective range will be 55.7 nm. Equation (7) also allows us to calculate the maximum
strength of the interaction, i.e. the value of the potential when for instance the two ions are in
contact. Assuming the ions are sodium ions with an ion radius of 0.098 nm, we find that
rmin =0.196 nm, and V (rmin ) = 1.18 x 10-18 J, which at 300 K amounts to about 284 kT . We
see that the Coulomb force between two ions is a very strong interaction.

Molecular interactions: Van der Waals interaction between molecules

Consider two molecules. In general these molecules may have a permanent dipole, and an
induced dipole (recall that even two uncharged molecules can induce a dipole moment in
each other). The two molecules will attract each other as a result of the permanent and
induced dipoles of the molecules. There are three different contributions to the overall
attractive interaction, which all show a dependence on the distance according to 1/r6

C
V (r)   (9)
r6
where C is a constant that depends on the magnitude of the permanent and induced dipole of
the molecules. The van der Waals interaction is an extremely important interaction in Foods
and other systems. As we shall show in the next section it is basically responsible for the
existence of liquid phase and liquid-gas coexistence.

Molecular interactions: Lennard-Jones potential

In general molecules will not have just one single interaction, but will have combinations of
the interactions. Consider for example two non-polar molecules. Between these molecules
there will be an attractive interaction as a result of the Van der Waals interactions. However,
at very small separation distances the electron clouds of the molecules will start to overlap,
and hard sphere repulsion will occur at these short distances. This repulsive interaction
depends on the distance between the particles according to 1/r12. The resulting interaction
potential (also called Lennard-Jones potential) is the sum of these two types of interactions

B C
V (r )  12
 6 (10)
r r
Note that the first term in this expression is the contribution from the repulsive interactions,
and is therefore positive, whereas the second term represents the attractive van der Waals
interactions, and is negative. In figure 7 we plot the value of the total potential and its
individual contributions as a function of r . We see that the van der Waals interaction has a
much longer range than the hard sphere repulsion (also referred to as Born repulsion). For
separation distances larger than about 0.45 nm the hard sphere repulsion is completely
negligible, the total potential closely follows that of the van der Waals interaction, and is
negative (attractive). As the separation distance decreases below a value of 0.45 nm, the hard
sphere repulsion becomes increasingly dominant. After reaching a minimum at a distance of
approximately 0.35 nm the total potential starts to increase again, and eventually, at about
0.32 nm becomes positive (repulsive). For this particular case the depth of the minimum is
about –0.5 kT , and thus of the same order of magnitude as the thermal energy of the system.

21
 Figure 7. Hard sphere potential, van der
5 Hard sphere repulsion
Waals potential, and total interaction
4
Van der Waals attraction potential in units kT as a function of
3
Total potential separation distance r (nm). The value of the
2
van der Waals coefficient C equals 1 x 10-77
Potential (kT)

0
Jm6, and the value of the coefficient B in Eq.
-1
0.2 0.3 0.4 0.5 0.6 0.7 0.8 (10) is set to 1 x 10-134 Jm12. The temperature
r equals 300 K. We see that there is a
-2
minimum in the potential curve.
-3

-4

15

Figure 8. Total potential in units kT as

10
a function of separation distance r (nm),
for several values of the van der Waals
5
coefficient C. From top to bottom we
Total Potential (kT)

0
have plotted the curves for C  1, 2, 3.5,
0.2 0.3 0.4 0.5 0.6 0.7 0.8 and 4 x 10-77 Jm6. We see that the
r
-5 minimum in the potential curve shifts to
smaller r values, and the depth of the
-10 minimum increases from about –
0.5 kT to almost –10 kT . The
-15 temperature equals 300 K.

In Figure 8 we see that the depth and location of the minimum in the total potential energy
depends on the value of the van der Waals coefficient C . For increasing C the minimum
shifts to lower r values, and the depth of the minimum increases form approximately –0.5 kT
for C = 1 x 10-77 Jm6, to almost –10 kT for C = 4 x 10-77 Jm6. From experimental
observations we know that some compounds form liquid or solid phases at room temperature,
and others remain in a gaseous state. In the beginning of this chapter we already stated that
condensed phases only form when there are attractive interactions in the system. Figure 12
illustrates how these attractive forces can induce the formation of a condensed phase. When
two freely moving molecules are placed at a distance of about 0.5 nm, they will attract each
other and start to approach each other, until they become “trapped” in the minimum of the
potential curve. When the minimum in the curve is of same order or smaller than kT , the
thermal energy in the system will be able to separate the two molecules, and they will not
stay trapped in the minimum. The molecules will be dispersed, and the system will be in a
gaseous state. However, when the minimum in the curve is sufficiently deep, the thermal
motion in the system will not be able to disperse the molecules. The molecules will cluster,
and when more molecules are added to the system, a liquid phase can be formed.

22
Molecular and colloidal scale: Hydrogen Bonding
Hydrogen bonding occurs in systems where hydrogen atoms are bound to strongly
electronegative atoms with one or more free electron pairs, like O, N, F, or Cl. The hydrogen
bond is not a covalent bond, but electrostatic in nature. It is an unusually strong form of
orientation or permanent dipole - permanent dipole interaction. Its strength is about 10-40
kJ/mol, much weaker than covalent bonds, but stronger than most van der Waals interactions
(0.05-40 kJ/mol). It is a short-range interaction, with an effective range less than 0.2 nm. Its
strength depends strongly on the orientation of the molecules, and is therefore temperature
dependent. When temperature increases, the thermal motion in the system will disturb the
preferential orientation, and the strength of the hydrogen bond decreases.
Hydrogen bonding can produce both intramolecular as well as intermolecular bonds,
and this together with the directional nature of the bond allows it to form networks (Figure
14), or three-dimensional structures. Some examples of these 3-d structures are the -helix
and -sheet found in globular proteins. In gelatin gels triple helix structures are formed, also
stabilized by hydrogen bonding. In certain polysaccharides, like for example Xanthan,
Carboxy Methyl Cellulose (CMC), or Locust Bean Gum (LBG) hydrogen bonding induces
the formation of double helix structures. The double helix formed by DNA molecules is also
stabilized by this interaction. Since the strength of the interaction is temperature dependent,
the formation and stability of these structures is also temperature dependent. From experience
we know that gelatin gels melt, and that the -helix and -sheet in globular proteins unfold
upon heating. This can be explained (in part) in terms of the weakening of the hydrogen
bonds that stabilized these structures.

H H
O

H H H H
O O
Figure 9. Hydrogen bonding can lead to network formation
H H or formation of three-dimensional structures like for
example a helix (gelatin, CMC, Xanthan, LBG, DNA).
O

Hydrophobic interaction
Hydrophobic interactions are closely related to the hydrogen bonding, and hence are also
based on orientation interactions. When a nonpolar molecule or particle is dispersed in a
water phase, the network formed by the water dipoles will locally be disturbed (Figure 10a).
The water dipoles surrounding the nonpolar particle can not form hydrogen bonds with the
nonpolar molecule, which results in a loss of hydrogen bond energy. To compensate for this
effect, the dipoles at the surface of the particle reorient themselves, to restore the number of
hydrogen bonds. By doing so they form a cage structure around the particle (Figure 10b).

23
 H H
O

H H H H
O O

Figure 10a) Close to the surface of a particle the water dipoles cannot form hydrogen bonds with the
particle, and have fewer bonds than dipoles in the bulk phase.
Figure 10b) By reorienting themselves the dipoles restore the number of hydrogen bond. This process
results in a cage structure around the particle. Here we show a dodecaeder shaped cage.

The size and shape of the cage are determined by the size of the particle. In Figure 10b the
dipoles are ordered in a dodecaeder structure. By forming these cages the number of
hydrogen bonds is restored, and the enthalpy of the system is lowered. But the dipoles lose
some translational and rotational entropy, because they are in a state with a higher degree of
order. So cage formation is entropically unfavorable. The Gibbs free energy of cage
formation is given by

G  H  TS (11)

where G is the change in Gibbs free energy, H the change in enthalpy, and S the change
in entropy. The change in enthalpy is negative, since hydrogen bonds are restored in this
process. Since the change in entropy is also negative, and for most nonpolar particles
H  TS we find that G  0. This explains why most nonpolar compounds are only
poorly soluble in water. When two nonpolar particles approach each other to a short distance
the system can increase its entropy by removing part of the cages in between the two
particles, and building a new cage that contains both particles (Figure 11). In this new cage
the number of dipoles per nonpolar particle is smaller than in the original cages. So S  0
for this process, and in view of Eq. (11) and the fact that H  TS we see that G  0.
We see that the system can lower its Gibbs free energy by this process. This leads to an
attractive interaction between the nonpolar particles, which, when not balanced by repulsive
forces, can lead to aggregation of the particles.

Figure 11. Origin of the hydrophobic

interaction: when two nonpolar
particles approach, part of the
entropically unfavorable cage
between the particles can be removed.
The result is an attractive interaction
between the particles.

The hydrophobic interaction has an effective range of about 2 nm, and is usually described
with an exponential potential of the form
V (r )  ae r / r0 (12)

24
where a is a constant, and r0 is a characteristic length of the order of 1 nm. Its strength is
about 10 kJ/mol.
The hydrophobic interaction is temperature dependent. When the temperature increases, the
strength of the interaction increases, in contrast to the strength of hydrogen bonding, which
decreases with increasing temperature. We see that for increasing temperatures the term
TS becomes increasingly positive, and G becomes increasingly negative, making the
process of cage removal energetically more favorable. As a result the strength of the
hydrophobic interaction increases.
The hydrophobic interaction is extremely important in food systems. We will see that this
interaction is an important factor in the stabilization of the hydrophobic core of globular
proteins, and plays an important role in the aggregation of denatured proteins.

Colloidal scale: Electrostatic interactions

At the start of this section we have discussed the electrostatic interaction on a

molecular scale, between two permanently charged ions (the Coulomb force), and found that
the interaction potential decays as r 1 . In this section we will discuss the interactions
between permanently charged colloidal particles, dispersed in an aqueous solution of an
electrolyte. These interactions are important in for example the stability of emulsions
stabilized by proteins, or the production of yoghurt from milk by acidification (the pH affects
the charge of the casein micelles in the milk, and as we will see in the following paragraphs,
this affects the electrostatic interactions between the micelles).
Colloidal particles can be charged in many ways. One possibility is the dissociation or
association of acid and base groups at the surface of the particles. Examples of such groups
are the carboxyl (-COOH), amine (-NH2), and sulfhydryl groups (-SH) found in proteins, but
also sulfate (-HSO3), or phosphate groups (-HPO4), and their salts (-SO3Na, -PO4Na). The
degree of dissociation or association of these groups depends on the pH of the solution. As a
result the charge and therefore also the strength of the interaction between the colloidal
particles will depend on pH.
Colloidal particles can also be charged by the adsorption of charged molecules at the
surface. An example is the adsorption of charged protein molecules or ionic surfactants at the
interface of an emulsion droplet.
When a colloidal particle is charged, the charges at the particle surface will interact
with the electrolyte ions in the surrounding medium. When the sign of the charge of the ions
is the opposite of the sign of the particle charge, the ions are called counter-ions. When the
sign of the ionic charge is equal to the sign of the particle charge, they are called co-ions. The
counter-ions will be attracted by the particle, and the co-ions ions will be repelled (Figure
12). The electrostatic interactions between the surface charges and these electrolyte ions
results in a distribution of the co- and counter-ions close to the particle, that differs
significantly from their distribution in the bulk phase, far away from the particle surface.
When the attractive interactions between the particle and counter-ions are sufficiently strong
(i.e. > kT ) an ordered layer of counter-ions will form, bound to the particle surface. This layer
is called the Stern or Helmholtz layer. Outside this layer the interactions are weaker, and the
thermal motion in the system will (partially) randomize the order imposed by the electrostatic
interactions. This balance between the thermal motion and electrostatic interactions results in
the formation of a diffuse electric double layer (Figure 13).

25
 + + Figure 12. The counter-ions are attracted by
- - the charges on the surface of the particle, and
+ + - Co-ions
- + the co-ions are repelled.
+ + -
- + +
+ -
- + +
+ + + - Counter-ions
- -
+ +

n(r)
ns
Counter-ions
nb
Figure 13. Concentration of counter- and
Co-ions co-ions in the diffuse electric double layer
as a function of distance to the particle
r surface.

The number of counter ions as a function of the distance x to the surface is given by the
Boltzmann distribution

 ze ( x) 
n( x)  nb exp    (13)
 kT 

We see that the number distribution decays exponentially from its maximum value at the
surface of the wall, to the value in the bulk phase, nb , far away from the wall. If we want to
calculate the distribution of counter ions and determine the screening distance, we still have
to figure out the potential field as a function of the distance x to the wall. Here we only
present the result for the approximate potential, assuming that the surface is completely flat
(this will be a good approximation in cases where the radius of curvature of the surface is
much larger than the Debye screening length). When the surface potential is small (i.e. < 25
mV), one finds

 ( x)   s ex (14)

where  -1 denotes the Debye screening length

 r 0 kT
 1  (15)
2 z 2e 2 nb
which is a characteristic dimension for the thickness of the diffuse double layer (the screening
length, or the length over which the potential has decreased significantly). The potential at the
wall is related to the surface charge density  [C/m2] of the wall. When the surface potential
is sufficiently low, (< 25 mV), one finds that

26
 
s  (16)
r 0 
So we now have an expression for the surface potential as a function of the thickness of the
diffuse double layer and the charge density of the wall.
Up to this point we have discussed only the interaction of a single charged flat wall
with the counter-ions and co-ions in the surrounding electrolyte. We have seen that the
competition between electrostatic interactions between the wall and ions, and the thermal
motion in the system, leads to the formation of a diffuse double layer close to the wall. We
are now in a position to answer the question what will happen if we bring two charged walls
into close proximity of each other. In Figure 14 we see two charged walls, separated by a
perpendicular distance D , with their diffuse double layers. When D is small the two double
layers will start to overlap, and the walls will start to repel each other.

s s Figure 14. Two charged walls at a distance D

with overlapping double layers. We will assume
m that the potential at the mid-plane is simply the
sum of the two potential fields evaluated at
x  D / 2.

0 X/D 1

The origin of this repulsion is entropic. When the double layers overlap, the ions between the
two walls are exposed to two electrostatic fields, and the total strength of the electrostatic
interactions acting on the ions increases. When the temperature, and hence the thermal energy
of the system, is kept constant, the ions will be in a state with higher order. So when the two
walls approach, the entropy of the ions is decreased. The system will oppose this, and this
results in a repulsive interaction between the two walls. From the discussion in the first part
of this section it is immediately obvious that the range of this interaction is determined by the
value of  1 , and according to Eq. (15), a function of the concentration of electrolyte in the
bulk phase, nb . For low concentrations the Debye length will be large, and the electrostatic
repulsion between the walls will have a long range. When we increase the value of nb , the
value of the Debye length will decrease, and the range of the electrostatic repulsion between
the walls will also decrease. We say that the repulsive interactions between the walls are
screened by the electrolyte ions in solution. The strength of the repulsive interaction is
determined by the value of the surface potential  s , which according to Eq. (16) is in large
part determined by the surface charge density  . As we noted at the start of this section the
surface charge density often depends on the pH of the system, so we can expect the repulsive
interactions between the walls to be pH dependent.
For a symmetric electrolyte and small values of the surface potential (< 25 mV) we
find that the disjoining pressure (force per unit area acting against bringing the plates closer
than distance D)

27
 P( D)  4nb ( ze s ) 2 e D / kT (17)

From the disjoining pressure we can calculate the repulsive interaction potential per unit area
W (D)
4nb ( ze s ) 2 D
D
W ( D)    P( D)dD  e (18)
 kT
This gives us the interaction potential per unit area for the repulsive interaction between two
infinite charged flat walls. This expression is applicable to the interaction of flat particles,
like for example clay particles or tobacco mosaic virus particles, or thin flat films in foams.
We can also use this expression to arrive at an expression for the interaction potential for the
interaction between two spherical particles with radius R . According to a so-called Derjaguin
approximation, which is valid when D  R and  1  R , the repulsive force between two
charged spheres is given by (see Figure 21)

Fsphere( D)  RW ( D)  4nb ( ze s ) 2 R(kT ) 1 e D (19)

Integrating this expression gives us the interaction potential for two charged spheres:

Vsphere( D)  4nb ( ze s ) 2 R 2 (kT ) 1 e D (20)

figure 15. Two charged spheres of radius R , at a distance D .

The above equations can be used to calculate the electrostatic repulsion between emulsion
droplets, or spherical colloidal particles. After substitution of (15) we find that

Vsphere( D)  2 r 0 R s2eD (21)

From this expression we see that the interaction potential does indeed depend on the surface
charge density, through a quadratic dependence on  s . As stated above this dependence on
the surface charge density introduces a dependence on pH in the expression for the
interaction potential. The range of the interaction is clearly determined by the value of  ,
which is determined by the concentration of electrolyte in the bulk phase. These observations
explain for example the destabilization of milk when it is acidified. When the pH of milk is
lowered towards the iso-electric point of the casein micelles, the surface charge of the
micelles is reduced, and the electrostatic repulsion between the micelles decreases. This leads
to aggregation of the micelles, and the formation of yoghurt.

28
Van der Waals interaction between colloids
In a previous section we discussed the van der Waals interactions between two molecules,
scaling with separation distance r as r 6 . When considering the van der Waals interactions
between colloidal particles, which consist of large clusters of molecules, the approach of
summing discrete interactions has to be followed. In summing we will use a continuum
approximation, which is equivalent to saying that a summation may be replaced by an
integration. For the total van der Waals interaction potential of per unit area between two
parallel infinite walls we find
A 1
W AB ( D)   AB 2 (22)
12 D
where AAB is generally referred to as the Hamaker constant. From this expression for two flat
walls we can calculate the interaction between two spheres of radius R A and R B , using the
Derjaguin approximation again. We find for the interaction potential
A  R R 
V AB ( D)   AB  A B  (23)
6 D  R A  RB 
For spheres of equal radius this reduces to

AAB R
V AB ( D)   (24)
12 D
From the expression for spheres with different radii we can derive another interesting
expression. When the radius of one of the particles goes to infinity (say for example R A ), we
obtain the interaction potential for a sphere with a semi-infinite wall:
A R
V AB ( D)   AB B (25)
6D
All these expressions find their use in practical food systems. The expression for two semi-
infinite walls can be applied to for example thin foam films, and the expression for two
spheres can be applied to stability calculations of emulsions or particle dispersions.

Derjaguin-Landau-Verwey-Overbeek –theory (DLVO theory)

In food systems we usually have a combination of the colloidal interactions we discussed in
the previous section. Consider as a first example the system below, which is an oil-in-water
emulsion, stabilized by adsorption of globular proteins (for example a whey protein).

29
In this example the pH of the water phase is below the isoelectric point of the protein, so the
protein molecules are positively charged. The adsorbed layer of proteins therefore gives the
emulsion droplets a net positive charge. If the ionic strength of the water phase is low, and
the pH is far below the isoelectric point we will have strong electrostatic repulsion between
the droplets. The thick layer of protein at the oil-water interface also induces strong steric
repulsion between the droplets (i.e. Born repulsion). Finally, since van der Waals interactions
are present in all systems, we also have an attractive contribution to the total interaction
potential from these interactions.

Derjaguin, Landau , Verwey and Overbeek were the first to discuss the combined effect of
the above mentioned three types of colloidal interactions on the stability of a system. After
their initials, the theory they developed is referred to as the DLVO theory. They considered
the interactions between two spherical particles of equal radius, and limited themselves to a
system with only Born repulsion, electrostatic repulsion, and van der Waals interactions. So
the total potential between two colloidal particles in the DLVO theory is written as

VTOT ( D)  VBORN ( D)  VER ( D)  VVDW ( D) (26)

In the figure below we see a typical example of the shape of the total potential as a function
of the separation distance.

VER
VTOT

VBORN

VMAX

D
VTOT

VVDW

Figure 17. Typical example of the total interaction potential as a function of separation distance for
two spheres. The dashed lines represent the contributions to the total potential: Born repulsion,
electrostatic repulsion, and van der Waals interaction.

When we let two particles approach each other from infinite separation distance, they will
first be exposed to the attractive van der Waals interactions, since this interaction has the
longest range of the three interactions. So initially the total potential will be negative, and
will become more negative as the separation distance decreases. When the separation
distance approaches about 5 to 6 times the Debye length, the electrostatic repulsion starts to
dominate, and the total potential starts to rise again, creating a minimum in the potential

30
curve. This minimum is referred to as the secondary minimum. When the separation
decreases further, and the electrostatic repulsion in the system is sufficiently strong, the total
potential may even become positive. The electrostatic repulsion has a finite value at contact,
whereas the van der Waals attraction goes to negative infinity, since it depends on the
separation distance as 1 / D [see Eq. (24)]. So at very small separation distances the van der
Waals interaction will become stronger than the electrostatic repulsion, and the total potential
will start to decrease again. The result is a maximum in the potential curve. When we
decrease the separation distance even further the potential becomes negative, until we reach a
point where the Born repulsion becomes important. At that point the total potential starts to
increase again, creating a minimum in the potential curve. This minimum is referred to as the
primary minimum. Upon further decreasing the separation distance the potential will rise
steeply, and eventually go to positive infinity at zero separation distance.
The particular shape of the potential curve determines if a system is stable against
aggregation. When the maximum in the energy curve is very high, i.e. much higher than kT ,
and the secondary minimum is less or equal to kT , the system will be stable. The maximum
in the potential curve acts as an energy barrier against aggregation. When VMAX  kT , but
the energy of the secondary minimum VMIN II
 kT , the system will aggregate in the
secondary minimum. The particles will not touch each other, but instead stay at a fixed
distance and orbit around each other. When VMAX  kT , and the energy of the primary
minimum VMIN I
 kT . The system will be unstable and aggregate in the primary minimum.
The magnitude of the energy maximum and the primary and secondary minimum is
determined by the experimental conditions, like temperature, pH, and ionic strength. These
conditions affect mainly the electrostatic repulsion, and have only a marginal effect on the
van der Waals attraction and Born repulsion. The pH affects the surface charge and therefore
the surface potential of the particles, whereas the ionic strength mainly affects the Debye
length.

Let us consider a second example of a globular protein solution with low ionic strength and a
pH below the isoelectric point of the protein, is heated above the denaturation temperature.
Because upon heating the hydrogen bonding that stabilizes the secondary and tertiary
structure of the globular protein decreases in strength, the protein partially unfolds, and some
of the hydrophobic parts of the protein are exposed. As a result there will be an attractive
hydrophobic interaction between the protein molecules. Because the pH is below the
isoelectric point, the proteins will be positively charged, resulting in electrostatic repulsion
between the proteins. When the proteins are very close together, such that the electron clouds
of the outer amino acid segments start to overlap, the Born repulsion will prevent the proteins
from merging. Of course there will also be an attractive contribution from the van der Waals
interaction. So the total interaction potential for this system is equal to

VTOT ( D)  VBORN ( D)  VER ( D)  VHYDROPHOBI C ( D)  VVDW ( D) (27)

The balance between the contributions to this potential determines if the protein molecules
remain in solution or form aggregates. Depending on the protein concentration and the
interactions between the aggregates the system will stay in a liquid state, or form a gel.

31
Exercises

1. We are given a system of molecules interacting through a Lennard-Jones potential,

B C
V (r )  12
 6
r r

with B  1.0  10 134 Jm12 and C  4.0  10 77 Jm6. The Boltzmann constant
k  1.38  10 23 J/K

a) Determine the point in the potential curve where the potential is equal to zero.
b) Determine the location of the minimum in this curve.
c) How large is the force acting on the molecules when they are in the minimum of the
potential curve?
d) Calculate the value of the potential in the minimum.
e) The temperature of the system is 300 K. Is the system in a gas state or in a liquid
state?

2. Consider two charged particles with equal radius of 1m, in a 0.5 mol/m3 NaCl
solution, at a temperature of 298 K. The wall potential of the particles is 2.0x10 -2 J/C.
The relative dielectric constant of water is 78.5, and the dielectric constant of vacuum
is equal to 8,854x10-12 C2/Jm. The Boltzmann constant is equal to 1.38x10-23 J/K,
Avogadro’s number is equal to 6.0x1023 mol-1, and the elementary charge is 1.602x10-
19
C.

a) Calculate the Debye length for this system.

b) Calculate the effective range of the electrostatic repulsion, and express this range in
multiples of the Debye length.
c) Calculate the maximum value of the electrostatic repulsion, and express this value in
multiples of kT.
d) How many grams of sodium chloride do we need to add to 1 liter of the dispersion, to
decrease the range of the interaction by a factor 2. (The molecular weight of sodium
chloride is 58.44 g/mol)

3. Consider two charged spherical particles, with a radius of 50 nm, in a 10.0 mol/m3
NaCl solution at pH 2. The pH of the solution was set using a concentrated HCl
solution. The temperature of the system is 298 K. The wall potential of the particles at
pH 2 equals 2.0x10-2 J/C (20 mV). The relative dielectric constant of water is 78.5,
and the dielectric constant of vacuum is equal to 8,854x10-12 C2/Jm. The Boltzmann
constant is equal to1.38x10-23 J/K, Avogadro’s number equals 6.0x1023 mol-1 and the
elementary charge of an electron is 1.602x10-19 C. The iso-electric point of the
particles is at pH 5.4.

a) Calculate nb , the total ion concentration in the bulk.

b) Calculate the Debye length for this system.
c) Calculate the effective range of the electrostatic repulsion between the two charged
particles.

32
Apart from electrostatic repulsion the particles also interact through hydrophobic
attraction. The expression for this interaction is given by

VHYD ( D)  a exp(  D / D0 )

The parameter a in this expression equals 4.0  10 20 J, and D0 equals 0.4 nm. The
contribution of the van der Waals attraction to the total interaction potential can be
neglected.

d) Give an approximate plot of the total interaction potential as function of the distance
between the particles.
e) Calculate the location of the maximum in the potential curve and calculate the value
of the total potential in this maximum.

f) Do you expect a dispersion of these particles to be stable, or will the dispersion

aggregate? Explain your answer.

33
 Chapter 3B

Physics Tools: Steric interaction

(effectively caused by excluded volume of polymers attached to a surface)

Colloidal food systems like solid-liquid dispersion or emulsions, are sometimes stabilized by
adsorbing polymers at the liquid-solid or liquid-liquid interface. When two particles coated
with a thin polymer layer approach to a distance smaller than the combined thickness of the
layers, the polymer segments at the outside of the layers will start to intertwine. As a result of
hard core repulsion these polymer segments cannot overlap, and are forced into a more dense
and ordered state. The entropy of the segments therefore decreases, which results in an
effective repulsive interaction between the particles. This interaction is often referred to as
steric repulsion, and as we will show later, it is able to stabilize dispersions and emulsions
against flocculation or coalescence.

a) b) c)

loop
train

tail
d) e) f)
Several examples of how polymer chains can adsorb on a solid wall: a) single chain, grafted to the
wall at one end of the chain. b) Chains grafted to a wall when the surface density is low and the
chains are not overlapping. c) Grafted chains at high surface density: as a result of crowding the
chains extend and orient themselves perpendicular to the solid-liquid interface. The structure formed
is often referred to as a polymer brush. d) Physically adsorbed chain on a wall. At low surface
densities and high affinities of the chain for the interface the chain can spread over the surface and
adsorb at multiple locations, called trains. In between the trains we find free loops of non-adsorbed
polymer segments. e) Surface layer at low surface density for physically adsorbed polymers. f)
Surface layer for physically adsorbed polymers at high surface densities. Again a brush is formed.

The range and strength of the steric repulsive interaction depends on the density and
thickness of the adsorbed layer. In the figure above we see several examples how the
polymers can be adsorbed. In a) through c) we see polymer chains attached to a solid surface
by a chemical bond. In this case we speak of chemical adsorption or grafting. The chain is

34
usually bound to the surface at one end, the other end can move freely. One of the parameters
that determine the density and thickness of the adsorbed layer is the number of chains
adsorbed on the surface per unit area,  . When  is low the individual chains will not
overlap and the dimension of the adsorbed polymer coil will be close to the dimensions of the
random coil conformation the polymers have in the bulk phase, far away from the surface.
The random coil conformation is discussed in Physics Tools Macromolecules. When  is
high, as depicted in 23c), the chains will extend and orient themselves perpendicular to the
surface. The structure formed is called a brush. The segment density will in general be much
higher in this type of film, and the film will also be thicker than in the case where  is low.
Part d) through f) represent systems where the polymer is physically adsorbed to the surface.
In general the polymer has more than one site that can adsorb to the surface. When  is low
the polymer will assume a configuration in which it has multiple sites along the backbone of
the chain adsorbed to the surface ( d) ). These sites are referred to as trains. In between the
trains we find loops of non-adsorbed segments. When  is sufficiently low and the affinity of
the polymer for the surface is not too strong, the individual chains will have a size
comparable to the dimensions of the random coil conformation in the bulk phase (see d)).
When  is low and the affinity for the surface is high, the coil may completely unfold and
spread itself over the surface. Such a film will usually be very thin and the stabilizing effect
of the steric repulsive forces will be small. At high  the system will again form a brush, with
high segment density and a thickness much larger than the dimensions of the random coil
conformation.

The picture presented above on polymer adsorption is far from complete, and limits itself to
two simple cases of single layer adsorption. In practical systems adsorption is much more
complex. As a result very few theories exist that adequately describe the strength of the
repulsive interactions as a function of separation distance, layer thickness, and surface
coverage  .
For the repulsive interaction between two semi-infinite walls we find in most systems that for
sufficiently small separation distances the potential energy per unit area is given by

W ( D)  e  D L D  2L

where  is a proportionality constant that depends on temperature and surface coverage  ,

and L is a characteristic dimension of the thickness of the polymer film. We will now discuss
two special cases. First we will consider end-grafted chains at low surface coverage. As
mentioned above, in this case the characteristic dimension for the thickness of the polymer
layer is equal to the dimension of the random coil conformation in the bulk phase. As we see
in Physics Tools Macromolecules this dimension is given by the radius of gyration, R g . So
the average thickness of the film is 2 R g . The thermal fluctuations of the random coil
conformation are of the same order as R g (see also Physics Tools Macromolecules), so L is
actually equal to 4 R g . As a result the chains will start overlapping when D is equal to 8R g .
When this occurs we find

W ( D)  36kT exp  D Rg  2 R g  D  8Rg

At high  , the characteristic length scale is much larger than R g , and is given by (in a good
solvent)

35
 nl 5 / 3
L
s2/3
where n is the number of segments in a single chain, l is the length of a single segment, and
s is the distance between to attachment points on the surface, which is related to  by
  1 / s 2 . When 0.2  D / 2L  0.9 , we find that the interaction potential per unit area is
approximately given by

kT exp  D L 
100 L
W ( D) 
s

where L must be calculated from the equation above.

36
 Chapter 3C
Physics Tools
Depletion Interaction

(originating form excluded volume effects of small objects versus that of large objects)

The interaction we will discuss here is the depletion interaction. This attractive interaction
occurs in systems that contain mixtures of large and small colloids, or mixtures of large and
small polymer coils. The origin of the interaction is illustrated in the figure below for a
mixture of small and large spheres.

D2
D1
D1

Illustration of excluded volume effect of small spheres that lead to an effective attractive interaction,
i.e. so called depletion interaction

When two large particles approach each other to a distance smaller than the size of the small
particles, there will be a region between the large particles, no longer accessible to the small
particles. Due to the excluded volume of the small particles, this region is depleted of small
particles. This is why one also calls this region the depletion zone. Since the concentration of
small particles is lower in the depletion zone than in the region outside this zone, there is a
difference in osmotic pressure between these two regions. The system tries to equalize the
osmotic pressure inside and outside this so called depletion zone by drawing solvent from the
low concentration region to the high concentration region. The liquid film between the large
particles therefore has to shrink, and as a result one finds that the particles are drawn closer
together. The net result of this process of depletion (which is caused by the excluded volume
of the small particles) is an attractive interaction between the large particles. Therefore this
interaction also has an excluded volume origin, like is also the case with the steric interaction
described above.

37
Depletion interaction can occur in many other systems. In the figure below one has a number
of examples where depletion interaction can be important.

Schematic situations where depletion effects exist. a) Mixture of large spheres and small polymers. b)
Mixture of large polymers and small spherical particles. c) Large random coil polymers mixed with
small polymers. d) Rod-like or cylindrical particles mixed with small polymers. e) Mixture of rod-like
particles and small spheres. f) Mixture of long and short rod-like particles.

In a) we see a mixture of large spherical particles and small random coil polymers. This
situation can for example be encountered in emulsions with polysaccharides added as a
thickening agent. In b) depicts a mixture of large random coil polymers and small spherical
particles, as seen in mixtures of small globular proteins and high molecular weight
polysaccharides. In c) we see a mixture of large and small random coils, like we encounter in
for example mixtures of gelatin and high molecular weight polysaccharides. Figure d) and e)
show mixtures of long rod-like or cylindrical particles with small polymers and small
spherical particles respectively. Examples of such systems are mixtures of f3-lactoglobulin
fibrils with polysaccharides or globular proteins. Finally, in f) we see a mixture of short and
long fibrils, which we find for example in fibril solutions with a high degree of
polydispersity.
The effect of the attractive interaction may vary for each system, and depends on the strength
of the depletion interaction and the strength of other (repulsive) interactions in the system.
When the depletion interaction is strong, and there are no other interactions in the system, the
depletion interaction may induce aggregation of the larger particles, leading to the formation
of large clusters, which may eventually precipitate. At high particle concentrations a gel
could be formed.
When the depletion interactions are insufficiently strong to cause aggregation, or there are
repulsive interactions that prevent the large particles from aggregating, the depletion
interactions may induce phase separation. The system separates in two phases, separated by
an interface. One phase will be rich in large particles, the other rich in small particles. When
the system consist of spherical particles or random coil polymers, both phases will in general
be liquid phases. When one of the species is rod-like or cylindrical the system can also form
liquid crystalline phases.
Theoretical descriptions of depletion interactions are in general quite complicated. For the
system of hard spheres mixed with small non-adsorbing polymer molecules a simple theory is
available, developed by Vrij (1976). Vrij showed that the interaction potential for two spherical
particles of diameter D1 in a solution of polymer molecules with diameter D2, is given by

38
   for 0  D  D1

V ( D)    Vdep ( D) for D1  D  D1  D2
 forD  D1  D2
 0

where  is the osmotic pressure, Vdep is the volume of the depletion zone, and D is equal to the
separation distance between the centers of the spheres.
The osmotic pressure can be calculated from the concentration cp and molar weight M of the polymer,
using the expression

c p RT

M

where R is the gas constant. The depletion volume can be calculated using [Vrij (1976)]

1  3D D3 
Vdep(D) =  ( D1  D2 ) 3 1   3
6  2( D1  D2 ) 2( D1  D2 ) 

39
 Chapter 4

Physics Tools: Proteins

1 The role of proteins in food

Proteins are added to foods for the following reasons:

1. Nutritional value: Source of Amino acids

2. Thickening agent: increased viscosity
3. Structuring: foams, emulsions, gels

The second and third above are functional applications: the protein is added to give the
system a certain physical property. In the case of thickening agents the protein is added to
increase the viscosity of the product, in order to influence its sensory perception.
When the protein is added as a structuring agent, the protein is added to give the product a
particular structure (for example the creation of a gel using gelatine) with specific physical
properties. The physical properties of a protein system are mainly determined by the structure
of the protein. The structure is determined by temperature, pH, ionic strength, presence of
surfactants, presence of reacting agents (e.g. breaking up -S-S- bridges), and quality of the
solvent. See also Physics Tools Macromolecules.

2 Protein structures
Based on the length scale, we can distinguish five different levels of structure in protein
systems. In order of increasing length scale and increasing complexity these are:

1. Primary Structure
2. Secondary Structure
3. Tertiary Structure
4. Quaternary Structure
Primary Structure
The primary structure, or configuration, describes the sequence of amino acids and the
location of the -S-S- bridges (Figure 1).

Figure 1. Example of the primary structure of a protein

So, with primary structure we actually mean the chemical structure formula of the protein.
The primary structure is formed by covalent chemical bonds, like for example peptide bonds
(Fig. 2).

40
 Figure 2. Segment of a peptide chain
Consequently, it is hard to break up the primary structure. Breaking up takes place by adding
reactants (e.g. reactants that break up -S-S- bridges), or enzymes.

Figure 3. Alpha helix structure.

This structure makes room for
large side chains -R. It is a right-
handed helix with 3.6 residues per
turn. The thin dashed lines indicate
hydrogen bonding within the chain
(H-----O).

Secondary Structure
With secondary structure we mean the local arrangement of chain segments that are close to
each other. These chains segments can arrange themselves to form: a) an -helix, and b) a -
sheet. In the -helix the chain segments assume a three-dimensional helix structure in order
to create more space for large side groups (R) (Fig. 3).

41
Figure 4. Pleated sheet structure proposed by Pauling for silk fibroin. Chains are contracted to make
room for small side chains. Adjacent chain segments head in opposite directions; the dashed lines
indicate hydrogen bonding between adjacent chain segments (O---H).

In the -plate structure two or more chain parts arrange themselves stretched and parallel in a
plate structure (Fig 4). The -helix as well as the -sheet are stabilized by hydrogen bonds
(within the chain). The energy of these hydrogen bonds is approximately 5-10 kcal per mol
per bond or 20-40 kJ per mol per bond. Figures 3 and 4 clearly show that the number of
hydrogen bonds is extremely high. So the secondary structure is stable (however less stable
than the primary structure, because covalent bonds are of course stronger than hydrogen
bonds).

Tertiary Structure
The tertiary structure is the three-dimensional arrangement of -helices and -sheets (Fig. 5).

Figure 5. Example of the tertiary structure of a protein.

42
The secondary and tertiary structures together are called the conformation of the protein. In
general we find that the stability of the conformation is less than the stability of the
configuration.

Classification of proteins based on conformation

Based on the conformation of a protein we can distinguish two kinds of proteins. In order of
decreasing complexity we can distinguish:

I) Globular proteins
II) Random coil proteins

Globular Proteins
Globular proteins are characterised by a lot of secondary and tertiary structure. They are
folded into a roughly spherical shape with the hydrophobic amino acid residues located in the
center of the sphere, forming the hydrophobic core. The hydrophilic residues with polar
groups and charges are located on the outer layer of the sphere, forming a hydrophilic shell
(Fig. 6). Globular proteins are very compact, and show little swelling in water. As a result
globular proteins, when dissolved as monomers, give only a small increase in viscosity, and
are hence not very effective as thickening agents.

Figure 6. Schematic representation of a globular protein.

Globular proteins with high molecular weights often fold themselves into chains of several
spheres (Fig. 7).

Figure 7. Ordering of a single large protein molecule

in a string of multiple spheres.

Figure 8 shows some examples of the tertiary structure of a number of globular proteins:

43
Figure 8. Tertiary
structures of beta-
lactoglobulin (A) and
apo-alpha-lactalbumin
(B).

In order to make a stable solution of a globular protein, the individual molecules must be
dispersed in the solvent (Fig. 9). A solution of a globular protein is a colloidal system and
therefore the stability of the system can be described with the DLVO theory (see Physics
Tools Interactions). The interaction potential between two dissolved globular proteins VTOT, is
given by

VTOT  V ATT  VREP (4.1)

with VATT the attractive interaction potential and VREP the repulsive potential. For native
globular protein solutions the attraction potential, VATT, is mainly determined by the Van der
Waals interactions. Other contributions to the attractive potential are from hydrophobic
interactions, hydrogen bonding, and sulphur and salt bridges. For reasons of simplicity we
will neglect these interactions here. The repulsion is mainly determined by electrostatic
repulsion and Born repulsion. The electrostatic repulsion is the result of the charges in the
“shell” of the globular protein.

Figure 9. Several (charged) globular proteins in solution.

As we can see in Chapter 9, the strength of the electrostatic repulsion depends on the surface
potential of the proteins, and the Debye length (thickness of the diffuse double layer). The
surface potential of the protein is mainly determined by the pH of the solution, and the Debye
length is determined by the salt concentration. The total interaction potential for a globular
protein and its various contributions are displayed in Fig. 10.

44
 Figure 10. Example of the total interaction potential between two globular proteins.

If VMAX is high enough, the protein solution will not aggregate. So in order to make a solution
of a globular protein, one has to make sure that the electrostatic repulsion is strong.
Therefore, a high surface potential and a large double layer thickness enhance the solubility
of the protein.
The pH influences the solubility of a globular protein through its charge. The charge of the
protein is minimal in the iso-electric point. At this point the value of surface potential is low
and the protein will not dissolve easily. Far from the iso-electric point the charge of the
protein will be at a maximum (positively charged for acidic conditions, negatively charged
for basic conditions). The surface potential will be high under these conditions, and the
protein will dissolve easily.
The Influence of salt on the solubility is more subtle: We restrict ourselves to the case of
higher salt concentrations. At high salt concentrations, the additional salt decreases the size of
the double layer. The electrostatic repulsion decreases and the protein precipitates. This is
called salting out.
Examples of globular food proteins are glycinin, -conglycinin (both soy proteins), -
lactoglobulin (whey protein), ovalbumin (protein from chicken egg whites), glyadin (wheat
protein), actin and myosin (muscle proteins).

Random coil proteins

Random coil proteins are long linear proteins, without secondary and tertiary structure.
Examples of random coil proteins are casein, gelatine or completely denatured globular
proteins ( T  100 0 C , or extremely alkaline conditions). Random coil proteins are rolled up
into a roughly spherical shape. See also Physics Tools Macromolecules. The dimension of the
sphere depends on the length of the chain, the stiffness of the chain, the temperature, and the
quality of the solvent. The radius of the sphere is proportional to the root mean square end-to-
end distance Rrms , defined by Rrms  beff N eff , where beff is the effective segment length or
persistence length of the chain (the length for which the chain can be considered rigid). N eff
is the number of effective chain segments, and  the linear expansion coefficient. The linear
expansion coefficient is a function of temperature, the number of effective chain segments,
and the solvent quality. The solvent quality is determined by the type of solvent (hydrophobic
or hydrophilic), pH, ionic strength, and presence of other substances like (poly-)saccharides
or surfactants. We distinguish three types of solvents: good solvents, marginal solvents, and

45
bad solvents. In a good solvent the affinity of the protein chain for the solvent is larger than
the chain-chain affinity. This results in a swelling of the protein coil, and hence in an increase
in the radius of the coil. The solubility of the protein is high in a good solvent. In a marginal
solvent  is equal to 1, and the dimension of the coil is equal to the dimensions of an ideal
chain (no swelling, no compaction). This type of solvent is also referred to as a theta solvent.
Finally, in a bad solvent, the chain-chain affinity is higher than the chain-solvent affinity.
This results in a compaction or collapse of the coil. Under these conditions the solubility of
the protein is usually very low.
When dissolved in a good solvent, random coil proteins are excellent thickening agents. As a
result of the swelling of the polymer coil, the coil encloses a large fraction of the solvent
molecules. The effective volume fraction of coils is therefore very high, even at small mass
fractions of dissolved protein. This results in a large increase of the viscosity of the solution.
Proteins like gelatine are also used as structuring agents .The structuring is a consequence of
how the protein molecules aggregate and thereby form larger structures.

Quaternary Structure

Quaternary structures are formed when several protein molecules form mesoscopic building
blocks. The mesoscopic building blocks are formed by aggregation or self-assembly.
Depending on the experimental conditions, several types of structures can form:

 Rigid rods (Fig. 12b)

 Semi-flexible chains (Fig. 12c)
 Linear flexible chains (Fig. 12d)
 Branched flexible chains (Fig. 12e)
 Micelles and fractal aggregates (Fig. 12f)
The two most important parameters that determine the type of structure are protein charge,
and salt concentration (ionic strength). The protein charge is of course mainly determined by
the pH.

Figure 12. Various structures formed by globular

proteins as a function of pH and ionic strength: A.
monomers, B. rigid rods, C. semi-flexible chains, D.
flexible chains, E. branched flexible chains, F.
aggregates

The formation of the various structures can again be explained using DLVO theory. The total
interaction potential between individual globular proteins consists of a repulsive and an
attractive contribution. The repulsive contribution originates from electrostatic repulsion,
whereas the attractive contribution consists mainly of hydrophobic and van der Waals
interactions. Other contributions to the attractive potential are from salt bridges, sulphur
bridges and hydrogen bonds. These last three contributions are short ranged, and will not be
considered here.
In the native state, the hydrophobic parts of the protein are located mainly in the interior of
the globular molecule. In this state the hydrophobic interaction is weak. Let us define the
parameter   pH  pI , where pI is the iso-electric point of the protein. At high values for 

46
(i.e far from the iso-electric point) and low ionic strength, the electrostatic repulsion is
dominant, and little or no aggregation takes place. Take for example lactoglobulin. This
protein has an iso-electric point of 5.2. At a pH between 5.2 and 6.7 the protein has very little
charge and exists as a stable dimer. At a pH between 5.2 and 3.5 it exists as stable octamers.
At pH values below 3.5 and above 6.7 the protein molecules exist as monomers. The protein
has its maximum positive charge at a pH of 2 (21 positive charges). At a pH of 8 the protein
has 9 negative charges. At a pH of 14 it has its maximum negative charge: 36 negative
charges. The electrostatic repulsion is dominant over almost the entire pH range, and
hydrophobic interaction is too weak to induce substantial aggregation. Screening the
electrostatic repulsion by adding salt will result in precipitation, and not in the formation of
quaternary structures.
When the protein is heated above its denaturation temperature, it will partially unfold. Part of
the hydrophobic amino acids will be exposed, and become available for aggregation. The
hydrophobic interaction increases. At low ionic strengths and a high value for  , the protein
is highly charged, and no aggregation will occur. When the ionic strength is increased, the
electrostatic repulsion will be partially screened, and aggregation may occur.

VTOT VER

increasing I

VMAX
x

VATT

figure 13. Total interaction potential for two protein particles. Total potential as a function of ionic
strength.

The type of aggregate that forms depends among other things on the balance between
electrostatic repulsion and hydrophobic interaction. Let us consider the aggregates depicted in
Figure 14.

Figure 14. Types of aggregates and their respective magnitudes of the electrostatic repulsive energy.

47
The total electrostatic repulsive energy for the rod-like structure is given by:

1,TOT
VER  VER ( D)  VER (2D)  VER (3D) (4.2)

while for structure 2 we find:

2,TOT
VER  VER ( D)  VER ( D2 )  VER ( D3 ) (4.3)

Since D2 < 2D, and D3 < 3D, we find that VER1, TOT < VER2, TOT. This can be explained by the
fact that in structure 2 the particles are closer to each other and their double layers will
overlap more, which results in a higher total energy of the cluster. Note that the specific form
of the potentials in Eqs (4.2) and (4.3) is not given by the simple expression for interaction
between two spheres. The potentials must include multi-body interactions as a result of
partial screening of the charges at 2D and 3D by the particle at distance D. In aggregates of
the third type we find:

3,TOT
VER  VER ( D)  2VER ( 2D) (4.4)

From equations (4.3) and (4.4) it is clear that VER3, TOT > VER2, TOT. Finally, for aggregates of
type 4 we find:

3,TOT
VER  VER ( D)  2VER ( 2D)  VER (2D) (4.4)

from which we conclude that VER4, TOT > VER3, TOT. When the ionic strength increases, the total
repulsive energy of all aggregates depicted in Figure 14 decreases. The hydrophobic
interactions are nearly unaffected by  and the ionic strength. Aggregation can occur when
the hydrophobic interaction is strong enough to overcome the repulsive interaction. As a
function of the repulsion and attraction energy we can distinguish the following structures
(Figure 15): At high  and low ionic strength (I1) no aggregation occurs. At slightly higher
ionic strength (I2 > I1) rigid rod-like aggregates are formed, since only VER1, TOT < |VATT|,
while the electrostatic repulsion of the other structures in Fig. 14 is still higher than |VATT|. At
even higher salt concentrations (I3 > I2) semi-flexible chains are formed, since at these
concentrations VER2, TOT is also smaller than |VATT|.

I1 I2 > I1 I3 > I2 I4 > I3 I5 > I4 I6 > I5

monomers in solution rod-like chain semi-flexible chain flexible coil branched flexible coil aggregate or micel

VER > |VATT | VER1, TOT < |VATT | VER1, TOT VER2, TOT << |VATT| VER3, TOT < |VATT| VER3, TOT << |VATT|
< |VATT |
VER2, TOT VER4, TOT < |VATT|
VER3, TOT > |VATT| VER4, TOT > |VATT|
VER2, TOT
> |VATT|
VER3, TOT VER3, TOT > |VATT |

Figure 15. Structures formed as a function of their respective total electrostatic repulsive energy.

48
When the ionic strength is increased slightly (I4 > I3) flexible coils are formed, since VER2, TOT
<< |VATT|. The chains can form loops, but no branches, because VER3, TOT is still higher than
|VATT|. At even higher ionic strength (I5 > I4), branched flexible chains are formed, since at
these salt concentrations VER3, TOT is also smaller than |VATT|. When even more salt is added (I6
>I5), aggregates or micelles are formed. At these concentrations even VER4, TOT is smaller than
|VATT |, and the protein molecules can form bonds over their entire surface area. Figure 16a
gives an example of semi-flexible structures formed by -lactoglobulin, at pH 2 and low
ionic strength, after heating for 10 hours at 80oC. Compare these to the short flexible
branched structures formed after heating for 30 minutes at the same temperature, but now at
pH 7.

aa. a.
b

0.5 m 0.6m

Figure 16. a) -lactoglobulin fibrils formed after heating for 10 hours at 80oC, at pH 2 and 10 mM
ionic strength. b) branched flexible aggregates formed from the same protein at pH 7, heated for 30
minutes at 80oC.

Note that the DLVO theory as presented in this section does not include ion-specific effects.
In some cases these effects are an important factor in the formation of meso structures. For
example, at a pH above the iso-electric point, at equal ionic strengths, -lactoglobulin gives
stronger gels in a solution with calcium ions than in a solution with sodium ions. Since the
proteins are negatively charged under these conditions, the di-valent positively charged
calcium ions can form salt bridges between the protein molecules, which leads to additional
branching or cross-linking of chain structures. This leads to more compact structures, causing
an increase in gel strength. Another example of ion-specific effects is the formation of fibrils
from globular actin, which is highly accelerated in the presence of divalent ions (specifically
magnesium), whereas self-assembly is slow in the presence of monovalent ions (like
potassium). So in general, the assembly of proteins is a complex subject, but there are several
general laws which still apply, as illustrated above.
An important issue is that the theory presented above suggests that the boundaries between
the various structures are sharp. These boundaries are in fact continuous. From statistical
mechanics we learn that the probability for the existence of an arbitrary structure x 1 is given
by

  V (x ) 
P(x1 )  exp  TOT 1  (4.5)
 kT 

49
where the total energy term is equal to the sum of repulsive and attractive interactions. The
exponential factor is of course the well known Boltzmann factor. We see that the probability
for a structure increases when the total energy becomes increasingly negative, i.e when VER
< |VATT |. The probability is low when the total energy is positive, i.e. when VER > |VATT |. The
probability is however not equal to zero. This means that when the electrostatic repulsion is
still at a level where the DLVO theory would predict only linear rod-like particles, there is in
fact a small fraction of flexible or even branched structures present in the solution.

Primary Structure Secondary Structure Tertiary Structure Quaternary Structure

T1.Globular Q1. Rods

s-s
Q2. Semi-
flexible
T2. Random Q3.
coil flexible

Q4.
Branched

Overview of the various structure levels encountered in protein systems. Q5.

Random

3. Transitions between protein structures: denaturation

The globular structure of a protein is stabilised by:

* Covalent bonds: -S-S-bridges

* Hydrogen bonds
* Hydrophobic interaction: stabilisation of the hydrophobic core
* Van der Waals interactions
* Electrostatic interactions (in the hydrophilic shell)

The denaturation reaction of a globular protein is given by:


k
f

N D 
ki
I (4.6)

kb

where N is the native globular structure, D the denatured structure, and I the irreversibly
denatured structure. The activation energy G for the denaturation reaction is given by:

G = H - TS (4.7)

50
where H is the enthalpy difference between initial and final structure, and S the entropy
difference between initial and final structure. H has several contributions: the first
contribution is the bond enthalpy HBOND. This is the enthalpy change as a result of
breaking up the covalent bonds, hydrogen bonds, hydrophobic interaction, and salt bridges.
This requires energy, and therefore HBOND is positive. The second contribution is the
hydration enthalpy HHYDRATION. This is the enthalpy change as a result of the hydration of
polar groups. By unfolding, a greater number of polar groups can make contact with the
solvent water. During this process energy is released, and hence HHYDRATION is negative.
The final contribution is the deformation enthalpy HDEFORMATION. In the globular state, a
great number of covalent bonds are “bent” by the compact structure. Therefore, from an
energy point of view, the bond angles do not have the most favourable value. Unfolding
decreases the bending, deformation energy is released, and therefore HDEFORMATION is
negative.
S has one contribution, the conformational entropy SCONFORMATION. The globular structure
is a compact structure with many internal bonds, and as a result the number of conformations
which the molecule can assume is relatively low. Therefore, this structure has a relatively low
conformational entropy. Unfolding causes the internal structure to break up and the molecule
will behave more like a random coil molecule. The number of possible conformations
increases and so does the conformation entropy. Therefore SCONFORMATION is positive.
Combining all these contributions we find

G = HBOND + HHYDRATION + HDEFORMATION - TSCONFORMATION

The reaction occurs when

G  0

From this we conclude that denaturation may happen when:

1. The temperature is increased: the contribution TSCONFORMATION becomes more important

when the temperature increases.
2. The temperature is decreased: the strong hydrophobic bond decreases, HBOND becomes
less positive, and consequently G decreases.
3. The pH is at extreme values: the electrostatic repulsion in the shell increases, the outer
shell of the globule becomes more stretched, and can even be broken open. This implies
that HBOND is less positive at extreme pH.
4. The quality of the solvent is altered. If a solvent is added that is a good solvent for the
hydrophobic core, the core of the globule may swell. Again HBOND becomes less positive
due to the fact that the hydrophobic interaction decreases. Moreover, we get back the
interaction of the nucleus with a good solvent. This provides an additional enthalpy-
change that is negative.
5. Surfactants are added that attach themselves to the hydrophobic core. As a result the
hydrophobic interaction within the core decreases and HBOND will be less positive. In
addition, we will gain the enthalpy change due to core-surfactant interactions, which is
negative. Therefore G decreases when surfactants are added.
6. Reactants are added, for instance for breaking S-S bonds. When the strength of the
internal structure decreases as a result of eliminating covalent bonds, HBOND becomes
less positive and G decreases.

51
Subsequent reactions which make the denaturation irreversible are:

Aggregation: the denatured proteins can aggregate to form micelles of filaments

Trans-Cis transition of the peptide bond
S-S Bridge reshuffling
Chemical cross-linking

Exercises

1. Which structural changes occur during heat-set gelling of a globular protein solution?
Explain these changes in terms of molecular interactions.

2. When an ovalbumin solution is heated at pH 3, a transparent gel is formed at ionic

strength equal to 1 mM, a slightly turbid gel at 30 mM, and a completely turbid gel at 50
mM ionic strength. Explain these observations. Note that the isoelectric point of
ovalbumin is about 5.1.

3. At a pH very far from the isoelectric point, a globular protein can denature without
heating. Explain this phenomenon in terms of molecular interactions, and discuss its
effect on the viscosity of the protein solution.

4. How does the viscosity of a solution of a random coil protein vary with pH? Explain your
answer in terms of molecular interactions.

5. At a pH of 2 and an ionic strength of 10 mM -lactoglobulin forms long rigid fibrillar

aggregates, with a contour length between 2 and 7 m. When the ionic strength is
increased, the stiffness of the aggregates decreases, and branching may occur. Explain
these observations in terms of molecular interactions.

6. A Bovine Serum Albumin (BSA) solution at pH 4, ionic strength of 100 mM, and a
protein concentration below the minimum gelling concentration, is heated at 70oC, for 60
minutes. Make a plot of turbidity and viscosity as a function of time for this process.
Explain your answer. Note that the isoelectric point of BSA is equal to 4.8.

52