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The Thermodynamics of Molecular Recognition

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University of New Orleans, New Orleans, LA, USA

1 Preamble

2 Basic Concepts

3 Practicalities

4 Conclusion

References

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PREAMBLE

Fischers lock and key hypothesis,1 then there was an

eighty-year induction period between this genesis and the

pioneering work of Cram, Lehn, and Pedersen, which ultimately led to their winning the Nobel Prize in 1987.24 As

Schalley has eloquently pointed out in his excellent book,

Analytical Methods in Supramolecular Chemistry,5 two of

the more important reasons for this induction period were

the time required for chemists to appreciate the importance

of weak intermolecular forces, and the time required for

chemists to develop techniques that could actually measure them. By the late 1960s, both these obstacles were

beginning to fall, and this was instrumental in heralding

in what can be called the first phase in the development

of supramolecular chemistry. In more ways than one, this

first phase has been a time of equilibration; it has been

a period during which the field has equilibrated and figuratively, found its feet, and a period in which the field

has literally been dominated by the measurement of twoentity systems at equilibrium. Now that it has found its

feet, the field has been busy working towards phase two:

applying what it now knows to topics such as sensing,6

while simultaneously exploring many-bodied systems that

may or may not be at equilibrium; the latter in the short term

is fundamental to the development of systems chemistry7

and in the long term may contribute to the development of

synthetic complex systems (i.e., those possessing emergent

phenomena).811 Regardless of exactly how this second

phase evolves, the ability to measure equilibria will always

be a part of supramolecular chemistry, and hence a thorough

understanding of the underlying concepts and the practical

aspects of measuring equilibria is of fundamental importance. This chapter summarizes the important concepts,

techniques, and protocols in studying systems at equilibrium. We begin with the basic concepts, and, where appropriate, relate these to the common methods of determining the thermodynamics of complexation [NMR (nuclear

magnetic resonance), UVvis spectroscopy, and isothermal titration calorimetry (ITC)]. In the second section, we

examine practical aspects of determining this data using the

aforementioned techniques.

2

2.1

BASIC CONCEPTS

Association constants and Gibbs free energy

association of two or more molecules is the embodiment

of supramolecular chemistry. The simplest supramolecular

systems therefore involve two molecules interacting with

each other, and frequently these are defined as host and

guest. The host, the larger of the two chemical entities, possesses curvature or concavity, which results in the inward

projection of functional groups or moieties (the recognition

motif) that define the binding site. In contrast, the smaller

Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.

This article is 2012 John Wiley & Sons, Ltd.

This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.

DOI: 10.1002/9780470661345.smc005

Concepts

which is recognized by the binding site. However, to understand the fundamentals behind the interaction of a host and

guest, we must first discuss simpler systemsspecifically

the simple case where only one species is dissolved in solutionand then the case were two species in solution are

in equilibrium. Once we have accomplished this, we will

be able to discuss the case of a host and guest being in

equilibrium with a hostguest complex. Readers who wish

to go deeper than the following discussion are directed to

two excellent texts.12, 13

For the simplest of cases, where solute A is dissolved

in solvent S, the Gibbs free energy (GFE) is the energy of

the entire system at constant pressure. The GFE of such a

solution is an intricate sum of the free energy of the solute

and solvent, as well as factors that take into account the

combining of the two, such as solvation of the former and

the overall entropy of mixing. To determine the GFE of a

solution, let us first consider solute A. The chemical potential of this solute (A ) is the extent to which the total GFE

(Gt ) of the solution changes as a function of the amount of

solute A. Mathematically, this is expressed by (1):

A =

Gt

nA

(1)

where nA is the mole fraction of solute A. Chemical potential is analogous to potential energy, and thus assuming

there is no (kinetic) barrier of impediment, a system of

higher chemical potential will change so that a lower potential can be attained. Note that (1) relates how the free energy

of a system changes upon a change in its composition, that

is, Gt = A nA . Building on this, the total GFE (Gt ) of

the solution of A is the sum of the products of the mole

fraction and chemical potential of each of the constituents.

Thus in the case of solute A dissolved in solvent (S), we can

write (2), where nA and nS are the mole fractions of solute

and solvent, and A and S are their respective chemical

potentials:

Gt = nA A + nS S

(2)

Law, each solute particle A is fully solvated, and there

is no aggregation occurring that could otherwise influence

the behavior of the solution. In such cases, the chemical

potential of solute A is given by (3):

A = A + RT ln nA

(3)

where A is the chemical potential of the standard (reference) state, R is the gas constant, T is the temperature, and

nA is, again, the mole fraction of A. However, most solutions do not behave ideally, and in these cases (3) can be

of the activity of solute A (aA ) rather than its mole fraction:

A = A + RT ln aA

(4)

fraction. It is introduced in order to preserve the form of

equations derived from ideal solution in nonideal situations.

All of the deviations from ideality are contained within the

activity. For nonideal solutions, we can further isolate

this deviation from ideality by invoking the activity coefficient ( A ), which relates the activity and the mole fraction

of the solute (5):

aA = A nA

(5)

activity coefficient, and since solutes obey Henrys law as

their concentrations approach zero, aA nA and A 1

as nA 0. The activity coefficients of a solute can be estimated by DebyeHuckel theory using interionic forces to

estimate aggregation. That said, this is an exceedingly difficult if not impossible process for solutions containing multiple species. As a result, it is normally assumed that A = 1,

and the conditions under study amount to an ideal dilute

solution. This assumption also negates the practical difficulties with measuring activities of solutions, because under

ideal, dilute conditions the activity of the solute is numerically equal to its (more readily measured) concentration.

Moving up to a slightly more complicated system,

consider now a system composed of two solutes (A and

B) in equilibrium (6).

AB

(6)

turns into B. In such a situation, the change in the amount

of A is dnA = d , and the change in the amount of B is

dnB = +d . The quantity is called the extent of reaction

(units, moles), and when the extent of reaction changes, the

initial amount of A (nA,0 ) decreases to nA,0 and the

amount of B increases (nB,0 ) to nB,0 + . The reaction

Gibbs energy Grct of this system is defined as the slope

of the line in the plot of Gibbs energy against the extent of

reaction [(7) and Figure 1]:

= Grct

(7)

p,T

of a difference in free energies. Here, however, Grct

represents the derivative, that is, the slope of the line at a

particular value of . There is, however, a close relationship

Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.

This article is 2012 John Wiley & Sons, Ltd.

This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.

DOI: 10.1002/9780470661345.smc005

state molar free energy of reaction, Grct , the change in

free energy when reactants in their molar standard state

change to products in their standard states (11):

G r < 0, reaction is

spontaneous

Grct = GB GA = 0B 0A

Gt

G r > 0, reverse

reaction is

spontaneous

Slope = G rct

B A = 0B 0A + RT (ln aB ln aA )

Grct =

nB = 1

Extent of reaction (z)

Grct

of reaction ( ).

Building from (2), while appreciating that dnA = d and

dnB = +d , for a small change in the amount of A ( d )

the corresponding change in free energy (dG) is given by

dG = A dnA + B dnB = A d A + B d B

= d (B A )

which can be rearranged to

G

= B A

p,T

Grct

+ RT (ln aB ln aA )

aB

= Grct + RT ln

aA

(12)

(13)

(14)

reaction to the activities of the two solutes. Furthermore, the

assumption that the system under study is an ideal and dilute

solution allows us to relate the concentration of solutes to

the free energy of reaction (15):

[B]

(15)

Grct = Grct + RT ln

[A]

(8)

At any point of a reaction, the ratio of products to reactants,

in this case [B]/[A], is defined by the reaction quotient Q.

Formally, and more generally, we can define Q as

(9)

and hence

Grct = B A

(11)

B in their standard molar states. If we expand (4) to

accommodate the additional solute B, we can write (12),

and noting (11) and (12), (13) and (14) readily follow:

G rct = 0

nB = 0

(10)

potentials (or partial molar free energies) of A and B:

but only at the particular composition of the mixture.

For contemporary supramolecular chemistry, determining

Grct at a particular point on the way to equilibrium is

not vital. However, in general terms it is important to note

the following three different cases for Grct = B A

in which A is equilibrating into a mixture of A and B:

(i) the forward reaction (A B) is spontaneous when

A > B (Gr is negative and the reaction is exergonic);

(ii) it is spontaneous in the reverse direction (B A) when

A < B , (Gr is positive, and the forward reaction is

endergonic); and (iii) the reaction is spontaneous in neither

direction when A = B (Grct = 0, i.e., the reaction is at

equilibrium).

So what of our more usual understanding of G terms,

that is, that they represent the free energy differences

Q = J aJ J

(16)

where denotes the product of what follows it (as

denotes the sum), aJ is the activity of component J, and

J is the stoichiometric number. The reader will recall

that stoichiometry numbers are different from stoichiometry

coefficients, and are negative for the reagents and positive

for the products of a reaction. Thus, in the case at hand,

Q = aA1 aB1 = aB /aA . Hence, (14) can be written in the

more general form (17)

Grct = Grct + RT ln Q

(17)

as the equilibrium constant K (K = Qeq ), and as Grct = 0

at equilibrium, we can write

Grct = RT ln K

(18)

between a solution of solute A in its standard state and

a solution of solute B in its standard state. Hence, with

the assumption that the system under study is an ideal

Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.

This article is 2012 John Wiley & Sons, Ltd.

This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.

DOI: 10.1002/9780470661345.smc005

Concepts

concentrations, (18) allows us to determine the free energy

difference between A and B in their standard states by

measuring the equilibrium concentration ratios of the two

solutes.

We can now take what we have learned from these simple

systems and apply it to the case when a host and guest are

in equilibrium with their complex (19).

H + G HG

(19)

the free energy difference between the reactants (H and G)

and the product (the HG complex) in this equilibrium. Here,

however, the thermodynamic equilibrium (association) constant Ka , again defined by (16), is formally the product

() of the activities (aJ ) of the host, guest, and hostguest

complex raised to the respective stoichiometric number

( J ). Thus, in the case at hand, Ka = aH 1 aG 1 aHG .

Again, assuming we are dealing with an ideal dilute solution, we can replace these activities with concentrations and

define the very familiar (20) and (21):

[HG]

[H ][G]

[H ][G]

Kd =

[HG]

Ka =

(20)

(21)

and guest, and Kd the reciprocal of Ka is the dissociation constant for the same process. For historical reasons, association constants (units, M1 ) have been preferred

by supramolecular chemists, whereas dissociation constants

(units, M) are routinely used by biochemists and medicinal

chemists. For Ka values, the larger the number, the stronger

the binding of the guest to the host, while for Kd values the

opposite is true. One of the reasons that many researchers

prefer dissociation constants is that a Kd value is numerically equal to the concentration of the guest [G] at which

the binding site of the host is half occupied, that is, when

[H ] = [HG]. In other words, a Kd value equates to the

concentration of a drug required to begin to significantly

inhibit an enzymatic target.

A major component of determining the thermodynamic

parameters for a complexation event is the determination

of the association constant (or dissociation constant). A

number of spectroscopic techniques (NMR, UVvis, fluorescence), as well as ITC, are used to make these determinations, and as we continue to expand on the basic concepts

and subsequently discuss the practicalities involved in such

determinations, we intrude these analytical techniques in

more detail.

molecularity of their respective forward and backwards

reactions. In a simple A B system, the equilibrium constant is defined by two unimolecular processes, whereas in

the H + G HG system the forward process is bimolecular and the reverse is unimolecular. This reaction asymmetry has ramifications that are both theoretical and practical.

Regarding the former, if concentrations are used to determine the Ka for an A B system, the Ka value has no

units and (18) can be directly applied to determine Grct

(G as we refer to it from now on) for the equilibrated

system. However, by the same approach the Ka for the system H + G HG has the units of reciprocal molar, and so

by the application of (18) we find ourselves trying to take

the natural logarithm of units! This issue stems from the

assumption that the system is behaving as an ideal dilute

solution and that we can replace the dimensionless activities of the solutes with concentrations (which in the case

of molarity has units M). As this assumption is generally

valid in most hostguest systems (or at least assumed to

be!), the only option is to ignore the units of Ka values

when applying (18). It is, however, important to appreciate

this very convenient omission.

The practical issue arising from the molecularity difference between the A B and H + G HG systems

concerns the composition of the systems at equilibrium and

how this varies with concentration. In the more straightforward A B system, the Ka value is defined as the ratio

of the two solutes, and this is independent of the actual

concentrations of the solutes. For example, if Ka = 5 and

the initial concentration of solute A is 6 mM, the concentration of solutes A and B at equilibrium would be 1 and

5 mM, respectively. If, however, the starting concentration

of A is 12 mM, then the concentration of solutes A and B at

equilibrium would be 2 and 10 mM, respectively. Again, a

5 : 1 ratio is observed between the concentration of reactants

and products. In other words, the Ka value is the gradient

of the straight line obtained in a graph of [B] against [A].

It is more complicated in the H + G HG system. Here,

the association constant is defined by a ratio in which the

numerator is a concentration to the first power, and the

denominator is related to concentration squared. The net

mathematical effect of this is that, with Ka a constant in

(20), for a series of complexations with increasingly dilute

initial concentrations of H and G, the change in [H ] and

[G] terms must decrease more slowly than the change in the

numerator [HG] term. In other words, if [H ] = [G], then

the Ka value is the gradient of the straight line obtained

in the plot of [HG] against [H ]2 . We discuss the practical

aspects of this phenomenon in more detail later, but a specific numerical example may illuminate this important point

further. For a system with Ka = 10, at 1 M starting concentrations of H and G, the equilibrium values of [H ], [G],

This article is 2012 John Wiley & Sons, Ltd.

This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.

DOI: 10.1002/9780470661345.smc005

and [HG] are respectively 0.27, 0.27, and 0.73 M. The host

and guest are 73% complexed. However, the same system is

only 1% complexed at 1 mM (approximate values for [H ],

[G], and [HG] are 0.99, 0.99, and 0.01 mM, respectively).

These changes can also be rationalized thermodynamically

(see above), but to do so, we need to embrace two topics

we have avoided so far: enthalpy and entropy. We discuss

these next.

2.2

is of course just the tip of the thermodynamic iceberg.

Under the surface are (among other things) the important

thermodynamic parameters: enthalpy and entropy. A lot of

chemical insight can be gained by determining the standard

enthalpy change (H ) and standard entropy change (S )

associated with a binding event. The link between free

energy, enthalpy, and entropy of a reaction is of course

the famous GibbsHelmholtz equation (22):

G = H TS

(22)

defined as the change in heat between reactants and products in their standard (1 M) state at constant pressure if no

work is done. The easiest way to view a change in enthalpy

is to associate it with changes in bond strength. From

a supramolecular perspective, we are primarily involved

with how noncovalent bonds change. However, because

of small changes in bond angle, strain, and torsion angle

arising through steric interactions between host and guest,

there are always subtle changes to the strengths of covalent

bonds within both host and guest as a complex is formed.

Although enumerating these different effects is difficult, at

a more general level we can of course classify a hostguest

complexation (or any reaction) as being endothermic (H

is positive) or exothermic (H is negative).

From a thermodynamic perspective (as opposed to a

statistical viewpoint), the entropy of a system is a measure of its disorder. The more the disorder, the lower the

overall free energy of the system. The standard change

in entropy for a chemical process is a measure of the

differences in atomic movements, that is, degrees of freedom, for reactants and products. The term degree of freedom encompasses three types of molecular motion, the

symmetry (point group) of the molecule(s), as well as

entropy changes arising through the mixing of chemical

entities. For a small molecule at 1 M concentration, these

entropic contributions can be ranked in the order of impor

) rotational entropy

tance: translational entropy (Strans

(Ssym

) entropy of mixing (Smix

). As a rule of thumb,

(entropy units, kcal mol1 K1 ). Both will be lost if a

small molecule undergoes a reaction. In contrast, a loss of

Svib

which can be mostly attributed to changes in internal

bond rotationsis usually an order of magnitude smaller.

It should be noted that of these three, only translational

entropy is concentration dependent. The additional entropy

and Smix

are generally quite small. Thus, the

factors Ssym

entropy of symmetry is defined by Ssym = R ln , where

is the symmetry number characteristic of the point group

of the molecule. For molecules of low symmetry (e.g.,

the C1 point group) = 1, whereas for higher symmetry molecules, for example, dodecahedrane (Ih point group)

= 60. In other words, Ssym

8.3 eu, with a distinct bias toward zero. The entropy

of mix

ing of i components is defined by, Smix

= R i ni ln ni ,

where n is the mole fraction. Thus, for an equimolar two

component system Smix

= R(0.5 ln 0.5 + 0.5 ln 0.5) =

R ln 2 = 1.38 eu. So again, any change in these types of

entropy as a result of reaction (or complexation) is usually

small. Hence, for a hostguest complexation event, it is the

changes in translational and rotational entropy that dominate, although in some cases a loss of the other forms of

entropy for the complexed host and guest may also play a

role. As a final note on entropy, it is also worth recalling

that as a solution is made more dilute, the entropy of the

system increases. In the case of the dilution of a solution

of host, guest, and hostguest complex (19), this entropic

change will be larger if the distribution of species shifts

toward free host and guest (two species) rather than the

hostguest complex. Hence the observed decomplexation

of a hostguest complex as a solution is diluted.

How do we determine the enthalpy and entropy contributions to the overall free energy change of a binding event?

We should recall that there are two general approaches.

The most accurate one is to determine the enthalpy change

directly using a calorimetric approach. ITC measures the

amount of heat liberated by a binding event as aliquots of

the guest are added to the host (or vice versa). As the titration proceeds, the amount of free host decreases and so the

amount of heat liberated with each addition of guest also

decreases. The result of an ITC experiment is therefore the

overall enthalpy change for complexation and, equally as

important, a curve of how the amount of heat liberated or

consumed decreases as a function of the host/guest ratio.

This latter curve defines the equilibrium constant for the

process, and hence using (18) and (22) the complete thermodynamic profile (G , H , S ) at constant pressure

is obtained. Errors in H can be as low as 1%, with

attendant errors in free energy and entropy changes ideally

<5%.

This article is 2012 John Wiley & Sons, Ltd.

This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.

DOI: 10.1002/9780470661345.smc005

Concepts

directly provides Ka , such as NMR, UVvis, or fluorescence spectroscopy, and takes advantage of the fact that

the binding constant changes as a function of temperature.

The relationship between Ka and temperature is apparent if

we (replacing K with Ka ) rearrange (18) in terms of ln Ka ,

and combine this with (22) giving (23):

S

H

+

RT

R

Guest 5

H (kcal mol1)

ln Ka =

+ve

(23)

is determined at several different temperatures, plotting

ln Ka versus 1/T gives a so-called vant Hoff plot, which

is a straight line whose slope is H /R and intercept

is S /R. With ITC being a relatively recent addition

to the list of tools commonly utilized in supramolecular

chemistry, spectroscopically derived vant Hoff plots are

very common. It should, however, be noted that the errors

inherent in this approach are larger than those using

ITC, primarily because the enthalpy change is determined

directly using ITC but is a first derivative when determined

using a vant Hoff plot. Additionally, in the latter there is

an inherent assumption that the enthalpy change does not

vary with temperature and that the obtained straight line

is just thata straight line. In other words, there is no

change in heat capacity associated with complexation. In

many systems studied in organic solvents, this is often a

reasonable assumption, but in water this is not normally

the case (see above). Hence, to err on the safe side, the

range of temperature used to determine the association

constant should be kept small (20 C). Correspondingly,

the number of determinations of K should be quite high

(five or more) so that the error in the extrapolation of the

straight line to the intercept is minimal.

There is another important issue that can arise with vant

Hoff plots. It is frequently the case that, when enthalpy

and entropy changes for guest complexation are carried out

for a series of guests, enthalpyentropy compensation is

observed.14a By way of example, it is frequently observed

that for a series of guests, as the enthalpy of complexation

becomes more exothermic, there is a corresponding increase

in the entropy penalty for each binding. The thermodynamic

data for a series of hypothetic guests is shown in Figure 2.

This plot fits the simple equation y = mx + C, where

y = H and x = S , and the slope of this line (m),

usually referred to as or the compensation temperature

Tc , is the temperature at which any variation in the H

for a series of guests is compensated by a change in S

such that the overall free energy change remains constant.

In Figure 2, G corresponds to the point on the line when

x = 0: that is, G is negative. There are two possibilities

as to the cause of such a trend: they reflect a real chemical

Guest 4

Guest 3

ve

+ve

Guest 2

Guest 1

ve

S (kcal mol1 T1)

guests binding to a common host.

the fact that in a vant Hoff plot H and S are

not measured independently. Krug and coworkers have

defined two important tests that differentiate between these

two possibilities.14b First, if there is a chemical cause to

the observed trend, then the compensation temperature Tc

must be significantly different from the average of the

experimental temperatures used to determine Ka . Second,

a true chemical effect can also be assumed when the vant

Hoff plots for the different reactions (in this case the

reaction between host and guest) are graphed together

and intersect at a common temperature. If either or both

of these scenarios are not observed, then the observed

enthalpyentropy compensation is most likely caused by

the statistical handling of the obtained data.

Both these approachesITC and the use of vant

Hoff plots in spectroscopic determinationsare commonly

used to measuring the enthalpy and entropy changes of

complexation; therefore, a practitioner of supramolecular

chemistry needs to be aware of the strengths and limitations

of the different approaches.

2.3

although the focus here is on thermodynamics we must

also touch on the topic of kinetics. In particular, we have

to discuss (i) the exchange rate between the free and bound

states in the system in question and how this relates to

the timeframe of the analytical technique being used to

investigate it; (ii) the timeframe by which equilibrium in

a system is attained, which is closely related to (i); and

This article is 2012 John Wiley & Sons, Ltd.

This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.

DOI: 10.1002/9780470661345.smc005

(iii) the overall timeframe (lifetime) of the system under

investigation. These points illustrate three different kinds

of systems that cover the gamut of chemical equilibria

possibilities: those that are equilibrated, those that not yet

equilibrated, and nonequilibrium systems. Considering all

three phenomena gives different perspectives on the notion

and importance of timeframes. The major fundamental to

note here is that all systems are tending toward equilibrium,

and it is just a matter of how long the system takes to attain

this state in relation to the experiment we are performing.

Let us first briefly consider the second and third

examples. In 10 min or so it may take to prepare a sample and run a NMR experiment, most hostguest systems

have attained equilibrium. The rate of the forward reaction

and the rate of the reverse process are so fast relative to

our actions that what is observed when the data is collected

is an equilibrated system. This is not, however, always the

case, particularly with the increasing structural complexity

of contemporary supramolecular systems. Regardless of the

structure of the host and/or guest, it is therefore prudent to

double-check that the system has equilibrated by running

the same experiment on the original sample after another

time period to confirm that the system has not changed. If

the system is still changing as a function of time, then the

system has not yet equilibrated and it may be possible to

use an analytical technique to study how the system changes

in a temporal manner. The term nonequilibrium systems

is normally reserved for those that are not at equilibrium

because some input of potential (chemical or otherwise)

is holding the system out of, or far from, equilibrium. In

other words, the system is subject to the laws of nonequilibrium thermodynamics.15 In chemistry, these types of systems are exemplified by oscillation reactions such as the

BelousovZhabotinsky (BZ) reaction.16, 17 The BZ reaction was the first of now many known nonlinear chemical

reactions that display periodic or chaotic temporal oscillation and spatial pattern formation. Although still often

viewed as a chemical oddity, we should note in passing

that nonequilibrated systems are apparent everywhere, from

the smallest life form to the largest hurricane (and beyond).

All nonequilibrium systems come into existence, exist for

a defined period of time, and then come to an end; and

in all cases the timeframe of our analytical measurements

is generally much shorter than the timeframe (lifespan) of

the system itself. Nonequilibrium systems are antithetical

to contemporary supramolecular system, but their appreciation bolsters the notion that we need to be mindful when

determining a binding constant that the system in question

is in equilibrium.

Closely related to the timeframe in which equilibrium is

established is the exchange rate between the free and bound

states in the system in question and how this compares to

the timeframe of the experimental technique being used.

the free and the bound states under examination faster or

slower than the timescale of the analytical technique? The

actual rate constants for the forwards and reverse reactions

of a simple hostguest system, k1 and k1 (24), correspond

to the bimolecular complexation (units = M1 s1 ) and the

unimolecular decomplexation (units = s1 ).

H +G

k1

k1

HG

(24)

system, we are observing its free and the bound states. For

example, for an NMR analysis we may be examining the

free and bound signals for a particular guest proton. Thus,

we are directly concerned with the observed rate constants

(ka and kb ) for the exchange between the free and bound

states (25).

Free

ka

kb

Bound

(25)

The rate constants for both the forward and reverse processes are those of unimolecular processes (units s1 ), and

it is the slowest of these two processes that we must compare with the rate at which the system is interrogated. For

spectroscopic determinations, the timescale of the analytical

technique is dictated by the Heisenburg uncertainty principle. The energy gap (E) between the two states connected

by the absorption is related to the time difference t by (26)

E t = 2

(26)

t is small, whereas the very small energy differences

between spin states in NMR means that t is relatively

large. To appreciate the timescale of NMR, consider the

situation in which two resonance signals from a proton in

the guestfor the bound and free statesare separated by

1 ppm. We must first recall that chemical shift () and the

change in chemical shift () are usually expressed as parts

per million so that the quoted values are independent of the

frequency of spectrometer. For our purposes here, however,

we must consider the resonance frequency (, units Hz) of

a nucleus, and recall that this is dependent on the applied

field strength (27).

=

B0

2

(27)

of the applied magnetic field. The value of in parts

per million is given by (28), where S and TMS are

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Concepts

question and the reference tetramethylsilane.

=

s TMS

Operating Frequency (MHz) 106

(28)

to 100 Hz on a 100 MHz instrument but 800 Hz on an

800 MHz instrument. As we will see, this is important

to note because the timescale of a 100 MHz instrument

is not the same as that of an 800 MHz. Focusing for

now on the smaller spectrometer, two peaks separated by

100 Hz are separated by 100 s1 . Returning to (25), if kb

(which for hostguest complexation formation is less than

ka ) is greater than this, say 400 s1 , then the exchange

process between the free and bound states is faster than the

100 MHz NMR timescale. In such a situation, the weighed

average of the two signals is observed. If, on the other hand,

kb = 20 s1 , then exchange is slow on the NMR timescale.

In this scenario both the free and the bound states are

distinct, that is, separate signals are observed. This situation

is more informative than when exchange is fast on the NMR

timescale because it is possible to determine the chemical

shift difference between the free and bound state of each

proton. Furthermore, with a slow exchanging system, the

Ka value can be determined directly from integration of the

two signals, whereas when the process is fast on the NMR

timescale, it is necessary to perform a titration experiment

in which the guest is added to the host (or vice versa) and

the shift in the target signal monitored as a function of

host/guest ratio (see below). Fast exchanging systems not

only require more work when it comes to spectroscopic

determinations, but the attendant errors are also larger: a

fact that should not be forgotten if a vant Hoff plots is

being constructed.

It is also possible that the exchange process being investigated occurs on the NMR timescale. Between the situations

when two peaks are observed (slow exchange) and one peak

is observed (fast exchange), signal coalescence occurs. This

scenario can lead to very broad signals, or signals disappearing into the baseline, a very uninformative situation.

Thankfully, the exchange rate is temperature dependent and

so by increasing or decreasing the temperature it is possible

to speed up or slow down exchange so that NMR data can

be gathered. Indeed, this is a common approach by which

kinetic barriers to exchange can be probed. Returning to

our larger NMR spectrometer, with a peak separation of

800 s1 , both the exchange processes discussed above will

be slow relative to this spectrometer. Hence, moving up to a

larger spectrometer is one, albeit expensive, way in which a

relatively fast exchanging system can be interrogated more

successfully.

NMR spectrometry is quite distinct from other spectroscopic techniques in regard to its timescale. Fluorescence,

timescales because they interrogate electronic absorption

(emission) and nuclear vibrational motion: processes that

are much faster than the complexation or decomplexation of a hostguest system. In other words, all binding

events are slow on the timescale of these techniques. Unfortunately, although all hostguest exchange processes are

slow, experiments with these techniques are not normally

as structurally informative as NMR.

Another way to visualize exchange processes, literally

the reciprocal of the viewpoint just given, is to consider the

lifetime of the species under consideration. The lifetime of

a species is defined as the reciprocal of the rate constant

for the first-order reaction forming it. Thus, in the case

of bound species in (25), its lifetime is defined by (1/ka ).

Hence, in the most general of terms, it can be asked

if the lifetime of a species is long enough such that it

holds together for the duration of the measurement being

carried out. This viewpoint allows us to move beyond

spectroscopy and ask, for example, if the lifetime is larger

than the high performance liquid chromatography (HPLC)

timescale (about 30 min). If it is, then HPLC will reveal

separate peaks for the free and bound species. If not, then

only one peak will be observed if host and guest were

combined in equimolar amounts. Supramolecular exchange

processes that are slow on the HPLC timescale are relatively

rare in synthetic systems,18 because the corresponding

association constant for the formation of the complex must

be very high (typically Ka > 1010 M1 ).

As we have discussed, ITC does not spectroscopically

examine the free and the bound states in a hostguest

system but instead determines the overall enthalpy change

upon addition of multiple aliquots of guest to a solution of

the host. Hence, the aforementioned discussion of experimental timeframes and their relationship to the exchange

rates do not apply to ITC. Instead, it is important to determine whether upon addition of each aliquot the mixture has

equilibrated before an addition aliquot has been added. In

other words, in ITC it is important that the time to equilibration for the system be faster than the pause between

each injection.

2.4

overstated, and in supramolecular chemistry we are particularly interested in how solvent interacts with the free

and bound solutes, and consequently how this influences

the noncovalent interactions between them. The bulk of

research in the field carried out thus far has been in organic

solvents, with a bias toward nonpolar and aprotic solvents that maximize electrostatic interactions between the

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DOI: 10.1002/9780470661345.smc005

molecules. This has given practitioners a firm understanding of both the relationship between molecular structure

and noncovalent interactions, and how the different properties of solvents influence noncovalent forces. That stated,

in the natural world most supramolecular chemistry occurs

in water. Consequently, aqueous supramolecular chemical

research that has also focused on water as a solvent, particularly with cyclodextrins,1923 cyclophanes,24 and, most

recently, dynamic molecular capsules.25 This research has

highlighted many differences between aqueous and nonaqueous supramolecular chemistry which are important to

appreciate.

At a very general level, working in water rather than

organic solvent affects the thermodynamics of binding in

two important ways. First, all other things being equal,

binding in water is stronger than binding in organic

solvents.14a Second, whereas heat capacity changes of binding processes in organic solvents are usually small or not

observed at all, this is generally not the case in water. In

other words, in aqueous solution it is frequently observed

that the H and S for complexation changes as a function of temperature. We will expand on these points in due

course, but for now let us briefly discuss the root cause of

these two phenomena, the hydrophobic effect. Most simply stated, the hydrophobic effect2630 is the reason why

oil and water do not mix. Many details of the hydrophobic effect are still to be identified and enumerated, but the

key noncovalent interaction behind it is hydrogen bonding.

More specifically, the strength of the waterwater interaction gives it a high cohesive energy and a high surface

tension, which leads to a sizable energetic penalty when

forming a cavity in water. Hence, the dissolution of a

solute, particularly a hydrophobic one, involves significant

changes to the local dynamical structure of the water. Key

to the hydrophobic effect is therefore the dynamical structure of the hydration shell around solutes, how this changes

according to the size, shape, and nature of the solute, and

how these changes alter the enthalpy and entropy of the

solute/solvation shell. Broadly speaking, these hydration

shells vary from solute to solute, but at a fundamental level

the hydration of solutes is still poorly understood. Some

of the best insight has come about from in silico studies

examining how the solvation shell changes as a function

of the size27 and shape31, 32 of the hydrophobe. In addition,

empirical studies have also been of immense importance.

For example, the state of the art in studying solvation shells

is quite advanced for protons and small hydrocarbons such

as methane,33, 34 and is improving both for inorganic salts35

and organic molecules such as -cyclodextrin.36

Whatever the rules that govern the hydration of solutes, a

key point is that when considering a binding event in water

we should be mindful that in water the hostguest equilibrium depicted in (19) is a considerable oversimplification.

The host, guest, and hostguest complex each has a particular solvation shell, and the change in solvation in forming

the complex plays a large part in the overall thermodynamics of binding. A much more accurate equation would therefore account for the hydrating water molecules. Regardless

of our poor understanding of these hydration shells, a comparison of many hostguest complexes reveals that the

complexation of most organic molecules receives a thermodynamic boost from the hydrophobic effect. That said,

because of the shielding properties of highly polar water,

noncovalent interactions that primarily involve electrostatic

forces cannot be relied upon to the same extent as they

can be in organic solvents. Hence, although the strongest of

noncovalent interactionsmetal coordinationhas proven

to be effective drivers of complexation (and assembly) in

water, hydrogen bonding has proven so far to be of limited

utility. However, more often than not, the thermodynamic

boost from the hydrophobic effect more than compensates

for any loss of attractive electrostatic interactions between

molecules and, as a result, binding constants in water tend

to be at least 12 orders of magnitude larger. That this is

true is perhaps not so interesting in its own right. After

all, there are many situations where strong binding is not

a requirement, and many systems where strong binding

is a detriment. However, that binding is usually stronger

means that by and large any particular host is able to

bind a wider range of potential guests; and if selectivity is required, it is always easier to prevent binding than

create it.

In addition to stronger binding, the desolvation of surfaces on the host and guest as they form a complex also

leads to a characteristic decrease in the heat capacity of

the solution. The standard heat capacity of a substance at

constant pressure (Cp ), is the amount of energy a substance

absorbs per unit change in temperature (29):

Cp =

H

T

(29)

event in water demonstrates that the free host and the guest

are able to absorb more energy per unit temperature than

the corresponding hostguest complex. Much is still to be

learned why this is so, but the current understanding is that

the ordered (and this word is used in the loosest possible

terms) solvation shells around the hydrophobes can act as

heat sinks because, as the temperature is raised, many less

ordered states become available in which energy can be

stored. However, desolvation of the hydrophobic surfaces

on the host and guest reduces the total number of salvationshell waters and attenuates this sink.29, 37 As a result,

the heat capacity of the solution decreases. Indeed, this

decrease in the heat capacity is one of the best hallmarks

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10

Concepts

hallmark than the often observed increase in entropy upon

binding usually attributed to the release of ordered water

molecules from the solvation shell.

A change in heat capacity for a binding event indicates

that its associated enthalpy as well as entropy change

changes as a function of temperature, and this can lead

to considerable inaccuracy in vant Hoff plots if the

thermodynamic parameters for complexation are being

sought. The relationship between the standard enthalpy

change (H ) and the standard heat capacity change (Cp )

of a reaction or a complexation is given by (30), where H

is the reference enthalpy at 298 K:

H = H + Cp

(30)

A similar equation (31) can be derived for the relationship between the standard entropy change (S ) and the

standard heat capacity change:

S = S + Cp ln T

(31)

(30) and (31) with (23) leads to (32):

(32)

This equation allows us to carry out vant Hoff plots even

when H and S change as a function of temperature.

By fitting the equation using the three variables H , S ,

and Cp and then using (30) and (31) we obtain H and

S at the temperatures sought.

It is useful to recall that Cp is a second derivative when

determined with NMR and other spectroscopic techniques;

Ka values must be determined at different temperatures and

vant Hoff plot performed in order to get the enthalpy

change, and it is how this enthalpy change changes as

a function of temperature that gives Cp . On the other

hand, Cp is a first derivative of an ITC experiment.

The enthalpy change is determined directly, and a series

of experiments at different temperatures yields Cp as

the gradient of the graph of H versus T . Hence,

ITC is a much more accurate technique for determining

Cp .

We have now introduced the necessary basics for determining the association constant (Ka ), standard free energy

change (G ), standard enthalpy change (H ), standard

entropy change (S ), and standard heat capacity change

(Cp ) for the complexation of a host and a guest. In

subsequent sections, we detail practical aspects of these

measurements.

PRACTICALITIES

Beyond the basic thermodynamics that we have just discussed, there are many considerations regarding how we

actually perform experiments to determine Ka , G , H ,

S , and Cp of complexation in 1 : 1 hostguest systems

(19). In determining thermodynamic data for a complexation event, there are, as there are with any physical chemistry problem, two goals. The first goal is to define the

system mathematically with a basic mathematical model;

the second is to fit the obtained data to the mathematical

model. The first goal is, of course, independent of the analytical technique we are going to use, whereas the second

is very much dependent on it. Irrespective of the technique

used, the overall aim is to quantify the formation of the

hostguest complex for a given initial concentration of host

and guest. This can be accomplished by directly measuring

the amount of hostguest complex, or indirectly by measuring the remaining free host or guest and using mass balance

equations to calculate the concentration of the complex.

Many analytical techniques are available to the experimentalist for this task, but we focus here on the most widely

used techniques, NMR, UV, and fluorescence spectroscopy,

and ITC, and give a succinct account on how to conduct

such experiments with these techniques. For more detailed

descriptions of the individual techniques, as well as details

of other techniques used, the reader is directed to some of

the many excellent reviews available in the literature.5, 3841

Each technique has its advantages and limitations, and our

intent here is to provide enough information to allow the

experimentalist to choose the most suitable technique for

his/her particular research.

This section begins with highlights of how the timeframe

of a technique and the concentration of a sample have

important practical ramifications. Subsequently, we discuss

the base mathematical model for 1 : 1 complexations before

looking at how this model is tailored to each analytical

technique. Finally, we discuss a common approach sometimes used to confirm 1 : 1 binding, as well as very briefly

highlight higher stoichiometry systems.

3.1

Timeframe of analysis

We have discussed the importance of timeframe of analysis with regard to both the timeframe by which equilibrium

in a system is attained and the timeframe of the exchange

process of the complexation under investigation. Practical

aspects of the former simply involve double-checking that

equilibrium has in fact been attained. Practical aspects of

the latter are a little bit more complex. As we have discussed, different techniques operate at different timeframes,

and it is important to appreciate how this relates to the

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exchange rates between the free and bound states (25). Is

the instrumental technique detecting an average signal of

the species involved, or are the individual signals from

each species apparent? In most spectroscopic techniques

including UVvis and fluorescence, the exchange between

free and bound species is much slower than the timeframe

of the technique. In other words, the binding event under

study is slow on the instrument timescale and the instrument

detects each of the species individually. In contrast, this is

not the case with NMR spectrometry where complexations

that are slow as well as fast on the NMR timescale are both

observed. We will return to this point when discussing the

individual techniques.

3.2

Concentration range

under which the complexation is being studied. Each technique has its own limitations defined by its sensitivity, and

we discuss these as we examine the individual approaches.

In addition, there is the common factor arising from the

asymmetry inherent to the hostguest equilibrium (19) and

how, for a fixed binding constant, the initial concentration

of the host and guest dictates the extent of complexation.

Figure 3 shows the state of affairs with a total (initial)

concentration of host (Ht ) and guest (Gt ) of 1 mM and

a range of binding constants (Ka ) from 10 to 108 M1 . Ideally, for accurate measurements, equilibrium should result

in somewhere between 20 and 80% complexation. If the

11

of host and guest will be too low and sizable errors will

ensue. Likewise, if the initial concentrations are too low,

then there will be an insufficient amount of the complex

formed and again large errors will arise. In the case shown

in Figure 3, the optimum range of Ka is observed to be

between 5 102 and 2 104 M1 . Of course, the distribution of species can be shifted easily by changing the ratio

of Ht /Gt ; for example, if Ka = 10, and Ht = 1 mM and

Gt = 100 mM, then 50% complexation is attained (compare

with first column in Figure 3).

The best way to determine the concentration at which an

experiment needs to be run is to know the binding constant;

but this is of course the first step of a circular argument.

The only option therefore is to take an educated guess at

the strength of association and then to perform the required

experiment. A second experiment can subsequently be run

if binding was weaker or stronger than anticipated.

An alternative viewpoint is expressed in Figure 4, which

shows percentage complexation against total host and guest

concentration (Ht and Gt ) for a hostguest complexation

(Ka = 1 103 ). This graph represents the effect of dilution upon complex formation. At high guest concentration

(1 M), the system is close to full saturation with 97% complexation. In contrast, if the working concentration is too

low (Ht = Gt < 1 104 M) essentially no complexation

is observed. We can define a concentration range for this

model system of between 0.5 to 50 mM, and whether a particular technique is suitable for the task at hand depends on

its inherent sensitivity. Furthermore, there may be solubility

1E 03

Concentration (M)

1E 03

8E 04

6E 04

4E 04

2E 04

0E +00

1E + 01

1E + 02

1E +03

1E +04

1E +05

1E + 06

1E + 07

1E + 08

K a (M1)

Figure 3 Graph of the concentration of host [H ] (blue), guest [G] (red), and hostguest complex [HG] (green) against equilibrium

constant (Ka ) where the total (initial) concentration of host and guest (Ht = Gt ) = 1 mM.

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12

Concepts

100

% complexation

80

60

40

20

0

1E + 00

1E 01

1E 02

1E04

G t (M)

1E 03

1E 05

1E 06

1E 07

Figure 4 Graph of the percentage complexation against total guest concentration (Gt = Ht ) where the equilibrium constant (Ka ) is

set to 1 103 M1 .

be considered.

3.3

With the exception of just one scenario, all of the techniques commonly utilized by supramolecular chemists to

determine thermodynamic data yield too many unknowns

if just a single host/guest ratio is studied. The exception,

slow exchanging systems studied by NMR, allows the direct

measurement of the individual components of the mixture (see below) from just one Ht : Gt ratio. For the other

approaches, we must carry out a titration in which the

Ht : Gt ratio is varied systematically and construct a mathematical model based on the mass balance equations for

the equilibrium and (20). The corresponding mass balance

equations are (33) and (34):

Ht = [H ] + [HG]

(33)

Gt = [G] + [HG]

(34)

guest in solution. For each of the analytical techniques

we are going to discuss, we define equations that relate a

measurable to three unknowns: the equilibrium constant;

a constant specific to the complex and the technique

used ( max , , and H for NMR, UVvis, and ITC

respectively); and the concentration of free guest [G]. We

therefore need an expression of [G] that relates it to a series

of known quantities and the equilibrium constant for the

process. To do this we will combine (20), (33), and (34) to

give (35):

Ka [G]2 + (1 Ka Gt + Ka Ht )[G] Gt = 0

(35)

equation for 1 : 1 complex formation, which we will combine with equations tailored for each analytical technique

relating a measurable to Ka , max //H , and the concentration of free guest [G]. It is useful to note that the

right-hand side of (36) is composed of only Ht and Gt

(which can be calculated) and the unknown Ka .

[G] =

(1 Ka Gt + Ka Ht )

(1 Ka Gt + Ka Ht )2 + 4Ka Gt

2Ka

(36)

3.4

NMR spectroscopy

chemistry have been performed using NMR, and, in particular, 1 H NMR. Protons are almost ubiquitous to organic

chemistry, and the 1 H nucleus is of high abundance. This

means a particularly fast analysis time relative to other

popular nuclei such as 13 C. In general, NMR will permit

the determination of binding constants of between 0.1 and

104 M1 , although stronger binding constants can be determined if a longer acquisition time is possible or a competition experiment is performed (see below). In these cases,

binding constants of up to 1 106 M1 represent the absolute upper limit of the technique before analyses are beset

by large errors. The normal limit of Ka 104 M1 in NMR

spectroscopy arises from the techniques relative insensitivity. As a result, most proton NMR spectra will be recorded

at sample concentrations of 15 mM. If the binding constant is high, then it will be necessary to run the sample

at concentrations of 100 M or less, and in such cases the

signal-to-noise ratio is small and an extended acquisition

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time is required. This may be possible in slow exchanging

systems (see below), but in fast exchanging systems this

approach becomes unwieldy.

A critical point in NMR determinations of thermodynamic data is to have a proton whose chemical environment changes sufficiently upon binding to lead to a change

in chemical shift, that is, Hfree = Hbound . As has been

discussed above, binding determinations by NMR can be

divided into two cases: those that are slow on the NMR

timeframe and those that are fast. The observed complexation and decomplexation rates (25) are those of unimolecular processes (units s1 ), and it is the slower of

these two processes that we must compare with the rate of

NMR data acquisition. Our previous discussion emphasized

how the timeframe of the NMR experiment was dependent on the external field strength of the instrument, but

it is also important to note that whether a complexation is

observed to be fast or slow on the NMR timescale depends

on the chemical shift difference between the free and the

bound state. The key equation is (37), which defines the

boundary between fast and slow timescales, that is, the

observed rate constant for coalescence (kcoal ) of the signals for the proton in question in the free and the bound

state:

kcoal = 2.22

(37)

constant [kb in (25)] needs to be to switch from slow to

fast on the NMR timescale. In certain cases, where there

is a spread in values ranging from the very small to

very large (e.g., 3 ppm or more), it is entirely possible for

some pairs of signals to be fast on the NMR timeframe

while others appear slow. Furthermore, in such cases,

intermediately shifted signals may be close to coalescence,

resulting in a broad or unobserved signal. In most cases,

however, all the signals are usually either fast or slow on

the NMR timeframe.

If an equilibrium process is slow on the NMR timescale,

the spectrum will show distinct peaks for the free and the

bound state. Hence, by knowing the initial concentration

of Ht and Gt , it is then straightforward to determine the

concentration of HG, H , and G by integration. In such

cases, the Ka values (and using (18) the G value) are

quickly determined, and if enthalpy and entropy change

or, indeed, heat capacity changes are also sought, it is

simply a matter of recording NMR spectra of the same

sample at different temperatures. That said, for a number

of reasons it is advisable to perform the determination of the

Ka and G at two or three different ratios of Ht and Gt .

Changing this ratio will help confirm which signals belong

to the formed complex (increase in intensity with increased

titrant), as well as confirm slow kinetics (the peaks will

13

major error since the Ka and G values obtained should

be identical. Determinations obtained this way and repeated

3 times with new stock solutions will give determinations

with errors of less than 10%.

During the course of the development of the field, hosts

(as well as guests) have tended to become structurally

more elaborate, in which case they often exchange slowly

on the NMR timescale. Nevertheless, structurally more

open hosts that exchange guests rapidly on the NMR

timescale still account for the majority of host molecules.

The determination of binding constants in these systems

involves more effort, both in terms of data collection and

fitting. Regarding the latter, we will need (36) to build our

mathematical model for Ka determinations with NMR.

We begin by first noting that, in fast exchanging systems,

the observed frequency Hobs of the proton of interest

becomes the weighed average of the free and bound

states (38):

Hobs = xfree Hfree + xbound Hbound

(38)

free component and the complex, respectively. In our

determination, we are going to change the ratio of host

and guest by titrating in the guest (or host) and monitor

how Hobs changes. In other words, we are going to plot a

binding isotherm. Typically, a host solution is titrated with

a stock guest solution in the NMR tube, and the shift of

the proton most affected by binding and not obscured by

other signals is then recorded for a minimum of 10 different

ratios. It is critical to cover a large range of host/guest ratios

to ensure that the system is close to saturation at the end of

the titration. In other words, the resulting binding isotherm

should reach a plateau. Now that we have our data, if we

set obs = Hobs Hfree and max = Hbound Hfree ,

then from the mass balance equations (33) and (34) and the

equilibrium equation (20), the NMR binding isotherm can

be expressed as

obs =

max Ka [G]

1 + Ka [G]

(39)

obs =

max

1

Ka [G]

+1

(40)

proton, and max is the maximum shift of the observed

proton at full complexation. Note that we have too many

unknowns in this equation (Ka , [G], and max ) to solve

for Ka with a single Ht : Gt ratio. To solve this problem,

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14

Concepts

[G] (35) to get a theoretical expression of obs versus

Gt (41):

obs =

to a step rather than a curve. In such cases, too few

points define the binding event and a large error ensues.

max

2

+1

Ka Gt Ka Ht 1 + (1 Ka Gt + Ka Ht )2 + 4Ka Gt

spreadsheet software such as Origin or the solver in

Excel will fit the experimentally derived binding isotherm

for obs versus Gt and will yield the remaining two

unknowns Ka and max .

As mentioned, the titration must cover the full range of

complexation and ultimately bring about the saturation of

the host. In addition, to avoid large errors, the obtained

binding isotherm must be neither too shallow nor too sharp.

Figure 5 shows three theoretical binding isotherms, where

a guest is titrated to a host solution of 1 mM, inducing

a max of 0.2 ppm. The binding isotherm obtained for

the Ka = 1000 M1 system is ideal. It covers the full

complexation range, and will allow a good fit of the data.

With the weaker binding guest (Ka = 50 M1 ) the binding

isotherm is more akin to a straight line and insufficient

guest has been added to saturate the host. In such cases,

there are two options: (i) titrate the host further with more

guest to reach a plateau close to the max = 0.2 ppm,

in which case unless a highly concentrated guest solution

can be made the dilution of the host must be taken into

account39 ; (ii) work at higher concentrations of the host,

which may or may not be possible depending on its

solubility. On the other hand, when the binding is too

strong for the working concentration (Ka = 104 M1 ), each

addition of guest results in it being fully complexed. The

(41)

working concentration, which is often not possible with

NMR because of its relatively low sensitivity. Alternatively,

a less direct approach with larger attendant errors is to

perform a competition experiment in which a complex of

the host and a relatively weakly bound guest is titrated with

the stronger binding guest (see below).

Once a Ka value has been determined, this leads directly

(18) to the corresponding G of complexation, but to

ascertain the H and S values of a fast exchanging

system it is necessary to determine a vant Hoff plot by

repeating the titration procedure at different temperatures.

Note that, because measured Ka values in fast exchanging

systems have larger intrinsic errors than analogous determinations in slowly exchanging systems, errors in ascertaining

the H and S values via a vant Hoff plot can be quite

substantial.

In summary, the relative insensitivity of NMR spectroscopy means that the solution under study must be

relatively concentrated, a factor that attenuates the range

of binding constants that can be determined. In NMR

examinations of supramolecular events, the exchange process can be faster than, slower than, or on the NMR

timescale. Slow exchanging systems provide more structural information and allow a more straightforward and

accurate determination of Ka , G , H , and S than

0.20

d (ppm)

0.15

0.10

0.05

0.00

0

0.002

0.004

0.006

0.008

G t (M)

0.01

0.012

0.014

0.016

Figure 5 Three theoretical binding isotherms for 1 mM host titrated with guests of association constants, Ka = 50 ( ), 1000 ( ), and

104 M1 ( ).

Supramolecular Chemistry: From Molecules to Nanomaterials, Online 2012 John Wiley & Sons, Ltd.

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DOI: 10.1002/9780470661345.smc005

fast exchanging systems. Finally, it should be noted the sensitivity weakness of NMR is countered by the large amount

of structural information provided by the technique, particularly when the exchange process between free host and

guest and the hostguest complex is slow on the NMR

timescale.

which expresses the total observed absorbance as a function

of the respective molar absorptivities of the host, guest, and

complex at a given wavelength (H , G , and HG ) and their

respective concentrations:

Aobs = H [H ] + G [G] + HG [HG]

3.5

(43)

upon complex formation:

probably the most widely used techniques for thermodynamic data collection. Both require UVvis absorbance

(and in the case of fluorescence, emission) of the host

and/or the guest. It is also important that upon complex

formation the absorbance or emission change. Both analytical techniques can be approached in a very similar fashion:

because of its wider popularity, we emphasize here UVvis

spectroscopy.

In UV spectrometry, the excitation of electrons by

absorption is accompanied by changes in vibrational and

rotational quantum numbers. As a consequence, a broad

signal of vibrational and rotational fine structure is produced rather than a single absorption line corresponding

to a particular electronic transition. Furthermore, interactions between the solute and the solvent further complicate

the data and smooth this broad absorption band. As a result,

the amount of structural information provided by these techniques is less than that provided by NMR. One consequence

of broad absorption bands is that there is inevitably extensive band overlap from the different species in solution: a

fact that can sometime complicate data interpretation. Even

if this is not the case, data gathering must rely on titration

studies analogous to those described for the NMR analysis of fast exchanging systems; there is no direct analogy

to systems exchanging slowly in the NMR timescale even

though exchange is slow on the UVvis timescale. The reason for this is that, with UVvis determinations, it is signal

intensity that we are measuring as a function of hostguest

ratio and this amplitude is dependent on the unknown

of the solution. Hence, there are too many unknowns to

determine Ka values from a single mixture of the host and

the guest.

For a hostguest complexation event, there are potentially three species that can absorb and, because the

absorbance of each is additive, we can write (42):

= HG H G

(42)

and AHG are the respective absorbances of H , G, and HG.

(44)

Equation (44) actually pertains to a relatively rare system, because in most cases we select the absorption to

monitor so that one of the species does not absorb. In

rare cases where all species do absorb at the wavelength

examined, it is necessary to determine G and H in a

separate experiment to reduce the number of unknowns.

Hiroses excellent practical guide on binding constant

determinations describes the methods to treat the collected data (Aobs vs Gt ) of this more complex regression

analyses.39

For the majority of cases where only one species

absorbs at the observed wavelength (H or G = 0), we

can write simplified equations (45 and 46). In the case of

these equations, we assume it is the guest that does not

absorb:

Aobs = AH + AHG

(45)

= HG H

(46)

(45), we can express (47):

Aobs = H Ht + [HG]

(47)

A = Ka [H ][G]

(48)

obtain the binding isotherm (49):

A =

Aobs = AH + AG + AHG

15

Ka Ht [G]

1 + K[G]

(49)

NMR (39) and we can insert (49) into our expression of

[G] for 1 : 1 complexations (36) to give (50):

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DOI: 10.1002/9780470661345.smc005

16

Concepts

Aobs =

Ht

2

+1

Ka Gt Ka Ht 1 + (1 Ka Gt + Ka Ht )2 + 4Ka Gt

must iterate with the experimentally determined data. We

can see that we are dealing with an equation similar to that

obtained for NMR in fast exchanging systems (41). The

experimental data will in this case be A versus Gt (rather

than obs vs Gt ), but the fitting of the binding isotherm

is exactly the same except that now Ka and are iterated

to fit the curve.

Remember that we are dealing with changes in absorption

intensity rather than a shift in absorption (as we were with

NMR). As a result, we cannot change the host/guest ratio

simply by adding one to the other because this will change

the concentration of the host and hence the absorption

intensity. Rather, the experimentalist must prepare separate

solutions with increasing amounts of guest (or host) at a

constant concentration of host.

Although these determinations require more effort,

because we are typically working at lower concentrations

(about 0.01 mM), UVvis spectrometry allows the determination of thermodynamic data that cannot be measured

by NMR because the binding constant is too high. The

typical range of Ka that UVvis experiments cover is

11 106 M1 , but higher binding constants can be determined if the extinction coefficient of the molecules is

high. As an example, if = 3 105 , this would give an

absorbance of 0.3 at 1 M, allowing Ka values as high as

1 107 M1 to be ascertained. On the other hand, the determination of small binding constants by UVvis is facilitated by weakly absorbing species. Of course, the general

point that studies with UVvis require working at lower

concentrations also means that each set of experiments

require less host and guest: a fact that reduces pressure

on synthesis to form the molecules in question.

The study of binding by fluorescence spectrometry is

performed with the same experimental and data analyses as

UVvis spectrometry. Thus experiments usually involve

either a fluorescent host or a fluorescent guest, and the

change in fluorescence is monitored at different host/guest

ratios. The UVvis equations described above can easily be

adapted to fluorescence to provide the association constant

for a binding event.5

In summary, UVvis and fluorescence spectroscopies

do not provide as much structural detail of a formed

hostguest complex. They do, however, allow a wider

range of binding constants to be determined. In addition,

although each determination requires more practical work,

the amount of material required for a determination isall

(50)

experiments.

3.6

The development of sensitive isothermal titration calorimeters first impacted the biological sciences. However, over

the last decade or so the technique has become increasingly utilized within supramolecular chemistry. One of the

major reasons behind its proven popularity is the fact that

Ka , G , H , and S are obtained in a single automated experiment. In a typical ITC titration, small aliquots

of a concentrated solution of the guest are added to a solution of the host in the ITC cell. Upon each addition, a

measured amount of heat is given off, and this decreases

during the titration and reaches zero upon saturation of

the host. The total heat liberated in this titration yields

the enthalpy change, while the shape of the curve for

heat release as a function of host/guest ratio provides the

equilibrium constant and hence the free energy of binding. As a result, the entropy change for complexation can

be directly calculated. This more direct approach to garnering thermodynamic data has much smaller associated

errors, particularly for H , and avoids the issues associated with vant Hoff plots such as the possibility that H

varies with temperature, or whether the obtained data has

chemical roots or is simply artifactual. ITC also allows

a more direct and accurate determination of heat capacity

changes. For straightforward cases of 1 : 1 complex formation, there is a linear relationship between H and T ,

the gradient of which is the Cp associated with binding. Thus, a series of five or more experiments run at

different temperatures accurately yields any heat capacity

change.

An ITC titration yields an amount of heat released or

absorbed (Q) for each aliquot of added guest solution.

The sum of these heats (Q) can be defined by (51):

(51)

is the molar heat of the ligand binding (in cal mol1 ).

This expression is similar to that obtained by NMR (39)

and we can insert it into our expression of [G] for 1 : 1

complexations (36) to give (52):

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DOI: 10.1002/9780470661345.smc005

Q = Gt V0 H V0 H

(1 Ka Gt + Ka Ht )

versus molar ratio of host and guest using a spreadsheet

software that takes into account the change in volume in

the cell as the titrant is added. This plot is equivalent to

the binding isotherm of NMR or UV. Thus, the theoretical expression of Q (52) can be fitted to a number of

preset models of differing stoichiometry to generate Ka

(and hence G ) and H and calculate S .40 In addition to the goodness of the fit of the obtained curve, ITC

also generates the stoichiometry number (N), which, in a

1 : 1 complex, should be close to 1 (0.95 < N < 1.05). Any

major deviation from this, for example, N = 0.5, suggests

another binding stoichiometry, in which case the fitting

model should be reconsidered. If the stoichiometry of complexation can be confirmed by another method, then the

ITC instrument allows the N value to be numerically fixed,

which can reduce the error of the obtained thermodynamic

data, especially in weakly binding systems. If the stoichiometry is in doubt, it is often a good idea to confirm it by NMR

or another spectroscopic technique.

Ideally an S-shaped titration curve should be obtained.

If binding is too weak, then the obtained curve will tend

toward linear, whereas if it is too strong, a step will

instead be observed. The steepness in the titration curve

can be described by the Wiseman parameter c, defined as

c = Ka [X], where [X] is the concentration of titrate.40

It is often quoted that, in order to obtain an isotherm

corresponding to a smooth S-shaped curve and minimal

errors in both H and Ka (23%), the Wiseman

parameter should lie in the range of 100500. However,

most recently it has been shown that it is possible to obtain

accurate determinations of Ka with c values as low as

5.42

ITC is capable of determining a wide range of binding constants, typically between Ka = 5 and 109 M1 .

Of course, the spectroscopic character of the molecules

involved is not important in such experiments. Instead, the

ability to measure Ka is dictated by the extent that the

complexation liberates or absorbs heat. For the lower limit,

the closer a complexation is to being enthalpically neutral,

the larger the error in both H and Ka , and events that

are truly enthalpically neutral across a temperature range

cannot be measured. Such events are relatively rare, and

consequently the lower practical limit of ITC is more often

than not defined by insufficient solubility of the host and

guest, which limits the amount of heat generated and results

in a flat curve. The upper limit of Ka determination in ITC

(1 Ka Gt + Ka Ht )2 + 4Ka Gt

2Ka

17

(52)

so strong that a smooth S-curve in not obtained but rather a

step is observed, and this cannot be remedied by dilution

because insufficient heat is given out by complexation, then

although the H of complexation can be determined, the

obtained Ka will have an unacceptably large error. For c

values >500, the steepness of the curve is such that errors

in Ka ensue.

In summary, ITC is capable of determining a wider range

of binding constants than either NMR or UVvis spectrometry, and generally does so with much smaller errors

in Ka , G , H , S , and Cp : the only prerequisite

being that the binding event is not enthalpically neutral at

the temperature range being studied. One drawback of the

approach that has perhaps inhibited the growth of the technique in supramolecular chemistry is the relatively large

volume of the ITC cell (1.4 ml). However, recently sample requirements have decreased considerably, with most

recent instruments having a cell volume of only 200 l.

Automated ITC instruments can also be purchased, which

makes an in-house ITC a rapid and accurate means to determine thermodynamic data.

3.7

measurements give the same results?

event using multiple analytical techniques will not necessarily give the same thermodynamic data. Yes, they are

measuring the same complexation process, but their viewpoints are not the same. In ITC, for example, even if heats

of dilution or heats of protonation are taken into account

by the subtraction of data from reference titrations, what

is being measured is a global change in enthalpy. Thus,

the data obtained will include, for example, changes in

the solvation of the host and guest. In contrast, a technique such as NMR relies on measuring subtle changes

in electron distribution in and around one atom. This more

local viewpoint is sometimes apparent from thermodynamic

data generated by monitoring the shift of different protons in a host (or guest). In such instances, the obtained

binding constants may differ somewhat from proton to proton. The different viewpoints of the various techniques

noted, the obtained thermodynamic data should be similar.

If not, problems with the determinations must be examined

for.

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18

3.8

Concepts

stoichiometry?

3.9

summarized by (19), and the tacit assumption has been

that the stoichiometry of the binding event is known. For

binding events that are slow on the NMR timescale, it

is trivial to determine the ratio of host and guest in the

complex. However, in fast exchanging systems, and when

using UVvis spectrometry and ITC, we rely on fitting

programs to confirm the stoichiometry of complexation

from the binding isotherm. But what do we do if a poor

fit is obtained? Are we dealing with a 1 : 1 complexation

and poor data, or are we dealing with a higher order

process? One way for ascertaining the stoichiometry of

binding is to perform a so-called Job plot (also called

the continuous variation method). The theory behind, and

practice of, this approach has been described elsewhere,38, 39

so we only briefly discuss the practicalities of the method.

The approach is straightforward: a series of at least 10

experiments are carried out in which a metric proportional

to [HG]chemical shift, absorption, enzyme activityis

measured as a function of different ratios of host and guest

at constant concentration. The plot of this metric against

the mole fraction (of either host or guest) is hyperbolic in

the case of 1 : 1 complexation, with a maximum at the 1 : 1

ratio of the host and guest (Figure 6).

If a higher stoichiometry is under investigation, the

hyperbolic curve will be asymmetric, with a maximum

for the plot of the mole fraction of the host (xH )

at xH /[xH + xG ]. Thus for a 1 : 2 complex or a 2 : 1

complex, the maxima will be at 0.666 and 0.333,

respectively.

Arbitrary units

3E 05

2E 05

2E 05

1E 05

5E 06

Competition experiments

upper limit for determining a binding constant (104 , 106 ,

and 109 M1 for NMR, UVvis, and ITC, respectively).

What can be done if a host and guest are found to associate

more strongly than this limit? The strategy to overcome this

problem is to perform a competition experiment, whereby

the guest that binds too strongly to the host (GA ) is titrated

to a complex between the host and a more weakly binding

guest (GB ) of known association constant (Ka(B) ). Such a

titration will allow an apparent reduction of the binding

constant of GA (Ka(A) ). We can write (53) and (54):

H + GA

ka(A)

HGA

(53)

H + GB

ka(B)

HGB

(54)

where Ka(A) > Ka(B) and the latter is known. The mass balance equations then become: Ht = [H ] + [HGA ] + [HGB ],

GAt = [GA ] + [HGA ], and GBt = [GB ] + [HGB ], where Ht

is the total host and GAt and GBt are the totals of GA

and GB , respectively. If we take the example of slow

exchanging system by NMR, the determination of Ka(A)

is accomplished using these mass balance equations, the

relative integration of HGA and HGB , and the following

equation (55):

[HGA ][GB ]

Ka(A)

=

Ka(B)

[HGB ][GA ]

(55)

from a competition experiment utilizing a fast exchanging

process in NMR or UVvis spectroscopy or ITC is

considerably more involved. These have been extensively

described in the literature,43, 44 particularly in regard to

the use of displacement assays for the determination of

association constants of spectroscopically silent guests.45

As a rule of thumb, competition experiments will extend

the upper binding constant limit of a technique by at

most 3 orders of magnitude. Put another way, they define

the upper limits of NMR, UVvis, and ITC respectively

to 107 , 109 , and 1012 M1 , respectively. The down side

of competition experiments is that the errors associated

with the determination of the weaker binding guest are

propagated into the competition experiment such that errors

in the determination of the larger association constant are

typically at least 1015% (and often higher).

0E + 00

0.0

0.2

0.4

0.6

Mole ratio

0.8

1.0

Figure 6 Continuous variation method (Job Plot) for a hypothetical 1 : 1 complex formation.

3.10

involving a host and two guests, are also frequently

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DOI: 10.1002/9780470661345.smc005

encountered in supramolecular chemistry. Assuming one is

interested in the microscopic (individual) binding events

rather than the macroscopic (overall) binding process,

these systems are intrinsically more complicated than

1 : 1 binding systems: not only because of the higher

stoichiometries but also because there are variations within

each system of defined stoichiometry. For example, in a 1 : 2

hostguest system the two binding sites may be the same or

different, may be independent of each other or coupled (the

system possesses cooperativity), and may bind their guest

at the same time or sequentially. This means that there is

no standard mathematical model for each binding/assembly

of defined stoichiometry, but rather models have to be

customized for each variation. The result is a considerable

broadening of the range of base mathematical models which

is beyond the scope of this introduction. Nevertheless, it is

instructive to consider some of the general principles.

In the 1 : 1 binding systems, we devised a base mathematical model (36) that gave an exact solution to the association

constant determination. Such a mathematical model is often

called a closed-solution approach. There is in fact an alternative to this, and that is to carry out a open-form approach.

We have not mentioned this up until this point because

the mathematics of the 1 : 1 complexation is simple enough

that an exact solution can be readily sought. In the case of

higher stoichiometries, however, an exact (closed) solution

is often difficult or impossible. As we discuss below, the

only option therefore is to take an open approach to such

systems.

Let us briefly consider stoichiometry of higher order

systems. Not unexpectedly, the overarching theme here is

that, the greater the number of entities involved in the

final chemical entity, the more complicated the mathematics

needed to describe the system in question. Let us begin by

expanding (19) to give a general equilibrium equation for

higher order assembly:

mH + nG Hm Gn

(56)

balance equations are: Ht = [H ] + m[Hm Gn ] and Gt =

[G] + n[Hm Gn ], where Ht and Gt are respectively the total

host and guest present in solution. It is possible to again

obtain an expression of free guest ([G]) such as was carried

out for the 1 : 1 complexes (36), but as the stoichiometry

increases, the order of the polynomial generally increases as

well. For example, in most ternary hostguest systems, the

base mathematical model, or closed-form expression of [G],

takes the form of a cubic equation (general form: f (x) =

ax3 + bx2 + cx + d), whereas a binding event involving

four species generally takes the form of a quartic function

(general form: f (x) = ax4 + bx3 + cx2 + dx + e). Hence,

19

even for a simple ternary complex, the exact (or closedform) solution of the required cubic equation is relatively

involved.

As just mentioned, in a host with two binding sites, the

nature of the binding sites, whether there is cooperativity

in binding, and whether there is an order to guest complexation, all modify the base mathematical model. The nature

of the recognition sites is straightforward; they can either

be identical or different. Regarding cooperativity in the system, there are three possibilities: (i) the two binding pockets

are independent of one another and there is no cooperativity; (ii) the net free energy change of binding for the

overall process is more negative than the sum of the individual free energy changes arising from binding each single

guest: in other words the system displays positive cooperativity; (iii) the system is negatively cooperative system,

that is, the net free energy change for the overall process

is less negative than the sum of the free energy changes

of the individual binding events. Finally, binding can occur

randomly or sequentially. The simplest combination of all

these variations is where the two binding sites are identical and independent of each other, in which case the exact

solution base model simplifies to a quadratic equation. If

the binding sites are different, and/or there is cooperativity

involved, and/or there is an order to complexation, then the

base mathematical models are generally cubic equations. As

with 1 : 1 complexations, the next step after a base model

has been chosen is to modify it in order for the model to fit

the chosen analytical technique. Software that models the

different systems arising from combinations of these factors is available in modern ITC instruments, and a number

of researchers have provided their own in-house software

for those techniques such as NMR that are not specifically

designed for binding constant determinations.46, 47

As alluded to above, an alternative to closed solutions of

higher polynomials is to use an open, or iterative, approach.

This more general strategy to parsing out the thermodynamic data for each microscopic binding event within

higher order systems requires that each equilibrium constant expression and corresponding mass balance equation

be identified. From this, the concentration of each species at

set Ka values can be formulated by iteration, and this process layered on top of the normal iteration process that

fits the species distribution to the experimentally obtained

data.48 The advantage of an open solution approach is that

it can be readily expanded for higher stoichiometric systems that are too difficult, if not impossible, to analyze

via closed-form solutions. It is worth noting that many

researchers make available in-house software for these calculations.4951 A significant disadvantage of this approach,

however, is that this dual iterative approach requires an

initial estimate of the association constants at each microscopic step; and the more complicated the system is, the

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DOI: 10.1002/9780470661345.smc005

20

Concepts

more the accuracy required for these estimations. The reason for this increased accuracy requirement is that different

initial Ka estimates can lead to different final Ka values. It

is therefore prudent to begin with a good estimate of the

Ka values and make sure that the final Ka values represent

a global minimum by changing the starting point to see

if this affects the outcome. It is also prudent to use chemical intuition when considering the final data. For example,

the garnering of four association constants from a binding isotherm possessing only one inflection point should be

treated with considerable suspicion. Likewise, if the magnitude of the obtained data seems overtly high or low relative

to published data, the practitioner should be cautious.

Of the numerous practical issues that can arise in

studying higher order systems, perhaps the most important

is the strength of cooperativity. The degree or strength of

cooperativity can be determined using the Hill equation.12

For practical purposes, however, we simply need to be

mindful of cases where binding is strongly cooperative

because this will affect our ability to observe selected

intermediates. For example, if it is strongly positively

cooperative (Ka2 Ka1 ), then the addition of host to guest

may not allow the observation of the intermediate HG

because the excess guest present will promote the full

conversion of HG to HG2 . In cases where exchange is

slow on the NMR timescale, this would mean that no

peaks corresponding to HG would be apparent; whereas

if exchange is fast on the NMR timescale (or we are

using UVvis or ITC), no inflection point corresponding

to this complex would be apparent in the binding isotherm.

If this is the case, then only the overall (macroscopic)

thermodynamic data can be obtained. One possible way to

obtain the microscopic data is to perform a reverse titration

of guest into host, but only if the positive cooperativity is

not too strong. Similarly, if strong negative cooperativity is

observed, it may not be possible to observe the formation

of HG2 even if the host is titrated into excess guest. These

influences of cooperativity mean that it is often instructive

to perform both a forward and a reverse titration when

studying higher order systems.49

CONCLUSION

aspects of determining the thermodynamic profiles of 1 : 1

hostguest complexations. The determination of binding

constants, the change in free energy, the change in enthalpy,

and the change in entropy for complexation processes has

had a profound influence on our understanding of how

structure and solvent effects influence intermolecular reactions. Quantifying these thermodynamic parameters has

also allowed researchers to identify previously underappreciated noncovalent forces, and how they can play a role in

the properties of molecules. As a result, en masse, these

studies have been responsible for defining and uniting the

field of supramolecular chemistry. As the field moves forward and begins to address new challenges of a second

phase, it is likely that binding constant determinations will

continue to play a significant role. That role may change,

but the need to quantify thermodynamic parameters of association is of such importance in both synthetic and biological contexts that a good grounding will always be essential.

With that in mind, we hope that readers have found this

introduction to the topic useful.

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DOI: 10.1002/9780470661345.smc005

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