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Binding Constants and Their Measurement

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You are on page 1of 36

Pall Thordarson

The University of New South Wales, Sydney, Australia

1 Introduction

2 Equations, Equilibria, and Experimental

Techniques

3 Determining Stoichiometry

4 Data Analysis and Software

5 Conclusions

Acknowledgments

References

1

1

2

26

28

34

35

35

INTRODUCTION

**In supramolecular chemistry, usually the first question that a
**

researcher might want to ask is: “how strong is this complex

or interaction?” In other words, what is the free energy

(G) associated with this interactions. In supramolecular

chemistry, the most straightforward way of measuring this

free energy difference is through the equilibrium constant

(K) for the system of interest since:

G = −RT ln K

Binding constants are a special case of equilibrium constants such as acid–base equilibrium constants (best known

as pKa and pKb ) and solubility equilibrium constants (Ks ).

Binding constants measure the bonding affinity between

two or more molecules at equilibrium. In supramolecular

chemistry, binding constants for host–guest complexation

or host–host aggregation (e.g., dimerization) are usually

**the subject of interest. Binding constants are also used in
**

biochemistry to describe the affinity of an enzyme to a

substrate and in pharmacology to describe the affinity of a

ligand (drug) to a receptor. Researchers from all these disciplines therefore need to deal with the same challenges but

using different terminology and approaches—a fact that

can be the source of endless frustration and mistakes to

newcomers. There are many excellent textbooks, reviews,

and software packages available for use in biochemistry

and pharmacology, showing how binding constants can be

obtained. These are extremely useful sources of information but need to be treated carefully in the supramolecular

chemistry context. In biochemistry, it is frequently possible

to determine directly the concentration of the complex of

interest, for example, by electrophoreses or filter-binding

assays,1 simplifying the data treatment considerably. This

is usually not possible in supramolecular chemistry where

methods such as NMR or UV–vis are used to indirectly

detect complexation. Another problem in this field is that

the fundamental work was done at a time when computational power was scarce or nonexisting, which meant that

researchers often had to make shortcuts or rely on a statistically questionable approach such as linear regression (e.g.,

Lineweaver–Burk) for what are really nonlinear problems.

With the power of modern day computers and software

packages, there is absolutely no reason for anyone to use

these outdated methods,2 yet they propagate in the literature adding another layer of confusion to newcomers to the

field.

This chapter covers the fundamentals of determining

binding constants in supramolecular chemistry. Rather than

trying to cover all the different methods that have been

applied to determine binding constants in supramolecular

chemistry, the emphasis is on the basic equations used.

Illustrative examples focus on the most important methods used in determining binding constants including NMR,

**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.smc018

2

Techniques

UV–vis, fluorescence, and calorimetric titrations. It is probably safe to say that over 90% of all experimentally determined binding constants in supramolecular chemistry are

now determined using one of these four techniques. Other

older or more specialized techniques such as solubility

methods,3, 4 potentiometric,3–5 and mass spectrometry5 have

been discussed in earlier reviews and are not covered here.

It should, however, be noted that the basic equations shown

here can usually also be readily adapted to fit these older

or more specialized techniques.

2.1

2

**The choice of “host” and “guest” is arbitrary but in
**

supramolecular chemistry the host is usually the larger

molecule (e.g., crown ether) and the guest the smaller

molecule or ion (e.g., potassium). Receptor (usually host),

substrate (usually guest), and ligand (guest) are other definitions that are frequently found in the supramolecular

chemistry literature, some having been borrowed for biochemistry or pharmacology as discussed above.

When m = 1, for each step of the equilibrium process

shown in Figure 1, we can define stepwise equilibrium

binding constants Km according to (1).

**If we consider the formation of a complex in solution
**

between two species, we can define:

Host = H with the concentration of the free host = [H]

Guest = G with the concentration of the free guest = [G]

Complex = Hm Gn with the concentration of the complex

= [Hm Gn ] and n, m = 1, 2, 3, . . .

**EQUATIONS, EQUILIBRIA, AND
**

EXPERIMENTAL TECHNIQUES

**Irrespective of the method used, the key step in determining
**

binding constants involves defining some sort of a model

that relates to the underlying equilibria. The model(s) is

then compared to the data obtained. If the data and model

are in reasonably good agreement, data analysis (fitting)

can be used to extract valuable information, including the

association constant (Ka ). Depending on the situation, this

process can also be used to verify if the model is truly

reasonable for the data obtained from the system under

investigation. Note, however, that just because there is a

good fit between data and a model, it does not automatically

imply that this particular model is the best one for that

system.2 Conversely, one cannot use a poor agreement

between data and a model as the sole criteria to reject

a model, especially if the raw data is of poor quality.

The following section describes some of the most common

binding models and data analysis strategies encountered in

supramolecular chemistry.

The general m:n system

mH + nG

bmn

HmGn

**The simple 1 : 1 system
**

H+G

Ka

HG + G

(a)

(b)

Kn =

Kn =

K1

K2

The 2 : 1 system

H+G

HG

HG + H

HG2

(c)

A+A

A2 + A

K3

A3 + A

K4

A4 + A

K5

A5 + A

K1

K2

HG

H2G

(d)

**The general aggregation system
**

K2

[HGn ]

[HGn−1 ][G]

(1)

**The stepwise equilibrium constants are also related to
**

the kn = forward and k−n = the backward rate of kinetics

of complexation according to (2).

The 1 : 2 system

H+G

HG

Basic definitions

K6

Ki

...

Ai

kn

k−n

(2)

The 2 : 2 system

2H + 2G

H2G2 + 2G

KF

KB

H2G2

2HG2

(e)

Dimerization model:

KD = K2, K3, K4, K5...Ki = 0

EK-aggregation model:

KE = K2 = K3 = K4 = K5... = Ki

**coEK-aggregation model: KE = K2/ρ = K3 = K4 = K5... = Ki
**

with r = K2/KE

(f)

Figure 1 The different equilibria discussed in this chapter. (a) The general m/n binding model. (b) The 1 : 1 equilibria. (c) The 1 : 2

equilibria. (d) The 2 : 1 equilibria. (e) The 2 : 2 equilibria. (f) The general aggregation system, including dimerization, equal-K linear

aggregation (EK model), and cooperative equal-K linear aggregation (coEK model).

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.smc018

**Binding constants and their measurement
**

We can also define the overall stability constant = β mn

according to (3).

β mn

[Hm Gn ]

=

[H]m [G]n

(3)

**As discussed below, these quantities can be measured
**

using a range of techniques including spectroscopic and

thermodynamic ones. Binding constants, similar to most

thermodynamic and kinetic properties, are highly dependent

on the solvent and temperature conditions. Even a change

from nondeuterated to deuterated solvent may give slightly

different outcomes. Given the sensitivity of thermodynamic

quantities to temperatures, it is essential to control and

record the temperature of any experiments used to determine binding constants, otherwise the results will be of

little value.

It should be noted that, strictly speaking, one should

also include activity coefficients in (1) and (3) to account

for deviations from ideal behavior in a mixture. This is

usually not done in practice—the usual assumption used

to justify this is that activity coefficients have a negligible

effect in the dilute regime used in typical supramolecular

chemistry experiments. Notable exceptions are experiments

conducted in aqueous buffer or salt solutions where activity

coefficients may have a significant effect on the final

outcome.6

It should also be noted that comparing binding constants that have different units (e.g., M−1 vs M−2 ) can be

fraught with complications as one then needs to consider

the reference concentrations of the reactants.7 The reason

behind this can be traced back to the fact that the fundamental relation between free energy (G) and the equilibrium constant (K) : G =

−RT ln K should be written

as G = −RT ln K + RT

ν ln c, were ν ln c represents

the reference concentrations of the reactants and products.8

Usually, the reference concentration is taken

as 1 for the

unit used (e.g., M), rendering the term +RT

ν ln c zero

but using such an arbitrary assigned value (e.g., 1 M) as

the reference state complicates the analysis of many binding phenomena, including the “chelate” effect and effective molarities. This has resulted in a lively debate to

date,7–11 on what the chelate effect and effective molarities mean, but this topic is outside the scope of this chapter

(The Thermodynamics of Molecular Recognition, Concepts).

The most important specific cases of the equilibria shown

in Figure 1(a) are discussed in detail below; n = m = 1

(Figure 1b, 1 : 1 equilibria), n = 1, m = 2 (Figure 1c, 1 : 2

equilibria), n = 2, m = 1 (Figure 1d, 2 : 1 equilibria), and

n = m = 2 (Figure 1e, 2 : 2 equilibria) as well as three

selected examples of self-aggregation (Figure 1f).

2.2

3

The 1 : 1 equilibria

**The 1 : 1 equilibria is perhaps the most commonly observed
**

and studied equilibria in supramolecular chemistry. The

binding constant or association constant = Ka for these

equilibria is defined by (4) which is just a special case

of (1).

Ka =

[HG]

[H][G]

(4)

**Here Ka = K1 in (1) but the former notion is chosen
**

to distinguish the 1 : 1 binding constant from the stepwise

binding constant K1 in the 1 : 2 and more complex equilibria

discussed below.

Measuring Ka according to (4) requires knowledge of at

least one of quantities [HG], [H], and [G]. This may sound

simple but it needs to be remembered that these are free

concentrations of these species in solution and generally

their concentration cannot be measured directly. The total

concentration of the host = [H]0 and the guest = [G]0 ,

however, are usually known and relate to [H], [G], and

[HG] with the mass balance (5) and (6).

[H]0 = [H] + [HG]

(5)

[G]0 = [G] + [HG]

(6)

**Taking (5) and (6) and isolating for [H] and [G] and
**

inserting into (4), then gives us (7).

[HG]

([H]0 − [HG])([G]0 − [HG])

[HG]

=

[H]0 [G]0 − [HG]([H]0 + [G]0 ) + [HG]2

Ka =

(7)

**Isolating for [HG] and rearranging (7) gives us the
**

quadratic (8).

1

[HG] − [HG] [G]0 + [H]0 +

Ka

2

+ [H]0 [G]0 = 0 (8)

**The real solution of (8) then gives us [HG] according
**

to (9).

1

[HG] =

2

−

1

[G]0 + [H]0 +

Ka

1

[G]0 + [H]0 +

Ka

2

− 4[H]0 [G]0

(9)

**The power of (9) cannot be overstated as the only
**

unknown quantity here is the binding constant Ka —the

**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.smc018

Ka and YHG . we finally obtain (17). the observed physical change is a function of the mole fractions = f of the host = fH . that is. Y = YH fH + YG fG + YHG fHG Rearrangement of (14) gives a quadratic equation that has one real relevant solution in the form of (15). while increasing [G]0 . The most commonly used method in supramolecular chemistry is the titration method. such as in the case of UV–vis spectroscopy. we can simplify (10) considerably using also [H] = [H]0 − [HG] from (5) to obtain (16). Using a variety of software packages or programs. Online 2012 John Wiley & Sons. such as for NMR. [G]0 + [H]0 + 1 Ka 2 1 Ka (18) + 4[H]0 [G]0 To obtain the two unknown variables. a titration of some sort is usually carried out. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. In some situation. The physical property of interest can be anything from an NMR resonance = δ to absorbance in UV–vis spectroscopy = A or heat absorbed/released in calorimetry = Q. as discussed further below. and [HG] in solution. and Ka . [HG] = 1 [G] = 2 (10) In other cases. but rather mole fraction. the problem with measuring [HG] directly still remains. Ltd. To address this issue.4 Techniques key quantity of interest! However. Further. The physical change of interest can usually be described as the aggregate of the physical property for the host = YH . while the concentration of the host [H]0 is kept fixed. Y = YH ([H]0 − [HG]) + YHG ([HG]) = Y0 + (YHG − YH )[HG] (16) Moving Y0 to the left and defining Y = Y − YH and YHG = YHG − YH . This article is 2012 John Wiley & Sons. keeping [H]0 constant. guest = YG . Y = YHG 1 2 [G]0 + [H]0 + − (13) (12) Equation (12) is known as the general binding isotherm for a simple 1 : 1 equilibria. If it is a function of the concentration of [H]. [G]0.smc018 (19) . approaches to monitor changes in [HG] indirectly have been developed. Y = YHG ([HG]) (17) Equations (9) and (17) can now be combined to give (18). if we recognize that Y0 = [H]0 YH . fHG = Ka [G] [HG] = [H] + [HG] 1 + Ka [G] [H]0 Ka [G] 1 + Ka [G] Expanding on (6) with (13) then yields (14). It shows that the mole fraction of the complex follows a hyperbolic relation3 with the free guest [G] concentration. Equation (12) can also be used as a starting point to obtain an expression for [G] that only depends on [H]0 . dependent with the mole fraction fx depends on the total concentration of species X = [X]0 according to (19).1002/9780470661345. Y = YH [H] + YG [G] + YHG [HG] (11) The mole fraction notion is of special interest as (4) can be used to define the mole fraction of the complex according to (12). and complex = fHG according to (11). the physical property is not concentration. the observed physical property can be described by (10). Starting from the fact that we can also write fHG = [HG]/[H]0 or [HG] = fHG [H]0 and combine with (12) to obtain (13). YG = 0—for instance in UV–vis titrations with a simple noncolored monoatomic ion such as chloride (Cl− ). [G]. guest = fG . which describes the experimentally observed change in a physical property (Y ) as a function of two known ([H]0 and [G]0 ) variables and two unknowns (Ka and YHG ). NMR. Ltd. for example. Equation (18) can be modified for any method where the physical property of interest is depended on concentration. fX = [X] [X]0 Supramolecular Chemistry: From Molecules to Nanomaterials. and complex Y = YHG and their abundance in solution.2 Here changes in a physical property = Y of the system are monitored on addition of the guest [G]0 . DOI: 10. [G]0 = [G] + [H]0 Ka [G] 1 + Ka [G] (14) [G]0 − [H]0 − 1 Ka [G]0 − [H]0 + + 1 Ka 2 [G]0 +4 (15) Ka We now first consider the situation where the guest is “silent”. it is then possible to fit the resulting binding isotherm to (18) by nonlinear regression methods to obtain the best estimates for Ka and YHG . Ltd.

we can define the change in physical property—the change in resonance as δ = δ − δ 0 . 2. UV–vis spectroscopy). we can also define the change in resonance for the host–guest complexation as δ HG = δ HG − δ H . and coworkers on DDDAAA hydrogen-bonded arrays formed among compounds 2. If we define the NMR resonance for the host as δ H . YG can be determined independently. We can now write the NMR version of our simple 1 : 1 equilibria according to (26). there are two ways in which one can obtain binding constants from NMR experiments and these depend on whether the system can be described as being in slow or fast exchange on the NMR timescale. Ltd.) and availability being the most important ones (see also NMR Spectroscopy in Solution. To illustrate this.2 that are dependent on the relative mole fractions of species of interest. 6. etc. nOe-contacts. to deal with 5 but this does not complicate the fitting process significantly.. as the molar absorptivity of the free guest = εG and the resulting value used in (25) to simplify the fitting process. Alternatively. Ka and YHG . [HG] (21) Y = YHG [H]0 Combining this with (9) then gives us (22).5 This implies that we can immediately apply equations such as those discussed in Section 2. and the host–guest complex as δ HG .2. Equations (18). DOI: 10. 1 1 Y = YG [G]0 + YHG [G]0 + [H]0 + 2 Ka 1 2 + 4[H]0 [G]0 (25) − [G]0 +[H]0 + Ka Equation (25) can then be treated exactly as (18) except we now have one more unknown variable. δ HG 1 1 δ = [G]0 + [H]0 + [H]0 2 Ka 2 1 + 4[H]0 [G]0 (26) − [G]0 + [H]0 + Ka Recent work by Leigh. and 8 in Figure 2(a) illustrates the application of (26) to determine the relevant association constants from the NMR-binding isotherms shown in Figure 2(b). 1 YHG 1 Y = [G]0 + [H]0 + [H]0 2 Ka 1 2 + 4[H]0 [G]0 (22) − [G]0 + [H]0 + Ka Equation (22) is very similar to (18) and the two unknown variables. Online 2012 John Wiley & Sons. The most commonly used approach assumes that the species of interest are in fast exchange. Y = YG [G]0 + YHG ([HG]) (24) This equation then yields (25) after substitution of [HG] with the right-hand side of (9). where. we rearrange (20) and define Y = Y − YH and YHG = YHG − YH to obtain (21). YG . Several factors contribute to the popularity of NMR. fluorescence.1002/9780470661345. and (25) can be modified for almost any form of a titration or a similar study where the observed physical changes are proportional to the change in concentration or mole fraction of the 1 : 1 complex.smc018 .12 Supramolecular Chemistry: From Molecules to Nanomaterials. can be obtained in a similar manner by titration and fitting the results data to (22) using a nonlinear regression program. the guest as δ G . (22).g.1 NMR measurements of 1 : 1 equilibria The most important technique for determining binding constants in modern supramolecular chemistry is undoubtedly NMR. In essence. 5. for example. Y = YH ([H]0 − [HG]) + YG ([G]0 − [HG]) + YHG ([HG]) = Y0 + YG [G]0 + (YHG − YH − YG )[HG] (23) Here we again used Y0 = [H]0 YH but now we define YHG = YHG − YH − YG and as before Y = Y − YH to obtain (24). the quality of information regarding the host–guest interactions (shifts in spectra. This article is 2012 John Wiley & Sons. Ltd. we start again from (10) using the same approach as with (16) to obtain (23). the application of these equations in NMR. and calorimetry is detailed below. UV–vis. If we then define δ 0 = NMR resonance of the host before the guest is added (before the start of titration).Binding constants and their measurement Starting from (11) and (19) and assuming YG is “silent. particularly 1 H NMR. [H]0 − [HG] [HG] Y = YH + YHG [H]0 [H]0 [HG] (20) = YH + (YHG − YH ) [H]0 As before. What happens if the guest is not “silent” (YG = 0)? As this is only going to be likely in cases where the physical property is concentration dependent (e. Ltd. Techniques).” we use [H] = [H]0 − [HG] again from (5) to obtain (20). the observed NMR resonance (δ) is the weighted average of the bound (HG) and unbound host (H) species. 7. McNab. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. in the case of host–guest equilibria.

6 2.4 × 104 M−1 (CDCl3. The lines indicate best-fitting Ka s for 5·2 (red). equally importantly. from the sensitivity of modern NMR spectrometers but measurements can now routinely be performed on samples with [H]0 < 10−4 M and. 8·2.) For systems in true slow exchange. equilibria monitored by UV–vis is always in the slow exchange region as the timescale for UV–vis transitions (<10−12 s) is much smaller than the lifetime of the complex(es) of interest. Concepts). The former restriction arises. one could expect up to 35–75% uncertainty in the Ka value obtained.6 OEt N H O N H 2 H N N 5·2 O H N O H N H 6·7 0. 15 If the dissociation constant is fairly large (in other words. large excesses of guest may be required to obtain useful binding data. Online 2012 John Wiley & Sons. 8·2 (blue).3 M−1 .13 This situation is. This implies that Supramolecular Chemistry: From Molecules to Nanomaterials. Wayland and coworkers recently used this approach to determine the binding constant for methanol to a tetramesityl rhodium(II) porphyrin. 2. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons.2. Given what was said above. and 6·7.12 (Reproduced with permission from Ref.Techniques NO2 NO2 OEt H DDD AA(A) O H CH3 OEt H OEt O N H N H N N H H 2 N O H N H 0. and 6·7 (green). 1 H NMR titration analyses performed in CDCl3 using the change in chemical shift (δ) of the amino NH2 groups of 2 (10−3 M) upon addition of 5 or 8 and the hydroxyl groups of 7 (10−3 M) upon addition of 6. it is possible to observe and measure the relative ratios of the free [H] and bound host–guest species [HG] by integration directly and then use the mass balance (5) and (6) and then (4) to determine the binding constant Ka . For the purpose of determining binding constants.0 O 0. For instance.smc018 . and (b) binding isotherms of receptor pairs 5·2.2 N 8 5 8·2 0. NMR works well provided (i) the concentration of the host solution is within one or two orders of magnitude from the millimolar (mM) region and (ii) the association constants of interest does not greatly exceed Ka = 105 M−1 . relatively rare as complications arising in the slow-to-intermediate exchange region complicate the analysis14 along with the inherent difficulties in obtaining accurate quantitative integration from NMR spectra. UV–vis spectroscopy is probably the second most important method for determining binding constants.2. association constants. with Ka = 3.4 7 O ∆d 6 6 N 0. This article is 2012 John Wiley & Sons.1 M). assumptions regarding fast exchange may break down once Ka > 105 M−1 . such as porphyrins. simulations have also shown that once the dissociation constant is more than two orders of magnitudes smaller than the host concentration.2 The main limitations of UV–vis spectroscopy include the need for a chromophore and that UV–vis titrations need to be carried out within a concentration range where the absorbance follows the Beer–Lambert law (A < 1). Additionally. the uncertainty from fitting the data becomes too high to yield any valuable information. Ltd. 2009. 12. In contrast to NMR. provided they are nonabsorbing. Ltd. 1H NMR) (CDCl3. It has the advantage of being cheap. As an example. however. DOI: 10.2 UV–vis measurements of 1 : 1 equilibria After NMR.4 Figure 2 (a) Structures. it is possible to use concentrations in the submicromolar region.4 0. the association constant is very small). Ltd. on the other. however.0 2. With chromophores.8 1.14 Competition experiments16 can circumvent this limitation but this approach is outside the scope of this chapter (Competition Experiments. 1H NMR) (b) 0. it has been noted that ideally the host concentration in supramolecular experiments should be as close as possible to the dissociation constant Kd = 1/Ka . on the one hand.1002/9780470661345. American Chemical Society.0 (a) Ka = 8 × 104 M−1 Ka = 6 × 104 M−1 Ka = 2. simple.2 [A]/[D] 1. this implies that binding constants up to Ka = 108 –109 M−1 can be accurately determined by UV–vis spectroscopy. from practical NMR issues with highly concentrated solutions (>0. which is the inverse of the association constant.2 This is one reason why Ka 105 M−1 obtained by NMR is unreliable. 1H NMR) (CDCl3. More importantly. with a [H]0 = 10−4 M (dilute NMR) and the “real” Ka = 106 M−1 (Kd = 10−6 M). and relatively insensitive to minor impurities.

the work of Sessler and coworkers on a tetrathiafulvalene diindolylquinoxaline receptor that binds to anions such as dihydrogen phosphate (Figure 3). Ltd.17 NH N NH N S S Ka 1 1 [G]0 + [H]0 + A = ε HG 2 Ka 2 1 − + 4[H]0 [G]0 (27) [G]0 + [H]0 + Ka S the observed absorption spectra are a sum of the spectra of the species in solution. We now recognize that the change in absorbance depends on two factors. For a recent example. lmax ~ 470 nm N NH + NH N S 1 Ka S [G]0 + [H]0 + + 4[H]0 [G]0 (28) S − 2 S S 1 1 A = εG [G]0 + ε HG [G]0 + [H]0 + 2 Ka S There is a myriad of examples in the literature where UV–vis titrations are used to determine binding constants in 1 : 1 equilibria systems. H2PO4− Binding constants and their measurement Supramolecular Chemistry: From Molecules to Nanomaterials. Figure 3 The binding strength of tetrathiafulvalene diindolylquinoxaline to phosphate was determined by a UV–vis titration by monitoring the gradual increase in absorbance at around 480 nm.17 If the guest is also absorbed at the wavelength of interest to us. the observed absorbance (A) is therefore the sum of the absorbance of the host. see for instance. the physical change we observe is the change in absorbance (A). we see that (18) and (24) could be applied to describe the change in absorbance if we define the change in molar absorptivity as ε HG = ε HG − ε H .lmax ~ 480 nm S S Ka = 6500 M−1 (as the TBA salt in CH2Cl2) S H2PO4− The molar absorptivity of the guest (εG ) can either determine independently in a separate experiments or it can be added to the list of unknown parameters (Ka and ε HG ) that will be obtained from the fitting process.smc018 7 . At a given wavelength. Ltd. defined as the difference between the observed (Aobs ) and initial (AH0 ) absorbance of the host or A = Aobs − AH0 . we obtain (27). Depending on whether the guest is nonabsorbing (silent) or not. DOI: 10. Online 2012 John Wiley & Sons. If we titrate a solution of a host with a guest solution. When the guest is nonabsorbing. the guest. the concentration and the molar absorptivity of species in solution. we get (28). This article is 2012 John Wiley & Sons. where ε HG and ε H are the molar absorptivities of the complex and the host. respectively. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. and the complex. Each of these in turn depends on the relative molar absorptivity (ε) of these species as well as the concentration (c) and cell path length (b) according to the Beer–Lamberts law (A = bcε).1002/9780470661345. Ltd.

both the guest and the host are also nonfluorescent and only the complex is fluorescent—in other words. If it is quenched. In fact.05). if the guest completely quenches the host fluorescence and dynamic quenching does not play a role.2. path length. we could simplify (29) to give us (31). we define the change in proportionality constant (kHG ) as the difference between the proportionality constants of the complex (kHG ) and host (kH ) or kHG = kHG − kH . dynamic (collisional) and static quenching. molar absorptivity. we need to modify this analysis.18 The application of the general binding equations above is complicated by the fact that one needs to consider whether the fluorescence is quenched or not on addition of a guest. If we wanted to describe static quenching or binding in the same way. as a function of the guest/quencher concentration and the fluorescence intensity ratio F0 /Fobs . and quantum yield of the species of interest. the fluorescence follows a Beer–Lamberts-type relation with the observed fluorescence F = kx [X] with kx = the proportionality constant for species X but kx in turn depends on the incoming light. where only the latter has any real significance in supramolecular chemistry. Techniques) is perhaps the most sensitive of all the methods used to determine binding constants.3 Fluorescence measurements of 1 : 1 equilibria Fluorescence spectroscopy (see also Luminescent Spectroscopy in Supramolecular Chemistry. allowing binding constants in excess of 109 M−1 to be determined with good accuracy. it is essential that the host concentration is kept low in fluorescence spectroscopy titrations as fluorescence response is only linear to the concentration provided that the absorption of the species at the wavelength used for excitation is low (A < 0.4 . two possible types of quenching are possible. A range of fluorescent hosts allow the detection of binding in the submicromolar concentration range. fluorescence is “turned on” by addition of the guest to form a fluorescent complex. [Q] = free concentration of the quencher. We shall first consider the situation when there is no dynamic quenching and where the change in fluorescence (F ) on addition of a guest can be described as the difference between the observed (Fobs ) and initial (F0 ) fluorescence (F = Fobs − F0 ). 1 1 [G]0 + [H]0 + Fobs = kH 2 Ka 1 2 − + 4[H]0 [G]0 (32) [G]0 + [H]0 + Ka However. which states that the fluorescence intensity ratio F0 /Fobs = 1 + KSV [Q] where KSV is the Stern–Volmer constant and as always. We start by noting that pure dynamic quenching is usually described by the Stern–Volmer relation. we obtain (30). on the other hand.8 Techniques 2.2. we would get (32).2 1 1 [G]0 + [H]0 + F = kHG 2 Ka − 1 [G]0 + [H]0 + Ka 2 + 4[H]0 [G]0 (29) When the guest is also fluorescent (and there is no dynamic quenching). We now obtain (29). 18 In this region. that is. F0 = 1 + Ka [G] Fobs (33) Inserting [G] from (20) into the static quenching expression above would then give us (34). we would get an almost identical expression in the form (34). 1 1 Fobs = kHG [G]0 + [H]0 + 2 Ka 1 2 + 4[H]0 [G]0 (31) − [G]0 + [H]0 + Ka Likewise. 1 1 [G]0 + [H]0 + F = kG [G]0 + kHG 2 Ka 2 1 + 4[H]0 [G]0 (30) − [G]0 + [H]0 + Ka If. if dynamic quenching is suspected to play a role. Assuming first that the quest is nonfluorescent (silent) and using the same approach as with UV–vis spectroscopy.

DOI: 10. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. This article is 2012 John Wiley & Sons.1002/9780470661345. Online 2012 John Wiley & Sons. Ltd. we can write a Stern–Volmer-like description of the observed fluorescence intensity ratio Fobs /F0 according to (35). Ltd. Ltd. 1 F0 1 [G]0 − [H]0 − = 1 + Ka Fobs 2 Ka 1 2 [G]0 +4 + [G]0 − [H]0 + (34) Ka Ka When the host is fluorescent and the addition of a guest results in both dynamic and static quenching.4 kH /kH0 + (kHG /kH0 )Ka [G] Fobs = F0 1 + Ka [G] Supramolecular Chemistry: From Molecules to Nanomaterials.smc018 (35) .

please note that we now use the Fobs /F0 ratio in (36) instead of the F0 /Fobs ratio used in the Stern–Volmer equations for dynamic and static quenching (29). We also see that when the host–guest complex fluorescence is fully quenched (kHG = 0).5(CF3 )2 C6 H3 )4 − ] (0 → 2. Online 2012 John Wiley & Sons. the heat (Q) formed or absorbed on adding the guest is measured.5 equivalents). if it is.0 2. The above-mentioned paper by Leigh.5-(CF3)2C6H3)4− ] 9 8 6 4 400 200 0 2 0 Mole fraction of 10+ 1. we can assume kH = kH0 and we get (36). and S (the latter from the relation S = (H − G)/T in one experiment. see Isothermal Titration Calorimetry in Supramolecular Chemistry. In calorimetry.52 10 Fluor@406 nm H Fluorescence@406 nm (104) 10+[B(3.smc018 . The main reasons that it is not used more widely than it is are the equipment cost and real or imagined issues regarding compatibility with organic solvents.12 1 + (kHG /kH0 )Ka [G] kG Fobs + 0 [G] = F0 1 + Ka [G] kH (37) This equation should give the same results as (30). Owing to complication arising from dynamic quenching.4 Calorimetric measurements of 1 : 1 equilibria In supramolecular calorimetric titrations. while the former refers to the proportionality constant of the host after a guest has been added. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. This change in the system is proportional to the concentration of the host–guest complex ([HG]). Ltd.12 Fobs 1 + (kHG /kH0 )Ka [G] = F0 1 + Ka [G] (36) The conditions for this equation are exactly the same as those for (29) and fitting data to (29 and 36) should give the same results—which equation should be used is just a matter of preference or fitting program. Hunter and coworkers used calorimetry to determine the association constant for certain zinc(II) porphyrin–pyridine complexes (Figure 5). that is.19 The reason why Ka = 3 × 1010 M−1 12 0. Equation (36) can also be modified to include the situation when the guest is fluorescent to give (37). we can (after correcting for dilution by the guest) therefore use (38). This makes calorimetry. using a 1 : 1 complexation model. This article is 2012 John Wiley & Sons.0 3. 12. kH and kH0 . 2009.12 2.0 0 0 1. (Inset) Job plot under the same conditions as in the titration experiment. Ltd.) Supramolecular Chemistry: From Molecules to Nanomaterials. and coworkers also illustrates the power of fluorescence titration as they were able to use (36) to measure Ka > 1010 in their cationic DDD-AAA array (Figure 4).Binding constants and their measurement Note here the two different proportionality constants for the host. the enthalpic (H ) changes on adding a guest to a host are measured and 14 N H N+ H N H N N N H 6 Ka = 3 × 1010 M−1 (CH2Cl2. McNab. For calorimetry titrations with 1 : 1 equilibria. maintaining the concentration of 6 constant. American Chemical Society. DOI: 10. the total volume of the solution (V ). We immediately see that this is just a special case of the absolute concentration depending equations for binding (18) and (19). However. in the absence of any guest. the two constants kH and kH0 may not be equal. one of the most powerful methods that exists for determining binding constants in supramolecular chemistry. usually in the form of isothermal calorimetry (ITC. 1 1 [G]0 + [H]0 + Q = HHG V 2 Ka 1 2 − + 4[H]0 [G]0 (38) [G]0 + [H]0 + Ka In their work on cooperative multipoint binding interactions. Ltd. Calorimetry can therefore yield all the key thermodynamic parameters G.12 (Reproduced with permission from Ref. Techniques). with the latter referring to the initial proportionality constant for the host. The sensitivity of the method also means that special care has to be taken to eliminate or correct for dilution and other interfering environmental factors. 298 K) the resulting binding isotherm then fitted to obtain the binding constant (Ka ) and hence the free energy change (G) in the system.2.1002/9780470661345. we obtain the Stern–Volmer (33) for static quenching back.0 [10+] nM Figure 4 Fluorescence intensities of 6 (1 × 10−10 M) at 406 nm in CH2 Cl2 at 293 K (λexcitation = 395 nm) on addition of 10+ [B(3. and the molar enthalpy for the complex formation (HHG ). Also. H .

[H] = [H]0 1 + K1 [G] + K1 K2 [G]2 [G] = [G]0 − K1 [H][G] − 2K1 K2 [H][G]2 (45) (46) We have deliberately chosen two slightly different approaches here to isolate for [H] and [G]. of the 1 : 2 binding system. it is easier to use the overall binding constant than the stepwise one. for example. it must be remembered that it is extremely unlikely that a termolecular complex HG2 can be formed by simultaneous collision of one host and two guest molecules. Ltd.3. as a stepwise process of two guest molecules binding to one host (Figure 1c) or as an overall equilibrium between the two guest molecules and a host (Figure 1a).g. 2.10 Techniques O R NH O O N N Zn O N H R N Ka O O N R O O + N R NH N R HN O HN N N Zn N N H N HN HN O R R= Figure 5 Ka = 790 M−1 in O R R O CHCl3 Ka = 940 M−1 in CHCl2CHCl2 The binding strength of the above porphyrin host to a pyridine guest in different solvents as determined by ITC. respectively. Ltd. The key issue is that we cannot usually determine the concentration of the species in (39) and (40) directly. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. The overall stability constant and the stepwise binding constants are linked by (42). (40) Here we made use of [HG] = K1 [H][G] from (39) on the right side of (40). We now substitute Supramolecular Chemistry: From Molecules to Nanomaterials. titration). We start with the mass balance (43) and (44).19 these authors used calorimetry was to overcome problems due to aggregation-induced NMR spectra broadening of the host in some of the solvents used. β 12 = K1 K2 2.1 Basic 1 : 2 equilibria The problem here is analogous with the situation described for 1 : 1 complexation.smc018 . There are of course two ways of viewing it.1002/9780470661345. we can now rewrite (43) and (44) and obtain (45) and (46).3 The 1 : 2 equilibria The 1 : 2 equilibria is another very important special case of (1) with n = 1 and m = 2. Ltd. Hence. Isolating for [H] and [G]. the 1 : 1 intermediate complex [HG]. The expressions for the stepwise equilibrium constants with first = K1 and second = K2 binding constants are shown in (39) and (40). The overall stability constant from (3) is then defined by (41). we can write the two latter terms in (43) and (44) as [HG] = K1 [H][G] and [HG2 ] = K1 K2 [H][G]. the stepwise binding constants are a much better physical description (42) [H]0 = [H] + [HG] + [HG2 ] (43) [G]0 = [G] + [HG] + 2[HG2 ] (44) Using (39) and (40).. however. DOI: 10. This article is 2012 John Wiley & Sons. (41) In some circumstances. Online 2012 John Wiley & Sons. [HG] [H][G] [HG2 ] [HG2 ] K2 = = [HG][G] K1 [H][G]2 K1 = (39) β 12 = [HG2 ] [H][G]2 We further discuss the relationship between K1 and K2 in terms of cooperative binding below but first we define the key equations that link K1 and K2 to a physical change in a typical supramolecular chemistry experiment (e.

) Supramolecular Chemistry: From Molecules to Nanomaterials. Ltd. YHG and YHG2 . we obtain [G] from the solution to the cubic (47). the product in both case is HG2A 2B . 2. and the 1 : 2 complex = YHG2 according to (48). respectively.2 Cooperativity and α-value How do the stepwise binding constants K1 and K2 relate to the binding constant for 1 : 1 equilibria Ka ? To explain this. 2011. with the equilibria described by the binding constants K1A and K1B .2. Y = [G]3 (K1 K2 ) + [G]2 {K1 (2K2 [H]0 − K2 [G]0 + 1)} + [G]{K1 ([H]0 − [G]0 ) + 1} − [G]0 = 0 (47) Equation (47) is a cubic equation containing two known ([H]0 and [G]0 ) variables and two unknowns (K1 and K2 ).2 When the first guest (G) binds to this host. we try to link the stepwise processes to 1 : 1 equilibria by relabeling a host (H) with two identical binding sites with the labels A and B (Figure 6) as they were two hosts with one binding site each. This article is 2012 John Wiley & Sons. noting that K1 = 4K2 as seen in the following discussion on cooperativity and α-value. described by the binding constants K2A and K2B (Figure 6). We can obtain these again by carrying out a titration and fit. the concentration of [H] could also be calculated from (45). we use a similar approach as used above to obtain (20). Assuming first that the guest is “silent. YHG = YHG − YH . K2A . we substitute for [H] from (45) to obtain (50). UV–vis). (48) Defining Y = Y − YH . instead of using (47) to obtain [G] and then [H] from (45). Equations (50) and (51) contain two additional unknown variables. In the case of statistical 1 : 2 binding. we obtain (49). Online 2012 John Wiley & Sons. 2. First. This cubic equation has three solutions that may or may not include complex numbers. Many software packages use this approach instead of solving the cubic (47).1002/9780470661345. resulting in a binding isotherm by nonlinear regression methods to (47) and (50) or (51) to obtain the best estimates for the unknown variables. and YHG2 = YHG2 − YH in (48) and making use of (39) and (40). (50) If the physical property depends on absolute concentration (e. 5 Finally. It should be noted that. it can bind either to site A to form a HG1A B (the prime indicates which site is occupied) complex or to site B to form HG1AB .g. Once the concentration of [G] is known.” we can describe the observed physical properties that depend on the mole fraction of individual species in solution as a function of the physical property of the host = YH . Ltd. To link (43) and (47) to the observed physical changes.smc018 . to the other two unknown (K1 and K2 ) and known ([H]0 and [G]0 ) from (47). it is possible to use the method of successive approximation with (45) and (46).. + K1A 11 [HG2 ] [H]0 G (49) HG1A′B G HGA1B′ G K2A HG2A′B′ + K1B YHG K1 [H][G] + YHG2 K1 K2 [H][G]2 [H]0 HAB + K2B Figure 6 A schematic explaining the microscopic (K1A . it is also possible to simplify the process a little further. Ltd.2 (Reproduced from Ref. Royal Society of Chemistry. [H]0 − [HG] − [HG2 ] Y = YH [H]0 [HG2 ] + YHG2 [H]0 + YHG [HG] [H]0 YHG K1 [G] + YHG2 K1 K2 [G]2 1 + K1 [G] + K1 K2 [G]2 Y = [H]0 (YHG K1 [G] + YHG2 K1 K2 [G]2 ) 1 + K1 [G] + K1 K2 [G]2 Y = YHG = [HG] [H]0 + YHG2 (51) In both (50) and (51). and it can be obtained by certain algorithms in a number of software packages. the only change we need to make is to multiply through (50) with [H]0 to obtain (51). an initial guess of [G] (and of course K1 and K2 ) by some approximation of (46) is made and the corresponding [G] is then used to calculate [H] from (45). DOI: 10.Binding constants and their measurement for [H] in (46) using (37) to obtain after rearranging terms (47). however. this requires a fairly good initial estimate of the parameters used in these iteration processes.3. The smallest positive real solution is the only one of relevance here. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. When the second G binds to the remaining sites in HG1A B and HG1AB . the 1 : 1 complex = YHG . The new [H] value would then be used to refine [G] in (46) and the process then repeated a couple of times or until [H] and [G] converge to a steady value. K1B . and K2B ) association constants involved in the stepwise formation of a 1 : 2 complex.

we can now use (40) to obtain (53). we cannot distinguish between HG1A B and HG1AB and. Online 2012 John Wiley & Sons. it follows that K1m = K2m and the binding is noncooperative. it is clear that K1m = K2m . determine K1A and K1B (or K2A and K2B ) as the physical changes analogous to. it follows that the second microscopic binding constant can be defined as = K2m with K2m = K2A = K2B . hence. it is possible to quantify the observed cooperativity according to three possible scenarios: • • • If α = 1. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. We can.4. This article is 2012 John Wiley & Sons. and K2B to the overall stepwise constants K1 and K2 . Combining these facts. If α > 1. Further. the interaction parameter = α simply reflects the ratio of the microscopic binding constants K1m and K2m according to (58). YHG in (17) for sites A and B in HG are identical. We define this quantity as the first microscopic binding constant = K1m with K1m = K1A = K1B . then it follows immediately that K1A = K1B and K2A = K2B . however. Using (54) and (55) above and noting that K1m = K2m and K1m = K1m /2 and we obtain (56).smc018 .3 Likewise. 23 He defined the interaction parameter = α. Ltd. (54) (55) The factor of 2 in (55) and (56) can also be explained on kinetic grounds in relation to (2) as there are two ways for the guest G to bind to the empty HAB host so that the observed on-rate (k1 ) appears twice as fast for the first binding. K1 4K2 (57) Alternatively.12 Techniques When the binding sites A and B are truly identical. Because of the stepwise free energy changes upon 1 : 2 binding. [HG2 ] = [HG2A B ]. From (39). In this case. K2 = (56) Connors pointed out that (56) can be used to define cooperativity or the lack of it. then K1m < K2m and the system displays positive cooperativity. Ltd. We first consider the situation where there is no change in the empty remaining site in HG1A B or HG1AB when the first guest G binds and no specific interaction (e.. we start with [HG] = [HG1A B ] + [HG1AB ] to obtain (52).20 = α= (53) If the binding sites A and B are identical. relate the binding constants K1A . K1 = 2K1m K2m K2 = 2 K1m K1 K1 K2m = = = 2 2 2×2 4 α= [HG2 ] K2 = ([HG1A B ] + [HG1AB ])[G] [HG2 ] = [HG2A B ] [HG2A B ] + [G] K2A [G] K2B [G] [HG2 ] [HG2A B ](K2A + K2B ) K2A K2B K2A K2B = K2A + K2B can also explain the statistical factors in (47) and (48) from symmetry numbers20–22 based on the degeneracy of the intermediates HG1A B and HG1AB . as Anderson and Hunter pointed out. Likewise. It is not possible to measure these microscopic binding constants directly but it is easy to link them to the macroscopic stepwise binding constants K1 and K2 using (52) and (53) to obtain (54) and (55). electrostatic) between two molecules of G bound to HG2A B . DOI: 10. for example. then K1m > K2m and the system display negative cooperativity.24 G1 = −RT ln K1m = −RT ln(K1 /2) (59) G2 = −RT ln K2m = −RT ln(2K2 ) (60) Supramolecular Chemistry: From Molecules to Nanomaterials. K1B . [HG1A B ] [HG1AB ] [HG1A B ] + [HG1AB ] = + [H][G] [H][G] [H][G] (52) = K1A + K1B K1 = We now make of use of (1) to show that the equilibrium constants K2A and K2B can also be used to give [HG1A B ] = [HG2A B ]/(K2A [G]) and [HG1AB ] = [HG2A B ]/(K2B [G]).1002/9780470661345. It is important to realize the difference between the macroscopic K1 and K2 and the microscopic binding constants K1m and K2m in (57) and (58) and that for noncooperative binding we would expect the latter to be identical.g. This situation describes classical noncooperative binding in a 1 : 2 system. Alternatively. we K1m K2m (58) The power of the interaction parameter α as defined by either (57) or (58) is that by determining K1 and K2 (or K1m and K2m ). it is useful to compare the stepwise binding constants in terms of free energy changes (G) according to (59) and (60). K2A . we can see that just as [HG] = [HG1A B ] + [HG1AB ]. We are now ready to look at the possible connection between the macroscopic binding constants K1 and K2 . If α < 1. Ltd. there are two ways for the guest G to come off (k−2 ) the complex HG2A B . according to (57).

Ltd. G12 > 0 for negative cooperativity. This article is 2012 John Wiley & Sons. and G12 < 0 for positive cooperativity.25 In many cases. in NMR titration experiments where the addition of the second guest influences the observed chemical shift of the system in a nonlinear manner due to ring-current or induction effects. √ YHG2 [G]( β 12 + β 12 [G]) Y = (65) √ 1 + 2 β 12 [G] + β 12 [G]2 √ YHG2 [H]0 [G]( β 12 + β 12 [G]) Y = (66) √ 1 + 2 β 12 [G] + β 12 [G]2 In principle. and (72). can be reduced by 1 by using K1 = 4K2 . the number of unknown parameters in (47). or both. √ δ HG 2 β 12 [G] + δ HG2 β 12 [G]2 δ = √ 1 + 2 β 12 [G] + β 12 [G]2 √ δ HG2 [G]( β 12 + β 12 [G]) δ = √ 1 + 2 β 12 [G] + β 12 [G]2 δ = δ HG K1 [G](1 + 2K2 [G]) 1 + K1 [G] + K1 K2 [G]2 Supramolecular Chemistry: From Molecules to Nanomaterials. we first define δ HG2 as the difference between the NMR resonances between the 1 : 2 host–guest complex (δ HG2 ) and the host NMR resonance (δ H ). One consequence of the above discussion is that if a system is considered to display noncooperativity. 2.3.24 G12 = G2 − G1 (61) Here. (71). As discussed below. Online 2012 John Wiley & Sons. that the NMR resonance changes are additive (δ HG2 = 2δ HG ). that is. (50). (63) and (64) would be simplified to yield (65) for systems with Y depending on mole fractions and (66) for system with Y depending on absolute concentrations. DOI: 10. It is possible to simplify (62)–(64) even further if we assume that the changes in the physical property Y of interest are additive. that is δ HG2 = δ HG2 − δ H . regardless of cooperativity or lack of it. Ltd.smc018 (70) (71) (72) . it is good practice to fit suspected 1 : 2 equilibria to both scenarios and then compare the outcomes in order to determine if a cooperative model with K1 = 4K2 is justified. and (51) as (62)–(64) with (63) describing systems where the observed physical change depends on mole fractions and (64) describing systems where it depends on absolute concentrations. [G]3 (β 12 ) + [G]2 (2β 12 [H]0 − β 12 [G]0 + β 12 ) + [G]{2 β 12 ([H]0 − [G]0 ) + 1} − [G]0 = 0 √ YHG 2 β 12 [G] + YHG2 β 12 [G]2 Y = √ 1 + 2 β 12 [G] + β 12 [G]2 √ [H]0 (YHG 2 β 12 [G] + YHG2 β 12 [G]2 ) Y = √ 1 + 2 β 12 [G] + β 12 [G]2 (62) (63) (64) It should be noted here that any 1 : 2 equilibria can be fitted to (62)–(64) instead of (47) and (50) or (51). Noncooperative systems (K1 = 4K2 ) also quite often show YHG2 = 2YHG .Binding constants and their measurement We can now also define cooperativity or the lack of it in terms of the free energy difference = G12 for the stepwise binding energies according to (61). This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons.3 NMR measurements of 1 : 2 equilibria Equation (50) provides the foundation for measuring 1 : 2 equilibria by NMR. G12 = 0 for noncooperative 1 : 2 binding. Using δ HG = δ HG − δ H for the change in NMR resonance for the 1 : 1 complex formation and the observed change in resonance as δ = δ − δ 0 similar to the 1 : 1 equilibria. we obtain (69). In this case. hence. Y = Y = YHG K1 [G](1 + 2K2 [G]) 1 + K1 [G] + K1 K2 [G]2 YHG K1 [H]0 [G](1 + 2K2 [G]) 1 + K1 [G] + K1 K2 [G]2 (67) (68) Note that here we used YHG instead of YHG2 in (65) and (66) to simplify the outcome in (67) and (68) further. We can use this to rewrite (47). K1 = 2 β 12 and K2 = ( β 12 )/2.2. Ltd. one could also apply the simplification YHG2 = 2YHG in (50) and (51) that explicitly use the stepwise binding constants K1 and K2 using (67) with Y 13 depending on mole fraction and (68) for Y depending on absolute concentrations. YHG2 = 2YHG . and hence (50) and (51). where fitting the titration data into a 1 : 2 binding model gave much better results than a 1 : 1 binding model. for example. it is appropriate to simplify the 1 : 2 binding equations in NMR titration by assuming that the binding is statistical (K1 = 4K2 ). we can use (70).1002/9780470661345. 5 δ = δ HG K1 [G] + δ HG2 K1 K2 [G]2 1 + K1 [G] + K1 K2 [G]2 (69) Here the guest [G] concentration is obtained from (47) as illustrated in the example below of nitrate binding to a ditopic pyromellitamide host (Figure 7). even in the absence of cooperativity. As with the 1 : 1 equilibria. It should be noted that this situation is probably rare as it is quite common for YHG2 = 2YHG in cooperative systems where K1 = 4K2 . It is then easy to see from (42) that β 12 = K1 /4 = √ √ 4K2 and. In these cases.

This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. and (76). the free guest concentration [G] is obtained from (47).1002/9780470661345. Ltd.3. (66). The change in absorbance is then defined as for 1 : 1 equilibria as A = Aobs − AH0 . This gives (73). (51).3 8.smc018 . Ltd.4 8.6 Fitted to a 1 : 2 equilibria: (Black isotherm) 8.4 UV–vis measurements of 1 : 2 equilibria For analysis of 1 : 2 equilibria by UV–vis spectroscopy.25 (b) 0 10 20 30 Equivalent NO3− added 40 Figure 7 Comparison of the fitted isotherms for the binding of nitrate to a pyromellitamide host. First we define the molar absorptivity change for the formation of a 1 : 2 complex (εHG2 ) as the difference between the molar absorptivity of the 1 : 2 complex (ε HG2 ) and that of the free host (εH ) or ε HG2 = ε HG2 − ε H .57 8. (75). and (68) can be applied after minor modifications.35 Fitted to a 1 : 1 equilibria: (Red isotherm) Ka = 131 ± 32 M−1 8.45 a = 0. Note the difference in estimated uncertainty for the 1 : 1 and 1 : 2 equilibria. Here (70) describes statistical 1 : 2 binding. 2. The choice between the above equations then depends on whether the binding is expected Supramolecular Chemistry: From Molecules to Nanomaterials. Online 2012 John Wiley & Sons. (71) statistical binding where the NMR resonances are additive and (72) where the 1 : 2 binding is not statistical but NMR resonances are additive. This article is 2012 John Wiley & Sons.14 Techniques O O O O O O O N N H H H H N N O O O O O N K1 O O N H H H H N N O O O O NO3− O O O O O O + + − NO3 K2 O NO3− O O O O − N N H H H H N NO3− O3N O N O O O O (a) O O 8. For (70) and (71). Ltd. (64). DOI: 10.55 K1 = 457 ± 41 M−1 dNH 8. we use (62) to obtain the free guest [G] concentration instead of (47). (74). (b) The fitted binding isotherms and the resulting binding isotherms for 1 : 1 (red dotted line) and 1 : 2 equilibria (black solid line).5 K2 = 65 ± 3 M−1 8.25 (a) The structures of the pyromellitamide host and the resulting 1 : 1 and 1 : 2 complexes. A = [H]0 (ε HG K1 [G] + ε HG2 K1 K2 [G]2 ) 1 + K1 [G] + K1 K2 [G]2 √ [H]0 (ε HG 2 β 12 [G] + ε HG2 β 12 [G]2 ) A = √ 1 + 2 β 12 [G] + β 12 [G]2 √ εHG2 [H]0 [G]( β 12 + β 12 [G]) A = √ 1 + 2 β 12 [G] + β 12 [G]2 A = εHG K1 [H]0 [G](1 + 2K2 [G]) 1 + K1 [G] + K1 K2 [G]2 (73) (74) (75) (76) In all these cases.

(b) The UV–vis titration of a 5.15 K2 = 1600 M−1 0. Ltd. This article is 2012 John Wiley & Sons.5 Fluorescence measurements of 1 : 2 equilibria Ignoring the complications arising from dynamic quenching.05 0 450 (b) 500 550 600 650 Wavelength (nm) Figure 8 UV–vis spectroscopic determination of a 1 : 2 equilibria.smc018 . As before. (a) The structures of the dizinc(II) bisporphyrin host and the 4pyridyldiphenylphosphine guest used and the corresponding stepwise equilibria. (79). (64). a 1 : 2 equilibria can be analyzed by fluorescence spectroscopy in a manner similar to the case of UV–vis spectroscopy using the corresponding versions of (51). (66). The change in fluorescence then defines on 1 : 1 equilibria as F = Fobs − F0 .1 a = 1. and (80).35 Fitted to a 1 : 2 equilibria: Absorbance 0. (73) and (74)]. Online 2012 John Wiley & Sons. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons.) Supramolecular Chemistry: From Molecules to Nanomaterials. we use (62) instead of (47) to obtain the free guest [G] concentration for (74) and (75).2 K1 = 6300 M−1 0.3.4 0. Ltd. (78). American Chemical Society.26 15 the molar absorptivity of the 1 : 2 complex (kHG2 ) and that of the free host (kH ) or kHG2 = kHG2 − kH .1002/9780470661345. We start by defining the change in molar proportionality constant (kHG2 ) as the difference between [H]0 (kHG K1 [G] + kHG2 K1 K2 [G]2 ) (77) 1 + K1 [G] + K1 K2 [G]2 √ [H]0 (kHG 2 β 12 [G] + kHG2 β 12 [G]2 ) F = √ 1 + 2 β 12 [G] + β 12 [G]2 (78) √ kHG2 [H]0 [G]( β 12 + β 12 [G]) F = √ 1 + 2 β 12 [G] + β 12 [G]2 (79) F = kHG K1 [H]0 [G](1 + 2K2 [G]) (80) 1 + K1 [G] + K1 K2 [G]2 R1 R 1 N N Zn N Ph N Zn Ph R1 Zn Zn P N N Ph = H N + N Ph Ph K1 + P N Ph Zn Zn N N Zn R1 K2 P Ph O P Ph O N H N Zn N Ph P Ph R1 N R1 = N R C5H11 1 (a) 0.25 0. nonstatistical [(73) and (76)].01 0. Ballester and coworkers used UV–vis titrations to determine the 1 : 2 binding constants for a dizinc-bisporphyrin host and a 4-pyridyldiphenylphosphine guest (Figure 8). or not [εHG2 = 2ε HG . concluding that it was statistical (α ≈ 1).16 × 10−5 M toluene solution of the dizinc(II) bisporphyrin with 4-pyridyldiphenylphosphine (0–135 equivalents). and (68). 2009. and if the molar absorptivities are additive [εHG2 = 2ε HG (75) and (76)]. DOI: 10. This gives (77). 27.Binding constants and their measurement to be statistical [(74 and (75)].3 0.26 (Reproduced with permission from Ref. Ltd. F = 2.

(79) and (80)] or not [kHG2 = 2kHG . As before. in all the cases.07 50 0 450 500 (b) 550 600 650 Wavelength (nm) 700 750 Intensity at 518 nm (a.u. we again use (62) instead of (47) to obtain the free guest [G] concentration. (c) The corresponding binding isotherm showing the change in emission at 518 nm as a function of [Na+ ]/[COPV]. and if the molar absorptivities are additive [kHG2 = 2kHG . Royal Society of Chemistry.) 190 180 170 160 150 140 0. the free guest concentration [G] is obtained from (47).) Supramolecular Chemistry: From Molecules to Nanomaterials. DOI: 10.4 × 10−6 M−1 ) on addition of NaPF6 . Ltd.u. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons.5 [Na+]/[COPV] 2.27 (Reproduced from Ref.5 × 107 M−1 K2 = 2. For (78) and (79). The resulting binding constants are also shown. Ltd.5 1.5 Figure 9 Fluorescence titration of a 1 : 2 equilibria.5 × 105 M−1 100 a = 0.0 1.) 150 K1 = 1. The choice between the above equations then depends on whether the binding is expected to be statistical [(78) and (79)] or nonstatistical [(77) and O O O O O O O O + 2Na+ O O O O O COPV (a) O O O 200 Fitted to a 1 : 2 equilbria: Intensity (a. (b) Emission spectra (λex = 453 nm) in chloroform of COPV (4.0 (c) 0. (a) The structure of the chiral ditopic oligo(p-phenylenevinylene) crown ether (COPV) binding to sodium (Na+ ). (77) and (78)].0 2. 2007. 18.smc018 .16 Techniques (80)].1002/9780470661345. Ltd. This article is 2012 John Wiley & Sons. Online 2012 John Wiley & Sons.

29 Supramolecular Chemistry: From Molecules to Nanomaterials. If the species that we keep at fixed concentration is actually the one binding two times to the other species. (82). Ltd. and (84). there are also situations where a “host” could form either a 1 : 2 or a 2 : 1 complex (and in extremely complicated cases both). the free guest concentration [G] is obtained from (47) and the choice between (81). In principle. This is because the concentration of [G] in (47). This article is 2012 John Wiley & Sons. we can run into some problems if we insist on defining the system in such a way that we can use the above 1 : 2 equations. Ko´zbiał and Pozna´nski used ITC to determine the association constants of various amino acids to a mannonapto-crown-6-ether (Figure 10). in all the cases. (50). This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons.2.1002/9780470661345. (83). we use (62) instead of (47) to obtain the free guest [G] concentration for (82) and (83). and (51) would be fixed within a fairly narrow range during the course of titration. We only focus on the key results here and skip most of the details on how these equations are derived at. Ltd. Ltd. we obtain (81). formed a 1 : 2 complex with this host with considerable positive cooperativity (α > 10).28 The ITC measurements showed that several of the amino acids studied.27 17 including L-tryptophan. However.smc018 .Binding constants and their measurement The 1 : 2 binding constants for a ditopic biscrown ether chiral oligo(p-phenylenevinylene) host (COPV) toward sodium (Na+ ) was determined by a fluorescence spectroscopy titration (Figure 9) in chloroform. it is possible to use the equations discussed in Section 2. Thus. We start by defining C6H13 Si t-Bu N t-Bu t-Bu H N Si O OH + H OH t-Bu 2 O O O C6H13 N HN O O t-Bu N C6H13 Si O N Zn N O Mannonaptho-crown-6-ether OH H2N (S ) N t-Bu O L-Tryptophan N Zn N t-Bu N t-Bu Si Figure 10 The host (left) and the guest (right) used by Ko´zbiał and Pozna´zski. 2. simply by defining the host as the species that binds to two guests. (66).28 C6H13 Porphyrin2 :DABCO Figure 11 Structure of the 2 : 1 porphyrin host–DABCO guest complex reported by Taylor and Anderson. (64).3. for calorimetric titrations (after correcting for dilution effects as mentioned in Section 2.29 The resulting equations are analogous but not identical to the one described for 1 : 2 complexation.4).5 It is for these reasons that it is often worthwhile to define the system so that the “host” forms a 2 : 1 complex with the “guest. except that we use the enthalpy changes for the 1 : 1 (HHG ) and 1 : 2 (HHG2 ) complexes and need to add the total volume (V ) to these equations. [H]0 (HHG K1 [G] + HHG2 K1 K2 [G]2 ) (81) Q=V 1 + K1 [G] + K1 K2 [G]2 Q=V √ [H]0 (HHG 2 β 12 [G] + HHG2 β 12 [G]2 ) √ 1 + 2 β 12 [G] + β 12 [G]2 (82) √ HHG2 [H]0 [G]( β 12 + β 12 [G]) Q=V √ 1 + 2 β 12 [G] + β 12 [G]2 HHG K1 [H]0 [G](1 + 2K2 [G]) Q=V 1 + K1 [G] + K1 K2 [G]2 (83) (84) As before. Yet again. (83). in supramolecular titrations. Online 2012 John Wiley & Sons.” An early illustrative example comes from the work of Taylor and Anderson on the binding of a zinc(II) porphyrin host to the ditopic guest DABCO (Figure 11).3 for any 1 : 2 interaction. Isothermal calorimetric titration in methanol gave K1 = 407 ± 162 M−1 and K2 = 1285 ± 307 M−1 for the formation of a 1 : 2 complex between the mannonapto-crown-6ether and L-tryptophan. and (68) in the same fashion as illustrated above in the case of UV–vis and fluorescence spectroscopy titration. and (84) depends on whether the binding is statistical and/or the enthalpy changes are additive or not. the concentration of one component is usually kept fixed while the other is varied.6 Calorimetric measurements of 1 : 2 equilibria Analysis of 1 : 2 binding equilibria by calorimetric titration is based on (51). Additionally. (82).4 The 2 : 1 equilibria 2. DOI: 10.

Ltd. [H]0 = [H] + [HG] + 2[H2 G] (87) [G]0 = [G] + [HG] + [H2 G] (88) Consequently. DOI: 10. In this case. 3 Y = or in fact any 2 : 1 binding. Likewise. we can write (95)–(98) instead of (65)–(68) for a 2 : 1 equilibria. excluding (93)–(98) from further discussion. we define the changes in the observed physical property as YH2 G = YH2 G − 2YH . if the property depends on absolute concentration (e. that is. as with 1 : 2 equilibria.1 NMR measurements of 2 : 1 equilibria In a manner analogous to the above discussion with the 1 : 2 equilibria. as was done for 1 : 2 equilibria with (62)–(68).29.30 while α < 1 suggests that the 1 : 1 dominates. 30 For practical examples of 2 : 1 equilibria. which are of course derived from (1). we could also use the overall binding constant β 12 to describe the system with (92)–(94). Equations (92)–(94) correspond to the similar (62)–(64) for 1 : 2 equilibria. we have (91). where the physical changes depend on mole fractions (e.18 Techniques the stepwise equilibria according to (85) and (86).g.g. [H] (K1 K2 ) + [H] {K1 (2K2 [G]0 − K2 [H]0 + 1)} + [H]{K1 ([G]0 − [H]0 ) + 1} − [H]0 = 0 (89) We now again use the same approach as with 1 : 2 equilibria to develop equations that link (90) to changes in physical properties. we limit the discussion to NMR and UV–vis titrations as most of the examples in the literature regarding 2 : 1 equilibria are concerned with either of the two of these techniques. an α > 1 implies that the formation of the 2 : 1 complex is favorable over the 1 : 1 complex. except when [H]0 is in excess of [G]0 . at the beginning of a titration. we now move from these equations to develop (89) for the free concentration of the host in 2 : 1 equilibria. we obtain (90). for the formation of the 2 : 1 complex. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. assuming that the changes in physical properties are additive with YH2 G = 2YHG √ YH2 G [G]0 [H]( β 12 + 2β 12 [H]) Y = (95) √ [H]0 (1 + 2 β 12 [H] + β 12 [H]2 ) Y = √ YH2 G [G]0 [H]( β 12 + 2β 12 [H]) √ 1 + 2 β 12 [H] + β 12 [H]2 (96) Y = YHG K1 [G]0 [H](1 + 4K2 [H]) [H]0 (1 + K1 [H] + K1 K2 [H]2 ) (97) Y = YHG K1 [G]0 [H](1 + 4K2 [H]) 1 + K1 [H] + K1 K2 [H]2 (98) 2 YHG K1 [G]0 [H] + 2YH2 G K1 K2 [G]0 [H]2 [H]0 (1 + K1 [H] + K1 K2 [H]2 ) (90) Comparing this with (50) reveals notable differences as both [H]0 and [G]0 feature in this equation as well as a factor of 2 that can be traced back to (87). [HG] [H][G] [H2 G] [HG2 ] K2 = = [HG][H] K1 [H]2 [G] K1 = (85) (86) The mass balance equations for a 2 : 1 equilibria are slightly different from the corresponding equations for the 1 : 2 equilibria as we can see with (87) and (88). Online 2012 John Wiley & Sons. respectively. Ltd. We also limit the NMR and UV–vis discussions to nonstatistical binding where the physical changes are not additive. NMR).. This key cubic equation is similar to (47) for 1 : 2 equilibria except that we have swapped H and G around..1002/9780470661345. Ltd. This explains why the so-called 2 : 1 “sandwich” complexes are most easily observed when the ratio of host-to-guest is 2 : 1 (0. Y = [G]0 (YHG K1 [H] + 2YH2 G K1 K2 [H]2 ) 1 + K1 [H] + K1 K2 [H]2 (91) These key equations can also be simplified to account for noncooperative binding and/or assuming that the observed changes in physical properties are additive. This article is 2012 John Wiley & Sons. [H]3 (β 12 ) + [H]2 (2β 12 [G]0 − β 12 [H]0 + β 12 ) (92) +[H]{2 β 12 ([G]0 − [H]0 ) + 1} − [G]0 = 0 √ √ 2[G]0 β 12 [H](YHG + YH2 G β 12 [H]) (93) Y = √ [H]0 (1 + 2 β 12 [H] + β 12 [H]2 ) √ √ [G]0 2 β 12 [H](YHG + YH2 G β 12 [H]) (94) Y = √ 1 + 2 β 12 [H] + β 12 [H]2 Here (93) and (94) are for systems with the physical changes depending on mole fraction and absolute concentration. Likewise.5 equivalents of guest added). we can define cooperativity in 2 : 1 equilibria using the interaction parameter α according to (57) and (58). we use (90) as the starting point for NMR Supramolecular Chemistry: From Molecules to Nanomaterials.4. First we need to realize that. for example. 2. For noncooperative binding. It should be noted here that. For 2 : 1 equilibria. UV–vis).smc018 . although the process is similar to that described for a 1 : 2 equilibria.

0 1.29 2. there are no points in the range 0 < mole ratio < 0.) O N Zn N N N N DABCO O N N N Viologen ZnPorClip Figure 13 The zinc(II) porphyrin clip (ZnPorClip) host used by Thordarson and Rowan to form a 2 : 1 complex with DABCO in the presence of viologen as an allosteric inducer. We also use δ HG = δ HG − δ H for the change in NMR resonance for the 1 : 1 complex and δ = δ − δ 0 for the observed change in resonances as before with 1 : 1 and 1 : 2 equilibria to give us (99). δ H2 G = δ H2 G − δ H . Using (101) from Sanders and coworkers.31 Supramolecular Chemistry: From Molecules to Nanomaterials.9 × 104 M–1 2.5 1.smc018 . A = [G]0 (ε HG K1 [H] + 2ε H2 G K1 K2 [H]2 ) 1 + K1 [H] + K1 K2 [H]2 (100) In their work on allosteric assembly.0 Mole ratio (DABCO)/(Porphyrin) (101) (a) Fitted to a 2 : 1 equilibria: N Toluene (PhCH3) Chloroform (CHCI3) K1: 1.006 0. that is. the interaction parameter (α) and hence the ratio between K1 and K2 was determined by an integration of the corresponding NMR spectra. the 2 : 1 complex formation is clearly observed in both toluene and chloroform from the “dip” around 0. Ltd.3 0.5 3. as all the species of interest were observable with the exchange process being slow on the NMR timescale. This gives (100).7 × 103 M–1 4. In this case.5 because the system is in slow exchange in this region. Ltd.1 –0. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. δ HG K1 [G]0 [H] + 2δ H2 G K1 K2 [G]0 [H]2 δ = [H]0 (1 + K1 [H] + K1 K2 [H]2 ) (99) In their seminal paper on porphyrin ladder formation. They then used the K1 value obtained from the 1 : 1 fitting of the UV–vis titration in the fitting process with (99) to obtain (i) PhCH3 0. Thordarson and Rowan used (100) to determine the binding constant of a zinc(II) porphyrin clip (ZnPorClip) host to DABCO in the presence of viologen (Figure 13) bound to the internal cavity of ZnPorClip.29 It should be noted that as the first binding constant (K1 ) was too large to be determined by an NMR.0 19 K2 from the NMR titration data.20 (b) O O O O O N O O N N Figure 12 (a) 1 H NMR titration of porphyrin (Figure 11) with DABCO in (i) d8 -toluene and (ii) CDCl3 . Online 2012 John Wiley & Sons. American Chemical Society. (b) The resulting binding constants in toluene and chloroform.29 (Reproduced with permission from Ref.5 equivalents of DABCO added (Figure 12).0 2. This article is 2012 John Wiley & Sons.32 the resulting concentration then yielded the K1 to K2 ratio and hence α.2 –0. where the concentration of the 2 : 1 complex would be negligible throughout the titration experiment. The smooth curves are calculated from (99). 1999.0 (ii) CHCI3 ∆d (ppm) –0. DOI: 10.2 UV–vis measurements of 2 : 1 equilibria For analysis of 2 : 1 equilibria by UV–vis spectroscopy. to obtain a good fit from (100). Taylor and Anderson used (99) to analyze the formation of the 2 : 1 porphyrin2 :DABCO complex shown above (Figure 11). (91) can be applied after defining the molar absorptivity change for the formation of a 2 : 1 complex (εH2 G ) as the difference between the molar absorptivity of the 2 : 1 complex (ε H2 G ) and that of the free host (εH ) or ε H2 G = ε H2 G − ε H . As before.Binding constants and their measurement analysis of 2 : 1 equilibria.4.8 × 106 M–1 K2: 9. We first need to define δ H2 G as the difference between NMR resonances between the 2 : 1 complex (δ H2 G ) and the host NMR resonance (δ H ).31 It should be noted that. Ltd. the change in absorbance is then defined as for 1 : 1 and 1 : 2 equilibria as A = Aobs − AH0 . K1 [HG]2 = K2 [H2 G][G] 0. dashed lines show the δ HG values of the 1 : 1 porphyrin:DABCO complex.9 × 103 M–1 a: 0.5 2.δ is the average change in chemical shift of the two β-protons.1002/9780470661345. 29. In curve (ii). these authors determined K1 independently from a UV–vis titration at a low concentration of the host.

An illustrative example comes from studies on 2 : 2 complexes29. Ltd. Online 2012 John Wiley & Sons. The equilibria for the formation (KF ) and breaking (KB ) constants of the 2 : 2 complex are shown. We start with the defining two binding constants: the formation constant = KF and breaking constant = KB for the H2 G2 sandwich formation. . 33 that have sometimes been referred to as sandwich complexes30 The analysis of this system is simplified by assuming that there are only two key steps: the formation of the 2 : 2 complex H2 G2 (Figure 1e) and its subsequent breakup when excess ligand is added (Figure 14). KF = Similar approaches can be used to develop equations for more complex equilibria but not only do these become computationally difficult (solving a quadric or even quintic equations) but because of the increased number of unknown parameters (K1 . simplifications become a necessity. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. Ltd.smc018 .5 Techniques The 2 : 2 equilibria and other related cyclic system balance (104) and (105).29 Supramolecular Chemistry: From Molecules to Nanomaterials.1002/9780470661345. [H]2 (2KF [G]2 ) + [H](1 + [G]2 KF KB ) − [H0 ] = 0 (106) [G]2 ([H] KF KB + 2[H]2 KF ) + [G] − [G0 ] = 0 (107) Ar C6H13 Si [H2 G2 ] [H]2 [G]2 Ar N N N N Ar Zn N Si C6H13 N Ar N 2× N Ar = KF Ar N C6H13 Si N C6H13 Si Zn N Ar N N N N Zn Ar N Ar N N N Ar Ar Zn N N N N N Ar N N Zn Si C6H13 N N Si C6H13 Ar N 2× N N C6H13 Si KB N N N N Ar Ar Zn N Ar N N N N Zn N N Si C6H13 Ar Figure 14 The formation of a 2 : 2 sandwich from a dizinc(II) porphyrin host with DABCO. . 30. These binding constants are defined by (102) and (103)29 and the corresponding mass KB = N Zn N [HG2 ]2 [H2 G2 ][G]2 (102) (103) [H]0 = [H] + 2[H2 G2 ] + [HG2 ] (104) [G]0 = [G] + 2[H2 G2 ] + 2[HG2 ] (105) Using (104) and (105) and rearranging (102) and (103) gives two related quadratic equation for the free host ([H]) and guest ([G]) concentrations according to (106) and (107). it also becomes difficult to get any meaningful results from the fitting process. DOI: 10. K2 .). This article is 2012 John Wiley & Sons. Here. Ltd. respectively. K3 .20 2.

Here. The binding constants for dimerization and selfassociation equilibria can be determined in a manner similar to the host–guest complexation equilibria discussed above. and Y = Y − YH to obtain (108). Ltd.29 20 80 A = ε H2 G2 KF [H]2 [G]2 + ε HG2 KF KB [H][G]2 (109) 15 (DABCO) / µM (a) 2.1002/9780470661345. including supramolecular gels and polymers. In the case of changes that depend on absolute concentration (e. American Chemical Society.5. (b) mole fractions of H. 0. YHG2 = YHG2 − YH . and H(DABCO)2 during the course of the titration. After a few iterative cycles. it is possible to solve these by the method of successful approximation starting by making an initial guess for [G] and using that to solve for [H] in (107).29 21 (b) 1 2 3 4 5 6 Mole ratio (DABCO) / (H)0 Figure 15 UV–vis spectrophotometric titration of the dizinc(II) porphyrin host (H) shown in Figure 14 with DABCO in toluene: (a) change in absorption at two wavelengths (668 and 725 nm) fitted to the calculated curve for the equilibria in Figure 14. Online 2012 John Wiley & Sons.g. 33 Note the different units between the two constants (M−3 vs M−1 ). H2 (DABCO)2 . we limit the discussion to UV–vis as it is the most practical method that could be applied to this type of equilibria. DOI: 10. the UV–vis titration data obtained (Figure 15a) was fitted to (109). the choice of reference state7. the reader is referred to the excellent work of Ercolani11 and a recent review by Hunter and Anderson. the mole fraction of the “sandwich” species H2 G2 is as high as 0.8 (Figure 15b).11 For a more detailed discussion about this and other more complex cyclic systems.1 UV–vis measurements of 2 : 2 equilibria We start here by modifying (108) by defining the change in molar absorptivity for the 2 : 2 complex (εH2 G2 ) as the difference between the molar absorptivity of the 2 : 2 complex (ε H2 G2 ) and that of the free host (εH ) or ε H2 G2 = ε H2 G2 − ε H . calculated from global factor analysis of binding curves at all wavelengths in the region 350–850 nm at 1-nm intervals. UV–vis).29 (Reproduced with permission from Ref.Binding constants and their measurement 2. has been and still is of significant interest to researchers in supramolecular chemistry.) Supramolecular Chemistry: From Molecules to Nanomaterials.15 0. 29.25 A725 Absorbance The solutions to (106) and (107) depend on each other and cannot be solved analytically. we define A = Aobs − AH0 to give us (109). This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons.29 Y = YH2 G2 KF [H]2 [G]2 + YHG2 KF KB [H][G]2 (108) It is not unusual to obtain formation constants (KF ) from (108) in the range of 1016 –1020 M−3 while the breaking constant (KB ) is around 103 –106 M−1 . 8 mentioned earlier (Section 1) becomes crucial—as Ercolani pointed out.10 0 5 10 H 100 25 30 H2(DABCO)2 H(DABCO)2 Mole fraction (%) 60 40 20 0 0 In the above-mentioned paper by Taylor and Anderson.smc018 . Ltd. Even the simplest of these systems. Under these conditions ([H] ≈10−6 M). the formation of the 2 : 2 sandwich is promoted when the concentration of the available binding sites is low.20 For examples of how the equations above can be applied. We also define the change in molar absorptivity for the HG2 complex (ε HG2 ) formed on breaking the H2 G2 as the difference between the molar absorptivity of the HG2 complex (εHG2 ) and that of the free host or ε HG2 = ε HG2 − ε H . Finally.27. we define YH2 G2 = YH2 G2 − 2YH . 29.6 Self-association: from dimerization to aggregates The association of a molecular (A) with itself is the fundamental step in the formation of many self-assembled materials. the self-associated dimer (A2 ). [H] and [G] are then used to calculate the expected changes in the physical property (Y ). This article is 2012 John Wiley & Sons.. They found KF = 5 × 1019 M−3 and K B = 2 × 105 M−1 in toluene. however.20 A668 0. Ltd. We start this discussion by describing how the binding constants for dimerization are derived at before turning to 0. 1999.

√ 4KD [A]0 + 1 − 1 + 8KD [A]0 Y = YD (119) 4KD [A]0 Note that we could also use (118) to write an alternative version of (117) in the form of (120). we do not simplify this equation further as the physical property of interest for the monomer (Yα ) and the dimer (Yλ ) are usually both unknown. Ltd. we use [A] = α[A]0 from (112) and rearrange (113) to make it equal to zero to obtain (114). and (120).g.1 Dimerization equilibria In most cases.. √ −1 + 1 + 8KD [A]0 (122) [A] = 4KD Dividing through (122) with [A]0 would give us A.34 KD = [A2 ] [A]2 (110) The mass balance equation for this equilibria is then described by (111). (114) This quadratic equation has only one relevant solution according to (115). [A] 2[A2 ] + Yλ Y = Yα [A]0 [H]0 2. Ltd. The discussion here is limited to linear (noncyclic aggregates). We therefore substitute for α using (115) in (116) to obtain (117).g. which then can be rearranged to a classical quadratic equation form. (119). Note that it is also possible to Supramolecular Chemistry: From Molecules to Nanomaterials. [A] 2KD [A]2 [A](1 + 2KD [A]) = + [A]0 [A]0 [A]0 = α + α(2KD [A]) = α(1 + 2KD [A]) 1= (113) Finally. √ −1 + 1 + 8KD [A]0 α= (115) 4KD [A]0 To link (115) to a change in physical property (Y ) that depends on the mole fraction (e. the reader is referred again to the work of Ercolani10.6.34 properties associated with the monomer as Yα and that of the dimer as Yλ . Y − Yα = (Yλ − Yα )(1 − α) (118) We now define Y = Y − Yα and YD = Yλ − Yα and substitute for α from (115) in (118) to give us (119). We then use this to give us (116). This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. we initially follow the approach outlined by Martin.smc018 .22 Techniques larger aggregates. which all describe physical changes that depend on mole fractions (e. but for self-association involving cyclic aggregates. 11 and a review by Hunter and Anderson. Online 2012 John Wiley & Sons. We start by defining the mole fraction of the dimer (λ) as λ = 1 − α. NMR). we use a similar approach as in the earlier sections on 1 : 1 and 1 : 2 equilibria. We then define the physical 0 = 2KD [A]2 + [A] − [A]0 (121) This equation has one relevant real solution in the form of (122). √ 4KD [A]0 + 1 − 1 + 8KD [A]0 Y − Yα = (Yλ − Yα ) 4KD [A]0 (120) Starting from the mass balance (111). Ltd. 0 = α(1 + 2KD [A]) − 1 = α(1 + 2KD [A]0 α) − 1 = 2KD [A]0 α 2 + α − 1 = Yα α + Yλ (λ) = Yα α + Yλ (1 − α) (116) √ 1 + 8KD [A]0 Y = Yα 4KD [A]0 √ 4KD [A]0 + 1 − 1 + 8KD [A]0 + Yλ (117) 4KD [A]0 −1 + If the physical property of the nonaggregated monomer (Yα ) is known (which is rare) before we can simplify the above equations further. We start by considering the monomer (A) to dimer (A2 ) equilibrium where the dimerization constant (KD ) is defined according to (110).20 To derive these binding constants. This article is 2012 John Wiley & Sons. taking us back to (115) and eventually to (117). which can be expanded by substitution from (110). then subtracting Yα from both sides of (116) and rearranging gives us (118). α= 1 [A] [A] = = [A]0 [A] + 2[A2 ] 1 + 2KD [A] (112) Dividing by [A]0 through (111) and using the substitution α = [A]/[A]0 from (112) gives us (113).1002/9780470661345. NMR). DOI: 10.. it is also possible to derive a different set of equations to analyze dimerization by rearranging it into a quadratic equation in the form of (121). [A]0 = [A] + 2[A2 ] = [A] + 2KD [A]2 = [A](1 + 2KD [A]) (111) We now define the mole fraction of (nonaggregated) monomer as alpha (α) according to (112). which can be expanded using (111).

4 × 104 M−1 ) in 99 : 1 CDCl3 /DMSO-d6 (v/v) but.Binding constants and their measurement NMR has been reached (<0. Chen and coworkers used the NMR dilution method to determine the dimerization of their amidourea compound (Figure 16). derive a quadratic equation from (111) that is expressed in terms of the concentration of the dimer [A2 ]. 2010. Using these definitions. We define the NMR resonance of the monomer as δ α and that of the dimer as δ λ . the dimerization constant dropped to KD = 15 M−1 with 20% DMSO-d6 present.6.5) × 104 M−1 in 1% DMSO-d6 /CDCl3 O C8H17 Figure 16 The self-complementary amidourea compound reported by Chen and coworkers forms strong dimers in 99 : 1 CDCl3 /DMSOd6 (v/v). This article is 2012 John Wiley & Sons. and (128). UV–vis).2 NMR measurements of dimerization equilibria 2. (119). (127). (125) We limit our discussion to the application of these equations in NMR and UV–vis dilution experiments for the determination of monomer–dimer equilibria. and then performing a series of dilution step until the detection limit for C8H17 C8H17 O O R For measurements of a dimerization equilibria by UV–vis. λ Y = Yα [A] + Yλ [A2 ] = [A]0 Yα α + Yλ 2 1−α = [A]0 Yα α + Yλ (123) 2 (124) Multiplying by [A]0 through (1 − α)/2 and using (115) for α then gives us (125) from (124). we start by defining (123) for the change in that physical property that depends on absolute concentration noting that the concentration of the dimer [A2 ] = λ[A]0 /2.) Supramolecular Chemistry: From Molecules to Nanomaterials. we can apply (102) with slight modifications. For (119). The change in the observed NMR resonance (δ) of interest can then be described by a modification of (117).1 mM in some cases). DOI: 10. 2.1002/9780470661345.35 This compound formed strong dimers (KD = 4. Online 2012 John Wiley & Sons. after defining the molar absorptivities of the dimer as ελ and the O H N H N O O N H N H O N H O H H N N O C8H17 H N O −1 + (128) Y − ([A]0 Yα ) = (2Yα + Yλ ) √ 4KD [A]0 + 1 − 1 + 8KD [A]0 × 8KD [A]0 H N √ 1 + 8KD [A]0 δ = δα 4KD [A]0 √ 4KD [A]0 + 1 − 1 + 8KD [A]0 (126) +δ λ 4KD [A]0 √ 4KD [A]0 + 1 − 1 + 8KD [A]0 (127) δ = δ D 4KD [A]0 √ 4KD [A]0 + 1 − 1 + 8KD [A]0 δ − δ α = (δ λ − δ α ) 4KD [A]0 If we now subtract ([A]0 Yα ) from both sides of (123) and rearrange as we did with (118). 35. Ltd. we get (124). or (120).g. we now obtain (126). Ltd.. that is. depending on individual preferences (or the program used).smc018 . To obtain equations that describe changes in physical properties upon dimerization that depend on absolute concentration (e. as the percentage of DMSO-d6 was increased. measuring the NMR.35 (Reproduced with permission from Ref.4 ± 0. Ltd. This involves taking a fairly concentrated sample (>10 mM if possible) of molecule A. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons.3 UV–vis measurements of dimerization equilibria The most commonly used method for analyzing dimerization equilibria is the method of NMR dilution. (1 − α) Y − ([A]0 Yα ) = [A]0 (2Yα + Yλ ) 2 23 O N H N H R O N H R = C6H13 Ka = (4.6. American Chemical Society. we then define the change in NMR resonance (δ D ) upon dimerization as δ D = δ λ − δ α .

it aggregates by forming an X–Y:(X–Y)n :X–Y.= [Ai ] (K2 K3 K4 K5 . This article is 2012 John Wiley & Sons.15-diphenyl-10. . Ltd. the association constant KE according to (130).1 × 106 M−1 at 25 ◦ C in the presence of 0.smc018 (139) . Ki ). . i-long stacks of A. we get (137). whereby all the association constants are equal (KE ). linear supramolecular polymer. Ki (132) We also define two dimensionless quantities x and L based on KE and the monomer [A] and the total [A]0 concentration of the aggregating molecule according to (133) and (134).20-bis[4-(N-methyl) pyridinium]porphyrin compound by a UV–vis dilution study in water in the presence of various salts. Ltd. . L = x(1 + ρ(2x + 3x 2 + 4x 3 + . Ercolani has also derived similar expressions for linear aggregation.e. .) = [A](1 + ρ(2KE [A] + 3KE 2 [A]2 + . Online 2012 John Wiley & Sons.01 M KNO3 (aq) for this compound.34 KE = K2 = K3 = K4 = K5 = . 3 . where the first association constant (K2 ) differs from all the subsequent association constants (K3 .. use 4KE below). linear supramolecular polymer. . We start our discussion by focusing on the coEK model as the EK model is just a simpler version of the coEK model. x = KE [A] (133) L = KE [A]0 (134) We can now write a mass balance equation for the aggregating molecule according to (135). . We also limit our discussion to how this equilibria can be analyzed by NMR as it is probably the most commonly used method used to analyze this form of aggregation. we assume σ = 1 based on the X–Y motif. [A]0 = [A] + 2K2 [A]2 + 3K2 K3 [A]3 + 4K2 K3 K4 [A]4 + . the ith equilibrium constant is defined according to (131) Ki = [Ai ] [Ai ] = [Ai−1 ][A] Ki−1 [Ai−2 ][A]2 =. then the symmetry number σ = 4. . the aggregation is cooperative once the initial dimer is formed. . . the equal K model (EK). then the symmetry number (σ )20. .) (138) Provided that x < 1 (as we always find in these systems).1002/9780470661345. .22 If the molecule has an X–X symmetry and aggregates by forming an X–X:(X–X)n :X–X. that is. . L = x(1 − ρ) + ρx (1 − x)2 Supramolecular Chemistry: From Molecules to Nanomaterials. We start by defining for the EK equilibria shown in Figure 1(f). . the equations below can be corrected by multiplying KE by σ (i. Ltd. . . . DOI: 10.34 which are not discussed here. we modify (130) by defining the cooperativity constant rho (ρ) as ρ = K2 /KE so that (130) becomes (132). [A]0 = [A](1 + 2K2 [A] + 3K2 K3 [A]2 + 4K2 K3 K4 [A]3 + . . . [A]0 = [A](1 + 2ρKE [A] + 3ρKE 2 [A]2 + .. .22 pointing out the relationship between K2 and the dimerization constant KD . however. . and the cooperative equal K model (coEK). which are all equal—in other words. showing that K2 = 2KD . 2. Ki ). . . K5 . We only consider two cases here (Figure 1f). Martin has also described other models for linear aggregation. . 2.36 They obtained KD = 1. .)) (137) We now use (133) and (134) for substitution in (137) to get (138). if σ = 4. This gives us then (129). K3. If the aggregating molecular has an X–Y symmetry.. we can now use a Maclaurin series expansion of (138) to get (139). This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. Ki−1 )[A]i (131) In the case of the coEK model.24 Techniques monomer as εα . KE = K2 /ρ = K3 = K4 = K5 = . A − ([A]0 ε α ) = (2ε α + ε λ ) √ 4KD [A]0 + 1 − 1 + 8KD [A]0 × 8KD [A]0 (129) Kano and coworkers applied (129) to study the dimerization of a cationic 5.6. This can explained on symmetry grounds. For the analysis below.)) = L = x(1 − ρ) + ρx(2x + 3x 2 + 4x 3 + . . 22 for aggregation is σ = 1. (135) Rearranging (135) gives us (136).) (136) After substituting for KE and ρ instead of the stepwise constants (K2 . K4 . Ercolani also pointed out that the symmetry of the aggregating molecule is important. = Ki (130) Here. .4 Aggregation (linear) equilibria and its analysis by NMR We again follow the approach outlined by Martin34 to derive the necessary equations to describe the linear aggregation of A into 1.

DOI: 10. while the latter two are needed to complete the solution to (152). which can then be expanded by substitution for L using (134). √ 2L + 1 − 4L + 1 2L2 √ 2KE [A]0 + 1 − 4KE [A]0 + 1 = 2KE 2 [A]0 2 (143) In the more complex coEK model. (137) and (138) are simplified to (140) and (141). and δ ε —the first two are required to solve (146). followed by Maclarin series expansion to get (149) and (150). (1 − x)2 (1 − αL)2 = (1 − x(2 − x)(1 − ρ)) (1 − αL(2 − αL)(1 − ρ)) (144) Multiplying from (144) and rearranging gives us the following cubic (145). (149). This article is 2012 John Wiley & Sons.3. and (150). Ltd. (146) The smallest real solution to cubic (146) is the only one of interest and it can be obtained by some software packages as was the case with the cubic (47) in Section 2. we start by defining the mole fractions for the three different environments within the aggregate that a molecular A can be in: as a free monomer (α). we make use of the equilibria shown in Figure 1(f) and (131) to obtain (147) and (148). where ρ = 1 (no cooperativity). Thordarson and coworkers found that the data fitted significantly better (Figure 17) to the coEK model using (152) than the EK model or the Supramolecular Chemistry: From Molecules to Nanomaterials. KE . α= α 3 L2 (ρ − 1) + α 2 L(L − 2(ρ − 1)) − α(2L + 1) − 1 = 0 (145) Substituting for L using (134) would then give us (146).smc018 . ρ. (149) and (150) can also be simplified as ρ = 1. Ltd. ρ = 1 and we have one less parameter to fit and we then use (143) instead of (146). ε= [A3 ] + 2[A4 ] + 3[A5 ]5 + 4[A6 ]6 + · · · + (i − 2)[Ai ]i [A]0 λ= 2[A2 ] + 2[A3 ] + 2[A4 ] + 2[A5 ] + · · · + 2[Ai ] [A]0 (147) 5 i (148) We now expand these equations in a similar manner as with (135) and (136) and then use (133) and (134) for substitution to x and L. α = (1 − x)2 = (1 − αL)2 (142) Multiplying from the bracket terms. we further assume that the NMR resonance for the molecule at the end of a stack is the average of the NMR resonances for the molecule inside the stack and that of the free monomer according to (151). respectively. δ α . δλ = δα + δe 2 (151) We can now write an equation that describes the observed NMR resonance (δ) according to (152). To apply the above equations for the analysis of linear aggregation by an NMR dilution study. inside the stack (δ ε ).2 by (112) as the mole fraction of the free monomer A. α 3 KE 2 [A]0 2 (ρ − 1) + α 2 KE [A]0 (KE [A]0 − 2(ρ − 1)) −α(2KE [A]0 + 1) − 1 = 0 two mole fractions. or at the end of a stack (δ λ ).) (140) x (141) L= (1 − x)2 We now make use of the quantity α defined in Section 2. (142) gives us a quadratic equation that has one relevant solution in the form of (143). Ltd. For the EK binding model. .1.1002/9780470661345.34 The solutions of (146) and (143) provide us with α or the mole fraction of the free monomer in solution for the coEK and EK models. To reduce the number of parameters that need to be fitted.6. ε= ρα 2 KE 2 [A]20 ραx 2 = (1 − x)2 (1 − αKE [A]0 )2 (149) 2ρα 2 KE [A]0 (1 − αKE [A]0 ) (150) λ= α= 25 Note that all the parameters we need to solve (149) and (150) are either experimentally accessible or obtained in the same fitting process required to obtain α from (143) or (146). we see that α = x/L. For the other δ = αδ α + λδ λ + εδ e (152) In the case of the coEK model. Online 2012 John Wiley & Sons. We first consider the simple EK model and substitute x/L by α in (141) to get (142). within a stack (ε). [A]0 = [A](1 + 2KE [A] + 3KE 2 [A]2 + . In the case of the EK model. Comparing (112) with (133) and (134). substitution of x/L by α into (139) would give us (144). and at the end of a stack (λ). We now define the NMR resonances for the aggregating molecule A depending on whether it is a free monomer (δ α ). which can be further simplified using x = αKE [A]0 . This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons.Binding constants and their measurement In the case of the EK model. In an NMR dilution study on the aggregation of a pyromellitamide Pyro in acetone. we therefore have four unknown parameters to deal with. .

7 ppm from the dimerization model. acetone-d6 ) dilution study at 300 K showing the change in resonance for the δN-H (blue open squares) and δAr-H (red solid circles) of the pyromellitamide Pyro at different concentrations of Pyro. that is.4 7. Unfortunately. Interestingly. 6.5 4 4.25 Fitting the data to the coEK model gives KE = 232 M−1 and ρ = 0. In many cases.98 ppm according to the coEK model compared to 21. Equal K aggregation models.1 8. Taken together. 3.2 8. 2.2 8 7.5 1 1. Prior knowledge of the stoichiometry helps in planning.2 M−1 .1002/9780470661345. Online 2012 John Wiley & Sons. UV–vis).5 5 Figure 17 (a) The structure of the aggregating pyromellitamide molecule Pyro. executing. 2007. Constancy of stability concentration as the concentration is varied.37 O O O O N O H H H H N N O N O O O O O Pyro (a) 3 d (ppm) 8. The mole ratio method. this knowledge is not available and the determination of stoichiometry can then become a challenge that needs to be addressed before any further data analysis (or experimentation) takes place. Connors suggested that a good starting point is to assume a simple 1 : 1 stoichiometry and then look for evidence to support or dispute that model. 3. In fact. the mole fraction of the guest (fG ) is varied while ensuring that the total concentration of the host + guest ([H]0 + [G]0 ) remains constant Supramolecular Chemistry: From Molecules to Nanomaterials. the data above suggest that the coEK model is the best model of the three to describe the aggregation of Pyro in acetone-d6 .8 7. American Chemical Society. The method of continuous variations (Job’s method).5 2 2. Ltd.5 3 3.6 8 8. The fact that Pyro aggregates DETERMINING STOICHIOMETRY One of the most important questions in studying host–guest complexation is the determination of stoichiometry for the system of interest. there is no magical “one-size-fits-all” solution to this challenge. the success of a stoichiometric model to account for the data. Ltd. 4.4 In their reviews.3 M−1 and the dimerization model KD = 5. The insert on the top left corner of (b) shows an enlargement of the region between 0 and 2 mM for the δN-H . This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons.4 0 1 [Pyro] (mM) 2 dN-H d (ppm) 8. Ltd. whereas the EK model gives KE = 10. the latter also being equal to the simple dimerization binding model. and analyzing the data obtained from a titration experiment.38 5. however. it has become so popular that other more appropriate methods are now often ignored as researchers follow the results obtained from Job’s method without realizing its considerable limitations and shortcomings. Consistency with the host structure and available information on the host–guest complex structure.smc018 . Connors4 and Tsukube5 listed several methods that could be used to test the 1 : 1 (or other assumed) stoichiometry hypothesis including the following2 : 1. Also shown are the calculated aggregation isotherms for the cooperative (coEK.7 ppm from the EK model and δAr-HD = 21.25 (b) Data from a 1 H NMR (400 MHz.25 It is noteworthy that the coEK model was the only model that gave reasonable values for the NMRs of the δAr-H resonances in Pyro for the free monomer versus inside the stack (or in the dimer for the dimerization model). 26. broken lines).6 dAr-H 7. This article is 2012 John Wiley & Sons.26 Techniques O in a positive cooperative manner in acetone are not surprising as Pyro readily forms supramolecular gels in cyclohexane. DOI: 10. solid line) and noncooperative (EK. The method is based around the assumption that the concentration [Hm Gn ] of a host–guest complex Hm Gn (Figure 1a) is at a maximum when the ratio between the free host and guest ([H]/[G]) is equal to m/n. The fitted difference between the δAr-H resonances for the free monomer (δAr-Hα ) versus inside the stack (δAr-Hε ) was 2.22 (K2 = 51 M−1 ). (Reproduced with permission from Ref. Comparison of stability constants evaluated by different experimental methods (NMR.3. The quality of fit for the coEK model was 12 times better than for the EK and dimerization model.) dimerization model according to (126). 5 To experimentally find this maximum.2 (b) 0 0. related pyromellitamide compounds also show exponential kinetics in terms of aggregation in cyclohexane.1 Job’s method Job’s method is undoubtedly the most commonly used of the above listed methods. Specific experimental evidence such as isobestic point(s).

this maxima would be expected at fG = 1/(1 + 1) = 0.27 It needs to be noted here that. simply applying one’s chemical knowledge and intuition based on the structural information about the host and the guest to determine the stoichiometry (Method 3) is perhaps the powerful and straightforward method available. Instead.g. needs to be defined with care.2 Taking the above into consideration. DOI: 10. If the host or the guest also aggregate.. researchers therefore make the assumption that the changes in the physical property of interest (e.2 Other methods for determining stoichiometry In general. This method sometimes works well. there are more than one type of complexes present—each inflection point can then point to the key complexes present. it is important to keep the host concentration constant throughout the course of the titration to avoid dilution effects. which corresponds to fG = n/(m + n) for the stoichiometry of the system. In most cases.2 Of all the methods listed above. δ in NMR) is proportional to the concentration of [Hm Gn ] in a linear manner may break down. it can be unreliable. The most straightforward method to achieve this is to use the same host solution that will be titrated to make up the solution of the guest that will then be used in the titration experiment..4 (Figure 17) Supramolecular Chemistry: From Molecules to Nanomaterials.1002/9780470661345.6. The wealth of background knowledge and the structural information available from NMR studies (including various two-dimensional methods) and X-ray structures as well as molecular modeling methods usually allow supramolecular chemistry researchers to make fairly accurate assumptions about the expected stoichiometry of the host–guest system of interest. that have quite different physical properties. that is YHG2 = 2YHG (see also Section 2. as in all host–guest titrations. the assumption that the observed physical property change (e. This curve then usually shows a maxima at a certain fG value. for instance. This article is 2012 John Wiley & Sons. it is advisable to use a combination of the methods listed above to determine the stoichiometry.26 3. NMR resonance or increase in a fluorescence signal—Figure 4) are directly proportional to [Hm Gn ]. not as straightforward as it sounds as the real concentration of [Hm Gn ] is usually not directly accessible in supramolecular chemistry experiments.1). simply looking at the quality of the fit is not enough as increasing the number of fitting parameters per se will always increase the quality of fit. testing if the expected stoichiometry model fits to the data at different concentrations is perhaps the most robust method available to confirm the expected stoichiometry in supramolecular chemistry (Method 6). In the case of a 1 : 1 stoichiometry. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. corresponding to the HG and HG2 present in this system. The example in Section 2. Ltd.39 [Hm Gn ] = δ[H]0 δ Hm Gn − δ H (153) Equation (153) highlights the problem with this approach as the NMR resonance for the value for the Hm Gn complex (δ Hm Gn ) can only be determined directly when the total concentration of the guest [G]0 approaches infinity. we have to make the assumptions about the value of δ Hm Gn based on extrapolation of the observed resonances (δ) at high [G]0 /[H]0 ratios. Job’s method usually works well with only one type of complex present. 40 This includes 1 : 2 and 2 : 1 equilibria as they usually include two different type of complexes.3. Other indications of “good fit” should be considered. The point where these two lines intersect usually corresponds to the main inflection point on the binding isotherm and the corresponding [G]0 /[H]0 27 ratio then corresponds to the n/m ratio of the complex. The mole ratio method (No. Another underutilized method is the comparison of the results obtained from different experimental methods. HG and HG2 .4..smc018 . The example in Figure 9 illustrates this clearly as there are two clear inflection points at [G]0 /[H]0 = 1 and also [G]0 /[H]0 = 2.4 When there are more than one type of complexes present. 1 : 2 equilibria).5 The apparent linear portions of the beginning and end of the titration curve are extrapolated. Note that what the “best fit” is. 2 above) is very straightforward as it usually involves inspecting the titration isotherm obtained upon titration of a guest into a host solution. including the scatter of the residual plot and whether all the fitted parameters (and not just the binding constants) make physical sense.g.Binding constants and their measurement and the concentration of the host–guest complex [Hm Gn ] is then plotted against fG .3 The converse is not necessarily true. Online 2012 John Wiley & Sons. The determination of [Hm Gn ] is. it strongly suggests that the underlying assumptions about the stoichiometry are valid. however. as the absence of more than one isobestic point cannot be used to rule out more complex stoichiometry’s. Ltd.2 Finally. If the physical properties are not additive in a simple manner. however. for example.39 It is sometimes also possible to look for specific evidence to rule out certain stoichiometry models—the presence of more than one isobestic points in UV–vis titration can be used to rule out simple 1 : 1 stochiometry (Method 5).4 If the titration data obtained at two or more different concentrations consistently shows the best fit to a particular binding model (e. especially where cooperative interactions play a role. including kinetic and solubility studies (Method 4). applied (153) to plot [Hm Gn ] versus fG .g. Crabtree and coworkers.5 (see Figure 4 for an example). the assumptions behind Job’s method usually also break down. Ltd.

Instead. 25 which is defined by (158) as the (co)variance of residual or yfit from (154). choosing initial values for the fitting process. divided by the co(variance) Supramolecular Chemistry: From Molecules to Nanomaterials. and δ HG2 ) to fit in (69). 41 yfit = ydata − ycalc (154) This equation provides the starting point for other methods to quantitate the quality of the fit. despite the fact that it had one additional parameter compared to the other models under consideration. how to display the results. three (β 12 . Most of the software programs used to fit binding data do so by minimizing the sum of the squared residuals (ssy ) according to (155). Therefore. In many circumstances. 4.1002/9780470661345. Ltd. or the slightly different standard mean of the y estimate (SEy ) value according to (157). There are a number of software programs available that can aid with this process (see below) but the fact remains that. transformation/correction of the raw data.2. and (63)–(68). ssy = (ydata − ycalc )2 (155) It might be tempting to use the ss y values obtained to compare different binding models.smc018 . This can be done by defining the chi-squared (χ 2 ) value according to (156). Taking NMR titrations as an example. there are four unknown parameters (K1 . Ltd. 2. including the choice of model to fit the data into. it would not be surprising if we find that the best fit of our data is to (69) as it has the largest number of parameters. choice of minimization algorithm. but this is not advisable without making some corrections for the number of data points used (N) and the number of parameters used (k) in the fitting process. Issues to consider in terms of choosing software for data analysis. decision on whether and then how to apply global analysis. Covering all of these topics is beyond the scope of this chapter—here the focus is therefore on the following: 1. and overall evaluation of the results. Model selection versus the number of fitted parameters We start by discussing the selection of the binding model versus the number of parameters to fit the data into. The situation highlighted in Section 2. it can be difficult to evaluate the results obtained—simply pressing a button on a computer program rarely does the trick. it is desirable or necessary to fit the data to more than the binding model and then compare the results obtained to select the best one. What we need to consider carefully here is that the more parameters there are to fit. δ HG . Many factors need to be considered. 4. K2 . The advantages of using global analysis methods. estimation of uncertainty of the fitting process. and two (β 12 and δ HG2 ) in (71). and δ HG2 ) in (70) and (72). The same would happen if we were to compare a simple binding to a more complicated one.1 is a case in point—1 : 2 equilibria can usually be fitted to four different types of model depending on whether one assumes the binding is statistical or not and whether the physical properties are additive in a simple manner or not as illustrated by (50). δ HG . DOI: 10.3.42 χ2 = SEy = (ydata − ycalc )2 N −k−1 (156) (ydata − ycalc )2 N −k (157) Another quantitative parameter that can be used to compare the quality of fit is the covariance (cov) of the fit (covfit ). A short review of a few selected commercial and noncommercial software packages for analyzing binding data and their relative strengths and weaknesses. that is. 5. the plot of yfit against the [G]0 /[H]0 ratio according to (154) where ydata is the raw data and ycalc is the calculated data according to our model.28 Techniques provides a good example—only the coEK model gives sensible results for the changes in the NMRs of interest. This means that when we cannot rely solely on comparing the “goodness-of-fit” parameter given by most programs to compare different binding models. the better the fit will be in general.2. especially if they do not contain the same number of fitted unknown parameters. we should also compare the scatter of the residuals. Online 2012 John Wiley & Sons. for example.1 The decision on which model to use versus the number of parameters to fit. suggesting that this model was the “best fit” to the data obtained. 3.25 4 DATA ANALYSIS AND SOFTWARE Analyzing results from supramolecular titration experiments and fitting the data to the binding models detailed in Section 2 is not always as a simple task as it seems. This article is 2012 John Wiley & Sons. without good understanding of these programs and how they relate to the equations above. adjusting the model. Methods to estimate uncertainty in the fitting progress. a 1 : 1 binding to a 1 : 2 binding model—the latter would almost certainly give a better fit. Ltd. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. (51).

1002/9780470661345. Then. the smaller and more random the scatter is. The following simulated example by Thordarson.smc018 . each time we can add the corresponding number of parameters corresponding to the physical changes to fit but not any additional binding constants.29 (158) The covfit from (158) is a numerical estimation of how small and random the scatter plot is—the lower the covfit value.g. every time we add another data set to our global analysis process. the quality of fit to both data sets is clearly much better by global analysis than if they are fitted separately. Comparing the results obtained (Figure 18c) reveals some striking differences between global and local analysis. On the other hand. Ltd. 41 The global analysis method is based on taking a related set of data and fit it simultaneously to the same binding model. if we add enough terms to it)? We need to make it clear that at the same time we are increasing the number of parameters in global analysis as illustrated by (158) and (159) and we have also increased the number of “raw” data points in our fitting process considerably. we often find that more than proton resonance is significantly affected (shifted) by the addition of our guests. We can go on adding more data sets. a random scatter of ±1% was added to these calculated A values (Figure 18a). In their work on 2 : 2 sandwich formation (Figures 14 and 15. especially in light of what was said earlier about the risks of adding extra parameters as it usually “inflates” the goodness of fit (any curve can be fitted to a polynomial y = 1 + x + x 2 + x 3 . Two UV–vis binding isotherms (with different εHG and ε HG2 values) for a 1 : 2 equilibria.43 This technique is therefore exceptionally powerful when it comes to fitting more complex equilibria such as 1 : 2 and 2 : 2 equilibria. More importantly. 4. and then averaged the results. were generated on the basis of (73). First.. we do not double the number of parameters to fit—the key parameters of interest (e. global analysis therefore does not give the same result as that if we had taken two different δNH and δArH or 500 different UV–vis isotherms in the examples above.26 Instead of fitting just one of these resonances.5). On a fundamental level. fitted them one-by-one to (69) or (109). we now fit both the δNH and δArH resonances simultaneously to (69). Ltd.2. Section 2. The two different data sets were then (i) fitted separately (red and blue curves) by local analysis to (73) and (ii) fitted by global analysis (black curves) using the corresponding modified version of (73) as shown in Figure 18(b). . where K1 = 100 000 M−1 and K2 = 10 000 M−1 .2 It is also important to consider if the fitted parameters make physical sense (e. The covfit value is also fairly insensitive to the number of parameters used in the fitting process. Online 2012 John Wiley & Sons. it is possible (and frequently done) to fit the whole UV–vis spectra to the binding equation of interest. DOI: 10. If we take an NMR titration analysis of a 1 : 2 equilibria as an example. global analysis tightens the so-called error surface in our fitting process. compared to local (simple) analysis as explained in an excellent review by Beechem. Taylor and Anderson used (160) to fit their UV–vis spectra from the region of n=350−850 nm n=350−850 nm j =350 nm j =350 nm A(i) = KF [H]2 [G]2 ε(i)H2 G2 n=350−850 nm + KF KB [H][G]2 ε(i)HG2 j =350 nm (160) In (160). This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons.Binding constants and their measurement of the raw data. Taking the pyromelliamide host titrated with nitrate guest in Figure 7 as an example.g.2 Global analysis Global analysis is perhaps one of the most powerful yet underutilized tools available in the analysis of binding data. The quality of these fits was then compared by (i) the calculated asymptotic error (see also below)41 and (ii) the standard error of the y estimate (SEy ) according to (130). Ltd. . K1 and K2 ) stay the same. we now have 502 parameters to fit (500 ε values + KF and KB ) to 500 data sets! Why would we want to add so many parameters to our fitting process. both the δNH and δArH resonances are significantly shifted upon addition of a nitrate. In the case of a UV–vis spectra. For this reason.. This article is 2012 John Wiley & Sons. Supramolecular Chemistry: From Molecules to Nanomaterials. no negative binding constants or 1 H NMRs changes of >100 ppm).2 clearly illustrates the power of global analysis. the model with larger number of parameters can be accepted as the best one provided the above conditions are fulfilled and that it provides a significantly better goodness of fit compared to other competing models. covfit = cov(yfit ) cov(ydata ) 350–850 nm at 1-nm intervals to the model based on (108) for 2 : 2 equilibria. which now becomes (159) δNH +δArH = 29 (δNHHG + δArHHG )K1 [G] +(δNHHG2 +δArHHG2 )K1 K2 [G]2 1 + K1 [G] + K1 K2 [G]2 (159) We now have six parameters to fit into two different data sets (δNH and δArH ). One also needs to consider if the models are chemically plausible or fit with the other information that we may have about the equilibria.

analysis is useless. SEy = standard error of the y estimate—see (157). K1 is within 2%.3 0. Even the calculated changes in molar absorptivities are closer to the “real” values by global analysis than they are when the data sets are fitted individually. 49 or Scathard50 transformation of binding equation were developed at a time when computer power was scarce or nonexisting. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons.02 0 (b) 0 2 4 6 8 10 Equivalent total guest added ([G]0/[H]0) Local fit for data 1: K1 = 21072 (±75%). Online 2012 John Wiley & Sons. data fitting is just an estimate of the real system and cannot therefore be 100% accurate. First. as the underlying Supramolecular Chemistry: From Molecules to Nanomaterials. and K2 within 5% of the “real” value while for Estimation of uncertainties Exactly how reliable or accurate are the results we obtain from analyzing binding data? This is perhaps the most important question we can ask about the results obtained—without any information about the reliability of our data.04 0. Ltd. Ltd. Most researchers carry out their titration experiments in triplicate and the variation in the results obtain from the three experiments gives us some idea of how reliable the results are and allow a very basic or conservative estimation of the uncertainties. Data 2: e∆HG = 30327 (±2%).44 This is not always practical or doable for economic reasons. See also text for details. K2 = 7392 (±21%). unless there is 0% random scatter in the underlying data but that is not likely to happen. e∆HG = 3012 (±72%). The data points (red and blue squares) are generated from (73) using the parameters shown here with an additional random noise of ±1%.16 data set 2 (which in local analysis mode gave a better fit than data set 1). e∆HG2 = −26282 (±20%). Royal Society of Chemistry. Here we need to remember that the fitting processes used by most or all modern computer programs are based on nonlinear regression methods. to obtain correct quantitative description about the variation of the data (parameters such as standard deviation and so on).14 4. Last but not the least. global analysis comes much closer to returning the initial values used to generate the two data sets. Data 1: e∆HG = 1009 (±9%). linear-regression methods such as Benesi–Hildebrand.) based on the SEy and asymptotic error values. K1 is 19% off.45 Lineweaver–Burk. However. SEy = 6 x 10−4 Local fit for data 2: K1 = 118900 (±9%). SEy = 1 x 10−3 (c) Global fit for data 1 and 2: K1 = 101800 (±5%).smc018 . (c) The results from the local and global fitting process with the asymptoting standard error in parentheses.2 (Reproduced from Ref. Unfortunately.08 0.48. Absorbance change (∆A) 0. and K2 26% off the “real” values. The enlarged area (red shaded square) shows the similarities of the local and global fitted isotherms. K2 = 10492 (±9%). 2.30 Techniques For both data: [H]0 = 10−5 M K1 = 100 000 K2 = 10 000 (a) Data 1: e∆HG = 1 000. Ltd.12 0. 2011. the difference between global and local analysis is not that clear if we just look at how well the curves fit into the data (see insert in Figure 18b). e∆HG = −20 000 2 Global fit Random error in ∆A = 1% 0.46 Scott. Second.1 0. (b) The resulting binding isotherms with the local and global fits (solid lines) superimposed. If both are known. highlighting the power of global analysis in data analysis. e∆HG2 = −18991 (±8%). the results obtained by global analysis are not the simple average of the results obtained from fitting the two data sets individually. namely the experimental repeatability uncertainty and the data analysis (fitting process) uncertainty. albeit this would have to be done in a subjective manner. For instance. Old fashioned. This article is 2012 John Wiley & Sons. Third and interestingly.47 Hanse–Woolf. e∆HG = 28104 (±4%). by global analysis. DOI: 10. Estimation of the uncertainty arising from the data analysis or fitting process itself is equally important—after all. e∆HG2 = 18894 (±4%).1002/9780470661345. e∆HG2 = 14384 (±11%). there are still some researchers that use them probably not knowing about the underlying fundamental problems associated with using these transformation. SEy = 1 x 10−3 Figure 18 (a) Local versus global fit for a hypothetical UV–vis titration for a 1 : 2 complexation equilibria (a). The initial conditions and the color coding for the two different data and the global fit.44 The estimation of uncertainty can and should be approached from at least two directions as there are the two components that usually have the largest contribution to the estimation of uncertainties in binding studies. e∆HG = 20000 2 Data 2: e∆HG = 30 000.06 0. we should probably carry out the experiment at least six to eight times. The estimation of uncertainty with regards to the repeatability of the experimental data is obtained by repeating the experiment n times under (near)-identical conditions and analyzing the data in the same manner. the overall uncertainty of the experiment can be estimated by comparing and possibly combining them. K2 = 22735 (±43%).

The only reason one would use plots such as the Scathard plot according to (161) is to plot the data after it has been calculated by nonlinear regression methods as it is often easier for the human eye to spot deviation from straight lines than from nonlinear plots. the ± errors are the same. This article is 2012 John Wiley & Sons. the key problem with nonlinear regression methods is that there are no straightforward methods for estimating the uncertainty in the fitting process. we could add ±1% random scatter to the measured absorbance (A). Ka from 100–1000 M−1 in the example above).g. Online 2012 John Wiley & Sons. this results in a unstable or a singular matrix3 that in turns gives a meaningless results in terms of the asymptotic errors. that is. ±1%) of random scatter to it (Figure 18). only an approximate as it relies on a number of assumptions about the nonlinear regression analysis process.1002/9780470661345. these methods frequently make highly questionable shortcuts such as [G]0 ≈ [G] or that the final observed physical change Yfinal equals the one from the fully formed complex. including grid search methods43 and Monte Carlo simulations. and 4% to our host concentration ([H]0 ).e. the 95% confidence limits based on the asymptotic error for K1 = 97 000–140 000 M−1 (around the average of 112 980 M−1 ).41 Y = −Ka [G] + Ka YHG [G] (161) Putting outdated linear regression methods aside. the [G]0 /[H]0 ratio.Binding constants and their measurement data comes from nonlinear processes. 2% to our guest concentration ([G]0 ). more complex equilibria such as the 1 : 2 binding model. DOI: 10. Perhaps the best method for estimating uncertainties from our fitting process is the Monte Carlo simulation approach. in reality.2 This is what one would expect—if the binding constants is too high for a given concentration. 41 In the former. consider again the data shown in Figure 18. we would vary Ka from 100–1000 M−1 in steps of 100 M−1 in (26)]. while the remaining parameter(s) are determined from a fitting process in the usual manner. The technical details are beyond the scope of this chapter but in essence the process involves a series of matrix manipulations resulting in a so-called variance–covariance matrix.. in an UV–vis titration. a Monte Carlo simulation on data from (26) with Ka = 1000 larger than 1/[H]0 gave the 95% confidence limits as—67% and +1014 %. we would then determine the range for the fixed parameters required to increase the fit (ssy ) above the minimum at a predetermined confidence interval (e. computational rounding errors can start to play a role here as the numbers used in these matrix manipulations involve the use of very small numbers. YHG = Yfinal . This process is then repeated n times and each data set then fitted to the binding equation(s) of interest. In a more extreme case. but this is probably not true in most cases.. 41 This computationally intensive method involves taking the raw initial data and adding a certain level (e.2.41 The asymptotic standard errors is. The only downside of this method is of course the fact that the magnitude of the uncertainty we add to the data could be quite subjective. while the Monte Carlo simulation gave the 95% confidence limits as K1 = 95 000–150 000 M−1 . one parameter is kept fixed at a range of values [(e. The resulting residuals ssy according to (155) are then plotted as a function of the variable that we kept fixed (i. Sometimes. Ltd. The key strength of this method is that it allows us to include the estimated errors for the raw x. Focusing on data set 2. This gives us an n times number of results that we can analyze in terms of confidence intervals (95%). Another problem is the actual method used to calculate the asymptotic error. the physical property (Y ) observed. this problem is particularly noticeable with larger. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. More intensive but rigorous uncertainty would involve some form of data mapping. using tools such as a box plot52 to get a better idea of how reliable the results are. Using an F statistics test.2. which in most cases is the socalled asymptotic standard error. Ltd. two runs of n-data simulations will usually not give us exactly the same results. including that the experimental error is only in the y-estimate.g. there is really no justification for using these lineartransformation methods for determining binding constants2 when nonlinear methods that provide exact solutions to the equations in Section 2 are now readily available in a number of software packages (including freeware). 67%). For these reasons and with the power of modern computers. 51 Second. hence. the asymptotic error often provides a good starting point for our uncertainty estimation. This allows us to overcome the key problem with the asymptotic error estimation method discussed above. For instance. they violate some of the fundamental assumptions behind linear regression by distorting the experimental error.smc018 . For an example of a Monte Carlo simulation approach.41 Additionally.2 Note that the confidence limits obtained with a Monte Carlo simulation are unsymmetrical around the calculated K1 value.g. Note that we use a random number generator to create our n-data. Ltd. it can easily be argued that the biggest experimental error is in the x-estimate.41. In this author’s experience. 31 Taking the above into account and provided we do not have problems with unstable matrix inversions. as the uncertainties in the concentrations used are often quite considerable. we Supramolecular Chemistry: From Molecules to Nanomaterials. but this assumes that the confidence interval is symmetrical around the parameter. Most of the software packages available will report an error estimate or the 95% confidence interval on the results obtained.. This is of course not true in most cases—in fact.43 This process can then be repeat for all the other parameters of interest.and y-data.3 The asymptotic error is then calculated from this matrix..

smc018 . Finally. DOI: 10. To start with. and is it possible the ability to edit these equations are also important factors when it comes to choosing a program. trying to tell the program to restrict the search for Ka values for positive values between 0 and 106 M−1 ). there are many factors to consider.. Christopher Hunter58 would also deserve mention but as it requires out-dated Apple Macintosh computers to run it. UV–vis. it may not always converge to solve the equation(s) of interest.5 Few selected software packages available for binding data analysis The short review given below of some available software packages is by no means comprehensive. Again. Documentation on how the program works.g. The steepest-gradient methods are generally more unforgiven in this respect than the Simplex or genetic algorithm methods. and so on) that one might want to use. In some cases. This article is 2012 John Wiley & Sons. Ltd. Simplex usually does not work with boundary values (e. and then perform minimization using one of the above methods (usually steepest-gradient type) from the best starting point(s). The once popular commercial program SPECFIT is no longer supported or sold as the company owning it seems to have folded. Currently. calorimetry. On the other hand. Many of these old legacy programs are based on MS-DOS or Apple II Macintosh operational system environments and do not compile or run well with modern operational systems (we know of at least one lab that keeps an old Apple II running just for this purpose!). Early legacy programs such as HYPERQUAD53 and EQNMR54 are still in use in some laboratories but the use of these old programs is now somewhat hampered by compatibility problems with modern operational system. it is worth considering whether features such as global analysis or data simulation are included. One related issue here is how to handle the starting values for the fitting process. This is particularly the cases with most of the custom-written (free) software packages available—usually they have been written by an enthusiastic researcher that has only been using some of these techniques herself/himself. which can be a problem (it keeps finding negative Ka values). neither should be ruled out as the successive approximation method is simpler and faster to implement—however. Mathematica . First. For researchers working mostly with relatively simple 1 : 1 equilibria. 4. Ltd. Ltd. or Origin as an underlying “engine” to perform most of the required functions. for example. biochemistry or pharmakinetics. the legacy program. Often user interface (e. The excellent curve-fitting program for NMR data from Prof. Current users of these programs will be aware of their strengths and limitations but given the above-mentioned problems with legacy program it is not likely that novice users would want to commit themselves to learning the necessary syntax. Some of these are reviewed in more detail below but in general one can divide these into two classes: standalone programs and those that build on or use commonly available data software packages such as Excel . it needs to be acknowledged that there is probably no program that covers all the different methods (NMR. the Simplex method or genetic algorithm methods may be required as the steepestgradient methods may not find the minima as easily.32 Techniques could expect a reasonable estimate on the lower (but not the higher) limit on what the binding constant is. When it comes to deciding on which program to use. it may not have a strong appeal for new users. data handling. 4. Most of the minimization algorithms require reasonable starting values or at least starting value boundaries.4 Issues to consider when choosing software for data analysis Currently.g. compatibility with Excel ) and availability (is it free?) are the deciding factors but other issues should always be considered. which are essential to fit 1 : 2 and other complex equilibria or if the method of successive approximation is used. Online 2012 John Wiley & Sons. It is also worth considering if the program uses methods that directly solve cubic equations such as (47).57 which create a scattered grid of starting values.1002/9780470661345. what equations are used for each equilibria. there are a number of software packages available for analyzing binding data. does the program use steepestgradient methods such as a Gauss–Newton or Marquardt algorithm? Or does it also allow or use other methods such as the Simplex method55 or genetic algorithm?56 The steepest-gradient methods are usually very fast (and they also give the asymptotic error directly) but for more complex or difficult problems. fluorescence. The minimization algorithm used is one factor that needs to be considered. there is also very little documentation available and the programs are only distributed Supramolecular Chemistry: From Molecules to Nanomaterials. making it sometimes difficult for supramolecular chemists to adapt them into their research. Commercial packages may also be focused on a different application field. they are probably not necessary but for others these should be considered seriously when choosing (or writing) software for data analysis. which is perhaps not surprising given its ubiquitous use in data handling. Matlab . and plotting. calculate the fit from these points. the preference seems to be for the latter category with programs using Excel being among the most popular ones. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. although still in use in some laboratories such as HYPERQUAD53 and EQNMR54 are not discussed here. It is also possible to use global search methods..

Online 2012 John Wiley & Sons. cubic equations for 1 : 2 equilibria). it uses the Simplex algorithm to solve or simulate 1 : 1.g.g..com) is a new global analysis software package from Dr Peter King and A/Prof. Adding your own equations (e.. for NMR) is relatively straightforward. DOI: 10.au/research/groups/ thordarson/fittingprogram) from the author of this chapter accompanying his recent review2 is a free collection of Matlab m-files for analyzing data from supramolecular titration experiments.12 Weaknesses: Apart from being limited to NMR and fluorescence titrations in its current version. Strengths: For a freeware. applies to most software packages).Binding constants and their measurement by “word of mouth” between laboratories. if the user has the original data in Excel or another program. Documentation on how equations are solved is somewhat sparse but the program seems to use the method of successive approximation to solve higher-order equations (e.g. however. It is based around using a genetic algorithm approach for fitting the data. the program also does not allow the user to modify the existing equations.. it is exceptionally well suited for the analysis of complex equilibria by UV–vis spectroscopy as illustrated in a recent paper by Leigh and coworkers on DDDD-AAAA arrays.g. The program does not give any estimation of the errors and it does not offer any simulation options.edu. and 2 : 1 equilibria for NMR and UV–vis data (the author has developed programs for other methods/equilibria but these are yet to be released). Many of the commercial programs are also of limited use as mentioned earlier as they have been written for a different user (e. Weaknesses: Being a commercial program. It is also a “standalone” program in terms of data input—that is. especially when analyzing 1 : 2 equilibria.41 which is also written by Motulsky.com) is a comprehensive software package for biostatistics.smc018 .. ReactLab (www.graphpad. methods to directly Supramolecular Chemistry: From Molecules to Nanomaterials. including a Monte Carlo approach. which means that the standard deviation error given is almost certainly the asymptotic error. This article is 2012 John Wiley & Sons.unsw.chem. Strengths: With what was said earlier in mind. Ltd. the interface is easy to use and the equations used are clear to the users. Ltd. The graphical interface also provides a clear illustration of the global residual plot and the distribution of components in solution at different stages of the titration. and they are too rigid to be modified for use in supramolecular chemistry (many programs ignore equations based on mole fractions but without them you cannot analyze NMR data). Ltd.org. The program offers global analysis functions and simulations. With its global analysis features. It is unclear if it could handle other equilibria based on absolute concentrations (e. Leigh and McNab used this program to analyze the binding data from the DDD-AAA complex shown in Figure 4. The documentation suggests that the Marquardt steepest-gradient method is used for optimization. the price might be an obstacle for some users.g.. The binding models are simply entered in via an Excel spreadsheet and the program also considers pH measurements and equilibria when it is desired or necessary. The price of the program will undoubtedly also be a barrier for some users. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. global vs local analysis). The program is written to allow the user to do all the data input and adjustment of equations via the Excel interface. Dr Harvey Motulsky has written an excellent book on biostatistics59 and the manual on GraphPad.uk/) is a standalone freeware written by Dr Dusan Djurdjevic. Strengths: Offers a range of relevant models for 1 : 1 and 1 : 2 binding for supramolecular chemistry. Running within the Matlab package. The documentation is second to none in terms of how the nonlinear regression works and the different options available (e. is one of the best references available on data analysis by nonlinear regression. It also offers simulation 33 features and factor analysis. pharmacology). Marcel Maeder that is based on Matlab but runs as a standalone addition to Excel using a free downloadable Matlab component runtime (MCR) module from Matlab to execute ReactLab. The documentation is also sparse as is usually the case with freeware. the fact that this program is built around global analysis of spectroscopic data is undoubtedly its strongest feature. Fittingprogram: (www. The founder of GraphPad.djurdjevic. Here the discussion is therefore limited to two examples of commercial programs and a handful of noncommercial programs that are well adapted for analysis of supramolecular binding constants. as is the fact that it really runs as a shell on top of Excel and Matlab without allowing the users access to the underlying programs as would be the case with VBA and Macro’s in Excel or m-files in Matlab but this is of course unavoidable for a commercially viable enterprise. it will have to be pasted across (this small problem. The inclusion of both 1 : 1 and 1 : 2 binding equilibria for fluorescence and NMR is important as these two methods are often ignored in other packages. GraphPad (www. which might become a problem is some complex situations. It offers fitting BET and other surface absorption data as well as fluorescence and NMR binding. Strengths: Resting on the power of Matlab to use a combination of the Simplex algorithm.1002/9780470661345. 1 : 2. Gas-Fit (http://gasfit. The genetic algorithm is the key strength of this program as it can greatly assist in finding “difficult” solutions.jplusconsulting. fluorescence) but NMR titrations are not included.60 Weaknesses: The program seems to have been written focusing around UV–vis spectroscopic titration of inorganic complexes.

Ltd. Section 4 then highlighted the importance of choosing and comparing the appropriate binding model(s).uk/j. This article is 2012 John Wiley & Sons. UV–vis. Another very powerful feature of these programs is that it allows one to fit NMR simultaneously to a dimerization and 1 : 1 host–guest complexation. Weaknesses: Although the documentation provided has some helpful comments. By explaining in detail how the equations for the most commonly encountered equilibria such as 1 : 1 and 1 : 2 host–guest complexation and host–host dimerization are obtained and then used.zip) is a standalone program written by Dr Chris Marjo. The global analysis function is also limited to up to four different data sets (NMRs or wavelengths) as each variation in the number of data sets requires a separate m-file. Weaknesses: The documentation is very sparse—only the equations for the equilibria are shown. sanderson/science/downloads. The program is limited to NMR and fluorescence titrations and does not provide any simulation features or estimation of the errors on the results obtained. It also does 1 : 1 and 1 : 2 fluorescence titrations as well as analysis of 1 : 1 equilibria by a fluorescence competition method (see also Competition Experiments. the common availability of software packages should hopefully once and for all stop users from using outdated linear transformation methods. it also provides simulation functions (although not for global analysis) that can also be used to perform Monte Carlo estimation of the fitting error. A simple program to help in importing and exporting data from Excel is also provided. The more complex or unusual examples and methods covered should also help the reader to tackle systems with similar complexity in their own work.34 Techniques solve cubic equations and global analysis. 31 In addition to giving the asymptotic error. dimerization.ac.2. Weaknesses: These programs only work for dimerization and 1 : 1 complexation NMR data. and then concluded with a review on a few selected software packages. it should be noted that it was written for Excel 2004 on Mac OS X—Windows users may experience some problems in running it (this author’s attempts to run it on Excel 2010 on a Win 7 computer were not successful). this program has been shown to tackle difficult 1 : 2 and 2 : 1 equilibria. Global analysis options are also not available in this program. which could cause problems with more complex systems. such as fluorescence.html) are two related Excel add-on programs by Dr J.au/Equilibria. even for beginners. Ltd. Concepts).3 Strengths: The program is simple and quite straightforward to use. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. how the uncertainties can be estimated. Looking ahead. Ltd. 25. The search algorithm is quick and fairly robust. and global search methods will also help more researchers in tackling Supramolecular Chemistry: From Molecules to Nanomaterials. is also an issue.dur.edu. NMR Fit HH and NMR Fit HG: (www. The minimization algorithm is not explained but seems to use a combination of a global search method57 using a grid of initial starting values. Additionally this program does global analysis.m. The program does not give any estimation of the uncertainties of the fitting process. it is the author’s opinion that the field is reaching a transition point on at least a couple of fronts. the author has attempted to give a broad overview of how binding constants are derived in supramolecular binding studies and how the data from supramolecular titration experiments is usually analyzed focusing on commonly used methods such as NMR. what factors to consider when choosing a computer program for data analysis. it would probably be difficult to modify them for more complex systems. 26. In Section 3. the power of global analysis. Equilibria (www. Online 2012 John Wiley & Sons.smc018 . and fluorescence spectroscopy. The program does fitting of NMR data into 1 : 1 host–guest complexation. DOI: 10. First.unsw. 5 CONCLUSIONS In this chapter. Unusually. The lack of other methods than NMR and UV–vis. The recent increase in the number of available software packages that use powerful methods such as global analysis. Sanderson for analyzing dimerization and 1 : 1 host–guest complexation and the combination of these two. it does not require the users to provide some initial guesses for the parameters that are optimized in the fitting programs. Finally. The 1 : 2 and competition fluorescence options are quite unique. it would be difficult for users that do not have some familiarity with Matlab to fully utilize this program—especially when it comes to troubleshooting. the fact that there is no method (including Job’s method) that provides a bulletproof definition of stoichiometry and that the researchers own chemical intuition should not be ignored has been highlighted. There is no excuse for anyone to use these often-inaccurate methods. Matlab is also not as readily available in many laboratories as Excel . as they are written around the Solver routine in Excel . Strengths: The Excel interface means that these programs are easy to use. The starting values for the grid search are liberal but cannot be edited by the users. The Matlab m-files can easily be edited by the user. and the combination of these two. and reviewers should make a note of pointing this out to authors when reviewing papers. genetic algorithm. the chapter may help the reader to develop their own equations for more complex equilibria. followed by a steepest-gradient method. The documentation regarding the equations used and how the software works relies on the above-mentioned review2 and the Matlab manual itself respectively. The documentation is also clear and concise. and its cost might be a barrier.1002/9780470661345. M.sseau. In addition.

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