Received: 2 March 2021 Revised: 2 April 2021 Accepted: 26 April 2021

DOI: 10.1002/jmr.2901

REVIEW

Applications of isothermal titration calorimetry in pure and

applied research from 2016 to 2020

Robert J. Falconer1 | Boelo Schuur2 | Anthony K. Mittermaier3

1
School of Chemical Engineering & Advanced
Materials, University of Adelaide, Adelaide,
South Australia, Australia
2
Faculty of Science and Technology,
University of Twente, Enschede, Netherlands
3
Department of Chemistry, McGill University,
Montreal, Quebec, Canada
Correspondence
Robert J. Falconer, School of Chemical
Engineering & Advanced Materials, University
of Adelaide, Adelaide, SA 5005, Australia.
Email: robert.falconer@adelaide.edu.au
Abstract
The last 5 years have seen a series of advances in the application of isothermal titra-
tion microcalorimetry (ITC) and interpretation of ITC data. ITC has played an invalu-
able role in understanding multiprotein complex formation including proteolysis-
targeting chimeras (PROTACS), and mitochondrial autophagy receptor Nix interaction
with LC3 and GABARAP. It has also helped elucidate complex allosteric communica-
tion in protein complexes like trp RNA-binding attenuation protein (TRAP) complex.
Advances in kinetics analysis have enabled the calculation of kinetic rate constants
from pre-existing ITC data sets. Diverse strategies have also been developed to study
enzyme kinetics and enzyme-inhibitor interactions. ITC has also been applied to
study small molecule solvent and solute interactions involved in extraction, separa-
tion, and purification applications including liquid-liquid separation and extractive dis-
tillation. Diverse applications of ITC have been developed from the analysis of
protein instability at different temperatures, determination of enzyme kinetics in sus-
pensions of living cells to the adsorption of uremic toxins from aqueous streams.

KEYWORDS
allostery, distillation, entropy, enzyme kinetics, kinITC, kinITC-ETC, liquid-liquid extraction,
supramolecular

1 | I N T RO DU CT I O N cell biology. From a societal or commercial perspective, it was the use

The applications for analytical technology are driven by the pressing
research questions of the day. The first high sensitivity isothermal
titration calorimetry (ITC) instruments to be commercially available
were released in 1990 and the early publications focused on biological
1,2
applications of this technology. In subsequent years, our biochemi-
cal knowledge underwent massive growth as the human genome was
sequenced and structural genomics determined the 3D structures of
an enormous library of proteins. ITC played an ever-increasing role in
characterizing proteins' interactions with other proteins, other bio-
macromolecules, and with small molecules including drugs. This was
aided by the publication of a series of papers explaining the method,
the mathematics, the interpretation of the data, and methods for
3-7
overcoming technical limitations of the technology. From a scien-
tific perspective, it was the use of ITC to study multi-protein com-
plexes that has made the greatest contribution to our knowledge in
of ITC in drug discovery and understanding drug-target interactions
that has arguably made the greatest contribution.
Since 1990, there has been a steady rise in publications citing iso-
thermal titration calorimetry until 2018 when the output of publica-
tions peaked (Figure 1). Protein chemistry continues to dominate ITC
use in research (Figure 2). In the period 2016 to 2020, 30 years after
ITCs release, it might be expected that microcalorimetry would be
stagnating. This is not the case. In protein chemistry, there have been
significant advances in data analysis of ITC study of multi-protein
complexes, and the use of ITC to study interaction and reaction kinet-
ics. In synthetic chemistry, ITC has played an ongoing role in deter-
mining the functionality of supramolecular structures and has future
scope to determine the mechanisms for binding interactions and the
role of solvents in this process. The application of ITC, previously used
to study biomacromolecules to studying synthetic macromolecules is
relatively straightforward extrapolation. The application of ITC to

J Mol Recognit. 2021;e2901. wileyonlinelibrary.com/journal/jmr © 2021 John Wiley & Sons Ltd. 1 of 17
https://doi.org/10.1002/jmr.2901
2 of 17
study interactions between small molecules is less obvious. It is worth
remembering that the ITC is simply a titration method that will detect
any interaction where there is a sufficient change in enthalpy (ΔH) for
the instrument to measure. ITC is not limited to studying macromole-
cules or even aqueous systems. ITC can study interactions between
small molecules, interactions that play a key part in determining the
effectiveness of processes like liquid-liquid extraction and extractive
distillation. For the cell biologist, interactions between small molecules
are often overlooked and phenomena like molecular crowding and
osmolyte control of osmotic pressure in halophiles are poorly under-
stood. Interestingly, despite 30 years of undertaking ITC research
using the very sensitive microcalorimetry instruments, the physical
chemistry of intermolecular interactions still presents some chal-
lenges. The apparent enthalpy/entropy compensation observed for
binding interactions between a target and closely related ligands are
still subject to debate.8-11
There were more than 3000 articles reporting the use of ITC
between January 2016 and December 2020 so the authors have
selected approximately 180 that they feel best to represent the field
and acknowledge many excellent papers were omitted from the selec-
tion. These references have been classified into the following broad
categories:
1-11
1. Introduction,
12-19
2. Review and Perspective Articles,
3. Methods Papers,20-29
4. Peptides, Proteins including Enzymes,30-85
5. Lipids, Micelles, and Membranes,86-94
6. Nucleic Acids including Aptamers,95-116
7. Supramolecular Structures and Nanoparticles,17,117-157
158-167
8. Small Chemicals, Polymers, and Minerals,
26,43,168-174
9. Binding and Reaction Kinetics,
10. Process Development,175-188 and
FALCONER ET AL.
F I G U R E 1 Articles written with isothermal
titration calorimetry content since 1990 sourced
from the Web of Science, search terms
“isothermal and titration”
11. Early ITC and non-ITC references.189-223
The authors' hope by providing a survey of the published litera-
ture the readers will appreciate the diverse applications of this useful
analytical technique.
2 | P R O T E I N CH E M I S T R Y
2.1 | Complex formation and allostery
ITC has played a role in understanding the formation of multi-protein
complexes, confirming binding interactions, determining the stoichi-
ometry and order of assembly. The field of biomolecular self-assembly
has benefitted from an orthogonal approach wherein ITC was the gold
standard for determining binding, while NMR, SAXS, and X-ray crys-
tallography were used to generate a detailed understanding of mul-
tiprotein complex structure, dynamics, and function. The last 5 years
have yielded a series of articles that are the culmination of many years
of research on multi-protein complex formation. Endosomal transport
is reliant on a series of multi-protein complexes and has been the sub-
ject of a series of excellent reviews including caveolae formation,189
retromer and retriever function in endosomal trafficking,190 and
retromer-independent endosomal cargo recycling.191
Three recent studies nicely illustrate the role ITC can play in teas-
ing out the details of protein complex formation. Proteolysis-targeting
chimeras (PROTACS) are involved in selective protein degradation by
bringing the target protein into proximity with E3 ubiquitin ligases,
which in turn attach a ubiquitin molecule to the target, leading to sub-
sequent degradation in proteasomes. ITC demonstrated that the for-
mation of the ligase-PROTAC-target complex occurs cooperatively.41
The second example is the use of ITC alongside NMR to observe the
role of phosphorylation of the mitochondrial autophagy receptor Nix
FALCONER ET AL.
in promoting the formation of complexes with LC3B. This is an impor-
tant step in Nix interaction with LC3/GABARAP, which leads to the
degradation of mitochondria in autophagosomes.70 In a third study,
ITC was used alongside NMR, x-ray crystallography, and molecular
dynamic simulation to study the tetradecameric caseinolytic protease
(ClpP) complex. The proteasome inhibitor bortezomib mimics a pep-
tide substrate by binding to the serine in the ClpP active-site inducing
allosteric activation of the enzymatic complex.40
The methodology for performing ITC analysis of multiprotein
complex formation has also advanced. Protein complexes and their
interactions with ligands are often substantially more complicated
than the simple 1:1 binding reactions frequently studied by ITC. For
example, the energetic dissection of protein complex formation usu-
ally requires multiple ITC experiments focusing on different steps in
the assembly pathway. Interpretation of these experiments in terms
of a global model of assembly is best achieved by fitting all the
datasets simultaneously to a single set of thermodynamic parameters
that describe the overall process. There are a number of different
software packages capable to some extent of globally fitting multiple
ITC datasets, including the native software provided with MicroCal
(Malvern Panalytical) instruments, and the AFFINImeter package
(S4SD).26 Notable among these is a suite of open-source software for
simultaneous analyses of ITC and multi-modal datasets developed by
Schuck and co-workers at the NIH. In 2016, they published an article
in Nature Protocols20 with step-by-step instructions for combined
analysis of ITC data using NITPIC (which performs an analysis of ITC
peak shapes to improve the accuracy of integrated heats),192
SEDPHAT (which performs the simultaneous analysis),193,194 and
GUSSI (which analyses the uncertainties in the extracted thermody-
namic parameters).195 Together, these articles have been cited more
than 500 times in 2016-2020, according to Clarivate Analytics Web
of Science, pointing to considerable uptake by the community
(Figure 2).
F I G U R E 2 Articles written on ITC for the
years 2000, 2005, 2010, 2015 and 2020 sourced
from the Web of Science, search terms
“isothermal and titration” plus the term on the
x-axis
3 of 17
SEDPHAT-based simultaneous analysis was recently applied to
better understand T cell activation in the immune response.64 One of
the early steps in transducing the signal from T cell antigen receptors
is the activation of the enzyme phospholipase-Cγ1, which is recruited
to the plasma membrane in complex with three other proteins. This
hetero-tetramer is of interest as a drug target for suppressing T cell
activation with potential applications in treating autoimmune diseases
and transplant rejection. Samelson and co-workers combined analyti-
cal ultracentrifugation data together with three binary ITC titrations
(monomer into monomer), four ternary titrations (monomer into
dimer), and one quaternary titration (monomer into trimer). They
found that the last step in the formation of the quaternary complex
was accompanied by a large favorable binding enthalpy that was
almost completely compensated by a large unfavorable binding
entropy. This is a hallmark of coupled folding and binding in intrinsi-
cally disordered regions (several of the components have large
unfolded regions in the monomeric state) and is thought to help pro-
tein/protein interactions achieve high specificity without becoming
overly tight, among other advantages.196,197 ITC was uniquely able to
detect this assembly-driven folding event since it directly gives the
heats of binding; the phenomenon would be virtually invisible to
methods that measure affinity only, because the enthalpic and entro-
pic components cancel out leaving a net free energy change of
almost zero.
Similarly, there have been recent advances in using ITC to unravel
complex allosteric communication networks underlying the binding
interactions of high-order multimers. Foster et al used ITC to investi-
gate interactions of the trp RNA-binding Attenuation Protein (TRAP)
with L-tryptophan.24 TRAP forms a ring-shaped complex of 11 identi-
cal subunits, which binds up to 11 molecules of tryptophan and inter-
acts with RNA to suppress transcription of the trp operon when
tryptophan is in abundance. Previous work had shown that TRAP
binds tryptophan with moderately positive cooperativity, raising the
4 of 17
question of what kind of binding model is appropriate for this system.
Combinatorial calculations show that there are 125 unique patterns
of bound and unbound subunits in a circular complex with 11 binding
sites.24
According to binding polynomial theory, 197
the equilibrium is
described with as many as 11 sets of change in enthalpy (ΔH) and
change in entropy (ΔS) binding parameters. Thus, at first glance, the
system could appear recalcitrant to detailed characterization. Never-
theless, Foster and co-workers showed that ITC data collected over a
range of temperatures could be well fit with a simple model compris-
ing three distinct types of binding event: binding to a subunit flanked
by either two empty subunits, one bound, and one empty subunit, or
two bound subunits. Values of ΔH, ΔS, and change in heat capacity
(ΔCp) were obtained for each of the three types of reaction. Binding to
subunits adjacent to one or two bound subunits occurred with about
5-fold higher affinity than when both adjacent subunits were empty.
Furthermore the magnitude of ΔCp was about 6-fold larger and nega-
tive when at least one of the adjacent subunits was already bound,
compared to the case when both adjacent subunits were empty,
pointing to the existence of allosterically-modulated conformational
changes linked to tryptophan binding that could trigger interactions
with RNA.
2.2 | Drug discovery
The last 30 years have seen major changes in the way drugs are devel-
oped. X-ray crystallography enabled the three-dimensional structures
of thousands of proteins to be determined with atomic resolution.
This in turn enabled drugs to be designed to interact with structurally
well-defined binding sites. Biophysical techniques were applied to
study the resulting target/drug binding. X-ray crystallography is rou-
tinely used with crystals of the target/drug complex to derive the
geometry of the binding interaction. Nuclear magnetic resonance
(NMR) is also used to determine the structures of the drug targets
with the candidates, in-situ. ITC gives the association constants and
the thermodynamic parameters for the binding interactions and sur-
face plasmon resonance (SPR) yields the association and dissociation
constants for the binding interaction.18 Both ITC and SPR determine
the equilibrium constant of the binding interaction from which the
change in free energy (ΔG) of binding can be calculated [Equation (1)].
ITC provides the observed ΔH and the calculated
TΔS of binding
[Equation (2)].
Δ G ¼
RTlnK a ð1Þ
ΔG¼ Δ H
T ΔS ð2Þ
Historically, enthalpy-driven binding was preferred over entropy-
driven binding and was seen as the key to better drug candidates. The
theory was that ΔH is due to hydrogen bonding or similar non-
covalent bonding and quite location specific, entropy on the other
hand was due to water displacement and quite nonspecific.199,200 In
principle, this is correct but in practice, it is dependent on the binding
FALCONER ET AL.
site. An apolar part of the drug candidate that localizes to an apolar
portion of the target binding site can be beneficial but contributes to

TΔS not ΔH. In a paper from 2010, Ladbury et al200 (all eminent ITC
scientists) defined the ΔH of binding as “a direct measure of the net
change in the number and/or strength of the non-covalent bonds on
going from the free to bound state.” The paper then went on to illus-
trate how statins and HIV protease inhibitors had been improved over
time by shifting from entropy-driven to enthalpy-driven binding. If the
interpretation of ΔH was simply a measure of the number and/or
strength of the non-covalent bonds, then modifying the drug candi-
date to enable additional or stronger non-covalent bonds would result
in greater ΔG of binding and stronger association. A complicating fac-
tor was the observation of entropy-enthalpy compensation (see later
section) where the improvement in ΔH was offset by a corresponding
change in
TΔS. ITC data are used during the later stages of the drug
development pathway where they are combined with X-ray crystal-
lography, NMR, and SPR to aid in the choice of lead candidates from
the panel of hits identified during screening, and subsequently during
the lead optimization process. Decision-making based on ITC data in
isolation would be a mistake.
One factor that is often overlooked when studying interactions
like drug candidate binding to a target is the role of the solution com-
position. Intriguing drug binding data produced at different tempera-
tures and in the presence of different buffers demonstrates that the
ΔG of a binding may be unchanged, while both ΔH and
TΔS can be
affected.16 Entropy-enthalpy compensation was evident in all of these
cases. It serves as a useful reminder to ensure solution conditions
should be as “natural” as practical to make rational decisions on drug
selection based on ΔH and
TΔS values. The challenge of interpreting
the thermodynamic data is worth meeting. Protonation states and
water effects have been overlooked in the past but play a role in bind-
ing that cannot be ignored. The beauty of the ITC is that it illustrates
the differences between apolar and polar interactions as it can detect
the change in water molecules and the transfer of protons, elements
in drug binding that are invisible using X-ray crystallography, NMR,
and SPR.
2.3 | Interaction and reaction kinetics
ITC is predominantly used to measure the thermodynamics of binding
and assembly processes, however, there is growing interest in using it
to measure the kinetics of reactions as well. These applications are
based on the idea that the raw ITC output gives a nearly (but not
exactly) real time readout of the rate that heat is being generated or
absorbed in the sample cell. The heat flow due to a chemical reaction
is directly proportional to its rate multiplied by the change in enthalpy.
Thus, ITC datasets can be readily interpreted in terms of reaction
velocities. As most reaction enthalpies are non-zero, ITC represents,
in principle, a nearly universal way to characterize the rates of chemi-
cal and biological processes. The primary complication in these types
of analyses is that there are several factors that contribute to the
shape of the ITC signal in addition to the kinetics of the reactions
FALCONER ET AL.
themselves. These include the rates of heat transfer within the sample
cell and its contents, the electronic response of the feedback circuitry,
7,169,201
and physical mixing of the injected titrant with the analyte.
a result, a rapid burst of heat in the sample, generated for example by
a rapid (sub-second) injection of ligand into a concentrated solution of
binding partner, is detected as a peak lasting tens of seconds. The
instrument response is often represented by the single time constant,
τITC, which describes the rate of exponential decay of the ITC signal
following a reference heater pulse or calibration injection.7,202 Note
that these methods can give slightly different results, likely because
the heat transfer steps differ in the two cases: heat is generated in the
wall of the sample cell during a heater pulse and in the sample solu-
tion itself for the injection.203 While this description holds for longer
time points, the shape of the ITC response at short times is more com-
169
plicated. Regardless of the level of detail of the description used,
the delayed response of the ITC instrument places an upper limit on
the rates of reactions that can be characterized by ITC and must be
accounted for quantitatively in any rigorous analyses of ITC
kinetic data.
One particularly interesting aspect of ITC kinetics analysis is that
a wealth of potentially informative kinetic data has been hiding in
plain sight. Many ITC datasets for 1:1 binding interactions contain the
distinctive feature wherein peaks become broader near the equiva-
lence point of the titration (equal mole fractions of binding partners),
with all other injection parameters held constant. Egawa et al202 and
subsequently Dumas et al6 showed that this peak broadening could
be quantitatively explained in terms of the association and dissocia-
tion rate constants, kon and koff. For a simple reaction A(cell) +
B(syringe) $ AB, the A component is in excess early in the titration
and B is consumed with a pseudo-first order rate constant kon[A]. The
concentration of free component A drops throughout the titration as
it is converted to AB, leading to successive reductions in the rate of
binding and broader peaks. Near the end of the titration, the B com-
ponent is in excess and A is consumed with the pseudo-first order
rate constant kon[B]. As free [B] accumulates, the forward rate
increases once again and peaks narrow. Thus, conventional ITC exper-
iments can encode kinetic information that is untapped in typical ther-
modynamics analyses in which only the peak integrals are considered.
The researchers provided two alternative approaches to
extracting kinetic information, which they refer to as kinITC6 and
kinITC-ETC (Equilibration Time Curve).21 In the first, raw ITC data are
fitted directly to chemical kinetic equations together with those
accounting for reagent mixing and the instrument response. This
approach has the advantage of being completely general in the type
of reactions that can be studied. For example, kinITC was used to
study thiamine binding to two different E. coli TPP riboswitches and
was able to resolve the kinetics of both of the binding step and a sub-
sequent RNA folding event.6,106 The drawback of this method is that
the mixing and the instrument response must be well accounted for in
order to faithfully reproduce experimental ITC peak shapes, which can
present considerable challenges, as mentioned above. Possibly due to
the relative complexity of the analysis, the examples of full kinITC
peak fitting are thus far limited to HIV protease/Nevirapine and TPP
5 of 17
riboswitch/thiamine interactions.6,21 Alternatively, the simplified
kinITC-ETC method analyses only the lengths of time required for ITC
As peaks to return to baseline following the injections (τend). For systems
giving useful kinetic data, the values of τend are shorter for the early
and late injections and longest near the middle of the titration, for the
reasons mentioned above.21 The τend vs injection number profile is
governed exclusively by kon (or koff) and KD (along with concentrations,
injection volumes, etc.) The value of KD is obtained from the tradi-
tional thermodynamic analysis. Thus fitting of the τend profile gives
one of the rate constants while the other is calculated from KD=koff/
kon. The advantage of this approach is that the complicated effects of
mixing and instrument response primarily affect the early portions of
ITC peaks, while the overall lengths of the peaks are dominated simply
by the binding kinetics (provided they are sufficiently slow). As a
rough guide, the authors recommend that in order for kinITC-ETC to
be successful, the following inequality should hold:
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
τITC koff kon ½A0 < 5, where [A]0 is the total amount of binding partner,
that is, [A] + [AB].6 Representative values of τITC vary between about
3.5 seconds for the Malvern ITC200 to 15 seconds for the Malvern
VP-ITC. For a Wiseman c value of 10 ([A]0 = 10koff/kon), this means
that koff should be slower than about 0.5 to 0.1 s
1. Importantly,
kinITC-ETC results are generally in good agreement with those of
SPR6,21,29,39,106 and fluorescence spectroscopy,99 providing validation
for the approach. The main disadvantage of this approach is that is
only applies to simple 1:1 (or 1 to multiple identical sites) binding reac-
tions, as much of the granular detail in the data is deliberately
discarded.
Recently, Dumas has partnered with the S4SD company to
include kinITC-ETC analysis as a standard feature of the
AFFINImeter-ITC software package, extending the accessibility of the
method. The program has been used to study the binding kinetics of
aptamers,99 carbonic anhydrase inhibitors,21,43 HIV reverse transcrip-
tase inhibitors,21 the nickel binding to the NikR transcription factor,39
aminopolycarboxylate ion binding,166 and the lectin domain of FimH,
an E. coli virulence factor.29,49 This last case is notable in that ITC data
had already been obtained for a panel of potential mannosidic inhibi-
tors and used to elucidate structure-activity relationships based on
binding affinity. Ernst et al re-mined the existing data to extract bind-
ing kinetic information, discovering that slow on-rates correlated with
H-bond formation while rapid on-rates correlated with long-range
electrostatic forces and conformational restrictions.29 There is grow-
ing awareness in the drug development field that efficacy is related to
both the strength and the kinetics of drug binding,204 thus structure-
kinetics relationships, such as those uncovered here, are of great
interest. The fact that both thermodynamic and kinetic information
can be obtained simultaneously in a straightforward manner has impli-
cations for the future role of ITC in drug development campaigns.
2.4 | Enzyme catalysis of single reactions
Another area where the use of ITC to measure chemical kinetics has
grown is in the characterization of enzyme kinetics. According to our
6 of 17
search of the literature, there were about 10, 8, 28, and 37 papers
published on this topic in the 5-year periods beginning in 2001, 2006,
2011, and 2016. In these applications, the heat measured by the calo-
rimeter is generated by the chemical reaction catalyzed by the enzyme
rather than by binding and concomitant structural changes, as is the
case for the majority of ITC experiments. This has several implications
for experimental design. Firstly, the fact that each enzyme typically
performs multiple catalytic turnovers during the ITC experiment effec-
tively amplifies the heat signal, and the amount of enzyme required is
far less (as low as picomolar)168 than that required for binding experi-
ments. In addition to the advantages of requiring less material and dis-
couraging enzyme self-association, this also means that the heats
generated by association and dissociation of substrates, products, and
inhibitors can be safely ignored, as they are far smaller than the heats
of catalysis. Secondly, the fact that an enzyme provided with suffi-
cient substrate can continue to generate heat signals almost indefi-
nitely opens the door to a wide variety of experimental designs. All
ITC enzyme kinetics measurements rely on the simple relationship
wherein the displacement of the ITC signal from the no catalysis base-
line is directly proportional to the velocity of the reaction. Thus, the
ITC output represents a (nearly) real-time readout of enzyme activity,
subject to the caveats of mixing and instrument response times
described above. Beyond this commonality, a diversity of approaches
is possible.
In many cases, enzyme kinetics are well described by the follow-
ing modified Michaelis-Menten equation [Equation (3)]205
½E0 kcat ½S
ν¼     ð3Þ
K m 1 þ K½Ii þ ½S 1 þ K½I0i
where ν is the rate of the reaction (-d[S]/dt), [S], [E]0, and [I] are the
concentrations of substrate, enzyme, and inhibitor, respectively. kcat is
the maximum number of reactions catalyzed by each molecule of
enzyme per unit time, Km is the concentration of substrate required to
reach half-maximal velocity in the absence of inhibitors, and Ki and K'I
are dissociation equilibrium constants for inhibitor binding to the
enzyme and enzyme-substrate complex. Most enzyme assays aim to
determine the values of these kinetic parameters. In their seminal
2001 paper, Todd and Gomez describe two general strategies, which
are now generally referred to as “multiple injection” and “single injec-
tion” experiments.4 In a multiple injection ITC enzyme kinetic assay,
the sample cell contains an enzyme solution and the syringe contains
the substrate. The enzyme concentration is chosen to be sufficiently
low so that substrate depletion during the experiment is negligible. As
a result, the ITC signal is ideally constant (horizontal) between sub-
strate injections and resembles a series of steps, one per injection.
The injections are designed such that early steps have [S]<<Km and
the final injections have nearly saturated the enzyme with [S]>>Km.
The displacement of each step relative to the initial baseline is directly
proportional to the reaction velocity, such that each step is smaller
than the one preceding it as the enzyme is gradually saturated with
substrate. The concentration of substrate present in the sample cell
FALCONER ET AL.
after each injection is known from the concentration of substrate in
the syringe and volumes of all injections. The reaction velocity can be
read directly from the vertical position of each step, tracing out a
complete ν versus [S] Michaelis-Menten curve, which can be fitted to
the modified Michaelis-Menten equation. In practice, we find that the
condition of negligible substrate consumption is met when [E]0 ≤
(10
4 s)  Km/kcat.
In a single injection ITC kinetic assay, the amount of enzyme is
typically chosen to be large enough so that the substrate can be fully
converted to product on the timescale of minutes or tens of minutes.
The concentration of substrate is chosen so that enzyme is initially
saturated following (each) injection ([S]>>Km).206 Single injection
assays can be initiated either by injecting substrate (syringe) into the
enzyme (cell) or by injecting enzyme (syringe) into the substrate (cell).
In either case, the ITC signal exhibits a large deflection immediately
after the injection with large heat flows continuing as long as the
enzyme remains saturated with substrate. The signal gradually returns
to the pre-injection baseline as the substrate is consumed. When the
substrate is present in the syringe, this procedure may be repeated
several times within the same experiment. When enzyme is in the
syringe, only a single injection can be made, as all the substrate is con-
sumed after the first injection. Once again, the velocity of reaction (ν)
can be read directly from the displacement of the ITC signal as a func-
tion of time. The amount of substrate at each time point is known
from partial integration of the ITC peak (the fraction of total substrate
remaining at time t is equal to the fraction of the total area of the
peak lying to the right of time t). Together the ν and [S] values trace
out a complete Michaelis-Menten curve. We find that substrate is
consumed sufficiently rapidly for this technique to be applied when
[E] ≥(10
2 s)Km/kcat. When the concentration of enzyme is too low,
the heat spike persists for such a long time (several hours or more)
that the return to baseline is difficult to distinguish. However, the
enzyme must be at a low enough concentration so that the return to
baseline takes at least seconds to tens of seconds. More rapid reac-
tions start to become obscured by the instrument response, as men-
tioned above.169 Compared to multiple-injection experiments, the
single injection method is more rapid and allows kcat and Km to be
extracted from as little as tens of seconds of raw ITC data. As well, a
great deal more product is generated in a single-injection experiment,
which can be advantageous for researchers seeking to characterize
product inhibition but is a disadvantage for those seeking to avoid this
complicating factor.77
The multiple-injection (dilute enzyme) and single-injection (con-
centrated enzyme) approaches provide frameworks for developing
new types of ITC kinetic assays. In a recent example, dilute enzyme
and substrate were both placed in the sample cell with an inhibitor in
the syringe, leading to a constant reaction velocity and horizontal ITC
signal prior to the first injection. Each injection of inhibitor reduced
the velocity and heat flow, but the change in reaction rate occurred
over a period of several minutes as the inhibitor gradually bound to
the active site of the enzyme. These time traces were fit to extract
the inhibitor association and dissociation rate constants, providing an
efficient method for rapidly determining inhibitor binding kinetics.168
FALCONER ET AL.
In another example, in a modified single injection experiment, the sub-
strate was placed together with inhibitor in the syringe and injected
multiple times into concentrated enzyme in the sample cell.170 Each
injection generated a peak, each of which was fitted to obtain values
of kcat and Km. The inhibitor accumulated in the sample cell with each
injection, thereby generating datasets of kcat and Km as functions [I],
allowing determination of Ki and K'I (modified Michaelis-Menten
equation) that is, completely characterizing the strength and mode of
inhibition. The method has since been applied to inhibitors of epoxide
hydrolase, which is of interest as a drug target.30 Notably, the previ-
ous (non-ITC) assay for this enzyme involved hyphenated liquid chro-
matography and mass spectrometry, which involves considerably
more time and experimental uncertainty than the ITC-based method.
ITC offers many advantages compared to traditional enzyme
assays. Firstly, ITC offers real-time monitoring of enzymatic reactions
in cases where other types of continuous assays are unavailable, such
as for epoxide hydrolase, above.30 It is also unique in that traditional
enzyme assays measure concentrations of substrates and products,
with rates determined indirectly. ITC directly detects the reaction
velocity, enabling new applications such as the inhibitor binding
kinetic experiments described above.168 The ability to employ natural
substrates is another large asset for ITC, as traditional assays often
involve modified colorigenic or fluorogenic substrate analogs that do
not necessarily have the same kinetic parameters as the native sub-
strate.58 ITC also offers advantages for enzymes where the standard
assay involves indirect readout with a coupled-enzyme system.207,208
This is particularly true when adding co-solutes or inhibitors that
affect enzymatic activity since the secondary enzymes can be affected
as well as the enzyme of interest. In addition, testing spectroscopically
active inhibitors or other effector molecules can become a challenge
when using spectrophotometric assays, that is, with chromogenic or
fluorogenic probes, or with coupled assays. In contrast, deeply colored
inhibitors are fully compatible with ITC inhibition assays.209 Further-
more, ITC's ability to characterize opaque samples further extends the
reach of this technique beyond spectroscopically-accessible systems.
One particularly interesting example involves using ITC to perform
enzyme kinetics experiments on suspensions of living cells.59,63,79,85
For instance, Yang et al used ITC to study the metallo-β-lactamase
NDM-1 in living cultures of E. coli. NDM-1 cleaves carbapenems, pro-
viding bacterial resistance to these “last resort” β-lactam antibiotics.85
Development of NDM-1 inhibitors has the potential to resensitize
resistant bacteria and offers an avenue for treating these kinds of seri-
ous drug-resistant infections. Single injection experiments with
cefazolin as a test substrate in the syringe and either purified NDM-1
or live E. coli bacteria expressing NDM-1 in the sample cell gave very
similar ITC heat signals. The authors went on to show that ITC could
diagnose the presence or absence of active NDM-1 in clinical strains
85
of pathogenic bacteria and used the in vivo ITC experiment to dis-
cover a new inhibitor scaffold.53 A similar approach was used to iden-
tify the presence of extended-spectrum β-lactamase activity in a panel
of 69 clinical Enterobacteriaceae samples, yielding results in just an
hour, compared to the 32 to 48 hours typically required for traditional
growth inhibition assays.59
7 of 17
2.5 | Enzyme catalysis of multiple reactions
To understand the kinetics of hydrolytic enzymes (glycoside hydro-
lase) that digest homopolymers like starch, cellulose, and chitin, you
need to consider the sequence of events that occur during cataly-
sis. Non-processive glycoside hydrolases usually follow the
sequence, (a) attach to the polymeric substrate, (b) hydrolyze the
bond, (c) separate from the polymer, (d) release the cleavage prod-
uct, then reattach to the polymer and start the process again. These
enzymes usually follow classic Michaelis-Menten kinetics, an exam-
ple being the hydrolytic cleavage of maltoheptose by human saliva
α-amylase.58 Substrates that are crystalline homopolymers, such as
chitin or cellulose are often structurally difficult to attack chemi-
cally or enzymatically due to the tightly packed polymers locked
together by hydrogen bonds. The ability of processive enzymes to
attach to the polymer prevents them from diffusing away from their
target and makes them more efficient than non-processive enzymes
at digesting these difficult substrates. Attachment of a processive
enzyme to a substrate can be studied using ITC. Serratia marcescens
chitinases, ChiA and ChiB binding to (GlcNAc)6 was demonstrated
by ITC using catalytically inactivated enzyme.50 Processive
enzymes generally don't follow classic Michaelis-Menten kinetics as
the rate of catalysis is partly determined by the level of crystallinity
of the substrate, which can be heterogeneous across different
regions of a single sample. Hydrolytic enzymes that break down
heteropolymers can follow Michaelis-Menten kinetics acting on
homogenous model substrates (eg, trypsin/Nα-Benzoyl-L-arginine
ethyl ester BAEE). Digestion of heterogeneous substrates is more
complicated. A single injection assay using ITC to study trypsin
hydrolysis of casein predictably deviates from Michaelis-Menten
kinetics.67 Small trypsin substrates with a single cleavage site (eg,
BAEE) are the method of choice for studying phenomenon like
molecular crowding as the interpretation of the results is easier
than a complex substrate like casein.67
Digestion of phytate (myo-inositol hexakisphosphate) by
Citrobacter braakii 6-phytase has five discrete cleavage steps and the
reaction deviates radically from Michaelis-Menten kinetics.54 The
thermogram produced using a single injection of phytate into phytase
had two peaks consistent with two periods of high enzyme activity
with a bottleneck at the third cleavage step.
2.6 | Thermostability of proteins
One of the more innovative uses of ITC published in the last 5 years
was a paper describing the analysis of the thermostability of lysozyme
using the ITC in isothermal mode without titration.71 Traditional calo-
rimetric measurements of thermostability employ differential scanning
calorimetry (DSC) where the instrument measures the heat flow
required to increase the temperature of a protein sample at a constant
rate. As the temperature rises, the protein unfolds, leading to an
increased heat absorption (or heat capacity, Cp) over the transition
region. The temperature corresponding to the peak (maximum Cp
8 of 17
value) in the unfolding thermogram (Tm) is used as a measure of pro-
tein stability.
The ITC research studied the irreversible unfolding and subse-
quent aggregation of hen egg lysozyme in a buffer at pH 9.0, using a
TA Instruments TAM IV.72 The temperature in the ITC was set at
57 C, 58 C, and 59 C, that is, below the Tm of 71 C, and followed the
heat flow over 6 days. The experiment demonstrated an alternative
method for studying the thermal stability of protein formulations at
temperatures that are more representative of physiological or ambient
conditions than those used in a DSC. The technique has merit for the
development of stable protein formulations and has also been demon-
strated for immunoglobulins36 which are the leading family of thera-
peutic proteins.
3 | S Y N T H E T I C CH E M I S T R Y
3.1 | Supramolecular structures
Great advances have been made in the field of supramolecular chem-
istry and an incredible diversity of supramolecular structures have
been synthesized. The molecular cages are an important family within
the supramolecular structures. These three-dimensional hollow struc-
tures include the crown ethers, cryptands, calixarenes, resorcinarenes,
curcurbiturils, cyclodextrins, pillararenes, cryptophanes, and hemi-
cryptophanes.155 The cavities within these structures provide envi-
ronments with specific dimensions and electric fields that will only
allow a limited number of molecules to enter. This ability to interact
with specific atoms or molecules provides a degree of specificity that
makes the supramolecular structures potentially useful as sensors,
drug delivery vehicles, catalytic sites, and as smart interlocking
materials.
A range of analytical techniques are used to study supramolecular
structures including X-ray crystallography to determine the molecular
structure, NMR to ascertain the positioning of binding partners within
the supramolecular structure, and ITC to study the stoichiometry and
thermodynamics of the interaction. ITC is usually confined to deter-
mining the association constant, the stoichiometry, and the change in
enthalpy and entropy for the interaction although in principle it also
could be used to study the kinetics of interactions and reaction kinet-
ics if the molecule has the catalytic capability (as described above).
Early work with ITC tended to focus on macromolecules of bio-
logical origin such as proteins, nucleic acids, lipids, and complexes,
which made it difficult to interpret experimental data at the molecular
level. The chemical simplicity of supramolecular structures enabled
them to be used as research tools to study intermolecular interactions
and study specific interactions. Research using supramolecular struc-
tures provides a unique opportunity to study the energies associated
with hydrogen bonding, electrostatic interactions, π π-stacking, cation
π and anion π interactions, and water displacement (hydrophobic
210
effect). The cavities formed in supramolecular structures represent
excellent model systems for studying phenomena like the exclusion of
145
water from hydrophobic pockets or constricted spaces or
FALCONER ET AL.
interactions between low charge density ions and hydrophobic
pockets.144 For example, the presence of low charge density ions in a
hydrophobic pocket is not driven by electrostatic interactions, since
the interacting partners are charge neutral, but is due to the energeti-
cally favorable displacement of water from this interface. The knowl-
edge gained by studying interactions with non-biological model
structures can be transferred to more complex systems like proteins,
nucleic acids, and macromolecular complexes.
Supramolecular structures that are luminescent or fluorescent
with specific recognition capabilities for molecules such as gasses,
ions, metabolites, drugs, etc. have potential applications as sen-
sors.136,211 ITC is a useful in these efforts when the analyte is soluble
and can be employed to characterize closely related molecules and to
ascertain the selectivity molecular recognition.
Drug delivery is an attractive application for supramolecular
structures. Many therapeutic agents are relatively hydrophobic and
have low solubility in water or blood. Encasing the therapeutic agent
in a molecular cage has been used to improve drug solubility in an
aqueous environment.212 For example, a new, highly insoluble
antidiabetes drug was successfully solubilized by forming inclusion
complexes with β-cyclodextrin and hydroxypropyl-β-cyclodextrin.146
Similar cyclodextrin inclusion strategies have been used with a range
of drugs, terpenes, and steroids.134 Cyclodextrins have also been pro-
posed to deliver drugs for release in the intestine where bile salts help
displace the drug from the complex, as demonstrated in vitro,137 and
have also been developed for siRNA delivery.143
Supramolecular structures have been used to create artificial
enzymes.148,150 ITC was used to study oxidase-like catalysis using the
multiple injection technique developed for studying enzyme kinetics
(see above).152 The possibility of designing synthetic enzymes with
better stability and efficacy than biological enzymes is an obvious goal
with great potential benefits.
Supramolecular polymer networks (SPNs) are polymers cross-
linked by non-covalent interactions.213 These structures differ from
typical covalently linked polymer networks in being capable of revers-
ible self-assembly and disassembly, as dictated by physical stress and
environmental changes. This leads to interesting properties such as
superior toughness, durability, and self-healing. ITC is uniquely suited
to measuring the stoichiometry of polymers cross-linked within the
supramolecular structures.
4 | P H Y S I CA L C H E M I S T R Y
4.1 | Enthalpy/entropy compensation
There are many examples where chemical changes to either the host
or guest in a binding interaction have resulted in an increase in ΔH
but was accompanied with a corresponding increase in TΔS that mini-
mized the change in ΔG.9 This enthalpy-entropy compensation is an
interesting phenomenon that has puzzled scientists since it was
observed in the 1970s.214 The introduction of ultrasensitive ITCs in
the 1990s made studying entropy-enthalpy compensation easier and
FALCONER ET AL.
raised its prominence within the scientific community.8,9 The consen-
sus is that the solvent plays an important role in entropy-enthalpy
10,11
compensation but definitive evidence for this hypothesis has yet
to be found.
Schönbeck and Holm published research in 2019, where they
used a relatively simple synthetic model system to experimentally
140
study entropy-enthalpy compensation. They used adamantane
derivatives binding to the cavity in cyclodextrins with hydroxypropyl
groups attached to the rim of the cyclodextrin to increase hydropho-
bic contacts made during the binding interaction. The interaction was
enthalpy-driven partly due to the semipolar nature of the cyclodextrin
and possibly due to the release of water constrained within the tight
cavity. The experiment determined the ΔH, TΔS, ΔG, and ΔCp of the
different binding interactions between the adamantane derivatives
and the modified cyclodextrins. While the experiment did not provide
a definitive confirmation that dehydration of apolar moieties is the
cause of observed entropy-enthalpy compensation, it does illustrate a
rational approach to this research. When combined with molecular
dynamic simulation this approach could well solve the puzzle of
entropy-enthalpy compensation.
5 | N E W A R EA S O F I T C R E S E A R C H
5.1 | ITC analysis of small molecule solvent and
solute interactions
For historic reasons, there is a tendency to think of ITC as being useful
for studying interactions involving macromolecules usually of biologi-
cal origin. This is partially due to the release of the first commercially
available microcalorimeters at the time when structural genomics was
emerging as an important area of research. The importance of interac-
tions between small molecules is obvious to process engineers where
these interactions affect processes like distillation and liquid-liquid
extraction.
We have dedicated a section of this review on the research into
small molecule interactions. For the cell biologist or protein chemist
small molecules are important and often overlooked. An obvious
example is the action of osmolytes in regulating in vivo osmotic pres-
sure in halophiles, without disrupting cell function. In mammalian cells,
small molecules play a role in regulating in vivo ionic strength and vis-
cosity that helps maintain cell integrity, and they play a role in molecu-
lar crowding. Protein chemists are aware that small molecules alter
protein solubility and stability, and have created theories like prefer-
ential hydration to explain their observations without studying small
molecule-water interactions to verify these theories' validity. It is
interesting that chemical engineers have taken the lead in using ITC to
investigate small molecule interactions where interactions like hydro-
gen bonding, electrostatic interaction or proton exchange can influ-
ence the success of distillation and liquid-liquid extraction processes.
In chemical engineering, the field of separations is an important
field, responsible for approximately half of the energy demand of the
entire chemical industry.215 Next to traditional distillations, solvent-
9 of 17
based separations are common unit operations applied in many appli-
cation areas, including oil refineries, bulk chemical production plants,
the mining industries, and for purification of fine chemicals and phar-
maceuticals. For any solvent-based separation, the separation perfor-
mance depends largely on intermolecular interactions between the
solvent and the various solutes in the mixture to separate.
Historically, information on intermolecular interactions has been
mostly derived indirectly from biphasic equilibrium measurements and
assumed interaction mechanisms, sometimes supported by spectro-
scopic evidence. For example, by measuring compositions in vapor-
liquid equilibria (VLE) or liquid-liquid equilibria (LLE), information on
the activity coefficients of the solutes in the solvent phase can be
obtained. VLE measurements are labor intensive, and don't provide
enough information to describe the interactions within the system.
For example, when strong complexes are formed between the solvent
and solute, additional insights are useful to aid solvent selection. Spec-
troscopic approaches (such as NMR or FT-IR) have been used to aid
understanding of the interactions within these mixtures but it is here
that ITC can play an invaluable role.
5.2 | Liquid-liquid extraction and extractive
distillation of organic chemicals
ITC is a technique that has been used to study fluid separations, as
direct measurement of heat effects arising from titrating a solute into
a solvent provides valuable information on the affinity between the
solvent and the solute. Two types of solvents may be considered in
fluid separations, single molecule solvents, and composite solvents
comprised of multiple types of molecules. Typically, single molecule
solvents are physical solvents, and although hydrogen bonding and
polarity interactions may occur, strong complexations are hardly
found. In composite solvents on the contrary, often an extractant is
dissolved in a diluent, and the extractant is added with the purpose to
form complexes with a solute that is aimed to be extracted or
entrained (the typical term in extractive distillation). In the past few
years, attempts have been made with ITC to characterize both com-
posite solvents and single molecule solvents.183,184
The use of ITC to characterize liquid-liquid separations and link
experimentally determined interaction energies directly to extraction
performance was pioneered by Cuypers et al in 2008.216 They applied
ITC to investigate on the thermodynamics of hydrogen bonding com-
plexations of various phenolics with tributyl phosphate (TBP) and
trioctyl phosphine oxide (TOPO) extractants. Importantly, they inves-
tigated complexation of TBP and TOPO in neat diluents, but also in
water-saturated diluents. The impact of water was obvious, and inter-
pretation of the role of water in the complexation thermodynamics, as
well as the impact that this has on the liquid extraction performance
was studied. Especially, considering that the ITC was used to under-
stand liquid-liquid extraction to remove phenols from aqueous solu-
tions, it is key to understand the impact of water that is being co-
extracted, even when it is only present in the organic solution in small
concentrations. They found that water interfered in hydrogen bonding
10 of 17
between the extractant and the solute, the fitted complexation con-
stant was significantly lower than in the neat solvent. The constant
obtained with ITC corresponded well with the observed complexation
constant based on fitting models to data from liquid-liquid extraction
experiments.
In liquid-liquid separations, simple 1:1 interactions often seen in
ITC biological applications are atypical. ITC data is ideal for determin-
ing the stoichiometry of interactions.183 The work of Sprakel and
Schuur183 investigated the solute concentration range in the molar
range, which is appropriate for bulk chemical separations. This con-
216
centration range differentiates this work from Cuypers et al and
ITC experiments undertaken by protein chemists or synthetic chem-
ists. In combination with allowing for complex stoichiometry, this
research applied a sequential reaction according to A + B ⇆ AB and
A + AB ⇆ A2B and found that obtaining equilibrium constants for the
sequential reaction model is possible in the molar concentration
range.
Having established and validated the application of ITC to the
183
area of fluid separations, Sprakel and Schuur have used the tech-
nique to investigate on the impact of diluents on the interactions
between extractants and solutes.186 For decades, extraction studies
have appeared claiming both hydrogen bonding complexes and proton
transfer from the acid to the base216 and often it was unclear which
of the mechanisms should be applied. Using a combination of ITC and
molecular modeling,186 for each of the diluents applied it could be
very clearly seen when ion exchange did occur and when it did not.
Active diluents capable of supporting hydrogen bonds such as
1-octanol clearly showed 1:1 stoichiometry and proton transfer,
resulting in the larger values of ΔH1,1 observed by ITC, was confirmed
by molecular modeling. Such understanding of extractant – solute
interactions can also be extended to study the impact of variations in
the extractant molecular structure. By investigating on the impact of
the molecular structure on the binding of the solute,185 insights were
obtained on the temperature dependency of liquid-liquid extraction,
which aids process design. Another example where ITC helped under-
standing phenomena observed in liquid-liquid extraction systems
180
involved a phosphonium phosphinate ionic liquid. In order to
understand why it was so hard to recover acetic acid from the ionic
1 31
liquid, a multitude of techniques including H-NMR, P-NMR, FT-IR,
and ITC were applied. ITC confirmed earlier suggested multi-acid to
phosphinate interactions,218 as well as a clear trend in interaction
energies, showing strong binding of the first acid.
Aiming at understanding also the impact of entrainers (solvents)
in extractive distillation, Sprakel et al184 performed ITC studies on a
wide range of entrainers for a variety of binary distillation systems.
Although good insights were obtained on the energies required to
yield interactions that were sufficiently strong to modify the relative
volatility in the extractive distillation, but sufficiently weak to allow
regeneration of the complexes, quantification of complexation con-
stants was mostly not possible, because the typical S-shaped curves in
the ITC data were not obtained. Not being able to quantify complexes
is primarily due to the absence of strong complexes when a single
molecular solvent is applied. As no complex constants could be fitted,
FALCONER ET AL.
also information on the Gibbs energy was lacking, and as a result, it
was not possible to determine a quantitative relation to the VLE with
ITC results only.
In conclusion, for the standard fluid separation operations in the
chemical industry, using ITC, possibly in combination with other tech-
niques such as molecular modeling or spectroscopic techniques, can
yield very valuable insights in intermolecular interactions, whether
interactions are exothermic or endothermic, and the magnitude of the
overall heat effect. Quantification of models describing phase equilib-
ria is not always directly possible, and depends on the presence of the
characteristic S-shaped ITC curve. For the specific situation that affin-
ity separations are addressed, with clear attractive interactions involv-
ing the solutes to separate (and typically exothermic heat effects), the
typical S-shaped curve allows one to fit one or more complexation
constants. From these, the Gibbs energies are easily calculated all-
owing quantitative comparisons with phase behavior. If the S-curve is
not clear enough, quantification of the Gibbs energy is difficult, and
thus far, only qualitative insights have been obtained.
5.3 | Liquid-liquid extraction for mineral
processing
To recover metal ions from leachates, liquid-liquid extraction is com-
monly applied. Mechanistically, liquid-liquid metal extractions differ
from liquid-liquid extraction of most organics, because in contrary to
organics, the physical solubility of metal ions organic solvents is typi-
cally negligible. Because of the negligible solubility in the organic dilu-
ent, doing homogeneous organic phase ITC is thus not an option.
Perhaps this limitation to biphasic systems and the corresponding
additional degree of complexity is the reason that only a very limited
number of studies have appeared to date. As early as 1973, Marcus
and Kolarik219 published an article on the use of an ITC for characteri-
zation of lanthanide extractions, the machine was based on an
800 mL measurement cell. In subsequent years, their equipment
reduced in size so that approximately 100 mL was sufficient.220
Although this early work showed that determination of thermody-
namic information including the enthalpy of the complexation reaction
in the extraction, the Gibbs energy, and corresponding entropy is pos-
sible, most other researchers continued to use other approaches,
including several spectroscopic techniques. Around the same time
that Cuypers et al216 reported the coupling of ITC with liquid-liquid
extraction of phenolics, the topic was picked up again, now with a
micro-ITC described in a paper by Zalupski and Nash, who applied a
biphasic ITC analysis.221 One key aspect for carrying out ITC in liquid-
liquid biphasic systems is to pre-saturate the solvent with the appro-
priate aqueous solution (eg, aqueous nitrate solution if the metal ion
is leached using nitric acid) to avoid additional heat effects interfering
with the measurement.
An important mechanism in metal extractions is chelation,
because to overcome the strong endothermic dehydration of the
metal ions in their aqueous solution,221 a strong interaction in the
organic phase is necessary. A recent study showed that also in the
FALCONER ET AL.
aqueous phase, chelation of metal ions can take place, and the ther-
modynamics including enthalpy, entropy, and equilibrium constant for
rare earth ion complexations with linear poly(ethylenimine
13
methylenephosophonate) were determined. The work by Archer
and Schulz13 very nicely displayed that the aqueous phase complexa-
tions are entropically driven, and depending on the degree of func-
tionalization with phosphonate groups, a stoichiometry of between
two and five phosphonate groups per rare earth element ion was
observed, the maximum value being reported for Eu(III) and the lowest
for Dy(III). Although in this work no extraction was studied, it clearly
demonstrated that ITC can be of great value in characterization of the
nature and strength of complexes formed with rare earth elements
and other metals, and the work may be of use for extending these
findings to aqueous two-phase systems. Among other applications,
aqueous two-phase systems comprising a polymer that binds metals
may be useful for extraction of rare earth elements. This is certainly of
interest for the process engineering community related to metals
extraction and recycling, and is likely to become a subject of studies in
the coming years.
5.4 | Adsorption processing for toxin removal
A totally different application field is adsorbing toxins from aqueous
streams, an important field in process engineering. Kato et al177 reported
an ITC study on the adsorption of uremic toxins (potassium cresyl sulfate
and potassium indoxyl sulfate) on Zr-based metal organic frameworks
(Zr-MOFs). It was found by ITC that the complexation of the Zr-MOFs
with the uremic toxins was enthalpic in nature while a positive entropic
term TΔS was observed. The ITC study was supported by density func-
tional theory (DFT) calculations, identifying π-π interactions between the
pyridine functionality in the Zr-MOF and the indoxyl groups of the cresyl
moiety. This study clearly shows how powerful ITC can be also for char-
acterization of adsorbent – adsorbate interactions.
6 | C LO S I N G R E M A R K
6.1 | Strengths and weaknesses in published
research
Publication of the thermograms in the manuscript or in the supple-
mentary data has increased over the last 5 years enabling appraisal of
the quality of the data and building confidence in the accuracy of the
analysis. Quality of much of the published ITC research is very good
and the authors have sensibly limited the interpretation of the data.
Probably the most common published error in ITC analysis is failure to
let the signal equilibrate before the next injection. This mistake builds
a systematic error into the calculated thermodynamic constants. The
criticism by Brian Pethica of published ITC research is a cautionary
warning to ITC users.222 It also can serve as guidance to help improve
the quality of ITC research.15
11 of 17
Studying weaker interactions continue to present a challenge to
some research groups. The authors refer the reader seeking assistance
in this field to Joel Tellinghuisen's 2008 paper on low c binding.223
The take home message from this paper is ITC can be used to
understand interactions between a range of molecules from
nanoparticles down to relatively small solutes and solvents. ITC is not
only able to detect interactions but also measure their kinetics. ITC's
capacity to answer research questions is more versatile than most
researchers currently utilize. It will be interesting to see if the next
5 years will see yet more innovative uses for ITC.
DATA AVAILABILITY STAT EMEN T
Data derived from public domain resources.
OR CID
Robert J. Falconer https://orcid.org/0000-0002-9912-9036
Boelo Schuur https://orcid.org/0000-0001-5169-4311
Anthony K. Mittermaier https://orcid.org/0000-0002-3767-1225
RE FE RE NCE S
(1) Introduction (2) Reviews and Perspective Articles (3)-
Methods Papers (4) Peptides and Proteins including Enzymes (5)-
Lipids, Micelles and Membranes (6) Nucleic Acid including
Aptamers (7) Supramolecular Structures and Nanoparticles (8)-
Other Small Chemicals, Polymers and Minerals (9) Binding and
Reaction Kinetics (10) Process Development (11) Early ITC
and non-ITC References
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How to cite this article: R. J. Falconer, B. Schuur, and A.
K. Mittermaier, Applications of isothermal titration calorimetry in
pure and applied research from 2016 to 2020, J Mol Recognit
(2021), e2901. https://doi.org/10.1002/jmr.2901