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Ground state spectroscopy of hydroxyquinolines:

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2014, 16, 61
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evidence for the formation of protonated species
in water-rich dioxane–water mixtures†
Osama K. Abou-Zied,*a John Husband,a Najla Al-Lawatiaa and
Thomas B. Steinbrecherb

Received 4th July 2013,
Accepted 5th October 2013
DOI: 10.1039/c3cp52811a
www.rsc.org/pccp
We have recently used 6-, 7-, and 8-hydroxyquinolines (HQs) as fluorescent probes to study the binding
mechanism in one of the drug binding sites of human serum albumin. In the present work we study the
absorption spectra of the HQ molecules in neat and binary mixtures of dioxane and water in order to identify
the different tautomeric species in the ground state. This study should help in identifying the environment in
nanocavities of macromolecules when HQs are used as local reporters. The enol form is shown to be the
only tautomer for the three HQs in dioxane and water, with the exception of 7HQ in which both the enol
and the zwitterion tautomers exist in equilibrium in water. The results are confirmed by the density functional
theory (DFT) calculations using the B3LYP method with a 6-311++G(2d,p) basis set. In water-rich dioxane
mixtures, all HQs are protonated. The results were confirmed by comparing the absorption spectra in binary
solvents with those in acidic and basic aqueous solutions, and by DFT calculations of the Franck–Condon
S1 ’ S0 transitions. The number of water molecules solvating the polar sites in each HQ molecule was
estimated from the spectral change in the binary solvent mixtures, and structures were calculated by DFT.
Mapping the water density around the polar sites in each HQ using molecular dynamics (MD) simulations
shows well-defined hydrogen bonds around the N-heteroatom in each HQ molecule. Water density is only
well-defined around the hydroxyl group in 8HQ. The MD simulations indicate free rotation of the OH
group in 6HQ and 7HQ, and the stability of the cis-isomer in 8HQ. The results point to the unique spectral
signatures of 7HQ in water which make this molecule a potential probe to detect the presence of water in
nanocavities of macromolecules, and to the ability of the three HQs to detect acidic media in binding sites.

1 Introduction molecules containing both acidic and basic functional

Characterization of the binding sites in biological systems is
important in correlating their interior properties to their structure
and functionality.1 These properties play a crucial role in the
specificity and affinity of protein–ligand binding which is important
to understand critical biological functions such as the role of drugs
when bound to enzymes and receptors.2,3 Such clarification is an
essential step towards optimizing drug formulae and maximizing
drug potency and efficacy.
One effective way to study binding sites in proteins is by
using small molecular probes as ligands. Several small organic
a
Department of Chemistry, Faculty of Science, Sultan Qaboos University,
P.O. Box 36, Postal Code 123, Muscat, Sultanate of Oman.
E-mail: abouzied@squ.edu.om; Fax: +968-2414-1469; Tel: +968-2414-1468
b
Department for Theoretical Chemical Biology, Institute for Physical Chemistry,
Karlsruhe Institute of Technology, Kaiserstr. 12, 76131 Karlsruhe, Germany
† Electronic supplementary information (ESI) available: Absorption spectra of
HQs in dioxane–water binary mixtures; absorption spectra of 6HQ in dioxane–
methanol and methanol–water binary mixtures. See DOI: 10.1039/c3cp52811a
groups show high sensitivity in their spectra to the change in
polarity and the hydrogen bonding nature of their immediate
environment.4,5 They can thus be used to report on their local
environment after binding to a protein, and may serve as models
to study proton transfer in enzymes and other proteins. We have
recently used three hydroxyquinolines (6-hydroxyquinoline
(6HQ), 7-hydroxyquinoline (7HQ) and 8-hydroxyquinoline
(8HQ)) as local reporters to examine one of the drug binding
sites of human serum albumin.6–8 The results point to the
existence of water inside the binding site and the role of water
in molecular recognition and ligand binding. HQs are useful in
this regard due to the high sensitivity of their spectra to protic
solvents. The small size of the HQ backbone is not expected to
induce large perturbations at the protein binding sites. The
absorption spectra of most HQs extend to a region which is
transparent to proteins. This fact is expected to provide selective
excitation of the probes while encapsulated in the macro-
molecular binding sites, and the ability to study the effect of
binding on the spectra of the probes.

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Fig. 1 Structures of mono-hydroxyquinolines.
In the present work, we characterize the spectra of 6-, 7-, and
8HQ (structures of all mono-hydroxyquinolines are shown in
Fig. 1) in different solvents, particularly in aqueous medium, in
order to understand the photophysics of the reversible inter-
action of HQs with water molecules. The current study is an
extension to our recent report on the spectroscopy of 7HQ.9
The chemistry of HQs has attracted special interest due to
their therapeutic properties and the fact that they are well-known
bidentate ligands.10 HQs belong to the class of molecules that can
exist in enol as well as keto forms by enol–keto tautomerization. The
most stable structure of HQs in their ground state is the enolic
form.11 It has been shown that direct intramolecular proton transfer
is possible only in 2HQ and 8HQ, whereas solvent-assisted proton
transfer is clearly needed for the other HQs owing to the large
distance between the hydroxyl group and the N-heteroatom.12
The different OH locations in HQs provide an opportunity to study
the mechanisms of intramolecular versus intermolecular proton
transfers. These two different mechanisms may be useful in
detecting water in binding sites of macromolecules.
HQs can exist in five different forms, the enol molecule (E),
the protonated (C), the deprotonated (A), the keto (K), and the
zwitterion (Z) species.13,14 Aqueous solutions of 7HQ, as well as
of 3HQ and 6HQ, are reported to undergo stepwise excited-state
tautomerization by forming a deprotonated intermediate species.
Photoinduced excited state proton transfer (ESPT) in 7HQ is a
unique reaction system.11 In a neutral aqueous solution, 7HQ
exists mostly as E (67%) and Z (29%) with minor species of C (3%)
and A (1%).15 In other solvents, 7-HQ exists in equilibrium
between the E and the K tautomers which is solvent dependent.
Z is stabilized in aqueous medium only which represents a
valence bond resonance structure of the K tautomer that empha-
sizes its large dipole moment. Considering the structure of 7HQ,
direct intramolecular proton transfer is not possible, and thus
solvent-assisted proton transfer is the only mechanism linking
together different tautomers. Solvent-assisted ESPT was proposed
in protic solvents16,17 in which the enol and imino groups become
62 | Phys. Chem. Chem. Phys., 2014, 16, 61--70
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more acidic and basic, respectively, in the excited state than in
the ground state.13 As a result, the K resonance species was
reported to form in nonaqueous protic solvents through a
solvent chain,18 whereas the Z resonance species was proposed
to form in aqueous solvents via a deprotonated intermediate
species.19,20 Fluorescence was observed from the E tautomer
only in nonprotic solvents, whereas in aqueous solution
fluorescence was observed from the Z tautomer only.7,9,21,22
The formation of the Z tautomer was proposed to be through a
cyclical complex between 7HQ and water molecules.23 We
found the number of water molecules in this network to be
three.8 Dual fluorescence was observed in alcohols.16,22,24 In
the ground state, the E tautomer is the only species that exists
in nonprotic and nonaqueous protic solvents, while both the E
and the Z tautomers are stable in comparable amounts in
aqueous solution.9,21
2HQ is also reported to exist in two tautomeric forms, which
are nearly equal in energy. The two tautomers are the lactim (E)
and the lactam (K) forms and are interconvertable by simple
hydrogen atom transfer between the OH group and the ring
nitrogen. Nimlos et al. studied the time of flight mass spectra,
fluorescence excitation and dispersed emission spectra for 2HQ
and could not observe any evidence of ESPT (K ’ E).25 Rautela
et al. concluded that in non-polar solvents, both the E and K
forms of 2HQ exist while in polar protic solvents the K form is
predominant. In water, the E along with the Z/K forms are
proposed to exist.26
In 3HQ, the photoacidic and photobasic groups are in close
proximity to each other, making this molecule a suitable candidate
for the study of proton migration in water. 3HQ in water exists as
one of the four prototropic species: (E, C, A, and Z). The Z species
undergoes fast electron rearrangement in the excited singlet state
to form the quinonoid tautomer E. The ground-state equilibrium
constants indicate that 3HQ in water exists almost exclusively as C,
E, and A at pH values of 2, 6, and 10, respectively; in addition to
that, 6% of 3HQ exists as Z at pH 6.13 The A species transforms
rapidly into Z in the pH region of 4–6. The proton migration is
suggested to proceed by the continuous formation and breakage of
hydrogen bonds involved in the hydrated proton cluster. However,
the Z tautomer is found to undergo rearrangement immediately to
form the more stable E tautomer.27
The 6HQ molecule is reported to exist in the trans-E form in
neutral medium, the C form in acidic solution, and in the A
form in basic solution.13,28 Similar to 7HQ, the OH group in
6HQ is more acidic and the ring nitrogen atom is more basic in
the excited state than in the ground state.13 Garcı́a-Ochoa et al.
reported the absorption and emission spectra of 6HQ and 7HQ
in ethylene glycol and glycerol.29 Unlike 7HQ, 6HQ is unable to
form the K species in ethylene glycol via a hydrogen bond
bridge, whereas in glycerol, a solvent molecule that is longer
than the ethylene glycol molecule, the fluorescence spectra
exhibit two bands, one for the E tautomer (380 nm) and one
for the Z tautomer (575 nm). Both 6HQ and 7HQ have the acidic
(H-bond-donating) and the basic (H-bond-accepting) sites at a
relatively large distance. Thus a single solvent molecule cannot
form a H-bonded bridge between the donor and the acceptor.28
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In 8HQ, similar to 2HQ, the close proximity between the OH
group and the N-heteroatom enhances the efficiency of intra-
molecular proton transfer and the ability of a single protic
solvent molecule to bind to both sites simultaneously. Previous
studies on 8HQ have shown that this molecule is fluorescent in
concentrated acidic media but not in dilute acidic, neutral, or
basic aqueous solutions.30–33 8HQ has a low fluorescence
quantum yield in many solvents which can be explained as a
consequence of the quenching action of the strong hydrogen
bond that leads to active intramolecular proton transfer in the
excited state.31,34 The fluorescence quantum yield is slightly
higher in solvents of intermediate polarity such as THF and
CHCl3.35 Water can be a possible environment for 8HQ iso-
merization to trans-E as predicted by molecular dynamics and
ab initio computations. The presence of trans-E in H2O suggests
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thermodynamic integration formalism in Amber, using 19 evenly
spaced l-points with independent 1 ns equilibration and 2 ns
data collection phases for each sub-step. The simulation setup
represents the removal of the solute from a box of water
molecules, using soft core potentials41 to facilitate an efficient
single step transformation.
3 Results and discussions
3.1 Absorption spectra and structures of different tautomers
The absorption spectra of HQs in water and dioxane are shown
in Fig. 2. The peak in the region 310–340 nm is due to transition
to the lowest 1(p,p*) excited state of the E tautomer.18 This peak
is structured in dioxane for 6HQ and 7HQ which can be

an alternative fluorescence quenching reaction path in which a
direct participation of solvent molecules induces intermolecular
proton transfer.35
We examine in this paper the spectra of 6HQ, 7HQ, and 8HQ
in different solvents with an emphasis on aqueous solvents in
order to better understand the change in their spectra when
incorporated into macromolecules. We investigate the mechanism
of water solvation of the polar centers in HQs, experimentally in
mixtures of binary solvents, and theoretically by density functional
theory (DFT) and molecular dynamics (MD) simulations.
attributed to weak solute–solvent interaction. The peak is broad
in water indicating a strong interaction between the solvent and
each of the OH group and the N heteroatom of the HQ
molecules. For 8HQ, the E peak is broad in both solvents which
can be explained by the presence of a strong intramolecular
hydrogen bond. In water, a second peak at 400 nm is observed
in 7HQ which is due to absorption of the Z tautomer that gains
its intensity at the expense of the E tautomer.9,23

2 Experimental and theoretical methods

6HQ (98%) and 7HQ (99%) were obtained from Acros Organics.
8HQ (99%) was purchased from Sigma-Aldrich. All HQs were
recrystallized from ether and the purity was checked via thin
layer chromatography and from their absorption and fluorescence
spectra in different solvents. Anhydrous dioxane was obtained
from Sigma-Aldrich Chemical Co. Deionized water (Millipore) was
used. For acidic media, fuming HCl (36.5–38.0%, analytical grade
from Sigma-Aldrich) was used to adjust the pH of the solution,
while 1.0 M NaOH (Pellets, >99%, Sigma-Aldrich) was used for
the basic media. The concentration of HQs in all solvents was
0.01 mM. For the study in binary mixtures, a stock solution of HQ
in dioxane (5 mM) was prepared. Equivolume amounts (50.0 mL)
of HQ in dioxane were pipetted into separate test tubes and
diluted with the appropriate amounts of dioxane–water to make
volume:volume (v/v) mixtures.
Absorption spectra were obtained using an HP 845 Diode
Array spectrophotometer. In all the experiments, samples were
contained in a 1 cm path-length quartz cell and the measure-
ments were conducted at 23  1 1C.
Geometry optimization and time-dependent calculations for
the different tautomers of HQs uncomplexed and complexed
with water molecules were carried out using the GAMESS
program.36 Polarizable continuum model (PCM) calculations
were carried out using Gaussian 03.37 MD simulations were
Fig. 2 Absorption spectra of HQs in water and dioxane. The spectra are
performed using the AMBER 11 program.38 The gaff forcefield vertically spaced for clarity. Vertical lines represent the DFT calculations for
with AM1BCC charges and the TIP4PEW water model were the Franck–Condon S1 ’ S0 transitions (dashed lines for gas phase, dotted
used.39,40 Free energies of solvation were computed using the lines for PCM).

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Table 1 Summary of DFT calculations Table 2 Calculated differences (Z–Ecis) in selected properties

6HQ 7HQ 8HQ E |EPCM  EBare| |DGsolv| N charge N charge

DFT DFT MD DFT-PCM AM1BCC
Bare PCM Bare PCM Bare PCM
(kcal mol1) (au)
Normalized DE (kcal mol1)
cis-E 0 0 0 0 0 0 2HQ 4.19 3.65 4.29 0.47 0.30
trans-E 0.43 0.34 1.10 0.35 8.00 1.82 4HQ 3.40 4.19 5.41 0.50 0.22
Z 20.32 6.22 11.56 1.63 15.36 4.66 7HQ 11.56 9.93 5.77 0.52 0.45
3HQ 14.67 10.53 10.10 0.55 0.62
S1 ’ S0 (nm) 6HQ 20.32 14.10 9.92 0.58 0.65
cis-E 310 315 303 310 340 331 8HQ 15.36 10.70 10.59 0.64 0.69
Z 596 484 440 413 655 536

The structures of the E and Z tautomers in each HQ
molecule were calculated in their ground states using the
DFT-B3LYP method with a 6-311++G(2d,p) basis set. The calcu-
Published on 10 October 2013. Downloaded on 26/12/2013 08:03:34.
and MD calculations for 2-, 3-, 4-, 6-, 7-, and 8HQ, summarized
in Table 2, show two distinct sets of behaviors. For 3-, 6-, and
8HQ, hydrogen transfer from oxygen to nitrogen can only yield
the Z-tautomer, and the energy differences between the E and Z

lations were carried out for the bare molecules (gas phase) and
including the PCM with water as the solvent. The structures
were fully optimized without any symmetry constraint. The
results are summarized in Table 1. For the gas phase tauto-
mers, the optimized structures were found to be planar in all
cases, with the cis-E tautomers more stable than the Z tauto-
mers in 7HQ and 8HQ. The trans-E isomer (in which the O–H
bond points away from the N heteroatom) is calculated to be 1.1
and 8.0 kcal mol1 less stable than the cis-E isomer in 7HQ and
8HQ, respectively. Similar results were obtained at the HF/
6-31G(d,p) level of calculations for 7HQ.42 Experimental results
in supersonic jets indicate that the trans-rotamer is barely
populated for the free 7HQ molecule, as well as for 7HQ
complexed with one water molecule.42 In 6HQ, although our
calculated total energy for the trans-E rotamer is more stable
than the cis one (only by 0.4 kcal mol1), the difference is such
that both rotamers are expected to contribute to the total
population through a near free-rotation of the OH group
(kT = 0.6 kcal mol1 at 298 K). The existence of both rotamers
was observed in jet-cooled spectra of the bare 6HQ molecule, as
well as for 6HQ complexed with one H2O molecule.28
It is observed that the energy difference between the cis and
the trans isomers decreases when including the PCM. In the
case of 8HQ, a dramatic decrease in the energy of the trans-E
narrows the gap between the two isomers which may indicate
that the trans isomer gains stability in water. This is as expected
with the breaking of the intramolecular hydrogen bond, some-
what compensated for by the solvation of the polar centers.
Table 1 also shows that the energy difference between the cis-E
and the Z tautomers in each HQ decreases dramatically when
applying the PCM (compared to gas phase). The relative popu-
lation of the Z tautomer to the cis-E tautomer is estimated from
the calculated energies for each tautomer using the PCM to
be >0.003% for 6HQ, 6.4% for 7HQ, and >0.04% for 8HQ. The
values are consistent with the observation of the Z tautomer in
7HQ (as evidenced by the absorbance at 400 nm in Fig. 2) and
the absence of any absorption due to the Z tautomer in 6HQ
and 8HQ. While the Z resonance form is likely to be favored
over that of the K form in water, the contribution of the K
resonance form appears to remain an important factor in the
relative stability of the Z/K tautomer vs. the E tautomer. DFT
forms are very large. For 2-, 4-, and 7HQ, both Z and K
resonance structures can be drawn. In this case the energy
difference between the E and Z/K forms is calculated to be
considerably smaller for 7HQ and reversed (Z/K more stable
than E) for 2HQ and 4HQ. When the PCM is applied the Z(/K)
forms are all preferentially stabilized with respect to the E, with
3-, 6-, and 8HQ (charged-separated Z form only) stabilized to a
greater extent. However, without the extra stability imparted by
the contribution of the K form, 3-, 6-, and 8HQ are not
sufficiently stabilized to expect any significant population of
the Z tautomer in water. Calculation of the nitrogen charge
(DFT-PCM or AM1BCC atomic partial charges from MD para-
meterization) and DGsolv (MD) also support the premise that the
contribution of the K form to the resonance is not insignificant:
the changes in charge (Z–E) and DGsolv (Z–E) are consistently
larger when the K form cannot be drawn.
Returning to our discussions of the spectra of 6-, 7-, and
8HQ, the calculated Franck–Condon S1 ’ S0 transitions in the
cis-E and Z tautomers are included in Table 1 and are displayed
in Fig. 2 as vertical lines. The values are in good agreement with
the measured absorption spectra shown in Fig. 2. The results
also indicate that the peak at B330 nm in each HQ is due only
to the corresponding cis-E tautomer with no contribution from
the Z tautomer. The peak at B400 nm for 7HQ is also due solely
to the Z tautomer.
3.2 Solvation of HQs by water
The above results indicate a strong interaction between HQs
and water. The selective stabilization of the Z tautomer in 7HQ
in the presence of water is due to solute–solvent interactions
that not only determine the relative stability of the tautomeric
forms but can also influence the interconversion mechanism
(E " Z). A protic solvent like water, a strong hydrogen bond
donor–acceptor, can accept a proton from the donor site of the
solute molecule and transfer a different proton to the acceptor
site of the solute. Water-assisted proton transfer mechanism
studies have shown that the assistance of a water molecule
significantly lowers the free energy barriers in proton-transfer-
related reactions.43,44 The dynamics of such proton transfer
reactions can be greatly influenced by the presence of water

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molecules through short-range hydrogen-bonding interactions. In
this case, an explicit interaction with a limited number of water
molecules could influence the whole reaction path by lowering the
energy barrier due to the direct participation of water molecules in
the proton transfer process. The absence of the Z absorption in
other protic solvents such as methanol and ethanol7,9,15,21,22 may
indicate that, unlike water, short range interaction between solvent
molecules and the polar centers in 7HQ is not enough to stabilize
the Z tautomer in the ground state.
The tendency of water molecules to strongly associate with
each other through intermolecular hydrogen bonds allows
more than one molecule of water to form a solvent network
or a solvent wire along which proton transfers can take place to
and from the solute. Such proton transfer mechanisms through
a water bridge have been formulated theoretically.44–47 We have
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recently estimated the number of water molecules solvating the
hydrogen bonding centers of 7HQ and several other systems
experimentally in binary mixtures of dioxane–water48–50 and
theoretically using ab initio methods.48,49 The choice of dioxane
is convenient because it is miscible with water in all proportions
and thus provides an opportunity to study the effect of a broad
range of solvent polarity. Mixtures of dioxane and water were
also proposed as media to study probes in nanoenvironments
similar to those encountered in vesicles and at interfaces.48,50–53
3.2.1 6HQ:H2O adducts. Fig. 3 shows the absorption spectra
of 6HQ in dioxane, water, and in 80% water in dioxane. Unlike
the spectra in neat dioxane and water, binary mixtures of the two
solvents show a broad peak at B350 nm which increases in
intensity for water volume fraction (Vw) >0.2 as shown in the
inset of Fig. 3. The peak reaches a maximum intensity when Vw =
0.6–0.8, then drops sharply in intensity and vanishes in pure
water. This peak is not likely to correspond to a zwitterionic or a
ketonic species since both are absent in water and do not show
any appreciable population theoretically in the ground state as
Fig. 3 Absorption spectra of 6HQ in different media as indicated in the
graph. Vertical lines represent the DFT calculations (PCM) for the Franck–
Condon S1 ’ S0 transitions in the protonated (dotted line) and deproto-
nated (dashed line) species. The inset shows the absorbance change of the
peak at 350 nm as a function of water volume fraction in the binary
mixtures of dioxane and water. The solid line represents the best nonlinear
regression fit to eqn (3).
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discussed above. In order to assign the origin of this peak, we
measured the absorption spectra of 6HQ in acidic (pH 2.0) and
basic (pH 13.0) aqueous solutions. The spectra are displayed in
Fig. 3. It is clear that the spectrum in 80% water in dioxane
shows a large similarity with that of acidified 6HQ. The calculated
Franck–Condon S1 ’ S0 transitions for the protonated and
deprotonated forms using the PCM are included in Fig. 3 as vertical
lines. The calculated transitions imply that the new peak in the
binary mixtures is most likely due to the protonated form. The
position of the calculated transition for the protonated species
(362 nm) falls within the observed peak in binary solvents, whereas
that for the deprotonated species (400 nm) is almost outside the
experimental range.
Formation of the protonated species is a consequence of
specific 6HQ–water H-bonding interaction that is absent in
neat water solvent. (Dioxane alone is not a hydrogen bond
donor solvent according to its Kamlet–Taft solvatochromic
parameter (a = 0).54) The formation of the protonated form in
a water-rich dioxane mixture must then stem from the local
solvation of the N-heteroatom of 6HQ by water molecules that
behave in an acidic manner in the presence of dioxane. We have
shown recently that a molecule such as 2-(20 -hydroxyphenyl)-
benzoxazole experiences a basic environment in its excited state
when dissolved in dioxane or methanol containing high water
contents.48 Dioxane and methanol solvents that contain high
water contents are reported to have higher ionization strength
than solvents containing low water contents.55 To our knowl-
edge, there have been no reports on either acidic or basic effects
on the ground state of any dye dissolved in binary mixtures of
dioxane or methanol with water. This may be due to undetect-
able or no effects on the ground state because of little to no
molecular response. The present study may be the first to report
an acid-like influence by a dioxane–water binary mixture on
the ground state of a solute molecule. This effect could be
a consequence of the amphoteric character of water that
can behave as either an acid or a base.54 In order to confirm
this behavior, we studied 6HQ in binary mixtures of dioxane–
methanol and methanol–water (data are included in the ESI†).
The results show no acidic or basic effect of the binary solvents,
which emphasizes the unique behavior of water in the presence
of dioxane.
The acidic effect of the binary mixtures of dioxane and water
on 6HQ can be explained by selective solvation of the polar sites
of 6HQ by water. In pure dioxane no absorption for the
protonated species is observed. As the water content is
increased the absorbance at 350 nm steadily increases until
dropping-off sharply as the solvent mixture approaches pure
water. This behavior can be used to investigate the solvation of
the protonated species. Increasing the water content leads to
local solvation of the polar sites by n water molecules which can
be expressed by the following equilibrium:
Keq
6HQ þ nH2 O Ð 6HQHþ : ðH2 OÞn1 þOH (1)
Water molecules in the first solvation shell will only participate
with a certain number (n) and the rest of the water molecules
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will have the same effect as bulk water. The formation of the
protonated species is then a consequence of solvation by n
confined water molecules. In this equation, Keq = [BS]/[B][S]n,
where Keq is the equilibrium binding constant, [BS] represents
the concentration of 6HQH+, [B] is the concentration of unsol-
vated 6HQ, and [S] is the water concentration. An expression
relating the relative concentrations to the observed absorbance
(Aobs), and to the absorbance of solvated 6HQH+ (ABS) and
unsolvated 6HQ (AB) can be written as:48–50,56
Fig. 4 Structures of the most stable ground-state minimum configura-
½BS ðAobs  AB Þ tions of the cis-E and Z tautomers of 6HQ with networks of five water
¼ (2) molecules. The structures were obtained from DFT calculations described
½B ðABS  Aobs Þ
in the text. The values in parentheses are relative energies in kcal mol1.
Using eqn (2) to rewrite the equilibrium constant, one obtains The lengths of the hydrogen bonds are shown (in Å).
the following expression:
AB þ Keq ABS ½Sn module of Amber was used to produce the water density maps
Published on 10 October 2013. Downloaded on 26/12/2013 08:03:34.

Aobs ¼ (3)
1 þ Keq ½Sn shown in Fig. 5. The maps show well-defined hydrogen bonds
about the N-heteroatom and much less defined hydrogen bonds
Eqn (3) represents a binding isotherm between species in
about the hydroxyl group in the E tautomer. For the Z tautomer,
equilibrium. The measured change in the absorbance of
the results show well-defined hydrogen bonds around both polar
6HQH+ as a function of water volume fraction is displayed in
sites. The results show clearly that the OH group adapts an
the inset of Fig. 3 (assuming the density of water = 1.0 g mL1,
average position to the H atom in which no clear cis/trans isomer
Vw = [S]  molecular weight of water). The best fit to eqn (3) is
is favored. This result agrees with the above DFT calculations in
also shown. The calculations from the best fit show that five
which the OH group possesses a free rotation.
water molecules locally solvate the polar groups in the 6HQ
Additionally, H-bond networks can be analyzed directly from
molecule (n = 5). The estimated value for Keq from the fit is
MD trajectory structures. We count the number of structural
(1.5  0.2)  109 M5. It should be noted here that Keq
snapshots in which one to four water molecules connect the
represents an equilibrium constant for the binding of five
polar sites of 6HQ by a continuous H-bond network, based on
water molecules. Further, the solvent polarity also changes with
standard structural parameters of the Amber ptraj module. Very
increasing the water concentration in the binary mixture which
few such water wires were found, with a water chain of length
may affect Keq.
4 occurring in 3% of snapshots and 0% of shorter chains
The inset of Fig. 3 indicates that the absorbance of 6HQH+
observed. This is in good agreement with the above results,
drops sharply after water volume fraction >80% (equivalent to
where five water molecules are needed to connect the polar sites.
[H2O] > 44 M). The behavior of binary mixtures of dioxane and
3.2.2 7HQ:H2O adducts. Fig. 6 shows the absorption spectra
water was investigated using the Kamlet–Taft parameter a and was
of 7HQ in water, dioxane, and in 80% water in dioxane. Similar
found to approach that of pure water above 25 M concentration of
to the case of 6HQ, the formation of 7HQH+ is evidenced in
water.57–59
water-rich binary solvents in which the spectrum is similar to
Our calculations, using the DFT-B3LYP method with a
that in aqueous pH 2.0. The calculated Franck–Condon S1 ’ S0
6-311++G(2d,p) basis set, show that 5 water molecules are
sufficient to link the polar sites in 6HQ. The structures shown
in Fig. 4 were produced by adding water molecules in close
proximity to the polar sites and then fully optimizing the
geometries. The produced water network could provide a
means to facilitate hydrogen transfer from oxygen to nitrogen.
However, in line with the PCM calculations, the relative
energies of these structures suggest that even if such a network
were formed, hydrogen transfer is still energetically prohibited:
the cis-E:5H2O complex was calculated to be more stable than
the Z:5H2O complex by 11.25 kcal mol1.
Further insight into the solvation of 6HQ by water can be
obtained from MD simulations. Simulations were performed in
explicit solvent for both the E and Z tautomers embedded in a
12 Å deep layer of TIP4PEW water molecules and equilibrated Fig. 5 Mapping the water density around the polar sites of the E tautomer
(left) and the Z tautomer (right) of 6HQ. The calculations were performed
to room temperature and pressure using standard procedures.
using MD simulations as described in the text. Dark (red) areas indicate
Parameterization of the 6HQ was done using the gaff forcefield increased oxygen density (H-bond acceptor position), and light (white)
and AM1BCC charges were assigned. After equilibration, 10 ns areas indicate hydrogen density (H-bond donor regions). The average
of unrestrained MD simulations were conducted and the ptraj structures of E and Z are superimposed onto the maps.

66 | Phys. Chem. Chem. Phys., 2014, 16, 61--70 This journal is © the Owner Societies 2014

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Fig. 6 Absorption spectra of 7HQ in different media as indicated in
the graph. Vertical lines represent the DFT calculations (PCM) for the
Franck–Condon S1 ’ S0 transitions in the protonated (dotted line) and
deprotonated (dashed line) species. The inset shows the absorbance
change of the peaks at 350 and 405 nm as a function of water volume
fraction in the binary mixtures of dioxane and water. The solid line
represents the best nonlinear regression fit to eqn (3).
transitions for the cationic and anionic forms using the PCM
(shown as vertical lines in Fig. 6) also support this conclusion.
Fitting the absorbance change of the protonated species to
eqn (3) gives n = 4 (Keq = (1.7  0.2)  107 M4), which is an
estimate for the number of water molecules needed to solvate
the polar sites (the fit is shown in the inset of Fig. 6). This may
indicate that the local solvation of the neutral and protonated
forms is subtly different as we have estimated recently the value
of n to be three for 7HQ from the fluorescence change of the Z
tautomer as a function of water concentration in the binary
mixtures.9 Our DFT calculations indicate that three water
molecules are needed to maintain the planar structures of the
cis-E and the Z tautomers with a stretched water network that
reaches the two polar regions in each tautomer. The optimized
structures are shown in Fig. 7 with the corresponding hydrogen
bonding lengths (I for cis-E:3H2O and II for Z:3H2O). As shown
in the figure, the water network (or water wire) forms strong
hydrogen bonds with each polar center in each tautomer. The
dihedral angles between the molecular plane and the hydrogen
bonds at each polar center in 7HQ are less than 21 in all cases
and all XOX angles are close to linearity (Z1761).The cis-E:3H2O
complex was calculated to be more stable than the Z:3H2O
complex by 6.44 kcal mol1.
The results of mapping the water density around the polar
sites in 7HQ are shown in Fig. 8. The maps show well-defined
hydrogen bonds about the N-heteroatom, but no directional
hydrogen bonds around the hydroxyl group. Free rotation of the
OH group is evident from the average structure location of the
H-atom being nearly collinear with the C–O bond, with no
preference for either the cis- or the trans-isomer. Although the
OH group is free to rotate during simulations, interestingly,
conducting the 7HQ parameterization with the cis-isomer gives
atomic partial charges that are slightly different from those
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Fig. 7 Structures of the most stable ground-state minimum configura-
tions of the cis-E and Z tautomers of 7HQ with three water molecules. The
structures were obtained from DFT calculations described in the text.
The values in parentheses are relative energies in kcal mol1. The lengths
of the hydrogen bonds are shown (in Å).
Fig. 8 Mapping the water density around the polar sites of the E tautomer
(left) and the Z tautomer (right) of 7HQ. The calculations were performed
using MD simulations as described in the text. Dark (red) areas indicate
increased oxygen density (H-bond acceptor position), and light (white)
areas indicate hydrogen density (H-bond donor regions). The average
structures of E and Z are superimposed onto the maps.
obtained when parameterizing the trans-isomer. In our previous
results, we started parameterization from the trans-isomer and
while the results showed essentially the same average molecular
structure a well-defined hydrogen bond in one direction was
observed.9 Since the cis-isomer of 7HQ was found to be the
more stable here, we reparameterized 7HQ using this starting
structure for the present work. No other HQ isomers exhibited
this strong starting structure bias effect.
For the Z tautomer, the maps show well-defined hydrogen
bonds around the N-heteroatom and slightly less well-defined
hydrogen bonds around the oxygen atom.
An H-bond network analysis as described above found that
water chains of three and four molecules connected the polar
groups of 7HQ in 4% and 11% of structural snapshots (no
shorter chains were found), again in good agreement with the
values of n = 3 or 4 found here and in previous work.
Finally, the absorption signature of the Z tautomer starts
to emerge at 405 nm (Fig. 6) in the binary mixtures only when
Vw > 0.70, and increases sharply for Vw Z 0.90. Both E and Z
are not formed when the protonated form is present in high
concentration in the binary mixtures. This is expected because any
Phys. Chem. Chem. Phys., 2014, 16, 61--70 | 67
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E or Z tautomers will be protonated in an acidic environment. It is

only when the local environment is neutral that both should be
stabilized.
3.2.3 8HQ:H2O adducts. Fig. 9 shows the absorption spec-
tra of 8HQ in the same media. Similar to 6HQ and 7HQ, the
absorbance of the protonated form is clearly observed at
360 nm in the binary mixtures. Fitting the absorbance increase
of the protonated form to eqn (3) as a function of Vw yields n = 4
and Keq = (9.1  0.3)  108 M4.
Four water molecules seem to be a large number if the E
tautomer is in the cis form. We calculated structures of both the
Fig. 10 Structures of the most stable ground-state minimum configura-
cis-E and the Z tautomers with one water molecule linking the
tions of the cis-E and Z tautomers of 8HQ with one water molecule linking
polar sites using the DFT method (Fig. 10). The results show the polar sites. The structures were obtained from DFT calculations
that one water molecule is enough to maintain the planarity of described in the text. The values in parentheses are relative energies in
the molecule and to connect the polar sites in each tautomer. kcal mol1. The lengths of the hydrogen bonds are shown (in Å).
Published on 10 October 2013. Downloaded on 26/12/2013 08:03:34.

The calculations show that there are two intermolecular H-bonds

where H2O acts as a bridge between the ring nitrogen atom and
the OH group. The cis-E:(H2O) complex was calculated to be
more stable than the Z:(H2O) complex by 8.74 kcal mol1.
Similar results were obtained by B3LYP calculations in which
the intermolecular hydrogen bond distances are found to be
shorter (stronger) in the 8HQ:H2O complex than the intra-
molecular hydrogen bonds, indicating that the proton transfer
process takes place more easily in the complex.60
MD simulation results clearly indicate that the E tautomer is
stable in the cis isomer (Fig. 11). Mapping the water density
around the polar sites in 8HQ shows well-defined hydrogen
bonds near the polar sites in each of the tautomers (cis-E and Z).
For the neighboring polar groups of 8HQ, the analysis of the
H-bond network from MD simulations indicated that a single
water molecule often connects the two sites (8% of snapshots)
with longer water chains observed frequently as well, in 3%, 6%
and 11% of structural snapshots respectively. The differences
in these water H-bond chains found for the three HQ molecules
Fig. 9 Absorption spectra of 8HQ in different media as indicated in the
graph. Vertical lines represent the DFT calculations (PCM) for the Franck–
Condon S1 ’ S0 transitions in the protonated (dotted line) and deproto-
nated (dashed line) species. The inset shows the absorbance change of the
peak at 360 nm as a function of water volume fraction in the binary
mixtures of dioxane and water. The solid line represents the best nonlinear
regression fit to eqn (3).
Fig. 11 Mapping the water density around the polar sites of the cis-E
tautomer (left) and the Z tautomer (right) of 8HQ. The calculations were
performed using MD simulations as described in the text. Dark (red) areas
indicate increased oxygen density (H-bond acceptor position), and light
(white) areas indicate hydrogen density (H-bond donor regions). The
average structures of E and Z are superimposed onto the maps.
correlate nicely with the increasing distance between O and N.
Few water molecules are needed to form a chain for 8HQ, 3–4
molecules for 7HQ and more than four for 6HQ.
Participation of two water molecules in the intermolecular
proton transfer process is shown to increase the fraction of the
trans-E tautomer.35 The existence of higher aggregates of
8HQ:(H2O)n has been postulated for low concentrations of
8HQ in chlorinated solvents.12 Amati et al. reported the prob-
ability distribution of the torsion angle between the OH group
and the aromatic rings of 8HQ and observed a doublet around
the cis-isomer.35 The two peaks of the doublet are shifted by
about +31 and 31 from 1801. The authors correlate this
observation with their calculated cis-conformer that shows a
similar torsion angle only when the 8HQ is solvated by four
water molecules. Our experimental estimate of four water
molecules (vide supra) may then be correlated with the existence
of this conformer.
4 Conclusions
In this work, we studied the ground state tautomeric equilibria
of 6, 7, and 8HQs in neat water and dioxane, and in their binary

68 | Phys. Chem. Chem. Phys., 2014, 16, 61--70 This journal is © the Owner Societies 2014

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mixtures. The results indicate that the enol form is the only
tautomer for the three HQs in dioxane and water, with the
exception of 7HQ in which both the enol and the zwitterion
tautomers exist in equilibrium in water. The different tautomers
were assigned using DFT calculations performed in the gas
phase and using PCM. The existence of a protonated species
in each molecule is detected in the absorption spectra when the
water concentration is high in the mixture (maximum protona-
tion was observed for Vw between 60–80%). The existence of
protonated species was confirmed by comparing the absorption
spectra in binary solvents with those in acidic and basic aqueous
solutions, and by DFT calculations of the Franck–Condon
S1 ’ S0 transitions. We determined the number of water
molecules solvating the polar sites in each HQ molecule by
fitting the absorbance change to a binding isotherm, and the
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structures were calculated by DFT. MD simulations indicate that
the water density is well-defined around the N-heteroatom in all
HQs, whereas the water density is only well-defined around the
hydroxyl group in 8HQ. The results from the MD simulations
point also to the presence of free rotation of the OH group in
6HQ and 7HQ, and the stability of the cis-isomer in 8HQ.
The current study clarifies the effect of various polarities of
the environment on the formation and stability of different
tautomers of HQs in the ground state and should help in
understanding the nature of binding sites in macromolecules
when HQs are used as local reporters. One possible application
of HQs is utilizing them to detect acidic media in binding sites.
The unique spectral signatures of 7HQ in water also make this
molecule a potential probe to detect the presence of water in
nanocavities of macromolecules.
Acknowledgements
The authors would like to thank Sultan Qaboos University
for financial support (Grant No. IG/SCI/CHEM/12/01), and
Dr Muataz Al-Barwani for assistance with calculations carried
out at the University’s High Performance Computing Facility.
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