Solvatochromic dyes detect the presence of homeopathic potencies

Abstract

A systematic approach to the design of simple, chemical systems for investigating the nature of homeopathic medicines has led to the development of an experimental protocol in which solvatochromic dyes are used as molecular probes of serially diluted and agitated solutions. Electronic spectroscopy has been used to follow changes in the absorbance of this class of dyes across the visible spectrum in the presence of homeopathic potencies.Evidence is presented using six different solvatochromic dyes in three different solvent systems. In all cases homeopathic potencies produce consistent and reproducible changes in the spectra of the dyes.Results suggest that potencies influence the supramolecular chemistry of solvatochromic dyes, enhancing either dye aggregation or disaggregation, depending upon dye structure. Comparable dyes lacking the intramolecular charge transfer feature of solvatochromic dyes are unaffected by homeopathic potencies, suggesting potencies require the oscillating dipole of solvatochromic dyes for effective interaction.The implications of the results presented, both for an eventual understanding of the nature of homeopathic medicines and their mode of action, together with future directions for research in this area, are discussed.

Full text

ORIGINAL PAPER
Solvatochromic dyes detect the presence
of homeopathic potencies
Steven J Cartwright*
DiagnOx Laboratory, Cherwell Innovation Centre, Upper Heyford, Oxon, OX25 5HD, UK
A systematic approach to the design of simple, chemical systems for investigating the
nature of homeopathic medicines has led to the development of an experimental proto-
col in which solvatochromic dyes are used as molecular probes of serially diluted and
agitated solutions. Electronic spectroscopy has been used to follow changes in the
absorbance of this class of dyes across the visible spectrum in the presence of homeo-
pathic potencies.
Evidence is presented using six different solvatochromic dyes in three different solvent
systems. In all cases homeopathic potencies produce consistent and reproducible
changes in the spectra of the dyes.
Results suggest that potencies influence the supramolecular chemistry of solvatochro-
mic dyes, enhancing either dye aggregation or disaggregation, depending upon dye
structure. Comparable dyes lacking the intramolecular charge transfer feature of solva-
tochromic dyes are unaffected by homeopathic potencies, suggesting potencies require
the oscillating dipole of solvatochromic dyes for effective interaction.
The implications of the results presented, both for an eventual understanding of the na-
ture of homeopathic medicines and their mode of action, together with future directions
for research in this area, are discussed. Homeopathy (2015) -,1e11.
Keywords: Homeopathic potencies; Solvatochromism; Aggregachromism;
Solvatochromic dyes; Intramolecular charge transfer; Supramolecular chemistry;
Dye aggregation and disaggregation
Introduction
There is no doubt that a plausible and testable hypothesis
for the mode of action of homeopathy and, by implication,
an understanding of the physico-chemical nature of ho-
meopathic potencies, would profoundly enhance homeop-
athy, both as an area of legitimate scientific study and as an
effective medical approach.
Research at the molecular level has the advantage over
other approaches in that it can ask the kinds of searching
and detailed questions necessary to arrive at fully testable
hypotheses as to the modus operandi of homeopathy.
With this view in mind a programme of investigation
aimed at developing well-defined chemical systems
capable of detecting consistent and replicable effects of
serially diluted and agitated solutions has been initiated.
Specifically, a simple chemical system utilising environ-
ment sensitive solvatochromic dyes
1
has been developed.
Solvatochromic dyes are sensitive to, and can be used to
follow, a range of solution dynamics through changes in
their absorbance spectra which, conveniently, occur in
the visible portion of the electromagnetic spectrum.
The system described below demonstrates not only that
homeopathic potencies have in vitro effects which can be
measured, but also because the system is both simple and
versatile, very specific questions can be asked about what
molecular effects potencies are having in solution and
what their ultimate nature might be.
Whilst a range of chemical and physical systems have
been employed in the past in the study of homeopathic
medicines, including UV-spectroscopy,
2
nuclear magnetic
resonance spectroscopy,
3
thermoluminescence,
4
high
*Correspondence: Steven J Cartwright, DiagnOx Laboratory,
Cherwell Innovation Centre, Upper Heyford, Oxon, OX25 5HD,
UK.
E-mail: steven.cartwright@oxford-homeopathy.org.uk
Received 12 March 2015; revised 18 June 2015; accepted 12
August 2015
Homeopathy (2015) -,1e11
Ó2015 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.homp.2015.08.002, available online at http://www.sciencedirect.com
Please cite this article in press as: Cartwright SJ, Solvatochromic dyes detect the presence of homeopathic potencies, Homeopathy (2015), http://dx.doi.org/
10.1016/j.homp.2015.08.002

voltage plasma visualisation,
5
solution conductivity
6
and
micro-calorimetry,
7
together with theoretical studies,
8
little
consensus has emerged as to the nature of the homeopathic
stimulus. Results suggest potencies may be electromag-
netic in nature,
9
or that they may involve and exploit the
intrinsic ability of water to form complex hydrogen
bonding networks.
10
They may have their origins in quan-
tum electrodynamics,
11
quantum entanglement,
12
complexity theory
13
or stochastic resonance.
14
The present approach has grown out of a recognition that
a number of criteria need to be fulfilled if homeopathic po-
tencies are to be studied in a way that provides results that
are (i) significantly above background noise and (ii) allows
the development of systems that can be manipulated to
reveal the effect of one variable at a time. The criteria are
essentially two fold. The first involves the stability of ho-
meopathic potencies. Whilst firm evidence is lacking, it
is a commonly held belief that potencies are sensitive to ul-
traviolet light, X-rays and strong magnetic and electrical
fields.
15
For this reason it was felt that any putative detec-
tion system should avoid UV-spectroscopy, thermolumi-
nescence, NMR and high voltage plasma visualisation.
Conversely, little is known about how potencies can be
effectively destroyed. Heat is commonly held to be effec-
tive, but again evidence is lacking.
16
The destruction of po-
tencies is important if one is to avoid cross-contamination
where glassware and other container materials are re-used.
For this reason the current study has employed disposable
containers in all situations where cross-contamination is a
potential problem.
The second group of criteria revolves around the issue of
the control of variables. Ideally any detection system
should be one in which the variables involved can be ad-
dressed individually. In this way specific questions can
be asked and specific answers obtained. A well-defined
and simple detection system is therefore highly desirable,
especially if the system is capable of providing different
types of information.
With these criteria in mind a detection system involving
solvatochromic dyes has been developed. In brief, changes
have been found to occur in the absorbance spectra of these
dyes in the presence of homeopathic potencies. In turn it
has then been possible to make certain inferences as to
the specific action of potencies in solution.
A system in which homeopathic potencies are added to
solutions of solvatochromic dyes is simple, versatile, and
involves a very small number of components and opera-
tional steps. In addition, the system is sensitive to changes
in a wide range of solution dynamics including solvent po-
larity, solvent-solute binding patterns, and supramolecular
interactions between solute molecules.
To date six different solvatochromic dyes have been
investigated, three positively solvatochromic and three
negatively solvatochromic (Figure 1).
An overview of solvatochromic dyes
Solvatochromic dyes are characterised by possessing an
electron donating group and an electron accepting group
with an electron delocalised system in- between.
17
For
negatively solvatochromic dyes the ground or resting state
is zwitterionic with a formal charge at either end of the
molecule. On absorption of light an electron travels from
one end of the molecule to the other to form an excited po-
lar, but uncharged, state (Figure 2). Whilst the lifetime of
the excited state is of the order of picoseconds this rapid
electron oscillation occurs constantly under the influence
of absorbed light. Importantly the wavelength of the ab-
sorbed light is influenced by the environment in which
the dye is placed. Conversely, for positively solvatochro-
mic dyes the ground or resting state is uncharged. On ab-
sorption of light an electron travels from one end of the
molecule to the other to form an equally short-lived
charged excited state (Figure 2). Again the wavelength of
the light absorbed is dependent upon the nature of the envi-
ronment in which the dye is placed. Negatively and posi-
tively solvatochromic dyes behave differently and
complementarily to each other in a number of ways. These
are important in relation to the results obtained with po-
tencies reported below.
Negatively solvatochromic dyes absorb at longer and
longer wavelengths (bathochromically shifted) as solvent
polarity decreases. For example ET30 (Figure 1) absorbs
at 450 nm in water, 550 nm in ethanol and 650 nm in
tert-butyl alcohol.
18
In addition these dyes tend to increas-
ingly aggregate as solvent polarity decreases producing ag-
gregates that are bathochromically shifted with respect to
monomer (unaggregated material), a phenomenon known
as aggregachromism.
19
These bathochromically shifted
species take the form of ‘steps’ in solution and are known
as J-aggregates
20
(Figure 3).
Conversely, positively solvatochromic dyes absorb at
shorter and shorter wavelengths (hypsochromically
shifted) as solvent polarity decreases. For example BDN
(Figure 1) absorbs at 614 nm in water, 564 nm in ethanol
and 537 nm in tert-butyl alcohol.
21
These dyes tend to
increasingly aggregate as solvent polarity decreases pro-
ducing aggregates that are hypsochromically shifted with
respect to monomer,
22
in contrast to that seen with nega-
tively solvatochromic dyes. These hypsochromically
shifted species take the form of ‘stacks’ in solution and
are known as H-aggregates
23
(Figure 3).
Dyes ET33, ET30 and BM produce J-aggregates in solu-
tion; dyes BDN, NR and PB produce H-aggregates.
24
Results reported below exploit these complementary
properties of positively and negatively solvatochromic
dyes.
In addition, dyes ET30, ET33 and BDN bind divalent
cations
25
to produce optical changes which can also be uti-
lised to demonstrate effects of homeopathic potencies.
Precautions taken in this study
The experimental protocols followed in the course of
this study have focused on a number of different practical
issues which need to be discussed before results are pre-
sented (for details see Materials and methods section).
The first involves the use of disposable cuvettes. Whilst
Solvatochromic dyes and homeopathic potencies
SJ Cartwright
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quartz cuvettes have been found to produce greater differ-
ences between controls and potency assays, so too have the
size of variations between assays, producing large error
bars. In part this has been found to arise from the necessary
re-use of quartz cuvettes. Whilst cuvettes were placed in
boiling water between assays it is possible that such a pro-
cedure is insufficient to destroy all the potency present.
Cross contamination is therefore a real possibility between
assays. For this reason the use of single-use polystyrene
(PS) and Brand UV (UV) cuvettes has been adopted.
Although differences between controls and potency assays
are smaller than those obtained with quartz cuvettes this
has been offset by using much higher potencies (50 M)
than those used originally with quartz cuvettes (200).
Choice of homeopathic potency
Another consideration has been that of the homeo-
pathic medicines chosen for assay. It was felt that rem-
edies derived from complex plant, mineral and animal
materials should be avoided on the basis of the desire
for simplicity throughout. Many remedies at different
potencies have been investigated during the course of
the present study and the results obtained are broadly
comparable to the results presented in this paper. How-
ever, for the purposes of keeping system components
to a minimum, and with an ever present desire for
simplicity, potencies of glycerol have been chosen as
the primary source of homeopathic remedy for assay.
Glycerol is a low molecular weight compound that can
Figure 1 Structures of ET33 (2,6-Dichloro-4-(2,4,6-triphenyl-pyridinium-1-yl)-phenolate); ET30 (2,6-Diphenyl-4-(2,4,6-triphenyl-pyrinidium-
1-yl)-phenolate); BM (4-[(E)-2-(1-methylpyridinium-4-yl)ethenyl]phenolate/Brooker’s merocyanine); BDN ((4-(Bis-(4-(dimethylamino)
phenyl)methylene)-1(4H)-naphthalenone); NR (9-diethylamino-5H-benzo[a]phenoxazime-5-one/Nile Red); PB (N,N-dimethylindoaniline/
Phenol Blue); ET33, ET30 and BM are negatively solvatochromic dyes; BDN, NR and PB are positively solvatochromic dyes. See text
for an explanation of negative and positive solvatochromism.
Solvatochromic dyes and homeopathic potencies
SJ Cartwright
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be obtained at very high purity levels, is pharmacologi-
cally inactive in material doses and is fully miscible
with both water and ethanol. In addition it is very closely
related structurally to both water and ethanol. It was
therefore considered that glycerol may be more likely
than most substances to produce results that represent
the potentisation process itself, free of any specific ho-
meopathic effects that might have derived from the start-
ing material, especially where that substance is
chemically reactive or pharmacologically active.
Considerations regarding the solvent in which assays
are performed
The final practical consideration in relation to experi-
mental protocol has centred on the choice of solvent in
which to carry out assays. A question from the beginning
of this study has been whether bulk water is essential in
order to observe any effects of homeopathic medicines.
The idea that water in some way can carry the memory
of substances placed in it is an enduring one,
26
but has
ignored the fact that pharmacies produce, and practi-
tioners use, potencies that are in 90% ethanol. For this
reason three different solvents have been chosen in
which to carry out assays. These are water, ethanol and
tert-butyl alcohol (see Materials and methods). These
three solvents form a series in which there is decreasing
hydrogen bonding capacity, increasing hydrophobicity
and decreasing solvent polarity in going from water to
tert-butyl alcohol. As such it was felt this series could
provide valuable information with respect to how po-
tencyeffectsmayvaryinrelationtotheseparticularsol-
vent parameters.
Results
Results with dye ET33
Figure 4 shows a difference spectrum obtained on add-
ing an aliquot of glycerol 50 M (sample cuvette) and an
equivalent volume of control (reference cuvette), to solu-
tions of the negatively solvatochromic dye ET33 dissolved
in ethanol. Scans were then repeatedly made from 400 nm
to 800 nm over several hours (see Materials and methods).
The scan shown was taken at 120 min after mixing. There is
Figure 3 Schematic representation of H-aggregates (A) and J-aggregates (B) in solution.
Figure 2 Diagram showing the structure of ground and excited states in negatively and positively solvatochromic dyes. For the positively
solvatochromic dyes used in this study electron donating groups (D) are of the form =Neand electron accepting groups (A) are of the
form =C=O. For the negatively solvatochromic dyes used in this study electron donating groups are of the form eO

and acceptor groups
are of the form ^N
+
e. (see text for more details).
Figure 4 Difference spectrum of ET33 in ethanol with 50 mlof
control added to the reference cuvette and 50 ml of glycerol
50 M added to the sample cuvette to make a total volume of
3 ml in each cuvette (see Materials and methods). ET33 is at a
concentration of 245 mM. Difference spectrum consists of the
sample spectrum minus the reference spectrum. Difference spec-
trum shown is that at t = 120 min after mixing. UV cuvettes used.
Insert shows the change in DOD442 nm over time.
Solvatochromic dyes and homeopathic potencies
SJ Cartwright
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a relative decrease at c.542 nm and a relative increase at
c.442 nm which builds up over time.
The difference is minimal at t = 0 but slowly reaches a
maximum at 60e120 min (see Figure 4 insert). This differ-
ence spectrum should be considered in relation to the spec-
trum of ET33 in ethanol which has a broad peak at 472 nm.
ET33 aggregates in solution to produce stepped aggregates
(J-aggregates) that absorb at longer wavelengths than
monomer (bathochromically shifted).
20
Solutions of
ET33 are therefore an equilibrium mixture of monomer
and aggregates ehence the broad nature of the absorption
spectrum of ET33. What Figure 4 appears to be showing
therefore is that potency is causing ET33 to disaggregate
to produce more monomer.
Support for this proposition comes from assays utilising
divalent cations. Strontium ions interact with the phen-
oxide moiety of ET33
25
(Figure 1) resulting in a loss of
absorbance. This loss occurs in two stages: a rapid first
phase due to the direct interaction of strontium ions with
free dye, followed by a second phase arising from rate-
limiting disaggregation of dye. If potency causes a greater
level of disaggregation of dye then one might expect more
rapid loss of absorbance in the presence of potency. This is
indeed what is seen. Figure 5 shows assays in which either
control plus strontium ions or glycerol 50 M plus strontium
ions are added to solutions of ET33 in ethanol. Loss of
absorbance is more rapid in the presence of potency, sup-
porting the idea that potency enhances disaggregation of
dye.
Difference spectra of ET33 in tert-butyl alcohol with and
without potency show a decrease at c.615 nm and an in-
crease at c.490 nm (the absorbance peak of ET33 in tert-
butyl alcohol is at 548 nm). As with assays in ethanol, glyc-
erol 50 M appears to be causing enhanced disaggregation
of dye. What these results in ethanol and tert-butyl alcohol
show is that bulk water is not essential to manifest a po-
tency effect. More specifically, as tert-butyl alcohol very
poorly hydrogen bonds, it is likely that hydrogen bonding
is not essential in order to manifest a potency effect.
Results with dye ET30
Difference spectra of ET30 (Figure 1) in water, ethanol
and tert-butyl alcohol indicate enhanced disaggregation in
the presence of glycerol 50 M in all three cases. The differ-
ence spectrum in tert-butyl alcohol is one of the largest
seen in any of the dye/solvent combinations examined
(Figure 6). As with ET33, difference spectra tend to
develop over time, although on some occasions evidence
of a difference spectrum is present even from t = 0. Scans
in most of the dye/solvent combinations tested tend to show
a return to base line with loss of any difference spectrum
overnight, indicating that the potency effect is one that
slowly builds up over time, reaching a maximum some-
where between 1 and 2 h and then slowly disappears over-
night.
Consistent with the results seen with ET33, glycerol
50 M also increases the rate of complexation of strontium
ions with ET30, through enhanced disaggregation.
Results with dye BDN
Complementary evidence to that with ET33 and ET30 is
seen with the positively solvatochromic dye BDN. Figure 7
shows a difference spectrum of BDN in ethanol with and
without glycerol 50 M. Conditions are exactly the same
as in Figure 4 except BDN has been substituted for
ET33. Scans were performed at the same time intervals
as for ET33. The scan shown was performed at
t = 30 min after mixing. There is a relative decrease at
c.615 nm and a relative increase at c.480 nm. This differ-
ence spectrum should be seen in relation to the absorption
spectrum of BDN in ethanol which shows two poorly
differentiated peaks at 484 nm and 564 nm corresponding
to aggregate and monomer peaks respectively. BDN in so-
lution produces aggregates (H-aggregates) that absorb at
shorter wavelengths than monomer. As with ET33, solu-
tions of BDN are equilibrium mixtures of monomer and ag-
gregates. However, in distinction to ET33 the aggregates in
Figure 5 Absorbance loss over time at 472 nm for 245 mM ET33
in ethanol with 70 mM SrCl2 and 50 ml control (upper curve) or
70 mM SrCl2 and 50 ml glycerol 50 M (lower curve). Assays carried
out in both PS and UV cuvettes. Details of assay conditions are
given in Materials and methods. N = 20; error bars are to first stan-
dard deviation; p < 0.0001, indicating high statistical significance.
Figure 6 Difference spectrum of ET30 in tert-butyl alcohol with
50 ml of control added to the reference cuvette and 50 ml of glycerol
50 M added to the sample cuvette to make a total volume of 3 ml in
each cuvette (see Materials and methods). ET30 is at a concen-
tration of 245 mM. Difference spectrum consists of the sample
spectrum minus the reference spectrum. Difference spectrum
shown is that at t = 120 min after mixing. UV cuvettes used.
Solvatochromic dyes and homeopathic potencies
SJ Cartwright
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BDN are hypsochromically shifted. The difference spec-
trum in Figure 7 therefore appears to show that glycerol
50 M is promoting dye aggregation in BDN.
Divalent cations interact with BDN to produce a rise in
absorbance at 615 nm and a decrease in absorbance at
484 nm. As with ET33 and ET30, this complexation has
a second slower phase arising from rate-limiting disaggre-
gation of dye. If potency is enhancing dye aggregation then
one would expect potency to reduce the rate of increase in
absorbance at 615 nm compared with that in the absence of
potency. This is indeed what is seen. Figure 8 shows assays
in which either control plus strontium ions or glycerol 50 M
plus strontium ions are added to solutions of BDN in
ethanol. Gain of absorbance is slower in the presence of po-
tency, supporting the idea that potency protects BDN from
complexation with strontium ions through enhanced dye
aggregation.
Difference spectra of BDN in water, ethanol and tert-
butyl alcohol with glycerol 50 M all show increases at
shorter wavelengths and decreases at longer wavelengths
corresponding to enhanced dye aggregation, irrespective
of solvent. Solvent does not therefore appear to be playing
a direct part in the action of glycerol 50 M. Furthermore, if
potency were acting on either ET33 or BDN through
changes in solvent polarity or hydrogen bonding capabil-
ities the dyes should not be affected in opposite ways.
Both dyes tend to aggregate more strongly as solvent polar-
ity decreases and disaggregate as solvent hydrogen
bonding capability increases. What is a more likely expla-
nation is that glycerol 50 M is acting directly on both ET33
and BDN. If this is the case then the charge transfer feature
of both dyes may be the significant factor in determining
interaction.
Results with non-solvatochromic dyes
In support of the idea that the charge transfer feature of
dyes ET33, ET30 and BDN may be the significant factor in
determining interaction with potencies, a number of non-
solvatochromic dyes were investigated. Dyes were chosen
which are zwitterionic, as structurally analogous as
possible to BDN and ET33, and display aggregation in so-
lution with monomer and aggregate spectra separated by at
least 40 nm. Dyes included Patent Blue VF, Green S and
Sulforhodamine 101. No evidence of any changes in their
spectra on addition of glycerol 50 M were seen under con-
ditions identical to those used for ET33 and BDN. This
suggests that it is indeed the intramolecular electron trans-
fer feature of solvatochromic dyes that is interacting with
glycerol 50 M. This interaction then appears to result in
changes in the supramolecular dynamics of solvatochromic
dyes.
Results with other solvatochromic dyes
Further evidence of interactions between glycerol 50 M
and solvatochromic dyes comes from experiments using
the negatively solvatochromic dye BM and the positively
solvatochromic dyes NR and PB (Figure 1). All these
dyes produce significant difference spectra in the presence
of potency. BM is particularly interesting as in all three sol-
vents glycerol 50 M promotes aggregation. This is in
contrast to that seen with ET30 and ET33. Aggregates
are bathochromically shifted with respect to monomer in
BM, so difference spectra show increases at longer wave-
lengths and decreases at shorter wavelengths. Figure 9
shows the difference spectrum in tert-butyl alcohol.
The difference spectra of NR and PB with glycerol 50 M
in water, ethanol and tert-butyl alcohol indicate potency
promotes disaggregation of both dyes in all three solvents.
Table 1 lists the effects of glycerol 50 M on the spectra of
all the solvatochromic dyes examined in this study, giving
the positions of maximum absorbance changes on addition
of potency. It should be noted that for dyes ET30, ET33 and
BM in water only single difference maxima are given. This
is because all dyes absorb strongly and non-specifically
below c. 400 nm, rendering accurate difference spectra
beyond the capabilities of the instrument below this wave-
length.
Figure 7 Difference spectrum of BDN in ethanol with 50 ml of con-
trol added to the reference cuvette and 50 ml of glycerol 50 M
added to the sample cuvette to make a total volume of 3 ml in
each cuvette (see Materials and methods). BDN is at a concentra-
tion of 80 mM. Difference spectrum consists of the sample spec-
trum minus the reference spectrum. Difference spectrum shown
is that at t = 30 min after mixing. UV cuvettes used.
Figure 8 Absorbance gain over time at 615 nm for 80 mM BDN in
ethanol with 70 mM SrCl2 and 50 ml control (upper curve) or 70 mM
SrCl2 and 50 ml glycerol 50 M (lower curve). Assays carried out in
both PS and UV cuvettes. Details of assay conditions are given in
Materials and methods. N = 20; error bars are to first standard de-
viation; p < 0.0001, indicating high statistical significance.
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Table 2, in turn, gives a summary, derived from differ-
ence spectra, of the deduced supramolecular effects of
glycerol 50 M on all dyes tested in all three solvents.
Discussion
Evidence has been presented which demonstrates it is
possible to develop a simple chemical system for the detec-
tion and investigation of homeopathic potencies. Solvato-
chromic dyes provide a versatile means of probing the
nature and action of potencies in solution, providing
several levels of information. Although results given are
with glycerol 50 M only, a large number of homeopathic
remedies in different potencies have been tested and their
effects found to be similar to those presented here.
The intramolecular electron transfer feature of solvato-
chromic dyes appears to be essential for detecting the pres-
ence of potencies, as non-solvatochromic dyes do not
display any spectroscopic changes when potencies are
introduced. The interaction of potency and solvatochromic
dye leads to supramolecular changes in the solution dy-
namics of all solvatochromic dyes tested, in the form of
enhanced dye aggregation or disaggregation. Where po-
tency causes a dye to exhibit enhanced aggregation this
Figure 9 Difference spectrum of BM in tert-butyl alcohol with 50 ml
of control added to the reference cuvette and 50 ml of glycerol 50 M
added to the sample cuvette to make a total volume of 3 ml in each
cuvette (see Materials and methods). BM is at a concentration of
125 mM. Difference spectrum consists of the sample spectrum
minus the reference spectrum. Difference spectrum shown is
that at t = 40 min after mixing. UV cuvettes used.
Table 1 Table showing the positions of maximum absorbance changes (difference spectra) on addition of glycerol 50 M to solvatochromic dyes
used in this study in three different solvent systems. Absorbance maxima for dyes in respective solvents are given in plain type whilst the
positions of maximum absorbance change on addition of potency are given in italics
Dye Solvent system
Water Ethanol Tert-butyl alcohol
Negatively solvatochromic
ET33 Absorbance maxima 409 nm (broad)
Decrease at c.380e420 nm with potency
Absorbance maxima 472 nm (broad)
Increase at 442 nm and decrease
at 542 nm with potency
Absorbance maxima 548 nm (broad)
Increase at 490 nm and decrease
at 615 nm with potency
ET30 Absorbance maxima 450 nm (broad)
Decrease at c.450 nm with potency
Absorbance maxima 550 nm (broad)
Increase at 450 nm and decrease
at 580 nm with potency
Absorbance maxima 650 nm (broad)
Increase at 580 nm and decrease at
715 nm with potency
BM Absorbance maxima
380 nm (monomer)
442 nm (aggregate)
Increase at 450 nm with potency
Absorbance maxima
400 nm (monomer)
513 (aggregate)
Decrease at 390 nm and increase
at 510 nm with potency
Absorbance maxima
402 nm (monomer)
c.496 nm (minor peak) (aggregate)
576 nm (aggregate)
Decrease at 404 nm and increase
at 500 nm and 570 nm with potency
Positively solvatochromic
BDN Absorbance maxima
490 nm (aggregate)
614 nm (monomer)
Increase at 460 nm and
decrease at 620 nm with potency
Absorbance maxima
484 nm (aggregate)
564 nm (overlapping aggregate
and monomer peaks?)
Increase at 480 nm and decrease
at 615 nm with potency
Absorbance maxima
459 nm (aggregate)
537 nm (aggregate)
609 nm (monomer)
Increase at 460 nm and 535 nm and
decrease at 620 nm with potency
NR Absorbance maxima
530 nm (aggregate)
593 nm (monomer)
Decrease at c.480 nm and increase
at c.560 nm with potency
Absorbance maxima 550 nm (broad)
Decrease at c.475 nm and increase
at c.560-570 nm with potency
Absorbance maxima 538 nm (broad)
Decrease at c.465 nm and increase
at c.560 nm with potency
PB Absorbance maxima 658 nm (broad)
Decrease at c.520 nm and increase
at c.670 nm with potency
Absorbance maxima
608 nm (broad)
Decrease at c.540 nm and increase
at c.680 nm with potency
Absorbance maxima 601 nm (broad)
Decrease at c.520 nm Increase at
c.620e660 nm with potency
Table 2 Table showing the deduced effect of glycerol 50 M on the
supramolecular chemistry (enhanced aggregation or enhanced
disaggregation) of all solvatochromic dyes used in this study
Dye Effect of glycerol 50 M potency
Water Ethanol Tert-butyl alcohol
Negatively solvatochromic
ET33 Disaggregation Disaggregation Disaggregation
ET30 Disaggregation Disaggregation Disaggregation
BM Aggregation Aggregation Aggregation
Positively solvatochromic
BDN Aggregation Aggregation Aggregation
NR Disaggregation Disaggregation Disaggregation
PB Disaggregation Disaggregation Disaggregation
Solvatochromic dyes and homeopathic potencies
SJ Cartwright
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occurs in all three solvents tested, and similarly where a po-
tency causes enhanced disaggregation, this occurs in all
three solvents. This is in marked contrast to the situation
in the absence of potency where aggregation occurs more
strongly as solvent polarity decreases and disaggregation
increases as solvent hydrogen bonding capability increases
for all dyes tested. In addition the observation that potency
causes some dyes to aggregate and others to disaggregate
in the same solvent strongly suggests solvent does not
mediate in the potency effects seen and that potency is in-
teracting directly with the dyes.
Tabl e 2 shows the aggregation/disaggregation effect of
potency on all dyes tested in all three solvents. No
pattern is immediately apparent. Whether a dye is posi-
tively or negatively solvatochromic does not in itself
determine whether enhanced aggregation or disaggrega-
tion occurs. However, a more detailed examination of
thedatainTabl e 1 and the following observations may
be relevant.
BM displays well-defined monomer and aggregate
peaks in all solvents at the wavelengths shown in Table
1, indicating that dye solutions consist of two or three
distinct species. The effect of potency appears to be to in-
crease or decrease the relative proportions of these species.
For instance, when glycerol 50 M is added to solutions of
BM in tert-butyl alcohol difference spectra display three
maxima ea decrease at c.404 nm, coincident with the
absorbance maximum for BM monomer (402 nm), an in-
crease at c.570 nm, coincident with the absorbance
maximum for aggregate (576 nm) and a substantial in-
crease at c.500 nm, coincident with a minor peak at
c.496 nm, which may represent a smaller aggregate species
than that at 576 nm (Figure 9). BM therefore seems to exist
as distinct aggregate and monomer species in solution and
this appears to be reflected in the difference spectra ob-
tained with potencies.
In contrast other dyes such as ET33 and ET30 have
spectra which are broad in all solvents, indicating a spec-
trum of populations from monomer through small aggre-
gates to much larger aggregates. Despite this, when
potency is added to these dyes, the difference maxima pro-
duced are distinct and, moreover, tend to lie well outside
the absorbance peak of the dye. For instance, this can be
clearly seen from the difference spectrum of ET30 in
tert-butyl alcohol. ET30 has its absorbance peak at
650 nm, yet the difference maxima obtained on addition
of potency occur at 580 nm and 715 nm (Figure 6). This
would suggest that potency is causing highly aggregated
dye to disaggregate to form monomer, rather than causing
moderately aggregated dye to disaggregate to less moder-
ately aggregated dye (which would lead to difference max-
ima that were closer together). In other words potency is
acting discontinuously to cause the system to polarise,
rather than simply shifting the aggregate equilibrium
slightly. A slight shift in equilibrium rather than polarisa-
tion is what one might expect from an energetic point of
view, so potencies appear to be doing something markedly
unexpected in solution. Whilst the conversion of highly
aggregated dye to monomer constitutes only some 1e2%
of the total dye in the system based on absorbance values,
this is a remarkable achievement given the supposedly
ultra-weak nature of homeopathic potencies.
Some discussion is required at this point with regard to
the reproducibility of the difference spectra given in
Figures 4, 6, 7 and 9. Difference spectra for ET30 in tert-
butyl alcohol have been performed in excess of 40 times
over the course of several months and a composite
spectra of all these runs is given in Figure 10, showing
mean absorbance values at set wavelengths across the
dye’s spectrum and associated error bars. This composite
curve should be seen in relation to control difference
spectra (50 ml of control solution added to both cuvettes;
n = 20) where no discernible difference spectrum is
observed (ie 0.000 0.000) within the sensitivity and pre-
cision limits of the spectrophotometer used in this study
(see Materials and methods). This difference between sam-
ples and controls is statistically highly significant (t-test,
p#0.0001). Similar composite difference spectra can
also be derived for ET33, BDN and BM, also giving highly
significant p values of <0.0001.
For dye BDN absorbance peaks are not as well separated
or as distinct as in BM and it is likely that BDN exists as
more than two, or even three, species in solution, but still
fewer than is the case for ET30 and ET33. In the presence
of potency, increases in levels of highly aggregated species
are seen in all solvents, combined with a decrease at
615e620 nm, corresponding to that of monomer. As with
ET30 and ET33 therefore potency seems to be acting
discontinuously, in this case by preferentially converting
Figure 10 Combined difference spectra (n = 20) of ET30 in tert-
butyl alcohol with 50 ml of control added to the reference cuvette
and 50 ml of glycerol 50 M added to the sample cuvette to make
a total volume of 3 ml in each cuvette (see Materials and
methods). ET30 is at a concentration of 245 mM. Difference spec-
trum consists of the sample spectrum minus the reference spec-
trum. UV cuvettes used. Error bars are to the first standard
deviation. Control ‘difference’ spectra (n = 20) where 50 ml of con-
trol are added to both cuvettes display no discernible difference
across the spectrum from 350 to 800 nm (ie 0.000 0.000) giving
a p value of <0.0001, indicating high statistical significance.
Solvatochromic dyes and homeopathic potencies
SJ Cartwright
8
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10.1016/j.homp.2015.08.002

monomer to highly aggregated dye, rather than stimulating
a slight shift in overall equilibrium.
Dyes NR and PB both display enhanced disaggregation
in the presence of potencies, with decreases in levels of
hypsochromic species and increases in levels of bathochro-
mic species relative to the dyes’ absorbance maxima (Table
1). These two dyes are significantly less strongly solvato-
chromic than ET33, ET30, BM and BDN however and
consequently it is not possible to determine in any more
detail solution dynamics using current instrumentation.
Returning to the issue of why some dyes display
enhanced aggregation, and some disaggregation, in the
presence of potencies, additional explanations may be
down to particular structural features of the dyes in ques-
tion and related dye dissociation constants (K
D
). As yet
no exact figures are available for K
D
values but plots of
absorbance peaks versus dye concentration (a standard
method of determining K
D
) indicate both BDN and BM
aggregate weakly whereas ET30, ET33, NR and PB aggre-
gate more strongly. Examination of further solvatochromic
dyes should clarify which of the above factors determine
whether enhanced aggregation or disaggregation occurs
in the presence of potencies.
Much work needs to be done to confirm and extend the
results described in this paper. In particular the detailed ef-
fects of a range of potencies of glycerol need to be estab-
lished. Preliminary results indicate that degree of effect is
not smoothly related to the degree of potentisation. A
similar discontinuity in the effect of potency scales has
been observed by workers using other systems.
27
Two of the dyes employed in this study (BM and NR) are
fluorescent and fluorescence spectroscopy should add valu-
able information about the way in which potencies are
affecting aggregation and disaggregation dynamics, partic-
ularly for BM which displays a marked dependence of fluo-
rescence intensity on aggregation levels.
It is also hoped that the identification of the essential fea-
tures of solvatochromic dyes necessary to interact with po-
tencies will allow the synthesis of derivatives of dyes
displaying ever greater spectroscopic changes. This in
turn will mean more searching questions can be asked.
All solvatochromic dyes are essentially oscillating di-
poles. Under the influence of light an electron travels from
one end of the dye to the other and back again several hun-
dred million times a second. It is conceivable that potencies
are interacting with solvatochromic dyes because they them-
selves are oscillating dipoles. It is hoped that it will in time
be possible to elucidate the nature of homeopathic potencies
using the chemical system described herein.
Conclusions
Solvatochromic dyes appear to provide a simple and ver-
satile method for the detection of homeopathic potencies.
Six different solvatochromic dyes have been tested in three
different solvents with and without potency present. In all
cases the visible spectra of the dyes are affected by the
presence of potency. Evidence has been presented which
indicates potencies are interacting directly with the dyes
themselves rather than through the solvent in which the
dyes are dissolved. Potencies have no effect on non-
solvatochromic dyes, suggesting it is the intramolecular
electron transfer feature of solvatochromic dyes which is
essential in determining dyeepotency interaction. In turn
this interaction results in supramolecular effects in the
form of enhanced dye aggregation or disaggregation.
This evidence has taken the form of difference spectra
together with assays involving the interaction of dyes
with strontium ions.
As solvatochromic dyes are oscillating dipoles it is
possible that homeopathic potencies share some common-
ality with this feature. It is hoped that future research utilis-
ing solvatochromic dyes will allow specific questions to be
asked regarding the physico-chemical nature and mode of
action of homeopathic potencies.
Materials and methods
Materials
Solvatochromic dyes 2,6-Dichloro-4-(2,4,6-triphenyl-
pyridinium-1-yl)-phenolate (ET33), 2,6-Diphenyl-4-
(2,4,6-triphenylpyridinium-1-yl)-phenolate (ET30), N,N-
dimethylindoaniline/Phenol Blue (PB), 9-diethylamino-
5H-benzo[a]phenoxazime-5-one/Nile Red (NR) and 4-
(Bis-(4-(dimethylamino)phenyl)methylene)-1(4H)-naph-
thalenone (BDN) were obtained from Sigma Aldrich UK
and used as provided. 4-[(E)-2-(1-methylpyridinium-4-yl)
ethenyl]phenolate/Brooker’s merocyanine (BM) was syn-
thesised and provided by Innovapharm Ltd., Kiev, Ukraine
and its structure and purity confirmed by NMR. Non-
solvatochromic dyes Patent Blue VF, Green S and Sulfo-
rhodamine 101 were obtained from Sigma Aldrich or
Fisher Scientific, UK. Strontium chloride, ethanol and
tert-butyl alcohol were obtained from SigmaeAldrich,
UK unless specified otherwise and were of the highest pu-
rity available.
Reverse osmosis water (ROW) was used throughout this
study and had a resistivity of 15 M Ucm (checked daily).
Disposable high purity optical PS cuvettes 4.5 ml capac-
ity/10 mm pathlength with polyethylene (PE) air-tight
stoppers were obtained from Elkay Laboratory Products,
UK. Disposable high purity UV 4.5 ml capacity/10 mm
pathlength cuvettes (UV) with PE air-tight stoppers were
obtained from Brand GMBH, Germany. High purity/low
leachable trace element Nalgene PE, and fluorinated ethyl-
enepropylene (FEP) bottles were obtained from Fisher Sci-
entific, UK.
Solution storage
All solutions were made up and stored in FEP or PE bot-
tles. Ethanol and tert-butyl alcohol were used from the bot-
tles in which they were provided or transferred to PE
bottles. Concentrated dye stocks in dimethylsulfoxide
(DMSO) or dye solutions in ROW, ethanol or tert-butyl
alcohol were stored in FEP or PE bottles at room tempera-
ture. Working dye solutions were made by dissolving dye
directly into solvent (ROW, ethanol or tert-butyl alcohol)
Solvatochromic dyes and homeopathic potencies
SJ Cartwright
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or by adding an aliquot of concentrated dye stock in DMSO
to solvent. In both cases dye solutions were left to equili-
brate overnight before use. Concentrations of dyes used
for difference spectra and assays with strontium ions
were as follows: ET33 e245 mM in ROW, ethanol and
tert-butyl alcohol; ET30 e245 mM in ethanol and tert-
butyl alcohol. ET30 is poorly soluble in ROW. Solutions
were made up in 20 mM borate buffer pH 10 and centri-
fuged to remove any precipitate; BDN e80 mMinROW,
ethanol and tert-butyl alcohol. BDN slowly precipitates
in ROW so solutions were made up and used within an
hour; BM e125 mM in ethanol, 245 mM in ROW and
125 mMintert-butyl alcohol; NR e16 mMinROW,
ethanol and tert-butyl alcohol; PB e62 mMinROW,
ethanol and tert-butyl alcohol. Dye concentrations were
chosen so as to give absorbances of c.1.0 at their absor-
bance maxima. At this absorbance level the Unicam UV-
500 spectrophotometer used in this study can comfortably
handle difference spectra. No light or temperature induced
degradation (determined by daily monitoring of visible
spectra) of ET33, ET30, PB, BDN, BM or NR was
observed over the period of this study. As a precaution so-
lutions of BM and NR were kept in the dark as both dyes
are fluorescent and potentially subject to light-induced
degradation.
Homeopathic potencies
Serially diluted and succussed solutions (homeopathic
potencies) of a range of compounds, including glycerol
50 M, were obtained from Helios Homeopathy Ltd Tun-
bridge Wells, UK or Ainsworths Homeopathic Pharmacy,
London, UK and were diluted 10 fold into 90% ethanol/
10% ROW to ensure consistent solvent composition. Po-
tencies are sold as made in 90% ethanol/10% ROW, but
the above step was taken as a further precaution to ensure
solvent equivalence with control solutions. Glycerol 50 M
is a homeopathic potency prepared by the Korsakoff
method.
28
Glycerol is serially diluted and succussed by
hand up to the 200c potency. Thereafter potentisation is
performed mechanically. At each step a portion of solution
is first diluted 100 fold, and then subjected to 10 succussion
strokes. 50 M means the homeopathic medicine has gone
through 50,000 such cycles. A total of 500,000 succussion
strokes have therefore been imparted with an effective dilu-
tion factor of 100
50000
. ROW is used throughout the poten-
tisation process.
Control solutions
Controls consisted of 90% ethanol/10% ROW alone.
Both potencies and controls were kept in the same 3.5 ml
amber moulded glass bottles used by Helios Homeopathy
Ltd (obtained from the Homeopathic Supply Company
Ltd, code MPB) under the same conditions at room temper-
ature. Any impurities leaching out of the glassware should
therefore be present at the same levels in both control and
potency solutions. Independent analysis by ICP-OES for
levels of Aluminium, Boron, Calcium, Iron, Potassium,
Magnesium, Sodium, Silicon and Titanium by LGC Health
Sciences, UK confirmed the same level of each element in
both control and potency bottles (kept at room temperature
for 6 months prior to analysis). Furthermore, at the levels of
elements detected (all <2 mg/ml) no effect on either dye dif-
ference spectra or assays involving dyes and strontium ions
(see Experimental procedures below) was observed in an
independent study in which salts of the above elements
were deliberately added to assay solutions. Levels of other
elements in control and potency solutions were below the
detection limits of the ICP-OES instrumentation.
Instrumentation
Assays and spectra were recorded on a Unicam UV500
UV/VIS double-beam spectrophotometer run with pre-
loaded Visionlite software capable of analysing curves
and providing data points at set time intervals as they are
generated. Manufacturer’s specifications state an accuracy
of 0.001 at an absorbance of 1.0, with a wavelength accu-
racy of 0.5 nm and resolution of 2 nm.
Experimental procedures
Assays were performed in single-use 4.5 ml capacity PS
cuvettes (assays in ROW and ethanol) or UV cuvettes (as-
says in ethanol and tert-butyl alcohol) as described above.
Tests showed no evaporation of contents occurred over the
time periods of assays from either cuvette type using the
stoppers described.
Difference spectra of dyes were performed as follows.
2.95 ml of dye solution at concentrations giving an absor-
bance of c.1.0 (see Solution storage above) was dispensed
into sample and reference cuvettes and the spectrophotom-
eter set to zero across the range of wavelengths to be as-
sessed (generally 350 or 400 nm to 700 or 800 nm
depending upon the dye being investigated). 50 ml of con-
trol solution (90% ethanol/10% ROW) was then added to
the reference cuvette and 50 ml of potency solution (90%
ethanol/10% ROW) added to the sample cuvette. Solutions
in both cuvettes are therefore materially identical. Solu-
tions were scanned at time intervals up to 3 h to give differ-
ence spectra of dye with potency minus dye with control.
Scans of solutions left overnight were also performed.
‘Difference’ spectra in which 50 ml of control solution
was added to both cuvettes demonstrated no effect over
comparable time scales compared with dye solutions to
which potency had been added.
Assays with strontium ions were carried out as follows.
To 2.95 ml of dye solution in PS or UV cuvettes was added
50 ml of either control solution or potency solution together
with, in both cases, 1 ml of a 210 mM solution of strontium
chloride. Absorbance was then followed at a set wave-
length to monitor changes in the interaction of dye with
strontium ions. The spectrophotometer was zeroed with
solvent alone in the respective cuvette type prior to assay.
All presented data show average values of at least 20 assays
under any one set of conditions.
PS or UV cuvettes have been used throughout this study.
Primarily this is because they are single-use and therefore
remove any possibility for cross-contamination by residual
Solvatochromic dyes and homeopathic potencies
SJ Cartwright
10
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potency potentially encountered with reusable quartz cu-
vettes (see text). Laboratory temperature was maintained
at 21 1C for assays involving ROW and ethanol and
24 1C for assays involving tert-butyl alcohol, using a
combination of background heating and air conditioning.
The highest purity ROW, ethanol and tert-butyl alcohol
were used in all assays. It is perhaps worth noting however
that on addition of 50 ml of either control or potency solu-
tions to 2.95 ml of dye solution the relative levels of host
solvent drop slightly because of the introduction of 45 ml
of ethanol and 5 ml of ROW. Bulk solvent is therefore at
a level of 98.5% in the case of those assays performed in
ROW, 99.8% in the case of those assays performed in
ethanol and 98.33% in the case of those assays performed
in tert-butyl alcohol. As these small changes occur in both
control and sample solutions they have not been taken into
account, especially as potency effects appear to be solvent-
independent (see text).
Conflict of interest statement
No source of funding had any influence on the design,
analysis, interpretation or outcome of the research con-
tained within this manuscript, nor on the writing of the
manuscript.
Acknowledgements
Primary funding for this work is gratefully acknowl-
edged from The Homeopathy Research Institute.
Additional funding from the following individuals, insti-
tutions and companies in alphabetical order is also grate-
fully acknowledged eAlliance of Registered
Homeopaths, European Central Council of Homeopaths,
Helios Homeopathy Ltd., Jane Milburn, The Tanner Trust
and Charles Wansbrough.
Grateful acknowledgement also to Alexander Tournier
for helpful discussions and comments during the compila-
tion of this manuscript.
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Solvatochromic dyes and homeopathic potencies
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