Sensors and Actuators B 49 (1998) 226 – 234

Development of chromogenic reactands for optical sensing

of alcohols
Gerhard J. Mohr *, Daniel Citterio, Ursula E. Spichiger-Keller
Centre for Chemical Sensors, ETH Technopark, Technopark St. 1, CH-8005 Zurich, Switzerland

Received 26 June 1997; received in revised form 26 February 1998; accepted 27 February 1998

Abstract

A new class of chromogenic reactands (or ‘chromoreactands’) has been synthesised that reversibly interact with alcohols
resulting in a change in absorbance. When embedded in plasticised PVC membranes, 4-(N,N-dioctylamino)-4%-trifluoroacetyl-
azobenzene (ETHT 4001) shows a significant signal change on exposure to aqueous ethanol solution with a decrease in absorbance
at around 490 nm and an increase in absorbance at around 430 nm wavelength. The sensor layer exhibits a dynamic range from
2 to 50 vol% ethanol with highest sensitivity in the 5–35 vol% range. The limit of detection is 1.5 vol%. The absorbance of the
sensor membrane is virtually insensitive to changes in pH, however, the magnitude of the relative signal change between plain
buffer and buffer containing ethanol is pH dependent. A similar response is observed for 1-(N,N-dioctylamino)-4-(4-tri-
fluoroacetylphenylazo)-naphthalene (ETHT 4002), however, with the absorbance shifted to longer wavelengths. The behaviour of
the reactands dissolved in alcohols correlates with the selectivity of the dyes in the sensor membranes. © 1998 Elsevier Science
S.A. All rights reserved.

Keywords: Alcohol sensor; Chromogenic reactand; ETHT 4001; ETHT 4002

1. Introduction the NADH concentration added as a cosubstrate [5,6]

The determination of alcohol has always been an
important task for clinical and industrial analysis as
well as for biochemical applications. Simple irreversible
methods have found wide-spread application such as
the Draeger test tube based on the reduction of
chromium (VI). However, they are not suitable for
on-line monitoring in bioreactors or industrial areas. In
order to carry out accurate evaluations, chromato-
graphic methods or distillation with subsequent deter-
mination of density and of refractometry are used [1,2].
Again these methods do not lend themselves to contin-
uous determination of analytes. Since there is an in-
creasing demand for on-line measurements during
industrial and biotechnical processes, reversible sensors
are required. In the last decade sensor devices have
been developed that are based on enzymatic recognition
of ethanol. The signal transduction is carried out am-
perometrically [3,4] or by optically detecting changes of
* Corresponding author. Tel.: +41 1 4451350; fax: + 41 1
4451223; e-mail: gerhard@chemsens.pharma.ethz.ch
or by monitoring the consumption of oxygen [7,8].
Another enzymatic approach is based on the chemilu-
minescence produced by the enzymatically catalysed
oxidation of ethanol to hydrogen peroxide [9]. Sensors
have been presented that are based on the solva-
tochromism of polarity-sensitive dyes [10–12] and on
infrared analysis using a multivariate statistical calibra-
tion [13].
Similarly, optical sensors for alcohols have been in-
troduced which, upon exposure to alcohols, show
fluorescence enhancement of fluorescein [14] or mala-
chite green [15] and fluorescence quenching of polyaro-
matic-substituted 1,3-oxazoles and -thiazoles [16].
Recently, trifluoroacetophenone derivatives have
been reported to selectively interact with alcohols
[17,18]. They have been shown to be sensitive and
selective and, although the recognition process is cou-
pled to a chemical reaction, to be very rapid. In addi-
tion, they have been successfully applied for optical
monitoring of ethanol in a bioreactor [19]. However,
these compounds have their wavelength of maximum
absorbance in the UV spectral range and they exhibit

0925-4005/98/$19.00 © 1998 Elsevier Science S.A. All rights reserved.

PII S0925-4005(98)00132-4
 G.J. Mohr et al. / Sensors and Actuators B 49 (1998) 226–234
low extinction coefficients (typically around 6000 l
mol − 1 cm − 1). The resulting sensing membranes have
to be used in the vapour phase to prevent interferences
from the absorbance of biomolecules. Therefore, the
authors developed a synthetic strategy to obtain chro-
mogenic reactands (or ‘chromoreactands’) with com-
parable sensitivity and selectivity but absorbance
shifted by more than 150 nm into the visible spectral
range. The name ‘reactand’ was coined, in analogy with
the term ‘ligand’, for the class of host compounds
which react reversibly with an analyte or target com-
pound [20].
In this work, the authors present a simple route to
novel chromogenic reactands which are sensitive for
alcohols and show reversible and rapid signal changes
in the visible spectral range.
2. Experimental
2.1. Apparatus
Melting points were measured on a Büchi melting
point apparatus (Flawil, Switzerland) and are uncor-
rected. 1H-NMR spectra were acquired on a Varian XL
200 Gemini spectrometer with chemical shifts given in d
(ppm) relative to TMS. Elemental microanalyses were
carried out on a Carlo Erba 1106 CHN microanalyser.
The pH of buffer solutions was measured with an
Orion 920A pH-meter. The absorbance spectra of dis-
solved dyes and of sensor layers were recorded on an
Uvikon 942 spectrophotometer at 229 1°C. The mea-
surements were carried out in a flow-through cell fixed
in the spectrophotometer by pumping the sample solu-
tions at a flow rate of 2.5 ml min − 1 using a Perpex
peristaltic pump (Jubile, Switzerland) [21]. The noise of
the photometer amounted to 0.00003 – 0.00006 ab-
sorbance units and the noise of the sensor membrane
on exposure to buffer was between 0.0008 and 0.00014
absorbance units. The detection limit (LOD) of the
sensor membranes was determined by alternatively ex-
posing the sensor layer five times to plain buffer and
low concentrations of ethanol and calculating the
ethanol concentration corresponding to the signal
change of six times the standard deviation of the plain
buffer signal.
2.2. Reagents
All reagents were of analytic reagent grade. Flash
chromatography was carried out with silica gel 60
(63 – 200 mm) from Fluka (Buchs, Switzerland). For
membrane preparation, poly(vinyl chloride) (PVC, high
molecular weight), bis(2-ethylhexyl)sebacate (DOS), tri-
dodecylmethylammonium chloride (TDMACl), silo-
prene K1000, siloprene cross-linking agent K-11 and
227
tetrahydrofuran (THF) were obtained from Fluka. The
synthesis of 4-ethylphenyldodecyl-1-nitrophenylether
(ETH 8045) has already been described in detail [22]
and the compound was re-synthesised in the laboratory.
Dimethylsiloxane bisphenol A carbonate block copoly-
mer was obtained from Ventron (Karlsruhe, Germany).
The preparation of trifluoroacetylaniline (TFAA) was
described in detail [23,24].
Phosphate buffered aqueous solutions (67 mM) of
pH 6.8 were used for the determination of the sensitiv-
ity and the selectivity of the sensor layers. In order to
measure the ethanol content in the beverages, the sam-
ples were diluted 1:1 (v/v) with the phosphate buffer.
The red wine was decolourised by addition of 1 g of
active carbon to 50 ml of the diluted sample solution
and subsequent filtration. The white wine samples were
adjusted to pH 6.8 by addition of 2.0 M sodium
hydroxide solution before diluting with the phosphate
buffer. The effect of pH on the sensor characteristics
was investigated by using a universal buffer which was
6.6 mM in citric acid, 21.5 mM in sodium borate and
10 mM in sodium dihydrogen phosphate. The pH was
adjusted with 1.0 M sodium hydroxide solution and 2.0
M sulphuric acid.
2.3. Synthesis
2.3.1. N,N-Dioctylaniline
A mixture of 4.47 g (0.048 mol) of aniline, 32.8 g
(0.17 mol) of 1-bromoctane, 21.9 g (0.17 mol) of N-
ethyldiisopropylamine and 30 ml of dimethylformamide
was stirred at 110°C for 20 h. After cooling to room
temperature, the product was poured on 200 ml of
distilled water leading to a separation of the product
from the aqueous phase. It was dissolved in 100 ml of
trichloromethane, washed two times with distilled wa-
ter, dried over magnesium sulphate and the solvent
removed. The resulting dark brown oil contained small
fractions of N-octylaniline and N,N,N-trioctylanilinium
bromide. It was purified by flash chromatography using
hexane/ethyl acetate (95:5 =v/v) as the eluent yielding
12.6 g of a yellow liquid.
1
H-NMR (CDCl3): d (ppm) 7.19 (m, 2 H, CH),
6.64 (m, 3 H, CH), 3.19 (t, 4 H, CH2), 1.58 (m, 4
H, CH2), 1.30 (m, 20 H, CH2), 0.88 (t, 6 H, CH3).
Calculated for C22H39N (317.56): C, 83.21; H, 12.38;
N, 4.41; found: C, 83.47; H, 12.15; N, 4.32.
2.3.2. N,N-Dioctylaminonaphthalene
The compound was prepared in analogy to N,N-
dioctylaniline by stirring a mixture of 6.87 g (0.048 mol)
of 1-aminonaphthalene, 32.8 g (0.17 mol) of 1-bromoc-
tane, 21.9 g (0.17 mol) of N-ethyldiisopropylamine and
30 ml of dimethylformamide at 110°C for 30 h.
1
H-NMR (CDCl3): d (ppm) 8.33 (m, 1 H, CH),
7.82 (m, 1 H, CH), 7.35–7.60 (m, 4 H, CH), 7.18
228 G.J. Mohr et al. / Sensors and Actuators B 49 (1998) 226–234

(d, 1 H, CH), 3.13 (t, 4 H, CH2), 1.52 (m, 4 H,
CH2), 1.25 (m, 20 H, CH2), 0.87 (t, 6 H, CH3).
Calculated for C26H41N (367.62): C, 84.95; H, 11.24;
N, 3.81; found: C, 84.57; H, 11.39; N, 3.62.
2.3.3. 4 -(N,N-Dioctylamino) -4 %-trifluoroacetylazo-
benzene
TFAA (1.42 g) was suspended in 3.8 ml of 6 M
hydrochloric acid and cooled to 5°C in an ice bath.
Then, 0.48 g of sodium nitrite in 2 ml of distilled water
was added and the resulting solution filtered. The solu-
tion was added dropwise to 2.22 g of N,N-dioctylani-
line in 40 ml of ethanol containing 0.6 ml of
concentrated hydrochloric acid. The reaction mixture
was stirred for 3 h at 5°C and 4 h at room temperature.
Afterwards, the solvent was evaporated. The resulting
purple oil was dissolved in 50 ml of trichloromethane,
washed once with a saturated solution of aqueous
sodium hydrogen carbonate and three times with dis-
tilled water, dried over magnesium sulphate and again
reduced to dryness yielding 2.1 g of a dark red oil. The
oil was purified by flash chromatography on 60 g of
silica gel with hexane/ethyl acetate (95:5= v/v) as the
eluent yielding 0.95 g of pure N,N-dioctylaminophenyl-
4%-trifluoroacetyl-azobenzene (ETHT 4001), m.p.=
43°C.
1
H-NMR (CDCl3): d (ppm) 8.18 (d, 2 H, CH), 7.88
(m, 4 H, CH), 6.70 (d, 2 H, CH), 3.38 (t, 4 H,
CH2), 1.68 (m, 4 H, CH2), 1.33 (m, 20 H, CH2),
0.89 (t, 6 H, CH3).
Calculated for C30H42F3N3O (517.68): C, 69.61; H,
8.18; N, 8.12; found: C, 69.87; H, 8.38; N, 8.20.
2.3.4.
1 -(N,N-Dioctylamino) -4 -(4 -trifluoroacetylphenylazo) -
naphthalene
The compound was obtained in analogy to ETHT
4001 by diazotation of 2.5 g of N,N-dioctylaminonaph-
thalene for 5 h resulting in a dark red oil. (Fig. 1).
1
H-NMR (CDCl3): d (ppm) 9.03 (d, 1 H, CH),
8.25 (t, 3 H, CH), 8.12 (d, 2 H, CH), 7.98 (d, 1 H,
CH), 7.62 (m, 2 H, CH), 7.15 (d, 1 H, CH), 3.33
(t, 4 H, CH2), 1.58 (m, 4 H, CH2), 1.25 (m, 20 H,
CH2), 0.86 (t, 6 H, CH3).
Calculated for C34H44F3N3O (567.74): C, 71.93; H,
7.81; N, 7.40; found: C, 71.84; H, 7.64; N, 7.48.
0.2 ml of the solution was transferred onto the rotating
glass support. The resulting membranes were placed in
ambient air for drying. The sensor membrane M5 was
obtained by spin coating of a solution composed of 2.0
mg of ETHT 4001, 0.44 mg of TDMACl and 200 mg of
carbonate blockcopolymer in 1.5 ml of dichloro-
methane. M6 was obtained by dissolving the same
amount of dye and TDMACl together with 140 mg of
siloprene K1000 and 20 mg of cross-linking agent K-11
in 1.5 ml of dichloromethane and spin-coating the
solution on the glass plates. All sensor membranes were
conditioned in plain buffer for 30 min. The composi-
tions of sensor membranes were compiled in Table 1.
2.5. Calculations
The ratio of the concentration of the free reactand
[R] to the total amount of reactand RT was described
via the measured absorbance at a fixed wavelength:
a= [R]/RT = (A− A0)/(A1 − A0) (1)
where A was the measured absorbance value on expo-
sure to aqueous alcohol, A1 the absorbance of the
membrane on exposure to dry nitrogen (a= 1) and A0
the absorbance on exposure to 40 vol% 1-propanol
giving a full signal change (a= 0).
The activities of alcohols in the aqueous (buffered)
solutions were calculated by the empirical model of
Margules according to Gmehling and Onken [25].
The term ‘relative signal change’ (RSC) was used
here to describe the signal changes at a certain wave-
length when changing from plain buffer to 40 vol% of
aqueous ethanol. It served as a measure for the sensitiv-
ity of the sensor membranes and simplified comparison.

2.4. Preparation of the sensor layers

Sensor membranes M1 – M4 were obtained by dis-

solving 80 mg of PVC, 160 mg of the plasticiser, 2.0 mg
of ETHT 4001 and the respective quantity of TDMACl
(0.44 mg for M1 and M4 – M7, 2.20 mg for M3) in 1.5
ml of THF. A dust-free glass plate was placed in a spin
coating device with a THF-saturated atmosphere. Then Fig. 1. Synthesis of chromoreactands ETHT 4001 and ETHT 4002.
 G.J. Mohr et al. / Sensors and Actuators B 49 (1998) 226–234 229

Table 1
Membrane compositions and sensor characteristics

Membrane Polymer Plasticiser Dye TDMACl (mol %) lmax. (nm) RSC

M1 PVC DOS ETHT 4001 20a 433/495 50%; 500b

M2 PVC DOS ETHT 4001 0 492 n.d.c
M3 PVC DOS ETHT 4001 100 432/485 50%; 500
M4 PVC ETH 8045 ETHT 4001 20 507 24%; 510
M5 Blockcopolymer ETHT 4001 20 495 n.d.
M6 Silicone ETHT 4001 20 465 n.d.
M7 PVC DOS ETHT 4002 20 438, 485 59%; 520

a
Amount of TDMACl relative to the reactand.
b
Wavelength (in nm) where the RSC was determined.
c
n.d., not determined.

3. Results It is interesting to notice that there was a signifi-

3.1. Optical properties of the chromogenic reactands
The investigated chromogenic reactands are repre-
sentatives of azo dyes, exhibiting the well-known elec-
tron acceptor–donor system [26] which shifts the
absorbance of the trifluoroacetylaniline moiety from
the UV into the visible spectral region. This fact is
important for the development of low-cost sensing
devices composed of LEDs, photodiodes and optical
waveguides. The alkylamino group acts as an electron
donor and, in addition, the long alkyl chains attached
to the nitrogen atom render the reactand highly
lipophilic. This enabled dissolution in lipophilic poly-
mer matrices and prevented from leaching on exposure
to aqueous samples of alcohol. The acceptor part of
the chromophore was provided by the trifluoroacetyl
group which exhibited a strong electron-withdrawing
capacity due to both the carbonyl and the trifl-
uoromethyl group. At the same time, the trifl-
uoroacetyl group acted as a selective reactand for
alcohols and, on chemical reaction with alcohols,
changed its acceptor capacity. The conversion of the
trifluoroacetyl group into a hemiacetal resulted in a
significant decrease in its acceptor capacity and, there-
fore, in a blue-shift of the absorbance spectrum. Fig. 2
shows reactand ETHT 4001 in various solvents indicat-
ing that the dye is solvatochromic. A shift in the
absorbance maximum from 467 to 497 nm was ob-
served in going from cyclohexane to acetonitrile.
However, this solvatochromism affected the shift of
the absorbance maximum to a lower extent than the
conversion to the hemiacetal caused by alcohols. As a
result, the absorbance maxima of methanol, ethanol,
1-propanol, 1-butanol and iso-butanol were found in
the range of 420–430 nm. The conversion of the trifl-
uoroacetyl group already started on dissolving the dye
in alcohol, however, when dissolving the dye in di-
ethylether and adding ethanol, the conversion was eas-
ily followed by absorption spectroscopy (Fig. 3).
cantly different behaviour of the dye in different alco-
hols. Whereas in the case of primary alcohols, similar
absorbance maxima at around 425 nm were observed,
the absorbance maxima for 2-propanol and t-butanol
were found at 468 nm and 484 nm, respectively. The
spectra of both 2-propanol and t-butanol were very
similar to the spectra of the reactand in non-alcoholic
solvents. The authors assume sterical hindrance of the
sterically more demanding secondary and tertiary alco-
hols when attaching the trifluoroacetyl group. Conse-
quently, the hemiacetal was not formed and the
absorbance band was not shifted to the blue region as
observed with primary alcohols.
3.2. Choice of polymer
Variation of the polymer is known to have a very
strong effect on the response of ion-sensitive electrode
membranes. Plasticised PVC has been widely used in
ion-selective electrode and optode membranes due to
the ease of membrane preparation and the good solu-
bility of ionophores and indicator dyes in PVC. In
addition, the selectivity and sensitivity of PVC-based
Fig. 2. Absorbance spectra of ETHT 4001 in different solvents
showing the positive solvatochromism of ETHT 4001 and its specific
interaction with ethanol.
230 G.J. Mohr et al. / Sensors and Actuators B 49 (1998) 226–234
Fig. 3. Absorbance spectra of a 18 mM solution of ETHT 4001 in
diethylether after mixing with ethanol (2:1 = v/v) showing the de-
crease of absorbance at 487 nm and the increase at 423 nm on
formation of the hemiacetal. (Spectra were recorded at intervals of 5
min).
sensor membranes may be tailored by the use of the
appropriate plasticiser. However, the plasticiser may
evaporate at elevated temperatures and the membranes
do not exhibit good mechanical stability. In contrast,
silicone membranes offer the use of a mechanically
more stable matrix without the need for plasticisers.
The same holds true for carbonate blockcopolymer
membranes which exhibit good mechanical stability and
also allow less toxic solvents to be used. However, in
initial tests, a significant leaching of ETHT 4001 from
both the silicone and the blockcopolymer membranes
was observed, preventing the applicability in optical
sensors for alcohols.
Consequently, plasticised PVC, although probably
not the ideal matrix in terms of mechanical and thermal
stability, was used as the polymeric support. It formed
fairly stable films and, in combination with the plasti-
ciser, acted as a good solvent for both the dyes and
long-chain quaternary ammonium ions.
3.3. Sensor response
On exposure to buffer solutions containing ethanol,
membrane M1 showed a distinct response by giving a
decrease of the absorbance at around 490 nm. At the
same time a new maximum was formed at around 430
nm which corresponded to the hemiacetal derivative of
the dye (Fig. 4). Fig. 4 also shows the diol formation
which occurred when the dry sensor membrane was
exposed to plain buffer. Both reactions, namely the diol
and the hemiacetal formation were fully reversible.
The relative absorbance values as a function of the
ethanol concentration are shown in Fig. 5. The RSC of
M1 when changing from 0 to 40 vol% ethanol at pH
6.8 was as high as 50%. A linear calibration function
was established by plotting a against the ethanol activ-
ity (a) with a = −1.3688 (9 0.037) a+ 0.6178 (9
0.001) and R2 = 0.994, (n =5). This fact allowed a two
point calibration of the sensor membrane. The LOD
Fig. 4. Absorbance spectra of M1 in contact with dry nitrogen and
different concentrations of aqueous ethanol. When changing from
nitrogen to buffer, the diol is formed, whereas, when changing from
plain buffer to aqueous ethanol, formation of the hemiacetal occurs.
Both types of reactions are fully reversible.
was found to be 1.5 vol% ethanol. The relative standard
deviation for 4 and 20 vol% ethanol (n = 10) was
determined to be 1.07 and 1.45%, respectively.
The forward response time t95 (for 95% of the total
signal change to occur) was in the range of 10–15 min,
whereas the time for the reverse response was in the
range of 20–30 min. M1 was used to measure the
alcohol content of several beverages and the results are
shown in Table 2. During 1 week of continuous mea-
surements with aqueous ethanol and methanol (up to
concentrations of 40 vol% alcohol), no leaching of the
dye could be observed. Even on exposure to the more
lipophilic 1-propanol, no significant leaching was ob-
served. However, after exposure to 1-propanol, the
reverse response time of M1 to plain buffer was pro-
longed up to 4–5 h which was attributed to a leaching-
out of the quaternary ammonium salt (see next
paragraph). The shelf lifetime of the membranes ex-
ceeded 2 months when stored in the dark at room
temperature.
In order to investigate the effect of TDMACl on the
sensor response, membranes without TDMACl (M2)
and with 100 mol% of TDMACl (M3) were prepared.
The presence of TDMACl turned out to be crucial for
Fig. 5. Work function of M1 on exposure to aqueous ethanol and
respective plots for interfering alcohols at pH 6.8.
 G.J. Mohr et al. / Sensors and Actuators B 49 (1998) 226–234
Table 2
Measurement of ethanol content in wine and spirits using sensor
membrane M1
Sample Measured (vol%) Reported (vol%)
(n=3)
Chasselas de Genève 10.6 90.6 11.2
Chai de Bordes-Quan- 10.8 90.8 11.5
card
Rioja Glorioso 12.2 90.7 12.5
Baselbieter Kirsch 37.3 90.5 37.5
Vodka Moskovskaya 39.4 90.3 40.0
a fast response. Without TDMACl, the response time
for changing from plain buffer to 40 vol% ethanol was
longer than 3 h. The reverse response was in the range
of 4 –5 h. Sensor membranes containing 20 mol% TD-
MACl showed fast response. During measurements,
however, a continuous increase in response time was
observed when cycling between plain buffer and
ethanol solutions, which was attributed to leaching-out
of the ionic TDMACl. Consequently, 100 mol% of
TDMACl were used in the membrane composition
(M3). However, TDMACl catalysed the reaction of the
trifluoroacetyl group with water to form the diol. As a
result, only a small further signal change occurred on
exposure to 40 vol% ethanol. Despite this fact, the
sensor might be used for measurements, because after
cycling several times between plain buffer and 40 vol%
ethanol, the dye was reconverted into the trifluoroacetyl
derivative and on exposure to plain buffer the maxi-
mum at around 490 nm was observed again.
In order to investigate the effect of the plasticiser on
the response characteristics, DOS was replaced by the
highly polar ETH 8045. The resulting sensor membrane
M4 showed smaller relative signal changes toward
ethanol (24% RSC on changing from plain buffer to 40
vol% ethanol) compared with the DOS plasticised
membranes (50% RSC). Both the forward and the
reverse response times, as well as the selectivity for
interfering alcohols remained unchanged.
Incorporation of ETHT 4001 into a carbonate block-
copolymer as well as into siloprene (membranes M5
and M6, respectively) did not provide sensor mem-
branes useful for alcohol sensing. M5 showed signifi-
cant dye-leaching on exposure to 40 vol% ethanol
whereas, in the case of the M6, the dye was completely
washed out.
Sensor membrane M7 incorporating ETHT 4002
showed a similar sensitivity and response behaviour to
M1, however, with changes of absorbance at longer
wavelengths. Although the relative signal change was
somewhat larger than for M1, ETHT 4002 was disad-
vantageous because of its significantly lower extinction
coefficient compared with dye ETHT 4001. Whereas
ETHT 4001 had an o of 38300 l mol − 1 cm − 1 (lmax. 467
231
nm) in cyclohexane, ETHT 4002 had an o of 18700 l
mol − 1 cm − 1 (lmax. 475 nm) only. Consequently, a
higher amount of ETHT 4002 has to be used in order to
obtain the same signal to noise ratio, which then could
result in longer response times or crystallisation of dye
and additive in the polymer matrix.
3.4. Selecti6ity toward interfering alcohols and
carbonate
Fig. 5 shows the relative signal changes of M1 caused
by methanol, 1-propanol, 2-propanol and t-butanol in
comparison to ethanol. The sensor exhibited compara-
ble selectivity for ethanol and methanol, but signifi-
cantly higher selectivity for the more lipophilic
1-propanol. The secondary and tertiary alcohols 2-
propanol and t-butanol, although being more lipophilic
than ethanol, were discriminated by the sensor
membrane.
Despite the fact, that the selectivity of M1 against
various alcohols was rather poor for primary alcohols,
the sensor is useful for applications in bioreactors and
beverages. In such areas, only methanol and ethanol
are expected to be found in substantial amounts. Cur-
rently, reactands are investigated that exhibit improved
selectivity for ethanol over methanol.
The sensor membrane M1 was also investigated with
respect to its sensitivity to carbonate ion. Trifluoroace-
tophenone derivatives are well-known for their selective
interaction with carbonate but not with hydrogen car-
bonate or carbon dioxide. At pH 6.8 (the pH value
used for the characterisation of M1, virtually all car-
bonate is present in the form of hydrogen carbonate
and carbon dioxide. Therefore, the pH value of the
phosphate buffer was adjusted to pH 8.8. M1 did not
show significant response to 0.01 M carbonate. The low
content of cationic sites (20 mol% TDMACl relative to
the reactand) prevented the motion of the divalent
anion into the sensor layer. The carbonate anion can
only move into the plasticised PVC layer when the
double-negative charge of the anion is compensated by
positive ionic sites (due to reasons of electroneutrality
within the polymer membrane).
3.5. Effects of pH
The absorbance of reactand ETHT 4001 in M1 was
only slightly pH-dependent and decreased by approxi-
mately 4% on increasing from pH 3.0 to 10.5. (Fig. 6).
However, the magnitude of the relative signal change
(e.g. on changing from plain buffer to buffer containing
10 vol% ethanol) was significantly pH-dependent. There
was almost no signal change (around 1%) when going
from plain buffer to 10 vol% ethanol at pH 3.0,
whereas the relative signal change was around 18% at
pH 10.5. Consequently, changes of pH affected both
232 G.J. Mohr et al. / Sensors and Actuators B 49 (1998) 226–234
Fig. 6. pH-dependence of the absorbance of M1 in plain buffer and
in buffer containing 10 vol% ethanol.
the sensitivity and the detection limit of M1 in that
sensitivity was lower at lower pH values.
The authors attribute the effect of pH on the re-
sponse to be caused by the anion exchange properties
of TDMACl which, at higher pH, provides a more
basic environment for the base-catalysed reaction of the
trifluoroacetyl group into the hemiacetal.
4. Discussion
4.1. Performance of ethanol-sensiti6e membranes
The trifluoroacetyl azo dyes embedded in plasticised
PVC show a significant change of absorbance on expo-
sure to aqueous samples of alcohols. This response is
catalysed by the presence of TDMACl (Fig. 7), but also
takes place without catalyst. It is due to a nucleophilic
reaction of alcohols with the highly electrophilic trifl-
uoroacetyl group. The conversion of the trifluoroacetyl
group into the hemiacetal changes the optical proper-
ties of the dye. The change of absorbance is also
observed in solution, where the absorbance maxima in
non-alcoholic solvents are located around 490 nm. Ex-
Fig. 7. Proposed mechanism of the base-catalysed alcohol recogni-
tion. Only a small fraction of TDMACl is assumed to be converted
into the hydroxide during conditioning in buffer [27].
posure to alcohols results in the formation of the
hemiacetal and a shift of the maxima to around 425
nm.
It is striking that only primary alcohols give a signifi-
cant shift in the absorbance maximum. In the case of
2-propanol and t-butanol, that are sterically hindered
to approach the trifluoroacetyl group, this shift is not
observed. The fact that iso-butanol, although having a
sterically demanding structure, reacts similarly to the
primary alcohols, allows the assumption that only alco-
hols with sterically demanding groups at the a-carbon
are hindered in the approach. Alcohols with two alkyl
chains at the b-carbon such as iso-butanol, however,
may react with the trifluoroacetyl group. The selectivity
of M1 for alcohols is very similar to the selectivity of
ETHT 4001 for alcohols in solution, showing high
sensitivity for primary and significantly lower sensitivity
for secondary and tertiary alcohols. The sensitivity
order within the primary alcohols is dependent on the
extraction into the polymer matrix which correlates
with the lipophilicity of the alcohols.
Although sensor membrane M1 exhibits significantly
lower selectivity than sensors based on enzymatic
recognition [3–9,18], the preparation and use of enzy-
matic sensors often is tedious. Enzymes require special
storage, the sensors should be prepared immediately
before application and their shelf- and operational life-
time are generally low. Furthermore, co-substrates may
be required which, if co-immobilised in the sensor, are
consumed after several measurements. If the reagents
are added to the sample solution, a FIA set-up is
necessary. Finally, enzymes require optimum pH condi-
tions in order to function properly.
In contrast to enzyme-based sensors, M1 is com-
posed of stable components. Consequently, both the
shelf-lifetime and the operational stability are superior.
However, the sensitivity of M1 is significantly lower
than that of enzymatic sensors and too low to use M1
in clinical applications, but it is sufficient to monitor
ethanol in beverages and bioreactors. Furthermore,
both applications do not require a high selectivity for
ethanol over more lipophilic alcohols since the content
of propanol and butanol in beverages and bioreactors is
found to be low (in the range of ppm).
4.2. Effect of additi6es on the response
The ion exchanger TDMACl has a significant effect
on the response of the sensor membranes composed of
plasticised PVC and trifluoroacetyl azo dyes. As has
been shown already for humidity sensors based on
trifluoroacetyl derivatives [27], the response time is
tremendously improved in the presence of TDMACl.
Without TDMACl, the response is in the range of h,
whereas by adding small amounts of TDMACl, it
changes to min. The presence of TDMACl results in
 G.J. Mohr et al. / Sensors and Actuators B 49 (1998) 226–234
the formation of the diol (Fig. 7) which shows ab-
sorbance similar to the hemiacetal [27,28]. Only a small
amount of TDMACl relative to the dye is necessary in
order to decrease the response time but not to lose the
sensitivity for alcohols. Typically, 20 mol% TDMACl
are appropriate. If more TDMACl is used (such as in
M3) then the dye is completely converted into the diol
and exposure to alcohols does not lead to further signal
changes. Since the membrane is more selective for
alcohol than for water [27,28], the sensor may be
reactivated by alternating exposure to alcohol and
buffer.
A problem that has been observed with the sensor
membranes is the continuous increase in response time
after exposure to high concentrations of alcohols (espe-
cially lipophilic alcohols). This drift is probably due to
leaching or decomposition of the quaternary ammonium
salt which, unlike the leaching of the dye, can not be
monitored optically. In future, this problem must be
addressed by either making use of more lipophilic
cationic sites or by using polymers with covalently
immobilised cationic sites.
5. Conclusions
The present work shows the synthesis of chromogenic
reactands for alcohols. The synthon used for the prepa-
ration of the dyes (trifluoroacetylaniline) not only allows
diazotation of aromatic amines, but also of aromatic
phenols and CH-acids. Consequently, a wide range of
reactands with different optical properties may become
available. Furthermore, by reacting trifluoroacetylani-
line with aromatic aldehydes, polymethine dyes may be
accessible which are advantageous because of their
fluorescence. When embedded in plasticised PVC mem-
branes, the reactands presented here show fast and
reversible response to alcohols, however, with significant
cross-sensitivity to pH. This may be overcome by using
fluorescent dyes in combination with an ion-imperme-
able but gas-permeable coating on the sensor layer or by
using azo dyes in ATR-mode [29].
Acknowledgements
This work was supported by the Austrian Science
Foundation within Erwin-Schroedinger fellowship
J01260-CHE which is gratefully acknowledged. We
would also like to thank Drs. Luzi Jenny and Ines
Oehme for the support during synthesis.
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Biographies
Gerhard J. Mohr received his Ph.D. in chemistry
(1996) at the Karl-Franzens University, Graz. His the-
sis focused on the development of optical sensors,
in particular for anions. During the thesis he has car-
ried out research in the groups of Professors P.R.
Coulet (Lyon) and U.-W. Grummt (Jena) to work on
biosensors and near-infrared dyes. In 1996 he has
moved to the Centre for Chemical Sensors of ETH
Zurich and is currently developing a new concept for
optical sensors based on chromogenic and fluorogenic
reactands.
Daniel Citterio graduated in chemistry in 1992 (ETH
Zurich) with special emphasis on organic and analytical
chemistry. His diploma work was entitled ‘Magnesium-
selective measurements under physiological conditions’.
In 1998, he has received his Ph.D. at the Centre for
Chemical Sensors of ETH Zurich in the field of NIR-
dyes for optical sensing.
Ursula E. Spichiger-Keller is the head of the Centre
for Chemical Sensors/Biosensors and bioAnalytical
Chemistry of the Swiss Federal Institute of Technology
(ETH) in Zurich which is funded entirely through na-
tional grants, private funds and cooperations with in-
dustrial partners. She performed permanent
collaboration with Prof. Wilhelm Simon since 1988 and
her habilitation work entitled ‘Chemical sensors and
biosensors for medical and biological applications: a
developing field in analytical chemistry’ was handed to
the Department of Pharmacy at ETH Zurich in Janu-
ary 1994; as from September 1995 the ‘Venia legendi’ at
ETH Zurich was granted.