development of chromogenic reactands for optical sensing

8/8/2019 Development of Chromogenic Reactands for Optical Sensing

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Sensors and Actuators B 49 (1998) 226234

Development of chromogenic reactands for optical sensingof 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 535 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 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

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

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* Corresponding author. Tel.: +41 1 4451350; fax: +41 1

4451223; e-mail: [email protected]

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

PII S0925-4005 98 00132-4


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G.J. Mohr et al. /Sensors and Actuators B 49 (1998) 226234 227

low extinction coefficients (typically around 6000 l

mol1 cm1). 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 withthe 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 Buchi 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 l

(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 2291C. 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 min1 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 theethanol 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

(63200 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

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) ofpH 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 was6.6 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 110C for 20 h. After cooling to roomtemperature, 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): l (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 110C for 30 h.

1H-NMR (CDCl3): l (ppm) 8.33 (m, 1 H , CH),7.82 (m, 1 H, CH), 7.357.60 (m, 4 H, CH), 7.18


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G.J. Mohr et al. /Sensors and Actuators B 49 (1998) 226234228

(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 5C 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 5C 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 againreduced 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.=

43C.1H-NMR (CDCl3): l (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).1H-NMR (CDCl3): l (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.

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 M4M7, 2.20 mg for M3) in 1.5

ml of THF. A dust-free glass plate was placed in a spincoating device with a THF-saturated atmosphere. Then

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-11in 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:

h= [R]/RT= (AA0)/(A1A0) (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 (h=1) and A0the absorbance on exposure to 40 vol% 1-propanol

giving a full signal change (h=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.

Fig. 1. Synthesis of chromoreactands ETHT 4001 and ETHT 4002.


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Table 1

Membrane compositions and sensor characteristics

Plasticiser Dye TDMACl (mol %) umax. (nm) RSCMembrane Polymer

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

M2 PVC DOS ETHT 4001 0 492 n.d.c

M3 DOSPVC ETHT 4001 100 432/485 50%; 500

ETH 8045 ETHT 4001 20PVC 507M4 24%; 510

BlockcopolymerM5 ETHT 4001 20 495 n.d.

SiliconeM6 ETHT 4001 20 465 n.d.

DOS ETHT 4002 20PVC 438, 485M7 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

3.1. Optical properties of the chromogenic reactands

The investigated chromogenic reactands are repre-sentatives of azo dyes, exhibiting the well-known elec-

tron acceptordonor 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 ofthe 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 ETH

T

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 420430 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).

It is interesting to notice that there was a signifi-

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-butanolwere 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.


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Fig. 3. Absorbance spectra of a 18 vM 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).

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.

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 formedfairly 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 430nm 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 h against the ethanol activ-

ity (a) with h=1.3688 (90.037) a+0.6178 (9

0.001) and R2=0.994, (n=5). This fact allowed a twopoint calibration of the sensor membrane. The LOD

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 1015 min,

whereas the time for the reverse response was in the

range of 2030 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 45 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.


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Table 2

Measurement of ethanol content in wine and spirits using sensor

membrane M1

Measured (vol%)Sample Reported (vol%)

(n=3)

Chasselas de Geneve 11.210.690.6

Chai de Bordes-Quan- 10.890.8 11.5

card

12.5Rioja Glorioso 12.290.7

37.537.390.5Baselbieter Kirsch

Vodka Moskovskaya 40.039.490.3

nm) in cyclohexane, ETHT 4002 had an m of 18700 l

mol1 cm1 (umax. 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 againstvarious 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% atpH 10.5. Consequently, changes of pH affected both

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 45 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. WhereasETHT 4001 had an mof 38300 l mol1 cm1 (umax. 467


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Fig. 6. pH-dependence of the absorbance of M1 in plain buffer and

in buffer containing 10 vol% ethanol.

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 theprimary alcohols, allows the assumption that only alco-

hols with sterically demanding groups at the h-carbon

are hindered in the approach. Alcohols with two alkyl

chains at the i-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 correlateswith the lipophilicity of the alcohols.

Although sensor membrane M1 exhibits significantly

lower selectivity than sensors based on enzymatic

recognition [39,18], 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 isfound 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, itchanges to min. The presence of TDMACl results in

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 morebasic 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 iscatalysed 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].


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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 foralcohol 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 lipophiliccationic 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 becomeavailable. 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.

References

[1] A. Caputi, D.P. Mooney, Gas-chromatographic determination

of ethanol in wine-a collaborative study, J. Assoc. Off. Anal.

Chem. 66 (1983) 11521157.

[2] K. Helrich, Official Methods of Analysis of the Association of

Official Analytical Chemists, vol. 2, 15, Association of Official

Analytical Chemists, Arlington, VA, 1990, p. 739.

[3] E. Dominguez, H.L. Lan, Y. Okamoto, P.D. Hale, T.A.

Skotheim, L. Gorton, B. Hahn-Hagerdal, Reagentless chemi-

cally-modified carbon-paste electrode based on a phenothiazine

polymer derivative and yeast alcohol-dehydrogenase for the

analysis of ethanol, Biosens. Bioelectron. 8 (1993) 229237.[4] M. Varadi, N. Adanyi, Application of biosensor with ampero-

metric detection for determining ethanol, Analyst 119 (1994)

18431847.

[5] B.S. Walters, T.J. Nielson, M.A. Arnold, Fiber-optic biosensor

for ethanol based on an internal enzyme concept, Talanta 35

(1988) 151155.

[6] S.M. Gautier, L.J. Blum, P.R. Coulet, Fiberoptic biosensor

based on luminescence and immobilised enzymesmicrodeter-

mination of sorbitol, ethanol and oxaloacetate, J. Biolumin.

Chemilumin. 5 (1990) 5763.

[7] O.S. Wolfbeis, H.E. Posch, A fibre optic ethanol biosensor,

Fresenius Z. Anal. Chem. 332 (1988) 255257.

[8] K.P. Volkl, D.W. Lubbers, N. Opitz, Continuous measurement

of concentrations of alcohol using a fluorescence-photometricenzymatic method, Fresenius Z. Anal. Chem. 301 (1980) 162

163.

[9] X. Xie, A.A. Suleiman, G.G. Guilbault, Z. Yang, Z. Sun,

Flow-injection determination of ethanol by fiber-optic chemilu-

minescence measurement, Anal. Chim. Acta 266 (1992) 325

329.

[10] F.L. Dickert, H. Kimmel, G.R. Mages, E.H. Lehmann, S.K.

Schreiner, Substituted 3,3-diphenylphthalides as optochemical

sensors for polar-solvent vapors, Anal. Chem. 60 (1988) 1377

1380.

[11] M.E. Kessler, O.S. Wolfbeis, On-line optical determination of

water in ethanol, Proc. Int. Soc. for Optical Engineering 1510

(1991) 218223.

[12] M.E. Kessler, J.G. Gailer, O.S. Wolfbeis, On-line determina-

tion of solvent mixtures based on a fluorescent solvent polarity

probe, Sens. Actuators B 3 (1991) 267272.

[13] D. Picque, D. Lefier, R. Grappin, G. Corrieu, Monitoring of

fermentation by infrared spectrometry, Anal. Chim. Acta 279

(1993) 6772.

[14] H.-H. Zeng, K.-M. Wang, D. Li, R.-Q. Yu, Development of

an alcohol optode membrane based on fluorescence enhance-

ment of fluorescein derivatives, Talanta 41 (1994) 969975.

[15] D.N. Simon, R. Czolk, H.J. Ache, Doped sol-gel films for the

development of optochemical ethanol sensors, Thin Solid

Films 260 (1995) 107110.

[16] G. Orellana, A.M. Gomez-Carneros, C. de Dios, A.A. Garcia-

Martinez, M.C. Moreno-Bondi, Reversible fiber-opticfluorosensing of lower alcohols, Anal. Chem. 67 (1995) 2231

2238.

[17] K. Seiler, K. Wang, M. Kuratli, W. Simon, Development of

an ethanol-selective optode membrane based on a reversible

chemical recognition process, Anal. Chim. Acta 244 (1991)

151160.

[18] U.E. Spichiger, M. Kuratli, W. Simon, ETH 6022: an artificial

enzyme? A comparison between enzymatic and chemical recog-

nition for sensing ethanol, Biosens. Bioelectron. 7 (1992) 715

723.

[19] R. Wild, D. Citterio, J. Spichiger, U.E. Spichiger, Continuous

monitoring of ethanol for bioprocess control by a chemical

sensor, J. Biotechnol. 50 (1996) 3746.

[20] U.E. Spichiger-Keller, Chemical Sensors and Biosensors for

Medical and Biological Applications, VCH, Weinheim, 1998.


9/9


[21] O. Dinten, U.E. Spichiger, N. Chaniotakis, P. Gehrig, B. Ruster-

holz, W.E. Morf, W. Simon, Lifetime of neutral-carrier-based

liquid membranes in aqueous samples and blood and the

lipophilicity of membrane components, Anal. Chem. 63 (1991)

596603.

[22] R. Eugster, T. Rosatzin,B. Rusterholz, B. Aebersold, U. Pedrazza,

D. Ruegg, A. Schmid, U.E. Spichiger, W. Simon, Plasticisers for

liquid polymeric membranes of ion-selective chemical sensors,

Anal. Chim. Acta 289 (1994) 113.

[23] K.T. Dishart, R. Levine, A new synthesis of ketones containing

one perfluoroalkyl group, J. Am. Chem. Soc. 78 (1956)2268 2270.

[24] K.J. Klabunde, D.J. Burton, The synthesis of amino-substituted

h,h,h-trifluoroacetophenones, J. Org. Chem. 35 (1970) 1711 1712.

[25] J. Gmehling, U. Onken, Vapor-liquid equilibrium data collection.

Aqueous-organic systems, in: D. Behrens, R. Eckermann, (Eds.),

Chemistry Data Series, vol. 1, part 1, DECHEMA, Deutsche

Gesellschaft fur Chemisches Apparatewesen, Frankfurt a. M.,

Germany, 1977.

[26] H. Zollinger, (Ed.), Azo dyes and pigments. In: Color Chemistry,

VCH, Weinheim, Germany, 1991.

[27] K. Wang, K. Seiler, J.P. Haug, B. Lehmann, S. West, K. Hartman,

W. Simon, Hydration of trifluoroacetophenones as the basis for

an optical humidity sensor, Anal. Chem. 63 (1991) 970974.

[28] C. Behringer, B. Lehmann, J.P. Haug, K. Seiler, W.E. Morf, K.Hartman, W. Simon, Anion selectivities of trifluoroacetophenone

derivatives as neutral ionophores in solvent-polymeric mem-

branes, Anal. Chim. Acta 233 (1990) 4147.

[29] R.E. Dohner, U.E. Spichiger, W. Simon, Evanescent-wave spec-

troscopy on bulk-response optode membranes, Chimia 46 (1992)

215217.

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 bioAnalyticalChemistry 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.

.

development of chromogenic reactands for optical sensing

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