Note: Descriptions are shown in the official language in which they were submitted.
~119~
FL/6- 19505/A
Optical sensor for the detennination of cations
The invention relates to a sensor for the oplical determination of cations from the group
consisting of metal and ammonium cations (referred to as cations below) in aqueous
samples by the fluorescence method, which sensor contains certain highly basic dyes from
the group consis~ing of rhodamines and acridines as fluolophores in the acdve coating, and
to a process for the qualitative or quantitative deterrnination of cations, in particular in
aqueous solutions, using the optical sensor. The invention furthermore relates to certain
acridines and rhodamines and to the use thereof as fluorophores in sensors for the optical
determination of cations in aqueous samples.
The optical determination of metal cations has recendy achieved increased impo~tance, - -
the presence or concentration of metal cations being measured, for example, via the
change in absorption or fluorescence of a suitable dye. The sensors known as optrodes -
generally comprise transparent support material and an active coating. This active coating
generally contains a transparent hydrophobic polymer and a lipophilic plasticizer for
achieving adequate ion diffusion and solubility of the active consdtuents. The acdve
constituents are a specific ionophore as complexing agent for metal cations, a lipophilic
salt as counterion for maintaining electrical neutra1ity, and an indicator substanse which,
due to a chemical change or a physical change in the environment, generates a measurable
optical signal.
US-A-4 645 744 describes systems of this type in which ihe indicator substance is a
neutral compound, for example a dye (p-nitrophenol), which interacts with an
ionophore/metal cation complex, causing a colour change as the optically measurable
signal. The interaction can cause, ~or example, the elimination of a proton from the dye,
causing a change in the electron state. Suitable compounds include fluorescing compounds
(for example fluorescein), whose fluorescence changes due to the change in the electron
state and can be determined optically by means of fluorescence measurements.
H. He et al. in Chemical, Biochemical and Environmental Fiber Sensors II, SPIE Vol.
l368, pages 165 to 174 ~1990), describe systems containing a proton canier (Nile Blue) as
.3 '~ 4 (~
indicator substance, in which the lransport of potassium into the active coating by means
of valinomycin as ionophore causes dissociation of the proton carrier and diffusion of a
proton into the aqueous phase. The proton carrier changes its colour from blue to red and,
depending on the choice of wave1ength, either the reduction in fluorescencc of the b1ue
dye or the corresponding increase in the fluorescence of the red dye can be determined.
Due to the higher sensitivity and selectivity, measurement of the fluorescence is preferred.
A significant disadvantage of the process is the low sensidvity of the system, due to the
low fluorescence quantum yield of the indicator dye used.
. ~. ..
J.N. Roe in Analyst, Vol. 1 lS, pages 353 to 358 (1990), describes a system based on
energy transfer due to complex formadon of the fluorescence dye used with the anionic ~ - -
form of a certain indonaphthol, which itself forms a ternary complex with the
potassium-charged ionophore. The potassium is determined by measuring the change in
absorpdon after charging with potassium or from the change in fluorescence. The
sensidvity and response speeds of this system are regarded as unsadsfactory.
Y. Kawabata in Anal. Chem. Vol. 62, pages 1528-1531 and 2054 to 2055, describes a
membrane system for the optical determinadon of potassium using a hydrophobic ion
exchanger, namely 3,6-bis(dimethylamino)-10-dodecyl- or -10-hexadecylacridinium ~ -
bromide. A change in fluorescence is achieved by changing the polarity in the
microenvironment of the sample, since the acridinium salts diffuse at the interface with ~ ~ -
the aqueous phase due to ion exchange with the potassium ion.
W.E. Morf et al. in Pure ~ Appl. Chem., Vol. 61, No. 9, pages 1613 to 1618 (1989),
describe the use of pH-sensitdve chtomo- or fluoroionopho~es for the optical determination
of cadons based on ion exchange reacdons. The sensitivity of these systems is reladvely
low, the measurement is hindered in optical1y dense systems, and relatively highconcentrations of chromo- or fluoroionophores in the membrane are required.
K. Wang et al. in Analytical Science, Vol. 6, pages 715 to 720 (1990), describe
membranes containing an absorption dye (Nile Blue) as indicator substance for the opdcal
measurement of metal cations. The system is based on an ion exchange mechanism which
reduces the absorpdon by protonadon of the dye. The sensitivity of the system is regarded
as too low.
Hitherto, no systems having an ion exchange mechanism for the optical measurement of
21~984~
` , .
metal cations has been disclosed which are based on the determination of the reduction in
fluorescence of a fluorophore and have high sensitivity, since the fluorescence quantum
yields and basicities of the known pH-sensidve fluorophores are too low.
It has now been found that certain acridine dyes and rhodlamine dyes surprisingly satisfy
these high requirements and are lipophilic, pH-sensitive and highly basic fluorophorcs
which are highly suitable, in a neutral polymer membrane together with an ionophore and
a lipophilic salt of a borate, for the determination of metal cations, in pardcular potassium,
by the ion exchange mechanism and have a fluorescence which is highly dependent on the
corresponding metal cadon concentradons. These fluorophores are disdnguished by a high
fluorescence quantum yield, high basicity, a large difference between the fluorescence
signals of the protonated and deprotonated forms, high lipophilicity, adequate
photostability and suitable absorption and emission wavelengths. Highly sensidve systems
for the optical determination of metal cations on the basis of ~uorescence measurements
can be provided.
The invention relates to compounds of the formulae I and II -
R1R2N~NHR3 (I), -
R4R5N~o~NR6
~ ' '
(II), - --
CO2H
in which Rl and R3, and R4 and R~ are Cl-C30a1kyl or C1-C30alkyl-CO-, and R2 and Rs are
H or Cl-C30alkyl, with the proviso that the total number of carbon atoms in the alkyl
groups is at least 12, and salts thereof with inorganic or organic acids.
In a preferred embodiment, R2 is H. ~ ~ ~
" ~ :
' :
The a1kyl groups can be linear or branched and preferably contain 1 to 22 carbon atoms.
Linear alkyl groups are prcferred. Examples of a1kyl are methyl, ethyl and the isomers of
propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,
tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl,
docosyl, tricosyl, tetracosyl and triacontyl. In a preferred embodiment, Rl and R3 are
C6-C24alkyl or C6-C24alkyl-C0-, particu1arly preferably C~0-C24alkyl or
C10-C24alkyl-CO-, especially prefcrably Cl4-C22alkyl or Cl4-C22alkyl-C0-, while R2 is H.
In another embodiment, R5 is preferably H, and R4 and R6 are preferably C6-C24alky1,
particularly preferably C1O-C24alkyl, especially preferably Cl4-C22alkyl. In a fur~her
embodiment, R4 and Rs are preferably Cl-C6alkyl, particularly preferably Cl-4alkyl,
especially preferably methyl or ethyl, and R6 is C10-C24alkyl or C10-C24aL~cyl-C~, - -
preferably Cl4-C22alkyl or C14-C22alkyl-C0-, especially preferably Cl6-C22alkyl or
Cl6-C22alkyl-C0-.
The salts of the compounds of the formulae I and II can be derived, for example, from HF,
HCI, HBr, HI, H2SO3, H2SO4, H3P03, H3P04, HNO2, HN03, HCI04, HBF4, HPF6,
HSbF6, CF3S03H, toluenesulfonic acid, Cl-C4alkyl- or phenylphosphonic acid, formic
acid, acedc acid, propionic acid, benzoic acid, mono-, di- or trichloroacedc acid, or ~-
mono-, di- or trifluoroacetic acid. ~reference is given to HCI, HBr, H2S04, HCl04, HBF4,
HPF6 and HSbF6-
The compounds of the formula I can be prepared in a manner known per se from
commercial 3,~diaminoacridine by stepwise alkylation by means of various alkyladng
agents or alkyladon using one alkyladng agent or acylating agent. Examples of suitable
alkyladng agents are dialkyl sulfates or monohaloalkanes, in pardcular chloro, bromo-
and iodoalkanes. Examples of suitable acyladng agents are carboxylic anhydrides and in
particular carboxylic acid halides, for example carboxylic acid chlorides. This reacdon can
be carried out in the presence of inert polar and aprotic so1vents, for example ethers, !
alkylated acid amides and lactams, sulfones or sulfoxides, and at elevated temperatures,
for example from 50 to 150C. It is expedient to add a halogen scavenger, for example - -
alkali metal carbonates. ;
The compounds of the formula II can be obtained, for example, by reacting phthalic
anhydride with 2 mol equivalents of 2-monoalkylaminophenol. Another possible
preparation comprises reacdng 2-monoalkylaminophenol with I mol equivalent of
2-hydroxy-4-dialkylamino-2'-caboxybenzophenone. These reactions are described, for
8~
example, in US-A-4 622 400. The reaction is expediently carried out in an inert solvent,
for example hydrocarbons or ethers. Molar amounts of a condensadon agent, for example
Lewis acids, concentrated sulfuric acid, perchloric acid or phosphoric acid, areadvantageously added. The reaction temperatures can be, for example, from S0 to 250C.
The compounds of the fonnula I can be isolated in a convendonal manner by precipitation,
crystallization or extraction and purified, if necessary, by recrystallization or
chromatography. They are crystalline, red, red-brown or red-violet compounds.
The compounds of the formulae I and II are highly suitable as fluorophoric dye indicators
for the optical determination of cations in an aqueous environment, in particular by
measurement of the change in fluorescence.
The invention furthermore relates to a composition comprising
(a) a transparent support,
(b) which is coated on at least one side with a transparent coating which comprises
(bl) a hydropho~ic polymer,
(b2) a plasticizer,
(b3) the salt of a lipophilic anion,
(b4) an ionophore which forms a complex with the ion to be determined, and
(bS) a compound of the formula I or II as fluorophore.
The compounds of the formulae I and II preferably have a pKa value of at least 8,
particularly preferably at least 10.
The support can be forrned, for example, from a plastic material, mineral materials or
glass and can have any desired shape, for example plates, cylinders, tubes, tapes or fibres.
Glasses are preferred.
The thickness of the coating on the support can be, for example, from 0.01 to 100 llm, -
preferably from 0.1 to S0 ~,lm, more preferably from 0.1 to 30 llm, and particularly
preferably from 0.1 to 10 ~Lm.
'"' :
Various types of polymer are suitable for the composition. They expediently have a mean
molecular weight of at least 100 000 daltons, for example from 100 000 to
2 000 000 daltons, preferably from 200 000 ~o I 000 000 daltons. The polymers must have
"~ " ;"~ ~ "~ "~ " ~
8~
- 6 -
adequate solubility in organic solvents so that they mix with the other components and can
be converted into coatings by conventional coating methods. Some examples of
homopolymers and copolymers are those of olefins, acrylates, methacrylates, vinyl esters,
acrylonitrile, dienes, styrene, methylstyrene, vinyl chloride, vinyl fluoride, vinylidene
chloride and vinyl ethers. Some specific examples are polyvinyl chloride, polyvinylidene
chloride, polyvinyl fluoride, polyacrylonitrile, polystyrene, poly(methylstyrene),
polyacrylates and polymethacrylates containing Cl-Cl8alkyl radicals in the ester groups,
vinyl chloride-vinyl acetate copolymers, vinyl chloride-vinyl acetate-vinyl alcohol
copolymers, vinyl chloride-vinyl acetate-acrylonitrile copolymers, vinyl
chloride-vinylidene chloride copolymers, vinylidene chloride-acrylonitrile copolymers,
and acrylonitrile-butadiene-styrene copolymers. Preferred polymers are homopolymers of
vinyl chloride and vinylidene chloride and copolymers of vinyl chloride and/or vinylidene
chloride and acrylates, methacrylates, vinyl esters, vinyl alcohol, acrylonitrile and styrene.
Particular preference is given to polyvinyl chloride.
It is advantageous to incorporate a plasticizer into the polymers in order to optimize the
diffusion coefficients of the membrane components for ion exchange. Depending on the
type of po1ymer and plasticizer, ~rom 10 to 90 % by weight, preferably from 20 to 70 % by
weight, particularly preferably from 20 to 50 % by weight, of polymer and from 90 to
10 % by weight, preferably from 80 to 30 % by weight, in particular from 8~ to 50 % by
weight, of plasticizer may be present. Suitable plasticizers are known in large number.
They can be, for example, higher alkanols or esters thereof, esters of fatty acids with diols
or alkanols, ethers with higher alkanols, esters of di- and polycarboxylic acids and esters
of higha alkanols and phosphoric acid or phosphorous acid. The higher alkanols can be,
for example, linear or branched C6-C22alkanols or phenols substituted by 1 to 3 linear or
branched C3-Cl8alkyl or C3-CI8alkoxy groups. Some specific examples are octadecanol,
phthalic diesters, glutaric diesters, adipic diesters, azelaic diesters and sebacic diesters
with C8-CI8alkanols, such as 2-ethylhexanol, n-octanol, n-decanol, n-dodecanol,
tetradecanols, hexadecanols, heptadecanols and octadecanols~ tris(2-ethylhexyl) phosphate
or tris(2-ethylhexyl) phosphite, tris(nonylphenyl) phosphite, ethylene glycol monostearate,
ethylene glycol dibenzoate and 2-nitrophenylbutyl or -octyl ether. The type and amount of
plasticizers are expediently selected so that they are compatible with the polymer, so that
phase separations do not occur and so that the hydrophobic properties of the polymer
undergo little or no change. The plasticizers should be lipophilic and contribute to
dissolution and distribution of the components in the coating (matrix).
,, ,- , . ~ -
~, . , . . , - . . .. .. .
9~0
Examples of suitable salts with lipophilic anions are alkali metal, alkaline earth metal and
ammonium salts with substituted or unsubstituted tetraphenylborates. Preferred cations are
Li~E3, Na~, K~3, Mg233, Ca2~, NH4~, and the ammonium cations of primary, secondary
and tertiary amines and quaternary ammonium cations which can contain from 1 to 60
carbon atoms. Some examples of ammonium cations are methyl-, ethyl-, propyl-, butyl-,
hexyl-, octyl-, decyl-, dodecyl-, tetradecyl-, hexadecyl-, octadecyl-, dimethyl-, diethyl-,
dibutyl-, butylmethyl-, dioctyl-, didoceyl-, dodecylmethyl-, trimethyl-, triethyl-, tripropyl-,
tributyl-, trioctyl-, tridodecyl-, dodecyldimethyl-, didodecylmethyl-, tetramethyl-,
tetraethyl-, tetrapropyl-, tetrabutyl-, tetrahexyl-, tetraoctyl-, tetradecyl~, tetradodecyl-,
dodecyltrimethyl-, octyltrimethyl-, didodecyldimethyl-, tridodecylmethyl-,
tetradecyltrimethyl- and octadecyltrimethylammonium. Quaternary ammonium salts are
preferred, in particular those having 4 to 48 carbon atoms.
An example of a suitable borate anion is tetraphenylborate, whose phenyl groups may be
substituted by one or more, preferably 1 to 3, particularly preferably 1 or 2, Cl-C4alkyl,
Cl-C4alkoxy, halogen, for example F or Cl, or trifluoromethyl groups. Some specific
examples are sodium tetraphenylborate, sodium tetra(3,5-bistrifluoromethylphenyl)~orate,
potassium tetra~4-chlorophenyl)borate, tetrabutylammonium tetraphenylborate and
tetradodecyl(4-chlorophenyl)borate. The salts with lipophilic anions serve as negative
charge compensation for the metal cations diffusing into tbe active coating and to be
measured therein in complexed forrn.
The amount of salts with lipophilic anions can be, for example, from 0.01 to 10 % by
weight, preferably from 0.1 to 5 % by weight, particularly preferably from 0.1 to 2 % by
weight, based on the amount of polymer and plasticizer.
The polymer coating (also referred to as membrane) contains an ionophore in, forexample, an amount of from 0.01 to 10 % by weight, preferably from 0.1 to 5 % by ~ -
weight, particularly preferably from 0.1 to 2 % by weight, based on the amount of polymer
and plasticizer. Ionophores are natural or synthetic organic compounds which contain a
plurality of, usually alternating, electron-rich heteroatoms, for example S, N and in
particular 0, in linear or cyclic carbon chains and which are capable of selectively
complexing the metal cations to be measured. The natural compounds are frequently
macrocyclic compounds, for example valinomycin, which is capable of selectively
binding potassium cations. Another example is nonactin. A large group of ionophores -
comprises macrocyclic polyethers (crown ethers), which are capable of complexing
.
1 .~,.. ' ,
'~! J ~
- 8 -
various metal cations, depending on the geometry and size. Further examples of
ionophores are coronandenes, kryptandenes and calixarenes. Examples of linear
ionophores are podandenes. Such ionophores are described, for example, in US-A-4 645
744.
The amount of compounds of the formulae I and II can be, for example, from 0.01 to 10 %
by weight, preferably from 0.1 to 5 % by weight, particularly preferably from 0.1 to 2 %
by weight, based on the amount of polymer and plasticizer. The compounds of the
formulae I and II can also be bonded to the polymers via suitable bridging groups.
The fluorophores to be used according to the invention have very suitable absorption and
emission wavelength ranges which allow the use of known and inexpensive light sources
and detectors, for example halogen or xenon lamps or light-emitting diodes. Examples of
detectors which can be employed are photodiodes. The fluorophores furthermore have ~ -
high absorption coefficients and high quantum yields can be achieved. The high
lipophilicity, high basicity and the large dynamic range of the change in fluorescence --between the protonated and deprotonated forms satisfy, in particular, the high
requirements for opdcal determination of cations based on fluorescence measurements.
Examples of suitable cations are metal cations of metals from the first to fifth main groups
and the ~Irst to eighth sub-groups of the Periodic Table of the Elements, the lanthanides
and actinides. Some examples of metals are Li, Na, K, Rb, Cs, Mg, Ca, Sr, Ba, B, Al, Ga,
In,Tl,Sn,Pb,Sb,Bi,Cu,Ag,Au,Zn,Cd,Hg,Sc,Y,Ti,Zr,Hf,Cr,Mo,W,Mn,Fe,Co,
Ni, Ru, Os, Rh, Ir, Pt, Pd, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Yb, Lu, Ac,
Th, Pa, U, Np and Pu. Preferred metal cations are the alkali and alkaline earth metal ions, b
in particular Li~3, Na~3, K~, Mg2~3, Ca2~3 and Sr2~, very particularly K~33, Na~3 and
Ca~. Examples of ammonium cations are NH4~ and the cations of protonated primary,
secondary and tertiary amines and quaternary ammonium. The amines can contain from 1
to 40, preferably from 1 to 20, particularly preferably from 1 to 12, carbon asoms. The
quaternary ammonium can contain from 4 to 40, preferably from 4 to 20, particularly
preferably from 4 to 16, carbon atoms.
The composition according to the invention is highly suitable as an optical sensor for the
quansitative determination of cations, in particular metal cations, very particularly
potassium cations, in an aqueous environment, preferably by means of fluorescence
spectrometry. The determinations can be carried out quickly with high accuracy even for
~1 ~9840
low concentrations (for example in the micromolar range to the nanomolar range), since
the pH-dependent equilibria of the complexing reactions and of proton exchange become
established quick1y and the fluorophores are characterized by a high fluorescence quantum
yield and sensidvity. The analyses can be carried out, for examp1e, directly in body fluids
(blood, urine, serum), natural water or waste water, where it may be possible for any
interfering cadons to be specifically bound or removed in advance. The composition
according to the invention is particularly suitable for the determination of physiological
amounts, for example in the range from QS to 10 mmol, of cadons in aqueous mcdia.
In addition to the preferred method of fluorescence spectroscopy, otha opdcal ~ -
measurement methods may also be used, for example surface plasmoresonance - ~ -~
spectroscopy, absorption spectroscopy, refle tion spectroscopy, interferometry or
surface-amplified Raman or fluorescence spectroscopy~
The invention further nore relates to an opdcal sensor for tho determinadon of cations in --
aqueous measurement samples, in particular by means of fluorescence sp~ctrometry,
whichcomprises ;;
(a) a transparent support,
(b) which is coated on at least one side with a transparent coadng comprising ~ ~ -
(bl) a hydrophobic polymer,
(b2) a plasticizer,
(b3) the salt of a lipophilic anion,
(b4) an ionophore which forms a complex with the ion to be determined, and -~
(bS) a compound of the fonnula I or II as the fluorophore.
The invendon furthermore relates to a method for the opdcal determinatdon of catdons in
aqueous measurement samples, in which a composition according to the invention is - ~ -
brought into contact with said aqueous measurement sample, and then, in particular, the
reducdon in fluorescence in the acdve polymer coatdng is measured. '~
',
The invendon furthermore relates to the use of the compounds of the formulae I and II as -
fluorophores for the opdcal determinadon of cations in aqueous measurement samples.
The process according to the inventdon can be carried out, for example, by immobilizing
the composition according to the invention comprising support and acdve polymer coating
in an optical cell in which the active coating is brought into contact with the measurement ~ -
- 10-
sample. The optical cell furthermore contains a window through which the active coating
can be excited by irradiation and the emitted fluorescence radiation can be measured by
means of a spectrophotometer. The wavelengths are adjusted so that the absorption is at a
maximum for the irradiadon and the emission is at a maximum for the fluorcscencemeasurement. The decrease in intensity as a function of time is measured. Thc
measurement system can be designed so that the measurement is carried out
discontinuously or continuously, for exarnple by pumping the measurement soludonthrough the measurement cell. In order to determine unknown concentratdons of cations,
the system is first calibrated by means of moasurement samples of known concentratdon, -
and the concentradons are plotted as a function of the fluorescence intensity. It is
expedient to add pH buffers to the measurement sample, since the sensitivity of the
measurement, and consequently also the fluorescence intensity of the fluorophore, ~;
depends on the pH of the measurement soludon due to the pH-dependence of the
absorptdon spectrum. The pH range of the measurement sample can be, for example, from
4 to 8, more preferably from 5.5 to 6.5. Examples of suitable buffers are citrate buffers and
phosphate buffers. Further buffer systems are described in US-A-4 645 744, in pardcular
including those which are incorporated direcdy into the acdve coating in ordér to avoid -
additdon to the measurement sample.
The examples below illustrate the invendon in greater detail.
A) Preparation of fluorophores
Example Al: Preparadon of 3,~bis(n-octylamino)acAdine.
6.33 g of anhydrous potassium carbonate are added to a soludon of 2.5 g of
3,6-diaminoacridine hydrochloride and 3.55 ml of 1-bromooctane in 50 ml of dimethyl
sulfoxide, and the mixture is sdrred at 80C for 48 hours. The cooled reaction mixture is
subsequently poured onto ice, and the brown suspension is extracted with methylene
chloAde. The organic phase is washed with saturated aqueous NaCI solution and dried
over sodium sulfate. After evaporadon, the red-brown oil is chromatographed on silica gel
using methylene chloAde/methanol (9:1). After evaporation of the solvent, the residue is
taken up in diethyl ether/methanol (10:1) and chromatographed on aluminium oxide. The
eluate is taken up in methanol, 2N HCI is added, the mixture is extracted with diethyl
ether, and the ether phase is dried and evaporated. The residue is dissolved in methylene
chloride, n-hexane is added, and the red crystalline precipitate fo,rmed is filtered off.
Further product can be isolated from the mother liquor after evaporadon. The melting
2 ~
point of the title compound is 245C. Absorption spectrum (ethanol): ~ma~= 472 nm;
~ = 51 400.
Example A2: Preparation of 3,6-bis(n-eicosylamino)acridine.
2.53 g of anhydrous potassium carbonate are added to a solution of 2.5 g of
3,6-diaminoacridine hydrochloride and 2.95 g of l-eicosyl bromide in 20 ml of
N,N'-dimethylethyleneurea, and the mixture is stirred at 50C for 86 hours. The cooled
reaction mixture is subsequently poured into water, and the orange-brown suspension is
extracted with methylene chloride. The organic phase is washed with water and dried over - - ~ -~
sodium sulfate. After evaporation, 2N HCI is added to the brown oil. The red precipitate
formed is filtered off, washed with water and then dried in a high vacuum. The resultant
red-brown crystals are taken up in methylene chloride/methanol (10:1) and ~ -
chromatographed on silica gel. After evaporation, the residue is taken up in diethyl
ether/methanol (10:1) and re-chromatographed on silica gel, giving the title compound as ~ -
red crystals, absorption spectrum (ethanol): ~ = 472 nm; ~ = 42 200.
,
Exarnple A3: Preparadon of 3,6-bis(n-hexylamino)acridine.
298 mg of ground potassium hydroxide are added to a solution of 500 mg of ;
N,N'-bistosyl-3,~diaminoacridine and 797 mg of l-bromohexane in 25 ml of
dimethylformamide, and the mixture is stirred at 60C for 22 hours. The cooled reaction
mixture is subsequently po~red into water and extracted with ethyl acetate, and the '
organic phase is separated off, washed with aqueous NaCI solution and dried over sodium
sulfate. Evaporation gives a dark-red oil, which is taken up in toluene/ethyl acetate (20:1)
and chromatographed on silica gel. Evaporation of the solvent gives a yell~w, viscous oil,
which is dissolved in 11.5 ml of glacial acetic acid, 4.6 ml of 97 % sulfuric acid are added
with water cooling, and the mixture is then stirred at room temperature for 15 hours. The
red reaction mixture is poured into ice water and adjusted to pH 11 by means of 30 %
NaOH. The mixture is extracted with ethyl acetate, and the organic phase is washed wi~h
2N HCi and saturated aqueous NaCI solution and then dried over sodium sulfate. After
evaporation, the dark-red, viscous oil is taken up in t-butyl methyl ether/methanol (5:1)
and chromatographed on silica gel, giving the title compound as orange-red crystals
having a melting point of > 200C (decomposition). IH-NMR (CDCI3): 8.1 [s, lH, C(9)];
7.44 [d, 2H, C(8)]; 6.93 [s, 2H, C(S)]; 6.82 [d, 2H, C(7)]; 3.20 [t, 4H, N-CH2]; 1.68 [m,
6H, CH3].
:
~ ,~
~.lg8~0
- 12-
Example A4: Preparation of 3,6-bis(n-heptylcarbonylamino)acridine.
3.1 ml of heptanoyl chloride are slowly added dropwise to a suspension of 2.5 g of
3,6-diaminoacridine hydrochloride in 50 ml of pyridine, and the mixture is then stirred for
30 minutes. The reaction mixture is subsequendy poured into water. The yellow
suspension is extracted with methylene chloride, and the organic phase is washed with
aqueous saturated NaCI solution and dried over sodium sulfate. After evaporation, the
dark-red oil is taken up in methylene chloridefmethanol (10:1) and chromatographed on
silica gd. The evaporated eluate is taken up in medhylene chloride and added dropwise to
cyclohexane. The yellow precipitate formed is filtered off, washed with cyclohexane and
dried in a high vacuumj giving the dde compound as yellow crystals having a meldng
point of 243-244C. Absorption spectrum (ethanol): ~ = 384 nm; ~ = 2 300.
Example A5: Preparation of
(HsC2)2N~N-(n-c2oH4l)
~COOH
a) A solution of 12.8 g nf phthalic anhydride and 13.2 g of 3-N,N-diethylaminophenol is
sdrred at 110C for 16 hours in 75 ml of toluene. The precipitated product is filtered off
and recrystallized from ethanol, giving brick-red crystals of
1-carboxy-1'-hydroxy-3'~iethylaminobenzophenone (product A) having a meldng point
of 214C.
b) A solution of S.S g of 3-aminophenol and 21.6 g of 1-bromoeicosane in 250 ml of
1,4-dioxane is stirred at 100C for 48 hours. The mixture is evaporated in vacuo, and! the
brown, gelatinous residue is taken up in toluene/ethyl acetate (10:1) and chromatographed
on silica gel, giving 3-N-eicosyla ninophenol as white crystals having a meldng point of
80C.
c) 626 mg of product A and 790 mg of 3-N-eicosylaminophenol are stirred for 2 hours at
170C in S ml of phosphoqic acid (85 %). After cooling, a solution of 1 ml of concentrated
HCl in 1 ml of methanol is added, and the mixture is subsequently extracted withmethylene chloride. After removal of the solvent, the residue is taken up in methylene
chloride/methanol (85:15) and chromatographed on silica gel, giving the title compound as
,
8 ~ ~
- 13-
, . ..
red-violet crystals having a melting point of 115C. Absorption spectrum (ethanol): -
= 532 nm; ~ = 90 000. : : :
Example A6: Preparation of ~ :
(n-c8H17)HN~f HCI
COOH
a) A solution of 5.45 g of 3-aminophenol and 11.6 g of 1-bromooctane in 250 ml of -,
dioxane is stirred at 100C for 80 hours, the solvent is then evaporated, and the residue is . ~:
then taken up in toluene/ethyl acetate (10:1~ and chromatographed on silica gel, giving -
N-octylaminophenol as beige crystals, melting point 75C.
b) 1.1 g of N-octylaminophenyl and 0.37 g of phthalic anhydride are melted together at ~:-
100C. 1 ml of phosphoric acid (85 %) is added to the melt, which is then heated to
170C. After 1 hour, the mixture is allowed to cool, and 2N HCl is added. The mixture is
extracted with methylene chloride, the solvent is removed, and the red residue is taken up
in methylene chloride/methanol (85:15). Chromatography on silica gel gives the tide .
compound as red crystals having a melting point of 183C. Absorption spectrum (ethanol): : :
= 522 nm; ~ = 73 7~
Preparadon of
(c2Hs)2N~oN-co-n-c17H3s
HCI04
~COOH
W ...
a) 1.57 g of product A from Example ASa, O.SS g of 3-aminophenol and 10 ml of
phosphoric acid (85 %) are stirred for 30 minutes at 170C. 6.7 ml of perchloric acid
(50 %) and 100 ml of methanol are then added, the mixture is re-heated, and the solvent is
~1~98~0
- 14-
then removed in vacuo. The residue is ~aken up in methy1ene chloride, the solution is
washed with water, and the solvent is removed again. The residue is taken up in methylene
chloride/methanol (10:1) and chromatographed on silica ge1, giving red crystals of
compound B of the formula
(C2H5)2N ~OX~NH
T HCI04
[~COOH
having a melting point of 175C.
b) 0.1 g of compound B is dissolved in 1 ml of methylene chloride and 0.3 ml of pyridine,
and 100 mg of stearoyl chloride are added. After 3 hours, dhe mixture is evaporated to
dryness in vacuo, and the residue is dissolved in methylene chloride/methanol (85:15) and
chromatographed on silica gel, giving the tide compound as red crystals having a melting
point of 145C. Absorption spectrum (edlanol): ~m8x= 56 nm; ~ = 10 900.
B) ~roduction of coated supports
Examples B1-B6:
a) The support material used is pretreated glass. Circular glass sheets (diameter 18 mm,
thickness 0.17 mm) are immersed for one hour in a solution of 10 % by ~olume of
dimethyldodecylchlorosilane in isopropanol. The glass sheets are then each washed one
after the other with 200 ml of isopropanol, ethanol and methanol and dried at 110C for
1 hour. The hydrophobicized surface has better adhesion of the membrane coating.b) Preparation of the coating solution.
The following consdtuents are introduced into a 2 ml bottle together with 1.5 ml of
tetrahydrofuran and shaken until the components have dissolved:
1. 80 mg of polyvinyl chloride (Fluka, 81392)
2. 160 mg of bis(2-ethylhexyl) sebacate (plasticiur)
3. 5 mg of valinomycin
4. 13 mg of potassium tetrakis(4-fluorophenyl)borate
5. 2 mg of fluo~ophore
~l~g~O
- 15-
Example No. Fluorophore
B 1 Example A5
B2 l~xample A6
B3 Example A7 ,
B4 Example A1
B5 Example A2
B6 Example A4 -
c) Broduction of coated glass supports.
The glass supports are clamped in the chamber of a spin-coating apparatus (Optocoat OS
35var, Willer Company, CH-8484 Weisslingen). The chamber is rinsed with 10 ml oftetrahydrofuran and rotated for 2 minutes at 3 800 revolutions/minute. 50 11l of the
particular coating solution are then pipetted onto the glass support, and the glass support is ~ - -
rotated for a further 10 seconds. The glass support coated with a membrane is then - ~ --
removed and dried for 10 minutes in air. - -
:
d) Spectral properties of the membranes. ~ ~
The coated glass supports are clamped in an optical cell in which the membrane is in -
contact with the measurement liquid. The membrane can be opdcally excited in the optical
cell and the fluorescence radiation measured. The optical cell is introduced into a
spectrophotometer (Perkin-Elmer LS-50), and the absorption and emission spectra are
measured. ~e relative fluorescence quantum yield is furthermore determined by the ~ -
method described by Calvert and Pitts in Photochemistry, pages 799 to 805 (1966),-John
Wiley and Sons Inc., using fluorescein as standard. The results are shown in Table 1.
Table 1:
Example AbsorptionEmission Absorbance Quantum
(~max~ nm)(~lmaX, nm) coefficient vield
Bl 540 570 90 000 0.14
B2 525 555 73 700 0.33
B3 500 560 20 000 0.08
B4 470 505 5 1400 0.75
B5 470 505 42 200 0.75
B6 380 460 2 300 0.10
119~0
- 16 -
Examples B7 to B12: Determination of pK, values and the change in fluorescence.
The fluorophores are dissolved in a mixture of 30 % by volume of methanol and 70 ~6 by
volume of phosphate buffer and adjusted to various pH values between 6 and 13. The
measurement solutions a~e introduced into a cell, and the fluorescence intensity is
measured using a spectrophotometer. The fluorescence intensity is plotted as a funcdon of
pH, and the pK. value is determined from the point of inflexion in the curve. The ratio
between the protonated and deprotonated forms of the fluorophore is furthermore
determined from the change in fluorescence by dissolving the fluorophore in
tetrahydrofuran, adjusdng the pH by addition of lM HCI or 1.0M NaOH and determining
the change in fluorescence intensity in percent. The concentration of the fluorophores is
lo-7 molll in each case. The results are shown in Table 2.
Table 2: -
Fluorophore of Example PK,, value Chan~e in fluorescence intensitv
A1 10.0 44
A2 11.1 41
A4 10.0 24
A5 10.3 33
A6 12.0 20
A7 10.1 15
, ::
C) Use examples:
,~
Example C1: The same experimental set-up as in Example Bld is used and the absorption
and emission wavelengths are adjusted to the corresponding maxima of the fluo~ophores
employed in the membrane. The membrane is brought into contact with an aqueous KCI
solution of defined concentration by pumping the solution through the cell at a rate of
1 mVmin and determining the change in fluorescence intensity. Before the measurement
and after each measurement, the cell is rinsed with potassium ion-free buffer soludons and
the fluorescence intensity is determined in order to define the base line. The reducdon in -
fluorescence intensity in percent at the respecdve potassium concentrations for the
fluorophore of Example A5 (membrane B1) is shown in Table 3.
- 17-
Table 3:
Potassium concentration (mM) % reduction in fluorescence intensitv
0.01 5
0.1 20
0.5 35
l.0 45
5.0 53
10.0 60
100.0 68
It can be seen from the table that the fluorescence intensity decreases with increasing
potassium concentration and, after calibradon, unknown concentrations can be determined
with high reliability. In particular in the physiological range from about 0.5 to 10 mM of
potassium, high sensitivity is guaranteed. - - -
Exarnple C2: The procedure is as in Example Cl using the fluorophore of Example A6. -
The reduction in fluorescence intensity in percent and the respective potassium
concentrations for the fluorophore of Example A6 (membrane B2) are shown in Table 4.
Table 4:
Potassium concentration (mM) . % reduction in fluorescence intensity
0.1 36
0.5 41
1.0 43
5.0 48
10.0 52 ~ ~ :
Example C3: The procedure is as in Example C1 using the fluorophore of Example Al.
The reduction in fluorescence intensity in percent and the respective potassium : :
concentrations for the fluorophore of Example A1 (membrane B4) are shown in Table 5.
.9~
- 18-
Table 5:
Potassium co ce_tration (mM) % reduction in fluorescence intensitY
0.1 5
0.5 15
l.0 25
5.0 39
10.0 45
100.0 75
Example C4: The procedure is as in Example C1 and the sodium ion concentration is
determined using a membrane comprising 2 mg of fluorophore of Example A5, 30 sng of
sodium ionophore (4-octadecanoyloxymethyl-N,N,N,N-tetracyclohexyl-
1,2-phenylenedioxydiacetamide, Fluka sodium ionophore V, Catalogue No. 71738), 2 mg
of potassium tetrakis(4-chlorophenyl)borate, 75 mg of polyurethane (Tecoflex~), 160 mg
of bis-2-ethylhexyl sebacate as plasticizer and 1 ml of tetrahydrofuran. The sensor is
exposed to a buffer solution at pH 5 (0.1 mol of trishydroxymethylaminomethane and lM
HCl) with different sodium concentrations. The results are shown in Table 6.
:
Table 6~
Sodium concentration (mM) % reduction in fluorescence intensitY
O O : :.
3 15
41
54
72
150 7~ - -
210 83
300 86
Example C5: The procedure is as in Example Cl and the calcium ion concentration is
determined using a membrane compnsing 2 mg of fluorophore of Example AS, 30 mg of
calcium ionophore ((-)-(R,R)-N,N-[bis(1 1-ethoxycarbonyl)undecyl]-N,N-4,5-
tetrarnethyl-3,6-dioxaoctanediamide, Fluka calcium ionophore I, Catalogue No. 21192),
6 mg of potassium tetrakis(4-chlorophenyl)borate, 75 mg of polyurethane (Tecoflex(~),
. : ~., . . : .
~119~40
- 19-
160 mg of bis-2-ethylhexyl sebacate as plasticizer and 1 ml of tetrahydrofuran. The sensor
is exposed to a buffer solution at pH 5 (0.02M NaOH and acetic acid) with different
calcium concentrations. The results are shown in Table 7.
Table 7:
alcium concentration (mM) % reduction in fluol~scence intensity
O
0.1 26.5
0.5 43.1
1.0 50.6
61.0
5.0 - 65.8
7.0 68.1 -:
0.0 71.0
:
":.'
, , , ,, . ,, .".. ,~ j.- ", ji
