Lipophilic derivative of rhodamine 19: characterization and spectroscopic properties

Analytica Chimica Acta 468 (2002) 13–25

Lipophilic derivative of rhodamine 19: characterization andspectroscopic properties

Snežana Miljanica,∗, Zvjezdana Cimermana, Leo Frkanecb, Mladen Žinicb

a Laboratory of Analytical Chemistry, Faculty of Science, University of Zagreb, Strossmayerov trg 14, 10000 Zagreb, Croatiab Laboratory of Supramolecular and Nucleoside Chemistry, Ru−der Boškovic Institute, Bijenicka 54, 10002 Zagreb, Croatia

Received 12 February 2002; received in revised form 20 June 2002; accepted 4 July 2002

Abstract

A new octadecylamide derivative of rhodamine 19 was synthesized by reaction of rhodamine 19 ethyl ester (rhodamine 6G) with octadecylamine. This lipophilic dye exists in two forms: as a cationic species containing a secondary amide groupand as an electrically neutral molecule containing an additional ring closed between the oxygen of the amide group and thecentral carbon of the xanthene part. Both forms were isolated and characterized in detail by means of elemental analysis, massspectrometry, IR, NMR and UV–VIS spectroscopy. Depending on the hydrogen ion activity in solution and the availability ofsolvent hydrogen for hydrogen bond formation, the cationic form easily converted into a neutral one and vice versa. On thebasis of absorption and emission UV–VIS spectroscopic data, the equilibrium of the two forms was studied in various solvents.An increase in dye concentration in acidified methanol/water mixture (volume fraction of water,ϕ = 80%) led to aggregationof the cationic molecules. Using the UV–VIS absorption data, a corresponding dimerization constant was evaluated.© 2002 Elsevier Science B.V. All rights reserved.

Keywords:Rhodamine 19 octadecylamide; Cationic form; Neutral form; Dimer; Fluorescence

1. Introduction

Rhodamines belong to the class of xanthene dyes,which are among the oldest and most commonly usedof all synthetic dyes. Initially, they were used forcloth and food coloring[1]. Owing to their uniqueoptical properties, the scope of their application hasbeen extended. Nowadays, they serve as water tracingagents[2], fluorescent markers for microscopic stud-ies of complex cellular processes and structure[3]and for detection of specific nucleic acid sequences[4], as photosensitizers[1], laser dyes[5], and since

∗ Corresponding author. Tel.:+385-1-4819283,fax: +385-1-4818458.E-mail address:[email protected] (S. Miljanic).

recently, as chromoionophores in optical chemicalsensors[6–14].

Rhodamine dyes have a large molar absorptivity(≈105 l mol−1 cm−1) in the visible region of the spec-trum where a corresponding absorption band is at-tributed to a�∗ ← � transition[15]. Absorption andemission are influenced by substituents on the nitro-gen of the amino groups of the xanthene moiety[16].When the phenyl carboxylic group is not esterified,the acid–base equilibrium has an effect on the spectro-scopic properties of the dye[17]. Since the probabilityof the intersystem crossing is very small, in additionto fluorescence, internal conversion is also a way ofdeactivation[18].

Depending on proton activity, solvent, temperatureand concentration, rhodamine dyes exist in solution in

0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.PII: S0003-2670(02)00627-X

14 S. Miljanic et al. / Analytica Chimica Acta 468 (2002) 13–25

several forms: as neutral species, ionized forms, lac-tones[19–21] or as aggregates[22–25]. Each formis characterized by appropriate absorption and emis-sion spectra which are further influenced by specificmedium effects. Therefore, spectroscopic propertiesof rhodamines are still the subject of numerous re-search studies and controversies. On the other hand,their equilibria can be very useful for analytical ap-plication in optical sensors. The equilibrium betweenthe “zwitterion” and the lactone form of rhodamine Bhas been used as a sensing mechanism for ammoniadetermination[6,7]. Octadecylester derivative of rho-damine B was applied for sensing of anions[8–10].In that case, one of the suggested response mecha-nisms included aggregation of a dye[8]. Nafion filmimpregnated with rhodamine 6 G has been found suit-able for metal ion sensing[11,12]. By immobilizationof the same compound and ethylene glycol/water mix-ture into a gelatin matrix a sensor for humidity hasbeen designed[13,14]. The underlying principle stillremains a topic of discussions, but aggregation of rho-damine 6 G also seems to play an important role withthese sensors[8].

Rhodamine 19 has the highest quantum yield (φ =0.95 in ethanol) among rhodamine dyes[17]. In thiswork, its new lipophilic amide derivative (R19OA)was synthesized as a chromoionophore for possibleuse in optical sensors. Equilibria of its molecular formsin solution were studied on the basis of the UV–VISabsorption and emission measurements. The aim wasto analyze the behavior of the synthesized dye in var-ious media and to evaluate the equilibria, which couldbe applied as sensing mechanisms in optical chemicalsensors.

2. Experimental

2.1. Chemicals

Rhodamine 6 G and octadecylamine were of an-alytical purity grade (Fluka, Buchs, Switzerland).Methanol (Fluka, Buchs, Switzerland), dioxane(Merck, Darmstadt, Germany), chloroform and di-ethyl ether (Kemika, Zagreb, Croatia) were of spec-troscopic grade. Water was doubly deionized and freefrom carbon dioxide. All solutions were prepared bydilution of a methanolic stock solution of rhodamine

19 octadecylamide hydrochloride salt (1.00×10−3 M)with an appropriate solvent. The pH was adjusted byaddition of adequate amounts of aqueous solutionsof HCl and NaOH. The pH dependent equilibriumof the cationic and the neutral forms was studied in5.00×10−5 M solutions, unless otherwise stated. Dyeaggregation was examined in the concentration rangefrom 5.0× 10−6 to 1.00× 10−4 M.

2.2. Synthesis of rhodamine 19 octadecylamide

A mixture of 1.083 g (2.26 mmol) of rhodamine 6 Gdissolved in 5 ml of toluene and 1.173 g (4.35 mmol) ofoctadecylamine was stirred at 100◦C for 7 days underargon atmosphere, followed by filtration and evapora-tion of solvent. The orange residue was dissolved indichloromethane and purified by column chromatog-raphy (silica gel 60, methanol/dichloromethane 1/19,volume fraction of methanol,ϕ = 5%). Pale orangeoil, a neutral form of rhodamine 19 octadecylamide,was obtained: C44H63N3O2 (Mr = 666.010). Calcu-lated: C, 79.33; H, 9.53; N, 6.31. Found: C, 79.39; H,9.36; N, 6.04.

1H NMR (CDCl3) δ (ppm): 7.92 (d, 1H (C2)), 7.43(t, 1H (C3)), 7.42 (t, 1H (C4)), 7.02 (d, 1H (C5)),6.35 (s, 2H (C2′, C2′′)), 6.23 (s, 2H (C5′, C5′′)),3.50 (s, 2H (C1′-NHCH2CH3, C1′′-NHCH2CH3)),3.21 (q, 4H (C1′-NHCH2CH3, C1′′-NHCH2CH3)),3.07 (t, 2H (C=NCH2CH2(CH2)15CH3)), 1.89 (s, 6H(C6′-CH3, C6′′-CH3)), 1.32 (t, 6H (C1′-NHCH2CH3,C1′′-NHCH2CH3)), 1.25 (m, 30H (C=NCH2CH2(CH2)15CH3)), 1.02 (m, 2H (C=NCH2CH2(CH2)15CH3)), 0.88 (t, 3H (C=NCH2CH2(CH2)15CH3)).

IR spectrum (KBr)ν (cm−1): 3425m, 3395m (stN–H), 1690s (st O–C=N), 1230m, 1210m (st C–O–C),1645m, 1630 s (δ N–H). Mass spectrumm/z: M•+666.5 (100%), 638.4 (7.2%), 426.2 (8.8%), 396.2(2.8%).

The orange substance was dissolved in methanol,acidified with hydrochloric acid (volume fraction ofHCl, ϕ = 3%). After removing the solvent under re-duced pressure, dark pink crystals, a chloride salt ofthe cationic form of rhodamine 19 octadecylamide,were obtained: C44H64N3O2Cl (Mr = 702.468). Cal-culated: C, 75.23; H, 9.18; N, 5.98. Found: C, 75.29;H, 9.15; N, 5.80.

1H NMR (CDCl3) δ (ppm): 8.04 (s, 1H (NH+CH2CH3)), 7.86 (d, 1H (C2)), 7.40 (t, 1H (C4)), 7.38 (t, 1H

S. Miljanic et al. / Analytica Chimica Acta 468 (2002) 13–25 15

(C3)), 6.92 (s, 1H (C5′′)) 6.91 (d, 1H (C5)), 6.77 (s, 1H(CONHCH2CH2(CH2)15CH3)), 6.54 (s, 1H (C5′)),6.30 (s, 2H (C2′, C2′′)), 3.40 (s, 1H (NHCH2CH3)),3.24 (q, 4H (NH+CH2CH3, NHCH2CH3)), 3.01(t, 2H (CONHCH2CH2(CH2)15CH3)), 2.14 (s, 3H (C6′′-CH3)), 2.05 (s, 3 H (C6′-CH3)), 1.34(t, 6H (NH+CH2CH3, NHCH2CH3)), 1.18 (m,30H (CONHCH2CH2(CH2)15CH3)), 0.97 (m,2H (CONHCH2CH2(CH2)15CH3)), 0.80 (t, 3 H(CONHCH2CH2(CH2)15CH3)).

IR spectrum (KBr)ν (cm−1): 3640–3120m,b (stN–H, N–H+), 1610s (st C=O), 1530s (st sy N–C=O),1505s (δ N–H+). Mass spectrumm/z: 666.6 (100%),638.6 (9.4%), 426.2 (15.0%), 396.2 (7.4%).

2.3. Instrumentation

Absorption and fluorescence spectra were recordedon a Varian spectrophotometer (model CARY 3) anda Perkin-Elmer spectrofluorimeter (model LS 50), re-spectively. Conventional quartz cells (10 mm×10 mm)were used throughout. Spectra were always recordedat 2 min, 30 min, and 24 h after the preparation of so-lutions.

For pH measurements, a Radiometer pH/mV meter,PHM 64 with a Radiometer combined glass–calomelelectrode GK 2401 C was used. The pH meter was cal-ibrated with standard aqueous buffer solutions as wellas with methanol/water 1/4 buffers (volume fraction ofmethanol,ϕ = 20%) prepared in the laboratory[26].

NMR spectra were recorded on a Varian XL Gemini300 spectrometer using tetramethylsilane as the inter-nal standard. IR spectra were taken on a Perkin-Elmer783 spectrometer. A vacuum generator mass spectro-

Scheme 1. Chemical structures of the cationic form (1) and the electrically neutral form (2) of rhodamine 19 octadecylamide.

meter (model ZAB-SEQ) was used for recording massspectra.

3. Results and discussion

A new lipophilic derivative of rhodamine 19 wasisolated in two forms: a cationic one (1), containinga secondary amide group, and an electrically neutralone (2), containing an additional ring closed betweenthe oxygen of the amide group and the central carbonof the xanthene part. Both forms were easily mutuallyconvertible in solution (Scheme 1). A correspondingequilibrium was found to depend on the hydrogen ionactivity in solution as well as on the availability of thesolvent hydrogen for the hydrogen bond formation.

3.1. Absorption spectra

3.1.1. Equilibrium of the cationic and the neutralforms

The solutions in which the cationic form waspreferably present absorbed in the visible region(525–533 nm). The absorption in this part of thespectrum was assigned to a�∗ ← � transition ofelectrons of the xanthene part of the molecule[15].The shape, wavelength and intensity of the absorptionmaximum depended on the solvent and on the timeof measurement (Fig. 1).

A decrease in absorbance with time was noted forall used solvents and could be explained by a shiftof equilibrium from the cationic to the neutral form(Scheme 1, Table 1). A bond between the oxygenand the central carbon atom of the xanthene group


Fig. 1. Absorption spectra of R19OA in various solvents at (a) 2 min, (b) 30 min and (c) 24 h after sample preparation,c(R19OA) =5.00× 10−5 M.


Fig. 1. (Continued).

Table 1Absorption and emission characteristics of R19OA in various solvents

Solventa εe (104 l mol−1 cm−1) (λabs (nm)) RIF (λex (nm)) RIF (λem (nm))

Water 1.85 (533) 6.5 (528) 6.6 (548)Methanol 5.76 (528) 755.7 (528) 762.1 (552)Chloroform 5.94 (525) 597.4 (528) 618.7 (551)Diethyl ether 5.52 (525) 580.2 (528) 584.0 (550)Dioxane 4.18 (527) 439.5 (528) 418.4 (554)

Methanol/water mixturesb

(ϕwater (%))10 6.32 (529) 63.2 (528) 130.0 (561)20 6.21 (529) 65.1 (528) 138.9 (561)30 6.13 (529) 59.7 (528) 136.0 (562)40 6.06 (529) 61.2 (528) 144.8 (562)50 5.84 (530) 61.4 (528) 162.6 (562)60 5.45 (531) 54.2 (528) 112.3 (562)

70 2.98 (503) 10.9 (528) 17.7 (567)3.46 (534)

80 3.05 (502) 1.8 (528) 3.2 (568)3.05 (535)

90 3.04 (502) 0.7 (528) 0.8 (568)2.93 (536)

εe—effective molar absorptivity at the absorption maximum; RIF—relative fluorescence intensity.a The concentration of R19OA was 5.00× 10−5 M except by fluorescence measurements of solutions prepared in pure solvents

(1.00× 10−5 M).b c(HCl) = 0.01 M.


caused a restriction of the electron flow of the xanthene�-system and produced a change in the chromophoreabsorption ability. Neutral molecules did not absorbin the visible region.

The conversion to the neutral form was more rapidin diethyl ether and dioxane than in methanol and chlo-roform. Specific solvent interactions should be takeninto consideration to explain this behavior. Solventswith a slightly acidic proton, methanol and chloro-form, stabilize cationic molecules. Namely, a hydro-gen bond between the available lone electron pair ofthe amino nitrogen and the hydrogen atom of the sol-vent reduces the participation of the aminon-electronsto the �-system of the chromophore, leading to alower contribution of the resonance structure with thepositive charge on the central carbon atom and there-fore, the lesser possibility of conversion to the neutralform. On the other hand, in aprotic solvents, diethylether and dioxane, the neutral form is preferred[21].

Although water hydrogen is favorably capable forforming a bond with the lone pair of electrons ofthe amino nitrogen of R19OA, fast decrease in ab-sorbance of aqueous solution was noted (Fig. 1).It could be assumed that intermolecular hydropho-bic interactions make these nitrogen atoms hardly

Fig. 2. Absorption spectrum of R19OA in methanol, in acidic medium,c(HCl) = 2.00 × 10−3 M, and in alkaline medium,c(NaOH) = 2.00× 10−3 M, c(R19OA) = 5.00× 10−5 M.

accessible and sterically hinder hydrogen bond forma-tion between strongly lipophilic molecule and watermolecule leading to its conversion to the neutral form.

The addition of hydrochloric acid to the methanolicsolution of R19OA did not produce significant changesin the absorption spectrum (Fig. 2). Absorbance ofthe dye in acidic solution remained constant, indicat-ing that protons stabilized the cationic form of themolecule. Conversely, in an alkaline medium an im-mediate equilibrium shift to the neutral form and lossof absorbance in the visible region were noted.

3.1.2. AggregationDespite the amphiprotic properties of water, a much

weaker absorption of the cationic molecules in aque-ous solution in comparison with the organic mediumwas observed and attributed to dye aggregation.

The influence of pH value on the spectral changescaused by aggregation was studied in detail in amethanol/water 1/4 mixture. In acidic solutions, twoabsorption bands in the visible region were observed(Fig. 3). This spectral characteristic should be due tothe splitting of the excited singlet state of the molecule,caused by aggregation of cationic monomers. Theabsorption band at a lower wavelength was assigned


Fig. 3. Absorption spectra of R19OA at different pH values in methanol/water 1/4 mixtures,c(R19OA) = 5.00× 10−5 M.

to the aggregates and the band at a higher wavelengthwas mainly the result of monomer absorption. An in-crease in pH caused a decrease in intensity of the lowerwavelength absorption band and finally, at pH 5.2, theband transformed to a shoulder. By a further increasein pH of the solution the cationic form was convertedto the neutral one. In alkaline solution, the conversionto the neutral form took place immediately. Since theneutral form did not absorb in the visible region, itwas not possible to confirm the existence of neutralmolecule aggregates in solution.

The aggregation equilibrium was further studied inacidified (HCl) methanol/water mixtures of variousvolume ratios. An intense absorption of monomer at529–531 nm was observed in the systems of water vol-ume fraction,ϕ ≤ 60% (Table 1). By increasing thevolume of water in the mixture above 60%, a red shiftand a significant decrease in intensity of this absorp-tion band were noted. Besides, a new absorption max-imum appeared at 502–503 nm as a consequence ofdye aggregation. The position of the new band and itsintensity, which was nearly half the intensity of themonomer band in pure methanol, suggested that thedominant species were dimers rather than trimers andtetramers[25].

The influence of dye concentration on the dimerformation in methanol/water 1/4, pH 3.2, is shown inTable 2and inFig. 4. The curves of effective molarabsorptivity versusλ shown inFig. 4were obtained bydividing the absorption spectra by the concentrationof dye in solution. An increase in dye concentrationwas followed by an increase in intensity of the lowerwavelength absorption band reflecting a formation ofaggregates. The isosbestic point of the curves was ob-served at 520 nm. A deviation of the curve of the mostdiluted solution (5.0×10−6 M) was the result of rapidconversion of molecules to the neutral form.

Aggregation was not observed in methanol. An in-crease in dye concentration in the range from 5.0×10−6 to 1.00×10−4 M did not cause appreciable spec-tral changes (Table 2). It could be assumed that inter-molecular hydrophobic interactions play a dominantrole in R19OA aggregation and that lipophilic deriva-tive of rhodamine 19 will not aggregate in pure organicsolvents.

From changes in absorption, caused by increasein dye concentration in methanol/water 1/4, pH3.2, both the formation constant (Kd) and the ab-sorption spectrum of the dimer can be evaluated[27,28]. Assuming that the absorption spectrum of


Table 2Absorption and emission characteristics of solutions of various R19OA concentrations

Solvent c (10−6 mol l−1) A (λabs (nm)) RIF (λex (nm)) RIF (λem (nm))

Methanol 5.0 0.2988 (528) 655.3 (528) 667.3 (550)10.0 0.5342 (528) 750.3 (528) 758.6 (550)30.0 1.8370 (528) 408.2 (528) 411.4 (555)50.0 2.8823 (528) 195.7 (528) 196.2 (560)70.0 3.2069 (526) 122.7 (528) 125.7 (561)

100.0 3.2891 (522) 75.7 (528) 77.0 (563)

Methanol/water 1/4 (pH 3.2) 5.0 0.1439 (533) 160.9 (528) 162.6 (548)10.0 0.2571 (504) 165.1 (528) 165.6 (552)

0.3468 (533)30.0 0.8843 (502) 37.8 (528) 8.4 (560)

0.9872 (534)50.0 1.5348 (502) 14.3 (528) 14.6 (567)

1.5878 (534)70.0 2.1564 (502) 4.7 (528) 5.1 (572)

2.2683 (534)100.0 3.1279 (502) 0.7 (528) 1.3 (578)

3.2193 (535)

A—absorbance; RIF—relative fluorescence intensity.

the diluted solution,c = 5.0× 10−6 mol l−1, was themonomer spectrum, 533 nm can be considered as thewavelength of the monomer absorption maximum.Subtracting the monomer absorption spectrum fromthe concentrated solution absorption spectrum (c =

Fig. 4. Effective molar absorption coefficients as a function ofλ by various dye concentration in methanol/water mixtures 1/4, pH 3.2.

1.00× 10−4 mol l−1) gave a dimer absorption maxi-mum at 502 nm (Fig. 5). By dividing the intensity ofabsorption at the wavelength of the dimer absorptionmaximum by the intensity at the wavelength of themonomer absorption maximum, the parameterR can


Fig. 5. Calculated dimer spectrum of R19OA in methanol/water mixtures 1/4, pH 3.2.

be calculated. The ratio of parameters for diluted so-lution (c = 5.0× 10−6 mol l−1) and for concentratedsolution gave the monomer fraction (x) in the concen-trated solution. Knowing the monomer molar fractionis necessary for calculating the dimerization constantdefined by the relation

Kd = 1− x

2cx2(1)

The obtained equilibrium dimerization constant of5050 was in good agreement with the reported valuesfor dimerization of rhodamine 6 G (Kd = 6200)[24]and its ester derivative (Kd = 5400) [29] in aqueoussolution.

In the calculated dimer spectra (Fig. 5) two max-ima at 502 and 544 nm are present. The splitting wascaused by an interaction between the xanthene groupsof two monomer units. The lower wavelength dimerabsorption band could be assigned to a geometricstructure of dimer in which the xanthene groups ofthe monomers are placed in parallel planes at a certaindistance, forming an angle >54.7◦ [30]. Some au-thors state that the hydrophobic interactions betweenmonoethylamino groups of the monomers as well asbetween long alkyl chains play an important role indimer formation[22,31]. However, the stacking inter-

actions of the�-system of the xanthene part of twomolecules and a possible hydrogen bond between thehydrogen atom of the amide group of one monomermolecule with the oxygen atom of the xanthene partof the other, could also be responsible for the sameeffect.

3.2. Fluorescence spectra

Fluorescence spectra of the dye were measured inwater, methanol, chloroform, diethyl ether and diox-ane (Table 1, Fig. 6). Although the wavelength of thelowest energy absorption maximum varied with thesolvent, the position of the excitation maximum wasnot solvent dependent. The excitation maximum al-ways appeared at 528 nm regardless of the solvent.This can be explained by a higher sensitivity of fluo-rescence measurements compared to absorption mea-surements[31]. While all light-absorbing species insolution contributed to the absorption spectrum, onlyfluorescent species contributed to the excitation spec-trum. The fluorescence of dye in organic solvents wasascribed to the cationic monomers.

A strong emission of the dye was observed inmethanol and chloroform solutions, where the cationicform was preferably present. On the contrary, a fast


decrease in dye fluorescence intensity was observedin diethyl ether and dioxane (Fig. 6). These spec-tral changes reflected conversion of the cationic intothe neutral form. Furthermore, in accordance withabsorption measurements, no significant changes influorescence spectra were observed after the addition

Fig. 6. Emission spectra of R19OA in various solvents at (a) 2 min, (b) 30 min and (c) 24 h after sample preparation,c(R19OA) = 1.00× 10−5 M.

of HCl to the methanolic solution (λex = 528 nm,λem= 552 nm). An addition of NaOH, causing a shiftof equilibrium to the neutral form, was followed bythe loss of fluorescence (Fig. 7).

Some authors have attempted to correlate emissionproperties of rhodamine dyes with the viscosity and


Fig. 6. (Continued).

polarity of the medium[17,21,32]. The rotation of mo-noethylamino groups of rhodamine dyes is hinderedin viscous solvents and the contribution of radiation-less transfer by internal conversion should be lower.

Fig. 7. Emission spectrum of R19OA in methanol, in acidic medium,c(HCl) = 2.00 × 10−3 M, and in alkaline medium,c(NaOH) = 2.00× 10−3 M, c(R19OA) = 5.00× 10−5 M.

Therefore, a stronger emission should be observed inviscous as compared to non-viscous solvents. LópezArbeloa et al.[17], however, claimed that the rotationalmotion was not related to solvent viscosity in a simple


Fig. 8. Emission spectra of R19OA at different pH values in methanol/water 1/4 mixtures,c(R19OA) = 5.00× 10−5 M.

way. Namely, the rate constant of the internal conver-sion of rhodamine B in glycerol (η = 1260) was foundto be similar to that obtained in ethanol (η = 1.2).

Furthermore, in the excited state rhodamines form atwisted conformation including a transfer of electroniccharge from the donor (amino groups) to the acceptor(xanthenyl cation), resulting in a highly polar structure[32]. In the case of weak electron donors (monoethy-lamino groups), the excited state of the non-planarmolecule has a higher energy than the excited stateof the planar molecule and will not be populated. Us-ing polar solvents the energy of the excited state ofthe non-planar molecule can be lowered below thatof the planar molecule, leading to internal conversionand decrease in fluorescence. Drawing a parallel be-tween emission properties of the cationic form of rho-damine 19 octadecylamide and solvent characteristics,like viscosity and polarity, would no doubt provideuseful data, but solvent dependent interconversion ofthis form to the non-fluorescent neutral form is a se-rious hindrance for such correlation.

A weak emission in water and acidified methanol/water systems of water volume fraction,ϕ > 60%was attributed to dye aggregation in solution (Table 1,Figs. 6 and 8). Namely, the splitting of the excitedsinglet state, caused by aggregate formation, leads to

an electron transition to a triplet state[30]. Conse-quently, the fluorescence is quenched.Fig. 8illustratesthe dependence of the dye fluorescence intensity onpH in a methanol/water 1/4 mixture. The aggregationwas promoted in a strong acidic medium. The dyeemission was very weak in solutions of pH 1.3, wheremost cationic molecules formed non-fluorescent ag-gregates. Increase in pH (pH 3.2) resulted in a shift ofequilibrium to monomers and to a stronger emission.Since aggregates, which were still present, reabsorbedthe light at 502 nm, a minimum appeared in the spec-trum at this wavelength. A further increase in pH re-sulted in the conversion of molecules to the neutralform and in the loss of fluorescence.

Increase in dye concentration in the systemmethanol/water 1/4, pH 3.2, was followed by a de-crease in emission intensity and a shift of the emissionband to a higher wavelength (Table 2). Dimers were ahighly dominant species in solutions of concentrationsc > 1.00× 10−5 M.

4. Conclusion

An unavoidable characteristic of the dyes that areused in conventional membranes of optical sensors


is an adequate lipophilicity. Another prerequisite foroptimal sensors is an understanding of structural andspectroscopic properties of a dye. Such approachleads to analytical devices that are based on predictiveparameters rather than on trial and error assump-tions. In this work, an octadecylamide derivative ofrhodamine 19 was synthesized and its spectroscopicproperties were studied in detail. Time, solvent, pHand concentration dependent spectral changes werefound to be the result of two equilibria in solution:one, between the cationic and the neutral form of thedye, and the other, between the monomer and theaggregate.

Owing to synthetically introduced lipophilicityand unraveled optical properties, the new dye canbe used as an attractive chromoionophore in opticalsensors based on absorbance or fluorescence mea-surements. The two equilibria studied—the acid–baseand aggregation—can serve as working principles.The corresponding experiments are in progress andwill be discussed in our forthcoming report.

Acknowledgements

This research was supported by the Ministry ofScience and Technology of the Republic of Croatia,Project No. 119410.

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Lipophilic derivative of rhodamine 19: characterization and spectroscopic properties

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