Ultrasensitive 4-methylumbelliferone fluorimetric determinationof water contents in aprotic solvents

Katarzyna Kłucińska, Rafał Jurczakowski, Krzysztof Maksymiuk, Agata Michalska n

Department of Chemistry, Warsaw University, Pasteura 1, 02-093 Warsaw, Poland

a r t i c l e i n f o

Article history:Received 14 July 2014Received in revised form9 September 2014Accepted 12 September 2014Available online 19 September 2014

Keywords:Water determinationKarl Fischer4-methylumbelliferoneAliphatic solventsFluorometry

a b s t r a c t

A novel approach to the quantification of relatively small amounts of water present in low polarity, aproticsolvents is proposed. This method takes advantage of protolitic reaction of 4-methylumbelliferone dissolvedin the solvent with water, acting as a base. The low emission intensity neutral 4-methylumbelliferone istransformed in reaction with water to a highly fluorescent anionic form. Thus the increase in emissionintensity is observed for increasing water contents in aprotic solvents. For low water contents and highlylipophilic solvents, this method yields (in practical conditions) higher sensitivity compared to biampero-metric Karl Fischer titration method in volumetric mode.

It is also shown that organic compounds of protolitic character (amines, acids) not only interfere withwater contents determination but increase the sensitivity of emission vs. water contents dependence.Introduction of aqueous solution of strong acid or base (HCl or NaOH) has similar effect on the system asintroduction of pure water.

& 2014 Elsevier B.V. All rights reserved.

1. Introduction

Determination of water contents, particularly in the case oforganic solvents is essential for many practical applications, espe-cially in industrial processes. Clearly, there is an interest in methodsallowing quick and easy access to information on water contents indifferent organic media. The golden standard for water determina-tion is coulometric Karl Fischer titration, for example [1]. In general,this is the most reliable method, however, in the case of minutewater concentrations and especially highly lipophilic, aprotic solventsproblems related to sampling and especially tendency of water tospontaneously accumulate close to more hydrophilic surfaces – e.g.glassware, may result in inaccurate determination results [1,2]. Inaprotic and non-alcoholic solvents Karl Fischer method is lesssensitive because the Bunsen reaction is favored and water/iodinemolar ratio changes toward 2:1 in contrast to the 1:l molar ratiofound in the protic Karl Fischer environment [3]. For oil samples theresults obtained by volumetric and coulometric Karl Fischer methodsusually considerably differ even after correcting for the instrumentalbias. Thus it was suggested that the volumetric method should bepreferred over coulometric method to measure all of the watercontent in oil samples [4,5]. Moreover, Karl Fischer titration canhardly be performed as an integral part of technological process, orbe used to continuously monitor any occurrence of water in the

sample media. Thus, different alternative methods of water quanti-fication have been proposed, including advanced techniques likeNMR [6], infrared spectrometry [7,8], UV/Vis spectrometry [9,10].Many of them tried to apply fluorometry for determination of watercontents in organic solvents as well for example [11–14]. The latterapproach, however, requires application of fluorophore of emissionspectra dependent on the presence of water in the studied system.Different compounds have been proposed/tested in this respect,most of them were tailor made (synthesized) for water determina-tion purpose. Moreover, in most cases the increase of water contentsin the sample was accompanied with decrease (quenching) ofemission signal (e.g. [11,13]). For practical analytical applications,however, commercially available fluorophores probes, and optimallyleading to increase of the emission signal with increasing analyte(water) concentrations are preferred. Among widely available, cost-effective ligands 4-methylumbelliferone (4-MU), pH-sensitive, widelyused, bright fluorophore is known to be soluble in variety of organicsolvents. It was also shown that the emission spectrum of 4-MUprobe is dependent on the solvent used and on the presence of acid/bases in the sample, for example [15,16]. The dependence of emissionintensity on water contents in ethanol in the presence of constantconcentration of hydrochloric acid was also reported [15]. Never-theless, to our best knowledge, the above-mentioned reports havenot led to analytical application of 4-MU as water-sensitive probe fororganic media.

The herein proposed approach is based on assumption that inthe lipophilic solvents (even in the absence of acid or bases inthe system) dissolved neutral 4-MU can spontaneously undergo

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Talanta

http://dx.doi.org/10.1016/j.talanta.2014.09.0180039-9140/& 2014 Elsevier B.V. All rights reserved.

n Corresponding author. Tel.: þ4822 8220211; fax: þ48 22 8225996.E-mail address: agatam@chem.uw.edu.pl (A. Michalska).

Talanta 132 (2015) 392–397

protolitic reaction with water leading to formation of highlyfluorescent species, especially deprotonated 4-MU, a bright fluor-ophore [15]. It is rational to expect that fluorescence intensity inthis case should be dependent on the amount of water present,thus allowing quantification of water in the sample, especially inemulsion. The herein proposed approach was used to quantifyamount of water present in the sample of a model organicmedium: hexadecane. The effect of other (model) protolitic agentspresent in the sample on the resulting emission signal was alsostudied. The proposed method was also applied for toluene andcyclohexane model solvents. Application of 4-MU to quantifyminute contents of water in aprotic, lipophilic solvents to our bestknowledge has not been proposed in literature.

Results of 4-MU-based water quantification using herein pro-posed method have been compared with water determinationresults performed using the classical approach – biamperometricKarl Fischer titration in volumetric mode, showing that especiallyfor low amounts of water present in the sample, herein proposedmethod offers higher sensitivity.

2. Experimental

2.1. Reagents

Hexadecane (HD), 4-methylumbelliferone (4-MU), HYDRA-NALs - Composite 5 K (Titration), HYDRANALs - Medium K,HYDRANALs - Water Standard 10.0, toluene, cyclohexane, hex-anoic acid, palmitic acid, octanoic acid, isopentyl amine, octadecylamine and molecular sieves were from Aldrich (Germany). Octy-lamine, hydrochloric acid and NaOH were of analytical grade andwere obtained from POCh (Gliwice, Poland).

Doubly distilled and freshly deionized water (resistance18.2 MΩcm, Milli-Qplus, Millipore, Austria) was used throughoutthis work. All solvents were dried over freshly activated molecularsieves (MS 4A).

2.2. Apparatus

Fluorimetric experiments were performed using a spectro-fluorimeter Cary Eclipse (Varian). After exposure at an excitationwavelength, unless otherwise stated, equal to 320 nm, emissionintensity was observed within the range from 350 nm (or 370 nm)

to 600 nm. The slits used were 5 nm both for excitation andemission, while the detector voltage was maintained at 800 V.

Biamperometric Karl Fischer titration in volumetric mode wasperformed using Titration Equipment: 716 DMS Titrino (Metrohm)and 728 Stirrer (Metrohm), platinum electrodes (400 mV) wereused. Chloroformwas added to the working solution as a solubilitypromoter so that the KF titration was conducted in the homo-geneous solution. This procedure was found to give more repro-ducible results. The requirements of complete dissolution in KFmethod was also discussed earlier [5]. Emulsions were preparedusing homogenizer Hielscher, model UP 200 S.

2.3. Preparation of 4-MU stock solutions

4-MU stock solutions in tested solvents were prepared bydissolving 2 mg of 4-MU in 80 ml of a solvent (either hexadecane,toluene or cyclohexane) under sonication (cycle 0.5, power 70%)yielding a concentration 0.025 mg/ml. Sonication was continuedfor another 5 min. Then the mixture was left for 24 hours tocomplete dissolution of the fluorophore. Thus prepared solutionswere used for fluorimetric measurements.

2.4. Preparation of the samples

Water was introduced to one of the tested solvents (hexade-cane, cyclohexane, toluene) containing 4-MU. To assure thatintroduced water is mixed with dry solvent, the sample wassonicated for 60 seconds. Samples for Karl Fischer titration controlexperiment were prepared in the same manner.

3. Results and discussion

4-MU is a well-known fluorescent pH indicator, showing strongemission at about 450 nm in aqueous alkaline solutions, thisemission is characteristic for deprotonated form of 4-MU – whenit is present as anion, Fig. S1 [15,16]. Analytical usefulness of thisreagent has been proven in numerous applications, includingthose where the enzymatic reaction leads to formation of highlyfluorescent 4-MU anion, from optically silent enzymatic subst-rates (for example using alkaline phosphatase, glucuronidase,β-glucosidase and other enzymes).

Fig. 1. presents excitation and emission spectra obtained for4-MU dissolved in concentration 0.025 mg/ml in hexadecane. As it

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Fig. 1. (A) Excitation and (B) emission spectra (excitation wavelength 320 nm) recorded for 4-MU (0.025 mg/ml) in HD, in the absence and in the presence of model additivestested: (green line) 4-MU in HD, (navy line) 4-MU in HD in the presence of 0.16% v/v water in HD, (red line) 4-MU in HD in the presence of 10�6 M octylamine and 0.16% v/vwater in HD, (black line) 4-MU in HD in the presence of 10�3 M octylamine and 0.16% v/v water in HD, (grey) 4-MU in HD in the presence of 10�4 M octanoic acid, (magenta)4-MU in HD in the presence of 10�4 M octanoic acid and 0.16% v/v water in HD.

K. Kłucińska et al. / Talanta 132 (2015) 392–397 393

can be seen in Fig. 1, for 4-MU dissolved in HD weak absorption (atabout 320–330 nm) and practically no emission is observed. Itshould be stressed that 0.025 mg/ml concentration of 4-MU is thehighest practically achievable for HD solutions. The emissionintensities observed at about 380 nm for the same concentrationof 4-MU in toluene were yet lower compared to HD, Fig. S2, andwere very weak for cyclohexane (the latter are not included inFig. S2). It should be stressed that the presence of emission peak at380 nm confirms, that as expected, neutral 4-MU is present in drytested solvents [15] (structure A in Fig. S1).

As 4-MU is known to be sensitive to the presence of protoliticagents in sample, the effect of presence of model compounds ofthis nature was tested. The introduction of protolitic reagents tothe model solvent tested, HD, results in only small changes in theemission spectra, Fig. S3. The presence of tested acids: hexanoic,octanoic or palmitic in 10�3 M concentration results in 4-MUemission observed at about 380 nm, suggesting that still neutral4-MU is present in the system, regardless acid added. For higherconcentrations of acid added to HD, 10�1 M, apart from significantincrease of peak at 380 nm, additional peak is formed at about490 nm, Fig. S3, inset. The latter is typical for neutral tautomerform of 4-MU, structure C, Fig. S1 [15]. This behavior is similar tothat reported earlier for strong acid (HCl) introduced to protoliticsolvents as water or ethanol [15]. Thus, it is probable that, in theabsence of other protolitic agents, even relatively weak (in aqu-eous environment) acids are able to induce the change of thestructure of neutral 4-MU molecule, leading to formation of abovementioned neutral tautomer characterized with emission at about490 nm.

On the other hand, introduction of a base: isopentylamine,octylamine or octadecylamine in low concentrations,o10�6 M,did not affect the emission spectra obtained. Regardless of theamine used, similar behavior was observed, the exemplary resultsobtained for octylamine are shown, Fig. 2. For low concentration ofamine present in the sample, both excitation and emission spectrarecorded resemble those of 4-MU in HD recorded in the absence ofamine. However, for higher concentration of amine (4 10�5 M)the intensity of emission of 4-MU dissolved in HD was significantlydependent on concentration of amine, as shown on example ofoctylamine, Fig. 2. For the amine concentration equal to 10�5 M asmall shoulder is formed on 4-MU emission spectra with amaximum at 450 nm, at wavelength typical for deprotonated formof 4-MU, Fig. S1 B. Further increase of amine contents to 10�4 M

and 10�3 M results in formation of a well-developed emissionpeak with maximum at 450 nm (excitation 330 nm), clearlypointing out to deprotonation of 4-MU as expected for presenceof a base in the sample [15].

Introduction of water to (net) HD containing 4-MU (5 ml to 3 ml,corresponding to 0.17% v/v or 0.093 M concentration) leads tosimilar change of 4-MU emission spectra as introduction of anyamines in 10�4 M concentration, Fig. 1. On the excitation spectra apronounced absorption peak is formed with maximum at about320 nm. On the emission spectra a strong peak is formed with amaximum at about 450 nm, suggesting the presence of deproto-nated form of 4-MU in HD. Thus, it can be assumed that in theabsence of other deprotonating agents, water as a base reacts with4-MU and this reaction leads to deprotonation of 4-MU andformation of structure depicted in Fig. S1 B. This process can beaccompanied by partition of 4-MU anion to the water phase, due toits higher (compared to neutral 4-MU) solubility in water. Thus theprotolitic reaction of 4-MU with water in aliphatic environment ispromising for the analytical, fluorimetric water determination.

Fig. 3. presents emission spectra of 4-MU dissolved in HD,recorded for changing water concentration in the sample from1.7.10�2% v/v (9.3.10�3 M) to 0.6% v/v (0.37 M). As it can be seenfrom Fig. 3 introduction of even relatively small amount of water,reaching 1.7.10�2% v/v, to 4-MU containing HD results in formationof emission peak at wavelength typical for deprotonated form of4-MU, at 450 nm (excitation wavelength 320 nm). Increase of theamount of water introduced to the sample results in clear increaseof the intensity of emission observed at 450 nm. The lineardependence of emission intensity on water contents was observedwithin the range from 1.7.10�2% to 0.33% v/v, that is up to 0.185 Mwater in HD, Fig. 4. Within this range the correlation coefficient,R2, of the dependence of emission intensity on water contents wasequal to 0.987, Table 1. Further increase of water contents in thesample to 0.67% v/v (0.37 M) resulted in some increase in therecorded emission, yet not linear. In this range also the magnitudeof error increases, Fig. S4. The plateau of the signal obtained can beattributed to different effects; however, it should be stressed thatfor these water contents the amount (mole) of water was morethan 2000 times higher compared to amount (mole) of 4-MUpresent in the sample. It can also result from the protoliticreaction, and from acid–base equilibrium prevailing in the sample.

The advantage of herein proposed approach is clearly seenwhen comparing the results of fluorimetric evaluation of water

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Fig. 2. Emission spectra recorded in different concentrations of octylamine in HD:(black line) 4-MU in HD, and gradually increasing octylamine concentration (greyline) 10�8 M, (red line) 10�7 M, (green line) 10�6 M, (cyan line) 10�5 M, (magentaline) 10�4 M and (blue line) 10�3 M. Inset – magnification of spectra obtained forlow concentration of octylamine in HD, excitation wavelength 330 nm.

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Fig. 3. Emission spectra recorded in different concentrations of water in HD: (blackline) 4-MU in HD and gradually increasing water contents from 1.7.10�2% v/v(9.3.10�3 M) to 0.6% v/v (0.37 M). Inset: magnification of spectra obtained for lowconcentration of water in HD: (black line) 4-MU in HD, (red line) 1.7.10�2% v/v(9.3.10�3 M) water, (green line) 3.3.10�2% v/v (1.8.10�5 M), (blue line) 5.10�2% v/v(2.8.10�2 M).

K. Kłucińska et al. / Talanta 132 (2015) 392–397394

contents changes using 4-MU as indicator, with results of KarlFischer titration, Fig. 4. For Karl Fischer titration, as it can be seenfrom Fig. 4, introduction of relatively small amounts of water tothe sample, below 0.1% v/v, yields negligible changes of estimatedwater amount pointing out to significant negative error of watercontents estimation. It should be stressed that Karl Fischer titra-tion is dependent on the sampling procedure, and in the case ofrelatively low water contents, e.g. accumulation of water intro-duced to lipophilic HD at the interface of the sample and usedvessel, significantly affects obtained results. Herein describedapproach takes the advantage of emission changes, resulting from4-MU spontaneous protolitic reaction with water. As 4-MU isdissolved in whole volume of HD, any contact of the sample withwater results in change of the emission spectra. Thus, as seen inFig. 4, fluorimetric approach is significantly more sensitive forrelatively low water contents. On the other hand, for higher watercontents, 40,5% v/v, Karl-Fischer method offers higher sensitivity,without limitation on high water contents.

Taking into account that fluorimetric response of 4-MU dye fordifferent water contents in HD originates from protolitic reactionwith water acting as a base, it seems justified to check how thepresence of base or acid in the sample influences the analyticalperformance of this system.

As discussed above (Fig. 2), the presence of base (amine) at theconcentration 10�6 M, in the absence of water, does not affect theemission spectra of 4-MU in HD. Similarly the presence of 10�6 Moctylamine in HD did not affect the dependence of emission onchanges of water contents in the sample. Both the excitation andemission spectra were comparable within the range of experi-mental error with that recorded in the absence of amine insolution (Fig. S5). Similarly, the dependence of emission intensity

at 450 nm on water contents in the sample was linear within therange from 1.7.10�2% to 0.33% v/v with R2 equal to 0.988, Table 1.

Increase of octylamine concentration in HD to 10�3 M results,in the presence of water, in a significantly broader peak observedon the excitation spectra of 4-MU, Fig. 1. Thus in this case theexcitation wavelength used was equal to 360 nm. The emissionintensities recorded in the presence of water (0.17% v/v) weresignificantly higher compared to that recorded for water contain-ing samples in the absence of amine or in the presence of 10�6 Moctylamine. The emission intensity vs. water contents dependencerecorded in the presence of 10�3 M octylamine, Fig. S6, for lowwater contents (o 0.3% v/v) was linear with R2¼0.991, Table 1.The sensitivity of determination was not only higher than for KarlFischer titration, but also it was the highest among tested modelsystems. The sensitivity obtained for 10�3 M amine present wasmore than two times higher compared to the system tested in theabsence or in the presence of low octylamine concentration in HD,Fig. S5 and Fig S6. Moreover, in the presence of amine in HD theerror measured as SD of three replicates is significantly smaller,except for the highest water content tested, which is already atplateau of the dependence, Fig. S4. Thus introduction of amine in10�3 M gives possibility of fine tuning of sensitivity of fluorimetricwater contents determination in aprotic solvents, offering signifi-cantly higher sensitivity within this region compared to KarlFischer titration, Fig. 4. However, the linear range of the depen-dence of emission on contents of water was shorten compared toabove-described cases, for higher water contents the decrease offluorescence intensity was observed, Fig. 4.

The significant increase of sensitivity of water contents deter-mination, especially at low water contents, observed for the10�3 M amine presence in the sample results from the properties

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Fig. 4. Dependence of intensity of emission read at 450 nm on the change of water contents in HD sample obtained (A) from fluorimetric experiment, in HD sample (■) in theabsence of octylamine, ( ) in the presence of 10�6 M octylamine, ( ) in the presence of 10�4 M octanoic acid, ( ) in the presence of 10�2 M octanoic acid and ( ) in thepresence of 10�3 M octylamine (using excitation equal to 360 nm); (B) amount of water determined plotted against amount of water added for Karl Fischer titration of HDsamples, inset: magnification of the low water contents range of Karl Fischer titration.

Table 1Summary of analytical performance of 4-MU as water sensitive fluorescent indicator

System Hexadecane Hexadecane þ10�2 Moctanoic acid

Hexadecane þ10�6 M octylamine Hexadecane þ10�3 M octylamine Toluene Cyclohexane

λexcitation (nm) 320 320 320 360 320 320λemission (nm) 450 450 450 450 450 450Water contents(%, v/v)

1.7.10�2–0.33 o 0.2 1.7.10�2–0.33 o 0.3 1.7.10�2–20 o 0.2

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of amine – which is stronger base than water. Thus amineinfluence on 4-MU deprotonation and concomitant formation ofemission signal is potentially stronger than that of water. Withincreasing water contents in the sample hydrophilicity of themedium increases, protolitic reaction between 4-MU and amine,resulting in protonation of amine on expense of creating of 4-MUanion, is facilitated, resulting in higher sensitivity of water con-tents determination. The strong emission signal observed can bealso to some extent related to formation of structures resemblingreversed micelles, containing water in their cores and beingstabilized by protonated amine oriented with protonated aminegroup toward hydrophilic environment of water and with the alkylchain oriented toward HD. It is probable that the anionic, depro-tonated and water-soluble form of 4-MU formed will be presentinside the reverse micelles resembling structures, being stabilizedwith positive charges of the protonated amine, this will ultimatelyyield higher emission intensity. This effect is probably of lowimportance for low (10�6 M) amine contents in the sample as thepopulation of positive charges that may be created is relativelylow, whereas at higher amine contents the enhancement ofsensitivity of water determination is clearly seen.

Introduction of acid, as a model compound octanoic acid wasused, in 10�4 or 10�2 M concentration, did not affect the emission orabsorption peak positions. The excitation and emission spectra andtheir maxima, recorded in the presence of octanoic acid, at twoconcentrations used, were not affected compared to spectra recordedfor similar system, yet in the absence of acid, Fig. 1. Introduction ofwater to HD-containing octanoic acid resulted in increase of emissionat 450 nm, similarly as for net HD-containing 4-MU (the maximumof excitation peak was observed at about 320 nm), Fig. 1. The effect ofpresence of octanoic acid in the system predominantly was observedon the emission intensity vs. water contents dependence. Althoughfor increase of water contents o0.1% v/v the increase of intensitywas slightly higher compared to HD system containing 4-MU in thepresence of 10�3 M octylamine, the further increase of watercontents resulted in much smaller increase of intensity of emissionand the dependence was reaching plateau, regardless the concentra-tion of acid present in the HD. Formation of plateau can be relatedto the fact that octanoic acid is stronger acid than 4-MU, thusdeprotonation of 4-MU in this case is not enhanced by wateraddition. On the other hand, dissociation of octanoic acid, if occurs,leads to formation of water containing reversed micelles resemblingstructures with anionic charge at the water/HD interface. Thus due to

electrostatic repulsion, incorporation of negatively charged 4-MUanion is less likely compared to positively charged water/HD inter-face in the case of amine reversed micelle resembling structuresformed.

Thus although the presence of carboxylic acid in the modelsolvent HD limits possibility of analytical quantification of thewater contents to the low amounts only, the proposed method isrobust enough to clearly distinguish samples containing waterfrom those free from water additions.

For analytical application of herein proposed system theinformation on response time of the system for water additionas well as stability of emission signal in time was collected, Fig. S7,using HD as model solvent. As shown above the intensity ofemission recorded in the presence of 10�3 M octanoic acid wasmuch lower compared to signals recorded in the presence ofwater, moreover the intensity of emission was fluctuating in timeespecially in the presence of octanoic acid in HD.

Introduction of water to the sample leads to pronouncedincrease of emission, Fig. S7, regardless of the system tested. Inthe absence of protolitic compounds in HD stable signal wasobtained after about 3 min. For sample containing amine or acidemission signal was increasing faster, the stable signal was achievedafter about 2 min time. The fastest response was observed forsystem containing octylamine, regardless of concentration of watertested (3.4.10-2% v/v or 1.7.10�1% v/v), the maximum intensity ofemission signal was observed after 1 min. This result is supportingthe thesis that presence of amine and possible formation ofreverse micelle-like structures favors reaction of 4-MU with wateradded to the system. It should be stressed that regardless of thesystem studied (either without additives or containing acid or base)emission signal in the presence of water was stable in experi-ment time.

Last, but not the least, the above-described experiments con-firm, that the proposed system is not only highly sensitive for lowwater contents but also the results are not interfered by thepresence of organic impurities of protolitic character in thesample. This effect is due to spontaneous and beneficial effect ofreverse micelles resembling structures formation (provided thatthe concentration of impurities is high enough) and was notobserved for introduction of aqueous solution of acid (HCl) orbase (NaOH) solution to the sample. In the latter case, the effectobserved was equivalent within the range of experimental error, tothat of addition of (neutral) water to the sample, Fig. S8.

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Fig. 5. Dependence of intensity of emission read at 450 nm on the change of water contents in (A) toluene and (B) cyclohexane: (■) 4-MU in tested solvent, (□) in thepresence of 10�6 M octylamine in tested solvent.

K. Kłucińska et al. / Talanta 132 (2015) 392–397396

The herein proposed simple method of water quantificationusing protolitic reaction of 4-MU was also tested on example ofother solvents – toluene and cyclohexane.

For 4-MU dissolved in toluene introduction of water resulted information of emission peak at 450 nm, of intensity dependent onwater contents, Fig. 5. As it can be seen in Fig. 5, similarly as forHD, a linear dependence of intensity of emission on contents ofwater in the sample was recorded; however, for toluene the linearrange of responses was much broader compared to HD. For testedrange 0.017% v/v to 20% v/v of water in toluene, it was character-ized with R2 equal to 0.991, Table 1. As it can be seen in Fig. 5,especially for low water contents,o0.7% v/v, a further increase ofsensitivity is observed if the octylamine is present in the toluene inconcentration 10�6 M, that is at the concentration of no beneficialeffect in the case of hexadecane. Probably the advantageous effectof already low concentration of amine present in toluene is relatedto higher polarity of this solvent compared to HD. Toluene partiallyparticipates in stabilization of water droplets within, thus evenlow contents of amine actively increases deprotonation of 4-MUmolecules and their partition into aqueous phase.

For cyclohexane containing 4-MU, similarly as for above dis-cussed examples, introduction of water resulted in formation ofemission peak at 450 nm, of intensity dependent on water con-tents in the sample, Fig. 5. The linear dependence of emissionintensity on water contents was however, limited to about 0.2% v/vof water (R2¼0.967), Table 1. Similarly as for toluene, introductionof amine in 10�6 M concentration to the cyclohexane has resultedin increased emission, however recorded dependence was notlinear. Increase of water contents above 0.33% v/v resulted indecrease of recorded emission.

4. Conclusions

The herein presented results clearly show that minute watercontents, especially in highly lipophilic, nonpolar solvents likehexadecane, can be determined with high sensitivity due toprotolitic reaction of 4-methylumbelliferone with water, actingas a base. The emission intensity of deprotonated 4-MU anion wellcorrelates with water contents in the nonpolar solvent. For lowconcentrations, this system offers higher sensitivity compared to

Karl Fischer titration. Moreover, presence of organic acid andespecially organic base leads to further increase of sensitivity,with increasing concentration of this additive in the solvent. Thiseffect can be attributed to formation of structures resemblingreversed micelle within the low-polarity solvent – with water coreand stabilized with protonated (in the case of amine) or deproto-nated (in the case of acid) organic ion located at the water –

solvent interface. This spontaneous process in the case of addedbase (amine) supports partition of ionized 4-MU into the minutewater phase.

Acknowledgment

Financial support from National Centre of Science (NCN,Poland), project 2011/03/B/ST4/00747, in the years 2012–2015, isgratefully acknowledged.

Appendix A. Supporting information

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.talanta.2014.09.018.

References

[1] N. Dantan, W. Frenzel, S. Küppers, Talanta 52 (2000) 101–109.[2] I. Gergen, F. Radu, D. Bordean, H.-D. Isengard, Food Control 17 (2006) 176–179.[3] S.K. MacLeod, Anal. Chem. 63 (1991) 557A–566A.[4] S.A. Margolis, Anal. Chem. 69 (1997) 4864–4871.[5] S.A. Margolis, Anal. Chem. 67 (1995) 4239–4246.[6] H. Sun, B. Wang, S.G. DiMagno, Org. Lett. 10 (2008) 4413–4416.[7] M. Li, G.E. Paecy, Talanta 44 (1997) 1949–1958.[8] S. Garrigues, M. Gallignani, M. de la Guardia, Anal. Chim. Acta 281 (1993)

259–264.[9] H. Langhals, Anal. Lett. 23 (1990) 2243–2258.[10] C. Pinheiro, J.C. Lima, A.J. Parola, Sens. Actuat. B 114 (2006) 978–983.[11] C. Yunxiang, J. Xin, Talanta 31 (1984) 556–558.[12] L. Ding, Z. Zhang, X. Li, J. Su, Chem. Commun. 49 (2013) 7319–7321.[13] F. Gao, F. Luo, X. Chen, W. Yao, J. Yin, Z. Yao, L. Wang, Microchim. Acta 166

(2009) 163–167.[14] W. Liu, Y. Wang, W. Jin, G. Shen, R. Yu, Anal. Chim. Acta 383 (1999) 299–307.[15] M. Nakashima, J.A. Sousa, R.C. Clapp, Nat. Phys. Sci. 235 (1972) 16–19.[16] E. Bardez, P. Boutin, B. Valeur, Chem. Phys. Lett. 191 (1992) 142–148.

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