Talanta 132 (2015) 392–397
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Ultrasensitive 4-methylumbelliferone fluorimetric determination of 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
art ic l e i nf o
a b s t r a c t
Article history: Received 14 July 2014 Received in revised form 9 September 2014 Accepted 12 September 2014 Available online 19 September 2014
A novel approach to the quantification of relatively small amounts of water present in low polarity, aprotic solvents is proposed. This method takes advantage of protolitic reaction of 4-methylumbelliferone dissolved in the solvent with water, acting as a base. The low emission intensity neutral 4-methylumbelliferone is transformed in reaction with water to a highly fluorescent anionic form. Thus the increase in emission intensity is observed for increasing water contents in aprotic solvents. For low water contents and highly lipophilic solvents, this method yields (in practical conditions) higher sensitivity compared to biamperometric Karl Fischer titration method in volumetric mode. It is also shown that organic compounds of protolitic character (amines, acids) not only interfere with water 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 as introduction of pure water. & 2014 Elsevier B.V. All rights reserved.
Keywords: Water determination Karl Fischer 4-methylumbelliferone Aliphatic solvents Fluorometry
1. Introduction Determination of water contents, particularly in the case of organic solvents is essential for many practical applications, especially in industrial processes. Clearly, there is an interest in methods allowing quick and easy access to information on water contents in different organic media. The golden standard for water determination is coulometric Karl Fischer titration, for example [1]. In general, this is the most reliable method, however, in the case of minute water concentrations and especially highly lipophilic, aprotic solvents problems related to sampling and especially tendency of water to spontaneously accumulate close to more hydrophilic surfaces – e.g. glassware, may result in inaccurate determination results [1,2]. In aprotic and non-alcoholic solvents Karl Fischer method is less sensitive because the Bunsen reaction is favored and water/iodine molar ratio changes toward 2:1 in contrast to the 1:l molar ratio found in the protic Karl Fischer environment [3]. For oil samples the results obtained by volumetric and coulometric Karl Fischer methods usually considerably differ even after correcting for the instrumental bias. Thus it was suggested that the volumetric method should be preferred over coulometric method to measure all of the water content in oil samples [4,5]. Moreover, Karl Fischer titration can hardly be performed as an integral part of technological process, or be used to continuously monitor any occurrence of water in the
n
Corresponding author. Tel.: þ 4822 8220211; fax: þ 48 22 8225996. E-mail address: [email protected] (A. Michalska).
http://dx.doi.org/10.1016/j.talanta.2014.09.018 0039-9140/& 2014 Elsevier B.V. All rights reserved.
sample media. Thus, different alternative methods of water quantification have been proposed, including advanced techniques like NMR [6], infrared spectrometry [7,8], UV/Vis spectrometry [9,10]. Many of them tried to apply fluorometry for determination of water contents in organic solvents as well for example [11–14]. The latter approach, however, requires application of fluorophore of emission spectra 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 determination purpose. Moreover, in most cases the increase of water contents in the sample was accompanied with decrease (quenching) of emission signal (e.g. [11,13]). For practical analytical applications, however, commercially available fluorophores probes, and optimally leading to increase of the emission signal with increasing analyte (water) concentrations are preferred. Among widely available, costeffective ligands 4-methylumbelliferone (4-MU), pH-sensitive, widely used, bright fluorophore is known to be soluble in variety of organic solvents. It was also shown that the emission spectrum of 4-MU probe is dependent on the solvent used and on the presence of acid/ bases in the sample, for example [15,16]. The dependence of emission intensity on water contents in ethanol in the presence of constant concentration of hydrochloric acid was also reported [15]. Nevertheless, to our best knowledge, the above-mentioned reports have not led to analytical application of 4-MU as water-sensitive probe for organic media. The herein proposed approach is based on assumption that in the lipophilic solvents (even in the absence of acid or bases in the system) dissolved neutral 4-MU can spontaneously undergo
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protolitic reaction with water leading to formation of highly fluorescent species, especially deprotonated 4-MU, a bright fluorophore [15]. It is rational to expect that fluorescence intensity in this case should be dependent on the amount of water present, thus allowing quantification of water in the sample, especially in emulsion. The herein proposed approach was used to quantify amount of water present in the sample of a model organic medium: hexadecane. The effect of other (model) protolitic agents present in the sample on the resulting emission signal was also studied. The proposed method was also applied for toluene and cyclohexane model solvents. Application of 4-MU to quantify minute contents of water in aprotic, lipophilic solvents to our best knowledge has not been proposed in literature. Results of 4-MU-based water quantification using herein proposed method have been compared with water determination results performed using the classical approach – biamperometric Karl Fischer titration in volumetric mode, showing that especially for low amounts of water present in the sample, herein proposed method offers higher sensitivity.
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to 600 nm. The slits used were 5 nm both for excitation and emission, while the detector voltage was maintained at 800 V. Biamperometric Karl Fischer titration in volumetric mode was performed using Titration Equipment: 716 DMS Titrino (Metrohm) and 728 Stirrer (Metrohm), platinum electrodes (400 mV) were used. Chloroform was added to the working solution as a solubility promoter so that the KF titration was conducted in the homogeneous solution. This procedure was found to give more reproducible results. The requirements of complete dissolution in KF method was also discussed earlier [5]. Emulsions were prepared using homogenizer Hielscher, model UP 200 S. 2.3. Preparation of 4-MU stock solutions 4-MU stock solutions in tested solvents were prepared by dissolving 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 continued for another 5 min. Then the mixture was left for 24 hours to complete dissolution of the fluorophore. Thus prepared solutions were used for fluorimetric measurements.
2. Experimental 2.4. Preparation of the samples 2.1. Reagents Hexadecane (HD), 4-methylumbelliferone (4-MU), HYDRANALs - Composite 5 K (Titration), HYDRANALs - Medium K, HYDRANALs - Water Standard 10.0, toluene, cyclohexane, hexanoic acid, palmitic acid, octanoic acid, isopentyl amine, octadecyl amine and molecular sieves were from Aldrich (Germany). Octylamine, hydrochloric acid and NaOH were of analytical grade and were obtained from POCh (Gliwice, Poland). Doubly distilled and freshly deionized water (resistance 18.2 MΩcm, Milli-Qplus, Millipore, Austria) was used throughout this work. All solvents were dried over freshly activated molecular sieves (MS 4A).
2.2. Apparatus Fluorimetric experiments were performed using a spectrofluorimeter Cary Eclipse (Varian). After exposure at an excitation wavelength, unless otherwise stated, equal to 320 nm, emission intensity was observed within the range from 350 nm (or 370 nm)
Water was introduced to one of the tested solvents (hexadecane, cyclohexane, toluene) containing 4-MU. To assure that introduced water is mixed with dry solvent, the sample was sonicated for 60 seconds. Samples for Karl Fischer titration control experiment were prepared in the same manner.
3. Results and discussion 4-MU is a well-known fluorescent pH indicator, showing strong emission at about 450 nm in aqueous alkaline solutions, this emission is characteristic for deprotonated form of 4-MU – when it is present as anion, Fig. S1 [15,16]. Analytical usefulness of this reagent has been proven in numerous applications, including those where the enzymatic reaction leads to formation of highly fluorescent 4-MU anion, from optically silent enzymatic substrates (for example using alkaline phosphatase, glucuronidase, β-glucosidase and other enzymes). Fig. 1. presents excitation and emission spectra obtained for 4-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 additives tested: (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/v water 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.
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can be seen in Fig. 1, for 4-MU dissolved in HD weak absorption (at about 320–330 nm) and practically no emission is observed. It should be stressed that 0.025 mg/ml concentration of 4-MU is the highest practically achievable for HD solutions. The emission intensities observed at about 380 nm for the same concentration of 4-MU in toluene were yet lower compared to HD, Fig. S2, and were very weak for cyclohexane (the latter are not included in Fig. S2). It should be stressed that the presence of emission peak at 380 nm confirms, that as expected, neutral 4-MU is present in dry tested solvents [15] (structure A in Fig. S1). As 4-MU is known to be sensitive to the presence of protolitic agents in sample, the effect of presence of model compounds of this nature was tested. The introduction of protolitic reagents to the model solvent tested, HD, results in only small changes in the emission spectra, Fig. S3. The presence of tested acids: hexanoic, octanoic or palmitic in 10 3 M concentration results in 4-MU emission observed at about 380 nm, suggesting that still neutral 4-MU is present in the system, regardless acid added. For higher concentrations of acid added to HD, 10 1 M, apart from significant increase of peak at 380 nm, additional peak is formed at about 490 nm, Fig. S3, inset. The latter is typical for neutral tautomer form of 4-MU, structure C, Fig. S1 [15]. This behavior is similar to that reported earlier for strong acid (HCl) introduced to protolitic solvents as water or ethanol [15]. Thus, it is probable that, in the absence of other protolitic agents, even relatively weak (in aqueous environment) acids are able to induce the change of the structure of neutral 4-MU molecule, leading to formation of above mentioned neutral tautomer characterized with emission at about 490 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 the amine used, similar behavior was observed, the exemplary results obtained for octylamine are shown, Fig. 2. For low concentration of amine present in the sample, both excitation and emission spectra recorded resemble those of 4-MU in HD recorded in the absence of amine. However, for higher concentration of amine ( 4 10 5 M) the intensity of emission of 4-MU dissolved in HD was significantly dependent on concentration of amine, as shown on example of octylamine, Fig. 2. For the amine concentration equal to 10 5 M a small shoulder is formed on 4-MU emission spectra with a maximum at 450 nm, at wavelength typical for deprotonated form of 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 emission peak with maximum at 450 nm (excitation 330 nm), clearly pointing out to deprotonation of 4-MU as expected for presence of 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 to similar change of 4-MU emission spectra as introduction of any amines in 10 4 M concentration, Fig. 1. On the excitation spectra a pronounced absorption peak is formed with maximum at about 320 nm. On the emission spectra a strong peak is formed with a maximum at about 450 nm, suggesting the presence of deprotonated form of 4-MU in HD. Thus, it can be assumed that in the absence of other deprotonating agents, water as a base reacts with 4-MU and this reaction leads to deprotonation of 4-MU and formation of structure depicted in Fig. S1 B. This process can be accompanied by partition of 4-MU anion to the water phase, due to its higher (compared to neutral 4-MU) solubility in water. Thus the protolitic reaction of 4-MU with water in aliphatic environment is promising 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 from 1.7.10 2% v/v (9.3.10 3 M) to 0.6% v/v (0.37 M). As it can be seen from Fig. 3 introduction of even relatively small amount of water, reaching 1.7.10 2% v/v, to 4-MU containing HD results in formation of emission peak at wavelength typical for deprotonated form of 4-MU, at 450 nm (excitation wavelength 320 nm). Increase of the amount of water introduced to the sample results in clear increase of the intensity of emission observed at 450 nm. The linear dependence of emission intensity on water contents was observed within the range from 1.7.10 2% to 0.33% v/v, that is up to 0.185 M water in HD, Fig. 4. Within this range the correlation coefficient, R2, of the dependence of emission intensity on water contents was equal to 0.987, Table 1. Further increase of water contents in the sample to 0.67% v/v (0.37 M) resulted in some increase in the recorded emission, yet not linear. In this range also the magnitude of error increases, Fig. S4. The plateau of the signal obtained can be attributed to different effects; however, it should be stressed that for these water contents the amount (mole) of water was more than 2000 times higher compared to amount (mole) of 4-MU present in the sample. It can also result from the protolitic reaction, and from acid–base equilibrium prevailing in the sample. The advantage of herein proposed approach is clearly seen when comparing the results of fluorimetric evaluation of water
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Wavelength (nm) Fig. 2. Emission spectra recorded in different concentrations of octylamine in HD: (black line) 4-MU in HD, and gradually increasing octylamine concentration (grey line) 10 8 M, (red line) 10 7 M, (green line) 10 6 M, (cyan line) 10 5 M, (magenta line) 10 4 M and (blue line) 10 3 M. Inset – magnification of spectra obtained for low concentration of octylamine in HD, excitation wavelength 330 nm.
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Wavelength (nm) Fig. 3. Emission spectra recorded in different concentrations of water in HD: (black line) 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 low concentration 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).
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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 the absence 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 the presence 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 HD samples, inset: magnification of the low water contents range of Karl Fischer titration. Table 1 Summary of analytical performance of 4-MU as water sensitive fluorescent indicator System
Hexadecane
Hexadecane þ 10 2 M octanoic acid
Hexadecane þ 10 6 M octylamine
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λexcitation (nm) λemission (nm) Water contents (%, v/v)
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contents changes using 4-MU as indicator, with results of Karl Fischer titration, Fig. 4. For Karl Fischer titration, as it can be seen from Fig. 4, introduction of relatively small amounts of water to the sample, below 0.1% v/v, yields negligible changes of estimated water amount pointing out to significant negative error of water contents estimation. It should be stressed that Karl Fischer titration is dependent on the sampling procedure, and in the case of relatively low water contents, e.g. accumulation of water introduced to lipophilic HD at the interface of the sample and used vessel, significantly affects obtained results. Herein described approach takes the advantage of emission changes, resulting from 4-MU spontaneous protolitic reaction with water. As 4-MU is dissolved in whole volume of HD, any contact of the sample with water results in change of the emission spectra. Thus, as seen in Fig. 4, fluorimetric approach is significantly more sensitive for relatively low water contents. On the other hand, for higher water contents, 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 for different water contents in HD originates from protolitic reaction with water acting as a base, it seems justified to check how the presence of base or acid in the sample influences the analytical performance of this system. As discussed above (Fig. 2), the presence of base (amine) at the concentration 10 6 M, in the absence of water, does not affect the emission spectra of 4-MU in HD. Similarly the presence of 10 6 M octylamine in HD did not affect the dependence of emission on changes of water contents in the sample. Both the excitation and emission spectra were comparable within the range of experimental error with that recorded in the absence of amine in solution (Fig. S5). Similarly, the dependence of emission intensity
at 450 nm on water contents in the sample was linear within the range 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 observed on the excitation spectra of 4-MU, Fig. 1. Thus in this case the excitation wavelength used was equal to 360 nm. The emission intensities recorded in the presence of water (0.17% v/v) were significantly higher compared to that recorded for water containing samples in the absence of amine or in the presence of 10 6 M octylamine. The emission intensity vs. water contents dependence recorded in the presence of 10 3 M octylamine, Fig. S6, for low water contents ( o 0.3% v/v) was linear with R2 ¼0.991, Table 1. The sensitivity of determination was not only higher than for Karl Fischer titration, but also it was the highest among tested model systems. The sensitivity obtained for 10 3 M amine present was more than two times higher compared to the system tested in the absence or in the presence of low octylamine concentration in HD, Fig. S5 and Fig S6. Moreover, in the presence of amine in HD the error measured as SD of three replicates is significantly smaller, except for the highest water content tested, which is already at plateau of the dependence, Fig. S4. Thus introduction of amine in 10 3 M gives possibility of fine tuning of sensitivity of fluorimetric water contents determination in aprotic solvents, offering significantly higher sensitivity within this region compared to Karl Fischer titration, Fig. 4. However, the linear range of the dependence of emission on contents of water was shorten compared to above-described cases, for higher water contents the decrease of fluorescence intensity was observed, Fig. 4. The significant increase of sensitivity of water contents determination, especially at low water contents, observed for the 10 3 M amine presence in the sample results from the properties
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of amine – which is stronger base than water. Thus amine influence on 4-MU deprotonation and concomitant formation of emission signal is potentially stronger than that of water. With increasing water contents in the sample hydrophilicity of the medium increases, protolitic reaction between 4-MU and amine, resulting in protonation of amine on expense of creating of 4-MU anion, is facilitated, resulting in higher sensitivity of water contents determination. The strong emission signal observed can be also to some extent related to formation of structures resembling reversed micelles, containing water in their cores and being stabilized by protonated amine oriented with protonated amine group toward hydrophilic environment of water and with the alkyl chain oriented toward HD. It is probable that the anionic, deprotonated and water-soluble form of 4-MU formed will be present inside the reverse micelles resembling structures, being stabilized with positive charges of the protonated amine, this will ultimately yield higher emission intensity. This effect is probably of low importance for low (10 6 M) amine contents in the sample as the population of positive charges that may be created is relatively low, whereas at higher amine contents the enhancement of sensitivity of water determination is clearly seen. Introduction of acid, as a model compound octanoic acid was used, in 10 4 or 10 2 M concentration, did not affect the emission or absorption peak positions. The excitation and emission spectra and their maxima, recorded in the presence of octanoic acid, at two concentrations used, were not affected compared to spectra recorded for similar system, yet in the absence of acid, Fig. 1. Introduction of water to HD-containing octanoic acid resulted in increase of emission at 450 nm, similarly as for net HD-containing 4-MU (the maximum of excitation peak was observed at about 320 nm), Fig. 1. The effect of presence of octanoic acid in the system predominantly was observed on the emission intensity vs. water contents dependence. Although for increase of water contents o0.1% v/v the increase of intensity was slightly higher compared to HD system containing 4-MU in the presence of 10 3 M octylamine, the further increase of water contents resulted in much smaller increase of intensity of emission and the dependence was reaching plateau, regardless the concentration of acid present in the HD. Formation of plateau can be related to the fact that octanoic acid is stronger acid than 4-MU, thus deprotonation of 4-MU in this case is not enhanced by water addition. On the other hand, dissociation of octanoic acid, if occurs, leads to formation of water containing reversed micelles resembling structures with anionic charge at the water/HD interface. Thus due to
electrostatic repulsion, incorporation of negatively charged 4-MU anion is less likely compared to positively charged water/HD interface in the case of amine reversed micelle resembling structures formed. Thus although the presence of carboxylic acid in the model solvent HD limits possibility of analytical quantification of the water contents to the low amounts only, the proposed method is robust enough to clearly distinguish samples containing water from those free from water additions. For analytical application of herein proposed system the information on response time of the system for water addition as well as stability of emission signal in time was collected, Fig. S7, using HD as model solvent. As shown above the intensity of emission recorded in the presence of 10 3 M octanoic acid was much lower compared to signals recorded in the presence of water, moreover the intensity of emission was fluctuating in time especially in the presence of octanoic acid in HD. Introduction of water to the sample leads to pronounced increase of emission, Fig. S7, regardless of the system tested. In the absence of protolitic compounds in HD stable signal was obtained after about 3 min. For sample containing amine or acid emission signal was increasing faster, the stable signal was achieved after about 2 min time. The fastest response was observed for system containing octylamine, regardless of concentration of water tested (3.4.10-2% v/v or 1.7.10 1% v/v), the maximum intensity of emission signal was observed after 1 min. This result is supporting the thesis that presence of amine and possible formation of reverse micelle-like structures favors reaction of 4-MU with water added to the system. It should be stressed that regardless of the system studied (either without additives or containing acid or base) emission signal in the presence of water was stable in experiment time. Last, but not the least, the above-described experiments confirm, that the proposed system is not only highly sensitive for low water contents but also the results are not interfered by the presence of organic impurities of protolitic character in the sample. This effect is due to spontaneous and beneficial effect of reverse micelles resembling structures formation (provided that the concentration of impurities is high enough) and was not observed for introduction of aqueous solution of acid (HCl) or base (NaOH) solution to the sample. In the latter case, the effect observed was equivalent within the range of experimental error, to that 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 the presence of 10 6 M octylamine in tested solvent.
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The herein proposed simple method of water quantification using protolitic reaction of 4-MU was also tested on example of other solvents – toluene and cyclohexane. For 4-MU dissolved in toluene introduction of water resulted in formation of emission peak at 450 nm, of intensity dependent on water contents, Fig. 5. As it can be seen in Fig. 5, similarly as for HD, a linear dependence of intensity of emission on contents of water in the sample was recorded; however, for toluene the linear range of responses was much broader compared to HD. For tested range 0.017% v/v to 20% v/v of water in toluene, it was characterized with R2 equal to 0.991, Table 1. As it can be seen in Fig. 5, especially for low water contents,o 0.7% v/v, a further increase of sensitivity is observed if the octylamine is present in the toluene in concentration 10 6 M, that is at the concentration of no beneficial effect in the case of hexadecane. Probably the advantageous effect of already low concentration of amine present in toluene is related to higher polarity of this solvent compared to HD. Toluene partially participates in stabilization of water droplets within, thus even low contents of amine actively increases deprotonation of 4-MU molecules and their partition into aqueous phase. For cyclohexane containing 4-MU, similarly as for above discussed examples, introduction of water resulted in formation of emission peak at 450 nm, of intensity dependent on water contents in the sample, Fig. 5. The linear dependence of emission intensity on water contents was however, limited to about 0.2% v/v of water (R2 ¼0.967), Table 1. Similarly as for toluene, introduction of amine in 10 6 M concentration to the cyclohexane has resulted in increased emission, however recorded dependence was not linear. Increase of water contents above 0.33% v/v resulted in decrease of recorded emission. 4. Conclusions The herein presented results clearly show that minute water contents, especially in highly lipophilic, nonpolar solvents like hexadecane, can be determined with high sensitivity due to protolitic reaction of 4-methylumbelliferone with water, acting as a base. The emission intensity of deprotonated 4-MU anion well correlates with water contents in the nonpolar solvent. For low concentrations, this system offers higher sensitivity compared to
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Karl Fischer titration. Moreover, presence of organic acid and especially organic base leads to further increase of sensitivity, with increasing concentration of this additive in the solvent. This effect can be attributed to formation of structures resembling reversed micelle within the low-polarity solvent – with water core and stabilized with protonated (in the case of amine) or deprotonated (in the case of acid) organic ion located at the water – solvent interface. This spontaneous process in the case of added base (amine) supports partition of ionized 4-MU into the minute water phase.
Acknowledgment Financial support from National Centre of Science (NCN, Poland), project 2011/03/B/ST4/00747, in the years 2012–2015, is gratefully acknowledged.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2014.09.018. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16]
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Ultrasensitive 4-methylumbelliferone fluorimetric determination of 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
art ic l e i nf o
a b s t r a c t
Article history: Received 14 July 2014 Received in revised form 9 September 2014 Accepted 12 September 2014 Available online 19 September 2014
A novel approach to the quantification of relatively small amounts of water present in low polarity, aprotic solvents is proposed. This method takes advantage of protolitic reaction of 4-methylumbelliferone dissolved in the solvent with water, acting as a base. The low emission intensity neutral 4-methylumbelliferone is transformed in reaction with water to a highly fluorescent anionic form. Thus the increase in emission intensity is observed for increasing water contents in aprotic solvents. For low water contents and highly lipophilic solvents, this method yields (in practical conditions) higher sensitivity compared to biamperometric Karl Fischer titration method in volumetric mode. It is also shown that organic compounds of protolitic character (amines, acids) not only interfere with water 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 as introduction of pure water. & 2014 Elsevier B.V. All rights reserved.
Keywords: Water determination Karl Fischer 4-methylumbelliferone Aliphatic solvents Fluorometry
1. Introduction Determination of water contents, particularly in the case of organic solvents is essential for many practical applications, especially in industrial processes. Clearly, there is an interest in methods allowing quick and easy access to information on water contents in different organic media. The golden standard for water determination is coulometric Karl Fischer titration, for example [1]. In general, this is the most reliable method, however, in the case of minute water concentrations and especially highly lipophilic, aprotic solvents problems related to sampling and especially tendency of water to spontaneously accumulate close to more hydrophilic surfaces – e.g. glassware, may result in inaccurate determination results [1,2]. In aprotic and non-alcoholic solvents Karl Fischer method is less sensitive because the Bunsen reaction is favored and water/iodine molar ratio changes toward 2:1 in contrast to the 1:l molar ratio found in the protic Karl Fischer environment [3]. For oil samples the results obtained by volumetric and coulometric Karl Fischer methods usually considerably differ even after correcting for the instrumental bias. Thus it was suggested that the volumetric method should be preferred over coulometric method to measure all of the water content in oil samples [4,5]. Moreover, Karl Fischer titration can hardly be performed as an integral part of technological process, or be used to continuously monitor any occurrence of water in the
n
Corresponding author. Tel.: þ 4822 8220211; fax: þ 48 22 8225996. E-mail address: [email protected] (A. Michalska).
http://dx.doi.org/10.1016/j.talanta.2014.09.018 0039-9140/& 2014 Elsevier B.V. All rights reserved.
sample media. Thus, different alternative methods of water quantification have been proposed, including advanced techniques like NMR [6], infrared spectrometry [7,8], UV/Vis spectrometry [9,10]. Many of them tried to apply fluorometry for determination of water contents in organic solvents as well for example [11–14]. The latter approach, however, requires application of fluorophore of emission spectra 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 determination purpose. Moreover, in most cases the increase of water contents in the sample was accompanied with decrease (quenching) of emission signal (e.g. [11,13]). For practical analytical applications, however, commercially available fluorophores probes, and optimally leading to increase of the emission signal with increasing analyte (water) concentrations are preferred. Among widely available, costeffective ligands 4-methylumbelliferone (4-MU), pH-sensitive, widely used, bright fluorophore is known to be soluble in variety of organic solvents. It was also shown that the emission spectrum of 4-MU probe is dependent on the solvent used and on the presence of acid/ bases in the sample, for example [15,16]. The dependence of emission intensity on water contents in ethanol in the presence of constant concentration of hydrochloric acid was also reported [15]. Nevertheless, to our best knowledge, the above-mentioned reports have not led to analytical application of 4-MU as water-sensitive probe for organic media. The herein proposed approach is based on assumption that in the lipophilic solvents (even in the absence of acid or bases in the system) dissolved neutral 4-MU can spontaneously undergo
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protolitic reaction with water leading to formation of highly fluorescent species, especially deprotonated 4-MU, a bright fluorophore [15]. It is rational to expect that fluorescence intensity in this case should be dependent on the amount of water present, thus allowing quantification of water in the sample, especially in emulsion. The herein proposed approach was used to quantify amount of water present in the sample of a model organic medium: hexadecane. The effect of other (model) protolitic agents present in the sample on the resulting emission signal was also studied. The proposed method was also applied for toluene and cyclohexane model solvents. Application of 4-MU to quantify minute contents of water in aprotic, lipophilic solvents to our best knowledge has not been proposed in literature. Results of 4-MU-based water quantification using herein proposed method have been compared with water determination results performed using the classical approach – biamperometric Karl Fischer titration in volumetric mode, showing that especially for low amounts of water present in the sample, herein proposed method offers higher sensitivity.
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to 600 nm. The slits used were 5 nm both for excitation and emission, while the detector voltage was maintained at 800 V. Biamperometric Karl Fischer titration in volumetric mode was performed using Titration Equipment: 716 DMS Titrino (Metrohm) and 728 Stirrer (Metrohm), platinum electrodes (400 mV) were used. Chloroform was added to the working solution as a solubility promoter so that the KF titration was conducted in the homogeneous solution. This procedure was found to give more reproducible results. The requirements of complete dissolution in KF method was also discussed earlier [5]. Emulsions were prepared using homogenizer Hielscher, model UP 200 S. 2.3. Preparation of 4-MU stock solutions 4-MU stock solutions in tested solvents were prepared by dissolving 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 continued for another 5 min. Then the mixture was left for 24 hours to complete dissolution of the fluorophore. Thus prepared solutions were used for fluorimetric measurements.
2. Experimental 2.4. Preparation of the samples 2.1. Reagents Hexadecane (HD), 4-methylumbelliferone (4-MU), HYDRANALs - Composite 5 K (Titration), HYDRANALs - Medium K, HYDRANALs - Water Standard 10.0, toluene, cyclohexane, hexanoic acid, palmitic acid, octanoic acid, isopentyl amine, octadecyl amine and molecular sieves were from Aldrich (Germany). Octylamine, hydrochloric acid and NaOH were of analytical grade and were obtained from POCh (Gliwice, Poland). Doubly distilled and freshly deionized water (resistance 18.2 MΩcm, Milli-Qplus, Millipore, Austria) was used throughout this work. All solvents were dried over freshly activated molecular sieves (MS 4A).
2.2. Apparatus Fluorimetric experiments were performed using a spectrofluorimeter Cary Eclipse (Varian). After exposure at an excitation wavelength, unless otherwise stated, equal to 320 nm, emission intensity was observed within the range from 350 nm (or 370 nm)
Water was introduced to one of the tested solvents (hexadecane, cyclohexane, toluene) containing 4-MU. To assure that introduced water is mixed with dry solvent, the sample was sonicated for 60 seconds. Samples for Karl Fischer titration control experiment were prepared in the same manner.
3. Results and discussion 4-MU is a well-known fluorescent pH indicator, showing strong emission at about 450 nm in aqueous alkaline solutions, this emission is characteristic for deprotonated form of 4-MU – when it is present as anion, Fig. S1 [15,16]. Analytical usefulness of this reagent has been proven in numerous applications, including those where the enzymatic reaction leads to formation of highly fluorescent 4-MU anion, from optically silent enzymatic substrates (for example using alkaline phosphatase, glucuronidase, β-glucosidase and other enzymes). Fig. 1. presents excitation and emission spectra obtained for 4-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 additives tested: (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/v water 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.
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can be seen in Fig. 1, for 4-MU dissolved in HD weak absorption (at about 320–330 nm) and practically no emission is observed. It should be stressed that 0.025 mg/ml concentration of 4-MU is the highest practically achievable for HD solutions. The emission intensities observed at about 380 nm for the same concentration of 4-MU in toluene were yet lower compared to HD, Fig. S2, and were very weak for cyclohexane (the latter are not included in Fig. S2). It should be stressed that the presence of emission peak at 380 nm confirms, that as expected, neutral 4-MU is present in dry tested solvents [15] (structure A in Fig. S1). As 4-MU is known to be sensitive to the presence of protolitic agents in sample, the effect of presence of model compounds of this nature was tested. The introduction of protolitic reagents to the model solvent tested, HD, results in only small changes in the emission spectra, Fig. S3. The presence of tested acids: hexanoic, octanoic or palmitic in 10 3 M concentration results in 4-MU emission observed at about 380 nm, suggesting that still neutral 4-MU is present in the system, regardless acid added. For higher concentrations of acid added to HD, 10 1 M, apart from significant increase of peak at 380 nm, additional peak is formed at about 490 nm, Fig. S3, inset. The latter is typical for neutral tautomer form of 4-MU, structure C, Fig. S1 [15]. This behavior is similar to that reported earlier for strong acid (HCl) introduced to protolitic solvents as water or ethanol [15]. Thus, it is probable that, in the absence of other protolitic agents, even relatively weak (in aqueous environment) acids are able to induce the change of the structure of neutral 4-MU molecule, leading to formation of above mentioned neutral tautomer characterized with emission at about 490 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 the amine used, similar behavior was observed, the exemplary results obtained for octylamine are shown, Fig. 2. For low concentration of amine present in the sample, both excitation and emission spectra recorded resemble those of 4-MU in HD recorded in the absence of amine. However, for higher concentration of amine ( 4 10 5 M) the intensity of emission of 4-MU dissolved in HD was significantly dependent on concentration of amine, as shown on example of octylamine, Fig. 2. For the amine concentration equal to 10 5 M a small shoulder is formed on 4-MU emission spectra with a maximum at 450 nm, at wavelength typical for deprotonated form of 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 emission peak with maximum at 450 nm (excitation 330 nm), clearly pointing out to deprotonation of 4-MU as expected for presence of 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 to similar change of 4-MU emission spectra as introduction of any amines in 10 4 M concentration, Fig. 1. On the excitation spectra a pronounced absorption peak is formed with maximum at about 320 nm. On the emission spectra a strong peak is formed with a maximum at about 450 nm, suggesting the presence of deprotonated form of 4-MU in HD. Thus, it can be assumed that in the absence of other deprotonating agents, water as a base reacts with 4-MU and this reaction leads to deprotonation of 4-MU and formation of structure depicted in Fig. S1 B. This process can be accompanied by partition of 4-MU anion to the water phase, due to its higher (compared to neutral 4-MU) solubility in water. Thus the protolitic reaction of 4-MU with water in aliphatic environment is promising 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 from 1.7.10 2% v/v (9.3.10 3 M) to 0.6% v/v (0.37 M). As it can be seen from Fig. 3 introduction of even relatively small amount of water, reaching 1.7.10 2% v/v, to 4-MU containing HD results in formation of emission peak at wavelength typical for deprotonated form of 4-MU, at 450 nm (excitation wavelength 320 nm). Increase of the amount of water introduced to the sample results in clear increase of the intensity of emission observed at 450 nm. The linear dependence of emission intensity on water contents was observed within the range from 1.7.10 2% to 0.33% v/v, that is up to 0.185 M water in HD, Fig. 4. Within this range the correlation coefficient, R2, of the dependence of emission intensity on water contents was equal to 0.987, Table 1. Further increase of water contents in the sample to 0.67% v/v (0.37 M) resulted in some increase in the recorded emission, yet not linear. In this range also the magnitude of error increases, Fig. S4. The plateau of the signal obtained can be attributed to different effects; however, it should be stressed that for these water contents the amount (mole) of water was more than 2000 times higher compared to amount (mole) of 4-MU present in the sample. It can also result from the protolitic reaction, and from acid–base equilibrium prevailing in the sample. The advantage of herein proposed approach is clearly seen when comparing the results of fluorimetric evaluation of water
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Wavelength (nm) Fig. 2. Emission spectra recorded in different concentrations of octylamine in HD: (black line) 4-MU in HD, and gradually increasing octylamine concentration (grey line) 10 8 M, (red line) 10 7 M, (green line) 10 6 M, (cyan line) 10 5 M, (magenta line) 10 4 M and (blue line) 10 3 M. Inset – magnification of spectra obtained for low concentration of octylamine in HD, excitation wavelength 330 nm.
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Wavelength (nm) Fig. 3. Emission spectra recorded in different concentrations of water in HD: (black line) 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 low concentration 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).
Fluorescence intensity @ 450 nm
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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 the absence 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 the presence 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 HD samples, inset: magnification of the low water contents range of Karl Fischer titration. Table 1 Summary of analytical performance of 4-MU as water sensitive fluorescent indicator System
Hexadecane
Hexadecane þ 10 2 M octanoic acid
Hexadecane þ 10 6 M octylamine
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contents changes using 4-MU as indicator, with results of Karl Fischer titration, Fig. 4. For Karl Fischer titration, as it can be seen from Fig. 4, introduction of relatively small amounts of water to the sample, below 0.1% v/v, yields negligible changes of estimated water amount pointing out to significant negative error of water contents estimation. It should be stressed that Karl Fischer titration is dependent on the sampling procedure, and in the case of relatively low water contents, e.g. accumulation of water introduced to lipophilic HD at the interface of the sample and used vessel, significantly affects obtained results. Herein described approach takes the advantage of emission changes, resulting from 4-MU spontaneous protolitic reaction with water. As 4-MU is dissolved in whole volume of HD, any contact of the sample with water results in change of the emission spectra. Thus, as seen in Fig. 4, fluorimetric approach is significantly more sensitive for relatively low water contents. On the other hand, for higher water contents, 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 for different water contents in HD originates from protolitic reaction with water acting as a base, it seems justified to check how the presence of base or acid in the sample influences the analytical performance of this system. As discussed above (Fig. 2), the presence of base (amine) at the concentration 10 6 M, in the absence of water, does not affect the emission spectra of 4-MU in HD. Similarly the presence of 10 6 M octylamine in HD did not affect the dependence of emission on changes of water contents in the sample. Both the excitation and emission spectra were comparable within the range of experimental error with that recorded in the absence of amine in solution (Fig. S5). Similarly, the dependence of emission intensity
at 450 nm on water contents in the sample was linear within the range 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 observed on the excitation spectra of 4-MU, Fig. 1. Thus in this case the excitation wavelength used was equal to 360 nm. The emission intensities recorded in the presence of water (0.17% v/v) were significantly higher compared to that recorded for water containing samples in the absence of amine or in the presence of 10 6 M octylamine. The emission intensity vs. water contents dependence recorded in the presence of 10 3 M octylamine, Fig. S6, for low water contents ( o 0.3% v/v) was linear with R2 ¼0.991, Table 1. The sensitivity of determination was not only higher than for Karl Fischer titration, but also it was the highest among tested model systems. The sensitivity obtained for 10 3 M amine present was more than two times higher compared to the system tested in the absence or in the presence of low octylamine concentration in HD, Fig. S5 and Fig S6. Moreover, in the presence of amine in HD the error measured as SD of three replicates is significantly smaller, except for the highest water content tested, which is already at plateau of the dependence, Fig. S4. Thus introduction of amine in 10 3 M gives possibility of fine tuning of sensitivity of fluorimetric water contents determination in aprotic solvents, offering significantly higher sensitivity within this region compared to Karl Fischer titration, Fig. 4. However, the linear range of the dependence of emission on contents of water was shorten compared to above-described cases, for higher water contents the decrease of fluorescence intensity was observed, Fig. 4. The significant increase of sensitivity of water contents determination, especially at low water contents, observed for the 10 3 M amine presence in the sample results from the properties
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of amine – which is stronger base than water. Thus amine influence on 4-MU deprotonation and concomitant formation of emission signal is potentially stronger than that of water. With increasing water contents in the sample hydrophilicity of the medium increases, protolitic reaction between 4-MU and amine, resulting in protonation of amine on expense of creating of 4-MU anion, is facilitated, resulting in higher sensitivity of water contents determination. The strong emission signal observed can be also to some extent related to formation of structures resembling reversed micelles, containing water in their cores and being stabilized by protonated amine oriented with protonated amine group toward hydrophilic environment of water and with the alkyl chain oriented toward HD. It is probable that the anionic, deprotonated and water-soluble form of 4-MU formed will be present inside the reverse micelles resembling structures, being stabilized with positive charges of the protonated amine, this will ultimately yield higher emission intensity. This effect is probably of low importance for low (10 6 M) amine contents in the sample as the population of positive charges that may be created is relatively low, whereas at higher amine contents the enhancement of sensitivity of water determination is clearly seen. Introduction of acid, as a model compound octanoic acid was used, in 10 4 or 10 2 M concentration, did not affect the emission or absorption peak positions. The excitation and emission spectra and their maxima, recorded in the presence of octanoic acid, at two concentrations used, were not affected compared to spectra recorded for similar system, yet in the absence of acid, Fig. 1. Introduction of water to HD-containing octanoic acid resulted in increase of emission at 450 nm, similarly as for net HD-containing 4-MU (the maximum of excitation peak was observed at about 320 nm), Fig. 1. The effect of presence of octanoic acid in the system predominantly was observed on the emission intensity vs. water contents dependence. Although for increase of water contents o0.1% v/v the increase of intensity was slightly higher compared to HD system containing 4-MU in the presence of 10 3 M octylamine, the further increase of water contents resulted in much smaller increase of intensity of emission and the dependence was reaching plateau, regardless the concentration of acid present in the HD. Formation of plateau can be related to the fact that octanoic acid is stronger acid than 4-MU, thus deprotonation of 4-MU in this case is not enhanced by water addition. On the other hand, dissociation of octanoic acid, if occurs, leads to formation of water containing reversed micelles resembling structures with anionic charge at the water/HD interface. Thus due to
electrostatic repulsion, incorporation of negatively charged 4-MU anion is less likely compared to positively charged water/HD interface in the case of amine reversed micelle resembling structures formed. Thus although the presence of carboxylic acid in the model solvent HD limits possibility of analytical quantification of the water contents to the low amounts only, the proposed method is robust enough to clearly distinguish samples containing water from those free from water additions. For analytical application of herein proposed system the information on response time of the system for water addition as well as stability of emission signal in time was collected, Fig. S7, using HD as model solvent. As shown above the intensity of emission recorded in the presence of 10 3 M octanoic acid was much lower compared to signals recorded in the presence of water, moreover the intensity of emission was fluctuating in time especially in the presence of octanoic acid in HD. Introduction of water to the sample leads to pronounced increase of emission, Fig. S7, regardless of the system tested. In the absence of protolitic compounds in HD stable signal was obtained after about 3 min. For sample containing amine or acid emission signal was increasing faster, the stable signal was achieved after about 2 min time. The fastest response was observed for system containing octylamine, regardless of concentration of water tested (3.4.10-2% v/v or 1.7.10 1% v/v), the maximum intensity of emission signal was observed after 1 min. This result is supporting the thesis that presence of amine and possible formation of reverse micelle-like structures favors reaction of 4-MU with water added to the system. It should be stressed that regardless of the system studied (either without additives or containing acid or base) emission signal in the presence of water was stable in experiment time. Last, but not the least, the above-described experiments confirm, that the proposed system is not only highly sensitive for low water contents but also the results are not interfered by the presence of organic impurities of protolitic character in the sample. This effect is due to spontaneous and beneficial effect of reverse micelles resembling structures formation (provided that the concentration of impurities is high enough) and was not observed for introduction of aqueous solution of acid (HCl) or base (NaOH) solution to the sample. In the latter case, the effect observed was equivalent within the range of experimental error, to that 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 the presence of 10 6 M octylamine in tested solvent.
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The herein proposed simple method of water quantification using protolitic reaction of 4-MU was also tested on example of other solvents – toluene and cyclohexane. For 4-MU dissolved in toluene introduction of water resulted in formation of emission peak at 450 nm, of intensity dependent on water contents, Fig. 5. As it can be seen in Fig. 5, similarly as for HD, a linear dependence of intensity of emission on contents of water in the sample was recorded; however, for toluene the linear range of responses was much broader compared to HD. For tested range 0.017% v/v to 20% v/v of water in toluene, it was characterized with R2 equal to 0.991, Table 1. As it can be seen in Fig. 5, especially for low water contents,o 0.7% v/v, a further increase of sensitivity is observed if the octylamine is present in the toluene in concentration 10 6 M, that is at the concentration of no beneficial effect in the case of hexadecane. Probably the advantageous effect of already low concentration of amine present in toluene is related to higher polarity of this solvent compared to HD. Toluene partially participates in stabilization of water droplets within, thus even low contents of amine actively increases deprotonation of 4-MU molecules and their partition into aqueous phase. For cyclohexane containing 4-MU, similarly as for above discussed examples, introduction of water resulted in formation of emission peak at 450 nm, of intensity dependent on water contents in the sample, Fig. 5. The linear dependence of emission intensity on water contents was however, limited to about 0.2% v/v of water (R2 ¼0.967), Table 1. Similarly as for toluene, introduction of amine in 10 6 M concentration to the cyclohexane has resulted in increased emission, however recorded dependence was not linear. Increase of water contents above 0.33% v/v resulted in decrease of recorded emission. 4. Conclusions The herein presented results clearly show that minute water contents, especially in highly lipophilic, nonpolar solvents like hexadecane, can be determined with high sensitivity due to protolitic reaction of 4-methylumbelliferone with water, acting as a base. The emission intensity of deprotonated 4-MU anion well correlates with water contents in the nonpolar solvent. For low concentrations, this system offers higher sensitivity compared to
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Karl Fischer titration. Moreover, presence of organic acid and especially organic base leads to further increase of sensitivity, with increasing concentration of this additive in the solvent. This effect can be attributed to formation of structures resembling reversed micelle within the low-polarity solvent – with water core and stabilized with protonated (in the case of amine) or deprotonated (in the case of acid) organic ion located at the water – solvent interface. This spontaneous process in the case of added base (amine) supports partition of ionized 4-MU into the minute water phase.
Acknowledgment Financial support from National Centre of Science (NCN, Poland), project 2011/03/B/ST4/00747, in the years 2012–2015, is gratefully acknowledged.
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