BY-NC-ND 3.0 license Open Access Published by De Gruyter April 10, 2018

Micelle-enhanced flow injection analysis

Dalia T. Abdeldaim and Fotouh R. Mansour ORCID logo


Surface-active agents are organic compounds of amphiphilic nature. When the concentration of surfactants is higher than a certain value, the monomers adhere to form well-defined aggregates known as micelles. These micelles have been employed in flow injection analysis (FIA) for various purposes. In chemiluminescence-based FIA, micelles can improve sensitivity by changing the chemical structures of the reagents, facilitating intramicellar energy transfer, accelerating the reaction kinetics, or stabilizing the excited singlet states. Micelles can improve sensitivity in FIA/ultraviolet-visible by electrostatic or solubilization effects. In FIA/fluorescence, emission intensity is considerably enhanced in micellar media due to the protective environment that restricts nonemissive energy transfer processes. If FIA is coupled with flame atomic absorption, the signals are enhanced with surfactants due to the decreased surface tension that results in the generation of smaller droplets during the aspiration and nebulization processes. In addition, surfactants promote the enrichment of the analyte in the double layer at the air-water interface. The FIA/cloud point extraction technique is based on using surfactants as alternatives to organic solvents. This review discusses the different roles of micelles in FIA methods.


Micelles are organized assemblies of amphiphilic molecules (surface-active agents, detergents, phospholipids, etc.; Sorrenti, Illa, and Ortuño 2013). These amphiphilic molecules possess groups of both hydrophilic and hydrophobic features (Ramanathan et al. 2013). The term “micelle” (normal micelle) commonly describes an aggregated system of surfactants dissolved in aqueous media; the nonpolar portion of the surfactant is labeled as the hydrophobic tail, whereas the polar portion of the amphiphile is known as the hydrophilic head (Mondal et al. 2015). As Figure 1A illustrates, the tails of the amphiphiles are packed together in the core (interior) of the micelles, whereas the polar heads form a borderline zone between the nonpolar core of the micelle and the polar aqueous solution beyond (Mclntire 1990). This arrangement can be reversed to form what is known as “reverse micelles” (Figure 1B; Lawler and Fayer 2015). Surfactants can be categorized as anionic, cationic, nonionic, or zwitterions depending on the nature of the polar group of the amphiphile. Cationic surfactants typically enclose quaternary nitrogen head groups (Li, Thomas, and Penfold 2014), whereas anionic surfactants include alkali and alkaline earth metal salts of carboxylic, sulfuric, or phosphoric acid (ShamsiJazeyi, Verduzco, and Hirasaki 2014). Table 1 summarizes the main characteristics of some commonly used surfactants of different types.

Figure 1: Schematic diagram of normal micelle (A) and reverse micelle (B).

Figure 1:

Schematic diagram of normal micelle (A) and reverse micelle (B).

Table 1:

Characteristics of some commonly used ionic and nonionic surfactantsa (Armstrong, 1985; El-Shaheny, El-Maghrabey & Belal, 2015).

Surfactant’s nameMolecular formulaTypeMolecular weightCMC (mm)Aggregation number at 25°C (monomer/micelle)Krafft or cloud point (°C)

thylammonium bromide
Lithium dodecyl sulfateCH3(CH2)11OSO3LiAnionic272.338.86216
Triton X-100C14H22O(C2H4O)n (n=9–10)Nonionic647.000.2–0.9100–15564
Tween 20C58H114O26Nonionic1228.000.0676

mmonium-3-propane-1-sulfonic acid



  1. aOther data are provided by Sigma-Aldrich (

When the concentration of surfactants is elevated above a distinctive value known as the critical micelle concentration (CMC) and the temperature of the system is greater than the critical micelle temperature, the monomers adhere to form comparatively well-defined aggregates known as micelles (Mclntire 1990). The size of micelles diverges greatly among surfactants. “Aggregation number” is the term used to indicate the number of monomer surfactants in the micellar form. The CMC and aggregation numbers are affected by various factors including temperature, pH, ionic strength, and presence of cosolvents (El-Shaheny, El-Maghrabey, and Belal 2015). The CMC is frequently denoted as a single value. However, it is a narrow range of concentrations over which the physical and chemical properties of the solution change. The formation of micelles does not preclude the presence of individual surfactant molecules in solution. In fact, at a concentration higher than the CMC, the concentration of amphiphiles existing in a free form remains quite constant upon the addition of new surfactants, a property that can be tested through the almost constant conductance of ionic micellar solutions above the CMC (Mclntire 1990). That gives micelles a dynamic nature originating from the rapid exchange of surfactants between the micellar and nonmicellar states (Roy et al. 2016). Micelles have been frequently used to enhance the performance of various analytical techniques such as titration (Söderman, Jönsson & Olofsson, 2006; Perspicace et al., 2013; Morrow et al., 2014), solid phase microextraction (Boyacı & Pawliszyn, 2014; Haftka et al., 2016), dispersive liquid-liquid microextraction (DLLME; Roosta, Ghaedi & Daneshfar, 2014; Meeravali, Madhavi & Kumar, 2016; Campillo et al., 2017), cloud point extraction (CPE; El-Shahawi et al., 2013; Jalbani & Soylak, 2015; Khan et al., 2017), liquid chromatography (Mansour, Kirkpatrick & Danielson, 2013; Kalyankar et al., 2014; El-Shaheny, El-Maghrabey & Belal, 2015; Fasciano, Mansour & Danielson, 2016; Khodabandeh et al., 2017), micellar electrokinetic chromatography (D’Orazio et al., 2014; Franze & Engelhard, 2014; Liu et al., 2015), capillary electrophoresis (Grochocki, Markuszewski & Quirino, 2015; Xu et al., 2015; Thang, See & Quirino, 2016), and different spectroscopic techniques including ultraviolet (UV; Ulusoy 2015), colorimetry (Pourreza & Golmohammadi, 2014; Kwon et al., 2015), nuclear magnetic resonance (NMR; Dodevski et al., 2014; Nucci, Valentine & Wand, 2014; Valentine et al., 2014), fluorescence (Caruana, Camilleri Fava & Magri, 2015; Song et al., 2015), chemiluminescence (CL; Chen et al., 2014; Pan et al., 2015), atomic absorption (Ghaedi et al. 2013), and flow injection analysis (FIA). To the best of our knowledge, the role of micelles in FIA has not been comprehensively reviewed yet (Růžička, 1992; Fletcher et al., 2001; Růžička & Hansen, 2008; Pharr, 2011; Memon & Tzanavaras, 2014). The aim of this work is to discuss the mechanisms of enhancement and the different applications of micellar FIA.

Micelle-enhanced FIA with CL detection

CL is a type of light emission resulting from a chemical reaction in which chemically excited molecules decline to the electronic ground state and emit photons. CL reactions form products in excited states capable of producing visible light. This CL is usually generated from exothermic reactions such as those of redox systems. As a principle in CL reactions, at least two reagents X and Y react to form a product Z, some portion of which is present in an electronically excited state Z*, which subsequently drops down to the ground state releasing photons as follows (Baeyens et al. 1998):


The intensity of CL can be affected by other reagents in the medium. For example, fluorescent compounds can act as sensitizers by being involved in the energy transfer process. These sensitizers help to reserve energy and make the light emission longer and more quantifiable. On the contrary, if the added reagents facilitate nonradiative processes, the CL signals will decrease and the added substance will be considered as a quencher.

The measurement of light from a chemical reaction is very advantageous from an analytical point of view because, under appropriate experimental conditions, the strength of CL signals is directly proportional to the analyte concentration. This allows for accurate and sensitive quantitative analysis. In addition, CL reactions follow steady-state kinetics, which makes the sample handling and measurement procedures straightforward (Roda et al. 2000). The main advantages of CL are the excellent detection limits and the wide dynamic ranges (Baeyens et al. 1998). This sensitivity can be further enhanced using surfactants as carrier solutions in FIA. The mechanisms by which surfactants enhance CL signal are described in the next section.

Mechanism of micelle-enhanced CL

CL is the most common detection technique in FIA (Table 2). There are various mechanisms by which surfactants enhance CL signal and they include the following.

Table 2:

Selected applications of micelle-enhanced FIA.

AlbuminBiological fluidsBis[2,4,6-trichlorophenyl]oxalate-H2O2-imidazoleSDSCL1.02–12 mg/l0.38 mg/l(Gámiz-Gracia et al. 2003)
AtenololPharmaceutical tabletsKMnO4-CdS QDsCTABCL0.001–4.0 mg/l0.0010 mg/l(Khataee et al. 2016)
Au nanoparticlesEnvironmental waterSodium borohydride and luminol (in 0.1 m NaOH)Triton X-114CL1.0–50 pM1.0 pM(Tsogas, Giokas, and Vlessidis 2014)
Ag nanoparticlesEnvironmental waterSodium borohydride and luminol (in 0.1 m NaOH)Triton X-114CL2.5–80 pM1.9 pM(Tsogas, Giokas, and Vlessidis 2014)
Fe2O3Environmental waterSodium borohydride and luminol (in 0.1 m NaOH)Triton X-114CL2.0–100 nm0.8 nm(Tsogas, Giokas, and Vlessidis 2014)
L-thyroxinePharmaceutical preparationsLuminol-KMnO4CTMABCL5.0×10−8–3.0×10−6m1.0×10−6m(Cao, Wang, and Liu 2015)
AdenineLuminol-K2Cr2O7SDBSCL2.92×10−6–4.38×10−10m2.46×10−10m(Erbao and Bingchun 2006)
Cu(II)Environmental water1,10-Phenanthroline-H2O2-NaOHCEDABCL0.015 ng/l(Yamada and Suzuki 1984)
SulfadiazineTablets and ampoulesBis[2,4,6-trichlorophenyl]oxalate-H2O2-imidazole-fluorescamineSDSCL126–2000 μg/l379 μg/l(Lattanzio et al. 2008)
Epinephrine Human urineo-PhenylenediamineTriton X-114FL1×10−11–5×10−7m3×10−12m(Davletbaeva et al. 2016)
Cr(III)Seawater8-Hydroxyquinoline-Na2SO3Triton X-114FL0.5–10.0 μg/l0.2 μg/l(Paleologos et al. 2001)
Sulfonylurea herbicidesEnvironmental waterNaOH (0.01 m)SDS/CTACFL0.1–360 μg/l0.1–1 μg/l(Coly and Aaron 1999)
Al(III)Seawater Lumogallion Brij-35FL0.15 nm(Resing and Measures 1994)
Mecoprop and 2,4-dichlorophenoxyacetic acidEnvironmental waterBuffer solution (pH 3)CTACFL0.1–5.0 mg/l73.2 and 33.5 μg/l(García-Campaña, Aaron, and Bosque-Sendra 2001)
Hg(II)Commercial cosmetics1,5-Diphenylthiocarbazone SDSVis0.05–1.50 mg/l0.03(Prasertboonyai et al. 2016)
CyromazineMilkDiperiodatocuprate(III)Triton X-100Vis5×10−4–10 mg/l1.5×10–4 mg/l(Asghar, Yaqoob, and Nabi 2017)
Se(IV)Selenium tabletsResorufin-Na2SCPCVis5–1000 μg/l1 μg/l(Safavi and Mirzaee 2000)
PdEnvironmental waterDimethylglyoximeTriton X-114AAS3.0–250 μg/l1.0 μg/l(Bakircioglu 2012)
PbEnvironmental waterDimethylglyoximeTriton X-114AAS4.5–250 μg/l1.4 μg/l(Bakircioglu 2012)

Structural transformations

Surfactants can change the chemical structures of CL reagents, which in turn affect the emission spectrum. These changes can be tracked from the shifts in the wavelength of maximum emission and the CL intensity. Huang et al. (2007) found that the cationic surfactant cetyltrimethylammonium bromide (CTAB) could cause the structural transformation of fluorescein from a highly fluorescent, open quinone form to a nonfluorescent, closed spirolactone form (Figure 2). CTAB could also substantially improve the CL of the fluorescein-human serum albumin (HSA) complex (Huang et al. 2007). This observation was used to develop a rapid and sensitive FIA/CL method for the determination of HSA. Because the structural transformation of fluorescein from the quinone to the spirolactone form is highly sensitive to pH, the pH of the final solution was maintained at pH 8.6 by carbonate buffer solution. The fluorescence intensity of fluorescein decreased gradually with increasing concentrations of CTAB, and there was a slight shift in the emission wavelength upon CTAB addition. This suggests that a possible structural transformation of fluorescein from the quinone to the spirolactone form occurred in the presence of CTAB (Huang et al. 2007).

Figure 2: Structural transformation of fluorescein in micellar media from quinone form (left) to spirolactone form (right).Redrawn with permission from Huang et al. (2007).

Figure 2:

Structural transformation of fluorescein in micellar media from quinone form (left) to spirolactone form (right).

Redrawn with permission from Huang et al. (2007).

Fluorescein was also used as an energy transfer reagent for the assay of isoxicam using diperiodatoargentate(III) (Zhao and Si 2013). The luminescence of the diperiodatoargentate(III)-fluorescein-isoxicam system was significantly enhanced by the presence of CTAB. The authors claimed that the possible mechanism of CL enhancement by CTAB was the changed kinetics of energy transfer, which decreased the nonradiative internal processes and increased the quantum yield. However, the observed red shift in the CL spectra when CTAB was added (Figure 3) suggests the involvement of structural transformations, especially that they used the same surfactant and the same energy transfer reagent employed by Huang et al. (2007). Although this explanation could be acceptable in the case of fluorescein-based systems, there is not enough evidence on the structural transformations of other CL systems. In addition, these chemical changes were not investigated by analytical techniques suitable for structure elucidation such as NMR or infrared. This indicates that other mechanisms could be involved in micelle-enhanced CL systems.

Figure 3: CL spectra of diperiodatoargentate(III)-isoxicam-fluorescein in the absence (A) and presence of CTAB (B).Reprinted with permission from Zhao and Si (2013).

Figure 3:

CL spectra of diperiodatoargentate(III)-isoxicam-fluorescein in the absence (A) and presence of CTAB (B).

Reprinted with permission from Zhao and Si (2013).

Intramicellar energy transfer

Lasovský and Grambal (1986) studied the CL of the luminol-fluorescein system in the presence of cationic surfactants. The oxidation rate, the quantum yields, and the possibility of energy transfer were all enhanced from 10 to 100 times. The intensity of the emitted radiation was related to the concentrations of micellar complexes. The donors (aminophthalate anion) and the acceptors (fluorescein anion) of the energy could be located at distances approximately corresponding to the diameter of the micelle (1–3 nm). This small diameter facilitates the transfer of electron energy, which can occur up to a distance of 10 nm. The dynamic concentration of the donors and acceptors in the Stern region induced several-fold increases in the intensity of the radiation (Lasovský and Grambal 1986). As an extension of this work, Lasovskf, Rypka, and Slouka (1995) found that the micellar phase of aliphatic cationic surfactant is an ideal medium for enhanced CL. Intramicellar processes of energy transfer can be optimized by changing the surfactant concentration to maximize the yield of energy conversion.

Yamada and Suzuki (1984) studied the micellar-enhanced CL of 1,10-phenanthroline for the determination of ultratraces of Cu(II) by FIA. The copper-10-phenanthroline complex catalyzed hydrogen peroxide (H2O2) decomposition in the alkaline solution to produce superoxide radical anion (O2-). This radical oxidized 1,10-phenanthroline through formation of a dioxetane intermediate, which decomposed via an exoenergic route producing an emitter. Several surfactants were added to the 1,10-phenanthroline solution to investigate their effects on the CL reaction. The increases in CL signal were observed in the presence of cationic surfactants, whereas anionic and nonionic surfactants did not increase the CL emission. The extent of enhancement observed with cationic surfactant was related to the hydrophobic group, which explained why cetylethyldimethylamonium bromide (CEDAB) provided the highest signal. The CL enhancement was also induced in alcohol solutions and increased with the alcohol content and with the decrease in dielectric constants of alcohol. This illustrates the role of hydrophobicity in the enhancement process either with the micelles or with the alcohol. The authors postulated two explanations for this enrichment. First, 1,10-phenanthroline, which existed as a nonionic species in the alkaline solution, migrated to the Stern region on micellar surfaces because of the significantly low polarity compared to water. At the same time, the O2- migrated easily on the positively charged micellar surface. Consequently, the nonionic species could react more effectively with O2 to form a dioxetane intermediate that decomposed via an exoenergic route, producing an emitter. The higher CL enhancement was therefore attributed to the higher excitation efficiency of the dioxetane decomposition in the less polar environment of the Stern region. The second explanation is that the emitter was solubilized inside the micelle, that is, in the micelle core that was more hydrophobic than the Stern region (Figure 4). This protective environment restricted nonemissive energy transfer. These two explanations were based on the formation of micelles. However, the CL was observably increasing with the surfactant concentration even if it was below the CMC. This suggests that other mechanisms were collaborating to induce this CL enhancement.

Figure 4: CL mechanism of 1,10-phenanthroline-Cu(II) system in micellar medium.Reprinted with permission from Lin and Yamada (2003).

Figure 4:

CL mechanism of 1,10-phenanthroline-Cu(II) system in micellar medium.

Reprinted with permission from Lin and Yamada (2003).

Changing the reaction kinetics

Surfactants can affect the rate of reactions either by acting as sensitizers or by electrostatic effects. Zhang and Chen (2000) studied the CL enhancement in the presence of the three kinds of surfactants: cationic [cetylpyridinium bromide (CPB) and CTAB], anionic [sodium dodecyl benzene sulfonate (SDBS) and sodium dodecyl sulfate (SDS)], and nonionic (Triton X-100 and Tween 20). The enhancing effect of the anionic surfactants was higher than that of the cationic and nonionic surfactants. The strongest CL among the various surfactants was obtained in the presence of SDBS, and the intensity of CL increased with the increase in SDBS concentration. It is worth mentioning that the enhancement effect of SDBS was more pronounced after the CMC. To study the mechanism of SDBS-induced CL enhancement, Zhang and Chen (2000) tried using sodium benzene sulfonate instead of SDBS. The CL was improved to the same extent as SDBS when their concentrations were below the CMC of SDBS. That could be because of the fluorescent characteristics of SDBS and sodium benzene sulfonate, which can act as sensitizers in the CL reaction. The CL mechanism went through the formation of singlet oxygen and intermolecular energy transfer (Slawinska and Slawinski 1975). When the concentration of SDBS reached its CMC value (1×10−4 g/l), the CL increased more rapidly with the SDBS concentrations (Zhang and Chen 2000) because of the protective effect of micelles.

Saitoh et al. (1998) used the catalytic action of Fe(II) on luminol-H2O2 reaction in alkaline media to determine Fe(II) and total iron. The addition of tetradecyltrimethylammonium bromide (TTAB) as a cationic surfactant with citric acid to the reaction reagents improved the method sensitivity. The rate of CL reaction was affected not only by the presence of surfactant but also by the surfactant concentration. Zhao et al. (2015) found that the time required to reach the maximum CL intensity was 11 s at polyoxyetylene 23 lauryl ether (Brij-35) concentrations below the CMC and it increased to 15 s when the surfactant concentration exceeded the CMC. This modified reaction kinetics may explain the gain in CL peaks. The effect of surfactant concentration on the micelle-induced enhancement is discussed later.

Other mechanisms

Micelles can enhance CL by stabilizing the excited state via host-guest interaction or by hindering oxygen-based quenching. Dissolved oxygen molecules have paramagnetic properties, which promotes the nonradiative intersystem crossing and formation of triplet states. Micelles can form a protective environment to prevent oxygen from quenching the radiative emission. This mechanism was proposed by Huang et al. (1999) to explain the strong enhancement effect of Tween 80 on the weak CL induced by the oxidation of sulfite-containing drugs (menadione sodium bisulfite and analgin) dissolved in oxygen in the presence of acidic rhodamine 6G. The compartmentalization due to the formation of micelle prevents oxygen-based quenching, leading to CL increase. Using a similar mechanism, Li, Feng, and Lu (1998) could determine the concentration of uric acid using FIA. When a mixture of uric acid and octylphenyl polygylcol ether (OP) was injected into a flowing stream of acidic KMnO4, strong CL occurred. The characteristics of several different sensitizers, including OP, β-cyclodextrin, Tween 20, Triton X-100, and SDS, were studied. It was found that only OP could enhance CL emission intensity and that CL was hardly observable without OP. On reaching the CMC of OP, which is 1.3×10−4m, amphiphilic surfactant molecules aggregated to form micelles, which made the system significantly different from that experienced in a bulk homogeneous solvent system. Consequently, CL enhancement was accredited to the favorable alteration of solvent properties such as micropolarity, microfluidity, viscosity, and dielectric constant. The net result is that the micelle provides a protective environment for the excited singlet state, which was ideal for the observation of enhanced CL (Li, Feng, and Lu 1998).

As an application, the food additive maltol was determined by its redox reaction with KMnO4 in sulfuric acid medium at 80°C using FIA/CL. The method sensitivity was enhanced by hexadecylpyridinium chloride (HDPC) and formic acid (Sanfeliu Alonso, Lahuerta Zamora, and Martínez Calatayud 2001). Organized media (micelles) could have a marked influence on the CL emission, as HDPC in H2SO4 solution enhanced CL signal by 20% compared to surfactant free medium, by the protection of the excited state through host-guest interactions (Sanfeliu Alonso, Lahuerta Zamora, and Martínez Calatayud 2001). FIA/CL was also used for the determination of cyromazine in milk by enhancing the Cu(III) chelate-Triton X-100 emission in alkaline media (Asghar, Yaqoob, and Nabi 2017). The method was linear over a wide concentration range (5×10−4–10 μg/ml) with a limit of detection (LOD) of 0.15 ng/ml. The results were comparable to the reported high-performance liquid chromatography method, but the micelle-enhanced FIA/CL method offered a higher sample throughput (160 samples/h).

Cao, Wang, and Liu (2015) introduced a novel FIA/CL method for the analysis of L-thyroxine in the presence of cethyltrimethylammonium bromide (CTMAB). The presence of surfactant in the CL reaction system offered many advantages, such as the promotion of dissolubility, changing the dielectric constant of the solution, increasing the stability, and further potentiating the CL intensity. The effect of diverse surfactants, including CPB, dodecyltrimethylammonium bromide (DTAB), Brij-35, (SDS), cetyltrimethylammonium chloride (CTAC), and CTMAB, on CL intensity was investigated. Results showed that only CPB and CTMAB could enhance the CL intensity, and the CL intensity produced by CTMAB was greater than that of CPB. Therefore, CTMAB was selected. The previously mentioned mechanisms will more or less work together to improve the CL peaks.

Determination of the CMC using FIA/CL

Using FIA/CL, surfactants can be studied as analytes rather than vehicles. The CMC of surfactants can be determined using FIA/CL. The CL intensity of the luminol-H2O2 system was gradually increasing with surfactant concentration to reach a maximum at the CMC (Zhao et al. 2015). Several surfactants were determined using this method including that of Brij-35. The CL peak signal was obtained at the concentration value of 1.2×10–4m, which was in good agreement with the CMC values of Brij-35 (Figure 5). The CL emission spectra of luminol-H2O2 reaction in the presence of the surfactants showed a maximum at 425 nm. The results were consistent with a previous report (Jurgensen and Winefordner 1984) for the oxidation of luminol, indicating that the surfactant did not change the nature of the luminescent substance (3-aminophthalate ion). The FIA/CL method was found to be simpler and faster than the traditional methods of determining the CMC such as electrical conductivity, surface tension, and dye solubilization (Zhao et al. 2015).

Figure 5: CL intensity of the luminol-H2O2-surfactant system as a function of concentration.Reprinted with permission from Zhao et al. (2015).

Figure 5:

CL intensity of the luminol-H2O2-surfactant system as a function of concentration.

Reprinted with permission from Zhao et al. (2015).

Reverse micelles in FIA/CL measurements

As defined previously, normal micelles are formed of lipophilic core and hydrophobic heads. If the micelle-forming surfactants were dissolved in an organic-rich media, this arrangement will be reversed to decrease the contact surface between the hydrophilic groups and the organic solvent. The core of reversed micelles is full of water enclosed with polar head groups. This aqueous center can act as a microreactor for chemical reactions. The incorporation of reverse micelles into the detection system provides many advantages, including sensitivity and improved selectivity in the reversed micellar system. The significance of reverse micelles in CL analysis emerges from their unique structure (size/shape) and composition. In addition to the improved sensitivity and selectivity, these microreactors can transfer species of experimental interest quantitatively into water (Figure 6).

Figure 6: Snapshot cross-section from a molecular dynamic trajectory of a w = 2 reverse micelle.Atoms are as follows: oxygen (red), hydrogen (white), sodium (blue), sulfur (yellow), and carbon (green).Reprinted with permission from Pieniazek et al. (2009).

Figure 6:

Snapshot cross-section from a molecular dynamic trajectory of a w = 2 reverse micelle.

Atoms are as follows: oxygen (red), hydrogen (white), sodium (blue), sulfur (yellow), and carbon (green).

Reprinted with permission from Pieniazek et al. (2009).

Reverse micelles have been applied to determine both organic and inorganic analytes by FIA. A method for the FIA/CL determination of iodine was reported (Fujiwara et al. 2006). The CL emission was based on the reaction of iodine and luminol in the reversed micellar medium of the surfactant (CTAC), which is well characterized among other quaternary ammonium salts for several reaction systems in reversed micellar media. A mixture of 1-hexanol-cyclohexane was used as a bulk solvent (Fujiwara et al. 2006).

Another FIA/CL method was proposed for the determination of quinine (Tang, Huang, and Shi 2005). The reaction was based on the dichloromethane solvent extraction of the ion pair complex of tetrachlorourate(III) with protonated quinine sulfate and luminol. CL emission detection was carried out in CTMAB reversed micellar medium. The ion pair complex of tetrachlorourate(III) with quinine sulfate emitted CL signal as it entered the reversed micelle pool (Tang, Huang, and Shi 2005).

Similarly, the chemical principles of ion-pair formation and liquid-liquid extraction were applied for the FIA/CL determination of p-toluidine and 2-methyl-5-nitroaniline in reversed micellar media (Mohammadzai, Ashiuchi, and Tsukahara 2005). Another study showed that the CL emission from the oxidation reaction of rhodamine B with Ce(IV) was found to be stronger in the CTAC reversed micellar solution compared to an aqueous solution (Hasanin et al. 2011). An ion-pair formation and liquid-liquid extraction method coupled with CL detection based on the reversed micelle-mediated oxidation of rhodamine B with Ce(IV) was developed for the determination of Sb(V), where rhodamine B was used not only as an extracting agent but also as a CL reagent. The modification of this technique allowed for the analyses of Sb(V) only, Sb(III) + Sb(V), or Sb(III) and Sb(V) in the same sample using Ce(IV) oxidant cycling (Yamamoto et al. 2013).

Yet, using reversed micelles in CL measurements is not as common as normal micelles due to the recent trends to use green analytical chemistry in various biomedical applications (Mansour & Danielson, 2017; Mansour & Khairy, 2017).

Effect of surfactant concentration

The enhancement in CL induced by surfactant is highly dependent on the surfactant concentration. Upon studying the effect of increasing the surfactant concentration on the CL intensity, three different patterns were obtained. The first was the gradual increases in CL with concentrations up to the CMC, after that the CL started to decline due to self-quenching or the increased viscosity, which affected the diffusion of the reactants. This effect was observed when TTAB was added to luminol-H2O2 systems as shown in Figure 7A (Saitoh et al. 1998). On the contrary, the CL could keep increasing even after the CMC was reached as shown in Figure 7B. This behavior is usually observed if the CL was affected not only by the micelle formation but also by the surfactant itself. SDBS, for example, enhanced the CL of IO4-H2O2 system not only by the protective effect of micelles but also by photosensitization (Zhang and Chen 2000). In this case, a sharp increase in CL intensity is observed after the CMC due to the dual mechanism. The CL intensity can be kept constant after reaching the CMC of the surfactant (Figure 7C). This behavior is common with nonionic surfactants due to the lack of electrostatic effects. Regardless of the type of surfactant, the concentration of surfactant has to be considered as one of the optimization parameters in FIA/CL.

Figure 7: Effect of surfactant conentration on micelle-induced CL enhancement.Redrawn with permission from Saitoh et al. (1998), Li, Feng, and Lu (1998), and Sanfeliu Alonso, Lahuerta Zamora, and Martínez Calatayud (2001).

Figure 7:

Effect of surfactant conentration on micelle-induced CL enhancement.

Redrawn with permission from Saitoh et al. (1998), Li, Feng, and Lu (1998), and Sanfeliu Alonso, Lahuerta Zamora, and Martínez Calatayud (2001).

Micelle-enhanced FIA with UV-visible (UV-vis) detection

UV-vis spectrophotometry is one of the most common detection methods in flow-based techniques (Mansour & Danielson, 2012; Mansour, Shafi & Danielson, 2012). Micelles that are formed by the aggregation of charged organic molecules can improve the sensitivity of UV-vis detection. If micelles have the same charge at the outer sphere as the reactants, the rate of reactions of charged species undergoes enhancement by the presence of micelles. This enhancement is caused by increasing the effective collisions of reacting molecules (Keyvanfard and Abedi 2011). Besides electrostatic repulsion, other enhancement mechanisms have been proposed such as ternary complexes and the solubilization effect. Surfactants were used to improve UV-vis spectrophotometric determinations of metal ions with metallochromic indicators (dyes; Pelizzetti and Pramauro 1986). Ternary complexes formed between the reactants and the surfactant molecules could show bathochromic shifts and hyperchromic effects compared to binary complexes. Moreover, the solubilizing power of micelles improved the molar absorptivity of metal complexes that are not reasonably soluble in water. One- to 10-fold increase in the absorbance of metal complexes in the presence of cationic surfactants improved the sensitivity of UV-vis detection (Pelizzetti and Pramauro 1986).

As an application, triphenylmethane dyes (TPM) could be used in the presence of cationic surfactants because of the oxygen donor atoms in TPM, which could form five-membered chelate rings with the metal ions. This complex formation implied a delocalization of the π-electrons in the dye molecule, with a corresponding red-shifted absorption maximum. When the surfactant concentration was lower than the CMC, only slightly soluble ion-associates were formed between anionic chelates and surfactant monomers, and turbidity was observed. At a concentration slightly higher than the CMC, solubilization of these aggregates occurred and the sensitization was maximal, whereas a further decrease of surfactant concentration decreased the absorbance and a hypsochromic shift was obtained. This observation could be ascribed to a decrease in the chelating affinity of the negatively charged ligand, which interacted strongly with the cationic micelles (Pelizzetti and Pramauro 1986).

As an application, an FIA/UV method for the determination of vanadium in micellar media was presented (Keyvanfard and Abedi 2011). Experimental results showed that the nonionic surfactant Triton X-100 was more effective than other surfactants when tested at concentrations higher than the CMC (Keyvanfard and Abedi 2011). Similarly, cetylpyridinium chloride (CPC) and DTAB were studied to determine their effect on the enhancement of the reaction rate of Ce(IV)-As(III) (Rubio and Pérez-Bendito 1989). The performance of DTAB was superior compared to that of CPC with respect to the rate of enhancement, which was explained by the structural features of both surfactants; the positive charges on the amino groups of the two surfactants were looser and less dense in CPC than in DTAB. That made the electrostatic interactions stronger with DTAB. Also, the dissolution power of DTAB was higher than that of CPC. The stronger the interaction and the solubilizing ability of the cationic surfactant, the greater the enhancements (Rubio and Pérez-Bendito 1989). However, small surfactant concentrations were enough to exert the solubilization effect, otherwise absorbance could decline due to the micellar dilution effect or the increased viscosity of the system (Memon et al. 2012).

Capella-Peiró et al. (2001) could develop an FIA/visible (FIA/Vis) method for the assay of nicotinic acid in N-CPC (NCPC) surfactants through the formation of a polymethine dye at ambient temperature. The absorbance of NCPC increased the sensitivity by a factor of 3.3 at 440 nm. This improvement was explained by the increased concentrations of the glutaconic aldehyde (the transformation product of nicotinic acid) and the arylamines on the micelle surface due to the association to the NCPC micelles by electrostatic and/or hydrophobic forces (Capella-Peiró et al. 2001). The sensitivity of Hg(II) determination by dithizone was improved in the presence of a mixture of SDS, ascorbic, and sulfuric acids (Prasertboonyai et al. 2016). The orange-colored complex was monitored at 490 nm by FIA/Vis detection. The method was successfully applied for the measurement of Hg(II) in cosmetic products and traditional medicines without prior extraction.

Determination of surfactants using FIA/spectrophotometry

Because UV-vis absorbance is dependent on the surfactant concentration, FIA/spectrophotometry can be used to determine the surfactant concentrations. An FIA equipped with UV-vis spectrophotometer was introduced (Patel and Singh Patel 1999) to measure cationic surfactants (CS), including DTAB, TTAB, CTAB, and CPC. The assay principle was based on the increased intensity of the Fe(III)-SCN signal in the presence of cationic surfactants due to the formation of higher thiocyanate species with enhanced kinetics.


where the value of n may vary from 2 to 6. Fe(III)-SCN-CS+ complexes exhibited a sharp absorption maximum around 475 nm. The position of λmax did not change when different CS was used but absorptivity was affected. This hyperchromic effect in the presence of CS was due to the formation of a higher thiocyanate complex. Various types of surfactants were found to remarkably affect the color intensity of the complex. The highest sensitivity was recorded with the cationic surfactant CPC due to the higher basic character and the lower steric hindrance (Patel and Singh Patel 1999).

Micelle-enhanced fluorescence in FIA

The fluorescence intensity of a molecule can be considerably enhanced when bound to a micellar system (Hinze et al. 1984). The increased fluorescence is due to (i) the protection of the fluorescent molecule from vibrational quenching by the hydrogen-bonded structure of water and (ii) the relatively high viscosity of the micellar media, which can inhibit quenching by molecular oxygen. This high viscosity can decrease the diffusion rates and reduce the accessibility of solvent-borne quenchers to get in close proximity with the fluorescent molecules. The measurement of fluorescence in micellar solutions has various advantages: lower detection limits can be attained using surfactant at concentrations equal to or higher than the CMC. In addition, using micelles eradicates the need for oxygen removal (Hinze et al., 1984; Pelizzetti & Pramauro, 1986) because of the diminished quenching effects of oxygen by micelle formation (Hinze et al., 1984; Al-Kindy, Suliman & Salama, 2003). The only requirement for micelle-enhanced FIA/fluorescence is that a surfactant concentration must be above the CMC to ensure micellization (Hinze et al. 1984), as micelles act as light-harvesting units focusing the exciting radiation on the luminescent molecule. The decrease in fluorescence after reaching the CMC is due to the self-quenching effects.

The fluorescence properties of α1-blockers in various surfactant media were studied (Mohamed, Ahmed, and El Zohny 2013) using different surfactant types, including SDS, which produced maximum enhancement of the fluorescence intensities for all the studied α1-blockers. Tween 20 was used to enhance the fluorescence of aluminum-morin complex and the emission was exploited to measure the concentration of aluminum (Al-Kindy, Suliman, and Salama 2003). Less enhancement was observed with Tween 80, which is more hydrophobic due to the favorable competition for morin, compared to aluminum. Similar results were obtained with other hydrophobic surfactants, such as CTAB or Triton X-100, which led to the quenching of the fluorescence. This quenching could be due to the decomposition of the complex by the favorable dissolution of morin in the hydrophobic core of the micelle. These results should encourage researchers to use micellar systems as carrier solutions in FIA/fluorescence applications.

Micelle-enhanced atomic absorption in FIA

The role of surfactants in atomic absorption is quite different from its role in the previous techniques. For flame atomic absorption spectrometry (FAAS), the flame acts as the sample cell. In order for atoms in a solution to be introduced in the flame, they must go through several processes. The sample is first aspirated into the pneumatic nebulizer where it breaks the liquid into small drops. This is often followed by one or more aerosol modifiers that break the drops into finer droplets and remove the larger ones so that only droplets of a specific size range enter the flame. Once in the flame, the solvent evaporates, the analyte vaporizes and atomization step proceeds (Pharr 1994).

Surfactants can enhance signals in FAAS. There have been several models proposed to explain the enhancement of absorbance signals in FAAS with surfactants. The surfactant’s ability to reduce surface tension at the CMC was first used to explain the enhancement. Surfactants could lower the surface tension, which resulted in the generation of smaller droplets during the aspiration and nebulization process. This increase in smaller droplet count resulted in a higher efficiency of the laminar flow burner so that a larger amount of the analyte would go into the flame. In FAAS, only 2%–3% of the analyte reached the burner flame. If atomic absorption was plotted against surfactant concentration, the signal intensity would increase at the CMC and then levels off. Similar trend was observed with different metals such as Co, Fe, Mn, Ni, Sn, Ti, V, and Zn (Pharr 1994). The theory also suggested that the observed absorbance would be independent on the type of surfactant used (cationic, anionic, or nonionic) or the counterion (Pharr 1994).

Another explanation was based on the aerosol ionic redistribution (AIR) theory (Kornahrens, Cook, and Armstrong 1982). The AIR theory suggested that surfactants in an FAAS sample can promote the enrichment of the analyte in the double layer at the air-water interface on drop surface. As these drops subdivide in the nebulization process, smaller droplets form from the stripping of the drop surface, whereas larger droplets arise from the analyte-depleted bulk drop center. Because smaller droplets are more efficiently sampled by the burner, a net enrichment results and enhancement of the analytical signal is observed. Surfactants therefore are expected to enhance analyte surface concentrations significantly, provided that analyte and surfactant head groups are oppositely charged. This hypothesis explains the decreasing absorbance beyond the CMC by the depletion of the analyte at the surface because of the binding to micelles in bulk. Armstrong (1985) used this theory to explain the enhancement of SDS on the absorbance of copper. The mechanism proposed allowed analyte transport to the hottest part of the flame by the interaction of negatively charged surfactant head group with the positively charged analyte (Armstrong 1985). The enrichment occurs at the double layer on the outside surface of the large drops. Micellar flow injection equipped with an AAS was used for the determination of iron and copper in food samples (Durukan et al. 2011), speciation analysis of chromium in water samples (Ezoddin, Shemirani, and Khani 2010), determination of free and bound iron in wines (Paleologos et al. 2002), measurement of Cd, Ni, and Zn in hemodialysis solutions and tuna fish samples (Galbeiro, Garcia, and Gaubeur 2014), analysis of Ni(II) in food and water samples (Rahnama and Najafi 2016), determination of Au(III) and Ga(III) in industrial water (Hasanin, Okamot, and Fujiwara 2016), and determination of Pb and Cd (Silva and Roldan Pdos 2009). Using surfactant as carrier solution in these applications improved the sensitivity. More attention should be directed to micelle-enhanced FIA-AAS in the future.

Micelle-enhanced preconcentration: CPE

Flow injection/CPE (FI/CPE) is a promising separation and extraction technique that is based on the use of surfactants as alternatives to organic solvents (Durukan et al., 2011; Melchert & Rocha, 2016). The ability of micelles to solubilize normally water-insoluble metal chelates and organic analytes has been discussed earlier regarding spectrophotometric and fluorometric methods of detection. Such in situ solubilization offers several advantages over conventional mixed solvent schemes, including experimental convenience, cost, ease of waste disposal, and, in some cases, enhanced spectroscopic signals. The analytical utility of these systems can be further increased when they are used to extract the insoluble complex or substrate from the bulk aqueous medium into a much smaller volume phase consisting almost entirely of surfactants. The resulting concentration of the molecular system of interest provides increased sensitivity of analysis. These separations depend on a particular physical property of the surfactant. For example, nonionic surfactants go through a critical point upon increasing the solution temperature known as the cloud point. Above this temperature, the nonionic micelle phase separates from the bulk aqueous medium into two phases, surfactant-rich phase (SRP) of small volume and a diluted aqueous phase (AQP), taking the already solubilized analyte into the SRP phase. Assuming a 100:1 ratio of water volume to surfactant volume, this results in a 100-fold increase in analyte concentration with the obvious benefit of increased sensitivity. Ionic surfactants can also phase separate, although the effect is normally generated with solution additives such as high levels of salts (Kumar et al. 2002).

The hydrophobic compounds initially present in the solution are bound to the micelles and extracted to the SRP. A successful CPE should maximize the extraction efficiency by minimizing the phase volume ratio (Vorg/Vaqueous), thus improving its concentrating ability. The CPE has been applied to extract and preconcentrate a wide range of metal ions from aqueous solution using nonionic surfactants. Using the FI/CPE method, a series of bulky steps in traditional CPE including incubation, centrifugation, and separation of the SRP from AQP and dilution of SRP could be omitted. All these steps can cause poor reproducibility, low preconcentration factor, and time-consuming procedures. The combination of online systems with CPE puts together the advantages of both systems: high velocity and analytical reproducibility, reduced sample and reagent consumption, and minimized risk of analyte loses and matrix contamination (Paleologos et al. 2001; 2002; Silva & Roldan Pdos, 2009; Ezoddin, Shemirani & Khani, 2010; Durukan et al., 2011). Davletbaeva et al. (2016) developed an automated method for the estimation of epinephrine in human urine using stepwise injection coupled to CPE and fluorescence detection; o-phenylenediamine was used as a fluorogenic agent and Triton X-114 was used for CPE. The fluorescent derivative was mixed with Triton X-114 in a ratio 6:1 (v/v); then, salting out was induced by the addition of 1% Na2CO3 to induce phase separation. The method was highly sensitive with an LOD as low as 3 pM.

Future trends

The incorporation of quantum dots in the micellar structure has been recently introduced to stabilize the micelle and to modulate the microenvironment (Fang et al., 2012; Lu et al., 2017). Dong, Zhong, and Lu (2014) used CTAB confined on the surface of carbon dots in a bilayer form to amplify the ultraweak CL responses of H2O2 in peroxymonocarbonate, peroxynitrous acid, and Fenton-like reactions; this confinement made the central carbon core more accessible for reactive intermediates.

An alternative approach was introduced by Shi et al. (2013) using polymer-micelle aggregates. In these aggregates, the polymer strand connected the spherical micelles in a “necklace model” as shown in Figure 8 (Shi et al. 2013). The polyethylene glycol (PEG) 1500 (Mansour, Zhou, and Danielson 2015) was passivated with fluorescent carbon dots before mixing with the surfactant (CTAB). The transition metal-induced enhancement in the fluorescence intensity was employed to measure the concentrations of Co(II) by FIA in the range from 1.0 to 1000 nm (Shi et al. 2013).

Figure 8: Schematic illustration for the concentration of hydroperoxyl anions in CTAB-PEG microenvironment.Reprinted with permission from Shi et al. (2013).

Figure 8:

Schematic illustration for the concentration of hydroperoxyl anions in CTAB-PEG microenvironment.

Reprinted with permission from Shi et al. (2013).

Micelles could also be employed to form supramolecular solvent for DLLME before FIA (Rastegar et al. 2016). In DLLME, an extracting solvent is mixed with the sample in the presence of a disperser to increase the contact surface between the analyte and the extractant. In supramolecular-assisted DLLME (SM-DLLME), the extractant and the disperser are sonicated with the sample to form reverse nanomicelles in which the analyte was extracted. SM-DLLME was successfully used for the determination of Pb ion in food samples by FIA using dithizone as a chelating agent, 1-decanol as an extractant, and tetrahydrofuran as a disperser (Rastegar et al. 2016).


The employment of micelles in FIA has been reviewed. Choosing the right surfactant is crucial to ensure enhanced performance in micellar FIA. In most applications, the enhancement was observed with a certain type of surfactants, which highlights the role that the charge could play in the micelle effect. The concentration of surfactant has to be optimized to achieve the maximum enhancement. The mechanism through which micelles affect the detection method in FIA has been discussed. Limited effort was exerted to confirm these pathways. More work is expected in the future to provide experimental evidence on the proposed mechanisms, to relate the enhancement to the chemical structures of the analyte as well as the surfactant, and to explore other possible pathways.


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Received: 2017-04-23
Accepted: 2018-03-01
Published Online: 2018-04-10

©2018 Walter de Gruyter GmbH, Berlin/Boston

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