Micellar Liquid Chromatography from Green Analysis Perspective

Abstract Micellar liquid chromatography (MLC) is a simple well-established branch of high-performance liquid chromatography. The applications of MLC for the determination of numerous compounds in pharmaceutical formulations, biological samples, food, and environmental samples have been growing very rapidly. MLC technique has several advantages over other techniques, such as simultaneous separation of charged and uncharged solutes, rapid gradient capability, direct on-column injection of physiological fluids, unique separation selectivity, high reproducibility, robustness, enhanced luminescence detection, low cost, and safety. This review is devoted to the evaluation of the agreement of MLC with the principles of green chemistry which recently represents a universal trend. Also, it provides an overview on the basics of MLC, in addition to a survey of MLC methods published in the past five years for the assay of various compounds in different matrices. Graphical Abstract


Introduction
Micellar liquid chromatography (MLC) is one of the modes of reversed-phase liquid chromatography (RPLC) in which the mobile phases are aqueous solutions of a surfactant at a concentration above the critical micelle concentration (CMC). Over the past fifteen years, the popularity of MLC has grown rapidly. Micelles have also been used in many other separation techniques; such as electrokinetic chromatography [1,2], ultrafiltration, and cloud point extraction [3].
Although some publications have reported on the basics and applications of MLC for the analysis of drugs in pharmaceutical preparations and biological samples [4][5][6][7][8][9][10][11], the focus of this review is to highlight the importance of MLC for green analytical chemistry. Green chemistry has evolved from the pollution prevention approach developed within the USA's Environmental Protection Agency as a conceptual framework that minimizes the undesirable effects of chemistry. Many efforts have been made in the field of analytical chemistry to avoid the hazards of the analytical methodologies and to reduce the costs of analysis [12]. In this review article, we present a brief overview of the basics of MLC as well as a comprehensive discussion about its importance from green analytical chemistry perspective. In addition, details about the analytical applications of MLC in the past five years will be presented. It is our hope that this review will provide many fertile ideas to the readers.

Basics of micellar liquid chromatography 2.1 Critical micelle concentration and Krafft point
Surfactants are amphiphilic molecules that consist of a hydrophobic moiety and a polar head group. Above their CMC, surfactants form aggregates that are known as micelles. Micelles have a dynamic structure that is the result of the rapid exchange of surfactants in the aggregated and monomeric forms. The number of monomer surfactants in the aggregate form (called aggregation number) and the size of micelles vary greatly between surfactants. The CMC and aggregation number are the result of many factors, such as ionic strength, presence of a co-solvent and temperature. A suitable surfactant for MLC should have a low CMC. A high CMC would result in working at a high surfactant concentration, which would create viscous solutions, giving undesirable high system pressure and background noise in UV detectors [5]. The selection is often limited to the following surfactants: the anionic sodium dodecyl sulphate (SDS), the cationic cetyltrimethylammonium bromide (CTAB), and the nonionic polyoxyethylene 23 lauryl ether (Brij-35) whose main characteristics are summarized in Table 1.
Another property of ionic surfactants is the Krafft point, which is defined as the temperature at which the solubility of surfactant is equal to its CMC. Below the Krafft point temperature, the solubility of surfactant is quite low and the solution appears to contain no micelles. Chromatographic work in MLC should be conducted above this temperature to avoid surfactant precipitation. This means that the Krafft point should be well below room temperature [5].
Non-ionic surfactants also have a specific temperature, called the cloud point, above which phase separation occurs [5]. Chromatographic work with these surfactants should be conducted below this temperature (e.g. for aqueous 1-6% solutions of Brij-35, it is 100°C, whereas for Triton X-100 this value is 64°C).

Principles of separation by MLC
MLC shares the basic components of RPLC systems: a nonpolar stationary phase and a polar aqueous mobile phase. However, hydro-organic mobile phases in conventional RPLC are homogeneous, whereas micellar solutions are heterogeneous, being consisted of two distinctive media: the amphiphilic micellar aggregates (micellar pseudophase) and the surrounding bulk water or aqueousorganic solvent that contains surfactant monomers in a concentration nearly equal to the CMC. On the other hand, the stationary phase is modified by the adsorption of surfactant monomers, creating a structure similar to an open micelle, and reducing silanophilic interactions. In micellar solutions, the concentration of monomer surfactants is essentially constant and equals the CMC. Thus, the composition of the stationary phase will not change with differences in the micelle concentration in the mobile phase. This is a different behavior than that found in RPLC, where the composition and conformation of the alkyl-bonded phase depend on the composition of the hydro-organic eluents [5,7].
Retention behavior in MLC is controlled by solute partitioning from the bulk solvent into micelles and into stationary phase as well as on direct transfer from the micelles in the mobile phase into the stationary phase. While retention of more polar compounds is determined by their partitioning from the bulk aqueous phase into micelle and alkyl stationary phase, the more hydrophobic compounds might be directly transferred from micelles in the mobile phase into the stationary phase [13,14].
The main drawback of MLC is the decreased column efficiency due to slow mass transfer from the stationary phase. Slow stationary phase mass transfer can be attributed to the poor "wetting" of the stationary phase with a purely aqueous mobile phase as well as to the adsorption of monomer surfactants that change the characteristics of the alkyl-bonded stationary phases [15]. To enhance the efficiency in MLC three main approaches have been adopted: addition of small concentrations of organic modifiers to the micellar mobile phase, increasing the column temperature, and decreasing the flow rate. For this reason, most procedures for the determination of compounds by MLC make use of micellar mobile phases containing an organic modifier, which is usually a shortchain alcohol (methanol, propanol, butanol or pentanol) or acetonitrile, so-called "hybrid micellar mobile phase". These modifiers increase the elution strength and often improve the shape of the chromatographic peaks. The modifiers act by solvation of the bonded stationary phase and decreasing the amount of surfactant adsorbed, such effect increases as the concentration and the hydrophobicity of the alcohol increases [15,16]. Meanwhile, the addition of triethylamine to a micellar mobile phase in combination with organic modifier enhances the efficiency over organic modifier added alone. This observation provides further evidence that efficiency and surfactant adsorption are linked by the effect of the latter on diffusion in the interfacial region between the mobile phase and stationary phase. Moreover, higher temperatures increase the kinetics of mass transfer. In general, operating under these conditions would enhance the column efficiency such that it becomes comparable with conventional RPLC. An interesting example is the case of basic compounds, which produce symmetrical peaks with high efficiencies in MLC, with columns that yield highly tailed peaks in RPLC without additives [16]. On considering the essential aspects of the analytical work, the analytical parameters emerge as the key factors to be considered. Accuracy, traceability, sensitivity, selectivity, and precision are the essential and basic goals which must be assured in order to provide to the industries, consumers, and strategy makers the appropriate tools to do their determinations. In the frame of analytical chemistry, MLC is an analytical technique that accomplishes the main analytical goals, in addition to the green parameters of the method as discussed below.

Green evaluation of MLC
In terms of ecological aspects, RPLC techniques are characterized by a large consumption of organic solvents. Developing a greener process in chromatography is a challenge. MLC constitutes a good alternative to RPLC which improves both economic and ecological aspects. MLC is greener than RPLC in all steps of the analysis from sample collection and preparation to separation and final determination. A discussion of the different points that contribute to the greenness of MLC is presented here:

Safety of reagents
Most MLC methods use hybrid mobile phases consisted of aqueous solutions of a surfactant above its CMC and a small portion of organic modifier (mostly 3-15%, v/v). As we stated earlier, SDS is the most commonly used surfactant in MLC, but CTAB and Brij-35 are also used. Micellar mobile phases are safer for both the operator and environment. Considering safety of the used surfactants, SDS is not carcinogenic when either applied directly to the skin or consumed [17]. A review of the scientific literature revealed that SDS was negative in an Ames (bacterial mutation) test, a gene mutation and sister chromatid exchange test in mammalian cells, and in an in-vivo micronucleus assay in mice. The negative results from in-vitro and in-vivo studies indicate that SDS does not interact with DNA. SDS has LD 50 of 0.8-1.1 g kg -1 in rats [17]. Based on the available data [18], CTAB is also considered safe. It is poorly absorbed from the intestine and is excreted in feces. It is absorbed into the skin, but not rapidly. Dermal exposure to 2% CTAB produced no evidence of teratogenicity. In addition, all mutagenesis tests and sensitization reactions were negative [18]. Fatty alcohol ethoxylate surfactants (e.g. Brij-35) were not found to cause genetic or reproductive damages. Also, no carcinogenic effects were noted in chronic studies either after oral or dermal exposure. Further, the fatty alcohol ethoxylates do not irritate the skin or eyes [19].
In comparison with the RPLC methods that employ aqueous-organic mobile phases, the micellar mobile phases have the advantage of using small amounts of organic modifier. Furthermore, propanol, butanol, and pentanol, the most common organic modifiers used in MLC, are retained in the micellar solution, thus reducing the risk of evaporation and making the micellar mobile phases more stable. In addition, they are also less toxic than methanol or acetonitrile, which are commonly used in conventional RPLC. The low content of organic solvents in the micellar mobile phases provides also the advantage of non-flammability and safety for laboratory work [9].

Waste generation
Since the possible contamination of the environment with wastes arising from analytical chemistry practice is an essential aspect for green chemistry, waste generation from MLC and its impacts should be discussed. Another important advantage of micellar mobile phases is the biodegradable character of surfactants used. SDS is a fatty alcohol sulfate that is aerobically degraded. Thomas and White [20] observed that 70% of 14 C SDS was degraded to CO 2 and the remaining 30% was incorporated into the microbial biomass, i.e., 100% of the SDS was utilized for either energy or biomass production. On the other hand, Brij-35 is one of the fatty alcohol ethoxylate derivatives, developed as an eco-friendly alternative to alkyl phenol ethoxylates [21]. A large number of reports have dealt with the biodegradability of these compounds. Linear fatty alcohol ethoxylate (e.g. Brij-35) are considered readily biodegradable. Kravetz et al. [22] observed 80% primary degradation in 28 days for such compounds. Meanwhile, CTAB belongs to the quaternary ammonium compounds which are also biodegradable through different pathways. One of these pathways is N-dealkylation, which involves monooxygenase activity with the production of trimethylamine and an alkyl residue [23,24]. Thus, waste generated from MLC could be considered a clean waste.
It is also worth noting that, mobile phase recycling is possible in case of MLC because of the low evaporation risk of organic solvents in hybrid micellar eluents. So, the micellar mobile phase can be recycled during the analysis, as long as a small number of injections are made.
On the other hand, due to the toxicity of methanol and acetonitrile, the most frequently used solvents in RPLC, safe disposal of the waste solvent is essential. Combustion in a hazardous waste plant, if available, is recommended. Otherwise, the waste can be degraded by chemical decomposition in laboratory through repetitive steps. Acetonitrile-water waste can be degraded to acetic acid and ammonia by treatment with excess sodium hydroxide. The waste must be diluted in water to 10% acetonitrile in order to prevent the formation of two-phase system upon addition of concentrated sodium hydroxide [25].

Determination of drugs in pharmaceuticals
MLC offers important benefits compared to conventional RPLC concerning sample treatment. For example, it allows a drug solution to be injected into the chromatographic system without any treatment other than filtration, reducing the sample preparation time. Drugs are easily extracted when the samples are treated with micellar solutions, since the excipients are usually not soluble in the micelles. The presence of a small amount of alcohol into the micellar media can improve the solubility of the drugs [6]. The solubilizing ability of micelles is one of their most important properties that allows the analysis of complex matrices without the need for extraction, while providing direct injection of untreated samples. The sample preparation is very simple and varies according to the kind of pharmaceutical formulation, whether solid (tablets, capsules, pills, and powders), liquid (drops, solutions, suspensions, sprays, oily injection, and syrups), ointment, cream, gel, or suppository. For solid dosage forms, a suitable number of units are weighed, (carefully emptied in case of capsules) and pulverized. Then, suitable amounts of powdered dosage forms are weighed and dissolved in the micellar mobile phase. For liquids, pretreatment is simpler and includes mixing with a small amount of alcohol and dilution with the micellar mobile phase or dilution directly with the micellar mobile phase. For more complex dosage forms such as ointment, cream, or gel, a suitable amount is weighed and mixed with the micellar mobile phase with the aid of sonication, or mechanical stirring [6]. For suppositories, one unit is dissolved in n-propanol, butanol, or pentanol (according to the mobile phase composition) with the aid of sonication and gentle heating, and then suitable volumes are diluted with the mobile phase then chromatographed [26].
Following this sample preparation and the selection of a suitable micellar mobile phase, the recoveries usually agreed well with the contents declared by the manufacturers within the tolerance limits. Another advantage of MLC is the sample preparation time, which is shorter than that required in conventional RPLC procedures where long, tedious extraction steps are often needed. Hence, it decreases error sources due to the minimized risks of losses and chemical changes in the analyte because of the reduced number of steps. Thus, MLC offers the advantages of reduced cost and time of the analyses and increased sample throughputs.

Determination of drugs in biological fluids
A major drawback of conventional RPLC methods for the routine analysis of protein-based biological samples is the need for repetitive sample preparation steps, prior to injection, to remove proteinaceous materials. This is essential to prevent irreversible adsorption to the packing and column plugging by the background proteins. Protein precipitation is tedious and time-consuming, and can cause sample dilution or loss of material.
A fascinating feature of certain types of micelles, such as SDS and Brij-35, is their ability to solubilize proteins. This capability has been effectively exploited for the direct injection of untreated biological fluids onto RPLC columns. The micelles tend to bind proteins competitively by releasing protein-bound drugs, so the substances are free to partition into the stationary phase, whereas the proteins, rather than precipitating into the column, are solubilized and eluted with or shortly after the solvent front. Dilution of the biological samples with the micellar mobile phase and filtration of samples prior to injection helps to decrease the width of protein band appearing at the beginning of the chromatograms, thus preventing interferences with drug determination. Dilution of samples also helps to extend the life time of the column [27].
Possibility of direct injection of physiological samples with MLC precludes elaborate multiple extraction steps through traditional liquid-liquid extraction procedures, hence decreasing the time and costs of the analysis and avoiding the consumption of large quantities of flammable, toxic organic solvents. Direct injection with MLC is also more advantageous than solid-phase extraction technique which requires more time, multiple steps, and special cartridges. Also, it is less complex than column-switching procedures, which require additional instrumentation (precolumns, switching valves, and additional pumps) and accurate timing of valve switching for a successful separation. Nevertheless, it is worth noting that cationic surfactants cause proteins to precipitate and cannot be usually used with physiological samples [27].

Compatibility with existing RPLC instruments
Another issue to be considered is the compatibility of MLC with existing RPLC instruments, since this matching would reduce the cost of MLC if there is no need for special instrumentations or resetting. Micellar mobile phases are compatible with RPLC stationary phases (C 18 , C 8 , cyanopropyl, phenyl, and monolith columns). In addition, gradient elution is also possible by increasing the concentration of micelles and/or organic modifier during the course of the separation [28,29]. However, the use of MLC would allow the simultaneous determination of hydrophobic and hydrophilic solutes in the same run with no need for gradient elution programs. MLC is also well-matched with several RPLC detection modes such as ultraviolet, fluorescence, phosphorescence, chemiluminescence, electrochemical, diode array detection (DAD), and inductively-coupled plasma mass spectrometry. Interestingly, micellar mobile phases could sometimes lead to improvements in the detection capabilities. The fluorescence intensity of certain compounds in micellar media can be dramatically increased due to micellar solubilization [5,[30][31][32]. Solutes that are localized in the anisotropic media of micelles experience a microenvironment with different polarity and higher viscosity than those of the bulk aqueous solvent. As a result, their freedom of movement is limited in the micelles and results in the shielding of compounds from non-radiation deactivation and/or an increase in quantum efficiency. Consequently, fluorescence signals are often intensified in the presence of micelles. Even room temperature liquid phosphorescence has been observed in ionic micellar solution with heavy atom counter-ions, which is attributed to the micelle stabilization effect of the triplet state of some molecules [5,32]. Moreover, the hydro-organic mobile phases used in conventional RPLC are detrimental to inductively-coupled plasma mass spectrometry analytical performance. Hydro-organic mobile phases may decrease sensitivity due to excessive solvent loading of the plasma, plasma instability, high background (due to the formation of molecular ions), and carbon deposition on the sampling cone. Using micellar mobile phases with this detection mode is therefore worthwhile [5]. Hence, we conclude that MLC does not require any modification of existing instrumentation; rather, it even has advantages such as lower detection limits.

Applications of MLC
The popularity of the applications of MLC in the determination of various compounds in pharmaceutical products, biological fluids, food samples, and environmental samples has grown rapidly in recent years. Many MLC methods were published in the past five years for the determination of various compounds in different matrices mostly using hybrid micellar mobile phases consisted of aqueous surfactant solution and small volume of organic modifiers. A survey of the MLC methods published in the past five years (2010 through early 2015) is presented in Table 2.
Additionally, some stability-indicating MLC methods were developed to study the degradation behavior of some pharmaceutical compounds including flavoxate HCl [36], nelfinavir mesylate [40], risedronate [43], and timolol maleate [47]. Recently, El-Shaheny developed a stabilityindicating MLC method for piroxicam, tenoxicam, and lornoxicam [41]. This method was also applied for the determination of these compounds in complex matrix formulations, including suppositories and gel by direct injection of samples without pretreatment steps other than dilution and filtration.
One of the main applications of MLC is the possibility of direct sample injection of biological materials onto the column due to the ability of micellar aggregates to dissolve sample proteins and other compounds. Many methods have been reported for the determination of several compounds in biological fluids such as plasma, serum, urine, gastric fluid, and intestinal fluid . Almost all      [68,71]. Only one method employed a micellar mobile phase containing Brij-35 together with acetonitrile as an organic modifier, a capillary column, and a microfluidic-based chemiluminescence detector for the direct analysis of buspirone in human plasma [52]. Raviolo and colleagues studied the stability of three new anti-HIV agents, which were obtained by the association of zidovudine with different amino acids, in different matrices including simulated gastric fluid and simulated intestinal fluid using MLC procedure [79]. Gualdesi et al. also developed an MLC method to study the stability of lamivudine and seven carbonate analogues in simulated gastric and intestinal fluids [62]. Such an approach represents an important addition to the applications of MLC.
Recently, MLC emerged as a promising separation technique for plasma metabolite analyses of short-lived radioligands, due to its potential to simplify and minimize sample processing time. This would in turn lead to less radioactive decay of the radionuclides and thus provide more accurate and precise determination. Nakao and team developed pioneering work in this field using MLC for the analysis of positron emission tomography radioligands and their radioactive metabolites in the plasma of humans and of monkeys [68][69][70][71].
Moreover, MLC has been used for the determination of harmful and dangerous compounds in environmental samples. Some banned toxic aromatic amines, namely benzidine, 1-amino-2-methylbenzene, and 2-methoxy-5-methylaniline, were identified in waste water by MLC method [96]. In addition, the antibiotic fungicides blasticidin S and kasugamycin were found in irrigation water using MLC [97]. Also, the synthetic insecticide carbaryl and its main metabolite 1-naphthol were identified in water, soil, and vegetables (lettuce) by MLC [98]. Beltrán-Martinavarro et al. have developed a MLC method for the detection of the synthetic chemical melamine in drinking water and wastewater [99].
Another important application of MLC is the detection of naturally occurring phytochemicals in herbal and plant extracts [100][101][102][103]. An MLC method was applied for the analysis of the skin whitening agent arbutin and hydroquinone in pear fruits as well as in creams [100]. Determination of disulfiram in illicit preparations (ayurvedic, herbal, divine ash, and traditional medicine), as well as in pharmaceuticals and urine has also been accomplished by an MLC method [101]. Ephedrine, pseudoephedrine, and methylephedrine have also been identified in Ephedra herb and in two traditional Chinese preparations adopting an MLC method [102]. Strychnine and brucine were also identified by an MLC method in several matrices: herbal preparations, homeopathic medicines, seeds of Nux-vomica, spiked serum, and real urine samples [103].
Finally, looking at an overview of the published papers on MLC in the 2010-early 2015 time period yields some useful statistical information. Fig. 1a illustrates the frequency of the different fields of applications of MLC in this period. The most frequent application of such technique is the analysis of biological fluids, which represents 31% of the total publications in this time period, with an additional 14% applied in the analysis of biological fluids as well as pharmaceutical preparations. This widespread application of MLC in the field of bioanalysis is attributed to possibility of direct sample injection with no need for any pretreatment other than filtration.
Surveying the scientific literature published on MLC in this time period also revealed that SDS is the most frequently used surfactant, being used in 84% of the published work. Brij-35 and Tween-20 were also used but to a much lesser extent (7 and 6%, respectively), with a few workers used mixed micelles of SDS and Triton X-100 (3%). Fig. 1b shows the frequency of use of these surfactants in this time period. The popularity of using SDS is due to its availability in high purity, relatively low cost, and efficiency in dissolving biological fluids (which is not possible for cationic surfactants). Moreover, SDS is also selected because the dynamic of its micelles is better known than that of other micellar systems [5].
In conclusion, the increasing number of published MLC methods reflects the tendency of the analytical chemistry community toward green methods, which improve safety to the analysts and the environment. We hope that bringing this comprehensive review of such a fascinating technique illuminates its importance as a green separation method and becomes a factor in its further dissemination in various fields of application.

Conclusion
Micellar Liquid Chromatography is a powerful separation technique that has been applied to different pharmaceutical, biomedical, and environmental studies of single and complex compounds. MLC analysis meets the requirements of green chemistry conception by using environment-friendly reagents; micellar mobile phases are less toxic, are non-flammable, and have lower environmental impact compared to conventional RPLC methods. Micellar mobile phases are also less expensive than hydro-organic mobile phases, they allow the direct determination of physiological samples without pretreatment steps, and they are well-matched with ordinary RPLC instrumentation, so they do not require special preparations. It is evident that many principles of green chemistry are already established in MLC which will improve safety to the operator and the environment.