Micellar LiquidChromatography

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MICELLAR LIQUID CHROMATOGRAPHY:

MICELLAR LIQUID CHROMATOGRAPHY Presented by: Ms. Ashritha Narikimalli, II M.Pharm., Pharmaceutical Analysis & Quality Assurance. Under the guidance of: Mr. D. Sathis Kumar, M.Pharm.,( Ph.D )., Associate Professor, Mrs. S. Namratha , M.Pharm., Assistant Professor, Aditya Institute Of Pharmaceutical Sciences & Research.

INTRODUCTION::

INTRODUCTION: Micellar liquid chromatography (MLC) is a reversed-phase liquid chromatographic (RPLC) mode with a mobile phase consisting in an aqueous solution of surfactant above its critical micellar concentration (CMC). In practice, however, the addition of a small amount of organic solvent to the micellar solution is needed to achieve retention in practical time windows and to improve peak efficiency and resolution.

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The stationary phase is modified with an approximately constant amount of surfactant monomers, and the solubilising capability of the mobile phase is altered by the presence of micelles, giving rise to a great variety of interactions (hydrophobic, ionic, and steric ) with major implications in retention and selectivity. The versatility of MLC is due to these wide variety of interactions that are established among the eluted solutes, the stationary phase, the aqueous phase and micelles. Their eluent characteristics allow the analysis of compounds with a wide range of polarities. MLC also provides a solution to the direct injection of real samples (physiological or food) by solubilising proteins . This simplifies and expedites treatment , which confers analytical procedures of greater accuracy and a lower cost .

GREEN CHEMISTRY:

GREEN CHEMISTRY The idea of using pure micellar solutions as mobile phases in RPLC is very attractive given their lower cost, less toxicity, and poorer environmental impact. Current concern about the environment also reveals MLC as an interesting technique for “green” chemistry because it uses mobile phases containing 90% or more water. These micellar mobile phases have a low toxicity and are not producing hazardous wastes.

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The presence of a surfactant not only modifies the interactions established inside the column but also reduces the necessary amount of organic solvent in the mobile phase, which can be recycled due to low evaporation . These characteristics are genuinely interesting given current concerns about reducing organic contaminant residues in laboratories.

SURFACTANT:

SURFACTANT The term surfactant is a  blend of   surface active agents. Surfactants are compounds that lower the surface tension  of a liquid, the interfacial tension between two liquids, or that between a liquid and a solid. Surfactants are usually organic compounds that are  amphiphilic , containing both  hydrophobic  groups (their  tails ) and  hydrophilic  groups (their  heads ). Surfactants reduce the surface tension  of water by adsorbing at the liquid-gas interface. The relation that links the surface tension and the surface excess is known as the  Gibbs isotherm .

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Most commonly, surfactants are classified according to polar head group. A non-ionic surfactant has no charge groups in its head. The head of an ionic surfactant carries a net charge. If the charge is negative, the surfactant is more specifically called anionic ; if the charge is positive, it is called cationic . If a surfactant contains a head with two oppositely charged groups, it is termed zwitterionic . The "tail" of most surfactants are fairly similar, consisting of a hydrocarbon chain, which can be linear, branch , or aromatic . Non ionic Anionic Cationic A mphoteric

MICELLE:

MICELLE In the bulk aqueous phase, surfactants form aggregates, micelles, where the hydrophobic tails form the core of the aggregate and the hydrophilic heads are in contact with the surrounding liquid. Other types of aggregates such as spherical or cylindrical micelles or bilayers can be formed. The shape of the aggregates depends on the chemical structure of the surfactants, depending on the balance of the sizes of the hydrophobic tail and hydrophilic head. This is known as the HLB, Hydrophilic- lipophilic balance.

MICELLAR MOBILE PHASE:

MICELLAR MOBILE PHASE Micellar mobile phases have been used with different bonded stationary phases (mostly C8, C18 and cyanopropyl ). The most common surfactants are : Anionic : sodium dodecyl sulphate (SDS), Cationic : cetyltrimethylammonium bromide (CTAB), Non-ionic : Brij-35. Several organic solvents have been used as modifiers, short/medium chain alcohols and acetonitrile being the most suitable. The presence of micelles contributes to keep these organic solvents in solution at concentrations well above their solubility in water. Also, the risk of evaporation is diminished.

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In the micelles, three sites of solubilisation can be identified: the core (hydrophobic), the surface (hydrophilic), and the palisade layer (the region between the surfactant head groups and the core). Solutes associated to micelles experience a microenvironment that is different from that of bulk solvent. Although the separation mode is still predominantly micellar in nature, the micelle is perturbed by the organic solvent. This can change micellar parameters, such as the CMC and surfactant aggregation number. The organic solvent decreases the polarity of the aqueous solution and alters the micelle structure . A high percentage of organic solvent can disrupt the micelle structure. The maximal allowable concentration depends on the type of organic solvent and surfactant.

Characteristics of the most common surfactants in MLC:

Characteristics of the most common surfactants in MLC Surfactant Molecular weight (g/mol) CMC (mol/L) Micellar Radius (nm) Molar Volume (L/mol) SDS 288.4 8.2 × 10 −3 2.5 0.246 CTAB 364.5 9 × 10 −4 3.2 0.364 Brij-35 1198 (avg.) 9 × 10 −5

Critical Micellar Concentration::

Critical Micellar Concentration: A suitable surfactant for MLC should have a low CMC. A high CMC would imply operating at high surfactant concentration, which would result in viscous solutions , giving undesirable high system pressure and background noise in UV detectors. The CMC is strongly affected by the presence of an organic solvent. The changes are related to the modification of the structure of the micelle, which also induces, at least partially, the reduced retention in MLC. Recently, some novel ionic liquid-based surfactants like 1-hexadecyl-3-butylimidazolium bromide have been used in MLC.

Krafft Point::

Krafft Point: The Krafft point is defined for ionic surfactants as the temperature at which the solubility of a surfactant monomer becomes equal to the CMC. Below the Krafft point temperature, the solubility 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. The Krafft point for SDS and CTAB is around 15 ◦ C and 20-25 ◦ C, respectively.

Cloud Point::

Cloud Point: Non-ionic surfactants also have a specific temperature, that if exceeded, phase separation occurs , which is called the cloud point. Chromatographic work with these surfactants should be conducted below this temperature (e.g., Brij-35, is ca. 100◦C for aqueous 1–6% solutions, whereas for Triton X–100 this value is 64◦C).

pH of the Mobile Phase::

pH of the Mobile Phase: MLC employs the same packing materials as classical RPLC, which for conventional columns have a limited working pH range of 2.5–7.5 . Appropriate pH values depend on the nature of the analytes and the surfactant selected. The pH of the micellar mobile phase is commonly fixed with phosphoric or citric acid buffers . For mobile phases containing SDS, potassium salts are not recommended as potassium dodecyl sulphate presents a high Krafft point and precipitates from aqueous solutions at room temperature

Organic Solvents: Types & Concentration:

Organic Solvents: Types & Concentration The selection of the appropriate organic solvent modifier in MLC should consider the polarities of the analytes . For polar compounds, sufficiently short retention times (below 20 min) are obtained with 1-propanol, 2-propanol, or acetonitrile. For nonpolar compounds or compounds with high affinity for the surfactant adsorbed on the stationary phase, stronger solvents as 1-butanol or 1-pentanol are needed. However, it should be noted that the two latter alcohols give rise to microemulsion formation at sufficiently high concentration. The amount of organic solvent that can be added is limited by its solubility .

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It should be noted that at high organic solvent concentration , the micelles disaggregate and the mobile phase contains only free surfactant molecules . The organic solvent contents that preserve the integrity of micelles are below 15% for propanol and acetonitrile, 10% for butanol , and 6% for pentanol . These contents are low in comparison with those needed in classical RPLC. The lower organic solvent consumption results in reduced cost and toxicity , which may become prominent for “green chemistry”. Also, the stabilization of the organic solvent in the micellar media decreases the risk of evaporation . This means that micellar mobile phases can be preserved in the laboratory for a long time without significant changes in their composition.

MODIFIED STATIONARY PHASE:

MODIFIED STATIONARY PHASE Surfactant Adsorption: The alkyl-bonded C18 is the stationary phase most widely used in MLC, but other columns can be selected (e.g., C8 and cyanopropyl ). Alkyl-bonded phase columns are strongly modified when SDS, CTAB, or Brij-35 is incorporated into the mobile phase. Surfactant adsorption on the porous RPLC packing affects drastically the chromatographic retention, owing to the change of diverse surface properties of the stationary phase ( e.g., polarity, structure, pore volume, and surface area). Surfactant molecules coat the stationary phase pores, reducing appreciably their volume.

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Ionic compounds are frequently added to micellar mobile phases for pH buffering and, eventually , ionic strength adjustment . Salt addition may change the amount of adsorbed ionic surfactant due to the reduction of both electrostatic repulsion and surfactant CMC, and the enhancement of hydrophobic interactions. Surfactant coating masks the bonded-stationary phase . This means that a full similar coating would render the stationary phases all similar. Solid-state nuclearmagnetic resonance studies for the most common used surfactant, SDS, reveal that the hydrophobic tail was found to be associated with the C18 alkyl-chain bonded to the silica stationary phase, the sulphate head group oriented away from the surface. This creates a negatively charged hydrophilic layer affecting the penetration depth of solutes into the bonded phase.

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Solute environment in a chromatographic system using octadecyl -bonded phase, and mobile phase containing the anionic SDS.

Presence of an Organic Solvent in the Mobile Phase:

Presence of an Organic Solvent in the Mobile Phase Organic solvents are added to micellar mobile phases to improve peak efficiencies and reduce retention times , giving rise to the so-called hybrid micellar mobile phases . Competition between alcohols and surfactant molecules for adsorption sites on the stationary phase explains the linear reduction in the amount of adsorbed surfactant with increasing concentration of alcohol in the mobile phase. Mobile phases rich in organic solvent can sweep completely the adsorbed surfactant molecules from the bonded phase.

SOLUTE-MICELLE & SOLUTE-STATIONARY PHASE INTERACTIONS:

SOLUTE-MICELLE & SOLUTE-STATIONARY PHASE INTERACTIONS The unique capabilities of micellar mobile phases are attributed to the ability of micelles to selectively compartmentalise and organise solutes at the molecular level . However, the association of the surfactant monomers to the bonded phase has deep implications with regard to retention and selectivity. The chromatographic behaviour in an RPLC system of a solute eluted with a mobile phase containing a surfactant above the CMC can be explained by considering three phases: stationary phase, bulk solvent, and micellar pseudophase ( micellar aggregates).

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Solutes are separated on the basis of their differential partitioning between bulk solvent and micelles in the mobile phase or surfactant- coated stationary phase. For water-insoluble species , partitioning can also occur via direct transfer of solutes between the micellar pseudophase and the modified stationary phase

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Direct transfer of highly hydrophobic solutes between micelle and surfactant-modified stationary phase

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The partitioning equilibria in MLC can be described by three coefficients: PWS (partition between aqueous solvent and stationary phase), PWM (between aqueous solvent and micelles), PMS (between micelles and stationary phase). The coefficients PWS and PWM account for the solute affinity to the stationary phase and micelles, respectively , and have opposite effects on solute retention: as PWS increases, the retention increases, whereas as PWM increases, the retention is reduced due to the stronger association to micelles.

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The retention behavior depends on the interactions established by the solute with the surfactant-modified stationary phase and micelles. Neutral solutes eluted with non-ionic and ionic surfactants and charged solutes eluted with nonionic surfactants will only be affected by nonpolar, dipole-dipole, and proton donor-acceptor interactions. Besides these interactions, charged solutes will interact electrostatically with ionic surfactants (i.e., with the charged surfactant layer on the stationary phase and the charged outer layer of micelles). In any case, the steric factor can also be important.

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With ionic surfactants , two situations are possible according to the charges of solute and surfactant: repulsion or attraction (by both surfactant-modified stationary phase and micelles). In the case of electrostatic repulsion , charged solutes cannot be retained by the stationary phase and elute at the dead volume , unless significant hydrophobic interaction with the modified bonded layer exists. In contrast, combined electrostatic attraction and hydrophobic interactions with the modified stationary phase may give rise to strong retention in MLC. Mixtures of polar and non polar solutes can be resolved, provided that an appropriate surfactant is chosen .

CARE OF THE CHROMATOGRAPHIC SYSTEM IN MLC:

CARE OF THE CHROMATOGRAPHIC SYSTEM IN MLC Mobile Phase Saturation: Pure and hybrid micellar solutions contain high amounts of water (usually more than 90% v/v) and are able to dissolve small amounts of silica, which could produce serious column damage. This is especially critical at 30 ◦ C and/or pH 6. For this reason, a saturating short column packed with 10 μm bare silica , or alternatively, the same packing as the analytical column , should be placed after the pump and before the injection valve to reduce pressure build-up .

Column Conditioning:

Column Conditioning A column for MLC is generally stored in 100% methanol . Before starting column conditioning, the solvent should be replaced by 100% water. For this operation, a low flow rate (≤0.5mL/min) should be selected at the beginning because of the high viscosity of the methanol-water mixture. Once the pressure decreases, the flow-rate may be raised. At least 30 column volumes of water are required to assure complete organic solvent removing. Now, the system is ready to be flushed with the micellar mobile phase. Different studies of column coating through surfactant breakthrough patterns have revealed that most surfactant adsorbs in less than one hour on the bonded stationary phase.

Mobile Phase Flushing:

Mobile Phase Flushing The micellar mobile phase should be continuously flushed through the system. If the chromatographic system is stopped during several hours, the micellar solution should not stay in contact with the bonded silica-based stationary phase to avoid surfactant precipitation. A static micellar mobile phase can also produce crystals around the pump plungers and seals. Such crystals may obstruct the system producing plugged connecting tubing and frits, seal failure, or scratched pistons . A micellar mobile phase can be kept inside the chromatographic system overnight if the pump is not off. This avoids daily cleaning and reequilibration .

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To reduce the cost, the mobile phase can be recycled, reducing the flow-rate to a minimal value ( often 0.1–0.25 mL /min ). However, it should be noted that in case of energy supply failure, column damage can occur. Mobile phase recycling is possible because of the low evaporation risk of organic solvents in hybrid micellar eluents . For the same reason, the micellar mobile phase can be recycled during the analysis, as long as a low number of injections are made.

Column Cleaning:

Column Cleaning In general, regeneration can be appropriately performed with methanol, where most surfactants are highly soluble . The cleaning protocol comprises a two-step procedure that takes about half an hour . First, the micellar mobile phase should be replaced by 100% pure water , by rinsing the chromatographic system with 10 to 20 column volumes of pure water . This step is necessary to avoid salt crystallization provoked by a brutal change from a buffered micellar mobile phase to 100% methanol.

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Next, water will be replaced by 100% methanol to remove the adsorbed surfactant on the stationary phase. The same caution commented under “column conditioning” about the initial use of a low flow-rate should be followed. To assure complete surfactant desorption, at least 10 column volumes of methanol should be passed through the column.

APPLICATIONS & ADVANTAGES::

APPLICATIONS & ADVANTAGES: A paper by N. Memon et al. studies the selectivity of a non ionic surfactant (Brij-35) in MLC separation of positional isomers. The effect of surfactant and organic solvent concentration on the separation of some selected isomers is studied and evaluated in terms of Linear Solvation energy Relationship (LSER). Besides basicity , dipolarizability and excess molar refraction are responsible for fine tuning of separation. This new face of non-ionic MLC opens field for many applications in separation of positional isomers.

APPLICATIONS & ADVANTAGES::

APPLICATIONS & ADVANTAGES: In the paper by A. U. Kulikov an MLC method was developed and validated according to ICH guidelines for the determination of sesquiterpenic acids in root and rhizome extract from Valeriana officinalis and valerian dry hydroalcoholic extract. Main advantage of MLC is that it allows separating compounds with different hydrophobicity in a single run without the gradient elution.

APPLICATIONS & ADVANTAGES::

APPLICATIONS & ADVANTAGES: In the paper by M.-L. Chin-Chen another applied work that determines the levels of the biogenic amine spermine in anchovy sauce after derivatization with 3,5-dinitrobenzoyl chloride is introduced. This methodology was validated in terms of linearity, sensitivity, limits of detection and quantification, accuracy, precision and recovery following the FDA guidelines. Direct injection of samples (after filtration) was used, thus avoiding any tedious extraction and purification step. This is another interesting advantage of the MLC technique.

REFERENCE::

REFERENCE: M.J. Ruiz- Ángel , S. Carda-Broch , J.R. Torres- Lapasió , M.C. García-Álvarez-Coque , “Retention mechanisms in micellar liquid chromatography”, Journal of Chromatography A, 1216 (2009) 1798–1814. Maria Rambla-Alegre , “ Basic Principles of MLC”, Chromatography Research International, Volume 2012, Article ID 898520. A. P. Boichenko, L. P. Loginova, A. U. Kulikov, ” Micellar liquid chromatography (Review). Part 1. Fundamentals, retention models and optimization of separation”, 2007, 92-116.

REFERENCE::

REFERENCE: Maria Rambla-Alegre , “ Retention Behaviour in Micellar Liquid Chromatography”, Chromatography Research International Volume 2012 (2012), Article ID 402635. Samuel Carda-Broch, Josep Esteve-Romero, Maria Rambla-Alegre, Maria Jose Ruiz-Angel, Alain Berthod , and Devasish Bose, “Micellar Liquid Chromatography: Recent Advances and Applications”, Chromatography Research International, Volume 2012, Article ID 573690.

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