Micellar liquid chromatography (MLC) is a form of reversed phase liquid chromatography that uses an aqueous micellar solutions as the mobile phase.
Theory The use of micelles in high performance liquid chromatography was first introduced by Armstrong and Henry in 1980.D.W. Armstrong, Sep. Purif. Methods 14 (1985) 213 The technique is used mainly to enhance retention and selectivity of various solutes that would otherwise be inseparable or poorly resolved. Micellar liquid chromatography (MLC) has been used in a variety of applications including separation of mixtures of charged and neutral solutes, direct injection of serum and other physiological fluids, analysis of pharmaceutical compounds, separation of enantiomers, analysis of inorganic organometallics, and a host of others.
One of the main drawbacks of the technique is the reduced efficiency that is caused by the micelles. Despite the sometimes poor efficiency, MLC is a better choice than ion-exchange LC or ion-pairing LC for separation of charged molecules and mixtures of charged and neutral species. Some of the aspects which will be discussed are the theoretical aspects of MLC, the use of models in predicting retentive characteristics of MLC, the effect of micelles on efficiency and selectivity, and general applications of MLC.
Reverse phase high-performance liquid chromatography (RP-HPLC) involves a non-polar stationary phase, often a hydrocarbon chain, and a polar mobile or liquid phase. The mobile phase generally consists of an aqueous portion with an organic addition, such as methanol or acetonitrile. When a solution of analytes is injected into the system, the components begin to partition out of the mobile phase and interact with the stationary phase. Each component interacts with the stationary phase in a different manner depending upon its polarity and hydrophobicity. In reverse phase HPLC, the solute with the greatest polarity will interact less with the stationary phase and spend more time in the mobile phase. As the polarity of the components decreases, the time spent in the column increases. Thus, a separation of components is achieved based on polarity. The addition of micelles to the mobile phase introduces a third phase into which the solutes may partition.
Micelles Micelles are composed of surfactant, or detergent, monomers with a hydrophobic moiety, or tail, on one end, and a hydrophilic moiety, or head group, on the other. The polar head group may be anionic, cationic, zwitterionic, or non-ionic. When the concentration of a surfactant in solution reaches its critical micelle concentration (CMC), it forms micelles which are aggregates of the monomers. The CMC is different for each surfactant, as is the number of monomers which make up the micelle, termed the aggregation number (AN). Table 1 lists some common detergents used to form micelles along with their CMC and AN where available. + Table 1 Type Name CMC (mM) AN Anionic Cholic acid, sodium salt 14 2-4 Deoxycholic acid, sodium salt 5 4-10 Glycocholic acid, sodium salt 13 2 Sodium dodecyl sulfate (SDS) 8.27 62 Taurocholic acid, sodium salt 10-15 4 Sodium tetradecyl sulfate 2.1 Cationic Cetyltrimethylammonium chloride 1 Cetyltrimethylammonium bromide (CTAB) 1.3 78 Dodecyltrimethlyammonium bromide (DTAB) 14 50 Hexadecyltrimethylammonium bromide 0.026 169 Zwitterionic 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) 8 10 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) 8 11 N-Dodecyl-N,N-dimethylammonio-3-propane sulfonate 3.3 Nonionic n-Decyl-b-D-glucopyranoside 2.2 Triton X-100 0.24 140 Polyoxyethylene (23) dodecanol (BRIJ 35) 0.1 Polyoxyethylene -sorbitane monooleate (Tween 80) 0.01 Polyoxyethylene -sorbitane monolaurate (Tween 20) 0.059
Many of the characteristics of micelles differ from those of bulk solvents. For example, the micelles are, by nature, spatially heterogeneous with a hydrocarbon, nearly anhydrous core and a highly solvated, polar head group. They have a high surface-to-volume ratio due to their small size and generally spherical shape. Their surrounding environment (pH, ionic strength, buffer ion, presence of a co-solvent, and temperature) has an influence on their size, shape, critical micelle concentration, aggregation number and other properties.Poole, C. Journal of Chromatography A, 1998, 807, 307–310
Another important property of micelles is the Krafft point, the temperature at which the solubility of the surfactant is equal to its CMC. For HPLC applications involving micelles, it is best to choose a surfactant with a low Krafft point and CMC. A high CMC would require a high concentration of surfactant which would increase the viscosity of the mobile phase, an undesirable condition. Additionally, a Krafft point should be well below room temperature to avoid having to apply heat to the mobile phase. To avoid potential interference with absorption detectors, a surfactant should also have a small molar absorptivity at the chosen wavelength of analysis. Light scattering should not be a concern due to the small size, a few nanometers, of the micelle.
The effect of organic additives on micellar properties is another important consideration. A small amount of organic solvent is often added to the mobile phase to help improve efficiency and to improve separations of compounds. Care needs to be taken when determining how much organic to add. Too high a concentration of the organic may cause the micelle to disperse, as it relies on hydrophobic effects for its formation. The maximum concentration of organic depends on the organic solvent itself, and on the micelle. This information is generally not known precisely, but a generally accepted practice is to keep the volume percentage of organic below 15–20%.
Research Fischer and Jandera studied the effect of changing the concentration of methanol on CMC values for three commonly used surfactants. Two cationic, hexadecyltrimethylammonium bromide (CTAB), and N-(a-carbethoxypentadecyl) trimethylammonium bromide (Septonex), and one anionic surfactant, sodium dodecyl sulphate (SDS) were chosen for the experiment. Generally speaking, the CMC increased as the concentration of methanol increased. It was then concluded that the distribution of the surfactant between the bulk mobile phase and the micellar phase shifts toward the bulk as the methanol concentration increases. For CTAB, the rise in CMC is greatest from 0–10% methanol, and is nearly constant from 10–20%. Above 20% methanol, the micelles disaggregate and do not exist. For SDS, the CMC values remain unaffected below 10% methanol, but begin to increase as the methanol concentration is further increased. Disaggregation occurs above 30% methanol. Finally, for Septonex, only a slight increase in CMC is observed up to 20%, with disaggregation occurring above 25%.
As has been asserted, the mobile phase in MLC consists of micelles in an aqueous solvent, usually with a small amount of organic modifier added to complete the mobile phase. A typical reverse phase alkyl-bonded stationary phase is used. The first discussion of the thermodynamics involved in the retention mechanism was published by Armstrong and Nome in 1981. In MLC, there are three partition coefficients which must be taken into account. The solute will partition between the water and the stationary phase (KSW), the water and the micelles (KMW), and the micelles and the stationary phase (KSM).
Armstrong and Nome derived an equation describing the partition coefficients in terms of the retention factor (formally capacity factor), k¢. In HPLC, the capacity factor represents the molar ratio of the solute in the stationary phase to the mobile phase. The capacity factor is easily measure based on retention times of the compound and any unretained compound. The equation rewritten by Guermouche et al. is presented here: 1/k¢ = [n • (KMW-1)/(f • KSW)] • CM +1/(f • KSW) Where: k¢ is the capacity factor of the solute KSW is the partition coefficient of the solute between the stationary phase and the water KMW is the partition coefficient of the solute between the micelles and the water f is the phase volume ratio (stationary phase volume/mobile phase volume) n is the molar volume of the surfactant CM is the concentration of the micelle in the mobile phase (total surfactant concentration - critical micelle concentration)
A plot of 1/k¢ verses CM gives a straight line in which KSW can be calculated from the intercept and KMW can be obtained from the ratio of the slope to the intercept. Finally, KSM can be obtained from the ratio of the other two partition coefficients: KSM = KSW/ KMW As can be observed from Figure 1, KMW is independent of any effects from the stationary phase, assuming the same micellar mobile phase.
The validity of the retention mechanism proposed by Armstrong and Nome has been successfully, and repeated confirmed experimentally. However, some variations and alternate theories have also been proposed. Jandera and Fischer developed equations to describe the dependence of retention behavior on the change in micellar concentrations. They found that the retention of most compounds tested decreased with increasing concentrations of micelles. From this, it can be surmised that the compounds associate with the micelles as they spend less time associated with the stationary phase.
Foley proposed a similar retentive model to that of Armstrong and Nome which was a general model for secondary chemical equilibria in liquid chromatography. While this model was developed in a previous reference, and could be used for any secondary chemical equilibria such as acid-base equilibria, and ion-pairing, Foley further refined the model for MLC. When an equilibrant (X), in this case surfactant, is added to the mobile phase, a secondary equilibria is created in which an analyte will exist as free analyte (A), and complexed with the equilibrant (AX). The two forms will be retained by the stationary phase to different extents, thus allowing the retention to be varied by adjusting the concentration of equilibrant (micelles).
The resulting equation solved for capacity factor in terms of partition coefficients is much the same as that of Armstrong and Nome: 1/k¢ = (KSM/k¢S) • [M] + 1/k¢S Where: k¢ is the capacity factor of the complexed solute and the free solute k¢S is the capacity factor of the free solute KSM is the partition coefficient of the solute between the stationary phase and the micelle [M] may be either the concentration of surfactant or the concentration of micelle
Foley used the above equation to determine the solute-micelle association constants and free solute retention factors for a variety of solutes with different surfactants and stationary phases. From this data, it is possible to predict the type and optimum surfactant concentrations needed for a given solute or solutes.Foley, J.P. Analytica Chimica Acta, 1990, 231, 237-247
Foley has not been the only researcher interested in determining the solute-micelle association constants. A review article by Marina and Garcia with 53 references discusses the usefulness of obtaining solute-micelle association constants. The association constants for two solutes can be used to help understand the retention mechanism. The separation factor of two solutes, a, can be expressed as KSM1/KSM2. If the experimental a coincides with the ratio of the two solute-micelle partition coefficients, it can be assumed that their retention occurs through a direct transfer from the micellar phase to the stationary phase. In addition, calculation of a would allow for prediction of separation selectivity before the analysis is performed, provided the two coefficients are known.