Biotechnology: Micellar Electrokinetic Chromatography (MEKC)

Introduction

An analytical technique is a method used to determine a chemical compound or chemical element concentration. There is a wide range of analytical techniques which can be used, ranging from simple weighing and titrations to highly advanced procedures utilizing highly specialized instrumentation.

According to the International Union of Pure and Applied Chemistry (IUPAC), chromatography can be defined as: 'A physical separation method in which the components to be separated are distributed between two phases, one of which is stationary (stationary) while the other (mobile) is moving in a definite direction’

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Electrokinetic chromatography (EKC) is a family of electrophoresis techniques named after electrokinetic phenomena, which include electroosmosis, electrophoresis, and chromatography.

Micellar Electrokinetic Chromatography (MEKC) was developed as a mode of Capillary Electrophoresis (CE) particularly for neutral/non-charged molecules. It was first developed in 1982 (published 1984) by Terabe et al. At that time, capillary GC offered less than 100000 theoretical plates, HPLC only offered approximately 5000.

Micellar electrokinetic chromatography (MEKC) is an EKC method which utilizes surfactants (micelles) to fit the buffer solution. Surfactants are hydrophobic and hydrophilic structures. Surfactant reduces surface tension and form micelles at concentrations above critical micelle concentration. Most common surfactant in MEKC is SDS (sodium dodecyl sulfate)

Micelle has polar 'face' groups that can be cationic, anionic, neutral, or zwitterionic, with hydrocarbon tails that are nonpolar. Micellular formation or 'micellisation' is a direct result of the 'hydrophobic effect.' The hydrocarbon tails, the non-polar tails, are then pointed to the middle of the aggregated molecules, whereas the polar head groups, hydrophilic, is pointed outwards Micellar solutions can solubilize hydrophobic substances that would otherwise be insoluble in liquid. Each surfactant has a characteristic Critical Micelle Concentration (CMC) and aggregation number, i.e., the number of surfactant molecules constituting a micelle (typically within the 50-100 range).

Micellar formulations were used in a number of separation and spectroscopic techniquesThe use of micellar solutions for reverse-phase liquid chromatography (RPLC) as mobile phases was pioneered by Armstrong and Henry in 1980. MEKC is also often referred to as MECC (micellar electrokinetic capillary chromatography), as the separations using a capillary tube are most frequently performed. There are other types of EKC such as Cyclodextrin EKC (CDEKC), ion exchange EKC (IXEKC), and microemulsion EKC (MEEKC).

Theoretical Concept

Separation is the process of incorporating analytes onto/into the micelle based on the micellar solubilisation. Selectivity (α) can be easily manipulated by changing the type(s) of surfactant(s) used. For hydrophobic analytes, organic solvents can by added to the solution for better partitioning into the aqueous phase. Highly hydrophobic analytes are difficult to separate.

When an anionic surfactant such as sodium dodecyl sulfate (SDS) is used, the micelle migrates to the positive electrode through electrophoresis. Due to the negative charge on the surface of fused silica, the electroosmotic stream transfers the bulk solution to the negative electrode. Generally, the electroosmotic flow (EOF) is greater than the micelle's electrophoretic migration under neutrality. The electroosmotic flow (EOF) is usually stronger than the micelle's electrophoretic migration under neutral or alkaline conditions and thus the anionic micelle often moves at a delayed velocity towards the negative electrode.

In 17 minutes, eight electrically neutral compounds were resolved successfully. The size of the capacity factor is included in the figure to demonstrate the relationship between the time of migration and the capacity factor. The capacity factor of infinity means the analyte has the same time of migration as the micelle. Theoretical plate numbers calculated from the peak widths range from 200,000 to 250,000 which is typical for MEKC separations.

Capacity factor, sometimes called retention factor, is used to evaluate the relative hydrophobicity of the analyte. The term capacity factor is written in k ' and is the ratio of moles analyte in the micelle phase-to-moles of analyte in the aqueous component of running buffer.

Instrumentation and Methodology

The separation is accomplished by micelles formation. During migration, micelle interact with analyte as chromatographic manner and separation is brought about.

The micellar solution is prepared by dissolving a surfactant at a concentration higher than its CMC into a buffer solution. To keep the pH constant, the buffer solution is required. Usually, concentrations of 30 to 100 mM are used for the components of the surfactant and buffer. To remove particulates, the separation solution has to be filtered through a membrane filter. A disposable, cartridge-type membrane filter can be used with a syringe as the solution needed for a MEKC run is typically less than 10 m. The direction of the electroosmotic flow is reversed when a cationic surfactant (e.g. CTAB) is used at a sufficiently high concentration. In this case it is important to reverse the polarity of the power supply.

Since the micellar solution has a relatively high conductivity, prevention of excessive Joule heating is preferred by a capillary with a small diameter. The capillary length is typically not very significant, but at the expense of time a longer one can handle a larger amount of sample solution. As relatively large i.d. capillaries is used for increased sample capacity, effective capillary cooling (such as the P / ACETM liquid cooling system) is essential. Numerous ionic surfactants are commercially available. The surfactants suitable for MEKC should meet the following criteria:

1. The surfactants must have enough solubility in the buffer solution to form micelles.
2. The micellar solution must be homogeneous and UV transparent.
3. The micellar solution must have a low viscosity

To avoid excessive current, the applied voltage must be held to a point that is not too high. Controlling the capillary temperature is also desirable because the migration time in MEKC is even more temperature-sensitive than in CZE.

Application in Biotechnology

Medical Analysis

This is applied for the determination and quantification of Paclitaxel, morphine, and codeine in urine sample of patients with different cancer types (breast, head and neck, and gastric) It separates both neutral and ionic compounds at once, provides quick sample preparation such as centrifugation and filtration, and direct injection method with biological samples

Environmental Analysis

It is applied for the determination of anti-inflammatory drugs in river water. Some sewage treatment plants are unable to eliminate hydrophilic and stable compounds entirely, resulting in the compounds remaining in water bodies. MEKC is one form in which these anti-inflammatory drugs can be detected in water. Since MEKC at low concentrations is not very sensitive, pre-concentration procedures are required before the separation can take place. The sample stacking and sweeping are two main methods for pre-concentration.

Pharmaceutical Analysis

The MEKC has the ability to separate complex mixtures (natural products, crude drugs) with high resolution. The pharmaceutical industry uses micellar electrokinetic chromatography to isolate enantiomers, to distinguish amino acids and closely related peptides, to separate very complex mixtures, to assess drugs in biological samples and to separate electrically neutral products.

Food analysis

Procyanidins and their monomers are compounds found in several species of plants and are frequently found in fruit peels and legume seed coat. Several procyanidins displayed anti-HIV, anti-ulcer and anti-allergy activity, which attracted a great deal of attention to this molecular group. Aside from other instrumentations such as high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), and nuclear magnetic resonance (NMR) spectroscopy, MEKC has proved to be the superior method of analyzing procyanidins. In order to concentrate the sample or make it optically transparent, spectroscopic methods require sample pre-treatments, and HPLC takes more than thirty minutes. In less than five minutes MEKC was able to separate seven compounds.

Separation of vitamins and antibiotics

MEKC method has seen higher selectivity with MEKC method than CZE. Shorter analysis time is required with MEKC than CZE. Separation of the enantiomer can be achieved when the mobile phase uses a chiral surfactant. Drug standards require 0.1% of impurities to be identified, and after using a preconcentration procedure such as sample stacking, this can be done using MEKC.

Conclusion

Micellar Electrokinetic Chromatography (MEKC) is a flexibleseparation technique. It is an effective separation of neutral molecules with usage of inexpensive equipment. Selectivity easily manipulated through various combinations of surfactants and organic solvents. However, finding the right combination can be difficult but this method could be more widely used if better detector interfaces are developed.

This analytical technique or instrumentation is beneficial due to its limited elution time which means of relatively short separation time. Moreover, it is useful for biological samples because it can separates ionic and neutral compounds at high efficiencies. It is advantageous due to its high separation power and it only requires a few nanoliters of sample. As it can separate small molecules, it remains cheaper than HPLC. Thus, it can separate chiral compounds.

Nevertheless, limited elution time limits peak capacity of technique. It is generally limited to compounds which are reasonably soluble in the mobile phase. If it has low concentrations, it will cause low sensitivity.

References

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