Microemulsion Electrokinetic Chromatography and its Applications

By - 08/08/2007

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Zahid Zaheer

Zahid Zaheer

Microemulsion electrokinetic capillary chromatography (MEEKC) is an electrodriven separation technique, which offers the possibility of highly efficient separations of both charged and neutral solutes covering a wide range of water solubilities.

The technique uses microemulsion buffers to separate solutes based on both their hydrophobicities and electrophoretic mobilities. Microemulsions are solutions containing a dispersion of nanometre droplets of an immiscible liquid. The microemulsions used in MEEKC are oil droplets dispersed in an aqueous buffer. The oil and water components are totally immiscible and do not mix together as there is a high surface tension between them.

The oil droplets are coated with a surfactant to reduce the surface tension between the two liquid layers and which allows the emulsion to form¹. The surface tension is further lowered to approach zero by the addition of a short-chain alcohol such as butan-l-ol which stabilises the microemulsion system. If the microemulsion system remains unstable then it will revert to individual layers of oil and water after a short period of time. The diameter of the oil droplets are below 10nm so they do not scatter white light which means that the microemulsion is optically transparent.

Use of a microemulsion containing ionic surfactants allows chromatographic separation to be obtained as solutes can partition between the oil droplet and the aqueous buffer phase. Water-insoluble compounds will favour inclusion into the oil droplet rather than into the buffer phase. This situation allows partitioning of the solute between the oil and water phases in a chromatographic fashion. Hydrophobic solutes will reside more frequently in the oil droplet than water-soluble solutes. The separation basis is similar to that involved in micellar electrokinetic chromatography (MEKC) where the surfactant monomers group together to form micelles². Solutes chromatographically interact with the micelles to achieve separation. Solutes are more easily able to penetrate the surface of the droplet than the surface of a micelle which is much more rigid. This ability allows MEEKC to be applied to a wider range of solutes.

Sodium dodecyl sulphate (SDS) is the most widely used emulsifier surfactant in MEEKC. The oil droplet is coated with SDS surfactant molecules making the droplet negatively charged. The C₁₂ alkyl chain of the surfactant penetrates into the oil droplet whilst the negatively charged hydrophilic sulphate groups reside in the surrounding aqueous phase. Charge repulsion of the negatively charged sulphate group on the SDS prevents highly efficient packing and prevents formation of an emulsion as the surface tension cannot be sufficiently reduced. A co-surfactant, usually a medium chain length alkyl alcohol such as butan-1-ol, is therefore essential in the formation and improved stability of the microemulsion. The co-surfactant bridges the oil and water interface and further reduces the surface tension of the system to zero. Figure 1 provides a schematic representation of the emulsion droplet showing the short chain alcohol, SDS, the octane droplet and the sodium ions surrounding the droplet.

Figure: 1

High pH buffers such as borate or phosphate are generally used in MEEKC. These buffers generate a high electro-osmotic flow (EOF) when a voltage is applied across a capillary filled with the buffer. This flow is relatively rapid and is towards the cathode situated near the detector. The surfactant-coated oil droplets are negatively charged and therefore attempt to migrate towards the anode when the voltage is applied. However, the EOF is sufficiently strong to eventually sweep the oil droplets through the detector to the cathode. Highly water-soluble neutral solutes such as methanol will reside predominantly in the aqueous phase and will sweep rapidly to the detector by the EOF giving a solvent front (t_o) measure.

Schematic representation of MEEKC process

Schematic representation of MEEKC process

Conversely a highly water insoluble solute such as dodecylbenzene will predominately favour partitioning into the negatively charged droplet and will be strongly retained with an infinitely high capacity factor. If a moderately soluble solute has a capacity factor (k^’) then it spends equal amounts of time in both the aqueous phase and the oil droplet. The MEEKC migration time, or capacity factor, of a neutral solute can be directly related to solubility (hydrophobicity) of the solute.

MEEKC is a relatively recent technique and it has not been widely applied to a range of application types. However, there are currently sufficient applications to demonstrate the wide potential uses of MEEKC. The reported application range will be summarised to provide an appreciation of the separation possibilities that MEEKC may offer.

Applications:

Solubility (hydrophobicity) Assessments

The solubility (log P) of a neutral solute can be directly assessed from migration time data obtained in MEEKC. This is the most frequently reported application of MEEKC where solubility data has been obtained for neutral, anionic and cationic solutes. Typically the migration times of a number of solutes with known log P are determined to generate calibration graph or migration index. The migration index (MI) is calculated by MI = c log K’ + d, where c and d are the slope and intercept of the calibration line.

Derivatised Sugars

MEEKC has been applied to the separation of highly insoluble diphenyl hydrazine derivatives of a 10 component carbohydrate test mixture was resolved by MEEKC using a SDS – octane – butan-l-ol microemulsion system. However, the borate – SDS MEKC system only permitted resolution of the test mixture into 3 multi-component peaks.

Polyaromatic Hydrocarbons

Polyaromatic hydrocarbons (PAH’s) are generally difficult to analyse by CE as they are neutral and possess low water solubilities. Simple aromatic solutes such as naphthol and toluene have also been separated using SDS – heptane – butan-l-ol microemulsions.³

Proteins

MEEKC has been used to separate a range of proteins⁶. The proteins were separated, largely based on their hydrophobicities, using an SDS – heptane – butan-l-ol microemulsion in 2.5mM borate buffer. Resolution of the separated proteins was strongly affected by the SDS concentration with maximum resolution obtained at 120mm SDS. The resolution obtained for ribonuclease A, carbonic anhydrase II, b-lactoglobulin A and myoglobulin by MEEKC was better than conventional CE using a borate buffer or MEKC. Proteins are generally too large to partition into a micelle but can partition into the microemulsion droplet which has a larger volume. The MEEKC method could resolve both basic and acidic proteins and was applied to the analysis of a range of injection formulations containing various protein mixtures.

Hop Bitter Acids

Hop bitter acids are present in the hops used to manufacture beer. The levels and composition of these acids affects the quality of the hop quality and is therefore tested before the hops are used in beer manufacture. MEEKC has been shown to give accurate and precise data for this analysis⁵. Resolution of the 6 major hop acids was achieved within 10 minutes with separation efficiencies in the order of 280-480,000 theoretical plates.

Agrochemicals

Resolution of 6 phenylurea herbicides and chlorsulfuron was achieved using a SDS – octane – butan-l-ol microemulsion system⁵. The highly insoluble herbicides were dissolved in N, N-dimethl formamide. The effect on the resolution was assessed for a range of operating parameters such as voltage, and the concentration of the butan-l-ol, SDS and oil.

Vitamins

Vitamins are classified into water- or fat-soluble. The water-soluble acidic vitamins such as nicotinic acid and Vitamin C possess an acidic function and these can be analysed using CE with high pH borate or phosphate buffers. However, the fat-soluble vitamins such as Vitamins A and E are neutral and have poor water-solubility and require use of a chromatographic based method. MEEKC has been shown to be useful for the simultaneous determination of water- and fat-soluble vitamins. A vitamin formulation containing both water-soluble and insoluble vitamins has been resolved using MEEKC.⁸ 1ml of the liquid formulation was diluted to 5ml with the microemulsion buffer and directly injected.

Ketones and Beta-Diketones

Test mixtures of various ketones such as acetylacetone, benzoylacetone, acetophenone and benzyoyltrifluoroacetone, were separated⁹ using a high pH carbonate buffer containing SDS, heptane and butan-l-ol. The MEEKC method was shown to give both improved resolution and analyse solubility range than a micellar method.

Pharmaceuticals

Analgesics/Cold Medicine Ingredients

A test mixture of 7 cold medicines were separated using a heptane – SDS – butan-l-ol microemulsion.¹⁰ Antipyrene analgesics were separated using MEEKC. A liquid cold medicine formulation was diluted with a microemulsion buffer and directly injected into the capillary⁷. The method was used to determine the quantity of active ingredients and the parahydroxybenzoate preservatives.

Steroids

A range of different microemulsion compositions were compared for the separation of 10 steroids.¹¹ Six different oils were assessed including hexane, cyclohexane, hexanol and octanol. Hexanol was considered to be optimal. Various surfactants were assessed with SDS being the optimal. The MEEKC method was applied to the measurement of 11-b-hydroxysteroid dehydrogenase activity in rat intestine. Results were comparable to those obtained by a HPLC method.

Basic Drugs

Basic drugs can interact with the surface silanols on the stationary phases used in HPLC and CE which can lead to tailing and loss of separation efficiencies of efficient separation of basic drugs by CE using low pH buffers is possible. Highly efficient MEEKC separations of a range of water-soluble and insoluble basic drugs including terbutaline, bupivacaine, salmeterol and amitrypline have been demonstrated with no evidence of peak tailing⁷.

Acidic Drugs

A range of water-soluble and insoluble acidic drugs has been resolved by a high pH MEEKC method.⁷ These included a range of related cephalosporins, acetylsalicylic acid and insoluble drugs such as ibuprofen, indomethacin and troglitazone. The method was used to quantify levels of troglitazone in a tablet formulation. An assay result of 199.4 mg/tablet was obtained compared to the label claim of 200 mg/tablet.

Cardiac Glycosides

This class of natural products compounds includes highly insoluble, neutral digoxin which is extracted from foxglove plants. MEEKC has been used to separate a range of related glycoside with detection at low UV wavelength.¹²

Natural Product Analysis

MEEKC has been applied to the separation and identification of the active components in Rheum plant extracts.¹³ The highly insoluble components were extracted into chloroform or ethanol. A microemulsion comprising of ethylacetate-SDS and butan – l – ol were used for separation. Resolution was further increased by the addition of acetonetrile. The method was used to quantify components in plant extracts. Recovery data in the range of 95-104% were reported. Acceptable linearity data of greater than 0.99 was demonstrated for detector response for the 5 active components.

Chiral separation

To-date there has been only one report of chiral resolution employing MEEKC. This paper showed resolution of ephedrine enantiomers using a microemulsion buffer containing a chiral oil (2R, 3R)-di-n-butyl tartrate.²

References

1. Mertzman, M.D. and J.P. Foley. Effect of Low-Interfacial-Tension Oil Substitution in Chiral Microemulsion Electrokinetic Chromatography. Electrophoresis 2004; 25: 723-732.
2. Pascoe, R. and J.P. Foley. Rapid separation of pharmaceutical enantiomers using electrokinetic chromatography with a novel chiral microemulsion. Analyst (2002); 127:710-714.
3. Pedersen-Bjergaard, S., O. Naess, S. Moestue, and K.E. Rasmussen. Microemulsion electrokinetic chromatography in suppressed electroosmotic flow environment: Separation of fat-soluble vitamins, J. Chromatogr. A (2000); 876:201-211.
4. F Sicoli and D Langevin, Shape fluctuations of microemulsion droplets, J.Phys.Chem., 99 (1995) 14819-14823
5. S Terabe, N Matsubara, Y Ishihama and Y Okada, Microemulsion electrokinetic chromatography: comparison with micellar electrokinetic chromatography, J. Chromatogr. , 608 (1992) 23-29)
6. Zhou, G., G. Luo, and X. Zhang. Microemulsion electrokinetic chromatography of proteins. J. Chromatogr. A. (1999); 853:277-248.
7. L Song, Q Ou, W Yu and Ganzuo Li, Separation of six phenylureas and chlorsulfuron standards by micellar, mixed micellar and microemulsion electrokinetic chromatography J. Chromatogr. A,, 699 (1995) 371-382
8. RL Boso, MS Bellini, I Miksik, Z Deyl, Microemulsion electrokinetic chromatography with different organic modifiers: separation of water- and lipid-soluble vitamins J. Chromatogr. A, 709 (1995) 11-19,
9. Altria KD, Application of microemulsion electrokinetic chromatography to the analysis of a wide range of pharmaceuticals and excipients, J.Chromatogr.A, 844 (1999) 371-386
10. X Fu, J Lu and A Zhu, Microemulsion elektrokinetic chromatographic separation of antipyretic analgesic ingredients, J. Chromatogr. A, 735 (1996) 353-356
11. L Vomastova, I Miksik, Z Deyl, Microemulsion and micellar electrokinetic chromatography of steroids, J. Chromatogr. B, 681 (1996) 107-113
12. L Debusschère, C Demesmay, JL Rocca, G Lachatre and H Lofti, Separation of cardiac glycosides by micellar electrokinetic chromatography and microemulsion electrokinetic chromatography, J. Chromatogr. A, 779 (1997) 227-233
13. G Li, Z Chen, M Liu and Z Hu, Separation and identification of active components in the extract of Rheum natural products by microemulsion electrokinetic chromatography Analyst, 123, (1998) 1501-1505

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