MEKC buffer solutions

Because micelles are negatively charged, they migrate toward the cathode with a velocity less than the electroosmotic flow velocity. Neutral species partition themselves between the micelles and the buffer solution in much the same manner as they do in HPLC. Because there is a partitioning between two phases, the term chromatography is used. Note that in MEKC both phases are mobile. ... 

The elution order for neutral species in MEKC depends on the extent to which they partition into the micelles. Hydrophilic neutrals are insoluble in the micelle s hydrophobic inner environment and elute as a single band as they would in CZE. Neutral solutes that are extremely hydrophobic are completely soluble in the micelle, eluting with the micelles as a single band. Those neutral species that exist in a partition equilibrium between the buffer solution and the micelles elute between the completely hydrophilic and completely hydrophobic neutrals. Those neutral species favoring the buffer solution elute before those favoring the micelles. Micellar electrokinetic chromatography has been used to separate a wide variety of samples, including mixtures of pharmaceutical compounds, vitamins, and explosives. 

In this experiment the enantiomers of cyclobarbital and thiopental, and phenobarbital are separated using MEKC with cyclodextran as a chiral selector. By adjusting the pH of the buffer solution and the concentration and type of cyclodextran, students are able to find conditions in which the enantiomers of cyclobarbital and thiopental are resolved. 

Weber, P. L. Buck, D. R. Capillary Electrophoresis A Past and Simple Method for the Determination of the Amino Acid Composition of Proteins, /. Chem. Educ. 1994, 71, 609-612. This experiment describes a method for determining the amino acid composition of cyctochrome c and lysozyme. The proteins are hydrolyzed in acid, and an internal standard of a-aminoadipic acid is added. Derivatization with naphthalene-2,3-dicarboxaldehyde gives derivatives that absorb at 420 nm. Separation is by MEKC using a buffer solution of 50 mM SDS in 20 mM sodium borate. 

MEKC is a CE mode based on the partitioning of compounds between an aqueous and a micellar phase. This analytical technique combines CE as well as LC features and enables the separation of neutral compounds. The buffer solution consists of an aqueous solution containing micelles as a pseudo-stationary phase. The composition and nature of the pseudo-stationary phase can be adjusted but sodium dodecyl sulfate (SDS) remains the most widely used surfactant. 

Micellar electrokinetic capillary chromatography (MEKC or MECC) is a more versatile technique than CZE due to its ability to separate neutral as well as ionic species. The term chromatography is used because a surfactant added to the buffer solution forms spherical aggregates of molecules... 

Different types of EKC have been developed. Cy-clodextrins (CDEKC) have been used to form inclusion complexes with solutes to effect their separation. Other examples of EKC include microemulsion electrokinetic chromatography (MEEKC). The MEKC technique (for a detailed treatise, the reader is referred to Ref. 4) utilizes the presence of micelles in the electrolyte buffer solution to influence the migration time of solutes. In this case, the separation carrier is the micelle [5]. 

The technique is therefore particularly suited to water-soluble species which possess a charge, which includes most dyes. Variations from the main technique (often referred to as capillary zone electrophoresis, CZE) do exist and these include the use of surfactants in the buffer to create micelles (often thought of as creating a pseudo-stationary phase inside the capillary) which then improve the separation of neutral species (and frequently charged molecules) owing to hydrophobic interactions. The term micellar electrokinetic chromatography (MECC or MEKC) is often used for CE separations carried out with the aid of a micellar buffer solution. 

A set of 25 barbiturates was analyzed using CZE and MEKC. Buffers consisting of 90 mM borate, pH 8.4 (CZE), and 20 mM phosphate, 50 mM sodium dodecylsulfate (SDS), pH 7.5 (MEKC). The methods were evaluated for their suitability in systematic toxicological analysis (STA), especially when a combination of methods having a low correlation is used (305). A solid-phase microextraction device in combination with CE for the determination of barbiturates was described (see 306 and Sec. VII). The detection limit for 10 barbiturates was 0.1 ppm in urine, while the limit of detection was about 3 times poorer in bovine serum (306). Polyacrylamide-coated columns have been used for barbiturates and benzodiazepines. Seven kinds of barbiturates were sucessfully separated with the coated columns without further additives (307). The benzodiazepines, which are electrically neutral solutes, were separated in the presence of SDS. The CE method offered fast and efficient separations of the more hydrophobic solutes. 

Fig. 4 Le/i PMMA-based 2D electrophoresis chip made for the multidimensional separation of proteins using SDS p-CGE and MEKC. The solution reservoirs were a) sample reservoir, (h) sample waste reservoir, (c) SDS p-CGE buffer reservoir, d) SDS p-CGE buffer waste reservoir, (e) MEKC buffer reservoir, if) MEKC buffer waste reservoir. SDS p-CGE channel Injection length 10 mm, separation length 40 mm, effective separation length = 30 mm MEKC channel separation length 25 mm, effective separation length 10 mm. Right. 2D separation of a fecal calf serum proteome using SDS p-CGE/MEKC. The 2D SDS p-CGE x MEKC were performed at 300 V/cm and 400 V/cm, respectively. A 10 s separation time was utilized in the first dimension prior to performing the serial 10 s MEKC cycles. A total of 159 MEKC cycles was used with a 1 s transfer time from the first to second dimension. All of the proteins were labeled with a fluorescent dye prior to the separation. Reprinted with permission from Shadpour and Soper [81] and Osiri et al. [82]...

Monomeric nucleosides and nucleotides, which appear as impurities in oligonucleotide synthesis, may be analyzed using the method of micellar electrokinetic chromatography (MEKC). With addition of SDS to the buffer solution, the surfactant concentration is above the critical micellar concentration. Movement of the micelles towards the anode against the EOF involves distribution processes, in which also nucleosides, nucleobases and nucleotides participate. Typical conditions are 50 mM phosphate buffer 40 mM SDS pH 6.5 20 kV (116 mA) 21 °C [186]. 

The composition of the micellar buffer solution can be changed in many ways in order to optimize the separations. The nature of the surfactant, that is, charge and concentration, the use of additives such as organic solvents, urea, and CDs can be altered in order to manipulate the separation of an analyte mixture. Books by Pyell [51] and Baker [31] dedicate chapters to more detailed discussions of MEKC theory and practice. Review papers by Pappas et al. [52], Molina and Silva [53], and Pyell [54] also cover many MEKC applications and development options. 

Procedure. A vitamin B complex tablet Is crushed and placed In a beaker with 20.00 mL of a 50% v/v methanol solution that Is 20 mM In sodium tetraborate and contains 100.0 ppm of o-ethoxybenzamIde. After mixing for 2 min to ensure that the B vitamins are dissolved, a 5.00-mL portion Is passed through a 0.45- xm filter to remove Insoluble binders. An approximately 4-nL sample Is loaded Into a 50- xm Internal diameter capillary column. For CZE the capillary column contains a 20 mM pH 9 sodium tetraborate/sodlum dIhydrogen phosphate buffer. For MEKC the buffer Is also 150 mM In sodium dodecylsulfate. A 40-kV/m electric field Is used to effect both the CZE and MEKC separations. 

In order to separate neutral compounds, Terabe et al. [13] added surfactants to the buffer electrolyte. Above their critical micellar concentration (cmc), these surfactants form micelles in the aqueous solution of the buffer electrolyte. The technique is then called Micellar electrokinetic capillary chromatography, abbreviated as MECC or MEKC. Micelles are dynamic structures consisting of aggregates of surfactant molecules. They are highly hydrophobic in their inner structure and hydrophilic at the outer part. The micelles are usually... 

Fig. 2.166. Separation of xanthomonasin-A and remaining Trp-P-2(NHOH) in the short-contact mode (a) and in the long-contact mode (b). (a) In the short-contact mode, the MEKC conditions were as follows capillary, 50 pm i.d. X 36 cm running solution, 10 mM SC solution in 25 mM phosphate buffer at pH 7 applied voltage, 15 kV temperature, 20°C detection, 264 nm concentration, O.lmM xanthomonasin A and O.lmM Trp-P-2(NHOH). (b) In the long-contact mode, the MEKC conditions were running solution, 10 mM SC solution in 25 mM phosphate buffer containing O.lmM xanthomonasin A at pH 7. The negative shift of the baseline (indicated by the arrow) shows that the zone of xanthomonasin-A passed the detection window. The other conditions are the same as those in the short-contact mode. Reprinted with permission from T. Watanabe el al. [338]...

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