Micellar mobile phase anions

High-Performance Liquid Chromatography of Organic and Inorganic Anions Use of Micellar Mobile Phase... 

The separation of anions by the use of a cationic micellar mobile phase results in a high degree of flexibility not available from other methods of ion chromatography. The importance of micelles in the mobile phase lies in their ability to participate in the partitioning mechanism. The three equilibria involved in micellar chromatography are schematically represented in Figure 1. The elution behaviour of the anionic solute depends on three partition coefficients K p, the partition coefficient between the bulk mobile phase and and the micelle K nn the partition coefficient between the bonded phase and the micelle and K mpi the partition coefficient between the bonded phase and the bulk mobile phase. 

Mullins and Kirkbright (38) reported separation of the UV absorbing anions iodate, nitrite, bromide, nitrate and iodide using a micellar mobile phase containing hexadecyltrimethylammonium chloride above its CMC. Figure 2 illustrates this separation with two different concentrations of micellar reagent. Increasing the concentration of hexadecyltrimethylammonium chloride decreases the retention time (38) on the column. 

The retention of the anions on a loaded octadecyl-bonded silica column in the presence of hexadecyltrimethylammonium chloride micellar mobile phase follows the order... 

This order is similar to the anion selectivity order found on a typical strongly basic anion exchanger. Figure 2(b) illustrates a separation of five inorganic anions with a 1.36 x 10 1M hexadecyltrimethylammonium chloride micellar mobile phase. No buffer salts or organic modifier were used in order to accomplish this separation. 

The above results show that conventional HPLC can be used with UV detection for the determination of inorganic anions, namely 103, N02-, Br-, N03 and I" with a cationic micellar mobile phase. One of the attractive features of this procedure is the ability to control retention by control of the concentration of aqueous micellar hexadecyltrimethylammonium chloride rather than by the use of organic modifiers. 

It is always recommended to use the same column with the same type of surfactant. A column should be dedicated to the anionic surfactants, a second one to the cationic surfactants, etc. The reproducibility of the results in MLC depends on the column equilibration. The adsorbed layer of surfactant should be done correctly. It was shown that the time to reach the equilibrium between the stationary phase and the mobile phase could be very long in ion-pair chromatography with sub-micellar mobile phases. Two days at 1 mL/min were necessary to equilibrate a 15 cm x 4.6 mm i.d. column of Hypersil ODS with a mobile phase containing 0.0003 M CTAB [19]. These low surfactant concentration solutions do not contain micelles. So, they are not used in MLC. With a micellar phase, the equilibration time is reduced. It is possible to use the rapid gradient capability just mentioned above. Typically, a mobile phase containing a high surfactant concentration (10 to 100 cmc) can be used to quickly saturate the column with surfactant. Then 5 to 10 column volumes are used to rinse the column with the mobile phase containing the desired amount of surfactant. 

F.G.P. Mullins and G.F. Kirkbright, Determination of Inorganic Anions by HPLC using a Micellar Mobile Phase, Analyst, 109 1217 (1984). 

Micellar mobile phases of anionic, cationic, nonionic and zwitterionic surfactants are used in conjunction with different bonded stationary phases (including C8, Cl8 and cyano). Considerably less surfactant (usually <0.2 M) is used compared to the organic modifier content in an analogous traditional separation. A variation in the concentration of surfactant is translated into an increase in the concentration of micelles in the solution, whereas the number of monomers of surfactant remain constant. As a consequence, the characteristics of the stationary phase modified by the adsorption of surfactant are very stable, and usually, a regular retention behavior is observed as a function of the concentration of surfactant. 

The retention of weak organic acids and bases is affected by the pH of the micellar mobile phase. Solute-micelle binding constants of the dissociated and undissociated forms of a compound with anionic or cationic micelles are... 

For monoprotic systems, the apparent protonation constant can be determined by measuring the retention factor at several pH values and fitting the experimental data to eq. 8.12, via nonlinear regression. A similar equation can be used for diprotic systems. Figure 8.2 shows micellar-induced shifts of the apparent protonation constmits, with increasing anionic micelles. The logarithm of the first protonation constant (log Kh = pK ) for tryptophan, phenylalanine and lysine in water is approximately 2.4. For the micellar mobile phase, log Kh are between 3.60 and 4.56. Therefore, a... 

The interactions of the amino acid derivatives with a modified C18 column and micellar mobile phases of the anionic SDS are mainly of hydrophobic and electrostatic nature. The basic structure of the amino acid isoindoles is the same. Consequently, in the absence of electrostatic interactions, the hydrophobic character of the Rj substituent should be responsible of the retention. The nitrogen atom in the isoindole heterocycle is nonprotonated at pH 3, and thus, electrostatic interactions can only exist with ionizable groups giving a positive charge to R in the molecule. This occurs with arginine and histidine, which have amino groups that do not react with OPA. 

Surprisingly, almost all the applied work in MLC to date appears to have involved only ionic micellar mobile phases composed of either anionic sodium dodecyl sulfate (SDS), or cationic hexadecyltrimethyl-ammonium bromide or chloride (CTAB or CTAC), and dodecyltrimethyl-ammonium bromide (DTAB). Reports on the use of unclmrg nonionic surfactants, such as Neodol 91-6 and polyoxyethylene(23) dodecanol (Brij 35) are exceptions. 

The pH of the micellar mobile phase is an important factor for the analysis of ionizable drugs using nonpolar column stationary phases. Fig. 11.7 shows the chromatograms of acetylsalicylic acid (log Kh = 3.5) in a serum sample at pH 3.0 and 6.5, eluted from a CIS column with a 0.08 M SDS mobile phase [20]. It can be seen that at the higher pH, the peak for this drug is not evident. The anionic solute probably eluted with the serum proteins. However, when pH was reduced to 3.5, the neutral form of the drag eluted at approximately 3.5 min, appreciably distinct from the serum components. 

Anions will be separated by cationic micellar phases. Kirkbright and Mullins obtained the elution order T> N03 > Br > N02 > lOj with a 0.14 M cetyltrimethylammonium chloride (CTAC) micellar mobile phase and a C18 Spherisorb column [29]. This is the usual eluotropic order obtained with strong basic anion exclmngers. They showed that the ion retention decreased with the increase of both the ionic strength, p, and the surfactant concentration. Plots of 1/k vs. and 1/k vs. [CTAC] were linear. The first linear relationship is typical of ion-exchange mechanisms, the second plot means that the anion-micelle interaction obeys the Armstrong-Nome model for molecule-micelle partition. 

If the cations are separated directly with an anionic micellar mobile phase, the equations derived for the MLC anion separation (eqs. 13.6, 13.7 and 13.S) can be used. Most often a complexing agent, a ligand H2L, is added to the micellar solution forming the following equilibria with the cation, M ... 

In MEKC, the supporting electrolyte medium contains a surfactant at a concentration above its critical micelle concentration (CMC). The surfactant self-aggregates in the aqueous medium and forms micelles whose hydrophilic head groups and hydrophobic tail groups form a nonpolar core into which the solutes can partition. The micelles are anionic on their surface, and they migrate in the opposite direction to the electroosmotic flow under the applied current. The differential partitioning of neutral molecules between the buffered aqueous mobile phase and the micellar pseudostationary phase is the sole basis for separation as the buffer and micelles form a two-phase system, and the analyte partitions between them (Smyth and McClean 1998). 

More importantly, the use of heavy metal anionic micellar media has been shown to allow for observation of analytically useful room-temperature liquid phosphorescence (RTLP) (7.484.487). There are several examples in which phosphorescence has been employed as a LC detector with the required micellar assembly being present as part of the LC mobile phase (482) or added post column (485). More recently, metal ions have been determined in a coacervate scum by utilizing the micellar-stabilized RTLP approach (498). Thus, the future should see further development in RTLP detection of metal ions in separation science applications. 

The decrease in retention of the anions as the concentration of hexadecyltrimethylammonium chloride is increased (Figure 2) can be attributed to anion interaction with micelles in the mobile phase. The retention time of the selected anions can be reduced by increasing the concentration of the micellar reagent (Figure 2(a),... 

A linear behavior between k and n was found for n-alkylbenzenes eluted with pure micellar eluents of the anionic sodium dodecyl sulfate (SDS), cationic cetyltrimethylammonium bromide (CTAB), and nonionic poly[oxyethylene(10 or 23)]dodecanol (Brij 22 or Brij 35), and with hybrid eluents containing SDS or CTAB and 2-propanol. The homologues of n-alkylphenones eluted with pure and hybrid mobile phases of CTAB showed the same behavior, but linear relationships were observed between log k and nc for these compounds eluted with SDS mobile phases, the same type of correlation found in aqueous-organic systems. It seems that the... 

A method was described for the RPLC determination of recombinant methionylaspartyl-human growth hormone (MD-HGH) in Escherichia coli (E. coli) fermentation broth [7], which utilizes mobile phases containing the anionic surfactant SDS and 1-propanol, under micellar conditions. A C4 column was used at 60°C for the separation. The methodology is directly applicable to the analysis of samples solubilized via sulfitolysis in the presence of SDS, and offers superior resolution in comparison with chromatography in the absence of the surfactant. 

---