The development of a novel monolithic column inapillary electrochromatography and on-line pre-concentration for the determination of phenolic acids in barley by micellar electrokinetic chromatography
Date Issued
2009
Date
2009
Author(s)
Wang, Guan-Ren
Abstract
A novel polymer-based capillary electrochromatography (CEC) monolithic column was prepared using an o-phthalaldehyde-L-phenylalanine Schiff base complex as the reactive center and a mixture of methanol and n-propanol as the porogen. This monolithic column was employed for the separation of a metal ion mixture. Tetrabutylammonium bromide (TBAB) was used as a mobile phase additive to enhance the separation efficiency of metal ions by EDTA precomplexation. Using a phosphate buffer (20 mM, pH 3.0), TBAB (10 mM), MeOH (15%, v/v) an applied voltage of -15 kV, and detection at 220 nm, the metal ion mixture was satisfactorily resolved. The theoretical plate number was between 10,500-30,800 plates/m. The separation was also carried out in the absence of TBAB, leading to a dissimilar elution order and shorter retention times. The separation behavior of the monolithic column was also compared with that of the blank polymer. The unique properties of the monolithic column might be mediated by a combination of electrophoretic behavior and chromatographic retention, involving hydrophobic and hydrophilic interactions, as well as ligand exchange. Different mobile phases have also been investigated to study their effects on retention behavior. Using EDTA was found to be superior to phosphate buffer. Under the condition of EDTA (20 mM, pH 3.5) and TBAB (10 mM), the theoretical plate number was between 20,300-64,100 plates/m. The results imply that the separation needs a low dielectric constant solvent when a weak ligand is used as the mobile phase. The established method was applied to determine the metal ions in lake water sampled from the NTU campus. The sample was analyzed directly and without any preliminary treatment; baseline separation indicated that the monolithic column was highly promising for the separation of metal ions in this complex matrix sample. Another application of the novel column was to the separation of hemoglobin (Hb) and myoglobin (Mb). Here, note that 3-sulfopropyl methacrylate potassium salt was added as a second functional monomer for the in situ polymerization to help prevent irreversible adsorption of proteins on the stationary phase. Several kinds of buffer have been tested as the mobile phase. Baseline separation of Hb and Mb could be achieved with borate buffer (20 mM, pH 9.0), Tris buffer (40 mM, pH 9.0) or EDTA buffer (10 mM, pH 9.0) at 214 nm and 15 kV. The separation mechanism might be attributable to π-π interactions and hydrophobic forces between the proteins and the L-phenylalanine and the functional monomers. Phenolic acids are plant metabolites that are wide spread throughout the plant kingdom. Recent interest in phenolic acids stems from their potential protective role against oxidative damage and related diseases. Micellar electrokinetic chromatography (MEKC), seldom operated at acidic conditions, was adopted for the separation of phenolic acids. Using an SDS (100 mM)/phosphate buffer (50 mM, pH 3.0), an applied voltage of -15 kV and detection at 214 nm, the baseline separation of a mixture of ten phenolic acids was satisfactorily achieved within 25 min. The relative standard deviations (RSD) of the retention times were less than 1.94%. The number of theoretical plates was 10,500-11,900 plates/m. The established method was applied for the determination of phenolic acids in barley extract. With the addition of methanol (20%, v/v), only 4-hydroxybenzoic acid was found. Many sample peaks were concealed by the large sample matrix effect. By using 280 nm as the analytical wavelength, the matrix effect can be overcome; seven peaks could then be detected. The elution was in the following order: ferulic acid, coumaric acid, chlorogenic acid, caffeic acid, vanillic acid, p-hydroxybenzoic acid and protocatechuic acid. For analysis of phenolic acids in barley, a pH-junction for on-line pre-concentration was explored. The capillary was filled with phosphate buffer (100 mM, pH 2.5). The analytes, in borate buffer (50 mM, pH 9.0), were then injected in electrokinetic mode (-15 kV, 20 min). With SDS/phosphate buffer (60/50 mM, pH 2.5) as the mobile phase, phenolic acids in barley extract could be detected at an applied voltage of -15 kV, and detection at 280 nm. A sensitivity enhancement of ~300-760-fold could be achieved and regression coefficients (r2) are above 0.997.The theoretical plate number was 24,900-35,800 plates /m.
Subjects
CEC
monolithic column
MEKC
on-line pre-concentration
Type
thesis
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