Marcus Öhman

Bonded dimethylacrylamide as a permanent coating for capillary electrophoresis

A method for coating capillaries for capillary electrophoresis with chemically bonded polydimethylacrylamide has been developed, and the properties of the capillaries have been evaluated. The coated capillaries provided high separation efficiency, 12 x 10(5) theoretical plates/m was obtained for cytochrome c. The electroosmotic flow at pH 8.0 was 10 x 10(-10) to 6 x 10(-10) m2 V(-1) s(-1). The coated capillaries were quite stable at high pH. At least 150 runs could be done at pH 10 without appreciable performance deterioration. The excellent performance of the coated capillaries was illustrated by separation of basic proteins, acidic proteins, 9-fluorenylmethyl chloroformate-derivatized neurotransmitter amino acids, peptide reference mixtures and peptides digested from a bacteria protein.

Separation of conjugated trienoic fatty acid isomers by capillary electrophoresis.

A method for direct resolution of conjugated trienoic fatty acid isomers by capillary electrophoresis has been developed. To obtain complete separation a dual cyclodextrin system was used. This contained heptakis-(6-sulfo)-beta-cyclodextrin (charged). Beta-cyclodextrin (uncharged) and sodium dodecylsulfate. Under optimized conditions, all seven isomers were well separated. On average, separation efficiency was 2.9 x 10(5) plates/m.

Methacrylate monolithic capillary columns for gradient peptide separations

For the separation of peptides with gradient-elution liquid chromatography a poly(butyl methacrylate-co-ethylene dimethacrylate) (BMA) monolithic capillary column was prepared and tested. The conditional peak capacity was used as a metric for the performance of this column, which was compared with a capillary column packed with C18-modified silica particles. The retention of the peptides was found to be smaller on the BMA column than on the particulate C18 column. To obtain the same retention in isocratic elution an approximately 15% (v/v) lower acetonitrile concentration had to be used in the mobile phase. The retention window in gradient elution was correspondingly smaller with the BMA column. The relation between peak width and retention under gradient conditions was studied in detail. It was found that in shallow gradients, with gradient times of 30min and more, the peak widths of the least retained compounds are strongly increased with the BMA column. This was attributed to the fact that these compounds migrate and elute with an unfavorable high retention factor. More retained compounds are eluted later in the gradient, but with a lower effective retention factor. With shallow gradients the peak capacity of the BMA column ( approximately 90) was clearly lower than that of a conventional packed column ( approximately 150). On the other hand, with steep gradients, when components elute with a low effective retention factor, the performance of the BMA column is relatively good. With a gradient time of 15min similar peak widths and thus similar peak capacities ( approximately 75) were found for the packed and the monolithic column. Two strategies were investigated to obtain higher peak capacities with methacrylate monolithic columns. The use of lauryl methacrylate (LMA) instead of butyl methacrylate (BMA) gave an increase in retention and narrower peaks for early eluting peptides. The peak capacity of the LMA column was approximately 125 in a 60min gradient. Another approach was to use a longer BMA column which resulted in a peak capacity of approximately 135 could be obtained in 60min.

Separation of conjugated linoleic acid isomers and parinaric fatty acid isomers by capillary electrophoresis

Separation of conjugated linoleic acid isomers and parinaric fatty acid isomers by capillary electrophoresis

Chemometric modeling of neurotransmitter amino acid separation in normal and reversed migration micellar electrokinetic chromatography

A chemometric experimental design has been applied for the optimization of neurotransmitter amino acid separation in capillary electrophoresis. The optimizations were carried out for normal micellar electrokinetic chromatography (N-MEKC) and reversed migration micellar electrokinetic chromatography (RM-MEKC). In order to optimize three separation factors and study the interaction between factors, a response function was optimized via searching its optimum (minimum/maximum). For this purpose a central composite design with multivariate linear regression (MLR) analysis was utilized. Modeling with good regression coefficients from the MLR adequately described the interaction of factors such as background electrolyte and sodium dodecylsulfate concentrations which had a large impact on selectivity and migration behaviors. Similar optimal conditions regarding resolution and number of theoretical plates but different retention behaviors as a function of background electrolyte and micellar concentrations were observed for N-MEKC and RM-MEKC. Improved overall performance from the RM-MEKC separation of five neurotransmitter acids, superior to N-MEKC, is demonstrated in terms of repeatability, peak symmetry, sensitivity, and in particular, impurity determination in an overloaded separation system.

Separation of divinyl ether fatty acid isomers by micellar electrokinetic chromatography

A micellar electrokinetic chromatography (MEKC) method has been developed for the direct resolution of divinyl ether type of hydrophobic fatty acid isomers. The fatty acid isomers resolved include colneleic acid (CL), colnelenic acid (CLn), 14(Z)‐etheroleic acid (14(Z)‐EL), 14(Z)‐etherolenic acid (14(Z)‐Eln), 11(Z)‐etheroleic acid (11(Z)‐EL), 11(Z)‐etherolenic acid (11(Z)‐Eln), etheroleic acid (EL) and etherolenic acid (Eln). These fatty acid isomers differ in number, position and spatial arrangement of the double bonds and the position of the ether oxygen. A central composite design was employed for the optimization of the key variables of the separation, namely the concentrations of sodium dodecyl sulfate (SDS) and organic modifiers. The use of micelles combined with an organic modifier in the background electrolyte made it possible to dissolve and separate relatively hydrophobic fatty acid isomers, and to achieve high separation efficiency. Using heptakis‐(2,3‐dimethyl‐6‐sulfato)‐β‐cyclodextrin (HDMS‐β‐CD) as a buffer additive, complete separation of the examined eight divinyl ethers was achieved. Separation efficiencies up to 5 × 105 theoretical plates/m were achieved under optimized conditions. Direct UV was applied for detection of the fatty acids. The results were compared with those obtained from high‐performance liquid chromatography (HPLC) separation.

Methacrylate monolithic stationary phases for gradient elution separations in microfluidic devices

Methacrylate monolithic stationary phases were produced in fused-silica chips by UV initiation. Poly(butyl methacrylate-co-ethylene dimethacrylate) (BMA) and poly(lauryl methacrylate-co-ethylene dimethacrylate) (LMA) monoliths containing 30, 35 and 40% monomers were evaluated for the separation of peptides under gradient conditions. The peak capacity was used as an objective tool for the evaluation of the separation performance. LMA monoliths of the highest density gave the highest peak capacities (≈40) in gradients of 15 min and all LMA monoliths gave higher peak capacities than the BMA monoliths with the same percentage of monomers. Increasing the gradient duration to 30 min did not increase the peak capacity significantly. However, running fast (5 min) gradients provides moderate peak capacities (≈20) in a short time. Due to the system dead volume of 1 μL and the low bed volume of the chip, early eluting peptides migrated over a significant part of the column during the dwell time under isocratic conditions. It was shown that this could explain an increased band broadening on the monolithic stationary phase materials used. The effect is stronger with BMA monoliths, which partly explains the inferior performance of this material with respect to peak capacity. The configuration of the connections on the chip appeared to be critical when fast analyses were performed at pressures above 20 bar.