Katalin Ganzler

Microwave extraction. A novel sample preparation method for chromatography.

The applicability of microwave irradiation to the extraction of various types of compounds from soil, seeds, foods and feeds as a novel sample preparation method for chromatography was investigated. Samples were ground and mixed with an appropriate solvent, methanol or methanol-water for polar compounds and hexane for non-polar compounds. The suspensions were irradiated for 30 s, but they were not allowed to boil. After cooling, the irradiation was repeated several times. The samples were then centrifuged, and aliquots of the supernatant were injected into a chromatographic column. The yields of the extracted compounds obtained by microwave irradiation were compared with those obtained by the traditional Soxhlet or shake-flask extraction methods. The microwave extraction method was more effective than the conventional methods. Due to the considerable savings in time and energy, this novel method is suitable for fast extractions of large sample series.

Comparative study of new shell-type, sub-2 μm fully porous and monolith stationary phases, focusing on mass-transfer resistance

Today sub-2 microm packed columns are very popular to conduct fast chromatographic separations. The mass-transfer resistance depends on the particle size but some practical limits exist not to reach the theoretically expected plate height and mass-transfer resistance. Another approach applies particles with shortened diffusion path to enhance the efficiency of separations. In this study a systematical evaluation of the possibilities of the separations obtained with 5 cm long narrow bore columns packed with new 2.6 microm shell particles (1.9 microm nonporous core surrounded by a 0.35 microm porous shell, Kinetex, Core-Shell), packed with other shell-type particles (Ascentis Express, Fused-Core), totally porous sub-2 microm particles and a 5 cm long narrow bore monolith column is presented. The different commercially available columns were compared by using van Deemter, Knox and kinetic plots. Theoretical Poppe plots were constructed for each column to compare their kinetic performance. Data are presented on polar neutral real-life analytes. Comparison of a low molecular weight compounds (MW=270-430) and a high molecular weight one (MW approximately 900) was conducted. This study proves that the Kinetex column packed with 2.6 microm shell particles is worthy of rivaling to sub-2 microm columns and other commercially available shell-type packings (Ascentis Express or Halo), both for small and large molecule separation. The Kinetex column offers a very flat C term. Utilizing this feature, high flow rates can be applied to accomplish very fast separations without significant loss in efficiency.

Determination of biogenic amines by capillary electrophoresis

A method for determining biogenic amines in food using micellar electrokinetic capillary chromatography has been developed. Derivatization of the amines was performed with AccQ (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate; Waters, Milford, MA, USA) reagent. The influence of buffer composition on the separation (including pH, SDS concentration and various additives) was investigated. The separation of seven biogenic amines (histamine, tyramine, tryptamine, spermine, spermidine, cadaverine and putrescine) could be achieved within 25-30 min with good repeatability. The biogenic amine profiles in three different food samples (wine, salami and chive) were determined and quantitated.

Effective sample preparation method for extracting biologically active compounds from different matrices by a microwave technique

A method is described for extracting lupin alkaloid (sparteine) and drug metabolites from different matrices (seeds and rat faeces) using microwave energy and for checking the homogeneity of the electric field in the microwave oven. The high-performance liquid chromatographic separation and determination of the extracted compounds showed that the microwave extraction method is more advantageous than other traditional extraction methods with regard to the yield of extraction and the time and cost of the procedure. The potential degradation of the extracted compounds may be considerably reduced.

Efficiency of the new sub-2 μm core-shell (Kinetex™) column in practice, applied for small and large molecule separation.

At present sub-2 μm packed columns are very popular to accomplish rapid and efficient separations. Applying particles with shortened diffusion path to improve the efficiency of separation performs higher efficiency than it is possible with the totally porous particles having the same size. The advantages of sub-2 μm particles and shell particles are combined in the new Kinetex 1.7 μm particles. In this study a systematical evaluation of the efficiency and achievable analysis time obtained with 5 cm long narrow bore column packed with sub-2 μm core-shell particles (1.25 μm core diameter and 0.23 μm porous silica layer), and other type very efficient columns is presented. The efficiency of separation was investigated also for small pharmaceutical and large molecules (proteins). Van Deemter, Knox and kinetic plots are calculated. The results obtained with low molecular weight polar neutral analytes (272 g/mol, 875 g/mol), with a polypeptide (4.1 kDa) and with different sized proteins (18.8 kDa, 38.9 kDa and 66.3 kDa) are presented in this study. Moreover, particle size distribution, and average pore size (low-temperature nitrogen adsorption, LTNA) of the new very fine core-shell particles were investigated. According to this study, increased flow rates can be applied on sub-2 μm core-shell columns to accomplish very fast separations without significant loss in efficiency. The new sub-2 μm shell particles offer very high efficiency both for small and large molecule separation.

Use of cyclodextrins and cyclodextrin derivatives in high-performance liquid chromatography and capillary electrophoresis

Carboxymethyl- and carboxyethyl-β-cyclodextrin have proved to be effective chiral mobile phase additives in HPLC for the resolution of enantiomers of hexobarbital, ephedrine and an aminomethylbenzodioxane derivative. The separations are influenced by a combination of hydrophobic and electrostatic interactions. The type and concentration of the cyclodextrin derivative and the pH and type of base used to adjust the pH can be varied in order to optimize the method. In addition, a coating procedure was developed for capillary electrophoresis. The enantiomers of epinephrine were resolved on a γ-cyclodextrin-coated capillary. The length of the capillary has a significant effect on the resolution. β-Cyclodextrin was found to give no resolution.

Shell and small particles; Evaluation of new column technology

The performance of 5 cm long columns packed with shell particles was compared to totally porous sub-2 microm particles in gradient and isocratic elution separations of hormones (dienogest, finasteride, gestodene, levonorgestrel, estradiol, ethinylestradiol, noretistherone acetate, bicalutamide and tibolone). Peak capacities around 140-150 could be achieved in 25 min with the 5 cm long columns. The Ascentis Express column (packed with 2.7 microm shell particles) showed similar efficiency to sub-2 microm particles under gradient conditions. Applying isocratic separation, the column of 2.7 microm shell particles had a reduced plate height minimum of approximately h=1.6. It was much smaller than obtained with totally porous particles (h approximately = 2.8). The impedance time also proved more favorable with 2.7 microm shell particles than with totally porous particles. The influence of extra-column volume on column efficiency was investigated. The extra-column dispersion of the chromatographic system may cause a shift of the HETP curves.

Effect of the degree of substitution of cyclodextrin derivatives on chiral separations by high-performance liquid chromatography and capillary electrophoresis

Optical isomers of some basic racemic drugs (oxprenolol, AMEBD, ephedrine) were separated by high-performance liquid chromatography (HPLC) and/or capillary electrophoresis (CE) using carboxymethyl-beta-cyclodextrin (CMBCD) with various degree of substitution (DS). The effects of the separation conditions (pH, concentration and DS of CMBCD) were studied and compared using CE and HPLC. The degree of substitution had a significant effect on the resolution of the optical isomers and the ionic strength of the separation media, hence the use of well characterized CD derivatives is crucial. Different optimum DS values for the same test samples were obtained when HPLC or CE was used.

Facts and myths about columns packed with sub-3 μm and sub-2 μm particles

Increasing the separating efficiency enhances the separation power. The most popular solution for improving chromatographic performance is to employ columns packed with small particle diameters (i.e., sub-2 microm particles) to induce a simultaneous improvement in efficiency, optimal velocity and mass transfer, albeit the cost of pressure. In this study a systematic evaluation of the possibilities and limitations of the separations obtained with 5 cm long narrow bore columns packed with 1.5-3.0 microm particles is presented. Several commercially available different sub-3 microm and sub-2 microm packed columns were evaluated by using van Deemter, Knox and kinetic plots. Theoretical Poppe plots were constructed for each column to compare their kinetic performance. Data are presented on different polar neutral real life analytes, to show that the separation time is not obviously shorter if the particle size is reduced. Comparison of low-molecular weight compounds (one steroid and one non-steroid hormone, with molecular weights lower than 500) and a high-molecular weight one (MW approximately 1000) was conducted. Same efficiency can be achieved with columns packed with 1.9-2.1 microm particles as with smaller particles. The column packed with 3 microm particles had the lowest reduced plate height minimum (h=2.2) while the column with the smallest particles (1.5 microm) gave the highest reduced plate height minimum (h approximately 3.0). According to this study, the theoretically expected efficiency of very fine particles (diameter <2 microm) used in practice today is compromised. Investigation of this phenomenon is presented.

Characterization of new types of stationary phases for fast liquid chromatographic applications

The performance of a narrow bore silica based monolith column (5 cm x 2 mm) was compared to 5 cm long narrow bore (internal diameter < or = 2.1 mm) columns, packed with shell particles (2.7 microm) and totally porous sub-2 microm particles (1.5 microm, 1.7 microm and 1.9 microm) in gradient and isocratic elution separations of steroids. The highest peak capacity could be achieved with the column packed with 1.5 microm totally porous particles. The columns packed with porous 1.7 microm and shell 2.7 microm particles showed very similar capacity. The monolith column provided the lowest capacity during gradient elution. The plate height (HETP) of the 2.7 microm Ascentis Express column was very similar to the HETP obtained with 1.5 microm and 1.7 microm totally porous particles. The Chromolith monolithic column displayed an efficiency that is comparable to that of columns packed with spherical particles having their diameter between 3 microm and 4 microm. A kinetic plot analysis is presented to compare the theoretical analysis speed of different separation media. At 200 bar, the monolith column provided the highest performance when the required plate number was higher than 5000 (N>5000), however the efficiency drifted off faster in the range of N<5000 than in the case of packed columns. If the possibility of maximum performance was utilized (1000 bar for sub-2 microm particles, 600 bar for shell particles and 200 bar for monolith column) the monolith column would provide the poorest efficiency, while the column, packed with 1.5 microm particles offered the shortest impedance time.