Vladislav Kahle

Zwitterionic silica-based monolithic capillary columns for isocratic and gradient hydrophilic interaction liquid chromatography

This study introduces zwitterionic monolithic capillary columns intended for isocratic and gradient HILIC separations. Silica-based monolithic capillary columns (100 μm × 150 mm) prepared by acidic hydrolysis of tetramethoxysilane in the presence of polyethylene glycol and urea were modified by a sulfoalkylbetaine zwitterion ([2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide) to HILIC stationary phase. The prepared columns were evaluated under the isocratic and gradient separation conditions using a standard mixture containing nucleic acid bases, nucleosides, and 2-deoxynucleosides. Mobile phases contained high concentration of acetonitrile (95-85%, v/v) and 5-50mM ammonium acetate or ammonium formate in the pH range of 3-6. The synthesized columns showed a long-term stability under the separation conditions while the high permeability and efficiency originating from dual structure of the silica monolith were preserved. The relative standard deviations (RSDs) for the retention times of tested solutes were lower than 2% under the isocratic conditions and lower than 3.5% under the gradient conditions.

Short monolithic columns for purification and fractionation of peptide samples for matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis in proteomics.

This study records a novel application of methacrylate-based monolithic columns for MALDI-TOF/TOF MS analyses in proteomics for pre-concentration and separation of peptides derived from protein digestion. Reversed-phase monolithic capillary columns (30mm x 0.32mm i.d.) were created inside the fused silica capillary via thermal-initiated free-radical polymerization of ethylene glycol dimethacrylate and lauryl methacrylate monomers in the presence of 1-propanol and 1,4-butandiol as a porogen system. The elution of peptides was achieved using a linear gradient of acetonitrile from 0 to 60% in water with 0.1% trifluoroacetic acid formed in a microsyringe. Individual fractions of separated peptides were collected on the MALDI target spots covered with alpha-cyano-4-hydroxycinnamic acid used as a matrix and then they were analyzed using MALDI-TOF/TOF mass spectrometry. The developed method was tested with a mixture of tryptic peptides from bovine serum albumin and its applicability was also tested for tryptic in-gel digests from barley grain extracts of water soluble proteins separated using SDS gel electrophoresis. The number of detected peptides was approximately three to four times higher compared to the analysis without previous separation. These results show an improved quality of sample information with the higher amount of identified peptides which increased protein sequence coverage and improved sensitivity of mass spectrometry measurements.

Capillary electromigration separation of proteins and microorganisms dynamically modified by chromophoric nonionogenic surfactant.

A chromophoric nonionogenic surfactant poly(ethylene glycol) 3-(2-hydroxy-5-n-octylphenylazo)-benzoate, HOPAB, has been prepared and used as a buffer additive for a dynamic modification of proteins and/or microorganisms including Escherichia coli , Staphylococcus epidermidis (biofilm-positive and biofilm-negative), and the strains of yeast cells Candida albicans and Candida parapsilosis (biofilm-positive and biofilm-negative) during a capillary electrophoresis and a capillary isoelectric focusing (CIEF) with UV detection at 326 nm. Values of isoelectric points of labeled proteins and microorganisms have been calculated using UV-detectable pI markers and have been found comparable with pI of the native compounds. Minimum detectable amount has been assessed lower than picograms of proteins and lower than a hundred cells injected into a separation capillary. The introduced labeling method facilitates CIEF separation of microorganisms from the clinical sample of the infected urine at their clinically important levels in the pH gradient pH range of 2-5 and their subsequent cultivation. At the same time, it has enabled the determination of albumin in human urine as a major clinical marker of urinary tract infections and kidney diseases.

Automated microgradient system for capillary electrochromatography

A microprocessor controlled gradient elution system suitable for capillary electrochromatography has been developed and tested. It is based on a liquid handling device described previously which is capable of liquid transport with both low and high fluid dispersion. The low dispersion region formed by stainless steel needle 250 microm I.D. serves for sample injection, while the high dispersion region, created by steep extension of tube diameter, is used for continuous mobile phase gradient generation. A homologous series of seven alkylphenones was electrochromatographically separated on a monolithic polyacrylic column under gradient conditions. An S-shaped acetonitrile gradient (30-70%) was applied. A high reproducibility of retention times (RSD about 0.1%) was obtained, indicating accuracy of automated gradient operations.

Silica-based monolithic capillary columns—Effect of preparation temperature on separation efficiency

The temperature effects during the sol-gel process and ageing of the silica-based monolith on the structure and separation efficiency of the capillary columns (100microm i.d., 150mm) for HPLC separations were studied. The tested columns were synthesized from a mixture of tetramethoxysilane, polyethylene glycol and urea under the acidic conditions. The temperature was varied from 40 degrees C to 44 degrees C and formation of bypass channels between the silica mold and the capillary wall was examined. The temperature of 43 degrees C was estimated as optimal for preparation of efficient silica capillary columns which were subsequently modified by octadecyldimethyl-N,N-diethylaminosilane or covered by poly(octadecyl methacrylate) and tested using standard mixture of alkylbenzenes under the isocratic conditions.

Automated instrumentation for miniaturized displacement electrophoresis with on-column photometric detection

Abstract This paper describes an automated equipment for capillary displacement electrophoresis (isotachophoresis). The equipment employs the advantages of a separation channel with a nonuniform cross-section. The system works in a closed mode, the analytes are determined as anions in the examined arrangement. The pH gradient is within the range 4.4–11.0, the total voltage over the separation channel is from 150 V to 900 V at a constant current of 20 μA or 10 μA, respectively. Cetyltrimethylammoniumbromide and either hydroxypropylcellulose or hydroxypropylmethylcellulose are used in the leading electrolyte to control the electroosmotic flow. The reproducibility and the minimum detectable concentrations are determined and calibration curves are measured using a model mixture of p1 markers and myoglobin as analytes and either low-molecular-mass ampholytec buffers or synthetic carrier ampholytes as spacers. Both Gaussian peaks and the square-wave zones were evaluated.

A portable device for fast analysis of explosives in the environment

A novel portable device for fast and sensitive analysis of explosives in environmental samples is presented. The developed system consists of miniaturized microcolumn liquid chromatograph, photolytic converter and chemiluminescence detector. The device is able to determine selectively nitramine- and nitroester- and most of nitroaromates-based explosives as well as inorganic nitrates at trace concentrations in water or soil extracts in less than 8 min. The device allows to analyze various environmental samples such as soils or water materials without previous preconcentration. Because of internal power supply, the device ensures 12h of continuous operation. Limits of detection of compounds of interest are in the range of concentrations from 5.0 × 10(-9)M to 8.0 × 10(-5)M for a signal-to-noise ratio of 3. Limits of quantification are in the range of concentrations from 1.7 × 10(-8)M to 2.7 × 10(-4)M for a signal-to-noise ratio of 10. The repeatability of the method (RSD=2.9-5.6%) was determined by repeated injections (n=10) of the standard samples during 4h.

Simple automated liquid chromatographic system for splitless nano column gradient separations

A simple splitless gradient liquid chromatographic system for micro and nano column separations has been assembled and tested. It consists of an OEM programmable syringe pump equipped with a glass microsyringe and ten-port selector valve. Gradient of mobile phase was created in the syringe barrel due to turbulent mixing. Capability of suggested system to create various gradient profiles was verified using 50-μl, 100-μl, and 250-μl glass syringes. Acetone, thiourea, and uracil were tested as gradient markers and finally, uracil was proved to be an excellent way of water-acetonitrile gradient tracing. It was found that up to 80% of the total syringe volume is available as a linear gradient section. In context to micro and nano column chromatography, the best results were obtained using the 100-μl syringe. Separations were performed on the capillary monolithic column Chromolith CapRod RP-18e (150mm×0.1mm) and system performance was evaluated using a test mixture of six alkylphenones as well as tryptic digest of bovine serum albumin. Results proved that suggested system is able to create uniform gradients with high repeatability of retention times of test solutes (RSD<0.3%). Repeatability of injection of sample volumes in the range of 0.1-3μl was evaluated using on-column preconcentration technique which means that sample was diluted in mobile phase of low eluting strength. Repeatability of the peak areas was measured and statistically evaluated (RSD<5%).

Combination of micropreparative solution isoelectric focusing and high-performance liquid chromatography for differentiation of biofilm-positive and biofilm-negative Candida parapsilosis group from vascular catheter.

This study utilizes the high-performance liquid chromatography technique in combination with the new micropreparative solution isoelectric focusing fractionation on non-woven fabric strip for the characterization and differentiation of biofilm-positive and biofilm-negative forms of Candida parapsilosis sensu stricto on the basis of the changes in the composition of their cell-surface. Treatment of yeasts by boiling in distilled water relased surface substances from yeasts cells. Consequently, the optimized procedure has been used for fast identification of the highly pathogenic biofilm-positive Candida parapsilosis group in real clinical material - sonicate from vascular catheters. Moreover, the capillary isoelectric focusing was used as supporting and control technique. Obtained results suggest that this new method can be used to distinguish between biofilm-positive and negative forms of Candida parapsilosis sensu stricto.

Performance of reversed-phase parallel-current open-tubular liquid chromatography columns and comparison with theory

Abstract The capillary column performance in reversed-phase parallel-current open-tubular liquid chromatography (RP-PC-OTLC) was studied with the use of cyclohexanol-water. The reduced retention and the height equivalent to a theoretical plate were studied as a function of the liquid velocity and the content of cyclohexanol in the capillary at constant column temperature. The effect of microemulsion formation on band broadening and retention is discussed. The plate heights obtained were compared with the Golay equation.