Search PubMed⌕ Search

Biomedical subjects

J Pawliszyn

Publications and source records attributed to J Pawliszyn.

65 records · Page 4Linked to original sources
PubMed1995

Application of capillary isoelectric focusing with absorption imaging detection to the quantitative determination of human hemoglobin variants.

The capillary isoelectric focusing (CIEF)-absorption imaging detector is a multicapillary separation instrument. Several protein samples can be separated and detected in a single run. The analysis time for one run is only 3 min. Because an imaging detector is used, the mobilization process of conventional CIEF is not necessary, eliminating such drawbacks as long analysis time and poor reproducibility in mobilization speed. Human hemoglobin variants were quantitatively determined using the instrument. The peak areas of the analytes were proportional to their concentrations in the concentration range of 0-200 micrograms/mL. Hemoglobin variant A2, which only comprises 2% of the whole hemoglobin, can be quantitatively determined with a standard deviation of less than 10%. For the high concentration variants, such as variant S, the deviation is less than 1%.

Equipment Design↗
PubMed1995

Fluorescence imaging detection for capillary isoelectric focusing.

A simple laser-induced fluorescence (LIF) imaging detector and an ultrasensitive LIF imaging detector are described for capillary isoelectric focusing (CIEF). An argon ion laser beam of 496.5 nm is used as excitation source. In the simple LIF imaging detector, the excitation beam is directed into a capillary column by an optic fiber array. In the ultrasensitive LIF imaging detector, the laser beam is first expanded, then is focused into the 4.5 cm long capillary column by a cylindrical lens. Fluorescence emission is detected by a charge-coupled device (CCD) camera. The feasibility and performance of the LIF imaging detector system for CIEF are first verified with a naturally fluorescent protein, b-phycoerythrin. Then, the ultrasensitive LIF imaging system is used as a detector for CIEF of proteins labeled with fluorescein isothiocyanate (FITC). Three FITC-labeled proteins (i) alpha-D-galatosylated FITC-albumin, (ii) insulin-FITC, and (iii) casein-FITC, are used as model samples. Fluorescence images of the model samples are measured during the CIEF process. The focusing of the protein samples is complete in about 1.5 min. The ultrasensitive detector's detection limits for the FITC-labeled proteins are at the level of 10(-10) M, and the mass detection limits are about 4.5 x 10(-17) mole, even though only 10% of the fluorescence emission is collected. Therefore, the method is capable of separating and detecting 10(-11) M or amole (10(-18) mole) level protein samples with a band-pass filter more specific to the fluorescence light.(ABSTRACT TRUNCATED AT 250 WORDS)

Caseins↗
PubMed1994

Application of capillary isoelectric focusing with absorption imaging detection to the analysis of proteins.

A capillary isoelectric focusing instrument with an on-line optical absorption imaging detector was used to analyse protein samples. The separation column was a 4 cm x 100 microns I.D. capillary. The light source of the imaging detector was a argon ion laser. The light beam from the laser was focused into the capillary by a cylindrical lens. An 1024-pixel charge-coupled device (CCD) measured intensity of light beam passing through the capillary. The optical alignment of the detector was optimized to eliminate interference produced by the refractive index gradient created by sample zones inside the capillary. The signal-to-noise ratio of the detector was enhanced by averaging 30 scans of the CCD every 3 s. The on-line imaging detector allows simultaneous separation and detection so that the analysis time for a sample is only 2-4 min. Several protein samples were analyzed by the instrument, including human hemoglobin variants, cytochrome c, myoglobin and transferrin.

Absorption↗
PubMed1994

Peptide mapping of bovine and chicken cytochrome c by capillary isoelectric focusing with universal concentration gradient imaging.

Capillary isoelectric focusing with universal concentration gradient imaging detection was used to separate and detect tryptic peptides from bovine and chicken cytochrome c. For a desalted sample of peptide angiotensin 2, the isoelectric point (pI) measured by the instrument agreed well with the pI calculated from amino acid pK values. For the cytochrome digests, correlations between measured and calculated pI values were imprecise because peak positions shifted slightly from test to test. This problem is thought to be caused by the inefficient desalting process used on the samples, leaving salt residues which caused distortion in the pH gradient during the focusing process. However, this system differentiated between the two cytochrome c's. The concentration gradient imaging detected peptides which contain no tyrosine and no tryptophan amino acids, which a UV absorption detector operating at 280 nm could not. The separation and detection steps took only 5-7 min because no mobilization was necessary after the focusing process.

Amino Acid Sequence↗
PubMed1992

Detection of substituted benzenes in water at the pg/ml level using solid-phase microextraction and gas chromatography-ion trap mass spectrometry.

Solid-phase microextraction (SPME) is combined with gas chromatography-ion trap mass spectrometry (GC-IT-MS) for the analysis of benzene, toluene, ethyl benzene and xylene isomers (BTEX) in water. SPME is a recent technique for extracting organics from an aqueous matrix into a stationary phase immobilized on a fused-silica fiber. The analytes are thermally desorbed directly in the injector of a gas chromatograph. The wide linear dynamic range (five orders of magnitude) and pg sensitivity of the ion trap mass spectrometer in its full scan mode is an ideal detector for identifying and quantifying the analytes extracted with an SPME device. The combined method SPME-GC-IT-MS, using fibers coated with a 100-microns polydimethylsiloxane coating, showed a limit of quantitation (LOQ) of 50 pg/ml benzene in water. This corresponds to 5 pg of benzene absorbed onto the fiber. The limit of detection (LOD) was 15 pg/ml benzene. For o-xylene spiked at 50 pg/ml in water 50 pg were absorbed by the fiber indicating an LOQ and LOD 10 times better than for benzene. The detection limits obtained exceed the requirements of both the United States Environmental Protection Agency method 524.2 and the Ontario Municipal/Industrial Strategy for Abatement program, which range from 30 to 80 pg/ml and 500 to 1100 pg/ml, respectively. The linearity of the method extended over five orders of magnitude. Relative standard deviation ranged from 2.7 to 5.2% for 15 ng/ml BTEX in water and from 5.5 to 7.5% for 50 pg/ml BTEX in water. SPME-GC-IT-MS was used to evaluate the contamination level in laboratory, potable and wastewater sources.

Benzene Derivatives↗
PubMed1992

Application of capillary isoelectric focusing with universal concentration gradient detector to the analysis of protein samples.

The design of a new capillary isoelectric focusing (cIEF) instrument, composed of a rugged cartridge holding a short piece of capillary and a universal, inexpensive concentration gradient detector, was optimized and applied to the analysis of various protein samples. High-efficiency cIEF separations with sub-femtomole detection limits for absolute amounts were obtained using 10 microns I.D. capillaries with large O.D.-to-I.D. ratios. An electric field strength of 1 kV/cm applied in the focusing step resulted in a 10(-8) M on-column concentration detection limit, which corresponded to 10(2) amol absolute amount of proteins. The detection volume was estimated to be 2 pl, which is among the smallest values reported to date for any optical or spectroscopic detector. When a 6-cm long capillary was used, proteins with isoelectric points ranging from 4.7 to 8.8 could be analyzed in about 5 min, the shortest analysis time ever reported for cIEF. Compared with commercial cIEF instruments with UV-visible absorbance detectors, the instrument is easier to use and has lower detection limits and better resolution. Several protein mixtures and real samples were separated with this instrument.

Antibodies, Monoclonal↗
PubMed1992

Solventless determination of caffeine in beverages using solid-phase microextraction with fused-silica fibers.

Caffeine concentrations in beverages were determined using a simple and rapid method based on microextraction of caffeine onto the surface of a fused-silica fiber. The uncoated fiber was dipped into the beverage sample for 5 min after the addition of isotopically labeled (trimethyl 13C)caffeine. The adsorbed caffeine was then thermally desorbed in a conventional split/splitless injection port, and the concentration of caffeine was determined using gas chromatography with mass spectrometric detection. Quantitative reproducibilities were ca. 5% (relative standard deviation) and the entire scheme including sample preparation and gas chromatographic analysis was completed in ca. 15 min per sample. The potential of the microextraction technique for the analysis of flavor and fragrance compounds in non-caffeinated beverages is also demonstrated. Since no solvents or class-fractionation steps are required, the method has good potential for automation.

Beverages↗
PubMedDate not supplied

Fast analysis of proteins by isoelectric focusing performed in capillary array detected with concentration gradient imaging system.

Isoelectric focusing of several protein samples is performed simultaneously in an array of 2-4 short capillaries (4-10 cm long). The separated proteins are detected by either an on-line concentration gradient imaging detection system, or a single point concentration gradient detector which requires a mobilization step following the focusing process. The application of the capillary array increases the throughput of the capillary isoelectric focusing (CIEF) technique, and makes the technique comparable in sample throughput to the gel slab isoelectric focusing technique, but with much faster speed of separation and quantitation. The analysis is completed in 2-3 min with the imaging detection system, which is about 100 times faster than the conventional gel slab isoelectric focusing and 10 times faster compared to conventional CIEF. The resolution of the separation is 0.02 pH units or better. All separated proteins in the capillary array are detected by the universal concentration gradient detector, which eliminates the need for sample derivatization. The concentration limit of detection for the inexpensive instrument described in this work is about 30 micrograms/mL and can be reduced by using higher quality components in the system, such as a more stable laser, higher optical quality capillaries and a faster computer A/D board. The sample consumption is 400 nL for the 4 cm long, 100 microns ID square capillary. The isoelectric point (pI) values of the samples can be directly determined without internal pI markers from their positions inside the capillary after focusing when the imaging detection system is used.

Costs and Cost Analysis↗
PubMedDate not supplied

Simple and rapid determination of amphetamine, methamphetamine, and their methylenedioxy derivatives in urine by automated in-tube solid-phase microextraction coupled with liquid chromatography-electrospray ionization mass spectrometry.

A simple and rapid method for the determination of amphetamine, methamphetamine, and their 3,4-methylenedioxy derivatives in urine samples was developed using automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). In-tube SPME is an extraction technique for organic compounds in aqueous samples in which analytes are extracted from the sample directly into an open tubular capillary by repeated draw/eject cycles of sample solution. LC-MS analyses of stimulants were initially performed by liquid injection onto an LC column to determine spectra. Five stimulants tested in this study gave very simple ESI mass spectra, and strong signals corresponding to [M+H]+ were observed for all stimulants. The stimulants were well separated with a Supelcosil LC-CN column using acetonitrile/50mM ammonium acetate (15:85) as a mobile phase. In order to optimize the extraction of stimulants, several in-tube SPME parameters were examined. The optimum extraction conditions were 15 draw/eject cycles of 35 microL of sample in 50mM Tris-HCI (pH 8.5) at a flow rate of 100 microL/min using an Omegawax 250 capillary column. The stimulants extracted by the capillary were easily desorbed by mobile phase flow, and carryover of stimulants was not observed. Using in-tube SPME-LC-ESI-MS with selected ion monitoring, the calibration curves of stimulants were linear in the range from 2 to 100 ng/mL with correlation coefficients above 0.9985 (n = 18) and detection limits (S/N = 3) of 0.38-0.82 ng/mL. This method was successfully applied to the analysis of human urine samples without interference peaks. The recoveries of stimulants spiked into urine samples were above 81%.

Amphetamines↗
PubMedDate not supplied

Automated in-tube solid-phase microextraction coupled with liquid chromatography-electrospray ionization mass spectrometry for the determination of selected benzodiazepines.

A simple, rapid, and sensitive method, which allowed us to simultaneously determine seven benzodiazepines (diazepam, nordiazepam, temazepam, oxazepam, 7-aminoflunitrazepam, N-desmethylflunitrazepam, and clonazepam) in buffer solution and in urine and serum samples, was investigated by automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). In-tube SPME, in which the analytes were extracted from the sample directly into an open tubular capillary column by repeated draw/eject cycles of sample solution, is an extraction technique for organic compounds in aqueous samples. The separation of benzodiazepines was carried out under ion-suppressed reversed-phase conditions by using methanol/50mM ammonium acetate in water (60:40) as a mobile phase with a Supelco LC-18 column. The optimal extraction condition was 10 draw/eject cycles of 30 mL of sample in 100mM Tris-HCl (pH 8.5) at a flow rate of 0.3 mL/min using a piece of 60-cm length Supelco-Q plot capillary column as the extraction capillary. The quantitative study was explored by operating in selected-ion monitoring (SIM) mode. The calibration curves were linear in the range from 0.5 ng/mL or 2 ng/mL to 500 ng/mL. The detection limits were from 0.02 ng/mL to 2 ng/mL. At the optimized capillary and fragmentor voltages, the characteristic ions for each compound clearly showed up in the spectra and it is possible to use the LC-MS to identify these compounds. The method was applied to the analysis of biological samples without interfering peaks. However, the recoveries for some of the compounds in serum samples need to be further improved.

Benzodiazepines↗