Since its inception, sequential injection chromatography (SIC) has evolved through several stages. Key moments including introduction of the novel technique combining sequential injection analysis and monolithic column, the first generation of commercial SIC system employing robust pump, the utilization of columns packed with fused-core particles, the on-line hyphenation of extraction and separation steps in SIC, are now followed by the second generation of commercial SIC system employing stainless steel syringe pump and parts optimized for chromatographic separation. The key developments always mean acceleration of the evolution by opening new avenues and reduction of compromises in automated analytical methods based on the flow analysis. The updates, new features, and prospects of the novel instrument are described and discussed on perspective of the method developed for extraction and separation of selected phenolic acids (gallic, protocatechuic, caffeic, p-coumaric and ferulic). The method hyphenates miniaturized on-line solid phase extraction using strong anion exchange sorbent in commercial cartridge for HPLC (20 × 1 mm) and liquid chromatography using chromatographic column (C18 50 × 4.6 mm, 5 μm particles) packed with fused-core particles in the SIC manifold. The separation in gradient mode used acetonitrile: aqueous formic acid pH 2.0 mobile phase and spectrophotometric detection at 270, 300, and 320 nm. Injected sample volumes were 200 and 500 μL. The performance of the extraction step was characterized by the recovery 94.0-107.8%, enrichment factors about 20 or 50, and the separation by peak capacities 13-34, peak symmetries 1.17-1.64, and resolutions 0.82-3.75). While using a sample volume of 200 μL, our method was characterized by the following validation parameters: LODs of 0.0075-0.03 mg L-1, LOQs of 0.025-0.10 mg L-1, calibration ranges 0.025-2.50 mg L-1 (r > 0.999), repeatability of signal at 0.50 mg L-1of RSD ≤ 1.46% (n = 6), and overall time of analysis 7.1 min. The results including pilot analysis of white and red wines demonstrated the capability of novel SIC instrument to enable fast, selective, and sensitive analysis.
- Publication type
- Journal Article MeSH
Food analysis demands fast methods for routine control and high throughput of samples. Chromatographic separation enables simultaneous determination of numerous compounds in complex matrices, several approaches increasing separation efficiency and speed of analysis were involved. In this work, modern types of column with monolithic rod or superficially porous particles were employed and compared for determination of eight synthetic food dyes, their chromatographic performance was evaluated. During method optimization, cyano stationary phase Chromolith Performance CN 100 × 4.6 mm and Ascentis Express ES-CN 100 × 4.6 mm, 5 µm were selected for the separation of polar colorants. The separation was performed by gradient elution of acetonitrile/methanol and 2% water solution of ammonium acetate at flow rate 2.0 mL min-1. Mobile phase composition and the gradients were optimized in order to enable efficient separation on both columns. The method using fused-core particle column provided higher separation efficiency, narrow peaks of analytes resulted in increased peak capacity and shortening of analysis time. After the validation, the method was applied for analysis of coloured beers, soft drinks and candies.
The presented work describes the development and validation of a rapid UHPLC-UV method using a fused core particle column with an RP-Amide stationary phase for the separation and quantitative analysis of caffeoylquinic and di-caffeoylquinic acids in green coffee extracts. Three caffeoylquinic acids (3-caffeoylquinic acid, 4-caffeoylquinic acid, and 5-caffeoylquinic acid) and two di-caffeoylquinic acids (1,3-di-caffeoylquinic acid, and 3,5-di-caffeoylquinic acid) were separated and analyzed in 8 min. That was possible due to the unique selectivity of the RP-Amide stationary phase for the analyzed acids. The retention behavior of all analytes under different compositions of the mobile phase on different columns was evaluated in this study. The optimal chromatographic separation was performed using an Ascentis Express RP-Amide (100 × 2.1 mm) fused-core column with a particle size of 2.7 μm at a temperature of 30 °C. For validation of the newly developed method, acetonitrile was used as mobile phase B and 5% formic acid, filtrated through a 0.22 μm filter, was used as mobile phase A. They were delivered at a flow rate of 0.9 mL min-1 according to the elution gradient program. The detection wavelength was set at 325 nm. A solid-liquid extraction with a solution of methanol and a 5% water solution of formic acid (25 + 75 v/v) using an ultrasonic bath was chosen for the preparation of the available commercial samples of food supplements containing a green coffee extract. Recoveries for all analyzed acids were 98.2-101.0% and the relative standard deviation ranged from 0.3% to 1.4% for intra-day and from 0.3% to 3.0% for inter-day repeatability. The limits of detection were in the range of 0.30-0.53 μg mL-1.
- MeSH
- Acetonitriles chemistry MeSH
- Amides chemistry MeSH
- Time Factors MeSH
- Liquid-Liquid Extraction instrumentation methods MeSH
- Chemistry, Pharmaceutical instrumentation methods MeSH
- Coffee chemistry MeSH
- Quinic Acid analogs & derivatives analysis MeSH
- Limit of Detection MeSH
- Porosity MeSH
- Plant Extracts analysis chemistry MeSH
- Chromatography, High Pressure Liquid instrumentation methods MeSH
- Publication type
- Journal Article MeSH
- Validation Study MeSH
A new high-performance liquid chromatography method using fused-core column for fast separation of resveratrol and polydatin has been developed and used for quality control of nutraceuticals with resveratrol and polydatin content. Retention characteristics (log k) were studied under different conditions on C-18, RP-Amide C-18, Phenyl-hexyl, Pentafluorophenyl (F5) and Cyano stationary phases for both compounds. The effect of the volume fraction of acetonitrile on a retention factors log k of resveratrol and polydatin were evaluated. The optimal separation conditions for resveratrol, polydatin and internal standard p-nitrophenol were found on the fused-core column Ascentis Express ES-Cyano (100×3.0mm), particle size 2.7μm, with mobile phase acetonitrile/water solution with 0.5% acetic acid pH 3 (20:80, v/v) at a flow rate of 1.0mL/min and at 60°C. The detection wavelength was set at 305nm. Under the optimal chromatographic conditions, good linearity with regression coefficients in the range (r=0.9992-0.9998; n=10) for both compounds was achieved. Commercial samples of nutraceuticals were extracted with methanol using ultrasound bath for 15min. A 5μL sample volume of the filtered solution was directly injected into the HPLC system. Accuracy of the method defined as a mean recovery was in the range 83.2-107.3% for both nutraceuticals. The intraday method precision was found satisfactory and relative standard deviations of sample analysis were in the range 0.8-4.7%. The developed method has shown high sample throughput during sample preparation process, modern separation approach, and short time (3min) of analysis. The results of study showed that the declared content of resveratrol and polydatin varied widely in different nutraceuticals according the producers (71.50-115.00% of declared content).
- MeSH
- Acetonitriles chemistry MeSH
- Glucosides chemistry MeSH
- Indicators and Reagents chemistry MeSH
- Nitrophenols chemistry MeSH
- Dietary Supplements analysis MeSH
- Quality Control MeSH
- Solutions chemistry MeSH
- Stilbenes chemistry MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
A new fast and sensitive method based on on-line solid-phase extraction on a fused-core precolumn coupled to liquid chromatography with fluorescence detection has been developed for ochratoxin A (OTA) and citrinin (CIT) determination in lager beer samples. Direct injection of 100 μL filtered beer samples into an on-line SPE-HPLC system enabled fast and effective sample extraction including separation in less than 6 min. Preconcentration of OTA and CIT from beer samples was performed on an Ascentis Express RP C18 guard column (5 × 4.6 mm), particle size 2.7 μm, with a mobile phase of methanol/0.5% aqueous acetic acid pH 2.8 (30:70, v/v) at a flow rate of 2.0 mL min(-1). The flow switch from extraction column to analytical column in back-flush mode was set at 2.0 min and the separation was performed on the fused-core column Ascentis Express Phenyl-Hexyl (100 × 4.6 mm), particle size 2.7 μm, with a mobile phase acetonitrile/0.5% aqueous acetic acid pH 2.8 in a gradient elution at a flow rate of 1.0 mL min(-1) and temperature of 50 °C. Fluorescence excitation/emission detection wavelengths were set at 335/497 nm. The accuracy of the method, defined as the mean recoveries of OTA and CIT from light and dark beer samples, was in the range 98.3-102.1%. The method showed high sensitivity owing to on-line preconcentration; LOQ values were found to be 10 and 20 ng L(-1) for OTA and CIT, respectively. The found values of OTA and CIT in all tested light, dark and wheat beer samples were significantly below the maximum tolerable limits (3.0 μg kg(-1) for OTA and 2000 μg kg(-1) for CIT) set by the European Union.
On-line sample pretreatment (clean-up and analyte preconcentration) is for the first time coupled to sequential injection chromatography. The approach combines anion-exchange solid-phase extraction and the highly effective pentafluorophenylpropyl (F5) fused-core particle column for separation of eight sulfonamide antibiotics with similar structures (sulfathiazole, sulfanilamide, sulfacetamide, sulfadiazine, sulfamerazine, sulfadimidine, sulfamethoxazole and sulfadimethoxine). The stationary phase was selected after a critical comparison of the performance achieved by three fused-core reversed phase columns (Ascentis(®) Express RP-Amide, Phenyl-Hexyl, and F5) and two monolithic columns (Chromolith(®) High Resolution RP-18 and CN). Acetonitrile and acetate buffer pH 5.0 at 0.60 mL min(-1) were used as mobile phase to perform the separations before spectrophotometric detection. The first mobile phase was successfully used as eluent from SPE column ensuring transfer of a narrow zone to the chromatographic column. Enrichment factors up to 39.2 were achieved with a 500 µL sample volume. The developed procedure showed analysis time <10.5 min, resolutions >1.83 with peak symmetry ≤1.52, LODs between 4.9 and 27 µg L(-1), linear response ranges from 30.0 to 1000.0 µg L(-1) (r(2)>0.996) and RSDs of peak heights <2.9% (n=6) at a 100 µg L(-1) level and enabled the screening control of freshwater samples contaminated at the 100 µg L(-1) level. The proposed approach expanded the analytical potentiality of SIC and avoided the time-consuming batch sample pretreatment step, thus minimizing risks of sample contamination and analyte losses.
- MeSH
- Anti-Bacterial Agents analysis isolation & purification MeSH
- Water Pollutants, Chemical analysis isolation & purification MeSH
- Equipment Design MeSH
- Solid Phase Extraction instrumentation MeSH
- Limit of Detection MeSH
- Flow Injection Analysis instrumentation MeSH
- Rivers chemistry MeSH
- Sulfonamides analysis isolation & purification MeSH
- Chromatography, High Pressure Liquid instrumentation MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
A new on-line SPE-HPLC method using fused-core columns for on-line solid phase extraction and large volume sample injection for increasing the sensitivity of detection was developed for the determination of insecticides fenoxycarb and cis-, trans-permethrin in surface waters. The separation was carried out on fused-core column Phenyl-Hexyl (100×4.6 mm), particle size 2.7 µm with mobile phase acetonitrile:water in gradient mode at flow rate 1.0 mL min(-1), column temperature 45°C. Large volume sample injection (1500 µL) to the extraction dimension using short precolumn Ascentis Express RP C-18 (5×4.6 mm); fused-core particle size 2.7 µm allowed effective sample preconcentration and efficient ballast sample matrix removal. The washing mobile phase consisting of a mixture of acetonitrile:water; 30:70, (v/v) was pumped at flow rate of 0.5 mL min(-1) through the extraction precolumn to the waste. Time of the valve switch for transferring the preconcentrated sample zone from the extraction to the separation column was set at 3rd min. Elution of preconcentrated insecticides from the extraction precolumn and separation on the analytical column was performed in gradient mode. Linear gradient elution started from 40% of acetonitrile at time of valve switch from SPE column (3rd min) to 95% of acetonitrile at 7th min. Synthetic dye sudan I was chosen as an internal standard. UV detection at wavelength 225 nm was used and the method reached the limits of detection (LOD) at ng mL(-1) levels for both insecticides. The method showing on-line sample pretreatment and preconcentration with highly sensitive determination of insecticides was applied for monitoring of fenoxycarb and both permethrin isomers in different surface water samples in Czech Republic. The time of whole analysis including on-line extraction, interferences removal, chromatography separation and system equilibration was less than 8 min.
- MeSH
- Water Pollutants, Chemical analysis MeSH
- Solid Phase Extraction MeSH
- Phenylcarbamates analysis MeSH
- Insecticides analysis MeSH
- Lakes analysis MeSH
- Environmental Monitoring MeSH
- Online Systems MeSH
- Permethrin analysis MeSH
- Rivers chemistry MeSH
- Chromatography, High Pressure Liquid MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Czech Republic MeSH
The presented work describes the development of a simple, fast and effective on-line SPE-UHPLC-UV/vis method using fused core particle columns for extraction, separation and quantitative analysis of the nine illegal dyes, most frequently found in chilli-containing spices. The red dyes Sudan I-IV, Sudan Red 7B, Sudan Red G, Sudan Orange G, Para Red, and Methyl Red were separated and analyzed in less than 9 min without labor-consuming pretreatment procedure. The chromatographic separation was performed on Ascentis Express RP-Amide column with gradient elution using mixture of acetonitrile and water, as a mobile phase at a flow rate of 1.0 mL min(-1) and 55°C of temperature. As SPE sorbent for cleanup and pre-concentration of illegal dyes short guard fused core column Ascentis Express F5 was used. The applicability of proposed method was proven for three different chilli-containing commercial samples. Recoveries for all compounds were between 90% and 108% and relative standard deviation ranged from 1% to 4% for within- and from 2% to 6% for between-day. Limits of detection showed lower values than required by European Union regulations and were in the range of 3.3-10.3 µg L(-1) for standard solutions, 5.6-235.6 µg kg(-1) for chilli-containing spices.
- MeSH
- Food Analysis methods MeSH
- Azo Compounds chemistry MeSH
- Coloring Agents chemistry isolation & purification MeSH
- Capsicum chemistry MeSH
- Solid Phase Extraction methods MeSH
- Indicators and Reagents chemistry MeSH
- Spices analysis MeSH
- Naphthols chemistry MeSH
- Online Systems MeSH
- Food Coloring Agents chemistry MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
