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HPLC Method Development for Determination of Vitamin E in Geladrink Preparation
Švestková, Petra ; Chocholouš, Petr (referee) ; Šatínský, Dalibor (advisor)
A HPLC method was developed for the determination of content of vitamine E acetate in preparation Geladrink Forte pulverized drink - pineapple. The method is based on using Chromolith Performance RP-18e column (100 x 4.6 mm) and UV detection at 290 nm. The vitamine E acetate was separated in the presence of internal standard tocol from ballast matrix. It was used isocratic elution of the mobile phase methanol:water (98:2) at a flow-rate of 1.5 ml/min. There was temperature 30 řC during the measurement. The system enabled successful separation in time less than 5 min. The retention time of tocol was 2.337 min and the retention time of vitamine E acetate was 4.271 min. The chromatographic resolution between tocol and vitamine E acetate was 7.515. The method was applied to analysis of vitamine E acetate in preparation Chondrotin - MSM 2600. Developed concentrations of vitamine E acetate were re-count on content of vitamine E that the producer declared. Keywords: vitamine E acetate, HPLC, Geladrink, Chondrotin - MSM 2600
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Determination of vitamins A, D and E using sequential injection chromatography method.
Trefilová, Kateřina ; Chocholouš, Petr (referee) ; Sklenářová, Hana (advisor)
The working isntruction was created for separation vitamins A, D and E by means of monolithic column engaged in system sequentional injectional analysis. Following conditions of separation were chosen after performance of optimalization: Analysis at monolithic column 25 mm long Chromolith Flash RP-18e column, 4,6 mm I.D. (Merck, Germany), with speed (10 µl/s) with applied mobile phase acetonitril metanol (1:1, v/v). Monolithic column was in SIA system and was engaged between moreway valve and flow Z cell. Spectrophotometric detection was performed at wave length for absorbing maximum for vitamins A - 324 nm, D - 265 nm, E - 285. Total SIA analysis time was 400 s (from that 230 s lasted separation process). From basic chromatographic validation parameters were evaluated the following symetric factor As, resolution Rs, amount theoretical trays and repeatability. Symetric factor for vitamin A - 1.309; vitamin D2 - 1.302; vitamin D3 - 1.286; vitamin E - 1.158. Peak resolution for vitamins A-D2 je 3.607; D2-D3 4.232; D-E 1.532. Amount theoretical stages for vitamin A 998, vitamin D2 1621, vitamin D3 2091 and vitamin E 2376. Repeatability was worked out from 6 sprays, and relative standard deviation of particular substances was calculated from the highest point of the graph peak. Repeatability was for...
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Optimisation of SIA decarbazine determination
Horáková, Markéta ; Chocholouš, Petr (referee) ; Sklenářová, Hana (advisor)
Dacarbazine was determinated by sequential injection analysis with chemiluminiscence detection during reaction of sample and KMnO4 in sulfuric acid. Reagent volumes, their concentrations, flow rate and structure of cycle were optimised. Also the influence of solvents (water, 60% ethanol, 60% methanol) and enhancers of CL reaction (sodium polyphosphate, formic acid, glutaraldehyde and formaldehyde) was tested. Final optimal conditions are: 40 μl 1 M H2SO4, 3 μl 10 mM KMnO4, 30 μl 10 mM solution of dacarbazine, 40 μl 1 M H2SO4, eventually aspiration of 50 μl enhancer. Aspiration of the solutions proceeded in flow rate 100 μl/s, mixing and flow rate to the detector was 40 μl/s. Washing with oxalic acid was used after each measurement cycle to prevent KMnO4 settling on tubes. Voltage at the photomultiplier of the detector was 420 V. The calibration relations of CL intensity to concentration of the samples were nonlinear with possibility of selection of linear areas. The linear area for the solution of dacarbazine without enhancers wasn't found with correlation coefficient high enough, for the solution of dacarbazine with 60% methanol was 0.5 - 10 mmol/l, for the solution of dacarbazine with 1.39 M formic acid 4 - 10 mmol/l. Limit of detection for the solution of dacarbazine without enhancers was 0.1474...
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Determination of ascorbic acid by HILIC approach - a comparison of HILIC stationary phases.
Bieleszová, Kateřina ; Chocholouš, Petr (referee) ; Nováková, Lucie (advisor)
This thesis was focused on the determination of ascorbic acid (AA) using Hydrophilic Interaction Chromatography (HILIC) approach and the comparison of HILIC stationary phases. Determination was carried out using three types of detection. To optimize the method ultraviolet (UV) detection was used. For the determination of AA and DHA (dehydroascorbic acid) on different stationary phases aerosol based detectors Charged Aerosol Detector (CAD) and Nano Quantity Analyte Detector (NQAD) were used. During method optimization suitable conditions for the determination of AA and DHA have been defined. Aqueous part of the mobile phase was formed by water, acetic acid or ammonium acetate buffer of increasing strength and different pH. Organic part was formed by acetonitrile. During the optimization the influence of gradual changes of water and organic part has been monitored. Compared stationary phases were Atlantis T3 and those specially developed for the determination by HILIC method - Luna HILIC and ZIC-HILIC. In the case of Luna HILIC and Atlantis T3 both analytes were not separated from the dead retention time. On ZIC-HILIC column separation of substances occurred, but peaks were too broad. None of these columns has been evaluated as suitable for the simultaneous determination of AA and DHA. A part of this...
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Study of temperature factor in gradient and isocratic HPLC separation
Habart, Miroslav ; Nováková, Lucie (advisor) ; Chocholouš, Petr (referee)
5 Abstract This thesis was focused on the determination of the temparature influence upon analysis of phenolic acids and fluoroquinolone antibiotics using high performance liquid chromatography. Analysis was performed on Zorbax SB C18 (50 x 4.6 mm; 1.8 µm) analytical column using ultra - viollet detection (UV) for both phenolic acids and fluoroquinolon antibiotics. The optimization of separation conditions was carried out for determination of phenolic acids and fluoroquinolone antibiotics. The effect of composition and concentration of aqueous part of mobile phase and especially the effect of temperature was investigated. The gradient elution was used for determination of phenolic acids. The mobile phase was formed by 0.1% formic acid and methanol. The isocratic elution was used for determination of fluoroquinolone antibiotics and the mobile phase was also formed by 0.1% formic acid and methanol. Accelerating of analysis and saving time and used solvents by using higher temperature were the main goals of this thesis. This premise came true in the analysis of phenolic acids. The analysis was faster with good peak resolution. The results for fluoroquinolone antibiotics were not better than basic 25žC measurement. Contrariwise, lower than laboratory temperature was more advantageous for their analysis. System...
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Sledování norfloxacinu, ciprofloxacinu a enrofloxacinu pomocí HPLC-FD ve vzorcích odpadních vod a jejich vliv na životní prostředí
Spurná, Marie ; Solich, Petr (advisor) ; Chocholouš, Petr (referee)
8 Abstract Fluoroquinolones (FQs) are used for therapy and prophylaxis of human and animal diseases and they are also used as growth promoters of animals. The usage of antibiotic is in connection with development of antibiotic resistance among bacterial populations. Since swine production industry is considered to be a relevant source of antibiotic resistant bacteria, measures to control the use of antimicrobial agents in animal husbandry have been adopted in recent years (1). The presence of FQs in wastewater from swine farms can introduce antibiotics in surface waters through agricultural runoff. FQs are rather resistant to microbial degradation and these compounds may be persisting within environmental waters because of their sorption properties, favouring the accumulation in sewage sludge, manure and soil (2). To trace the sources and ways of contamination, and evaluate the environmental impact of an outdoor swine production system, the occurrence norfloxacin (NOR), ciprofloxacin (CIP) and enrofloxacin (ENRO) residues were evaluated in wastewater and surface water samples. A LC-FD method based on the application of monolithic column (Chromolith Performance RP-18e (100 x 4.6 mm) successfully developed in previous studies (3,4,5) was applied. FQs were isocratically eluted using a mobile phase consisting...
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Separation and Determination of Betamethasone and chloramphenicol in multicomponent pharmaceutical preparations using a monolithic columns coupled to sequential injection system
Salabová, Markéta ; Šatínský, Dalibor (advisor) ; Chocholouš, Petr (referee)
SEPARATION AND DETERMINATION OF BETAMETHASONE AND CHLORAMPHENICOL IN MULTICOMPONENT PHARMACEUTICAL PREPARATIONS USING A MONOLITHIC COLUMNS COUPLED TO SEQUENTIAL INJECTION SYSTEM Markéta Salabová Abstract A new separation method based on a reversed-phase sequential injection chromatography (SIC) technique was used for simultaneous determination of chloramphenicol and betamethasone in pharmaceutical eye drops in this contribution. Short monolithic column coupled with a sequential injection analysis (SIA) system enabled separation of two compounds in one step. A Chromolith Flash RP-18e, 25-4.6 mm column with 5 mm precolumn (Merck, Germany) and a FIAlab 3000 system (USA) with a 6-port selection valve and 5 ml syringe were used for sequential injection chromatographic separations in study. The mobile phase used was acetonitrile-water (30:80, v/v), flow rate 0.48 ml min-1 ; UV detection was at two wavelengths 241 nm and 278 nm, (absorption maximum of betamethasone and chloramphenicol). The basic validation parameters showed good results: linearity of determination for both compounds including internal standard (propylparaben) > 0.999; repeatability of determination (RSD) in the range 0.8 - 1.7% at two different concentration levels, and detection limits in the range 0.5 - 1.0 µg ml-1 . The chromatographic...
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Sequential injection chromatography
Chocholouš, Petr ; Solich, Petr (advisor) ; Polášek, Miroslav (referee) ; Blešová, Marie (referee)
This work brings extending of applications of sequential injection analysis in area of pharmaceutical analysis. It is focused on integration of monolithic columns into SIA system that enables simultaneous determination of few analytes in samples with matrix (biologic samples) or simple mixtures (pharmaceutical preparations). A new created method was called Sequential Injection Chromatography (SIC) and represents a new generation of flow methods. Integration of short monolithic column with low back-pressure leads into creation of low pressure flow separation method. Commercially produced monolithic columns with reverse phase sorbent C18 are suitable for determination of active compounds and adjuvants in pharmaceutical preparations. This enables fast determination of samples without or only with simple off-line pre-treatment of sample. Employment of various lengths of monolithic columns used in SIC was studied. First experimental work (appendix I) was first SIC experiment. The aim of it was trouble-free connection of monolithic column with SIA manifold and application on determination of pharmaceutical preparation Diclofenac HBF emulgel (ointment contains diclofenac nitrate, methylparaben and propylparaben). All parts of system were optimized for higher work pressure. Sample of ointment was...
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