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HPLC Method Development and Validation for Determination of Active Substances and Impurity in Valetol Preparation
Maroušková, Alena ; Sklenářová, Hana (referee) ; Šatínský, Dalibor (advisor)
This work extends the graduation thesis which found optimal chromatographic conditions for a separation of paracetamol, caffeine and propyphenazon, and the impurity p-aminophenol in the pharmaceutical preparation Valetol by HPLC method. At first, the benzoic acid was found as a suitable internal standard for the quantification. Next, the analytical method validation was carried out, including the suitability test of the chromatographic system. Within the suitability test of the chromatographic system the following attributes were tested: column efficiency expressed as a number of theoretic plates N assymetry of chromatographic peaks expressed as factor T resolution of chromatographic peaks reproducibility expressed as a relative standard error Within the analytic method validation were tested: precision linearity accuracy selectivity robustness stability The robustness test confirmed the analysis sensitivity to the change in the character of the mobil phase. As a part of the robustness test two parameters of mobile phase were tested: pH and triethylamine concentration changes. It was confirmed that the change of pH of the mobile phase influences mostly the retention times of p-aminophenol and benzoic acid. The change of triethylamine amount in the mobile phase influences mostly...
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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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HPLC separation of p-aminophenol, paracetamol, caffeine and propyphenazone on polar stacionary phase.
Maroušková, Alena ; Sklenářová, Hana (referee) ; Šatínský, Dalibor (advisor)
This thesis deals with finding optimal chromatographic conditions for a separation of p- aminophenol, paracetamol, caffeine and propyphenazon by HPLC. These analytes have different polarity and acidobazic properties. The application of traditional octadecylsilane stationary phases for their simultaneous determination is problematical. The PEG column have proved to be suitable for an efficient separation of the analytes with different polarities, i.e. with different chromatographic behavior. The resulting method brings shortening of analysis time, which is important for analysis of big series . Within the optimization of chromatographic conditions: a wide range of mobile phases has been tested (various organic solvents, various ratio of organic components to water phase components) a various level of acidity of the mobile phase has been tested ranging from 4 to 7 pH units various buffers have been tested behavior of the analytes has been tested under an enhanced temperature two PEG columns with different lengths have been tested Optimal chromatographic conditions for a separation of the analytes mentioned above, as well as the fulfillment of requirements for simplicity, analysis time and high degree of reliability have been found. The final chromatographic conditions: Stationary phase:...
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HPLC application in pharmaceutical analysis
Bajcurová, Lucie ; Sklenářová, Hana (referee) ; Matysová, Ludmila (advisor)
The HPLC method for determination of ketoprofen in therapeutic preparations "PRONTOFLEX" - a 10% skin spray and "Ketonal" - a 5% cream has been developed. This method was based on already developed and validated method for analyzing the preparation "KETOPROFEN" - a 2.5% gel. Both of the methods have been developed in the same department. The chromatographic separation was performed on SUPELCO Discovery C18 column (125 mm x 4.6 mm, 5 µm). The mobile phase consisted of a mixture of acetonitrile, water and a phosphate buffer pH 3.5 (40:58:2, v/v/v). At a mobile phase flow rate of 1.0 ml/min, injection volume of 10 μl and UV detection at a wavelength of 233 nm, the total time of analysis was less than 10 minutes. Ethylparaben was used as an internal standard.
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Determination of nitrate and nitrite by SIA method.
Kymlová, Helena ; Sklenářová, Hana (referee) ; Šatínský, Dalibor (advisor)
New SIA method was developed for simultaneous determination of nitrites and nitrates. Nitrites were analyzed by Griess reaction. Nitrates were determined after their reduction to nitrites in cadmium column. Absorbance was measured at maximum of absorption 540 nm. Optimal conditions for analysis: speed of peristaltic pump is 40 % of its maximal speed, speed of syringe pump is 50 µl.s-1 , sample volume of nitrites and nitrates is 150 µl. Optimal time for reduction of nitrates is 0 sec, it means during their flow through cadmium column. Method was used for analysis of nitrites and nitrates in samples of waters: Fromin, Bonaqua, Rajec, Mattoni and tap water in Hradec Králové. Obtained values were compared with values declared by producers. New SIA method for simultaneous determination of nitrites and nitrates is used in subject Special methods of instrumental analysis as a new practical task.
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Determination of biologically active substances by sequential injection analysis with chemiluminescence detection.
Štěpánková, Lucie ; Polášek, Miroslav (advisor) ; Sklenářová, Hana (referee)
1.1 Abstrakt A simple and rapid automated method for the assay of norepinephrine by sequential injection analysis (SIA) with chemiluminescence detection was devised and optimised. The optimised parameters were aspirated volumes of the norepinephrine bitartrate sample and reagents, flow rate, composition of the carrier stream and the photomultiplier voltage. Optimum sample volume was 80 μl and the optimum reagent concentrations (and aspirated volumes) were - 25 mM sodium polyphosphate (44 μl), 0,1 M sulphuric acid (24 μl) and 1 mM potassium permanganate (21 μl)., The optimum flow rate (for pushing the luminescent zone to the detector) was 91 μl/s and the photomultiplier voltage was 450 V. The carrier stream was ultrapure water. The calibration curve was rectilinear in the range 1 - 40 µmol/l of norepinephrine bitartrate. The parameters of the calibration curve y = kx + q (where y is the intensity of chemiluminescence in nA and x stands for the concentration of norepinephrine bitartrate in µmol/l) were calculated by the linear regression method. The slope k was 33.923 and the intercept q was 34.839. The correlation coefficient was 0.9997. The relative standard deviations were 2.78 or 1.18 % (n = 10) when determining 4.75 or 18.98 µmol/l norepinephrine bitartrate in pure standard solutions respectively. The...
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SIA determination of nitrites, nitrates and chlorides using valid ISO norms.
Dundová, Aneta ; Sklenářová, Hana (advisor) ; Šatínský, Dalibor (referee)
This diploma work deals with determination of nitrite, nitrate and chloride based on Sequential Injection Analysis methodology following valid ISO norms. CSN EN ISO 11 732 sets method suitable for determination of ammonia nitrogen based on the FIA and CFA techniques in water samples. Determination is based on injecting sample with nitrite and nitrate content into continual flow of the carrier stream, where it is mixed with alkaline solution. Ammonium is separated in diffusion segmentor unit through semi-permeable membrane and is introduced into the flow with pH indicator. In consequences changes of pH values change the indicator color, which is scanned by spectrophotometer. Transfer of the mentioned FIA determination into the SIA system was carried out meeting calibration ranges of the ISO norm. Nitrite was determined directly using the Griess diazo- coupling reaction and the formed azo dye was measured at 540 nm in the flow cell of the spectrophotometer. The calibration curves were linear over the ranges 0,01 - 0,1 mg/l and 0,1 - 1,0 mg/l; correlation coefficients were 0,9979 and 0,9962. Nitrate solution was aspirated to reducing column containing copperised cadmium. After reduction of nitrate into nitrite the sample was aspirated to the holding coil, where it was mixed with the reagent and passed...
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Vývoj optického sensoru pro salicylát
Soukupová, Marie ; Matysová, Ludmila (advisor) ; Sklenářová, Hana (referee)
We dealt with the development of optical sensor for salicylate. Sensing layer of sensor was based on metalloporphyrins. Metalloporphyrins embody great optical features. Metalloporphyrins are commonly employed as ionophores in ion-selective electrodes (ISEs). Observations from work with ISEs are applied to optical sensors. At first, membrane was constructed, based on tetraphenylporphyrin copper(II) (Cu(TPP)) and it did not show significant sensitivity towards salicylate. Further there were membranes created from tetraphenylporphyrin manganese(III)chloride (Mn(TPP)) and gallium(III)phthalocyanine chloride (Ga(PC)). Reactivity of Mn(TPP) was comparable with reactivity of Cu(TPP). Membrane from Ga(PC) displayed considerable reactivity and sensitivity towards salicylate. Later the membrane's capability was tested to detect valproate. We discovered that the limit of detection for valproate is much lower than for salicylate. Due to great reactivity and variability in membrane response in spectrophotometric assay we were not able to set the working conditions for application to flow system and to dissolution as well. Ga(PC) has thus very good potential in use for detection of valproate. Valproate is one of the most used antiepileptic drugs.
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Verapamil determination using sequential injection analysis with chemiluminescence detection
Homolová, Zuzana ; Sklenářová, Hana (advisor) ; Šatínský, Dalibor (referee)
The verapamil was determined by the technique of sequential injection analysis (SIA) with chemiluminescence (CL) detection. The CL was emitted during the oxidation of the analyte by permanganate in aqueous sulphuric acid in the presence of CL enhancer polyphosphate (PFS). Concentration, order, volume of reagents and flow rate were optimised. Also influence of solvents 60% methanol and 60% ethanol was proved. The optimum order, volumes and concentrations of reagents for aqueous solution of verapamil were: 30 μl of 10 mM KMnO4, 50 μl of 2% PFS, 50 μl of 1 mM verapamil, 30 μl of 1 M H2SO4 and for 60% ethanolic solution of verapamil were: 20 μl of 10 mM KMnO4, 30 μl of 2% PFS, 50 μl of 1 mM verapamil, 30 μl of 2 M H2SO4, 30 μl of 2% PFS and 30 μl/s flow rate. The transient CL signal was recorded at the wavelength 390 nm. Calibration curves relating the intensity of CL (peak heights) to the concentration of the analyte were curvilinear with rectilinear sections in the range 510-5 - 510-3 M for aqueous solution and 110-5 - 110-3 M for 60% ethanolic solution of verapamil. The limits of detection were 210-5 M for aqueous solution and 110-5 M for 60% ethanolic solution. Repeatability of peak heights (RSD, n = 10) ranged between 4.43 % (110-4 M) and 1.83 % (110-3 M) for aqueous solution, and...
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