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Determination of Heparin by Sequential Injectin Analysis with Spectrophotometric Detection
Lišková, Růžena ; Hraníček, Jakub (advisor) ; Nesměrák, Karel (referee)
In this work the determination of heparin using Sequential Injection Analysis (SIA) technique with spectrophotometric detection was studied. The principle of this method was measuring the decrease of absorbance of phenothiazine dyes in its maximum wavelength caused by its interaction with heparin. The decrease is proportional to concentration of heparin in sample. The aim of this work was to find optimum conditions for determination of heparin by the SIA with spectrophotometric detection and to try to apply this measurement to real sample under these conditions. Three phenothiazine dyes were used: Methylene Blue, Azure A and Azure B and for each of them an optimum concentration was found. Their values were for methylene blue 4.0 ∙ 10-5 mol/dm3 and 5.0 ∙ 10-5 mol/dm3 for Azure A and Azure B. 150 µl was chosen as an optimum amount of heparin and reagent aspirated. Optimum reaction time was 0 s and flow rate during measurement 1.5 ml/min. Working concentration range was found between 0.23 and 15 mg/dm3 for methylene blue, 1.2 and 13 mg/dm3 for Azure A and 0.67 and 12 mg/dm3 for Azure B. Limit of detection varied between 0.09 - 0.35 mg/dm3 and limit of quantification between 0.30 and 1.2 mg/dm3 for different dyes. Relative standard deviation was determined as a measure of repeatability. Its values were...
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Determination of Nadroparine in Fraxiparine injection solution using flow techniques of anlysis
Miklošinová, Aneta ; Hraníček, Jakub (advisor) ; Křížek, Tomáš (referee)
This thesis was focused on a determination of nadroparin using sequential injection analysis and flow injection analysis with fluorimetric and spectrophotometric detection. The principle of determination was based on the interaction of nadroparin with phenothiazine dye. Methylene blue was used as phenothiazine dye. The determination was performed on a laboratory made FIA instrument and on the commercial SIA instrument. Optimal conditions for SIA were concentration of dye 6 ∙ 10-5 mol dm-3 , injected volume 100 µl, flowrate 1,5 ml min-1 , for FIA: concentration of dye 3 ∙10-5 mol dm-3 flowrate 2 ml ∙ min-1 , injected volume= 100 µl, for fluorimetric detection was sensivity of the detector 1000 V, Emission was measured by 2 nm and excitation wavelenght 621 nm. For spectrophotometric detection, absorbance was detected at the absorption maximum wavelength. Repeatability ranged from 2.01 to 2.85%. The limit of detection for FIA was 0.05-0.09 IU ml-1 , for SIA were limits of detection in range 0,25 - 1,63 IU ml-1 , limits of quantification in range 0,83 - 5,44 and linear range was from 0,5 - 20 IU ml-1 . The limits of detection, limits of determination and the linear range could be corrected for the SIA by the injected volume of phenothiazine dye.
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Determination of Heparin by Sequential Injectin Analysis with Spectrophotometric Detection
Lišková, Růžena ; Hraníček, Jakub (advisor) ; Nesměrák, Karel (referee)
In this work the determination of heparin using Sequential Injection Analysis (SIA) technique with spectrophotometric detection was studied. The principle of this method was measuring the decrease of absorbance of phenothiazine dyes in its maximum wavelength caused by its interaction with heparin. The decrease is proportional to concentration of heparin in sample. The aim of this work was to find optimum conditions for determination of heparin by the SIA with spectrophotometric detection and to try to apply this measurement to real sample under these conditions. Three phenothiazine dyes were used: Methylene Blue, Azure A and Azure B and for each of them an optimum concentration was found. Their values were for methylene blue 4.0 ∙ 10-5 mol/dm3 and 5.0 ∙ 10-5 mol/dm3 for Azure A and Azure B. 150 µl was chosen as an optimum amount of heparin and reagent aspirated. Optimum reaction time was 0 s and flow rate during measurement 1.5 ml/min. Working concentration range was found between 0.23 and 15 mg/dm3 for methylene blue, 1.2 and 13 mg/dm3 for Azure A and 0.67 and 12 mg/dm3 for Azure B. Limit of detection varied between 0.09 - 0.35 mg/dm3 and limit of quantification between 0.30 and 1.2 mg/dm3 for different dyes. Relative standard deviation was determined as a measure of repeatability. Its values were...
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Determination of heparine by SIA with spectrofluorimetric detection
Bár, Ladislav ; Rychlovský, Petr (advisor) ; Nesměrák, Karel (referee)
This thesis was focused on a determination of heparin using sequential injection analysis with spectrofluorimetric and spectrophotometric detection. The principle of determination was based on the interaction of heparin with phenothiazine dye. A decrease of fluorescence intensity of dye in its emission maximum was detected. In the case of spectrophotometric detection a decrease of the absorbance of dye was measured. Azure A, azure B and methylene blue were used as representantive selection of phenothiazine dyes. The determination was performed on a laboratory made SIA apparatus, for which a control software in LabVIEW 7.1 graphical programming was created. Two types of flow configuration for spectrofluorimetric detection were implemented. Type 1: For deionized water as a carrier stream with a injection of heparin and dye zones there were found the following optimal conditions: cdye = 1×10-5 mol dm-3 ; vflow = 2500 µl min-1 ; reaction coil length of 0 cm; injected volume of dye 150 µl and injected volume of sample 150 µl. Dynamic range of calibration curves with an exponential course for the individual dyes in the range of LOQ - 1200, eventually up 1500 IU dm-3 were detected. Limits of detection between 7.6 - 39.1 and the limits of quantification between 58.8 - 124.5 IU dm-3 were found. Type 2: For...
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