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HPLC determination of gallic acid as a possible product of enzymatic reaction of shikimic acid, NADP+ and SDH.
Smolejová, Jana ; Červený, Václav (advisor) ; Nesměrák, Karel (referee)
This diploma thesis deals with the development of an HPLC method for the determination of selected compounds participating in enzymatic reaction leading to the formation of gallic acid. The analysed reaction mixture contains the following reagents: shikimic acid, NADP+ and shikimatedehydrogenase (SDH) extracted from parsley; the presumed product of the reaction is gallic acid. Two chromatographic methods for the determination of the above mentioned compounds were developed using C18 HPLC column and porous graphitic carbon Hypercarb column. Molecular absorption spectrometric detection in the UV range was used in all measurements. Separation on the C18 column was found particularly suitable for analysing the composition of the end products of the reaction. Because of the NADP+ and shikimic acid peak overlap it is necessary to observe absorbance at 212 and 260 nm. Shikimic acid and NADP+ can be quantified due to the fact that shikimic acid does not absorb at 260 nm while NADP+ absorb radiation at both wavelengths. Separation via Hypercarb column was found particularly suitable for analysing the process of the reaction; additional products or intermediates can be seen in chromatograms compared to the C18 method. Determination with Hypercarb column is characterized by higher sensitivity and lower limit...
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