Immobiliseret fluorescerende pH indikator til HPLC

Simon Hockenhull

Studenteropgave: Speciale


By using an immobile pH indicator it is possible to detect pH in media monitored in fluorescence microscope or confocal laser scanning microscope. This principle makes it possible to monitor shifts in pH under circumstances where it would otherwise be impossible to monitor such shifts. In response to a request from FEF Chemicals A/S the work behind this thesis will develop a pH indicator, the purpose of which is to constitute a stationary phase in a column. Detection of pH shifts takes place directly in the column with eluent and solute flowing by. The work involves fluorescein, anthraquinone-2-carboxylic acid, amino-propyl silane, and a protocol for developing amide compounds will be introduced. Amide compounds are synthesized through reaction with oxalyl chloride and with DMF as the catalyst, and the products are characterised. Characterisation takes place by comparing data for the pure starting compounds with data for the synthesized product. The NMR method is used to prove the correctness of the product and is supported by LC-MS, HPLC and IR analyses. HPLC is the key method and experiments are made with eluent compounds. A spectrofluorometer is used for analysing excitation and emission maximum for fluorescent reactant and for synthesized product. The immobile product is tested for pH sensitivity, pH reversibility and chemical binding to the stationary phase by using the Fluorescence Recovery After Photobleaching technique (FRAP). Based on test results the conclusion is that an immobile and fluorescent pH indicator is detected, and that a shift in intensity can be observed in the pH range of 8 to 2.

UddannelserKemi, (Bachelor/kandidatuddannelse) Kandidat
Udgivelsesdato4 nov. 2008
VejledereJohn Mortensen


  • fluorescein
  • immobil pH indikator
  • Flourescence Recovery After Photobleaching (FRAP)
  • pH reversibilitet
  • oxalylchlorid
  • confocal laser scanning microscope
  • Spektrofluorometer
  • anthraquinon-2-carboxylsyre
  • aminopropylsilan
  • HPLC