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Development of a Method for Separation of Adenine Nucleotides

Miegel, Erika LU (2020) KASM01 20192
Centre for Analysis and Synthesis
Abstract
Adenosine tetraphosphate, or Ap4A, is a nucleotide with very interesting proposed biological effects. Previous studies have indicated that blood glucose levels in mammals might increase in the presence of Ap4A, and that Ap4A might antagonize pancreatic
ATP channels. In order to study these effects further, a reliable method of separating Ap4A from other nucleotides in biological samples is required. This is made difficult by the high polarity and the high structural resemblance of different nucleotides. The
aim of this study was to develop a method for separation of Ap4A from ATP and AMP on the Agilent Poroshell 120 HILIC-Z column, using conditions compatible with mass spectrometry.

A method previously employed to separate simple... (More)
Adenosine tetraphosphate, or Ap4A, is a nucleotide with very interesting proposed biological effects. Previous studies have indicated that blood glucose levels in mammals might increase in the presence of Ap4A, and that Ap4A might antagonize pancreatic
ATP channels. In order to study these effects further, a reliable method of separating Ap4A from other nucleotides in biological samples is required. This is made difficult by the high polarity and the high structural resemblance of different nucleotides. The
aim of this study was to develop a method for separation of Ap4A from ATP and AMP on the Agilent Poroshell 120 HILIC-Z column, using conditions compatible with mass spectrometry.

A method previously employed to separate simple nucleotides, using 10 mM ammonium acetate buffer at pH 9, a column temperature of 25℃, a flow rate of 300 µL/min, and a re-equilibration time of 33.3 minutes was used as a starting point. [1] In order
to investigate the retention mechanism of the separation, an array of parameters (column temperature, mobile phase flow rate, re-equilibration time, buffer concentration, and mobile phase pH) were varied one by one while all the other parameters were held constant.

Out of all evaluated parameters, increasing the buffer concentration to 20 mM was the only adjustment that had a positive effect on the separation. In this method two peaks were observed; one corresponding to ATP + AMP and the other corresponding to Ap4A, with a resolution of about 1.3. Furthermore, it was found that reproducible results could be achieved with a re-equilibration time as short as 8.3 minutes. (Less)
Popular Abstract (Swedish)
På den ryske botanikern Mikhail Semyonovich Tsvets gravsten står det "Han uppfann kromatografin, separerade molekyler men förenade folk". Hans metod för att separera molekyler används än idag - till exempel i detta arbete.

Tsvets försöksuppställning bestod av en glascylinder fylld med kolsyrad kalk, samt en lösning som innehöll olika färgpigment från växter. En stund efter att han hällt pigmentblandningen i den kalkfyllda cylindern visade det sig att de olika färgerna hade delat upp sig som på bilden. Pigmenten hade nämligen olika kemiska egenskaper och hann därför rinna olika långt genom cylindern innan de fastnade i kalken. Tsvet kallade sin teknik för kromatografi efter grekiskans färgskrift. Idag utförs kromatografi med avancerade... (More)
På den ryske botanikern Mikhail Semyonovich Tsvets gravsten står det "Han uppfann kromatografin, separerade molekyler men förenade folk". Hans metod för att separera molekyler används än idag - till exempel i detta arbete.

Tsvets försöksuppställning bestod av en glascylinder fylld med kolsyrad kalk, samt en lösning som innehöll olika färgpigment från växter. En stund efter att han hällt pigmentblandningen i den kalkfyllda cylindern visade det sig att de olika färgerna hade delat upp sig som på bilden. Pigmenten hade nämligen olika kemiska egenskaper och hann därför rinna olika långt genom cylindern innan de fastnade i kalken. Tsvet kallade sin teknik för kromatografi efter grekiskans färgskrift. Idag utförs kromatografi med avancerade apparater under högt tryck, och kalk används inte längre som packningsmaterial, men principen är fortfarande densamma.

Ibland kan ämnen med väldigt olika biologiska funktioner ha liknande kemiska egenskaper. Olika nukleotider, som till exempel adenosintrifosfat (ATP), är sådana molekyler. ATP minns du säkert som kroppens energivaluta, men det finns även mindre välkända nukleotider, exempelvis diadenosintetrafosfat (Ap4A). Ap4A liknar ATP i sin kemiska struktur, men tros till skillnad från ATP påverka utvecklingen av diabetes. För att undersöka denna molekyl måste man först separera den från andra nukleotider med hjälp av kromatografi, men eftersom kromatografi bygger på molekylers olika egenskaper är detta en utmaning. I mitt arbete har jag använt en speciell kromatografisk metod som kallas för HILIC - ungefär hydrofil kromatografi - för att utnyttja små skillander mellan olika nukleotider för att separera dem. Efter mycket arbete och en gnutta kromatografisk magi i labbet lyckades jag - nästan. (Less)
Please use this url to cite or link to this publication:
author
Miegel, Erika LU
supervisor
organization
course
KASM01 20192
year
type
H2 - Master's Degree (Two Years)
subject
keywords
analytical chemistry, HPLC, HILIC, chromatography, technical analytical chemistry, teknisk analytisk kemi
language
English
id
9005987
date added to LUP
2020-03-26 08:42:30
date last changed
2020-03-26 08:42:30
@misc{9005987,
  abstract     = {Adenosine tetraphosphate, or Ap4A, is a nucleotide with very interesting proposed biological effects. Previous studies have indicated that blood glucose levels in mammals might increase in the presence of Ap4A, and that Ap4A might antagonize pancreatic
ATP channels. In order to study these effects further, a reliable method of separating Ap4A from other nucleotides in biological samples is required. This is made difficult by the high polarity and the high structural resemblance of different nucleotides. The
aim of this study was to develop a method for separation of Ap4A from ATP and AMP on the Agilent Poroshell 120 HILIC-Z column, using conditions compatible with mass spectrometry.

A method previously employed to separate simple nucleotides, using 10 mM ammonium acetate buffer at pH 9, a column temperature of 25℃, a flow rate of 300 µL/min, and a re-equilibration time of 33.3 minutes was used as a starting point. [1] In order
to investigate the retention mechanism of the separation, an array of parameters (column temperature, mobile phase flow rate, re-equilibration time, buffer concentration, and mobile phase pH) were varied one by one while all the other parameters were held constant.

Out of all evaluated parameters, increasing the buffer concentration to 20 mM was the only adjustment that had a positive effect on the separation. In this method two peaks were observed; one corresponding to ATP + AMP and the other corresponding to Ap4A, with a resolution of about 1.3. Furthermore, it was found that reproducible results could be achieved with a re-equilibration time as short as 8.3 minutes.},
  author       = {Miegel, Erika},
  keyword      = {analytical chemistry,HPLC,HILIC,chromatography,technical analytical chemistry,teknisk analytisk kemi},
  language     = {eng},
  note         = {Student Paper},
  title        = {Development of a Method for Separation of Adenine Nucleotides},
  year         = {2020},
}