By Anonymous on Tuesday, February 18, 2003 - 09:16 am:
hello all-
i am trying to run a basic HPLC seperation with ATP and a reverse phase column, hopefully using a methanol/H2O mobile phase. the ATP, however, is not being retained and is appearing in the solvent front. please help- thanks
By Greg Kovac on Tuesday, February 18, 2003 - 09:41 am:
your compound is neutral,acid or base?
when ionic compounds are present pH can have a marked influence on retention and selectivity.
By Anonymous on Tuesday, February 18, 2003 - 10:43 am:
thanks greg- i havent touched the pH although my next step was to lower the pH- what pH do you think i should use?
By Anonymous on Tuesday, February 18, 2003 - 11:28 am:
Please supply more info.... what column are you using? .... gradient or isocratic flow? Is your analyte adenosine triphospate (ATP)? This is water soluble and may be best separated on a column specific for hydophilic compounds.
By Anonymous on Tuesday, February 18, 2003 - 11:42 am:
c18 reverse phase column, 1ml/min isocratic flow of 20%MeOH in water, analyte is ATP only. i am getting the analyte in the solvent front- will pH increase the retention time enough or should i use an ion-pairing agent?
By Anonymous on Tuesday, February 18, 2003 - 12:16 pm:
Maybe you should try tetrabutylammoniumhydrogensulphate.
I have good experiences with this modifier.
By Einar Pontén - SeQuant AB on Tuesday, February 18, 2003 - 02:40 pm:
The free amino group and the phosphate groups of ATP (Adenosine triphosphate) will make this molecule highly influenced by pH. When charged the retention on a C18 is none.
Ion-pairing is one alternative by adding a counter-ion into the eluent.
I also suppose that hydrophilic interaction chromatography (HILIC) is an alternative.
By Patrik Appelblad on Tuesday, February 18, 2003 - 02:50 pm:
Dear Anonymous,
Change your mode of separation to Hydrophilic Interaction Liquid Chromatography (HILIC).
Polar compounds, such as ATP are well suited for hydrophilic interaction chromatography.
Below you'll find a link to a test chromatogram showing the separation of uracil and cytosine.
http://www.sequant.com/pdf/2700-05A2.pdf
I've performed a few tests on ATP as well, and it is easily retained on our ZIC-HILIC phase.
Unfortunately,I do not have those data as a pdf.
However, if you would like to discuss the use of HILIC for your application, please drop me an email.
Kind Regards,
PAtrik
By Anonymous on Monday, March 29, 2004 - 04:36 am:
Which is the best separation method for nucleotides (ATP, ADP and AMP)