HPLC of Nucleotide Triphosphates

bates bates at liv.ac.le
Tue Feb 21 07:25:30 EST 1995


Stephen Marshalko writes:

>I realize that this is not strictly a molecular biology question, but I was 
>wondering if anyone out there who had experience with the purification of 
>nucleotide triphosphates would be able to help. Ideally, I would like to 
>purify some triphosphates using HPLC, but use a volatile buffer as opposed 
>to the standard high salt phosphate buffer system that is routinely employed
>on a strong anion exchange column (SAX). If anyone had any suggestions for 
>me I would really appreciate it. Thanks,


Stephen,

One way is to elute your ion exchanger with a gradient of
triethylammonium bicarbonate (TEAB), made by bubbling CO2 through an
appropriate concentration solution/suspension of triethylamine until it
gets to about pH 8.5.  The eluted product will now freeze-dry to leave
the triethylammonium salt of your NTP, and nothing else.  I was doing
this just this week, and it works well.  If you need a different salt
in the end, then you will need to make the free acid, using a cation
exchanger, then neutralise with eg NaOH.

Hope this is helpful.

Andy Bates.

____________________________________________________________
Dr. Andrew D. Bates             Tel:   (+44) (0)151 794 4322
Department of Biochemistry      Fax:   (+44) (0)151 794 4349
University of Liverpool         Email: bates at liv.ac.uk
PO Box 147
Liverpool
L69 3BX, UK



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