HILIC seperation of short chain acyl CoAs and nucleotides
Posted: Thu Jun 23, 2005 12:54 pm
I am developing methods to separate short-chain acyl-CoAs (free CoASH, acetyl-CoA, malonyl-CoA, acetoacetyl-CoA, etc) and nucleotides (ATP, ADP, dATP, etc) using HILIC. These are polar phosphorylated molecules that are ususally separated by RP HPLC using a gradient from ~97% 0.1M PO4 buffer pH 5.3 to 40% ACN over ~60 min with UV detection at 260nm. I want to develop the separation to be compatible with ESI- MS, and HILIC seems to be the way to go.
So far I've tested PolyLC Aspartamide A and TSK Amide80 columns with isocratic and gradient separations of ACN or MeOH versus water, ammonium acetate pH 6.5, TEAP pH 2.8, or ammonium bicarbonate pH 8.8. The outcome is that the best separation uses a decreasing gradient of ACN vs ammonium bicarbonate pH 8.8 - all the other conditions either result in no elution or elution of all components in the standard mix as a single unresolved peak (mix is always dissolved in 80-90% ACN).
It's not surprising to me that the high pH gives the best separation, as this will result in the maximum charge (and hydrophilicity) on the CoA phospho groups. Unfortunately, this pH will also seriously degrade the abovementioned silica-based columns. To solve this problem, I was thinking about purchasing a Sequant ZIC-pHILIC column, which is polymeric and supposedly high pH-stable. There is even an application note on the Sequant website that describes a high pH HILIC method for nucleotides with this column:
http://www.sequant.com/products/zicphil ... 00-03A.pdf
I'd like to try this column, but it is rather pricey. Does anyone have experience in using this new polymeric HILIC column, or others like it, for nucleotide and/or small phosphorylated molecule separations?
Thanks
Tony
So far I've tested PolyLC Aspartamide A and TSK Amide80 columns with isocratic and gradient separations of ACN or MeOH versus water, ammonium acetate pH 6.5, TEAP pH 2.8, or ammonium bicarbonate pH 8.8. The outcome is that the best separation uses a decreasing gradient of ACN vs ammonium bicarbonate pH 8.8 - all the other conditions either result in no elution or elution of all components in the standard mix as a single unresolved peak (mix is always dissolved in 80-90% ACN).
It's not surprising to me that the high pH gives the best separation, as this will result in the maximum charge (and hydrophilicity) on the CoA phospho groups. Unfortunately, this pH will also seriously degrade the abovementioned silica-based columns. To solve this problem, I was thinking about purchasing a Sequant ZIC-pHILIC column, which is polymeric and supposedly high pH-stable. There is even an application note on the Sequant website that describes a high pH HILIC method for nucleotides with this column:
http://www.sequant.com/products/zicphil ... 00-03A.pdf
I'd like to try this column, but it is rather pricey. Does anyone have experience in using this new polymeric HILIC column, or others like it, for nucleotide and/or small phosphorylated molecule separations?
Thanks
Tony