I am analyzing nucleotides from cells and I have then found that the separation is very good on C18 columns with tetrabutylammonium ions as ion pairing agent. But, as I have discussed in an earlier thread, the loading capacity is very limited (I can anly load a limited amount) and a conclusion then was that this is typical when analyzing ions on C18 (the loading capacity can according to literature decrease up to 50 times), and one of the reasons for this is charge repulsion. In the case of the nucleotides I am studying (NTPs and dNTPs), the charge is -3 to -4 depending on the pH (I am analying at pH 5.5.-6.5) and I was thinking how this separation is affected by the ion pairing agent. Tetrabutylammonium is quite bulky and I guess it is difficult for many of them to bind to a single nucleotide. Is there anyone that has experience of trying different types of ion pairing agents? I could imagine that having one longer chain and the other ones much shorter (e.g. N,N,N-trimethyl-N-octylammonium ion) would create more space to fit several of them to the same nucleotides and thereby neutralize the charge better and perhaps increase the loading capacity.
Thanks for the suggestion. I got the feeling that this has not been studied for nucleotides and decided to order some other ion pairing agents to get a better understanding of this.
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