It was also in the 1980's that many of the HPLC systems became more reliable and added fully computerized automation for data acquisition and data analysis which were huge improvements over what we had before (strip chart records and cutting peaks out with scissors is not missed by me). Low, solvent saving flow rates were used with super high-efficiency columns (1.0, 2.1 and 3.0 mm ID columns with 3u particles) to produce some great separations, but the market was just not ready for it. Back then, I used one of the early HP 1090M HPLC systems with a DAD and DR5 pumps with great success that many modern HPLC systems have only recently equaled and surpassed). We first starting using small particles packed in narrow diameter commercial HPLC columns coupled to low delay volume HPLC systems in the early to mid 1980's (e.g. So called " UHPLC" is nothing more than HPLC, by a different marketing name. The technique of HPLC is mature (~ 53 years old) with few major advances in the past years. This question comes up frequently and progress has been slow. 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. 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. Tetrabutylammonium is quite bulky and I guess it is difficult for many of them to bind to a single nucleotide. 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. 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. 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.
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