Chromatography Forum

Been trying to work with the limited columns we have and can't get the results. PI says I can look into purchasing a new column to get the job done. Still sorta new at this so I come to you.

I want to inject an entire 1 umol oligonucleotide synthesis and have ideal resolution of product from N+/-1. Pump is capable of 10 mL/min ( PI doesn't want it up that high ) and it's connected to a computer running Clarity Lite ( blegh ).

Any and all recommendations/suggestions welcome.

How long are your oligos? Any modifications?

Oligonucleotides Separation Technology Columns are offered by several HPLC column manufacturers. For sure you can use any superior C18 column, but reproducibility is some times difficult for such tricky application.
If you are working in an diagnostic lab I would look for suppliers like Chromsystems or Recipe for a complete Kit.
Good luck.

How long are your oligos? Any modifications?

As of this moment 10-20 nts. Yes, 2'-propargyl. Though I couldn't tell you what modification I might need to synthesize next or the one after. I'd like it to be able to handle up to 50, but if i had to lower my length threshold of switching to PAGE that can be done.

Maybe I'm asking for the world here, but still new and trying to pick up as much as I can as fast as I can. Typically use A. 20 mM ph 7 TEAA B. 50% A 50% ACN. 5-100% B over 20 min.

10-20mer- "DMT-on" SPE-type purification, I would have to believe, would leave you with a very high purity product (so long as your synthesizer is working well)

We're a crystallography lab, I would like the highest purity possible to facilitate high quality single crystals.

Anion-exchange can separate a 21-mer from a 20-mer by a peak width and a half. See the example here: http://www.polylc.com/Oligo.v.2.htm

Of course, you would have to desalt the collected peak afterwards, but that's more straightforward than separating an oligonucleotide from its homologues +/- 1 base.

Anion-exchange can separate a 21-mer from a 20-mer by a peak width and a half. See the example here: http://www.polylc.com/Oligo.v.2.htm

Of course, you would have to desalt the collected peak afterwards, but that's more straightforward than separating an oligonucleotide from its homologues +/- 1 base.

Could you provided the literature and suggest the exact column size for the application I am looking for. I'm only familiar with anion-exchange with our analytic system.

Sorry for the delay in my response.

What literature are you asking me to provide? Clicking on the link in my previous message will take you to a web page showing the separation via anion-exchange that I described. It also provides the running conditions. The column is 100x4.6-mm in size. I estimate that the loading capacity is about 4 mg. You can scale up or down from that with columns of different sizes. If you'd like to discuss this in greater detail, then it would be a good idea to do so off-list.