Well, "the best" conditions will nobody be able to tell, as the coupling efficacy and impact of side reactions is a multiple variants depending complex system. For example, you will apply very different conditions depending on making a ~20nt siRNA or a 50-70nt aptamer or something even longer.

Please specify the trouble you experience, then one will be able to give solid advice what to look into.

As you named coupling time let me tell you the following. Long coupling time will raise the efficacy of the coupling, but the slight acidity of the activator can lead to early dmt cleavage of rG and rA. Hence statiscally distributed M+1 products will appear. However, this might be tunable with another activator.

Best,

Lucas

 First of all thank you for you answer Lucas, that's true I wasn't too precise. I recently started to work with AKTAoligopilot 10 which was disregulated. I am trying to optimize method for RNA 1umol scale using 2'OTBS protecting group and preparing 8mer. First of all I experience loss of efficacy during first attachment (usually about 30%) but than it stabilize on relative constant level. Secondly, I noticed rise of yield by changing recycling speed from 275 to 350 cm/h and I am wondering how further increases in recycling velocity with influence the each cycle yield for shorter (8-20mers) and longer (50-75mers) RNAs. For me it is hard to compare batch synthesizer (e.g.K&A) and times used for couplings, capping and oxidizing with continuous flow synthesizer (like AKTA). I would appreciate any tips on that.

If you are using CPG the efficacy drop in the first few couplings are normal. This is more a property of the CPG, the linker used for the loaded base and the loaded base itself.

Keep in mind, that you probably do not record full peaks in detrit, but haircuted Peaks. This means the Signal you see is more a kintetic, not an area under the curve being linear to the activity. Make sure you record full peaks first.

The recycle can be followed by conductivity readout. Conductivity should have a nicely oscillating shape with decreasing amplitude over the time (bit like the FID in NMR before FT). Every high peak in the oscillation means that the reagent front of activator and amidite is at the conductivity detector. So you now know how often the reagent front hits the solid phase. The reagents are not homogenously distributed over the coupling volume. Recycling faster at constant coupling time means that you will push the reagent front more often to the solid support. As soon as the oscillation is dissipated, the reagents are uniformly distributed (which also means diluted by the ACN push volume).

Rising the recycle speed will increase back pressure. If the backpressure is okay you might raise the recycle speed further, thus making longer contact of reagents to the solid phase. However, the coupling reaction is limited by diffusion into the pores, a high speed will at some point hamper this. My suggestion would be to stay with the default recycle speed and elongate the recycle time.