Chromatography Forum

I am currently in the process of developing a reverse phase separation for a synthetic oligonucleotide. I have tried several different columns and procedures supplied by the column manufacturer but in all cases do not obtain a separation just a broad ill-defined peak. Any suggestions?

Hard to say without more detail, but one thought is that if your "oligo" is reasonably big and not very pure, you may be seeing the "envelope" of a large number of slightly separated peaks.

Andy,
RPLC separations of oligonucleotides often have problems when the oligos have secondary structures. Elevated temperatures may help, but anion-exchange columns like the DNAPac PA100/200 [Dionex] series offer more versatility for breaking the hydrogen bonds that cause the poor chromatography. They also offer better resolution. If your application requires high purity oligo, I would recommend the DNAPac columns.
Cheers,
Jim Thayer

If Tom is right, a further flattening of the gradient will help, and you will get multiple peaks.

We currenly have an anion exchange procedure which offers minimal resoultion between the main components and the impurities. In addition, I have reduced the slope to as low as 0.1% Acetonitrile/minute and have not achieved any resolution between the main peak and the impurities.

Andy,

In your last post you refer to an Anion-Exchange separation, then mention using a slope of 0.1% MeCN/minute gradient. Do these refer to the same method?

For the AEC method, if you are obtaining "minimal resolution between the main component and the impurities", then you may need to adjust the pH. As the eluent pH increases above pH 9, the tautomeric oxygens on each T (or U) and G will begin to ionize, thus increasing the net charge on the oligo in a base-composition dependent manner. At pH ~12, the tautomeric oxygens on both bases will be fully ionized. This often helps resolve difficult to separate components.

In some cases, modification of the oligo (e.g, phosphorothioates) the subtitutions can impede the separations. In such cases altogether different methods may be needed. Are your oligos derivatized?

Cheers,
Jim Thayer

Andy,

Oligonucleotides with secondary structures (does your oligo have secondary structures?) can give poor chromatography at ambient temperatures. When you run at higher temperatures (60 to 80ºC) you should easily get better resolution. I am going to dig up some literature that show the effect of temperature on separation performance of "difficult" oligos in RP mode (on polymeric RP columns).

Best regards,

Robert Haefele
Hamilton Company
rhaefele_at_hamiltoncompany.com

They were separate methods. The anion exchange method uses Tris buffer at pH 8.0 with 1 M LiCl as the eluent. The acetonitrile gradient referred to the reverse phase method which we a trying o develop. Both methods are run at elevated temperature (80 C for anion exchange and 60 C for reverse phase).

Dear Andy,

Assuming that you are using a non-porous anion exchanger like the DNAPac columns, you may drop the temperature to 25-27°C, and replace the tris buffer with 25 mM NaOH (resulting pH will be 12.4 where Watson-Crick H-bonds are fully disrupted). Do NOT employ both high temperature and high pH with these columns as that combination is known to degrade methacrylate-based phases.

Use of these conditions should dramatically improve your separation, unless you are using either a weak anion exchanger (no capacity at high pH) or a porous strong anion exchanger (with these columns, a much lower flow rate will be needed to recover resolution lost by the poorer mass transfer properties of porous ion-exchange phases).

Cheers,
Jim Thayer

Just to update everyone. The magic ingredient to obtain the separation was hexafluoro-2-propanol. The papers that I had read said that this improved the ionization in LCMS but nothing about improving the separation.