Chromatography Forum

Hi all!

I have a bit of a challenge for you folks:

We are working on a multi-component liposome for delivery of siRNA, and we have been struggling with a chromatography method for quantification of all lipid components (siRNA is handled separately). We have tried C8 and C18 modalities (both referred to in literature), high temperature, and multiple organic solvents. The problems we have are thus:

1. The column to column variability is quite high, even within the same lot. The lipids shift in relative retention times.
2. There appears to be a fairly rapid column fouling, which is seemingly impossible to deal with.

We have been generally successful, but we are moving to qualification activities, and I am afraid our robustness is terrible. Our analytes include a permanently charge (quaternized amine) cationic lipid, cholesterol, a "helper" lipid (think DSPC, but a touch different), and a targeting compound (which we really don't have trouble with). The analysis difficulty is compounded by the need to use evaporative light scattering detection, which has proven to be a bit finicky.

What kind of approach would you take to tackle this? I can certainly respond with non-proprietary details, as necessary. Thanks!

I think you might want to investigate SFC (Supercritical Fluid Chromatography). Look up "SFC lipids" on Google and you will find a large number of hits. SFC uses liquid or supercritical carbon dioxide [CO2] as the main component in the mobile phase. A lot of the early work has been done with capillary SFC but most commercial instruments are now packed column systems. They use regular HPLC columns typically with polar [normal phase] stationary phases like silica, diol, amino and cyano. The technique can handle an extremely wide dynamic range of polarities when combined with polar organic modifiers [co-solvents], so your quat salt won't be left behind. On the other hand, your RNA may be a bit too polar for many phases.

A great test is to see if you can get the lipids to move on TLC plates [don't worry about tailing at this point]. If you can, you likely have a reasonably good shot with SFC

You will likely be most successful with packed column SFC which is easily interfaced to your ELSD. Mass Spec is another good choice for molecular weight determination. SFC is available from many mainstream vendors. There is even an implementation that converts a standard HPLC into an SFC.