Prep LC from Analytical Scouting Runs


Prep LC from Analytical Scouting Runs

Which standard operating procedure in a preparative chromatography lab is more efficient: routinely running generic full-range gradients to purify mixtures, or taking the extra time and trouble to develop focused gradient methods? The answer may depend, in general or on a case-by-case basis, on how long it takes to develop the method, the purification run time, the quantity of sample and solvent consumed, and your output quality requirements (level of purity, yield).

Simple full-range (0 to 100%) gradients require minimal time to develop and are useful for mixtures with a wide range of structures. To improve resolution the analyst may extend the run time to 'flatten' the gradient. The benefit of not having to develop a more detailed method, however, is substantially offset by the longer run time and added solvent required to achieve resolution between compounds.

Consider as well that in many prep LC applications the goal is not to resolve multiple compounds, but instead to separate one target alone, as quickly as possible, and with the highest practical purity and yield. Optimizing a prep LC method for single-target purification takes additional effort, which is often justified by faster purification, reduced solvent consumption, and higher productivity based on the ability to load more compound per run.

When comparing the pros and cons of generic vs. focused gradients, it helps to envision the changing behaviour of a compound during a 0% to 100% gradient. Once the injected sample has adsorbed onto the stationary phase, before the target compound has fully eluted, it will be in one of three states (below) in which k*represents the retention factor under specific gradient conditions.

  1. Strong retention on the stationary phase; compound not moving (k* >> 10)
  2. Weak retention on the stationary phase; beginning to travel down the column (1 < k* < 10)
  3. Zero retention on the stationary phase; compound traveling at solvent front (k* < 1)

These three pre-elution states each correspond to a specific %B zone within the gradient. There is a fourth zone post-elution where the process continues to run while the compound is already off the column.

From a purification standpoint, the simplest case would one in which all non-target compounds are completely retained while the target is fully eluted. The reverse occurs when the target compound is retained on the column, while the impurities fully elute. The target is then eluted using a second strong solvent, that is, a catch and release purification.

The most difficult, by contrast, would involve two or more compounds having weak retention in the same %B range, in which case they will easily co-elute unless an optimized gradient is employed.

Generic gradients are relatively inefficient as compared to focused gradients. In a generic method, the most effective segment of the 0-100% gradient range is the 5-15 %B portion in which the k* value for the target compound is between 1 and 10, with the compound actively migrating down the column. In a generic gradient, therefore, 85% to 95% of the run is wasted because the compound is a) still sitting at the head of the column and not moving at all; or b) moving with the solvent front and having no further interaction with the stationary phase; or c) already eluted and collected into your test tube.