In-column dilution: Improving volume loadability in reverse phase gradient chromatography through the use of a silica pre-column.

Volume overload is a critical limitation in Reversed Phase (RP)-HPLC purification of pharmaceutical compounds. Limited solubility of these materials in most injection solvents leads to large injection volumes in order to maximize throughput. However, peak distortion due to volume overload limits injection amounts, and results in suboptimal use of chromatographic instruments. Volume loading for RP gradient separations was significantly increased by inserting a silica column ahead of the RP separation column. The sole purpose of this column is to dilute the plug of strong injection solvent so that the actual sample constituents are retained when the diluted injection plug arrives at the RP column. This is similar to the concept of a "retention gap" in GC, yet this has never been applied to liquid chromatographic separations. Injection volumes were increased by almost a factor of 3 when using appropriately sized silica columns. A discussion of critical parameters that determine the effectiveness of this approach is provided. The concept is easily applied and does not require any system modifications. It is therefore well suited for open access applications where more instrument intensive approaches, such as "At-Column Dilution", would be less desirable. We will also show that the generic concept which we have titled "In-Column Dilution" can easily be applied to increase the detection sensitivity for analytical application as well by allowing injection of larger sample volumes without peak deterioration for purifications.