Reverse-Phase High Performance Liquid Chromatography (RP-HPLC) operates on the same principle as a reverse-phase cartridge. However, the higher resolution allows for higher purity levels. HPLC is an efficient purification method for oligos with fluorophores, as their intrinsic lipophilicity provides excellent separation of product from its contaminants. Furthermore, RP-HPLC is a method of choice for larger scales due to the capacity and resolving properties of the column. The resolution based on lipophilicity will decrease with the length of the oligo. Therefore, RP-HPLC is usually not recommended for purifying products longer than 50 bases. Although longer oligos (up to 80 bases) can be purified using this method, the purity and yields may be adversely affected. However, RP-HPLC (HPLC) still remains as the HPLC method of choice than cartridge purification.
Reverse-Phase (RP) purification (cartridge purification) use the difference in hydrophobicity between full-length product (which contains a 5’-DMT group) and truncated sequences (without DMT groups). While the full-length DMT oligo is retained on the column, the truncated sequences are washed off . After cleaving the DMT on the cartridge, the expected product is recovered.
As the oligo length increases, the proportion of uncapped products (truncated sequences bearing the DMT) tends to increase. These impurities will not be removed by reverse-phase cartridge and thus for longer oligos, HPLC or PAGE is recommended. Oligos modified with certain dyes at the 5’-end (for example Cyanine® dyes or WellRED dyes), are compatible with this type of purification due to the increased lipophilicity imparted by the dye molecule. These cartridges are quite costly, not reusable, and generally of relatively low capacity, thus obviating their use in many laboratories.