Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) works similarly to a reverse-phase cartridge. However, its higher resolution results in higher purity levels. HPLC is an efficient purification method for oligonucleotides containing fluorophores since their intrinsic lipophilicity provides excellent product separation from contaminants. RP-HPLC is the method of choice for larger-scale purification due to the capacity and resolving properties of the column. The resolution based on lipophilicity will decrease with the length of the oligonucleotide. Therefore, RP-HPLC is usually not recommended to purify products longer than 50 nucleotide bases. Although longer oligonucleotides (up to 80 nucleotide bases) can be purified using this method, the purity and yields may be adversely affected. However, RP-HPLC (HPLC) remains the HPLC method of choice rather than cartridge purification.
Reverse-Phase (RP) cartridge purification uses the difference in hydrophobicity between full-length product (which contains a 5’-DMT group) and truncated sequences (without DMT groups). The full-length DMT oligonucleotide binds to the column, and the truncated sequences are washed off. The product is recovered after cleaving the DMT on the cartridge.
As the oligonucleotides length increases, the proportion of uncapped products (truncated sequences bearing the DMT) also tends to increase.
These impurities will not be removed by reverse-phase cartridge and thus, for longer oligonucleotides, HPLC or PAGE is recommended. Oligonucleotidess 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 preventing their use in many laboratories.