The challenges associated with the reversed-phase liquid chromatography (RPLC) analysis of intact proteins are due to the issues, such as carryover or “ghosting”, and poor recovery. These issues arises due to the adsorption of proteins on the column frit, packing material, and column walls.
In a study published in 2006, Eschelbach and Jorgenson have shown that increasing the column pressure eliminates carryover, and improves protein recovery. For this study, they used four model proteins (ribonuclease A, bovine serum albumin, myoglobin, and ovalbumin) and subjected them to RPLC separation at conventional and ultrahigh pressure. They used two separate columns that have approximately same dimensions: 360-µm outer diameter, 50-µm inner diameter, and 35 cm lengths; but, packed with C18 particles of different diameters.
The column used for conventional RPLC and run at a pressure of 160 bar was packed with 5-µm diameter particles, while the ultrahigh pressure column run at 1600 bar was packed with 1.4-µm diameter particles. The flow rate used in each column was ~130 nL/min. The eluted proteins were detected with electrospray time-of-flight mass spectrometry (ESI-TOF-MS).
The results show that the upper limit of pressure that eliminates carryover of proteins from the column used in this study is 1600 bar.
Protein recovery was determined from the peak area after UV detection at 215 nm. Figure 1 shows recovery curve of RNase A at conventional (160 bar) and ultrahigh (1600 bar) pressures. The slopes show that recovery of RNase A at ultrahigh pressure is ~60% greater than at the conventional low pressure RPLC.
|Figure 1. Recovery curve for ribonuclease A at conventional, 160 bar, and ultrahigh, 1580 bar, pressure.|
The proposed mechanisms for improved recovery of proteins by ultrahigh pressure are partial unfolding, and increased solubility. The partial unfolding of proteins caused by ultrahigh pressure exposes their hydrophobic core favoring better interaction with the hydrophobic stationary phase and possibly improving protein recovery. The ultrahigh pressure may also cause deaggregation and increase the solubility of proteins at the head of the column, enabling efficient recovery.
Eschelbach JW and Jorgenson JW (2006). Improved protein recovery in reversed-phase liquid chromatography by the use of ultrahigh pressures. Anal. Chem.