Top 10 Misunderstandings of HPLC and UHPLC Columns (part3)
The guard column is not necessary
There are many benefits to using a guard column. First, the guard column prevents chemicals or particles from damaging the analytical column. Second, replacing a 5mm guard column requires only a small cost compared to replacing an expensive analytical column. Modern guard columns have the advantage of virtually no dead volume, fast replacement, and high pressure for UHPLC.
You can’t reverse the HPLC column to flush out the particles.
In fact, the HPLC column fill pressure is much higher than the maximum use pressure (usually 2 times higher). If a proper homogenizer is used for the column and a time is allocated to stabilize the bed, a well-filled column can be used in both directions.
An exception to the reverse use of the column is that the manufacturer uses a larger aperture plate at the injection end of the column, and reverse use may flush the packing out of the bed. If the manufacturer uses a high porosity frit at the inlet of the column, then backflushing the column may flush the column packing from the packed bed. When the column is packed at the factory, the sieve opening at the outlet end must be smaller than the smallest particle size in the column. For example, the average particle size of the chromatographic packing is 5 μm, the particle size distribution range is 3-7 μm, and the pore size of the outlet end sieve plate must be less than 3μm, so that the filler is not likely to run from the bed to the outside of the column. Most manufacturers choose a sieve plate with a pore size of 2 μm. As for the column screens at both ends of some manufacturers, the aperture is not the same. Generally, the injection end is larger and the sample end is smaller. Therefore, some manufacturers place an arrow indication at the column label indicating that it must be used in only one direction. There is certainly a possibility, so a chromatographer should read the column manual or instructions, or determine with the manufacturer whether the column can be backflushed.
The smaller the particle size of the column packing, the higher the pressure, the better the separation effect.
Ultra-small particle size and ultra-high column pressure are not necessarily the best choice for chromatographers!
Column characteristics studies of modern columns have led to new methods to evaluate the performance of a column. For example, a column with a new superficially porous material has the same column efficiency as the sub-2μm UHPLC column, but the column pressure is very low compared to conventional LC packed columns.
Column pressure does not affect chromatographic separation
Many parameters of the chromatogram are affected by column pressure, including the molar volume of some solute, stagnation volume, column porosity, retention factor, mobile phase density, dielectric constant, stationary phase structure, pH and ionization constant. Why does column pressure cause more and more attention? The reason is that many commercially available chromatographs are ultra-high pressure chromatographs and columns. When the column is operated at a pressure of about 2000 psi (13.789 MPa), even if there is a small difference in retention time, it does not attract our attention; especially if the repeatability is good and the quantification is not affected. However, when the column pressure is close to 2000 psi (13.789 MPa), the effect of column pressure may be quite significant.