Top 10 Misunderstandings of HPLC and UHPLC Columns (part 2)
4. At least 10 column volumes are required to re-balance the LC column
Equilibration time is very important for gradient chromatography because it is a limiting factor for the entire technology. There are two types of balance: repeat balance and full balance. Repeated balancing also means a balance that can be achieved without achieving complete balance. In fact, if the repeatability of the retention time is less than 0.002 min in subsequent runs, then the unbuffered eluent and basic compound for the non-ionized solute are supplemented with the usual trifluoroacetic acid and formic acid as additives. The balance is achieved within the range of two column volumes.
5. Surface porous particles can significantly reduce sample capacity compared to pure porous particles
The capacity of the HPLC column packing to the sample is proportional to its surface area, which is related to the amount of chemically bonded phase formed by the silanol group bonding through the monomer. However, the results of the study indicate that under the same experimental conditions, the surface porous particles and the porous particles have substantially the same capacity for the sample.
6. UHPLC packed columns are more susceptible to blockage than common HPLC packed columns
As the particle size of the column packing porous material continues to decrease, the main hardware design of the column changes. Blockage of the sub-2 μm UHPLC column is the result of sample and mobile phase material lodging at the column inlet. If you clean the sample before injection, such as using solid phase extraction, filtration or centrifugation, you can avoid any contamination problems.