Common problems occurred in the use of HPLC columns
HPLC column is the “HEART” of the chromatographic system. Due to the wide variety of samples and the complex matrix of the sample, HPLC column is easily contaminated by strong retained substances. These strongly retained substances are usually difficult to elute. After multiple sample loadings, they accumulate at the end of the column, block the frit, and may even interact with the packing materials to cause it to denature, which leads to a decrease in column efficiency, a decrease in resolution, and an abnormal peak shape and other abnormal phenomenon. Therefore, the maintenance of the column is very necessary.
Maintenance of HPLC columns
HPLC columns require maintenance, but column maintenance varies from manufacturer to manufacturer. Welch Column is recommended as “forward use and back flushing”. Because the pore size of the frits used at both ends of Welch’s column is the same, there will be no case where the packing materials is flushed out during backflushing. When the sample is analyzed, most of the contaminants are enriched at the end of the column. At this time, if backflushing is immediately performed, column lifetime can be greatly extended.
How to back flush the HPLC columns?
The principle of column back flushing is to flush with a solvent which has a higher elution capacity than the mobile phase.
Reversed-phase columns (C18, C8) can be backflushed with less polar solvents. The following flushing methods are recommended:
Water → methanol → acetonitrile → isopropanol → dichloromethane → isopropanol → acetonitrile
Normal phase columns (SiO2, NH2, CN, Diol) can be backflushed with more polar solvents. The flushing method is as follows:
If the mobile phase contains buffer salts and abnormal column pressure rises during the running sequence, buffer salts are precipitated on the column bed. The following maintenance methods are recommended:
• back Flush the column with 60 column volumes using methanol/water = 10/90 at analytical flow rate, column temperature: 35 °C;
• back flush the column overnight with methanol/water = 10/90 at a flow rate of 0.2 ml/min.
Correct use flow of mobile phase containing buffer salts:
Example: There is buffer salt in the mobile phase, try to back flush with 10% methanol 0.2ml/min. After one night, the column efficiency returns to normal.
Column: Ultisil XB-C18, 4.6 x 250 mm, 5 μm.
Wavelength: 254 nm;
Mobile phase: methanol/water = 75/25;
Temperature: 25 ° C;
Flow rate: 1.0 ml/min;
Injection volume: 20 μL.
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