Common Problems and Solutions of HPLC Columns

When Using Hawach HPLC column, such as You also can choose C18 Alkaline HPLC Columns, Phenyl-Ether HPLC Columns, C18 Standard HPLC Columns, and other used HPLC columns, you may encounter the following problems.

1. What are the most basic indicators for evaluating an HPLC column?
The basic indicators include: number of plates, peak asymmetry factor, column pressure, range of application and concentration of bonded phase, and peak capacity.

2. When doing HPLC analysis, the column pressure is unstable, what is the reason? How to solve it?
a. There is air in the pump, and the solution is to remove the air in the pump and degas the solvent.
b. Because of the proportional valve failure, it should be replaced a proportional valve.
c. Pump gasket is damaged, and replace the gasket.
d. There are air bubbles in the solvent, the solution is to degas the solvent, change the degassing method if necessary.
e. Find out the leak point, and seal it.
f. Gradient elution: the pressure fluctuation is normal.

3. Why is the column pressure too high during HPLC column acceptance test?
There are many reasons for this, and often it is not the problem of the column itself, check the cause of the problem based on the followings .
a. Remove the protective pre-column, see if the column pressure is still high, otherwise it is the problem of the protective column; if the column pressure is still high, check again.
b. Remove the column from the instrument and see if the pressure drops, otherwise it is a blockage of the pipeline and needs to be cleaned; if the pressure drops, check again.
c. Reverse the inlet and outlet of the column and connect it to the instrument, and flush the column with 10 times the column volume of mobile phase (do not connect the detector at this time to prevent solid particles from entering the mobile cell). At this point, if the column pressure still does not drop, check again; use only for used columns.
d. Replace the column inlet sieve plate. If the column pressure drops, it means that the solvent or sample contains particulate impurities and it is these impurities that are blocking the sieve plate causing the pressure to rise. Generally, an in-line filter between the inlet and the guard column can avoid the problem of high column pressure.

4. What other factors besides flow rate can cause pressure changes?
Changes in mobile phase composition and temperature; changes in column length, column I.D. and packing size; sudden column blockage pressure increase (all other things being equal the column pressure increases gradually under normal conditions).

5. Will the mobile phase in the HPLC column drain?
For operators who do chromatographic separation tests have encountered the situation that the mobile phase was not replenished in time and the pump drained the mobile phase from the solvent bottle, thus the HPLC system stopped working. Will this damage the column? Has the pump drained all the mobile phase from the column? Can the column still be used?

In fact, if the pump drains the mobile phase from the solvent bottle, it will not damage the column. Even if the pump is full of air, the pump will not drain the air into the column. The reason is that the pump can only deliver liquid, not air. In contrast, another situation more likely to happen is forgetting to close the sealing caps on the ends of the column or having the caps too loose and drying out the column. Again, it is less likely that the entire column will dry out, and most likely only a few millimeters at each end of the column will dry out, as it takes a considerable amount of time to evaporate all the solvent to dry out the column.
Even if the column does dry out, try flushing the column with a degassed, low surface tension solvent (e.g., methanol degassed with helium) to remove the gas. The lower surface tension helps to wet the packing surface; the degassed solvent should be able to dissolve and remove any gas trapped in the packing. The column needs to be flushed (at a flow rate of 1 mL/min) for approximately one hour or more to be wetted down and return to normal conditions.

6. What are the several most common causes of retention time drift?
a. Column equilibration
If we observe retention time drift, the first consideration should be whether the column has been equilibrated with the mobile phase. Usually equilibration requires 10-20 column volumes of mobile phase, but if small amounts of additives (e.g. ion-pair reagents) are added to the mobile phase it can take quite a long time to equilibrate the column. Mobile phase contamination may also be a cause. Small amounts of contaminants dissolved in the mobile phase may slowly enrich on the column and thus cause retention time drift. It should be noted that water is a mobile phase component that can be easily contaminated.

b. Stability of stationary phase
The stability of stationary phases are limited, and even when used in the recommended pH range, stationary phases will slowly hydrolyze. For example, silica matrix is hydrolytically stable at pH 4.  Bifunctional and trifunctional ligands are more stable than monofunctional ligands for bonded phases; long chain bonding is more stable than short chain bonding; and alkyl bonding is much more stable than cyano bonded phases. Frequent cleaning of the column also accelerates the hydrolysis of the stationary phase of the column. Other silica matrix bonded phases can also undergo hydrolysis in an aqueous environment, such as amino bonded phases.