15. My column isn’t working properly. How do I obtain the right performance?
Column performance can be measured by many parameters. These include column efficiency, selectivity and resolution, peak symmetry and column pressure drop. There are many possible reasons for the change of column performance while in operation. Some are the normal chromatographic problems which can occur with any column. Some, especially for coated Daicel columns, relate to specific properties of the columns and of the stationary phases.
In general, if a column problem is suspected, it should first be thoroughly flushed (see its operating instructions) and then tested under the QC conditions used when it was originally packed. The results of this test can usually help diagnose the problem.
Since the solution of such problems is simpler when immobilised columns—CHIRALPAK IA, IB, IC, ID, IE, IF and IG are used, the resolution of problems with these columns will be addressed first. Later we will note differences between the traditional coated columns and the immobilised columns.
Operating Pressure: With immobilised columns, the source of an increase in operating pressure is usually the inlet frit. This can be blocked either by solids in the sample or entrained in the mobile phase. They can also be blocked by the introduction of a sample which was dissolved in a solvent stronger than the mobile phase; as it mixes with the mobile phase, material can be precipitated from solution and is filtered out by the frit. This can be corrected by changing or cleaning the inlet frit. It is sometimes difficult to remove the inlet frit and such removal always comes with the danger of disturbing the packed bed of the column. One easy experiment is to reverse the flow direction through the column in the hope that the foreign matter will be washed from the frit. It is, of course, always better to prevent such problems by the use of (and regular replacement of) a guard cartridge.
Sudden increases in operating pressure with traditional coated columns can be due to the effects of solvents on the chiral stationary phase or to sample solubility issues. If the pressure increase is due to the introduction of a solvent which can damage the stationary phase, it is usually too late. To prevent such an occurrence, it is vital to ensure that the entire HPLC system is flushed of potentially harmful solvents before the column is connected to the system. Proper sample clean-up and preparation are also vital. Small amounts of non-allowed solvent in a sample preparation may seem insignificant, but these low-level residues often dissolve the chiral polymer, which leads to a rapid decay in column performance. In these cases, the test chromatogram will almost certainly show a marked drop in column efficiency and selectivity. Although in some cases prolonged flushing with 2-propanol may improve the situation, usually the column is most likely dead and will need to be replaced.
Column Efficiency: In most cases, changes in column efficiency are accompanied by changes in peak symmetry or peak shape. In rare cases, a reduction in efficiency accompanied by the appearance of shoulders on the peak trailing edge may be due to void formation at the head of the column. This could be due to dissolution of the silica support by the mobile phase conditions (usually in reverse-phase mode), over-pressurizing of the column, collapsing the silica particles or packing disturbances in the column. Most loss in efficiency problems are due either to partial blockage of the inlet frit (see above) or are due to the adsorption of material at the head of the column.
Adsorption of material at the head of the column can be seen where the samples are not pure and contain components which are strongly adsorbed on the stationary phase. This can often be resolved when using immobilised columns by flushing with a strong solvent such as THF or DMF (please refer to instruction manuals for detailed protocols). This approach cannot be realized with the coated columns and the best that can be done is to flush them with the strongest compatible solvent, often 2-propanol. For those cases in which recommended washing fails to restore performance, more drastic washing may be needed. Such washing procedures carry a significant risk of column damage, so they are best used as a measure of last resort.
For more information on such procedures, contact us: Chiral Technologies Europe (CTE), support@cte.daicel.com, Chiral Technologies, Inc. (CTI), questions@cti.daicel.com, or Daicel Chiral Technologies India (DCTI), chiral@chiral.daicel.com
This approach is rarely successful and represents another reason why we strongly recommend using the immobilised columns wherever possible.
Sometimes an established separation cannot be duplicated on a new column. We carefully ensure minimum lot-to-lot variation in column performance and this situation more often results because the established separation is dependent on some type of column conditioning that the new column has yet to be subjected to. The older column may have a “memory effect” in which additives used in the past history of the column have become adsorbed on the stationary phase, and are crucial to the current separation. With immobilised columns, a simple flush with DMF (followed by EtOH rinsing, as described in regeneration protocol) may be all that is necessary to “reset” the stationary phase. In many cases, the problem can be resolved by conditioning the new column for a few hours with mobile phase that contains the pertinent additive. In those cases in which the separation is still not restored, the method may need to be redeveloped with a different mobile phase, column, or temperature. For this reason, we recommended developing new separations on a new column, or one for which the mobile phase and sample history are documented. Validated methods should be established with more than one column.
E-mail questions concerning columns which are not working properly are always welcome. Contact us: Chiral Technologies Europe (CTE), support@cte.daicel.com, Chiral Technologies, Inc. (CTI), questions@cti.daicel.com, or Daicel Chiral Technologies India (DCTI), chiral@chiral.daicel.com
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