HAWACH to Talk About the 30 FAQs About HPLC Column (Chapter 3)

Q11: What are the reasons for retention time drift?
There are two different situations for retention time which does not reproduce: retention time drift and retention time fluctuation. The former means that the retention time only changes in one direction, while the latter means that there is no fixed fluctuation in retention time. Separating these two situations is often helpful in finding the cause of the problem.

For example, retention time drift is often caused by HPLC column aging, which is unlikely to cause irregular fluctuations in retention time. In fact, most of the reasons for the retention time drift are column aging of different mechanisms, such as loss of stationary phase (for example, through hydrolysis), HPLC column contamination (caused by the sample or mobile phase), etc.
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Several common reasons for retention time drift are as follows:

1. HPLC Chromatographic column equilibrium
If we observe retention time drift, we should first consider whether the HPLC column has been completely equilibrated with the mobile phase. Usually, 10-20 column volumes of mobile phase are required for equilibration, but if a small number of additives (such as ion-pair reagents) are added to the mobile phase, it takes a considerable amount of time to equilibrate the HPLC column.

Mobile phase contamination may also be one of the reasons. A small number of contaminants dissolved in the mobile phase may slowly accumulate on the HPLC column, causing drift in retention time.

2. The stability of the stationary phase
The stability of the stationary phase is limited, even if it is used within the recommended pH range, the stationary phase will slowly hydrolyze. For example, the silica gel matrix has good hydrolytic stability at pH 4.

The rate of hydrolysis depends on the type of mobile phase and the ligand. Bifunctional ligands and trifunctional ligands are more stable than monofunctional ligands; long-chain bonds are more stable than short-chain bonds; alkyl bonds are more stable than cyano bonds. Cleaning the column frequently will also accelerate the hydrolysis of the HPLC column stationary phase. Other silica-based bonded phases can also undergo hydrolysis in aqueous solutions, such as amino bonding.
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3. HPLC Chromatographic column pollution
Another common cause of retention time drift is column contamination. HPLC chromatographic column is a very effective adsorption filter, which can filter and adsorb any substance carried by the mobile phase. Sources of contamination can be the mobile phase itself, the mobile phase container, connecting tubes, pumps, injectors and instrument seals, and samples. The source of pollution can usually be determined through experiments.

If there is a strong component remaining on the HPLC column in the sample, it may be a potential source of retention time drift. These roots are usually the sample matrix. A simple way to avoid column contamination is to prevent it from happening. A strong solvent under the given chromatographic conditions is usually used, but not all contaminants can be dissolved in the mobile phase. Using guard columns is a very effective method. Backflushing the column is only a last resort.

4. Composition of the mobile phase
The slow change in mobile phase composition is also a common cause of retention time drift. For example, the volatilization of volatile components in the mobile phase is equal to the circulating flow.

5. Hydrophobic collapse
When a reversed-phase packing HPLC column with a small pore size and good end-group sealing uses nearly 100% water as the mobile phase, sometimes a sudden loss of separation occurs and the retention of the analyte is significantly reduced or not retained at all. This is called hydrophobic collapse.

This phenomenon is caused by the mobile phase not infiltrating the surface of the stationary phase. The salvage method is to infiltrate the stationary phase with a mobile phase containing a large number of organic components and then balance it with a mobile phase with high water content.
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This phenomenon can also occur with long-term column storage. The use of reversed-phase HPLC columns with embedded polar groups (such as Waters SymmetryShield RP HPLC columns) or non-end-sealed HPLC columns (such as Waters Resolve HPLC columns) can also avoid collapse.

Q12: Why are there shoulders or forks?
①The sample volume is too large: use the mobile phase to prepare the sample, the total sample volume is less than 15% of the first peak;
②Sample solvent is too strong: use a weak sample solvent;
③The HPLC column collapses or forms a short-circuit channel: replace the HPLC chromatographic column, using weaker corrosive conditions;
④The sintered stainless steel in the HPLC column fails: replace the sintered stainless steel, add an online filter, and filter the sample;
⑤The injector is damaged: replace the injector rotor.

Q13: Why is there a ghost peak?
①Residual peaks of the injection valve: clean the valve with a strong solvent after each use to improve the cleaning of the valve and the sample;
②Unknown in the sample: processing the sample;
③The column is not equilibrated: re-equilibrate the HPLC column, using the mobile phase as the sample solvent (especially ion pair chromatography);
④Trifluoroacetic acid (TFA) oxidation: freshly prepared every day, with antioxidants;
⑤Water pollution (reverse phase): check the water quality by changing the equilibrium time, and use HPLC grade water.

Q14: Why is there peak tailing?
①Column overload: reduce the sample volume, increase the column diameter and use a higher capacity stationary phase;
②Peak interference: clean the sample and adjust the mobile phase;
③The role of silicon hydroxyl group-add triethylamine: passivate the HPLC column with alkali, increase the concentration of buffer or salt, lower the PH value of the mobile phase, and purify the sample;
④The sintered stainless steel in the HPLC column fails: replace the sintered stainless steel, add an online filter, and filter the sample;
⑤The column collapses or forms a short-circuit channel: replace the HPLC chromatographic column, using weaker corrosive conditions;
⑥The dead volume or the volume outside the column is too large: the connection point is reduced to very low. Make appropriate adjustments to all the connection points, and use the connection pipe with a thin inner diameter as much as possible;
⑦Decreased column efficiency: replace columns with lower corrosion conditions and use protective columns.