- There could be many different causes for this. Let's go through them one by one. People most commonly assume that drifting retention times are an equilibration problem. If you are doing normal phase chromatography on unmodified silica columns this is also the most likely problem.
- The retention times in normal phase chromatography are very susceptible to the amount of water adsorbed on the silica surface, which in turn is a function of the water dissolved in the mobile phase. Since the solubility of water in solvents like hexane or methylene chloride is extremely low, it takes a long time for the columns to equilibrate. In many cases the retention times in very dry hexane were still shifting after a week of equilibration.
- It is recommended to avoid very dry solvents. A common solution to the equilibration problem of silica with water is to use solvents that are "half-saturated" with water. They are prepared by saturating a given volume of hydrophobic solvent with water and then mixing it 1:1 with a "dry" solvent.
- This approach speeds equilibration times up tremendously. In reversed-phase chromatography, equilibration is usually very fast. A few (5 to 10) column volumes of mobile phase are usually sufficient for equilibration. This is however not always the case.
- A notable exception is the equilibration of a column with ion-pairing reagent in ion-pair chromatography. The ion-pairing reagents are typically used at a concentration of about 2 to 5 mmol/L or less. They adsorb onto the surface of the reversed-phase packing at a surface concentration of between 0.5 to 2 μmol/m2.
- A 4.6mm x 250mm column contains about 3 g of packing with a surface area of 330 m2/g. That means that there are 1000 m2 of surface in the column. At a surface concentration of 2 μmol/m2 2 mmol of reagent need to be pumped into the column for complete equilibration.
- At a concentration of 2 mmol/L this takes a liter of mobile phase. This is clearly an extreme case, but don't be surprised if it takes a few hundred milliliters of mobile phase to equilibrate the column. It is therefore unwise to convert columns used in ion-pair chromatography to an organic solvent for overnight storage, because during this process the ion-pairing reagent is washed off and the next day you have to go through a lengthy re-equilibration procedure.
Other causes of shifting retention times
- It is possible that the mobile phase composition is changing with time. If you are not using the on-line mixing capabilities of today’s instruments, you may be looking at a slow evaporation of a component in the mobile phase.
- This is especially true when you are sparging the mobile phase with helium to avoid air-bubbles in the pump. One should keep the rate of sparging to a minimum.
- An often underestimated cause of shifts in retention is the hydrolysis of the bonded phase. The manufacturers usually specify a pH-range, outside which the bonded phase is "unstable". This range is typically from pH 2 to 8 or 9. However, one has to treat these limits with a lot of caution.
- There is not a sharp boundary, hydrolysis depends also on other factors like temperature and organic solvent, and slow hydrolysis occurs inside these limits as well.
- The hydrolytic stability of a bonded phase is best at intermediate pH-values, around pH 3 to 5 and at low temperature. Isocratic chromatographic conditions are better than gradients. While hydrolysis does occur in isocratic conditions as well, the bonded phase often adsorbs to itself and is in a local equilibrium. However, when a higher concentration of organic solvent is used - as in a gradient or during column cleaning procedures - this local equilibrium is interrupted and the hydrolyzed bonded phase is washed out of the column.
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