When detecting a normal phase system in liquid chromatography, it is common to observe retention time drift, where the retention time of several consecutive sample injections shifts either forward or backward. Some may attribute this to an equilibration issue, but upon closer examination, it is more accurately described as a problem with the stability of the mobile phase flow.
In normal-phase chromatography analysis, highly polar components, especially water in the mobile phase, can significantly affect retention time. Most organic solvents contain varying amounts of water, and the amount of water can be unstable. Therefore, if the organic phase is not specially treated, the differences in water content in the mobile phase will be significant, resulting in significant differences in retention time and irreproducibility of retention time in normal-phase chromatography. In addition, if the organic phase is not treated, there may be issues with long equilibration time and continuous injection retention time drift.
Therefore, when performing normal phase chromatography analysis, it is recommended to first saturate the organic phase with water for the major component, then mix the water-saturated organic phase with the unsaturated organic phase at a 1:1 ratio to form a semi-saturated organic phase, and finally prepare the mobile phase.
For example, in the case of a mobile phase consisting of 90% n-hexane and 10% isopropanol for normal phase chromatography, we can first saturate n-hexane with water, then mix 450 mL of the water-saturated n-hexane with 450 mL of unsaturated n-hexane, and finally add 100 mL of isopropanol, mix well, and sonicate to remove any air bubbles.
Condition 1: Tetrahydrofuran: n-Hexane: Acetonitrile: Trifluoroacetic acid = 175:750:75:1 (n-Hexane not treated with water saturation)
Condition 2: Tetrahydrofuran: n-Hexane: Acetonitrile: Trifluoroacetic acid = 175:750:75:1 (n-Hexane subjected to water half-saturation treatment)
From the graph, it can be seen that the flow rate prepared with non-saturated n-hexane has a retention time drift and is unstable during detection. However, after the n-hexane was semi-saturated before preparation, the flow rate was detected with stable retention time, which meets the detection requirements.