Impact of Mobile Phase Preparation Step

Due to significant volumetric contraction or expansion of the mixture of buffer solution or water with the organic solvent like methanol and acetonitrile, the way of mixing of solvent is very crucial. That’s why you can observe significant change in the retention time when the same composition of mobile phase prepared in different ways. As in different ways your composition will slightly differ from other ways hence elution strength will also change and it will have direct impact on the retention time. Methanol water mixtures represent the case where the contraction is more.

In the Figure, three ways of making mobile phase are presented and the impact on Retention time is displayed in all the three cases.

First Way

Take 50 mL water in measuring cylinder, Add the methanol in the measuring cylinder and make up the volume to 100 mL. Here, adding aqueous and organic directly in measuring cylinder. The volume of solvent which is added later will differ. Hence this method is not recommended.

Second Way

Use separate channel for both solvent
Channel A (Water) - 0.5 mL/ min,
Channel B (Methanol) - 0.5 mL/ min,
This is alternate method where premixing of solvents can be avoided. But there will be impact on RT if gradient mixing valve malfunctioned.

Third Way

Take 50 mL water in measuring cylinder and transfer in mobile phase bottle. Take 50 mL methanol in measuring cylinder and transfer in mobile phase bottle. Here, separate volumetric measurement is performed. As there is no influence of contraction and compression on the volume of both solvents, hence this method is considered as preferred method.

Figure illustrates the three possible ways of solvent mixing during mobile phase preparation and its impact on peak retention times.