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5. Temperature Difference Between the Column and the Mobile Phase

Mixing water and acetonitrile in a proportion of 1:1 at room temperature (25 ℃), the mixture becomes 20 ℃ (– 5 ℃). On the other hand, mixing water and methanol in the same proportion at room temperature, the mixture becomes 33 ℃ (+8 ℃). Because of the high flow rate in preparative HPLC, mixing solutions by the pump causes a temperature distribution in the column, which leads to peak distortion. The preheat mixer is used to prevent this phenomenon by making the mobile phase temperature the same as the column temperature.