Recently I tested the R_T of cinnamic acid standard sample by RP-HPLC C18. The column length is 150mm,200mm and 250mm respectively. The mobile phase is methanol:water:acetic acid, with the following methanol:water ratio, 72:22,72:28,72:32,72:34 and 72:88 Details are listed in the attached file.

fficeffice" />

It is easy to understand that the R_T varies as per the column length and the mobile ratio. But what perplex me is how to explain the fact that “ the area of the standard sample also varies as per the column length and the mobile ratio.” If that is the case, it means under different conditions the eluted amount of the standard sample varies, so how to determine the correct amount of the standard sample.

Any comments on this subject are welcome!

Different length of column have differengt number of theoretical plate.The longer of the column is ,the better of the column effect is.