Effect of Sample Load on Theoretical Plate

The relation between sample load and theoretical plate number for RSpak RP18-415 (a column for reversed phase chromatography) is shown. The theoretical plate number does not decrease when sample load is lower than 10μg. However, it suddenly decreases when the sample load exceeds 10μg. It is recommended to use the column with the sample load lower than 10μg.


Sample : Lysozyme

Column       : Shodex RSpak RP18-415 (4.6mmID*150mm)
Eluent       : (A);0.1% TFA aq./CH3CN=99/1
               (B);0.1% TFA aq./CH3CN=5/95
               Linear gradient: 0 to 25min : 20%(B) to 60%(B) 
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : Room temp.