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 10 micro-g. However, it suddenly decreases when the sample load exceeds 10 micro-g. It is recommended to use the column with the sample load lower than 10 micro-g.

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.