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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/CH3CN=99/1
(B);0.1% TFA/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.