Question

Reversed Phase gradient HPLC is characterised by the analyte behaviour under the influence of the gradient conditions. Which of the following statements are correct (may be more than one correct statement):

a. Retention factors are calculated in the same way as in conventional isocratic HPLC

b. Most analyte molecules will remain wholly partitioned in the stationary phase at the beginning of the gradient analysis.

c. The analyte hydrophobicity will dictate its rate of acceleration through the column

d. The retention factor k of all analytes constantly changes during gradient elution

Answer 1

C & D are correct statements.

In reverse phase column first we use water as eluent. So analyte's interaction with water determines the speed of column. If the analyte is hydrophobic in nature it easily comes out of the column. If it is hydrophilic in nature it stays in column.

In a gradient column the polariy of solvents changes continuously. So retention factor also changes. Retention factor depends on the distance travel by the analytes. And the distance t depends on the polarity.