Question

What is an isocratic separation? What is a gradient separation? Describe a gradient
elution in normal phase and in reverse phase.

Answer 1

Isocratic separation is the elution process where a single solvent or a solvent mixture of constant composition is employed, i.e., it is the separation in which mobile phase remains constant throughout the separation

Gradient separation is the process where the composition of mobile phase does not remain constant. In this process after the elution has begun, the ratio of solvents is varied in a programmed way to produce a gradient in the mobile phase. It greatly enhances the separation efficiency of elution.

Normal-phase chromatography utilises a stationary phase that is more polar than the mobile phase.
Typical stationary phases include bare silica as well as cyano, diol, and amino bonded phases. Typical mobile phase constituents include organic solvents such as hexane and
ethyl acetate. One example is a gradient starting at 10% methanol and ending at 90% methanol after 20 minutes. The two components of the mobile phase are typically termed "A" and "B"; A is the "weak" solvent which allows the solute to elute only slowly, while B is the "strong" solvent which rapidly elutes the solutes from the column.

Gradients in reversed-phase chromatography typically involve the on-line (dynamic) mixing of solvents to achieve a steady increase in the organic solvent (typically methanol or acetonitrile) over the course of the analysis, thus increasing the elution strength of the eluent over time. In reversed-phase chromatography, solvent A is often water or an aqueous buffer, while B is an organic solvent miscible with water, such as acetonitrile, methanol, THF, or isopropanol.