Question

HPLC question- Eluent strength Definition Isocratic vs gradient elution - when to use each Normal phase with gradient elution of MeOH/H20 Start with 80%MeOH/20%H2O and end with 50/50 or Start with 50/50 and end with 80/20 Why? Repeat but for reverse phase

Answer 1

In Gradient elution two or more solvent systems are employed which differ in polarity and mobile phase is programed to change in composition during elution.

Now What is Reverse phase chromatography?

The common technique is Normal phase chromatography in which Stationary Phase (Column) is use to be Hydrophilic where as Mobile phase (solvent) is Hydrophobic in nature. If we reverse it, means when Mobile Phase is Hydrophilic and Stationary phase is Hydrophobic then it becomes Reverse ohase chromatograhy.

Here, for Gradient elution Normal phase (means Hydrophilic Column) Polar compounds will bind to the column and elute at the end whereas Non-polar compounds will come out in initial fractions.

For normal phase starting is 80%MeOH/20%H2O and end 50/50 because here Mobile phase is Hydrophobic and MeOH is less polar than H2O so polarity of the solvent will gradually increase by increasing the %age of H2O for the better separation.

Whereas for Reverse phase Start with 50/50 (polar) and end with 80/20 (less polar) will work, because here running /mobile phase is polar as per the principle. Polar molecule will elute first, the decrese in polarity of mixture will elute the non-polar molecules at the end.