Question

Contrast the advantages and disadvantages of reversed-phase HPLC with normal-phase chromatographic techniques.

Answer 1

In RP-HPLC (reverse phase-high performance liquid chromatography), mobile phase is polar. The stationary phase is hydrophobic or non-polar. So, the molecules of mobile phase adsorb to the molecules of stationary phase. The hydrophilic molecules of mobile phase (sample) tend to pass through the column easily. So, this technique is a useful technique to separate hydrophilic or polar molecules. The hydrophobic stationary phase can be used in conjunction with hydrophobic, hydrophilic and ionic compounds to separate their components. This means that most of organic analytes can be separated via this technique. One more advantage is that water can be used as a mobile phase in reverse phase HPLC. So, the technique becomes a bit cheap and easy to operate in comparison to others. Gradient separation is also possible. There is a choice of pH selectivity during the separation process.

This is not the case with normal-phase chromatography.

In normal- phase chromatography opposite phenomenon happens. The mobile phase is 100% organic or non-polar. The stationary phase is silica gel which is polar. Molecules of mobile phase adsorb over stationary phase. But the hydrophilic molecules of the mobile phase (sample) will not be separated. They will bind with the molecules of mobile phase itself. So, resolution will not be very good in comparison to HPLC. Moreover normal-phase technique is tedious because lot of preparation is required in silica.

Some disadvantages of reverse-phase technique are:

• Low capacity
• Denaturing conditions
• There is an obligatory need of hazardous substances like acetonitrile in RP-HPLC