Introduction

The problems associated with the isocratic HPLC analysis can be overcome by using gradient HPLC. The mobile phase composition is altered due to the analysis normally by increasing the amount of organic modifier in this mode of analysis. The initial composition is chosen in order to assure that the strength is appropriate to retain and resolve early eluting analytes. In order to elute compounds with optimum resolution, the elution strength is increased in a predetermined way. The final elution of all compounds of interest comes from the column along with the mobile phase from the column within a reasonable time. In order to wash the strongly retained potentially contaminating components from the column, the concentration of organic modifier can be increased. Gradient elution is best suited to analysis carried out using RP-HPLC, normal phase using bonded stationary phases and for ion-exchange chromatography.

In order to carry out gradient HPLC analysis which allows on-line mixing of the mobile phase components, particular pumps are required.

Advantages of gradient elution

1. Improved Resolution
2. Increased detection
3. Ability to separate complex samples
4. Shorter analysis time
5. It causes decrease in column deterioration which are caused due to strongly retained components.

Disadvantages of gradient elution

1. Most expensive instrumentation
2. While using multiple proportion valves, there are chances of possible precipitation at interfaces.
3. Re-Equilibrium time adds to analysis time.
4. Instruments vary in their dwell volume which can cause method transfer problems.

Peak shape in gradient HPLC

The peaks are narrow with almost equal peak widths in gradient elution. The narrow peak shape is due to the velocity of the peak as the component leaves the column. During the gradient elution, all compounds accelerate through the column and thus elute at a high velocity. The percentage organic modifier at which each compound starts to accelerate leads to difference in retention times between the compounds. All the compounds should have approximately the same speed when they leave the column. The front and tail of a peak are residing in different concentrations of organic modifier which is the main reason for peak focussing. Peak focussing occurs when the tail will experience higher percentage of organic modifier than the heart of the peak. The asymmetric peaks in gradient elution are less frequently a problem. Narrow peaks obtained will provide better detection limits and higher loading capacities. As the gradient is applied:

1. Moderately disperse analyte band –begins to move down the column.
2. Front and rear of the analyte band are in slight different mobile phase compositions to the main middle region of the band.
3. Analytes at the front of the band migrate in slightly lower concentration of the organics whereas the analytes at the back of the band migrates slightly faster in a marginally higher organic concentration.

This has effect on ‘concentrating’ or ‘focussing’ the analyte band so improving the peak shape.

Limitations of gradient elution                        

Gradient Elution may not be suitable for-

1. Applications utilizing strongly retained additives.
2. Ion-pair applications
3. Normal phase liquid chromatography on base silica due to water adsorption and irreproducibility.