The choice of mobile phases, the number of sample components, the concentration of two different solvents used, sample solubility, detector efficiency (PDA or UV/VIS), and other factors may all affect resolution in HPLC separation.
High-performance liquid chromatography is a separation technique in chemistry, used for qualitative and quantitative analysis of components. In chromatographic separation, the resolution of elution is a quantitative measurement of how two peaks of analytes can be isolated.
Resolution is an important factor of each type of chromatographic technique, which is determined by three important parameters such as retention, efficiency, and selectivity. It is defined as the dissimilarity of RT between two peaks, divided by the width of the elution peaks.
To improve peak resolution of closely eluted molecules is an issue encountered by all chromatographers during the HPLC method development. The chemical differences between analytes can be used to get better separations. Chromatographers are used numerous parameters to develop the separation of closely related compounds.
Parameters that may be modified for the preferred separations consist of the stationary phase, mobile phase, flow rate, and temperature of the columns. Change the column particle size is another effective way used by many analyst. Small particle-sized columns generate high theoretical plate numbers by giving the Gaussian peak shape and are capable of resolving closely isolated peaks. Compounds that have a high molecular weight cannot separate on a small pore size column, therefore, they must resolve on a large pore size column.
The resolution equation states that three major parameters will influence the resolution in high-performance liquid chromatography.
1. Retention factor or capacity factor (k):
2. Separation factor (Selectivity):
3. Efficiency:
1. Retention factor or capacity factor (k):
2. Separation factor (Selectivity):
3. Efficiency:
Selectivity is the factor that highest impact on the resolution as compared with other factors.
- The particle size of the column
- Column dimension
- The activity of the adsorbent
- Column overload
- The temperature of the column
- Degradation of HPLC column
- Inadequate saturation of column
- Mobile phase pH
- The polarity of the solvents
- Composition of the mobile phase
- The viscosity of the mobile phase
- The polarity of the sample
- the pH of the sample
- Structure of the solute
- The flow rate of the system
- Pressure on the system
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