Learn about the theoretical plates N and their calculation in HPLC using the retention time and peak width. This is a helpful parameter to determine the system suitability.
Theoretical plates are known as a measuring tool of HPLC column efficiency. Any chromatography column doesn't have any physical plate but it is a result of a mathematical calculation.
The columns having a high number of theoretical plates are considered more efficient in HPLC separation than the columns having less number of theoretical plates. A more efficient HPLC column will have a narrower peak than a less efficient column with less theoretical plates at the same retention time.
Related: Principle of HPLC
High column efficiency is needed to resolve the narrow peaks in the drug analysis. Hence the resolution of peaks also depends upon the column efficiency i.e. theoretical plates. Theoretical plates are calculated per meter length of the column and often called as Nm. As per the United States Pharmacopoeia (USP) following formula is used to calculate the theoretical plates of HPLC columns.
N = 16(Ve/Wb)2
Where,
N = Theoretical plates
Ve = Retention Time
Wb = Peak Width
Theoretical plates should be determined under specific set conditions; specifically, temperature plays an important role that alters the number of theoretical plates. Retention factor (k) of test solute used to determine the theoretical plates should be more than 5. Less than 5 retention factor can give an inaccurate number of theoretical plates.
Related: Resolution Factor, Tailing Factor, Theoretical Plates and Capacity Factor in HPLC
When comparing the efficiency of two columns; there should be same temperature conditions and retention factor (k) for a valid evaluation of their performance.
All the columns don’t have the same number of theoretical plates. Generally, it ranges between 8000-12000 but it also depends upon the flow-rate, viscosity of mobile phase and molecular of the compound to be analyzed. In reverse phase chromatography it is determined using simple hydrophobic compounds, like toluene, naphthalene or acenaphthene and mobile phase contains a higher concentration of organic solvents having low viscosity.
Related: Relative Response Factor (RRF) and its Calculation in HPLC Analysis
In general HPLC analysis, most of the analyzed compounds are more polar and their mobile phase has a higher concentration of water. The mobile phase having water is more viscous than the mobile phase having organic solvents.
When viscosity increases the count of the column theoretical plates decrease and this is the reason due to which the theoretical plates are found lower in practical use than the standard testing conditions.
Also see: HPLC Column Receipt, Checking and Regeneration
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