HPLC is a kind of column chromatography having a wide range of uses in pharmaceuticals and analytical chemistry as an effective separation tool. The column, which acts as the stationary phase plays a key role in the separation of components in HPLC. Stationary phases are usually composed of polar or non-polar compounds as per column type. Either polar or non-polar columns are used to separate compounds depending on the nature of the compound to be analyzed.
The mobile phase is pumped into the system with the help of mechanical pumps and the sample is introduced into the mobile phase using the injector. The pumps maintain a constant flow rate of the mobile phase.
Upon entering the column, components get separated based on their polarity (also depends on the polarity of the stationary phase i.e. the column). If the column is non-polar then non-polar compounds get attached to the column and polar compounds elute first to reach the detector and vice versa. A compound is identified by calculating the retention time or Rt, which is the time required for a particular compound to reach the detector through the column after the injection is made.
HPLC column efficiency is measured using the theoretical plate (Tp) concept. There is no physical plate present in a column; rather it is based on mathematical calculation. Theoretical plates in HPLC can be regarded as a hypothetical zone consisting of two phases present in equilibrium with each other.
Columns with a higher number of theoretical plates are regarded as more efficient compared to columns with the lesser number of Tp. A column with more theoretical plates (i.e. more efficient) will give narrower peaks for the same compound in comparison to less efficient columns.
The theoretical plates can be calculated per meter length of the column. It is often known as N or Nm. According to USP or the United States Pharmacopoeia, the theoretical plate of a column is calculated using the following formula:
N = 16(Ve/Wb)2
N is the theoretical plates
Ve is the retention time
Wb is the width of the peak
The determination of Tp should always be made while maintaining specific set conditions for all the test columns. Column temperature, in particular, plays an important role in altering the theoretical plate number in a column.
Retention factor or Rf (ratio of the distance traveled by the component and the solvent) of the test solute to be used for the determination of Tp of a column should always be more than 5 to get an accurate value of Tp.
Therefore, while comparing the column efficiency across columns the temperature and retention factor should be maintained same. Theoretical plates in HPLC column also depend on the mobile phase viscosity, flow rate and molecular structure of the compound to be analyzed.
Mobile phases containing more percentage of water are more viscous in comparison to those having the higher concentration of organic solvents. Thus, the theoretical-plates of a column decrease with increase in mobile phase water percentage. This is a reason to have lower Tp for columns during practical use compared to the standard testing condition.