HPLC is a type of column chromatography that has a wide range of uses in pharmaceuticals and analytical chemistry as an efficient separation tool. The column, which acts as a stationary phase, plays a key role in the separation of components in HPLC. Stationary phases are usually composed of polar or nonpolar compounds depending on the type of column. Polar or nonpolar 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 through the injector. The pumps maintain a constant flow rate of the mobile phase.
Upon entering the column, the components separate according to their polarity (it also depends on the polarity of the stationary phase, i.e. the column). If the column is nonpolar, nonpolar compounds stick 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 injection is performed.
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 calculations. Theoretical plates in HPLC can be considered as a hypothetical zone consisting of two phases present in equilibrium with each other.
Columns with a higher number of theoretical plates are considered more efficient compared to columns with a lower number of Tp. A column with more theoretical (ie more efficient) plates will give narrower peaks for the same compound compared to less efficient columns.
Theoretical plates can be calculated per meter of column length. It is often referred to as N or Nm. According to the USP or the United States Pharmacopeia, the theoretical plate of a column is calculated by the following formula:
N = 16(Ve/Wb)2
Where,
N are the theoretical plates
Ve is the retention time
Wb is the width of the peak
The determination of Tp should always be carried out maintaining the established conditions specific to all test columns. Column temperature, in particular, plays an important role in altering the theoretical number of plates in a column.
The retention factor or Rf (ratio of the distance traveled by the component and the solvent) of the test solute to be used for the Tp determination of a column must always be greater than 5 to obtain an accurate Tp value.
Therefore, when comparing column efficiency between columns, the temperature and retention factor should be kept the same. Theoretical plates on the HPLC column also depend on the viscosity of the mobile phase, the flow rate and the molecular structure of the compound to be analyzed.
Mobile phases that contain a higher percentage of water are more viscous compared to those that have a higher concentration of organic solvents. Thus, the theoretical plates of a column decrease with the increase in the percentage of water in the mobile phase. This is one reason for having a lower Tp for columns in practical use compared to standard test conditions.