Compounds That Have The Same Molecular Formula But Different Structures Theoretical Plates in HPLC

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Theoretical Plates in HPLC

HPLC is a kind of column chromatography that is widely used as an effective separation tool in pharmaceuticals and analytical chemistry. A column acting as a stationary phase plays a key role in the separation of components in HPLC. Stationary phases usually consist of polar or non-polar compounds depending on the type of column. Depending on the nature of the compound being analyzed, either polar or non-polar columns are used to separate the compounds.

The mobile phase is pumped using mechanical pumps in the system and the sample is introduced into the mobile phase using an injector. The pumps maintain a constant flow rate of the mobile phase.

When entering the column, the components are separated based on their polarity (also depends on the polarity of the stationary phase, i.e. the column). If the column is non-polar, non-polar compounds will attach to the column and polar compounds will elute first to reach the detector and vice versa. To detect a compound, the retention time or Rt is calculated, which is the time it takes for a particular compound to reach the detector through the column after injection.

The efficiency of an HPLC column is measured using the theoretical plate (Tp) concept. There is no physical plate in the column; rather, it is based on a mathematical calculation. Theoretical HPLC plates can be thought of as a hypothetical zone consisting of two phases 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 slabs can be calculated per meter length of the column. This is often known as N or Nm. According to the USP or United States Pharmacopoeia, the theoretical plate of the column is calculated using the following formula:

N = 16 (Ve/Wb)2

where,

N are the theoretical plates

Ve is the storage time

Wb is the peak width

Tp should always be determined while maintaining specific set conditions for all test bars. In particular, the column temperature plays an important role in changing the theoretical plate number in the column.

The retention factor or Rf (ratio of the distance traveled by the component to the solvent) of the analyte used to determine the Tp of the column should always be greater than 5 to obtain an accurate Tp value.

Therefore, when comparing column efficiency across columns, the temperature and retention factor should be kept the same. The theoretical plates of an HPLC column also depend on the viscosity, flow rate and molecular structure of the mobile phase of the analyte compound.

Mobile phases containing a higher percentage of water are more viscous compared to those with a higher concentration of organic solvents. Thus, the theoretical plates of the column decrease with increasing percentage of water in the mobile phase. This is the reason why the Tp of the columns is lower in practical use compared to standard test conditions.

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