What Is The Molecular Formula For The Following Compound Theoretical Plates in HPLC

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Theoretical Plates in HPLC

HPLC is a type of column chromatography that has a wide range of applications in medicine 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 non-polar compounds depending on the type of column. Polar or non-polar columns are used to separate compounds depending on the nature of the compound to be analyzed.

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

On entering the column, the components are separated based on their polarity (it also depends on the polarity of the stationary phase, i.e. the column). If the compound is non-polar then the non-polar compounds get attached to the compound and the polar compounds elute first to reach the detector and vice versa. A compound is identified by calculating the retention time or Rt, the time required for a particular compound to reach the detector through the column after the injection is made.

HPLC column performance is measured using the theoretical plate (Tp) concept. There is no meat plate in the column; rather it is based on mathematical calculations. The theoretical plates in HPLC can be considered as a hypothetical medium consisting of two phases that exist 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 theoretical plates (ie efficiency) will give narrower peaks for the same compound compared to columns with less efficiency.

Theoretical plates can be calculated by meter length of the column. It is usually called 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

There,

N theoretical plates

Storage time

Wb is the peak width

Determination of Tp should always be done while maintaining certain fixed conditions in all test columns. Column temperature, in particular, plays an important role in changing the theoretical plate number in the column.

The retention or Rf (the ratio of the distance traveled by the component and the solvent) of the test solute to be used for the determination of the Tp of the column must always be greater than 5 to obtain the correct Tp value.

Therefore, while comparing the efficiency of the column to the columns the temperature and storage of the material should be kept the same. The theoretical plates in 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 containing higher percentages of water are more visible compared to those with higher amounts of organic solvents. Thus, the theoretical-plates of the column decrease with the increase of the percentage of water of the mobile phase. This is the reason for having a lower Tp in the columns during active use compared to the normal test condition.

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