HPLC is a Sort of column Chromatography having a wide assortment of applications in pharmaceuticals and analytical chemistry as an effective separation instrument. The column, which functions as the stationary phase plays an integral role in the separation of elements in HPLC. Stationary phases are often composed of polar or non-polar chemicals according to column type. Both polar and non-polar columns are utilized to separate compounds based on the nature of the chemical to be examined.
The mobile phase is pumped into the system with the support of mechanical pumps and the sample is introduced to the mobile phase using the injector. The pumps maintain a continuous flow rate of the mobile phase. Upon entering the column, Components becomes separated according to their polarity also depends upon the polarity of the stationary phase i.e. the pillar. If the column is non-polar then non-polar chemicals become connected to the column and polar compounds elute first to reach the sensor and vice versa. A chemical is identified by what is hplc calculating the retention period or RT that is the time necessary for a specific compound to get to the sensor through the column following the injection is made.
HPLC column efficiency is measured with the theoretical plate Tp concept. There’s absolutely not any physical plate within a column; instead it is based on mathematical calculation. Theoretical plates in HPLC can be considered a hypothetical zone comprising two phases present in balance with one another. Columns with a high number of Theoretical plates are considered more effective compared to columns with the lesser amount of Tp. A pillar with more theoretical plates i.e. more efficient provides narrower peaks to the same compound compared to less effective columns.
The theoretical plates can be calculated per meter length of this column. It is often called N or Nm. In accordance with USP or the United States Pharmacopoeia, the theoretical plate of a column is calculated with the following formula:
N = 16Ve/Wbtwo
N is the theoretical plates
Ve is the retention period
Wb is the width of this peak
The conclusion of Tp should always be made while keeping specific set requirements for all of the test columns. Column temperature, in particular, plays a significant role in changing the theoretical plate number in a column. Retention variable or RF ratio of the distance traveled by the part as well as the solvent of the test solute to be utilized for the conclusion of Tp of a column must be more than 5 for a precise value of Tp.