High Performance Liquid Chromatography (HPLC) is a cornerstone technique in the realm of pharmaceutical instrumental methods of analysis.
Introduction to HPLC:
High Performance Liquid Chromatography (HPLC) is an advanced form of liquid chromatography used to separate, identify, and quantify components in a mixture.
In the pharmaceutical industry, HPLC is extensively used due to its precision and predictability.
While traditional liquid chromatography techniques used gravity for elution, HPLC systems utilize high pressures to push solvents through columns, thus allowing for a much finer degree of separation and analysis.
Theory of HPLC:
The fundamental theory behind HPLC is similar to other chromatographic methods.
It's based on the partitioning of analyte between a mobile and a stationary phase.
Here's a breakdown:
1. Mobile Phase:
This is the solvent (or solvents in a gradient system) that carries the analyte through the column.
The mobile phase is pumped through the column under high pressure.
2. Stationary Phase:
This is usually a packed column where particles (often silica or other polymers) are coated with a particular phase.
This phase interacts with analytes as they pass through, causing each compound to elute at a different time (known as its retention time).
3. Partitioning:
Compounds in the sample mixture distribute themselves between the mobile phase and the stationary phase based on their respective affinities.
Compounds with a stronger affinity for the mobile phase will elute (come out) faster, while those with a higher affinity for the stationary phase elute later.
4. Separation:
As the sample mixture is pushed through the column, compounds separate based on their partitioning between mobile and stationary phases.
The difference in affinity leads to a difference in retention times for each compound, resulting in their separation.
5. Detection:
As individual compounds elute from the column, they pass through a detector.
The detector generates a signal proportional to the amount of compound present.
This produces a chromatogram, which is a plot of signal intensity versus time (or volume).
Types of Interactions:
Depending on the types of stationary and mobile phases used, several modes of HPLC can be employed:
1. Normal Phase HPLC (NP-HPLC):
The stationary phase is polar (like silica), and the mobile phase is less polar (like hexane).
Compounds are separated based on polarity.
2. Reverse Phase HPLC (RP-HPLC):
The stationary phase is non-polar or weakly polar, and the mobile phase is polar.
This is the most commonly used mode in pharmaceutical analysis.
3. Ion-Exchange HPLC:
The stationary phase has charged groups, and separation is based on the charge properties of the molecules.
4. Size Exclusion HPLC (SEC or GPC):
Separation is based on molecule size.
Larger molecules elute first as they're excluded from the porous stationary phase.
