High-Performance Liquid Chromatography (HPLC) is an advanced analytical technique widely used for the separation, identification, and quantification of components in a mixture. It operates based on the differential affinities of compounds toward the stationary and mobile phases, resulting in varying retention times (RTs) for each analyte.

​

​

Operating Modes of HPLC

​

HPLC primarily operates in two modes, based on the polarity of the stationary and mobile phases:

​

​

1. Reverse Phase HPLC (RP-HPLC)

​

Stationary Phase: Non-polar, typically composed of silica particles bonded with hydrophobic alkyl chains such as C8 or C18.

 

Mobile Phase: Polar, usually consisting of water (aqueous phase) mixed with organic solvents such as methanol or acetonitrile.

 

Separation Mechanism: Polar compounds have a stronger affinity for the polar mobile phase and therefore elute faster, while non-polar compounds interact more with the non-polar stationary phase and elute later.

​

Advantages:

​

•    Broad Applicability
Suitable for a wide range of compounds, especially non-polar and moderately polar substances.
•    Effective for Biological Samples
Ideal for analyzing proteins, peptides, and other biomolecules due to compatibility with water-based mobile phases.
•    High Sensitivity and Precision
Provides accurate quantification and high-resolution separation.
•    Excellent Separation of Non-Polar Compounds
Offers better resolution for non-polar and moderately polar analytes.
•    Scalable for Various Applications
Can be used for both small-scale analytical and large-scale preparative work.
•    Stable and Reproducible Stationary Phase
Commonly used phases like C18 are durable and deliver consistent results.
•    Improved Peak Shapes
Generally produces sharp, well-resolved peaks with minimal sample degradation.

​

2. Normal Phase HPLC (NP-HPLC)

​

Stationary Phase: Polar, typically composed of silica or alumina.

 

Mobile Phase: Non-polar solvents such as hexane or chloroform.

​

Separation Mechanism: Non-polar compounds exhibit a stronger affinity for the non-polar mobile phase and elute more quickly, whereas polar compounds interact more with the polar stationary phase, resulting in longer retention.

​

Advantages:

​

•    Effective for Polar Compounds
Well-suited for separating polar and highly polar compounds that may not perform well in reverse phase methods.
•    Useful for Chiral Separations
Particularly effective for separating chiral compounds and structural isomers.
•    High Sensitivity and Precision
Delivers accurate separation and quantification for compounds suited to this technique.
•    Stable Stationary Phase
Uses silica-based stationary phases that are robust and reliable under normal phase conditions.
•    Improved Peak Shapes
Typically produces sharp, well-defined peaks with low sample degradation.

 

Principle of Liquid Chromatography

​

The fundamental principle of liquid chromatography is the differential partitioning of compounds between the stationary and mobile phases. This separation is governed by the partition coefficient, which reflects a compound's relative affinity for each phase. Compounds with higher affinity for the stationary phase exhibit longer retention times, while those favoring the mobile phase elute earlier.

​

Analytical Applications

​

Our laboratory routinely employs HPLC techniques to support pharmaceutical development and quality control. The following analyses are performed to ensure the integrity, consistency, and stability of drug formulations: 

•    Assay
•    Blend Assay                                                                                                                                
•    Content Uniformity
•    Blend Uniformity
•    Related Substances
•    Dissolution Testing

These tests are critical in the development of new dosage forms as well as in conducting stability studies to evaluate the shelf life and performance of pharmaceutical products.