WHAT IT IS
Standard High-Performance Liquid Chromatography (HPLC) is the conventional format of liquid chromatography used to separate, identify, and quantify compounds in liquid mixtures. It operates at pressures up to about 40 MPa and employs columns packed with stationary-phase particles in the 3–10 µm range.
HOW IT WORKS
Standard HPLC involves a series of coordinated steps:
Solvent Delivery - A pump drives the mobile phase—usually a mixture of water and organic solvents—through the system at constant flow and controlled pressure.
Sample Introduction - A small, precise volume of liquid sample is injected into the mobile phase stream.
Column Separation - The sample enters a packed column. Compounds partition differently between the stationary and mobile phases depending on their polarity, hydrophobicity, or charge.
Elution - Separation may use isocratic conditions (constant solvent composition) or gradient conditions (changing solvent strength to improve resolution).
Detection - Eluted compounds are recorded by detectors such as UV absorption, fluorescence, refractive index, or mass spectrometry.
Data Output - The detector produces a chromatogram in which retention time indicates identity and peak area provides quantification.
TYPES
Within standard HPLC, several operational modes are distinguished by the stationary phase and separation mechanism:
Reversed-Phase HPLC (RP-HPLC): Uses non-polar stationary phases (commonly C18 silica) to separate analytes by hydrophobicity.
Normal-Phase HPLC: Employs polar stationary phases and non-polar mobile phases, suited for separating isomers and hydrophobic compounds.
Ion-Exchange HPLC: Utilizes charged stationary phases to separate ionic compounds such as amino acids, peptides, and inorganic ions.
Hydrophilic Interaction Chromatography (HILIC): Designed for highly polar analytes that are difficult to retain in reversed-phase systems.
Affinity HPLC: Uses biologically specific interactions (e.g., antibody–antigen) to isolate biomolecules.
ADVANTAGES
Separation Efficiency: Columns with optimized stationary phases provide reliable resolution of complex mixtures.
Quantitative Accuracy: Standard HPLC delivers precise and reproducible measurements, particularly when calibrated with external or internal standards.
Broad Applicability: Suitable for non-volatile, polar, and thermally labile compounds across pharmaceuticals, food, environmental, and biological matrices.
Flexible Detection: UV, fluorescence, refractive index, and mass spectrometry allow sensitive analysis of diverse analytes.
Coupling with Other Techniques: HPLC is frequently combined with MS (LC-MS, LC-MS/MS) for enhanced selectivity and structural characterization.
CHALLENGES AND LIMITATIONS
Analysis Time: Larger particle sizes limit efficiency, leading to slower runs and broader peaks compared with UHPLC.
Solvent Consumption: Standard HPLC uses relatively large volumes of high-purity solvents, increasing operating cost and waste.
Sample Preparation: Complex samples often require filtration or cleanup to protect the column and detector from fouling.
Column Overload: Excess sample volume or concentration can distort peak shapes and reduce resolution.
System Maintenance: Columns, seals, and pumps require regular servicing to maintain consistent performance.