HPLC

HPLC (high-performance liquid chromatography) is column chromatography performed under high pressure with small stationary phase particles, giving dramatically improved resolution. It handles samples that cannot be analyzed by GC: non-volatile molecules, thermally unstable compounds, ionic species, biological macromolecules.

The HPLC Setup

1. Pump: delivers solvent at high pressure (up to 400 bar).
2. Injector: introduces the sample (typically 1-100 μL) into the flowing solvent.
3. Column: packed with small (3-5 μm) silica particles, bonded with specific chemistry (C18 is most common).
4. Detector: measures compounds as they elute. Common: UV/Vis, fluorescence, MS, electrochemical.
5. Computer: records the chromatogram and controls the system.

Reverse-Phase HPLC (Most Common)

In reverse-phase HPLC, the stationary phase is NONPOLAR (typically C18 alkyl chains bonded to silica), and the mobile phase is POLAR (water + acetonitrile or methanol).

• Nonpolar compounds interact strongly with the stationary phase → retained longer.
• Polar compounds prefer the polar mobile phase → elute faster.

This is the OPPOSITE of TLC and normal-phase column chromatography.

Reverse-phase HPLC dominates pharmaceutical and biochemical analysis because water-based mobile phases are compatible with biological samples and drug molecules.

Normal-Phase HPLC

Rare today. Uses polar silica column + nonpolar mobile phase. Similar to column chromatography but with better resolution.

Isocratic vs. Gradient Elution

• Isocratic: constant mobile phase composition throughout the run. Simple but cannot resolve wide-polarity mixtures.
• Gradient: mobile phase composition changes during the run (e.g., from 90% water / 10% acetonitrile to 10% water / 90% acetonitrile over 30 minutes). Resolves complex mixtures.

Gradient elution is the standard for complex samples in pharmaceutical QC, proteomics, and drug discovery.

Detection

• UV/Vis detector: most compounds absorb in the UV (200-280 nm). Simple, sensitive, and the default for most HPLC.
• Fluorescence: for fluorescent molecules or those tagged with a fluorophore.
• Mass spectrometry (LC-MS): combines HPLC separation with MS identification. The go-to for proteomics, metabolomics, and drug metabolism studies.

Applications

• Drug purity analysis: verify that a pharmaceutical meets specification (99%+ purity).
• Pharmaceutical drug discovery: analyze compound libraries.
• Amino acid analysis: separate and quantify amino acids in protein hydrolysates.
• Metabolite profiling: identify small molecules in biological samples (metabolomics).
• Environmental analysis: pesticides, pollutants in water and soil.

Typical Retention Times

HPLC runs typically take 15-60 minutes. Peaks emerge at specific retention times characteristic of each compound. Standards are run in parallel to confirm identity.

HPLC in Drug Development

Every approved pharmaceutical drug is characterized by HPLC during development and manufacturing:

• Analytical HPLC: monitors purity (standard: ≥99% drug, ≤0.1% any single impurity).
• Preparative HPLC: purifies drug candidates at gram-to-kilogram scale.
• LC-MS: elucidates drug metabolism and degradation products.

LC-MS: The Power Combo

Coupling HPLC to mass spectrometry gives:

• Retention time from HPLC → one piece of identification.
• Mass spectrum from MS → second piece of identification + structural information.

LC-MS/MS (tandem MS) adds even more specificity: the first MS selects one mass, fragments it, and the second MS analyzes the fragments. Essential for clinical diagnostics, doping tests, and metabolomics.