Advanced Topic 908 of 1102 Analytical, Lab & Spectroscopy
HPLC
High-performance liquid chromatography separates compounds under high pressure.
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In-depth explanation of HPLC
High-performance liquid chromatography pumps a liquid mobile phase at high pressure, typically 100 to 400 bar, through a column packed with very small particles, which gives high resolution and fast analysis. In the common reversed-phase mode the stationary phase is nonpolar C18-bonded silica and the mobile phase is a mixture of water with acetonitrile or methanol, so more hydrophobic compounds are retained longer. Detectors such as UV-visible absorbance, fluorescence or mass spectrometry monitor the eluent continuously. HPLC handles nonvolatile, polar and thermally unstable compounds that cannot be analysed by gas chromatography, including drugs, proteins and sugars.
Going deeper: HPLC
Key ideas: Typical analytical setup: C18 column of 150 mm by 4.6 mm with 5 micrometre particles, flow 1 mL/min, injection of 10 to 20 microlitres. In reversed phase, increasing the organic fraction shortens retention. Isocratic runs hold the mobile phase constant, while gradients change it to speed late peaks. Filter and degas solvents and samples to protect pumps and columns. Use buffers for ionizable analytes.
Common mistakes and connections: Students confuse normal and reversed phase and predict the wrong elution order, since in reversed phase polar compounds elute first. Using a pH outside the column range dissolves silica. Air bubbles cause baseline noise and pressure changes. Injecting a sample in a much stronger solvent than the mobile phase distorts peaks. Links to retention time, polarity, calibration curves and GC.
Visualization of HPLC
In reversed-phase HPLC polar compounds like vitamin C elute early and hydrophobic ones later; peak area gives the amount of each component. The interactive lesson shows this as a live animation or chart.
Worked example and use case
Vitamin C content in a juice sample is quantified by reversed-phase HPLC with UV detection.
Frequently asked questions about HPLC
What is HPLC?
High-performance liquid chromatography separates compounds under high pressure.
Is HPLC a beginner, intermediate or advanced topic?
HPLC is a advanced topic in Analytical, Lab & Spectroscopy. It builds on beginner and intermediate topics, so review those first if needed.
What should I learn before HPLC?
Study these earlier topics in Analytical, Lab & Spectroscopy first: Thin Layer Chromatography, Column Chromatography, Gas Chromatography.
What comes after HPLC?
The next topics in Analytical, Lab & Spectroscopy are Retention Time and Mass Spectrometry Basics.
Can I practise HPLC with quizzes?
Yes. The interactive lesson includes a self-check question on HPLC, and the Analytical, Lab & Spectroscopy quiz draws on every topic in this area.
Where HPLC fits in Analytical, Lab & Spectroscopy
HPLC is topic 32 of 99 in Analytical, Lab & Spectroscopy.