HPLC (High-Performance Liquid Chromatography)
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What is HPLC?
High-Performance Liquid Chromatography (HPLC) is an advanced analytical chemistry technique used to separate, identify, and quantify each component within a liquid mixture. In the context of peptide science, HPLC is the gold-standard method for determining peptide purity. The technique works by pumping a sample dissolved in a solvent (the mobile phase) at high pressure through a column packed with a solid adsorbent material (the stationary phase). Different components in the mixture interact with the stationary phase at different rates, causing them to elute from the column at different times, thereby achieving separation.
How HPLC Works in Peptide Analysis
For peptide analysis, reversed-phase HPLC (RP-HPLC) is the most commonly employed variant. In RP-HPLC, the stationary phase is hydrophobic (typically C18-bonded silica), while the mobile phase is a polar aqueous-organic solvent mixture. Peptides are separated based on their hydrophobicity: more hydrophilic peptides elute first, while more hydrophobic peptides are retained longer on the column. A UV detector, typically set at 214 nm (the absorbance wavelength of the peptide bond), monitors the eluting fractions. The resulting chromatogram displays peaks corresponding to each component, and the area under the main peak relative to total peak area provides the purity percentage.
Why HPLC Matters for Research Peptides
HPLC analysis is essential for ensuring that research peptides meet the stringent purity requirements necessary for reliable experimental results. Without HPLC verification, researchers cannot be confident that their peptide preparations are free from synthesis by-products, deletion sequences, or other contaminants that could confound study outcomes. At Pure U.S. Peptides, every product undergoes rigorous HPLC testing, and the resulting chromatograms are included in the Certificate of Analysis provided with each order. This transparency allows researchers to verify that their peptides exceed 99% purity before beginning their experiments.
Key HPLC Parameters
Retention Time
The time a compound takes to travel through the column. Each peptide has a characteristic retention time under given conditions.
Resolution
The degree of separation between two adjacent peaks. Higher resolution means better distinction between the target peptide and impurities.
Mobile Phase Gradient
The programmed change in solvent composition over time, optimized to achieve the best separation for a given peptide mixture.
Detection Wavelength
Typically 214 nm or 220 nm for peptides, corresponding to the absorbance of the amide bond present in all peptides.