Liquid chromatography (LC)

Definition

Liquid chromatography is an analytical technique used to separate and quantify individual compounds in a mixture. The technique works by passing a sample (dissolved in a solvent called the mobile phase) through a column packed with material (the stationary phase). Different compounds interact differently with the stationary phase, causing them to move through the column at different speeds and exit at different times (retention times). As compounds exit the column, a detector measures their abundance, producing a chromatogram (a graph showing detection signal versus time). Each peak in the chromatogram represents a different compound; the peak area is proportional to the amount of that compound in the sample. For peptide analysis, high-performance liquid chromatography (HPLC) uses a narrow column, high pressure, and small-particle stationary phase to achieve excellent separation and sensitivity. LC is the primary method for assessing peptide purity; a pure peptide shows a single major peak (the target peptide) in the chromatogram, while impure samples show multiple peaks representing contaminants or degradation products. Regulatory agencies require HPLC analysis of therapeutic peptides to establish their purity and identity.

Different modes of liquid chromatography exist, depending on the stationary phase and the type of compounds being separated. Reversed-phase HPLC uses a non-polar stationary phase and is widely used for separating peptides and proteins. Ion-exchange chromatography separates compounds based on their charge. Gel-filtration chromatography separates compounds based on their size. For peptide analysis, reversed-phase HPLC coupled with UV detection is standard because it provides good separation and sensitive detection of the peptide (which absorbs light at 214 or 280 nm).

When HPLC is coupled to mass spectrometry (LC-MS), the output of the LC is fed directly into a mass spectrometer, which measures the mass of compounds exiting the column. This combination provides both the separation power of LC and the identification power of MS, allowing researchers to confirm that peaks represent the expected peptide (by measuring molecular weight) and to identify any impurities (by measuring their molecular weights). LC-MS is considered the gold standard for peptide characterization.