High-Performance Liquid Chromatography (HPLC)
High-Performance Liquid Chromatography (HPLC) distinguishes itself from traditional
liquid chromatography by operating at significantly higher pressures (around 50–1400
bar), while traditional methods rely on gravity. Typical HPLC columns are 2.1–4.6 mm in
diameter and 30–250 mm in length, packed with smaller adsorbent particles (1.5–50 μm),
giving HPLC superior resolving power.
An HPLC instrument typically includes solvent reservoirs, pumps, a solvent-degasser, a
sampler, a column, and a detector. Prepared solvents pass through the degasser to remove
dissolved gases, mix to form the mobile phase, and flow through the sampler, which
introduces the sample mixture. The pumps regulate the flow and composition of the mobile
phase through the column, leading to a detector that provides quantitative analysis by
measuring the emerging sample components.
The HPLC system is controlled by a digital microprocessor and software, allowing data
analysis. Some HPLC models can mix multiple solvents to create a gradient in the mobile
phase. Column ovens adjust the separation temperature.
A small volume of the sample mixture is introduced into the mobile phase stream, moving
through the column at different velocities based on interactions with the adsorbent. The
retention time, under specific conditions, identifies each analyte.
Columns are available with various particle sizes, porosity, and surface chemistry, requiring
higher operational pressure for smaller particle sizes, improving resolution. The most
common liquid chromatography mode is reversed phase, using water or buffers with
organic solvents like acetonitrile and methanol. Some techniques use water-free mobile
phases. The aqueous mobile phase may contain acids (e.g., formic, phosphoric,
trifluoroacetic acid) or salts to aid separation.
The mobile phase composition can be constant ("isocratic elution") or varied ("gradient
elution"). Isocratic elution is effective for simple mixtures, while gradient elution is used for
complex mixtures. In reversed phase chromatography, a gradient profile might start at 5%
acetonitrile and increase to 95% over 5–25 minutes. The chosen mobile phase composition,
additives, and gradient conditions depend on the column and sample components, often
determined by trial runs.
The partitioning process in the column is similar to liquid–liquid extraction but continuous.
In a water/acetonitrile gradient, more hydrophobic components elute later, with elution
speeding up as the mobile phase becomes richer in acetonitrile. The choice of mobile phase
components, additives, and gradient conditions depends on the column and sample
components. Often a series of trial runs is performed to find the HPLC method that gives
adequate separation.
