High Performance Liquid Chromatography (HPLC)

What Is HPLC?

High-Performance Liquid Chromatography (HPLC) is an analytical technique used to separate, identify, and quantify components in complex mixtures. Operating typically at pressures below 6,000 psi, HPLC systems pump a liquid mobile phase through a column packed with fine stationary-phase particles, usually 3–10 µm in diameter. These particles create extensive surface area for interaction between analytes and the stationary phase, enabling efficient and reproducible separations. By precisely controlling mobile-phase composition, flow rate, and temperature—and coupling the system with sensitive detectors—HPLC provides high-resolution and quantitative results across diverse applications, including drug identity confirmation, purity testing, and therapeutic monitoring.

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How Do HPLC Columns Work?

HPLC columns are engineered to separate complex mixtures into individual components with high precision. Inside each column, the stationary phase interacts with analytes as they are carried through by the mobile phase under high pressure. The effectiveness of this separation depends on selecting the right combination of column chemistry, particle size, and mobile phase conditions. By fine-tuning these variables, scientists can achieve optimal resolution, sensitivity, and reproducibility for a wide range of analytical applications.

HPLC Column Types

Reserved-Phase

Features a non-polar stationary phase and polar mobile phase. Commonly used for non-polar to moderately polar compounds.

Normal-Phase

Uses a polar stationary phase and non-polar mobile phase. Ideal for separating polar compounds or those with functional groups that interact with the polar phase.

HILIC

Uses a hydrophilic stationary phase with a mobile phase high in organic solvent. Effective for retaining and separating polar compounds.

Chiral LC

Separates mirror-image molecules (enantiomers) using chiral columns with specialized stationary phases. It's widely used in pharma and biotech to ensure the purity and effectiveness of

Size-Exclusion

Separates molecules by size. Larger molecules elute first, while smaller ones are retained longer due to pore interaction.

Ion Exchange

Separates analytes based on their ionic charge using charged stationary phases. Anion exchange columns retain negatively charged ions (anions), while cation exchange columns retain positively charged ions (cations).

Ion Exclusion

Separates components by excluding highly ionized species while retaining weakly ionized or neutral molecules. Using a sulfonated resin, IEC is ideal for analyzing organic acids, sugars, and carbohydrates.

Shop by HPLC Column Brands

Looking for high-performance columns tailored to your application? Phenomenex offers a leading portfolio of HPLC columns.

Biozen

Biologic Columns

Clarity

Oligonucleotide Columns

Gemini

High pH Columns

Kinetex

Core-Shell

Luna

High Performance Columns

Luna Omega

Omega Robust High Performance Columns

Lux

Chiral Columns

Synergi

Method Development HPLC Columns

Particle Types

Fully Porous

Traditional materials used in HPLC columns, featuring internal pores that allow analytes and mobile phase to diffuse throughout. This design provides a high surface area, resulting in strong separation efficiency. Their versatility makes them ideal for high-resolution analytical applications across various fields.

Core-Shell

Has a solid core with a thin, porous outer shell, reducing analyte diffusion paths. This design enables fast, high-efficiency separations with lower back pressure than fully porous sub-2 micron particles. Core-shell technology is ideal for rapid, high-resolution HPLC analyses with broad system compatibility.

Monolithic

Formed from a continuous porous rod rather than packed beads, offering high permeability. This allows faster flow rates with lower back pressure, making them ideal for high-speed separations. Monolithic columns are especially effective in bioanalytical applications like protein and peptide analysis.

Polymer-Based

Made from materials like polymethacrylate or polystyrene-divinylbenzene, offer excellent chemical stability across a wide pH range. They tolerate extreme acidic or basic conditions, making them ideal for analyzing organic acids, sugars, and non-water-soluble polymers. These columns are widely used in pharmaceutical, environmental, and food testing for reliable, high-efficiency separations.

Benefits of HPLC

Robustness & Reliability – HPLC systems are extremely durable, less sensitive to minor pressure fluctuations, and can handle “dirtier” samples (like food, environmental, or plant extracts) without clogging as easily.
Method Transferability – Most regulatory and compendial methods (USP, EP, JP) are written for HPLC, making it the default platform in QC and regulated environments.
Cost Efficiency – HPLC instruments, columns, and maintenance are generally less expensive. UHPLC columns and hardware can be more costly and cn wear faster under high pressure.
Sample Load Capacity – Because HPLC columns typically use larger particle sizes (3–5 µm), they often tolerate higher injection volumes, which can be useful for preparative work or when analytes are at low concentrations.
Ease of Use – HPLC has broader accessibility; analysts may prefer it for routine, high-throughput work where speed isn’t the primary concern.