Get the industry-leading Aminex HPLC and guard columns for all your carbohydrate, organic acid, and alcohol separations.
Type I Columns
For economical, flexible, high-resolution analytical or semipreparative purification of biomolecules including proteins, peptides and nucleic acids.
Resolution Size Exclusion Columns
Use ENrich size exclusion high performance liquid chromatography columns for high-resolution separation of proteins & biomolecules at fast flow rates.
Resolution Ion Exchange Columns
Need fast, high-resolution purification of biomolecules? Integrate high-capacity ENrich ion exchange columns into your workflow.
High performance liquid chromatography is the use of high pressure to generate flow through a packed chromatography column. The development of what was initially called high-pressure liquid chromatography, and is now called high performance liquid chromatography (both abbreviated HPLC), has increased both the speed and sensitivity of molecule separation for analytic and semipreparative scale applications.
In high performance liquid chromatography, the particles in the column packing have smaller diameters than in gravity or low-pressure systems, providing a greater surface area and thus increasing resolution. Additionally, the higher pressures yield faster run times. The level of pressure used depends on the capability of the pumps in the system and pressure rating of the column and packing.
Bio-Rad offers several high performance liquid chromatography columns including our industry-leading Aminex HPLC columns, ideal for analyzing small organic molecules like carbohydrates or organic acids, alcohols, and bases. Our Bio-Scale CHT Type I columns, featuring Bio-Rad's unique multimodal CHT ceramic hydroxyapatite media, are traditionally used for medium-pressure applications but are also ideal for high-performance liquid chromatography. ENrich is our newest line of HPLC columns and can be used for size exclusion or ion exchange chromatography, with either cationic or anionic media types. The new ENrich columns are uniquely designed for separating proteins and other biomolecules at fast flow rates.
The basics of a high performance liquid chromatography system include one or more reservoirs for solvent, a high-pressure pump, some form of sample injector, the column, a detection system, and a container for waste collection. Detectors are usually linked to a computer to generate a chromatogram and perform data analysis. Frequently, there is a fraction collector immediately downstream of the detection system to collect the separated molecules. The fraction collector can collect fractions continuously or may be linked to the detector and collect only peaks of interest.
Another component frequently added to a high performance liquid chromatography system is a guard column. This is placed after the sample injector but before the analytical column. A guard column will catch particulates in the sample and bind any compounds that could permanently bind to the main column and damage it. The guard column protects the HPLC column, both maintaining its high resolution and prolonging the lifetime of the column. For a high performance liquid chromatography column with silica-based packing, a silica-containing guard column can be used so that solvents that attack silica (such as water and methanol) will be adsorbed before the solvent reaches the main column, significantly increasing its lifetime.
The type of detector used depends on the samples and analytes being detected therein. The most common type of detection is by ultraviolet (UV) absorption, as many compounds absorb in the UV range. Other types of detectors include fluorescence and evaporative-light-scattering detector (ELSD). Some systems have more than one detector type linked in series, and a high performance liquid chromatography system is frequently linked to a downstream mass spectrometer to form a liquid chromatography–mass spectrometry (LC-MS) system.
An inline degasser between the solvent reservoirs and the pump is now present in most HPLC systems. This system is much more effective in preventing air bubbles in the pump than degassing solvents prior to use. Air bubbles in high pressure systems were a major problem until effective inline degassers were developed.
In high performance liquid chromatography, the particles in the column are called the stationary phase, and the solvent is known as the mobile phase. Separation of molecules in high performance liquid chromatography is usually based on one or more of three main chemical characteristics: polarity, charge, and size. The choice of the stationary phase is determined by the molecule(s) of interest in the sample and which type(s) of interaction with the stationary phase will give the highest resolution.
There are two types of elution modes, isocratic and gradient. During isocratic elution, the mobile phase is the same throughout. For gradient elution, the system must include two or more solvent reservoirs, a gradient valve, and a mixer to provide a gradient in the solvent profile over time. Solvent gradients usually involve a change in the concentration of an ion or an organic solvent.
The choice of stationary and mobile phases and isocratic or gradient elution requires a knowledge of the molecule(s) of interest in the sample. The most common choice for high performance liquid chromatography is called reversed phase chromatography, which uses a nonpolar stationary phase and a polar mobile phase.