High-performance liquid chromatography (HPLC) is a well-established technique for separating mixtures of substances into their constituent parts based on specific chemical and molecular properties. Separation of liquids is based on the distribution of analytes of interest between a mobile phase (eluent) and a stationary phase.
This stationary phase is contained within the HPLC column, and a sample containing the analyte is injected onto the column then pumped through with a flow of the mobile phase. As they pass through the column, the constituents interact differently with the stationary phase, causing them to separate and elute from the column at different times. These separated substances are typically detected with HPLC-detectors.
A basic analytical high-performance liquid chromatography system comprises several components, including an eluent reservoir, a pump, an injection valve, an HPLC column, a detector, and a PC with a liquid chromatography data system installed. In this blog post, Knauer explores the working principles of each of these essential liquid chromatography modules in more detail.
High Performance Liquid Chromatography Pumps and Eluent Reservoirs
There are two primary modes used for pumping the mobile phase through an HPLC system. Isocratic elution is used when the mobile phase will remain constant during a run, such as when the eluent is being recycled. Gradient elution systems are used when the composition of the eluent will change during separation.
The detection signal of eluted substances is called a peak. Applying a gradient enables late peaks in a chromatogram to be eluted faster than in isocratic mode, by changing the mobile phase composition as appropriate.
In a gradient elution system, it is possible to use both high- and low-pressure gradients (HPG/LPG). LPG enables solvents to be drawn from distinct reservoirs and mixed on the suction side of the pump, while HPG uses multiple pumps to supply individual streams of solvents for mixing on the discharge side.
High Performance Liquid Chromatography Columns
The HPLC column is the foundation of this instrument. It contains the stationary phase media required to separate sample ingredients into their constituent parts. Geometry is an important indicator of performance when it comes to HPLC columns. Inner diameter and length impact retention rates and elution times, as will the particle size and chemical composition of the stationary phase. Numerous separation mechanisms are supported by HPLC columns, including normal and reversed phase, size exclusion, ion exchange, hydrophilic interaction, affinity, and more.
Stable column temperature is another critical factor in achieving reproducible separation results and consistent retention rates. Column thermostats are used in order to maintain stable temperatures, and to ensure uniform temperature distributions between heating sleeves and eluent heaters.
High Performance Liquid Chromatography Detectors and Software
The type of detector used to acquire a peaks is determined by the analyte. Various HPLC detection principles are available, some are described here: UV detectors are common but require substance molecules to absorb light in the ultraviolet part of the spectrum. An RI detector is used when substances do not absorb UV light. These detectors are used to measure the signal peaks of separated analytes over time but can only be used for isocratic applications. The complete set of peaks is known as a chromatogram. This is generated by the chromatographic data system as the detector converts the RI or UV signal into an electrical value. Each distinct peak provides both qualitative and quantitative information about the analyte, and the data managements system can subsequently calculate the composition of the sample.
High Performance Liquid Chromatography with Knauer
Knauer has over fifty years’ experience in the development and manufacture of advanced scientific tools and liquid chromatography instruments. We understand the unique challenges of HPLC/UHPLC and can suggest suitable methods and techniques for analyzing your proprietary analytes.