HPLC Basics: What You Should Know Vol. 1

by Chris Tuczemskyi

You would be hard-pressed not to find a high-performance liquid chromatography (HPLC) instrument in today’s analytical laboratory, given its critical role in a range of fields, from pharmaceutical to food and beverage, manufacturing and environmental safety.

This blog post explains what HPLC is, how it works and the different techniques liquid chromatographs use to purify mixtures.

What is HPLC?

HPLC is a broad analytical chemistry technique used to separate, identify and quantify compounds in a chemical mixture. These separations utilize the pressure-driven flow of a mobile phase through a column packed with a stationary phase.

The mobile phase carries a liquid sample through the column to the detector, and compounds — or analytes — separate due to varying degrees of interaction with the stationary phase.

A detector measures the analytes after elution off the column, and a chromatography data system (CDS) translates the detected signal.

The translated data output of an HPLC analysis is called a chromatogram, where the x-axis shows time and the y-axis is a specific signal generated by the detector.

How does HPLC work?

An HPLC instrument generally has four major hardware components: a pump, autosampler, column and detector. Additional elements include solvents and a CDS package plus connective capillaries and tubing to allow the continuous flow of the mobile phase and sample through the system.

Every HPLC analysis includes the following steps:

1. Mobile phase begins to flow — The pump pushes the eluents through the system at a specified flow rate.
2. Sample injection — After injection into the mobile phase, the sample travels with the mobile phase from the injection point to the head of the column.
3. Compound separation — Physical separation of the compounds happens on the column stationary phase. After elution from the column, the separated sample components travel to the detector.
4. Analyte detection — Detection of specified analytes based on an electrical signal generated by specific properties.
5. Chromatogram generation — Translation of the detected analyte signal by the CDS into a chromatogram of analyte signal versus time.

What factors affect HPLC separations?

Many factors like the mobile phase composition, column chemistry, and temperature can influence HPLC separations. Successful separation only occurs if the analytes have differing affinities for the column, so selecting the appropriate stationary phase for your compounds is crucial.

The main factors influencing the overall separation process are:

• Physiochemical properties of the analyte, such as size, charge, polarity and volatility
• Physiochemical properties of the stationary phase, such as polarity, charge and viscosity
• Physiochemical properties of the mobile phase used and interaction with the analyte and stationary phases

Isocratic versus gradient separations

All HPLC separations are carried out in one of two modes — isocratic or gradient.

• Isocratic methodsseparatebyusing a consistent eluent composition duringanalysis, like 100 percent acetonitrile or a 50:50 mixture of acetonitrile to water.
• Gradient methods include a change in the mobile phase composition across a separation. These methods often employ two solvents, called A and B. The run will begin with a certain percentage of A to B, like 60 percent water to 40 percent acetonitrile, for instance, followed by a percentage change throughout a separation.