HPLC, high pressure liquid chromatography, is versatile and used in many industries.
Since the initial discoveries of the early to mid-twentieth century the science of chromatography has seen rapid growth and expansion into an important and fundamental tool of many analytical laboratories. The techniques have involved from classical wet chemistry techniques to more instrument driven measurements facilitating an explosion of instrumentation and methodologies. We take a look at HPLC chromatography in this blog post.
What is HPLC?
HPLC chromatography or high performance liquid chromatography, is a chromatographic technique used to separate, identify and quantify sample components in a liquid mixture, often characterized by high pressure.
HPLC chromatography process
All HPLC systems commercially available have different features and selling points, but all systems have some basic modules or components. The key to liquid chromatography is the mobile phase so all HPLC systems have one or more mobile phase pumps. Mobile phase is stored in reservoirs which are plumbed into a pumping module. Binary pumps can pump two separate channels or mobile phases at the same time. Quaternary pumps can pump up to four different channels or mobile phases at the same time to the system.
Modern HPLC chromatography systems
Modern HPLC systems contain solvent degassing modules which remove bubbles and dissolved gases in the mobile phases which can cause baseline noise or hinder the efficiency of the chromatography system. Solvent degassing can be accomplished by helium purging, vacuum degassing, sonication, or a combination of these methods. Older HPLC systems often lacked an online degassing module and relied on external helium sparging to dissolved air in the mobile phase while in the reservoir bottles.
Another more modern module to HPLC systems is an autosampler compartment instead of single manual injection port. Autosamplers allow for loading multiple samples which are injected without human involvement and reduce sampling errors.
Prior to widespread use of autosamplers, sample injections were made manually and were subject to higher error and variability.
Two HPLC components
The final two components of a liquid chromatography system are: the column compartment (or column oven) and the detector. The column compartment is a temperature-controlled compartment that contains the chromatography column. Many modern systems have the ability for a wide range of heating and cooling functions.
The next and sometimes final component is a detector module that records the separated analytes that elute from the column and produce a signal that a chromatograph records on a chromatogram. A chromatogram is the visual data record of the analyte responses recorded as they elute from the column while a chromatograph is the instrumentation that records the data (FIGURE 3). After the data is recorded the mobile phase and eluent usually ends up as waste for an analytical system or can be separately collected for use in a prep chromatography system.
HPLC system chromatogram
The data produced is often represented as peaks or patterns that correspond to separate components in a mixture expressed over time. The time which a selected analyte elutes is called its retention time. The retention time is the amount of time it takes for an analyte to pass through the system from injection to detection. Retention times change with different conditions such as pH, temperature and stationary phase type, column dimensions and mobile phase or solvent compositions.
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