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One of the most accurate and straightforward methods for testing contaminants and compounds in a liquid sample is high-performance liquid chromatography. But how does high-performance liquid chromatography work?

Our step-by-step guide will take you through the HPLC process from start to finish while also showing the benefits of the method and its typical applications.

What Is HPLC?

High-performance liquid chromatography (HPLC) separates components in a liquid mixture to detect and identify them. In essence, the compounds are introduced into a solvent that passes through a column steadily.

The components separate within the column as they pass through the packing materials and exit into a detector. The detector tracks and identifies the elements as they exit the column, and a chromatogram chart analyzes the compounds.

That’s a brief description of what happens in the HPLC process, but we’ll go much more in-depth on the components involved and how the sample moves from one to the other.

The HPLC Process From Start to Finish

The HPLC process begins with the solvent reservoir (mobile phase) before it’s introduced to the pump that will push the solvent through the HPLC process. An injector submits the sample to the solvent before entering the HPLC column.

The sample components exit the HPLC column into the detector, where the chromatogram analyzes the data before it’s dumped into waste. We’ll go through each step of the process and explain the role of each HPLC component.

Solvent (Mobile Phase)

The first step in the HPLC process is the solvent reservoir, otherwise known as the mobile phase, because it later moves through the HPLC column. It will carry the sample through the process as a liquid solvent and can hold anything that can be dissolved.

Pump (Solvent Delivery System)

To get the mobile phase into the HPLC column, push it through at a steady flow, and then exit the column, the mobile phase needs a pump. Also known as a solvent delivery system, the pump maintains a constant flow rate, typically milliliters per minute but not exclusively depending on the scale of the process and sample.

Injector (Sample Manager)

The solvent reservoir is connected to the HPLC column with high-pressure tubing. The injector will introduce the sample into the mobile phase stream from the pool to the column.

HPLC Column

The HPLC column is where the separation and accurate testing of the sample are done. Thanks to the pump, the mobile phase will enter the column and pass through packing material particles at a steady flow rate.

The sample is bunched together in one single band upon entering the column. But as the mobile phase flows through the particles, the components are slowly separated into different bands. The sample components move at different speeds because they have various attractions to the mobile phase rather than the stationary phase.

The components with more significant attraction will move through the column faster and exit first, while the less attracted will move slower. It varies how long this takes, but a general barometer is about ten minutes.

Detector

The components are separated and exit the column to enter the detector. The detector does what its name suggests—it detects the compounds against the mobile phase backdrop to sense which combinations are emerging first.

Once the detector senses the presence of a compound, it sends the corresponding electrical signal to the computer data station (chromatogram) for analysis. The sensor is not one-size-fits-all—there is a wide variety of detectors depending on what compounds are being tested.

Chromatogram

Once the sample compounds exit the HPLC column, and the detector identifies the compounds and sends the data to the chromatogram for analysis. The chromatogram is what plots the chemical separation of the components in the HPLC column.

The chromatogram charts the speed and concentration of the components using the time of the first injection as a baseline. A chromatogram chart shows a steady baseline of the mobile phase with a sharp, narrow spike of the first compound that exits the HPLC.

Because it’s the fastest compound, its peak will be the tallest and narrowest as it quickly exits the detector to waste. After the first, there’s some time to return to a steady baseline before a second spike. Since it’s slightly slower, this peak will be lower and wider, and this will continue as all the compounds pass through the column and detector.

The Benefits of HPLC

There are numerous benefits to HPLC testing compared to other alternatives. First, it’s a very affordable and manageable process on a standard, small scale, especially with used HPLC equipment that works just as well as new equipment.

HPLC is also adaptable and is used in various applications and industries. The entire process is also automated, from the sample to reporting results, so labs can be more productive while the HPLC process runs its course.

The Applications for HPLC

Because HPLC is affordable, adaptable, and automated, many industries use it for testing, from environmentalists to drug companies to medical researchers.

Drug Development

The pharmaceutical industry commonly uses HPLC during the manufacturing process of its drug products. HPLC is a simple and reliable method for testing the purity of products so they can further develop better and purer products.

HPLC is quite affordable for pharmaceutical companies on a small scale, but that benefit lessens in large-scale applications. Although it’s common in testing, drug companies don’t typically use it for large-scale, bulk product drugs.

Cannabis Testing

HPLC is also a common method for testing cannabis and synthetic drugs. HPLC is an excellent method for characterizing cannabinoids extracted from the cannabis plant so drugmakers can understand the drug’s potency.

HPLC has also been a valuable tool for advocates of the drug’s legalization as the method has helped to disprove unfounded claims about the plant and prove its medicinal qualities.

Medical Diagnosis and Research

HPLC testing is also helpful for the medical field and patients. HPLC can also separate components from mixtures to analyze nutrients precisely and reliably in a person’s blood.

Typically, HPLC is used to determine things like a vitamin deficiency. It’s also common in medical research to use HPLC to identify metabolites in patients with existing illnesses.

For instance, a researcher could use HPLC to identify metabolites in a patient with Parkinson’s disease, which will help them learn more about early diagnosis for future patients.

Illegal Drug Detection

Along with detecting what’s not in a person’s bloodstream (vitamins), HPLC can see when illegal agents are present, such as cannabinoids, cocaine, or LSD. It can also detect doping agents and opioids, so HPLC is a standard method for law enforcement and athletic leagues to test players for illegal substances.

Food Contamination

Chemicals are used now in agriculture and food production more than ever before. To ensure that the food we grow and consume is safe, HPLC is used to identify any possible contaminants or chemical residues before they’re released to the public.

Hopefully, you now understand how high-performance liquid chromatography works, its benefits, and its applications. If you have more questions about HPLC or its equipment, consult with the experts at GenTech Scientific.

How Does High-Performance Liquid Chromatography Work?