About HPLC

How Does HPLC Work?

HPLC purifies compounds and separates them according to their polarity. Water is a polar compound while Benzene is a non-polar compound. Molecules with similar chromatographic polarity are attracted to each other, while those with dissimilar polarity have a weaker attraction. This method passes a liquid mobile phase through a stationary phase within the column. When the sample is injected, each different analyte reacts differently with the absorbent material (stationary phase), providing the feedback you’re looking for.

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What Are Common HPLC Applications?

 HPLC can be used as an analytical device to assay environmental and biological samples to determine the absence or presence of known compounds. This process can help to determine the existence of pesticides, drugs, toxins, and more.  

It can also be used to monitor the purity of products as well as chemical reactions. Compounds of interest can also be purified for expanded use.  


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Components of HPLC Instrumentation

This sophisticated analytical technique and tool is strategically designed to provide the accurate results that you need. We have listed the different components and the role they play in HPLC analysis below. 

HPLC Column

Compact in nature and made from a variety of substances, mainly silica, the HPLC column is known as the stationary phase of HPLC. The carbon chains that are functionalized by the silica particles become longer the more apolar they are.  C18 columns are commonly used, these are columns with 18-carbon chains.

HPLC Sample

HPLC can be used to identify the compounds in biological specimens like urine, muscle, saliva, and blood.  It can also be used in environmental   microbiology to identify toxins in bacteria, and medicinal chemistry in drugs detection.  These sample types, or the compounds being assessed can vary.

Sample Injection

Once you determine your sample, it is injected into the column.  Automatic injection allows for higher throughput.  The software in HPLC equipment allows you to automate your samples in terms of how much and in what order they should be injected, making the process more efficient.  

Mobility Phase

The mobile phase can be comprised of a mixture of a polar solvent such as water and an organic solvent.  It is referred to as the mobile phase because this is the part of the process that moves through the column as it flushes the compounds from the column.  

These compounds can be eluted (flushed out) along a concentration gradient along the duration of the method.  This means that the percentage of the polar solvent in the mobile phase is decreasing over time as the percentage of apolor solvent is increasing. 


HPLC Run

The most commonly used HPLC method is reversed-phased (RP-HPLC).  This method separates the compounds beginning with the most polar and ending with the most apolar compounds.  

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What Are the Advantages of HPLC?

 HPLC can be used to analyze both raw materials and finished goods and can be used as a quality control check to determine the correct purity and grade of a product.

HPLC can also be beneficial in reverse engineering formulations to find errors and offer quick and accurate data for informed quality decisions.      


What is a HPLC Output?

The output is the result of the HPLC run. The result is displayed as a horizontal series of peaks that represent the compounds analyzed in the column. With detectors such as a diode array (DAD) detector or ultraviolet (UV) detector, you can look at the chromatogram of the separated compounds in wavelengths to determine what they are.



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What is HPLC?

HPLC is a chromatographic technique used to separate the components in a mixture, identify and quantify each component. HPLC stands for high performance liquid chromatography, which is a sophisticated analytical technique used to leverage information based on how different compounds behave differently in a mixture. The instruments that perform this technique separate the components in a sample to quantify and identify its parts. HPLC can be utilized in the medical, pharmaceutical, polymer, coatings, and many other industrial and commercial sectors.  

HPLC Questions

HPLC leak repair

HPLC Instrumentation for Analysis: Identifying and Repairing Leaks

By Ben Wartel, Overbrook Liquid Phase Engineer • November 16, 2020
Leaks are one of the most common issues you will encounter while operating an HPLC. Some leaks are easily observable as drops of liquid forming where a capillary nut meets a fitting. Discover the signs of a leak and ways to repair it.
HPLC Baseline Noise

HPLC Repair Services: Common Causes of Baseline Noise

By Overbrook Liquid Phase Engineering Team • August 31, 2020
The appearance of your HPLC’s baseline noise is one of the most useful indicators of its overall performance and cleanliness. The ratio of the detector’s signal response to a specified standard when compared to the background signal variation is known as the “signal to noise ratio.” When you notice abnormalities in the baseline noise, it can be attributed to a number of different factors.
HPLC Maintenance

HPLC Service Provider: Ideas to Get the Most Out of Your High Performance Liquid Chromatography

By Ted Palashis • January 24, 2020
Highly sensitive lab instruments need to be serviced and maintained appropriately and effectively. Avoiding contamination can help prevent signal suppression, reduce background noise, minimize adduct formation, and elevate spectrum complexity. In order to achieve the most accurate results, contamination prevention is crucial.
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