What is HPLC Analysis for?

In the field of new materials research, it is essential to have analytical techniques that provide precise information about complex mixtures that define the quality of a product formulation intended to stand out in the market. For this reason, the availability of separation and analytical detection techniques for this type of mixture is of great importance, such as HPLC Analysis or High Performance Liquid Chromatography (HPLC).

In this article, we will explain in detail HPLC Analysis, one of the most interesting analytical characterization tests that we use in our projects.

How HPLC equipment works

High Performance Liquid Chromatography, also known as High Pressure Liquid Chromatography (HPLC), is an analytical technique that enables the separation of complex mixtures. Its basic principle is the same as that of conventional liquid chromatography: the sample is introduced into a tube (called a chromatography column), which is filled with porous particles (known as the stationary phase) that interact physically and/or chemically with the components of the sample, separating them.

The separated components are collected at the outlet of the column and identified using an external measurement technique, such as a spectrophotometer that measures color intensity, or another device capable of quantifying their amount.

How an HPLC analysis is performed

As mentioned above, the operating principle of HPLC Analysis is the same as that of liquid chromatography, with the difference that HPLC offers higher speed, efficiency, and sensitivity.

In this case, a small volume of liquid sample is injected into a tube called a column, which is filled with micrometric particles (known as the stationary phase). The individual components of the sample are carried through the column by a liquid (known as the mobile phase) under the high pressure supplied by a pump.

During their movement through the column, the components undergo physical and/or chemical interactions with the microparticles that make up the column, leading to their separation. Afterward, the separated components are detected at the outlet by a detector that generates a signal called a chromatogram.

In the chromatogram, the chromatographic peaks corresponding to each separated component can be distinguished, with the signal represented as a function of time after injection. Each HPLC analysis typically takes between 10 and 30 minutes.

Types of HPLC

The key aspect of HPLC is the separation of the sample components. For this to occur, the sample must interact with the microparticles that make up the column. As a result of these physical or chemical interactions, separation is achieved.

Due to its importance, different separation modes have been developed. The most commonly used for the majority of compounds are:

• Size Exclusion Chromatography (SEC). In this type of separation, molecules diffuse through a porous medium. Separation depends on the relationship between molecular size and pore size. Molecules larger than the pore diameter do not diffuse, while smaller ones do. In this type of chromatography, larger molecules elute first. It is commonly used for the analysis of polymers and proteins.
• Ion Exchange Chromatography. In this case, the column is composed of particles with ionic groups, and the mobile phase is an aqueous buffer. This technique is used for the separation of anions and cations, both inorganic and organic, in aqueous solution. A typical example is the separation of amino acids and proteins.
• Adsorption or Normal Phase Chromatography. In this mode, the column packing is polar, and the mobile phase is non-polar. It is used to analyze water-sensitive compounds, geometric isomers, and chiral compounds.
• Reverse Phase Chromatography. This is the most widely used mode. In this type of separation, the column packing is non-polar, and the mobile phase is an organic solvent miscible with water. It can be used for non-polar, polar, ionizable, and ionic molecules. For very complex samples with many constituents, gradient elution is used.

Applications of HPLC

As mentioned, HPLC analysis is a powerful analytical tool that provides valuable information about complex mixtures, regardless of their nature.

Some of its most important applications include:

• Separation and analysis of non-volatile or thermally stable compounds. Common examples include pharmaceuticals, proteins, polymers, unstable compounds such as trinitrotoluene, and motor oils.
• Qualitative analysis of individual compounds. Based on retention time information from the chromatogram, compounds can be identified with high precision, relating them to their chemical structure or molecular weight.
• Quantitative analysis of individual compounds. When a standard (known quantities of a compound) is available, the exact amount can be determined by comparing the signals of the standard and the sample. Peak height and/or width provide quantifiable values.
• Preparation of pure compounds. Derived from the above applications, purity can be adjusted based on the relationship between compound concentration and signal, for example through solvent evaporation and signal monitoring.
• Trace analysis. Enables detection of substances at concentrations below 1% by weight. This is particularly useful for detecting toxic substances in pharmaceuticals or environmental samples.

Advantages of HPLC analysis

Main advantages:

• Quantitative and qualitative analysis of complex mixtures. HPLC allows the separation and analysis of components in complex mixtures, identifying each compound individually and accurately quantifying its concentration. This is especially useful in pharmaceuticals, food analysis, and environmental samples.

• Trace detection. Thanks to its high sensitivity, HPLC can detect even minute amounts of substances, enabling the identification of contaminants, impurities, or metabolites that might go unnoticed with other techniques.

• Automation. Modern HPLC systems allow automation of sample injection, elution, and data acquisition, increasing reproducibility and reducing the possibility of human error.

• High sensitivity and precision. By combining high-efficiency columns with advanced detectors, HPLC provides highly precise and reproducible results, even for compounds that are difficult to analyze, ensuring reliable measurements.

Our HPLC services

At ATRIA, we are aware of the potential of HPLC for the analysis of complex mixtures. It also plays a key role in quality control of industrial processes, where the presence of contaminants could reduce the market value of a product.

For this reason, ATRIA has a team of technical professionals capable of developing the best HPLC methodology for each case. From literature review of analytical procedures to troubleshooting product anomalies, ATRIA offers a wide range of high-value analytical tools such as HPLC.

Would you like to carry out HPLC in one of your projects? Contact us!