High-performance liquid chromatography or high-pressure liquid chromatography or HPLC is a chromatographic technique used to isolate a mixture of compounds in analytical chemistry and biochemistry to classify, measure or purify the individual components of the mixture.
Chromatography is a method of separation and the word chromatography originates from the words chroma meaning “color”, and graphein meaning “write”. Chromatograph is the equipment for separation and a chromatogram is the output chart that is obtained from the analysis done.
Reversed-phase HPLC or ultra-high-performance Liquid Chromatography (UHPLC) is a widely used separation mode. It provides complex preservation of hydrophobic and organic compounds. The combination of hydrophobic and van der Waals type interactions between all target compounds and both stationary and mobile phases enables the preservation of these compounds by reversed-phase.
Applications for HPLC
An HPLC analysis can be used to analyze both raw materials and finished products. When first identifying a raw material supplier or for regular quality control tests, it is necessary to test the raw materials to ensure that you have obtained the correct purity and quality of the product.
Many raw materials look and sound the same, but to fully understand if you’re getting exactly what you’re paying for, they should be checked and confirmed.
Manufacturers often use HPLC-enabled laboratories to also reverse engineer formulations, solve product failure problems, conduct competitor product analysis, and scan for pollutants or other impurities. These types of testing can be conducted on both the raw materials and the finished product.
Sometimes, after the manufacturing process is completed, the final product is checked again to check that it contains the correct amount of active ingredients as stated on the packaging. And because this method of testing will detect any pollutants or other product flaws that may have occurred in the batch during the manufacturing phase, conducting these tests will minimize the risk of the product being recalled. The applications of the industry include:
- Purification of water
- Detection of impurities in the pharmaceutical industry
- Pre-concentration of trace elements
- Ligand-Chromatography of Trade
- Ion-exchange protein chromatography
How It Works
In very small quantities, the sample mixture to be extracted and evaluated is sent through a column to a mobile phase stream. Various types of columns are available with sorbents of different particle sizes and surfaces.
The mixture passes through the column at varying speeds and interacts with the sorbent, also known as the stationary phase. The velocity of each component in the mixture depends on (1) its chemical structure, (2) the nature of the column, and (3) the composition of the mobile phase. The time at which a particular analyte emerges from the column is referred to as its retention time. The retention time shall be measured under particular conditions and shall be considered to be the distinguishing attribute of the analyte.
Sorbent particles can be hydrophobic or polar in nature. Mobile phases widely used involve any miscible mixture of water and organic solvents such as acetonitrile and methanol. Mobile water-free phases may also be used.
The aqueous portion of the mobile phase can contain acids such as formic, phosphoric, or trifluoroacetic acid or salts to allow the separation of the sample components. During the chromatographic analysis, the composition of the mobile phase is either preserved as constant or as varied.
The constant approach is efficient for the separation of the sample components which are not very different in their affinity to the stationary phase. In a varied approach, the composition of the mobile phase varies from low to high eluting power. The eluting strength of the mobile phase is expressed in the retention times of the analyte, where high eluting strength causes rapid elution.
The composition of the mobile phase is chosen based on the strength of interactions between several sample components and the stationary phase.
The HPLC partitioning process is somewhat similar to the liquid-liquid extraction process, except that the former is a continuous process, while the latter is a step-by-step process.
It is recommended that the procedure of partitioning of the test be carried out to evaluate the exact HPLC method that would provide an appropriate separation.
Liquid chromatography is a valuable analytical method for determining the components of the formulation of a drug, allowing researchers to quantify the formulation and find out if there are any impurities in the substance.
The other techniques that HPLC can be combined with further its capabilities, making it the perfect analytical tool for pharmacists to ensure the high quality of medicines.