High-performance liquid chromatography (HPLC)

DEFINITION: Technique used in the separation, detection, and identification of the pure compounds present in a mixture.

SIGNIFICANCE: Forensic scientists can use high-performance liquid chromatography to determine the identity of an unknown substance or to establish a match between two different samples. The technique is particularly useful in the forensic analysis of drugs.

High-performance liquid chromatography is one of the many subtypes of chromatography. Analysis by high-performance liquid chromatography (HPLC) begins with the injection of a liquid sample onto a chromatography column. The sample is then carried through the column by a high-pressure liquid solvent commonly referred to as the mobile phase. Because the individual pure compounds that are present in the sample have different chemical or physical properties, they travel through the column at different rates and exit the column at different times. Typically, the compounds are detected through the measurement of the absorption of a specific wavelength of ultraviolet light. However, the use of a diode array detector allows simultaneous measurement of absorption across the entire ultraviolet and visible spectra and is thus particularly useful for the identification of unknown compounds.

An HPLC instrument, which consists of the injector, pump, column, and detector, consumes relatively large amounts of solvent. For this reason, HPLC is generally not a field-portable technique, so samples to be analyzed by HPLC must be carefully collected and handled prior to analysis. The analysis itself typically requires less than one hour, but often a good deal of sample preparation and method optimization must be done before the sample can be injected. The process must be conducted by a knowledgeable and experienced forensic chemist.

HPLC is particularly useful for the identification of drugs because of its ability to detect a small amount of a specific chemical substance in a mixture of many other substances. It is thus useful in detecting small amounts of drugs in blood or urine. Because HPLC can also identify and quantify the other substances in the mixture, it is useful in establishing a match between two different samples. For example, HPLC can be used not only to identify a drug definitively but also to identify impurities that may link that drug to a specific laboratory.

Significant improvements have been made to the speed, resolution, and sensitivity of HPLC over time; advanced HPLC technology is sometimes known as ultra-high-performance liquid chromatography (UHPLC or UPLC). Its applications, however, are much the same as those of the older HPLC. Another advanced form of liquid chromatography is rapid resolution liquid chromatography (RRLC), which allows for high-resolution results and time-efficient testing. Nano liquid chromatography (NLC) is a quicker and cheaper alternative to HPLC that offers increased resolution power and produces less waste. Ultra-fast liquid chromatography (UFLC) is approximately ten times faster than traditional liquid chromatography methods, ensuring quick analysis without negatively affecting analytical reliability or precision.

Bibliography

Bell, Suzanne. Forensic Chemistry. 3rd ed., CRC Press, 2022

Fanali, Salvatore, et al. Liquid Chromatography: Applications. 3rd ed., Elsevier, 2023.

“High-Performance Liquid Chromatography Fundamentals.” Agilent, 2024, www.agilent.com/en/product/liquid-chromatography/hplc-fundamentals. Accessed 5 Dec. 2025.

Houck, Max M., and Jay A. Siegel. Fundamentals of Forensic Science. 3rd ed., Academic Press, 2015.

“HPLC Basics.” ThermoFisher Scientific, 2024, www.thermofisher.com/in/en/home/industrial/chromatography/chromatography-learning-center/liquid-chromatography-information/hplc-basics.html. Accessed 8 Dec. 2025.

Naushad, Mu, and Mohammad Rizwan Khan. Ultra Performance Liquid Chromatography Mass Spectrometry: Evaluation and Applications in Food Analysis. CRC Press, 2014.