High-performance liquid chromatography (HPLC)
High-performance liquid chromatography (HPLC) is a sophisticated analytical technique used for the separation, detection, and identification of pure compounds within a mixture. It is particularly significant in forensic science for identifying unknown substances and establishing connections between different samples, especially in drug analysis. The HPLC process begins with the injection of a liquid sample into a chromatography column, where it is propelled by a high-pressure liquid solvent known as the mobile phase. Due to the varying chemical and physical properties of the compounds, each travels through the column at different rates, allowing for their separation and detection.
Typically, a specific wavelength of ultraviolet light is used to measure the absorption of these compounds, although advanced detection methods such as diode array detectors can analyze a wider spectrum. While HPLC requires careful sample collection and preparation, the actual analysis can be completed in under an hour, though it necessitates the expertise of a skilled forensic chemist. Over the years, advancements have led to ultra-high-performance liquid chromatography (UHPLC) and other variations, which improve speed, resolution, and sensitivity, making HPLC a valuable tool across various scientific fields.
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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 chromotography (NLC) is a quicker and cheaper alternative to HPLC that offers increased resolution power and produces less waste. Ultra-fast liquid chomotography (UFLC) is approximately ten times faster than traditional liquid chomotography methods, ensuring quick analysis without negatively affecting analytical reliability or precision.
Bibliography
Bell, Suzanne. Forensic Chemistry. 2nd ed. Upper Saddle River: Pearson, 2014. Print.
Fanali, Salvatore, Paul Haddad, Colin Poole, Peter Schoenmakers, and David Lloyd. Liquid Chromatography: Applications. 2d rev. ed., Elsevier, 2017.
"High-Performance Liquid Chromatography Fundamentals." Agilent, 2024, www.agilent.com/en/product/liquid-chromatography/hplc-fundamentals. Accessed 15 Aug. 2024.
Houck, Max M., and Jay A. Siegel. Fundamentals of Forensic Science. 3d ed.,Elsevier/Academic Press, 2015.
"HPLC Basics." ThermoFisher Scientific, 2024, www.thermofisher.com/us/en/home/industrial/chromatography/chromatography-learning-center/liquid-chromatography-information/hplc-basics.html. Accessed 15 Aug. 2024.
Naushad, Mu, and Mohammad Rizwan Khan. Ultra Performance Liquid Chromatography Mass Spectrometry: Evaluation and Applications in Food Analysis. Boca Raton: CRC, 2014. Print.