Editing High-performance liquid chromatography (section)

{{Short description|Technique in analytical chemistry}}
[[File:HPLC Instrument.jpg|thumb|upright|A modern self-contained HPLC]]
[[File:HPLC apparatus.svg|thumb|Schematic representation of an HPLC unit
(1) solvent reservoirs, (2) solvent degasser, (3) gradient valve, (4) mixing vessel for delivery of the mobile phase, (5) high-pressure pump, (6) switching valve in "inject position", (6') switching valve in "load position", (7) sample injection loop, (8) pre-column (guard column), (9) analytical column, (10) detector (''i.e.'', IR, UV), (11) data acquisition, (12) waste or fraction collector.]]

'''High-performance liquid chromatography''' ('''HPLC'''), formerly referred to as '''high-pressure liquid chromatography''', is a technique in [[analytical chemistry]] used to separate, identify, and quantify specific components in mixtures. The mixtures can originate from [[food]], [[Chemical substance|chemicals]], [[pharmaceuticals]],<ref>{{Cite book |title=HPLC for pharmaceutical scientists |date=2007 |publisher=Wiley-Interscience |isbn=978-0-471-68162-5 |editor-last=Kazakevich |editor-first=Yuri |location=Hoboken, NJ |editor-last2=LoBrutto |editor-first2=Rosario}}</ref> [[Biology|biological]], [[Natural environment|environmental]] and [[agriculture]], etc., which have been dissolved into liquid solutions.{{citation needed|date=July 2024}}

It relies on high pressure pumps, which deliver mixtures of various solvents, called the [[Elution|mobile phase]], which flows through the system, collecting the sample mixture on the way, delivering it into a cylinder, called the column, filled with solid particles, made of [[adsorption|adsorbent material]], called the [[Chromatography|stationary phase]].<ref name=":3">{{Cite web |title=Chromatography |url=https://web.njit.edu/~kebbekus/analysis/4CHROMAT.htm |access-date=2024-08-05 |website=web.njit.edu}}</ref>

Each component in the sample interacts differently with the adsorbent material, causing different migration rates for each component.{{citation needed|date=April 2025}}{{Better source needed|reason=The current source is insufficiently reliable ([[WP:NOTRS]]).|date=January 2025}} These different rates lead to separation as the species flow out of the column into a specific [[Sensor|detector]] such as [[UV detectors]]. The output of the detector is a graph, called a chromatogram. Chromatograms are graphical representations of the signal intensity versus time or volume, showing peaks, which represent components of the sample. Each sample appears in its respective time, called its retention time, having area proportional to its amount.<ref name=":3" />

HPLC is widely used for manufacturing (''e.g.'', during the production process of pharmaceutical and biological products),<ref>{{Cite journal |last=Levin |first=Shulamit |date=January 2004 |title=Reversed Phase Stationary Phases in Pharmaceutical Sciences |url=https://www.tandfonline.com/doi/full/10.1081/JLC-120030606 |journal=Journal of Liquid Chromatography & Related Technologies |language=en |volume=27 |issue=7–9 |pages=1353–1376 |doi=10.1081/JLC-120030606 |s2cid=97490509 |issn=1082-6076|url-access=subscription }}</ref><ref>{{Cite journal |last1=Gerber |first1=F. |last2=Krummen |first2=M. |last3=Potgeter |first3=H. |last4=Roth |first4=A. |last5=Siffrin |first5=C. |last6=Spoendlin |first6=C. |year=2004 |title=Practical aspects of fast reversed-phase high-performance liquid chromatography using 3μm particle packed columns and monolithic columns in pharmaceutical development and production working under current good manufacturing practice |journal=Journal of Chromatography A |volume=1036 |issue=2 |pages=127–133 |doi=10.1016/j.chroma.2004.02.056 |pmid=15146913}}</ref> legal (''e.g.'', detecting performance enhancement drugs in urine),<ref>{{Cite book |last1=Bayne |first1=Shirley |title=Forensic Applications of High Performance Liquid Chromatography |last2=Carlin |first2=Michelle |publisher=CRC Press |year=2017 |isbn=9780429251962 |edition=1st}}</ref> research (''e.g.'', separating the components of a complex biological sample, or of similar synthetic chemicals from each other), and medical (''e.g.'', detecting vitamin D levels in blood serum) purposes.<ref>{{Cite journal |last1=Seger |first1=Christoph |last2=Salzmann |first2=Linda |date=2020-08-01 |title=After another decade: LC–MS/MS became routine in clinical diagnostics |journal=Clinical Biochemistry |series=Advancement and Applications of Mass Spectrometry in Laboratory Medicine |volume=82 |pages=2–11 |doi=10.1016/j.clinbiochem.2020.03.004 |pmid=32188572 |s2cid=213186669 |issn=0009-9120|doi-access=free }}</ref>

[[Chromatography]] can be described as a [[mass transfer]] process involving [[adsorption]] and/or [[Partition coefficient|partition]]. As mentioned, HPLC relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with adsorbent, leading to the separation of the sample components. The active component of the column, the adsorbent, is typically a granular material made of solid particles (''e.g.'', [[silica]], polymers, etc.), 1.5–50 μm in size, on which various reagents can be bonded.<ref>{{Citation |title=Chapter 5 Silica columns–packing procedures and performance characteristics |date=1979-01-01 |series=Journal of Chromatography Library |volume=16 |pages=169–186 |editor-last=Unger |editor-first=K. K. |url=https://www.sciencedirect.com/science/article/pii/S030147700860809X |access-date=2024-08-05 |publisher=Elsevier|doi=10.1016/S0301-4770(08)60809-X |isbn=978-0-444-41683-4 |url-access=subscription }}</ref><ref>{{Cite journal |last1=Xu |first1=Yan |last2=Cao |first2=Qing |last3=Svec |first3=Frantisek |last4=Fréchet |first4=Jean M.J. |date=2010-04-15 |title=Porous polymer monolithic column with surface-bound gold nanoparticles for the capture and separation of cysteine-containing peptides |journal=Analytical Chemistry |volume=82 |issue=8 |pages=3352–3358 |doi=10.1021/ac1002646 |issn=0003-2700 |pmc=2875083 |pmid=20302345}}</ref> The components of the sample mixture are separated from each other due to their different degrees of interaction with the adsorbent particles. The pressurized liquid is typically a mixture of solvents (''e.g.'', water, [[Buffer solution|buffers]], [[acetonitrile]] and/or [[methanol]]) and is referred to as a "mobile phase". Its composition and [[temperature]] play a major role in the separation process by influencing the interactions taking place between sample components and adsorbent.<ref>{{Cite journal |last1=Panella |first1=Cristina |last2=Ferretti |first2=Rosella |last3=Casulli |first3=Adriano |last4=Cirilli |first4=Roberto |date=2019-10-01 |title=Temperature and eluent composition effects on enantiomer separation of carvedilol by high-performance liquid chromatography on immobilized amylose-based chiral stationary phases |journal=Journal of Pharmaceutical Analysis |volume=9 |issue=5 |pages=324–331 |doi=10.1016/j.jpha.2019.04.002 |pmid=31929941 |issn=2095-1779|pmc=6951491 }}</ref> These interactions are physical in nature, such as hydrophobic (dispersive), dipole–dipole and ionic, most often a combination.<ref>{{Cite web |title=Molecular Interaction of HPLC Stationary Phase |url=https://www.imtakt.com/jp/Products/Interaction/indexE.htm |access-date=2024-08-08 |website=www.imtakt.com}}</ref><ref>{{Cite journal |last1=Kadlecová |first1=Zuzana |last2=Kalíková |first2=Květa |last3=Folprechtová |first3=Denisa |last4=Tesařová |first4=Eva |last5=Gilar |first5=Martin |date=2020-08-16 |title=Method for evaluation of ionic interactions in liquid chromatography |url=https://www.sciencedirect.com/science/article/pii/S0021967320305793 |journal=Journal of Chromatography A |volume=1625 |pages=461301 |doi=10.1016/j.chroma.2020.461301 |pmid=32709344 |issn=0021-9673|url-access=subscription }}</ref>