This is a working overview of reversed-phase, written for readers who want more than a one-paragraph summary but less than a textbook.
Reviewed 2026-02-21. Anything still debated is marked as such rather than presented as settled.
Laboratories apply HPLC testing across pharmaceutical, food, environmental, and industrial chemistry. The method can measure active ingredients, impurities, additives, preservatives, and degradation products. Sample preparation often includes dilution, filtration, and sometimes extraction or derivatization. The choice of column, mobile phase, pH, temperature, and detector depends on the analytes and matrix. Results are compared with reference standards to assign identity and concentration. Method suitability is judged by resolution, precision, and accuracy.
HPLC testing is not a single fixed procedure; it is a family of separation modes. Reversed-phase, normal-phase, ion-exchange, size-exclusion, and affinity chromatography each suit different analyte properties. Reversed-phase methods dominate because they handle many neutral and moderately polar compounds. Detection can be optical, electrochemical, or mass spectrometric, and the detector dictates what information is available. Coupling with mass spectrometry increases selectivity and enables identification when standards are unavailable. The technique cannot separate every mixture without adjustment.
Quality control samples are inserted at intervals to monitor accuracy and precision throughout a batch. Blank samples detect contamination, while spiked samples assess recovery from the sample matrix. Calibration standards establish the relationship between detector response and concentration, and control samples are prepared independently from them whenever possible. Laboratories also participate in proficiency testing and maintain audit trails, instrument logs, and reagent records. Ongoing review of control charts can reveal trends before they cause out-of-specification results.
Method validation demonstrates that an HPLC procedure is suitable for its intended purpose. Common validation parameters include accuracy, precision, specificity, linearity, range, limit of detection, limit of quantification, and robustness. Accuracy reflects agreement with a reference value, while precision describes repeatability under defined conditions. Specificity shows whether the method can measure the analyte in the presence of impurities or matrix components. Validation documents are reviewed before a method is used for routine testing or regulatory submissions.
| Property | Value | Notes |
|---|---|---|
| Abbreviation | HPLC | Also called high-performance liquid chromatography |
| Separation mechanism | Differential partitioning | Compounds distribute between mobile and stationary phases |
| Typical column chemistry | C18 (octadecylsilane) | Used in reversed-phase separations |
| Typical detector | UV-Vis or photodiode array | Mass spectrometry is common for trace and confirmatory work |
| Typical particle size | 1.8–5 µm | Smaller particles require higher pressure and can improve speed |
Separation modes differ by the chemistry of the stationary phase and the composition of the mobile phase. Reversed-phase testing uses a nonpolar column and polar solvents, making it common for pharmaceutical, environmental, and food analytes. Normal-phase testing uses a polar column and nonpolar solvents for compounds that are poorly retained in reversed-phase systems. Ion-exchange and ion-pair methods separate charged species, while size-exclusion methods sort molecules by hydrodynamic volume. Gradient elution changes solvent strength over time to resolve complex mixtures, and isocratic elution holds solvent composition constant for simpler assays.
Key performance measures include retention time, peak area, peak height, resolution, tailing factor, and plate count. Retention time helps identify a peak under fixed conditions, but confirmation often requires a second method or detector. Peak area and height relate to concentration through calibration curves, which may be linear or nonlinear depending on the detector response. Resolution describes separation between adjacent peaks, while tailing factor and plate count describe peak shape and column efficiency. Performance checks verify these values before and during a run to confirm that the instrument is performing within limits.
High-performance liquid chromatography is an analytical technique that separates components of a liquid sample by passing it through a packed column under pressure. A pump delivers a mobile phase at a controlled flow rate, and an injector introduces the sample into the stream. Differences in interaction with the stationary phase cause analytes to migrate at different rates. Detectors record elution as peaks, and a data system converts signals into a chromatogram. The method is suited to compounds that dissolve in a liquid and are not volatile enough for gas chromatography.
Reversed-phase chromatography dominates modern HPLC testing, using a nonpolar stationary phase such as chemically bonded octadecyl groups and a polar mobile phase of water mixed with organic solvent. Analytes partition between the mobile and stationary phases according to hydrophobicity. Gradient elution changes the mobile phase composition over time to separate compounds with a wide range of retention. Isocratic elution keeps the composition constant and is simpler for routine assays. Column temperature, pH, and flow rate influence selectivity, peak shape, and retention time, so these parameters are controlled during a validated method.
In quality control laboratories, HPLC testing supports batch release, raw material checks, stability studies, and impurity profiling. A validated method defines sample preparation, instrument settings, calibration, and acceptance criteria. Analysts compare results with specifications and investigate out-of-specification outcomes before a batch is approved. Documentation includes chromatograms, integration records, audit trails, and reagent details. Because results influence product decisions, laboratories follow formal quality systems and data integrity rules. The exact tests and limits depend on the material, its intended use, and the applicable regulatory framework.
Method validation examines whether an HPLC procedure is suitable for its intended purpose. Common parameters include accuracy, precision, specificity, linearity, range, detection limit, quantification limit, and robustness. Accuracy describes closeness to a true or accepted value, while precision describes agreement among repeated measurements. Specificity shows whether the method can measure the analyte without interference from related substances. Robustness tests small deliberate changes in flow, temperature, or solvent composition. Validation is not a one-time event; methods may need partial revalidation after changes to instruments, columns, sample handling, or specification limits. Regulatory guidance provides frameworks, but some details remain method-specific.
Regulatory and pharmacopeial texts shape how HPLC testing is performed and documented. The International Council for Harmonisation provides validation guidance, while pharmacopeias publish general chromatography chapters and monographs for specific materials. Accreditation standards such as ISO/IEC 17025 address laboratory competence and traceability. Inspectors may review instrument qualification, analyst training, reference material control, and electronic records. Open questions include how best to validate methods for new complex products and how to handle automated data processing. Laboratories generally resolve these issues through risk assessment, method lifecycle management, and documented scientific justification.
18 October — The Primary Principals Collective Bargaining Union accepts a 2.5% pay rise offer following four months of negotiations with the Ministry of Education. This will be followed by another 2.1% pay rise in 2026. 20 October: The New Zealand Government introduces two new requirements for job seeker beneficiaries who have failed a welfare obligation: engaging in three job search activities weekly and attending at least one employment-training course per month. A measles case linked to international travel is reported in Auckland. 21 October: Strong winds cause power outages, fallen trees and disrupt road and rail travel across the Wairarapa region. Lines company Powerco reports that 10,000 houses in the area have lost electricity due to fallen power lines. Strong winds and heavy rains disrupt road, rail and air travel in the Wellington Region. One person is killed by a fallen tree branch in Mount Victoria during a storm that hits Wellington. Strong winds cause four vegetation fires in the Hawke's Bay Pōrangahau area, leading to the evacuation of vie homes. Strong winds and dry conditions cause six fires in the Kaikōura District, leading to the destruction of 14 buildings and local evacuations. Three new measles cases are reported in Manawatū and Nelson. 22 October: Fire and Emergency New Zealand deploys numerous firefighters and helicopters to combat fires in the Kaikōura District and Pōrangahau. Strong winds and fires cause power outages and flight disruptions through the lower North Island and upper South Island.
Ernst on developing the first two-dimensional NMR experiments, and established the nuclear Overhauser effect as a convenient way of measuring distances within proteins. This research later led to the complete assignment of resonances for among others the bovine pancreatic trypsin inhibitor and glucagon. In October 2010, Wüthrich participated in the USA Science and Engineering Festival's Lunch with a Laureate program where middle and high school students will get to engage in an informal conversation with a Nobel Prize–winning scientist over a brown-bag lunch. Wüthrich is also a member on the USA Science and Engineering Festival's Advisory Board and a supporter of the Campaign for the Establishment of a United Nations Parliamentary Assembly, an organisation which campaigns for democratic reform in the United Nations. Wüthrich is a member of the Executive Advisory Board of the World.Minds Foundation, where he contributes to international dialogue on science, research, and innovation policy.
His crimes, which targeted around 3,500 children across 30 countries, are described by Mr Justice O'Hara as ones of "sadism and depravity" which "scarred" the childhoods of his victims. Appearing at the Old Bailey, Dylan Earl pleads guilty to carrying out an arson attack on a Ukrainian-owned business in east London on behalf of Russia. A Scottish couple are given permission to proceed with a legal case that will attempt to overturn the scrapping of universal winter fuel payments for pensioners. 27 October – Labour MP Mike Amesbury is suspended from the party and has the whip withdrawn, after CCTV footage emerges appearing to show him punching a man to the ground. 28 October Far-right activist Tommy Robinson is sentenced to 18 months in prison for contempt of court for repeating false allegations against a Syrian refugee, in breach of an injunction. Manchester United sack manager Erik ten Hag less than 24 hours after a 2–1 defeat to West Ham United, leaving the club in 14th place after nine games. Assistant Ruud van Nistelrooy takes charge on an interim basis until a permanent replacement is found. 30 October October 2024 United Kingdom budget: Rachel Reeves unveils her first budget as chancellor, which includes £40bn in tax rises, intended to offset what she alleges is a £22bn "black hole" in the nation's finances, while enabling the largest hike in NHS funding since 2010. The first case of Clade Ib mpox, a more spreadable variant linked to the outbreak in Africa, is detected in the UK.
== Relation to humans == Arapaima is exploited in many ways by local human populations. Its tongue is thought to have medicinal qualities in South America. It is dried and combined with guarana bark, which is grated and mixed into water. Doses are given to kill intestinal worms. The bony tongue is used to scrape cylinders of dried guarana, an ingredient in some beverages, and the bony scales are used as nail files. Arapaima produce boneless steaks and are considered a delicacy. In the Amazon region, locals often salt and dry the meat, rolling it into a cigar-style package that is then tied and can be stored without rotting, which is important in a region with little refrigeration. Arapaima are referred to as the "cod of the Amazon", and can be prepared in the same way as traditional salted cod. Designers have begun using the skin of the arapaima as leather to make jackets, shoes, and handbags, and to cover furniture. In July 2009, villagers around Kenyir Lake in Terengganu, Malaysia, reported sighting A. gigas. The "Kenyir monster", or "dragon fish" as the locals call it, was claimed to be responsible for the mysterious drowning of two men on 17 June. In August 2018, India Times reported that arapaima has been spotted in the Chalakudy River, following floods in Kerala; their presence in India is attributed to illegal importation for fish farming. The arapaima is depicted on both the flag and the seal of the Department of Ucayali, Peru.
=== Eugenics === Crick occasionally expressed his views on eugenics, usually in private letters. For example, Crick advocated a form of positive eugenics in which wealthy parents would be encouraged to have more children. He once remarked, "In the long run, it is unavoidable that society will begin to worry about the character of the next generation ... It is not a subject at the moment which we can tackle easily because people have so many religious beliefs and until we have a more uniform view of ourselves I think it would be risky to try and do anything in the way of eugenics ... I would be astonished if, in the next 100 or 200 years, society did not come round to the view that they would have to try to improve the next generation in some extent or one way or another."
Sources: en.wikipedia.org
==== United States ==== In December 2012, the US Drug Enforcement Administration proposed classifying lorcaserin as a Schedule IV drug because it has hallucinogenic properties at higher than approved doses and users hypothetically might develop psychiatric dependencies on the drug. On 7 May 2013, the US Drug Enforcement Administration classified lorcaserin as a Schedule IV drug under the Controlled Substances Act.
While it is usually considered that the driver has the responsibility when collisions occur, vehicle can also contribute to collisions, up to 3% to 5% of crashes. Two kinds of programs exist: new car assessment program for new cars, and vehicle inspections for other ones. A New Car Assessment Program is a government or institutional car safety program tasked with evaluating new car designs for performance against various safety threats. Two well known NCAP are United States New Car Assessment Program since 1978 and European New Car Assessment Programme since 1997.
Abortive initiation, also known as abortive transcription, is an early process of genetic transcription in which RNA polymerase binds to a DNA promoter and enters into cycles of synthesis of short mRNA transcripts which are released before the transcription complex leaves the promoter. This process occurs in both eukaryotes and prokaryotes. Abortive initiation is typically studied in the T3 and T7 RNA polymerases in bacteriophages and in E. coli. Abortive initiation occurs prior to promoter clearance. RNA polymerase binds to promoter DNA to form an RNA polymerase-promoter closed complex RNA polymerase then unwinds one turn of DNA surrounding the transcription start site to yield an RNA polymerase-promoter open complex RNA polymerase enters into abortive cycles of synthesis and releases short RNA products (contains less than 10 nucleotides) RNA polymerase escapes the promoter and enters into the elongation step of transcription
14N + p → 11C + 4He − 2.92 MeV It can also be produced by fragmentation of 12C by shooting high-energy 12C at a target. Carbon-11 is commonly used as a radioisotope for the radioactive labeling of molecules in positron emission tomography. Among the many molecules used in this context are the radioligands [11C]DASB and [11C]Cimbi-5. Due to the short half-life, the chemical reactions used to manipulate the radioisotope as generated and incorporate it into a biomolecule must be efficient. Popular key intermediates include [11C]phosgene, for carboxylation-related reactions, though other synthons, such as [11C]carbonyl fluoride and [11C]carbon dioxide, are also being explored. For methylation, [11C]iodomethane and related synthons are used.
Sources: en.wikipedia.org
It measures the presence and amount of one or more compounds in a liquid sample. Separation occurs in a column, and detection produces a signal proportional to concentration. Identification usually requires comparison with a known reference standard under the same conditions.
In most cases the sample is consumed or altered during analysis, though some detectors are non-destructive. Fractions can be collected after separation for further study. Repeated testing therefore requires additional sample.
Run times range from under a minute for fast methods to over an hour for complex separations. Sample preparation, equilibration, and data review add time. Throughput depends on instrument configuration and method requirements.
Method validation is the documented process of showing that an HPLC procedure produces reliable results for a defined purpose. It examines parameters such as accuracy, precision, specificity, linearity, and robustness. Regulators and quality systems often require validation before routine use.