A practical reference on Chromatogram: what it is, how it behaves, what the literature reports, and where the honest uncertainties sit.
Reviewed 2026-02-27. Anything still debated is marked as such rather than presented as settled.
High-performance liquid chromatography is an analytical technique that separates components in a liquid sample. A pump moves a liquid mobile phase through a column packed with a solid stationary phase. Compounds interact differently with both phases and travel at different rates, leaving the column at distinct retention times. A detector records these arrivals as peaks on a chromatogram. The resulting pattern supports identification and quantification of substances in mixtures. Modern instruments use high pressure to force solvent through small particles, which improves speed and resolution compared with older low-pressure liquid chromatography methods.
Separation in HPLC depends on the chemistry of the stationary phase, the composition of the mobile phase, and the physical properties of the column. Reverse-phase separations use a nonpolar stationary phase and a polar mobile phase, and they are common for many organic compounds. Ion-exchange, size-exclusion, and normal-phase modes serve other classes of analytes. Gradient elution changes solvent strength over time, while isocratic elution holds it constant. Flow rate, temperature, particle size, and column length all influence peak shape and resolution. Detection may use ultraviolet absorbance, fluorescence, refractive index, or mass spectrometry, depending on the analyte and the required sensitivity.
Routine HPLC testing compares a sample result with a calibration curve prepared from known reference standards. Peak area or peak height is plotted against concentration, and the curve is used to estimate unknown amounts. Retention time supports tentative identification when compared with a standard, though mass spectrometry or another confirmatory method may be needed for definitive identification. Pre-run checks verify repeatability, resolution, and peak symmetry before sample analysis. Limits of detection and quantification describe the smallest amounts that can be reliably observed or measured. Sample preparation, filtration, and degassing help prevent column damage and inconsistent results.
Instrumentation includes a solvent delivery system, an autosampler, a column oven, and one or more detectors. Reversed-phase columns with chemically modified silica are widely used, but normal-phase, ion-exchange, size-exclusion, and affinity modes exist for specific separations. Detectors may rely on ultraviolet absorbance, fluorescence, refractive index, or mass spectrometry. Column temperature, mobile phase composition, and flow rate are adjusted to improve resolution. System pressure is monitored because rising pressure can indicate column blockage or deteriorating packing.
Separation performance depends on particle size, pore size, column length, and the chemistry of the stationary phase. Smaller particles generally improve efficiency but require higher pressure and suitable instrumentation. The mobile phase often contains buffers and organic solvents that influence retention and selectivity. Testing labs select conditions based on the analytes, sample matrix, and required sensitivity. Method development frequently involves screening several columns and solvent mixtures before a final set of conditions is chosen.
High-performance liquid chromatography is an analytical technique that separates components in a liquid sample by passing them 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 how analytes partition between the mobile phase and the stationary phase cause them to exit the column at different times. Detection then records a signal proportional to the amount of each separated substance. The resulting chromatogram provides retention times and peak areas for identification and quantification.
| Property | Value | Notes |
|---|---|---|
| Common abbreviation | HPLC | High-performance liquid chromatography |
| Separation basis | Differential partitioning | Between liquid mobile phase and solid stationary phase |
| Common mode | Reverse phase | Nonpolar column, polar mobile phase |
| Typical detector | UV-Vis absorbance | Widely used for compounds with chromophores |
| Typical column particle size | 2–5 µm | Smaller particles can improve resolution |
Practical HPLC testing depends on careful sample preparation and instrument maintenance. Samples may require filtration, dilution, pH adjustment, or extraction to avoid column damage and matrix interference. Mobile phases are degassed and filtered, and columns are equilibrated before injection. Common problems include peak tailing, baseline drift, ghost peaks, carryover, and co-elution of analytes. Documentation of instrument logs, calibration records, and electronic audit trails supports data integrity and traceability. Ongoing training and routine maintenance help reduce variability between analysts and laboratories.
Quality control laboratories use HPLC to check identity, purity, concentration, and stability of raw materials and finished products. A validated method specifies the column, mobile phase, flow rate, detection wavelength, injection volume, and run time. Samples are prepared and compared against reference standards of known concentration. The resulting chromatogram provides quantitative data, such as assay values and impurity levels. This approach is common in pharmaceutical, food, environmental, and industrial testing where consistent measurements are required.
Method validation demonstrates that an analytical procedure is suitable for its intended purpose. Typical validation characteristics include accuracy, precision, specificity, linearity, range, limit of detection, limit of quantitation, and robustness. Regulatory guidance from bodies such as the International Council for Harmonisation and the United States Pharmacopeia outlines expectations, though specific criteria depend on the product and method. System suitability tests are run before sample analysis to confirm resolution, peak symmetry, column efficiency, and injection repeatability. Failure of these checks can invalidate a batch of measurements.
Validation demonstrates that a method is suitable for its intended use. Typical performance characteristics include accuracy, precision, specificity, linearity, range, limit of detection, limit of quantitation, and robustness. Regulators and standards organizations provide frameworks, but specific requirements depend on the application and jurisdiction. System suitability tests are run before sample analysis to confirm resolution, peak symmetry, retention time repeatability, and sensitivity. A validated method is not permanently fixed; changes may require partial or full revalidation.
Routine HPLC testing depends on controlled reagents, calibrated instruments, and documented procedures. Columns degrade over time, so retention times and peak shapes are monitored for drift. Mobile phases are filtered and degassed to prevent pump damage and detector noise. Reference standards must be traceable and stored under suitable conditions. Data handling systems record injections, calculations, and audit trails. Quality control samples interspersed with unknowns help detect errors during a run.
== Epidemiology == Beriberi is a recurrent nutritional disease in detention houses, even in this century. In 1999, an outbreak of beriberi occurred in a detention center in Taiwan. High rates of illness and death from beriberi in overcrowded Haitian jails in 2007 were traced to the traditional practice of washing rice before cooking; this removed a nutritious coating which had been applied to the rice after processing (enriched white rice). In the Ivory Coast, among a group of prisoners with heavy punishment, 64% were affected by beriberi. Before beginning treatment, prisoners exhibited symptoms of dry or wet beriberi with neurological signs (tingling: 41%), cardiovascular signs (dyspnoea: 42%, thoracic pain: 35%), and edemas of the lower limbs (51%). With treatment, the rate of healing was about 97%. Populations under extreme stress may be at higher risk for beriberi. Displaced populations, such as refugees from war, are susceptible to micronutritional deficiency, including beriberi. The severe nutritional deprivation caused by famine also can cause beriberi, although symptoms may be overlooked in clinical assessment or masked by other famine-related problems. An extreme weight-loss diet can, rarely, induce a famine-like state and the accompanying beriberi. Workers on Chinese squid ships are at elevated risk of beriberi due to the cheap refined grain and raw fish diet they are fed and the long period of time between shoring. Between 2013 and 2021, 15 workers on 14 ships have died with symptoms of beriberi.
(1933), first African-American basketball player to be selected as All-American Alfred Skrobisch (1933), Olympic fencer Cliff Montgomery (1934), led the Columbia Lions football team to victory in the Rose Bowl John O'Brien (1938), basketball player for the Akron Wingfoots Ben Johnson (1938), sprinter who rivaled Jesse Owens Sid Luckman (1939), NFL Hall of Fame Chicago Bears quarterback Ken Germann (1943), football coach, athletic director of Columbia University, and former Southern Conference commissioner Paul Governali (1943), football player for the Boston Yanks and New York Giants Walt Budko (1948), basketball player for Baltimore Bullets and Philadelphia Warriors Bruce Gehrke (1948), football player for New York Giants Bill Swiacki (1948), player for New York Giants, member of the College Football Hall of Fame Lou Kusserow (1949), football player for Hamilton Tiger-Cats and New York Yanks John Azary (1951), basketball player, recipient of the Haggerty Award Jack Molinas (1953), NBA player for the Fort Wayne Pistons Jack Rohan (1953), head coach of the Columbia Lions men's basketball team 1961–1974, and 1990–1995 George Shaw (1953), Olympic triple jumper Richard Ballantine* (1967), cyclist and cycling advocate; son of Ian Ballantine '38 of Ballantine Books James Margolis (1958), Olympic fencer James Melcher (1961), Olympian fencer, president of Fencers Club and hedge fund manager Robert Contiguglia (1963), soccer player, former president of the United States Soccer Federation Peter Salzberg (1964), head coach of Vermont Catamounts men's basketball 1972–1981 Archie Roberts (1965), former football player for the Miami Dolphins and cardiac surgeon Jim McMillian (1968), NBA player for the Los Angeles Lakers, Buffalo Braves, New York Knicks and Portland Trail Blazers Dave Newmark (1968), NBA player for the Chicago Bulls; also played for Israeli team Hapoel Tel Aviv B.C. Marty Domres (1969), football player for San Diego Chargers and Baltimore Colts Heyward Dotson (1970), basketball player George Starke (1971), offensive lineman for the Washington Redskins Henry Bunis (1975), two-time All-American tennis player, runner-up in 1977 Chilean Open Rick Fagel (1975), professional tennis player Vitas Gerulaitis* (1975), champion tennis player Thomas Losonczy (1975), Olympic fencer, winner of the Congressional Gold Medal Alton Byrd (1979), basketball player Eric Fromm (1980), tennis player John Witkowski (1983), football player for Detroit Lions and Houston Oilers Gene Larkin (1984), member of the Minnesota Twins 1987 and 1991 World Series championship teams Amr Aly (1985), soccer player who won the Hermann Trophy as the top college player of the year 1984; member of the 1984 U.S. Olympic Soccer Team and indoor soccer team Los Angeles Lazers Stephen Trevor (1986), Olympic fencer Kyra Tirana Barry (1987), team leader for U.S. women's national wrestling team Caitlin Bilodeaux (1987), Olympic fencer Howard Endelman (1987), tennis player Phil Williamson (1987), tennis player for Antigua and Barbuda Bob Cottingham (1988), Olympic fencer Jon Normile (1989), Olympic fencer Frank Seminara (1989), Major League Baseball pitcher for the San Diego Padres and the New York Mets Tom Auth (1990), Olympic rower Christine Vardaros (1991), professional cyclist Ann Marsh (1994), Olympic fencer Ríkharður Daðason (1996), Icelandic soccer player Marcellus Wiley (1997), football player for the Buffalo Bills, San Diego Chargers and Dallas Cowboys Dan Kellner (1998), fencer Pellegrino Matarazzo (1999), head coach of VfB Stuttgart Matt Napoleon (1999), Olympic soccer goalkeeper Cristina Teuscher (2000), Olympic gold medalist swimmer Jedediah Dupree (2001), NCAA Champion fencer Veljko Urošević (2003), Serbian Olympic rower Fernando Perez (2004), outfielder for the Tampa Bay Rays Jeremiah Boswell (2005), professional basketball player for BC Sliven, KK Strumica, and KK Torus Delilah DiCrescenzo (2005), long-distance runner, inspiration and subject of the Grammy-nominated song Hey There Delilah Michael Quarshie (2005), Finnish American football player who played for the Oakland Raiders and Frankfurt Galaxy Lisa Nemec (2006), Croatian long-distance runner Miloš Tomić (2006), Serbian Olympic rower Erison Hurtault (2007), Dominican sprinter James Leighman Williams (2007), fencer who won silver in the 2008 Summer Olympics Emily Jacobson (2008), fencer İhsan Emre Vural (2008), Turkish rower for Galatasaray S.K. Sherif Farrag (2009), Egyptian-American Olympic fencer Nicholas la Cava (2009), Olympic rower Jeff Spear (2010), Olympic fencer Daria Schneider (2010), fencer Jeff Adams (2011), Houston Texans offensive tackle Nicole Ross (2011), Olympic fencer Isadora Cerullo (2013), Brazilian-American Olympic rugby player Katie Meili (2013), Olympic swimmer, Pan American Games and 2016 Summer Olympics gold medalist Josh Martin (2013), Kansas City Chiefs linebacker John Gregorek Jr. (2014), middle-distance runner David Najem (2014), American soccer player for New Mexico United and the Afghanistan national football team Nadia Eke (2015), Ghanaian triple jumper, African Championships gold medalist in 2016 Kristine Musademba (2015), figure skater Max Schnur (2015), tennis player playing on the ATP Challenger Tour Nzingha Prescod (2015), Olympic fencer Ramit Tandon (2015), professional squash player Jakub Buczek (2016), Canadian Olympic rower Sasha DiGiulian (2016), world champion climber Jacqueline Dubrovich (2016), Olympic fencer Maodo Lô (2016), German basketball player for Brose Bamberg Robb Paller (2016), American-Israeli Olympic baseball player Jeff Coby (2017), American basketball player for Xuventude Baloncesto Cameron Nizialek (2017), football player for Atlanta Falcons Akua Obeng-Akrofi (2018), Ghanaian sprinter Charlotte Buck (2018), Olympic rower Osama Khalifa (2018), #1 ranked college squash player in the U.S. for the 2016–17 season Camille Zimmerman (2018), American basketball player for Norrköping Dolphins Yasmeen Al-Dabbagh (2019), Saudi Arabian sprinter Jessica Antiles (2019), swimmer who won silver and bronze medals in the 2017 Maccabiah Games Dylan Castanheira (2019), soccer player, goalkeeper for Fort Lauderdale CF Sophie Whitehouse (2019), goalkeeper for Republic of Ireland women's national football team Mike Smith (2020), basketball player Anthony Jackie Tang (2020), Hong Kong tennis player John Tanguay (2020), rower who won a silver medal in the 2020 Summer Paralympics Dylan Geick* (2021), wrestler and internet personality Velavan Senthilkumar (2021), British Junior Open Squash champion and Asian Junior Squash champion Nastasya Generalova (2023), gymnast and model Olivia Giaccio (2024), Olympic freestyle skier Evita Griskenas (2024), rhythmic gymnast Camden Pulkinen (2024), figure skater Abbey Hsu (2024), basketball player
== Limitations == The cytocentrifugation process can cause cells to appear distorted. Cells located at the centre of the smear may look compressed compared to cells at the periphery. Cell nuclei may develop artifactual clefts, lobes, or holes, and the cytoplasm may appear vacuolated or develop irregular projections. Cytoplasmic granules may be pushed to the periphery of the cell. If the cell count is high, cells may be distorted due to crowding; therefore, samples with high cell counts are diluted prior to smear preparation.
=== Biological fluorescence === Chemical biologists often study the functions of biological macromolecules using fluorescence techniques. The advantage of fluorescence versus other techniques resides in its high sensitivity, non-invasiveness, safe detection, and ability to modulate the fluorescence signal. In recent years, the discovery of green fluorescent protein (GFP) by Roger Y. Tsien and others, hybrid systems and quantum dots have enabled assessing protein location and function more precisely. Three main types of fluorophores are used: small organic dyes, green fluorescent proteins, and quantum dots. Small organic dyes usually are less than 1 kDa, and have been modified to increase photostability and brightness, and reduce self-quenching. Quantum dots have very sharp wavelengths, high molar absorptivity and quantum yield. Both organic dyes and quantum dyes do not have the ability to recognize the protein of interest without the aid of antibodies, hence they must use immunolabeling. Fluorescent proteins are genetically encoded and can be fused to your protein of interest. Another genetic tagging technique is the tetracysteine biarsenical system, which requires modification of the targeted sequence that includes four cysteines, which binds membrane-permeable biarsenical molecules, the green and the red dyes "FlAsH" and "ReAsH", with picomolar affinity. Both fluorescent proteins and biarsenical tetracysteine can be expressed in live cells, but present major limitations in ectopic expression and might cause a loss of function.
Sources: en.wikipedia.org
== January 8, 1982 (Friday) == To end a federal antitrust suit that had been brought in 1974 by the U.S. Department of Justice, the telephone monopoly American Telephone & Telegraph (AT&T) agreed to sell its 22 individual Bell System companies that had controlled most U.S. telephone lines. On the same day, the Department of Justice dropped its 12-year-old antitrust suit against International Business Machines (IBM), ending a trial that had started on May 19, 1975. The Umkhonto we Sizwe paramilitary force of South Africa's anti-apartheid African National Congress, carried out an attack on the construction site of the Koeberg Nuclear Power Station, planned to be the white South African government's first nuclear power plant, causing 500 million rands worth of damage and setting back the completion date by 18 months. The first White House meeting concerning Project Excalibur, the proposed Strategic Defense Initiative (nicknamed the "Star Wars defense") for the United States against an enemy missile attack, took place as Edward Teller, Karl Bendetsen, William Wilson and Joseph Coors made a one-hour presentation to U.S. President Ronald Reagan for a satellite with lasers that could destroy nuclear missiles after their launch.
== Childhood obesity == Childhood obesity and adolescent obesity have also increased in Brazil. National surveys and international reports have identified Brazil as one of the countries experiencing a rapid rise in obesity among children and adolescents. Public health experts have expressed concern that increasing childhood obesity may contribute to future increases in diabetes, cardiovascular disease, and healthcare expenditures. The World Obesity Federation has highlighted childhood obesity as an emerging public health challenge in Brazil and throughout Latin America.
== Diagnosis == Affected patients may have normal, low, or slightly elevated TSH depending on the spectrum and phase of illness. Total T4 and T3 levels may be altered by binding protein abnormalities, and medications. Reverse T3 levels are generally increased, while FT3 is decreased. FT4 levels may have a transient increase, before becoming subnormal during severe illness. Correspondingly, in the majority of cases calculated sum activity of peripheral deiodinases (SPINA-GD) is reduced. Generally the levels of free T3 will be lowered, followed by the lowering of free T4 in more severe disease. Several studies described elevated concentrations of 3,5-T2, an active thyroid hormone, in NTIS. 3,5-T2 levels were also observed to correlate with concentrations of rT3 (reverse T3) in patients with euthyroid sick syndrome. NTIS is a component of a complex endocrine adaptation process, so affected patients might also have hyperprolactinemia and elevated levels of corticosteroids (especially cortisol) and growth hormone. NTIS can be difficult to distinguish from other forms of thyroid dysfunction in the hospital setting. Both NTIS and primary hypothyroidism may have reduced fT3 and fT4, and elevated TSH (which is common in the hospital, during the recovery phase of NTIS). Prescribing thyroxine to treat this may lead to lifelong thyroid overtreatment. Hyperthyroidism may be assumed due to decreased TSH and a transient fT4 increase. In some cases, this can be distinguished from NTIS by a thyroid ultrasound, which is commonly available in the hospital intensive care unit.
=== tRNA === While consensus secondary and tertiary structures have been observed in tRNAs, there has not been evidence of tRNAs creating a quaternary structure thus far. Of note, it has been observed through high resolution imaging that tRNA interacts with the quaternary structure of bacterial 70S ribosome and other proteins.
The first 7-Eleven in Australia opened on 24 August 1977, in the Melbourne suburb of Oakleigh. The majority of stores are located in metropolitan areas, particularly in central business district areas. Stores in suburban areas often operate as petrol stations and most are owned and operated as franchises, with a central administration. 7-Eleven bought Mobil's remaining Australian petrol stations in 2010, converting them to 7-Eleven convenience store / petrol stations. In South Australia all Mobil petrol stations were later sold to Peregrine Corporation and rebranded to OTR convenience store / petrol stations. In April 2014, 7-Eleven announced plans to start operating stores in Western Australia, with 11 stores planned to operate within the first year and a total of 75 stores established within five years. The first store was opened on October 30, 2014, in the city of Fremantle. The country has 675 stores as of January 2018. In April 2022, 7-Eleven Australia settled a class-action lawsuit from its franchisees for A$98 million, amid claims that it had misled franchisees about the profitability of its business model. In December 2023, Seven & I Holdings of Japan agreed to purchase the Australian 7-Eleven franchise from its original franchise owners for A$1.71 billion. The purchase was finalized in April 2024.
Sources: en.wikipedia.org
HPLC testing measures the presence and amount of one or more compounds in a liquid sample. It separates mixture components and records detector responses as peaks, which are compared with reference standards. Results are usually reported as concentrations or relative percentages.
Retention time is the interval between sample injection and the detector response for a given compound. It depends on the compound's interactions with the stationary and mobile phases under set conditions. Matching a retention time to a standard supports tentative identification but is not always unique.
HPLC alone can separate unknown compounds and provide retention times, but it often cannot identify them with certainty. Coupling HPLC to mass spectrometry gives mass information that improves identification. Confirmation usually requires comparison with reference standards or complementary techniques.
HPLC separates and detects individual compounds in a liquid sample, producing peaks at characteristic retention times. Peak area or height can be used to estimate concentration when calibrated with known standards. It does not identify unknown compounds with certainty unless additional detectors or reference materials are used.