title: Tamerlan Tsarnaev

text: According to a 2010 photo essay about him in "The Comment", the graduate student magazine of Boston University College of Communication, Tsarnaev said that he was working to become a naturalized citizen in time to be selected for the U.S. Olympic boxing team. He added that he would "rather compete for the United States than for Russia", while remarking that he "didn't understand" Americans and did not have any American friends. A later FBI report recorded Tamerlan stating that was a misquote, and that most of his friends were American. He added that he abstained from drinking and smoking, because "God says no to alcohol" and that "there are no values anymore. People can't control themselves".


title: Tamerlan Tsarnaev

text: Pro-super middleweight Edwin Rodriguez sparred with Tsarnaev in 2010, and later said that, although Tsarnaev hit hard, he lacked competitiveness and immediately complained of stomach pain and rib pain. He described Tsarnaev as arrogant but "a coward". Tsarnaev's landlord said the boxer's aspirations were never met because "his back was in really bad shape and he couldn't get into the Olympics".


title: Tamerlan Tsarnaev

text: His coach and another boxer described him as talented but cool and arrogant. Rule changes disqualified all non-US citizens from Golden Gloves boxing, ending Tsarnaev's boxing career and Olympic hopes.


title: Tamerlan Tsarnaev

text: According to an aunt in Dagestan, "He started to be really interested in Islam about three years ago [April 2010], but he was never a radical."


title: Tamerlan Tsarnaev

text: In the spring of 2010, his girlfriend Katherine Russell became pregnant with their child and dropped out of college at the end of her junior year to marry Tsarnaev on June 21, 2010, in a 15-minute ceremony in an office at the Masjid Al Quran in the Dorchester area of Greater Boston. Imam Taalib Mahdee said that he had not met the couple before the ceremony, and Katherine was the one who had called and asked to be married there. Katherine Russell had converted to Islam after starting dating Tsarnaev and had adopted the Muslim name Karima. The couple had a daughter, Zahara Tsarnaev, born in October 2010.


title: Tamerlan Tsarnaev

text: Tsarnaev first came to the attention of Russian security forces in December 2010 when William Plotnikov was briefly detained in Dagestan and forced to disclose his social networking contacts in North America with ties to Russia.


title: Tamerlan Tsarnaev

text: In early 2011, Russia's Federal Security Service (FSB) told the Federal Bureau of Investigation that Tsarnaev was a follower of Islamic extremism and a strong believer. The FSB said that he was preparing to leave the United States to travel to the Russian region to join unspecified underground groups. The FBI initially denied that it had contacted Tsarnaev, but then said that it actually had after Tsarnaev's mother talked about the FBI's contacts with her son on RT. The FBI said that it interviewed him and relatives of his, but did not find any terrorist activity, and that it provided the results in the summer of 2011. At that point, the FBI asked the FSB for more information, but the Russians did not respond to the American request, and the FBI officially closed the case.


title: Tamerlan Tsarnaev

text: Tsarnaev's mother said that FBI agents had told her they feared her son was an "extremist leader", and that he was getting information from "extremist sites". She said Tsarnaev had been under FBI surveillance for at least three years and that "they were controlling every step of him". The FBI flatly denied this accusation. Tsarnaev "vaguely discussed" "jihad" during a 2011 phone call with his mother that was taped by the FSB, and intelligence officials also discovered text messages in which his mother discussed how he was ready to die for Islam. In late 2011, the Central Intelligence Agency put both Tsarnaev and his mother on its Terrorist Identities Datamart Environment database.


title: Tamerlan Tsarnaev

text: Two Jewish men, Erik Weissman and Raphael Teken, as well as their roommate Brendan Mess, were killed in a triple homicide in Waltham, Massachusetts, on September 11, 2011, the 10-year anniversary of the 9/11 attacks. Each victim's throat had been slit with such great force as to be nearly decapitated. Thousands of dollars worth of marijuana and cash were left covering the victims' bodies, and $5,000 was left at the scene. The local district attorney said that it appeared that the killer and the victims knew each other. It was reported on April 23, 2013, that local authorities believed Tsarnaev may have been responsible for the triple homicide, and they and the FBI were investigating the possibility. In May, forensic evidence connected the two brothers to the scene of the killings, and their cell phone records appeared to place them in the area. The officials cautioned that until more definitive DNA testing is complete, it is still too early to consider bringing an indictment against the younger of the two brothers.


title: Tamerlan Tsarnaev

text: ABC reported on April 23, 2013, that authorities linked Tsarnaev to the Waltham unsolved triple homicide. A search warrant affidavit partially unsealed in November 2019 provided further details to Tsarnaev's link to the triple homicide.


title: Tamerlan Tsarnaev

text: Tsarnaev traveled to Russia through Moscow's Domodedovo International Airport in January 2012, and returned to the U.S. in July 2012. He and his wife received public assistance and food stamps from September 2011 to November 2012, which included all the time Tsarnaev was in Russia. Zubeidat Tsarnaeva said her son had wanted his wife and their child to move to Dagestan with him, and that: "She herself agreed; she said she wanted to study a different culture, language."


title: Tamerlan Tsarnaev

text: Tsarnaev's father said that he was with him in Makhachkala, the capital of Dagestan, for six months and that they had done ordinary things, such as visiting relatives. His father also said that they visited Chechnya twice, to see relatives there and to receive his son's new Russian passport. While Tsarnaev arrived in Russia in January 2012, however, he only arrived in Dagestan around March, and his father arrived there in May. U.S. House Homeland Security Chairman Michael McCaul said he believed that Tsarnaev received training during his trip, and became radicalized. In an early report, Dagestan's interior minister Abdurashid Magomedov said through a spokesman that Tsarnaev "did not have contact with the [Islamist] underground during his visit".


title: Tamerlan Tsarnaev

text: The Tsarnaev brothers' mother's second cousin, Magomed Kartashov, is a figure in Dagestan's Islamist community. Zubeidat confirmed that they "became very close." Kartashov's Islamist organization, "The Union of the Just," advocates Islam as a political system under sharia law. Kartashov later stated the Boston bombing was "good" in that it would increase converts to Islam, similar to the attacks of September 11.


title: Tamerlan Tsarnaev

text: According to media reports, Tsarnaev was seen by Dagestan police, who were conducting surveillance, making six visits to a known Islamic militant in a Salafi mosque in Makhachkala founded by an associate of Ayman Zawahiri. According to Russian investigative newspaper "Novaya Gazeta", quoting unnamed Russian security sources, Tsarnaev was linked to 23-year-old William Plotnikov, an ethnic Russian-Tatar Islamic militant and Canadian citizen, with whom he communicated via online social networking sites. Tsarnaev had also visited Toronto, where Plotnikov lived with his parents. Once in Dagestan, Tsarnaev is said to have met on several occasions with Makhmud Mansur Nidal, a 19-year-old Dagestani-Palestinian man. Nidal was under close surveillance by Dagestan's anti-extremism unit for six months as a suspected recruiter for Islamist insurgents, before the police killed him in May. According to "Novaya Gazeta", Tsarnaev had sought to join the local insurgency, and was put on a period of 'quarantine' – a clearance check by insurgents looking for infiltrating double agents, taking several months for a recruit to be verified. After Tsarnaev's alleged contacts were both killed, he "got frightened and fled". He left Russia in July two days after Plotnikov was killed, in an apparent hurry that Russian authorities considered suspicious, not waiting to pick up his new Russian passport – ostensibly one of his main reasons for coming to Russia.


title: Tamerlan Tsarnaev

text: In an interview, Tsarnaev's father later said he had to force his son to return to the United States to complete his U.S. citizenship application, after Tsarnaev tried to convince his family to allow him to stay in Dagestan for good.


title: Tamerlan Tsarnaev

text: Tsarnaev returned to the U.S. on July 17, 2012, having grown a long, thick beard and wearing kohl around his eyes as a sign of his religious devotion to the Sunni of Islam and the example of Muhammad. His life took on an "increasingly puritanical religious tone" with "Islamist certainty". He appeared, to some family members, to have become an "extremist".


title: Tamerlan Tsarnaev

text: After his return to the U.S., Tsarnaev created a YouTube channel with playlist links to two videos which were tagged under a category labeled "Terrorists", including one to Dagestani Islamic militant Amir Abu Dujana (Gadzhimurad Dolgatov, also known as 'Robin Hood', a commander of a small group in the Kizilyurt district, who was killed in battle in late December 2012); the videos were later deleted. CNN and the SITE Institute found a screen grab of one of the videos, which featured members of the militant Islamist group Caucasus Emirate from the North Caucasus. He also linked to jihadi videos on YouTube, including ones by radical cleric Feiz Mohammad; in one video, voices can be heard singing in Arabic as bombs explode. He frequently read extremist sites, including Al-Qaeda in the Arabian Peninsula's "Inspire" online magazine.


title: Tamerlan Tsarnaev

text: Tsarnaev applied for U.S. citizenship on September 5, 2012, but Homeland Security held up the application for "additional review" because they found a record of the 2011 FBI interview of him.


title: Tamerlan Tsarnaev

text: Tsarnaev and his wife were receiving state welfare benefits as late as November 2012, but not at the time of the Marathon Bombings in April 2013. His wife's lawyer said that Tsarnaev was unemployed prior to the bombing and had been helping take care of their daughter, while his wife worked over 70 hours a week as a home health care aide, to support her family.


title: Tamerlan Tsarnaev

text: Tsarnaev was pulled over by police in Boston, Brookline, and Cambridge at least nine times in four years. The source does not state which years these were exactly.


title: Tamerlan Tsarnaev

text: In November 2012, Tsarnaev reportedly confronted a shopkeeper at a Middle Eastern grocery store in Cambridge, near a mosque where he sometimes prayed, after seeing a sign there advertising Thanksgiving turkeys. He said "This is kuffar"—an Arabic reference to non-Muslims—"that's not right!". Also in November 2012, Tsarnaev stood up and challenged a sermon in which the speaker said that, just like "we all celebrate the birthday of the Prophet, we can also celebrate July 4 and Thanksgiving," according to Yusufi Vali, a mosque spokesman. Vali said Tsarnaev stated he "took offense to celebrating anything," be it the Prophet's birthday (which not all Muslims celebrate) or American holidays. In January 2013, Tsarnaev again disrupted a Martin Luther King Jr. Day sermon at a mosque in Cambridge. He objected to the speaker's comparison of Muhammad to Martin Luther King Jr. Tsarnaev was shouted down by members of the congregation and was later asked not to return to the mosque unless he was willing to refrain from shouting during sermons. The mosque said Tsarnaev had disrupted a sermon previously.


title: Tamerlan Tsarnaev

text: Tamerlan Tsarnaev, along with his brother Dzhokhar, committed the Boston Marathon bombing on April 15, 2013. After the bombing, both were involved in the murder of MIT police officer Sean Collier. In the process of escaping, the brothers committed a carjacking on April 18, 2013. Dun "Danny" Meng, the carjacking victim, was held hostage in his car until his escape at a gas station. A subsequent shootout in Watertown between the brothers and police would result in Tamerlan's death.


title: Tamerlan Tsarnaev

text: In the early hours of April 19, 2013, in Watertown, a suburb of Boston, Tsarnaev was apprehended by police after being shot multiple times. The exact sequence of events remains clouded in confusion, as do key details. According to police, Tsarnaev's younger brother ran him over with an SUV and dragged him with the vehicle for , which is substantiated by the federal indictment.


title: Tamerlan Tsarnaev

text: According to paramedic Michael Sullivan, who treated him after the shootout, Tsarnaev angrily resisted efforts by paramedics to treat him as he was being driven to the hospital, lifting himself from the stretcher and screaming loudly.


title: Tamerlan Tsarnaev

text: He was taken to Beth Israel Deaconess Medical Center, where, despite efforts to revive him by emergency medical personnel, he was pronounced dead from several critical injuries, massive blood loss, and cardiac and respiratory arrest. Emergency physicians said that he did not appear to have been run over. An eyewitness says that he was struck by a police SUV before he was shot multiple times.


title: Tamerlan Tsarnaev

text: Tsarnaev's parents continue to proclaim his innocence. His mother is quoted as saying, "America took my kids away from me. I'm sure my kids were not involved in anything."


title: Tamerlan Tsarnaev

text: The imam of a prominent Boston mosque condemned the violence and distanced himself and his mosque from the suspects, refusing to give Tsarnaev a Muslim burial. His body was released to the funeral service hired by the family at 5:30 pm. EDT May 2, 2013, by the Massachusetts Office of the Chief Medical Examiner.


title: Tamerlan Tsarnaev

text: His death certificate gives cause of death as gunshot wounds to the torso and extremities, as well as blunt trauma to the head and torso. It is confirmed that he was struck and dragged by a vehicle, in addition to being shot.


title: Tamerlan Tsarnaev

text: Tsarnaev's body was moved to a funeral home in North Attleborough; after protesters picketed the building, it was handed over to Graham, Putnam, and Mahoney Funeral Parlor in Worcester. Officials in Boston, Cambridge, at a state prison, and in over 120 other U.S. and Canadian locations refused to allow Tsarnaev's body to be buried in their jurisdictions.


title: Tamerlan Tsarnaev

text: On May 9, Worcester police announced that Tsarnaev's body had been buried in an undisclosed location. It was later reported that Tsarnaev was buried in a small Muslim cemetery, Al-Barzakh Cemetery, in Doswell, Virginia. The burial was set in motion by Martha Mullen of Richmond, Virginia, who said she was appalled by the protests at the funeral home, which she said "portrayed America at its worst" and wanted to find a way to end the impasse. She contacted Islamic Funeral Services of Virginia, which agreed to provide an unmarked plot in their cemetery. The funeral agency released a statement saying "What Tsarnaev did is between him and God. We strongly disagree with his violent actions, but that does not release us from our obligation to return his body to the earth." Caroline County Sheriff Tony Lippa said the burial was legal. Locals, as well as the imam of the Virginia Islamic Centre, condemned the secretive burial.


title: Tamerlan Tsarnaev

text: On June 19, 2013, Tsarnaev's name was read aloud (in the context of a victim of gun violence) during a "No More Names" event held in Concord, New Hampshire. In response, Michael Bloomberg's Mayors Against Illegal Guns issued a statement explaining that they were using a list compiled by "Slate", and apologized saying that his name was "a mistake" and should have been removed.


title: Tamerlan Tsarnaev

text: Ruslan Tsarni is the paternal uncle of Tamerlan Tsarnaev, and the brother of Anzor Tsarnaev. He was trained as a lawyer, and moved to Washington State in 1995. He returned to Kyrgyzstan by the end of the decade, and then returned to the United States, settling in Montgomery County, Maryland.


title: Tamerlan Tsarnaev

text: During the manhunt for the brothers, he was interviewed by the FBI. When the media arrived at his home, he denounced the actions of his nephews and called on them to turn themselves in. He also buried the remains of Tsarnaev.


title: Tamerlan Tsarnaev

text: Zubeidat Tsarnaeva is the mother of Tsarnaev and his brother. In photos of her as a younger woman, she wore western-style clothing. After she arrived in the U.S. from Russia in 2002, she took classes at the Catherine Hinds Institute of Esthetics before becoming a state-licensed aesthetician and getting a job at a suburban day spa. After deciding she could no longer work in a business that served men, she started working from home, where clients saw her become more radical and promote 9/11 conspiracy theories.


title: Tamerlan Tsarnaev

text: Tsarnaev's mother has been quoted as saying she urged him to embrace Islam in 2008 because she was concerned about his drinking, smoking, and pursuit of women. She said he began to read more about it on the Internet. She also urged him to quit boxing because Islam prohibits hitting someone in the face. She also praised Russell, saying, "She is a serious, good, American girl who converted to Islam as if she had always been a Muslim. We all love her a lot."


title: Tamerlan Tsarnaev

text: Tsarnaev's mother discussed "jihad" during a 2011 phone call with him that was taped by a Russian government agency, and intelligence officials also discovered text messages in which she discussed how Tsarnaev was ready to die for Islam. His mother was also recorded suggesting that Tsarnaev go to Palestine.


title: Tamerlan Tsarnaev

text: Both Tsarnaev and his mother were the subject of a Russian Intelligence inquiry to the U.S. government in 2011 because of what the Russians perceived as extremist Islamic views. She was interviewed by the FBI who found nothing to pursue at the time. In late 2011, the CIA put both Tsarnaev and his mother in its Terrorist Identities Datamart Environment database.


title: Tamerlan Tsarnaev

text: Ruslan Tsarni told the AP from his home in Maryland that he believed his former sister-in-law had a "big-time influence" on Tsarnaev's growing embrace of his Muslim faith and decision to quit boxing and school.


title: Tamerlan Tsarnaev

text: In early 2012, Zubeidat Tsarnaeva was arrested for shoplifting $1,624 worth of women's clothing from Lord and Taylor in Natick, Massachusetts. She left the U.S. for Russia and did not appear in court. Anzor and Zubeidat Tsarnaev divorced in 2011 after twenty-five years of marriage. The couple had no personal property or real estate to divide and listed no retirement or pension benefits. They gave the reason for their split as "irretrievable breakdown of the marriage" with "no chance of reconciling our differences". The mother's move toward more Islamic extremism was reportedly a factor in the breakdown of the marriage. They may have reconciled in Dagestan.


title: Tamerlan Tsarnaev

text: She has strongly expressed in TV interviews that her sons are innocent and that they were framed by the FBI.


title: Tamerlan Tsarnaev

text: Tamerlan Tsarnaev's widow, Katherine Russell (a.k.a. Karima Tsarnaeva or Katherine Tsarnaeva), was born on February 6, 1989, in Texas. She was raised in Rhode Island; her father is an emergency room doctor and her mother is a nurse. Their home has been described as nominally Christian and Russell reportedly was not religious "at all" in high school. She attended North Kingstown High School, and graduated in 2007. Her yearbook entry lists her plans as college and the Peace Corps. She was remembered for her talent in painting and drawing.


title: Tamerlan Tsarnaev

text: Tsarnaev and Russell met in 2007 in a nightclub, soon after she started as a communications major at Suffolk University. They started dating on and off, and at one point in 2009, Tsarnaev was living with another woman. Tamerlan was known to cheat on Russell, and a friend of Russell's told her mother that the relationship was abusive. Russell's mother did not like Tamerlan from the first time she met him. At Tsarnaev's insistence, Russell converted to Islam in 2008, adopted the hijab, and chose the name Karima after her conversion.


title: Tamerlan Tsarnaev

text: Russell dropped out of college in the Spring of 2010 after she became pregnant in her junior year, and the couple married on June 21, 2010, in a 15-minute ceremony in a Dorchester mosque. According to the officiant, it was Russell who called and made the arrangements. Only two witnesses attended the wedding.


title: Tamerlan Tsarnaev

text: She moved into her husband's apartment in Cambridge and gave birth to their daughter Zahara in late 2010. At times, she worked as a home health aide. From September 2011 to November 2012, she and her husband had their income supplemented by public assistance and food stamps. When Tsarnaev was in Russia for six months, she and their daughter stayed in Cambridge.


title: Tamerlan Tsarnaev

text: At the time of the bombings on April 15, 2013, Russell was living with her husband and daughter in the Norfolk Street family home in Cambridge. The younger brother also officially lived there, but in practice stayed in a dorm at UMass Dartmouth.


title: Tamerlan Tsarnaev

text: After her husband died, Russell retreated to her parents' home in Rhode Island. Her parents released a statement saying "[o]ur daughter has lost her husband today, the father of her child. We cannot begin to comprehend how this horrible tragedy occurred. In the aftermath of the Patriots' Day horror, we know that we never really knew Tamerlan Tsarnaev. Our hearts are sickened by the knowledge of the horror he has inflicted." She refused to take custody of her husband's remains and has reverted to using her maiden name.


title: Tamerlan Tsarnaev

text: Investigators found bomb-making instructions, downloaded from "Inspire" magazine, on Russell's computer. Dzhokhar Tsarnaev told the FBI that he and his brother had learned to make bombs by reading "Inspire", but it was not clear who downloaded the files. Her web history included searches for "If your husband becomes a shahid, what are the rewards for you?" and "the rewards for the wife of Mujahedeen." The FBI collected Russell's DNA after female DNA was found on bomb fragments; neither her DNA nor her fingerprints matched those on the bombs.


title: Tamerlan Tsarnaev

text: No charges have been filed against Russell and "there’s been no suggestion at all by the government that she’s going to be indicted," according to her lawyer, Amato DeLuca. He insists that Russell was not aware of her husband's criminal activity, and says she has since "provided them [the FBI] with a lot of information, many, many, many many hours. I don’t know what else she could have done."


title: Action role-playing game

text: An action role-playing game (often abbreviated action RPG or ARPG) is a subgenre of video games that combines core elements from both the action game and role-playing genre.


title: Action role-playing game

text: The games emphasize real-time combat where the player has direct control over the characters as opposed to turn or menu-based combat while still having a focus on character's Stats in order to determine relative strength and abilities. These games often use action game combat systems similar to hack and slash or shooter games. Action role-playing games may also incorporate action-adventure games, which include a mission system and role-playing game mechanics, or MMORPGs with real-time combat systems.


title: Action role-playing game

text: Allgame listed the following games released prior to 1984 as action RPGs: "Temple of Apshai" (1979) and its sequel "Gateway to Apshai" (1983), "Beneath the Pyramids" for the Apple II (1980), "Bokosuka Wars" (1983), and "Sword of Fargoal" (1983). Jeremy Parish of USgamer claimed that "Adventure" (1980) was an action RPG. Bill Loguidice and Matt Barton claimed that the Intellivision games "" (1982) and "Treasure of Tarmin" (1983) were action RPGs. Shaun Musgrave of TouchArcade notes that "Adventure" lacked RPG mechanics such as experience points and permanent character growth, and argues that "Gateway to Apshai" is "the earliest game I'd feel comfortable calling an action-RPG" but notes that "it doesn't fit neatly into our modern genre classifications", though came closer than "Bokosuka Wars" released the same year.


title: Action role-playing game

text: Jeremy Parish of 1UP.com argues that Japanese developers created a new brand of action role-playing game; these new Japanese games combined the role-playing genre with arcade-style action and action-adventure elements. Shaun Musgrave of TouchArcade also traces the genre's roots to Japan, noting that the "Western game industry of the time had a tendency to treat action games and RPGs as separate things for separate demographics".


title: Action role-playing game

text: Jeremy Parish argues that action RPGs were popularized in Japan by "The Tower of Druaga". It was released for arcades in June 1984, and was intended as a "fantasy version of "Pac-Man", with puzzles to solve, monsters to battle, and hidden treasure to find". Its success in Japan inspired the development of "Dragon Slayer" (1984) and "Hydlide" (1984). "Dragon Slayer", "Hydlide" and "Courageous Perseus" (1984) "vie for position as genre precedent" according to John Szczepaniak, and there was an ongoing rivalry developing between the "Dragon Slayer" and "Hydlide" series over the years. "The Tower of Druaga", "Dragon Slayer" and "Hydlide" were influential in Japan, where they influenced later action RPGs such as "Ys", as well as "The Legend of Zelda".


title: Action role-playing game

text: Falcom's "Dragon Slayer", created by Yoshio Kiya, is "the very first action-RPG ever made" according to GameSetWatch. Originally released for the PC-8801 computer in September 1984, it abandoned the command-based battles of earlier role-playing games in favor of real-time hack-and-slash combat that required direct input from the player, alongside puzzle-solving elements. In contrast to earlier turn-based roguelikes, "Dragon Slayer" was a dungeon-crawl role-playing game using real-time, action-oriented combat, combined with traditional role-playing mechanics. "Dragon Slayer's" overhead action role-playing formula was used in many later games.


title: Action role-playing game

text: T&E Soft's "Hydlide", released in December 1984, was created by Tokihiro Naito, who was influenced by "The Tower of Druaga". It was the first action RPG with an overworld. The game was immensely popular in Japan, selling 2 million copies across all platforms. According to John Szczepaniak, it "cannot be overstated how influential Hydlide was on the ARPGs which followed it". The same year, "Courageous Perseus" was also one of the earliest action RPGs.


title: Action role-playing game

text: "", released in 1985 (billed as a "new type of real-time role-playing game"), was an action role-playing game including many character stats and a large quest. It also incorporated a side-scrolling view during exploration and an overhead view during battle, and an early "Karma" morality system where the character's Karma meter will rise if he commits sin (killing "good" enemies), which in turn causes the temples to refuse to level him up. "Xanadu Scenario II", released in 1986, was an expansion pack, created to expand the content of "Dragon Slayer II: Xanadu". "" (1985) also featured a morality system. Eurogamer cites "Fairlight" (1985) as an early action RPG.


title: Action role-playing game

text: An important influence on the action RPG genre was the 1986 action-adventure "The Legend of Zelda", which served as the template for many future action RPGs. In contrast to previous action RPGs, such as "Dragon Slayer" and "Hydlide," which required the player to bump into enemies in order to attack them, "The Legend of Zelda" featured an attack button that animates a sword swing or projectile attack on the screen. It was also an early example of open-world, nonlinear gameplay, and introduced new features such as battery backup saving. These elements have been used in many action RPGs since.


title: Action role-playing game

text: In 1987, "" implemented a more traditional RPG-esque system, including experience points and levels with action game elements. Unlike its predecessor, "Zelda II" more closely fits the definition of an action RPG.


title: Action role-playing game

text: Another "Metroidvania"-style action RPG released that year was System Sacom's Sharp X1 computer game "Euphory", which was possibly the only "Metroidvania"-style multiplayer action RPG produced, allowing two-player cooperative gameplay. The fifth "Dragon Slayer" title, "Sorcerian", was also released that year. It was a party-based action RPG, with the player controlling a party of four characters at the same time in a side-scrolling view. The game also featured character creation, highly customizable characters, class-based puzzles, and a new scenario system, allowing players to choose from 15 scenarios, or quests, to play through in the order of their choice. It was also an episodic video game, with expansion disks later released offering more scenarios. Falcom also released the of its "Ys" series in 1987. While not very popular in the West, the long-running "Ys" series has performed strongly in the Japanese market, with many sequels, remakes and ports in the decades that followed its release. Besides Falcom's own "Dragon Slayer" series, "Ys" was also influenced by "Hydlide", from which it borrowed certain mechanics such as health-regeneration.


title: Action role-playing game

text: "The Faery Tale Adventure" offered one of the largest worlds at the time, with over 17,000 computer screens without loading times.


title: Action role-playing game

text: In 1988, Telenet Japan's "Exile" series debuted, and was controversial due to its plot, which revolves around a time-traveling Crusades-era Syrian assassin who assassinates various religious/historical figures as well as 20th-century political leaders, The gameplay of "Exile" included both overhead exploration and side-scrolling combat, and featured a heart monitor to represent the player's Attack Power and Armor Class statistics. Another controversial aspect of the game involved taking drugs (instead of potions) that increase/decrease attributes, but with side effects such as heart-rate increase/decrease or death. Origin Systems, the developer of the "Ultima" series, also released an action RPG in 1988, titled "Times of Lore", which was inspired by various NES titles, particularly "The Legend of Zelda". "Times of Lore" inspired several later titles by Origin Systems, such as the 1990 games "Bad Blood" (another action RPG based on the same engine) and "", based on the same interface.


title: Action role-playing game

text: Also in 1989, the enhanced remake "Ys I & II" was one of the first video games to use CD-ROM, which was utilized to provide enhanced graphics, animated cut scenes, a Red Book CD soundtrack, and voice acting. Its English localization was also one of the first to use voice dubbing. The game received the Game of the Year award from "OMNI Magazine" in 1990, as well as other prizes. Another 1989 release, Activision's "", attempted to introduce "Nintendo-style" action combat to North American computer role-playing games.


title: Action role-playing game

text: Action RPGs were far more common on consoles than computers, due to gamepads being better suited to real-time action than the keyboard and mouse. Though there were attempts at creating action-oriented computer RPGs during the late 1980s and early 1990s, very few saw any success. "Times of Lore" was one of the more successful attempts in the American computer market, where there was a generally negative attitude towards combining genres in this way and more of an emphasis on preserving the purity of the RPG genre. For example, a 1991 issue of "Computer Gaming World" criticized several computer role-playing games for using "arcade" or "Nintendo-style" action combat, including "Ys", "Sorcerian," "Times of Lore," and "Prophecy".


title: Action role-playing game

text: In 1991, Square released "Seiken Densetsu: Final Fantasy Gaiden", also known as "Final Fantasy Adventure" or "Mystic Quest" in the West, for the Game Boy. Like "Crystalis", the action in "Seiken Densetsu" bore a strong resemblance to that of "Legend of Zelda", but added more RPG elements. It was one of the first action RPGs to allow players to kill townspeople, though later "Mana" games removed this feature. "Arcus Odyssey" by Wolf Team (now Namco Tales Studio) was an action RPG that featured an isometric perspective and co-operative multiplayer gameplay.


title: Action role-playing game

text: In 1993, the second "Seiken Densetsu" game, "Secret of Mana", received considerable acclaim, for its innovative pausable real-time action battle system, and its innovative cooperative multiplayer gameplay, where the second or third players could drop in and out of the game at any time, rather than players having to join the game at the same time. The game has remained influential through to the present day, with its ring menu system still used in modern games and its cooperative multiplayer mentioned as an influence on games such as "Dungeon Siege III" (2011).


title: Action role-playing game

text: Most other such games, however, used a side-scrolling perspective typical of beat 'em ups, such as the "Princess Crown" series, including "Odin Sphere" and "". Princess Crown had a more cartoon-like visual appeal. It still had quality visuals due to the George Kamitani style.


title: Action role-playing game

text: "LandStalker's" 1997 spiritual successor "Alundra" is considered "one of the finest examples of action/RPG gaming", combining platforming elements and challenging puzzles with an innovative storyline revolving around entering people's dreams and dealing with mature themes.


title: Action role-playing game

text: "Ultima Underworld"'s influence has been found in "BioShock" (2007), and that game's designer, Ken Levine, has stated that "all the things that I wanted to do and all the games that I ended up working on came out of the inspiration I took from ["Ultima Underworld"]". "Gears of War" designer Cliff Bleszinski also cited it as an early influence, stating that it had "far more impact on me than "Doom"". Other games influenced by "Ultima Underworld" include "", "Deus Ex", "", "", and "Half-Life 2".


title: Action role-playing game

text: FromSoftware's "Demon's Souls" (2009) emphasized unforgiving enemies and environments, combined with risk-and-reward mechanics such as limited checkpoints, collecting "souls" that can be consumed as experience points to increase the player's stats, or as a currency to purchase items, and penalizing player deaths without imposing an outright failure state. It also incorporated online features allowing players to leave messages in the overworld that can be read by other players, to temporarily join other players' sessions to assist them cooperatively, or "invade" another player's session to engage in player versus player combat. Especially after the release of its spiritual successor "Dark Souls" (2011) and its sequels, other action RPGs emerged in the 2010s that incorporated mechanics influenced by those of "Demon's Souls", which have been popularly referred to as "Soulslike" games.


title: Action role-playing game

text: In 2013, "Vanillaware" released the fantasy beat 'em up ARPG "Dragon's Crown", a spiritual successor to "Princess Crown" and a "deeply moving product" of Vanillaware director George Kamitani. Kamitani cites many classic RPGs as his inspiration, stating in the "Dragon's Crown Artworks" foreword: "The motif within Dragon's Crown is all the fantasy works that has affected me until now: the PC RPG Wizardry that I first came into contact with as a student; Ian Livingstone's gamebooks; games like "Tower of Druaga", "Golden Axe" and "The King of Dragons"." He also cites his early 20s work on "" as "truly something that I had aspired for". "Dragon's Crown" was re-released with a PS4 "Pro" edition in 2018.


title: Action role-playing game

text: "Assassin's Creed," a long-running Ubisoft franchise, also shifted towards the action RPG formula, inspired by the successes of "The Witcher 3" and the "Dark Souls" series, with its titles "Origins" (2017), "Odyssey" (2018) and "Valhalla" (2020).


title: Action role-playing game

text: In late 1987, FTL Games released "Dungeon Master", a dungeon crawler that had a real-time game world and some real-time combat elements (akin to Active Time Battle), requiring players to quickly issue orders to the characters, setting the standard for first-person computer RPGs for several years. It inspired many other developers to make real-time dungeon crawlers, such as "Eye of the Beholder" and "".


title: Action role-playing game

text: "", released in 1992, has been cited as the first RPG to feature first-person action in a 3D environment. "Ultima Underworld" is considered the first example of an immersive sim, a genre that combines elements from other genres to create a game with strong player agency and emergent gameplay, and has influenced many games since its release. The engine was re-used and enhanced for "Ultima Underworld"s 1993 sequel, "". Looking Glass Studios planned to create a third "Ultima Underworld", but Origin rejected their pitches. After Electronic Arts (EA) rejected Arkane Studios' pitch for "Ultima Underworld III", the studio instead created a spiritual successor: "Arx Fatalis". Toby Gard stated that, when designing "Tomb Raider", he "was a big fan of ... "Ultima Underworld" and I wanted to mix that type of game with the sort of polygon characters that were just being showcased in "Virtua Fighter"". "Ultima Underworld" was also the basis for Looking Glass Technologies' later "System Shock".


title: Action role-playing game

text: The 1988 Origin Systems title "Times of Lore" was an action RPG with an icon-based point-and-click interface. "Bad Blood", another Origin Systems game from 1990, would use the same interface. The designers were inspired by console titles, particularly "The Legend of Zelda", to make their interface more accessible. The 1994 title "Ultima VIII" used mouse controls and attempted to add precision jumping sequences reminiscent of a "Mario" platform game, though reactions to the game's mouse-based combat were mixed. In 1997 Blizzard's "Diablo" was released and became massively successful. It was an action RPG that used a mouse oriented point-and-click interface and offered gamers a free online service to play with others that maintained the same rules and gameplay.


title: Action role-playing game

text: "Diablo"s effect on the market was significant, inspiring many imitators. Its impact was such that the term "action RPG" has come to be more commonly used for "Diablo"-style games, with "The Legend of Zelda" itself slowly recategorized as an action-adventure. Very commonly, these games used a fixed-camera isometric view of the game world, a necessity of the limitations of 2D graphics of early computers; even with 3D graphic engines, such point-and-click games are still presented from a similar isometric view, though providing options to rotate, pan, and zoom the camera to some degree. As such, these are often grouped with other "isometric RPGs".


title: Action role-playing game

text: The popularity of The Diablo series spawned such franchises like "Divinity", "Torchlight", "Dungeon Siege" and "Sacred". Commonly, these games used a fixed-camera isometric view of the game world, a necessity of the limitations of 2D graphics of early computers; The Diablo series spawned many terms like being referred to as "dungeon crawler" "slasher RPG" "hack and slasher", the series was also heavily criticized by players and media for not being a proper RPG due to it being focused more on fighting enemies and creating character builds than following a proper narrative and dialogue-heavy journey. After its success many other games tried to mix its influences with different structures and narratives, there are multiple games like "Divine Divinity" that were an attempt to have a more dialogue-heavy experience akin to the "Baldurs Gate" games and even older series like Falcom's "Dragon Slayer/Xanadu" series had its outing "Xanadu Next" with similar Diablo influences. The influences also come full circle when the first Diablo game was inspired by rogue-likes Umoria and Angband and in more recent years many games in the rogue-like genre are inspired by more classic dungeon crawler ARPGs that Diablo helped spawn.


title: Action role-playing game

text: In this sub genre there are such recent titles as Grim Dawn (2016), "Zenonia S: Rifts In Time" (2015), "Book of Demons" (2018), "Shadows: Awakening" (2018), "Snack World: The Dungeon Crawl Gold" (2017), "Titan Quest: Anniversary Edition" (2016) and its expansions "Titan Quest: Ragnarök" (2017) and "Titan Quest: Atlantis" (2019), "" (2020) and "Minecraft Dungeons" (2020).


title: Action role-playing game

text: The prominence of Diablo 2 in the gaming market and its influence on the MMORPG genre later popularized the strongly used mouse-oriented point and click combat. While in the Diablo series this type of combat does not have a lock-on key, World of Warcraft and most MMO games uses some kind of key to target an enemy, usually TAB, to lock into it, usually referred to as "tab-target". In tab-target combat the player's character automatic do attack animations with some kind of regular attack, while the player can focus on activating other skills and items by pressing other keys. Usually this type of combat is not heavily based on aiming or hit boxes thus the player can hit enemies from different distances and even from a far. Some tab-target MMOs have other targeting options such as an "Action mode".


title: Action role-playing game

text: Shooter-based action RPGs include "Strife" (1996), "System Shock 2" (1999), the "Deus Ex" series (2000 onwards) by Ion Storm, Bungie's "Destiny" (2014), Irem's "Steambot Chronicles" (2005), Square Enix's third-person shooter RPG "" (2006), which introduced an over-the-shoulder perspective similar to "Resident Evil 4", and the MMO vehicular combat game "Auto Assault" (2006) by NetDevil and NCsoft. Other action RPGs featured both hack and slash and shooting elements, with the use of both guns (or in some cases, bow and arrow or aerial combat) and melee weapons, including Cavia's flight-based "Drakengard" series (2003 to 2005), and Level-5's "Rogue Galaxy" (2005).


title: Action role-playing game

text: Other RPS games include the "Mass Effect" series (2007 onwards), "Fallout 3" and subsequent "Fallout" titles (2008 onwards), "" (2008), and "Borderlands" (2009). "Borderlands" developer Gearbox Software has dubbed it as a "role-playing shooter" due to the heavy RPG elements within the game, such as quest-based gameplay and also its character traits and leveling system. "Half-Minute Hero" (2009) is an RPG shooter featuring self-referential humour and a 30-second time limit for each level and boss encounter. Other action role-playing games with shooter elements include the 2010 titles "Alpha Protocol" by Obsidian Entertainment and "The 3rd Birthday", the third game in the "Parasite Eve" series, features a unique blend of action RPG, real-time tactical RPG, survival horror and third-person tactical shooter elements. Shooter-based RPGs include Imageepoch's post-apocalyptic "Black Rock Shooter" (2011)"," which employs both first-person and third-person shooter elements, and Square Enix's "Final Fantasy XV" (2016)"," which features both hack and slash and third-person shooter elements.


title: Action role-playing game

text: The online live service version gained a lot of popularity on the 2010s with titles such as "Warframe" (2013), "Destiny" (2014) and "Destiny 2" (2017), "Anthem" (2019), "The Division" (2016) and "The Division 2" (2019).


title: Paranormal Activity: Next of Kin

text: Paranormal Activity: Next of Kin is a 2021 American found footage supernatural horror film directed by William Eubank, written by Christopher Landon, and produced by Jason Blum and Oren Peli. Serving as the seventh installment of the "Paranormal Activity" series, the film stars Emily Bader, Roland Buck III, Dan Lippert, Henry Ayres-Brown, and Tom Nowicki, and follows a group who attempt to make a documentary on an Amish community, only for them to discover the horrific secrets the town holds.


title: Paranormal Activity: Next of Kin

text: Although "" was promoted as the final installment in the original series, Paramount Pictures announced in June 2019 that a seventh installment and stand-alone sequel was in development, with Blum and franchise creator Peli. Landon was hired to write the script in early 2020, while Eubank was put as film's director in February 2021. Principal photography was wrapped by July 2021.


title: Paranormal Activity: Next of Kin

text: "Paranormal Activity: Next of Kin" was released in the United States on Paramount+ on October 29, 2021. It received negative reviews from critics, with a further sequel, subtitled "The Other Side", in active development.


title: Paranormal Activity: Next of Kin

text: Margot and her cameraman friend Chris want to make a documentary about Margot's past. Her mother, Sarah, abandoned her outside a hospital, and she wonders what drove Sarah to make such a drastic decision. Through a genetics site, she and Chris meet her blood relative Samuel, who is an Amish currently going through his rumspringa. After the three fly to Buffalo, NY, they are joined by Dale, their documentary's soundman. Samuel leads them to Beiler Farm, where he and Margot's mother came from.


title: Paranormal Activity: Next of Kin

text: Jacob, the patriarch of the commune and Sarah's father, welcomes the team, and they find lodging in a rustic room. Late at night, Margot wakes up and discovers red lights moving in the distance; Samuel dismisses it as a hunting party. Margot visits the barn and finds a little girl brushing her doll's hair. The doll is named Sarah, and when Margot tells the girl that that was her mother's name, the girl cryptically says that Sarah is "still there." Come night time, Margot hears sounds from her mother's old room in the attic and sees a spirit.


title: Paranormal Activity: Next of Kin

text: The next morning, they interview Jacob on camera. He tells them about the free-spirited Sarah, who defied their practices by sleeping with a boy from the neighboring village and getting pregnant. Instead of following custom by being forced to give up Margot to an adoptive family, she instead left her at a hospital, and Sarah is now assumed dead. Through drone footage, Margot's team later finds a church which holds a warning to stay away, and Jacob quickly arrives to prevent them from entering. That night, Margot and the team discover a strange ritual in the barn in which a newborn two-headed goat is sacrificed.


title: Paranormal Activity: Next of Kin

text: Determined to find out the commune's secrets, Margot and Chris break into the church while Dale distracts Samuel. They discover murals depicting the demonic figure Asmodeus, as well as a deep tunnel which Margot descends into before retreating when she hears a strange noise. Margot sneaks into Jacob's room the next day and finds his computer, where she discovers that he and Samuel had known about her being Sarah's daughter and orchestrated her arrival. She becomes upset and Chris suggests that they leave, but a shadow-like ghoulish being attacks Margot that night. The next day, Chris and Dale find a catatonic Margot with her sheets soaked with blood; despite assurance from the local doctor, the two become suspicious.


title: Paranormal Activity: Next of Kin

text: Chris and Dale hitch a ride into town with the mailman to get a new car battery, and are informed by the mailman that the people at Beiler Farm are not Amish. Using the internet, the two discover that Beiler Farm is a community of demon worshippers. According to myth, the Norwegian village of Beskytter suffered a massacre believed to be caused by Asmodeus. They trapped the demon inside the body of a woman, and the demon continues to be passed from mother to daughter in the bloodline. Chris and Dale realize that Margot is next in line and this is why she was brought to the settlement.


title: Paranormal Activity: Next of Kin

text: When Chris and Dale return from their trip to the store, Margot has disappeared. While Dale leaves to install the battery, Chris enters the church to search for Margot. After encountering and killing Jacob, Chris finds Margot at the bottom of the pit and rescues her, but they are pursued by a skeletal creature as they flee the church. The creature kills Dale and pursues Margot and Chris in the barn, but Margot, realizing that the creature is Sarah possessed by Asmodeus, calls her by her name, which causes the creature to relent. Taking advantage of her mother's weakened state, Margot then pushes Sarah, sending her falling to her death onto a spiked row of farm tools.


title: Paranormal Activity: Next of Kin

text: With the death of Sarah, Asmodeus wreaks havoc in Beiler Farm, with the residents killing each other and the livestock and the homes burning. Margot and Chris retrieve the car keys from Dale's body and flee the farm. Later, police officers arrive on the scene and are attracted to a child's crying in the barn. Upon investigating, they realize the noises are caused by the demonically-possessed Samuel. Using his demonic possession, Samuel forces the police officers to die by suicide before driving away in a police cruiser.


title: Paranormal Activity: Next of Kin

text: On June 19, 2019, Paramount Pictures announced that a seventh installment of the film series was in development, with Jason Blum and franchise creator Oren Peli. In February 2020, Blum revealed that Christopher Landon was writing the script. On February 12, 2021, it was announced that William Eubank was attached to direct the film, with the film also confirmed to be a reboot rather than a continuation of the previous films. On February 14, 2021, the film was announced to be in production with "The In Between" and an untitled "Pet Sematary" film for Paramount+. In March 2021, Emily Bader, Roland Buck III, Dan Lippert, and Henry Ayres-Brown were cast in undisclosed roles. By October 2021, Tom Nowicki was revealed as part of the cast.


title: Paranormal Activity: Next of Kin

text: Principal photography concluded in July 2021. In September 2021, the film's title was officially revealed to be "Paranormal Activity: Next of Kin".


title: Paranormal Activity: Next of Kin

text: "Paranormal Activity: Next of Kin" was released in the United States on Paramount+ on October 29, 2021. The film was originally set for a theatrical release on March 19, 2021, but was delayed to March 4, 2022, due to the COVID-19 pandemic. Instead of the theatrical release, the film was released exclusively on Paramount+. ViacomCBS CEO Robert Bakish said the film would premiere before the end of 2021. The film's release was officially moved up to October 29, 2021.


title: Paranormal Activity: Next of Kin

text: On Rotten Tomatoes, the film has an approval rating of 30% based on 46 reviews from critics and an average rating of 4.80/10. The website's critical consensus reads: ""Paranormal Activity: Next of Kin" gives the long-running found-footage franchise a new level of visual polish; unfortunately, effective scares are few and far between". On Metacritic, the film has a score of 37 out of 100 based on reviews from 13 critics, indicating "generally unfavorable reviews".


title: Paranormal Activity: Next of Kin

text: On October 30, 2021, the day after "Next of Kin"s release to the Paramount+ streaming service, "Screen Rant" confirmed that an eighth film had been greenlit for an October 2022 release on the service, tentatively entitled "Paranormal Activity: The Other Side". Featuring a script written by franchise creator Oren Peli, his first since writing-directing the original 2007 "Paranormal Activity" film, the film would be directed by Wincent Sommers, and star Sonja Gerhardt, Chethrin Schulze, Nika Sofie, Tamara Hitz, Chany Dakota, and Leon Pelz. After then announcing the film would feature a "return to Katie Featherston's story with Tob[i]" the following November, by January 2022, the film was specified by "The 25th Frame" to be set for an October 14, 2022 release date; however, on February 28, 2022, British listings service "Radio Times" reported that the film (described as an action-horror, roughly 89 minutes long) had been delayed to an unspecified 2023 release date. "The Other Side" will feature four young friends who move into Katie and Kristi's childhood house together before college starts, only to discover the gruesome past of their home and its connection to the 1988 incidents. Set in the 1990s, it will reportedly address the fire that destroyed the property which has only been mentioned briefly in past installments.


title: High-performance liquid chromatography

text: 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 each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column.


title: High-performance liquid chromatography

text: HPLC has been used for manufacturing ("e.g.", during the production process of pharmaceutical and biological products), legal ("e.g.", detecting performance enhancement drugs in urine), 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.


title: High-performance liquid chromatography

text: Chromatography can be described as a mass transfer process involving adsorption. 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.), 2–50 μm in size. 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, 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. These interactions are physical in nature, such as hydrophobic (dispersive), dipole–dipole and ionic, most often a combination.


title: High-performance liquid chromatography

text: HPLC is distinguished from traditional ("low pressure") liquid chromatography because operational pressures are significantly higher (50–350 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. Due to the small sample amount separated in analytical HPLC, typical column dimensions are 2.1–4.6 mm diameter, and 30–250 mm length. Also HPLC columns are made with smaller adsorbent particles (2–50 μm in average particle size). This gives HPLC superior resolving power (the ability to distinguish between compounds) when separating mixtures, which makes it a popular chromatographic technique.


title: High-performance liquid chromatography

text: The schematic of an HPLC instrument typically includes a degasser, sampler, pumps, and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the desired flow and composition of the mobile phase through the column. The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample components. A digital microprocessor and user software control the HPLC instrument and provide data analysis. Some models of mechanical pumps in an HPLC instrument can mix multiple solvents together in ratios changing in time, generating a composition gradient in the mobile phase. Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or based on mass spectrometry. Most HPLC instruments also have a column oven that allows for adjusting the temperature at which the separation is performed.


title: High-performance liquid chromatography

text: The sample mixture to be separated and analyzed is introduced, in a discrete small volume (typically microliters), into the stream of mobile phase percolating through the column. The components of the sample move through the column at different velocities, which are a function of specific physical interactions with the adsorbent (also called stationary phase). The velocity of each component depends on its chemical nature, on the nature of the stationary phase (column) and on the composition of the mobile phase. The time at which a specific analyte elutes (emerges from the column) is called its retention time. The retention time measured under particular conditions is an identifying characteristic of a given analyte.


title: High-performance liquid chromatography

text: Many different types of columns are available, filled with adsorbents varying in particle size, porosity, and surface chemistry. The use of smaller particle size packing materials requires the use of higher operational pressure ("backpressure") and typically improves chromatographic resolution (the degree of peak separation between consecutive analytes emerging from the column). Sorbent particles may be hydrophobic or polar in nature.


title: High-performance liquid chromatography

text: Common mobile phases used include any miscible combination of water with various organic solvents (the most common are acetonitrile and methanol). Some HPLC techniques use water-free mobile phases (see normal-phase chromatography below). The aqueous component of the mobile phase may contain acids (such as formic, phosphoric or trifluoroacetic acid) or salts to assist in the separation of the sample components. The composition of the mobile phase may be kept constant ("isocratic elution mode") or varied ("gradient elution mode") during the chromatographic analysis. Isocratic elution is typically effective in the separation of sample components that are very different in their affinity for the stationary phase. In gradient elution the composition of the mobile phase is varied typically from low to high eluting strength. The eluting strength of the mobile phase is reflected by analyte retention times with high eluting strength producing fast elution (=short retention times). A typical gradient profile in reversed phase chromatography might start at 5% acetonitrile (in water or aqueous buffer) and progress linearly to 95% acetonitrile over 5–25 minutes. Periods of constant mobile phase composition may be part of any gradient profile. For example, the mobile phase composition may be kept constant at 5% acetonitrile for 1–3 min, followed by a linear change up to 95% acetonitrile.


title: High-performance liquid chromatography

text: The chosen composition of the mobile phase depends on the intensity of interactions between various sample components ("analytes") and stationary phase ("e.g.", hydrophobic interactions in reversed-phase HPLC). Depending on their affinity for the stationary and mobile phases, analytes partition between the two during the separation process taking place in the column. This partitioning process is similar to that which occurs during a liquid–liquid extraction but is continuous, not step-wise. In this example, using a water/acetonitrile gradient, more hydrophobic components will elute (come off the column) late, once the mobile phase gets more concentrated in acetonitrile ("i.e.", in a mobile phase of higher eluting strength).


title: High-performance liquid chromatography

text: The choice of mobile phase components, additives (such as salts or acids) and gradient conditions depends on the nature of the column and sample components. Often a series of trial runs is performed with the sample in order to find the HPLC method which gives adequate separation.


title: High-performance liquid chromatography

text: Prior to HPLC scientists used standard liquid chromatographic techniques. Liquid chromatographic systems were largely inefficient due to the flow rate of solvents being dependent on gravity. Separations took many hours, and sometimes days to complete. Gas chromatography (GC) at the time was more powerful than liquid chromatography (LC), however, it was believed that gas phase separation and analysis of very polar high molecular weight biopolymers was impossible. GC was ineffective for many biochemists because of the thermal instability of the solutes. As a result, alternative methods were hypothesized which would soon result in the development of HPLC.


title: High-performance liquid chromatography

text: Following on the seminal work of Martin and Synge in 1941, it was predicted by Calvin Giddings, Josef Huber, and others in the 1960s that LC could be operated in the high-efficiency mode by reducing the packing-particle diameter substantially below the typical LC (and GC) level of 150 μm and using pressure to increase the mobile phase velocity. These predictions underwent extensive experimentation and refinement throughout the 60s into the 70s. Early developmental research began to improve LC particles, and the invention of Zipax, a superficially porous particle, was promising for HPLC technology.


title: High-performance liquid chromatography

text: The 1970s brought about many developments in hardware and instrumentation. Researchers began using pumps and injectors to make a rudimentary design of an HPLC system. Gas amplifier pumps were ideal because they operated at constant pressure and did not require leak-free seals or check valves for steady flow and good quantitation. Hardware milestones were made at Dupont IPD (Industrial Polymers Division) such as a low-dwell-volume gradient device being utilized as well as replacing the septum injector with a loop injection valve.


title: High-performance liquid chromatography

text: While instrumentational developments were important, the history of HPLC is primarily about the history and evolution of particle technology. After the introduction of porous layer particles, there has been a steady trend to reduced particle size to improve efficiency. However, by decreasing particle size, new problems arose. The practical disadvantages stem from the excessive pressure drop needed to force mobile fluid through the column and the difficulty of preparing a uniform packing of extremely fine materials. Every time particle size is reduced significantly, another round of instrument development usually must occur to handle the pressure.


title: High-performance liquid chromatography

text: Partition chromatography was one of the first kinds of chromatography that chemists developed. The partition coefficient principle has been applied in paper chromatography, thin layer chromatography, gas phase and liquid–liquid separation applications. The 1952 Nobel Prize in chemistry was earned by Archer John Porter Martin and Richard Laurence Millington Synge for their development of the technique, which was used for their separation of amino acids. Partition chromatography uses a retained solvent, on the surface or within the grains or fibers of an "inert" solid supporting matrix as with paper chromatography; or takes advantage of some coulombic and/or hydrogen donor interaction with the stationary phase. Analyte molecules partition between a liquid stationary phase and the eluent. Just as in Hydrophilic Interaction Chromatography (HILIC; a sub-technique within HPLC), this method separates analytes based on differences in their polarity. HILIC most often uses a bonded polar stationary phase and a mobile phase made primarily of acetonitrile with water as the strong component. Partition HPLC has been used historically on unbonded silica or alumina supports. Each works effectively for separating analytes by relative polar differences. HILIC bonded phases have the advantage of separating acidic, basic and neutral solutes in a single chromatographic run.


title: High-performance liquid chromatography

text: The polar analytes diffuse into a stationary water layer associated with the polar stationary phase and are thus retained. The stronger the interactions between the polar analyte and the polar stationary phase (relative to the mobile phase) the longer the elution time. The interaction strength depends on the functional groups part of the analyte molecular structure, with more polarized groups ("e.g.", hydroxyl-) and groups capable of hydrogen bonding inducing more retention. Coulombic (electrostatic) interactions can also increase retention. Use of more polar solvents in the mobile phase will decrease the retention time of the analytes, whereas more hydrophobic solvents tend to increase retention times.


title: High-performance liquid chromatography

text: Normal–phase chromatography was one of the first kinds of HPLC that chemists developed. Also known as normal-phase HPLC (NP-HPLC) this method separates analytes based on their affinity for a polar stationary surface such as silica, hence it is based on analyte ability to engage in polar interactions (such as hydrogen-bonding or dipole-dipole type of interactions) with the sorbent surface. NP-HPLC uses a non-polar, non-aqueous mobile phase ("e.g.", Chloroform), and works effectively for separating analytes readily soluble in non-polar solvents. The analyte associates with and is retained by the polar stationary phase. Adsorption strengths increase with increased analyte polarity. The interaction strength depends not only on the functional groups present in the structure of the analyte molecule, but also on steric factors. The effect of steric hindrance on interaction strength allows this method to resolve (separate) structural isomers.


title: High-performance liquid chromatography

text: The use of more polar solvents in the mobile phase will decrease the retention time of analytes, whereas more hydrophobic solvents tend to induce slower elution (increased retention times). Very polar solvents such as traces of water in the mobile phase tend to adsorb to the solid surface of the stationary phase forming a stationary bound (water) layer which is considered to play an active role in retention. This behavior is somewhat peculiar to normal phase chromatography because it is governed almost exclusively by an adsorptive mechanism ("i.e.", analytes interact with a solid surface rather than with the solvated layer of a ligand attached to the sorbent surface; see also reversed-phase HPLC below). Adsorption chromatography is still widely used for structural isomer separations in both column and thin-layer chromatography formats on activated (dried) silica or alumina supports.


title: High-performance liquid chromatography

text: Partition- and NP-HPLC fell out of favor in the 1970s with the development of reversed-phase HPLC because of poor reproducibility of retention times due to the presence of a water or protic organic solvent layer on the surface of the silica or alumina chromatographic media. This layer changes with any changes in the composition of the mobile phase ("e.g.", moisture level) causing drifting retention times.


title: High-performance liquid chromatography

text: Recently, partition chromatography has become popular again with the development of Hilic bonded phases which demonstrate improved reproducibility, and due to a better understanding of the range of usefulness of the technique.


title: High-performance liquid chromatography

text: A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities.


title: High-performance liquid chromatography

text: There are distinct differences between displacement and elution chromatography. In elution mode, substances typically emerge from a column in narrow, Gaussian peaks. Wide separation of peaks, preferably to baseline, is desired in order to achieve maximum purification. The speed at which any component of a mixture travels down the column in elution mode depends on many factors. But for two substances to travel at different speeds, and thereby be resolved, there must be substantial differences in some interaction between the biomolecules and the chromatography matrix. Operating parameters are adjusted to maximize the effect of this difference. In many cases, baseline separation of the peaks can be achieved only with gradient elution and low column loadings. Thus, two drawbacks to elution mode chromatography, especially at the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loadings. Displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than “peaks”. Because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentration.


title: High-performance liquid chromatography

text: Reversed phase HPLC (RP-HPLC) has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary phase is a silica which has been surface-modified with RMeSiCl, where R is a straight chain alkyl group such as CH or CH. With such stationary phases, retention time is longer for molecules which are less polar, while polar molecules elute more readily (early in the analysis). An investigator can increase retention times by adding more water to the mobile phase; thereby making the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase. Similarly, an investigator can decrease retention time by adding more organic solvent to the eluent. RP-HPLC is so commonly used that it is often incorrectly referred to as "HPLC" without further specification. The pharmaceutical industry regularly employs RP-HPLC to qualify drugs before their release.


title: High-performance liquid chromatography

text: RP-HPLC operates on the principle of hydrophobic interactions, which originates from the high symmetry in the dipolar water structure and plays the most important role in all processes in life science. RP-HPLC allows the measurement of these interactive forces. The binding of the analyte to the stationary phase is proportional to the contact surface area around the non-polar segment of the analyte molecule upon association with the ligand on the stationary phase. This solvophobic effect is dominated by the force of water for "cavity-reduction" around the analyte and the C-chain versus the complex of both. The energy released in this process is proportional to the surface tension of the eluent (water: 7.3 J/cm, methanol: 2.2 J/cm) and to the hydrophobic surface of the analyte and the ligand respectively. The retention can be decreased by adding a less polar solvent (methanol, acetonitrile) into the mobile phase to reduce the surface tension of water. Gradient elution uses this effect by automatically reducing the polarity and the surface tension of the aqueous mobile phase during the course of the analysis.


title: High-performance liquid chromatography

text: Structural properties of the analyte molecule play an important role in its retention characteristics. In general, an analyte with a larger hydrophobic surface area (C–H, C–C, and generally non-polar atomic bonds, such as S-S and others) is retained longer because it is non-interacting with the water structure. On the other hand, analytes with higher polar surface area (conferred by the presence of polar groups, such as -OH, -NH, COO or -NH in their structure) are less retained as they are better integrated into water. Such interactions are subject to steric effects in that very large molecules may have only restricted access to the pores of the stationary phase, where the interactions with surface ligands (alkyl chains) take place. Such surface hindrance typically results in less retention.


title: High-performance liquid chromatography

text: Retention time increases with hydrophobic (non-polar) surface area. Branched chain compounds elute more rapidly than their corresponding linear isomers because the overall surface area is decreased. Similarly organic compounds with single C–C bonds elute later than those with a C=C or C–C triple bond, as the double or triple bond is shorter than a single C–C bond.


title: High-performance liquid chromatography

text: Aside from mobile phase surface tension (organizational strength in eluent structure), other mobile phase modifiers can affect analyte retention. For example, the addition of inorganic salts causes a moderate linear increase in the surface tension of aqueous solutions (ca. 1.5 J/cm per Mol for NaCl, 2.5 J/cm per Mol for (NH)SO), and because the entropy of the analyte-solvent interface is controlled by surface tension, the addition of salts tend to increase the retention time. This technique is used for mild separation and recovery of proteins and protection of their biological activity in protein analysis (hydrophobic interaction chromatography, HIC).


title: High-performance liquid chromatography

text: Another important factor is the mobile phase pH since it can change the hydrophobic character of the analyte. For this reason most methods use a buffering agent, such as sodium phosphate, to control the pH. Buffers serve multiple purposes: control of pH, neutralize the charge on the silica surface of the stationary phase and act as ion pairing agents to neutralize analyte charge. Ammonium formate is commonly added in mass spectrometry to improve detection of certain analytes by the formation of analyte-ammonium adducts. A volatile organic acid such as acetic acid, or most commonly formic acid, is often added to the mobile phase if mass spectrometry is used to analyze the column eluant. Trifluoroacetic acid is used infrequently in mass spectrometry applications due to its persistence in the detector and solvent delivery system, but can be effective in improving retention of analytes such as carboxylic acids in applications utilizing other detectors, as it is a fairly strong organic acid. The effects of acids and buffers vary by application but generally improve chromatographic resolution.


title: High-performance liquid chromatography

text: Reversed phase columns are quite difficult to damage compared with normal silica columns; however, many reversed phase columns consist of alkyl derivatized silica particles and should never be used with aqueous bases as these will destroy the underlying silica particle. They can be used with aqueous acid, but the column should not be exposed to the acid for too long, as it can corrode the metal parts of the HPLC equipment. RP-HPLC columns should be flushed with clean solvent after use to remove residual acids or buffers, and stored in an appropriate composition of solvent. The metal content of HPLC columns must be kept low if the best possible ability to separate substances is to be retained. A good test for the metal content of a column is to inject a sample which is a mixture of 2,2'- and 4,4'-bipyridine. Because the 2,2'-bipy can chelate the metal, the shape of the peak for the 2,2'-bipy will be distorted (tailed) when metal ions are present on the surface of the silica...


title: High-performance liquid chromatography

text: Size-exclusion chromatography (SEC), also known as "gel permeation chromatography" or "gel filtration chromatography", separates particles on the basis of molecular size (actually by a particle's Stokes radius). It is generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of the purification. It is also useful for determining the tertiary structure and quaternary structure of purified proteins. SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of a particle. The larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time.


title: High-performance liquid chromatography

text: This technique is widely used for the molecular weight determination of polysaccharides. SEC is the official technique (suggested by European pharmacopeia) for the molecular weight comparison of different commercially available low-molecular weight heparins.


title: High-performance liquid chromatography

text: In ion-exchange chromatography (IC), retention is based on the attraction between solute ions and charged sites bound to the stationary phase. Solute ions of the same charge as the charged sites on the column are excluded from binding, while solute ions of the opposite charge of the charged sites of the column are retained on the column. Solute ions that are retained on the column can be eluted from the column by changing the solvent conditions ("e.g.", increasing the ion effect of the solvent system by increasing the salt concentration of the solution, increasing the column temperature, changing the pH of the solvent, etc.).


title: High-performance liquid chromatography

text: Types of ion exchangers include polystyrene resins, cellulose and dextran ion exchangers (gels), and controlled-pore glass or porous silica. Polystyrene resins allow cross linkage which increases the stability of the chain. Higher cross linkage reduces swerving, which increases the equilibration time and ultimately improves selectivity. Cellulose and dextran ion exchangers possess larger pore sizes and low charge densities making them suitable for protein separation


title: High-performance liquid chromatography

text: An increase in counter ion (with respect to the functional groups in resins) concentration reduces the retention time. A decrease in pH reduces the retention time in cation exchange while an increase in pH reduces the retention time in anion exchange. By lowering the pH of the solvent in a cation exchange column, for instance, more hydrogen ions are available to compete for positions on the anionic stationary phase, thereby eluting weakly bound cations.


title: High-performance liquid chromatography

text: This form of chromatography is widely used in the following applications: water purification, preconcentration of trace components, ligand-exchange chromatography, ion-exchange chromatography of proteins, high-pH anion-exchange chromatography of carbohydrates and oligosaccharides, and others.


title: High-performance liquid chromatography

text: This chromatographic process relies on the property of biologically active substances to form stable, specific, and reversible complexes. The formation of these complexes involves the participation of common molecular forces such as the Van der Waals interaction, electrostatic interaction, dipole-dipole interaction, hydrophobic interaction, and the hydrogen bond. An efficient, biospecific bond is formed by a simultaneous and concerted action of several of these forces in the complementary binding sites.


title: High-performance liquid chromatography

text: Aqueous normal-phase chromatography (ANP) is a chromatographic technique which encompasses the mobile phase region between reversed-phase chromatography (RP) and organic normal phase chromatography (ONP). This technique is used to achieve unique selectivity for hydrophilic compounds, showing normal phase elution using reversed-phase solvents.


title: High-performance liquid chromatography

text: A separation in which the mobile phase composition remains constant throughout the procedure is termed "isocratic" (meaning "constant composition"). (The example of these the percentage of methanol throughout the procedure will remain constant i.e. 10%) The word was coined by Csaba Horvath who was one of the pioneers of HPLC.,


title: High-performance liquid chromatography

text: The mobile phase composition does not have to remain constant. A separation in which the mobile phase composition is changed during the separation process is described as a "gradient elution". One example is a gradient starting at 10% methanol and ending at 90% methanol after 20 minutes. The two components of the mobile phase are typically termed "A" and "B"; "A" is the "weak" solvent which allows the solute to elute only slowly, while "B" is the "strong" solvent which rapidly elutes the solutes from the column. In reversed-phase chromatography, solvent "A" is often water or an aqueous buffer, while "B" is an organic solvent miscible with water, such as acetonitrile, methanol, THF, or isopropanol.


title: High-performance liquid chromatography

text: In isocratic elution, peak width increases with retention time linearly according to the equation for N, the number of theoretical plates. This leads to the disadvantage that late-eluting peaks get very flat and broad. Their shape and width may keep them from being recognized as peaks.


title: High-performance liquid chromatography

text: Gradient elution decreases the retention of the later-eluting components so that they elute faster, giving narrower (and taller) peaks for most components. This also improves the peak shape for tailed peaks, as the increasing concentration of the organic eluent pushes the tailing part of a peak forward. This also increases the peak height (the peak looks "sharper"), which is important in trace analysis. The gradient program may include sudden "step" increases in the percentage of the organic component, or different slopes at different times – all according to the desire for optimum separation in minimum time.


title: High-performance liquid chromatography

text: In isocratic elution, the selectivity does not change if the column dimensions (length and inner diameter) change – that is, the peaks elute in the same order. In gradient elution, the elution order may change as the dimensions or flow rate change.


title: High-performance liquid chromatography

text: The driving force in reversed phase chromatography originates in the high order of the water structure. The role of the "organic component of the mobile phase" is to reduce this high order and thus "reduce the retarding strength of the aqueous component."


title: High-performance liquid chromatography

text: HPLC separations have theoretical parameters and equations to describe the separation of components into signal peaks when detected by instrumentation such as by a UV detector or a mass spectrometer. The parameters are largely derived from two sets of chromatographic theory: plate theory (as part of Partition chromatography), and the rate theory of chromatography / "Van Deemter equation". Of course, they can be put in practice through analysis of HPLC chromatograms, although rate theory is considered the more accurate theory.


title: High-performance liquid chromatography

text: They are analogous to the calculation of retention factor for a paper chromatography separation, but describes how well HPLC separates a mixture into two or more components that are detected as peaks (bands) on a chromatogram. The HPLC parameters are the: efficiency factor("N"), the retention factor (kappa prime), and the separation factor (alpha). Together the factors are variables in a resolution equation, which describes how well two components' peaks separated or overlapped each other. These parameters are mostly only used for describing HPLC reversed phase and HPLC normal phase separations, since those separations tend to be more subtle than other HPLC modes ("e.g.", ion exchange and size exclusion).


title: High-performance liquid chromatography

text: Void volume is the amount of space in a column that is occupied by solvent. It is the space within the column that is outside of the column's internal packing material. Void volume is measured on a chromatogram as the first component peak detected, which is usually the solvent that was present in the sample mixture; ideally the sample solvent flows through the column without interacting with the column, but is still detectable as distinct from the HPLC solvent. The void volume is used as a correction factor.


title: High-performance liquid chromatography

text: Efficiency factor ("N") practically measures how sharp component peaks on the chromatogram are, as ratio of the component peak's area ("retention time") relative to the width of the peaks at their widest point (at the baseline). Peaks that are tall, sharp, and relatively narrow indicate that separation method efficiently removed a component from a mixture; high efficiency. Efficiency is very dependent upon the HPLC column and the HPLC method used. Efficiency factor is synonymous with plate number, and the 'number of theoretical plates'.


title: High-performance liquid chromatography

text: Retention factor ("kappa prime") measures how long a component of the mixture stuck to the column, measured by the area under the curve of its peak in a chromatogram (since HPLC chromatograms are a function of time). Each chromatogram peak will have its own retention factor ("e.g.", "kappa" for the retention factor of the first peak). This factor may be corrected for by the void volume of the column.


title: High-performance liquid chromatography

text: Separation factor ("alpha") is a relative comparison on how well two neighboring components of the mixture were separated ("i.e.", two neighboring bands on a chromatogram). This factor is defined in terms of a ratio of the retention factors of a pair of neighboring chromatogram peaks, and may also be corrected for by the void volume of the column. The greater the separation factor value is over 1.0, the better the separation, until about 2.0 beyond which an HPLC method is probably not needed for separation.


title: High-performance liquid chromatography

text: Resolution equations relate the three factors such that high efficiency and separation factors improve the resolution of component peaks in an HPLC separation.


title: High-performance liquid chromatography

text: The internal diameter (ID) of an HPLC column is an important parameter that influences the detection sensitivity and separation selectivity in gradient elution. It also determines the quantity of analyte that can be loaded onto the column. Larger columns are usually seen in industrial applications, such as the purification of a drug product for later use. Low-ID columns have improved sensitivity and lower solvent consumption at the expense of loading capacity.


title: High-performance liquid chromatography

text: Larger ID columns (over 10 mm) are used to purify usable amounts of material because of their large loading capacity.


title: High-performance liquid chromatography

text: Analytical scale columns (4.6 mm) have been the most common type of columns, though smaller columns are rapidly gaining in popularity. They are used in traditional quantitative analysis of samples and often use a UV-Vis absorbance detector.


title: High-performance liquid chromatography

text: Narrow-bore columns (1–2 mm) are used for applications when more sensitivity is desired either with special UV-vis detectors, fluorescence detection or with other detection methods like liquid chromatography-mass spectrometry


title: High-performance liquid chromatography

text: Capillary columns (under 0.3 mm) are used almost exclusively with alternative detection means such as mass spectrometry. They are usually made from fused silica capillaries, rather than the stainless steel tubing that larger columns employ.


title: High-performance liquid chromatography

text: Most traditional HPLC is performed with the stationary phase attached to the outside of small spherical silica particles (very small beads). These particles come in a variety of sizes with 5 µm beads being the most common. Smaller particles generally provide more surface area and better separations, but the pressure required for optimum linear velocity increases by the inverse of the particle diameter squared.


title: High-performance liquid chromatography

text: According to the equations of the column velocity, efficiency and backpressure, reducing the particle diameter by half and keeping the size of the column the same, will double the column velocity and efficiency; but four times increase the backpressure. And the small particles HPLC also can decrease the width broadening. Larger particles are used in preparative HPLC (column diameters 5 cm up to >30 cm) and for non-HPLC applications such as solid-phase extraction.


title: High-performance liquid chromatography

text: Many stationary phases are porous to provide greater surface area. Small pores provide greater surface area while larger pore size has better kinetics, especially for larger analytes. For example, a protein which is only slightly smaller than a pore might enter the pore but does not easily leave once inside.


title: High-performance liquid chromatography

text: Pumps vary in pressure capacity, but their performance is measured on their ability to yield a consistent and reproducible volumetric flow rate. Pressure may reach as high as 60 MPa (6000 lbf/in), or about 600 atmospheres. Modern HPLC systems have been improved to work at much higher pressures, and therefore are able to use much smaller particle sizes in the columns (<2 μm). These "ultra high performance liquid chromatography" systems or UHPLCs, which could also be known as ultra high pressure chromatography systems, can work at up to 120 MPa (17,405 lbf/in), or about 1200 atmospheres. The term "UPLC" is a trademark of the Waters Corporation, but is sometimes used to refer to the more general technique of UHPLC.


title: High-performance liquid chromatography

text: HPLC detectors fall into two main categories: universal or selective. Universal detectors typically measure a bulk property ("e.g.", refractive index) by measuring a difference of a physical property between the mobile phase and mobile phase with solute while selective detectors measure a solute property ("e.g.", UV-Vis absorbance) by simply responding to the physical or chemical property of the solute. HPLC most commonly uses a UV-Vis absorbance detector, however, a wide range of other chromatography detectors can be used. A universal detector that complements UV-Vis absorbance detection is the Charged aerosol detector (CAD). A kind of commonly utilized detector includes refractive index detectors, which provide readings by measuring the changes in the refractive index of the eluant as it moves through the flow cell. In certain cases, it is possible to use multiple detectors, for example LCMS normally combines UV-Vis with a mass spectrometer.


title: High-performance liquid chromatography

text: When used with an electrochemical detector (ECD) the HPLC-ECD selectively detects neurotransmitters such as: norepinephrine, dopamine, serotonin, glutamate, GABA, acetylcholine and others in neurochemical analysis research applications. The HPLC-ECD detects neurotransmitters to the femtomolar range. Other methods to detect neurotransmitters include liquid chromatography-mass spectrometry, ELISA, or radioimmunoassays.


title: High-performance liquid chromatography

text: Large numbers of samples can be automatically injected onto an HPLC system, by the use of HPLC autosamplers. In addition, HPLC autosamplers have an injection volume and technique which is exactly the same for each injection, consequently they provide a high degree of injection volume precision.


title: High-performance liquid chromatography

text: HPLC has many applications in both laboratory and clinical science. It is a common technique used in pharmaceutical development, as it is a dependable way to obtain and ensure product purity. While HPLC can produce extremely high quality (pure) products, it is not always the primary method used in the production of bulk drug materials. According to the European pharmacopoeia, HPLC is used in only 15.5% of syntheses. However, it plays a role in 44% of syntheses in the United States pharmacopoeia. This could possibly be due to differences in monetary and time constraints, as HPLC on a large scale can be an expensive technique. An increase in specificity, precision, and accuracy that occurs with HPLC unfortunately corresponds to an increase in cost.


title: High-performance liquid chromatography

text: This technique is also used for detection of illicit drugs in urine. The most common method of drug detection is an immunoassay. This method is much more convenient. However, convenience comes at the cost of specificity and coverage of a wide range of drugs. As HPLC is a method of determining (and possibly increasing) purity, using HPLC alone in evaluating concentrations of drugs is somewhat insufficient. With this, HPLC in this context is often performed in conjunction with mass spectrometry. Using liquid chromatography instead of gas chromatography in conjunction with MS circumvents the necessity for derivitizing with acetylating or alkylation agents, which can be a burdensome extra step. This technique has been used to detect a variety of agents like doping agents, drug metabolites, glucuronide conjugates, amphetamines, opioids, cocaine, BZDs, ketamine, LSD, cannabis, and pesticides. Performing HPLC in conjunction with mass spectrometry reduces the absolute need for standardizing HPLC experimental runs.


title: High-performance liquid chromatography

text: Similar assays can be performed for research purposes, detecting concentrations of potential clinical candidates like anti-fungal and asthma drugs. This technique is obviously useful in observing multiple species in collected samples, as well, but requires the use of standard solutions when information about species identity is sought out. It is used as a method to confirm results of synthesis reactions, as purity is essential in this type of research. However, mass spectrometry is still the more reliable way to identify species.


title: High-performance liquid chromatography

text: Medical use of HPLC can include drug analysis, but falls more closely under the category of nutrient analysis. While urine is the most common medium for analyzing drug concentrations, blood serum is the sample collected for most medical analyses with HPLC. Other methods of detection of molecules that are useful for clinical studies have been tested against HPLC, namely immunoassays. In one example of this, competitive protein binding assays (CPBA) and HPLC were compared for sensitivity in detection of vitamin D. Useful for diagnosing vitamin D deficiencies in children, it was found that sensitivity and specificity of this CPBA reached only 40% and 60%, respectively, of the capacity of HPLC. While an expensive tool, the accuracy of HPLC is nearly unparalleled.


title: Kyle Lowry

text: Kyle Terrell Lowry (born March 25, 1986) is an American professional basketball player for the Miami Heat of the National Basketball Association (NBA). He has been a six-time NBA All-Star and was named to the All-NBA Third Team in 2016. Lowry won an NBA championship with the Toronto Raptors in 2019, their first title in franchise history. He was a member of the U.S. national team that won a gold medal in the 2016 Summer Olympics.


title: Kyle Lowry

text: Lowry played two seasons of college basketball with the Villanova Wildcats before he was selected by the Memphis Grizzlies in the first round of the 2006 NBA draft with the 24th overall pick. He began his NBA career with Memphis and the Houston Rockets before being traded to Toronto. In his second season with the Raptors, he helped them reach the playoffs for the first time in seven years and win an Atlantic Division title during the 2013–14 season. In 2015–16, he led the Raptors to 56 wins in what was then the highest win total in franchise history, as well as helped the team make the Eastern Conference Finals for the first time.


title: Kyle Lowry

text: Lowry is widely regarded as the greatest Toronto Raptors player of all time due to his work in turning the franchise around, from the post-Vince Carter era to a legitimate NBA contender bringing the franchise its first championship in 2019. The Raptors had their greatest success under Lowry, from their two Eastern Conference Finals berths to all five of their 50 wins campaigns. The Raptors regularly improved their win totals with Lowry at the helm.


title: Kyle Lowry

text: Born and raised in North Philadelphia, Lowry is one of two sons of Marie Holloway and Lonnie Lowry Sr. alongside Lonnie Jr., his older brother by 5 years. His father lived 10 minutes away from the family's home, which also included Lowry's cousin, Laquita. Lonnie Sr. became estranged from the family when Kyle was about seven years old, leaving him and his siblings to be raised by his mom (who worked at the Internal Revenue Service and the U.S. Post Office) as well as his disciplinarian grandmother (who worked at Dunkin Donuts). Kyle attributes his survival and well-being to Lonnie Jr. for ensuring that he did not follow in the same path as his childhood friends who are no longer alive, including not allowing Kyle to have any tattoos and piercings. His brother was also instrumental in teaching him how to play basketball, playing pickup basketball together against older boys, and learning not to dwell on his estranged father. His brother also searched newspaper ads for AAU basketball tryouts and ensured that Lowry would get there and make their teams.


title: Kyle Lowry

text: Lowry attended Cardinal Dougherty High School in Philadelphia and played point guard for the school's varsity basketball team.