A practical reference on retention time: what it is, how it behaves, what the literature reports, and where the honest uncertainties sit.
Reviewed 2025-11-10. Anything still debated is marked as such rather than presented as settled.
High-performance liquid chromatography is an analytical technique that separates components in a liquid sample. A pump moves a liquid mobile phase through a column packed with a solid stationary phase. Compounds interact differently with both phases and travel at different rates, leaving the column at distinct retention times. A detector records these arrivals as peaks on a chromatogram. The resulting pattern supports identification and quantification of substances in mixtures. Modern instruments use high pressure to force solvent through small particles, which improves speed and resolution compared with older low-pressure liquid chromatography methods.
Separation in HPLC depends on the chemistry of the stationary phase, the composition of the mobile phase, and the physical properties of the column. Reverse-phase separations use a nonpolar stationary phase and a polar mobile phase, and they are common for many organic compounds. Ion-exchange, size-exclusion, and normal-phase modes serve other classes of analytes. Gradient elution changes solvent strength over time, while isocratic elution holds it constant. Flow rate, temperature, particle size, and column length all influence peak shape and resolution. Detection may use ultraviolet absorbance, fluorescence, refractive index, or mass spectrometry, depending on the analyte and the required sensitivity.
Reversed-phase chromatography dominates modern HPLC testing, using a nonpolar stationary phase such as chemically bonded octadecyl groups and a polar mobile phase of water mixed with organic solvent. Analytes partition between the mobile and stationary phases according to hydrophobicity. Gradient elution changes the mobile phase composition over time to separate compounds with a wide range of retention. Isocratic elution keeps the composition constant and is simpler for routine assays. Column temperature, pH, and flow rate influence selectivity, peak shape, and retention time, so these parameters are controlled during a validated method.
Detection in HPLC testing commonly relies on ultraviolet-visible absorbance, fluorescence, refractive index, or mass spectrometry. A diode array detector records full spectra across a wavelength range, which helps identify co-eluting peaks. Mass spectrometry provides mass-to-charge ratios and can confirm molecular identity at low concentrations. The choice of detector depends on analyte structure, required sensitivity, and whether quantitation or identification is the goal. No single detector works for every compound, and method development often compares responses before selecting one.
High-performance liquid chromatography is an analytical technique that separates components of a liquid sample by passing it through a packed column under pressure. A pump delivers a mobile phase at a controlled flow rate, and an injector introduces the sample into the stream. Differences in interaction with the stationary phase cause analytes to migrate at different rates. Detectors record elution as peaks, and a data system converts signals into a chromatogram. The method is suited to compounds that dissolve in a liquid and are not volatile enough for gas chromatography.
| Property | Value | Notes |
|---|---|---|
| Common abbreviation | HPLC | High-performance liquid chromatography |
| Separation basis | Differential partitioning | Between liquid mobile phase and solid stationary phase |
| Common mode | Reverse phase | Nonpolar column, polar mobile phase |
| Typical detector | UV-Vis absorbance | Widely used for compounds with chromophores |
| Typical column particle size | 2–5 µm | Smaller particles can improve resolution |
Documentation and traceability are central to regulated HPLC testing. Records typically include instrument logs, column history, mobile-phase preparation, sample preparation, injection sequences, raw chromatograms, and audit trails. Electronic systems may require user access controls, time-stamped changes, and backup procedures. Training records show that analysts are qualified for assigned methods. Audits and inspections check whether written procedures match actual practice and whether deviations are documented. These controls support reproducibility and allow results to be reconstructed if questions arise later.
Method validation establishes that an HPLC procedure is suitable for its intended purpose. Typical parameters include accuracy, precision, specificity, linearity, range, limit of detection, limit of quantitation, robustness, and solution stability. Accuracy reflects closeness to a reference value, while precision reflects agreement among repeated measurements. Specificity shows whether the method can measure the analyte without interference from matrix components. Validation is documented through protocols and reports, and the required extent depends on the method's use and regulatory context.
Routine quality control uses system suitability, blank injections, check standards, and control samples to detect drift or contamination. System suitability criteria may specify minimum resolution, maximum tailing factor, and a permitted range for repeated injections. Blank injections reveal carryover or solvent contamination, while check standards confirm calibration accuracy over a batch. Control samples with known analyte levels can show whether results remain within statistical limits. When a control result falls outside limits, the analyst investigates the cause and may invalidate affected results before repeating the batch.
Method validation evaluates accuracy, precision, specificity, linearity, range, detection limit, quantitation limit, and robustness. Regulatory guidance for pharmaceuticals, foods, and environmental testing defines expected documentation and acceptance criteria. Verification confirms that a validated method works in a specific laboratory with its own instruments and reagents. Calibration curves use reference standards with known purity and traceability, while measurement uncertainty is estimated from validation data, control charts, and collaborative studies. The scope of validation depends on the method's intended use.
Routine quality control monitors retention time shifts, baseline noise, system pressure, and peak shape. Trends can reveal column aging, mobile phase preparation errors, detector drift, or sample degradation. Corrective actions may include replacing the column, preparing fresh mobile phase, or recalibrating the detector. Stability testing often uses HPLC to measure parent compound loss and degradation product formation. Open questions remain about how accelerated stability results extrapolate to long-term storage under varied conditions.
Quality control samples are inserted at intervals to monitor accuracy and precision throughout a batch. Blank samples detect contamination, while spiked samples assess recovery from the sample matrix. Calibration standards establish the relationship between detector response and concentration, and control samples are prepared independently from them whenever possible. Laboratories also participate in proficiency testing and maintain audit trails, instrument logs, and reagent records. Ongoing review of control charts can reveal trends before they cause out-of-specification results.
Method validation demonstrates that an HPLC procedure is suitable for its intended purpose. Common validation parameters include accuracy, precision, specificity, linearity, range, limit of detection, limit of quantification, and robustness. Accuracy reflects agreement with a reference value, while precision describes repeatability under defined conditions. Specificity shows whether the method can measure the analyte in the presence of impurities or matrix components. Validation documents are reviewed before a method is used for routine testing or regulatory submissions.
Validation establishes that a method is suitable for its intended purpose. Typical parameters include accuracy, precision, specificity, linearity, range, limit of detection, limit of quantification, robustness, and stability of standards and samples. Acceptance criteria are defined in advance, and results are documented in a validation report. Regulatory guidance for pharmaceuticals, foods, and environmental testing differs, so the applicable framework must be identified. Ongoing verification uses control samples and trend charts after validation. Method transfer to another laboratory may require partial revalidation.
Routine quality control includes blanks, duplicates, spiked samples, and certified reference materials. Calibration curves are prepared with standards at several concentrations, and the detector response is checked for linearity. Carryover, column aging, mobile phase evaporation, and temperature drift can shift retention times or peak areas. Maintenance such as replacing seals, filters, and columns helps prevent failures. Records of injections, integration, and deviations support traceability. Audits may request raw data and instrument logs for each batch.
Opioids include opiates, an older term that refers to such drugs derived from opium, including morphine itself. Opiate is properly limited to the natural alkaloids found in the resin of the opium poppy although some include semi-synthetic derivatives. Other opioids are semi-synthetic and synthetic drugs such as hydrocodone, oxycodone, and fentanyl; antagonist drugs such as naloxone; and endogenous peptides such as endorphins. The terms opiate and narcotic are sometimes encountered as synonyms for opioid. Narcotic, derived from words meaning 'numbness' or 'sleep', originally referred to any psychoactive compound with numbing or paralyzing properties. As an American legal term, narcotic refers to cocaine and opioids, and their source materials; it is also loosely applied to any illegal or controlled psychoactive drug. In some jurisdictions all controlled drugs are legally classified as narcotics. The term can have pejorative connotations and its use is generally discouraged where that is the case.
is the concentration of the solid in the diffusion layer surrounding the solid. D is the diffusion coefficient. L is the diffusion layer thickness. As can be inferred from the Noyes–Whitney equation, the rate of dissolution may be modified primarily by altering the surface area of the solid by altering the particle size (e.g., with micronization). For many drugs, reducing the particle size reduces the dose needed to achieve the same therapeutic effect. The particle size reduction increases the specific surface area and the dissolution rate and does not affect solubility. The rate of dissolution may also be altered by choosing a suitable polymorph of a compound. Different polymorphs have different solubility and dissolution rate characteristics. Specifically, crystalline forms dissolve slower than amorphous forms since they require more energy to leave the lattice during dissolution. The stablest crystalline polymorph has the lowest dissolution rate. Dissolution also differs between anhydrous and hydrous forms of a drug. Anhydrous forms often dissolve faster but sometimes are less soluble. Esterification is also used to control solubility. For example, stearate and estolate esters of drugs have decreased solubility in gastric fluid. Later, esterases in the gastrointestinal tract (GIT) wall and blood hydrolyze these esters to release the parent drug. Coatings on a tablet or pellet may act as barriers to reducing the dissolution rate. Coatings may also be used to control where dissolution takes place.
=== Other medications === Bupropion, topiramate, metformin, zonisamide, and amphetamine are sometimes used off-label for weight loss. The usefulness of certain drugs depends upon the comorbidities present. Metformin is preferred in overweight diabetics and for those gaining weight, as it may lead to mild weight loss in comparison to sulfonylureas or insulin. The thiazolidinediones, on the other hand, may cause weight gain, but decrease central obesity. Diabetics also achieve modest weight loss with fluoxetine and orlistat over 12–57 weeks. Sibutramine (Meridia), which acts in the brain to inhibit deactivation of the neurotransmitters, thereby decreasing appetite was withdrawn from the UK market in January 2010 and United States and Canadian markets in October 2010 due to cardiovascular concerns. In 2010 it was found that sibutramine increases the risk of heart attacks and strokes in people with a history of cardiovascular disease. Recombinant human leptin is very effective in those with obesity due to congenital complete leptin deficiency via decreasing energy intake and possibly increases energy expenditure. This condition is, however, rare and this treatment is not effective for inducing weight loss in the majority of people with obesity. It is being investigated to determine whether or not it helps with weight loss maintenance. Though hypothesized that supplementation of vitamin D may be an effective treatment for obesity, studies do not support this. There is also no strong evidence to recommend herbal medicines for weight loss.
He soon determined that random X chromosome inactivation causes tissue mosaicism in female mammals, in that each somatic cell expresses one (but not both) of the alleles of X-linked genes with which it is endowed. This he accomplished by showing that two populations of erythrocytes exist in the blood of African American women who are heterozygous for G6PD deficiency. Mary F. Lyon independently hypothesized that variegated coat colors in mice might arise from random X chromosome inactivation. This, too, flowed from Ohno's observations. Beutler's seminal work on G6PD deficiency led him to further explore hemolytic anemias caused by various enzyme deficiencies. The systematic methodology that he developed became the standard approach to study of patients with these disorders. Beutler made major contributions to the understanding of Tay–Sachs disease. He purified the enzyme that is aberrant in this disease and demonstrated its multimeric structure. His group cloned the gene responsible for Gaucher disease and developed treatments for this disease, as well as diagnostic tests. Beutler also developed a screening test for galactosemia, which is used to this day to detect the disease in neonates, and prevent its severe consequences. Beutler was the first to attempt pharmacologic intervention in sickle cell disease by increasing methemoglobin levels, carboxyhemoglobin levels, and fetal hemoglobin levels. The latter approach depended on administration of estrogen, progesterone and human chorionic gonadotropin.
Sources: en.wikipedia.org
In September 2015, chairman Russ Withers and chief executive Warren Wilmot announced they were resigning from the company. Deputy chairman Michael Smith replaced Withers, while Bob Baily was appointed as interim chief executive. In December 2015, Stewart Levitt of law firm Levitt Robinson Solicitors, who featured prominently in the Four Corners program, announced a potential class action lawsuit against 7-Eleven head office on behalf of franchisees who had allegedly been lured into signing on with 7-Eleven by false representations. This announcement was made on the same day as a Court finding describing Levvit Robinson's "hellish bullying" of Dr Brendan French, miring the action in controversy. Also on that day, 7-Eleven offered to pay "the first $25 million of back-pay claims brought by current and former workers. Franchisees would then pay the next $5 million and any payments after that would be split 50-50 between head office and franchisees." Fels "described the $25 million offer from head office as a 'significant step forward'" but added that his panel's investigation would not be effected. 7-Eleven ultimately paid more than $173 million for "systematic wage theft" to workers employed between 2015 and 2020. Levvit Robinson was forced to retract misleading statements made in advertising to 7-Eleven franchisees in June 2018 by the Federal Court of Australia. This occurred only months after Levvit Robinson launched a new class action against 7-Eleven that included the ANZ Bank despite banks having stopped loans to 7-Eleven franchisees in 2015.
In 1917, before the revolution, health conditions were significantly behind those of developed countries. As Lenin later noted, "Either the lice will defeat socialism, or socialism will defeat the lice". The Soviet health care system was conceived by the People's Commissariat for Health in 1918. Under the Semashko model, health care was to be controlled by the state and would be provided to its citizens free of charge, a revolutionary concept at the time. Article 42 of the 1977 Soviet Constitution gave all citizens the right to health protection and free access to any health institutions in the USSR. Before Leonid Brezhnev became general secretary, the Soviet healthcare system was held in high esteem by many foreign specialists. This changed, however, from Brezhnev's accession and Mikhail Gorbachev's tenure as leader, during which the health care system was heavily criticized for many basic faults, such as the quality of service and the unevenness in its provision. Minister of Health Yevgeniy Chazov, during the 19th Congress of the Communist Party of the Soviet Union, while highlighting such successes as having the most doctors and hospitals in the world, recognized the system's areas for improvement and felt that billions of rubles were squandered. After the revolution, life expectancy for all age groups went up. These improvements continued into the 1960s when statistics indicated that the life expectancy briefly surpassed that of the United States; life expectancy started to decline in the 1970s, possibly because of alcohol abuse.
=== Pathophysiology of mutated erythrocytic membrane proteins === These proteins are necessary to maintain the normal shape of a red blood cell, which is a biconcave disk. The integrating protein that is most commonly defective is spectrin which is responsible for incorporation and binding of spectrin to the greater actin cytoskeleton. This dysfunction of cytoskeletal instabilities ensue, and leave the plasma membrane of the cell less supported and/or weakened.
Brazzein is a sweet-tasting protein that occurs naturally in oubli (Pentadiplandra brazzeana), a fruit native to the Atlantic coastal areas of Central Africa. Brazzein was named in 1994 by scientists at the University of Wisconsin–Madison. It is roughly 500 to 2000 times sweeter than sucrose. Brazzein is found in the extracellular region of oubli fruit, in the pulp tissue surrounding the seeds. It was first discovered and isolated in 1989 by Wel et al. under the name of pentadin. The corresponding author of both these publications has later opined that pentadin and brazzein are two different names for the same protein. Even though pentadin is the earlier proposed name, brazzein is the more commonly used name after more extensive characterisation of the sweet protein. Like other sweet proteins discovered in plants, such as monellin and thaumatin, brazzein is extremely sweet compared to commonly used sweeteners. The fruit tastes sweet to humans, monkeys, and bonobos, but gorillas have mutations in their sweetness receptors so that they do not find brazzein sweet, and they are not known to eat the fruit.
=== Analgesia === Dextropropoxyphene is generally considered a weak analgesic, with several studies finding its efficacy is no better than acetaminophen. Like codeine, it is a weak opioid. However, dextropropoxyphene has one-third to one-half of the analgesic activity of codeine.
Sources: en.wikipedia.org
HPLC testing measures the presence and amount of one or more compounds in a liquid sample. It separates mixture components and records detector responses as peaks, which are compared with reference standards. Results are usually reported as concentrations or relative percentages.
Retention time is the interval between sample injection and the detector response for a given compound. It depends on the compound's interactions with the stationary and mobile phases under set conditions. Matching a retention time to a standard supports tentative identification but is not always unique.
HPLC alone can separate unknown compounds and provide retention times, but it often cannot identify them with certainty. Coupling HPLC to mass spectrometry gives mass information that improves identification. Confirmation usually requires comparison with reference standards or complementary techniques.
HPLC testing separates and quantifies components in a liquid sample. It is used to check identity, purity, concentration, or stability. The technique works best for compounds that dissolve and are not easily vaporized.