If you have been reading about reversed-phase and want a single page that covers the useful parts, this is it: definitions, context, how it is studied, and the questions that come up repeatedly.
Last reviewed on 2025-10-18. Where a claim depends on a specific study, the study is described rather than over-claimed.
Most routine HPLC testing uses reversed-phase columns, where the stationary phase is nonpolar and the mobile phase is a polar mixture such as water with an organic solvent. Analytes partition between the two phases according to polarity, size, and charge. Gradients that change solvent composition over time can separate compounds with broad retention ranges. Isocratic conditions keep solvent composition constant and suit simpler mixtures. The choice of column chemistry, pH, and temperature affects selectivity and peak shape.
Detection in HPLC testing commonly relies on ultraviolet-visible absorbance, fluorescence, refractive index, or mass spectrometry. UV detection is widely used because many organic compounds absorb light, but it requires a chromophore. Mass spectrometry provides mass-based identification and high sensitivity for trace analytes. Each detector has trade-offs in selectivity, cost, and compatibility with mobile phases. Quantification typically uses calibration curves prepared from reference standards. Results are reported as concentration, purity, or presence above a limit.
High-performance liquid chromatography, or HPLC, separates dissolved compounds by passing a liquid mobile phase through a packed column. Components distribute differently between the stationary phase and the moving liquid, so they travel at different speeds and exit at different times. A detector records these eluting bands as peaks, and peak area or height relates to amount. The technique supports testing in pharmaceuticals, foods, environmental samples, and industrial chemicals. Quantification usually depends on calibration with known standards.
Several separation modes exist, including reversed-phase, normal-phase, ion-exchange, size-exclusion, and hydrophilic interaction liquid chromatography. Reversed-phase uses a nonpolar stationary phase with a polar mobile phase and is widely applied to small organic molecules. Gradient elution changes mobile phase composition during the run, while isocratic elution keeps it constant. Column chemistry, particle size, temperature, flow rate, and mobile phase pH all influence retention and resolution. Method development selects conditions that separate analytes from matrix components and from each other.
| Property | Value | Notes |
|---|---|---|
| Separation mode | Reversed-phase | Nonpolar stationary phase with polar mobile phase |
| Typical column particle size | 3–5 µm | Smaller particles improve resolution but raise pressure |
| Typical flow rate | 0.5–2.0 mL/min | Depends on column dimensions and pressure limits |
| Common detection | UV-Vis absorbance | Requires analytes with chromophores |
| Typical run time | 5–30 min | Varies with method, gradient, and sample complexity |
System suitability testing is performed before and during analytical runs to confirm that the instrument and method are working as expected. Common checks include retention time, peak area, resolution between critical pairs, tailing factor, and theoretical plate count. Results are compared with predefined limits, and a failed check requires investigation before sample results are reported. Quality control samples at low, middle, and high concentrations are injected at intervals to monitor accuracy and precision. Blank injections detect carryover and contamination, while control charts track performance over time.
Data handling and documentation are central to HPLC quality control. Electronic systems should have audit trails that record changes to methods, sequences, and results. Integration parameters, such as peak baseline and threshold, can affect reported areas and must be defined in advance. Out-of-specification results trigger a structured investigation that may include reanalysis, instrument checks, and review of sample preparation. Regulatory inspections often examine raw data, audit trails, and training records to verify that reported results are traceable and reliable.
Method validation establishes that an HPLC procedure is suitable for its intended use. Key parameters include accuracy, precision, specificity, linearity, range, limit of detection, limit of quantitation, and robustness. Accuracy measures agreement with a true or accepted value, while precision describes repeatability and intermediate precision. Specificity confirms that the method measures the analyte without interference from impurities, degradants, or excipients. Validation is documented in a protocol and report, and acceptance criteria are set before experiments begin. Regulatory guidance varies by region, but the general principles are widely harmonized.
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.
Developing an HPLC test begins with defining the analytes, matrix, and required reporting limits. Chemists select a separation mode, column chemistry, mobile phase composition, flow rate, and detection wavelength or mass transition. Experiments then adjust these variables to achieve adequate retention, resolution, and peak shape. System suitability tests confirm that the instrument and method perform consistently before sample analysis. Without suitable resolution, quantitative results may be unreliable. Preliminary runs often use scouting gradients to locate retention windows.
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 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.
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.
== ABC News report == An 11-segment series of reports in March 2012 from ABC News brought widespread public attention to and raised consumer concerns about the product. The product was described as "essentially scrap meat pieces compressed together and treated with an antibacterial agent". Lean finely textured beef (LFTB) was referred to as "an unappetizing example of industrialized food production". The product has been characterized as "unappetizing, but perhaps not more so than other things that are routinely part of hamburger" by Sarah Klein, an attorney for the food safety program at the Center for Science in the Public Interest. Nutritionist Andy Bellatti has referred to the product as "one of many symptoms of a broken food system". Food policy writer Tom Laskawy noted that ammonium hydroxide is only one of several chemicals routinely added to industrially produced meat in the United States. It was reported at that time that 70% of ground beef sold in US supermarkets contained the additive, and that the USDA considered it as meat. The USDA issued a statement that LFTB was safe and had been included in consumer products for some time, and its Under Secretary of Agriculture for Food Safety Elisabeth A. Hagen stated that "The process used to produce LFTB is safe and has been used for a very long time. And adding LFTB to ground beef does not make that ground beef any less safe to consume".
==== Medications ==== Following a heart attack, nitrates, when taken for two days, and ACE-inhibitors decrease the risk of death. Other medications include: Aspirin is continued indefinitely, as well as another antiplatelet agent such as clopidogrel or ticagrelor ("dual antiplatelet therapy" or DAPT) for up to twelve months. If someone has another medical condition that requires anticoagulation (e.g. with warfarin) this may need to be adjusted based on risk of further cardiac events as well as bleeding risk. In those who have had a stent, more than 12 months of clopidogrel plus aspirin does not affect the risk of death. Beta blocker therapy such as metoprolol or carvedilol is recommended to be started within 24 hours, provided there is no acute heart failure or heart block. The dose should be increased to the highest tolerated. Contrary to most guidelines, the use of beta blockers does not appear to affect the risk of death, possibly because other treatments for MI have improved. When beta blocker medication is given within the first 24–72 hours of a STEMI no lives are saved. However, 1 in 200 people were prevented from a repeat heart attack, and another 1 in 200 from having an abnormal heart rhythm. Additionally, for 1 in 91 the medication causes a temporary decrease in the heart's ability to pump blood. ACE inhibitor therapy should be started within 24 hours and continued indefinitely at the highest tolerated dose. This is provided there is no evidence of worsening kidney failure, high potassium, low blood pressure, or known narrowing of the renal arteries.
The two substrates of this enzyme are shikimate and oxidised nicotinamide adenine dinucleotide phosphate (NADP+). Its products are 3-dehydroshikimate, reduced NADPH, and a proton. This enzyme participates in phenylalanine, tyrosine and tryptophan biosynthesis.
Sources: en.wikipedia.org
Actin-binding proteins (also known as ABPs) are proteins that bind to actin. This may mean ability to bind actin monomers, or polymers, or both. Many actin-binding proteins, including α-actinin, β-spectrin, dystrophin, utrophin and fimbrin, do this through the actin-binding calponin homology domain. This is a list of actin-binding proteins in alphabetical order. 25kDa 25kDa ABP from aorta 30akDA 30bkDa 34kDA 45kDa 110 kD dimer ABP 110 kD (Drebrin) p53 p58gag p185neu p116rip a-actinin Abl ABLIM Actin-Interacting MAPKKK Ssk2p ABP120 ABP140 Abp1p ABP280 (Filamin) ABP50 (EF-1a) Acan 125 (Carmil) ActA Actibind Actin Actinfilin Actinogelin Actin-regulating kinases Actin-Related Proteins Actobindin Actolinkin Actopaxin Actophorin Acumentin (= L-plastin) Adducin ADF/Cofilin Adseverin (scinderin) Afadin AFAP-110 Affixin Aginactin AIP1 Aldolase Angiogenin Anillin Annexins Aplyronine Archvillin (isoform of Supervillin) Arginine kinase Arp2/3 complex Band 4.1 Band 4.9 (Dematin) b-actinin b-Cap73 Bifocal Bistramide A BPAG1 Brevin (Gelsolin)
AS9100 Revision C (2009), Quality Management System – Requirements for Aviation, Space and Defense Organizations The update of AS9100 from revision B to C is largely to address the following question: "Our supplier is repeatedly late on delivery and failing to meet our requirements, how is it possible that they still have their AS9100 certificate?" That is, the AS9100C changes are driven by repeated delivery of non-conforming product and repeated late delivery by organizations that held AS9100A/B certifications. Those organizations had documented controls that conformed to the standard, however, there were insufficient processes in place to assure effectiveness of those controls. The response in this AS9100 revision is to elevate the requirements for Risk Management and to make Risk Management an integrated theme throughout the standard. A major challenge to AS9100B-compliant organizations was the new AS9100 auditing standards defined in AS9101 Revision D, which eliminates the clause-based compliance checklist and requires organizations to provide evidence of effectiveness of their systems and processes. Summary of changes between AS9100B and AS9100C:
Thus tetravalent protactinium in solutions is obtained by the action of strong reducing agents in a hydrogen atmosphere. Tetravalent protactinium is chemically similar to uranium(IV) and thorium(IV). Fluorides, phosphates, hypophosphates, iodates and phenylarsonates of protactinium(IV) are insoluble in water and dilute acids. Protactinium forms soluble carbonates. The hydrolytic properties of pentavalent protactinium are close to those of tantalum(V) and niobium(V). The complex chemical behavior of protactinium is a consequence of the start of the filling of the 5f shell in this element. Uranium has a valence from 3 to 6, the last being most stable. In the hexavalent state, uranium is very similar to the group 6 elements. Many compounds of uranium(IV) and uranium(VI) are non-stoichiometric, i.e. have variable composition. For example, the actual chemical formula of uranium dioxide is UO2+x, where x varies between −0.4 and 0.32. Uranium(VI) compounds are weak oxidants. Most of them contain the linear "uranyl" group, UO2+2. Between 4 and 6 ligands can be accommodated in an equatorial plane perpendicular to the uranyl group. The uranyl group acts as a hard acid and forms stronger complexes with oxygen-donor ligands than with nitrogen-donor ligands. NpO2+2 and PuO2+2 are also the common form of Np and Pu in the +6 oxidation state. Uranium(IV) compounds exhibit reducing properties, e.g., they are easily oxidized by atmospheric oxygen. Uranium(III) is a very strong reducing agent.
== Adverse effects == Since acarbose prevents the degradation of complex carbohydrates into glucose, some carbohydrate will remain in the intestine and be delivered to the colon. In the colon, bacteria digest (ferment) the complex carbohydrates, causing gastrointestinal side-effects such as flatulence (78% of patients) and diarrhea (14% of patients). Since these effects are dose-related, in general it is advised to start with a low dose and gradually increase the dose to the desired amount. One study found that gastrointestinal side effects decreased significantly (from 50% to 15%) over 24 weeks, even on constant dosing. Sucrose is more likely to trigger GI side effects compared to starch. Acarbose is associated with very rare elevated transaminases (19 out of 500,000). Even rarer cases of hepatitis has been reported with acarbose use. It usually goes away when the medicine is stopped. Liver enzymes should be checked before and during use of this medicine as a precaution. A 2016 meta-analysis confirms that alpha-glucosidase inhibitors, including acarbose, have a statistically significant link to elevated transaminase levels.
Sources: en.wikipedia.org
==== Gas scrubbing ==== Aqueous [Fe(EDTA)]− is used for removing ("scrubbing") hydrogen sulfide from gas streams. This conversion is achieved by oxidising the hydrogen sulfide to elemental sulfur, which is non-volatile:
=== Nutrition === Appropriate nutrition may not always be provided to farmed insects. For instance, adult black soldier flies are generally not fed during rearing due to a common misconception that they cannot eat (which is true for some insects, such as adult silkworm moths). In reality, adult black soldier flies have functional mouthparts and a functional digestive system, can break down both carbohydrates and proteins, will gravitate towards and consume food when provided, have food preferences, and have a prolonged lifespan when fed. Insects may be fasted prior to slaughter (with unknown welfare impacts); this is generally to improve taste/quality of the resulting product or to reduce the likelihood of microbial contamination of the product due to the feed. More evidence is needed to know if fasting periods are necessary for product safety, as some research suggests they may not reduce microbial loads. Farmed insects may be provided with homogenous feeds that lack macronutrient profiles self-selected by the animals; this may play a role in promoting cannibalism in species like the yellow mealworm. Nutritional enrichments, such as dietary diversity, can support brain development in crickets but may not be provided depending on the farm's available feedstock. Poor hydration for some animals may result from an effort to control disease outbreaks/fungal growth in the feed.
Conventional insulin therapy is a therapeutic regimen for treatment of diabetes mellitus which contrasts with the newer intensive insulin therapy. This older method (prior to the development of home blood glucose monitoring) is still in use in a proportion of cases.
== Retirement == On 29 August 2013, Hille announced his intention to retire from AFL Football at season's end. Hille's career at Essendon included captaining the team in 2006 in the absence of injured forward and captain Matthew Lloyd, winning Essendon's best and fairest, the W.S. Crichton Medal, in 2008 and winning the Yiooken Award in 2010, awarded for being adjudged best on ground in the annual Dreamtime at the 'G match between Essendon and Richmond. Hille, along with 33 other Essendon players, was found guilty of using a banned performance-enhancing substance, thymosin beta-4, as part of Essendon's sports supplements program during the 2012 season. He and his team-mates were initially found not guilty in March 2015 by the AFL Anti-Doping Tribunal, but a guilty verdict was returned in January 2016 after an appeal by the World Anti-Doping Agency. He was suspended for two years which, with backdating, ended in November 2016, although by this stage had already retired from football at all levels.
Brenipatide (INNTooltip International Nonproprietary Name, USANTooltip United States Adopted Name; developmental code name LY-3537031) is a dual agonist of glucagon-like peptide-1 (GLP-1) receptors and gastric inhibitory polypeptide (GIP) receptors. Brenipatide is under development by Eli Lilly and Company for the treatment of alcoholism, bipolar disorder, asthma, smoking withdrawal, cardiovascular disorders, liver disorders, metabolic disorders, and obesity. It is taken by subcutaneous injection once per month. The drug has a longer elimination half-life than tirzepatide or retatrutide. As of December 2025, it was in phase 3 clinical trials for alcoholism and bipolar disorder, phase 2 trials for asthma and smoking withdrawal, and phase 1 trials for cardiovascular disorders, liver disorders, metabolic disorders, and obesity.
Sources: en.wikipedia.org
It separates components in a liquid sample and measures their amounts using a detector. Results can indicate concentration, purity, or identity based on retention time and detector response. The technique works for mixtures that can be dissolved and filtered.
It offers high resolution, reproducibility, and compatibility with many sample types. A single run can separate and quantify multiple analytes. It is common in pharmaceutical, food, environmental, and industrial laboratories.
Samples must be soluble in a suitable mobile phase and free of particles that can block the column. Detector response depends on analyte structure, so some compounds need derivatization or alternative detection. Complex matrices may require extensive sample preparation.
HPLC separates and quantifies compounds in a liquid sample. Detectors produce a response proportional to the amount of a compound passing through the flow cell. Identification by retention time requires comparison with a known standard.