Chromatogram comes up often in conversation and rarely with the context attached. Here we lay out the basics in order, then work through the practical considerations.
Last reviewed on 2026-02-25. Where a claim depends on a specific study, the study is described rather than over-claimed.
High-performance liquid chromatography is an analytical technique that separates components in a liquid sample. A pump moves a liquid mobile phase through a column packed with a solid stationary phase. Compounds interact differently with both phases and travel at different rates, leaving the column at distinct retention times. A detector records these arrivals as peaks on a chromatogram. The resulting pattern supports identification and quantification of substances in mixtures. Modern instruments use high pressure to force solvent through small particles, which improves speed and resolution compared with older low-pressure liquid chromatography methods.
Separation in HPLC depends on the chemistry of the stationary phase, the composition of the mobile phase, and the physical properties of the column. Reverse-phase separations use a nonpolar stationary phase and a polar mobile phase, and they are common for many organic compounds. Ion-exchange, size-exclusion, and normal-phase modes serve other classes of analytes. Gradient elution changes solvent strength over time, while isocratic elution holds it constant. Flow rate, temperature, particle size, and column length all influence peak shape and resolution. Detection may use ultraviolet absorbance, fluorescence, refractive index, or mass spectrometry, depending on the analyte and the required sensitivity.
Routine HPLC testing compares a sample result with a calibration curve prepared from known reference standards. Peak area or peak height is plotted against concentration, and the curve is used to estimate unknown amounts. Retention time supports tentative identification when compared with a standard, though mass spectrometry or another confirmatory method may be needed for definitive identification. Pre-run checks verify repeatability, resolution, and peak symmetry before sample analysis. Limits of detection and quantification describe the smallest amounts that can be reliably observed or measured. Sample preparation, filtration, and degassing help prevent column damage and inconsistent results.
Instrumentation includes a solvent delivery system, an autosampler, a column oven, and one or more detectors. Reversed-phase columns with chemically modified silica are widely used, but normal-phase, ion-exchange, size-exclusion, and affinity modes exist for specific separations. Detectors may rely on ultraviolet absorbance, fluorescence, refractive index, or mass spectrometry. Column temperature, mobile phase composition, and flow rate are adjusted to improve resolution. System pressure is monitored because rising pressure can indicate column blockage or deteriorating packing.
Separation performance depends on particle size, pore size, column length, and the chemistry of the stationary phase. Smaller particles generally improve efficiency but require higher pressure and suitable instrumentation. The mobile phase often contains buffers and organic solvents that influence retention and selectivity. Testing labs select conditions based on the analytes, sample matrix, and required sensitivity. Method development frequently involves screening several columns and solvent mixtures before a final set of conditions is chosen.
High-performance liquid chromatography is an analytical technique that separates components in a liquid sample by passing them through a packed column under pressure. A pump delivers a mobile phase at a controlled flow rate, and an injector introduces the sample into the stream. Differences in how analytes partition between the mobile phase and the stationary phase cause them to exit the column at different times. Detection then records a signal proportional to the amount of each separated substance. The resulting chromatogram provides retention times and peak areas for identification and quantification.
| Property | Value | Notes |
|---|---|---|
| Common abbreviation | HPLC | High-performance liquid chromatography |
| Separation basis | Differential partitioning | Between liquid mobile phase and solid stationary phase |
| Common mode | Reverse phase | Nonpolar column, polar mobile phase |
| Typical detector | UV-Vis absorbance | Widely used for compounds with chromophores |
| Typical column particle size | 2–5 µm | Smaller particles can improve resolution |
Practical HPLC testing depends on careful sample preparation and instrument maintenance. Samples may require filtration, dilution, pH adjustment, or extraction to avoid column damage and matrix interference. Mobile phases are degassed and filtered, and columns are equilibrated before injection. Common problems include peak tailing, baseline drift, ghost peaks, carryover, and co-elution of analytes. Documentation of instrument logs, calibration records, and electronic audit trails supports data integrity and traceability. Ongoing training and routine maintenance help reduce variability between analysts and laboratories.
Quality control laboratories use HPLC to check identity, purity, concentration, and stability of raw materials and finished products. A validated method specifies the column, mobile phase, flow rate, detection wavelength, injection volume, and run time. Samples are prepared and compared against reference standards of known concentration. The resulting chromatogram provides quantitative data, such as assay values and impurity levels. This approach is common in pharmaceutical, food, environmental, and industrial testing where consistent measurements are required.
Method validation demonstrates that an analytical procedure is suitable for its intended purpose. Typical validation characteristics include accuracy, precision, specificity, linearity, range, limit of detection, limit of quantitation, and robustness. Regulatory guidance from bodies such as the International Council for Harmonisation and the United States Pharmacopeia outlines expectations, though specific criteria depend on the product and method. System suitability tests are run before sample analysis to confirm resolution, peak symmetry, column efficiency, and injection repeatability. Failure of these checks can invalidate a batch of measurements.
Routine HPLC testing depends on controlled reagents, calibrated instruments, and documented procedures. Columns degrade over time, so retention times and peak shapes are monitored for drift. Mobile phases are filtered and degassed to prevent pump damage and detector noise. Reference standards must be traceable and stored under suitable conditions. Data handling systems record injections, calculations, and audit trails. Quality control samples interspersed with unknowns help detect errors during a run.
Developing an HPLC method begins with defining the purpose, such as quantifying a main component, measuring impurities, or confirming identity. Analysts select separation mode, column, mobile phase, detection, and sample preparation based on analyte properties and matrix. Experiments vary solvent strength, pH, buffer type, and temperature to achieve resolution between critical peaks. The goal is a robust method that produces reliable results across instruments and operators. Method development often involves trial runs and statistical optimization.
=== Assembly === Ribosomal RNA's integration and assembly into ribosomes begins with their folding, modification, processing and assembly with ribosomal proteins to form the two ribosomal subunits, the LSU and the SSU. In Prokaryotes, rRNA incorporation occurs in the cytoplasm due to the lack of membrane-bound organelles. In Eukaryotes, however, this process primarily takes place in the nucleolus and is initiated by the synthesis of pre-RNA. This requires the presence of all three RNA polymerases. In fact, the transcription of pre-RNA by RNA polymerase I accounts for about 60% of cell's total cellular RNA transcription. This is followed by the folding of the pre-RNA so that it can be assembled with ribosomal proteins. This folding is catalyzed by endo- and exonucleases, RNA helicases, GTPases and ATPases. The rRNA subsequently undergoes endo- and exonucleolytic processing to remove external and internal transcribed spacers. The pre-RNA then undergoes modifications such as methylation or pseudouridinylation before ribosome assembly factors and ribosomal proteins assemble with the pre-RNA to form pre-ribosomal particles. Upon going under more maturation steps and subsequent exit from the nucleolus into the cytoplasm, these particles combine to form the ribosomes. The basic and aromatic residues found within the primary structure of rRNA allow for favorable stacking interactions and attraction to ribosomal proteins, creating a cross-linking effect between the backbone of rRNA and other components of the ribosomal unit.
TMA-2 (2,4,5-trimethoxyamphetamine) is very similar in chemical structure to the monoaminergic neurotoxin 6-hydroxydopamine (2,4,5-trihydroxyphenethylamine). A variety of derivatives of TMA-2 (MMM) have been developed and studied, such as MEM among others.
==== Quarantine/isolation ==== Cats with suspected or diagnosed FPLV should be kept in isolation. This non-enveloped virus is very resistant to environmental conditions and many disinfectants, is highly contagious, and rapidly accumulates in the environment due to high shedding of virus from affected animals. Strict protocols for containment – with isolation, minimal handling, and disinfection of all potential sources of fomites – is warranted. Recovered cats can still shed the virus for up to six weeks and can carry it on their body for prolonged periods.
==== Hypogonadism ==== Insufficient or low levels of testosterone in the body can affect the proper functioning of the male reproductive structures and thus lead to decreased secretions or hypospermia. Long-term exposure to drugs with anti-androgen properties (e.g. spironolactone) can also lead to infertility or low volume of semen. Testosterone is produced by the testes and can be used to diagnose hypogonadism.
Sources: en.wikipedia.org
=== Solubility === Carboxylic acids are polar. Because they are both hydrogen-bond acceptors (the carbonyl −C(=O)−) and hydrogen-bond donors (the hydroxyl −OH), they also participate in hydrogen bonding. Together, the hydroxyl and carbonyl group form the functional group carboxyl. Carboxylic acids usually exist as dimers in nonpolar media due to their tendency to "self-associate". Smaller carboxylic acids (1 to 5 carbons) are soluble in water, whereas bigger carboxylic acids have limited solubility due to the increasing hydrophobic nature of the alkyl chain. These longer chain acids tend to be soluble in less-polar solvents such as ethers and alcohols. Aqueous sodium hydroxide and carboxylic acids, even hydrophobic ones, react to yield water-soluble sodium salts. For example, enanthic acid has a low solubility in water (0.2 g/L), but its sodium salt is very soluble in water.
{\displaystyle \tau _{ij}=\mu \left({\frac {\partial v_{i}}{\partial x_{j}}}+{\frac {\partial v_{j}}{\partial x_{i}}}-{\frac {2}{3}}\delta _{ij}\nabla \cdot \mathbf {v} \right)+\kappa \delta _{ij}\nabla \cdot \mathbf {v} }
== Self-report and 2013 investigations == On 5 February 2013, Essendon reported itself to the AFL and the Australian Sports Anti-Doping Authority (ASADA) over concerns about the program. Two days later, the Australian Crime Commission (ACC) released a broad report entitled "Organised Crime and Drugs in Sport", the culmination of a twelve-month investigation which outlined an increase in illegal activities in sport across Australia, including drug use, match-fixing, and links to organised crime; part of the report dealt with an increase in the seizure and use of steroids and illegal supplements, and included an anonymous reference to Essendon's program. The timings of these events led to speculation that the AFL, having been privy to a confidential briefing on the ACC report, had tipped off Essendon and encouraged the club to self-report before the report went public, but this was never proven and was denied by the AFL. Within days, Essendon removed banners and murals from the façade at Windy Hill bearing the words "whatever it takes", which was the slogan of the club's 2013 membership drive, but now carried unfortunate doping connotations; the club struggled to distance itself from the bad publicity associated with the slogan in the wake of the scandal. Following Essendon's self-reporting, ASADA and the AFL launched a joint investigation into the supplements program and conducted that investigation over the next sixteen months.
=== Positron emission tomography === Positron emission tomography (PET) imaging in theranostics provides insight into metabolic and molecular processes within the body. The PET scanner detects photons and creates three-dimensional images that enable visualization and quantification of physiological and biochemical processes. PET imaging uses radiotracers that target specific molecules or processes. For example, [18F] fluorodeoxyglucose (FDG) is commonly used to assess glucose metabolism, as cancer cells exhibit increased glucose uptake. Other radiotracers target specific receptors, enzymes, or transporters, allowing the evaluation of various physiological and pathological processes. PET imaging plays a role in both diagnosis and treatment planning. It aids in the identification and staging of diseases, such as cancer, by visualizing the extent and metabolic activity of tumors. PET scans can also guide treatment decisions by assessing treatment response and monitoring disease progression. Additionally, PET imaging is used to determine the suitability of patients for targeted therapies based on specific molecular characteristics, enabling personalized treatment approaches.
Tetramethylurea (TMU) is the organic compound with the formula (Me2N)2CO. It is a substituted urea. This colorless liquid is used as an aprotic-polar solvent, especially for aromatic compounds and is used e. g. for Grignard reagents. TMU has a mild aromatic odor and, unlike other compounds of urea, is a colorless liquid.
Sources: en.wikipedia.org
=== Mechanism of action === Phencyclidine is a noncompetitive NMDA receptor antagonist that blocks the activity of the NMDA receptor to cause anaesthesia and analgesia without causing cardiorespiratory depression. NMDA is an excitatory receptor in the brain, when activated normally the receptor acts as an ion channel and there is an influx of positive ions through the channel to cause nerve cell depolarisation. Phencyclidine inhibits the NMDA receptor by binding to the specific PCP binding site located within the ion channel. The PCP binding site is near the magnesium blocking site, which may explain the similar inhibitory effects. Binding at the PCP site is mediated by two non-covalent interactions within the receptor: hydrogen bonding and hydrophobic interaction. Binding is also controlled by the gating mechanism of the ion channel. Because the PCP site is located within the ion channel, a coagonist such as glycine must bind and open the channel for PCP to enter, bind to the PCP site, and block the channel.
The monoepoxide of treosulfan (EBDM) exhibited greater brain penetration than the parent compound, with tissue-to-plasma ratios of 0.25 in young adult rats and 0.50 in juvenile rats. The elimination half-life of treosulfan in patients is short, averaging 1.5–2.0 h. The apparent elimination half-life of the biologically active epoxides is similar to that of treosulfan, reflecting the phenomenon of formation rate-limited elimination. The most common side effects include infections, nausea (feeling sick), stomatitis (inflammation of the lining of the mouth), vomiting, diarrhea, and abdominal pain (belly ache). Tiredness, febrile neutropenia (low white blood cell counts with fever) and high blood levels of bilirubin (a breakdown product of red blood cells) are also seen in more than 1 in 10 adults, and rash also affects more than 1 in 10 children. The most common adverse reactions include musculoskeletal pain, stomatitis, pyrexia, nausea, edema, infection, and vomiting. Selected grade 3 or 4 nonhematological laboratory abnormalities include increased GGT, increased bilirubin, increased ALT, increased AST, and increased creatinine. Treosulfan was authorized for medical use in the European Union in June 2019, and approved for medical use in the United States in January 2025.
In the 5–10 years since 2010, there has been a change in the ways new antimicrobial agents are discovered and developed – principally via the formation of public-private funding initiatives. These include CARB-X, which focuses on nonclinical and early phase development of novel antibiotics, vaccines, rapid diagnostics; Novel Gram Negative Antibiotic (GNA-NOW), which is part of the EU's Innovative Medicines Initiative; and Replenishing and Enabling the Pipeline for Anti-infective Resistance Impact Fund (REPAIR). Later stage clinical development is supported by the AMR Action Fund, which in turn is supported by multiple investors with the aim of developing 2–4 new antimicrobial agents by 2030. The delivery of these trials is facilitated by national and international networks supported by the Clinical Research Network of the National Institute for Health and Care Research (NIHR), European Clinical Research Alliance in Infectious Diseases (ECRAID) and the recently formed ADVANCE-ID, which is a clinical research network based in Asia. The Global Antibiotic Research and Development Partnership (GARDP) is generating new evidence for global AMR threats such as neonatal sepsis, treatment of serious bacterial infections and sexually transmitted infections as well as addressing global access to new and strategically important antibacterial drugs. The discovery and development of new antimicrobial agents has been facilitated by regulatory advances, which have been principally led by the European Medicines Agency (EMA) and the Food and Drug Administration (FDA).
Psychological assessment usually includes determining a mental disorder diagnosis. Many countries use the International Statistical Classification of Diseases and Related Health Problems (ICD-10 or ICD-11) while the US most often uses the Diagnostic and Statistical Manual of Mental Disorders (DSM-5). Both are nosological systems that largely assume categorical disorders diagnosed through the application of sets of criteria including symptoms and signs. Several new models are being discussed, including a "dimensional model" based on empirically validated models of human differences (such as the five factor model of personality) and a "psychosocial model", which would take changing, intersubjective states into greater account. The proponents of these models claim that they would offer greater diagnostic flexibility and clinical utility without depending on the medical concept of illness. However, they also admit that these models are not yet robust enough to gain widespread use, and should continue to be developed.
This layer acts as a nucleation site during the next step, where a process of electroless deposition layers a coating of gold on the nanotubes to form metal-insulator-metal trilayer coaxial nanocables. Peptide nanotubes are able to produce nanowires of uniform size, and this is particularly useful in the nano-electric applications as electrical and magnetic properties are sensitive to size. Nanotubes' exceptional mechanical strength and stability makes them excellent materials for application in this area. Nanotubes have also been used in developing electrochemical biosensing platforms and have proved to have great potential. Dipeptide nanotubes deposited on graphite electrodes improved electrode sensitivity; thiol-modified nanotubes deposited on gold with a coating of enzymes improved sensitivity and reproducibility for the detection of glucose and ethanol, as well as a shortened detection time, large current density, and improved stability. Nanotubes have also been successfully coated with proteins, nanocrystals, and metalloporphyrin through hydrogen bonding, and these coated tubes have great potential as chemical sensors. Designed peptides with a known structure that would self-assemble into a regular growth template would enable the self-assembly of nanoscale electronic circuits and devices. However, one issue that has yet to be resolved is the ability to control the positioning of the nanostructures. This positioning relative to substrates, to each other, and to other functional components is crucial.
Sources: en.wikipedia.org
HPLC testing measures the presence and amount of one or more compounds in a liquid sample. It separates mixture components and records detector responses as peaks, which are compared with reference standards. Results are usually reported as concentrations or relative percentages.
Retention time is the interval between sample injection and the detector response for a given compound. It depends on the compound's interactions with the stationary and mobile phases under set conditions. Matching a retention time to a standard supports tentative identification but is not always unique.
HPLC alone can separate unknown compounds and provide retention times, but it often cannot identify them with certainty. Coupling HPLC to mass spectrometry gives mass information that improves identification. Confirmation usually requires comparison with reference standards or complementary techniques.
HPLC separates and detects individual compounds in a liquid sample, producing peaks at characteristic retention times. Peak area or height can be used to estimate concentration when calibrated with known standards. It does not identify unknown compounds with certainty unless additional detectors or reference materials are used.