If you have been reading about Chromatogram 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 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.
Biodiversity informatics deals with the collection and analysis of biodiversity data, such as taxonomic databases, or microbiome data. Examples of such analyses include phylogenetics, niche modelling, species richness mapping, DNA barcoding, or species identification tools. A growing area is also macro-ecology, i.e. the study of how biodiversity is connected to ecology and human impact, such as climate change. The enormous number of published literature makes it virtually impossible for individuals to read every paper, resulting in disjointed sub-fields of research. Literature analysis aims to employ computational and statistical linguistics to mine this growing library of text resources. For example: Abbreviation recognition – identify the long-form and abbreviation of biological terms Named-entity recognition – recognizing biological terms such as gene names Protein–protein interaction – identify which proteins interact with which proteins from text The area of research draws from statistics and computational linguistics.
The American Journal of Respiratory Cell and Molecular Biology is a monthly peer-reviewed medical journal and an official publication of the American Thoracic Society. It covers research on the structure and function of the respiratory system under physiologic and pathophysiologic conditions. It was established in July 1989. The founding editors-in-chief were Jerome S. Brody, Robert M. Senior, and Mary C. Williams. John A. Mcdonald served as editor from 1993 to 1998. Kenneth B. Adler (North Carolina State University) served as editor from 2009 to 2016. Paul Schumacker (Northwestern University) served as editor from October 1, 2016, to October 21, 2023. Andrew Halayko (University of Manitoba) assumed the editorship on November 1, 2023. The journal is abstracted and indexed in BIOSIS Previews, Current Contents/Life Sciences, Current Contents/Critical Care Medicine, Embase, Index Medicus/MEDLINE/PubMed, Science Citation Index Expanded, and Scopus. According to the Journal Citation Reports, the journal has a 2024 impact factor of 5.3. Official website
The protein is 82.3 kDa and has a 40 amino acid long LEM domain located at its amino-terminal region. In its carboxyl end it has a RNA recognition motif (RRM). The LEM domain is also common to two other integral proteins of the INM: lamina-associated polypeptide 2 (LAP2) and emerin. The LEM segment enables LEMD3 to attach to the barrier-to-autointegration factor (BAF), and therefore, indirectly interact with the chromatin. LEMD3 also has several implications in regulating the cytokine family such as the transforming growth factor beta (TGF-β) and bone morphogenic protein (BMPs). The RRM domain in its carboxylic region attaches to the SMAD (protein) proteins, which is involved in mediating TGF-β cellular signalling. Consequently, LEMD3 indirectly regulates downstream genes. LEMD3 seems to play an important role in regulating the expression of several fundamental genes. LEMD3 has been associated with laminopathies as well as osteopoikilosis. Mutations in the LEMD3 gene have been linked to several genetic diseases such as osteopoikilosis, melorheostosis and Buschke–Ollendorff syndrome.
Annals of Clinical Biochemistry is a bimonthly peer-reviewed scientific journal covering all aspects of clinical biochemistry. The editor-in-chief is Michael J Murphy (University of Dundee). It was established 1960 and is published by SAGE Publications on behalf of The Association for Clinical Biochemistry and Laboratory Medicine. The journal is abstracted and indexed in: According to the Journal Citation Reports, its 2012 impact factor is 1.922, ranking it 12th out of 31 journals in the category "Medical Laboratory Technology". Official website
Sources: en.wikipedia.org
Gs exerts its effects via two pathways. Firstly, it directly opens L-type calcium channels (LTCC) in the plasma membrane. Secondly, it renders adenylate cyclase activated, resulting in an increase of cAMP, activating protein kinase A (PKA) which in turn phosphorylates several targets, such as phospholamban, LTCC, Troponin I (TnI), and potassium channels. The phosphorylation of phospholamban deactivates its own function which normally inhibits SERCA on the sarcoplasmic reticulum (SR) in cardiac myocytes. Due to this, more calcium enters the SR and is therefore available for the next contraction. LTCC phosphorylation increases its open probability and therefore allows more calcium to enter the myocyte upon cell depolarisation. Both of these mechanisms increase the available calcium for contraction and therefore increase inotropy. Conversely, TnI phosphorylation results in its facilitated dissociation of calcium from troponin C (TnC) which speeds the muscle relaxation (positive lusitropy). Potassium channel phosphorylation increases its open probability which results in shorter refractory period (because the cell repolarises faster), also increasing lusitropy. Furthermore, in nodal cells such as in the SA node, cAMP directly binds to and opens the HCN channels, increasing their open probability, which increases chronotropy.
The molecular weight of c7orf26 is 50 kilodaltons. The isoelectric point is 7.61. The protein sequence is uniquely rich for leucine at 15.8% of its composition, this may indicate a leucine-zipper. Further analysis from PSORT indicates that a leucine-zipper region is found at amino acid 318 and lasts until position 340 (22 amino acids long). There are no extremes with regards to acidity and alkalinity. c7orf26 has a positive charge cluster from amino acid 245 – 275 and does not have any negative, or mixed charge clusters. An even distribution of amino acids compose c7orf26. The percent composition of each amino acid is fairly consistent throughout the orthologs of the protein. The most distant ortholog displays the most variance in amino acid composition. There is a higher percent composition of tyrosine, histidine and leucine and a lower composition of valine and alanine.
Adenosine triphosphate (ATP) is a nucleoside triphosphate that provides free energy of approximately 58 kJ/mol (0.6 eV) to drive and support many processes in living cells, such as muscle contraction, nerve impulse propagation, and chemical synthesis. Found in all known forms of life, it is often referred to as the "molecular unit of currency" for intracellular energy transfer. When consumed in a metabolic process, ATP converts either to adenosine diphosphate (ADP) or to adenosine monophosphate (AMP). Other processes, such as oxidative phosphorylation or substrate-level phosphorylation, regenerate ATP. ATP is also a precursor to DNA and RNA, and is used as a coenzyme. Daily, an average adult human recycles through synthesis and hydrolysis around 50 kilograms of ATP (about 100 moles). From the perspective of biochemistry, ATP is classified as a nucleoside triphosphate, which indicates that it consists of three components: a nitrogenous base (adenine), the sugar ribose, and the triphosphate.
Advanced product quality planning is a process developed in the late 1980s by a commission of experts who gathered around the 'Big Three' of the US automobile industry: Ford, GM, and Chrysler. Representatives from the three automotive original equipment manufacturers (OEMs) and the Automotive Division of American Society for Quality Control (ASQC) created the Supplier Quality Requirement Task Force for developing a common understanding on topics of mutual interest within the automotive industry. This commission worked five years to analyze the then-current automotive development and production status in the US, Europe, and especially in Japan. At the time, the Japanese automotive companies were successful in the US market. APQP is utilized by US automakers and some of their affiliates. Tier 1 suppliers are typically required to follow APQP procedures, techniques, and are also typically required to be audited and registered to IATF 16949. This methodology is also being used in other manufacturing sectors. The Automotive Industry Action Group (AIAG) is a non-profit association of automotive companies founded in 1982. The basis for the process control plan is described in AIAG's APQP manual These include:
Absorption is the journey of a drug travelling from the site of administration to the site of action. The drug travels by some route of administration (oral, topical-dermal, etc.) in a chosen dosage form (e.g., tablets, capsules, or in solution). Absorption by some other routes, such as intravenous therapy, intramuscular injection, enteral nutrition, is even more straightforward and there is less variability in absorption and bioavailability is often near 100%. Intravascular administration does not involve absorption, and there is no loss of drug. The fastest route of absorption is inhalation. Absorption is a primary focus in drug development and medicinal chemistry, since a drug must be absorbed before any medicinal effects can occur. Moreover, the drug's pharmacokinetic profile can be easily and significantly changed by adjusting factors that affect absorption.
Sources: en.wikipedia.org
The modern era of CCC began with the development of the planetary centrifuge by Dr. Yoichiro Ito which was first introduced in 1966 as a closed helical tube which was rotated on a "planetary" axis as is turned on a "sun" axis. A flow-through model was subsequently developed and the new technique was called countercurrent chromatography in 1970. The technique was further developed by employing test mixtures of DNP amino acids in a chloroform:glacial acetic acid:0.1 M aqueous hydrochloric acid (2:2:1 v/v) solvent system. Much development was needed to engineer the instrument so that required planetary motion could be sustained while the phases were being pumped through the coil(s). Parameters such as the relative rotation of the two axes (synchronous or non-synchronous), the direction of flow through the coil, and the rotor angles were investigated.
Because lipopolysaccharides are potent inflammatory agents, and the action of antibiotics can result in the release of these compounds, the binding capacity of BPI was explored as a possible means of reducing injury. Xoma Ltd. developed a recombinant 21kDa portion of the BPI molecule called rBPI21, NEUPREX, or opebecan. In a trial, it was found to decrease the mortality of Gram-negative bacterial-induced sepsis. Studies suggest that its binding activity is not the means by which it mediates its protective effect. Studies show biological effects with Gram-positive bacteria and even in infection by the protozoan, Toxoplasma gondii. The N-terminal portion of murine BPI (199 amino acids) genetically fused to Halobacterium sp. NRC-1 GvpC protein was bound to the surface of gas vesicle nanoparticles (GVNPs) and tested for protective activity using a murine model of endotoxic shock. Depending on the time of delivery and exposure to lethal concentrations of lipopolysaccharide (LPS) and D-galactosamine, the treatment resulted in increased survival and reduced symptoms of inflammation, including inflammatory anemia, recruitment of neutrophils, liver apoptosis as well as increased pro-inflammatory serum cytokine levels. When administered via footpad and before LPS exposure, there was 100% survival of the experimental cohort.
The urine test may be a chromatographic immunoassay or any of several other test formats, home-, physician's office-, or laboratory-based. Published detection thresholds range from 20 to 100 mIU/mL, depending on the brand of test. Early in pregnancy, more accurate results may be obtained by using the first urine of the morning (when urine is most concentrated). When the urine is dilute (specific gravity less than 1.015), the hCG concentration may not be representative of the blood concentration, and the test may be falsely negative. The serum test, using 2-4 mL of venous blood, is typically a chemiluminescent or fluorimetric immunoassay that can detect βhCG levels as low as 5 mIU/mL and allows quantification of the βhCG concentration. The hCG levels grow exponentially after conception and implantation. hCG levels typically peak around weeks 8-11 of pregnancy and are generally higher in the first trimester compared to the second trimester. The following is a list of serum hCG levels: LMP is the last menstrual period dated from the first day of the last menstrual period
Biochemistry laboratories often use in vitro studies to explore ATP-dependent molecular processes. ATP analogs are also used in X-ray crystallography to determine a protein structure in complex with ATP, often together with other substrates. Enzyme inhibitors of ATP-dependent enzymes such as kinases are needed to examine the binding sites and transition states involved in ATP-dependent reactions. Most useful ATP analogs cannot be hydrolyzed as ATP would be; instead, they trap the enzyme in a structure closely related to the ATP-bound state. Adenosine 5′-(γ-thiotriphosphate) is an extremely common ATP analog in which one of the gamma-phosphate oxygens is replaced by a sulfur atom; this anion is hydrolyzed at a dramatically slower rate than ATP itself and functions as an inhibitor of ATP-dependent processes. In crystallographic studies, hydrolysis transition states are modeled by the bound vanadate ion. Caution is warranted in interpreting the results of experiments using ATP analogs, since some enzymes can hydrolyze them at appreciable rates at high concentration.
One way to effectively deal with the inherent multi-physics and the control systems development that is involved when including intelligent systems, is to adopt the V-Model approach to systems development, as has been widely used in the automotive industry for twenty years or more. In this V-approach, system-level requirements are propagated down the V via subsystems to component design, and the system performance is validated at increasing integration levels. Engineering of mechatronic systems requires the application of two interconnected "V-cycles": one focusing on the multi-physics system engineering (like the mechanical and electrical components of an electrically powered steering system, including sensors and actuators); and the other focuses on the controls engineering, the control logic, the software and realization of the control hardware and embedded software.
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.