stationary phase 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.
Updated 2026-06-14. Numbers and descriptions here follow the published literature rather than marketing material.
HPLC testing is not a single fixed procedure; it is a family of separation modes. Reversed-phase, normal-phase, ion-exchange, size-exclusion, and affinity chromatography each suit different analyte properties. Reversed-phase methods dominate because they handle many neutral and moderately polar compounds. Detection can be optical, electrochemical, or mass spectrometric, and the detector dictates what information is available. Coupling with mass spectrometry increases selectivity and enables identification when standards are unavailable. The technique cannot separate every mixture without adjustment.
HPLC testing is an analytical technique used to separate, identify, and quantify components in a liquid sample. It relies on a pressurized mobile phase that carries the sample through a column packed with stationary phase. Different compounds travel at different rates because of interactions with the stationary and mobile phases. The resulting signal versus time is a chromatogram. Peak position indicates identity under specified conditions, while peak area or height relates to amount.
Laboratories apply HPLC testing across pharmaceutical, food, environmental, and industrial chemistry. The method can measure active ingredients, impurities, additives, preservatives, and degradation products. Sample preparation often includes dilution, filtration, and sometimes extraction or derivatization. The choice of column, mobile phase, pH, temperature, and detector depends on the analytes and matrix. Results are compared with reference standards to assign identity and concentration. Method suitability is judged by resolution, precision, and accuracy.
Regulatory and pharmacopeial texts shape how HPLC testing is performed and documented. The International Council for Harmonisation provides validation guidance, while pharmacopeias publish general chromatography chapters and monographs for specific materials. Accreditation standards such as ISO/IEC 17025 address laboratory competence and traceability. Inspectors may review instrument qualification, analyst training, reference material control, and electronic records. Open questions include how best to validate methods for new complex products and how to handle automated data processing. Laboratories generally resolve these issues through risk assessment, method lifecycle management, and documented scientific justification.
In quality control laboratories, HPLC testing supports batch release, raw material checks, stability studies, and impurity profiling. A validated method defines sample preparation, instrument settings, calibration, and acceptance criteria. Analysts compare results with specifications and investigate out-of-specification outcomes before a batch is approved. Documentation includes chromatograms, integration records, audit trails, and reagent details. Because results influence product decisions, laboratories follow formal quality systems and data integrity rules. The exact tests and limits depend on the material, its intended use, and the applicable regulatory framework.
| Property | Value | Notes |
|---|---|---|
| Abbreviation | HPLC | Also called high-performance liquid chromatography |
| Separation mechanism | Differential partitioning | Compounds distribute between mobile and stationary phases |
| Typical column chemistry | C18 (octadecylsilane) | Used in reversed-phase separations |
| Typical detector | UV-Vis or photodiode array | Mass spectrometry is common for trace and confirmatory work |
| Typical particle size | 1.8–5 µm | Smaller particles require higher pressure and can improve speed |
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.
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.
Key performance measures include retention time, peak area, peak height, resolution, tailing factor, and plate count. Retention time helps identify a peak under fixed conditions, but confirmation often requires a second method or detector. Peak area and height relate to concentration through calibration curves, which may be linear or nonlinear depending on the detector response. Resolution describes separation between adjacent peaks, while tailing factor and plate count describe peak shape and column efficiency. Performance checks verify these values before and during a run to confirm that the instrument is performing within limits.
High-performance liquid chromatography testing separates components of a liquid sample by forcing a mobile phase through a packed column. The stationary phase inside the column interacts with analytes to different degrees, so each compound exits at a characteristic retention time. A pump delivers solvent at controlled flow and pressure, while an injector introduces a precise sample volume. Detectors such as ultraviolet-visible, fluorescence, refractive index, or mass spectrometric instruments record the separated bands. The resulting chromatogram provides qualitative and quantitative information about the mixture.
Separation modes differ by the chemistry of the stationary phase and the composition of the mobile phase. Reversed-phase testing uses a nonpolar column and polar solvents, making it common for pharmaceutical, environmental, and food analytes. Normal-phase testing uses a polar column and nonpolar solvents for compounds that are poorly retained in reversed-phase systems. Ion-exchange and ion-pair methods separate charged species, while size-exclusion methods sort molecules by hydrodynamic volume. Gradient elution changes solvent strength over time to resolve complex mixtures, and isocratic elution holds solvent composition constant for simpler assays.
Degradation of an amino acid often begins with transamination, in which its amino group is transferred to α-ketoglutarate, forming glutamate. This process involves transaminases, often the same enzymes used in amino acid synthesis. In many vertebrates, the amino group is subsequently released as ammonia and converted to urea via the urea cycle for excretion. However, amino acid degradation can result in uric acid or ammonia instead, depending on the organism. For example, serine dehydratase converts serine directly to pyruvate and ammonia. After removal of one or more amino groups, the remaining carbon skeleton of an amino acid can serve as a precursor for synthesizing other amino acids, be further metabolized for energy after conversion into intermediates of glycolysis (typically via gluconeogenesis) or of the citric acid cycle, or be used for fatty acid synthesis and storage as triacylglycerol. Amino acids are bidentate ligands, forming transition metal amino acid complexes.
Ana María Muñoz Jauregui (born 1969, Lima) is a Peruvian pharmacist, biochemist and nutritionist. She has served as Rector and, since 2023, as the Vice Rector of research at San Ignacio de Loyola University (USIL). Muñoz has authored numerous publications and received national and international awards, such as silver, gold medals and semi-grand prize wins at the International KIWIE Award. Muñoz, was born in Lima, Peru. In 1996, she attended the National University of San Marcos (UNMSM), where she earned her pharmaceutical degree. While studying, she developed an interest in the field of nutrition. She earned a master's degree in biochemistry and nutrition, and earned a PhD in Pharmacy and Biochemistry from the National University of San Marcos (UNMSM) in 2006.
As mentioned above, G-proteins may terminate their own activation due to their intrinsic GTP→GDP hydrolysis capability. However, this reaction proceeds at a slow rate (≈0.02 times/sec) and, thus, it would take around 50 seconds for any single G-protein to deactivate if other factors did not come into play. Indeed, there are around 30 isoforms of RGS proteins that, when bound to Gα through their GAP domain, accelerate the hydrolysis rate to ≈30 times/sec. This 1500-fold increase in rate allows for the cell to respond to external signals with high speed, as well as spatial resolution due to limited amount of second messenger that can be generated and limited distance a G-protein can diffuse in 0.03 seconds. For the most part, the RGS proteins are promiscuous in their ability to deactivate G-proteins, while which RGS is involved in a given signaling pathway seems more determined by the tissue and GPCR involved than anything else. In addition, RGS proteins have the additional function of increasing the rate of GTP-GDP exchange at GPCRs, (i.e., as a sort of co-GEF) further contributing to the time resolution of GPCR signaling. In addition, the GPCR may be desensitized itself. This can occur as:
The inverted terminal repeat (ITR) sequences comprise 145 bases each. They were named so because of their symmetry, which was shown to be required for efficient multiplication of the AAV genome. The feature of these sequences that gives them this property is their ability to form a hairpin, which contributes to so-called self-priming that allows primase-independent synthesis of the second DNA strand. The ITRs were also shown to be required for both integration of the AAV DNA into the host cell genome (19th chromosome in humans) and rescue from it, as well as for efficient encapsidation of the AAV DNA combined with generation of a fully assembled, deoxyribonuclease-resistant AAV particles. With regard to gene therapy, ITRs seem to be the only sequences required in cis next to the therapeutic gene: structural (cap) and packaging (rep) proteins can be delivered in trans. With this assumption many methods were established for efficient production of recombinant AAV (rAAV) vectors containing a reporter or therapeutic gene. However, it was also published that the ITRs are not the only elements required in cis for the effective replication and encapsidation. A few research groups have identified a sequence designated cis-acting Rep-dependent element (CARE) inside the coding sequence of the rep gene. CARE was shown to augment the replication and encapsidation when present in cis.
Several approaches have been developed to analyze the location of organelles, genes, proteins, and other components within cells. A gene ontology category, cellular component, has been devised to capture subcellular localization in many biological databases. Microscopic pictures allow for the location of organelles as well as molecules, which may be the source of abnormalities in diseases. Finding the location of proteins allows us to predict what they do. This is called protein function prediction. For instance, if a protein is found in the nucleus it may be involved in gene regulation or splicing. By contrast, if a protein is found in mitochondria, it may be involved in respiration or other metabolic processes. There are well developed protein subcellular localization prediction resources available, including protein subcellular location databases, and prediction tools.
Sources: en.wikipedia.org
In March 2003, Cambridge Antibody Technology (CAT) stated its wish to "initiate discussions regarding the applicability of the royalty offset provisions for Humira" with Abbott Laboratories in the High Court of London. In November 2004, the trial began, and in December 2004, Justice Hugh Laddie ruled for CAT. A short version of the full statement of the proceedings was released. In it Justice Laddie remarked, "Abbott was in error when it made its first royalty payment to CAT calculated on the basis that only 2% of the Net Sales was due. It should have calculated on the basis of the full royalty of just over 5% and should have paid and continued to pay CAT accordingly." Justice Laddie went on to observe "...that the construction advanced by Abbott does violence to the language of the agreements, renders them obscure and makes little or no commercial sense. For this reason CAT wins the action." Abbott was required to pay CAT US$255 million, some of which was to be passed to its partners in development. Of this sum, the Medical Research Council received US$191 million, and in addition, Abbott was asked to pay the MRC a further US$7.5 million over five years from 2006, providing that Humira remains on the market. The MRC also is to receive a further £5.1 million (sterling) in respect of past royalties.
The Analytical Information Markup Language (AnIML) is an open ASTM XML standard for storing and sharing analytical chemistry and biological data. A main reason of using AnIML is that FAIR data (Findable, Accessible, Interoperable and Reusable) standards are automatically implemented. As AnIML's structure is human-readable, Accessibility is given. Interoperability, Reusability and Findability are secured by the AnIML Core and AnIML Technique Definitions. AnIML has been continuously worked on starting from 2003 up to 2020. The last AnIML Core Version update happened in 2010. So far, no standardisation document nor public example files have been published. The standard exists only in pre-release form. AnIML is a XML standard which consists of two logical layers:
A typical intracellular concentration of ATP is 1–10 μmol per gram of muscle tissue in a variety of eukaryotes. The dephosphorylation of ATP and rephosphorylation of ADP and AMP occur repeatedly in the course of aerobic metabolism. ATP can be produced by a number of distinct cellular processes; the three main pathways in eukaryotes are (1) glycolysis, (2) the citric acid cycle/oxidative phosphorylation, and (3) beta-oxidation. The overall process of oxidizing glucose to carbon dioxide, the combination of pathways 1 and 2, known as cellular respiration, produces about 30 equivalents of ATP from each molecule of glucose. ATP production by a non-photosynthetic aerobic eukaryote occurs mainly in the mitochondria, which comprise nearly 25% of the volume of a typical cell.
The cytoskeleton is found underlying the cell membrane in the cytoplasm and provides a scaffolding for membrane proteins to anchor to, as well as forming organelles that extend from the cell. Indeed, cytoskeletal elements interact extensively and intimately with the cell membrane. Anchoring proteins restricts them to a particular cell surface—for example, the apical surface of epithelial cells that line the vertebrate gut—and limits how far they may diffuse within the bilayer. The cytoskeleton is able to form appendage-like organelles, such as cilia, which are microtubule-based extensions covered by the cell membrane, and filopodia, which are actin-based extensions. These extensions are ensheathed in membrane and project from the surface of the cell in order to sense the external environment and/or make contact with the substrate or other cells. The apical surfaces of epithelial cells are dense with actin-based finger-like projections known as microvilli, which increase cell surface area and thereby increase the absorption rate of nutrients. Localized decoupling of the cytoskeleton and cell membrane results in formation of a bleb.
Affimer molecules, also known as adhiron, are small proteins that bind to target proteins with affinity in the nanomolar range. These engineered non-antibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in different applications. These affinity reagents have been optimized to increase their stability, make them tolerant to a range of temperatures and pH, reduce their size, and to increase their expression in E.coli and mammalian cells.
Sources: en.wikipedia.org
The linear amino acid sequence of a protein is called the primary structure. The primary structure can be easily determined from the sequence of codons on the DNA gene that codes for it. In most proteins, the primary structure uniquely determines the 3-dimensional structure of a protein in its native environment. An exception is the misfolded prion protein involved in bovine spongiform encephalopathy. This structure is linked to the function of the protein. Additional structural information includes the secondary, tertiary and quaternary structure. A viable general solution to the prediction of the function of a protein remains an open problem. Most efforts have so far been directed towards heuristics that work most of the time.
Beta-peptides (β-peptides) are peptides derived from β-amino acids, in which the amino group is attached to the β-carbon (i.e. the carbon two atoms away from the carboxylate group). The parent β-amino acid is β-alanine (H2NCH2CH2CO2H), a common natural substance, but most examples feature substituents in place of one or more C-H bonds. β-peptides usually do not occur in nature. β-Peptide-based antibiotics are being explored as ways of evading antibiotic resistance. Early studies in this field were published in 1996 by the group of Dieter Seebach and that of Samuel Gellman.
Generally speaking single-particle measurement instruments desorb particles one at a time using a pulsed laser. The process is called laser desorption/ionization (LDI) and is the primary ionization method used for single-particle measurements. The main advantage of using LDI over thermal desorption, is the ability to analyze both non-refractory and refractory (e.g., mineral dust, soot) components of atmospheric aerosols. Laser vaporization allows precise laser firing when individual particles fly through the vaporization zone, and the systems are thus dubbed single particle mass spectrometers (SPMS). Several versions of SPMS have been reported, including the aerosol time-of-flight mass spectrometer (AToFMS), the laser mass analyzer for particles in the airborne state (LAMPAS), particle analysis by laser mass spectrometer (PALMS), the rapid single-particle mass spectrometer (RSMS), the bioaerosol mass spectrometer (BAMS) b194 Steele et al., 2003), the nanoaerosol mass spectrometer (NAMS), the single-particle laser ablation time-of-flight mass spectrometer (SPLAT), the single-particle aerosol mass spectrometer (SPAMS), and laser ablation aerosol particle time-of-flight mass spectrometer (LAAP-ToF-MS). Among the most commons of these instruments is the aerosol time-of-flight mass spectrometer (AToFMS).
3K3A-Activated Protein C (3K3A-APC) is an experimental drug for the treatment of stroke developed by ZZ Biotech, a company founded by Berislav Zlokovic. It is a genetically engineered variant of activated protein C designed to reduce anticoagulant activity while preserving its cytoprotective and anti-inflammatory properties. This recombinant protein is created by substituting three lysine residues with alanines, thereby minimizing the risk of bleeding associated with native activated protein C. 3K3A-APC exerts its therapeutic effects by targeting protease-activated receptor 1 (PAR1) pathway. A Phase 3 clinical trial was scheduled to start when the NIH paused the trial on November 16, 2023, due to concerns about falsification of data in the studies supporting the clinical trial. The drug was also being assessed for the treatment of Alzheimer's disease, and is currently being evaluated by Dermatherix for wound care.
Wheeler was awarded the Lab on a Chip Pioneers in Miniaturization Award (2017), an E.W.R. Steacie Fellowship from the Natural Sciences and Engineering Research Council of Canada (NSERC) (2015), a Connaught Foundation McLean Fellowship (2014), the Joseph Black Award from the Royal Society of Chemistry (2012), the Arthur F. Findeis Award from the American Chemical Society (2012), the Young Innovator Award in Analytical Chemistry (2011), and a Sloan Research Fellowship (2009). In January 2024, Wheeler was honored with the Ricardo Aroca Award, recognizing his contributions to analytical chemistry and advancing lab-on-a-chip techniques, particularly through the use of digital microfluidics (DMF), which enables precise manipulation of liquid droplets on electrode arrays. The Award is bestowed upon a scientist living in Canada who has made a notable contribution to the discipline of analytical chemistry while conducting research within the country.
Sources: en.wikipedia.org
It measures the presence and amount of one or more compounds in a liquid sample. Separation occurs in a column, and detection produces a signal proportional to concentration. Identification usually requires comparison with a known reference standard under the same conditions.
In most cases the sample is consumed or altered during analysis, though some detectors are non-destructive. Fractions can be collected after separation for further study. Repeated testing therefore requires additional sample.
Run times range from under a minute for fast methods to over an hour for complex separations. Sample preparation, equilibration, and data review add time. Throughput depends on instrument configuration and method requirements.
System suitability is a set of checks performed before and during an HPLC run to confirm that the instrument and method are working as expected. It may include retention time repeatability, resolution between peaks, peak symmetry, and signal intensity. Failing suitability criteria usually invalidates the run.