Low Molecular Weight Compounds Research Articles

Rhodotorulic Acid and its Derivatives: Synthesis, Properties, and Applications.

Siderophores are low molecular weight compounds produced by microorganisms to scavenge iron in iron-deficient environments. Rhodotorulic acid, a natural hydroxamate siderophore, plays a vital role in iron acquisition for fungi and bacteria. As the simplest natural hydroxamate siderophore, it exhibits a high affinity for ferric ions, enabling it to form stable complexes that facilitate iron uptake and transport within microorganisms. This article provides a comprehensive analysis of this hydroxamate siderophore, rhodotorulic acid, its synthesis, physicochemical properties, and biological significance. It also explores its applications in antifungal and plant protection strategies. Insights into RA derivatives reveal distinct biological effects and applications with potential in various fields, from antioxidants to antifungals. Rhodotorulic acid and its derivatives show promise for novel therapies, plant protection strategies, and iron supplementation in agriculture. Understanding their properties could advance science and medicine with sustainable practices.

Calcium Phosphate Nanoparticles as Carriers of Low and High Molecular Weight Compounds

Nanoparticles could improve the bioavailability of active agents of various natures to human, animal, and plant tissues. In this work, we compared two methods on the synthesis of calcium phosphate nanoparticles (CaPs), differed by the synthesis temperature, pH, and concentration of the stabilizing agent, and explored the possibilities of incorporation of a low-molecular-weight peptide analogue enalaprilat, the enzyme superoxide dismutase 1 (SOD1), as well as DNA and dsRNA into these particles, by coprecipitation and sorption. CaPs obtained with and without cooling demonstrated the highest inclusion efficiency for enalaprilat upon coprecipitation: 250 ± 10 μg/mg of CaPs and 340 ± 30 μg/mg of CaPs, respectively. Enalaprilat sorption on the preliminarily formed CaPs was much less effective. SOD1 was only able to coprecipitate with CaPs upon cooling, with SOD1 loading 6.6 ± 2 μg/mg of CaPs. For the incorporation of DNA, the superiority of the sorption method was demonstrated, allowing loading of up to 88 μg/mg of CaPs. The ability of CaPs to incorporate dsRNa by sorption was also demonstrated by electrophoresis and atomic force microscopy. These results could have important implications for the development of the roots for incorporating substances of different natures into CaPs for agricultural and medical applications.

Accessing microbial natural products of the past

Abstract Microbial natural products—low molecular weight compounds biosynthesized by microorganisms—form the foundation of important modern therapeutics, including antibiotics, immunomodulators, and anti-cancer agents. This perspective discusses and contrasts two emerging approaches for uncovering natural products of the past. On the one hand, ancestral sequence reconstruction allows recreating biosynthetic pathways that date back hundreds of millions of years. On the other hand, sequencing and de novo assembly of ancient DNA reveals the biosynthetic potential of ancient microbial communities up to 100 000 years. Together, these approaches unveil an otherwise hidden reservoir of functional and structural molecular diversity. They also offer new opportunities to study the biological function and evolution of these molecules within an archaeological context.

Abstract 4135476: The Cardiomyocyte Hypertrophy Inhibitor RFN-409, Identified by High Throughput Screening Assay, Suppresses Pressure Overload-induced Systolic Dysfunction in Mice by Suppressing p38 Activity

Purpose: When the heart is exposed to stresses such as myocardial infarction or hypertension, it undergoes compensatory hypertrophy in response. However, continuation of the stress causes this compensatory mechanism to fail, and eventually systolic dysfunction or decompensated heart failure occur. As the hypertrophy of individual cardiomyocytes has been observed in this process, controlling cardiomyocyte hypertrophy is a potential target the prevention and treatment of heart failure. In this study, we constructed a high throughput screening (HTS) assay using cardiomyocyte hypertrophy as an index parameter. Compounds that inhibit cardiomyocyte hypertrophy were selected from our low molecular compound library. Methods and Results: In the primary screening, cultured rat primary cardiomyocytes were treated with each compound at a final concentration of 1 µM and then stimulated with 30 µM phenylephrine (PE) for 48 hours. These cells were subjected to fluorescent immunostaining with α-actinin, and cardiomyocyte area was measured using an ArrayScan™ system. The hypertrophy inhibition rate (%) of each compound was calculated as [(PE(+) - compound) / (PE(+) - PE(-))] × 100. The compounds with a hypertrophy inhibition rate greater than 50% and less than 150% were selected as hit compounds. In the secondary screening, these hit compounds were evaluated based on the dose-dependency of cardiomyocyte hypertrophy inhibition and the inhibition of the mRNA levels of the cardiac hypertrophy response genes ANF and BNP using real-time PCR. From the 269 low molecular-weight compounds in the original compound library, eight were selected through the primary and secondary screenings. Among them, we focused on Reference Number 409 (RFN-409). Western blotting indicated that RFN-409 inhibited PE-induced p38 activation. Next, we investigated the effect of RFN-409 on heart failure. Eight-week-old male C57 BL/6J mice were subjected to transverse aortic constriction (TAC) surgery and then randomly assigned to intraperitoneal treatment with RFN-409 (3, 10 mg/kg) or vehicle for eight weeks. RFN-409 at 10 mg/kg significantly prevented TAC-induced increase in left ventricular posterior wall thickness and decrease in left ventricular fractional shortening. Discussion: RFN-409 suppressed TAC-induced development of heart failure, at least partially by inhibiting p38 activity. These findings suggest that RFN-409 may be an effective agent for heart failure therapy.

How to Find a Fragment: Methods for Screening and Validation in Fragment-Based Drug Discovery.

Fragment-based drug discovery (FBDD) is a crucial strategy for developing new drugs that have been applied to diverse targets, from neglected infectious diseases to cancer. With at least seven drugs already launched to the market, this approach has gained interest in both academics and industry in the last 20 years. FBDD relies on screening small libraries with about 1000-2000 compounds of low molecular weight (about 300 Da) using several biophysical methods. Because of the reduced size of the compounds, the chemical space and diversity can be better explored than large libraries used in high throughput screenings. This review summarises the most common biophysical techniques used in fragment screening and orthogonal validation. We also explore the advantages and drawbacks of the different biophysical techniques and examples of applications and strategies.

Field and laboratory study on the distribution, fate and transport of polycyclic aromatic hydrocarbons in the loess porous medium at a refinery polluted site: Understanding the soil-groundwater interface

Soil and groundwater pollution from industrial and agricultural activities has become a global concern. This study therefore investigates the occurrence, sources, and transport mechanisms of polycyclic aromatic hydrocarbons (PAHs) in the soil-groundwater system of a closed refinery. Field investigations and laboratory column experiments revealed widespread PAH contamination in both groundwater and soil, with higher concentrations near the groundwater level (6.45–8.75 m). PAHs in soil and groundwater were predominantly low and medium molecular weight compounds (LMW and MMW), originated from petroleum leakage and coal combustion. The region’s soil, primarily composed of loess and gravel with low total organic carbon (TOC) content (mean 0.07 %), showed a positive correlation between PAH concentration and TOC/clay content, while sand content had no significant impact. Groundwater fluctuations influence PAH accumulation at the soil-groundwater interface (SGI), which may suggest potential for increased migration under varying hydraulic conditions. This vertical migration offers new insights into contaminant transport models in groundwater systems, particularly for arid regions, and could inform future remediation strategies for similar contamination scenarios.

Multi-defense Pathways against Electrophiles through Adduct Formation by Low Molecular Weight Substances with Sulfur Atoms.

There is a variety of electrophiles in the environment. In addition, there are precursor chemicals that undergo metabolic activation by enzymes and conversion to electrophiles in the body. Although electrophiles covalently bind to protein nucleophiles, they also form adducts associated with adaptive or toxic responses. Low molecular weight compounds containing sulfur are capable of blocking such adduct formation by capturing the electrophiles. In this review, we present out findings on the capture and inactivation of electrophiles by 1) intracellular glutathione, 2) reactive sulfur species and 3) extracellular cysteine (formed during the production of sulfur adducts). These actions not only substantially suppress electrophilic activity but also regulate protein adduct formation.

Graphite and multi-layer graphene from a low molecular weight carbon source

While carbonaceous structures formed by pyrolysis of macromolecular carbon sources with silicates are well described in the literature, information on the structure of carbons obtained in the similar way from low molecular weight (LMW) carbon sources is not sufficient. This study is therefore aimed at characterizing the material obtained by pyrolyzing (1300 °C; Ar atmosphere) montmorillonite containing ∼25–50 wt% LMW cations as a carbon source. Pyrolyzed samples contained ∼5–20 wt% carbon, and thermogravimetric and elemental analysis showed a higher yield for a higher original amount of the LMW carbon source. In addition to amorphous carbon, graphite was formed during pyrolysis, as confirmed by X-ray diffraction analysis, Raman spectroscopy, and X-ray photoelectron spectroscopy. The presence of graphene-based nanomaterial, specifically multi-layer graphene, was revealed by transmission electron microscopy (TEM). Electrical conductivity of the silicate/carbon material reached ∼35 S m−1. Raman spectra and TEM images are similar to those from studies describing the use of macromolecular carbon sources. LMW compounds are a sufficient source of carbon for the preparation of electrically conductive materials containing graphite and multi-layer graphene.

Effects of Secondary Amine and Molecular Weight on the Biological Activities of Cationic Amphipathic Antimicrobial Macromolecules.

Cationic amphipathic antimicrobial agents inspired by antimicrobial peptides (AMPs) have shown potential in combating multidrug-resistant bacteria because of minimal resistance development. Here, this study focuses on the development of novel cationic amphipathic macromolecules in the form of dendrons and polymers with different molecular weights that employ secondary amine piperidine derivative as the cationic moiety. Generally, secondary amine compounds, especially at low molecular weights, have stronger bacteriostatic, bactericidal, and inner membrane disruption abilities than those of their primary amine counterparts. Low molecular weight D2 dendrons with two cationic centers and one hydrophobic dodecyl chain produce outstanding synergistic activity with the antibiotic rifampicin against Escherichia coli, where one-eighth of the standalone dose of D2 dendrons could reduce the concentration of rifampicin required by up to 4000-fold. The low molecular weight compounds are also less toxic and therefore have higher therapeutic index values compared to compounds with larger molecular weights. This study thus reveals key information that may inform the design of future synthetic AMPs and mimics, specifically, the design of low-molecular-weight compounds with secondary amine as the cationic center to achieve high antimicrobial potency and biocompatibility.

Ecological and Geochemical Assessment of the State of Soils in the City of Baikalsk According to the Content of Polycyclic Aromatic Hydrocarbons

The pollution of the topsoils of the city of Baikalsk (Irkutsk region) under the influence of industrial emissions and wastes of the Baikal Pulp and Paper Mill (BPPM) was studied. The content of 16 individual PAH structures in samples of urban and background soils taken during the soil geochemical survey in the summer of 2019 was analyzed. Relatively low levels of PAH content were found in the lignin sludge from the BPPM and СHP ash. The concentration of total PAHs in CHP ash reaches 46 mg/ kg with a predominance of low molecular weight compounds (the proportion of naphthalene and its homologues is 24% and 34% of the total PAHs, respectively), among high molecular weight PAHs, 5-nuclear benzo(b)fluoranthene dominates (16%). In lignin sludge, the amount of PAHs is 7.16 mg/kg with the predominance of benzo(b)fluoranthene (83%). In the soils of Baikalsk, the average total content of PAHs (38.4 mg/kg) is 5 times higher than the background content. In urban soils, 4–5-nuclear fluoranthene (61.1%) and benzo(b)fluoranthene (29.4%) prevail. This makes it possible to attribute soil pollution to the fluoranthene type. The soils of the motor transport (total PAH 105 mg/kg) and industrial (59.5 mg/ kg) zones are the most polluted, where the most contrasting PAH anomalies were formed. In descending order of the amount of PAHs, the land use zones of the city form a series: motor transport industrial residential one-storey railway transport residential multi-storey recreational zone. Several local anomalies in the amount of PAHs are distinguished, forming two large pollution halos in the western and eastern parts of the city. The leading factors in the accumulation of high molecular weight PAHs in soils are acid-alkaline conditions and soil organic matter, while the accumulation of low molecular weight polyarenes is mainly controlled by pH. The environmental hazard of pollution of Baikalsk soils with polyarenes is due to benzo(b)fluoranthene, its contribution is 83.5%.

Immune checkpoint blockade lowers the threshold of naïve T-cell priming to drug-associated antigens in a dose-dependent fashion.

A growing body of clinical and experimental evidence indicates that immune checkpoint blockade enhances patient susceptibility to hypersensitivity reactions to co-administered medications. In this study, we utilized an in vitro T-cell priming assay to demonstrate one of the mechanistic hypotheses on how this occurs; through lowering of the threshold in patients to elicit aberrant T-cell responses. We outline the dependency of de novo T-cell priming responses to drug-associated antigens on dose at initial exposure. Findings support the aforementioned hypothesis and offer an experimental representation of fundamental parameters at play in hypersensitivity reactions to low molecular weight compounds.

Periodic membrane fractionation of freshwater organic matter reveals various reactivity patterns during chlorine/chloramine disinfection

Although drinking water disinfection has significantly reduced mortality from waterborne diseases, the formation of potentially harmful disinfection by-products (DBPs) remains a concern today. These DBPs emerge from the reaction of dissolved organic matter (DOM) with chlor(am)ine during disinfection. Identifying these DBP precursors from heterogeneous and complex DOM mixtures is challenging and to address this, a novel membrane fractionation protocol was developed to isolate and identify the organic DBP precursors from fresh water sources. A tight ultrafiltration and nanofiltration membrane were carefully selected to partition DOM into three molecular weight (MW) fractions: high (>20 kDa), medium (0.3–20 kDa) and low (<0.3 kDa) as defined by high performance-size exclusion chromatography- total organic carbon analysis. A mathematical tool was developed to optimize the fractionation protocol. Therefore, ultrafiltration was executed before nanofiltration and the concentration factor was maximized without inducing fouling to obtain the purest fractions. The mathematical tool was also set in place to predict the necessary diafiltration factor for each individual fractionation experiment based on the initial organic matter composition of the surface water allowing the fractionation protocol to be effective at all times. The protocol was applied to surface water samples collected six times across three seasons yielding a fraction enriched in high MW compounds (up to 50 %), a fraction having more than 80 % of medium MW compounds and a fraction only containing low MW compounds. Although the medium MW fraction showed the highest reactivity for the majority of the investigated DBPs, the low MW fraction showed high reactivity for iodinated trihalomethanes during chlorination and for haloacetonitriles during chloramination. The high MW fraction had the lowest reactivity towards DBPs, which can have important implications for a drinking water treatment since this fraction is generally the most effectively removed by e.g. coagulation, while the more important fractions for DBP formation such as the medium and low MW compounds remain in the water until the disinfection step.

Exploring the Distribution of Low Molecular Weight Compounds in Water-Based Two-Phase Systems with Various Salt Additives

The partition coefficients of seven low molecular weight compounds were tested in different aqueous two-phase systems. The ionic composition of each system included specific salt additives, and it was found that there is a linear relationship between the solute partition coefficients and the presence of different salt additives. The study suggests that the solute structure and the type of ions influence the solute response to the ionic environment. Additionally, it was observed that the solutes’ polar surface area and the solvent-accessible surface area are the essential structural features governing partitioning in aqueous two-phase systems.

Partitioning of proteins and small molecular weight compounds from alfalfa juice during ultrafiltration

Ribulose-1,5-biphosphate-carboxylase/oxygenase (RuBisCO) from alfalfa is a promising source of alternative food protein, but it is challenging to purify. Ultrafiltration shows potential for concentrating and fractionating RuBisCO from low molecular weight compounds present in the extracted juice, but there is limited information on their partitioning depending on the membrane pore size. Therefore, the objective of this study was to evaluate the partitioning of various compounds from the juice depending on membrane pore size. It was hypothesized that both membrane pore sizes will retain RuBisCO but might partition low molecular weight compounds within the matrix differently. RuBisCO was fully retained by both the 300 and 100 kDa polyethersulfone (PES) membranes, while ash (29.7%–35.4% dry matter (DM)), non-protein nitrogen (2.38%–2.53% DM), and free N-terminals (10.13–10.34 mg glutamic equivalent/100 mg DM) were transmitted through both membranes. The retentates showed a distinct yellow color when filtering with 100 kDa membranes, but not when using 300 kDa membranes. Although no significant differences in total polyphenol permeation were observed through a spectrophotometric assay, a metabolomics screening analysis showed that the 100 kDa membranes had less transmission of the flavonoids apigenin, chrysoeriol, tricin, and tricin glycosides, which might contribute to the yellow color. These results reiterate the importance of measuring individual polyphenols using hyphenated techniques as opposed to spectrophotometric total reducing capacity. Sixteen saponins were identified and their permeation was lower in the 100 than the 300 kDa membranes, indicating a better potential for higher degree of protein refinement for the latter. This study demonstrates for the first time the difference in partitioning of the small molecular weight components, using PES membranes of similar chemistry but different size, during ultrafiltration of alfalfa juice.

Oxygen-containing compounds in polydimethylsilanes and -carbosilanes

An analysis of fractions extracted from polydimethylsilanes, as well as distillates obtained during the synthesis of poly(oligo)carbosilanes was carried out. By the methods of gel permeation chromatography and chromatography-mass spectrometry, the range of molecular weights in extracts and distillates, the composition and structure of compounds, as well as the sequence of their retention times on the chromatographic column were determined. It was established that the compounds extracted from polydimethylsilanes are low molecular weight cycles containing methylsilane and/or methylsiloxane units. In distillates, compared with extracts, the number of low molecular weight compounds containing individual or mixed methylsilane, carbosilane and siloxane units increases.

Selective extraction of biomolecules from Saccharomyces cerevisiae assisted by high-pressure homogenization, pulsed electric fields, and heat treatment: Exploring the effect of endogenous enzymes

Saccharomyces cerevisiae contains valuable compounds sought by the industry, prompting the exploration of effective extraction methods. Three physical methods were compared—High-Pressure Homogenization (HPH), Pulsed Electric Fields (PEF), and Heat Treatment (HT)—for releasing diverse bioproducts (amino acids, glutathione, proteins, and mannoproteins) from S. cerevisiae. Treatments (HPH: 2 passes at 100 MPa, PEF: 15 kV/cm for 100 μs (44.9 kJ/kg), HT: 60 °C for 5 min) affected over 90% of S. cerevisiae cells. Despite permeabilization, HT exhibited low efficacy in releasing compounds. HPH led to complete cell disruption, extracting maximum glutathione (1.09 ± 0.04 g/100 gdw) and proteins (68.95 ± 0.96 g/100 gdw) after 2 h. PEF facilitated the gradual extraction of low molecular weight compounds while leaving high molecular weight compounds unreleased initially. After 48 h, 72% of the total protein and amino acid content was released through hydrolysis catalyzed by endogenous yeast proteases. Furthermore, 64.1% of mannoproteins were obtained after 72 h of incubation. This study highlights the efficacy, scalability, and industrial potential of HPH and PEF, demonstrating their ability to produce tailored extracts over time and underscoring the significant role of endogenous enzymes in optimizing extraction yields. These findings provide valuable insights for enhancing efficiency and sustainability in bioproduct extraction from S. cerevisiae.

Pyridazine Derivatives: Molecular Docking, ADMET Prediction, and Synthesis for Antihypertensive Activity.

The drug discovery and development domain has witnessed remarkable advancements due to the integration of computational methods, particularly Computer-Aided Drug Design (CADD). Discovering and creating new drugs involves structural modifications to enhance their effectiveness and physical attributes. This frequently includes employing semisynthetic techniques to investigate structure-activity relationships thoroughly. Noticeable progress in molecular biology, computational chemistry, combinatorial chemistry, and highthroughput screening is steering transformative changes in the pharmaceutical industry. High blood pressure or hypertension, a significant health issue, elevates the chances of heart, kidney, and brain complications, among other health concerns. It's a leading cause of untimely mortality globally. Therefore, it is important to search for new antihypertensive compounds that have fewer side effects and higher therapeutic activity. Following molecular docking of the pyridazine derivatives, compounds were subjected to In-silico ADMET analysis. Subsequently, a low molecular weight compound was synthesized. Among the synthesized compounds characterization procedures include TLC, FT-IR, 1HNMR, and LC-MS techniques. Compound 8 exhibited the most favorable molecular docking results with alpha A1 and beta 1 adrenergic receptors. Compounds 3, 5, and 6 fulfilled the essential ADMET criteria. Subsequently, Compounds 3, 4, and 5 underwent additional synthesis and characterization procedures, including TLC, FT-IR, 1H-NMR, and LC-MS techniques. Similar behavior was observed in compounds 6, 8, 10, and 11, all violating Pfizer's 3/75 rules in terms of TPAS. Hydrazinolysis of these b-benzoyl propionic acids produced pyridazine, which was utilized in synthesizing pyridazine derivatives. TLC, FT-IR, 1HNMR, and LCMS have characterized the compounds.

Phytoremediation of crude oil-contaminated soil using Vigna Unguiculata and associated rhizosphere bacteria

Persistent crude oil contamination poses a significant environmental challenge. In this study, the efficacy of Vigna unguiculata (L.) and associated rhizospheric microorganisms in remediating crude oil-contaminated soil within a microcosm setting was investigated. A randomized block design was employed, and soil samples were subjected to varying degrees of contamination: 0% (UR), 2.5% (CR2), 5.0% (CR5), 7.5% (CR7), and 10.0% (CR10) w/w crude oil. The investigation aimed to assess the potential of Vigna unguiculata (L.) in mitigating crude oil contamination across these defined contamination gradients. The plant growth and crude oil removal were monitored concurrently post-emergence. Plant emergence and growth were significantly affected due to contamination, especially among plants in CR5 and CR10. The bacterial population was higher in the rhizosphere, and the treatments with lower hydrocarbon contamination. It was shown that plant density encouraged the growth of bacterial communities. Significant reduction in soil TPH was observed in CR2 (76.61%) and CR7 (65.88%). There was a strong correlation between plant growth and oil-utilizing bacterial population (r2 = 0.966) and plant growth and hydrocarbon reduction (r2 = 0.956), signifying the role of plant-bacterial synergy. Saturate fractions (C30 – C32) were significantly degraded to lower molecular weight compounds (C11 – C14). Except in CR5 and CR10, the remediation within the cowpea rhizosphere was effective even at regulatory standards. Understanding the rhizosphere ecological dynamics would further highlight the role the bacteria played; hence, it is recommended.

Getting to the root of the problem: Soil carbon and microbial responses to root inputs within a buried paleosol along an eroding hillslope in southwestern Nebraska, USA

Large quantities of soil carbon (C) can persist within paleosols for millennia due to burial and subsequent isolation from plant-derived inputs, atmospheric conditions, and microbial activity at the modern surface. Erosion exposes buried soils to modern root-derived C influx via root exudation and root turnover, thus stimulating microbial activity leading to SOC decomposition and accumulation through organo-mineral stabilization of modern C. With this study we aim to quantify how modern root-derived C inputs impact paleosol C decomposition and stabilization across varying degrees of isolation from modern surface conditions in southwestern Nebraska, USA, where hillslope erosion is bringing a buried Late-Pleistocene-early Holocene paleosol (the “Brady Soil”) closer to the modern surface. We collected Brady Soil samples from 0.2 m, 0.4 m, and 1.2 m below the modern surface and conducted two lab-based incubations. Soils were amended with either (1) a lab-synthesized mixture of low molecular weight compounds (12 atom% 13C), or (2) 13C enriched root residues (92 atom% 13C), in 30-day and 240-day incubation experiments, respectively. We determined microbial responses to synthetic root exudates and residues by partitioning the 13C label from Brady Soil C, including measurements of total, root, and primed C respiration, microbial biomass C (MBC), microbial C use efficiency (CUE). To assess the capacity of isolated paleosols to accrue modern plant C, we used Nano-scale Secondary Ion Mass Spectrometry imaging. We found that: (1) adding root-derived C inputs primed Brady Soil C across all depths, and was mediated by depth and composition of root additions; (2) root-derived C inputs stimulated microbial biomass C (MBC) growth similarly across depths, but the magnitude of CUE and MBC varied by chemistry of root-derived additions; (3) new particulate organic matter was incorporated into mineral-associated pools over time; (4) material from the added root residues was found in association with bacterial cells and fungal hyphae as well as with soil aggregate and mineral surfaces. Our study shows that paleosols defy expectations of C content and reactivity with depth, and changes in land cover and climate will expose buried paleosols to modern surface conditions, increasing respired C. This work highlights the importance of evaluating the role resurfacing buried soils through landscape change plays in C cycle feedbacks to the climate system.

On physical meaning of van't Hoff equation and its applicability in chromatography

The van't Hoff equation is a widely used tool to study adsorption thermodynamics in chromatography. Recent experimental data and some theoretical arguments questioned the accuracy of thermodynamic characteristics determined by using this equation. The present report addresses these concerns and shows that if certain requirements are satisfied, the van't Hoff analysis provide reliable results. These requirements include isothermality of the column not perturbed by the percolation of the mobile phase, mass transfer kinetics allowing sufficient proximity to equilibrium, pressure below 200 bar for low molecular weight compounds and below 50 bar for macromolecules, and knowledge of the phase ratio at all experimental temperatures to allow accurate conversion of the retention factor to the equilibrium constant. Physical meaning of adsorption enthalpy and entropy obtained by means of the van't Hoff analysis particularly in the case of heterogeneous adsorbents is discussed and recommendations on how to perform experiments to obtain reliable results are given.