Base Hydrolysis Research Articles

Sustainable Hydrogen Production by Glycerol and Monosaccharides Catalytic Acceptorless Dehydrogenation (AD) in Homogeneous Phase.

In the quest for sustainable hydrogen production, the use of biomass-derived feedstock is gaining importance. Acceptorless Dehydrogenation (AD) in the presence of efficient and selective catalysts has been explored worldwide as a suitable method to produce hydrogen from hydrogen-rich simple organic molecules. Among these, glycerol and sugars have the advantage of being cheap, abundant, and obtainable from fatty acid basic hydrolysis (biodiesel industry) and from biomass by biochemical and thermochemical processing, respectively. Although heterogeneous catalysts are more widely used for hydrogen production from biomass-based feedstock, the harsh reaction conditions applied limit applicability due to deactivation of active sites due to coking of carbonaceous materials. Moreover, heterogeneous catalyst are more difficult to fine-tune than homogeneous counterparts, and the latter also allow for high process selectivities under milder conditions. The present Concept article summarizes the main features of the most active homogeneous catalysts reported for glycerol and monosaccharides AD. In order to directly compare hydrogen production efficiencies, the choice of literature works was limited to reports where hydrogen was clearly quantified by yields and turnover numbers (TONs). The types of transition metals and ligands is discussed, together with a perspective view on future challenges of homogeneous AD reactions for practical applications.

Synthesis of Aza‐S(VI) Fluorides and Primary Sulfonimidamides from Sulfinylamines

AbstractAza‐S(VI) fluorides are crucial compounds in the synthesis of various S(VI) derivatives, which find broad applications in drug discovery. However, the synthesis of sulfonimidoyl and sulfondiimidoyl fluorides have been relatively underexplored, often requiring lengthy reaction sequences and/or the use of hazardous gaseous reagents. In this study, we present a rapid one‐pot method for producing sulfonimidoyl fluorides from sulfinylamines via a nucleophilic addition/electrophilic fluorination sequence. Similarly, sulfondiimidoyl fluorides can be synthesized using the same sequence, preceded by the formation of unsymmetrical sulfurdiimides readily generated in situ from sulfinylamines. Whereas isolation of sulfondiimidoyl fluorides was not feasible, they could be efficiently converted into primary sulfonimidamides by base hydrolysis in a one‐pot process. Furthermore, we explored the reactivity of aza‐S(VI) fluorides with various nucleophiles, demonstrating a versatile synthetic platform for the synthesis of aza‐S(VI) compounds.

Development of LC/LCMS method for estimation of impurities in anti-HIV drug (Bictegravir) using Analytical Quality by Design (AQbD) approach.

Analytical Quality by Design (AQbD) was used to construct stability indicating linked compounds using the HPLC technique of an anti-HIV medicine (Bictegravir). Process-related and degrading impurities were separated on a Zorbax SB-C8 (150×4.6) mm, 3.5 m column. The buffer pH of the mobile phase was kept at 2.5 by employing potassium dihydrogen phosphate 0.05M. Mobile phase A contains 5% methanol and 95% buffer, whereas mobile phase B contains 50% acetonitrile, 10% methanol, 25% tetrahydrofuran, and 15% water. The completed chromatographic settings were a flow rate of 1.2 mL/min, a detector wavelength of UV at 250 nm, and an injection volume of 20 µL. This approach has been verified in accordance with ICH recommendations. The approach was discovered to be particular, sensitive, linear, exact, and accurate. The limit of quantification for all contaminants was determined to be less than 0.05%, the correlation coefficient for all impurities is between 0.9996 and 1.0000, and the percent recovery for all impurities is between 91% and 108% at the LOQ level and 97% to 113% at the specification level. Forced degradation experiments in chemical and physical stress tests were performed in accordance with regulatory criteria, and the molecule was discovered to be susceptible to acid and base hydrolysis. Imp-A was the most common degrading impurity in both acid and base hydrolysis.

A novel stability-indicating chromatographic quantification of the antiparkinsonian drug safinamide in its pharmaceutical formulation employing HPTLC densitometry and ion-pair HPLC–DAD

Parkinson's disease (PD) emerges as a notable health concern among the elderly population. Safinamide mesylate (SAF) is a novel and emerging add-on therapy in PD treatment. The stability of innovative drug formulations and the development of appropriate stability-indicating methods are of great importance to modern pharmaceutical analysis. The current work has established novel comprehensive stability-indicating chromatographic approaches, HPTLC coupled with densitometric quantification and HPLC–DAD, for the selective assay of SAF in pharmaceutical formulation along with its synthetic precursor impurity; 4-hydroxy benzaldehyde (4-HBD) in presence of its stress induced degradation products. The stability of SAF was investigated under different stress conditions. It was found that SAF is likely to undergo acid, base hydrolysis, and oxidative degradation. Using mass spectrometry and infrared spectroscopy, the structures of the forced degradation products were confirmed and elucidated. The dissolution behavior of Parkimedine® Tablets was also monitored in the FDA suitable medium. Multiple assessment tools were used to evaluate the environmental sustainability of the proposed methods and the reported one. The greenness tools included Complex-GAPI and AGREE metrics. In addition, the innovative concepts of "blueness" and "whiteness" evaluation were incorporated through the newly introduced BAGI and RGB12 algorithms, respectively.

Stability Indicating High‐performance Liquid Chromatography Method for Quantification of Amoxapine and Characterization of Forced Degradation Products Employing Quadrupole‐Time of Flight Mass Spectrometry

ABSTRACTAmoxapine, a tricyclic antidepressant, has been widely used for long‐term treatment of depression along with other psychiatric disorders involving neurosis, agitation, and anxiety. The drug may undergo degradation during manufacturing, transportation, and storage. To ensure drug safety, both identification and characterization of the degradation products (DPs) should be considered. Accordingly, a sensitive and selective stability‐indicating assay method was developed for amoxapine using high‐performance liquid chromatography (HPLC). The amoxapine drug was subjected to acidic hydrolysis, basic hydrolysis, and oxidation separately. The degradation susceptibility of the amoxapine was found to be in the order of Acid hydrolysis > Oxidation > Base hydrolysis. The developed method was validated as per the International Council for Harmonization Q2(R2) guideline. Further, LC‐electrospray ionization‐quadrupole‐time of flight‐tandem mass spectrometry was employed for the identification and characterization of the DPs formed under stress conditions. The study revealed that under accelerated stress conditions, three hydrolytic DPs and one oxidative DP were formed. Three of the four DPs were found to be novel and never reported before. In addition, a plausible mechanism representing the formation of all DPs has also been established.

A Cell-Free Kinetic Analysis of Ionizable Lipid Hydrolysis.

The prolonged retention of ionizable lipids within the body limits the repeated dosing of lipid nanoparticles (LNPs) for nucleic acid delivery. While most ionizable lipids are primarily metabolized in the liver via the enzymatic hydrolysis of ester bonds, elimination half-lives can range from several hours to days. The development of compounds that undergo rapid biodegradation remains a major engineering challenge in the absence of standardized biodegradability assessments in the early stages of drug discovery. Here, we analyze and compare the hydrolysis kinetics of well-known ionizable lipids (ALC-0315, DLin-MC3-DMA, LP-01, L319, and SM-102) using optimized cell-free reactions monitored by 1H NMR. Unlike conventional analytical techniques, these NMR-based methods are universal and suitable for high-throughput screening. We demonstrate that enzyme-catalyzed and base hydrolysis reactions can predict whether ionizable lipids undergo fast or slow liver elimination, as our results are in alignment with prior pharmacokinetic studies. Furthermore, we show that the hydrolysis kinetics of ionizable lipids vary by several orders of magnitude depending on steric effects. This study provides a framework to expedite the discovery of rapidly degradable ionizable lipids, with implications for improving the therapeutic index of LNP-based drugs.

Hydrogen Peroxide-Assisted Alkaline Defluorination of the Fumigant and Potent Greenhouse Gas, Sulfuryl Fluoride: Hydrogen Peroxide as a Nucleophilic Reagent.

Sulfuryl fluoride (SO2F2, SF) is an effective and increasingly popular fumigant for treating buildings and commodities in international trade but has come under scrutiny as a potent greenhouse gas. Passage of vent gases through an alkaline spray has been proposed for scrubbing SF, but base hydrolysis is insufficiently fast and generates equal yields of fluoride and fluorosulfate, the latter of unknown environmental hazard. We report here that alkaline hydrogen peroxide (H2O2) markedly accelerates SF removal and gives nearly quantitative yield of fluoride, with fluorosulfate produced in less than 3.5% yield. The other major products are sulfate, peroxymonosulfate, and oxygen. The oxidation state of S was unchanged. Hydroxyl and superoxide radical scavengers had no effect on the rate. The reaction proceeds by sequential nucleophilic displacement of fluoride by hydroperoxide ion (HO2-) to form a transient diperoxysulfate species that rapidly undergoes intramolecular redox rearrangement to give sulfate and singlet oxygen. Peroxymonosulfate, produced through side reactions, can fully defluorinate SF as well, although more slowly. Two new peaks were detected in the 19F-NMR spectrum corresponding to intermediates. Fluoride can be removed conventionally, and the other products are innocuous or short-lived. Thus, H2O2-assisted alkaline defluorination promises to be an effective method for scrubbing spent SF fumes and preventing SF from reaching the atmosphere. This study highlights the benefits of H2O2 and peroxymonosulfate as nucleophiles in remediation chemistry.

Nanocellulose from Mankamana-3 corncob biomass: Synthesis, characterization, surface modification and potential applications

This study characterizes corncob from the Nepali variety Mankamana-3, an abundantly available agricultural waste, extracts nanocellulose from it, modifies the nanocellulose with copper nanoparticles, and explores its potential application. The corncobs were mechanically processed into fine powder, delignified by base hydrolysis, bleached and then subjected to acid hydrolysis to obtain nanocellulose fibrils. The corncob contained ~38.86 % cellulose, ~22.02 % lignin, ~34.89 % hemicellulose, ~0.97 % moisture, ~3.33 % ash with minimal moisture content (0.97 %). Transmission electron microscopy imaging confirmed nanocellulose fibers with an average diameter of 28.2 ± 2.57 nm. X-ray diffraction results revealed the existence of both cellulose I and cellulose II polymorphs in nanocellulose with 66.55 % crystallinity index (CrI). Further, copper nanoparticles were grown on nanocellulose fibrils, which showed significant antimicrobial activity against E. coli, S. aureus and C. albicans, and prolonged stability in the ambient environment. The corncob biomass-derived nanocellulose could potentially replace conventional inorganic supports for growing various nanoparticles.

"Marriage" of Inorganic to Organic Chemistry as Motivation for a Theoretical Study of Chloroform Hydrolysis Mechanisms.

Incorporation of chlorides in coordination complexes, prepared by reactions in CHCl3, stimulated MP2 and DFT studies of its complete hydrolysis mechanisms. In excellent agreement with previous experimental results, the most important mechanism for CHCl3 basic hydrolysis at room temperature is the radical one producing :CCl2. The latter inserts into the H-O bond of H2O yielding dichloromethanol (1). The hydrolysis mechanism of α-H-lacking PhCCl3 to the corresponding dichloro(phenyl)methanol (3) was also studied. 1 decomposes by H2O to formyl chloride (2) and HCl. 2, following a variety of pathways, leads to known CHCl3 hydrolysis products, such as CO (4) and formic acid (6), via the intermediates chloromethanediol (5), s-cis, s-trans-dihydroxycarbene (ct-7), and s-trans, s-trans-dihydroxycarbene (tt-7). Interestingly, both ct-7 and tt-7 intermediates have recently been implicated in the reduction of CO2 with H2 to 6. The conversion of CO to HCOOH was studied. Most of the reactions studied are asynchronous concerted processes, the radical mechanism being a multistep one. The synthetic utility of this mechanism is briefly mentioned. To avoid chloride ions when performing reactions in CHCl3, we should use the solvent at room temperature even in the presence of water. This has been verified further by coordination chemistry reactions in progress.

Utilizing cutting-edge analytical techniques for the Identification, Isolation, and structural characterization of donepezil HCl forced degradation products

The current study elucidates the stress degradation behavior of donepezil HCl, a medication used to treat Alzheimer-type dementia. Using hydrolytic, oxidative, thermal, and photolytic conditions, the forced degradation investigation was conducted in accordance with ICH recommendations. In neutral hydrolysis, oxidative, thermal, and photolytic conditions, donepezil is comparatively stable; nevertheless, significant degradation was noted in acid and base hydrolysis. Using sophisticated analytical methods such as LCMS, Preparative-HPLC, NMR, and HRMS, the degradation products were identified, isolated, and their structural validity was confirmed. Degradation products 1, 2, and 3 are the three degradation products that were found and isolated during acid degradation. Conversely, products 1 and 3 generated during base hydrolysis degradation. Degradation product 1 and 3 were known products, while degradation product 2 is new compound that have not yet been documented. Using an advanced analytical approach, this is the first recorded comprehensive structural characterization of all degradation products. In the current work, high-resolution mass spectroscopy and nuclear magnetic resonance spectroscopy were employed to get specific confirmation of degradation product structures. In the future, degradation products with lower runtime will also be identified using the present LCMS technique.

Heterologous Biosynthesis of New Lanthipeptides Nocardiopeptins with an Unprecedented Bridging Pattern of Lanthionine and Labionin.

The class III lanthipeptide synthetase (LanKC) installs unusual amino acids, such as lanthionine and labionin, in lanthipeptides. Through genome mining, we discovered a new class III lanthipeptide synthetase coding gene (nptKC) and precursor peptide coding genes (nptA1, nptA2, and nptA3) in the genome of the actinobacterium Nocardiopsis alba. Coexpression experiments of the biosynthetic genes in Escherichia coli resulted in the production of new lanthipeptides named nocardiopeptins A1-A3. Analysis of two-dimensional NMR spectra after enzymatic degradation and partial basic hydrolysis of nocardiopeptin A2 revealed that labionin was located in lanthionine with opposite orientations, forming a nesting structure in nocardiopeptin A2. To the best of our knowledge, this bridging pattern in the lanthipeptides was unprecedented, indicating a novel reaction characteristic of the class III lanthipeptide synthetase NptKC.

HPTLC method for determination of anti-hypertensive drug combination metoprolol succinate and dapagliflozin

Combination of Metoprolol Succinate and Dapagliflozin is recommended in the treatment of Hypertension. Accurate and precise thin layer chromatographic method has been developed for the determination of Metoprolol Succinate and Dapagliflozin in combination. Pre-coated silica gel G60—F254 aluminium sheet (10 × 10 cm),0.2 mm layer thickness used as stationary phase and n-Butanol: Ethyl acetate: Triethylamine (6:4:0.1 v/v/v) used as mobile phase. The method was found to be linear with a range of 200–1200 ng/band for DAP and 1000–6000 ng/band for MET with correlation coefficient (r2) 0.996 for both DAP and MET. The proposed method was validated as per ICH Q2 (R2) guideline with respect to linearity, accuracy and precision. Stress degradation was carried out to find out the intrinsic stability of the molecules. Both the drugs were highly susceptible to acid and base hydrolysis and degradation products were well separated with drug peak. The method was successfully used for determination of metoprolol succinate and dapagliflozin in combination.

HPTLC – densitometric method of analysis for estimation of efonidipine hydrochloride and telmisartan used in treatment of hypertension

Precise, accurate, and sensitive high performance thin layer chromatographic method has been developed and validated for simultaneous determination of efonidipine hydrochloride (EFD) and telmisartan (TEL) in a tablet dosage form which is used in treatment of hypertension. The proposed method used aluminum plates pre-coated with silica gel 60 F254 as a stationary phase and n-butanol: toluene: acetic acid (3:7:0.2, v/v/v) as a mobile phase for separation purpose. Compact spots of TEL and EFD were obtained at Rf 0.73 and 0.37, respectively. Densitometric detection was carried out at 299 nm in UV. Linear responses were shown by the detector over the range of 200–1200 ng/band for both drugs. The proposed method was validated as per ICH guidelines. The method was successfully applied for estimation of both drugs in tablet formulations. Forced degradation study was carried and efonidipine was found to be susceptible to base hydrolysis and oxidative stress degradation while TEL was stable in acid-base hydrolysis, oxidative stress, dry heat and photo stability testing conditions. The developed method can be used for the analysis of stability samples. Greenness assessment of developed method was performed using AGREE software. The method was found to be greener having greenness score of 0.69.

The Preparation and Crystal Structures of Octaoxoketocalix[8]arene Derivatives: The Ketocalixarene Counterparts of the Largest "Major" Calixarene.

The purpose of this study was to synthesize and structurally characterize ketocalixarenes (i.e., calixarenes where the bridging methylene bridges are replaced by carbonyl groups) derived from the largest "major" calixarene, namely p-tert-butylcalix[8]arene 3a. Ketocalix[8]arenes were synthesized by the oxidation of protected p-tert-butylcalix[8]arene derivatives. Octamethoxy-p-tert-butylketocalix[8]arene 6b was prepared by the photochemical reaction of the calixarene 3b with NBS in a CHCl3/H2O mixture. The oxidation of the methylene groups of octaacetoxy-p-tert-butylcalix[8]arene 3c was conducted by a reaction with CrO3 in Ac2O/AcOH. The basic hydrolysis of the acetate groups of the oxidation product yielded octahydroxy-p-tert-butylketocalix[8]arene 6a. In the crystal, the molecule adopts a saddle-like conformation of crystallographic C2 and idealized S4 symmetry. Strikingly, the array of OH/OH intramolecular hydrogen bonds present in the parent 3a is completely disrupted in 6a.

Isolation, Identification, and Characterization of Forced Degradation Products of Bosentan by Using Advanced Analytical Techniques

ABSTRACTThe present study explains the degradation behavior of bosentan (BOS), active pharmaceutical ingredient (API) (BOS monohydrate), a Food and Drug Administration (FDA)–approved drug used for the treatment of chronic heart failure, subarachnoid hemorrhage, and Raynaud's syndrome pulmonary hypertension. The stress study was performed under various stress conditions such as acid hydrolysis, base hydrolysis, oxidation, thermal degradation, and photolysis as per International Council for Harmonization (ICH) guidelines and found three degradation products (DPs) under acid hydrolysis conditions (1 N HCl/4 h/60°C reflux conditions) (the net degradation of API ∼ 21.39%). The drug was found to be stable under the remaining conditions (thermal, photolytic, oxidative, and basic hydrolytic conditions). Mechanisms for forming the three acid DPs were also proposed and discussed. The DPs were initially detected by ultrahigh‐performance liquid chromatography–mass spectrometry analysis. These DPs were isolated by preparative HPLC and then characterized by high‐resolution mass spectrometry and nuclear magnetic resonance spectroscopy techniques. Based on the structural characterization study, all these DPs are new and not reported elsewhere in the literature to my knowledge. Many reports were found in the literature for the quantification of the drug in the APIs and formulation products. However, there is no report published yet for the identification and characterization of DPs of the BOS drug.

Photochemical [2 + 2] Cycloaddition Enables the Synthesis of Highly Thermally Stable and Acid/Base-Resistant Polyesters from a Nonpolymerizable α,β-Conjugated Valerolactone.

We report a simple strategy to transform a nonpolymerizable six-membered α,β-conjugated lactone, 5,6-dihydro-2H-pyran-2-one (DPO), into polymerizable bicyclic lactones via photochemical [2 + 2] cycloaddition. Two bicyclic lactones, M1 and M2, were obtained by the photochemical [2 + 2] cycloaddition of tetramethylethylene and DPO. Ring-opening polymerization (ROP) of M1 and M2 catalyzed by diphenyl phosphate (DPP), La[N(SiMe3)2]3, and 1-tert-butyl-4,4,4-tris(dimethylamino)-2,2-bis[tris (dimethylamino) phosphoranylide-namino]-2λ5, 4λ5-catenadi(phosphazene) (tBu-P4) were conducted. M1 is highly polymerizable, either DPP or La[N(SiMe3)2]3 could catalyze its living ROP under mild conditions, affording the well-defined PM1 with a predictable molar mass and low dispersity. M2 could only be polymerized with tBu-P4 as the catalyst, also generating the same polymer PM1. PM1 has high thermal stability, with a Td,5% being up to 376 °C. Ring-opening copolymerization (ROcP) of M1 and δ-valerolactone (δ-VL) catalyzed by La[N(SiMe3)2]3 afforded a series of random copolymers with enhanced thermal stabilities. Both PM1 and the copolymer containing 10 mol % M1 exhibited excellent resistance to acidic and basic hydrolysis. Our results demonstrate that direct photochemical [2 + 2] cycloaddition of α,β-conjugated valerolactone is not only a strategy to tune its polymerizability, but also allows for the synthesis of highly thermally stable aliphatic polyesters, inaccessible by other methods.

Stability indicating RP-HPLC method development and validation for the determination of pyrimethamine in an oral paediatric suspension

The aim of this study is to validate a high-performance liquid chromatography (HPLC) – UV assay method for a stability assay of an oral suspension of PYR developed for newborns. Analyses were performed by HPLC with a Kinetex Coreshell C18 column, thermostated at 40°C, coupled to a diode array (λ=230 nm, 280 nm, scan from 190 to 400 nm) with an acetonitrile/methanol/KH2PO4 buffer gradient (pH 3, 10 mM). A forced degradation study was performed to validate the stability-indicating character. PYR was subjected to acidic (pH 2) and basic (pH 12) hydrolysis stress, thermal hydrolysis stress at native pH (60°C and 4°C), H2O2 oxidation stress (3 % and 15 %) and photolysis stress (UV and natural light) at day 0, 3 and 16. Linearity, specificity, precision, and accuracy of this assay method have been tested. Forced degradation tests demonstrated the stability indicator character of the method and showed that PYR was very sensitive to oxidation, sensitive to light (UV) and insensitive to hydrolysis (thermal and pH). The method of quantification was linear, specific, precise, and accurate according to ICH recommendations. A stability study on the formulation under development will be carried out with this HPLC – UV method.

Synthesis, characterization and base hydrolysis of cobalt(III) complexes coordinated by substituted phenylthioether ligands

A synthetic route to the preparation of cobalt(III) complexes coordinated by bidentate phenylthioether ligands is described. Complexes of the type ((2-(X-phenylthio)ethylamine)-N,S)bis(ethylenediamine)cobalt(III) perchlorate, [(en)2Co(S(X-phenyl)CH2CH2NH2)](ClO4)3, where X = 4-methoxy (2a), 4-methyl (2b), 3-methyl (2c), nothing (2d), 3-methoxy (2e), 4-bromo (2f) and 3-bromo (2g) were prepared. The synthetic route involves reaction of trans-dichloridobis(ethylenediamine)cobalt(III) chloride with NH2CH2CH2SC6H4X to produce cis-[(en)2CoCl(NH2CH2CH2SC6H4X)]Cl2 (1a–g). Formation of the Co–S bond, completing the ring closure, is then accomplished by removing the coordinated chlorido by addition of AgClO4 in sulfolane. Complexes were characterized by 1H and 13C NMR spectroscopy, UV-Vis spectroscopy and elemental analysis. In addition, the solid-state structure (X-ray) of the monohydrate of 2d confirms the coordination mode of the ligand. Preliminary kinetic investigations in basic solution show that the Co–S bond is broken resulting in the formation of [(en)2Co(OH)(NH2CH2CH2SC6H4X)]2+. At 15.0°C, a Hammett Plot is linear (r2 = 0.981) with ρ = 2.24 ± 0.13.

Stability‐Indicating HPLC Method Development and Validation for the Quantification of Tofacitinib Citrate and Its Related Substances Using Hydrophilic Liquid Interaction Chromatography

ABSTRACTA new stability‐indicating RPHPLC method with short run time has been developed and validated for tofacitinib citrate and its related substances. The novel HPLC method integrates hydrophilic interaction liquid chromatography (HILIC) technology as the stationary phase and employs a mobile phase composed of phosphate buffer (pH 7.0) and acetonitrile (45:55, %v/v) at a flow rate of 0.5 mL/min under isocratic elution. Analytes were monitored via a UV detector at 210 nm and with the column oven temperature at 30°C for a 20‐min analysis. Precision (%RSD) for Impurity‐A and Impurity‐B and tofacitinib met specifications at 2.4%, 0.8%, and 0.0%, respectively. Accuracy ranged from 86% to 100% for impurities, with LOD at 0.03% and LOQ at 0.05%–0.06%. Correlation coefficients exceeded 0.999 for impurities and tofacitinib citrate. Solution stability was confirmed for 24 h at room temperature. The method range was extended from LOQ to ∼1.5% for impurities and LOQ to ∼150% for tofacitinib citrate. The method's stability was evaluated under acid and base hydrolysis, oxidative and water hydrolysis, and thermal and photolytic degradation. Introducing HILIC as the stationary phase for this work proved effective, eliminating the need of ion pair reagents, reducing analysis time, and ensuring consistent results.

Calibrating ITC instruments: Problems with weak base neutralization

Modern isothermal titration calorimetry instruments give great precision, but for comparable accuracy they require chemical calibration. For the heat factor, one recommended process is HCl into the weak base TRIS. In studying this reaction with a VP-ITC and two Nano-ITCs, we have encountered some problems, most importantly a titrant volume shortfall Δv ≈ 0.3 μL, which we attribute to diffusive loss of HCl in the syringe tip. This interpretation is supported by a mathematical treatment of the diffusion problem. The effect was discovered through a variable-v protocol, which thus should be used to properly allow for it in any reaction that similarly approaches completion. We also find that the effects from carbonate contamination and from OH− from weak base hydrolysis can be more significant that previously thought. To facilitate proper weighting in the least-squares fitting of data, we have estimated data variance functions from replicate data. All three instruments have low-signal precision of σ ≈ 1 μJ; titrant volume uncertainty is a factor of ∼2 larger for the Nano-ITCs than for the VP-ITC. The final heat factors remain uncertain by more than the ∼1 % precision of the instruments and are unduly sensitive to the HCl concentration.