Even-Odd Layer-Dependent Exchange Bias Effect in MnBi2Te4 Chern Insulator Devices.

Magnetic topological materials with coexisting magnetism and nontrivial band structures exhibit many novel quantum phenomena, including the quantum anomalous Hall effect, the axion insulator state, and the Weyl semimetal phase. As a stoichiometric layered antiferromagnetic topological insulator, thin films of MnBi2Te4 show fascinating even-odd layer-dependent physics. In this work, we fabricate a series of thin-flake MnBi2Te4 devices using stencil masks and observe the Chern insulator state at high magnetic fields. Upon magnetic field training, a large exchange bias effect is observed in odd but not in even septuple layer (SL) devices. Through theoretical calculations, we attribute the even-odd layer-dependent exchange bias effect to the contrasting surface and bulk magnetic properties of MnBi2Te4 devices. Our findings reveal the microscopic magnetic configuration of MnBi2Te4 thin flakes and highlight the challenges in replicating the zero magnetic field quantum anomalous Hall effect in odd SL MnBi2Te4 devices.

Validation of an HPLC-CAD Method for Determination of Lipid Content in LNP-Encapsulated COVID-19 mRNA Vaccines.

Lipid nanoparticles (LNPs) are widely used as delivery systems for mRNA vaccines. The stability and bilayer fluidity of LNPs are determined by the properties and contents of the various lipids used in the formulation system, and the delivery efficiency of LNPs largely depends on the lipid composition. For the quality control of such vaccines, here we developed and validated an HPLC-CAD method to identify and determine the contents of four lipids in an LNP-encapsulated COVID-19 mRNA vaccine to support lipid analysis for the development of new drugs and vaccines.

Ultrathin Solid Polymer Electrolyte Design for High-Performance Li Metal Batteries: A Perspective of Synthetic Chemistry.

Li metal batteries (LMBs) have attracted widespread attention in recent years because of their high energy densities. But traditional LMBs using liquid electrolyte have potential safety hazards, such as: leakage and flammability. Replacing liquid electrolyte with solid polymer electrolyte (SPE) can not only significantly improve the safety, but also improve the energy density of LMBs. However, till now, there is only limited success in improving the various physical and chemical properties of SPE, especially in thickness, posing great obstacles to further promoting its fundamental and applied studies. In this review, the authors mainly focus on evaluating the merits of ultrathin SPE and summarizing its existing challenges as well as fundamental requirements for designing and manufacturing advanced ultrathin SPE in the future. Meanwhile, the authors outline existing cases related to this field as much as possible and summarize them from the perspective of synthetic chemistry, hoping to provide a comprehensive understanding and serve as a strategic guidance for designing and fabricating high-performance ultrathin SPE. Challenges and opportunities regarding this burgeoning field are also critically evaluated at the end of this review.

Antinuclear antibodies in follicular fluid may be a risk factor in vitro fertilization and embryo transfer.

OBJECTIVE: To explore the effect of anti-nuclear antibodies (ANAs) on the outcome of in vitro fertilization-embryo transplantation (IVF-ET) and to study the effect of ANAs in follicular fluid (FF) on embryonic development. METHODS: The expression of ANAs in FF of patients treated with IVF-ET and healthy group. The patients were divided into ANAs-positive group and ANA-negative group. The age, duration of infertility, body mass index (BMI), basic follicle stimulating hormone (bFSH), anti-Mullerian hormone (AMH), number of retrieved oocytes, portion of metaphase II oocytes (MII), number of embryos in the cleavage stage, bipronuclear (2PN), number of embryos, number of high-quality embryos and the outcome of IVF-ET were compared between the two groups. In vitro, HTR8/SVneo trophoblast cells were cultivated and divided into ANAs-negative group (control group) and ANAs-positive group (ANAs-positive FF was added to cytotrophoblasts). The ANAs titer in the serum and FF of patients who treated with IVF-ET was detected using ELSIA method. CCK-8 assay and flow cytometry (at 24 h and at 48 h) were used to detect the cell proliferation and apoptosis frequency of the two groups, respectively. RESULTS: Among those who underwent IVF-ET treatment, the number of retrieved oocytes, the number of fertilization and the portion of MII oocytes in the FF-positive group were significantly lower than those in the FF-negative group. Furthermore, the implantation rate and the clinical pregnancy rate were decreased, and early miscarriage rate was increased in the FF-positive patients than those in the FF-negative patients. In vitro, the cytotrophoblasts proliferation activity in the ANAs group was significantly lower than that in the control group. Moreover, the cytotrophoblasts apoptosis rate in the ANAs group was significantly higher than that in the control group. CONCLUSIONS: Our data suggested that ANAs in FF might become an obstacle to embryonic development through promoting trophoblast apoptosis and inhibiting trophoblast proliferation. ANAs in FF might be an unfavorable factor for the outcome of those who undergo IVF-ET treatment.

Interleukin-33 promotes proliferation and inhibits apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis.

OBJECTIVES: The aim of our study was to determine the effect of interleukin (IL)-33 on the proliferation, apoptosis, and secretion of inflammatory cytokines by fibroblast-like synoviocytes (FLSs) in rheumatoid arthritis (RA) and to investigate the underlying mechanisms. METHODS: Cultured RA FLSs and osteoarthritis (OA) FLSs were cocultured with different concentrations of IL-33. TUNEL assay and flow cytometry were used to detect apoptosis. Western blotting and Real-time (RT)-PCR were used to detect the expression levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax), while the Cell Counting Kit-8 was used to determine cell proliferation in each cocultured group. Enzyme-linked immunosorbent assay was used to detect the expression levels of tumour necrosis factor (TNF)-α and IL-6 in the supernatant from each cell culture. Western blot analysis was used to determine the phosphorylated expression levels of the nuclear factor-kappa light chain enhancer of the activated B cells (NF-κB) pathway in each group. RESULTS: IL-33 inhibited RA FLS apoptosis, promoted FLS proliferation, increased Bcl-2 protein expression levels, and decreased Bax protein expression levels. It also increased the expression levels of inflammatory cytokines TNF-α and IL-6 and increased the expression levels of P-NF-κ B in FLSs. CONCLUSIONS: IL-33 inhibited apoptosis and promoted proliferation of FLSs; in addition, IL-33 increased the serum levels of inflammatory cytokines. The effect of IL-33 on RA FLSs was likely mediated via the NF-κB pathway.

TRIM32 Deficiency Impairs Synaptic Plasticity by Excitatory-Inhibitory Imbalance via Notch Pathway.

Synaptic plasticity is the neural basis of physiological processes involved in learning and memory. Tripartite motif-containing 32 (TRIM32) has been found to play many important roles in the brain such as neural stem cell proliferation, neurogenesis, inhibition of nerve proliferation, and apoptosis. TRIM32 has been linked to several nervous system diseases including autism spectrum disorder, depression, anxiety, and Alzheimer's disease. However, the role of TRIM32 in regulating the mechanism of synaptic plasticity is still unknown. Our electrophysiological studies using hippocampal slices revealed that long-term potentiation of CA1 synapses was impaired in TRIM32 deficient (KO) mice. Further research found that dendritic spines density, AMPA receptors, and synaptic plasticity-related proteins were also reduced. NMDA receptors were upregulated whereas GABA receptors were downregulated in TRIM32 deficient mice, explaining the imbalance in excitatory and inhibitory neurotransmission. This caused overexcitation leading to decreased neuronal numbers in the hippocampus and cortex. In summary, this study provides this maiden evidence on the synaptic plasticity changes of TRIM32 deficiency in the brain and proposes that TRIM32 relates the notch signaling pathway and its related mechanisms contribute to this deficit.

Corrosion Behavior of the As-Cast and As-Solid Solution Mg-Al-Ge Alloy.

The corrosion behavior of Mg-3Al-xGe (x = 1, 3, 5) alloy in as-cast and as-solid was investigated by virtue of microstructure, corrosion morphology observation, and electrochemical measurement. Among the as-cast alloys, the corrosion rate of Mg-3Al-1Ge with a discontinuous bar-morphology was the highest, which was 101.7 mm·a-1; the corrosion rate of Mg-3Al-3Ge with a continuous network distribution was the lowest, which was 23.1 mm·a-1; and the corrosion rate of Mg-3Al-5Ge of Ge-enriched phase with sporadic distribution was in-between, which was 63.9 mm·a-1. It is suggested that the morphology of the Mg2Ge phase changes with a change in Ge content, which affects the corrosion performance of the alloy. After solid solution treatment, the corrosion rate of the corresponding solid solution alloy increased-Mg-3Al-1Ge to 140.5 mm·a-1, Mg-3Al-3Ge to 52.9 mm·a-1, and Mg-3Al-5Ge to 87.3 mm·a-1, respectively. After investigation of the microstructure, it can be suggested that solid solution treatment dissolves the Mg17Al12 phase, which changes the phase composition of the alloy and also affects its microstructure, thus affecting its corrosion performance.

Determination of tropane alkaloids by heart cutting reversed phase - Strong cation exchange two dimensional liquid chromatography.

Current Chinese Pharmacopoeia (ChP) standards apply liquid extraction combined with one dimensional liquid chromatography (1DLC) method for determining alkaloids in herbal medicines. The complex pretreatments lead to a low analytical efficiency and possible component loss. In this study, a heart cutting reversed phase - strong cation exchange two dimensional liquid chromatography (RP - SCX 2DLC) approach was optimized for simultaneously quantifying tropane alkaloids (anisodine, scopolamine and hyoscyamine) in herbal medicines and herbal medicine tablets without further treatment of the filtered extract. The chromatographic conditions were systematically optimized in terms of column type, mobile phase composition and flow rate. To improve peak capacity and obtain symmetric peak shape of alkaloids, a polar group embedded C18 column combined with chaotropic salts was used in the first dimension. To remove the disturbance of non-alkaloids, achieve unique selectivity and acquire symmetric peak shape of alkaloids, an SCX column combined with phosphate buffer was used in the second dimension. Method validation was performed in terms of linearity, precision (0.54-0.82%), recovery (94.1-105.2%), limit of detection (LOD) and limit of quantification (LOQ) of the three analytes varied between 0.067-0.115mgL-1 and 0.195-0.268mgL-1, respectively. The method demonstrated superiority over 1DLC method in respect of resolution (less alkaloid co-eluted), sample preparation (no pretreatment procedure) and transfer rate (minimum component loss). The optimized RP - SCX 2DLC approach was subsequently applied to quantify target alkaloids in five herbal medicines and herbal medicine tablets from three different manufactures. The results demonstrated that the developed heart cutting RP - SCX 2DLC approach represented a new, strategically significant methodology for the quality evaluation of tropane alkaloid in related herbal medicines that involve complex chemical matrix.

[A highly sensitive high performance liquid chromatography-electrochemical detection method for the determination of five phenolic compounds from Salvia miltiorrhiza].

The determination of antioxidants continues to be interested, since the oxidative damage is thought to be one of the main mechanisms involved in nearly all chronic renal pathologies. A highly sensitive high performance liquid chromatography-electrochemical detection (HPLC-ECD) method was developed for evaluating the antioxidant properties of Salvia miltiorrhiza (Dan Shen). The method was optimized with respect to selectivity and sensitivity. Chromatographic conditions, including mobile phase pH value, buffer concentration, buffer type, organic solvent type, gradient profile and flow rate, were systematically investigated. Low pH value (2.8), low buffer concentration (20 mmol/L NaH2PO4), a shallow water-acetonitrile gradient, and a flow rate of 0.2 mL/min were the determined optimal conditions for the quantitative analysis of aimed five antioxidants from 14 batches of Dan Shen samples. The described method provided a good recovery (>95%), a very wide linear range (up to 104 for all analytes), a good precision (RSDs<4.01%), and a high sensitivity (LOQ of caffeic acid, 1.5 µ g/L). Compared with UV detection, the described ECD method was also more effective for evaluating the antioxidant properties of Dan Shen, as it provided highly selective detection of electro-active antioxidants.

[High sensitive non-derivative determination of cyclovirobuxin D by high-performance liquid chromatography-electrochemical determination].

A high-performance liquid chromatography-electrochemical detection (HPLC-ECD) method was developed to determine cyclovirobuxin D (CVB-D) levels in tablets and human blood samples. A column with a positive charge-modified C18 stationary phase, C18HCE, was selected to analyze CVB-D, because it provided a sharper and more symmetric peak for CVB-D than conventional C18 stationary phase. Two types of working electrode materials, glassy carbon (GC) and boron-doped diamond (BDD), were evaluated. BDD was found to provide better sensitivity than GC owing to its lower background current and baseline noise. Utilizing the BDD electrode, C18HCE column, and optimized mobile phase composition, the developed HPLC-ECD method showed a much better sensitivity. The limit of detection and limit of quantification of the HPLC-ECD method for CVB-D were 0.198 and 0.297 µg/L, respectively. It was approximately 12727, 11481, and 2630 times more sensitive than ultraviolet (UV), evaporative light scattering detection, and charged aerosol detection, respectively. The sensitivity of the developed HPLC-ECD method was comparable or even better (16.8 times) than reported mass spectrometry (MS) methods for the determination of CVB-D. Additionally, it offered a much wider linear dynamic range (up to 4 orders of magnitude, 0.297-1891 µg/L) and was much less complicated than MS methods for determination of CVB-D. The developed HPLC-ECD method can be used for determination of CVB-D at both high and low concentrations. Good intra-day (relative standard deviation (RSD) of peak area<5.08%) and inter-day (RSD of peak area<5.57%) reproducibilities of the developed HPLC-ECD method were obtained even for a low mass concentration (59.1 µg/L) sample. After the optimized parameters were acquired, this method was applied to the quantitative analysis of CVB-D in CVB-D tablets and human blood samples. With a slight modification, the current HPLC-ECD method can also be applied to analyze many other basic compounds including basic drugs and environmental pollutants.

A non-derivative method for the quantitative analysis of isosteroidal alkaloids from Fritillaria by high performance liquid chromatography combined with charged aerosol detection.

A non-derivative method was developed for the qualitative and quantitative analysis of isosteroidal alkaloids from Fritillaria thunbergii. During method development the performance of two universal detectors, the charged aerosol detector (CAD) and evaporative light scattering detector (ELSD), were evaluated. The CAD was found to be 30 to 55 times more sensitive than ELSD enabling the measurement of low levels of reference compound impurities that could not be detected by ELSD. The peak area percent of the reference compound, peimisine, obtained by CAD was 50.10%, but 91.66% by ELSD showing that CAD is suitable to estimate the presence of impurities. The CAD showed good reproducibility with overall intra- and inter-day peak area RSD values of less than 1.8% and 2.7%, respectively and had a linear dynamic range of up to 4 orders of magnitude (0.06-44mg/L) for peimine and peiminine. The optimized method was used for the quantitative analysis of peimine and peiminine from F. thunbergii.

A sensitive non-derivatization method for apramycin and impurities analysis using hydrophilic interaction liquid chromatography and charged aerosol detection.

A sensitive non-derivatization method was developed for the analysis of apramycin and impurites using hydrophilic interaction liquid chromatography (HILIC) and charged aerosol detection (CAD). Sample was pretreated with an effective SPE method (recovery >90%) to remove interference with apramycin impurities from sulfate, then analyzed with direct injection. Different chromatography modes of separation and choices of HILIC column were investigated in search of a direct analysis method. The HILIC-CAD method was optimized using a cysteine-bonded zwitterionic HILIC column and compared to the strong cation exchange-ultraviolet (SCX-UV) method with post-column derivatization recommended by the Chinese Pharmacopoeia (veterinary) 2010. The improved chromatographic resolution and peak shape with the HILIC-charged aerosol detection method allows for increase of sample load to 48.9 µg from only 2.8 µg with the SCX-UV approach. More than 16 impurities were detected with this method with improved resolution, and four were identified with MS, while only 7 impurities were detected with the SCX-UV method. Moreover, the current method has a good precision and reproducibility. The intra-day and inter-day of peak area variability was less than or equal to 4.760% RSD and 9.950%, respectively. The average limit of detection and quantization was 80 ng and 200 ng injected on the column, respectively. The overall results demonstrated that the presented method can be used as an alternative to SCX-UV method in the analysis of apramycin and impurities.

[Quantitative analysis of five antiviral drugs by hydrophilic interaction liquid chromatography-charged aerosol detection].

Antiviral drugs are widely used for human and animals. However, the analysis of the mixture of antiviral drugs is a challenge for high performance liquid chromatography, since some of the antiviral drugs have weak UV absorbance and poor retention in reversed phase liq- uid chromatography. A method of hydrophilic interaction liquid chromatography-charged aerosol detection (HILIC-CAD) was optimized for the qualitative and quantitative analysis of five antiviral drugs. In this study, Click TE-Cys was used as the stationary phase and CAD was used as the detector. Various chromatographic conditions including the kind of detector, chromatographic mode, column and mobile phase composition were investigated. Compared to UV-Vis, more antiviral drugs could be detected by CAD, since it is a universal detector. HILIC mode is an alternative to reversed phase liquid chromatography mode. HILIC provides higher sensitivity and unique selectivity to target compounds. After the optimized parameters were obtained, the developed method was used for the quantitative analysis of the five antiviral drugs. As a result, the current method has good repeatability, a wide linear range (0.07-2.28 mg/mL) and good sensitivity (LOQ ≤ 0.04 mg/mL). The RSDs of intra-day and inter-day peak areas were less than 3. 06% and 5. 38% respectively. The above results demonstrated that the current method is sensitive, robust and effective for the separation and determination of these five antiviral drugs.

Radical-scavenging activity and mechanism of resveratrol-oriented analogues: influence of the solvent, radical, and substitution.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene, 3,5,4'-THS) is a well-known natural antioxidant and cancer chemopreventive agent that has attracted much interest in the past decade. To find a more active antioxidant and investigate the antioxidative mechanism with resveratrol as the lead compound, we synthesized 3,5-dihydroxy-trans-stilbene (3,5-DHS), 4-hydroxy-trans-stilbene (4-HS) 3,4-dihydroxy-trans-stilbene (3,4-DHS), 4,4'-dihydroxy-trans-stilbene (4,4'-DHS), 4-hydroxy-3-methoxy-trans-stilbene (3-MeO-4-HS), 4-hydroxy-4'-methoxy-trans-stilbene (4'-MeO-4-HS), 4-hydroxy-4'-methyl-trans-stilbene (4'-Me-4-HS), 4-hydroxy-4'-nitro-trans-stilbene (4'-NO(2)-4-HS), and 4-hydroxy-4'-trifluoromethyl-trans-stilbene (4'-CF(3)-4-HS). The radical-scavenging activity and detailed mechanism of resveratrol and its analogues (ArOHs) were investigated by the reaction kinetics with galvinoxyl (GO(*)) and 2,2-diphenyl-1-picrylhydrazyl (DPPH(*)) radicals in ethanol and ethyl acetate at 25 degrees C, using UV-vis spectroscopy. It was found that the reaction rates increase with increasing the electron-rich environment in the molecules, and the compound bearing o-dihydroxyl groups (3,4-DHS) is the most reactive one among the examined resveratrol analogues. The effect of added acetic acid on the measured rate constant for GO(*)-scavenging reaction reveals that in ethanol that supports ionization solvent besides hydrogen atom transfer (HAT), the kinetics of the process is partially governed by sequential proton loss electron transfer (SPLET). In contrast to GO(*), DPPH(*) has a relatively high reduction potential and therefore enhances the proportion of SPLET in ethanol. The relatively low rate constants for the reactions of ArOHs with GO(*) or DPPH(*) in ethyl acetate compared with the rate constants in ethanol prove that in ethyl acetate these reactions occur primarily by the HAT mechanism. The contribution of SPLET and HAT mechanism depends on the ability of the solvent to ionize ArOH and the reduction potential of the free radical involved. Furthermore, the fate of the ArOH-derived radicals, i.e., the phenoxyl radicals, was investigated by the oxidative product analysis of ArOHs and GO(*) in ethanol. The major products were dihydrofuran dimers in the case of resveratrol, 4,4'-DHS, and 4-HS and a dioxane-like dimer in the case of 3,4-DHS. It is suggested from the oxidative products of these ArOHs that the hydroxyl group at the 4-position is much easier to subject to oxidation than other hydroxyl groups, and the dioxane-like dimer is formed via an o-quinone intermediate.

4,4'-Dihydroxy-trans-stilbene, a resveratrol analogue, exhibited enhanced antioxidant activity and cytotoxicity.

Resveratrol (3,5,4'-trans-trihydroxystibene) is a natural phytoalexin present in grapes and red wine, which possesses a variety of biological activities including antioxidant activity. In order to find more active antioxidant with resveratrol as the lead compound we synthesized 4,4'-dihydroxy-trans-stilbene (4,4'-DHS). The antioxidant activities of resveratrol and 4,4'-DHS were evaluated by the reaction kinetics with galvinoxyl radical or Cu(II) ions, and the inhibition effects against free-radical-induced peroxidation of human erythrocyte ghosts. It was found that 4,4'-DHS exhibits remarkably higher antioxidant activity than resveratrol. The oxidative products of resveratrol and 4,4'-DHS in the presence of Cu(II) in acetonitrile were identified as the dihydrofuran dimers by spectroscopic method, and the antioxidant mechanism for 4,4'-DHS was proposed. In addition, 4,4'-DHS exhibits remarkably higher cytotoxicity against human promyelocytic leukemia (HL-60) cells than resveratrol.

Mass transfer kinetics in the chromatography of insulin variants under nonlinear conditions.

Experimental data regarding the thermodynamics and kinetics of adsorption of lispro, an insulin variant, onto a YMC ODS-A column, from an aqueous solution of acetonitrile (31%) and TFA are reinterpreted, using a more complex model of the mass transfer kinetics. The adsorption behavior follows the Toth isotherm model, suggesting either a strongly heterogeneous surface or, rather, that when insulin molecules adsorb they contact the surface along different areas of the molecule. The lumped pore diffusion (POR) model of chromatography accounts well for the band profiles. The internal mass transfer resistances are higher than expected, which suggests that intraparticle diffusion is slower. Furthermore, the pore diffusion coefficient increases with decreasing sample size. That surface diffusion accounts for the mass transfer kinetics inside particles explains these results. Assuming that the gradient of the surface concentration is the driving force of surface diffusion, it is possible to account very well for the band profiles of samples of widely different sizes, using a single value of the surface diffusivity.

Modeling of the separation of two enantiomers using a microbore column.

A microbore column packed with Chiralcel OB (cellulose tribenzoate coated silica) was used for the measurement of the single and competitive equilibrium-isotherm data of the 1-indanol enantiomers by frontal analysis. The amount of sample needed for the isotherm data acquisition was about 20 times less than that required with a conventional column. The data obtained were fitted to different single and competitive isotherm models. Both the single and the competitive data sets fitted best to the same Bilangmuir (BL) isotherm model with small differences in the numerical values of the parameters. The best fitted Bilangmuir single and competitive isotherm models were used to predict the overloaded experimental profiles of both pure enantiomers, of the racemic mixture, and of different enantiomeric mixtures. All the calculated profiles were in excellent agreement with the experimental ones. This agreement confirms that in many chiral separations, the competitive isotherms can be derived from data acquired from the mere racemic mixture with a sufficient accuracy for a correct prediction of the band profiles of all kinds of enantiomer mixtures, making possible the computer-assisted optimization of the experimental conditions.

Influence of pressure on the retention and separation of insulin variants under linear conditions.

The effect of pressure on the retention behavior of insulin variants in RPLC on a YMC-ODS C18 column was investigated under linear conditions. The retention factors of these variants increase nearly 2-fold when the average column pressure is increased from 55 to 250 bar while their separation factors remain nearly unchanged. This effect is explained by a change of the partial molar volume of the insulin variants associated with their adsorption that decreases from -99 to -80 mL/mol for mobile-phase concentrations of acetonitrile increasing from 29 to 33% (v/v). This volume change is much larger than the one observed with low molecular weight compounds. For the same pressure variation, the average number Z of acetonitrile molecules displaced from the protein and the stationary phase upon adsorption increases from 22 to 23.3. The pressure-induced relative increase of the term b[S]/[D0]z (which corresponds to the initial slope of the adsorption isotherm) is approximately twice as large for Lispro than for porcine insulin. Because the binding constant of insulin decreases with increasing pressure, this suggests that the number of binding sites on the stationary phase increases even faster. Finally, it was observed that the column efficiency at flow rates higher than 0.6 mL/min increases slightly with increasing pressure. It is suggested that these observations are also valid for other proteins analyzed in RPLC.

Pressure-induced effects in the heterogeneous adsorption of insulin on chromatographic surfaces.

The effect of increasing the average column pressure (ACP) on the heterogeneous adsorption of insulin variants on a C18-bonded silica was studied in isocratic reversed-phase HPLC. Adsorption isotherm data of lispro and porcine insulin obtained for values of the ACP ranging from 57 to 237 bar were fitted to the Langmuir-Freundlich and the Tóth equation. The resulting isotherm parameters, including the equilibrium adsorption constant and the heterogeneity index, were next used for the calculation of distribution functions characterizing the energy of interactions between the adsorbed insulin molecules and the stationary phase. It was observed that increasing the pressure by 180 bar causes a broadening of the distribution functions and a shift of the position of their maximum toward lower interaction energies. These findings suggest that, under high pressures, the insulin molecules interact with the stationary phase in a more diversified way than under low pressures. Additionally, the most probable value of the energy of the insulin-surface interactions becomes lower when the ACP increases. The pressure-induced changes in the interaction of insulin variants with the hydrophobic surface are attributed to a possible conformational flexibility of the molecular structure of this protein.

Prediction of band profiles of mixtures of bradykinin and kallidin from data acquired by competitive frontal analysis.

The competitive adsorption isotherms of two closely related peptides, bradykinin and kallidin, were measured by frontal analysis on a Zorbax SB-C18 microbore column. An aqueous soluton at 20% acetonitrile (0.1% TFA) was used as the mobile phase. The competitive isotherm data were fitted to four different models: Langmuir, Bilangmuir, Langmuir-Freundlich, and Toth. These data fitted best to a Bilangmuir isotherm model. The influence of the pressure on the retention factors of the two peptides was found to be small and was not investigated in detail. The band profiles of large samples of the single components and of their mixtures were recorded. The overloaded profiles calculated using either the equilibrium-dispersive or POR model are in excellent agreement with the experimental profiles in all cases. Our results confirm that the competitive isotherm data derived from mixtures may suffice for a reasonably accurate prediction of the band profiles of all mixtures of the two components, provided their composition is close to 1/1.