Frontal Analysis Method Research Articles

Retention mechanism on phosphodiester stationary phases in hydrophilic interaction liquid chromatography and purely aqueous mobile phase part II: Overloading with limited soluble samples

The adsorption behaviour of caffeine and theophylline under hydrophilic interaction chromatography and purely aqueous conditions was investigated on four phosphodiester stationary phases. Solute adsorption isotherms were determined by frontal analysis or inverse method. The bi-Langmuir model was found to be the best choice to describe the behaviour of caffeine and theophylline adsorption in purely aqueous conditions, whereas the bi-Moreau model describes the adsorption phenomena in HILIC conditions. The results obtained demonstrate that the interaction of caffeine and theophylline with the stationary phase surface varies depending on the mobile phase composition. Both in pure aqueous mobile phase and in HILIC mode, the heterogeneity of the surface of the studied stationary phases is confirmed. In hydrophilic solutions, the sample molecules interact with the stationary phase only. In hydrophobic conditions, a lateral interaction occurs between caffeine or theophylline molecules, which are poorly soluble in acetonitrile-rich solvents. This confirms that the same compound on the same stationary phase can behave rather differently, depending on the mobile phase composition. Thus, the mobile phase may govern and control the retention mechanism.

THE 14TH INFANTRY DIVISION OF THE SOUTH-EASTERN FRONT IN OCTOBER-DECEMBER 1919

Purpose. The purpose of the article is to investigate the general nature and the main content of combat operations of the 14th Infantry Division, as well as the party-political work of its command to fulfill the combat tasks in the period from September 1919, when the division joined the newly formed Southeastern Front of the Red Army, and up to the forcing of the Don and the beginning of active combat operations on its right bank (December 1919).
 Method and methodology. The general methodological approach to the research is based on the basic principles and methods of scientific and historical cognition. The priority scientific methods in this study were the general scientific methods of analysis, synthesis, classification, analogy, as well as methods of historical analysis (descriptive, comparative-historical, logical, retrospective, method of frontal analysis of archival materials).
 Results. The results of the research showed that the 14th Division of the 9th Army of the Southeastern Front was able to recover from its almost complete defeat in early October 1919. It continued to fulfill the tasks on its section of the front. In the life of the division there was a place for party-political work. Going on the offensive to the Khoper River, the division with its actions restrained the enemy forces. Having accomplished the task, the division from the beginning of December went on the active offensive to the Don River. Having forded the Don River, the division continued the offensive on the right bank.
 The scope of applicability of results. The results of the research can contribute to a more in-depth study of the Civil War in the period from October to the end of December 1919.

Affinity Chromatographic Method for Determining Drug-Protein Interaction with Enhanced Speed Than Typical Frontal Analysis.

Revealing drug-protein interaction is highly important to select a drug candidate with improved drug-like properties in the early stages of drug discovery. This highlights the urgent need to develop assays that enable the analysis of drug-protein interaction with high speed. Herein, this purpose was realized by the development of an affinity chromatographic method with a two-fold higher speed than typical assays like frontal analysis and zonal elution. The method involved synthesis of a stationary phase by immobilizing poly(ADP-ribose) polymerase-1 (PARP1) onto macroporous silica gel through a one-step bioorthogonal reaction, characterization of mutual displacement interaction of two canonical drugs to the immobilized PARP1, determination of the interaction between three (iniparib, rucaparib, and olaparib) drugs and the protein, and validation of these parameters by typical frontal analysis. The numbers of binding sites on the column were (2.85 ± 0.05) × 10-7, (1.89 ± 0.71) × 10-6, and (1.49 ± 0.06) × 10-7 M for iniparib, rucaparib, and olaparib, respectively. On these sites, the association constants of the three drugs to the protein were (9.85 ± 0.56) × 104, (2.85 ± 0.34) × 104, and (1.07 ± 0.35) × 105 M-1. The determined parameters presented a good agreement with the calculation by typical frontal analyses, which indicated that the current continuous competitive frontal analysis method was reliable for determining drug-protein interaction. Application of the methods was achieved by screening tubeimosides I and II as the bioactive compounds against breast cancer in Bolbostemma paniculatum. Their mechanism may be the interference of DNA repair via down-regulating PARP1 and meiotic recombination 11 expressions, thus leading to oncogene mutations and death of cancer cells. The method was high speed since it allowed simultaneous determination of binding parameters between two drugs and a protein with a smaller number of experiments to be performed. Such a feature made the method an attractive alternative for high-speed analysis of drug-protein interaction or the other bindings in a binary system.

Identifying potential ligands specifically binding to beta1-adrenoceptor from Radix Aconiti Lateralis Praeparata extract by affinity chromatographic method

As expressed predominantly in cardiac tissue, beta1-adrenoceptor (β1-AR) is broadly accepted as one of the main targets for drugs against cardiovascular ailments. However, the discovery of β1-AR ligand is gravely challenged due to the lack of efficient screening method. This work developed a general strategy for pursuing β1-AR ligands from the herbal extract by immobilizing haloalkane dehalogenase (Halo)-tagged β1-AR onto microspheres coated with 6-chlorohexanoic acid, and applying the immobilized β1-AR in the analysis of ligand-receptor interaction. The morphology was characterized by scanning electron microscope (SEM) and X-ray photoelectron spectroscopy (XPS). The chromatographic specificity of the immobilized receptor column was evaluated by determining the association constants of atenolol, esmolol and metoprolol using stepwise frontal analysis plus injection amount-dependent method. The potential ligands binding to β1-AR was screened by collecting the peak with retention time longer than the void time, and identified the collection by reverse phase liquid chromatography coupled with tandem mass spectrometry. The association constants of the three drugs to β1-AR were (3.33 ± 0.29)× 106 M−1, (2.33 ± 0.23)× 106 M−1 and (2.06 ± 0.03)× 106 M−1, indicating a desired specificity of the immobilized receptor for recognizing its ligands. Molecular docking showed that van der Waals, hydrogen bonds, and hydrophobic interactions were the principal interaction forces for the receptor-drug complexes. Benzoylmesaconine was screened as the potential ligand of β1-AR in Radix Aconiti Lateralis Praeparata extract. The association constant of the ligand was (1.06 ± 0.02)× 105 M−1, hinting structural modification may be required before clinical application. The immobilized β1-AR is possible to provide a rapid method for screening potential ligands in herbal extract.

An Experimental and Modeling Combined Approach in Preparative Hydrophobic Interaction Chromatography

Chromatography is a technique widely used in the purification of biopharmaceuticals, and generally consists of several chromatographic steps. In this work, Hydrophobic Interaction Chromatography (HIC) is investigated as a polishing step for the purification of therapeutic proteins. Adsorption mechanisms in hydrophobic interaction chromatography are still not completely clear and a limited amount of published data is available. In addition to new data on adsorption isotherms for some proteins (obtained both by high-throughput and frontal analysis method), and a comparison of different models proposed in the literature, two different approaches are compared in this work to investigate HIC. The predictive approach exploits an in-house code that simulates the behavior of the component in the column using the model parameters found from the fitting of experimental data. The estimation approach, on the other hand, exploits commercial software in which the model parameters are found by the fitting of a few experimental chromatograms. The two approaches are validated on some bind-elute runs: the predictive approach is very informative, but the experimental effort needed is high; the estimation approach is more effective, but the knowledge gained is lower. The second approach is also applied to an in-development industrial purification process and successfully resulted in predicting the behavior of the system, allowing for optimization with a reduction in the time and amount of sample needed.

Monument to Taras Shevchenko in Galicia: analysis of architectonics and meaning

The purpose of the article is to describe and characterize the unknown monument to Taras Shevchenko, built in the village of Budylo near Sniatyn in honor of the 100th anniversary of the great Ukrainian poet birth and has not yet been included to the history of national memorials.
 The methodology. The author used the method of frontal examination and analysis of the rural memorial in the historical aspect, synthesizing its architectural elements and religious symbolism.
 The results. The monument to Taras Shevchenko in Galicia is a typical memorial phenomenon of the early XX century as a synthesis of civil and sacred styles. It is made purely by architectural means without sculptural decoration and focuses on the importance of the historical date. That’s we can see the balanced rhythm of the entire composition. The harmony of the lines of the monument embodies a deep philosophical meaning as the ratio of genius and eternity. The memorial reminds of the connection between history and modernity, inspires to enlighten united, free and independent Ukraine. The article expands the understanding of the geography and architectural forms of the Shevchenko memorials of the XX century.
 The scientific topicality. The memorial in Galicia, dedicated to the memory of Kobzar, deserves to be studied as a rare combination of architectural and decorative elements of different cultures — Orthodox and Catholic.
 The practical significance. Monument to Taras Shevchenko, opened in the village of Budylo, will certainly attract cultural historians by its memorial obelisks, columns as an example of Western Ukraine memorial architecture, which has its own features and differences.

Generation of a pH-blind/pH-sensitive alternating surface on a hydrophobic resin by mussel-inspired chemistry and investigating the effect of surface modification on the adsorption dynamics of some anionic colorants

The effect of a surface modification process known as "mussel-inspired modification" on the adsorption dynamics of hydrophobic adsorbent, namely XAD1180, was studied based on equilibrium adsorption data. In the study, three anionic colorants (i.e., Sunset yellow, SY; Allura red, AR; and Tartrazine, TZ) were used as the model compounds. The role of some experimental parameters, such as pH (4–7), ionic strength (0.01–0.50 mM) and colorant concentration (25–125 mg/L), were investigated to determine optimal conditions leading to the highest adsorption. Equilibrium adsorption data were obtained under optimal conditions by stepwise frontal analysis method (SFA) and modeled on the basis of some common adsorption isotherm models (i.e., Langmuir and Freundlich). The adsorption dynamics of the XAD1180 resin have been found to vary significantly after PD coating. After the PD coating process, it was found that the highly hydrophobic background material, XAD1180, gained a pH-sensitive character, thereby replacing a sharp decrease in the adsorption capacity at pH = 5.0–7.0 with an increase in pH = 4.0. Another dramatic effect of the PD coating was observed with Langmuir monolayer adsorption capacities, so that the PD layer was found to increase the monolayer saturation capacity of the XAD1180 (from 8.0 to 17 µmol/g for SY, from 7.7 to 9.9 µmol/g for AR, and from 5.3 to 9.1 µmol/g for TZ). It was also deduced from the Freundlich isotherm parameter, KF, that there was, in general, a clear increment in the overall effect of adsorption capacity and the average affinity after mussel-inspired surface modification process.

ФРОНТІРНИЙ АНАЛІЗ СОЦІАЛЬНО-ДЕМОГРАФІЧНИХ ДАНИХ

Determining the level of efficiency of the socio-economic system is a subjective process for each researcher, because the quantitative characteristics that determine the level of effective operation of the system are different. Successful selection of indicators and method of data processing provides an opportunity to conduct an assessment. The ability to determine the efficiency of processes within the system provides a number of advantages, such as functionality, flexibility, scaling, performance. The article proposes to use the method of frontal analysis, which is widely used in assessing the quality of banks. The proposed methodology provides a positive result when working with data from the health care system of different countries to identify applicants for analysis of differences from the existing health care system in Ukraine. The urgency of solving the problem of analyzing the quality of the health care system of countries with relatively effective indicators is a step towards a fundamental reform of the existing system and choosing the direction of further development and possible options for change in the wrong course of this development. Qualitative and quantitative indicators that shape the effectiveness of the health care system are proposed to use the costs of health care, life expectancy and the use of the health care system by citizens of the country. When analyzing medical indicators, it is very important to determine their effectiveness. With frontal analysis, this capability is realized on the basis of one input value – health care costs, and output values, such as life expectancy and health care use. As a result of the analysis, the open free software Frontier Analyst Application and the information base of the Organization for Economic Cooperation and Development were used to compare the performance of countries according to the benchmark in the group and determine which country and by how many deviations of efficiency indicators. The study allowed to process the important socio-economic experience of the countries, which serves as a basis for the reform of the health care system in Ukraine in order to improve and enhance it.

Behavioral imaging of serum albumin during matrine transport based on capillary electrophoresis

Matrine (MT) is an alkaloid widely used in the treatment of tumor diseases. It is the main medicinal ingredient in the dried roots of kuh-seng (Sophora flavescens Ait). However, there have been few studies on its transport mechanism. Serum albumin (SA) is the most abundant protein in blood. SA combines easily with many substances, including MT. MT and human serum albumin (HSA) were analyzed by capillary electrophoresis (CE) under in vitro conditions. The capillary tubing was 50 μm. The total length of the capillary was 60 cm, the total effective length was 50 cm. The interaction models of ligand-receptor binding were constructed by the mobility and frontal analysis (FA) methods. The purpose of establishing the interaction model was to study the binding of MT and SA. The phosphate buffer solution (PBS, 0.02 mol/L) was prepared in double distilled water. All solutions were prepared in PBS (0.02 mol/L). All solutions were filtered twice through a 0.45 μm microporous membrane, degassed for 5 min at a time. In the mobility method, different gradient MT solutions were used as running buffers. Their concentrations were 1.0×10-4-1.0×10-3 mol/L, with the gradient of 1.0×10-4 mol/L. And the HSA solution containing (0.5% (v/v)) acetone was used as test sample. Its concentration was 1.0×10-5 mol/L. The nonlinear fitting method was used to obtain the binding parameters of MT and HSA. In the FA method, different gradient MT-HSA solutions were used as test samples. Their concentrations were 1.0×10-4-1.0×10-3 mol/L, with the gradient of 1.0×10-4 mol/L. And the PBS solution (0.02 mol/L) was used as running buffer. Then three equations were used to obtain the binding parameters of MT and HSA. And the applicability of the models was analyzed using the binding parameters. These three equations were nonlinear regression equation, Scatchard linear equation, and Klotz linear equation. Using the mobility method, the apparent binding constant KB was 8.072×103 mol/L. According to the FA method, three apparent binding constants were obtained for MT and HSA. The apparent binding constant KB of HSA and MT by nonlinear regression equation, Scatchard linear equation and Klotz linear equation were 1.434×103, 1.781×103 and 2.133×103 mol/L. The comparison was as follows, KB(nonlinear regression equation) < KB(Scatchard linear equation) < KB(Klotz linear equation). The number of binding sites was about 1.0. It was indicating that MT had only a single type of binding site with HSA. By analyzing the applicability of the model, the correlation coefficients (r) of the three equations were obtained. The comparison was as follows, r(Klotz linear equations) > r(nonlinear regression equations) > r(Scatchard linear equations). The results showed that both the methods were all suitable for analyzing the MT-SA system. The FA method could calculate the apparent binding constants and the numbers of binding sites. Therefore, it was more suitable for the analysis of MT and HSA. And the Klotz linear equation was the best fit for the theoretical model among the three equations. The combined parameters indicated that the interaction of MT with HSA had only one binding site. And the binding of MT with HSA was stable. This experimental method could be used to determine the binding status of MT and HSA. It is useful to further explore the binding mechanism of MT and HSA. This work provides valuable information on the interaction mechanism of typical alkaloids with SA. It will be useful in studies of the blood transport mechanisms of alkaloids.

Dynamic adsorption/desorption of proteins with thermo-responsive polymer grafted sepharose fast flow sorbents

This investigation has focused on the dynamic adsorption and desorption behaviour of proteins with thermo-responsive co-polymer grafted chromatographic materials. To that end, the dynamic adsorption performance of bovine holo-lactoferrin (bhLf) with two thermo-responsive chromatographic sorbents, i.e. poly (N-isopropyl acrylamide-co-tert-butylacryl amide-co-acrylic acid) grafted Sepharose Fast Flow (ITA) and poly (NIPAAm-co-tert-butylacryl amide-co-2-(diethylamino)ethyl methacrylate)) grafted Sepharose Fast Flow (ITD) at two different column temperatures and different protein loading conditions was examined by frontal chromatographic analysis methods. The dynamic binding capacity (DBC), dynamic binding rate (DBR) and column efficiency of the ITA sorbent for bhLf at 50 °C were greater than those at 20 °C. The dynamic binding characteristics were observed to be influenced by the protein feed concentrations, showing an increase of binding capacity with an increase in the bhLf feed concentration. A large proportion (≥65%) of the bhLf molecules adsorbed onto the ITA sorbent could be eluted from the column by simply lowering the column temperature from 50 °C to 20 °C. The corresponding DBC values of ITA sorbent for bovine serum albumin (BSA) were much lower than those for bhLF at both 20 °C and 50 °C due to electrostatic repulsion effects between the negatively charged BSA and the negatively charged co-polymer modified surface of the ITA sorbent, whilst the opposite trend was found with positively charged co-polymer (i.e. poly (N-isopropyl acrylamide-co-tert-butylacryl amide-co-2-(diethyl-amino)ethyl methacrylate)) grafted Sepharose Fast Flow (ITD), indicative of different separation selectivity.

Cell membrane chromatography for the analysis of the interaction between chloroquine and hydroxychloroquine with ACE2 receptors

The recent emergence of the novel pathogenic coronavirus disease 2019 (COVID-19) is responsible for a worldwide pandemic. In sight of this, there has been growing interest in the use of chloroquine (CQ) and hydroxychloroquine (HCQ) as potential treatments. In this study, we use angiotensin converting enzyme 2 (ACE2) over-expressed cell membrane chromatography (CMC) to study the interaction of CQ and HCQ with ACE2 receptor. Both CQ and HCQ were retained on the ACE2/CMC column. Then we analyzed the binding character of CQ and HCQ to ACE2 by CMC frontal analysis, ionic force investigation and competitive binding experiment. Results showed that CQ and HCQ KD values obtained from the CMC frontal analysis method were 8.22(±0.61) × 10−7 M and 11.70(±2.44) × 10−7 M. Compare to CQ, HCQ has the weaker affinity with ACE2. The action force of CQ, HCQ and ACE2 is mainly ionic force. CQ and HCQ have different degrees of competitive binding relationship with ACE2. Our study revealed the interaction of CQ and HCQ with ACE2 receptor, which provides new insights for the use of CQ and HCQ in the treatment of COVID-19. Moreover, this biomimetic drug screening method is expected to open the door for rapid targeting and separating bioactive ingredients active towards ACE2 receptor.

Application of a stepwise frontal analysis method in cell membrane chromatography

Bio-affinity chromatography is used in the study of drug-receptor interactions. A stepwise frontal analysis (SFA) method was developed based on frontal analysis (FA). A high expression alpha 1A adrenergic receptor (α1A AR) cell membrane chromatography (CMC) method was then developed and combined with SFA to investigate the affinity of three model α1A AR-binding drugs towards α1A AR. Equilibrium dissociation constant (Kd) values for drug-receptor interactions were determined by FA and SFA; results showed that these methods were highly consistent. The results demonstrate that the CMC/SFA method is a time-saving and less wasteful method than traditional method for the evaluation of drug-receptor binding characteristics, and could be used to study the interactions between drugs and membrane receptors.

Measuring Binding Constants of His-Tagged Proteins Using Affinity Chromatography and Ni-NTA Immobilized Enzymes.

Affinity chromatography is one way to measure the binding constants of a protein-ligand interaction. Here, we describe a method of measuring a binding constant using Ni-NTA resin to immobilize a His-tagged enzyme and the method of frontal analysis. While other methods of immobilization are possible, using the strong affinity interaction between His-tagged proteins and Ni-NTA supports results in a fast, easy, and gentle method of immobilization. Once the affinity support is created, frontal analysis can be used to measure the binding constant between the protein and various analytes.

Discovery and analysis the anti-pseudo-allergic components from Perilla frutescens leaves by overexpressed MRGPRX2 cell membrane chromatography coupled with HPLC-ESI-IT-TOF system.

Screen and identify the anti-pseudo-allergic activity components of Perilla frutescens leaves that interacted with MRGPRX2 (a new reported pseudo-allergic reaction-related receptor). An overexpressed MRGPRX2 cell membrane chromatography (CMC) coupled with HPLC-ESI-IT-TOF system has been established to screen and identify the effective components from P.frutescens leaves. A frontal analysis method was performed to investigate the binding affinity between ligands and MRGPRX2. Their activity of relieving pseudo-allergic reaction was evaluated in vitro by histamine release assay, β-hexosaminidase release assay and intracellular Ca2+ mobilization assay. Extract of P.frutescens leaves was proved to be effective in anti-pseudo-allergic reaction by inhibiting MRGPRX2. Apigenin (API) and rosmarinic acid (ROS) were confirmed to be the potential anti-allergy compounds that could bind with MRGPRX2. The binding affinity (KD ) of ROS and API with MRGPRX2 was (8.79±0.13)×10-8 m and (6.54±1.69)×10-8 m, respectively. The IC50 of API inhibiting laboratory of allergic disease 2 cells degranulation was also determined to be (51.96±0.18)μm. A MRGPRX2/CMC coupled with HPLC-ESI-IT-TOF system was successfully established and applied to discover the effective components from P.frutescens leaves.

Measurements of Ion Binding to Lipid-Hosted Ionophores by Affinity Chromatography.

The binding affinity between antibiotic ionophores and alkali ions within supported lipid bilayers was evaluated using affinity chromatography. We used zonal elution and frontal analysis methods in nanovolume liquid chromatography to characterize the binding selectivity of the carrier and channel ionophores valinomycin and gramicidin A within different phosphatidylcholine bilayers. Distinct binding sensitivity to the lipid phase, both in affinity and selectivity, is observed for valinomycin, whereas gramicidin is less sensitive to changes in a membrane environment, behavior that is consistent with ion binding occurring within the interior of an established channel. There is good agreement between the chromatographic retention and the reported binding selectivity measured by other techniques. Surface potential near the binding site affects ion retention and the apparent association binding constants, but not the binding selectivity or enthalpy measurements. A model accounting for the surface potential contributions of retained ions during frontal analyses yields values close to intrinsic binding constants for gramicidin A (KA for K+ between 70 and 120 M-1) using reasonable estimates of the initial potential that is postulated to arise from the underlying silica.

Analysis of the Interaction Mechanisms of Polysaccharide Homologs Binding with Serum Albumin Using Capillary Electrophorsis

Different polysaccharide homologs (as the ligand) binding to serum albumin (bovine serum albumin (BSA)) as the receptor were investigated by capillary electrophoresis (CE). The Hummel–Dreyer method (HD), frontal analysis method(FA), and vacant peak method(VP) were used to measure the interaction parameters of the polysaccharide-BSA system. A workable CE method was selected by comparing different analytical methods and a feasible binding model was established by evaluating the different theoretical equations. The binding mechanism of polysaccharide-BSA was also explored under physiological conditions. The results showed that both HD and FA method can be used to analyze the polysaccharide-BSA system, with the most suitable CE method being the HD, and the double logarithmic equation gives the best fit to the data. The binding parameters indicate that the interaction between polysaccharides and BSA had moderate affinity, which had only one type of binding site. This work provides a reference for future studies of polysaccharide-protein interactions.

Column chromatography for separation and fractionation of flavor-active esters on hydrophobic resins and simulation of breakthrough behavior

For simulating an adsorption/elution step for separation and recovery of flavor-active esters in beer in the presence of ethanol at various temperatures, and validating the predicted breakthrough behavior, equilibrium data on concentration of each ester is required. This work evaluates the application of frontal analysis method (FA) for prediction of breakthrough behavior for adsorption of ethyl acetate, and determination of equilibrium concentrations and binding capacity for competitive adsorption of four major flavor-active esters in beer (i.e. ethyl acetate, isopentyl acetate, ethyl 4-methylpentanoate, and ethyl hexanoate), together with improvement of the obtained results, through fraction collection, and offline analysis, on columns packed with hydrophobic resins, Amberlite XAD16N and Sepabeads SP20SS. Single-component adsorption of ethyl acetate reveals a shorter breakthrough time, and higher slope of breakthrough curve for adsorption on SP20SS, due to smaller particle size, (50–100 µm), and enhanced mass transfer characteristics of this resin. Competitive frontal analysis tests, neatly demonstrate that increase in temperature is not favorable for adsorption but aids the elution step, 63–100% recovery of flavors at 333.15 K in comparison to 40–80% recovery at 298.15 K. Lower binding capacity of esters and shorter adsorption/elution cycle time is achieved at higher ethanol concentration and cyclic operation simulated under non-isothermal condition, exhibit higher accuracy between predicted and experimental breakthrough curves for XAD16N. A cyclic operation is simulated, for a larger scale column, for two scenarios, separation of ethyl acetate and complete separation of all flavor-active esters in the mixture. For more detailed prediction of breakthrough behavior, the influence of other components present in process streams needs to be investigated on competitive adsorption of esters.

Effect of Porogen Solubility Parameter on Structure of Chromatographic Supports with Large Pores

Abstract The macroporous microspheres were prepared through suspension polymerization based on a copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate. The effect of porogen on the microspheres structure was evaluated in terms of pore size and surface area. Porogen contained dichloromethane (δ = 9.7 (cal cm–3)1/2) and N-octanol (δ = 10.3 (cal cm–3)1/2) which were correspond to a good and poor solvent, respectively. The solubility parameter of porogen was controlled in the range of 9.89–10.09 (cal cm–3)1/2. The pore size of microspheres increased with the increase of difference value of solubility parameter between the polymer and the porogen. On the contrary, the surface area of microspheres decreased. The anion exchange media was prepared through coupling poly(ethylene imine) in the microspheres and the proteins transport was determined by frontal analysis method. The macroporous microspheres with 257 nm pore size could still afford a high proteins capacity (45.1 mg mL–1). These macroporous supports showed great potential in rapid separation of proteins.

Determine equilibrium dissociation constant of drug-membrane receptor affinity using the cell membrane chromatography relative standard method

The equilibrium dissociation constant (KD) of drug-membrane receptor affinity is the basic parameter that reflects the strength of interaction. The cell membrane chromatography (CMC) method is an effective technique to study the characteristics of drug-membrane receptor affinity. In this study, the KD value of CMC relative standard method for the determination of drug-membrane receptor affinity was established to analyze the relative KD values of drugs binding to the membrane receptors (Epidermal growth factor receptor and angiotensin II receptor). The KD values obtained by the CMC relative standard method had a strong correlation with those obtained by the frontal analysis method. Additionally, the KD values obtained by CMC relative standard method correlated with pharmacological activity of the drug being evaluated. The CMC relative standard method is a convenient and effective method to evaluate drug-membrane receptor affinity.

Confirming therapeutic target of protopine using immobilized β2 -adrenoceptor coupled with site-directed molecular docking and the target-drug interaction by frontal analysis and injection amount-dependent method.

Drug-protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized β2 -adrenoceptor (β2 -AR) by linkage of the receptor on macroporous silica gel surface through N,N'-carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site-directed molecular docking. Subsequent application of immobilized β2 -AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount-dependent method. The association constants of protopine to β2 -AR by the 2 methods were (1.00±0.06)×105 M-1 and (1.52±0.14)×104 M-1 . The numbers of binding sites were (1.23±0.07)×10-7 M and (9.09±0.06)×10-7 M, respectively. These results indicated that β2 -AR is the specific target for therapeutic action of protopine in vivo. The target-drug binding occurred on Ser169 in crystal structure of the receptor. Compared with frontal analysis, injection amount-dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high-throughput drug-receptor interaction analysis.