Specific DNA aptamer-immobilized cryogel membranes as novel bioaffinity supports and their potential for the purification of activated protein C.

Activated protein C (APC), a serine protease produced from zymogen protein C (PC), is the key enzyme of the protein C pathway. APC has anticoagulant, anti-inflammatory, and cytoprotective features. APC has recently been shown to significantly reduce coagulation as well as mortality in patients with severe sepsis. Herein, we aimed to develop an affinity support material that allows the purification of plasma APC for the first time. In this research, a novel APC-specific DNA aptamer-based poly(2-hydroxyethyl methacrylate-glycidyl methacrylate) (poly(HEMA-GMA/DNA-Apt)) macroporous cryogel membrane at different molar ratios was prepared using affinity binding method and their potential for purification and identification of APC was investigated. The DNA aptamer-immobilized cryogels were characterized to examine their structural and morphological properties. The effect of pH, initial concentration, temperature, ionic strength difference, and flow rate changes was examined. Selectivity studies were performed in the presence of APC and competitive proteins, and cryogel support materials were shown to have a very high affinity for APC. Adsorption capacity was found to be 89.02 mg/g. Finally, NaCl revealed efficiency for APC desorption and the reuse of cryogels was successfully tested for ten cycles.

Isothermal titration calorimetry binding properties of Cibacron Blue F3GA in complex with human serum albumin.

Binding interactions between Cibacron Blue-F3GA (CB-F3GA) and human serum albumin (HSA, at physiologically ten-fold lower concentration) was studied by isothermal titration calorimetry (ITC) and in-silico docking computations. ITC experiments revealed two separate binding sites on HSA with different binding affinities for CB-F3GA. The high-affinity binding site (PBS-II) on HSA binds CB-F3GA at nanomolar scale (KD1 = 118 ± 107 nM) with favorable binding enthalpy (ΔHo 1 = - 6.47 ± 0.44 kcal/mol) and entropy (-TΔSo 1 = -2.98 kcal/mol) energies. CB-F3GA binds to the low-affinity binding site (PBS-I) at µM scale (KD2 = 31.20 ± 18.40 µM) with favorable binding enthalpy (ΔHo 1 = - 5.03 ± 3.86 × 10-2 kcal/mol) and entropy (-TΔSo 1 = -1.12 kcal/mol) energies. ITC binding data strongly suggest that CB-F3GA binding to PBS-II site increases the formation of dimeric-HSA clusters (N1 = 2.43 ± 0.50), while binding to PBS-I leads to tetrameric-HSA clusters (N2 = 4.61 ± 0.90). These results suggest that a higher degree of HSA aggregation upon drug binding may be expected under physiological conditions, a notion that should be further investigated for the delivery and toxicity of drug-HSA interactions.

Recognition of human hemoglobin with macromolecularly imprinted polymeric nanoparticles using non-covalent interactions.

Hemoglobin (Hb) is the most abundant protein in the blood. It is vital for the living as oxygen carriers. Some of the very pure Hb-containing biological fluids are currently under clinical trial. However, the removal and purification of Hb from the blood are quite difficult, especially when it is at a low concentration level. In this study, the molecularly imprinted polymeric nanoparticles (MIPNs) were prepared using N-methacryloyl-histidine methyl ester (MAH) by mini-emulsion polymerization technique for specific binding of human hemoglobin (HHb). MIPNs in monosize form have a size of 152 ± 4 nm. They also have a high binding capacity (32.33 mg/g) of HHb. MIPNs retain 84% of the re-binding capacity for HHb after 10 cycles. The nanoparticles have 16 and 5 times higher binding capacity of HHb, respectively, in the presence of bovine serum albumin and lysozyme. Thanks to their high binding capacity and selectivity, MIPNs will allow them to be detected selectively for different target molecules. According to molecular docking, the main binding forces depend on hydrogen bonds and Van der Waals forces in the interaction within 5 Å around MAH molecule are observed through the amino acid residues of HHb at ß1 and ß2 subunit. The statistical mechanical analysis of docking showed that the free energy (ΔG) is -2732.14 kcal/mol, which indicates the interaction between MAH and HHb is energetically favorable at 298.15°K.

Molecular docking of metal ion immobilized ligands to proteins in affinity chromatography.

Immobilized metal ion affinity chromatography (IMAC) has become a widespread analytical and preparative separation method for therapeutic proteins, peptides nucleic acids, hormones, and enzymes. N-Methacryloyl-l-histidine Methyl Ester (MAH) monomer is recently used as a synthesized affinity ligand in IMAC. It is capable of chelating with many transition metal ions such as Zn2+ , Ni2+ , and Cu2+ ions through its histidine residue. In this way, proteins can bind selectively to these immobilized metal ions through MAH as a ligand in affinity chromatography. In this study, we applied the computational docking method on the interactions that occur between the MAH monomer and its complexes with Zn2+ ions as ligands and protein molecules as targets. MAH monomer was drawn and created using the Avogadro software as an optimization tool. Human insulin (Ins) molecule and horse heart cytochrome C (Cyt C) were selected as target proteins to interact with MAH monomer as affinity ligand. Automated docking software AutoDock v4.2 was used for docking of MAH monomer to Ins and Cyt C, respectively. The affinity ligand complexes with Zn2+ ions bound to one, two, and three moles of MAH were studied and compared separately. The lowest binding energies of Ins and Cyt C proteins in 1:1 mol ratio of MAH-Zn2+ were found as (-4.14) and (-4.92) kcal/mol, respectively.

S-citalopram imprinted monolithic columns for capillary electrochromatography enantioseparations.

In this study, the molecular imprinting method was used to separate enantiomeric forms of chiral antidepressant drug, R,S-citalopram (R,S-CIT) in aqueous solution by CEC system combining the advantages of capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). For that, an amino acid-based molecularly imprinted monolithic capillary column was designed and used as a stationary phase for selective separation of S-citalopram (S-CIT) for the first time. S-CIT was selectively separated from the aqueous solution containing the other enantiomeric form of R-CIT, which is the same in size and shape as the template molecule. Morphology of the molecularly imprinted (MIP S-CIT) and non-imprinted (NIP S-CIT) monolithic capillary columns was observed by scanning electron microscopy. Imprinting efficiency of MIP S-CIT monolithic capillary column used for selective S-CIT separation was verified by comparing with NIP S-CIT and calculated imprinting factor (I.F:1.81) proved the high selectivity of the MIP S-CIT for S-CIT. Cavities formed for S-CIT form enabled selective (α = 2.08) separation of the target molecule from the other enantiomeric R-CIT form. Separation was achieved in a short period of 10 min, with the electrophoretic mobility of 7.68 × 10-6 m2 /Vs for R,S-CIT at pH 7.0 10 mM PB and 50% ACN ratio. The performance of both MIP S-CIT and NIP S-CIT columns was estimated by repeating the R,S-CIT separations with intra-batch and inter-batch studies for reproducibility of retention times of R,S-CITs. Estimated RSD values that are lower than 2% suggest that the monolithic columns separate R,S-CIT enantiomers without losing separation efficiency.

Molecularly imprinted nanofilms for endotoxin detection using an surface plasmon resonance sensor.

In this study, a simple, fast, sensitive and selective surface plasmon resonance (SPR) sensor was prepared using molecular imprinting method for endotoxin detection. Endotoxin imprinted and non-imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine methyl ester) based nanofilms were synthesized on the SPR chip surfaces using ultraviolet polymerization. Endotoxin imprinted and non-imprinted SPR sensors were characterized by using contact angle, atomic force microscopy and ellipsometry. After characterization studies, kinetic studies was carried out in the concentration range of 0.5-100 ng/mL. The limit of detection and quantification were obtained as 0.023 and 0.078 ng/mL, respectively. The response time for the equilibration, adsorption and regeneration was approximately 14 min. The selectivity studies with cholesterol and hemoglobin of endotoxin imprinted SPR sensor were examined. Validation studies were carried out via limulus amebocyte lysate (LAL) in order to demonstrate the applicability of the SPR sensor.

Organic polymer-based monolithic capillary columns and their applications in food analysis.

In recent years, the use of organic polymer monolithic capillary columns in separation science has gained popularity due to the fact that they are easy to fabricate and do not require retaining frits. These materials have been applied in different fields including foods, proteomics, and pharmaceuticals. The interest in food analysis still needs to develop in order to increase the sensitivity towards micro/nano-scale food applications for food samples of < 5 µg (e.g., foodomics). In this regard, polymer monolithic capillary columns offer great separation capability in the food analytical separation science. We review the most important applications in food analysis using polymer monolithic capillary columns. In addition, several examples of the use of capillary separation methods combined with mass spectrometry detection in food analysis are summarized.

Combined protein A imprinting and cryogelation for production of spherical affinity material.

Cryogels have been demonstrated to be efficient when applied for protein isolation. Owing to their macroporous structure, cryogels can also be used for treating particle-containing material, e.g. cell homogenates. Another challenging development in protein purification technology is the use of molecularly imprinted polymers (MIPs). These MIPs are robust and can be used repeatedly. The paper presents a new technology that combine the formation of cryogel beads concomitantly with making imprints of a protein. Protein A was chosen as the print molecule which was also be the target in the purification step. The present paper describes a new method to produce protein-imprinted cryogel beads. The protein-imprinted material was characterized and the separation properties were evaluated with regard to both the target protein and whole cells with target protein exposed on the cell surface. The maximum protein A adsorption was 18.1 mg/g of wet cryogel beads. The selectivity coefficient of protein A-imprinted cryogel beads for protein A was 5.44 and 12.56 times greater than for the Fc fragment of IgG and protein G, respectively.

A nitrocellulose paper strip for fluorometric determination of bisphenol A using molecularly imprinted nanoparticles.

The authors describe a test stripe for fluorometric determination of the endocrine disruptor bisphenol A (BPA). Graphene quantum dots (GQDs) were immobilized on molecularly imprinted nanoparticles which then were placed on nitrocellulose paper. The GQDs display blue fluorescence (with excitation/emission peaks at 350/440 nm) which is reduced in the presence of BPA. The test stripe has a 43.9 ± 0.8 µg·L-1 limit of detection in case of water samples. The stripe was applied to the determination of BPA in (spiked) tap water and sea water, and the LODs were found to be 1.8 ± 0.2 µg·L-1 and 4.2 ± 0.5 µg·L-1, respectively. Structural analogs of BPA, such as aminophenol, phenol, hydroquinone and naphthol were found not to interfere. Graphical abstract Schematic presentation of graphene quantum dots immobilized on molecularly imprinted nanoparticles placed on nitrocellulose paper for the determination of Bisphenol A in tap water and seawater. The method is based on the fluorescence quenching due to binding of targets in specific recognition sites.

Molecularly Imprinted Polymer Based Sensors for Medical Applications.

Sensors have been extensively used owing to multiple advantages, including exceptional sensing performance, user-friendly operation, fast response, high sensitivity and specificity, portability, and real-time analysis. In recent years, efforts in sensor realm have expanded promptly, and it has already presented a broad range of applications in the fields of medical, pharmaceutical and environmental applications, food safety, and homeland security. In particular, molecularly imprinted polymer based sensors have created a fascinating horizon for surface modification techniques by forming specific recognition cavities for template molecules in the polymeric matrix. This method ensures a broad range of versatility to imprint a variety of biomolecules with different size, three dimensional structure, physical and chemical features. In contrast to complex and time-consuming laboratory surface modification methods, molecular imprinting offers a rapid, sensitive, inexpensive, easy-to-use, and highly selective approaches for sensing, and especially for the applications of diagnosis, screening, and theranostics. Due to its physical and chemical robustness, high stability, low-cost, and reusability features, molecularly imprinted polymer based sensors have become very attractive modalities for such applications with a sensitivity of minute structural changes in the structure of biomolecules. This review aims at discussing the principle of molecular imprinting method, the integration of molecularly imprinted polymers with sensing tools, the recent advances and strategies in molecular imprinting methodologies, their applications in medical, and future outlook on this concept.

Molecular Fingerprints of Hemoglobin on a Nanofilm Chip.

Hemoglobin is an iron carrying protein in erythrocytes and also an essential element to transfer oxygen from the lungs to the tissues. Abnormalities in hemoglobin concentration are closely correlated with health status and many diseases, including thalassemia, anemia, leukemia, heart disease, and excessive loss of blood. Particularly in resource-constrained settings existing blood analyzers are not readily applicable due to the need for high-level instrumentation and skilled personnel, thereby inexpensive, easy-to-use, and reliable detection methods are needed. Herein, a molecular fingerprints of hemoglobin on a nanofilm chip was obtained for real-time, sensitive, and selective hemoglobin detection using a surface plasmon resonance system. Briefly, through the photopolymerization technique, a template (hemoglobin) was imprinted on a monomeric (acrylamide) nanofilm on-chip using a cross-linker (methylenebisacrylamide) and an initiator-activator pair (ammonium persulfate-tetramethylethylenediamine). The molecularly imprinted nanofilm on-chip was characterized by atomic force microscopy and ellipsometry, followed by benchmarking detection performance of hemoglobin concentrations from 0.0005 mg mL-1 to 1.0 mg mL-1. Theoretical calculations and real-time detection implied that the molecularly imprinted nanofilm on-chip was able to detect as little as 0.00035 mg mL-1 of hemoglobin. In addition, the experimental results of hemoglobin detection on the chip well-fitted with the Langmuir adsorption isotherm model with high correlation coefficient (0.99) and association and dissociation coefficients (39.1 mL mg-1 and 0.03 mg mL-1) suggesting a monolayer binding characteristic. Assessments on selectivity, reusability and storage stability indicated that the presented chip is an alternative approach to current hemoglobin-targeted assays in low-resource regions, as well as antibody-based detection procedures in the field. In the future, this molecularly imprinted nanofilm on-chip can easily be integrated with portable plasmonic detectors, improving its access to these regions, as well as it can be tailored to detect other proteins and biomarkers.

Synthesis of hydrophobic nanoparticles for real-time lysozyme detection using surface plasmon resonance sensor.

Diagnostic biomarkers such as proteins and enzymes are generally hard to detect because of the low abundance in biological fluids. To solve this problem, the advantages of surface plasmon resonance (SPR) and nanomaterial technologies have been combined. The SPR sensors are easy to prepare, no requirement of labelling and can be detected in real time. In addition, they have high specificity and sensitivity with low cost. The nanomaterials have also crucial functions such as efficiency improvement, selectivity, and sensitivity of the detection systems. In this report, an SPR-based sensor is developed to detect lysozyme with hydrophobic poly (N-methacryloyl-(L)-phenylalanine) (PMAPA) nanoparticles. The SPR sensor was first characterized by attenuated total reflection-Fourier transform infrared, atomic force microscope, and water contact angle measurements and performed with aqueous lysozyme solutions. Various concentrations of lysozyme solution were used to calculate kinetic and affinity coefficients. The equilibrium and adsorption isotherm models of interactions between lysozyme solutions and SPR sensor were determined and the maximum reflection, association, and dissociation constants were calculated by Langmuir model as 4.87, 0.019 nM-1 , and 54 nM, respectively. The selectivity studies of SPR sensor were investigated with competitive agents, hemoglobin, and myoglobin. Also, the SPR sensor was used four times in adsorption/desorption/recovery cycles and results showed that, the combination of optical SPR sensor with hydrophobic ionizable PMAPA nanoparticles in one mode enabled the detection of lysozyme molecule with high accuracy, good sensivity, real-time, label-free, and a low-detection limit of 0.66 nM from lysozyme solutions. Lysozyme detection in a real sample was performed by using chicken egg white to evaluate interfering molecules present in the medium.

Dopamine-imprinted monolithic column for capillary electrochromatography.

A dopamine-imprinted monolithic column was prepared and used in capillary electrochromatography as stationary phase for the first time. Dopamine was selectively separated from aqueous solution containing the competitor molecule norepinephrine, which is similar in size and shape to the template molecule. Morphology of the dopamine-imprinted column was observed by scanning electron microscopy. The influence of the organic solvent content of mobile phase, applied pressure and pH of the mobile phase on the recognition of dopamine by the imprinted monolithic column has been evaluated, and the imprinting effect in the dopamine-imprinted monolithic polymer was verified. Developed dopamine-imprinted monolithic column resulted in excellent separation of dopamine from structurally related competitor molecule, norepinephrine. Separation was achieved in a short period of 10 min, with the electrophoretic mobility of 5.81 × 10-5  m2 V-1 s-1 at pH 5.0 and 500 mbar pressure.

Synthesis of hydroxyethyl-methacrylate-(L)-histidine methyl ester cryogels. Application on the separation of bovine immunoglobulin G.

In this study cryogels based 2-hydroxyethyl methacrylate (HEMA) functionalized with N-methacryloyl-L-histidine methyl ester (MAH) were synthesized and used for the adsorption and separation of bovine IgG. Two series of cryogels functionalized with 5 and 10 mg of MAH as pseudobioaffinity ligand were prepared and characterized by swelling test, FTIR and SEM analysis. The adsorption efficiency of the bovine immunoglobulin into cryogels is discussed with respect to the following chromatographic parameters: pH, flow rate, initial IgG concentration, adsorption time and ionic strength. Our results show good adsorption of bovine immunoglobulin under mild separation conditions at pH 7.4. The maximum binding capacity was determined (32.4 mg/g of cryogel) and demonstrates the efficiency of the used cryogels. This efficacy is clearly seen upon increasing the maximum binding capacity from 23.2 mg (obtained with cryogels with 5 mg MAH) to 32.4 mg/g (for cryogel with 10 mg MAH ligand concentration). The purity of separated fractions was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Together our observations highlights poly (HEMA-MAH) as an efficient adsorbent for bovine immunoglobulins G separation.

Protein C recognition by ion-coordinated imprinted monolithic cryogels.

The protein C imprinted monolithic cryogel was synthesized using 2-hydroxyethyl methacrylate by redox cryo-polymerization method. The prepared monolithic cryogel was characterized by Fourier transform infrared spectroscopy, swelling test, surface area measurements, and scanning electron microscopy. The nonimprinted cryogel was prepared as well for control. Adsorption of protein C from aqueous solutions was investigated in a continuous mode and several parameters affecting adsorption performance were optimized. The maximum protein C adsorption amount was 30.4 mg/g. The selectivity studies were performed by monolithic column studies and fast protein liquid chromatography, using hemoglobin and human serum albumin as competing proteins. The relative selectivity coefficients were 2.37 and 8.89 for hemoglobin and human serum albumin, respectively. Reusability was tested for ten consecutive adsorption-desorption cycles, and no significant change in adsorption capacity was recorded. A pseudo-second-order model was suitable to interpret kinetic data, and the Langmuir model suited the adsorption isotherms well.

Microcontact Imprinted Plasmonic Nanosensors: Powerful Tools in the Detection of Salmonella paratyphi.

Identification of pathogenic microorganisms by traditional methods is slow and cumbersome. Therefore, the focus today is on developing new and quicker analytical methods. In this study, a Surface Plasmon Resonance (SPR) sensor with a microcontact imprinted sensor chip was developed for detecting Salmonella paratyphi. For this purpose, the stamps of the target microorganism were prepared and then, microcontact S. paratyphi-imprinted SPR chips were prepared with the functional monomer N-methacryloyl-L-histidine methyl ester (MAH). Characterization studies of the SPR chips were carried out with ellipsometry and scanning electron microscopy (SEM). The real-time Salmonella paratyphi detection was performed within the range of 2.5 × 106-15 × 106 CFU/mL. Selectivity of the prepared sensors was examined by using competing bacterial strains such as Escherichia coli, Staphylococcus aureus and Bacillus subtilis. The imprinting efficiency of the prepared sensor system was determined by evaluating the responses of the SPR chips prepared with both molecularly imprinted polymers (MIPs) and non-imprinted polymers (NIPs). Real sample experiments were performed with apple juice. The recognition of Salmonella paratyphi was achieved using these SPR sensor with a detection limit of 1.4 × 106 CFU/mL. In conclusion, SPR sensor has the potential to serve as an excellent candidate for monitoring Salmonella paratyphi in food supplies or contaminated water and clearly makes it possible to develop rapid and appropriate control strategies.

Molecular Imprinting of Macromolecules for Sensor Applications.

Molecular recognition has an important role in numerous living systems. One of the most important molecular recognition methods is molecular imprinting, which allows host compounds to recognize and detect several molecules rapidly, sensitively and selectively. Compared to natural systems, molecular imprinting methods have some important features such as low cost, robustness, high recognition ability and long term durability which allows molecularly imprinted polymers to be used in various biotechnological applications, such as chromatography, drug delivery, nanotechnology, and sensor technology. Sensors are important tools because of their ability to figure out a potentially large number of analytical difficulties in various areas with different macromolecular targets. Proteins, enzymes, nucleic acids, antibodies, viruses and cells are defined as macromolecules that have wide range of functions are very important. Thus, macromolecules detection has gained great attention in concerning the improvement in most of the studies. The applications of macromolecule imprinted sensors will have a spacious exploration according to the low cost, high specificity and stability. In this review, macromolecules for molecularly imprinted sensor applications are structured according to the definition of molecular imprinting methods, developments in macromolecular imprinting methods, macromolecular imprinted sensors, and conclusions and future perspectives. This chapter follows the latter strategies and focuses on the applications of macromolecular imprinted sensors. This allows discussion on how sensor strategy is brought to solve the macromolecules imprinting.

Metal-immobilized magnetic nanoparticles for cytochrome C purification from rat liver.

Cu(2+) -immobilized magnetic poly(hydroxyethylmethacrylate-N-methacryloyl-(l)-histidinemethylester) (mPHEMAH) nanoparticles were prepared by surfactant-free emulsion polymerization for cytochrome C (cyt C) purification from rat liver. Elemental analysis, atomic force microscopy, zeta sizer, and vibrating sample magnetometer were used to characterize mPHEMAH nanoparticles. In addition to these characterization steps, surface area, average particle size, and size distribution of mPHEMAH nanoparticles were determined. Quantity of immobilized Cu(2+) was measured using atomic absorption spectrophotometry. N-Methacryloyl-(l)-histidinemethylester and Cu(2+) content of mPHEMAH nanoparticles were 0.18 mmol/g polymer and 0.11 mmol/g polymer, respectively. Specific surface area of Cu(2+) -immobilized mPHEMAH nanoparticles was 1180 m(2) /g. Effect of initial cyt C concentration, pH, temperature, and ionic strength on cyt C adsorption onto Cu(2+) -immobilized mPHEMAH nanoparticles was investigated. Maximum cyt C adsorption capacity of Cu(2+) -immobilized mPHEMAH nanoparticles was 311.9 mg/g polymer. Maximum adsorption was obtained at pH 8.0 and 4 °C. Cu(2+) -immobilized mPHEMAH nanoparticles were used ten times with 4.1% decrease in adsorption capacity. In the last stage, Cu(2+) -immobilized mPHEMAH nanoparticles were used to purify cyt C from rat liver tissue, and the purity of desorbed fractions was controlled by SDS-PAGE.

Catalase purification from rat liver with iron-chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-glutamic acid) cryogel discs.

In this study, iron-chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-glutamic acid) (PHEMAGA/Fe(3+)) cryogel discs were prepared. The PHEMAGA/Fe(3+) cryogel discs were characterized by elemental analysis, scanning electron microscopy, Fourier transform infrared spectroscopy, swelling tests, and surface area measurements. The PHEMAGA/Fe(3+) cryogel discs had large pores ranging from 10 to 100 µm with a swelling degree of 9.36 g H2O/g cryogel. Effects of pH, temperature, initial catalase concentration, and flow rate on adsorption capacity of the PHEMAGA/Fe(3+) cryogel discs were investigated. Maximum catalase adsorption capacity (62.6 mg/g) was obtained at pH 7.0, 25°C, and 3 mg/ml initial catalase concentration. The PHEMAGA/Fe(3+) cryogel discs were also tested for the purification of catalase from rat liver. After tissue homogenization, purification of catalase was performed using the PHEMAGA/Fe(3+) cryogel discs and catalase was obtained with a yield of 54.34 and 16.67 purification fold.

Triazine herbicide imprinted monolithic column for capillary electrochromatography.

Trietazine was selectively separated from aqueous solution containing the competitor molecule cyanazine, which is similar in size and shape to the template molecule. Structural features of the molecularly imprinted column were figured out by SEM. The influence of the mobile-phase composition, applied electrical field, and pH of the mobile phase on the recognition of trietazine by the imprinted monolithic polymer has been evaluated, and the imprint effect in the trietazine-imprinted monolithic polymer was demonstrated by an imprinting factor. The optimized monolithic column resulted in separation of trietazine from a structurally related competitor molecule, cyanazine. In addition, fast separation was obtained within 6 min by applying higher electrical field, with the electrophoretic mobility of 2.97 × 10(-8) m(2) V(-1) s(-1) at pH 11.0.