Toxicological screening of human plasma by on‐line SPE‐HPLC‐DAD: identification and quantification of acidic and neutral drugs

A multi‐analyte screening method for the quantification of 50 acidic/neutral drugs in human plasma based on on‐line solid‐phase extraction (SPE)–HPLC with photodiode array detection (DAD) was developed, validated and applied for clinical investigation. Acetone and methanol for protein precipitation, three different SPE materials (two electro‐neutral, one strong anion‐exchange, one weak cation‐exchange) for on‐line extraction, five HPLC‐columns [one C₁₈ (GeminiNX), two phenyl‐hexyl (Gemini C₆‐Phenyl, Kinetex Phenyl‐Hexyl) and two pentafluorophenyl (LunaPFP(2), KinetexPFP)] for analytical separation were tested. For sample pre‐treatment, acetone in the ratio 1:2 (plasma:acetone) showed a better baseline and fewer matrix peaks in the chromatogram than methanol. Only the strong anion‐exchanger SPE cartridge (StrataX‐A, pH 6) allowed the extraction of salicylic acid. Analytical separation was carried out on a Gemini C₆‐Phenyl column (150 × 4.6 mm, 3 µm) using gradient elution with acetonitrile–water 90:10 (v/v) and phosphate buffer (pH 2.3). Linear calibration curves with correlation coefficients r ≥ 0.9950/0.9910 were obtained for 46/four analytes. Additionally, this method allows the quantification of 23 analytes for therapeutic drug monitoring. Limits of quantitation ranged from 0.1 (amobarbital) to 23 mg/L (salicylic acid). Inter‐/intra‐day precisions of quality control samples (low/high) were better than 13% and accuracy (bias) ranged from ?14 to 10%. A computer‐assisted database was created for automated detection of 223 analytes of toxicological interests. Four cases of multi‐drug intoxications are presented. Copyright © 2015 John Wiley & Sons, Ltd.     

Quinine‐modified polymer monolithic column with reversed‐phase /strong anion‐exchange mixed‐mode for pressurized capillary electrochromatography

Via the facile ring‐opening reaction of epoxy groups with quinine, a novel polymer monolith with quaternary ammonium for reversed‐phase/strong anion‐exchange mixed‐mode has been fabricated for pressurized capillary electrochromatography (pCEC). Optimization on the preparation of quinine‐modified monoliths has been investigated, and characteristics including morphology, permeability, mechanical stability, reproducibility, and column performance have been also studied. Active quaternary ammonium groups were conveniently produced to generate cationic action sites and stable anodic electroosmotic flow. Multiple interactions including reversed‐phase, strong anion‐exchange, electrostatic repulsion and π–π stacking interactions were obtained. Satisfactory separation capability of various analytes such as alkylbenzenes, polycyclic aromatic hydrocarbons, benzoic acid and its homologs, and β₂‐receptor excitants has been achieved. Applied to the real sample, the good resolution of three alkaloids in Corydalis yanhusuo were achieved by pCEC with the quinine‐modified monolith. The results light a potential access to facilely fabricating quaternary ammonium‐functionalized polymer monolith with multiple interactions for efficient electrochromatography profiling of various compounds.     

Separation of cannabinoids on three different mixed‐mode columns containing carbon/nanodiamond/amine‐polymer superficially porous particles

Three mixed‐mode high‐performance liquid chromatography columns packed with superficially porous carbon/nanodiamond/amine‐polymer particles were used to separate mixtures of cannabinoids. Columns evaluated included: (i) reversed phase (C₁₈), weak anion exchange, 4.6 × 33 mm, 3.6 μm, and 4.6 × 100 mm, 3.6 μm, (ii) reversed phase, strong anion exchange (quaternary amine), 4.6×33 mm, 3.6 μm, and (iii) hydrophilic interaction liquid chromatography, 4.6 × 150 mm, 3.6 μm. Different selectivities were achieved under various mobile phase and stationary phase conditions. Efficiencies and peak capacities were as high as 54 000 N/m and 56, respectively. The reversed phase mixed‐mode column (C₁₈) retained tetrahydrocannabinolic acid strongly under acidic conditions and weakly under basic conditions. Tetrahydrocannabinolic acid was retained strongly on the reversed phase, strong anion exchange mixed‐mode column under basic polar organic mobile phase conditions. The hydrophilic interaction liquid chromatography column retained polar cannabinoids better than the (more) neutral ones under basic conditions. A longer reversed phase (C₁₈) mixed‐mode column (4.6 × 100 mm) showed better resolution for analytes (and a contaminant) than a shorter column. Fast separations were achieved in less than 5 min and sometimes 2 min. A real world sample (bubble hash extract) was also analyzed by gradient elution.     

Molecularly imprinted polymer‐based solid phase clean‐up for analysis of ochratoxin A in beer,red wine,and grape juice

A simple, reliable, and low‐cost method based on molecularly imprinted polymer as a selective sorbent of SPE was proposed for the determination of ochratoxin A (OTA) in beer, red wine, and grape juice by HPLC coupled with fluorescence detection (HPLC‐FLD). Samples were diluted with water and cleaned up with an AFFINIMIP® SPE OTA column. After washing and eluting, the analyte was analyzed by HPLC‐FLD. Under the optimized conditions, LOD and LOQ for OTA were 0.025 and 0.08 ng/mL, respectively. The recoveries of OTA from beer, red wine, and grape spiked at 0.1, 2, and 5 ng/mL ranged from 91.6 to 101.7%. Furthermore, after a simple regenerated procedure, the molecularly imprinted polymer based SPE column could be reused at least 14 times to achieve more than 80% recoveries of OTA in real samples. The developed method was applied to the detection of 30 beer, red wine, and grape juice samples and only four samples were contaminated by OTA with levels below the legal limits.     

Preparation of a weak anion exchange/hydrophobic interaction dual‐function mixed‐mode chromatography stationary phase for protein separation using click chemistry

In this study, 3‐diethylamino‐1‐propyne was covalently bonded to the azide‐silica by a click reaction to obtain a novel dual‐function mixed‐mode chromatography stationary phase for protein separation with a ligand containing tertiary amine and two ethyl groups capable of electrostatic and hydrophobic interaction functionalities, which can display hydrophobic interaction chromatography character in a high‐salt‐concentration mobile phase and weak anion exchange character in a low‐salt‐concentration mobile phase employed for protein separation. As a result, it can be employed to separate proteins with weak anion exchange and hydrophobic interaction modes, respectively. The resolution and selectivity of the stationary phase were evaluated in both hydrophobic interaction and ion exchange modes with standard proteins, respectively, which can be comparable to that of conventional weak anion exchange and hydrophobic interaction chromatography columns. Therefore, the synthesized weak anion exchange/hydrophobic interaction dual‐function mixed‐mode chromatography column can be used to replace two corresponding conventional weak anion exchange and hydrophobic interaction chromatography columns to separate proteins. Based on this mixed‐mode chromatography stationary phase, a new off‐line two‐dimensional liquid chromatography technology using only a single dual‐function mixed‐mode chromatography column was developed. Nine kinds of tested proteins can be separated completely using the developed method within 2.0 h.     

Anion Exchange-Adsorption on Low Capacity Anion Exchangers: Separation of Organic Acids,Amino Acids,Small Chain Peptides

Abstract

The variables that influence the retention of organic analyte anions on a macroporous, high surface area polystyrenedivinyl-benzene copolymer that is chemically modified by attaching tetraalkylammonium groups to the copolymer surface are identified and studied as a function of anion exchange capacity. A combined adsorption-anion exchange retention of the organic analyte anion is possible providing the analyte has both a hydrophophic center and an anionic charge site. As the column anion exchange capacity (0 to 173 μeq of anion exchange sites/column was studied) increases, analyte retention due to adsorption decreases and retention due to anion exchange increases. The factors influencing organic analyte anion retention by adsorption are low anion exchange capacity and mobile phase solvent composition, type of organic modifier, and pH for analytes that are weak organic acids. For anion exchange the major factors are a high anion exchange     

On.column pre‐concentration of alcohol dehydrogenase in capillary electrophoresis

The analysis of alcohol dehydrogenase (ADH) at low concentration using capillary electrophoresis is described. Several simple and effective ways to improve detection limits and sensitivity are investigated. These include large volume sample stacking, head column field amplified sample stacking, and sweeping. Results indicate that by using a combination of head‐column field amplified sample stacking and sweeping, fluorescently labelled alcohol dehydrogenase can be pre‐concentrated online by dissolving samples in water or other low conductivity matrices, and injecting into a high conductivity micellar buffer. The abrupt changes in conductivity cause narrowing of the analyte length and thus enhance the detection sensitivity. Combination of this approach with laser induced fluorescence detection yields a limit of detection of 5×10^–13 M. Both qualitative and quantitative aspects of this method are investigated.     

Determination of Glycolate Acid,Mono‐ and Dichloroacetic Acids in Synthetical Betaine by Anion‐exchange Chromatography

An anion‐exchange chromatography method combined solid phase extraction (SPE) was developed for the simultaneous analysis of glycolate acid (GL), monochloroacetic acid (MCA) and dichloroacetic acid (DCA) in synthetical betaine products. The analytes and unknown anionic impurities were well separated on a Metrosep A supp5 anion‐exchange column (150 mm×4 mm) with 2.0 mmol/L Na₂CO₃+2.0 mmol/L NaHCO₃ solution as eluent. Suppressed conductivity detection was used. A strong cation exchange (SCX) solid phase extraction (SPE) cartridge was used to reduce the concentration of matrix betaine and a Cleanert IC‐Ag pretreatment cartridge was used to remove high Cl^? concentration. The detection limits of GL, MCA and DCA were 0.09, 0.017 and 0.05 µg/L, respectively. The relative standard deviations (RSDs) of the retention times and peak areas were less than 0.09% and 0.49%, respectively. The recoveries of the three analytes were between 90.6% and 100.8%. The analytical results showed that GL and DCA were present in high concentration and no MCA was found when the proposed ion chromatography method was applied to three synthetical betaine samples. The proposed method is simple, sensitive and timesaving, and is also suitable for routine analysis in quality control of synthetical betaine products.     

Dicationic imidazolium ionic liquid modified silica as a novel reversed‐phase/anion‐exchange mixed‐mode stationary phase for high‐performance liquid chromatography

A dicationic imidazolium ionic liquid modified silica stationary phase was prepared and evaluated by reversed‐phase/anion‐exchange mixed‐mode chromatography. Model compounds (polycyclic aromatic hydrocarbons and anilines) were separated well on the column by reversed‐phase chromatography; inorganic anions (bromate, bromide, nitrate, iodide, and thiocyanate), and organic anions (p‐aminobenzoic acid, p‐anilinesulfonic acid, sodium benzoate, pathalic acid, and salicylic acid) were also separated individually by anion‐exchange chromatography. Based on the multiple sites of the stationary phase, the column could separate 14 solutes containing the above series of analytes in one run. The dicationic imidazolium ionic liquid modified silica can interact with hydrophobic analytes by the hydrophobic C₆ chain; it can enhance selectivity to aromatic compounds by imidazolium groups; and it also provided anion‐exchange and electrostatic interactions with ionic solutes. Compared with a monocationic ionic liquid functionalized stationary phase, the new stationary phase represented enhanced selectivity owing to more interaction sites.     

Use of short chain alkyl imidazolium ionic liquids for on‐line stacking and sweeping of methotrexate,flinic acid and folic acid: Their application to biological fluids

Methotrexate (MTX) is widely used for the treatment of many types of cancer. Folinic acid (FNA) and folic acid (FA) were usually simultaneously supplemented with MTX to reduce the side effects of a folate deficiency. This study, for the first time, included on‐line sample preconcentration by stacking and sweeping techniques under reduced or enhanced electric conductivity in the sample region using short chain alkyl imidazolium ionic liquids (ILs) as micelle forming agents for analyte focusing. Both analyte focusing by micelle collapse (AFMC) and sweeping‐MEKC had been investigated for the comparison of their effectiveness to examine simultaneously MTX, FNA and FA in plasma and urine under physiological conditions. In sweeping‐MEKC, the sample solution without micelles was hydrodynamically injected as a long plug into a fused‐silica capillary pre‐filled with phosphate buffer containing 3.0 mol/L of 1‐butyl‐3‐methylimidazolium bromide (BMIMBr). Using AFMC, the analytes were prepared in BMIMBr micellar matrix and hydrodynamically injected into the phosphate buffer without IL micelles. The conductivity ratio between BGE and sample (γ, BGE/sample) was optimized to be 3.0 in sweeping‐MEKC and 0.33 in AFMC resulting the adequate separation of analytes within 4.0 min. To reduce the possibility of BMIMBr adsorption, an appropriate rinsing protocol was used. The limits of detection were calculated as 0.1 ng/mL MTX, 0.05 ng/mL FNA and 0.05 ng/mL FA by sweeping‐MEKC and 0.5 ng/mL MTX, 0.3 ng/mL FNA and 0.3 ng/mL FA by AFMC. The accuracy was tested by recovery in plasma and urine matrices giving values ranging between 90 and 110%. Both stacking and sweeping by BMIMBr could be successfully used for the rapid, selective and sensitive determination of pharmaceuticals in complex matrices due to its fascinating properties, including high conductivity, good thermal stability and ability to form different types of interactions by electrostatic, hydrophobic, hydrogen bonding and π–π interactions. In sweeping‐MEKC, the using of BMIMBr enhanced the γ factor, k retention factor and the injected amount of sample. Consequently, this technique offers particular potential for higher sensitivity by giving 22‐ and 5‐fold sensitivity enhancement factors (SEFs) of MTX compared to CZE and AFMC, respectively.     

The degree of substitution affects the enantioselectivity of sulfobutylether‐β‐cyclodextrin chiral stationary phases

Three chiral stationary phases were prepared by dynamic coating of sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD) with different degrees of substitution, onto strong anion‐exchange stationary phases. The enantioselective potential and stability of newly prepared chiral stationary phases were examined using a set of structurally different chiral analytes. Measurements were performed in RP‐HPLC. Mobile phases consisted of methanol/formic acid, pH 2.10, and methanol/10 mM ammonium acetate buffer, pH 4.00, in various volume ratios. SBE‐β‐CDs with degrees of substitution (DS) 4, 6.3, and 10 proved suitable for the enantioseparation of 14, 11, and 8 analytes, respectively. The SBE‐β‐CD DS 4 based chiral stationary phase enabled the enantioseparation of the nearly all basic and neutral compounds. Chiral stationary phases with higher sulfobutylether‐β‐cyclodextrin substitution (especially DS 10) yielded higher enantioresolution values for acidic compounds.     

Sulfoalkylbetaine‐based monolithic column with mixed‐mode of hydrophilic interaction and strong anion‐exchange stationary phase for capillary electrochromatography

A novel monolithic stationary phase with mixed mode of hydrophilic and strong anion exchange (SAX) interactions based on in situ copolymerization of pentaerythritol triacrylate (PETA), N,N‐dimethyl‐N‐methacryloxyethyl N‐(3‐sulfopropyl) ammonium betaine (DMMSA) and a selected quaternary amine acrylic monomer was designed as a multifunctional separation column for CEC. Although the zwitterionic functionalities of DMMSA and hydroxy groups of PETA on the surface of the monolithic stationary phase functioned as the hydrophilic interaction (HI) sites, the quaternary amine acrylic monomer was introduced to control the magnitude of the EOF and provide the SAX sites at the same time. Three different quaternary amine acrylic monomers were tested to achieve maximum EOF velocity and highest plate count. The fabrication of the zwitterionic monolith (designated as HI and SAX stationary phase) was carried out when [2‐(acryloyloxy)ethyl]trimethylammonium methylsulfate was used as the quaternary amine acrylic monomer. The separation mechanism of the monolithic column was discussed in detail. For charged analytes, a mixed mode of HI and SAX was observed by studying the influence of mobile phase pH and salt concentration on their retentions on the poly(PETA‐co‐DMMSA‐co‐[2‐(acryloyloxy)ethyl]trimethylammonium methylsulfate) monolithic column. The optimized monolith showed good separation performance for a range of polar analytes including nucleotides, nucleic acid bases and nucleosides, phenols, estrogens and small peptides. The column efficiencies greater than 192 000 theoretical plates/m for estriol and 135 000 theoretical plates/m for charged cytidine were obtained.     

Graphene‐based solid‐phase extraction disk for fast separation and preconcentration of trace polycyclic aromatic hydrocarbons from environmental water samples

Graphene has great potentials for the use in sample preparation due to its ultra high specific surface area, superior chemical stability, and excellent thermal stability. In our work, a novel graphene‐based SPE disk was developed for separation and preconcentration of trace polycyclic aromatic hydrocarbons from environmental water samples. Based on the strong π–π stacking interaction between the analytes and graphene, the analytes extracted by graphene were eluted by cyclohexane and then determined by GC‐MS. Under the optimized conditions, high flow rate (30 mL/min) and sensitivity (0.84–13 ng/L) were achieved. The proposed method was successfully applied to the analysis of real environmental water samples with recoveries ranging from 72.8 to 106.2%. Furthermore, the property of anticlogging and reusability was also improved. This work reveals great potentials of graphene‐based SPE disk in environmental analytical.     

Simultaneous determination of four fluorescent whitening agents (including trans and cis isomers) in facial mask by dispersive solid‐phase extraction combined with ultra high performance liquid chromatography and diode array detection

A novel ultra high performance liquid chromatography with diode array detection method, based on the dispersive solid‐phase extraction by using polymer weak anion exchange as the sorbent, was established for the simultaneous determination of fluorescent whitening agents 85, 28, 351, and 71 in facial mask. The amounts of polymer weak anion exchange, adsorption time, and volume of elution solvent in the dispersive solid‐phase extraction technology were optimized, and the developed method was validated in terms of the method limit of detection, method limit of quantitation, linear range, recovery, accuracy, and precision. Results indicated that the standard curves were linear over the selected concentration ranges of 0.05–100 mg/L for four target analytes, with determination coefficients greater than 0.999. The method limits of quantitation of the target analytes were in the range of 0.6–2.8 mg/kg. Recoveries were calculated at the concentrations of 1.0–30 mg/kg spiked in negative samples and the values were between 89.3 and 102% with an RSD of 2.5–5.1% for intraday precision and 3.8–5.0% for interday precision. The method was successfully applied to analyze 20 facial mask samples and fluorescent whitening agent 85 was detected in one sample with the concentration of 4.7 mg/kg.     

Solid-phase extraction and sample stacking-micellar electrokinetic capillary chromatography for the determination of multiresidues of herbicides and metabolites

Micellar electrokinetic capillary chromatography (MEKC) with diode array detection was used for the separation of 13 compounds (eight herbicides widely used in agriculture: metribuzin, lenacil, ethofumesate, atrazine, terbutryn, isoproturon, chlorotoluron and linuron, and five of their principal degradation products; namely, deethylatrazine, 2-hydroxyatrazine, deethyl-2-hydroxyatrazine, deisopropylatrazine and 3-chloro-4-methylphenylurea). Peak separation for the 13 analytes was not successful when a single surfactant system was employed, neither sodium dodecyl sulfate (SDS) nor dioctyl sulfosuccinate (DOSS) sodium salt. However, a mixture of these herbicides was successfully separated using a mixed micellar system involving SDS–DOSS in less than 14 min. An application study of an on-line concentration technique for MEKC was carried out to enhance sensitivity. The optimized on-line stacking procedure consisted simply of the addition of 50 mM of sodium chloride to the injection sample, the stacking effect being more intensive as analyte polarity increased. When this stacking procedure was combined with an off-line sample preconcentration step, based on solid-phase extraction, analytes could be detected in the ppb range. The whole method was applied to ultra-high-quality and natural waters. Linear relationships between the analytical signal and the initial analyte concentration were found to be independent of the type of water, except for the more polar analytes for which small differences were observed.     

Facile Fabrication of Polymeric Ionic Liquid Grafted Porous Polymer Monolith for Mixed‐Mode High Performance Liquid Chromatography

A novel polymeric ionic liquid grafted porous polymer monolith has been facilely fabricated for mixed‐mode chromatography. The column is prepared from poly (glycidyl methacrylate‐co‐ethylene dimethacrylate) monolith through hydrolyzation of the epoxy moieties into hydroxyl groups, followed by "grafting from" polymerization of ionic liquid of 1‐vinyl‐3‐butylimidazolium chloride. Successful modification is characterized by scanning electron microscope, infrared spectroscopy, elemental analysis and mercury intrusion porosimetry. The HPLC performance of developed column is evaluated by separating acidic vitamin B analytes, neutral steroids and basic aromatic amines in mixed‐mode chromatography on a single column, respectively. The ionic liquid affords the monolith with both enhanced separation ability and improved column efficiency.     

Determination of chloramphenicol in aquatic products by graphene‐based SPE coupled with HPLC‐MS/MS

An effective analytical protocol using graphene‐based SPE coupled with HPLC‐MS/MS for determination of chloramphenicol (CAP) in aquatic products has been developed. In the present work, graphene was evaluated as SPE sorbents for the analytes enrichment and clean up. The target analytes were quantified by a triple‐quadrupole linear ion trap MS in multiple‐reaction monitoring mode. In addition, the proposed method was validated according to Commission Decision 2002/657/EC. The calibration curve was linear over the range of 0.5–100 ng/mL. The mean values of RSD of intra‐ and interday ranging from 1.48 to 4.29% and from 3.25 to 7.42% were obtained, respectively. In the three fortified levels, the recoveries of CAP ranging from 92.3 to 103.4% with RSDs ≤ 5.58% were obtained. The proposed method has been successfully applied to the analysis of CAP in several aquatic product samples, indicating that graphene was a potential SPE sorbent for the enrichment of trace residues in food samples.     

Preparation of a Hyper‐cross‐linked Polymer Monolithic Column and Its Application to the Sensitive Determination of Genomic DNA Methylation

A hyper‐cross‐linked polymer monolithic column, poly(methacrylatoethyl trimethyl ammonium‐co‐vinylbenzene chloride‐co‐divinylbenzene) (MATE‐co‐VBC‐co‐DVB) with phenyl and quaternary ammonium groups was successfully prepared in the current study. The prepared monolith possesses large specific surface area, narrow mesopore size distribution and high column efficiency. The poly(MATE‐co‐VBC‐co‐DVB) monolithic column was demonstrated to have strong anion exchange/reversed‐phase (SAX/RP) mixed‐mode retention for analytes on capillary liquid chromatography (cLC). By using this monolithic column, we developed a rapid and sensitive method for the detection of DNA methylation. Our results showed that six nucleobases (adenine, guanine, cytosine, thymine, uracil, and 5‐methylcytosine (5‐mC)) can be baseline separated within 15 min by electrostatic repulsion and hydrophobic interactions between nucleobases and the monolithic stationary phase. The limit of detection (LOD, signal/noise=3) of 5‐mC is 0.014 pmol and endogenous 5‐mC can be distinctly detected by using only 10 ng genomic DNA, which is comparable to that obtained by mass spectrometry analysis. Furthermore, by using the method developed here, we found that DNA methylation inhibitor 5‐azacytidine (5‐aza‐C) and 5‐aza‐2′‐deoxycytidine (5‐aza‐CdR) could induce a significant decrease of genome‐wide DNA methylation in human lung carcinoma cells (A549) and cervical carcinoma cells (HeLa).     

Ionic liquids based on imidazole for online concentration of catecholamines in capillary electrophoresis

Potential possibilities of long‐chain ionic liquids based on imidazole (1‐dodecyl‐3‐methylimidazolium chloride and 1‐cetyl‐3‐methylimidazolium chloride) for online sample concentration techniques (field‐amplified sample stacking, head‐column field‐amplified sample stacking, and sweeping) of catecholamines were studied in both capillary zone electrophoresis and micellar electrokinetic chromatography. The use of a high‐conductivity sample matrix in sweeping was found to significantly increase the separation efficiency of analyte up to 2 × 10⁶ theoretical plates per meter and remarkably reduce limits of detection for catecholamines up to 50 ng/mL. This approach was shown to be suitable for the determination of trace amounts of catecholamines in biological fluids.     

Monodisperse porous polymer particles with polyionic ligands for ion exchange separation of proteins

A new “grafting to” strategy was proposed for the preparation of polymer based ion exchange supports carrying polymeric ligands in the form of weak or strong ion exchangers. Monodisperse porous poly(glycidyl methacrylate-co-ethylene dimethacrylate), poly(GMA-co-EDM) particles 5.9 μm in size were synthesized by “modified seeded polymerization”. Poly(2,3-dihydroxypropyl methacrylate-co-ethylene dimethacrylate), poly(DHPM-co-EDM) particles were then obtained by the acidic hydrolysis of poly(GMA-co-EDM) particles. The hydroxyl functionalized beads were treated with 3-(trimethoxysilyl)propyl methacrylate to have covalently linked methacrylate groups on the particle surface. The selected monomers carrying weak or strong ionizable groups (2-acrylamido-2-methyl-1-propane sulfonic acid, AMPS; 2-dimethylaminoethylmethacrylate, DMAEM and N-[3-(dimethylamino)propyl] methacrylamide, DMAPM) were subsequently grafted onto the particles via immobilized methacrylate groups. The final polymer based materials with polyionic ligands were tried as chromatographic packing in the separation of proteins by ion exchange chromatography. The proteins were successfully separated both in the anion and cation exchange mode with higher column yields with respect to the previously proposed materials. The plate heights obtained for poly(AMPS) and poly(DMAEM) grafted poly(DHPM-co-EDM) particles by using proteins as the analytes were 80 and 200 μm, respectively. Additionally, the plate height exhibited no significant increase with the increasing linear flow rate in the range of 1–20 cm/min. The most important property of the proposed strategy is to be applicable for the synthesis of any type of ion exchanger both in the strong and weak form.