Effect Of Mobile Phase Composition Research Articles

Development of Response Surface Approach for Determination of Paracetamol, Chlorpheniramine Maleate, Caffeine and Ascorbic Acid by Green HPLC Method: A Desirability-Based Optimization.

Design of experiment is an efficient and cost-effective tool to optimize the chromatographic separation of a multicomponent mixture. The central composite design was conducted to develop and optimize a green high performance liquid chromatography (HPLC) method for simultaneous quantitation of a quaternary mixture of paracetamol, chlorpheniramine maleate, caffeine and ascorbic acid in their pharmaceutical dosage form as well as the determination of their dissolution profile. A five-level three-factor model was performed to investigate the effect of mobile phase composition, pH and flow rate on enhanced resolution and short run time. Analysis was performed using a Kinitex EVO C18 column and a mobile phase composed of methanol: 0.02M phosphate buffer pH3.3 (34:66, v/v) at 1.0mL/min using photodiode array detection. Optimum chromatographic separation was achieved in <6min with a desirability of 0.999. Linearity was achieved over a range of 1.00-300.00, 1.00-50.00, 2.00-50.00 and 2.00-100.00μg/mL for paracetamol, chlorpheniramine maleate, caffeine and ascorbic acid, respectively, with a limit of detection (<0.1μg/mL). The greenness profile was evaluated using the analytical eco-scale and Analytical GREEnness Metric Approach with values of 81 and 0.77, respectively.

Evaluation of size-exclusion chromatography, multi-angle light scattering detection and mass photometry for the characterization of mRNA

The recent approval of messenger ribonucleic acid (mRNA) as vaccine to combat the COVID-19 pandemic has been a scientific turning point. Today, the applicability of mRNA is being demonstrated beyond infectious diseases, for example in cancer immunotherapy, protein replacement therapy and gene editing. mRNA is produced by in vitro transcription (IVT) from a linear DNA template and modified at the 3′ and 5′ ends to improve translational efficiency and stability. Co-existing impurities such as RNA fragments and double-stranded RNA (dsRNA), amongst others, can drastically impact mRNA quality and efficacy. In this study, size-exclusion chromatography (SEC) is evaluated for the characterization of IVT-mRNA. The effect of mobile phase composition (ionic strength and organic modifier), pH, column temperature and pore size (300 Å, 1000 Å, and 2000 Å) on the separation performance and structural integrity of IVT-mRNA varying in size is described. Non-replicating, self-amplifying (saRNA), temperature degraded, and ribonuclease (RNase) digested mRNA, the latter to characterize the 3′ poly(A) tail, were included in the study. Beyond ultraviolet (UV) detection, refractive index (RI) and multi-angle light scattering (MALS) detection were implemented to accurately determine molecular weight (MW) of mRNA. Finally, mass photometry is introduced as a complementary methodology to study mRNA under native conditions.

Beta‐cyclodextrin covalent organic framework coated silica composite as chiral stationary phase for high‐performance liquid chromatographic separation

AbstractA beta‐cyclodextrin covalent organic framework coated silica composite, namely beta‐cyclodextrin covalent organic framework@SiO2, was prepared by coating the beta‐cyclodextrin covalent organic framework onto a spherical aminized silica matrix and used as the chiral stationary phase for high‐performance liquid chromatography separation in both normal‐ and reversed‐phase modes. The beta‐cyclodextrin covalent organic framework@SiO2 packed column showed good separation performance towards positional isomers such as nitrotoluene and aminophenol, and benzene homologs in the reversed‐phase high‐performance liquid chromatography mode. While chiral drug molecules including omeprazole and lansoprazole could be also enantioseparated on beta‐cyclodextrin covalent organic framework@SiO2 packed column in the normal‐phase high‐performance liquid chromatography mode. Besides, the effects of mobile phase composition, column temperature, and other factors of high‐performance liquid chromatography separation on beta‐cyclodextrin covalent organic framework@SiO2 packed column were studied in detail. Moreover, the stability test of the beta‐cyclodextrin covalent organic framework@SiO2 packed column was also investigated. The results of continuous injections and monthly detection were basically identical, suggesting the excellent durability of the obtained high‐performance liquid chromatography column. This work indicated that beta‐cyclodextrin covalent organic framework@SiO2 composite could effectively separate different compounds in high‐performance liquid chromatography normal/reversed‐phase modes, making it a promising chiral stationary phase in the field of high‐performance liquid chromatography separation.

Chiral LC method development: Stereo-selective separation, characterization, and determination of cabotegravir and related RS, RR, and SS isomeric impurities on coated cellulose-based chiral stationary phase by HILIC-LC and LC-MS

The first, sensitive, and rapid chiral method was developed for enatioseperations and determination of cabotegravir and its enantiomeric impurities by using HPLC and LC-MS. Cabotegravir is an antiretroviral medication used for the treatment of HIV/AIDS approved by the food and drugs administration (FDA) in the year 2021. The cabotegravir chiral separation was achieved on the coated cellulose-tris (4-chloro-3-methyl phenyl carbamate) (CHIRALCEL OX-3R) column in HILIC mode and the total run time is less than 15 min. The effects of mobile phase composition, elution mode, and percentage of organic modifier as well as the effect of mobile phase-additives and column temperature were investigated on selectivity, resolution, and peak symmetry. The mobile phase consisted of acetonitrile and water with 0.1% (v/v) addition of formic acid additive with the flow rate of 1 mLmin−1. UV detection was carried out at 220 nm. The calibration curves of cabotegravir and its enantiomers were linear over the concentration range of 0.04–1.125 µgmL−1. The limits of detection and quantification for cabotegravir and its enantiomer (RS isomer) were ≤ 0.02 and ≤ 0.06, and the RR and SS-isomers limits were ≤ 0.02 and 0.03 µgmL−1 respectively. It was demonstrated that the proposed method is selective, precise, and robust. Finally, the validated method was applied for the determination and identification of cabotegravir and its chiral enantiomers in the bulk drugs by using HPLC and LC-MS techniques.

Comparing the chromatographic performance of benzene and aniline end capped stationary phase synthesized by click chemistry

Two stationary phases named Sil-styrene and Sil-PAS were synthesized using styrene and p-aminostyrene as end capped functional monomers by the reaction of click chemistry, respectively. Intermediates and stationary phase materials were characterized by various methods. Four types of compounds including polycyclic aromatic hydrocarbons (PAHs), alkylbenzenes, positional isomers, nucleotides and nucleosides were chosen to assess reversed-phase and hydrophilic interaction separation modes of two prepared columns. Retention mechanism of these analytes was researched through investigating effects of mobile phase composition on retention. Prepared columns could achieve better separation effects including higher resolution, shorter retention time and less organic solvent compared with traditional commercial columns. Moreover, the PAHs in barbecued meat samples was achieved separation under the reverse-phase mode on the Sil-styrene column. The experimental results shows that amino groups on the benzene ring can affect the hydrophilic and hydrophobic interactions with analytes, and the prepared Sil-styrene column shows enormous potential for separation of PAHs in actual samples.

Optimization of gradient reversed phase high performance liquid chromatography analysis of acetaminophen oxidation metabolites using linear and non-linear retention model

Acetaminophen (paracetamol, APAP) is one of the most widely used drugs worldwide. Unfortunately, its overdose, which is caused by predominant oxidation of APAP, can lead to acute liver injury. In liver, oxidized APAP is conjugated with glutathione, leading to APAP-glutathione conjugate, which is metabolized to APAP-cysteine and APAP-N-acetylcysteine conjugates. Thus, all of those compounds could be used to monitor APAP metabolism in the overdosed patients. To date, only a limited number of rapid and accurate methods have been reported for the assessment of APAP oxidation metabolites using simple instrumentation, and thus this work was aimed at developing a fast and convenient gradient HPLC-UV/MS method. For this purpose, APAP conjugates with glutathione, cysteine, and N-acetylcysteine were synthesized, purified by preparative liquid chromatography, and characterized by NMR and high-resolution mass spectrometry. The gradient elution conditions were optimized using the window diagram approach and the effects of mobile phase composition and additives on separation and detection sensitivity were evaluated using two, i.e., linear and non-linear isocratic retention models. Quantitative parameters of the developed method were evaluated and the effectiveness, sensitivity, and specificity of the method were demonstrated on the analysis of human kidney HK-2 cell lysates, confirming the suitability of the method for routine use in studies on APAP toxicity.

Separation of multiphosphorylated cyclopeptides and their positional isomers by hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS)

Peptides are efficient models used in different fields such as toxicology to study the interactions of several contaminants at the molecular scale, requiring the development of bio-analytical strategies. In this context, Hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS) was used to separate synthetic multiphosphorylated cyclopeptides and their positional isomers at physiological pH. We assessed (i) the selectivity of eleven HILIC columns, from different manufacturers and packed with diverse polar sorbents, and (ii) the effect of mobile phase composition on the separation selectivity. The best selectivity and baseline resolution were achieved with the columns grafted by neutral sorbents amide and diol. Furthermore, we investigated the HILIC retention mechanism of these peptides by examining the effect of the number of phosphorylated residues in the peptide scaffold on their retention. The peptide behavior followed the classical hydrophilic partitioning mechanism exclusively on amide and diol columns. This trend was not fully respected on bare and hybrid silica due to the attractive/repulsive interactions of the deprotonated surface silanol groups with the Arginine or Glutamate residues in the peptide scaffold according to the peptide sequence. The position of the phosphorylated amino acid in the peptide backbone also showed to have an impact on the retention, making possible the separation of positional isomers of these multiphosphorylated cyclic peptides using HILIC.

How mobile phase composition and column temperature affect enantiomer elution order of liquid crystals on amylose tris(3-chloro-5-methylphenylcarbamate) as chiral selector.

A comprehensive study into the effects of mobile phase composition and column temperature on enantiomer elution order was conducted with a set of chiral rod-like liquid crystalline materials. The analytes were structurally similar and comprised variances such as length of terminal alkyl chain, presence of chlorine, number of phenyl rings, and type of chiral center. Experiments were carried out in polar organic and reversed-phase modes using amylose tris(3-chloro-5-methylphenylcarbamate) immobilized on silica gel as the chiral stationary phase. For all liquid crystals, reversal of elution order of enantiomers was observed based on type of used cosolvent and/or its content in the mobile phase; for some of the liquid crystals a temperature-induced reversal was also observed. Both linear and nonlinear dependencies of natural logarithm of enantioselectivity on temperature were found. Tested mobile phases comprised pure organic solvents and binary and tertiary mixtures of acetonitrile with organic solvents and/or water. Effect of acidic/basic mobile phase additives was also tested. Effect of structure of chiral selector is briefly discussed.

Preparation of value-added metal-organic frameworks for high-performance liquid chromatography. Towards green chromatographic columns

This work applies the concepts of green chemistry, where polyethylene terephthalate (PET) bottles were used as the acid-dicarboxylic linker source for the synthesis of MIL-53(Al) metal organic frameworks (MOFs) and then used as a stationary phase for the separation of various solutes and compared with MIL-53(Al) synthesized from traditional terephthalic acid. Both synthesized MIL-53(Al) MOFs were characterized by scanning electron microscopy (SEM), FT-IR, X-ray diffraction (XRD), thermogravimetric analysis (TGA), and specific surface area analysis. Eight groups of standard analytes in addition to real samples were tested to evaluate the separation performance of the MIL-53(Al) packed columns in HPLC under various chromatographic conditions. Based on elution order of the studied compounds and the effects of mobile phase composition, the working mechanism was reversed phase mode in the presence of size-exclusion effects for large molecules, which exceeded the dynamic diameter of MIL-53(Al) (~7.6 Å). The effects of stationary phase sieving, mobile phase flow rate and composition, injected sample mass, and temperature were investigated relative to the chromatographic behavior of MIL-53(Al). MIL-53(Al) particle sieving before packing reduced peak broadening and significantly enhanced the chromatographic performance of the prepared columns up to 2.26 times relative to the number of theoretical plates. The MIL-53(Al) packed columns offered high-resolution separation for all studied mixtures with Rs >2 and good stability and long-term durability. At optimal conditions, the prepared columns exhibited efficiencies between 5600−63200 plates m−1. Higher efficiencies were observed for alkylbenzenes and polyaromatic hydrocarbons as the organic linker in the MIL-53(Al) structure, which improved retention and separation of aromatics through π–π interactions. Thermodynamic parameters including ΔH, ΔS, and ΔG for the transfer of analyte from the mobile phase to the MIL-53(Al) stationary phase were studied. Compared with previously cited MOFs packed columns, the present MIL-53(Al) columns gave comparable selectivity and much better efficiency for most of the studied chemicals at optimum conditions, indicating the feasibility of MIL-53(Al) as a stationary phase for HPLC applications.

Enantioseparation performance of superficially porous particle vancomycin-based chiral stationary phases in supercritical fluid chromatography and high performance liquid chromatography; applicability for psychoactive substances

Novel psychoactive substances (NPS) are synthetic compounds that have been designed to produce the physiological and psychological effects of known recreational drugs, while circumventing current drug control laws and scheduling guidelines. Such “designer drugs” pose problems in detection and prevention of use, and they are no less dangerous than known controlled substances. Among the various classes of NPS, many are chiral. As they are synthetic products, most are racemates. Not unexpectedly, there is limited information about different the pharmacological and toxicological properties of their pure enantiomers. Hence, fast and reliable enantioselective methods are of great interest.In this work, superficially porous particle (SPP) vancomycin-based chiral stationary phases were used for development of fast enantioselective separation methods for various classes of NPS in supercritical fluid chromatography and liquid chromatography. The NPS tested included pyrovalerones, benzofurans, phenidines and phenidates. The effect of mobile phase composition on the retention and resolution of NPS in supercritical fluid chromatography was examined. The amount as well as the ratios of additives used is crucial for enantioseparation efficiency. Results showed the high enantioselective potential of vancomycin-based columns in both chromatographic techniques; 88% of NPS tested were enantioseparated in supercritical fluid chromatography and 69% of NPS tested were enantioseparated in liquid chromatography. Moreover, under optimized conditions, simultaneous enantioseparations of some NPS were achieved, which indicates great suitability of vancomycin-based columns for this purpose. The proposed methods can serve as guides for method development and for enantioseparation of further upcoming NPS.

Preparation and chromatographic evaluation of a polymer-based stationary phase for weak anion-exchange/hydrophilic interaction

a weak cationic exchange/hydrophilic interaction mixed-mode stationary phase (Sil-PolyCOOH) for high performance liquid chromatography was prepared by bonding polyethylene maleic anhydride to the surface of amino silica gel via a nucleophilic substitution reaction, followed by hydrolysis of the residual maleic anhydride. The new stationary phase material was characterized by solid-state13C nuclear magnetic resonance analysis and ζ-potential measurements. The chromatographic performance and retention mechanism of this stationary phase were evaluated using nucleosides and nucleic bases as probes. The effects of mobile phase composition, ion strength, and pH on the retention factor were investigated. The results show that the retention mechanism of the stationary phase involves both hydrophilic distribution interaction and multiple forces between the host and guests. The stationary phase also showed good separation performance for saccharides, diquat, and paraquat. These results show that the stationary phase has a good application prospect in the separation of polar compounds.

Application of supercritical fluid chromatography for separation and quantitation of 15 co-formulated binary anti-hypertensive medications using a single elution protocol.

A facile supercritical fluid chromatography method is proposed to analyse 15 co-formulated binary anti-hypertensive drug combinations using a customized elution procedure. The effect of mobile phase composition, column back pressure and temperature was suitably optimized for adequate retention, analyte response and resolution. The chromatographic separation of the different drug combinations was performed on a DCPak poly(4-vinylpyridine) column (250 × 4.6 mm, 5 μm) at 125-bar pressure and 40°C using a photodiode array detector. A linear gradient of CO2 and 0.1% formic acid in methanol provided the best elution conditions for all drug combinations. Baseline separation of the drugs was possible with resolution factor Rs ranging from 1.42 to 12.58. The method was validated for specificity, sensitivity, accuracy and precision, recovery and robustness. The limit of detection and limit of quantitation for aliskiren, amlodipine, atenolol, candesartan, hydrochlorothiazide, lisinopril, losartan, metoprolol, olmesartan, telmisartan and valsartan were in the range of 0.26-2.56 and 0.77-7.75 μg/mL, respectively. The thermodynamic study revealed that interactions of the drugs with the stationary phase were spontaneous as evident from the negative free energy values, and the separation process was enthalpy driven. The developed method was successfully employed to analyse these drugs in their co-formulated tablet formulations.

Recent applications of retention modelling in liquid chromatography.

Recent applications of retention modelling in liquid chromatography (2015–2020) are comprehensively reviewed. The fundamentals of the field, which date back much longer, are summarized. Retention modeling is used in retention‐mechanism studies, for determining physical parameters, such as lipophilicity, and for various more‐practical purposes, including method development and optimization, method transfer, and stationary‐phase characterization and comparison. The review focusses on the effects of mobile‐phase composition on retention, but other variables and novel models to describe their effects are also considered. The five most‐common models are addressed in detail, i.e. the log‐linear (linear‐solvent‐strength) model, the quadratic model, the log–log (adsorption) model, the mixed‐mode model, and the Neue–Kuss model. Isocratic and gradient‐elution methods are considered for determining model parameters and the evaluation and validation of fitted models is discussed. Strategies in which retention models are applied for developing and optimizing one‐ and two‐dimensional liquid chromatographic separations are discussed. The review culminates in some overall conclusions and several concrete recommendations.

Quality Risk Management Based: Analytical Quality by Design Approach to Eco-Friendly and Versatile Chromatography Method for Simultaneous Estimation of Multiple Fixed-Dose-Combination Products of Anti-Diabetic Drugs

The chromatography methods such as HPLC, HPTLC etc. were developed for simultaneous estimation of multiple fixed-dose combination products of metformin hydrochloride which need single chromatography condition to save solvent consumption. The method was developed by the implementation of analytical quality by design approach based on principles of quality risk management (QRM) and design of experiments (DoE) as per regulatory requirements. QRM was applied for the analysis of twenty potential method risk parameters using the Ishikawa diagram and quality risk assessment according to ICH Q9 guideline. DoE was applied by Taguchi OA screening design and Box-Behnken response surface analysis method. Method operable design range was navigated for optimisation of critical method risk parameters as per the analytical target profile for development of the method. The developed method was applied for the analysis of three different fixed-dose combination products of metformin hydrochloride. By the QRM process, seven method risk parameters were found critical for the development of the method. After the Taguchi screening design, the main effect of mobile phase composition, %w/v of ammonium acetate and saturation time was found to be significant. The results of the Box-Behnken design showed a quadratic relationship between CMPs and CMAs. The developed method was found to be specific, accurate and precise. The results of the assay were found in good agreement with the labelled claim. The developed method can analyse three different fixed-dose combination products of metformin hydrochloride which required three different chromatography methods. Hence, the method is versatile and eco-friendly.

Rapid determination of trace haloacetic acids in water and wastewater using non-suppressed ion chromatography with electrospray ionization-tandem mass spectrometry

A simple and rapid method employing non-suppressed ion chromatography with electrospray ionization tandem mass spectrometry has been developed for the direct determination of trace-level haloacetic acids (HAAs) in water samples. Using 70/30 (v/v) acetonitrile/1 M aqueous methylamine as the mobile phase, three IC columns – AS16, AS18 and AS24 from Thermo-Scientific – were tested, respectively, with the AS16 column exhibiting the best overall performance with respect to resolution and retention time. To assess the effects of mobile phase composition on retention time of HAAs, the AS16 column was further tested using (i) different proportions of acetonitrile to aqueous methylamine, (ii) different proportions of acetonitrile to aqueous solution at fixed methylamine concentrations, and (iii) different concentrations of methylamine at fixed proportions of acetonitrile to aqueous solution. With a low proportion of aqueous solution, van der Waals and/or hydrogen-bonding interactions appeared to play an important role in governing HAA retention, i.e., HAAs with relatively higher apparent logKow* caused by elevated solvent sspKa exhibited longer retention times; whereas with a high proportion of aqueous solution, ionic interactions appeared to dominate retention of HAAs, with the more polarizable HAAs exhibiting longer retention times. Using 70/30 (v/v) acetonitrile/1 M aqueous methylamine, the method detection limits were in the range of 0.090–0.216 μg/L for the 11 selected chloro-, bromo- and iodoacetic acids. Finally, this method was applied to monitor HAAs yields in laboratory chlorination experiments and to determine concentrations of HAAs in tap water and wastewater effluent samples.

Influence of mobile phase composition and temperature on the retention behavior of selected test substances in diol-type column

In this work the effect of mobile phase composition and temperature on the retention behavior of several test compounds (including non-polar and polar substances) on diol-Acclaim Mixed Mode HILIC-1 column in methanol-water systems with high (RPLC mode) and low (HILIC mode) water content was analyzed. In the studied systems both linear, slightly curved and curved van’t Hoff plots were observed. This indicates, that depending on solute physicochemical properties and mobile phase composition, both single and more complex retention mechanism(s) may influence the retention behavior of tested substances. The possibilities of different retention mechanisms existence (e.g. connected with adsorbent surface heterogeneity, hydrophobic and hydrogen bond interactions) in the analyzed systems have been also discussed.

Preparation and evaluation of a molybdenum disulfide quantum dots embedded C18 mixed-mode chromatographic stationary phase.

Molybdenum disulfide quantum dots (MoS2 QDs) were chosen as a functional two-dimensional material to improve the separation performance of a traditional C18 column. In this work, MoS2 QDs were synthesized by the combination of sonication and solvothermal treatment of bulk MoS2. The prepared MoS2 QDs were characterized by transmission electron microscope (TEM), Zeta potential measurement, UV-visible absorption and fluorescence spectroscopy. Then, a novel MoS2 QDs embedded C18 (Sil-MoS2-C18) stationary phase was prepared for performing mixed-mode liquid chromatography. The results of elemental analysis (EA), thermogravimetric analysis (TGA), Fourier transform infrared spectrometry (FT-IR), X-ray photoelectron spectroscopy (XPS), scanning electron microscope (SEM) and Brunauer-Emmett-Teller (BET) measurements indicated the stationary phase was prepared successfully. Five types of compounds including alkylbenzenes, polycyclic aromatic hydrocarbons (PAHs), nucleosides and nucleobases, anilines and flavonoids were utilized to evaluate reversed phase, weak cation exchange and hydrophilic interaction of the new column. To a certain extent, the column could achieve separation for different properties of samples on one column, with less organic solvent and shorter time than conventional alkyl and amino columns. Furthermore, the mechanism for separation was studied by investigating effects of mobile phase composition and pH on retentions. In summary, the Sil-MoS2-C18 stationary phase was deemed able to serve the performance of various types of phases, which revealed the prepared mixed-mode column could be potentially applied for the analysis of complex samples. Graphical abstract.

Liquid chromatographic chiral recognition of phytoalexins on immobilized polysaccharides chiral stationary phases. Unusual temperature behavior

Three immobilized polysaccharide chiral stationary phases, Chiralpak IA, Chiralpak IB and Chiralpak IC, were used for the study of enantioseparation of 36 derivatives of natural indole phytoalexins, in most cases bioactive, including racemic spirobrassinin, 1-methoxyspirobrassinin and 1-methoxyspirobrassinol methyl ether. Almost all analytes were baseline resolved at least on two different polysaccharide columns in normal phase mode. The effects of mobile phase composition, the analyte structure and the column temperature on the retention and enantioseparation were investigated. Evaluation of the corresponding thermodynamic parameters using van´t Hoff plots (ln k versus 1/T) in the temperature range -15 to 50 °C indicated that separations were enthalpy controlled in most cases, but some entropy controlled separations were also observed. Moreover, unusual phenomenon, an increase retention with increasing temperature accompanied with increased resolution was observed on the Chiralpak IC column. The elution order of enantiomers was determined in some cases and reversed elution order was also observed.

Separation of enantiomers of native amino acids with polysaccharide-based chiral columns in supercritical fluid chromatography

In this short communication preliminary results are reported on the separation of enantiomers of underivatized amino acids on polysaccharide-based chiral columns in sub/super-critical fluid chromatography (SFC).Racemic mixtures of 14 amino acids were used to evaluate the chiral recognition ability of 8 different polysaccharide-based chiral columns with coated or covalently immobilized chiral selectors. Among the studied columns, the one based on cellulose (3,5-dichlorophenylcarbamate) exhibited the most universal chiral recognition ability providing baseline resolution for the enantiomers of 10 studied amino acids. In addition, the effect of mobile phase composition (in particular, of the amount of additive used), as well as the potential contribution of evaporative light scattering detector (ELSD) to extra column band-broadening was evaluated.

Preparation, characterization, and application of a porous poly (TAIC-co-MMA-co-EDMA) monolithic column for small molecules of aromatics and Chinese herbal medicine notoginseng by HPLC

A novel porous monolithic column was synthesized via in situ free-radical polymerization within a stainless steel column (50 × 4.6 mm i.d.) by using triallyl isocyanurate and methyl methacrylate as co-monomers, ethylene glycol dimethacrylate as a crosslinking agent, 1,4-butanediol, 1- dodecanol, and N, N-dimethylformamide as tri-porogens, and azobisisobutyronitrile as an initiator. The optimized monolithic columns were investigated by scanning electron microscopy, a nitrogen absorption-desorption apparatus, and mercury intrusion porosimetry. The effect of mobile phase composition and temperature on chromatographic behavior was studied in detail as well. The results showed that homemade monolithic columns not only have relative uniform porosity structure, low back pressure, and high thermal stability, but also show high column efficiency, the theoretical plates number of the new polymer monolithic column exceeded 31,000 plates m−1. Good resolution was achieved for HPLC separation of small molecules, including polycyclic aromatic hydrocarbons, isomers of phenols, and the Chinese herbal medicine notoginseng. Methodological validation of six drugs in notoginseng show that correlation coefficients of the linear regressions were in the range of 0.9989–0.9997; the value of relative standard deviation for precision were in the range of 1.37–4.21% for intra-day and 0.34–5.31% for inter-day, respectively; the values of accuracy expressed by recovery were in the range of 100.57–102.51%, 95.18–102.68%, 101.61–102.82%, 100.94–102.40%, 100.99–103.15% and 99.49–103.91% for the six drugs in order, respectively. Taking aromatic hydrocarbons as samples, the relative standard deviation values of the run-to-run (n = 5) and column-to-column (n = 5) based on peak area were < 2.01% and 2.39%, and two RSDs based on retention time were < 2.56% and 2.23%, respectively. The result showed that the polymer monolithic column has good separation ability and high permeability for small molecular substances.