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.     

Poly(l‐lactic acid)‐modified silica stationary phase for reversed‐phase and hydrophilic interaction liquid chromatography

Poly(l ‐lactic acid) is a linear aliphatic thermoplastic polyester that can be produced from renewable resources. A poly(l ‐lactic acid)‐modified silica stationary phase was newly prepared by amide bond reaction between amino groups on aminopropyl silica and carboxylic acid groups at the end of the poly(l ‐lactic acid) chain. The poly(l ‐lactic acid)‐silica column was characterized in reversed‐phase liquid chromatography and hydrophilic interaction liquid chromatography with the use of different mobile phase compositions. The poly(l ‐lactic acid)‐silica column was found to work in both modes, and the retention of test compounds depending on acetonitrile content exhibited “U‐shaped” curves, which was an indicator of reversed‐phase liquid chromatography/hydrophilic interaction liquid chromatography mixed‐mode retention behavior. In addition, carbonyl groups included into the poly(l ‐lactic acid) backbone work as an electron‐accepting group toward a polycyclic aromatic hydrocarbon and provide π–π interactions.     

Tetra‐proline‐modified calix[4]arene‐bonded silica stationary phase for simultaneous reversed‐phase/hydrophilic interaction mixed‐mode chromatography

A new water‐soluble tetra‐proline‐modified calix[4]arene‐bonded silica stationary phase was prepared straightforwardly by an indirect method and characterized by elemental analysis, energy dispersive Spectrometry, solid‐state ¹³C NMR spectroscopy, Fourier‐transform infrared spectroscopy, and thermogravimetric analysis. Due to the simultaneous introduction of polar tetra‐proline and nonpolar calix[4]arene, the developed column possessing a double retention mode of reverse‐phase liquid chromatography and hydrophilic interaction liquid chromatography. A series of hydrophobic and hydrophilic test samples, including nucleosides and nucleotides, amines, monosubstituted benzenes, chiral compounds, and phenols, were used to evaluate the developed stationary phase. A rapid separation capability, high separation efficiency, and selectivity were achieved based on the multiple interactions between solutes and tetra‐proline‐modified calix[4]arene‐bonded silica stationary phase. Moreover, the developed stationary phase was further used to detect and separate hexamethylenetetramine in rice flour. All the results indicated the potential merits of the developed stationary phase for simultaneous separation of complex hydrophobic and hydrophilic samples with high selectivity.     

Preparation of a monolithic column with a mixed‐mode stationary phase of reversed‐phase/hydrophilic interaction for capillary liquid chromatography

A monolithic capillary column with a mixed‐mode stationary phase of reversed‐phase/hydrophilic interaction chromatography was prepared for capillary liquid chromatography. The monolith was created by an in‐situ copolymerization of a homemade monomer N,N‐dimethyl‐N‐acryloxyundecyl‐N‐(3‐sulfopropyl) ammonium betaine and a crosslinker pentaerythritol triacrylate in a binary porogen agent consisting of methanol and isopropanol. The functional monomer was designed to have a highly polar zwitterionic sulfobetaine terminal group and a hydrophobic long alkyl chain moiety. The composition of the polymerization solution was systematically optimized to permit the best column performance. The columns were evaluated by using acidic, basic, polar neutral analytes, as well as a set of alkylbenzenes and Triton X100. Very good separations were obtained on the column with the mixed‐mode stationary phase. It was demonstrated that the mixed‐mode stationary phase displayed typic dual retention mechanisms of reversed‐phase/hydrophilic interaction liquid chromatography depending on the content of acetonitrile in the mobile phase. The method for column preparation is reproducible.     

Comprehensive analysis of pharmaceutical products using simultaneous mixed‐mode (ion‐exchange/reversed‐phase) and hydrophilic interaction liquid chromatography

Liquid chromatographic assays were developed using a mixed‐mode column coupled in sequence with a hydrophilic interaction liquid chromatography column to allow the simultaneous comprehensive analysis of inorganic/organic anions and cations, active pharmaceutical ingredients, and excipients (carbohydrates). The approach utilized dual sample injection and valve‐mediated column switching and was based upon a single high‐performance liquid chromatography gradient pump. The separation consisted of three distinct sequential separation mechanisms, namely, (i) ion‐exchange, (ii) mixed‐mode interactions under an applied dual gradient (reversed‐phase/ion‐exchange), and (iii) hydrophilic interaction chromatography. Upon first injection, the Scherzo SS C₁₈ column (Imtakt) provided resolution of inorganic anions and cations under isocratic conditions, followed by a dual organic/salt gradient to elute active pharmaceutical ingredients and their respective organic counterions and potential degradants. At the top of the mixed‐mode gradient (high acetonitrile content), the mobile phase flow was switched to a preconditioned hydrophilic interaction liquid chromatography column, and the standard/sample was reinjected for the separation of hydrophilic carbohydrates, some of which are commonly known excipients in drug formulations. The approach afforded reproducible separation and resolution of up to 23 chemically diverse solutes in a single run. The method was applied to investigate the composition of commercial cough syrups (Robitussin®), allowing resolution and determination of inorganic ions, active pharmaceutical ingredients, excipients, and numerous well‐resolved unknown peaks.     

A reversed phase/hydrophilic interaction/ion exchange mixed-mode stationary phase for liquid chromatography

A new stationary phase demonstrated effective separation towards polar analytes or their counterions within a single run.     

Comparison of reversed‐phase liquid chromatography and hydrophilic interaction chromatography for the fingerprint analysis of Radix isatidis

Radix isatidis is a famous anti‐influenza virus herbal medicine traditionally taken as a water decoction. However, the chemical fingerprint analysis of Radix isatidis is dominantly based on RPLC, from which it is difficult to obtain fingerprint information of hydrophilic compounds. Here, we developed the separation of Radix isatidis by RPLC and hydrophilic interaction chromatography, comparing the traditional RPLC fingerprint with the hydrophilic interaction chromatography fingerprint. Besides, an anti‐viral assay of Radix isatidis was conducted to evaluate its efficacy. The fingerprint–efficacy relationships between the fingerprints and the anti‐viral activity were further investigated with principal component regression analysis. The results showed that the anti‐viral activity correlated better with the hydrophilic interaction chromatography fingerprint than with the RPLC fingerprint. This study indicates that hydrophilic interaction chromatography could not only be a complementary method to increase the fingerprint coverage of conventional RPLC fingerprint, but also can better represent the efficacy and quality of Radix isatidis.     

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.     

Evaluation and application of a mixed‐mode chromatographic stationary phase in two‐dimensional liquid chromatography for the separation of traditional Chinese medicine

In this study, two mixed‐mode chromatography stationary phases (C8SAX and C8SCX) were evaluated and used to establish a two‐dimensional liquid chromatography system for the separation of traditional Chinese medicine. The chromatographic properties of the mixed‐mode columns were systematically evaluated by comparing with other three columns of C8, strong anion exchanger, and strong cation exchanger. The result showed that C8SAX and C8SCX had a mixed‐mode retention mechanism including electrostatic interaction and hydrophobic interaction. Especially, they were suitable for separating acidic and/or basic compounds and their separation selectivities could be easily adjusted by changing pH value. Then, several off‐line 2D‐LC systems based on the C8SAX in the first dimension and C8SAX, C8SCX, or C8 columns in the second dimension were developed to analyze a traditional Chinese medicine—Uncaria rhynchophylla. The two‐dimensional liquid chromatography system of C8SAX (pH 3.0) × C8SAX (pH 6.0) exhibited the most effective peak distribution. Finally, fractions of U. rhynchophylla prepared from the first dimension were successfully separated on the C8SAX column with a gradient pH. Thus, the mixed‐mode stationary phase could provide a platform to separate the traditional Chinese medicine in practical applications.     

Serial coupling of reversed‐phase and zwitterionic hydrophilic interaction LC/MS for the analysis of polar and nonpolar phenols in wine

In the present study, an easy and efficient method based on the serial coupling of analytical reversed‐phase and zwitterionic hydrophilic interaction liquid chromatography was developed for the simultaneous separation of polar and nonpolar phenols occurring in wine. The zwitterionic hydrophilic column was connected in series to the reversed‐phase one via a T‐piece, with which the ACN content in eluent of the second dimension was increased, in order to cope the solvent strength incompatibility between the two columns. The final mobile phase at low‐flow rate (≤0.5 mL/min), high‐ACN content (90%), and low‐salt concentration was directed to an ESI‐TOF‐MS , for high accurate mass detections. The developed method was applied for the identification of target phenols in several wines. Retention time and peak width intra‐ and interday repeatability studies proved the reliability of the method for the simultaneous analysis of all the polar and nonpolar analytes in wine. The serial reversed‐phase/zwitterionic hydrophilic interaction liquid chromatography coupling offered the possibility to enlarge the number of identified compounds and it represents a valid approach for nontarget analysis of complex samples by a single injection.     

Silica modified with a thiourea derivative as a new stationary phase for hydrophilic interaction liquid chromatography

Nowadays there are limited types of commercially available stationary phases for hydrophilic interaction liquid chromatography and therefore new ones with unique selectivity are urgently in demand to meet the need of separations of various polar and hydrophilic analytes. The present study describes the preparation and evaluation of a new stationary phase based on thiourea derivative modified silica for hydrophilic interaction liquid chromatography. Thiourea derivative was bonded onto the surface of silica particles via a mild addition reaction between –NH₂ and –SCN, and the result of elemental analysis together with infrared analysis and solid‐state NMR spectroscopy proved that the synthesis method was feasible. The new stationary phase succeeded in fast separations of a wide range of polar and hydrophilic analytes and exhibited excellent separation performance, especially unique selectivity. Furthermore, the effects of water content, buffer pH, and salt concentration on retention indicated that a complicated separation mechanism rather than partitioning was involved in the stationary phase and hydrogen bonding interaction between analytes and thiourea functional group could play a very important role in its selectivity. For sure, the new stationary phase is of a great potential as a new type of hydrophilic interaction liquid chromatographic stationary phase.     

Enhanced‐fluidity liquid chromatography using mixed‐mode hydrophilic interaction liquid chromatography/strong cation‐exchange retention mechanisms

The potential of enhanced‐fluidity liquid chromatography, a subcritical chromatography technique, in mixed‐mode hydrophilic interaction/strong cation‐exchange separations is explored, using amino acids as analytes. The enhanced‐fluidity liquid mobile phases were prepared by adding liquefied CO₂ to methanol/water mixtures, which increases the diffusivity and decreases the viscosity of the mixture. The addition of CO₂ to methanol/water mixtures resulted in increased retention of the more polar amino acids. The “optimized” chromatographic performance (achieving baseline resolution of all amino acids in the shortest amount of time) of these methanol/water/CO₂ mixtures was compared to traditional acetonitrile/water and methanol/water liquid chromatography mobile phases. Methanol/water/CO₂ mixtures offered higher efficiencies and resolution of the ten amino acids relative to the methanol/water mobile phase, and decreased the required isocratic separation time by a factor of two relative to the acetonitrile/water mobile phase. Large differences in selectivity were also observed between the enhanced‐fluidity and traditional liquid mobile phases. A retention mechanism study was completed, that revealed the enhanced‐fluidity mobile phase separation was governed by a mixed‐mode retention mechanism of hydrophilic interaction/strong cation‐exchange. On the other hand, separations with acetonitrile/water and methanol/water mobile phases were strongly governed by only one retention mechanism, either hydrophilic interaction or strong cation exchange, respectively.     

Preparation of a silica‐based high‐performance hydrophobic interaction chromatography stationary phase for protein separation and renaturation

In this work, based on the structural characteristics of bio‐membrane molecules, a novel type of high‐performance hydrophobic interaction chromatography stationary phase was prepared using cholesterol as a ligand. Investigating the separation performance of this stationary phase, the effect of pH and salt concentration of the mobile phase on the retention time, the absorption capacity, and the hydrophobic ability revealed that this stationary phase had a high loading capacity and moderate hydrophobic interactions compared with four different hydrophobic interaction chromatography stationary phase ligands. Five types of standard proteins could be baseline separated with a great selection for protein separation. When 3.0 M urea was added to the mobile phase, it could be refolded with simultaneous purification of denatured lysozyme by one‐step chromatography. The mass recovery of lysozyme reached 89.5%, and the active recovery was 96.8%. Compared with traditional hydrophobic interaction chromatography, this new stationary phase has a good hydrophobic ability and a significant refolding efficiency.     

A reversed-phase/hydrophilic interaction mixed-mode C18-Diol stationary phase for multiple applications

A mixed-mode chromatographic packing material, C18 and diol groups modified silica (C18-Diol), was prepared with controllable hydrophobicity and hydrophilicity. It demonstrated excellent aqueous compatibility and stability in aqueous mobile phase; compared to the traditional C18 column, improved peak shape of basic analytes was also obtained. Additionally, it exhibited both reversed-phase liquid chromatographic (RPLC) and hydrophilic interaction chromatographic (HILIC) performance; the analyte separation scope was thus enlarged, demonstrated by simultaneous separation of twenty acids, bases and neutrals. More interestingly, a novel on-line two-dimensional liquid chromatography on the single column (2D-LC-1C) was established by modifying the high performance liquid chromatographic instrument only with the addition of an extra six-port two-position valve. The early co-eluted components of the extract of Lonicera japonica on the 1st-dimension (RPLC) were collected for the online re-injection to the 2nd-dimension (HILIC) by conveniently varying the mobile phase components. Six more peaks were obtained. The established system was simple, easy operation and low cost, which had advantages in analyzing complicated samples.     

Investigation of polar stationary phases for the separation of sympathomimetic drugs with nano-liquid chromatography in hydrophilic interaction liquid chromatography mode

In this study, the retention and selectivity of a mixture of basic polar drugs were investigated in hydrophilic interaction chromatographic conditions (HILIC) using nano-liquid chromatography (nano-LC). Six sympathomimetic drugs including ephedrine, norephedrine, synephrine, epinephrine, norepinephrine and norphenylephrine were separated by changing experimental parameters such as stationary phase, acetonitrile (ACN) content, buffer pH and concentration, column temperature. Four polar stationary phases (i.e. cyano-, diol-, aminopropyl-silica and Luna HILIC, a cross-linked diol phase) were selected and packed into fused silica capillary columns of 100 μm internal diameter (i.d.). Among the four stationary phases investigated a complete separation of the all studied compounds was achieved with aminopropyl silica and Luna HILIC stationary phases only. Best chromatographic results were obtained employing a mobile phase composed by ACN/water (92/8, v/v) containing 10 mM ammonium formate buffer pH 3. The influence of the capillary temperature on the resolution of the polar basic drugs was investigated in the range between 10 and 50 °C. Linear correlation of ln k vs. 1/T was observed for all the columns; ΔH° values were negative with Luna HILIC and positive with aminopropyl- and diol-silica stationary phases, demonstrating that different mechanisms were involved in the separation.To compare the chromatographic performance of the different columns, Van Deemter curves were also investigated.     

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.     

Sulfonated cyclofructan 6 based stationary phase for hydrophilic interaction chromatography

A stationary phase composed of silica-bonded sulfonated cyclofructan 6 (SCF6) was synthesized and evaluated for hydrophilic interaction chromatography (HILIC). The separation of a large variety of polar compounds was evaluated on different versions of the stationary phase and compared with the same separations obtained with commercially available HILIC columns. The new columns successfully separate polar and hydrophilic compounds including β blockers, xanthines, salicylic acid related compounds, nucleic acid bases, nucleosides, maltooligosaccharides, water soluble vitamins and amino acids. The separation conditions were optimized by changing the composition and the pH of the mobile phase. The dependence of analyte retention on temperature was studied using van't Hoff plots. The newly synthesized stationary phase showed broad applicability for HILIC mode separations.     

Hydrophilic interaction chromatography: A promising alternative to reversed‐phase liquid chromatography systems for the purification of small protonated bases

The overloaded band profiles of the protonated species of propranolol and amitriptyline were recorded under acidic conditions on four classes of stationary phases including a conventional silica/organic hybrid material in reversed‐phase liquid chromatography mode (BEH‐C₁₈), an electrostatic repulsion reversed‐phase liquid chromatography C₁₈ column (BEH‐C₁₈+), a poly(styrene‐divinylbenzene) monolithic column, and a hydrophilic interaction chromatography stationary phase (underivatized BEH). The same amounts of protonated bases per unit volume of stationary phase were injected in each column (16, 47, and 141 μg/cm³). The performance of the propranolol/amitriptyline purification was assessed on the basis of the asymmetry of the recorded band profiles and on the selectivity factor achieved. The results show that the separation performed under reversed‐phase liquid chromatography like conditions (with BEH‐C₁₈, BEH‐C₁₈+, and polymer monolith materials) provide the largest selectivity factors due to the difference in the hydrophobic character of the two compounds. However, they also provide the most distorted overloaded band profiles due to a too small loading capacity. Remarkably, symmetric band profiles were observed with the hydrophilic interaction chromatography column. The larger loading capacity of the hydrophilic interaction chromatography column is due to the accumulation of the protonated bases into the diffuse water layer formed at the surface of the polar adsorbent. This work encourages purifying ionizable compounds on hydrophilic interaction chromatography columns rather than on reversed‐phase liquid chromatography columns.     

Preparation of a mixed-mode hydrophilic interaction/anion-exchange polymeric monolithic stationary phase for capillary liquid chromatography of polar analytes

A novel cationic hydrophilic interaction monolithic stationary phase based on the copolymerization of 2-(methacryloyloxy)ethyltrimethylammonium methyl sulfate (META) and pentaerythritol triacrylate (PETA) in a binary porogenic solvent consisting of cyclohexanol/ethylene glycol was designed for performing capillary liquid chromatography. While META functioned as both the ion-exchange sites and polar ligand provider, the PETA, a trivinyl monomer, was introduced as cross-linker. The monolithic stationary phases with different properties were easily prepared by adjusting the amount of META in the polymerization solution as well as the composition of the porogenic solvent. The hydrophilicity of the monolith increased with increasing content of META in the polymerization mixture. A typical hydrophilic interaction chromatography mechanism was observed when the content of acetonitrile in the mobile phase was higher than 20%. The poly(META-co-PETA) monolith showed very good selectivity for neutral, basic and acidic polar analytes. For polar-charged analytes, both hydrophilic interaction and electrostatic interaction contributed to their retention. Peak tailing of basic compounds was avoided and the efficient separation of benzoic acid derivatives was obtained.     

Porous graphene decorated silica as a new stationary phase for separation of sulfanilamide compounds in hydrophilic interaction chromatography

Porous graphene (PG) was prepared by combustion method and then coated onto aminopropyl-silica in deep eutectic solvents (DESs). PG-modified silica was evaluated in hydrophilic interaction chromatography for the separation and determination of sulfonamides in human serum samples.