Recent trends in ultra‐fast HPLC: New generation superficially porous silica columns

New generation columns, i.e. packed with superficially porous silica particles are available as trade names with following manufacturers: Halo, Ascentis Express, Proshell 120, Kinetex, Accucore, Sunshell, and Nucleoshell. These provide ultra‐fast HPLC separations for a variety of compounds with moderate sample loading capacity and low back pressure. Chemistries of these columns are C₈, C₁₈, RP‐Amide, hydrophilic interaction liquid chromatography, penta fluorophenyl (PFP), F5, and RP‐aqua. Normally, the silica gel particles are of 2.7 and 1.7 μm as total and inner solid core diameters, respectively, with 0.5‐μm‐thick of outer porous layer having 90 Å pore sizes and 150 m²/g surface area. This article describes these new generation columns with special emphasis on their textures and chemistries, separations, optimization, and comparison (inter and intra stationary phases). Besides, future perspectives have also been discussed.     

C18‐bound porous silica monolith particles as a low‐cost high‐performance liquid chromatography stationary phase with an excellent chromatographic performance

Ground porous silica monolith particles with an average particle size of 2.34 μm and large pores (363 Å) exhibiting excellent chromatographic performance have been synthesized on a relatively large scale by a sophisticated sol–gel procedure. The particle size distribution was rather broad, and the d(0.1)/d(0.9) ratio was 0.14. The resultant silica monolith particles were chemically modified with chlorodimethyloctadecylsilane and end‐capped with a mixture of hexamethyldisilazane and chlorotrimethylsilane. Very good separation efficiency (185 000/m) and chromatographic resolution were achieved when the C₁₈‐bound phase was evaluated for a test mixture of five benzene derivatives after packing in a stainless‐steel column (1.0 mm × 150 mm). The optimized elution conditions were found to be 70:30 v/v acetonitrile/water with 0.1% trifluoroacetic acid at a flow rate of 25 μL/min. The column was also evaluated for fast analysis at a flow rate of 100 μL/min, and all the five analytes were eluted within 3.5 min with reasonable efficiency (ca. 60 000/m) and resolution. The strategy of using particles with reduced particle size and large pores (363 Å) combined with C₁₈ modification in addition to partial‐monolithic architecture has resulted in a useful stationary phase (C₁₈‐bound silica monolith particles) of low production cost showing excellent chromatographic performance.     

Characterization of the pigment fraction in sweet bell peppers (Capsicum annuum L.) harvested at green and overripe yellow and red stages by offline multidimensional convergence chromatography/liquid chromatography–mass spectrometry

Offline multidimensional supercritical fluid chromatography combined with reversed‐phase liquid chromatography was employed for the carotenoid and chlorophyll characterization in different sweet bell peppers (Capsicum annuum L.) for the first time. The first dimension consisted of an Acquity HSS C₁₈ SB (100 × 3 mm id, 1.8 μm particles) column operated with a supercritical mobile phase in an ultra‐performance convergence chromatography system, whereas the second dimension was performed in reversed‐phase mode with a C₃₀ (250 × 4.6 mm id, 3.0 μm particles) stationary phase combined with photodiode array and mass spectrometry detection. This approach allowed the determination of 115 different compounds belonging to chlorophylls, free xanthophylls, free carotenes, xanthophyll monoesters, and xanthophyll diesters, and proved to be a significant improvement in the pigments determination compared to the conventional one‐dimensional liquid chromatography approach so far applied to the carotenoid analysis in the studied species. Moreover, the present study also aimed to investigate and to compare the carotenoid stability and composition in overripe yellow and red bell peppers collected directly from the plant, thus also evaluating whether biochemical changes are linked to carotenoid degradation in the nonclimacteric investigated fruits, for the first time.     

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.     

Feasibility of ultra high performance supercritical neat carbon dioxide chromatography at conventional pressures

The implementation of columns packed with sub-2 μm particles in supercritical fluid chromatography (SFC) is described using neat carbon dioxide as the mobile phase. A conventional supercritical fluid chromatograph was slightly modified to reduce extra column band broadening. Performances of a column packed with 1.8 μm C18-bonded silica particles in SFC using neat carbon dioxide as the mobile phase were compared with results obtained in ultra high performance liquid chromatography (UHPLC) using a dedicated chromatograph. As expected and usual in SFC, higher linear velocities than in UHPLC must be applied in order to reach optimal efficiency owing to higher diffusion coefficient of solutes in the mobile phase; similar numbers of theoretical plates were obtained with both techniques. Very fast separations of hydrocarbons are presented using two different alkyl-bonded silica columns.     

Comparison of different types of outlet frits in slurry‐packed capillary columns

Fused‐silica capillary columns for high‐performance liquid chromatography with 320 and 250 μm inner diameter were prepared by slurry packing with 5 and 3 μm Nucleosil C₁₈ stationary phase. Different types of mechanical and monolithic outlet frits were used and their influence on the resulting column performance was evaluated. Columns with quartz wool exhibited symmetrical peaks and low theoretical plate height, and the preparation time was short. The performance of monolithic frits varied based on type of monolith, length of the frit, and silanization procedure. The best frit performed similarly to the quartz wool ones, but the preparation took several hours. Their main advantage lies in the possibility of on‐column detection, because the detection window can be burnt immediately behind the frit.     

硅胶基质高效液相色谱填料研究进展

高效液相色谱(HPLC)不仅是一种有效的分析分离手段,也是一种重要的高效制备分离技术。色谱柱是HPLC系统的核心,不同性能的填料是HPLC广泛应用的基础。硅胶是开发最早、研究最为深入、应用最为广泛的HPLC固定相基质,其制备方法主要有喷雾干燥法、溶胶-凝胶法、聚合诱导胶体凝聚法及模板法等。近年来,亚2μm小粒径硅胶、核-壳型硅胶、双孔径硅胶、介孔性硅胶、有机杂化硅胶等新型硅胶应用于HPLC并取得了色谱分离技术的飞速发展,例如基于亚2μm填料的超高压液相色谱技术、基于核-壳型填料的快速分离技术、基于杂化硅胶填料的高温液相色谱技术等。硅胶经表面化学键合、聚合物包覆等有机改性可制得先进的大分子限进填料、温敏性填料、手性填料等,大大扩展了HPLC的应用范围。本文对液相色谱用硅胶的制备方法、改性与修饰方法以及硅胶基质固定相的评价方法加以系统综述,概述了新型硅胶在HPLC中的应用进展,并对硅胶基质填料的发展方向与应用前景进行了展望。     

High‐efficiency sample preparation approach to determine acrylamide levels in high‐fat foods

An improved sample preparation method was developed to enhance acrylamide recovery in high‐fat foods. Prior to concentration, distilled deionized water was added to protect acrylamide from degradation, resulting in a higher acrylamide recovery rate from fried potato chips. A Chrome‐Matrix C₁₈ column (2.6 μm, 2.1 × 100 mm) was used for the first time to analyze acrylamide levels using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry, displaying good separation of acrylamide from interference. A solid‐phase extraction procedure was avoided, and an average recovery of >89.00% was achieved from different food matrices for three different acrylamide spiking levels. Good reproducibility was observed, with an intraday relative standard deviation of 0.04–2.38%, and an interday relative standard deviation of 2.34–3.26%. Thus, combining the improved sample preparation method for acrylamide analysis with the separation on a Chrome‐Matrix C₁₈ column (2.6 μm, 2.1 × 100 mm) using ultra high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry is highly useful for analyzing acrylamide levels in complex food matrices.     

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.     

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.     

基于硅胶整体材料研磨颗粒的多功能聚乙二醇键合固定相的制备及其在高效液相色谱多模式分离中的应用

研究通过对溶胶-凝胶法制备的硅胶整体材料进行研磨、浮选、假晶相转换和水热处理，最终获得了粒径为2~5 μm、孔径为20~60 nm的硅胶颗粒。利用部分含氟的阴离子表面活性剂Capstone FS-66和常用的阳离子表面活性剂十六烷基三甲基溴化铵（CTAB）组成的双胶束模板体系对硅胶基质进行假晶相转换处理；再采用碳酸钠溶液水热处理的方式，进一步扩大孔径。用扫描电镜（SEM）和N₂吸附-解吸等温线测量对扩孔处理前后的硅胶整体材料研磨颗粒进行表征，结果清楚地显示了处理前后的形貌变化和差异。随后将含有长链聚乙二醇（PEG）的硅烷键合到扩孔后的硅胶颗粒表面，分别利用元素分析、红外光谱以及热重分析对固定相进行表征，并对固定相进行色谱性能评价。对键合固定相的元素分析和热重分析数据进行分析表明，硅胶表面键合PEG的含量约为8%。研究揭示了利用假晶相转换法与碳酸钠溶液水热处理和长链PEG硅烷修饰的硅胶整体材料颗粒在尺寸排阻色谱分离蛋白质方面的良好分离效果。同时进一步的高效液相色谱评价结果表明，该键合固定相还可用于疏水作用色谱模式分离核糖核酸酶A和溶菌酶，以及可用于亲水作用色谱模式分离吡啶甲酸、左旋多巴、三聚氰胺和邻苯二酚等极性比较强的化合物。研究显示了PEG键合固定相具有多功能性，及其在多模式高效液相色谱分离中的应用潜力。     

Use of Chemically Bonded β‐Cyclodextrin Silica Stationary Phase for Liquid Chromatographic Separation of Structural Isomers

The retention behavior of five disubstituted benzene derivatives and two naphthalene derivatives is examined by using a chemically bonded β‐cyclodextrin silica stationary phase with the moiety containing the s‐triazine. The chromatographic results of five disubstituted benzene derivatives and two naphthalene derivatives show that effective separation is achieved on this stationary phase by high‐performance liquid chromatography. The results of the present investigation indicate that the formation of inclusion complexes plays a dominant role in the separation mechanism. However, the selectivity can be significantly enhanced by the n‐n interactions between the s‐triazine ring of the chemically bonded β‐cyclodextrin silica stationary phase and the aromatic ring of solutes. For example, the effective separation of the o‐, m‐, and p‐toluidine isomers on this stationary phase with the moiety containing the s‐triazine ring was better than on that of some β‐cyclodextrin bonded stationary phases without the moiety containing s‐triazine ring.     

Rapid determination of parabens in seafood sauces by high‐performance liquid chromatography: A practical comparison of core–shell particles and sub‐2 μm fully porous particles

In this work, the chromatographic performance of superficially porous particles (Halo core–shell C₁₈ column, 50 mm × 2.1 mm, 2.7 μm) was compared with that of sub‐2 μm fully porous particles (Acquity BEH C₁₈, 50 mm × 2.1 mm, 1.7 μm). Four parabens, methylparaben, ethylparaben, propylparaben, and butylparaben, were used as representative compounds for calculating the plate heights in a wide flow rate range and analyzed on the basis of the Van Deemter and Knox equations. Theoretical Poppe plots were constructed for each column to compare their kinetic performance. Both phases gave similar minimum plate heights when using nonreduced coordinates. Meanwhile, the flat C‐term of the core–shell column provided the possibilities for applying high flow rates without significant loss in efficiency. The low backpressure of core–shell particles allowed this kind of column, especially compatible with conventional high‐performance liquid chromatography systems. Based on these factors, a simple high‐performance liquid chromatography method was established and validated for the determination of parabens in various seafood sauces using the Halo core–shell C₁₈ column for separation.     

Preparation and evaluation of monodispersed,submicron, non‐porous silica particles functionalized with β‐CD derivatives for chiral‐pressurized capillary electrochromatography

Submicron, non‐porous, chiral silica stationary phase has been prepared by the immobilization of functionalized β‐CD derivatives to isocyanate‐modified silica via chemical reaction and applied to the pressurized capillary electrochromatography (pCEC) enantio‐separation of various chiral compounds. The submicron, non‐porous, cyclodextrin‐based chiral stationary phases (sub_μm‐CSP2) exhibited excellent chiral recognition of a wide range of analytes including clenbuterol hydrochloride, mexiletine hydrochloride, chlorpheniramine maleate, esmolol hydrochloride, and metoprolol tartrate. The synthesized submicron particles were regularly spherical and uniformly non‐porous with an average diameter of around 800 nm and a mean pore size of less than 2 nm. The synthesized chiral stationary phase was packed into 10 cm × 100 μm id capillary columns. The sub_μm‐CSP2 column used in the pCEC system showed better separation of the racemates and at a higher rate compared to those used in the capillary liquid chromatography mode (cLC) system. The sub_μm‐CSP2 possessed high mechanical strength, high stereoselectivity, and long lifespan, demonstrating rapid enantio‐separation and good resolution of samples. The column provided an efficiency of up to 170 000 plates/m for n‐propylbenzene.     

Evaluation of silica from different vendors as the solid support of anion‐exchange chiral stationary phases by means of preferential sorption and liquid chromatography

Chromatographic performance of a chiral stationary phase is significantly influenced by the employed solid support. Properties of the most commonly used support, silica particles, such as size and size distribution, and pore size are of utmost importance for both superficially porous particles and fully porous particles. In this work, we have focused on evaluation of fully porous particles from three different vendors as solid supports for a brush‐type chiral stationary phase based on 9‐O‐tert‐butylcarbamoyl quinidine. We have prepared corresponding stationary phases under identical experimental conditions and determined the parameters of the modified silica by physisorption measurements and scanning electron microscopy. Enantiorecognition properties of the chiral stationary phases have been studied using preferential sorption experiments. The same material was slurry‐packed into chromatographic columns and the chromatographic properties have been evaluated in liquid chromatography. We show that preferential sorption can provide valuable information about the influence of the pore size and total pore volume on the interaction of analytes of different size with the chirally‐modified silica surface. The data can be used to understand differences observed in chromatographic evaluation of the chiral stationary phases. The combination of preferential sorption and liquid chromatography separation can provide detailed information on new chiral stationary phases.     

Gold nanoparticle decorated graphene oxide/silica composite stationary phase for high‐performance liquid chromatography

In the initial phase of this study, graphene oxide (GO)/silica was fabricated by assembling GO onto the silica particles, and then gold nanoparticles (GNPs) were used to modify the GO/silica to prepare a novel stationary phase for high‐performance liquid chromatography. The new stationary phase could be used in both reversed‐phase chromatography and hydrophilic interaction liquid chromatography modes. Good separations of alkylbenzenes, isomerides, amino acids, nucleosides, and nucleobases were achieved in both modes. Compared with the GO/silica phase and GNPs/silica phase, it is found that except for hydrophilicity, large π‐electron systems, hydrophobicity, and coordination functions, this new stationary phase also exhibited special separation performance due to the combination of 2D GO with zero‐dimensional GNPs.     

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.     

A validated stability‐indicating ultra performance liquid chromatography method for the determination of potential process‐related impurities in eplerenone

A simple, sensitive, and accurate stability‐indicating analytical method has been developed and validated using ultra high performance liquid chromatography. The developed method is used to evaluate the related substances of eplerenone (EP). The degradation behavior of EP under stress conditions was determined, and the major degradants were identified by ultra high performance liquid chromatography with tandem mass spectrometry. The chromatographic conditions were optimized using an impurity‐spiked solution, and the samples, generated from forced degradation studies. The resolution of EP, its potential impurities, and its degradation products was performed on a Waters UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) by linear gradient elution using a mobile phase consisting of 10 mmol/L ammonium acetate adjusted to pH 4.5, methanol and acetonitrile. A photo‐diode array detector set at 245 nm was used for detection. The flow rate was set at 0.3 mL/min. The procedure had good specificity, linearity (0.02–3.14 μg/mL), recovery (96.1–103.9%), limit of detection (0.01–0.02 μg/mL), limit of quantitation (0.03–0.05 μg/mL), and robustness. The correction factors of the process‐related substances were calculated.     

Comparison of a new high‐resolution monolithic column with core‐shell and fully porous columns for the analysis of retinol and α‐tocopherol in human serum and breast milk by ultra‐high‐performance liquid chromatography†

The reduction of analysis time, cost, and improvement of separation efficiency are the main requirements in the development of high‐throughput assay methods in bioanalysis. It can be achieved either by ultra‐high‐performance liquid chromatography (UHPLC) using stationary phases with small particles (<2 μm) at high back pressures or by using opposite direction—monolithic stationary phases with low back pressures. The application of new types of monolithic stationary phases for UHPLC is a novel idea combining these two different paths. The aim of this work was to test the recently introduced second‐generation of monolithic column Chromolith® HighResolution for UHPLC analysis of liposoluble vitamins in comparison with core‐shell and fully porous sub‐2 μm columns with different particle sizes, column lengths, and shapes. The separation efficiency, peak shape, resolution, time of analysis, consumption of mobile phase, and lifetime of columns were calculated and compared. The main purpose of the study was to find a new, not only economical option of separation of liposoluble vitamins for routine practice.     

Separation of complex sugar mixtures on a hydrolytically stable bidentate urea‐type stationary phase for hydrophilic interaction near ultra high performance liquid chromatography

A stationary phase bearing both bridged bis‐ureido and free amino groups (USP‐HILIC‐NH₂–2.5SP) for high‐speed hydrophilic interaction liquid chromatography separations was prepared using a one‐pot two‐step procedure starting from 2.5 μm totally porous silica particles. Highly polar compounds, such as polyols, hydroxybenzoic acids, and sugars, were successfully analyzed in shorter times and with higher peak efficiency, when compared to results obtained with a bidentate urea‐type column packed with 5 μm particles. Increased sugarophilicity and better peak shape were attested for the USP‐HILIC‐NH₂–2.5SP column (100 × 3.2 mm id) when compared with two commercially available UHPLC columns, namely an acquity BEH amide packed with totally porous 1.7 μm microparticles and a HILIC Kinetex column packed with core–shell 2.6 μm particles. Finally, the new column was employed in the separation of complex mixture of sugars (mono‐, di‐, and oligosaccharides) and in the analysis of beer samples. The resulting chromatograms showed good selectivity and overall resolution, while the catalyzing effect of the free amino moieties resulted in excellent peak shapes and in the absence of split peaks due to sugar anomerization phenomena.