Mixed-mode Column Research Articles

Two-dimensional liquid chromatography based on different modulations for the determination of Aristolochic Acid Ⅰ in Asari Radix et Rhizoma

Asari Radix et Rhizoma (ARR) is a traditional Chinese herbal medicine derived from the genus Asarum in the Aristolochiaceae family, which has been widely used for years. Aristolochic acids in it significantly restrict the clinical application of ARR due to its nephrotoxic and carcinogenic properties, of which aristolochic acid I (AA I) is the most representative. The present paper describes two 2D-LC systems modulated by a sample loop and a trap column respectively for the detection of trace aristolochic acid I in ARR. An Inertsil ODS-3 C18 column and a Luna 3u Phenyl-Hexyl column were separately utilized in the first-dimension (1D) and second-dimension (2D) chromatography. A mixed-mode column with reversed-phase and ion-exchange was selected for trap column modulation of 2D-LC system. Method validation results showed that these two 2D-LC methods performed well in terms of repeatability, linearity, intra/interday precision, stability, and accuracy, which were demonstrated qualified for the simultaneous determination of trace AA I in ARR. The AA Ⅰ contents in three batches of ARR were 0.466∼0.812 μg/g by the two 2D-LC methods, which were below the limit requirement in the Chinese Pharmacopoeia. The comprehensive comparison between the two methods showed that a significant solute focusing effect was obtained by trap column modulation, which led to four-time lower LOD compared with loop modulation, while the loop-modulation-based method achieved higher analytical efficiency when analyzing a large batch of samples.

Investigation on the Retention and Separation of Glucosinolates With a Mixed-Mode Reversed-Phase/Weak Anion-Exchange Column.

Glucosinolates, a crucial group of secondary metabolites in Brassica vegetables, present significant chromatographic separation challenges due to their anionic form, structure diversities, and co-existence of other phenolic compounds. This study comparatively investigated the retention and separation of seven glucosinolates using a mixed-mode reversed-phase/weak anion-exchange column and a conventional reversed-phase C18 column. Separation factors for each glucosinolate with its adjacent peaks were over 1.0 on the mixed-mode column, while co-eluting was observed on the C18 column. The effects of mobile phase additives and pH on the separation and retention of glucosinolates were also investigated. Results showed that glucosinolate retention was inversely related to both buffer concentration and pH. The optimized method for the mix-mode column was applied to the complex Brassica vegetable samples. In addition to the 17 well-resolved glucosinolate peaks, 34 peaks for phenolic compounds were identified in broccoli microgreen, suggesting the successful application scenarios for qualitative analysis in comparison with the single mode reverse phase C18 column. This study demonstrates that the mixed-mode reversed-phase/weak anion-exchange column can be used as a promising separation tool for organic anions in a complex sample matrix.

Modelling the pH dependent retention and competitive adsorption of charged and ionizable solutes in mixed-mode and reversed-phase liquid chromatography

This study investigated the influence of pH on the retention of solutes using a mixed-mode column with carboxyl (-COOH) groups acting as weak cation exchanger bonded to the terminal of C18 ligands (C18-WCX column) and a traditional reversed-phase C18 column. First, a model based on electrostatic theory was derived and successfully used to predict the retention of charged solutes (charged, and ionizable) as a function of mobile phase pH on a C18-WCX column. While the Horváth model predicts the pH-dependent retention of ionizable solutes in reversed-phase liquid chromatography (RPLC) solely based on solute ionization, the developed model incorporates the concept of surface potential generated on the surface of the stationary phase and its variation with pH. To comprehensively understand the adsorption process, adsorption isotherms for these solutes were individually acquired on the C18-WCX and reversed-phase C18 columns. The adsorption isotherms followed the Langmuir model for the uncharged solute and the electrostatically modified Langmuir model for charged solutes. The elution profiles for the single components were calculated from these isotherms using the equilibrium dispersion column model and were found to be in close agreement with the experimental elution profiles. To enable modelling of two-component cases involving charged solute(s), a competitive adsorption isotherm model based on electrostatic theory was derived. This model was later successfully used to calculate the elution profiles of two components for scenarios involving (a) a C18 Column: two charged solutes, (b) a C18 Column: one charged and one uncharged solute, and (c) a C18-WCX Column: two charged solutes. The strong alignment between the experimental and calculated elution profiles in all three scenarios validated the developed competitive adsorption model.

Molecular Dynamics Simulation for the Acidic Compounds Retention Mechanism Study on Octyl-Quaternary Ammonium Mixed-Mode Stationary Phase.

With the widespread application of mixed-mode chromatography in separation analysis, it is becoming increasingly important to study its retention mechanism. The retention behavior of acidic compounds on mixed-mode octyl-quaternary ammonium (Sil-C8-QA) columns was investigated by computer simulation. Firstly, the benzoic acid homologues were used as the analytes, and the simulation model was constructed by the Materials Studio. Geometric optimization, annealing and molecular dynamics (MD) simulation of these complexes resulted in optimized conformations. The binding energy, mean square displacement (MSD) and torsion angle distribution generated by MD simulation were then analyzed. The results showed that the more negative binding energy, the greater the MSD and the narrower the torsion angle distribution, indicating that the stationary phase behaves with stronger interaction and retention. The retention behavior of five acidic drugs on the Sil-C8-QA column was then successfully explained by simulation. Acidic drugs are more retentive on the mixed-mode column due to the more substantial interaction brought by the reversed-phase/ion-exchange mixed-mode mechanism compared to other single-mode columns. This simulation method is expected to provide ideas for studying the separation mechanism and predicting the retention behavior of more complex samples.

Cation-exchange/hydrophilic interaction mixed-mode liquid chromatographic method for analyzing the trigonelline class of bioactives in coffee beans

The coffee phytochemical trigonelline and its roasting products nicotinic acid and N-methylpyridinium represent an important class of bioactives that mediate the health benefits of coffee consumption. However, there is a paucity of methods for their simultaneous analysis in coffee beans, especially those affordable and reliable for widespread routine use. To improve this situation, an easy-to-use HPLC−UV method was developed in this study based on a reversed-phase/cation-exchange mixed-mode column showing unique chromatographic selectivity toward the polar and ionic analytes over the matrix components of coffee. The selectivity was achieved by using a decreasing acetonitrile gradient, which tuned the column to a cation-exchange/hydrophilic interaction (CEX/HILIC) mixed separation mode whilst minimizing its hydrophobic interaction capability. The method was assessed for figures of merit, giving limits of detection in coffee beans at around the 0.1 mg/100 g wet matter level, and verified for its applicability to a heterogeneous set of coffee bean samples differing in roasting degree and origin.

Fabrication of ionic liquid functionalized silica with different anions and the application in mixed-mode and chiral chromatography

To realize the potential of ionic liquid functionalized silica to prepare mixed-mode and chiral stationary phases, two ionic liquid silane reagents with different anions were synthesized via a high-efficiency click reaction. Then they were decorated onto the surface of silica by a one-step bonding reaction. The functionalized silica was characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and elemental analysis (EA). Two stationary phases provided satisfactory performance when compared with a commercial mixed-mode column. Notably, Sil-C10Im-D-BCS with D-3-bromocamphor-8-sulfonate (D-BCS) as anion presented chiral separation capacity towards 1,2,3,4-Tetrahydro-1-naphthol. The separation mechanism was investigated through multiple pathways, and the results revealed that the prepared stationary phases can retain and separate solutes through multiple interactions, like hydrophobic effect, ion exchange, hydrogen-bond interaction, etc. Quantum chemical calculation (QC) was employed to obtain the optimized structures and the binding energy of anions to cations. The results provided some insights into the retention mechanism from a molecular perspective. This work demonstrated the superiority of ionic liquid functionalized silica as mixed-mode stationary phases and the potential of chiral ionic liquid as chiral selectors.

ACHIEVING LOW BACKGROUNDS DURING COMPOUND-SPECIFIC HYDROXYPROLINE DATING: HPLC COLUMN EFFECTS

ABSTRACT Accelerator mass spectrometry (AMS) radiocarbon (14C) dating is central to the development of robust chronologies in archaeological and paleoenvironmental contexts spanning the last 50,000 years. For dates to be accurate, samples must be free of exogenous carbon contamination. At the Oxford Radiocarbon Accelerator Unit (ORAU), considerable advancements in the dating of bone collagen have been made through the development of a high performance liquid chromatography (HPLC) method for the dating of the amino acid hydroxyproline, which can mitigate the effects of carbon contamination. However, recent changes in ligand manufacturing methods for the mixed-mode column used in the ORAU protocol (Primesep A, SIELC Technologies; IL, USA) have resulted in unacceptably high analytical backgrounds. Prior to the manufacturing change, backgrounds of > 50k BP were achievable. Since the manufacturing change, a mean background of 32.5k BP has been measured. Due to column bleed, the Primesep A is therefore no longer suitable for 14C measurement of hydroxyproline from older material. Here, we present background data and the chromatography conditions used to isolate hydroxyproline using an alternative column, a preparative-scale Newcrom AH, which shows promising potential as an alternative for the routine isolation and AMS dating of hydroxyproline—especially approaching the age and mass limits of the method.

Preparation of ionic liquid stationary phase copolymerized with anion and cation for mixed-mode liquid chromatography

Ionic liquids (ILs) are a promising material to develop stationary phases for mixed-mode chromatography (MMC). In order to investigate the effect of anion type and polymerization method of ionic liquid, this work prepared and compared two stationary phases (Sil-IM-Br and Sil-IM-SAG) based on the polymeric ionic liquid, one of which copolymerized cation and anion. Two stationary phases were characterized and provided satisfactory retention repeatability (RSD < 0.3 %) and column efficiency (84600 plates/m). Chromatographic conditions (ACN content, salt concentration, and pH) were varied to reveal the retention characters and interactions between stationary phases and solutes. The results showed that both two developed stationary phases can operate under the mixed mode of RPLC/HILIC/IEC, but diverse selectivity was provided. So, the retention mechanisms were studied by linear solvation energy relationship equation (LSER) and thermodynamic research. Two stationary phases showed totally different retention towards carboxylic acids, which resulted from the effect of anion bonded. Lastly, the developed stationary phases were applied to separate alkylbenzenes and polycyclic aromatic hydrocarbons (PAHs) under RPLC. Furthermore, 7 carboxylic acids and 7 nucleosides and bases and complex samples were successfully separated under HILIC mode, Sil-IM-Br and Sil-IM-SAG provide better performances and different selectivity when compared with commercial mixed-mode columns. This work proves that cationic and anionic copolymerization provides a new method for stationary phase design, and better selectivity can be obtained by changing the anions.

Mixed-mode column allows simple direct coupling with immobilized enzymatic reactor for on-line protein digestion

Liquid chromatography coupled with mass spectrometry is widely used in the field of proteomic analysis after off-line protein digestion. On-line digestion with chromatographic column connected in a series with immobilized enzymatic reactor is not often used approach. In this work we investigated the impact of chromatographic conditions on the protein digestion efficiency. The investigation of trypsin reactor activity was performed by on-line digestion of N-α-benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA), followed by separation of the digests on the mixed-mode column. Two trypsin column reactors with the different trypsin coverage on the bridged ethylene hybrid particles were evaluated.To ensure optimal trypsin activity, the separation temperature was set at 37.0 °C and the pH of the mobile phase buffer was maintained at 8.5. The on-line digestion itself ongoing during the initial state of gradient was carried out at a low flow rate using a mobile phase that was free of organic modifiers. Proteins such as cytochrome C, enolase, and myoglobin were successfully digested on-line without prior reduction or alkylation, and the resulting peptides were separated using a mixed-mode column. Additionally, proteins that contain multiple cysteines, such as α-lactalbumin, albumin, β-lactoglobulin A, and conalbumin, were also successfully digested on-line (after reduction and alkylation). Moreover, trypsin immobilized enzymatic reactors were utilized for over 300 injections without any noticeable loss of digestion activity.

HPLC method development using a mixed-mode column for following 68Ga-radiolabeling

The aim of our work was to develop a generally applicable liquid chromatographic method for the determination of the residue free 68Ga3+ content of labeling reaction mixtures, in order to facilitate comparative labeling experiments in the early stages of chelator development. We achieved the retention of free 68Ga3+ on a mixed mode cation exchanger HPLC column with a gradient program, which allowed the separation from the non-retained complexes. The effects of reaction conditions (time, temperature) and the application of non-coordinating buffers were examined. Furthermore, recovery of free 68Ga3+ and these complexes were investigated, values of which were 94.9–97.4% and 87.4–99.6%.

Design of Novel Mixed-Mode Columns for Analysis of Amino Acids Derivatized with 4-Fluoro-7-nitro-2,1,3-benzoxadiazole and the Evaluation of Retention Properties

Three-dimensional (3D) HPLC methods are useful tools for the selective analysis of biomolecules in complicated real-world matrices, however, the columns applicable for the second dimension are still limited. In the present study, two types of mixed-mode columns (Singularity MX-102 and 103) were newly designed/developed focusing on the separation of N-protected chiral amino acids. The retention profiles of the two mixed-mode columns were evaluated using 10 proteinogenic amino acids with various chemical properties, namely, alanine, asparagine (Asn), glutamine, glycine, lysine (Lys), phenylalanine, proline, serine (Ser), threonine and valine (Val), and compared to that of a mixed-mode column (Singularity MX-001) previously adopted for the 3D-HPLC analysis of chiral amino acids. The amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and detected by their fluorescence (ex. 470 nm, em. 530 nm). For the mobile phases, mixed solutions of acetonitrile/methanol containing formic acid were used. As a result, NBD-Val eluted the fastest for all the tested mixed-mode columns. However, the NBD-amino acids most strongly retained on the columns were different (Asn for MX-001, Ser for MX-102 and Lys for MX-103). Also, the elution orders of the 10 NBD-amino acids on the three mixed-mode columns were not the same as that on the reversed-phase C18 column, and characteristic elution orders and retention properties were observed for the individual mixed-mode columns. These results indicated that mixed-mode columns reported in the present study have different retention mechanisms, and the development of highly selective 3D-HPLC systems using these columns is expected.

Dilute-and-shoot coupled to mixed mode liquid chromatography-tandem mass spectrometry for the analysis of persistent and mobile organic compounds in human urine

In this work, a comprehensive method for the simultaneous determination of 33 diverse persistent and mobile organic compounds (PMOCs) in human urine was developed by dilute-and-shoot (DS) followed by mixed-mode liquid chromatography coupled with tandem mass spectrometry (MMLC-MS/MS). In the sample preparation step, DS was chosen since it allowed the quantification of all targets in comparison to lyophilization. For the chromatographic separation, Acclaim Trinity P1 and P2 trimodal columns provided greater capacity for retaining PMOCs than reverse phase and hydrophilic interaction liquid chromatography. Therefore, DS was validated at 5 and 50 ng/mL in urine with both mixed mode columns at pH = 3 and 7. Regarding figures of merit, linear calibration curves (r2 > 0.999) built between instrumental quantification limits (mostly below 5 ng/mL) and 500 ng/mL were achieved. Despite only 60% of the targets were recovered at 5 ng/mL because of the dilution, all PMOCs were quantified at 50 ng/mL. Using surrogate correction, apparent recoveries in the 70–130% range were obtained for 91% of the targets. To analyse human urine samples, the Acclaim Trinity P1 column at pH = 3 and 7 was selected as a consensus between analytical coverage (i.e. 94% of the targets) and chromatographic runs. In a pooled urine sample, industrial chemicals (acrylamide and bisphenol S), biocides and their metabolites (2-methyl-4-isothiazolin-3-one, dimethyl phosphate, 6-chloropyridine-3-carboxylic acid, and ammonium glufosinate) and an artificial sweetener (aspartame) were determined at ng/mL levels. The outcomes of this work showed that humans are also exposed to PMOCs due to their persistence and mobility, and therefore, further human risk assessment is needed.

Development of an enantioselective three-dimensional HPLC system for the determination of alanine, valine, isoleucine, allo-isoleucine and leucine in human plasma and urine

A three-dimensional (3D) HPLC system was designed/developed for the discriminative determination of aliphatic chiral amino acids, namely, alanine (Ala), valine (Val), isoleucine (Ile), allo-Ile (aIle) and leucine (Leu), in human physiological fluids. These aliphatic amino acid enantiomers are expected to be new physiologically active molecules and/or biomarkers in mammals. Among these aliphatic amino acids, the structural chain isomers of Leu (Ile/aIle/Leu) have similar chemical-physical properties, and the analytical method for these aliphatic amino acids is required to be highly enantio- and chemo-selective. In the present study, a reversed-phase column (Singularity RP18, first dimension) and a mixed-mode column (Singularity MX-103, second dimension) were utilized to separate these aliphatic amino acids as their scalemic mixtures, then chiral separations were performed using a Pirkle-type enantioselective column (Singularity CSP-001S) in the third dimension. By using the 3D-HPLC system, these aliphatic amino acid enantiomers were completely discriminated, and the analysis of these aliphatic chiral amino acids in the human plasma and urine was successfully carried out. The obtained amounts of the aliphatic amino acids (and %D value, the percentage of D-form to D + L forms) in the human plasma were 2.1 µM (0.5%) for D-Ala, and trace/not-detected for the other D-forms. In human urine, the values were 102.0 µM (24.7%) for D-Ala, 2.0 µM (3.4%) for D-Val, 2.3 µM (10.8%) for D-aIle (%D value of D-aIle was calculated using D-aIle and L-Ile) and 3.3 µM (5.5%) for D-Leu. The present 3D-HPLC system is a powerful and well validated tool for the simultaneous determination of aliphatic amino acid enantiomers, and further biological and clinical studies are expected.

The benefits of mixed-mode chromatography columns for separation of peptides and protein digests

In this work, the evaluation and comparison of mixed-mode chromatography and reversed-phase chromatography for separation of peptides and protein digests have been performed. The effects of pH of aqueous part of mobile phase as well as the effects of organic modifier on retention, resolution, and peak shape were investigated on several columns including three mixed-mode columns possessing reversed-phase/anion-exchange mechanism, two reversed-phase octadecyl columns, and one column with mixed-mode reversed-phase/anion-exchange character only in defined pH range. The set of peptides varying in their polarity, length, amino acid sequence, and charge state, namely dipeptides, N-blocked dipeptides, and oligopeptides, was selected to describe the chromatographic behavior under different conditions properly. These measurements showed the potential of mixed-mode chromatography columns for analysis of differently charged peptides in a single run. The applicability of the tested conditions has been verified by the analysis of cytochrome C digested fragments. Two types of samples were analyzed and compared, i.e., commercial cytochrome C digested standard and cytochrome C digested via trypsin spin columns. The obtained results point to the necessity of using mass spectrometry detection because of large number of unknown peaks in cytochrome C digested standard, probably originating from chymotryptic and miscleavage activities.Graphical abstract

Semi-empirical description of a retention of small organic probes on AtlantisTM Premier BEH C18 AX mixed-mode column under varying pH and organic modifier conditions

Mixed-mode chromatography is a widely used analytical method that utilizes multiple separation mechanisms in one chromatographic column; frequently a reversed-phase and ion-exchange interactions. Adequate understanding of how the individual interactions influence the overall retention of the analyte is important for fast and effective design of mixed-mode-based methods. Previous attempts to derive semi-empirical mathematical models of mixed-mode chromatography were limited to a narrow range of analytes and did not consider the effect of organic modifier in the mobile phase. In this paper, we attempt to improve upon the previous models by extending the range of classes of analytes to weak acids and bases and including the effect of organic modifier. An improved semi-empirical model was derived that captures all important retention trends of the combined effect of reversed-phase and ion-exchange retention mechanisms for all tested classes of analytes and up to 30% (v/v) organic modifier content.

Simultaneous determination of glyphosate, glufosinate, and their metabolites in honey using liquid chromatography-tandem mass spectrometry and solid-phase extraction.

Here, we developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of residual glyphosate, glufosinate, and their metabolites N-acetylglyphosate (Gly-A), 3-methylphosphinicopropionic acid (MPPA) and N-acetylglufosinate (Glu-A) in honey using a mixed mode column of reversed-phase and anion exchange without derivatization. The target analytes were extracted from honey samples using water, cleaned up on a reverse phase C18 cartridge column and an anion exchange NH2 cartridge column, and quantified using LC-MS/MS. Glyphosate, Glu-A, Gly-A, and MPPA were detected in negative ion mode based on deprotonation, whereas glufosinate was detected in positive ion mode. The coefficients of determination (R2) of the calibration curve, calculated in the range of 1-20µg/kg for glufosinate, Glu-A, and MPPA, and 5-100µg/kg for glyphosate and Gly-A, were higher than 0.993. The developed method was evaluated using honey samples spiked with glyphosate and Gly-A at 25µg/kg and glufosinate, and MPPA and Glu-A at 5µg/kg, based on the maximum residue levels. The validation results show good recoveries (86-106%) and precision (< 10%) for all target compounds. The limit of quantification of the developed method is 5µg/kg for glyphosate, 2µg/kg for Gly-A, and 1µg/kg for glufosinate, MPPA and Glu-A. These results suggest that the developed method is applicable for quantifying residual glyphosate, glufosinate, and their metabolites in honey in compliance with Japanese maximum residue levels. Moreover, the proposed method was applied to the analysis of honey samples and glyphosate, glufosinate, and Glu-A were detected in some samples. The proposed method will be a useful tool for the regulatory monitoring of residual glyphosate, glufosinate, and their metabolites in honey.

Development of a three-dimensional HPLC system for acidic amino acid enantiomers and determination of their amounts in mice lacking D-aspartic acid oxidase activity

A highly-selective three-dimensional (3D) HPLC system has been designed/developed for the determination of acidic amino acid enantiomers, i.e., aspartic acid (Asp) and glutamic acid (Glu). The 3D-HPLC system consisted of a reversed-phase column (Singularity RP18, 1.0 ×250 mm) in the first dimension, a mixed-mode column (Singularity MX-001, 1.5 ×250 mm) in the second dimension and a Pirkle-type enantioselective column (Singularity CSP-011S, 1.5 ×250 mm) in the third dimension. By using this system, the amounts of Asp and Glu enantiomers in 6 tissues (cerebrum, heart, lung, liver, kidney and testis) and 2 physiological fluids (plasma and urine) were determined in the control C57BL/6J mice and B6DDO-/- mice lacking D-aspartic acid oxidase activity. In the control C57BL/6J mice, a high amount of D-Asp (466.15 nmol/g) was observed in the testis, and relatively high amounts of D-Asp were observed in the cerebrum, lung, liver and kidney (21.40–92.87 nmol/g). In the heart and 2 physiological fluids, the amounts of D-Asp were low (trace–7.75 nmol/g or mL). In the B6DDO-/- mice, the amounts of D-Asp drastically increased in all the tissues and physiological fluids. In contrast to the results of D-Asp, the amounts of D-Glu did not change depending on the alteration of the DDO activity, and the amounts were extremely low and not detectable in most of the tested tissues and physiological fluids. Although the amounts of D-Glu were low, it is noteworthy that clear localization in the testis (8.60–9.53 nmol/g) was demonstrated in both the C57BL/6J and B6DDO-/- mice.

Harmonization of Experimental Methods Used to Measure the True Hold-Up Volume of Liquid Chromatography Columns

There are as many measurement values of the true column hold-up volume, V0, as techniques applied to evaluate this most important property in liquid chromatography (LC). The relative errors made on V0 measurements using conventional “non-retained” markers—such as acetone, uracil, or thiourea in reversed-phase liquid chromatography (RPLC), or benzene or acenaphthene in hydrophilic interaction chromatography (HILIC)—can be as large as ±30%. This situation is extremely confusing for LC users who wish to classify and predict the retention behavior of LC columns. In this work, along with advances in mass spectrometry (MS) instrumentation, the hold-up volume of any LC column—including, but not limited to, RPLC, HILIC, ion exchange chromatography (IEX), and mixed-mode columns—is accurately measured by injecting labeled deuterated acetonitrile (CD3CN) molecules and detecting them selectively by MS-single ion reaction (m/z = 45) using non-labeled and pure acetonitrile (CH3CN) as the eluent. This proposed harmonization of all conventional V0 measurement methods is illustrated and successfully applied to RPLC, HILIC, anion exchange (AEX), and RP-AEX mixed-mode chromatography, irrespective of the mobile phase composition selected.

Studying the possibilities of dual stationary phase gradients to explore alternative selectivities in liquid chromatography

Previous studies on stationary phase gradients have inspired a new phase of theoretical work and an expansion of the concept to include multiple retention mechanisms. The theoretical work presented here corroborates previous reports that a stationary phase gradient can produce selectivity in a separation as effectively as careful adjustments to a mobile phase gradient program, especially when such column is operated in mobile phase gradient elution mode. In reality, no column is singularly based on one type of interaction mechanism, and many columns are nowadays designed to solicit multiple solute to stationary phase interactions. These mixed-mode columns inspired us to give more careful consideration to the idea of dual stationary phase gradients. With the theory applied here, it is suggested that the modulation of two unique interaction mechanisms across a chromatographic column has the potential to open up previously unseen selectivities. With the increasing prevalence of mixed-mode columns, we believe there will be ample opportunity to explore these new concepts in experimental work.

One-Dimensional Simultaneous Achiral and Chiral Analysis

Most molecules with therapeutic value have at least one chiral center, requiring resolution of both achiral and chiral impurities. Separate achiral and chiral methods are the most used methodologies, but two-dimensional (2D) methods and columns in series have also been implemented. Having a mixed-mode column that can provide both achiral and chiral resolution in one-dimension (1D) would significantly improve impurity profile understanding and reduce testing workload.