Increasing the separation efficiency and understanding the interactions associated with the columns and the stationary phases are some of the major objectives of fundamental studies on liquid chromatography. The separation of isotopic compounds is significant in separation science for how it illustrates the extremely high ability of high performance liquid chromatography systems, and has its own significance, too, as a longtime target of scientific curiosity. Isotopic separation is an interesting subject for separation science by itself because of the high similarity in the solute structure. Fundamental information on molecular interactions will be helpful for understanding the retention behaviors of various solutes in combination with diverse stationary phases and mobile phases. Difficult separations have often been carried out by recycle chromatography using a series of several columns to allow the solute mixture band to go through the columns many times before being eluted out.

Herein, commercially available columns employed in hydrophilic interaction chromatography (HILIC) were characterized by determining their ability to selectively distinguish the minute structural differences between small molecules such as nucleosides and xanthines in complex sample matrices. Principal component analysis (PCA) was applied to the data obtained from structurally similar analytes, and the results showed that HILIC columns could generally be classified into two groups: (i) silane-modified columns that were prepared from either native silica particles or silica particles modified with low-molecular-weight silanes and (ii) polymer-modified columns obtained from silica particles functionalized with organic polymers. These two groups could be further subdivided based on the functionalities attached to the respective stationary phases. These results were confirmed via cluster analysis by preparing a dendrogram using the morphology-based selectivity parameters associated with the respective columns. We were able to determine the selectivity of columns for the OH groups, i.e., α(OH) and the prevailing pH conditions (cation- and anion-exchanging natures) on the surface of the respective stationary phases; α(theobromine/theophylline) was employed to obtain a similar two-dimensional plot. This test scheme, in which five compounds were analyze for each column, was helpful for understanding the impact of factors such as the hydrophilicity, degree of hydration, acidity/basicity, or the weak ion-exchange nature of the respective stationary phases on the separation characteristics of new HILIC stationary phases. The selectivity of columns for the CH2 group was also examined. The cation-exchange nature of the HILIC columns significantly influenced native silica columns and some polymer-modified columns. Herein, 45 commercially available HILIC columns were classified according to this method, and the results proved useful for understanding distinct separation characteristics of each HILIC column, enabling improved column selection.

Poly(acrylamide) (PAAm)-modified hydrophilic interaction chromatography (HILIC) columns were prepared via surface-initiated atom transfer radical polymerization (SI-ATRP) and free radical polymerization (FRP) to generate brush-like and mushroom-like polymer chains on silica particles, respectively. The maltose homologues (MHs) and cyclodextrins (CDs) were chosen as analytes to evaluate steric selectivity by the different polymer morphologies in the ATRP-PAAm and the FRP-PAAm columns. The ATRP-PAAm exhibited superior retention than the FRP-PAAm and three commercial HILIC columns. The house-made PAAm columns provided significant hydrophilicity that enabled to analysis the oligosaccharides even in 60:40 mixture of acetonitrile-aqueous buffer. In the case of three ATRP-PAAm columns characterized by different polymer lengths and the density on the silica particles, those are different thickness of the water-enriched layer, and phase ratio phi, based on hydrophilicity of them columns. The logarithm of the retention factor (ln k) displayed a non-linear dependence on the inverse of the temperature (1/T, T = 278-333 K). Notably, a similar correlation was observed to exist between the logarithm of the phase ratio (ln phi), and 1/T. A van't Hoff plot was used to determine the thermodynamic parameters of the partition process for each MH. The values of the Gibbs free energy (Delta G degrees) for the analytes partition on the ATRP-PAAm columns were smaller than their counterparts measured for the FRP-PAAm columns; by contrast, the opposite trend was observed for the Delta G degrees values measured for CDs. The standard entropy Delta S degrees for MHs and CDs were comparable for the two types PAAm columns, while, the standard enthalpy, Delta H degrees displays significant difference between the ATRP and the FRP PAAm columns. These findings indicate that the differences between PAAm morphology and polymer densities on the stationary phase surface affect analyte differentiation on the basis of molecular steric factors. The higher selectivity for MHs and CDs displayed by ATRP-PAAm columns with respect to their FRP-PAAm and commercial amide columns will be useful for the fine separation of oligosaccharides. (C) 2021 Elsevier B.V. All rights reserved.

A new commercially available HPLC column, poly-N-(1H-tetrazole-5-yl)-methacrylamide-bonded stationary phase (Daicel DCpak PTZ), was systematically evaluated for its carbohydrate isomer separation capability by hydrophilic interaction liquid chromatography (HILIC) with charged aerosol detection (CAD) or (tandem) mass spectrometry. Reducing sugars tend to split into two anomer peaks which makes carbohydrate isomer separations in non-derivatized form even more complicated. For practical purposes anomer separations are therefore ideally suppressed which can be accomplished by using high temperature or high pH that are both associated with fast interconversion kinetics leading to peak coalescence, or on the other hand by conditions with low chromatographic anomer selectivity. Four major hexoses (glucose, mannose, galactose, fructose), five main pentoses (ribose, ribulose, xylose, xylulose, arabinose) and five most important disaccharides (maltose, cellobiose, lactose, sucrose, trehalose) were analyzed as single carbohydrate standards by isocratic HILIC with 0.1% (v/v) formic acid and 2 mM ammonium acetate at various temperatures to study anomer interconversion equilibria in a pH-dependent manner. Rate constants of forward (alpha -> beta) and backward (beta -> alpha) anomerization and corresponding energy barriers were calculated. The energy barriers of anomerisation were in the range of around 83-91 kJ mol(-1) at 298 K and the difference between forward (alpha -> beta) and backward reaction (beta -> alpha) was typically between 1-3 kJ mol(-1). The systematic studies finally allowed to pick conditions for the simultaneous analysis of all 14 carbohydrates by HILIC-ESI-MS(/MS) with PTZ in gradient elution mode. A combination of carbohydrate isomer-selective LC (with PTZ), tandem MS (with carbohydrate group-selective MS1 and some species-specific SRM transitions) and a simple deconvolution strategy allowed the determination of all carbohydrates of the complex test mixture except for the disaccharide pair lactose and maltose (which can be determined as sum). Consequently, the proposed method represents a successful step towards a global glycometabolomics profiling method of mono- and disaccharides by HILIC-ESI-MS/MS. (C) 2020 Elsevier B.V. All rights reserved.

The role of individual functional groups has been assessed with regard to surface charge and chromatographic retention. Coatings were prepared from various fragments of the chiral zwitterionic materials Chiralpak ZWIX(+) and ZWIX(-). The different chromatographic ligands allowed fine tuning of the surface charge. Chiralpak ZWIX phases showed strongly negative zeta-potentials over the entire pH-range. Zwitterionic congeners with quinuclidine and sulfonic acid moieties but lacking the quinolone ring in the ligand structure exhibited shifted zeta-potentials of around + 5 to 20 mV depending on the surrounding residues. Capillary electrophoretic mobilitiy measurements with the chromatographic ligands and molecular dynamics simulations were carried out to offer some explanation of these surface charge differences of the distinct zwitterionic stationary phases. The new mixed-mode phases were also chromatographically characterized by simple RP and HILIC tests. The results allowed their positioning within a large variety of different commercially available RP, HILIC and mixed-mode phases, which were evaluated as well, by multivariate data processing using principal component analysis. The new mixed-mode phases overall exhibit reasonable hydrophilicity-lipophilicity balance and enable retention of ionic compounds by additional ionic interactions through weak anion-exchange (WAX-type), strong cation-exchange (SCX-type) or both (RP/ZWIX-type). Hence, the new RP/ZWIX phases can be flexible tools for selectivity tuning in RP and HILIC separations. (C) 2020 Elsevier B.V. All rights reserved.

Silica particles with various pore sizes were modified with poly(acrylamide) via surface-initiated atom-transfer radical polymerization (SI-ATRP) under different reaction conditions. Twenty different columns were prepared and characterized according to a test method for hydrophilic interaction chromatography (HILIC) columns. Hydrophilic retention by the SI-ATRP columns was much higher than that of poly(acrylamide) columns prepared via free-radical polymerization and many commercially available HILIC columns. The SI-ATRP columns displayed greater selectivity for -OH groups than any of the HILIC columns based on their α(U/2dU) values. SI-ATRP functionalization was used to increase the polymer chain density on the silica particles, which suggested a brush-type morphology, and improved hydrophilic selectivity. This indicated that hydrophilic retention and selectivity could be controlled by adjusting the morphology of the organic stationary phase. This stationary phase design strategy was validated experimentally by the effective separation of highly hydrophilic analytes. The findings of this study will greatly contribute to the creation of better separation media.

There is a huge, still increasing market for synthetic and therapeutic peptides. Their quality control is commonly based on a generic reversed-phase liquid chromatography (RPLC) method with C18 stationary phase and acetonitrile gradient with 0.1% trifluoroacetic acid in the mobile phase. It performs exceptionally well for a wide variety of impurities, yet structurally closely related impurities with similar sequences, not resolved in preparative RPLC, may easily coelute in the corresponding QC run as well. To address this problem an advanced generic 2D-LC impurity profiling method was developed in this work. It employs a selective comprehensive (high resolution sampling) RP×RP 2D-LC separation using a 100×2.1 mm ID column with the common acidic generic gradient in the first dimension, while RPLC under basic pH on a short 30×3 mm ID column is used in the second dimension. Recording data with a UV detector at 215 nm after 1D separation provides the common generic 1D chromatogram. However, after the 2D separation a flow splitter enabled recording of the signals of complementary detectors comprising a diode array detector (DAD) in-line with a charged aerosol detector (CAD) and a quadrupole-time-of-flight (QTOF) mass spectrometer (MS) with an electrospray ionization (ESI) source. Generic conditions of this 2D-LC method have been established through optimization of 2D stationary and mobile phase considering different pH values and buffer concentrations. The orthogonal separation principle has been documented by a number of therapeutic peptides including Exenatide, Octreotide, Cyclosporine A and Oxytocin as well as some other proprietary synthetic peptides. The information density can be further enhanced by using the QTOF-MS detector by data independent acquisition with SWATH. Through this sequential window acquisition of all theoretical fragment ion mass spectra it became possible to collect MS/MS data comprehensively in the high-resolution sampling window, thus enabling the extraction of 2D-EICs from fragment ions and the generation of 2D-contour plots of all product ions. Using Oxytocin as an example for an important therapeutic peptide, the ability of this advanced generic sRP-UV×RP-DAD-CAD-ESI-QTOF-MS/MS method with SWATH for peptide quality control is discussed.

A Poly(N-(1H-tetrazole-5-yl)methacrylamide)-bonded (PTZ) stationary phase has recently gained increased attention in hydrophilic liquid chromatography (HILIC) mode for separation of polar compounds and is now commercially available as DCpak PTZ. It is chromatographically characterized in this work. The property of the new column was proven to be the most hydrophilic and acidic by separation of mixtures of purines and pyrimidines (theophylline, theobromine, uridine and 2'-deoxyuridine) in comparison to other two commercial columns Luna HILIC and LiChrospher Diol column. The retention mechanism of the new column was investigated by design-of-experiment (DoE) approach using factorial design models. Water/acetonitrile ratio in the mobile phase, buffer salt concentration and buffer pH were considered factors employing the purine/pyrimidine mixture and glucose derivatives as test samples. The resultant retention model coefficients and contour plots documented the complementary retention and selectivity profiles of the new PTZ column as compared to Diol and Luna HILIC. Moreover, it became clearly evident that the PTZ column exhibits its best HILIC performance at eluent pH ≥ 5 because the NH group in tetrazolyl moiety is dissociated under these conditions. The applicability and great potential of the new HILIC column was proven by the chromatographic separation of complex mixtures of very hydrophilic glucose and glucose derivatives (sucrose, glucosamine, glucuronic acid, glucose-1-phosphate and trehalose, glucose, maltose, glucosamine-6-phosphate, glucose-6-phosphate and gluconic acid δ-lactone) as well as monosaccharides found in N-glycans. It is concluded that the new DCpak PTZ HILIC column could have good prospects for the separation of polar compounds e.g. in metabolomics and glycomics.

Applications of hydrophilic interaction chromatography for the analysis of biopharmaceutical drugs, i. e., glycosylated proteins represented by monoclonal antibodies are discussed in the manner of glycoproteomics. They can be analyzed using hydrophilic interaction chromatography in five different stages as (1) their intact forms, (2) their subunits, (3) N-and O-glycopeptides digested by proteases, (4) N-and O-glycans released from the glycoproteins or glycopeptides, and (5) monosaccharides. Hydrophilic interaction chromatography is a more useful tool in the order of (1) to (5). At the stages (4) and (5), quantitation of glycans and saccharides are also reported. Hydrophilic interaction chromatography is employed not only for analytical uses, but also pretreatment items as solid phase extraction, followed by reversed-phase liquid chromatography separations. Comprehensive search results of these application of hydrophilic interaction chromatography are summarized in tables to show what kind of hydrophilic interaction chromatography columns are suitable for each step of analysis. Relationship of favored and less favored hydrophilic interaction chromatography columns and their separation characteristics such as hydrophilicity, and selectivity for structural difference, is also discussed. Analysis of the therapeutic peptides (not glycosylated) using hydrophilic interaction chromatography is summarized, too.

8-Aminopyrene-1,3,6-trisulfonate (APTS) is one of the most frequently used reagent in capillary electrophoresis. Three sulfonate groups in APTS generate fast electrophoretic mobilities of derivatized glycans, therefore very suitable for CE-LIF applications. However, these groups also make separation with partition chromatography difficult. A novel column for hydrophilic interaction liquid chromatography (HILIC) with an anionic tetrazole functionalized polymer-based silica was examined for the separation of APTS-labeled glycans derived from specific glycoproteins. This separation mode has enhanced capability for the size resolution of neutral and acidic oligosaccharides. In addition, specific glycan isomers, which are usually difficult to separate with HILIC methods, were also separated. IgG-derived complex-type glycans that have an isomeric pair of monogalactosylated glycans, as well as differences in the number of galactose residues, are separable using this mode. We also utilized this column for the fractionation of APTS-labeled glycans from porcine thyroglobulin and examined their migration times with CE by co-migration with a mixture of glycoprotein glycans. Combinational modes of HILIC and anionic repulsion show promise for the separation and preparation of glycoprotein-derived glycans labeled with APTS.

A systematic method in hydrophilic interaction chromatography (HILIC) was developed for the separation of four monophosphate nucleotides using design of experiment (DOE) approaches. Three HPLC parameters, the buffer concentration (ammonium acetate concentration), gradient time, and temperature, were evaluated within the quality design framework, and the effects on chromatographic parameters were investigated. Four zwitterionic columns (ZIC-HILIC, ZIC-cHILIC, NUCLEODUR HILIC, and PC HILIC) were used to separate four nucleotides, and the HPLC conditions for each column were successfully optimized, although PC HILIC did not give peaks that were suitable for optimization. In addition, it was proved that optimized HPLC conditions differed from column to column even when the same types of zwitterionic sulfobetaine-functionalized columns were applied. This tendency was explained by differences in the separation characteristics of each column, the thickness of the water-enriched layer on the surface of the silica supports, and the pH. DOE for development of the HPLC method provides an effective explanation of the interactions among the variable parameters, especially in HILIC mode. Finally, a robust analytical method could be established by setting the optimum parameters. Among the employed columns, ZIC-cHILIC provided the widest range of suitable analytical conditions. NUCLEODUR HILIC was difficult to build a robust analytical method since the elution order of cytidine monophosphate and guanosine monophosphate was reversed.

Continuous rapid-flow electron spin resonance (ESR) was used to detect poly(vinyl alcohol) (PVA)-derived carbon-centered radicals, in order to characterize the short-lived intermediate radicals in the initial stage of the graft polymerization of PVA. ESR spectra were recorded by mixing PVA, hydrogen peroxide, and Ti2(SO4)3 at pH 2.0. The observed spectrum indicated three paramagnetic species: a minor species A, and two major species B1, and B2. Based on the observed proton hyperfine coupling constant (hfcc) values, species A was assigned to the carbon-centered radical of PVA produced by hydrogen atom abstraction from the methine carbon bearing a hydroxyl group. Species B1 and B2 were ascribed to two structural isomers of the PVA-derived, carbonyl-conjugated radical produced by acid-catalyzed radical reaction at the vicinal diol moiety (i.e., head-to-head structure) of PVA. Spin-trapping ESR measurements were performed at 80 °C using a water-soluble spin-trapping reagent 3,5-dibromo-4-nitroso-benzenesulfon...

Based on the results of rapid-flow electron spin resonance (RF-ESR) and spin-trapping (ST-ESR) measurements, carbonyl-conjugated radicals derived from the vicinal diol moiety (2, i.e., the head-to-head structure) of poly(vinyl alcohol) (PVA) have been proposed to be the most probable intermediate species involved in the initial stages of the graft reaction of PVA with methyl methacrylate (MMA). A modified poly(vinyl alcohol) (PPVA) bearing a 1,2-propanediol pendant moiety (3) with a molar ratio of 8% (based on the monomer unit) was prepared to clarify the role of the carbonyl-conjugated radicals of PPVA in the graft reaction with MMA. The RF-ESR spectra observed for the mixtures composed of PPVA, hydrogen peroxide (HPO) or ammonium persulfate (APS), and Ti2(SO4)3 (pH ca. 2) revealed the formation of four radical species (A, B1, B2, and C) derived from PPVA. By comparison of the RF-ESR spectra observed for PPVA and PVA, species A (g = 2.0041) was confirmed to be the pendant derived radical of PPVA, and spe...

Monolithic silica columns have greater (through-pore size)/(skeleton size) ratios than particulate columns and fixed support structures in a column for chemical modification, resulting in high-efficiency columns and stationary phases. This review looks at how the size range of monolithic silica columns has been expanded, how high-efficiency monolithic silica columns have been realized, and how various methods of silica surface functionalization, leading to selective stationary phases, have been developed on monolithic silica supports, and provides information on the current status of these columns. Also discussed are the practical aspects of monolithic silica columns, including how their versatility can be improved by the preparation of small-sized structural features (sub-micron) and columns (1 mm ID or smaller) and by optimizing reaction conditions for in situ chemical modification with various restrictions, with an emphasis on recent research results for both topics.

Separation of diastereomers of DL--tocopherol was studied by reversed-phase liquid chromatography using three types of stationary phases, polymeric ODS, polymeric C30, and monomeric ODS. Polymeric ODS stationary phase (Inertsil ODS-P, 3 mmID, 20 cm) was effective for the separation of the isomers created by the presence of three chiral centers on the alkyl chain of synthetic DL--tocopherol. Considerable improvement of the separation of isomers was observed on ODS-P phase at high pressure and at low temperature. Complete separation of four pairs of diastereomers was achieved at 12.0 oC, 536 bar, while three peaks were observed when the separation was carried out either at 12.0 oC at low pressure or at 20 oC at 488 bar. Higher temperature (30.0 oC) with the ODS-P phase resulted in only partial separation of the diastereomers even at high pressure. Only slight resolution was observed for the mixture of diastereomers with the C30 stationary phase (Inertsil C30) at 12.0 oC and 441 bar, although the stationary phase afforded greater resolution for and -tocopherol than ODS-P. A monomeric C18 stationary phase did not show any separation at 12.0 oC and 463 bar. The results suggest that the binding site of the polymeric ODS-P phase is selective for flexible alkyl chains that provided the longest retention for the natural form, (R,R,R) form, and the enantiomer, (S,S,S) form, of DL--tocopherol.

Separation of diastereomers of dl-α-tocopherol was studied by reversed-phase liquid chromatography using three types of stationary phases, polymeric ODS, polymeric C30, and monomeric ODS. Polymeric ODS stationary phase (Inertsil ODS-P, 3mmID, 20cm) was effective for the separation of the isomers created by the presence of three chiral centers on the alkyl chain of synthetic dl-α-tocopherol. Considerable improvement of the separation of isomers was observed on ODS-P phase at high pressure and at low temperature. Complete separation of four pairs of diastereomers was achieved at 12.0°C, 536bar, while three peaks were observed when the separation was carried out either at 12.0°C at low pressure or at 20°C at 488bar. Higher temperature (30.0°C) with the ODS-P phase resulted in only partial separation of the diastereomers even at high pressure. Only slight resolution was observed for the mixture of diastereomers with the C30 stationary phase (Inertsil C30) at 12.0°C and 441bar, although the stationary phase afforded greater resolution for β- and γ-tocopherol than ODS-P. A monomeric C18 stationary phase did not show any separation at 12.0°C and 463bar. The results suggest that the binding site of the polymeric ODS-P phase is selective for flexible alkyl chains that provided the longest retention for the natural form, (R,R,R) form, and the enantiomer, (S,S,S) form, of dl-α-tocopherol.

Fast Liquid Chromatography–Mass Spectrometry Methods in Food and Environmental Analysis, pp. 57-107 (2015) No AccessChapter 3: Monolithic Columns in Fast Liquid ChromatographyTakeshi Hara, Oscar Núñez, Tohru Ikegami, and Nobuo TanakaTakeshi HaraDepartment of Chemical Engineering, Vrije Universiteit Brussel, Belgium, Oscar NúñezDepartment of Analytical Chemistry, University of Barcelona, Spain, Tohru IkegamiDepartment of Biomolecular Engineering, Kyoto Institute of Technology, Japan, and Nobuo TanakaGL Sciences Inc., JapanUniversity of California, Davis, USAhttps://doi.org/10.1142/9781783264940_0003Cited by:0 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: The following sections are included: Features of Monolithic Silica Columns: Rapid Separation Using Monolithic Silica Columns in Rod and Capillary Formats Fabrication of columns The support structure regarding through-pores, skeletons, and amount of silica in a column Column permeability, column efficiency, and improvement of preparation method Current performance of monolithic silica columns Kinetic performance Functionalization of monolithic silica columns Advantages and disadvantages: roles of monolithic silica columns Features of Organic Polymer Monolithic Columns Food and Environmental Applications Summary and Conclusions References FiguresReferencesRelatedDetails Fast Liquid Chromatography–Mass Spectrometry Methods in Food and Environmental AnalysisMetrics History PDF download

The enantioselectivity of immobilized β-cyclodextrin phenyl carbamate-based silica monolithic capillary columns was compared to our previously described polymer counterpart. 2,3,6-Tris(phenylcarbamoyl)-β-cyclodextrin-6-methacrylate was used as a functional monomer for the preparation of β-cyclodextrin (β-CD)-based silica and polymer monoliths. The silica monoliths were prepared via the sol–gel technique in fused silica capillary followed by modification of the bare silica monoliths with an anchor group prior to polymerization with β-CD methacrylate using either 2,2′-azobis(isobutyronitrile) or benzoylperoxide as radical initiators. On the other hand, the polymer monoliths were prepared via the copolymerization of β-CD methacrylate and ethylene glycol dimethacrylate in different ratios in situ in fused silica capillary. The prepared silica/polymer monoliths were investigated for the chiral separation of different classes of pharmaceuticals namely; α- and β-blockers, anti-inflammatory drugs, antifungal drugs, dopamine antagonists, norepinephrine-dopamine reuptake inhibitors, catecholamines, sedative hypnotics, diuretics, antihistaminics, anticancer drugs and antiarrhythmic drugs. Baseline separation was achieved for alprenolol, bufuralol, carbuterol, cizolertine, desmethylcizolertine, eticlopride, ifosfamide, 1-indanol, propranolol, tebuconazole, tertatolol and o-methoxymandelic acid under reversed phase conditions using mobile phase composed of methanol and water. The silica-based monoliths showed a comparative enantioselectivity to the polymer monoliths.

A meter-scale monolithic silica capillary column modified with urea-functional groups for hydrophilic interaction liquid chromatography (HILIC) was developed for highly efficient separation of biological compounds. We prepared a ureidopropylsilylated monolithic silica capillary column with a minimum plate height of 12 μm for nucleosides and a permeability of 2.1 × 10(-13) m(2), which is comparable with the parameters of monolithic silica-C18 capillary columns. Over 300,000 theoretical plates were experimentally obtained in HILIC with a 4 m long column at 8 MPa; this is the best result yet reported for HILIC. A 2 m long ureidopropylsilylated monolithic silica capillary column was utilized to develop a HILIC mode LC-MS system for proteomics applications. Using tryptic peptides from human HeLa cell lysate proteins, we identified the comparable numbers of peptides and proteins in HILIC with those in reversed-phase liquid chromatography (RPLC) using a C18-modified monolithic silica column when shallow gradients were applied. In addition, approximately 5-fold increase in the peak response on average was observed in HILIC for commonly identified tryptic peptides due to the high acetonitrile concentration in the HILIC mobile phase. Since HILIC mode LC-MS shows orthogonal selectivity to RPLC mode LC-MS, it is useful as a complementary tool to increase proteome coverage in proteomics studies.

•Higher pressure was shown to increase selectivity between isomers in RPLC.•Pressure effect was observed for several types of compounds on two RPLC columns.•Polymeric C18 and C30 were useful for isomer separation especially at high pressure.•ODS-P was effective for both rigid and flexible solutes while C30 for rigid solutes.•Opposite elution orders were observed on the two columns in some cases.