Hydrophilic Interaction Liquid Chromatography Conditions Research Articles

Evaluating orthogonality between ion-pair reversed phase, anion exchange, and hydrophilic interaction liquid chromatography for the separation of synthetic oligonucleotides

The orthogonality of separation between ion-pair reversed phase (IP-RP), anion exchange (AEX), and hydrophilic interaction liquid chromatography (HILIC) was evaluated for oligonucleotides. A polythymidine standard ladder was first used to evaluate the three methods and showed zero orthogonality, where retention and selectivity were based on oligonucleotide charge/size under all three conditions. Next, a model 23-mer synthetic oligonucleotide containing 4 phosphorothioate bonds with 2′ fluoro and 2′-O-methyl ribose modifications typical of small interfering RNA was used for evaluating orthogonality. The resolution and orthogonality were evaluated between the three modes of chromatography in terms of selectivity differences for nine common impurities, including truncations (n-1, n-2), addition (n + 1), oxidation, and de-fluorination. We first evaluated different ion-pairing reagents that provided the best separation of the key impurities while suppressing diastereomer separation due to phosphorothioate linkages. Although different ion-pairing reagents affected resolution, very little orthogonality was observed. We then compared the retention times between IP-RP, HILIC, and AEX for each impurity of the model oligonucleotide and observed various selectivity changes. The results suggest that coupling HILIC with either AEX or IP-RP provide the highest degree of orthogonality due to the differences in retention for hydrophilic nucleobases and modifications under HILIC conditions. IP-RP provided the highest overall resolution for the impurity mixture, whereas more co-elution was observed with HILIC and AEX. The unique selectivity patterns offered by HILIC provides an interesting alternative to IP-RP or AEX, in addition to the potential for coupling with multidimensional separations. Future work should explore orthogonality for oligonucleotides with subtle sequence differences such as nucleobase modifications and base flip isomers, longer strands such as guide RNA and messenger RNA, and other biotherapeutic modalities such as peptides, antibodies, and antibody-drug-conjugates.

Influence of ion-pairing reagents on the separation of intact glycoproteins using hydrophilic-interaction liquid chromatography - high-resolution mass spectrometry

Hydrophilic-interaction liquid chromatography (HILIC) of intact proteins offers high-resolution separations of glycoforms of glycoproteins differing in the number of (neutral) glycans. However, to obtain efficient separations it is essential that the positively charged sites of the proteins are shielded by acidic (negative) ion-pair reagents (IPRs), so as to enhance the contribution of the hydroxyl groups of the (neutral) sugars in the glycoprotein. Here, we studied the influence of various IPRs that differ in physico-chemical properties, such as hydrophobicity and acidity, on the capillary-scale HILIC separation of intact (glyco)proteins. We evaluated the use of fluoroacetic acid (MFA), difluoroacetic acid (DFA), trifluoroacetic acid (TFA), and heptafluorobutyric acid (HFBA) as diluents for sample preparation, as solvents for sample loading on a reversed-phase trap prior to the HILIC separation, and as mobile-phase components for HILIC and HILIC-MS. To reduce the contribution of ion-exchange interaction with the (silica-based) stationary phase, we used an acrylamide-based monolithic column. We studied the influence of the different IPRs on each step of the separation of a mixture of proteins of different size and hydrophilicity and on the separation of the five glycoforms of ribonuclease B. The content of IPR in the sample was shown not to affect the separation and the MS detection. However, a low content of TFA and DFA in the mobile phase is favourable, as it reduces adduct formation and leads to higher signal intensity. The optimized HILIC conditions successfully resolved nine major glycoforms groups of a ∼40 kDa glycoprotein horseradish peroxidase (HRP), as an example of a complex glycoprotein.

Sub/supercritical fluid chromatography versus liquid chromatography for peptide analysis

The aim of this study was to evaluate the potential of ultra-high performance supercritical fluid chromatography (UHPSFC) for peptide analysis by comparing its analytical performance to several chromatographic approaches based on reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC) and mixed-mode liquid chromatography. First, the retention behavior of synthetic peptides with 3 to 30 amino acids and different isoelectric points (acid, neutral, and basic) was evaluated. For all the tested conditions (13 peptides in 8 conditions), only 4 results were not exploitable (not retained or not eluted), confirming that all the tested chromatographic conditions can be successfully applied when analyzing a wide range of diverse peptides. Average tailing factor were quite comparable across all chromatographic modes, while the best peak capacity values were obtained under mixed-mode LC conditions. Selectivity for each chromatographic mode was also evaluated for six closely related peptides having minor modifications on their structures. The LC-based chromatographic modes confirmed their superior selectivity over UHPSFC. By contrast, when analyzing short peptides (di- or tripetides), UHPSFC was the only technique allowing to simultaneously separate highly polar and less polar peptides within the same run confirming its unique versatility. In addition, the sensitivity of each chromatographic approach was accessed by for two representative peptides by both UV and MS detection. With UV detection, limit of detection (LOD) values were comparable among the different chromatographic modes, ranging from 0.5 to 2 µg mL−1. However, major differences were found when employing MS detection (LOD values ranged from 0.05 to 5 µg mL−1). The best results were obtained under HILIC conditions, followed by SFC, and finally mixed-mode LC and RPLC modes.

Separation of labeled isomeric oligosaccharides by hydrophilic interaction liquid chromatography – the role of organic solvent in manipulating separation selectivity of the amide stationary phase

The advantages of using mixtures of organic solvents for the separation of labeled oligosaccharides on the amide stationary phase under hydrophilic interaction liquid chromatography conditions are presented. The effect of the type of buffer as well as solvent or their mixtures on retention of uracil, saccharide labeling reagents (2-aminobenzoic acid, 2-aminobenzamide, ethyl 4-aminobenzoate, procainamide), and corresponding labeled saccharides were evaluated. The successful isocratic separation of labeled isomeric trisaccharides (maltotriose, panose, and isomaltotriose) was achieved in the mobile phase consisting of a 90% (v/v) mixture of organic solvents (methanol/acetonitrile 60:40) and 10% (v/v) 30 mM ammonium formate, pH 3.3. Changing the volume ratio between methanol/acetonitrile from 60:40 to 50:50 (v/v) allowed to obtain the separation of di-, tri-, and tetrasaccharides labeled by ethyl 4-aminobenzoate in less than 10.5 min.

Effect of Oxime Encapsulation on Acetylcholinesterase Reactivation: Pharmacokinetic Study of the Asoxime-Cucurbit[7]uril Complex in Mice Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry.

Oxime-based molecules are used for the treatment of patients to reactivate acetylcholinesterase (AChE) function after organophosphate intoxication. However, their efficacy is limited by low penetration through the blood-brain barrier and fast elimination. In this work, the cucurbit[7]uril (CB[7]) carrier was used for the encapsulation of the clinical agent asoxime to enhance brain bioavailability and the treatment window. We present a pharmacokinetic study of asoxime and the asoxime-CB[7] complex in an in vivo mouse model. Ultrahigh-performance liquid chromatography with electrospray ionization-mass spectrometry detection was developed to determine asoxime and CB[7] in biological fluids and tissues after thorough optimization of chromatographic conditions. The dihydroxypropane-silica stationary phase using hydrophilic interaction liquid chromatography conditions provided the best chromatographic performance. The final method was validated and applied for the pharmacokinetic study of mouse plasma, urine, bile, liver, kidney, and brain samples at different times after administration of asoxime and the asoxime-CB[7] complex. The results showed a greater than 3-fold increase in the area under the curve (AUC) in the brain for asoxime administered as a complex with CB[7] relative to that for the administration of asoxime alone. The effectiveness of the treatment strategy was evaluated using a reactivation study and a functional observatory battery. Protection of brain AChE activity is crucial for saving human lives or reducing the consequences of poisoning. The asoxime administered as a complex increased the brain activity by approximately 30% compared to that with atropine alone. CB[7] coadministration improved the AChE activity by 11%, which agrees with the higher asoxime AUC assessed in the pharmacokinetic study.

Study of enantioseparation of β-blockers using amylose tris(3-chloro-5-methylphenylcarbamate) as chiral stationary phase under polar-organic, reversed-phase and hydrophilic interaction liquid chromatography conditions

In this study, we describe the experimental variables influencing enantioseparation of twelve β-blockers when analyzed under polar-organic, reversed-phase and hydrophilic interaction liquid chromatography conditions on a column with immobilized amylose tris(3-chloro-5-methylphenylcarbamate) as chiral stationary phase. Regarding polar-organic mode, two component mobile phases consisting of methanol, ethanol or acetonitrile with the addition of basic additives such as diethylamine, triethylamine, mono-ethanolamine, ethylendiamine or trifluoroacetic acid/diethylamine mixture were evaluated. Studies of retention at different temperatures were also performed. In reversed-phase mode, mixtures consisting of methanol or acetonitrile with either aqueous boric acid-borate buffer or sodium hydrogen carbonate-carbonate buffer solutions were compared aiming to study the influence of organic modifier as well as buffer type and pH on resolution. In addition, a systematic evaluation of the retention factors of β-blockers enantiomers in hydro-organic eluents containing acetonitrile in presence of diethylamine as additive was carried out by increasing progressively the water content, in order to check for retention dependencies indicative of the interplay of both hydrophilic interaction liquid chromatography and reversed-phase modes.

Hydrophilic interaction liquid chromatography coupled to quadrupole time-of-flight mass spectrometry as a potential combination for the determination of sulfonamide residues in complex infant formula matrices

An accurate, sensitive and selective analytical method is proposed for sulfonamide residues analysis in infant formulas based on hydrophilic interaction liquid chromatography (HILIC) and quadrupole time-of-flight mass spectrometry in full scan mode. The sample preparation approach involves low-temperature lipid precipitation followed by dispersive solid-phase extraction with PSA and C18 sorbents, which was successfully optimized using Plackett-Burman design. In order to achieve high analytical sensitivity, the influence of HILIC conditions on sulfonamide ionization was investigated, such as the mobile phase composition, buffer concentration, and sample diluent for injection. The method performance characteristics, including linearity (range 5–120 µg kg–1), reliable limits of quantification (between 5 and 20 µg kg–1), recovery (72.9–109.2%) and precision (coefficient of variation values ≤ 19.8%) under repeatability and within-laboratory reproducibility conditions, were in accordance with the Codex Alimentarius Commission CAC/GL 71–2009 for quantitative analytical methods for veterinary drug residues in foods. Moreover, adequate identification of the compounds was provided with accurate mass measurement of both precursor and fragment ions in one single run. Finally, the developed method was applied to thirty-five powdered milk-based infant formula samples available in the Brazilian market.

Controlled Fabrication of Silica@Covalent Triazine Polymer Core-Shell Spheres as a Reversed-Phase/Hydrophilic Interaction Mixed-Mode Chromatographic Stationary Phase.

The unique properties of covalent triazine-based organic framework/polymers, including large surface area, hydrophilic-lipophilic-balanced adsorption, and economical preparation, make it a promising candidate as a stationary phase for high-performance liquid chromatography. However, irregular shapes and wide size distributions of such particles hinder column packing, resulting in a low column efficiency or a high back pressure. Herein, we describe the fabrication of SiO2@ covalent triazine-based organic polymer (CTP) core-shell microspheres with a distinct sphere-coating-sphere appearance using aminosilica as the supporting substrate to grow the CTP shell. By adjusting the amount of reactants, the thickness of the CTP shell, which consists of triazine and 1,3,5-triphenylbenzene monomers, was easily controlled. The developed core-shell microspheres were characterized via scanning electron microscopy, energy dispersive X-ray spectroscopy, transmission electron microscopy, solid-state 13C nuclear magnetic resonance analysis, and N2 adsorption experiments. The synergism of the triazine and aromatic moieties on CTP provides the new stationary phase with multiple retention mechanisms, including hydrophobic, π-π, electron donor-acceptor, hydrogen-bonding interactions, and so forth. On the basis of these interactions, successful separation and higher shape selectivity were achieved among several analytes that vary in polarity under both reversed-phase and hydrophilic interaction liquid chromatography conditions. Therefore, SiO2@CTP microspheres combine the advantages of good column packing properties of the uniform monodisperse silica microspheres and the recognition performance of CTP, generating flexible selectivity and application prospect for both hydrophilic and hydrophobic analytes.

Hydrophilic Interaction Chromatography

Hydrophilic interaction liquid chromatography (HILIC), which includes the distribution, adsorption, ion exchange, and exclusion of analytes, has gained active development recently. The review covers the principles of the method, types of stationary and mobile phases, mechanisms of retention of highly polar compounds under HILIC conditions, implementation of 2D chromatography, and also fields of application of the method. Keywords: hydrophilic chromatography, retention mechanism, reverse phase HPLC, normal phase mode, HILIC stationary phases, zwitterionic phases, high polar analytes

Insights into the Mechanism of Separation of Bisphosphonates by Zwitterionic Hydrophilic Interaction Liquid Chromatography: Application to the Quantitation of Risedronate in Pharmaceuticals

Bisphosphonates are used to treat various skeletal disorders, as they modulate bone metabolism by inhibition of the osteoclast-mediated bone resorption. These compounds are both polar and ionic, and therefore, by using reversed phase liquid chromatography are eluted rapidly. Hydrophilic interaction liquid chromatography (HILIC) is an advantageous technique for the separation and analysis of polar molecules. As the elution order in HILIC is reversed to reversed phase liquid chromatography, a reasonable retention and selectivity for polar compounds is expected. In this work the retention mechanism of three bisphosponates, namely risedronate, tiludronate and zoledronate, was investigated under zwitterionic HILIC conditions. The key factors influencing the retention of the analytes on a zwitterionic ZIC®-pHILIC column (150.0 × 2.1 mm i.d., 200 Å, 3.5 μm) have been systematically investigated. It was found that apart from partition, electrostatic repulsions play an important role in the retention of bisphosphonates. Peak tailing of risedronate and zoledronate was improved by the addition of sodium pyrophosphate in the mobile phase. A zwitterionic hydrophilic interaction liquid chromatography-photodiode array (HILIC-PDA) method was further optimized and fully validated to quantitate risedronate in commercial film-coated tablets. The calibration curves for risedronate showed good linearity (r > 0.9991) within the calibration range tested. The intra- and inter-day coefficient of variation (CV) values was less than 0.6%, while the relative percentage error (%Er) was less than −2.3%. Accelerated stability studies of risedronate conducted under several degradation conditions including hydrolysis, oxidation and heat demonstrated the selectivity of the procedure. A short-run analysis of not more than 6 min allowed the analysis of large samples per day. The applicability of the method for the quantitation of risedronate was demonstrated via the analysis of commercial tablets containing this compound.

HPLC Enantioseparations with Polysaccharide-Based Chiral Stationary Phases in HILIC Conditions.

In contrast to achiral hydrophilic interaction liquid chromatography (HILIC), which is a popular and largely applied technique to analyze polar compounds such as pharmaceuticals, metabolites, proteins, peptides, amino acids, oligonucleotides, and carbohydrates, the introduction of the HILIC concept in enantioselective chromatography has been relatively recent and scarcely debated. In this chapter, the HILIC enantioseparations carried out on polysaccharide-based chiral stationary phases are grouped and discussed. Another objective of this chapter is to provide a comprehensive overview and insight into the experimental conditions needed to operate under HILIC mode. Finally, to stimulate and facilitate the application of this chromatographic technique, a detailed experimental protocol of a chiral resolution on a chlorinated cellulose-based chiral stationary phase under HILIC conditions is described.

Synthesis and evaluation of a pentafluorobenzamide stationary phase for HPLC separations in the reversed phase and hydrophilic interaction modes.

A pentafluorobenzamide stationary phase was synthesized by an easy method with no intermediate purification steps. Physicochemical characterization (elemental analysis, fourier transform infrared spectroscopy, 29 Si and 13 C nuclear magnetic resonance spectroscopy) confirmed the presence of pentafluorobenzamide functionalization on the surface of the silica particles. The pentafluorobenzamide stationary phase proved to be quite versatile as it can be used in two different modes in liquid chromatography: reversed phase and hydrophilic interaction liquid chromatography. Chromatographic characterizations in both modes confirmed the multiple interactions established by the new stationary phase, such as hydrogen bonding and π-π and ion-exchange interactions. The pentafluorobenzamide stationary phase was successfully employed for the separation of nucleosides and antihypertensive drugs under hydrophilic interaction liquid chromatography conditions, as well as pesticides and benzodiazepine using reversed phase conditions. The stationary phase showed significant potential when compared with commercial columns.

Assessment of intra-particle diffusion in hydrophilic interaction liquid chromatography and reversed-phase liquid chromatography under conditions of identical packing structure

A recently developed stripping protocol to completely remove the stationary phase of reversed-phase liquid chromatography (RPLC) columns and turn them into hydrophilic interaction liquid chromatography (HILIC) columns with identical packing characteristics is used to study the underlying mechanisms of intra-particle diffusion in RPLC and HILIC. The protocol is applied to a column with a large geometrical volume (250×4.6mm, 5μm) to avoid extra-column effects and for compounds with a broad range in retention factors (k” from ∼0.6 to 8). Three types of behavior for the intra-particle diffusion (Dpart/Dm) in RPLC versus HILIC can be distinguished: for nearly unretained compounds (k”<0.6), intra-particle diffusion in HILIC is larger than in RPLC; for compounds with intermediate retention behavior (k”∼0.9–1.2), intra-particle diffusion in HILIC and RPLC are similar; and for well retained compounds (k”>1.8), intra-particle diffusion in RPLC is larger than in HILIC. To explain these observations, diffusion in the stationary phase (γsDs) and in the stagnant mobile phase in the mesopore zone (γmpDm) are deduced from experimentally determined values of the intra-particle diffusion, using models derived from the Effective Medium Theory. It is demonstrated that the larger intra-particle diffusion obtained for slightly retained compounds under HILIC conditions is caused by the higher mesopore diffusion in HILIC (γmp=0.474 for HILIC versus 0.435 for RPLC), while the larger intra-particle diffusion obtained for strongly retained compounds under RPLC conditions can be related to the much higher stationary phase diffusion in RPLC (γsDs/Dm=0.200 for RPLC versus 0.113 for HILIC).

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

Hydrophilic interaction liquid chromatography (HILIC) is a well-established technique for the separation and analysis of small polar compounds. A recently introduced widepore stationary phase expanded HILIC applications to larger molecules, such as therapeutic proteins. In this paper, we present some generic HILIC conditions adapted for a wide range of FDA and EMA approved recombinant monoclonal antibody (mAb) species and for an antibody-drug conjugate (ADC). Seven approved mAbs possessing various isoelectric point (pI) and hydrophobicity as well as a cysteine conjugated ADC were used in this study. Samples were digested by IdeS enzyme and digests were further fragmented by chemical reduction. The resulting fragments were separated by HILIC. The main benefit of HILIC was the separation of polar variants (glycovariants) in a reasonable analysis time at the protein level, which is not feasible with other chromatographic modes. Three samples were selected and chromatographic conditions were further optimized to maximize resolution. A commercial software was used to build up retention models. Experimental and predicted chromatograms showed good agreement and the average error of retention time prediction was less than 2%. Recovery of various species and sample stability under the applied conditions were also discussed.

Separation of substrates and closely related glucuronide metabolites using various chromatographic modes

The aim of this study was to assess the retention and selectivity of a cocktail of 10 substrates of uridine diphosphate glucuronosyltransferase enzymes (UGTs) and their respective glucuronides using four chromatographic approaches. For this purpose, seven different stationary phases were employed in reversed phase liquid chromatography (RPLC), two in hydrophilic interaction liquid chromatography (HILIC), one in aqueous normal phase chromatography (ANPC) and four in subcritical fluid chromatography (SFC). Highly orthogonal separations were achieved with these chromatographic modes. Hydrophobic interactions mainly governed the retention of the substrates and their polar glucuronides in RPLC despite the use of different chemical stationary phase bonding, involving additional possible interactions. In ANPC, atypical separations and poor peak shapes were observed with the selected compounds. In HILIC and SFC conditions, the metabolites were more retained than the substrates because of the polarity increase related to the glucuronic acid moiety. For the latter, a very high proportion of organic solvent (up to 80%) was required to elute the glucuronides that often displayed poor peak shapes. Finally, the selectivity of nine chromatographic systems was compared for the separation of isomeric and diastereoisomeric compounds. The stationary phases used in RPLC mode were more selective towards the two positional isomers of morphine glucuronides since they possess distinct lipophilicity. HILIC and SFC columns were found to be promising for the separation of a critical diastereoisomers pair, namely epitestosterone-glucuronide and testosterone-glucuronide.

Retention Study of Flavonoids Under Different Chromatographic Modes.

The goal of this study was to investigate the chromatographic behavior of selected flavonoids from their different subgroups (flavonols, flavanones, flavones and isoflavones) in hydrophilic interaction liquid chromatography (HILIC). Chromatographic measurements were made on two different HILIC columns: cross-linked DIOL (Luna HILIC) and zwitterionic sulfoalkylbetaine (SeQuant ZIC-HILIC). Separation parameters such as the content of acetonitrile and pH of an eluent were studied. On the ZIC column, the retention factors of flavonoids increased with decreasing water content in the mobile phase. The increase in pH of the aqueous component mainly affects the polarity of the analytes. DIOL stationary phase shows more or less apparent dual retention mechanism, HILIC at the acetonitrile (ACN) content ≥75% and reversed phase (RP) with lower content of organic modifier. In the presence of ammonium acetate in the mobile phase, the retention of flavonoids onto the DIOL column increases without change in the selectivity of the separations. The similar effect, but considerably smaller was observed for aglycones on the ZIC column. The retention of studied glycosides (hesperidin, rutin) decreases in the presence of salt in the mobile phase. The significantly higher mass spectrometry sensitivity was observed under HILIC conditions in comparison with the most often used RP LC due to much higher content of ACN in the mobile phase. Finally, under optimal chromatographic conditions, the method was validated and applied for the determination of flavonoids in chamomile (Matricaria chamomilla L.) infusion.

Evaluation of the Kinetic Performance Differences between Hydrophilic-Interaction Liquid Chromatography and Reversed-Phase Liquid Chromatography under Conditions of Identical Packing Structure.

A protocol using trifluoroacetic acid at a temperature of 60 °C is developed for the adequate removal of the stationary phase of reversed-phase liquid chromatography (RPLC) columns. This procedure allows for studying the same column first under RPLC and subsequently under hydrophilic-interaction liquid chromatography (HILIC) conditions to isolate intrinsic differences between mass transfer properties in HILIC and RPLC from differences in packing quality. The established procedure allows for a complete removal of the stationary phase (confirmed by retention studies and thermogravimetry analyses) while leaving the structure of the packing unaffected (witnessed by an unchanged external porosity and pressure drop). Accurate plate height analysis comparing compounds at the same zone retention factor indicates a significant difference in reduced c-term (typically 40-80% larger under HILIC conditions), despite the columns otherwise being identical. Correcting for the known contributions of longitudinal diffusion (b-term) and mass transfer (cm- and cs-term) to focus on band broadening originating from eddy dispersion, similar strong differences are observed (differences of some h = 0.3 up to 1.2). These findings show that the interior structure and retention mechanism of the particles have a very strong effect on the observed eddy dispersion, a factor typically ascribed to phenomena occurring outside the particles. This also implies that comparing the quality of packings of different particle types is virtually impossible without the availability of a sound model to correct for the intraparticle effect on the observed eddy dispersion.

Ultra-fast high-efficiency enantioseparations by means of a teicoplanin-based chiral stationary phase made on sub-2 μm totally porous silica particles of narrow size distribution

A new ultra-high performance teicoplanin-based stationary phase was prepared starting from sub-2μm totally porous silica particles of narrow size distribution. Columns of different lengths were packed at high pressure and a deep and systematic evaluation of kinetic performance, in terms of van Deemter analysis, was performed under different elution conditions (HILIC, POM, RP and NP) by using both achiral and chiral probes. For the achiral probes, the efficiency of the columns at the minimum of the van Deemter curves were very high leading to some 278000, 270000, 262000 and 232000plates/m in hydrophilic interaction liquid chromatography (HILIC), polar organic mode (POM), normal phase (NP) and reversed phase (RP) respectively. The lowest plate height, Hmin=3.59μm (h(/)=1.89), was obtained under HILIC conditions at a flow rate of 1.4mL/min. Efficiency as high as 200000–250000plates/m (at the optimum flow rate) was obtained in the separation of the enantiomers of chiral probes under HILIC/POM conditions. N-protected amino acids, α-aryloxy acids, herbicides, anti-inflammatory agents were baseline separated on short (2-cm) and ultra-short (1-cm) columns, with analysis time in the order of 1min. The enantiomers of N-BOC-d,l-methionine were successfully baseline separated in only 11s in HILIC mode. Several examples of fast and efficient resolutions in sub/supercritical fluid chromatography were also obtained for a range of chiral carboxylic acids.

Preparation, characterization and application of a reversed phase liquid chromatography/hydrophilic interaction chromatography mixed-mode C18-DTT stationary phase

A mixed-mode chromatographic stationary phase, C18-DTT (dithiothreitol) silica (SiO2) was prepared through “thiol-ene” click chemistry. The obtained material was characterized by fourier transform infrared spectroscope, nitrogen adsorption analysis and contact angle analysis. Chromatographic performance of the C18-DTT was systemically evaluated by studying the effect of acetonitrile content, pH, buffer concentration of the mobile phase and column temperature. It was demonstrated that the novel stationary phase possessed reversed phase liquid chromatography (RPLC)/hydrophilic interaction liquid chromatography (HILIC) mixed-mode property. The stop-flow test revealed that C18-DTT exhibited excellent compatibility with 100% aqueous mobile phase. Additionally, the stability and column-to-column reproducibility of the C18-DTT material were satisfactory, with relative standard deviations of retention factor of the tested analytes (verapamil, fenbufen, guanine, tetrandrine and nicotinic acid) in the range of 1.82–3.72% and 0.85–1.93%, respectively. Finally, the application of C18-DTT column was demonstrated in the separation of non-steroidal anti-inflammatory drugs, aromatic carboxylic acids, alkaloids, nucleo-analytes and polycyclic aromatic hydrocarbons. It had great resolving power in the analysis of various compounds in HILIC and RPLC chromatographic conditions and was a promising RPLC/HILIC mixed-mode stationary phase.

Evaluation of the Effects of Sample Dilution and Volume in Hydrophilic Interaction Liquid Chromatography

The effects of sample dilution and volume on the peak shapes in hydrophilic interaction liquid chromatography (HILIC) were evaluated using fluorescence-labeled thiols as model compounds and ZIC-HILIC as the HILIC column. The content of acetonitrile, which was selected as the diluent of the aqueous samples in this study, was varied 0−95 % in the injection samples, and the numbers of theoretical plates (NTPs) and retention times were compared using a mobile phase composed of ammonium formate buffer/acetonitrile (25:75, v/v). Although the NTPs and retention times decreased with decreasing acetonitrile content, the peak shapes were acceptable for samples with acetonitrile contents down to 50 % based on asymmetry factor. Furthermore, the sample volume had a serious effect for samples with low acetonitrile contents. Although a high content of acetonitrile in samples is still recommended, the capacity for the aqueous solution in the injection samples under HILIC conditions should vary with the composition of the mobile phase and may be larger than previously thought. These findings should be helpful in deciding the sample composition under HILIC conditions, particularly in bioanalysis, where aqueous solution is often contained in the injection samples and the sample dilution with an organic solvent may decrease the detection sensitivity.