Liquid Chromatography Mobile Phase Research Articles

Harnessing an elastic flow instability to improve the kinetic performance of chromatographic columns

Despite decades of research and development, the optimal efficiency of slurry-packed HPLC columns is still hindered by inherent long-range flow heterogeneity from the wall to the central bulk region of these columns. Here, we show an example of how this issue can be addressed through the straightforward addition of a semidilute amount (500 ppm) of a large, flexible, synthetic polymer (18 MDa partially hydrolyzed polyacrylamide, HPAM) to the mobile phase (1% NaCl aqueous solution, hereafter referred to as “brine”) during operation of a 4.6 mm × 300 mm column packed with 10μm BEHTM 125 Å particles. Addition of the polymer imparts elasticity to the mobile phase, causing the flow in the interparticle pore space to become unstable above a threshold flow rate. We verify the development of this elastic flow instability using pressure drop measurements of the friction factor versus Reynolds number. In prior work, we showed that this flow instability is characterized by large spatiotemporal fluctuations in the pore-scale flow velocities that may promote analyte dispersion across the column. Axial dispersion measurements of the quasi non-retained tracer thiourea confirm this possibility: they reveal that operating above the onset of the instability improves column efficiency by greater than 100%. These experiments thereby suggest that elastic flow instabilities can be harnessed to mitigate the negative impact of trans-column flow heterogeneities on the efficiency of slurry-packed HPLC columns. While this approach has its own inherent limitations and constraints, our results lay the groundwork for future targeted development of polymers that can impart elasticity when dissolved in commonly used liquid chromatography mobile phases, and can thereby generate elastic flow instabilities to help improve the resolution of HPLC columns.

Elucidating important factors and corresponding method optimization for sensitive detection of small peptide drugs in human urine by solid-phase extraction and UPLC-HRMS: The influence of MS scan modes, protein precipitants, and ammonium formate.

Small peptide hormones are widely used in sports as performance-enhancing substances, making it crucial to develop sensitive analytical methods for their detection in doping control analysis. Various factors significantly affect analytical sensitivity, such as the selection of ultra-performance liquid chromatography (UPLC) mobile phase, high-resolution mass spectrometry (HRMS) scanning modes, and extraction solvents for pretreatment. Herein, comparative study approach was utilized to investigate the sensitivity of each peptide analyte under both full scan and parallel reaction monitoring (PRM) modes of HRMS and assess the effects of some protein precipitants as a part of extraction solvents on solid-phase extraction (SPE). The results showed that full scan should be selected as the primary scan mode of HRMS, and the combination with PRM mode could effectively compensate for the limitations of full scan, and the addition of protein precipitants would adversely affect the detection of certain small peptide analytes. Meanwhile, influences of ammonium formate in reverse UPLC mobile phase on the charge state distribution of small peptides were investigated and elucidated. Based on these findings, a sensitive and reliable UPLC-HRMS analytical method combining full scan and PRM mode was validated for screening and confirmation of 63 small peptide analytes after SPE, with limits of detection (LODs) ranging between 0.010 and 0.473 ng/ml and limits of identification (LOIs) ranging between 0.015 and 1.512 ng/ml. Additionally, suggestions were provided for the detection of [Arg8]-vasopressin, dermorphin, and its analogues.

Quantitative investigation of drug-drug interaction between bergenin and vilazodone in rats through UPLC-MS/MS assay

In this study, we firstly established and verified a method by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for the analysis of vilazodone and its metabolite M10 in rat plasma, then this method was used to explore the pharmacokinetics of vilazodone and M10 present or absence of 80 mg/kg bergenin in rats. Protein precipitation with acetonitrile was used to prepare the samples in this research. The mobile phase for liquid chromatography was consisted of 0.1% formic acid aqueous solution and acetonitrile. Brexpiprazole was used as the internal standard (IS), and the multiple reaction monitoring (MRM) mode was used for detection. The verification items required by the US Food and Drug Administration (FDA) guidelines such as selectivity, sensitivity, linearity, stability, recovery and matrix effect of this method were all met the standards. Besides, rats were used to explore the drug-drug interaction between vilazodone and bergenin, which were divided into two groups, and separately gavaged with the same-volume of carboxymethyl cellulose sodium (CMC-Na) solution and 80 mg/kg bergenin, respectively. The results showed that bergenin significantly affected the metabolism of vilazodone. It suggested that there was a potential drug-drug interaction between bergenin and vilazodone in rats. In clinical application, we should pay attention to the dose of vilazodone when in combination with bergenin.

M-nitrobenzyl alcohol supercharging reagent enhances the chromatographic separation and the charging of disulfide bond linked and His-tag peptides

The linkages of disulfide bond (DSB) play important roles in protein stability and activity. Mass spectrometry-based (MS-based) techniques become accepted tools for DSB analysis in the recent decade. In the bottom-up approach, after enzyme digestion, the neighbouring amino acids of cysteines have great impacts on the physicochemical properties of resulting disulfide bond peptides, determining their retention behaviour on liquid chromatography (LC) and their MS ionization efficiency. In this study, the addition of supercharging reagent in LC mobile phase was used to examine the impact of supercharging reagent on the charge states of disulfide-bond peptides. The results showed that 0.1 % m-nitrobenzyl alcohol (m-NBA) in LC mobile phase increased the sensitivity and charge states of DSB peptides from our model protein, equine Interleukin-5 (eIL5), as well as the resolution of reversed-phase chromatography. Notably, also the sensitivity of C-terminal peptide with His-tag significantly improved. Our findings highlight the effectiveness of employing m-NBA as a supercharging reagent when investigating disulfide-linked peptides and the C-terminal peptide with a His-tag through nano-liquid chromatography mass spectrometry.

Nonenzymatic Posttranslational Modifications and Peptide Cleavages Observed in Peptide Epimers.

Posttranslational modifications (PTMs) play vital roles in cellular homeostasis and are implicated in various pathological conditions. This work uses two ion mobility spectrometry-mass spectrometry (IMS-MS) modalities, drift-tube IMS (DT-IMS) and trapped IMS (TIMS), to characterize three important nonenzymatic PTMs that induce no mass loss: l/d isomerization, aspartate/isoaspartate isomerization, and cis/trans proline isomerization. These PTMs are assessed in a single peptide system, the recently discovered pleurin peptides, Plrn2, from Aplysia californica. We determine that the DT-IMS-MS/MS can capture and locate asparagine deamidation into aspartate and its subsequent isomerization to isoaspartate, a key biomarker for age-related diseases. Additionally, nonenzymatic peptide cleavage via in-source fragmentation is evaluated for differences in the intensities and patterns of fragment peaks between these PTMs. Peptide fragments resulting from in-source fragmentation, preceded by peptide denaturation by liquid chromatography (LC) mobile phase, exhibited cis/trans proline isomerization. Finally, the effects of differing the fragmentation voltage at the source and solution-based denaturation conditions on in-source fragmentation profiles are evaluated, confirming that LC denaturation and in-source fragmentation profoundly impact N-terminal peptide bond cleavages of Plrn2 and the structures of their fragment ions. With that, LC-IMS-MS/MS coupled with in-source fragmentation could be a robust method to identify three important posttranslational modifications: l/d isomerization, Asn-deamidation leading to Asp/IsoAsp isomerization, and cis/trans proline isomerization.

Selection of Stationary Phase and Mobile Phase in High Performance Liquid Chromatography

Selection of ideal mobile phase and stationary phase is very important to get accurate separation of mixtures or impurities as a whole. There indeed are parameters which are explained in this article, important to be kept in mind while a method development of HPLC method. But as such there is no list of such parameters and their accurate limits can be applied while method development. In this article, there have been mention of certain parameters which are generally looked for, and not exact, but optimal application of such has been discussed. When we talk about stationary phase, parameters which we tend to optimize very often include mostly commonly the pH of the analyte as well the mobile phase used and stability of the column packing material over a range of temperature. Choosing of stationary phase greatly depends on the nature of analyte. Stationary phase will depend and vary simultaneously if analyte is lipophilic or an ionic compound. In order to increase separation and increase mass transfer there have been new addition to column packing material apart from the widely used silica. New advances result into greater column loading, better flow, reduced plate height and reduced back pressure. Leaving alone stationary phase, mobile phase selection is also a task of its own. Very commonly used mobile phase solvents include acetonitrile, methanol, THF, water and buffer of salts like acetate, phosphate, etc. Apart from these traditional choices, there have been use of pressurized hot water and ionic liquids as mobile phases which are termed as “Environmental-friendly” because these solvents are devoid of any organic counterpart or organic modifier in them, which produces harmful fumes on getting heated. Apart from the environmentally friendly mobile phase options, we have some specific chemicals to be used when chemical property of the analyte is specific.

Investigation of the potential of mixed solvent mobile phases in temperature-responsive liquid chromatography (TRLC).

Temperature-responsive liquid chromatography (TRLC) allows for extensive retention and selectivity tuning through temperature in HPLC. This is mainly achieved through the use of a stationary phases comprising of a temperature-responsive polymer which undergoes a reversible change from hydrophilic to hydrophobic behaviour upon increasing the temperature. The approach can allow for reversed phase type separations to be achieved with purely aqueous mobile phases, whereby the retention is controlled through temperature instead of mobile phase composition. Despite the promising nature of such form of retention control under isocratic mobile phase conditions, TRLC can suffer from excessive retention of highly apolar solutes even at lower column temperatures whereby the polymer is considered hydrophilic. This is related both to a residual apolarity of the polymer chain and due to the high log P's and low water solubility of higly apolar compounds. While it was known that elution in TRLC doesn't necessarily has to be performed under purely aqueous conditions and that the use of organic co-solvents to the water is possible, the impact thereof on the temperature responsive behaviour itself had not yet been investigated in a systematic way. Therefore in this work the advantages and drawbacks of the use of the organic co-solvents methanol and acetonitrile in TRLC is assessed on two types of temperature reponsive phases: poly-N-N-propylacrylamide (PNNPAAm) and poly-N-isopropylacrylamide (PNIPAAm). The influence of organic co-solvents is investigated with two representative test mixtures (comprising 4 parabens and 5 apolar steroids).

Development of an ammonium chloride-enhanced thermal-assisted-ESI LC-HRMS method for the characterization of chlorinated paraffins

Simultaneous quantification of short-, medium-, and long-chain chlorinated paraffins (CPs) in environmental matrices is challenging and has received much attention from environmental chemists. In this study, ammonium-chloride-enhanced liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) was developed for the first time to quantify CPs in sediments and aqueous samples. Three ionization sources, including atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), and thermal-assisted-ESI, were employed to examine the performance of ammonium chloride as the chloride ion supply reagent in comparison with traditional chloride ion supply reagent, dichloromethane. Ammonium chloride can be easily used with reversed-phase liquid chromatography (LC), whereas dichloromethane is not compatible with aqueous LC mobile phase. Furthermore, other anion-supply reagents, such as ammonium formate, ammonium acetate, and ammonium bromide, were also tested. It was concluded that the adducts of the CPs with the anions were reversible and could partially dissociate into deprotonated CP ions. The yield of deprotonated CP ions was associated with the gas-phase basicity of the deprotonated CP ions and the corresponding anions. Furthermore, collision-induced dissociation curves were drawn to quantify the stability of anionic CP adducts. The ammonium-chloride-enhanced LC-HRMS was further employed for identifying CPs in sediment samples and coupled with an online SPE method for detecting CPs in aqueous samples. This study may significantly contribute to the qualification and quantification of CPs in environmental matrices.

Green method using water for lactose and lactulose extraction and determination in milk by high-performance liquid chromatography with refractive index detection

Lactose is the main carbohydrate found in milk. However, the ultra-high temperature (UHT) process might convert lactose to lactulose by isomerization. Lactulose can be prebiotic; nevertheless, at high concentrations, it can be fermented in the gastrointestinal tract because it is not hydrolyzed by enzymes. In view of the increase in lactose intolerant consumers, it becomes necessary to determine the presence and amounts these compounds in UHT milk. Hence, this study aims to propose and validate a method with simple sample preparation to measure lactose and lactulose contents in milk. Following green chemistry principles, the proposed method is distinguished by the use of only water in the extraction step, with an effective sample clean-up step performed via ultracentrifugation; likewise, water was also employed as the mobile phase for high-performance liquid chromatography with refractive index detection analysis. Moreover, the proposed method allows for the verification of the efficiency of the lactose-free milk process by monitoring lactose residual levels in addition to measuring the degradation contents of glucose and galactose. Further, the developed method even represents an alternative for detecting counterfeit milk made with added sucrose, ensuring the quality of milk for consumers.

Current trends in supercritical fluid chromatography.

Supercritical fluid chromatography (SFC), which employs pressurized carbon dioxide as the major component of the mobile phase, has been known for several decades but has faced a significant resurgence of interest in the recent years, thanks to the development of modern instruments to comply with current expectations in terms of robustness and sensitivity. This review is focused on the recent literature, specifically since the introduction of modern systems but in relation to older literature, to identify the changing trends in application domains. Typically, natural products, bioanalysis, food science, and environmental analyses are all strongly increasing. Together with reduced extra-column volumes in the instruments, the advent of sub-2-μm particles and superficially porous particles in the stationary phases is favoring ultra-high-performance SFC (UHPSFC) allowing for improved resolution and faster analyses, but without the constraints of viscous liquids encountered in ultra-high-performance liquid chromatography (UHPLC). Hyphenation to mass spectrometry is also more frequent and opened the way to new application domains, and raises different issues from liquid chromatography mobile phases, especially due to decompression of carbon dioxide. It is also shown that the frontiers between SFC and HPLC are fading, as switching from one method to the other, even within the course of a single analysis, is facilitated my modern instruments. The present review is not intended to be exhaustive but rather giving a snapshot of recent trends in supercritical fluid chromatography, based on the observation of about 500 papers published in English-written peer-reviewed journals from 2014 to 2018. Graphical abstract ᅟ.

High throughput method to characterize acid-base properties of insoluble drug candidates in water

In drug design experimental characterization of acidic groups in candidate molecules is one of the more important steps prior to the in-vivo studies. Potentiometry combined with Yasuda-Shedlovsky extrapolation is one of the more important strategy to study drug candidates with low solubility in water, although, it requires a large number of sequences to determine pKa values at different solvent-mixture compositions to, finally, obtain the pKa in water (pwwKa) by extrapolation. We have recently proposed a method which requires only two sequences of additions to study the effect of organic solvent content in liquid chromatography mobile phases on the acidity of the buffer compounds usually dissolved in it along wide ranges of compositions. In this work we propose to apply this method to study thermodynamic pwwKa of drug candidates with low solubilities in pure water. Using methanol/water solvent mixtures we study six pharmaceutical drugs at 25 °C. Four of them: ibuprofen, salicylic acid, atenolol and labetalol, were chosen as members of carboxylic, amine and phenol families, respectively. Since these compounds have known pwwKa values, they were used to validate the procedure, the accuracy of Yasuda-Shedlovsky and other empirical models to fit the behaviors, and to obtain pwwKa by extrapolation. Finally, the method is applied to determine unknown thermodynamic pwwKa values of two pharmaceutical drugs: atorvastatin calcium and the two dissociation constants of ethambutol. The procedure proved to be simple, very fast and accurate in all of the studied cases.

Sol-gel niobia sorbent with a positively charged octadecyl ligand providing enhanced enrichment of nucleotides and organophosphorus pesticides in capillary microextraction for online HPLC analysis.

A niobia-based sol-gel organic-inorganic hybrid sorbent carrying a positively charged C18 ligand (Nb2 O5 -C18 (+ve)) was synthesized to achieve enhanced enrichment capability in capillary microextraction of organophosphorus compounds (which include organophosphorus pesticides and nucleotides) before their online analysis by high-performance liquid chromatography. The sorbent was designed to simultaneously provide three different types of molecular level interactions: electrostatic, Lewis acid-base, and van der Waals interactions. To understand relative contributions of various molecular level analyte-sorbent interactions in the extraction process, two other sol-gel niobia sorbents were also created: (a) a purely inorganic sol-gel niobia sorbent (Nb2 O5 ) and (b) an organic-inorganic hybrid sol-gel niobia sorbent carrying an electrically neutral-bonded octadecyl ligand (Nb2 O5 -C18 ). The extraction efficiency of the created sol-gel niobia sorbent (Nb2 O5 -C18 (+ve)) was compared with that of analogously designed and synthesized titania-based sol-gel sorbent (TiO2 -C18 (+ve)), taking into consideration that titania-based sorbents present state-of-the-art extraction media for organophosphorus compounds. In capillary microextraction with high-performance liquid chromatography analysis, Nb2 O5 -C18 (+ve) had shown 40-50% higher specific extraction values (a measure of extraction efficiency) over that of TiO2 -C18 (+ve). Compared to TiO2 -C18 (+ve), Nb2 O5 -C18 (+ve) also provided superior analyte desorption efficiency (96vs. 90%) during the online release of the extracted organophosphorus pesticides from the sorbent coating in the capillary microextraction capillary to the chromatographic column using reversed-phase high-performance liquid chromatography mobile phase.

DMSO as a mobile phase additive enhances detection of ubiquitination sites by nano-LC-ESI-MS/MS.

Large-scale detection of ubiquitination sites in whole cell proteomes using nano-liquid chromatography coupled to tandem mass spectrometry is a well-established technique that has deepened the understanding of protein degradation processes in eukaryotic cells. Ubiquitination sites are usually identified by detection of Lys-ɛ-Gly-Gly (K-ɛ-GG)-remnant peptides, which are generated by tryptic digestion of proteomes. We show in this application note that dimethyl sulfoxide addition to the liquid chromatography mobile phase enhances identification rates of K-ɛ-GG peptides by more than 100% due to an increase of peptide signal intensities. The gain in the number of ubiquitination site identifications exceeds by far the gain that has been published for other posttranslational modifications, namely, phosphorylation and acetylation.

Sustainable and Eco-Friendly Alternatives for Liquid Chromatographic Analysis

This Perspectives article covers the most relevant current strategies aimed at reducing the environmental fingerprint caused by mobile phases in liquid chromatography. Modern stationary phases, such as columns packed with core–shell particles or stationary phases allowing high-temperature separations, are discussed. Mobile-phase additives creating secondary partitioning equilibria (micelle-forming reagents, cyclodextrins, or ionic liquids) and alternatives to acetonitrile as organic solvent are critically compared in terms of separation efficacy and wastes’ toxicity. Advantages of CO2 and superheated solvents are also considered to complete the picture of the most recent developments in the field.

Quantitative analysis of acid-catalyzed levulinic acid product mixture from cellulose by mixed-mode liquid chromatography

A mixed-mode weak anion-exchange/reversed-phase liquid chromatography (LC) column was successfully applied for the analysis of levulinic acid (4-oxopentanoic acid, LA) product mixture derived from cellulose. Due to the existence of ionic and neutral byproducts, the analysis of the product mixture usually requires ion chromatography, LC and gas chromatography simultaneously. The new method enables accomplishment of the analysis in one LC run within 6min. LC mobile phase of 10mM phosphate buffer containing 5% acetonitrile with pH=5.5 was used. The linear regression coefficients for the UV signal of standard compounds with the corresponding mass concentrations were greater than 0.999. The method recoveries were between 98.57–103.48%. The limits of quantification were 5, 10,1000, 1500 and 3000ng/mL for 5-hydroxymethylfurfural, furfural, acetic acid, formic acid and LA respectively. The mixed-mode column exhibits comprehensive separation mechanism of both reversed-phase and ion-exchange interactions. The mobile phase with different pH, organic modifier solvent and buffer concentration provided flexible LC method for the sample with different complexity.

Determination of glyphosate in surface water with high organic matter content

In this paper, we investigate the sample preparation and analysis process in order to achieve adequate results for surface water collected from rivers that flow through swamps and are consequently rich in organic matter. We show that matrix effects in glyphosate determination can be reduced by optimizing sample volume, liquid chromatography (LC) mobile phase buffer concentration and pH as well as gradient speed. Also, aspects of derivatization procedure (borate buffer concentration, fluorenylmethyloxycarbonyl chloride concentration) and their influence on accuracy are considered in detail. We encountered a cross-talk effect in the mass spectra, interfering with quantization during analysis, which was removed by optimizing MS parameters. As a result it was demonstrated that isotope-labelled internal standard with just one 13C atom is sufficient for the analysis.All these aspects were found to strongly impact the accuracy of the glyphosate determination but have received little or no attention in earlier works. We propose a reliable solid phase extraction and LC/ESI/MS/MS method for determination of glyphosate in organic-rich waters and demonstrate that LoD can be decreased by about two times using an ESI nebulizer with a modified design.

UPLC-MS/MS determination of ptaquiloside and pterosin B in preserved natural water.

The naturally occurring carcinogen ptaquiloside and its degradation product pterosin B are found in water leaching from bracken stands. The objective of this work is to present a new sample preservation method and a fast UPLC-MS/MS method for quantification of ptaquiloside and pterosin B in environmental water samples, employing a novel internal standard. A faster, reliable, and efficient method was developed for isolation of high purity ptaquiloside and pterosin B from plant material for use as analytical standards, with purity verified by 1H-NMR. The chemical analysis was performed by cleanup and preconcentration of samples with solid phase extraction, before analyte quantification with UPLC-MS/MS. By including gradient elution and optimizing the liquid chromatography mobile phase buffer system, a total run cycle of 5min was achieved, with method detection limits, including preconcentration, of 8 and 4ng/L for ptaquiloside and pterosin B, respectively. The use of loganin as internal standard improved repeatability of the determination of both analytes, though it could not be employed for sample preparation. Buffering raw water samples in situ with ammonium acetate to pH ∼5.5 decisively increased sample integrity at realistic transportation and storing conditions prior to extraction. Groundwater samples collected in November 2015 at the shallow water table below a Danish bracken stand were preserved and analyzed using the above methods, and PTA concentrations of 3.8 ± 0.24μg/L (±sd, n = 3) were found, much higher than previously reported. Graphical abstract Workflow overview of ptaquiloside determination.

Comparison of liquid and supercritical fluid chromatography mobile phases for enantioselective separations on polysaccharide stationary phases

Analysis and production of enantiomerically pure compounds is a major topic of interest when active pharmaceutical ingredients are concerned. Enantioselective chromatography has become a favourite both at the analytical and preparative scales. High-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) are dominating the scene and are often seen as complementary techniques. Nowadays, for economic and ecologic reasons, SFC may be preferred over normal-phase HPLC (NPLC) as it allows significant reductions in solvent consumption. However, the transfer of NPLC methods to SFC is not always straightforward.In this study, we compare the retention of achiral molecules and separation of enantiomers under supercritical fluid (carbon dioxide with ethanol or isopropanol) and liquid normal-phase (heptane with ethanol or isopropanol) elution modes with polysaccharide stationary phases in order to explore the differences between the retention and enantioseparation properties between the two modes. Chemometric methods (namely quantitative structure-retention relationships and discriminant analysis) are employed to compare the results obtained on a large set of analytes (171 achiral probes and 97 racemates) and gain some understanding on the retention and separation mechanisms. The results indicate that, contrary to popular belief, carbon dioxide − solvent SFC mobile phases are often weaker eluents than liquid mobile phases. It appears that SFC and NPLC elution modes provide different retention mechanisms. While some enantioseparations are unaffected, facilitating the transfer between the two elution modes, other enantioseparations may be drastically different due to different types and strength of interactions contributing to enantioselectivity.

Application of solvation model to prediction of the solute retention in liquid chromatography over a wide range of mobile-phase compositions

Possibilities have been studied of using a solvation model to predict the retention behavior of solutes in liquid chromatography mobile phases of water-acetonitrile and water-methanol with the organic solvent content varying from 1 to 100 vol%. Twenty-one test solutes, both aliphatic and aromatic compounds, have been selected on the basis of two-level factorial designs. Using the multiple linear regression analysis, regression coefficients, which are characteristics of the stationary and mobile-phase system, were calculated for different mobile phases in the solvation model. Regression coefficients have been used for the prediction of the retention behavior. Unbiased results have been obtained by using two sets, one training and the other the testing set. The predicted retention has been compared with the experimental data. The methanol-water system provided good results at low and medium methanol concentrations; the retention prediction was unsatisfactory for mobile phases containing more than 90% of methanol. The acetonitrile-water system yielded similar results, but the retention prediction ceased to be at acetonitrile concentrations greater than 80%. The retention has primarily been determined by cohesive and acid-base interactions. The dependences of the regression coefficients on the mobile-phase composition were similar for the acetonitrile-water and methanol-water systems.

HYDROPHILIC INTERACTION CHROMATOGRAPHY (HILIC): STATE OF THE ART AND APPLICATIONS

Hydrophilic interaction liquid chromatography (HILIC) has been gaining increased attention for its effective separation of highly polar compounds, which include carbohydrates, amino acids, pharmaceutical compounds, proteins, glycoproteins, nucleosides, etc. Polar compounds are usually poorly retained on reverse-phase liquid chromatography (RP-HPLC) columns or have poor solubility in the apolar mobile phase of normal-phase high performance liquid chromatography (NP-HPLC). Since HILIC uses organic solvents such as ACN or MeOH ( > 70%), also used in RP-HPLC and polar stationary phases similar to NP-HPLC (bare silica, diol, amino, amide, saccharide, zwitterionic stationary phases, etc.), it represents a hybrid of the two separation modes. The high organic content in the MP leads to good compatibility with mass spectrometry (MS), increasing the detectivity. This review describes the fundamentals of HILIC and highlights some interesting applications.