Mixed-mode hydrophilic interaction/cation-exchange chromatography: separation of complex mixtures of peptides of varying charge and hydrophobicity

Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) was applied to the separation of two mixtures of synthetic peptide standards: (i) a 27-peptide mixture containing three groups of peptides (each group containing nine peptides of the same net charge of +1, +2 or +3), where the hydrophilicity/hydrophobicity of adjacent peptides within the groups varied only subtly (generally by only a single carbon atom); and (ii) peptide pairs with the same composition but different sequences, where the sole difference between the peptides was the position of a single amino acid substitution. HILIC/CEX is essentially CEX chromatography in the presence of high levels of organic modifier (generally ACN). The present study demonstrated the dramatic effect of increasing ACN concentration (optimum levels of 60-80%, depending on the application) on the separation of both mixtures of peptides. The greater the charge on the peptides, the better the separation achievable by HILIC/CEX. In addition, HILIC/CEX separation of both the peptide mixtures used in the present study was shown to be superior to that of the more commonly applied RP-HPLC mode. Our results highlight again the efficacy of HILIC/CEX as a peptide separation mode in its own right as well as an excellent complement to RP-HPLC.     

Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) of peptides and proteins

This review represents a summary of the development and application of a novel mixed-mode HPLC approach to the separation and analysis of peptides and proteins termed hydrophilic interaction/cation-exchange chromatography (HILIC/CEX). This approach combines the most advantageous aspects of two widely different separation mechanisms, i.e. a separation based on hydrophilicity/hydrophobicity differences between polypeptides overlaid on a separation based on net charge. Applications described include HILIC/CEX separations of cyclic peptides, alpha-helical peptides, random coil peptides and modified or deletion products of synthetic peptides. In addition, the excellent resolving ability of HILIC/CEX for modified histone proteins is described. This approach is shown to represent an excellent complement to RP chromatography (RPC), as well as being a potent analytical tool in its own right.     

Hydrophilic-interaction chromatography of peptides on hydrophilic and strong cation-exchange columns

Hydrophilic-interaction chromatography (HILIC) was recently introduced as a potentially useful separation mode for the purification of peptides and other polar compounds. The elution order of peptides in HILIC, which separates solutes based on hydrophilic interactions, should be opposite to that obtained in reversed-phase chromatography, which separates solutes based on hydrophobic interactions. Three series of peptides, two of which consisted of positively charged peptides (independent of pH at pH less than 7) and one of which consisted of uncharged or negatively charged peptides (dependent on pH), and which varied in overall hydrophilicity/hydrophobicity, were utilized to examine the separation mechanism and efficiency of HILIC on hydrophilic and strong cation-exchange columns.     

Temperature profiling of polypeptides in reversed-phase liquid chromatography. I. Monitoring of dimerization and unfolding of amphipathic alpha-helical peptides

The present study sets out to extend the utility of reversed-phase liquid chromatography (RP-HPLC) by demonstrating its ability to monitor dimerization and unfolding of de novo designed synthetic amphipathic alpha-helical peptides on stationary phases of varying hydrophobicity. Thus, we have compared the effect of temperature (5-80 degrees C) on the RP-HPLC (C8 or cyano columns) elution behaviour of mixtures of peptides encompassing amphipathic alpha-helical structure, amphipathic alpha-helical structure with L- or D-substitutions or non-amphipathic alpha-helical structure. By comparing the retention behaviour of the helical peptides to a peptide of negligible secondary structure (a random coil), we rationalize that "temperature profiling" by RP-HPLC can monitor association of peptide molecules, either through oligomerization or aggregation, or monitor unfolding of alpha-helical peptides with increasing temperature. We believe that the conformation-dependent response of peptides to RP-HPLC under changing temperature has implications both for general analysis and purification of peptides but also for the de novo design of peptides and proteins.     

Orthogonal method development using hydrophilic interaction chromatography and reversed-phase high-performance liquid chromatography for the determination of pharmaceuticals and impurities

A hydrophilic interaction chromatography (HILIC) method has been developed and validated as a secondary or orthogonal method complementary to a reversed-phase HPLC (RP-HPLC) method for quantitation of a polar active pharmaceutical ingredient and its three degradation products. The HILIC method uses a diol column and a mobile phase consisting of acetonitrile/water and ammonium chloride. The compounds of interest show significant differences in retention behaviors with the two very different chromatographic systems, which are desired in developing orthogonal methods. The HILIC method is validated and has met all validation acceptance criteria for the support of drug development activities.     

Novel two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography, an excellent orthogonal system for practical analysis

A novel two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography (2D-RPLC/HILIC) system is developed with the introduction of the click beta-cyclodextrin (beta-CD) stationary phase. The offline system shows excellent abilities for the separation of polar and medium-polarity components in traditional Chinese medicine (TCM). It facilitates us not only to separate components that cannot be resolved by uni-dimensional chromatography, but also to achieve much more efficient detection of components with low abundance. The orthogonality of this new system is excellent. A simple geometric approach is developed to characterize the practical orthogonality of 2D-LC in the analysis of complex unknown samples. The mathematical characterization results are in good accordance with the experimental findings, and both demonstrate that the proposed 2D-RPLC/HILIC system is a powerful tool for the separation of polar and medium-polarity complex samples. This system may have a great potential for the separation of peptides and metabolomic compounds with similar polarity.     

多羟基化合物键合亲水色谱固定相的制备及其分离性能

用硅胶与氨丙基三甲氧基硅烷反应,再与δ-葡萄糖酸内酯反应,制备了一种多羟基化合物键合的新型亲水色谱固定相。以水-有机溶剂(乙醇、乙腈、四氢呋喃)为流动相,通过改变流动相中有机溶剂的种类及浓度、缓冲盐浓度和柱温,考察了该固定相对6种强极性中药组分的保留行为和保留机理。当水的比例在0～40%(v/v)范围时,溶质的保留随着流动相中水的比例的增大而减小,属于典型的亲水色谱分离模式;而当流动相中水的比例在0～100%(v/v)范围内变化时,溶质的保留随着水的比例变化呈“U”形曲线,属于亲水色谱和反相色谱的混合保留机理。缓冲盐的浓度和pH效应说明,所选用的中药组分与所制备的固定相间还存在弱的静电作用。该固定相对6种中药组分以及丹参注射液具有良好的分离性能,表明其在强极性中药有效成分的分离及其他强极性物质的分离分析中具有一定的应用前景。     

Imidazoline type stationary phase for hydrophilic interaction chromatography and reversed-phase liquid chromatography

An imidazoline was prepared by solvent-free microwave-assisted organic synthesis and immobilized on porous silica particles by polymerization. The resulting material was composed of both hydrophobic alkyl ester chains and hydrophilic imidazoline rings, which gave it both hydrophilic interaction and reversed-phase characteristics. The titration curve suggests that the new material has buffering capacity and acquires increasing positive charge over the pH range 9-4, and is "zwitterionic" in the upper part of this pH range. Through investigating the effect of column temperature, the water content, pH and ion strength of mobile phase on the retention time of polar compounds in highly organic eluents, it was found that the new material could be used as a hydrophilic interaction liquid chromatography (HILIC) stationary phase which involved a complex retention process consisting of partitioning, surface adsorption and electrostatic interactions. In addition, the retention behavior of aromatic compounds in different mobile phase conditions was also studied, which showed the new material mainly exhibited a partitioning mechanism in the reversed-phase liquid chromatography (RPLC) mode. The separation of six water-soluble vitamins and five aromatic compounds were achieved by using the new material in the HILIC and RPLC modes, respectively.     

Hydrophilic interaction liquid chromatography (HILIC) in proteomics

In proteomics, nanoflow multidimensional chromatography is now the gold standard for the separation of complex mixtures of peptides as generated by in-solution digestion of whole-cell lysates. Ideally, the different stationary phases used in multidimensional chromatography should provide orthogonal separation characteristics. For this reason, the combination of strong cation exchange chromatography (SCX) and reversed-phase (RP) chromatography is the most widely used combination for the separation of peptides. Here, we review the potential of hydrophilic interaction liquid chromatography (HILIC) as a separation tool in the multidimensional separation of peptides in proteomics applications. Recent work has revealed that HILIC may provide an excellent alternative to SCX, possessing several advantages in the area of separation power and targeted analysis of protein post-translational modifications. Figure Artistic impression of the HILIC separation mechanism     

Separation with zwitterionic hydrophilic interaction liquid chromatography improves protein identification by matrix‐assisted laser desorption/ionization‐based proteomic analysis

Comprehensive proteomic analyses necessitate efficient separation of peptide mixtures for the subsequent identification of proteins by mass spectrometry (MS). However, digestion of proteins extracted from cells and tissues often yields complex peptide mixtures that confound direct comprehensive MS analysis. This study investigated a zwitterionic hydrophilic interaction liquid chromatography (ZIC‐HILIC) technique for the peptide separation step, which was verified by subsequent MS analysis. Human serum albumin (HSA) was the model protein used for this analysis. HSA was digested with trypsin and resolved by ZIC‐HILIC or conventional strong cation exchange (SCX) prior to MS analysis for peptide identification. Separation with ZIC‐HILIC significantly improved the identification of HSA peptides over SCX chromatography. Detailed analyses of the identified peptides revealed that the ZIC‐HILIC has better peptide fractionation ability. We further demonstrated that ZIC‐HILIC is useful for quantitatively surveying cell surface markers specifically expressed in undifferentiated embryonic stem cells. These results suggested the value of ZIC‐HILIC as a novel and efficient separation method for comprehensive and quantitative proteomic analyses. Copyright © 2009 John Wiley & Sons, Ltd.     

Grafting of silica with a hydrophilic triol acrylamide polymer via surface-initiated "grafting from" method for hydrophilic-interaction chromatography

A novel hydrophilic polymer-coated silica sorbent has been prepared using the radical "grafting from" polymerization method through surface-bound azo initiators for hydrophilic-interaction chromatography (HILIC). The azo groups were introduced to the surface of silica gel through the reaction with amino groups on the surface of silica gel with 4,4'-azobis(4-cyanopentanoic acid chloride) (ACVC). The resultant azo-immobilized silica gel served as surface initiator to polymerize hydrophilic triol acrylamide monomer N-acryloyltris(hydroxymethyl) aminomethane (NA) in methanol to get hydrophilic polymer-coated silica sorbent. The obtained poly(NA)-coated silica (pNA-sil) was characterized by Fourier transform infrared spectroscopy (FT-IR), elemental analysis (EA), and nitrogen sorption porosimetry (NSP). Then the pNA-sil was packed into the stainless-steel column and evaluated in high-performance liquid chromatography (HPLC). Good chromatographic performance for the separation of peptides and nucleosides was obtained under HILIC mode. The results indicated that the pNA-sil stationary phase behaved as mixed-mode retention mechanisms of hydrophilic and ionic interactions. Furthermore, the pNA-sil phase was used to separate tryptic digest of β-casein and our results showed that more than 12 peptides peaks were resolved and well distributed within the elution window. Finally, the pNA-sil stationary phase was demonstrated to possess remarkable reproducibility and stability. Taken together, the pNA-sil stationary phase prepared in the current study offers a potential application in proteomics study.     

Predictive Liquid Chromatography of Peptides Based on Hydrophilic Interactions for Mass Spectrometry-Based Proteomics

High-performance liquid chromatography (HPLC) is widely used for separation of complex peptide mixtures before mass spectrometry-based proteome analysis. In this analysis, reversed phase HPLC (RPHPLC) using non-polar stationary phases such as surface-modified silica containing alkyl groups (e.g., C18) is typically employed. Because of the high heterogeneity of proteomic samples, multidimensional separation approaches gained increasing attention recently to tackle this complexity and extremely high range of concentrations. In two-dimensional liquid chromatography, hydrophilic interaction chromatography (HILIC) is often a method of choice for combination with RP-HPLC because it uses reversed-phase type eluents and allows efficient separation of polar peptides. Due to the high degree of orthogonality in this two-dimensional separation space, it is tempting to develop approaches for predicting peptide retention times for HILIC-based separations similar to the ones for RP-HPLC. Recent successful efforts in this area were focused on developing retention coefficient (RC)-based approaches. Herein, we explored the feasibility of using a statistical thermodynamic model for prediction of peptide retention times in HILIC separations and determined the phenomenological parameters of the model for a bare silica column. The performance of the developed model was tested using HPLC-MS analysis of a set of synthetic peptides, as well as a tryptic peptide mixture.     

Capillary liquid chromatography using a hydrophilic/cation-exchange monolithic column with a dynamically modified cationic surfactant

A novel form of reversed-phase liquid chromatography (RPLC) by the dynamically modified hydrophilic interaction monolithic column has been described in this paper. A porous poly(SPMA-co-PETA) monolith with strong cation-exchange (SCX) was prepared and the resulting monolith showed a typical hydrophilic interaction chromatography (HILIC) mechanism at higher organic solvent content (ACN% > 50%). The good selectivity for neutral, basic and acidic polar analytes was observed in the HILIC mode. In order to increase the hydrophobic interaction, the monolith with SCX was dynamically modified with a long-chain quaternary ammonium salt, cetyltrimethylammonium bromide (CTAB), which was added to the mobile phase. CTAB ions were adsorbed onto the surface of the SCX monolithic material, and the resulting hydrophobic layer was used as the stationary phase. Using the dynamically modified SCX monolithic column, neutral, basic and acidic hydrophobic analytes were well separated with the RPLC mode.     

Online coupling of reverse-phase and hydrophilic interaction liquid chromatography for protein and glycoprotein characterization

We have developed a novel system for coupling reverse-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) online in a micro-flow scheme. In this approach, the inherent solvent incompatibility between RP and HILIC is overcome through the use of constant-pressure online solvent mixing, which allows our system to perform efficient separations of both hydrophilic and hydrophobic compounds for mass spectrometry-based proteomics applications. When analyzing the tryptic digests of bovine serum albumin, ribonuclease B, and horseradish peroxidase, we observed near-identical coverage of peptides and glycopeptides when using online RP-HILIC—with only a single sample injection event—as we did from two separate RP and HILIC analyses. The coupled system was also capable of concurrently characterizing the peptide and glycan portions of deglycosylated glycoproteins from one injection event, as confirmed, for example, through our detection of 23 novel glycans from turkey ovalbumin. Finally, we validated the applicability of using RP-HILIC for the analysis of highly complex biological samples (mouse chondrocyte lysate, deglycosylated human serum). The enhanced coverage and efficiency of online RP-HILIC makes it a viable technique for the comprehensive separation of components displaying dramatically different hydrophobicities, such as peptides, glycopeptides, and glycans.     

Selectivity differences in the separation of amphipathic alpha-helical peptides during reversed-phase liquid chromatography at pHs 2.0 and 7.0: effects of different packings, mobile phase conditions and temperature

In an ongoing effort to understand the effect of varying reversed-phase high-performance liquid chromatography (RP-HPLC) parameters on the retention behaviour of peptides, necessary for the rational development of separation/optimization protocols, we believe it is important to delineate the contribution of alpha-helical structure to the selectivity of peptide separations. The present study reports the effects of varying column packing, mobile phase conditions and temperature on RP-HPLC retention behaviour at pHs 2.0 and 7.0 of peptides based on the amphipathic peptide sequence Ac-EAEKAAKEXEKAAKEAEK-amide (with position X in the centre of the hydrophobic face of the alpha-helix), where position X is substituted by L- or D-amino acids. At pH 2.0, an increase in trifluoroacetic acid concentration or the addition of sodium perchlorate to a phosphoric acid-based mobile phase had the similar effect of improving peak shape as well as increasing peptide retention time due to ion-pairing effects with the positively-charged peptides; in contrast, at pH 7.0, the addition of salt had little effect save an improvement in peak shape. Temperature was shown to have a complex influence on peptide selectivity due to varying effects on peptide conformation. In addition, subtle effects on peptide selectivity were also noted based on the column packings employed at pHs 2.0 and 7.0.     

Separation efficiencies in hydrophilic interaction chromatography

Hydrophilic interaction chromatography (HILIC) is important for the separation of highly polar substances including biologically active compounds, such as pharmaceutical drugs, neurotransmitters, nucleosides, nucleotides, amino acids, peptides, proteins, oligosaccharides, carbohydrates, etc. In the HILIC mode separation, aqueous organic solvents are used as mobile phases on more polar stationary phases that consist of bare silica, and silica phases modified with amino, amide, zwitterionic functional group, polyols including saccharides and other polar groups. This review discusses the column efficiency of HILIC materials in relation to solute and stationary phase structures, as well as comparisons between particle-packed and monolithic columns. In addition, a literature review consisting of 2006-2007 data is included, as a follow up to the excellent review by Hemstr?m and Irgum.     

Retention behavior of peptides in hydrophilic-interaction chromatography

Selected hydrophilic interaction chromatography (HILIC) columns packed with bare silica, bridge-ethyl hybrid silica, or an amide sorbent chemistry were utilized for an investigation of chromatographic behavior and separation selectivity of tryptic peptides. Retention model was proposed allowing for retention prediction of peptides with correlation coefficient R(2)~0.92-0.97 for various columns. The values of optimized amino acid retention coefficients were compared to those obtained for reversed-phase liquid chromatography (Gilar et al., Anal. Chem. 2010, 82, 265-275) and used to elucidate the impact of different amino acid on peptide HILIC retention. In contrast to reversed-phase chromatography, where presence of Phe, Trp, Ile, and Leu amino acid residues in sequence strongly promoted, and presence of hydrophilic His, Lys and Arg residues strongly reduced peptide retention, the effects of these amino acid residues in HILIC were opposite (His, Lys and Arg promote, Phe, Trp, Ile and Leu demote peptide retention in HILIC). Retention coefficient optimized for pH experiments illustrated the impact of silanols on HILIC retention.     

Evaluation of hydrophilic interaction chromatography versus reversed-phase chromatography in a plant metabolomics perspective

The metabolomics goal, the unbiased relative quantification of all metabolites in a biological system, still lacks a universal analytical approach. In the LC-MS line of approach, one of the major problems encountered is the polar nature of a large group of (plant) metabolites. Here, we investigate the potential of hydrophilic interaction chromatography (HILIC) and compare its qualities with extended polarity RP chromatography. Two opposite LC phase compositions (Atlantis dC18 vs. TSKgel Amide-80) are compared in a plant metabolomics setting. Both performed equally well with regard to retentive capacities, but variation in peak area was about 5% higher for the HILIC approach. Focussing on matrix effects (ME) on the other hand, it was observed that this well-known problem in RP LC-MS metabolomics was not reduced on using hydrophilic interaction chromatography.     

Temperature selectivity effects in reversed-phase liquid chromatography due to conformation differences between helical and non-helical peptides

In order to characterize the effect of temperature on the retention behaviour and selectivity of separation of polypeptides and proteins in reversed-phase high-performance liquid chromatography (RP-HPLC), the chromatographic properties of four series of peptides, with different peptide conformations, have been studied as a function of temperature (5-80 degrees C). The secondary structure of model peptides was based on either the amphipathic alpha-helical peptide sequence Ac-EAEKAAKEX(D/L)EKAAKEAEK-amide, (position X being in the centre of the hydrophobic face of the alpha-helix), or the random coil peptide sequence Ac-X(D/L)LGAKGAGVG-amide, where position X is substituted by the 19 L- or D-amino acids and glycine. We have shown that the helical peptide analogues exhibited a greater effect of varying temperature on elution behaviour compared to the random coil peptide analogues, due to the unfolding of alpha-helical structure with the increase of temperature during RP-HPLC. In addition, temperature generally produced different effects on the separations of peptides with different L- or D-amino acid substitutions within the groups of helical or non-helical peptides. The results demonstrate that variations in temperature can be used to effect significant changes in selectivity among the peptide analogues despite their very high degree of sequence homology. Our results also suggest that a temperature-based approach to RP-HPLC can be used to distinguish varying amino acid substitutions at the same site of the peptide sequence. We believe that the peptide mixtures presented here provide a good model for studying temperature effects on selectivity due to conformational differences of peptides, both for the rational development of peptide separation optimization protocols and a probe to distinguish between peptide conformations.     

Polarity‐based fractionation in proteomics: hydrophilic interaction vs reversed‐phase liquid chromatography

During recent decades, hydrophilic interaction liquid chromatography (HILIC) ahs been introduced to fractionate or purify especially polar solutes such as peptides and proteins while reversed‐phase liquid chromatography (RPLC) is also a common strategy. RPLC is also a common dimension in multidimensional chromatography. In this study, the potential of HILIC vs RPLC chromatography was compared for proteome mapping of human peripheral blood mononuclear cell extract. In HILIC a silica‐based stationary phase and for RPLC a C₁₈ column were applied. Then separated proteins were eluted to an ion trap mass spectrometry system. Our results showed that the HILIC leads to more proteins being identified in comparison to RPLC. Among the total 181 identified proteins, 56 and 38 proteins were fractionated specifically by HILIC and RPLC, respectively. In order to demonstrate this, the physicochemical properties of identified proteins such as polarity and hydrophobicity were considered. This analysis indicated that polarity may play a major role in the HILIC separation of proteins vs RPLC. Using gene ontology enrichment analysis, it was also observed that differences in physicochemical properties conform to the cellular compartment and biological features. Finally, this study highlighted the potential of HILIC and the great orthogonality of RPLC in gel‐free proteomic studies. Copyright © 2015 John Wiley & Sons, Ltd.