Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds

Alpert, Andrew J.

doi:10.1016/s0021-9673(00)96972-3

Cited by 1,814 publications

(1,071 citation statements)

References 58 publications

Supporting

Mentioning

1,016

Contrasting

Unclassified

Order By: Relevance

“…Hydrophilic interaction chromatography (HILIC) is a separation technique that is used for the separation of polar biomolecules [97]. The principle of this method is that peptides in an organic solution (mobile phase) bind to a neutral, hydrophilic stationary phase by hydrogen bonding.…”

Section: Hydrophilic Interaction Chromatographymentioning

confidence: 99%

Analytical strategies for phosphoproteomics

2009

View full text Add to dashboard Cite

Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events in which the complex interplay between protein kinases and protein phosphatases strictly controls biological processes such as proliferation, differentiation, and apoptosis. Defective or altered signaling pathways often result in abnormalities leading to various diseases, emphasizing the importance of understanding protein phosphorylation. Phosphorylation is a transient modification, and phosphoproteins are often very low abundant. Consequently, phosphoproteome analysis requires highly sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phosphospecific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis will be on the affinity methods utilized specifically for phosphoprotein and phosphopeptide enrichment prior to MS analysis, and on recent applications of these methods in cell biological applications.

show abstract

Section: Hydrophilic Interaction Chromatographymentioning

confidence: 99%

Analytical strategies for phosphoproteomics

2009

View full text Add to dashboard Cite

show abstract

“…It can be utilized in optimizing information regarding the constituents of a polar mixture, especially of small molecules [1][2][3][4][5].…”

Section: Introductionmentioning

confidence: 99%

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

Bobály

D’Atri

Beck³

et al. 2017

Journal of Pharmaceutical and Biomedical Analysis

View full text Add to dashboard Cite

a b s t r a c tHydrophilic 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.

show abstract

“…The term HILIC was originally introduced to describe separations based on solute hydrophilicity [16]. Thus, separation by HILIC, in a manner similar to normal-phase chromatography (to which it is related), depends on hydrophilic interactions between the solutes and a hydrophilic stationary phase, i. e. solutes are eluted in order of increasing hydrophilicity (decreasing hydrophobicity).…”

Section: Principles Of Hilic/cex Versus Rp-hplcmentioning

confidence: 99%

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

Mant

Hodges²

2008

J of Separation Science

View full text Add to dashboard Cite

Mixed-mode hydrophilic interaction/cationexchange 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. IntroductionMixed mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) is an HPLC approach to peptide separations introduced by our laboratory over 15 years ago [1]. HILIC/CEX combined the most advantageous aspects of two widely different separation mechanisms: a separation based on hydrophilicity/hydrophobicity differences between peptides overlaid on a separation based on net charge [1 -8]. Characteristic of HILIC/ CEX separations is the presence of a high organic modifier concentration (generally, ACN) to promote hydrophilic interactions between the solute and the hydrophilic/charged CEX stationary phase, with peptides then eluted from the column with a salt gradient. Peptides are generally eluted in groups of peptides in order of increasing net positive charge; within these groups, peptides are eluted in order of increasing hydrophilicity (decreasing hydrophobicity). Indeed, HILIC/CEX is basically CEX in the presence of high concentrations of ACN (60 -80%, in general).The HILIC/CEX approach has proven to be very versatile for applications to peptide separations, representing an excellent complement to RP-HPLC and, indeed rivalling or even exceeding RP-HPLC for specific peptide mixtures [2,3,6,9]. For example, HILIC/CEX has successfully separated deletion products from crude synthetic peptides (not achievable by RP-HPLC) [3] as well as enabled the purification of synthetic peptides from closely related serine side-chain acetylated peptides (again not achievable by RP-HPLC) [6]. The complementary nature of HILIC/ CEX and RP-HPLC was also demonstrated by the ...

show abstract

Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds

Cited by 1,814 publications

References 58 publications

Analytical strategies for phosphoproteomics

Analytical strategies for phosphoproteomics

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

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

Contact Info

Product

Resources

About