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Electrostatic Repulsion Hydrophilic Interaction Chromatography (ERLIC)
Phosphopeptides, isolation, separations, hydrophilic interaction, electrostatic repulsion, mobile phase, nucleotides, nucleic acids, amino acids, peptides, proteins.
Electrostatic Repulsion Hydrophilic Interaction Chromatography is a new mode of chromatography. It permits some separations to be done isocratically that normally would require a gradient. It also permits the selective isolation of phosphopeptides from a tryptic digest. ERLIC, the column is of the same charge as the sample solutes.
The mobile phase contains enough organic solvent so that hydrophilic interaction keeps the solutes on the column despite the electrostatic repulsion. The pH of the mobile phase is selected to ensure that the solutes do have the same charge as the column. When the best-retained solutes in a mixture are highly charged, this combination can be used to selectively antagonize their retention.
ERLIC (electrostatic repulsion hydrophilic interaction chromatography) term is coined in 2008 by Alpert, for HILIC separations where an ionic column surface chemistry is used to repel a common ionic polar group on an analyte or within a set of analytes, to facilitate separation by the remaining polar groups.
Electrostatic effects have an order of magnitude stronger chemical potential than neutral polar effects. This allows one to minimize the influence of a common, ionic group within a set of analyte molecules; or to reduce the degree of retention from these more polar functional groups, even enabling isocratic separations in lieu of a gradient in some situations.
His subsequent publication further described orientation effects which others have also called ion-pair normal phase or e-HILIC, reflecting retention mechanisms sensitive to a particular ionic portion of the analyte, either attractive or repulsive.
ERLIC (eHILIC) separations need not be isocratic, but the net effect is the reduction of the attraction of a particularly strong polar group, which then requires less strong elution conditions, and the enhanced interaction of the remaining polar (opposite charged ionic, or non-ionic) functional groups of the analytes.
One could use a cation exchange (negatively charged) surface chemistry for ERLIC separations to reduce the influence on retention of anionic (negatively charged) groups (the phosphates of nucleotides or of phosphonyl antibiotic mixtures; or sialic acid groups of modified carbohydrates) to now allow separation based more on the basic and/or neutral functional groups of these molecules.
Modifying the polarity of a weakly ionic group (carboxyl) on the surface is easily accomplished by adjusting the pH to be within two pH units of that group's pKa. For strongly ionic functional groups of the surface (sulfates or phosphates) one could instead use a lower amount of buffer, so the residual charge is not completely ion paired.
An example of this would be the use of a 12.5mM (rather than the recommended >20mM buffer), pH 9.2 mobile phase on a polymeric, zwitterionic, betaine-sulfonate surface to separate phosphonyl antibiotic mixtures (each containing a phosphate group). This enhances the influence of the column's sulfonic acid functional groups of its surface chemistry over its, slightly diminished (by pH), quaternary amine.
Commensurate with this, these analytes will show a reduced retention on the column eluting earlier, and in higher amounts of organic solvent, than if a neutral polar HILIC surface were used. This also increases their detection sensitivity by negative ion mass spectrometry.
By analogy to the above, one can use an anion exchange (positively charged) column surface chemistry to reduce the influence on retention of cationic (positively charged) functional groups for a set of analytes, such as when selectively isolating phosphorylated peptides or sulfated polysaccharide molecules.
Use of a pH between 1 and 2 pH units will reduce the polarity of two of the three ionizable oxygens of the phosphate group, and thus will allow easy desorption from the (oppositely charged) surface chemistry. It will also reduce the influence of negatively charged carboxyl’s in the analytes, since they will be protonated at this low pH value, and thus contribute less overall polarity to the molecule.
Any common, positively charged amino groups will be repelled from the column surface chemistry and thus these conditions enhance the role of the phosphate's polarity (as well as other neutral polar groups) in the separation.
ERLIC has proved to be effective if not superior for tryptic peptide fractionation and has been applied successfully with various biological samples such as serum, milk, and tissue extracts. Peptides are fractionated according to isoelectric point and polarity.
It also allows the enrichment of phosphopeptides and has also been utilized to enrich other PTMs such as glycosylated and deamidated peptides. ERLIC is expected to be utilized further in studies of PTMs to identify novel modified sites as potential biomarkers for diseases. In addition, simultaneous analysis of the proteome and post translational modifications has been achieved. ERLIC has been shown to perform as well or better than methods currently in widespread use for various applications.
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