Schematic of the solvent range for partition systems.

HILIC is primarily a partition system which is useful for the retention and separation of polar analytes with neutral to negative distribution coefficients (LogD values) for both charged and uncharged species, or partition coefficients (LogP values) of uncharged species.

Schematic of the polarity range for effective HILIC separations.

ERLIC (also called eHILIC and Ion-Pair Normal Phase) is a subset of HILIC separations which employs charged interactions and their subsequent Orientation Effects. A charged functional group on a column's surface's chemistry has an order of magnitude greater free energy of interaction than that of an uncharged analyte.

Repulsive Subtractive eHILIC

Effects of pH and Buffer Strength

1) Many gradient separations become isocratic separations;
2) Phosphopeptides can be isolated selectively from tryptic digests and separated with high resolution.
3) Phospholipid-glycopeptides can be separated with high resolution of the glycopeptide portion.
4) Phosphonyl antibiotic mixtures can be resolved based on the non-phosphorylated portion of the molecule.

Ionizable biochemicals with a high charge are generally significantly better retained in chromatography than compounds of low charge (e.g. ATP vs. AMP), and those charges tend to dominate the mode of interaction with the surface chemistry to the detriment of other functional groups. This is true in both ion-exchange and hydrophilic interaction chromatography (HILIC). A gradient is necessary to make both types of compounds elute in the same time frame. However, if HILIC is performed using an ion-exchange column of the same charge as the most highly-charged functional groups, then their retention is selectively antagonized by electrostatic repulsion (ERLIC). This permits their elution in the same time frame as less highly-charged compounds using isocratic conditions, or with lower amounts of buffer, and it allows the chromatography to enhance the presence of oppositely charged or neutral polar functional groups within the molecule.

The polarity of the surface chemistry of the PolyHYDROXYETHYL A™ column ( poly-2-hydroxyethyl aspartamide) is modifiable by pH. In this respect it is analogous to a weak ion exchange chemistry, where elution can be effected by either a pH change or by solvent polarity (buffer strength or organic solvent content). By selecting a pH of 2.7, 4.4 or 6.5 one can select a surface polarity which can compliment the polarity of the functional group in one's molecule to enhance selectivity.

In contradistinction to the PolyHYDROXYETHYL A, the zwitterionic, ZIC®-HILIC, and the inverted zwitterion, ZIC®-cHILIC, stationary phases maintain their charge over a wider pH range, analogous to a strong ion exchange chemistry. This provides a stable surface chemistry against which the polarity of the analytes alone can be modified, when trying to affect selectivity by operating at different pH's.

Schematic illustation of the zwitterionic, ZIC-HILIC, betain sulfonate stationary phase.

Schematic illustation of the inverted zwitterionic, ZIC-cHILIC, cholinate stationary phase.

Schematic illustation of the slightly negative, iHILIC® FUSION® stationary phase.

Schematic illustation of the slightly positive, iHILIC® FUSION(+)® stationary phase.

Schematic illustation of the iHILIC® FUSION(P)® stationary phase.

Schematic illustation of the low buffer, diol polymer, charge balanced (i.e. zwitterionic), iHILIC®-(P) Classic stationary phase.

A. Isocratic and Gradient Separations:

Amino acids, peptides and proteins: The most polar groups in this family of molecules are the basic, amino residues. If ERLIC is performed using a column with an amine functional group, an anion-exchange column (e.g., PolyWAX LP™) at a pH ~ 2.0 (low enough for carboxyl groups to lose their negative charge), then amino acids, peptides and proteins will have a net positive charge and will thus experience some degree of electrostatic repulsion. The most basic compounds, normally the best-retained in HILIC, experience the most repulsion in ERLIC.





Nucleotides and nucleic acids: If ERLIC is performed with a negatively charged surface chemistry, a cation-exchange column (e.g. PolySULFOETHYL A™) with TEAP buffer at a pH < 3.5, a pH at which phosphate groups have just a single negative charge [note: electrostatic repulsion is too great at a higher pH where phosphate groups acquire a second negative charge], then the long eluting tri-phosphates can be eluted with significantly less buffer (even as little as 10mM). Use of triethylamine methyl phosphonate buffer salts (which aren't volatile either), significantly increases the retention of tri-phosphorylated species vs. triethylamine phosphate. While a HILIC separation of these molecules is possible using a gradient, it requires up to 250mM ammonium acetate to elute the tri-phosphates, and the phosphate groups dominate the separation order.



However, with the use of a polymer based, zwitterionic, HILIC chemistry (iHILIC® Fusion(P)) at a pH nearer the pKa of the amine of the surface, a more negative surface chemistry is formed. This permits a similar Adenosine Phosphate analysis (AMP, ADP, ATP, CoA, Acetyl-CoA, and Pantothenate) using gradient ERLIC LC/MS conditions, but with volatile buffers (80% ACN, 20% 10 mM Ammonium Carbonate, 0.2% NH₄OH).

ERLIC for 2-D Proteomics Fractionation
Comparison of SCX and ERLIC in conjunction with RPC. ERLIC Yields a More Uniform Separation of Peptides

B. Selective Isolation:

ERLIC (eHILIC) for Selective Isolation of Phosphopeptides:
At pH 2.0, phosphate groups in peptides retain some of their negative charge. This does not permit the isolation by anion-exchange chromatography of singlely phosphorylated peptides from tryptic digests, since the electrostatic attraction is not sufficient to overcome the electrostatic repulsion from the N-terminus and the C-terminal Lys- or Arg- residue toward the basic functional groups of the column. However, phosphate residues are quite hydrophilic.

In the eHLIC mode, the combination of electrostatic attraction and hydrophilic interaction suffices to pull singly phosphorylated peptides away from the non-phosphorylated peptides in tryptic digests, at pH 2. Also, unlike the situation with high-affinity media such as IMAC or titania, the phosphopeptides are well-resolved from each other. This permits their convenient separation into numerous fractions, an important tool in phosphoproteomics for identifying the sequences of thousands of phosphopeptides from a single sample.

Peptides with multiple phosphate groups are retained so strongly that a salt gradient is necessary for elution as shown in the following two examples:

ERLIC separation of phospho-peptides from HeLa cells using a PolyWAX LP column, P104WX0503. It should be noted that the recovery of phosphopeptides is highest if low-salt fractions are desalted with HyperCarb® media and high-salt fractions are desalted using RPC, C-18 media.

Subtractive Isolation of Phosphopeptides:
Use of an SCX chemistry (e.g., PolySULFOETHYL A™) for fractionation of a tryptic digest of a phosphorylated protein can segregate the phosphorylated peptides. They elute at the beginning of the gradient, while the high capacity SCX column holds up the more positively charged species. Take care to lyophillize the ammonium bicarbonate two to three times with methanol to reduce the buffer-salt content of the sample, otherwise doubly charged peptides can contaminate these single charged peptides in the first fraction.

Comparisons of ERLIC (eHILIC) Approaches for Phosphopeptides:

• Comparison of SCX, TiO, HILIC (HEA) and HILIC (WAX) for phosphopeptide segregation and/or fractionation shows the value of ERLIC surfaces for reducing the amount of buffer necessary for eluting phosphopeptides compared to using other HILIC surface chemistries.
• Comparison of SCX and ERLIC phospho enrichment strategies. Kehasse, A., et al., Boston University School of Medicine, ASMS-2011 Poster.
• ERLIC outperforms TiO₂ - MOAC in quantitative phospho-proteomics with low sample amounts. Comparison of PolyWAX LP® to titanium dioxide

ERLIC (eHILIC) for Glycopeptide Isolation:

• "Simultaneous Identification of Unmodified Tryptic Peptides, Phosphopeptides, Glycopeptides, and Deamidated Peptides" (2011 ASMS, Siu Kwan Sze Poster, Nanyang Technological University, Singapore).
• Comparison of Hydrazide Covalent Chromatography and ERLIC for Glycopeptide Separations
• Effect of Pore Size on Glycan Separations