How HILIC works
HILIC separates compounds by passing a hydrophobic or mostly organic mobile phase across a neutral hydrophilic stationary phase, causing solutes to elute in order of increasing hydrophilicity-the inverse of RPC. With neutral peptides one may use 15mM ammonium formate and reverse organic conditions. Highly charged molecules require low amounts (e.g., 10 mM) of salt for ion suppression, and a slight perchlorate or sulfate gradient (in a high organic solvent concentration) to effect desorption.
HILIC or RPC for Polar Molecules
If you are trying to increase retention of hydrophilic molecules by RPC, there is a versatile, effective alternative to consider: hydrophilic interaction chromatography (HILIC). A rival technique to RPC for separating polar peptides, HILIC is easy to use and works best where RPC works worst: with polar solutes which aren't retained well on RPC. HILIC has been used successfully with phosphopeptides, crude extracts, peptide digests, membrane proteins, carbohydrates, histones, oligonucleotides and their anti sense analogs, polar lipids and in preparative applications where changing the order of elution affects isolation yields.
HILIC using PolyCAT A™ or PolySULFOETHYL A™, PolyHYDROXYETHYL A™, and PolyWAX™ LP
There are many column surfaces to perform HILIC separations, and each offers alternative separation mechanisms for added versatility. The PolyHYDROXYETHYL Aspartamide™ column (HEA) will retain solutes solely through hydrophilic interactions when using mobile phase concentrations in the range of 40-85% acetonitrile. At low pH the column has a slight positive charge, at pH 4.5 it is neutral, and at pH 6.5-7.0 there is a slight negative charge. Under non-HILIC conditions (mobile phase concentrations less than 40% acetonitrile) the column will perform small-molecule size exclusion separation. 1
A second chemistry choice is either the PolySULFOETHYL Aspartamide™ SCX column or PolyCAT A™ column, which perform hydrophilic interaction separations superimposed upon electrostatic effects under HILIC conditions as above, or cation exchange mixed-mode separations 2 where resolution is enhanced for peptides with the same net positive charge under non-HILIC conditions.
The use of an anion exchange column, PolyWAX™ LP, provides a net positive surface at pH <7.5 for enhanced selectivity of acidic molecules where the differences are concentrated at the acidic sites of the molecules.
Operating Recommendations for HILIC Separations
Initial Use of PolyHYDROXYETHYL A (Mfg. Instruction Sheet): When using either column to perform HILIC separations, flush the new column with 30 mL water, and then condition with at least 60 mL of a buffer solution with a salt concentration of 0.2 - 0.4 M and a pH in the range of 3 - 6 (exact figures are not important here). Flush again with another 20 ml water, then equilibrate with 30 ml of the mobile phase before injecting samples. (These volumes relate to 4.6 mm ID columns. For 9.4 mm ID columns, the volumes above should be multiplied by a factor of four). To prepare the HEA column for size exclusion chromatography or the SCX column for ion exchange chromatography, see the appropriate section below).
Routine Use: Filter samples and mobile phases before use. Flush and store HILIC columns in water when not in use. Operation at room temperature is recommended, since elevated temperature shortens column lifetime.
General Mode of Operation: Salt is not required with solutes that are not electrolytes. In the case of electrolytes, use at least 25 mM buffer in the mobile phase. Gradient elution can be accomplished by a decreasing organic gradient (starting from 80-85% acetonitrile for peptides or 95% for phospholipids) or an increasing salt gradient (which typically gives flatter baselines). Solubility of salts can be a problem with mostly organic mobile phases, but sodium perchlorate works well and is transparent at low wavelengths (Fisher sells an HPLC grade). Buffer salts with reasonable solubility in 80% acetonitrile include triethylamine phosphate (TEAP) and sodium methylphosphonate (from methylphosphonic acid). Isocratic retention is typically several times greater with TEA salts than with the corresponding sodium or potassium salt. With 80% acetonitrile, concentrations of 75 mM (pH 5.0) or 100 mM (pH 2.8) TEAP are attainable. Gradient elution in HILIC generally requires one-fifth to one-tenth the concentration of salt required in ion-exchange chromatography.
A stock solution of TEAP can be prepared by making a concentrated aqueous solution of phosphoric acid, adding TEA until the desired pH is attained, then diluting to give a known final concentration (e.g., 2 M in phosphate). Similar methods are used with phosphonate buffers. Prepare stock solutions fresh monthly and store in the refrigerator. For preparation of the mobile phase, add the appropriate amount of stock solution and water to a volumetric flask. Next, add the acetonitrile to a level several mL short of the mark. Mix, then put the flask in a sonication bath for 5 minutes, this degasses and warms the solution. Finally, add acetonitrile to the mark and mix.
Organic solvents such as isopropanol can be used as alternatives to acetonitrile, but higher concentrations are usually required to attain the same degree of retention, and the resulting mobile phases are appreciably more viscous.
HILIC of Peptide and Proteins
A good mobile phase to try is 10 mM TEA, pH 2.8, containing 80% acetonitrile. Run gradients as described above. If retention in inadequate, try 85% acetonitrile.
The following factors affect retention of peptides in HILIC:
1. Retention is proportional to the hydrophilicity of a peptide: Basic groups are the most hydrophilic, followed by phosphorylated residues. Thereafter, retention follows the opposite trend seen with reversed-phase HPLC: Asn promotes retention the most, followed by Ser-, Gly-, etc., with Phe- and Leu- promoting retention the least.
2. Juxtaposition of an acidic and basic residue: An acidic and a basic residue, or an acidic residue as the N-terminus, largely eliminates the normal retention effects of a basic residue.
3. Change in polarity with a change in pH: At pH 2.8, only basic and phosphorylated groups will be charged. At pH 5.0, both acidic and basic residues will be charged. This factor can be used to manipulate selectivity.
4. Retention proportional to the number of basic residues: In general, at pH 2.8 peptides will elute in order of increasing number of basic residues, as do cation-exchange separations. However, unlike cation-exchange, a particularly hydrophilic peptide can be retained more strongly than a hydrophobic peptide with more basic residues. Thus, the selectivity of the two methods is complementary.
HILIC of Sugars and Oligosaccharides:
No salt is necessary unless the carbohydrate is charged. The mobile phase should contain 80-85% acetonitrile (with much lower levels used with amino- sugars). Anomeric forms of reducing sugars are resolved.
HILIC of Oligonucleotides
Try a salt gradient in 75% acetonitrile. C and G are retained much more than A and T, and may necessitate lower levels of acetonitrile.
HILIC of Phospholipids
Try a mobile phase of 15 mM ammonium formate pH 6.5 and 95% acetonitrile decreasing to 50%. Selectivity depends upon the pH and ionic strength.
HILIC of Drugs, Small Molecules and Metabolites
Retention will be the opposite of that with reverse-phase HPLC. Initially, try mobile phases with 80% acetonitrile. Some experimentation with the salt level and pH will be necessary in each case. Use of 60Å pore columns for underivatized amino acids and folic acid metabolites gives better retention.
Volatile Mobile Phases and Sequencing
The presence of 5-10 mM nonvolatile buffer salt does not interfere with many sequencing techniques for peptides. If a completely volatile mobile phase is needed, as for mass spectroscopy, then ammonium formate can be used as the buffer salt, with a descending acetonitrile gradient. Unfortunately, formate absorbs and gives baseline artifacts in gradient elution at low wavelengths. No such problems are encountered at 254 or 280 nm.
NOTE: If the mobile phase contains over 80% organic solvent, then the sample should contain at least 70%. Otherwise, pure solutes may elute in multiple peaks.
Operating Recommendations for SCX Separations
The PolySULFOETHYL Aspartamide SCX column in the ion exchange mode is useful for 2-D HPLC LC MS/MS in proteomics, n-terminal variant analysis, neuropeptides, growth factors, CNBr peptide fragments, and synthetic peptide isolation as a complement to RPC.
See PolyLC for Part Numbers and Prices.
1. Alpert, A.J., "Hydrophilic Interaction Chromatography (HILIC): A New Method for Separation of Peptides, Nucleic Acids, and Other Polar Solutes," J. Chromatogr. 499, 177-196 (1990).
2. Zhu, B., Mant, C., Hodges, R., "Mixed-Mode Hydrophilic and Ionic Interaction Chromatography Rivals Reversed-Phase Chromatography for the Separation of Peptides," J. Chromatog., 594, 75-86 (1992).
3. Boutin et al., HILIC of phosphorylated peptides and tyrosine kinase reaction mixtures, submitted to Anal. Biochem.
4. Fong et al., HILIC and SCX of Hydroxyproline-rich peptides from Douglas Fir cell wall proteins, submitted to Plant Physiol.
5. Kieliszewski et al., HILIC and SCX of Hydroxyproline-rich peptides from cell wall proteins of Zea maize (corn), submitted to Plant Physiol.
6. Przysiecki et al., HILIC, SEC and SCX of recombinant antistasin with a preproleader sequence, submitted to Arch. Biochem. Biophys. p> 7. Przysiecki et al., HILIC, SEC and SCX of recombinant antistasin with a preproleader sequence, submitted to Arch. Biochem. Biophys.
8. Przysiecki et al., Characterization of Recombinant Antistasin Secreted by Saccharomyces cerevisiae, "Protein Expression & Purification", 3,.185-195 (1992).
Last Updated: 02/25/08