What is HILIC?

Hydrophilic Interaction Liquid Chromatography (HILIC) is a version of normal phase liquid chromatography which can  be performed with partially aqueous mobile phases. The name HILIC was suggested by Dr. Andrew Alpert of PolyLC in his 1990 paper on the subject[1].
He described the chromatographic mechanism for it as liquid-liquid partition chromatography

When to use HILIC process?

The HILIC process is used increasingly as a normal phase separation method to  separate  some biomolecules including

• peptides, phosphopeptides, crude  extracts, peptide digests,
• carbohydrates,
• histones
• polar lipids
• oligonucleotides and their antisense analogs
• nucleic acids and
• many proteins.  membrane proteins, 

by their differences in polarity. Furthermore some organic and some inorganic molecules[2] can also be separated successfully with .

Its utility has increased due to the simplified sample preparation for biological samples, when analysing for metabolites, since the metabolic process generally results in the addition of polar groups to enhance elimination from the cellular tissue. For the detection of polar compounds with the use of electrospray-ionization mass spectrometry as a chromatographic detector, HILIC can offer a tenfold increase in sensitivity over reversed-phase chromatography[2] because the organic solvent is much more volatile

Stationery  phases to be used?

Any polar chromatographic surface can be used for HILIC separations. Even non-polar bonded silicas have been used with extremely high organic solvent composition, when the silica used for the chromatographic media was particularly polar. With that exception, HILIC phases can be grouped into five categories of neutral polar or ionic surfaces:

    * simple unbonded silica silanol or diol bonded phases
    * amino or anionic bonded phases
    * amide bonded phases
    * cationic bonded phases
    * zwitterionic bonded phases.

Mobile phase to be used

A typical mobile phase for HILIC chromatography includes acetonitrile ("MeCN", also designated as "ACN") with a small amount of water. However, any aprotic solvent miscible with water (e.g. THF or dioxane) can be used. Alcohols can also be used, however, their concentration must be higher to achieve the same degree of retention for an analyte relative to an aprotic solvent - water combination. See also Aqueous Normal Phase Chromatography
 

Additives

Ionic additives, such as ammonium acetate and ammonium formate, are usually used to control the mobile phase pH and ion strength. In HILIC they can also contribute to the polarity of the analyte, resulting in differential changes in retention. For extremely polar analytes (e.g. aminoglycoside antibiotics (gentamicin or Adenosine triphosphate), higher concentrations of buffer (ca. 100mM) are required to assure that the analyte will be in a single ionic form. Otherwise asymmetric peak shape, chromatographic tailing, and/or poor recovery from the stationary phase will be observed. For the separation of neutral polar analytes (e.g. carbohydrates), no buffer is necessary.

Use of other salts such as 100-300mM sodium perchlorate, which are soluble in high-organic solvent mixtures (ca. 70% acetonitrile), can be used to increase the mobile phase polarity to effect elution. These salts are not volatile, so this technique is less useful with a mass spectrometer as the detector. Usually a gradient (to increasing amounts of water) is enough to promote elution.

All ions partition into the stationary phase to some degree, so an occasional "wash" with water is required to ensure a reproducible stationary phase.

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.

.It is commonly believed that in HILIC, the mobile phase forms a water-rich layer on the surface of the polar stationary phase vs. the water-deficient mobile phase, creating a liquid/liquid extraction system. The analyte is distributed between these two layers. However, HILIC is more than just simple partitioning and includes hydrogen donor interactions between neutral polar species as well as weak electrostatic mechanisms under the high organic solvent conditions used for retention. This distinguishes HILIC as a mechanism distinct from ion exchange chromatography. The more polar compounds will have a stronger interaction with the stationary aqueous layer than the less polar compounds. Thus, a separation based on a compound's polarity and degree of solvation takes place.

 Dual process possibilities with  PolySULFOETHYL or PolyHYDROXYETHYL Aspartamide

The inventor of HILIC . PolyLC (Dr. Andrew Alpert ) has two stationery phases for this process as follows:There  are two column choices to perform HILIC separations, and can be extended to perform additional separation mechanism 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 . Under non-HILIC conditions (mobile phase  concentrations less than 40% acetonitrile) the column will perform  small-molecule size exclusion separation. (SEC)
The second column is  the PolySULFOETHYL Aspartamide SCX column which performs either  hydrophilic interaction separations  superimposed upon electrostatic  effects under HILIC conditions as above, or a cation exchange mixed-mode  separation where resolution is enhanced for peptides with the same  net positive charge under non-HILIC conditions.

Operating  Recommendations for HILIC Separations

 Initial Use
When using either column to perform HILIC separations, flush the  new  column with 25 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 5-10 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 . 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 is 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 Miscellaneous 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.

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 SEC Separations
 

Background on SEC Use
The PolyHYDROXYETHYL Aspartamide column was created specifically  to perform HILIC and retain solutes solely through hydrophilic interactions.  However, when used with a mobile phase which does not contain enough  organic solvent to induce hydrophilic interaction, then it  functions  in the size exclusion chromatography mode. Swelling the coating  with a suitable mobile phase causes the effective pore diameter  to become the spacing between polymer chains of the coating (~15Å),  allowing solutes as small as water to be separated by size.
The HEA column is available in three pore sizes, 200Å, 300Å and  1000Å. A column with 200Å pores has a fractionation range of 20-10,000  MW allowing resolution of the smallest of bioorganic  molecules.  The 300Å pore size accommodates a range of 20-80,000, and the 1000Å  pore material can fractionate over a size range of 1,000 - 2,000,000,  (over 5 orders of magnitude). The HEA columns may also be used with  volatile mobile phases.

Start-up
Flush new columns (4.6 mm ID) with 25 ml water, then condition with  at least 60 ml of a buffer solution with salt concentration of 0.2  M and a pH in the range  of 3-6 (exact figures are not important  here). Flush with another 20 ml water, then equilibrate for six  hours (flow rate 0.5 ml/min) using one of the mobile phases recommended  below before injecting samples. (For 9.4 mm ID columns, the above  volumes should be multiplied by a factor of 4, and the flow rate  for equilibration is 2 ml/min.) It is not necessary to repeat this  conditioning step thereafter unless the column is flushed with organic  solvent  for long-term storage or used under HILIC conditions.
HEA Columns will exhibit two different fractionation ranges, depending  on the mobile phase used. For a mobile phase of 0.2 M (Na)2SO4 +  5 mM K-PO4, pH 3.0, containing 25% acetonitrile, the fractionation  range will be approximately Mol. Wt. 400-10,000 for columns with  200Å pores. For HEA columns with 300Å pores, the fractionation range  is approximately MW 1000-200,000.
For a mobile phase of 50 mM formic  acid, the fractionation range  will be approximately MW 20 - 1000 for columns with 200Å pores.  For 300Å columns, MW 20 - 80,000. The same column can be used for  both fractionation ranges, simply by switching between these two  mobile phases. The formic acid mobile phase is volatile, but precludes  detection below 240 nm. The use of volatile mobile phases which  are transparent at 215 nm (e.g. hexafluoro-isopropanol, HFIP) is  experimental and hazardous,  although it has the same effect on the  fractionation range as formic acid.

Sample  Composition
The sample solvent should not differ greatly from the mobile phase  in ionic strength or organic solvent content, in order to prevent  a significant difference in viscosity of the two. With high viscosity,  solute molecules might not diffuse from the mobile phase to the  stationary phase before the sample passes  through the column. The  loading capacity of a 4.6 mm I.D. column is roughly 0.4 - 0.8 mg  peptide with no significant loss of resolution, but this number  depends on the composition of the sample.

Operating  Recommendations for SCX Separations

The PolySULFOETHYL Aspartamide SCX column in the ion exchange mode  is useful for n-terminal variant analysis, neuropeptides, growth  factors, CNBr peptide  fragments, and synthetic peptides as a complement  to RPC.

Initial  Use
Flush the methanol storage solvent from the column with at least  40 ml water before elution with salt solution to prevent salt precipitation.  Then elute the column with a strong buffer for at least one hour  prior to its initial use. A convenient solution to use is 0.2 M  monosodium phosphate + 0.3 M sodium acetate.

Routine  Use
Filter samples and mobile phases before use. Flush and store the  column in water when not in use. Operation at room temperature is  recommended, since elevated temperature shortens column life-time.  Use of 0.1% TFA or high concentrations of formic acid in the mobile  phase is not recommended. The conditioning process is reversed by  exposing the column to pure organic solvents. Accordingly, to minimize  the time  to start the column for a 1-2 day storage, the column should  be flushed with at least 40 ml of deionized water (not methanol),  and the ends should be plugged. For extended storage it is recommended  that a 100% methanol storage be used to prevent bacterial growth  and contamination. Exercise care when using organic solvents to  prevent precipitation of salts.

General  Operation in the  SCX Mode
By varying the pH, ionic strength or organic solvent concentration  in the mobile phase, selectivity can be significantly enhanced.  Mobile phase modifiers help to improve peptide solubility or to  mediate the interaction between peptide and stationary phase. For  more strongly hydrophobic peptides, a non-ionic surfactant (at a  concentration below its CMC) and/or acetonitrile or n-propanol as  mobile phase modifiers, can substantially improve  resolution and  recovery over conventional reverse phase methods. You may obtain  additional selectivity by simply changing the slope of the KCl or  (NH4)2SO4 gradient.
Using this column at pH 3 is better for retention of neutral to  slightly acidic peptides. Use of a higher pH may be considered for  basic hydrophobic peptides.
Adding MeCN or propanol to the A&B solvents changes the separation  mechanism and results in a separation based not only on  positive  charge, but also on hydrophobicity. Experimentation with organic  content is encouraged.
One set of operating conditions for these applications for an analytical  column would be:
Buffer A: 5 mM K-PO4 + 25% MeCN;
Buffer B: 5 mM K-PO4 + 25% MeCN + 300-500 mM KCl;
Linear gradient, 30 min at 1 ml/min.
Peptides are retained by the positive charge of at least the n-terminal  amine and eluted by a combination of total charge, charge  distribution  and hydrophobicity. If your peptide does not stick to the column,  be sure it is in a small amount of buffer, or decrease the concentration  of organic in the A&B solvents to 10 or 5%. (Organic solvent concentration  is empirically determined).
Since total binding capacity is on the order of 100 mg/gm of packing  (for nonresolved materials) there will be a considerable Donan effect  present. To prevent exclusion from the column put your  sample in  5-15 mM of salt or buffer. Additionally, the gradient at the outlet  of the column will be much more concave than that observed on the  chart paper. Consequently, if you have had no prior experience using  this column, we recommend following a standard methods development  protocol to be sure that your protein is eluting properly (request  publication P4). We recommend an upper load limit of 1 milligram  for an analytical column, although up to 40  mg of a soluble peptide  has been separated on one.
When using a guard column as a methods development column, we recommend  a load limit of one-tenth of a milligram with gradient times shortened  to 8-10 min at the same flow rate since the void volume is only  0.3 ml.

References
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).  doi:10.1016/S0021-9673(00)96972-3.
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.
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).
a b Eric S. Grumbach et al. (October 2004). "Hydrophilic Interaction Chromatography Using Silica Columns for the Retention of Polar Analytes and Enhanced ESI-MS Sensitivity". LCGC Magazine. http://www.lcgcmag.com/lcgc/issue/issueDetail.jsp?id=4734. Retrieved on 2008-07-14.