PolyHydroethyl

PolyHYDROXYETHYL

for Hydrophilic Interaction Chromatography (HILIC)  and

Size Exclusion Chromatography (SEC)

Hydrophilic Interaction Liquid Chromatography (HILIC)
What is HILIC?
HILIC is a variant of normal-phase chromatography which can be performed with partially aqueous mobile phases. This permits normal-phase separation of peptides, carbohydrates, nucleic  acids, and many proteins. The elution order is least to most polar, the opposite of that in reversed-phase HPLC (RPC). The stationary phase in HILIC must be extremely polar. PolyLC has developed a material specifically for this purpose: PolyHYDROXY-ETHYL Aspartamide (or PolyHYDROXYETHYL A). It retains solutes almost solely on the basis of hydrophilic interaction. Volatile mobile phases can be used.

How to use HILIC
 Retention is proportional to the amount of organic solvent in the mobile phase (the opposite of RPC). Typical HILIC mobile phases contain 65-80% acetonitrile or propanol. Gradient elution may be performed either with a decreasing organic or increasing salt gradient. 10 mM salt is necessary with charged solutes such as peptides but is not necessary with uncharged solutes such as carbohydrates. Salts with good solubility in HILIC mobile phases include potassium methylphosphonate,  triethylamine phosphate (TEAP), and sodium perchlorate. Ammonium formate and acetate are volatile but not transparent below 230 nm; they can be used for direct mass spec analysis.

For more details on the HILIC mode, see J. Chromatogr. (1990) 177-196.

HILIC works well with:

  1. Peptide mapping.
  2. Purification of synthetic and natural peptides.
  3. Glycopeptides and phosphopeptides.
  4. Complex carbohydrates.
  5. Oligonucleotides and their analogues.
  6. Membrane proteins.

When to use HILIC:

  1. When you need a volatile phase and RPC does not suffice.
  2. With solutes too weakly or too strongly retained in RPC.
  3. For HPLC of solutes which aggregate or aren't soluble in aqueous media (eg. amyloid peptides).
  4. To separate solutes differing in a hydrophilic residue (eg. Ser-).
  5. Purifications and quality control assays which require a complementary ("orthogonal") mode.
  6. Separating  electroeluted proteins from SDS, Coomassie blue, and salts.                                                                                                            

Size Exclusion Chromatography (SEC)
PolyHYDROXYETHYL Aspartamide columns can each be used in two different fractionation ranges, merely by changing the mobile phase. With conventional salt buffers, the fractionation range is determined by the pore diameter of the packing. Nonspecific interactions with polypeptides are generally lower than with other SEC columns. If the mobile phase contains a denaturing agent (eg. 50 mM formic acid or  hexafluoro-2- propanol (HFIP), then sieving occurs between the polymer chains of the coating. This results in a dramatic shift of the fractionation range to lower values; solutes as small as formic acid can be separated by size! Moreover, these separations can be effected with volatile mobile phases.

With 60-angstrom pore column, the fractionation range is 20-600 daltons. This permits SEC of small solutes not possible heretofore. Examples include desalting a dipeptide, or  separation of small solutes from a large excess of an even smaller derivatizing agent.

Use our SEC columns for:

  • Routine SEC of enzymes and other proteins.
  • SEC of polypeptides which exhibit nonspecific interaction or poor recovery from other SEC columns.
  • Resolution of the smallest peptides and other solutes by size.
  • SEC in a volatile mobile phase. This permits direct feed to a mass spectrometer.
  • Analysis of residual monomer content of a polymer.
  • Desalting of just about anything, including removal of derivatizing reagents present in great excess.
  • Peptide mapping, either before or after reversed-phase HPLC (RPC).

For routine SEC applications, we recommend the 200 x 9.4 mm columns, which offer optimal separations at @ 0.5 ml/min. Smaller columns can be used if the HPLC system can deliver low flow rates accurately (eg. 0.12 ml/min for 200 x 4.6 mm columns).
                                                                                                       

Papers   on PolyHYDROXYETHYL Aspartamide

Subject

H1.  Alpert, J. Chromatogr. 499 (1990) 177.

Introduction  of HILIC for HPLC of polar compounds.

H2.  Zhu et al., J. Chromatogr. 548 (1991) 13.

Peptide  HILIC on PolyHYDROXY- ETHYL A and PolySULFOETHYL A.

H3.  Fainsilber et al., Eur. J. Biochem. 202 (1991) 589.

Size-exclusion  HPLC of a conotoxin peptide.

H4.  Andrews & Alpert, Poster #110, 10th ISPP (10/90)

Size-exclusion  HPLC of small solutes.

H5.  Pike et al., Proc. Natl. Acad. Sci. 88 (1991) 11081.

Lactic  acid stimulates B-cell growth.

H6.  Przysiecki et al., Protein Expression Purif. 3 (1992) 185.

HILIC, SEC, and SCX of recombinant antistasin with a  preproleader sequence

H7.  Swiderek et al., Poster #M93, Protein SocietySymp. (7/92).

Isoln. of carcinoembryonic antigen glycopeptides by HILIC,  with mass spec. sequencing

H8.  Boutin et al., J. Chromatogr. 583 (1992) 137.

HILIC  of phosphorylated peptides and tyrosine kinase reaction  mixtures.

H9.  Fong et al., Plant Physiol. 99 (1992) 137.

SEC  of gymnosperm Ser-Hyp4 motif protein (same ref. as S26).

H10.  Hogrefe et al., Nucl. Acids Res. 21 (1993) 2031.

HILIC  of methylphosphonate oligonucleotides.

H11.  Bickel et al., Proc. Natl. Acad. Sci. 90 (1993) 2618.

Purif.  of biotinylated VIP analog.

H12.  Horst et al., EMBO Journal 12 (1993) 3035.

HILIC of mitochondria proteins from SDS-PAGE gel bands.

H13.  Clauser &  Burlingame, Poster M286, Protein Society Symp. (7/93).

Removal  of SDS from proteins by capillary HILIC.

H14.  Boutin et al., Drug Metab. Disp. 21 (1993) 1157.

HILIC  of glucuronides of diosmetin.

H15.  Barret et al., Chem. Biol. Interact. 86 (1993) 1157.

Assay  of ATPase activity by HILIC

H16.  Jenö et al., Anal. Biochem. 215 (1993) 292.

Removal  of SDS and salts from electroeluted proteins.

H17.  Soltysik et al., Ann. NY Acad. Sci. 690 (1993) 392.

Purif.  of saponins by HILIC.

H18.  Alpert et al., J. Chromatogr. A 676 (1994) 191.

PolyGLYCOPLEX for HILIC of complex carbohydrates.

H19.  Swiderek et al., Poster 268-M, Protein Society Symp. (7/94).

HILIC  for removal of SDS, Triton X-100, & Nonidet P40 from peptides  online for LCMS.

H20.  Alpert et al., Poster 269-M, Prot. Soc. Symp. (7/94).

HILIC  of intact proteins.

H21.  Harwig et al., Method Enzymol. 236 (1994) 160.

Purif.  by HILIC of defensins.

H22.  Emould et al., Int. J. Peptide Protein Res. 43 (1994) 496.

HILIC  of phosphorylated peptides from tyrosine kinase reactions.

H23.  Taylor et al., J. Am. Chem. Soc. 116 (1994) 10803.

Isoln.  of dihydroxyproline from mussel adhesive protein.

H24.  Silvestre et al., J. Agric. Food Chem. 42 (1994) 2778.

Fractionation  of casein hydrolyzates by SEC.

H25.  Silvestre et al., J. Agric. Food Chem. 42 (1994) 2783.

Characterization  by SEC of casein hydrolyzates.

H26.  Shushan, Perkin-Elmer "Views" (Fall 1994) 16.

SEC  desalting of proteins for ES-mass spec.

H27.  Furuya et al., Proc. Natl. Acad. Sci. 92 (1995) 12323.

Isoln.  by RPC/HILIC of a diuretic hormone polypeptide from mealworm  (T. molitor).

H28.  Kieliszewski et al., J. Biol. Chem. 270 (1996) 2541.

HILIC  of hydroxyproline-rich glycopeptides from Douglas Fir.

H29.  Sarg et al., Chromatographie 1/95 (1995) 50.

HILIC  of histones (using PolyCAT A).

H30.  Oyler et al., J. Chromatogr. A724 (1996) 378.

HILIC  of diastereomers of atosiban.

H31.  Pauly et al., Carbohydr. Res. 282 (1996) 1.

HILIC  of nitrobenzyl-derivs. of oligo- saccharides on PolyGLYCOPLEX.

H32.  Horst et al., Meth. Enzymol. 260 (1996) 232.

Isoln.  of yeast mitochondrial membrane proteins from 2-D gels by HILIC.

H33. Lindner et al., J. Chromatogr. A 743 (1996) 137.

HILIC  of acetylated histones (using PolyCAT A.

H34.  Nelson et al., Exp. Cell Res. 229 (1996) 20.

Isoln.  of a hepatic  growth stimulator by SEC; it's glycerophosphoryl-  ethanolamine.

H35.  Lindner et al., J. Chromatogr. A 782 (1997) 55.

HILIC  of phosphorylated histones using PolyCAT A (same ref.  as C64).

H36.  Swiderek et al., ABRF News 8(4) (1997) 17.

Analysis  of protein and peptide samples that contain detergents.

H37.  Lane et al., J. Cell Biol. 125 (1994) 929.

Analysis  of the peptides GHK and KGHK in digests of SPARC protein.

H38.  Swiderek et al., Meth. Enzymol. 271 (1996) 68.

Separation  of glycopeptides from digest of carcinoembryonic antigen by  RPC-HILIC.

H39.  Mant et al., Meth. Enzymol. 289 (1997) 426.

CEX-HILIC  of peptides (using PolySULF A).

H40.  Schmerr et al., J. Chromatogr. A 802 (1998) 125.

Purification  and analysis of the scrapie prion protein (using PolyWAX  LP) (same ref. as W5)

H41.  Lindner et al., J. Biol. Chem. 273 (1998) 13324.

HILIC  of deamidation and acetylation variants of H1 histone (using PolyCAT A).

H42.  Strege, Anal. Chem. 70 (1998) 2439.

HILIC-ES-MS  of small polar compounds in drug discovery screens.

H43.  Zhang and Wang, J. Chromatogr. B 712 (1998) 73.

HILIC  of coeluting glycopeptides from an RPC map of a tryptic  digest of recombinant gamma-interferon from a CHO cell culture.

H44.  Faull et al., Neuropeptides 32 (1998) 339.

RPC-HILIC-CEX  isolation of gamma-lipotropin and beta-endorphin from rat pituitary.

H45.  Mant et al., J. Chromatogr. A 816 (1998) 65.

CEX-HILIC  detects substitutions on the polar face of amphipathic alpha-helical  peptides; RPC, the non-polar face (PolySULF A used).

H46.  Mant et al., J. Chromatogr. A 816 (1998) 79.

CEX-HILIC  vs RPC for separation of variants of cyclic  peptides (Gramicidin  S analogues) (PolySULF A used).

H47.  Alpert, Size Exclusion HPLC of Small Solutes in Column Handbook  for Size Exclusion Chromatography (C.S. Wu, ed.), Academic Press,  1999.

Explains  the principles and gives applications.

H48.  Strege, Am. Pharmaceut. Rev. 2 (1999) 53.

HILIC-MS  combination for drug discovery.

H49.  Litowski et al., J. Pept. Res. 54 (1999) 1.

HILIC-CEX  can resolve peptides that vary in position of Ser-  acetylation (PolySULF A used).

H50.  Croes et al., Poster 134, 47th ASMS Conference (June 1999, Dallas)

HILIC  of underivatized amino acids in seeds with MS/MS detection,  run time of 6 minutes.

H51.  Dahiyat and Mayo, Proc. Natl. Acad. Sci 94 10172.

SEC  of designed proteins @ packing interactions.

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