PolyHYDROXYETHYL Aspartamide for Hydrophilic Interaction Liquid Chromatography(HILIC)

Transcription

1 PolyHYDROXYETHYL Aspartamide for Hydrophilic Interaction Liquid Chromatography(HILIC) Hydrophilic Interaction Liquid Chromatography(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. The stationary phase in HILIC must be extremely polar. PolyLC has developed a material specifically for this purpose. PolyHYDROXYETHYL A. It retains solutes almost solely on the basis of hydrophilic interaction. Volatile mobile phases can be used. HILIC exhibits retention porportional to the amount of organic solvent in the mobile phase(opposite of RPC). Typical HILIC mobile phases contain 65-80% organic in the background, and in the sample. Gradient elution may be performed either with a decreasing organic or increasing salt gradient(10mm salt is necessay with charged solutes such as peptides, but is not necessary with uncharged solutes such as carbohydrates. Salts with good solubility in HIILIC mobile phases include potassium methylphosphonate, trietylamine phosphate, and sodium perchlorate. Ammonium formate and acetate are volatile but not transparent below 230nm and can be used for direct LC/MS analysis. Notable application areas for the PolyHYDROXYETHYL A (HILIC) column. 1.) Isocratic Analysis by LC/MS of very Polar compounds( ie Amino acids). 2.) Hydrophilic peptide mapping. 3.) Purification of synthetic and natural peptides, where a different selectivity to Reversed-Phase in needed. 4.) Glycopeptides, and Phosphopeptides. 5.) Membrane Proteins& very hydrophobic peptides. 6.) Small molecule LC/Mass Spec. Ordering Information, Catalog Number and Prices Particle Size: 5um Particle Size 12um Column IDx Length 300A Pore Size 1000A Pore Size Price 300A 1000A Price 1.0x 50mm 051HY HY0510 $ x 150mm 151HY HY0510 $ x 100mm 102HY HY0510 $ x 200mm 202HY HY0510 $ x 35mm 3.54HY HY0510 $ x 100mm 104HY HY0510 $ x 200mm 204HY HY0510 $ HY HY1210 $ x 200mm 209HY HY0510 $ HY HY1210 $ x250mm 259HY HY0510 $ HY HY1210 $ x 250mm 2521HY HY0510 $ HY HY1210 $ x 250mm 5025HY HY0510 Inq. 5025HY HY1210 inq. Javalin Guard Columns 2.1x10mm J22GCHY0503 J22GCHY0510 $75.00 each 4.6x10mm JGCHY0503 JGCHY0510 $75.00each 10x10mm Cartridge G10-HY1203 G10-HY1210 $ each, requires holder C-1000, $ x20mm Cartridge G20-HY1203 G20-HY1210 $ each, requires holder F 1403, $ Bulk Material BMHY0503 BMHY0510 $60.00/gm BMHY1203 BMHY1210 $15./gm Solid Phase Extraction Cartridges Package/24 UltraMicro Spin MacroSpin Column SPEHY1203x $ UMSCHY1210 $55.00/10 MASCHY1210 $55.00/10 7

2 When to USE HILIC: Need a volatile mobile phase and Reversed-Phase does not suffice. Solutes too weakly or too strongly retained in Reversed-Phase. Solutes which aggregate or are not soluble in aqueous mobil phase( e.g. amyloid peptides). Separate solutes differing in a hydrophilic residue(e.g. Ser-). Complementary orthagonal mode of chromatography. Removing electroeluted proteins from SDS, Coomassie blue, and salts. Elute molecules into Mass detector at high levels of organic to increase sensitivity. Fast Isocratic analysis by HILIC-MS/MS of Amino Acids and Other Polar Compounds Column: Polyhydroxyethyl A, 200x4.6mm; 5um, 100A catalog number; 204HY0501 Lysine & Catabolites in developing Soy seed Mobile Phase: 0.2% formic acid + 10mM ammonium formate with 60%acetilnitrile. Flow rate: 0.8ml/min MS Detector: Finnigan LCQ(APCI positive ion mode) Data courtey of Robert Croes, Dupont Biotech/Newark, DE 8

3 HILIC of Glycopeptides after initial Reversed-Phase Separation HILIC of Very Hydrophobic Biotinylated Peptide Using Reversed-Phase HPLC, the components of this mixture were recovered with 5% yield. With HILIC, recovery was quantitative and resolution complete. Column: Polyhydroxyethyl A 200x4.6mm Catalog number 204HY0503 Gradient: Decreasing NECN in 50mM TEAP, ph 2.8 9

4 Peptide Selectivity using HILIC Peptide: Tyr-Asn-Arg-Thr-Arg-Arg-Ile-Ser(Po4)-Glu + ailure sequences Column: PolyHydroxyethyl A, 4.6x200mm, 5um, 300A Catalog number 204HY0503 Gradient: 10mM TEAP, ph % -45% MeCN in 50 min. Detection: A /225nm=0.1AUFS 10

5 Small Molecule Cosmetic Components HILIC on a very Hydrophilic Peptide 1) Urea 2) Allantoin 3) Lysidone PolyHydroxyethyl A 100A pore Column 204HY0501 Detection 200nm In the digest of whole SPARC protein, the levels of peptides of interest were too low to yield discrete UVpeaks. The use of volatile mobile phase permitted direct mass spec analysis, which confirmed their presence. 11

6 Aminoglycoside Antibiotics by HILIC using Mass Spec Detection. This study demonstrates the capability of Hydrophilic Interaction Chromatography for the separation of Aminoglycoside residues. The method is expecially suited to the Mass Spectrometric detection of these analytes, providing a high conclusive method with superior peak shape and sensitivity than most available methods. (1) Column: PolyHydroxyethyl A, 200x4.6, 1000A, 5um Catalog Number 204HY0510 Mobile Phase conditions: 80% Acetonitrile, 20% 250mM Ammonium Acetate, ph 4.0 to 25% Acetonitrile, 75% 250mM Ammonium Acetate, ph 4.0 in 5 min Hold for 10 min more back to initial conditions in 3 min. Flow rate; 1ml/min, Injection Volume; 50ul. (1) Poster present at Euroresidue IV (5/2000) Veldhoven, The Netherlands titled, A Novel LC-MS compatible method for the analysis at residue levels of Aminoglycoside antibiotics by M. McGrane et. al. RIKILT, Netherlands 12

7 HILIC of Deoxynucleosides(minus hydroxyl) dt, da, dc, dg Column: Polyhydroxyethyl A, 5um 100A Catalog number; 204HY0501 Isocratic; 90% MeCN, 10mM Ammonium acetate, ph ml/min A 254, time base e.g.5.96 divided by