PolyHYDROXYETHYL A™ Columns
- for Hydrophilic Interaction and Size Exclusion HPLC -
This neutral, polar material was developed specifically for Hydrophilic Interaction Chromatography (HILIC). HILIC is a variant of normal phase chromatography that is performed with a polar stationary phase and a partly aqueous mobile phase. This permits normal-phase separations of peptides, nucleic acids, carbohydrates, some proteins, and polar solutes in general. In comparison with other materials used for HILIC, PolyHYDROXYETHYL A™ affords sharper peaks and better selectivity and recovery. Its great polarity means less organic solvent is needed to get retention. Use PolyHYDROXYETHYL A™ for:
1) Analysis of polar small molecules for metabolomics or analysis via HILIC-MS/MS of specific small molecules (amino acids; methotrexate) in plasma or crude extracts.
2) Peptide separations and mapping involving differences in polar groups (Ser-; glycopeptides; etc.).
3) Multidimensional purification of synthetic and natural peptides or fractionation of really complex digests.
4) HPLC of solutes that aren't soluble in aqueous media (membrane proteins;phospholipids).
5) Eliminating detergents, lipids, and salts from a sample.
6) Oligonucleotides and their analogs.
In the absence of organic solvent, PolyHYDROXYETHYL A™ functions in the Size Exclusion Chromatography (SEC) mode.
For proteins and peptides, use 200- or 300-Å material. For polar small solutes, try our premium 3-µm, 100-Å material.
Oligonucleotides and PCR Products
Larger oligonucleotides, their analogs, and dsDNA fragments are analyzed and purified by anion-exchange chromatography. Our PolyWAX LP™ material affords excellent results in such applications, especially the 3-µm, 1500-Å version. G-rich nucleic acids cause problems with some separation methods but not with PolyWAX LP™ columns eluted with a NaClO4 gradient.
Order PolyWAX LP™ now.
PCR Reaction Products: Results from Raquel Hernandez' group (North Carolina St. Univ.) demonstrate that PCR reaction products can be purified on a column of this material much more conveniently than with a PAGE gel and with higher recovery [BELOW]. This is true even of GC-rich products.
Oligonucleotides: The 3-µm version of PolyWAX LP™ affords unusually good resolution of oligonucleotides [BELOW].
Mobile Phase: A) 25mM Tris-Cl, pH 8.0, with 30% ACN, B) Same +1 M NaCl
Gradient: 60-100% B in 50' Flow Rate: 0.5ml/min Temp: 60° C Detection: A250
Phosphorothioates: These elute in broader peaks than do regular oligonucleotides, since the phosphorus atoms are optically active centers. Thus, phosphorothioates consist of 2ndiastereomers (n = # of bases). An example is shown below.
Mobile Phase: A) 25mM Tris, pH 8.0, with 30% ACN, B) Same +1 M NaClO4
Gradient: 0-3': 0% B; 3-4': 0-60% B; 4-34': 60-100% B; 34-38': 100% B
Flow Rate: 0.5ml/min Temp: Ambient Detection: A260 Sample: 1.7µg
Hydrophilic Interaction Chromatography: This is an alternative to anion-exchange that can be used with volatile solvents. The following example consists of phosphorothioates with the same base sequence as the conventional oligonucleotide above. Peaks are broader, although it is unclear how much of this is due to:
a) The mode used;
b) The diastereomeric composition of phosphorothioates; and
c) The use of a 5-µm column for HILIC instead of 3-µm.