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What is a good column choice for HILIC analysis and/or purification of oligonucleotides? - WKB221820

Article number: 221820

ENVIRONMENT

  • Optimum bed density (OBD)
  • Preparative chromatography
  • Lab scale purification
  • Prep column
  • High Performance Liquid Chromatography (HPLC)
  • Bridged ethylene hybrid (BEH)
  • Hydrophilic interaction chromatography (HILIC)
  • Oligonucleotides

ANSWER

UPLC:

  • ACQUITY Premier BEH Amide (1.7um)
  • ACQUITY UPLC BEH Amide (1.7um)

UHPLC:

  • XBridge Premier BEH Amide (2.5um)

HPLC:

  • XBridge BEH Amide (3.5um, 5.0um)

Preparative (up to 30mm I.D.):

  • XBridge BEH Amide (5.0um)

ADDITIONAL INFORMATION

See: HILIC as an Alternative Separation Mode for Intact Mass Confirmation of Oligonucleotides on the BioAccord System

  • This method uses SKU: 186009504 - ACQUITY Premier BEH Amide Column, 1.7 µm, 2.1 mm X 50 mm, 1/pk
  • The ACQUITY Premier and XBridge Premier BEH Amide column hardware features MaxPeak High Performance Surfaces (HPS).  This technology makes the stainless steel surfaces inside the column inert.  This is an ideal hardware to prevent the phosphate groups in oligos from adsorbing to the stainless steel inside the column. 

Also see:

MaxPeak Premier Columns Web Page

MaxPeak Premier Columns with MaxPeak High Performance Surfaces - Ordering Information  (lit code 720007209EN)

Notes:

BEH Amide columns can resolve 7-10 kDa oligonucleotides (25-30 mer) reasonably well. (However, see the notes below.)

XBridge BEH Amide OBD Prep columns can be used for oligo purification. 

An XBridge BEH Amide, 130Å, 5 µm, 30 mm X 100 mm (or 50-150 mm length), for example, can be used for tens of µmoles injection in overloading mode.

But there are problems, as follows:

  1. It is hard to dissolve high concentrations of oligonucleotides in 70% acetonitrile solvent (precipitation of oligos). Oligos do not like highly organic solvents, but ~70% acetonitrile is necessary in HILIC; otherwise, peaks smear or there is a breakthrough of the sample.
  2. At-column dilution may fix this problem. See At-Column Dilution Application Notes to learn how at-column dilution works.
  3. Separation of oligos is OK up to ~20 mer; longer ones are more difficult to resolve in HILIC. Ion-pair reverse-phase is a better alternative for oligos if it can be used.
  4. If use of ion-pairing agents is OK for your method, ion-pair reverse-phase (IP-RP) LC is a better option overall.

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