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Tosoh Bioscience LLC: The Chemistry of Innovation
TSKgel Columns
Affinity
Hydrophobic Interaction
Ion Exchange
Normal Phase / HILIC
TSKgel Amide-80
TSKgel NH2-100
Reversed Phase
Size Exclusion
Custom Columns
Parts and Accessories


While reversed phase is the most widely used mode of retention in HPLC, very polar compounds are often not sufficiently retained in low percent organic, or even in 100% aqueous mobile phase.

By using an amide or amino bonded phase column, polar compounds can be retained by a normal phase or hydrophilic interaction chromatography (HILIC; see references) retention mechanism using a mobile phase mixture of acetonitrile and ammonium acetate buffer. In contrast to the retention behavior in reversed phase, in HILIC solutes will be retained longer when increasing the percent acetonitrile.


TSKgel Amide-80

Bonded phase

 

Amide-80 bonded phase does not react with reducing sugars

NH2-100 bonded phase is more stable than conventional amino phases due to a special endcapping prior to introduction of aminoalkyl groups.

Anomer formation can be prevented by raising mobile phase temperature up to 80°C.

Amino-bonded phase can react with a reducing sugar to form a Schiff base.

Detectors

Can be used with evaporative light scattering (ELS) and mass spec (MS) detectors

Mobile Phase

Stable in 100% organic eluents

Solutes

Retains very polar drug candidates and drug metabolites

Applications

saccharides

saccharides

polyols (polyalcohols)

polyols (polyalcohols)

drug metabolites

methotrexate polyglutamate derivatives

peptides

pyridylaminated oligosaccharides

melamine & cyanuric acid

water-soluble vitamins

oligonucleotides

nucleic acid fragments

polar compounds in fermentation broth

cytosine




The 'normal' or traditional way of doing liquid chromatography, normal phase liquid chromatography (NPC) uses a polar stationary phase and a non-polar mobile phase. The polar surface can either be obtained by derivatizing the support with a ligand containing a polar functional group, or it can be the original (polar) support, as in the case of silica gel.

The popularity of bare silica gel declined after the introduction of bonded phases, since bonded phases feature shorter equilibration times and allow the use of gradient elution. Cyano, amino, and diol bonded phases are the most common normal phase column types.

The order of elution in normal phase is opposite that found in RPC for the same mixture of compounds. Although non-polar organic mobile phases and a silica stationary phase were used traditionally in normal phase LC, today most normal phase separations are performed with aqueous-organic mobile phases and a more polar-bonded stationary phase. This mode of HPLC is now commonly referred to as HILIC, hydrophilic interaction chromatography.

 

Tosoh Bioscience LLC 3604 Horizon Drive, Suite 100, King of Prussia, PA, 19406 Tel: (800) 366-4875  Email: info.tbl@tosoh.com