TSKgel Amide-80 and TSKgel NH2-100 columns enable the analysis of hydrophilic biomolecules, such as peptides1,2, carbohydrates3,6, and small molecules for drug discovery4,5,8, that are either weakly retained or elute in the void volume of reversed phase chromatography (RPC) columns. Normal phase liquid chromatography (NPLC) and hydrophilic interaction chromatography (HILIC)7 on TSKgel Amide-80 columns1-6 have been employed to effectively separate and analyze these compounds.
Particle characteristics
The TSKgel Amide-80 columns are packed with 3, 5 and 10µm spherical silica particles covalently bonded with carbamoyl groups. The polar functional groups of the sample, such as hydroxy groups, form hydrogen bonds with the polar groups (amino groups) of the packing. The number of hydroxy groups, conformation and solubility in the mobile phase determines the order of elution.
The TSKgel NH2-100 columns are packed with 3µm silica particles bonded with alkylamino groups. A unique endcapping reaction provides protection against bonded phase hydrolysis at low pH.
Bonded phase stability
The bonded phase in TSKgel Amide-80 columns posseses superior chemical stability and will not react with reducing sugars in carbohydrate analysis. The excelllent stability of the TSKgel Amide-80 stationary phase enables detection with Evaporative Light Scattering (ELS) detectors.
The bonded phase in the TSKgel NH2-100 columns offers expanded stability9,10 under HILIC conditions. Amino groups are introduced step-wise after an initial endcapping procedure, which results in a better safeguard against hydrolysis of the underlying silica.
References:
1) Yoshida, T., Analytical Chemistry, 69 (1997) 3038-3043, Peptide Separation in Normal Phase Liquid Chromatography.Yoshida, T., Analytical Chemistry, 69 (1997) 3038-3043, Peptide Separation in Normal Phase Liquid Chrom.
2) Yoshida, T., Journal of Chromatography A (1998) 808:105-112, Calculation of
peptide retention coefficients in normal-phase liquid chromatography.
3) Tosoh Bioscience, Technical Report #33 (TosoHaas).
4) Strege, M.A., Presented at the 22nd International Symposium on High
Performance Liquid Phase Separations and Related Techniques, St. Louis, MO, May 2-8, 1998, Poster L-1305.
5) Strege, M.A., Analytical Chemistry, 70 (1998) 2439-2445, Hydrophilic Interaction Chromatography-Electrospray Mass Spectrometry Analysis of Polar Compounds for Natural Product Drug Discovery.
6) Kuraya, N. and S. Hase, Analytical Biochemistry (1996) 233:205-211, Analysis of Chains by Two-Dimensional Sugar Mapping.
7) Albert, A.J., Journal of Chromatography, 499 (1990) 177-196, Hydrophilic-Interaction Chromatography for the Separation of Peptides, Nucleic Acids and Other Polar Compounds.
8) Strege, M.A., LCGC, Vol 26, No 7 (July 2008) 632-642, A Hydrophilic Interaction Chromatography Method for the Purity of Cytosine.
9) Tosoh Bioscience, Application Note #20 (2009)
10) Moriyama, H., Presented at Pittcon, Chicago, IL, March 8-13, 2009, Poster 2730-8.