Saccharide Separation by Polymer-Based-Amino HILIC Column
Kanna Ito, Showa Denko America Inc.
Data provided by Showa Denko K.K.
Introduction:
Quantification of saccharides is required in a wide area of industries; from food, pharmaceutical, to
biofuel productions. Separation of saccharide by Hydrophilic Interaction Liquid Chromatography
(HILIC) is a simple and an effective method. The strength of HILIC mode is for the analysis of polar
hydrophilic molecules, including saccharides that cannot be analyzed by general reversed-phase
mode. With a HILIC column, desired separation can be achieved using a simple combination of
aqueous and organic solvents.
HILIC columns are filled with highly polar particles, and they are available in both silica and polymer
bases. Shodex Asahipak NH2P series is a polymer-based-amino column: A hydrophilic polyvinyl
alcohol gel is modified with polyamine (pentaethylenehexamine). The biggest advantage of polymerbased over silica-based columns is its durability. An excellent chemical stability and resistance to time
degradation have been observed. Although most analyses can be achieved at neutral pH conditions,
its wide working pH range (2~13) allows a large selection of usable eluents including different
cleaning solvents to remove built-up impurities. Another advantage of NH2P series column is its very
small “bleeding” of particles. Consequently, this reduces the back-ground noise level. This is
especially beneficial when coupling the HPLC with a corona charged aerosol detection (CAD) or an
evaporative light scattering detector systems. The capability of using high organic solvents is also
ideal for mass spectrometry detection.
Our recent addition to the NH2P series is the NH2P-40 3E, which is filled with smaller packed gel, 4m compared to 5-m gel in NH2P-50 series. The NH2P-40 gel is packed in a 3.0-mm internal
diameter (ID) column housing. The selected ID size allows the column to be used with a conventional
LC system, i.e., without a need of semi-micro or any other special LC system. In this article, the
advantages of this newly available NH2P-40 3E column are presented.
Experimental:
Two columns, Shodex Asahipak NH2P-40 3E (3.0 mm ID x 250 mm) and NH2P-50 4E (4.6 mm ID x
250 mm), were compared for their analytical performances. Mobile phase consisted of 75% (v/v)
CH3CN and 25% (v/v) water. A conventional HPLC system was used at a flow rate of 0.35 mL/min
and 1.0 mL/min for NH2P-40 3E and NH2P-50 4E columns respectively. The column oven
temperature was set at 25 oC. Shodex RI-101 (0.25 mm ID tube) was used for detection. Three
saccharide standards; fructose, glucose, and sucrose were analyzed. Saccharide standards were first
dissolved in water to prepare 1.0% (w/v) stock solution. The test sample contained final concentration
of 0.5% (w/v) each of three saccharide standards and 75% (v/v) CH3CN in water. The injection
volume was 4 L.
Results:
Figure 1 shows the separation of fructose, glucose, and sucrose using NH2P-40 3E and NH2P-50 4E
columns. Sucrose was used to calculate the theoretical plate number (TPN). The available TPN was
improved from 8,700 of NH2P-50 4E to 11,300 of NH2P-40 3E. The resolutions between
fructose/glucose and glucose/sucrose pairs were improved from 4.2 and 5.9 using a NH2P-50 4E
column, to 6.4 and 8.5 using a NH2P-40 3E column. Improved detection limit (S/N=3) for sucrose was
also obtained; from 110 mg/L (NH2P-50 4E) to 20 mg/L (NH2P-40 3E).
Another advantage using NH2P-40 3E column over NH2P-50 4E column is the reduced solvent
consumption. The solvent required for NH2P-40 3E column is approximately 1/3 of the NH2P-50 4E
column.
(a) NH2P-40 3E
(3.0 mm ID x 250 mm)
(b) NH2P-50 4E
(4.6 mm ID x 250 mm)
Figure 1. Sample chromatograms for the separation of three saccharides obtained by (a) NH2P-40
3E and (b) NH2P-50 4E. Sample contained 0.5 % (w/v) each of 1.Fructose, 2.Glucose, and
3.Sucrose. Mobile; CH3CN/H2O=75/25, Flow rate; (a) 0.35 mL/min and (b) 1 mL/min, Column oven;
25 oC, Injection volume; 4 L. Detected by Shodex RI-101 detector.
Conclusions:
Shodex Asahipak NH2P series has been a popular use in the separation of various saccharides. Now
with the new member of the series, NH2P-40 3E, provides an improved resolution while reducing the
solvent consumption. A conventional HPLC system is used, and thus does not require a semi-micro
system. Previously available applications for NH2P-50 series, such as saccharide analysis in various
food stuff and biomass can now be achieved with even higher resolution. Applications with potential
improved performance also include the analysis of sugar alcohols, amino sugars, oligopeptide,
vitamin C, and melamine. Meanwhile, the existing NH2P-50 4E column is suitable for use with
detectors, such as CAD, that requires flow rate >0.5 mL/min for the stable baseline.
Product Name
Plate Number
(TP/column)
NH2P-40 3E
≥8,500
Amino
4
NH2P-50G 3A
(guard column)
Amino
5
Column Size
(mm)
I.D. x L
Shipping Solvent
List
Price
Product Code
100
3.0 x 250
H2O/CH3CN=25/75
US $960
F7630007
-
3.0 x 10
H2O/CH3CN=25/76
US $385
F6710030
Functional Particle Pore Size
Group
Size (um)
(Å)
Showa Denko America Inc.
420 Lexington Avenue Suite 2850, New York, NY, 10170
Contact: [email protected]
Web: www.shodex.net