62
hilic
Oligosaccharides
T
he TSKgel Amide-80 can separate oligosaccharides very rapidly
and efficiently.
Figure 4
shows a separation of a ß-cyclodextrin
hydrolysate in less than 10 minutes. The labels indicate the number of
base sugars such as glucose in each oligomer.
Glycans
Glycosylation is one of the most common post-translational modifica-
tions in eukaryotic cells. Complex N- and O-linked structures compo-
sed of repeating sugar moieties form the so called glycans. HILIC with
fluorescence detection is the method of choice to effectively separate,
identify and quantify glycans after exoglycosidase cleavage and fluore-
scent labeling. In order to normalize retention times of complex glycan
structures a dextran ladder consisting of glucose oligomers is used as
calibration reference. The calculated numbers of glucose units (GU)
can be used in subsequent database queries (Glycobase, autoGU) to
predict the glycan structure. For years TSKgel Amide-80 columns have
been used successfully in glycan analysis. Amide-80 chemistry is ideal-
ly suited for the separation of carbohydrate structures.
Figure 5
shows
the high-resolution separation of a 2-aminobenzamide (2AB) labeled
dextran ladder within 30 minutes on a TSKgel Amide-80 3 μm column.
This ladder can be used as a calibration standard for HPLC and MS
analysis of glycans. The ladder contains glucose homopolymer species
from degree of polymerization (dp) 1 to dp 22 (i.e. the glucose monomer
GU1-2AB to GU22-2AB).
figure 4
Separation of ß-cyclodextrin hydrolysate on TSKgel Amide-80 column
Sepa ation of
β
-cyclodextrin hydrolysate on TSKgel
Amide-80 o umn
Minutes
0
10
Column:
TSKgel Amide-80, 4.6mm ID x 25cm
Sample:
2µL,
β
-cyclodextrin hydrolysate, 1-7 degrees
of polymerization (4.6mg/mL)
Elution:
ACN/water (55/45)
Flow Rate:
1.0mL/min
Detection:
refractive index detector
Temperature: 25ºC
1
2
3
4
5
6
7
Column: TSKgel Amide-80 (4.6 mm ID x 25 cm L); Sample: 2 μL,
b
-cyclodextrin
hydrolysate, 1 - 7 degrees of polymerization (4.6 mg/mL); Elution: ACN/water
(55/45); Flow rate: 1.0 mL/min; D tection: RI; Te perature: 25 ºC
figure 5
Separation of a 2-AB-labeled dextran ladder on TSKgel Amide-80
Column: TSKgel Amide-80 (3 μm, 2.0 mm ID × 15 cm L)
Eluent: A) 50 mM Ammonium formate (pH 4.3), B) Acetonitrile;
Gradient: 0 - 35 min (75 - 35 % B); Flow rate: 0.22 mL/min;
Detection: Fluorescence Ex @ 360 nm, Em @ 425 nm; Temperature: 50 °C;
Injection vol: 3 µl; Sample: CAB-GHP dextran ladder (Ludger; ~300 fmol for
GU2)
* Courtesy of K. Darsow & H. Lange, Institute of Bioprocessing, University of Nürnberg/Erlangen
Applications of TSKgel amide-80 columns
