4
– Applications
Thermo Scientific
Product Manual for CarboPac PA200 Columns
Page 20 of 37
065734-01
For Research Use Only. Not for use in diagnostic procedures.
4.
Applications
The CarboPac PA200 columns have been designed exclusively for isocratic or gradient separation
of oligosaccharides using electrolytically generated potassium methanesulfonate and potassium
hydroxide gradients with a combined concentration up to 200 mM. Certain separations require
only an isocratic separation. However, some groups of analytes will require a step or gradient
elution. Retention of carbohydrates can be varied with eluent concentration, in some cases
changing the elution order as the eluent concentration increases.
Depending upon your system, you may have to make small adjustments to your gradient
conditions or operating temperature to achieve resolution of all analytes.
4.1
Fetuin Oligosaccharide Alditol Profiling
The high resolution of the CarboPac PA200 is exemplified in the following example. CarboPac
columns separate mono-, oligo- and polysaccharides on the basis of fine structural differences in
branching, linkage isomerism, anomericity and sialylation. In the fetuin oligosaccharide alditol
standard shown below, peaks are separated according to branching, sialylation and linkage
isomerism. The disialylated biantennary peaks are eluted before the trisialylated triantennary
peaks which are eluted before the tetrasialylated tetraantennary peaks. In addition, within each
grouping, the
2-6
isomer is eluted before, and well resolved from, the
2-3
isomer.
The Dionex OligoStandard, Sialylalted N-Linked Alditols, Item # 043164 contains 25 nmol
oligosaccharides purified from bovine fetuin. Dilute the standard prior to use, by adding a known
volume of DI water. Dionex recommends running this standard every time a new column is
installed and subsequently anytime it becomes necessary to troubleshoot your system.
The CarboPac PA200, 3 mm column (Document No. 031992) is NOT compatible with the ICS-
6000 Dual EG mode but it is recommended to be used with manually-prepared sodium
acetate/sodium hydroxide eluents. The illustration of the application on the CarboPac PA200, 3
mm column is for comparison only, to demonstrate the comparable performance of the CarboPac
PA200, 1 mm and 0.4 mm columns with the ease of use.
Columns:
CarboPac PA200, 3-mm (guard + separator)
CarboPac PA200 1-mm (guard + separator)
CarboPac PA200 0.4-mm (guard + separator)
Gradient:
CarboPac PA200, 3-mm:
0-60 min: 20-150 mM NaOAc in 100 mM NaOH
60-65 min: 500 mM NaOAc in 100 mM NaOH
65-80 min 20 mM NaOAc in 100 mM NaOH
CarboPac PA200, 1-mm:
0-50 min: 15-64 mM KMSA in 136 mM KOH
50-60 min: 80 mM KMSA in 90 mM KOH
60-65 min: 100 mM KMSA in 100 mM KOH
65-80 min: 15 mM KMSA in 136 mM KOH
CarboPac PA200, 0.4-mm:
