The sensitive determination of sugars and other carbohydrates is of great interest because of its importance in ecological, biological, clinical and nutritional research.
There are a number of different determination methods in use, but these often require very expensive equipment such as HPLC-MS or are somewhat complicated to perform such as GC with derivatization.
It's not the separation's fault.
The separation works very well on an HPLC system using high-performance anion exchange chromatography (HPAEC).
Small differences in the pKa values of the carbohydrates' hydroxyl groups cause different retention of the saccharides on an anion exchange resin with strong alkaline eluents.
UV or fluorescence detection are both very common in liquid chromatography, but practically can't be used for sugars due to a lack of chromophores in the molecule. Refractive index detection is very easy to use, but not very sensitive and therefore cannot be used for the determination of low sugar contents.
With a derivatization step that forms a chromophore, the spectroscopic methods could be made more sensitive to carbohydrates. Derivatization, however, requires additional equipment, chemicals and work steps.
An alternative approach takes advantage of the fact that carbohydrates can react electrochemically. This allows them to be sensitively detected quite selectively by amperometry.
Application example: Analysis of wood sugars and uronic acids for reasearch on alternative fuels and raw materials.
In quantitative analysis, amperometry is known as a highly sensitive determination method.
An electrolysis current is measured at a working electrode under constant electrochemical potential.
The electrolysis current is proportional to the concentration of the oxidized/reduced analyte.
Classical amperometry works discontinuously: filling the reactor, measurement/titration, emptying reactor, cleaning, regenerating, next measurement ... and therefore cannot be used directly with HPLC in this form.
The sugar analysis chromatography system from KNAUER uses pulsed amperometric detection (PAD) including regeneration of the electrode, required for sugar detection. Using a very small measuring cell, this technique enables rapid measurements.
A cycle of measurement, desorption and regeneration of the electrode takes only half a second and is repeated continuously during the analysis. Therefore, as with other flow detectors, a chromatogram can be recorded in the HPLC.
Apart from the freshly prepared aqueous basic eluent, the procedure requires no additional chemicals.
Electrochemical detection is not just very suitable for carbohydrate determination.
Other oxidizable or reducible analytes, such as fat-soluble vitamins, can also be determined very well with it in an HPLC system.