Analysis methods
Methods used routinely by the Ferrier team to analyse oligo- and polysaccharides and glycoconjugates.
HPAEC with PAD detection
This method is used to identify and analyse monosaccharides and oligosaccharides. Monosaccharides may be free sugars or constituent sugars released from larger polysaccharide molecules by acid hydrolysis.
HP-SEC-MALLS
HP-SEC-MALLS is used for molecular weight determination of polymers in solution, typically polysaccharides and proteoglycans.
We can elucidate the weight average (Mw), number average (Mn) and Z-average (Mz) molecular weight, as well as the molecular weight profiles and polydispersity indices of such materials.
This technique involves separating samples on size-exclusion columns (TSK type) and utilising a triple detection system—UV, MALLS and DRI (differential refractive index).
Capillary electrophoresis with UV detection
Capillary electrophoresis (CE) is useful for separating charged molecules. Whole GAG preparations can be separated with only nanolitres of sample solutions. This technique is useful for process monitoring.
We also use CE to separate and quantify enzyme digests of GAGs, particularly heparins and heparan sulfates.
NMR spectroscopy
Both 1D and 2D spectroscopy techniques are used routinely to identify different GAGs, proteins, lipids and small organic molecules in natural product extracts.
Comparing1H–13C 2D NMR spectra of highly polydisperse GAG mixtures can identify levels of impurities in a sample. This is especially useful in the heparin manufacturing process.
GC and GC-MS
Since pioneering analytical methods in the 1980s, our team has remained a leader in the analysis of complex carbohydrates with GC and GC-MS.
To fully characterise a polysaccharide, its constituent sugars and glycosidic linkages are identified and quantified. We routinely carry out both these tasks.
Constituent sugar analysis
Acid hydrolysis breaks down a polysaccharide into its constituent sugars. This process is followed by derivatisation to the corresponding alditol acetates, which are quantified by GC. Alternatively, we can use methanolysis and convert the constituent sugars to their trimethylsilyl derivatives.
Glycosidic linkage analysis
To determine how sugar units are linked, a polysaccharide is permethylated then converted to partially methylated alditol acetates.
Analysis of these alditol acetates by GC-MS enables the types of sugars and their glycosidic linkages to be determined.
Acidic sugar-containing molecules
Molecules that are made up of both acidic and neutral sugars (such as pectic polysaccharides) must have the uronic acid and methyl-esterified uronic acid residues reduced with a two-step carboxyl reduction method before constituent sugar or linkage analysis.
Viscosity
We offer basic viscosity testing in house and can provide accredited viscosity analysis with our collaborators and external service providers.
Particle size
We have a laser diffraction particle size analyser for routine analysis.
UV/Vis spectrophotometry
We offer a full range of colorimetric or spectrophotometric assays for carbohydrate analyses, including total carbohydrate, hexoses, ketoses, uronic acids and hexosamines.
ESI-MS, LCMS and LCMSMS
These techniques are useful for analysing polar molecules ranging from less than 100 to more than 1 million Da, particularly large biomolecules such as carbohydrates, glycolipids or peptides.
LCMS enables complex carbohydrate mixtures to be separated and the individual compounds characterised.
LCMSMS enables low levels of analytes in a complex matrix to be identified and quantified, down to the low ng/mL range.
HPLC
We have a number of HPLC instruments and detection systems and routinely separate and purify a diversity of complex carbohydrate-containing molecules, such as glycolipids and glycoproteins. A key HPLC detection technique is charged aerosol detection (CAD), which allows near-universal quantification of analytes that are with or without a UV chromophore.