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Polysaccharide analysis

Industrial Research is one of only three laboratories worldwide that offers a full carbohydrate analysis facility for complex polysaccharide, lipopolysaccharide, glycolipid and glycosaminoglycan samples.

NMRIRL has an array of leading-edge equipment and expertise in analytical techniques including Nuclear Magnetic Resonance (NMR) spectroscopy.

The strength of the Industrial Biotechnologies Cluster at IRL lies in its leading-edge equipment and significant expertise in a wide variety of analytical techniques. These factors allow us to probe the most complex carbohydrates with routine analyses as well as undertaking more advanced R&D projects.

We specialise in the extraction, purification and analysis of oligo- and poly-saccharides, proteoglycans, glycosaminoglycans and glycolipids, and enjoy the challenge of new product development.

We have expertise in: 

HPAEC

We use HPAEC (High Performance Liquid Anion-Exchange Chromatography) (Dionex Corporation), with Pulsed Amperometric Detection (PAD) for the identification and analysis of carbohydrates, both mono and oligosaccharides. Monosaccharides may be free sugars, or constituent sugars released from larger polysaccharide molecules by acid hydrolysis.

HP-SEC-MALLS

High Performance Size Exclusion Chromatography-Multi Angle Laser Light Scattering (HP-SEC-MALLS) is used for molecular weight analysis of polymers in solution. These are typically polysaccharides and proteoglycans.
We can determine the weight average (Mw), number average (Mn) and Z-average (Mz) molecular weights, molecular weight profiles and polydisperisty indices, by separating samples on size-exclusion columns (TSK type) and using a triple detection system - UV, MALLS and DRI (differential refractive index).

HPLC

We have a number of High Performance Liquid Chromatography (HPLC) instruments with the following detection systems:

  • Coronal Aerosol Discharge (CAD)
  • Evaporative Light Scattering (ELS)
  • Fluorescence
  • Photodiode Array (PD)
  • UV.

Separation and purification of a wide range of complex carbohydrate-containing molecules (eg: glycolipids and glycoproteins) is possible using the chromatographic phases we have in-house, such as:

  • reverse-phase (C18, C8, C4)
  • hydrophilic interaction (HILIC)
  • strong anion-exchange (SAX)
  • graphitised carbon.

Capillary Electrophoresis

Capillary electrophoresis (CE) with UV detection enables the separation of charged molecules. IRL has capability in separating whole glycoaminoglycan (GAG[?]) preparations where only nanolitres of sample is analysed - a useful technique for process monitoring. We also use CE to separate and quantify enzyme digests of GAGs, particularly of heparins and heparan sulfates.

NMR spectroscopy

IRL has a suite of Nuclear Magnetic Resonance (NMR) instruments for carbohydrate analysis. We routinely use our two 500MHz NMR spectrometers for the identification and characterisation of purified polysaccharides and complex mixtures of polysaccharides, proteoglycans and glycolipids.

Both 1D and 2D spectroscopy enable the differentiation of various glucosaminoglycan (GAG), proteinaceous, lipid and small organic chemical natural product extracts. In particular, comparison of H-C 2-D spectra of highly polydisperse GAG mixtures can determine impurity levels - especially useful in the analyses of heparin manufacturing processes.  We also routinely carry out detailed full structural characterisation of small synthetic and natural product derived molecules.

GC and GCMS

Gas Chromatography (GC) and Gas Chromatography-Mass Spectrometry (GC-MS) are powerful routine methods for the analysis of complex carbohydrates, lipids and other natural products. Since pioneering analytical methods in the 1980s, our team has remained a leader in the analysis of complex carbohydrates with GC and GC-MS. Full analysis requires both the identification and quantification of constituent sugars in a polysaccharide molecule, as well as the identification of the glycosidic linkages that connect the sugars.

Constituent sugar analysis

The composition of a polysaccharide can be determined by first breaking it down into constituent sugars by acid hydrolysis, then forming corresponding alditol acetates in a derivatisation step, followed by quantification by GC. Alternatively, we can analyse a polysaccharide by methanolysis and conversion of its constituent sugars to their trimethylsilyl derivatives.

Glycosidic linkage analysis

To determine the positions of the glycosidic linkages between constituent sugars, polysaccharides are first permethylated, then converted to partially methylated alditol acetates. Analysis of the alditol acetates by GC-MS allows the types of sugars and their glycosidic linkages to be determined.

Acidic sugar-containing molecules

For molecules with both acidic and neutral sugars (such as pectic polysaccharides), uronic acid and methyl-esterified uronic acid residues must first be reduced with a two-step carboxyl reduction method prior to either constituent sugar or linkage analysis. 

UV/Vis spectrophotometry

We have a full range of colorimetric or spectrophotometric assays for carbohydrate analyses, including total carbohydrate, hexoses, ketoses, uronic acids and hexosamines. 

ESI-MS and LCMS

Electrospray ionisation mass spectrometry (ESI-MS) is an advanced technique well-suited to the analysis of polar molecules ranging from less than 100 to more than 1,000,000 Da, in particular to the analysis of large biomolecules such as carbohydrates, glycolipids or peptides. We have a Q-Tof Premier mass spectrometer equipped with standard electrospray ionisation (ESI) (or atmospheric pressure chemical ionisation APCI), a high performance platform for MS and MS/MS analysis incorporating high sensitivity, high resolution, high mass measurement capability, enhanced speed and wide m/z range. The built-in LockSpray capability enables routine exact mass measurement of precursor and fragment ions in MS and MS/MS modes to yield the highest confidence in structural elucidation and databank search results. The availability of combined high performance liquid chromatography with mass spectrometry (LCMS) enables both the separation of complex carbohydrate mixtures and the characterisation of individual compounds contained. Using ESI-MS and/or LC-ESI-MS we have analysed a large number of compounds/mixtures including synthetic oligosaccharides, heparin sulfates, glycoceramides, glycopeptides and dendrimers capped with sugars as well as natural PIMs (phosphorylinositolmannans) and GAGs (glycosaminoglycans).

 


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