Carbohydrates / Polysaccharides - Structure elucidation - Determening structure elucidation approchas
10 important questions on Carbohydrates / Polysaccharides - Structure elucidation - Determening structure elucidation approchas
What is the general approach to investigate the structure elucidation of carbohydrates?
- Sample preparation
- Analysis of mono- and oligosaccharides
- Extract the different polysaccharides.
Example of the Cell Wall Material (CWM) - Fractionate extracted polysaccharides
“Homogeneous polysaccharide fraction”
How does sample preparation form foods go to analyse carbohydrates?
- Grinding and immediate inactivation of endogenous enzymes (heat, organic solvent)
- Extraction of fat (hexane)
- Extraction of low Mw materials (ethanol)
● AIS (alcohol insoluble solids) - Removal of protein
● Proteases, Phenol/acetic acid/water (PAW) - Removal of starch
● gelatinization and extensive treatment with amylases, then put in 80% ethanol which gets the glucose and polysaccharides out and the polysaccharides are left)
What sample preparation would you recommend before investigation the cell wall of polysaccharides of a fresh apple?
- Grinding and immediately inactivation of enzymes by heating.
- Removal of low Mw materials with ethanol (sugars enz.)
- Removal of proteins, cutting them with proteases (and then?)
- Removal of starch by first gelatinization and then addition of amylases)
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What is the principle of polysaccharide extraction?
How does extraction of different types of pectin from Cell Wall Material go?
Bij the third step ester and hydrogen bonds are broken and the next set of pectins are removed.
What are the two alternative routes of pectin extraction?
Hereby you extract as much pectin as possible.
Destroying glycocitic linkages making the pectin soluble.
However you are destroying part of the structure.
How can you determine which type of hemicellulose you have?
On what differences can the extracted polysaccharides be fractionated?
- Charge
- size
- conformation
- polarity
What is the difference between SEC and AEC?
AEC (anion-exchange chromatography): neutral molecules are separated from the charged molecules; the latter will remain on the column. Charged molecules are eluted from the column with a salt of pH gradient: less charged molecules will elute before more charged molecules
How can you fractionate insoluble apple cell wall material? Mention which population is targeted in each case.
- First you do a pectin extraction, by adding hot and ocld water (water soluble pectins)
- Then you add sodium hexa methaposphate ammoniom oxaleta, at pH 5, for 4 hours at 20-25 degrees (calcium bound pectins)
- Then you add 0.05 M sodium carbonate and NaBH4 pH9.5 ( pectins bound by ester linkages and hydrogen bonding)
- 1M NaOH at 0 degrees for 1 to 6 hours (hemicellulose I: mainly xylans hydroysis of strong ester linkages disruption of hydrogen bonding)
- Diluted sodium hydroxide 4-5M NaOH 0 degrees for 1-6 hours (alkali soluble pectins)
- Your residue is lignin
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