Ch 1 Wet Methods of Carbohydrate Analyses

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Lecture 1.WET METHODS OF

CARBOHYDRATE ANALYSES

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Nomenclature of Carbohydrates• D, L Defines the configuration at C5

D has the OH at Right in Fischer projectionL has the OH at Left in Fischer projection

• Gluco defines the configuration of the OH at C2, C4, C5. TheseOH’s are on same side while the C3 -OH is opposite to others

• α ,β defines the configuration of the OH at C1, the anomeric carbon• Pyran indicates 6 member ring size• Furan indicates 5 member ring size

Examples follow

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In Glucuronic acid C2, C4, C5 OH’s areon same side

C

CO 2H

H

O

OHH

C

CO 2H

H

O

OHH

OH

H

OHH

HO

H OH

H

H

H

HO

HO

glucuronic acid galacturonic acid

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Alditols• In Mannitol C2, C4,

C5 OH’s are not atsame side in Fisher

Projection

CH2OH

CH2OH

H

H

OH

OH

H

H

HO

HO

Mannitol

CH2OH

OH

OH

H

CH2OH

H

HO

H

Xylitol

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Conformations

O

OH

OH

OHOH

CH 2 OH

-D glucopyranose

O

OH

OH

OH

OH

CH 2 OH

-D glucopyranose

[a ] 25D +19

o +112 o

Anomers

For aged solutions

[a ] 25D = +52.7 o

Rotations ofFreshSolutions

Reason: Mutarotation is the best evidence for the cyclichemiacetal structure of D-(+)-glucose

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Monosaccharides,Hemiacetal Formation II

C

C

C

O H

CH 2OH

C

C O

H

..HH

H

H

OH

OH

HO

OHC

C

C

O

CH 2OH

C

C

HH

H

H

OH

OH

HO H

O

C

C C

C

HC HHO

CH2OH

O

H

OHH

H OH H

.. O

C

C C

C

C HHO

CH2OH

OHH

H OH HOH

H

C5 OH attacks aldehyde giving a pyranose ring (6 member structure)

C4 OH attacks aldehyde giving a furanose ring (5 member structure)

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O

OH

OH

OH

OH

CH 2 OH

O

O

OH

OH

OH

OH

CH 2 OH

CHO

OH

OHOH

OH

CH 2 OH

OH

OH

OH

HO

CH 2 OH

O

OH

OH

OHHO

CH 2 OH

CHO

H OH

HO H

H OH

H OH

CH 2 OH

CHOOH

OH

OH

D glucose

OH

-D glucopyranose

CH 2 OH

-D glucofuranose

Mutarotation

-D glucofuranose

-D glucopyranose

Ring closure betweenC1 and C4 -OH

Ring closure betweenC1 and C5 -OH

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• Oligosaccharides – consist of several monosaccharide

residues joined together with glycosidic

linkages

– di, tri, tetrasaccharides

(depending on the number of monosaccharides)

– up to 10 - 20 monosaccharides (dependingon analytical techniques i.e GC vs LC/MS)

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• Polysaccharides – refer to polymers composed of a large

number of monosaccharides linked by

glycosidic linkagesex. Cellulose

oxygen bridge (ether-type or glycosidic bond)

anhydro-glucopyranose unit

Cellobiose

n = 1 -5000

OH

OH

HO

CH2OHOO

CH2OH

HO

OH

OOH

HOHO

CH2OHO

OOH

OHO

CH2OHO

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Cellulose

-D-anhydroglucopyranose unitslinked by (1,4)-glycosidic bonds

O O

O OO

OO

OH

CH2OHHO

HOHO

OH

CH2OHHO

OH

CH 2OH

CH 2OH

OH

OHHO

3'

4'n

1

2

3

4

5

6

2'5'

6'

1'

(potential aldehyde)

Non-ReducingEnd-Group

ReducingEnd-Group

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Polysaccharides

Polysaccharides can be divided into two classes – Homopolysaccharides

• consist of only one kind of monosaccharideex cellulose

– Heteropolysaccharides• consist of two or more kinds of

monosaccharidesex galactoglucomannans

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Polysaccharides

Polysaccharides can not only havedifferent sequences of monosaccharideunits, but also different sequences of

glycosidic linkages and different kinds ofbranching – a very high degree of diversity for

polysaccharides and their structure-function relationships

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Plant Polysaccharides

The conformation of individualmonosaccharide residues in a polysaccharideis relatively fixed, however, joined byglycosidic linkages, they can rotate to give

different chain conformations.

OOHO

HOO

OH

O

HOHO

O

OH

HO O

HOHO

O

OHOHO

HOO

OH1,4 glycosidiclinkage 1,6 glycosidic

linkage

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The different kinds of primary structuresthat result in secondary and tertiarystructures give different kinds ofproperties

– water solubility, aggregation andcrystallization, viscosity, gelation, etc.

Polysaccharides have a variety offunctions

– Storage of chemical energy in

photosynthesis

Plant Polysaccharides

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StarchStarch is composed completely of D-

glucose – found in the leaves, stems, roots,

seeds etc in higher plants

– stores the chemical energy producedby photosynthesisMost starches are composed of two types

of polysaccharides - amylose andamylopectin – amylose - a mixture of linear

polysaccharides of D-glucose units

linked -(1-4) to each other

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Starch Polymer Components

Amylose

Amylopectin (1 residue in every 20 is 1 6 linked to branch off)

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The Components of Starch

O

HOO

OH

OH

O

HO

HO

OH

OHO

O

OH

OH

O

O

OHO

HO OH

O

(1-4)

AmyloseAmylopectin

(1-4)

(1-6)O

HOO

OH

OH

O

HOO OH

OH

O

HOO

OH

OH

O

HOOH

OO

HOO OH

OH

O

O

HOO

HO

OH

O

Starch tertiary structure (Helix)

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QUALITATIVE ANALYSIS

There various tests that can be used todetect the presence or absence ofcarbohydrates or sugars. Some of theseare:

• Molisch Reaction• Anthrone Reaction• Iodine Test• Benedict Test

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MOLISCH REACTION

In this reaction the furfural that is formed fromthe carbohydrate by the sulfuric acidcondenses with the phenol to give the

characteristic color.PROCEDURETwo ml of a sample solution is placed in atest tube. Two drops of the Molisch reagent(a solution of α-napthol in 95% ethanol) isadded. The solution is then poured slowlyinto a tube containing two ml of concentratedsulfuric acid so that two layers form.

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MOLISCH REACTION

A positive test is indicated by the formationof a purple product at the interface of thetwo layers.

a negative test (left) and a positive test

(right)

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ANTHRONE REACTION

Anthrone, 9,10-dihydro-9-ketoanthracenereacts with many carbohydrates to give agreen color.

PROCEDURE1 ml of a sample solution is placed in a testtube. 2ml of a 0.2% of Anthrone in

conconcentrated sulfuric acid is added. In thepresence of carbohydrates a clear greencolor will appear and will rapidly increase inintensity until a dark blue-green solutionresults.

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QUANTITATIVE ANALYSIS

The quantitative methods for theestimation of sugars and carbohydratesdepend on the properties of reduction andoptical rotation that the sugars have.Some of the quantitative methods usedare;

• Munson and Walker Method• Iodide-Thiosulfate Method• Lane-Eynon Titrimetric Method

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LANE-EYNON METHOD

This is a short and rapid method and oftenthe most accurate method for theestimation of reducing sugars. It is basedon a determination of the volume of a testsolution required required to reducecompletely a known volume of alkaline

copper reagent. The end point is indicatedby the use of an internal indicator,methylene blue.

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LANE-EYNON METHOD

SAMPLE PREPARATION• 12.5g of the sample is dissolved in water.• 25ml of 10% neutral lead acetate solution is

added.• Some alumina cream is added and made upto 250ml in a volumetric flask.

• The solution is shaken thoroughly and

filtered.• 10ml 10% solution of potassium oxalate is to100ml of the filtrate and made up to 500ml,shaken and filtered

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LANE-EYNON METHODPROCEDURE

• 10ml of the mixed Fehling reagent is placed in a 250mlErlenmeyer flask.

• The sugar solution is transferred into a burette andsuspended over the Erlenmeyer flask.

• 15ml of the sugar solution is added to the flask and heated toboiling.

• The solution is boiled for about 15 seconds and portions ofthe sugar solution is added rapidly until only the faintestperceptible blue color remains.

• 2-5 drops of a 1% aqueous solution of methylene blue isadded and heating is continued.• The sugar solution is added dropwise until the titrtion is

complete which is shown by the reduction of the dye.

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LANE-EYNON METHOD

The amount of sugar may be calculated bythe formula;

The factor is obtained in Literature, inwhich the factor for each titration from 15to 50ml is given.