Ch 1 Wet Methods of Carbohydrate Analyses

8/10/2019 Ch 1 Wet Methods of Carbohydrate Analyses

http://slidepdf.com/reader/full/ch-1-wet-methods-of-carbohydrate-analyses 1/26

Lecture 1.WET METHODS OF

CARBOHYDRATE ANALYSES



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



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



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



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



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)



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



• 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)



• 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



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



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



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



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



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



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



Starch Polymer Components

Amylose

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



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)



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



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.



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)



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.



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



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.



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



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.



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.

Documents

Ch 1 Wet Methods of Carbohydrate Analyses