Download - 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 projection.

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Page 1: 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 projection.

Lecture 1.WET METHODS OF CARBOHYDRATE

ANALYSES

Page 2: 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 projection.

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. These OH’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

Page 3: 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 projection.

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

C

CO2H

H

O

OHH

C

CO2H

H

O

OHH

OH

H

OHH

HO

H OH

H

H

H

HO

HO

glucuronic acid galacturonic acid

Page 4: 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 projection.

Alditols• In Mannitol C2, C4,

C5 OH’s are not at same side in Fisher Projection

CH2OH

CH2OH

H

H

OH

OH

H

H

HO

HO

Mannitol

CH2OH

OH

OH

H

CH2OH

H

HO

H

Xylitol

Page 5: 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 projection.

Conformations

O

OH

OH

OHOH

CH2OH

-D glucopyranose

O

OH

OH

OH

OHCH2OH

-D glucopyranose

[a]25D

+19o +112o

Anomers

For aged solutions [a] 25D

= +52.7o

Rotations of Fresh Solutions

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

Page 6: 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 projection.

Monosaccharides,Hemiacetal Formation II

C

C

C

O H

CH2OH

C

C O

H

..HH

H

H

OH

OH

HO

OHC

C

C

O

CH2OH

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 H

OH

H

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

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

Page 7: 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 projection.

O

OH

OH

OHOH

CH2OHO

O

OH

OH

OH

OHCH2OH

CHO

OHOH

OH

OH

CH2OH

OH

OHOH

HOCH2OH

O

OH

OH

OHHOCH2OH

CHO

H OH

HO H

H OH

H OH

CH2OH

CHOOH

OH

OH

D glucose

OH

-D glucopyranose

CH2OH

-D glucofuranose

Mutarotation

-D glucofuranose

-D glucopyranose

Ring closure between C1 and C4 -OH

Ring closure between C1 and C5 -OH

Page 8: 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 projection.

• 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 (depending on analytical techniques i.e GC vs LC/MS)

Page 9: 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 projection.

• Polysaccharides – refer to polymers composed of a large

number of monosaccharides linked by glycosidic linkages

ex. Cellulose

oxygen bridge (ether-type orglycosidic bond)

anhydro-glucopyranose unit

Cellobiose

n = 1 -5000

OHOH

HO

CH2OHOO

CH2OH

HOOH

OOH

HOHO

CH2OHO

OOH

OHO

CH2OHO

Page 10: 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 projection.

Cellulose

b-D-anhydroglucopyranose units linked by (1,4)-glycosidic bonds

O O

O OO

OO

OH

CH2OH

HOHO

HOOH

CH2OHHO

OH

CH2OH

CH2OH

OHOH

HO

3'

4'n

1

2

3

4

5

6

2'5'

6'

1'

(potential aldehyde)

Non-ReducingEnd-Group

ReducingEnd-Group

Page 11: 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 projection.

PolysaccharidesPolysaccharides can be divided into two classes

– Homopolysaccharides• consist of only one kind of monosaccharide

ex cellulose

– Heteropolysaccharides• consist of two or more kinds of

monosaccharides

ex galactoglucomannans

Page 12: 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 projection.

Polysaccharides

Polysaccharides can not only have different sequences of monosaccharide units, but also different sequences of glycosidic linkages and different kinds of branching

– a very high degree of diversity for polysaccharides and their structure-function relationships

Page 13: 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 projection.

Plant PolysaccharidesThe conformation of individual monosaccharide residues in a polysaccharide is relatively fixed, however, joined by glycosidic linkages, they can rotate to give different chain conformations.

OOHO

HOO

OH

OHO

HOO

OH

HO OHO

HOO

OHOHO

HO O

OH

1,4 glycosidiclinkage 1,6 glycosidic

linkage

Page 14: 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 projection.

The different kinds of primary structures that result in secondary and tertiary structures give different kinds of properties

– water solubility, aggregation and crystallization, viscosity, gelation, etc.

Polysaccharides have a variety of functions

– Storage of chemical energy in photosynthesis

– Inducing Structural Integrity in plant cell walls

Plant Polysaccharides

Page 15: 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 projection.

StarchStarch is composed completely of D-

glucose – found in the leaves, stems, roots,

seeds etc in higher plants–stores the chemical energy produced

by photosynthesisMost starches are composed of two types

of polysaccharides - amylose and amylopectin–amylose - a mixture of linear

polysaccharides of D-glucose units linked a-(1-4) to each other• between 250-5,000 glucose residues

Page 16: 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 projection.

Starch Polymer Components

Amylose

Amylopectin (1 residue in every 20 is 16 linked to branch off)

Page 17: 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 projection.

The Components of Starch

O

HOO

OH

OH

O

HO

HO

OH

OHO

O

OH

OH

O

O

OHO

HO OH

O

(1-4)

Amylose Amylopectin

(1-4)

(1-6)O

HOO

OH

OH

O

HOO OH

OH

O

HOO

OH

OH

OHO

OH

OO

HOO OH

OH

O

O

HOO

HO

OH

O

Starch tertiary structure (Helix)

Page 18: 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 projection.

QUALITATIVE ANALYSIS

There various tests that can be used to detect the presence or absence of carbohydrates or sugars. Some of these are:

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

Page 19: 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 projection.

MOLISCH REACTION

In this reaction the furfural that is formed from the carbohydrate by the sulfuric acid condenses with the phenol to give the characteristic color.

PROCEDURETwo ml of a sample solution is placed in a test tube. Two drops of the Molisch reagent (a solution of α-napthol in 95% ethanol) is added. The solution is then poured slowly into a tube containing two ml of concentrated sulfuric acid so that two layers form.

Page 20: 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 projection.

MOLISCH REACTION

A positive test is indicated by the formation of a purple product at the interface of the two layers.

a negative test (left) and a positive test (right)

Page 21: 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 projection.

ANTHRONE REACTION

Anthrone, 9,10-dihydro-9-ketoanthracene reacts with many carbohydrates to give a green color.

PROCEDURE

1 ml of a sample solution is placed in a test tube. 2ml of a 0.2% of Anthrone in conconcentrated sulfuric acid is added. In the presence of carbohydrates a clear green color will appear and will rapidly increase in intensity until a dark blue-green solution results.

Page 22: 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 projection.

QUANTITATIVE ANALYSIS

The quantitative methods for the estimation of sugars and carbohydrates depend on the properties of reduction and optical rotation that the sugars have. Some of the quantitative methods used are;

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

Page 23: 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 projection.

LANE-EYNON METHOD

This is a short and rapid method and often the most accurate method for the estimation of reducing sugars. It is based on a determination of the volume of a test solution required required to reduce completely a known volume of alkaline copper reagent. The end point is indicated by the use of an internal indicator, methylene blue.

Page 24: 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 projection.

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 up

to 250ml in a volumetric flask.• The solution is shaken thoroughly and

filtered.• 10ml 10% solution of potassium oxalate is to

100ml of the filtrate and made up to 500ml, shaken and filtered

Page 25: 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 projection.

LANE-EYNON METHOD

PROCEDURE• 10ml of the mixed Fehling reagent is placed in a 250ml

Erlenmeyer flask.• The sugar solution is transferred into a burette and

suspended over the Erlenmeyer flask.• 15ml of the sugar solution is added to the flask and heated to

boiling.• The solution is boiled for about 15 seconds and portions of

the sugar solution is added rapidly until only the faintest perceptible blue color remains.

• 2-5 drops of a 1% aqueous solution of methylene blue is added and heating is continued.

• The sugar solution is added dropwise until the titrtion is complete which is shown by the reduction of the dye.

Page 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 projection.

LANE-EYNON METHOD

The amount of sugar may be calculated by the formula;

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