1. Lecture 1. WET METHODS OF CARBOHYDRATE ANALYSES

2. Nomenclature of Carbohydrates D, L Defines the configuration at C5 D has the OH at Right in Fischer projection L 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

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

4. Alditols In Mannitol C2, C4, C5 OH’s are not at same side in Fisher Projection

5. Conformations [a][a][a][a] 25 D +19 o +112 o Anomers For aged solutions [a][a][a][a] 25 D = +52.7 o Rotations of Fresh Solutions Reason: Mutarotation is the best evidence for the cyclic hemiacetal structure of D-(+)-glucose

6. Monosaccharides,Hemiacetal Formation II C5 OH attacks aldehyde giving a pyranose ring (6 member structure) C4 OH attacks aldehyde giving a furanose ring (5 member structure)

7. Ring closure between C1 and C4 -OH Ring closure between C1 and C5 -OH

8. 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)

9. Polysaccharides –refer to polymers composed of a large number of monosaccharides linked by glycosidic linkages

10. Cellulose  -D-anhydroglucopyranose units linked by (1,4)-glycosidic bonds

11. Polysaccharides Polysaccharides can be divided into two classes –Homopolysaccharides celluloseconsist of only one kind of monosaccharide ex cellulose –Heteropolysaccharides consist of two or more kinds of monosaccharides galactoglucomannans ex galactoglucomannans

12. 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

13. Plant Polysaccharides The conformation of individual monosaccharide residues in a polysaccharide is relatively fixed, however, joined by glycosidic linkages, they can rotate to give different chain conformations. 1,4 glycosidic linkage 1,6 glycosidic linkage

14. 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

15. Starch Starch is composed completely of D- glucose –found in the leaves, stems, roots, seeds etc in higher plants –stores the chemical energy produced by photosynthesis Most starches are composed of two types of polysaccharides - amylose and amylopectin –amylose - a mixture of linear polysaccharides of D-glucose units linked  -(1-4) to each other between 250-5,000 glucose residues

16. Starch Polymer Components Amylose Amylopectin (1 residue in every 20 is 1  6 linked to branch off)

17. The Components of Starch Amylose Amylopectin Starch tertiary structure (Helix)

18. 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

19. 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. PROCEDURE Two 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.

20. 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)

21. 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.

22. 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

23. 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.

24. 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

25. 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.

26. 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.