Estimation of Sugars | Plants

In this article we will discuss about the top two methods used for estimation of sugar in plants. The methods are: 1. Titrimetric Method 2. Colorimetric Method Using Anthrone Reagent.

Method # 1. Titrimetric Method:

Principle:

All the sugars with free reducing group (aldehyde or ketone) undergo enolisation in presence of alkali. As a result they behave as strong reducing agents and are easily oxidized to acids by various oxidizing agents including the ions of metals — such as copper, silver, bismuth etc.

The amount of reduced metal is directly equivalent to the amount of reducing sugar present in the sample solution. Finally, by iodo-metric assay method, residual available oxidized metallic salt is determined to denote the amount of reduced metallic salt formed by the reducing sugars. The free iodine is determined by sodium thiosulphate solution using starch indicator.

The reactions are:

Polysaccharides and other non-reducing sugars are, of course, relatively stable in alkaline solution. Thus by this estimation method one can estimate the amount of non-reducing sugar after hydrolysis.

Requirements:

(B) Glass apparatus:

Sugar-heating tube with lid.

Water-bath and burner.

Cold-chambers.

Pipettes, Burette.

Balance, measuring cylinder etc.

(C) Preparation of sample solution:

Except polysaccharides, sugars can be easily dissolved in water. Polysaccharides can be dissolved in hot water. Before estimation, non-reducing sugar is hydrolysed by acids. From plant materials, sugars are extracted by 80% alcohol.

The extract is centrifuged and boiled in water-bath to remove alcohol. Then the residue is suspended by Lead-acetate solution (5%) and the colloidal precipitate is removed by centrifugation.

Finally the supernatant liquid is taken and the excess lead is removed by the addition of Na₂HPO₄ soln. (10%). Then the mixture is centrifuged and the super­natant solution is taken in a volumetric flask and the volume is made up to 100 ml before estimation of sugar.

1. At first the copper reagent is prepared by mixing reagent solutions A and B in the ratio 2 : 5.

2. Then 5 ml of reducing sugars or hydrolysed non-reducing sugar sample is taken in a sugar heating tube and to it 5 ml of A and B mixture is added.

3. Five such identical sets are made along with 2 control sets (which do not contain sugar solution, instead they contain 5 ml of water).

4. All these sets are kept in a boiling water-bath for about 15-20 mins. During boiling the tubes are covered by lids.

5. When the solutions turn reddish and brick-red precipitation is deposited at the bottom of the tubes, then the tubes are placed in ice cold water to cool the contents.

6. Finally 5 ml of Soln. C and 5 ml of Soln. D are added to the contents of the tubes and thoroughly shaken for about 5 mins. to ensure complete reaction. 3-5 replica of each set is made.

7. The content of each tube is then titrated with 0.01N Sodium thiosulphate soln. using starch indicator. Thus blank reading and sugar reading of thiosulphate are obtained.

8. The strength of Na-thiosulphate soln. used is determined by standardisation against N/100 K₂Cr₂O₇ Soln. For such standardization 3 ml of KI-Na₂HCO₃ reaction mixture (1 gm. of NaHCO₃ and 1.5 gms of KI in 15 ml dist. water) and 0.6 ml conc. HCl and 10 ml of N/100 K₂Cr₂O₇ are mixed together in a tube with lid and kept in darkness for 10 mins. before titration against Na- thiosulphate, using starch indicator. About 5 separate readings are taken.

9. Certain conversion factors for thiosulphate titration are also determined from the Vender- Plank calibration chart, using the formula:

Thiosulphate value = Blank reading of thiosulphate – Sugar reading of thiosulphate = X ml.

Conversion factor for such thiosulphate value from chart = Y

10. Finally sugar content of the sample solution is determined by the formula:

Amount of Sugar = Thiosulphate value/ Factor × strength of thiosulphate soln.

Method # 2. Colorimetric Method Using Anthrone Reagent:

Principle:

The furfural derivate is produced by acid decomposition of sugar and such a derivative reacts with anthrone reagent to produce a blue-green coloured complex:

Sugar + Acid → Furfural compound + Acid + Water

Furfural Compound + Anthrone reagent → Blue-green coloured complex.

The intensity of the coloured complex is equivalent to the amount of sugar present in the sample. Thus, by colorimetric estimation, the colour intensity is determined and a standard curve for known concentrations of sugar is also prepared.

Requirements:

(a) Reagent:

Anthrone reagent (2 gm. anthrone in 100 ml of 50% H₂SO₄).

(b) Instrument:

Colorimeter.

(c) Standard sugar solution:

Known concentration of sugar (50 µ gms/ml) is prepared for the use of standard curve preparation.

Procedure:

The following experimental sets are prepared by using standard sugar soln. and anthrone reagent:

Set I — 0.5 ml of sugar + 1.5 ml of dist. water.

Set II — 1.0 ml of sugar + 1.0 ml of dist. water.

Set III — 1.5 ml of sugar + 0.5 ml of dist. water.

Set IV — 2.0 ml of sugar + no water.

Then in each set 4 ml of anthrone reagent is added. A replica of 5 for each set are prepared separately. Finally, the sets are kept in cold bath for 10-15 mins. to develop colour. The colour intensity thus developed is determined at 620 nm (red filter) against a control blank set containing 2 ml of water and 0.5 ml of anthrone reagent.

The results are plotted on mm graph paper for the preparation of standard curve of known quantity of sugar using ‘X’ axis as sugar concentration, ‘Y’ axis as corresponding colorimetric reading (optical density). Sugar samples of unknown concentration then can be used in the same way for determination of the concentration by comparing its colorimetric reading with the standard curve.