10. Spectrometric determination of glucose

THEORETICAL BACKGROUND

A number of chemical reactions may occur between sugars and various reagents producing coloured products. The intensity of colour is related to the concentration of sugar present. Absorbance can be measured and related to concentration of sugars in standard solutions. Only a limited number of reactions are known for polysaccharides and most involve the reduction of simple sugars. A method for the determination of glucose in jam consists of the hydrolysis of total sugars with H₂SO₄ and neutralisation of the sample with NaOH. After heating, the DNS reagent reacts with the sugars producing a red-brown product. The concentration of coloured complex can be determined with the spectrometer SpektraTM using the blue LED.

PRINCIPLE

In hot solution, the DNS reagent (3,5-dinitrosalicil acid) reacts with the reducing sugars producing the red-brown product 3-amin,5-nitosalicil acid. The concentration of the coloured complex is measured with the Spektra^TM spectrometer using the blue LED (540nm).

EQUIPMENT AND REAGENTS
 

Preparation of DNS reagent – 3,5-dinitrosalicil acid

Weigh 10 g of DNS and dissolve it in 200 mL NaOH solution  (c = 2 mol/L). Heat the solution and stir intensively. Add colour stabiliser Na-K-tartrate (disolve 300g of Na-K-tartrate in 500 mL of distilled water). Combine both solutions, stir well and add distilled water to 1 L.

SAMPLE PREPARATION

1. Jam – sugars
   
   Weigh 1 – 2 g of jam into an Erlenmeyer flask and then add 10 mL of H₂SO₄ (c = 1,5mol/L). Heat in boiling water bath for 20 minutes. Stir periodically untill hydrolysis is complete. Cool the sample and carefully add 12 mL of NaOH (w = 10 %). Stir and filter into 100 mL volumetric flask, fill with distilled water to 100 mL. Using a pipette, transfer 10 mL of solution into another 100 mL volumetric flask and fill with distilled water to 100 mL. Stir well.
2. Jam – reducing sugars
   
   Weigh ca. 3 g of jam into an Erlenmeyer flask, add 50 mL of distilled water, heat and stir for 10 minutes. Filter the contents into a 100 mL volumetric flask and fill the flask with distilled water to 100 mL. Using a pipette, transfer 10 mL of the solution into another 100 mL volumetric flask and fill with distilled water to 100 mL.

PRODUCING COLOUR AND MEASURMENTS

Stock standard glucose solution

Weigh 1.5 g of glucose, transfer it into a 100 mL volumetric flask, fill with distilled water to 100 mL and stir. γ( glu) = 15 mg/mL

Standard solutions for calibration curve

Prepare 5 (100 mL) volumetric flasks and mark them. Using a pipette, transfer different volumes of standard glucose solution and distilled water into each flask as specified below:

Mass concentrations of glucose in calibration solutions are:

Prepare 6 test tubes and fill them as specified below:

Heat the test tubes in boiling water for 5 minutes. DNS reacts with sugar while heated producing a red-brown product. Cool test tubes and add 6 mL of distilled water into each test tube. Shake well. Transmittance of solutions can now be measured using the blue LED (540nm).

CALCULATIONS

Glucose concentration in 100 g of sample is calculated as follows:

g glucose / 100 g sample =  γ(glucose) · 10 · 100

10 = dilution
100 = volume of 1g of sample

MEASUREMENTS AND RESULTS

Table 1: Calibration curve – Absorbance related to mass concentration

 
Graph 1: Calibration curve – absorbance related to mass concentration      

Developed and prepared by: Alma Kapun Dolinar in Irena Štrumbelj Drusany, Srednja agroživilska šola Ljubljana