10. Spectrometric determination of glucose

THEORETICAL BACKGROUND

PRINCIPLE

EQUIPMENT AND REAGENTS

SAMPLE PREPARATION

PRODUCING COLOUR AND MEASURMENTS

CALCULATIONS

MEASUREMENTS AND RESULTS

glucose

A number of chemical reactions may occur between sugars and various reagents producing coloured product. 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 simple sugars, usually reducing sugars. A method for determination of glucose in jam consists of hydrolysis of total sugars with H₂SO₄ and neutralisation of the sample with NaOH. After heating the DNS reagent reacts with reducing sugars producing red-brown product. The concentration of coloured complex can be determined with the spectrometer SpektraTM using the blue LED.

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

Spektra^TM spectrometer
jam
DNS reagent (3,5dinitrosalicil acid)
H₂SO₄ solution (c= 1,5mol/L)
NaOH solution (w=10%)
pipettes

100 mL volumetric flasks
test tubes
balance
water bath
funnel
filter paper

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.

Jam – sugars

Weigh 1 – 2 g of jam into Erlenmeyer flask. Add 10 mL of H₂SO₄ (c = 1,5mol/L). Heat in boiling water bath for 20minuts. Stir periodically unstill hydrolysis is completed. 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. Transfer with pipette 10 mL of solution into another 100 mL volumetric flask and fill with distilled water to 100 mL. Stir well.
Jam – reducing sugars

Weigh cca. 3 g of jam into Erlenmeyer flask add 50 mL of distilled water, heat and stir for 10 minuts. Filter into 100 mL volumetric flask. Fill the flask with distilled water to 100 mL. Transfer with pipette 10 mL of solution into another 100 mL volumetric flask and fill with distilled water to 100 mL.

Stock standard glucose solution

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

Standard solutions for calibration curve

Prepare 5 (100 mL) volumetric flasks and mark them. Transfer with pipette into each flask different volume of standard glucose solution and distilled water as specified below:

flask	1	2	3	4	5
st. glucose sol. (mL)	2	3	6	8	10
dist. water (mL)	98	97	94	92	90

Mass concentrations of glucose in calibration solutions are:

flask	1	2	3	4	5
mass conc. (mg/mL)	0.3	0.45	0.9	1.2	1.5

Prepare 6 test tubes and fill them as specified below:

Heat test tubes in boiling water for 5 minutes. DNS reacts with sugar while heated producing 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).

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

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

10 = dilution
100 = volume of 1g of sample

stand. sol. (mL)

st. sol. conc. (mg/mL)

DNS reagent (mL)

distilled water (mL)

Table 1: Calibration curve – Absorbance related to mass concentration

mass concentration (mg/mL)	absorbance
0	0.56
0.15	0.62
0.3	0.63
0.45	0.69
0.9	0.74
1.2	0.82
1.5	0.84

sample	A
1	0.73
2	0.67

Graph 1: Calibration curve – absorbance related to mass concentration

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