FINDING OUT THE LEVEL OF SUGAR FROM AN ORANGE USING THE GOD-PAP METHOD

Introduction Glucose and fructose are simple monosaccharides, which are found in all plants, with fructose and galactose, glucose is on of the three dietetics monosaccharides which are absorbed directly in blood during the digestion. Glucose is the main source of energy for organism. It is a solid substance, crystalised, incolor, soluble in water, sweet taste and its melting point is very high, because among its many hydroxy groups (-OH) are formed many hydrogen bonds. Glucose is obtained by hydrolysis of starch (from potatoes or grain), and by hydrolysis of cellulose under the action of dilute mineral acids (HCl or H2SO4), in autoclaves. Glucose is used in the preparation of ethyl alcohol and carbon dioxide in the manufacture of mirrors, in printing textile fabrics, the preparation of synthetically vitamin C. The so-called GOD-PAP assay (glucose oxidase peroxidase aminophenazone) is based on two coupled enzymes reactions with a colorimetric end-point. The characterizing ecuation is the following: D-Glucose+O2pH2O2 +gluconate 2H2O2+Phenol+AminophenazonepRed quinone+H2O

Glucose oxidase (GOD) turns the sample into gluconate. The hydrogen peroxyde (H2O2) produced in the reaction is degraded by peroxidase (POD) and gives a colored product which is measurable using the spectrophotometer at 500nm. The increase of absorbance is correlated with the glucose concentration of the sample. Precise spectrometric study on awide range of wavelengths and low concentrations of substance can be performed using devices called spectrometers. In such cases, the analyzed compound is introduce dinto a cuvettewith 1 cm side. The cuvettes are transparent optically cells that hold the substance under study and are used to introduce samples into the light path. A witness cuvette is always needed for setting

the spectrophotometer to read zero absorbance at each wavelength change. The source of light is often a tungsten lamp for the visible region of the spectrum. The absorption spectrum or the absolute absorption spectrum, of a compound may be shown as a plot of the light absorbed by that compound against wavelength. Such a plot for a colored compound will have one or more absorption max's in the visible region of the spectrum (400 to 700 nm). To obtain an absorption spectrum, the absorbance of a substance must be measured at a series of different wavelengths. Absorption in the visible and ultraviolet regions can be measured by a UV/visible spectrophotometer. Thisexperimentaimed toestimate the level ofthe glucoseinan orangeusing acalibrationcurveandthe GOD-PAP reducing method.

METHODS Preparation of an extract of an orange After being accurately weighed 2.5 grams oforange, were placed in a beaker that contained 75ml distilled water and homogenized for about 3minutes. Homogenate was transferred equally in two 50ml centrifuge plastic tubes, and centrifuged for 3minutes at 3000rpm. The supernant was carefully poured off through a funnel lined with glass wool into a 100ml volumetric flask, which was filled up to the mark with distiled water and mixed thoroughly.

Dilution of orange extract Because at that stage was known whether the concentration of glucose would give an absorbance of the top of the calibration curve, a range of dilutions of the extract were prepared before carrying out the GOD-PAP assay. Three Eppendorf tubes were labeled A, B and C and made three dilutions of the orange extract as follows:

Dilution: A Orange extract(ml): Distiled water(ml): Dilution factor: 0.1 0.9 10 B 0.2 0.8 5 C 0.5 0.5 2

Each microcentrifuge tube was mixed by vortexing. Assay for glucose by the GOD-PAP method

Cuvette Glucose conc (mM)

S0 a/b 0.0

S1 a/b 0.2

S2 a/b 0.4

S3 a/b 0.6

S4 a/b 0.8

S5 a/b 1.0

A a/b ?

B a/b ?

C a/b ?

Add the following reagents: 1mM Glucose standard ( l) Distilled water (Ql) Diluted extract (Ql) GOD-PAP reagent 0 100 20 80 40 60 60 40 80 20 100 0 100 100 100 -

Add 1.0 ml to ALL cuvettes using automatic dispenser

A series of cuvettes were set up, as shown below in Table1.

Table 1: Estimation of glucose by the GOD-PAP method-setting up your cuvettes Samples marked S0-S5 were the calibration standards and were set up in duplicate. The 1mM glucose standard provided was used to set up the calibration series. S0 represents the standard and served as reagent blank, it contained only the assay reagent and it was used to set the spectrophotometer reading to zero before the other solutions were measured. A cuvette stirrer was used for mixing all cuvettes, then were incubated at room temperature for 25 minutes.

Observation: After the incubation, the content of cuvettes turned in pink, the intensity of color was directly proportional with the glucose concentration.

The spectrophotometer was used for reading the absorbance at 500nm, and the absorbance readings were recorded in Table2.

Table 2: Absorbance readings for GOD-PAP glucose assay

Dilution factor: S5

x10

x5

x2

Cuvette No. Glucose concn. (Standards) (mM) Series (a) Absorbance at 500nm Glucose concn. (Unknowns) (mM) Series (b) Absorbance at 500nm Glucose concn. (Unknowns) (mM)

S0

S1

S2

S3

S4

A B

0.0

0.2

0.4

0.6

0.8

1.0 Fffffff f0.27 Sfdsf 0.28 Dsffd Dfwd 0.29 f0.72 Lkjiih 0.7

0.00

0.13

0.23

0.4

0.52

0.66

0.14

0.00

0.13

0.27

0.4

0.53

0.68

0.14

Observing the table it is obvious that the results obtained are very close, comparing samples Sa and Sb is clear that the results were accurate. Readings for S0 to S5 have been used to plot a calibration curve (Figure1), (attached to the end of this lab report).

Calculation of glucose content The average of the two B dilutions is calculated, but this concentration is currently in mM. To convert concentration in umoles/ml is necessary to multiply the average by the dilution factor, which is 5, for obtaining the concentration in umoles/ml of undiluted extract. The total volume of undiluted orange extract was 100ml, so to get the concentration in umoles of glucose per total volume (100ml) of orange extract, the answer was multiplyed by 100. To get umoles of glucose/g orange the number obtained before was divided by the used weight of orange which was recorded at the beginning of the laboratory (2,5 grams) and multiplyed by 100 to get umoles/100g orange.

The complex equation is the following:

Dilution factor x 100 (ml) Average [glucose] (mM) x Weight of orange (g) x

180 x 100 g/100 g orange 106

= __0.81__ g/100g orange

Discussion

The BeerLambertBouguer law is the linear relationship between absorbance of light and concentration of the material through which the light is travelling. This law is the basis in colorimetric and photometric analysis. It should be noted that othe rphysical phenomena are being ignored, such as: diffusion, refraction and polarization. As result of the BeerLambert law for a particular substanceand a precise thick layer, dependence between absorption and concentration is linear. This dependence is valid only for small concentrations of up to 10-20mole/l. In this case, Beer-Lambert law is obeyed, as the graph (Figure1), clearly shows the dependence between the absorption and concentration. Other factor sthat could influence the accuracy of results and cause deviations from linearity are of chemical and physical like: scattering of light due to particulates in the sample stray light reflection and absorption caused by the cuvette walls cloudy substance temperature variations, which in this case can be neglected air bubbles or any dirt on the cuvettes walls Other sources of error could be constituted by the fact that labskills, are not fully developed yet, and more hours spent in laboratory are needed. But the sugar level can vary from one orange to another.

Sugar is a carbohydrate, most often found in the form of sucrose, a white crystalline solid. Common sugar is composed of two simple sugars: glucose and fructose. As glucose, sugar is used as a biologica lreserve of energy in cells. The estimated sugar content of an

average orange (200grams), is 15grams of sugar. But only half of the 15 grams is glucose, because theother half is fructose.

This laboratory was designed to assess laboratory skills, and assimilation of new knowledge such as carrying out the GOD-PAP assay, using a blender and a whirlymixer and preparation of an orange extract.

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1. www.fiu.edu/spectrophotometry.pdf