Title : Determination of glucose by the Nelson-Somogyis method

Objectives :

1. To determine glucose concentration by using the Nelson-Somogyis method.

2. To learn how to calibrate the spectrophotometer for biological and chemical studies

purpose.

3. To constructs a standard curve of glucose concentration by measuring absorbance.

4. To determine the glucose concentration of the milk sample extract.

Principle :

By using the Nelson-Somogyis method, the concentration of glucose and other reducing
sugars can be determined. This can be done when the sample extract or test solution is heated
together with alkaline tartrate. This will result the reducing of copper from the cupric to
cuprous state. Hence, this causes cuprous oxide to be formed. When cuprous oxide is react
with arsenomolyybdate reagent, it will formed molybdenum blue which causes the
appearance of violet coloured complex. The colour develop is compared with a set of
standards in a colorimeter at 500nm.

Introduction :

Monosaccharide is a monomer of carbohydrate. In monosaccharide, all of the carbon

atoms except one have a hydroxyl group attached. Monosaccharides can either aldose or
ketose based on the position of carbonyl group. Sugar that have aldehyde group is called
aldose or aldo sugar while sugar with keto group is called ketose or keto sugar. Glucose is a
hexose sugar and from the aldose group and has a formula of C6H12O6.

Reducing sugars are sugar that contain aldehyde or ketone group. Therefore, all
monosaccharides are reducing sugars. When reducing sugars react with other substances, it
will go through the process of oxidation-reduction chemistry. Oxidation is the process where
electrons being removed while reduction is the process of gaining electrons. From the
reaction, it produced a reduced substance plus the oxidized sugar molecule. Hence, reducing
sugar that have an aldoses group acts as reducing agent where it can give up the electrons.
The oxidising agent is reduced by receiving the electrons. Carboxylic acid group will produce
from the oxidation of the aldehyde group. Ketoses act as weak reducing sugars under alkaline
conditions this is due to that they will partially isomerize to aldoses.

Nelson-Somogyis method is one of the classical and widely used for determined
the amount of reducing sugars. The Cu2+ ions that been used in this experiment is being
reduced to Cu+ ions by reducing sugar. The arsenomolybdate complex which is prepared by
reacting ammonium molybdate , (NH4)6Mo7O24 and sodium arsenate, Na2HAsO7 in sulphuric
acid is then being reduced by Cu+ ions. An intense, stable blue colour is produced from the
reduction of the arsenomolybdate complex. The colour develop is measured
spectrophotometrically with the absorbance value at 500nm. A standard curve of glucose
concentration with its absorbance value needs to be constructed because the reaction is not
stoichiometric.

The Nelson-Somogyis method need to be conducted under the proper conditions to

get a precise and accurate result.. Thus, D-glucose, D-galactose and maltose is accurate to
+ 0.01under proper conditions. Besides that, the experiment needs to carried out under inert
atmosphere and the measured concentration do not more than 1 mg/ml, so that consistent
result can be get. The method still can be performed qualitatively even though a
spectrophotometer is not available.

Materials and methods :

Materials :

- Boiling water bath

- Visible spectrophotometer

- 5% ZnSO4 solution

- 0.3 M Ba(OH)2

- Milk sample

- Micropipette

- Cuvette
- Parafilm

- Test tubes

- Test tube holder

- Alkaline copper reagent:

Solution A: Dissolve 25g anhydrous Na2CO3, 25g Na-K tartrate (Rochelle salt), 20g

NaHCO3 and 200g anhydrous sodium sulphate in 800ml distilled water and

make up to 1litre.

Solution B: Prepare 15% (w/v) CuSO4.5H2O and add one or two drops of concentrated

sulphuric acid.

Mix 25 parts of solution A and 1part of solution B before use.

- Arsenomolybdate reagent : Dissolve 25g of ammonium molybdate in 450ml of distilled

water. Add 21ml of concentrated sulphuric acid and mix well. Dissolved 3.0g of disodium

hydrogen arsenate in 25ml water and added the solution to the acidified molybdate solution.

The solution is mix well and kept at 37oC for about 24-48 hours.

- Standard glucose solution : 0.2g of glucose is added to 10-15ml of 0.2% benzoic acid and

the volume is make up to 100ml by adding distilled water.

Methods :

1. 4ml of milk sample is prepared.

2. 0.4ml of 0.3M Ba(OH)2 was added to 1.0ml of sample extract and the solution is mix well.

3. 0.2ml of ZnSO4 was added and the solution is shake gently.

4. The solution was filtered by using Whatman no.1 filter paper after 10minutes.

5. 1.0ml of alkaline copper reagent was added to 0.1ml aliquot of the filtrate. The mouth of

the test tube is covered with parafilm and heat in the water bath for 20minutes.

6. The tube was then allowed to cool after 20minutes.

7. 1.0ml of arsenomolybdate solution was added.

8. The wavelength of spectrometer is adjusted to 500nm and the reading of spectrometer is

 then zeroed by using the BLANK solution.

9. 1ml of solution is pipetted from tube into cuvette .The cuvette is put into the spectrometer

and the reading is recorded.

10. The procedure 6-8 is repeated with glucose containing 0.2 to 1.0 ml standard glucose

solution.

11. A standard graph of absorbance vs. glucose concentration is plotted.

Results :

Concentration Alkaline CuSO4 Arsemonolybdate Distilled Absorbance

(ml) (ml) (ml) water (ml) (nm)
Blank 1.0 1.0 3.0 0.000
Standard (ml)
0.2 1.0 1.0 2.8 0.216
0.4 1.0 1.0 2.6 0.575
0.6 1.0 1.0 2.4 0.579
0.8 1.0 1.0 2.2 0.682
1.0 1.0 1.0 2.0 0.720
Sample
0.1 1.0 1.0 2.9 0.112
 1.000

0.900

0.800

0.700

0.600
Absorbance value (500nm)

0.500

0.400

0.300

0.200

0.100

0.000
0 0.2 0.4 0.6 0.8 1 1.2
Glucose concentrtion (g/mL)
Calculation :

Concentration of stock standard : 100mg/100ml

Working standard concentration : 100g/ml (10ml of stock diluted to 100ml)

Absorbance corresponds to 0.1ml of sample = 0.12mg of glucose

Therefore, 5ml contains = (0.12/0.1)(5)

= 6.0% of reducing sugars
There are 0.12 mg glucose in 5 ml, thus there are (0.12/5)(100) = 2.4 mg glucose/100 ml

Discussion :

From the graph, it shows that the absorbance value increases as the concentration of
glucose solutions increases. The concentration of sample extract which is 0.12 mg/mL, was
determined using the plotted standard curve of absorbance vs. concentration of glucose. Thus,
in 5ml of solution, there is 6% of reducing sugars. Based on the graph, the points (0.20mL,
0.216), (0.40mL, 0.575), (0.80mL, 0.682) and (1.00mL, 0.720) do not fall on the standard
curve line. The points (0.20mL, 0.216) and (0.40mL) fall slightly above the line while the
points (0.80mL, 0.682) and (1.00mL, 0.720) fall below the line. This could be due to the bad
pipetting technique during dilution that occured in early steps. Besides, the cuvette might be
handled in an inaccurate way which may also affect the result.

Furthermore, systematic and/or random errors might occur during experiment.

Systematic errors include errors that arising from detector linearity, non-standard geometry,
polarization, bandwidth and wavelength. Systematic errors are caused by the characteristics
of the instrument that are similar for all measurements. Random errors can be cause by
inability to control the instrument which may be caused by sample presentation, drift and
electric noise.

Nelson-Somogyis method was used to determine the glucose concentration.

Glucose which is one of the reducing sugar was oxidised to Cu (I) ion from the solution of
Cu(II) ion during the first step. Then, in the next step, arsenomolybdate complex was used in
which Cu (I) ion is oxidised back to Cu (II) ions. When the arsenomolybdate react it will
reduced to give the characteristics of blue colour. Lastly, spectrophotometer was used to
measure the absorption of the arsenomolybdate blue. The graph is plot for compared to
standards prepared from reacting sugar solutions of known concentration, to determine the
amount of reducing-sugar present. Arsenomolybdate complex which is formed by the
reaction of ammonium molybdate, (NH4)6Mo7O24 with sodium arsenate, Na2HAsO7 was used
in the Somogyi-Nelsons method. Sodium sulphate is added in the copper reagent so that
reoxidation of cuprous oxide by atmospheric oxygen does not happened.

The sample that been used in this experiment is the milk sample. This sample is being
used because it is simple and the most important that this sample is easily available. Milk
contain lactose sugar which is it a reducing sugars.

In this experiment, the solutions need to be placed in boiling water bath for 20minutes
and cool down for 20minutes. This is to allow the colour to develop for 20 minutes. The
colour develops occured rapidly and will be completed by the time thorough mixing and
evolution of carbon dioxide is completed. The colour that formed is very stable and its
stability is absolute and not relative. Moreover, the density of the blanks as well as of the
more deeply colored solutions remains the same with time.

The wavelength for determination of glucose was 500nm. This wavelength was
chosen simply because it can portrayed a fulfilling expectations that compromise between the
sensitivity and the benefits that get by decreases to a minimum the effect of variation in such
circumstances as blank due to reagent and reoxidation of cuprous oxide.

The glucose solution is transferred to a clean cuvette after 20minutes and wiped with
a tissue before putting inside the spectrophotometer to get more accurate results. Measuring
and recording of absorbance value must be fast because the colour of glucose will continue to
develop and get darker with time. This will make the result inaccurate and subsequently the
concentration of the sample obtained from the standard curve will not be accurate. Cuvette
should be handled carefully so that they are not scratched. To avoid contaminating the glass
with skin oils, the cuvette is only held at the top edge and the cuvette is rinsed with distilled
water immediately after use to prevent materials from drying onto the inner surface.

The absorbance value must not exceed than 1.0 O.D. unit. This is because it can affect
the linearity of the absorbance versus concentration. If the absorbance that measured is more
than 1.0 O.D. unit or more, the sample needs to be diluted so that absorbance value is less
than 1.0 O.D unit. Moreover, insoluble residues can affect the experiment result.
Conclusion :

From the experiment, the concentration of sample can be found by plotting the
standard curve by absorbance versus glucose concentration. The graph of the glucose
absorbance is directly proportional to its concentration and this shows their relationship is
linear. Therefore, the sample extract concentration of which has the absorbance of 0.112 is
0.12mg/mL as the value can be determined from the standard curve drawn. Furthermore,
there is only 6% of reducing sugars that contain in 5ml of solution.

Literature cited :

1. Nelson, N. (1944). A Photometric Adaptation of the Somogyi Method for the

Determination of Glucose. Retrieved on 6 June 2011 from
http://www.jbc.org/content/153/2/375.full.pdf+html

2. E. J. King and R. J. Garne. (N.D). The Colorimetric Determination of Glucose. Retrieved

on 7 June 2011 from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1023202/pdf/jclinpath00001-0034.pdf

3. Somogyi, M. (1945). A New Reagent for the Determination of Sugars. Retrieved on 7June
2011 from http://www.jbc.org/content/160/1/61.full.pdf

4. Fales, F. W. ; Russell, J. A. and Fain, J. N. Some Applications and Limitations of the

Enzymic, Reducing (Somogyi), and Anthrone Methods for Estimating Sugars. Retrieved on
7June 2011 from http://www.clinchem.org/cgi/reprint/7/4/289.pdf
Acknowledgement :

First of all, I would like to thank God for blessing and guiding me to make my
practical and report possible. I would like to express my gratitude to several people because
of their willingness for lending me hands and spontaneously supported me in order to
complete my report.

First and foremost, I would like to wish big thanks to my practical lecturer, Ms. Anto
Cordelia for her invaluable assistance, patience, and unwavering support, priceless advises
guidance, and suggestions throughout the duration of this practical. Besides, not forgotten to
the rest of the lab officer from who have giving me a very good cooperation in preparing the
solution for used of the practical.

I also would not forget the assistance and encouragement from my practical group
members. With their help, I could solve the difficulties I faced in practical and report.

There are too many experiences that I have learned through this period of finishing
the project. I really appreciated all the support that has been done to make this practical and
report goes well.

Last but not least, I want to thank my friends, course mates and all the individuals
who have in one way or another to helped me in the completing of practical and this report.
Thanks for their patience, tolerance and moral support and the words of encouragement
which helps me a lot when facing difficulties and it also has always been a source of
motivation throughout my study.