Experiment 7: Quantitative determination of sugars in honey and milk

Part A: Enzymatic determination of glucose in honey

Results:

Groups Absorbance of Absorbance of Concentration of Concentration

standard solutions sample solutions at sample glucose of fructose
at 500 nm 500 nm (mg/mL) (mg/mL)

A 0.980 0.121 3.858 x 10

4
5.99 x 10-2

B 0.165 0.173 3.277 x 10

3
6.06 10-2

C 0.209 0.234 3.499 10-3 6.04 10-2

Calculation:

According to Beer-Lambert Law,

A
c=
l

Therefore,

A unknown Cunknown
=
A standard C standard

0.1mL
0.05 mg x
Concentration of standard glucose = 1.6 mL

3
=3.125 x 10 mg/mL

0.121 C unknown
=
0.98 3.125 x 103
 4
Concentration of unknown glucose = 3.858 x 10 mg/mL

Concentration of fructose

Reducing sugar = glucose + fructose

Based on the result in experiment 7b, the reducing sugar in honey is 6.03 x 10-2 mg/mL.

4
6.03 x 10-2 mg/mL=3.858 x 10 mg/mL + fructose

Concentration of fructose in honey = 5.99 x 10-2 mg/mL

Mean for the concentration of glucose in honey:

X 1+ X 2 + X 3 3.858 x 104 +3.277 10 +3.499 10

= =2.387 10 mg/mL
3 3

Standard deviation:

2
3.499 10 2.387 10

3.277 10 2.387 10 2 +
3.858 x 104 2.387 10 2 +

6.03344 106
2
=1.7369 103

Answer: 2.387 10 mg/mL 1.7369 10

3
Discussion

To calculate the concentration of glucose in the honey, Beer-Lambert Law was

applied in this experiment. Beer-Lambert Law states that the amount of light absorbed or
transmitted by a solution is a function of the concentration of the substance and the
sample path length (Csuros 1997).

There are three reactions occurring in glucose oxidase method of analysis. Firstly, with
the presence of glucose oxidase and oxygenglucose is oxidized to gluconic acid and
hydrogen peroxide. The resulting concentrations of gluconic acid and H2O2 are
proportional to the amount of glucose originally present. Secondly, hydrogen peroxide
was converted to water and the chromogen with the presence of peroxidase enzyme and a
chromagen. The chromogen produces a color (Estridge et al 2000). Oxidized o-
Dianisidine reacts with sulfuric acid to form a more stable colored product. The intensity
of the pink color measured at 540 nm is proportional to the original glucose concentration
(Batu & Solomon, 2014).

Based on the result, the concentration of glucose in the honey sample is 3.125 x 103

mg/mL. Another two more results were taken from the other two groups and the mean of
the concentration of glucose in honey was calculated which is 3.72210 -3 mg/mL. The
estimated absorbance value of standard solution is different with the experimental result
which shows us the absorbance value of standard solution (0.98) is higher than the
absorbance value of sample solution (0.121). This might due to human error, the solution
might cross contaminated between different solutions or reagent where the pipette was
not rinsed properly with distilled water. The concentration of sample glucose for group

A, B and C are 3.858 x 104 , 3.277 x 103 and 3.499 10-3 respectively. In

contrast, The concentration of sample fructose for group A, B and C are 5.99 x 10 -2, 6.06
10-2 and 6.04 10-2 respectively. It can be concluded that the concentration of fructose
is higher than glucose in honey (Ball 2007). Besides, the mean concentration of reducing

sugar in honey sample was 2.387 10 mg/mL with standard deviation,

1.7369 103 . The standard deviation value was 73.76% which is more than 10% of

the mean. This shows that the result had high dispersion, thus it is not reliable. This can
be due to some human error when carrying out this experiment.

The advantage of the method is it is highly specific. It is based upon the release of
hydrogen peroxide from glucose during its oxidation by glucose oxidase. The peroxide is
detected by a suitable indicator in the presence of enzyme (Eliot & Turner 1958).
Besides, the glucose oxidase method had the best repeatability and best reproducibility as
compared with hexokinase method and glucose dehydrogenase method (Dohnal et al
2010).

Most of the carbohydrates in honey are monosaccharides, with more fructose than
glucose. At a distant third place is sucrose; other disaccharides present in honey, albeit in
very small quantities, are maltose, isomaltose, nigerose, turanose, and maltulose. At about
1% or less of the total sugars, a small quantity of higher sugars, oligosaccharides, and
dextrins are present cosin honey. Besides, honey has low moisture content. Honey is
typically about 83 degrees Brix or about 83% sugar. Honey is deceptively acidic, as the
high sugar content tends to mask the acidity in the taste. The average pH of honey is 3.9.
In addition to diastase, invertase, and glucose oxidase, other enzymes found in honey
include catalase and an acid phosphatase. There is only a small quantity of protein in
finished honey as indicated by the low nitrogen content. Total amino acid rarely exceeds
300 ppm (Ball 2007).

Nevertheless, the composition of honey is not only carbohydrate but also mineral.
Potassium, sulfur, chlorine, calcium, phosphorus, magnesium, sodium, iron, copper and
manganese are between 50-fold ranges of values in honey. Trace element such as
chromium, lithium, nickel, lead, tin, and zinc also can be found in honey. The
concentration of vitamin in honey is low. Those detectable quantities of vitamin
contained in honey include riboflavin, pantothenic acid, niacin, thiamin, pyridoxin and
ascorbic acid. Honey has a high refractive index, 1.49 and high viscosity, 120 poise at
room temperature (Ball 2007).

There are some precaution steps that needed to take care throughout the experiment. The
precaution step including mixing the mixture thoroughly by using the Vortex to avoid the
existence of air bubbles inside the sample. Next, stopper was used for test tube to prevent
any cross contamination. To get the precise reading, the cuvette should be wash with
distilled water and wipe with tissue paper before measure.

Conclusion

The mean concentration of glucose in honey is 2.387 10 mg /mL . The

concentration of fructose is higher than glucose in honey.

Reference

Ball, D. W., 2007. The Chemical Composition of Honey. Journal of Chemical Education,
84(10), pp. 1643-1646.

Batu, D. W. & Solomon, T. W., 2014. Quantitative Determination of Sugar Levels in

Natural Plants of Cactus Pear (Opuntiaficus indica) and Votre-Coach Alimantaire
Cultivated in Adigrat, North of Ethiopia. International Journal of Innovation and
Scientific Research, 10(1), pp. 125-134.

Csuros, M., 1997. Environmental Sampling and Analysis Lab Manual. Boca Raton: CRC
Press.
Dohnal, L., Kalousova, M. & Zima, T., 2010. Comparison of Three Methods for
Determination of Glucose. Prague Medical Report, 111(1), pp. 42-54.

Eliot, F. B. and Turner, J. J., 1958. An Enzymatic Method for Glucose Determination in
Body Fluids, Clinical Chemistry, 4 (6), pp.462-475.

Estridge, B. H., Reynolds, A. P. & Walters, N. J., 2000. Basic Medical Laboratory
techniques. 4th ed. USA: Delmarn Thomson Learning.