Determination of the glucose concentration of an orange by using

spectrophotometer
Introduction
Concentration of glucose can be determined by using spectrophotometer.
Spectrophotometer works by measuring absorbance of a solution at a set
wavelength since concentration is proportional to absorbance based on BeerLamberts law. Therefore, glucose concentration of an orange can be determined.
(Glencross et al. 2011)
Glucose is a six-carbon monosaccharide found in fruits however fruits include
inverted sugar that is a combination of glucose and fructose. Therefore, glucose
was extracted by the use of GOP-PAP assay. GOD-PAP assay is an enzyme with
breaks down sucrose into glucose and fructose. (Coventry University 2014).
In this experiment, glucose oxidase breaks down glucose into gluconate and
hydrogen peroxide (HO). HO produced in the reaction is broken down by an
enzyme (peroxidase) that gives the coloured red dye. The red dye (quiononimin)
was used to determine the glucose concentration of an orange (Medichem). At
500nm, the intensity of red dye is measured, and the result will be proportional
to the concentration of glucose in the sample. GOD-PAP assay was used to break
down sucrose into fructose and glucose. Thus, glucose can be measured.
glucose
oxidase
D-glucose + O +Ho

HO + gluconate

Aminophenazone + phenol + HO

peroxidase

red dye + HO

The spectrophotometer was set at 500nm, and this was the max at which
glucose absorbs light best, because different molecules absorb light at different
wavelength. Thus, its important to have the right wavelength. In addition, BeerLambert theory states that the absorbance is proportional to the concentration
thus the absorbance of a known concentration can be compared to the
absorbance of an unknown concentration by reference to the calibration curve.
(Glencross et al. 2011)
The aim is to determine the glucose concentration of an orange by running
absorption spectrum of a series of known glucose concentration sample and
comparing the absorbance obtained of the unknown glucose sample from an
orange.
Method

In preparation to determine the glucose concentration of sample A, B and C, the

experiment was carried out as described in the laboratory schedule (Coventry
University, 2014).

Results
Spectrophotometer was set at 500nm, and the absorbance of known and
unknown concentration was recorded (table 1), and the spectrophotometer was
re-blanked as the absorbance was recorded for each concentration.
CHO
0
Concentrati
on (mM)
Absorbanc
0
e of A
Absorbanc
0
e of B
(Table 1)

20

40

60

80

100

0.124

0.235

0.330

0.415

0.526

0.111

0.386

0.595

0.124

0.229

0.346

0.436

0.577

0.128

0.457

0.542

The absorption spectrum was plotted to create a calibration curve (figure 1).
The standard concentration of glucose was used to construct a calibration curve.

0.7
0.6
0.5
0.4
Absorbance

0.3
0.2
0.1
0
0

10

20

30

40

50

60

70

80

90

100

Concentration of CHO (mM)

(Figure 1)Calibration curve showing the concentration of series A and

series B and unknown sample (A, B and C)

The calibration curve is passing through the origin, and the line is linear (figure
1). To determine the glucose concentration of A, B and C the absorbance of the
duplicates (1mM glucose standard solution) was used. Absorbance of series A
and series B was recorded to obtain a line of best fit, and to determine the
concentration of A, B and C, the calibration curve was used.
Absorbance of concentration 20 of series A and B was the same (0.124mM)
(Table 1). As the concentration of series A and series B increased absorbance
also increased for both series. Absorbance of concentration 100 (series A and B)
was not within the line of best fit compared to the other concentration (figure
1).
A
Concentration
(mM)
Duplicate 1 (D1)
Concentration
(mM) Duplicate 2
(D2)
Difference (D1-D2)

0.22

0.72

1.04

0.26

0.86

1.50

0.04

0.14

0.46

(Table 2)
Table 2 shows the values of unknown concentration sample A, B and C. The
calibration curve was used to determine the values by reading of the
absorbance.
An appropriate concentration was selected which was within the range of the
calibration curve, and the average was calculated of the two replicates. The
suitable concentration was sample A because the duplicates had a difference of
0.04 whereas the difference between the replicates in sample B and sample C
was greater.
Average glucose in dilution A = 0.1195 (0.120 1.dp)
Calculation:CHO /100g orange
Average [CHO ] (mM)x dilution factor x 100(ml) x 180 x 100
Weight of orange (g)

10^6

CHO in orange = 0.847 (0.85 1.dp) g/100g orange

Ratio = 1:1:2 Glucose:Fructose:Sucrose
Therefore: Mass of inverted sugar: 0.85 2 = 1.7
Discussion
Spectrophotometer was used to determine the concentration of sample A, B and
C. Spectrophotometer works by the use of visible light to absorb substances at
particular wavelength. Therefore, the concentration of the substance can be

determined (Meah et al. 2012). The dye (quiononimin) absorbed light at 500nm
(max) because the dye absorbs light efficiently at 500nm (Coventry University
2014). Having a suitable wavelength is important because it affects how much
light is absorbed and the accuracy of the results (Gore, M.G., 2000).
When light is absorbed molecules such as chromophore become excited, and
energy is transferred from the ground state to an excited state. The amount of
energy transferred is related to how much light is absorbed. Moreover, the
amount of light absorbed depends on how concentrated the solution is and the
number of molecules present. Thus, at high concentration more light is absorbed
because molecules can collide more, therefore, absorbance of glucose is high.
(Glencross et al. 2011)

The calibration curve is a straight line, starting from the origin and gradually
increasing linearly. The origin is the value of the blanks. The calibration curve
shows as glucose concentration increases absorbance also increases linearly.
Beer-Lambert law states concentration is proportional to absorbance hence if
concentration increases absorbance also increases. (Glencross et al. 2011). In
support, the calibration curve is a straight line indicating Beer-Lambert law was
met.
Concentration of A, B, C was obtained by plotting wavelength along the x-axis
and absorbance along the y-axis to create a calibration curve. The calibration
curve was used to obtain the concentration of A, B and C by reading of the
absorbance. The values of the duplicates were not consistent, and this indicates
the measurements were not accurate. For example, in sample C the
concentration in duplicate 1 is 1.04 whereas the concentration in duplicate 2 is
1.50, theres a difference of 0.46.
It could be because of, inaccurate pipetting and finger prints on the cuvettes.
The fingerprints on the cuvette can affect the amount of light being absorbed
because it may interfere with the path of light. Moreover, the angle of light may
change causing the molecules in the sample to absorb less light. Moreover,
absorbance is the difference between absorbed light and transmitted light. The
proportion of transmitted light is more likely to be high then the proportion of
absorbed light hence absorbance will be small (Gore, M.G., 2000). Also, some
light can be scattered instead of being absorbed. To prevent this, the sample
should be close to the light detector. (Gore, M.G., 2000)

Absorbance is the different of absorbed light and transmitted light: A =

log 10 1 /T (Gore, M.G., 2000)

Secondly, contamination of equipments can affect the concentration of the
sample. Cuvette lids can be used to prevent contamination (Gore, M.G., 2000).
Moreover, weighing out the orange was inaccurate due to rounding.
The aim was to find the glucose content in the orange sample. Oranges contain
sugars such as sucrose, glucose and fructose. The glucose content in the orange
sample was 0.85g. The average glucose content in an orange is 2.737g
(Livestrong). The glucose content in the sample is close to the average, showing
good accuracy.
The total sugar content of a standard orange is 9.2g (The Paleo Diet). Sugar
content of inverted sugar was 1.7g this was calculated by doubling the value of
glucose content because the proportion of glucose and inverted sugar is 1:2
(Thermo Scientific).
However, in regards to the ratio, glucose does not represent the total sugar
content in the orange sample because there are other sugars present such as
fructose and sucrose. Moreover, the concentration of fructose and sucrose is not
known. The concentration of both sugars may be different to glucose, and it can
affect the sugar content of fructose, sucrose and total sugar content. Therefore,
concentration of fructose can be measured by using an enzyme called
hexokinase (Steegmans et al. 2007). Therefore, the mass of fructose and glucose
is known and mass of inverted sugar can be determined. Secondly, the same
proportion of glucose and fructose is needed to make sucrose based on the ratio
2:1:1.
Overall, spectrophotometer was used to produce a calibration curve and the
calibration curve was used to determine the concentration of sample A, B and C.
The accuracy was affected by human errors such as inaccurate pipetting and
finger prints on cuvettes and systematic errors such as weighing out the sample.
Finally, it can be argued that glucose does not represent the total sugar content
in an orange since fructose and sucrose is present.
Words: 1489
Reference
Coventry University (2014) 116BMS Skills in Biomolecular Sciences schedule:
Determination of the Glucose Content of an Orange
Glencross, H., Ahmed, N. and Wang, Q. (2011) Biomedical Science Practice;
Oxford University Press
Gore, M.G. (2000) Spectrophotometry and Spectrofluorimetry a practical
approach; Oxford University Press

Meah, M.S. and Kebede-Westhead, E. (2012) Essential Laboratory Skills for

Biosciences; Wiley-Blackwell
Aleppo., Syria, (2010), Medichem Middle East, In vitro diagnostics,
http://www.medichem-me.com/old/Method/1-Substrate/13%20Glucose
%20PAP.pdf, ( Oct 30th, 2014)
Dionex,Thermo Scientific, Analysis of fruit juice adulterated with medium invert
sugar from beets, 1992, http://www.medichem-me.com/old/Method/1Substrate/13%20Glucose%20PAP.pdf, (Oct 28th, 2014)
Loren Cordian, The paleo diet, http://thepaleodiet.com/fruits-and-sugars/,(Oct
28th, 2014)
Steegmans M., Iliaens S., Hoebregs H, (2004), Enzymatic, spectrophotometric
determination of glucose, fructose, sucrose and inulin/oligofructose in foods.
Livestrong, Natural Sugars in Oranges, 2014,
http://www.livestrong.com/article/267094-natural-sugars-in-oranges/ (Nov 9 th,
2014)