Okonkwo et al. Int. J. Res. Chem. Environ. Vol.2 Issue 4 Oct.

2012(270-274)

International Journal of Research in Chemistry and Environment

Vol. 2 Issue 4 Oct. 2012(270-274)
ISSN 2248-9649

Research Paper

Comparative Analysis of Monosaccharide and Disaccharide Using Different Instrument

Refactometer and Polarimeter

Anyika L.C.1,*Okonkwo S.I.2 and Ejike E.N.3

1,3. Department of Chemistry, Federal University of Technology Owerri, Imo State, NIGERIA
2. Department of Pure and Industrial Chemistry Anambra State University Uli, Anambra State NIGERIA

Available online at: www.ijrce.org

(Received 05th April 2012, Accepted 07th July 2012)

Abstract: Comparative analysis of monosaccharide and disaccharides were carried out with the aid of
refractometer and polarimeter, statistical analysis were made using linear regression equation and analysis
of variance (ANOVA). Refractometry was more sensitive (R2=0.99) than polarimetry whose sensitivity is
(R2=0.94). The observed significant difference among the methods is (P ≤ 0.05). The results show that
retractometry with R2=0.99 is more sensitive than polarimeter.The sensitivity of R2=09.99 is similar for all
the sugars and their standard mixtures suggesting that without prior separation refractometry can be used
for the determination of any of these sugars and their mixtures Polarimeter was less sensitive in determining
the fructose level.

Keywords: Monosaccharide, disaccharides, refractometer and polarimeter

Introduction
Carbohydrate is any of a group of organic
compounds that include sugars, starches, celluloses, and
gums. It serves as a major energy source in the diet of
animals. These compounds are produced by photosynthetic
plants and contain only carbon, hydrogen, and oxygen,
usually in ration 1:2:1[1]. The class of compounds known as
carbohydrate includes poly- hydroxyaldehydes and
polyhydroxyketones or substances which when hydrolysed,
give these compounds as products.
They are normally broken down into five major
classifications, which are monosaccharide, disaccharides,
oligosaccharides, polysaccharides and nucleotides. The
common monosaccharide is glucose, fructose and
galactose. Disaccharide means the two sugars are
commonly found in nature as sucrose, lactose and maltose.
Any solution containing dissolved solid will refract
light rays. Refractometry measures the concentration of
many solution of dissolved solid based on the degree to
which the light rays bend, as rays of light. Past literature
shows that biodegradable polymers can be obtained from
monosaccharide and disaccharide[2]. A large number of
analytical technique have been developed to measure the
total concentration and type of carbohydrate present in
foods, industrial starch and additives etc[3].
Chemical methods, chromatographic methods,
titration methods, colorimetric methods, gravimetric
methods, Enzymatic methods, D-glucose/D fructose and
many physical methods such as polarimetry, infrared,
density, refractive index, refractometry and so on, are the
methods in use to determine the type and concentration of
sugars[3].
For sensitive analysis of mono and disaccharide,
carbopac(R) PAIO is an ideal choice for separation of mono
and disaccharide found in mammalian glycoprotein[4].
Refractomery and polarimetry are the common methods
used by various workers[5,6]. Comparative sugar recovery
yields for pretreatment of corn stover and poplar by leading
technologies followed by enzymatic hydrolysis of the
remaining solids carried out by Wyman et al. (2007) show
that pretreatment is essential for high yield and low cost of
biological processing of cellulosic biomass to fuel and
chemicals[7].
The concentration of carbohydrate in an unknown
sample is determined by measuring its angle of rotation and

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comparing it with calibration curve(8) the vast majority of Preparation of Standard

plastic products are made from petroleum based synthetic
polymers that do not degrade in a land fill in a compost like
environment. Therefore the disposal of these products
poses serious environmental problems. Conversion of
biomass to energy, cellulose and hemicelluloses are
normally reduced to mono and disaccharides. Fuel from
petroleum are discouraged because of the pollutants they
emit into the air.
Standard solutions of glucose, fructose, sucrose,
maltose and lactose were each prepared from the pure
substance. A standard solution of the mixture was also
prepared by mixing equal weight of glucose, fructose,
sucrose, maltose and lactose.
The concentrations of each of the sugars used were
2%, 4%, 6%, 8%, 10%, 15% and 20%.

Monosaccharide and disaccharides are very useful
in production of the bio-degradable polymer, therefore this
work aims at determining the percentage concentration of
monosaccharides and disaccharides.
This determination is a difficult one. Some
sophisticated methods have been introduced recently by
using ion exchange chromatographic columns with pulsed
amperometric detector for high performance liquid
chromatography (HPLC).
Also cabopac “PAIO” are used for separation of
monosaccharide and disaccharides. This facility is at
present a technology not available in Nigeria. Therefore
there is a need to use other common methods of
monosaccharide/disaccharide determination to estimate the
amount of sugar moieties in the cellulose degradative
slurring using different instruments: refractometer and
polarimeter.
Material and Methods
The instruments used for the analysis were Abbe
60 refractometer, Bellingham and Stanley limited England
and model D polarimeter Bellingham and Stanley limited,
England with polarimeter tubes.
A 2% standard solution of the mixture was
prepared by dissolving 0.2g each of glucose, fructose,
sucrose, maltose and lactose and mixing them to make a
50cm3 solution using distilled water. This procedure was
carried out using 0.4g, 0.6g, 0.8g, 1.0g, 1.5g and 2.0g of
each sugars (glucose, fructose, sucrose, maltose and
lactose) mixed and dissolved in 50cm3 of solution with
distilled water for the standard mixture of 4%, 6%, 7%, 8%,
10%, 15% and 20% respectively.
Methods Preparation
Each of the different concentrations of each of the
sugar standard was introduced between the two prisms of
the refractometer using a dropper. On closing the prisms,
the liquid spread evenly to make a thin film.
The light was turned on and the scale was
illuminated when viewed through to obtain a uniform field.
Once a uniform field was found, the coarse and fine
adjustments knobs were rotated until the dividing line
between the light and dark halves of the visual field
coincided with the centre of the gross hairs. The refractive
index was read through the scale eye piece. The above was
repeated for each of the concentration of the different
standards and the mixture.

Standard solutions of glucose, fructose, sucrose, maltose, Standard curves of each of the sugar standards and
lactose and a standard mixture of these sugars were freshly the standard mixture was obtained using common axis. In
prepared for the study. the use of polarimetry, care was taken to ensure that there
were no suspended particles or air bubbles in the solutions.
The chemical and reagents used were obtained The equipment was turned on, left for ten minutes to allow
from chemistry laboratory Federal University of the sodium to be properly warmed up.
Technology Owerri, Nigeria. The analysis was also carried
out at the same laboratory.
Table: 1
Refractive index with various sugar standard and standard mixtures at different concentration using refractometer
Concentration Refractive Index
Fructose Glucose Sucrose Maltose Lactose Standard
standard Standard standard standard standard mixture
2% 1.3355 1.3370 1.3360 1.3365 1.3360 1.3371
4% 1.3390 1.3392 1.3391 1.3399 1.3380 1.3391
6% 1.3417 1.3428 1.3417 1.3420 1.3410 1.3415
8% 1.3450 1.3460 1.3445 1.3457 1.3442 1.3451
10% 1.3479 1.3481 1.3483 1.3480 1.3460 1.3482
15% 1.3540 1.3550 1.3549 1.3541 1.3530 1.3562
20% 1.3604 1.3622 1.3629 1.3590 1.3570 1.3601
Unknown (10%): refractive index= 1.3492
Temperature at 29oC
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Figure 1: shows the plot of various sugar with concentration using refractometry

Table 2: ANOVA two-factor without replication refractometry

Summary Count Sum Average Variance
2 6 8.0181 1.33635 3.95E-07
4 6 8.0343 1.33905 3.71 E-07
6 6 8.0507 1.341783 3.58E-07
8 6 0.0705 1.345083 4.7E-07
10 6 8.0865 1.34775 7.55E-07
15 6 8.1272 1.354533 1.19E-06
20 6 8.1616 1.360267 4.6E-06
Fructose 7 9.4235 1.346214 7.56E-05
Glucose 7 9.4303 1.347186 7.92E-05
Sucrose 7 9.4274 1.346771 8.9E-05
Maltose 7 9.4252 1.346457 6.34E-05
Lactose 7 9.4152 1.345029 5.993E-05
Standard mixture 7 9.4273 1.346757 7.53E-05

ANOVA
Source of SS Df MS F P-value F crit
Variance
Row 0.00263 6 0.000438 623.8519 4.3E-30 2.420523

Columns 1.96E-05 5 3.92E-06 5.581904 0.000945 2.533555

Total 0.002671 41
Table 3: show the result of angle of rotation with various sugar standards and standard mixture at different
concentration using polarimeter
Angle of rotation
Concentration Fructose Glucose Sucrose Maltose Lactose Standard
standard standard standard standard standard mixture
2% -3.1 +1.0 +1.2 +1.1 +1.1 -0.7
4% -11.7 +2.8 +4.0 +3.3 +2.5 +1.1
6% -16.7 +5.3 +6.8 +5.8 +4.1 +3.2
8% -19.6 +7.9 +9.8 +6.2 +5.2 +4.8
10% -22.6 +9.5 +13.2 +8.2 +6.2 +5.2
15% -24.9 +11.7 +16.7 +10.2 +9.7 +7.5
20% -30.2 +14.7 +18.2 +13.7 +11.2 +8.7
Unknown (10%): Angle of rotation, 0 = +3.5 Temperature = 29OC
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 Okonkwo et al. Int. J. Res. Chem. Environ. Vol.2 Issue 4 Oct. 2012(270-274)

Figure 2: show the plot of various sugar with concentration using polarimetry

Table 4: Anova: Two-Factor without Replication Polarimetry

Summary Count Sum Average Variance

2 6 8.2 1.366667 0.7506
4 6 25.4 4.233333 14.310
6 6 41.9 6.9833 24.261
8 6 53.5 8.9166 30.817
10 6 64.9 10.816 41.193
15 6 80.9 13.483 40.481
20 6 96.7 16.116 57.941

Fructose 7 128.8 18.4 80.406

Glucose 7 52.9 7.5571 23.799
Sucrose 7 69.9 9.9857 41.014
Maltose 7 48.5 6.9285 17.852
Lactose 7 40 5.7142 13.451
Standard Mixture 7 31.4 4.4857 9.3580

ANOVA

Source of SS Df MS F P-value F crit

Variation
Rows 953.655 6 158.942 29.499 2.66E 2.4205
Columns 887.146 5 177.429 32.930 2.56E 2.5335
Errors 161.641 30 5.38804
Total 2002.444 41

The equipment was checked initially by making a
zero reading with the sample cell filled with only the
solvent (water), the zero reading corresponds with the zero
degree calibration mark. The reading was determined by
laying the sample tubes containing each of the standard
sugar solution with the cradle, closing the cover, and
turning the knob until the proper angle of the analyzer was
reached. In this instrument, the eye sees a split field whose
section must be match in light intensity.
The value of the angle through which the plane of
polarized light has been rotated was read directly from the

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 Okonkwo et al. Int. J. Res. Chem. Environ. Vol.2 Issue 4 Oct. 2012(270-274)
scale. The reading for the standard mixture was also
obtained in similar manner.
Treatment of Data
The method of linear regression and analysis of
variance (ANOVA) were used in the treatment of data. All
statistical analysis were at 95% confidence limit P≤ 0.05.
Results and Discussion
Table 1 presents the refractive index values of the
various sugar standards and standard mixtures at different
concentrations.
The regression results of the refractometry is R2 =
0.99, while polarimetry regression for fructose is R2 = 0.86.
This show that refractometry is more sensitive to all the
sugar.
The result suggests that standard mixture cannot be
used effectively for fructose in polarimeter. So the
refactometer showed more sensitivity than polarimeter.
The problem with polarimetric determination of
fructose may be due to the fact that while other sugars are
dextrorotatory,while fructose is levorotary. Fructose is also
ketohexose sugar while the others are either aldohexose or
Aldohexose containing sugars. This property of fructose
could affect the magnitude and direction of rotation. The
presence of fructose in sucrose could similarly account for
source being next in term of low sensitivity in polarimetry.
Table 2 and 4 shows the analysis of variance for
each of the two different methods refractometry and
polarimetry. This result shows that there is significant
difference in what? (P < 0.05) at different concentrations
and that there is also significant difference in what? (P <
0.05) among sugar for each of the methods. The result
shows that there is significant difference of (P < 0.05)
among sugars and that there is significant difference among
the methods for the determination of sample. The
individual variation among different concentrations and
different sugar for refractometry was however not
significant as can be seen from Table 2.
This shows that different sugars at different
mixtures can be determined without discrimination using
refractometry.
Conclusion
This study has revealed that refractometry can be
used to determine the sugars, fructose, glucose, sucrose,
maltose, lactose and their standard mixture with the highest
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sensitivity (R2 = 0.99) being the best out of the two. This
implies that any of the monosaccharide and disaccharides
can be determined with refractometer with the highest
degree of sensitivity.
Polarimetry was the less sensitive method (R2 =
0.94), to be used in the determination of these sugar. It was
possible to identify the higher percentage specific
monosaccharide. This was shown by the significant
difference among sugars for each method.
Fructose showed remarkable variation in
sensitivity (R2 = 0.88), for the polarimetric method. This
could be attributed to the difference in its direction of
rotation as well as its magnitude. The same fructose has
different response to chemical reagent used in sugar
concentration as opposed to all the other sugar which either
glucose or glucose containing unit.
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