Okonkwo et al. Int. J. J . Res. Chem. Environ. Vol.2 Issue Iss ue 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 1
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Anyika L.C. ,*Okonkwo S.I. and Ejike E.N.
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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 th
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(Received 05 April 2012, Accepted 07 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 R 2=09.99 is similar for all the sugars and their standard mixtures suggesting that without prior separation refractometry can be used for the determination deter mination of any of these thes e sugars and their mixtures Polarimeter was less sensitive se nsitive in determining deter mining the fructose level. Keywords: Monosaccharide, disaccharides, refractometer and polarimeter Introduction total concentration and type of carbohydrate present in [3] foods, industrial starch and additives etc .
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, [1] usually in ration 1:2:1 . The class of compounds known as carbohydrate includes poly- hydroxyaldehydes and polyhydroxyketones or substances which when hydrolysed, give these compounds as products.
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 refractometry and so on, are the methods in use to determine the type and concentration of [3] sugars .
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.
For sensitive analysis of mono and disaccharide, (R) carbopac PAIO is an ideal choice for separation of mono [4] and disaccharide found in mammalian glycoprotein . Refractomery and polarimetry are the common methods [5,6] used by various workers . 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) (2007) show that pretreatment is essential for high yield and low cost of biological processing of cellulosic biomass to fuel and [7] chemicals .
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 [2] monosaccharide and disaccharide . A large number of analytical technique have been developed to measure the
The concentration of carbohydrate in an unknown sample is determined by measuring its angle of rotation and (270)
Okonkwo et al. Int. J. Res. Chem. Environ. Vol.2 Issue 4 Oct. 2012(270-274) (8)
comparing it with calibration curve the vast majority of 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.
Preparation of Standard 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.
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 3 50cm 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 3 lactose) mixed and dissolved in 50cm of solution with , distilled water for the standard mixture of 4%, 6% 7%, 8%, 10%, 15% and 20% respectively.
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).
Methods Preparation
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.
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.
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. Standard solutions of glucose, fructose, sucrose, maltose, lactose and a standard mixture of these sugars were freshly prepared for the study.
Standard curves of each of the sugar standards and the standard mixture was obtained using common axis. In the use of polarimetry, care was taken to ensure that there were no suspended particles or air bubbles in the solutions. The equipment was turned on, left for ten minutes to allow the sodium to be properly warmed up.
The chemical and reagents used were obtained from chemistry laboratory Federal University of 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 o Temperature at 29 C (271)
Okonkwo et al. Int. J. Res. Chem. Environ. Vol.2 Issue 4 Oct. 2012(270-274)
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 4 6 8 10 15 20 Fructose Glucose Sucrose Maltose Lactose Standard mixture ANOVA Source of Variance Row
6 6 6 6 6 6 6 7 7 7 7 7 7
8.0181 8.0343 8.0507 0.0705 8.0865 8.1272 8.1616 9.4235 9.4303 9.4274 9.4252 9.4152 9.4273
1.33635 1.33905 1.341783 1.345083 1.34775 1.354533 1.360267 1.346214 1.347186 1.346771 1.346457 1.345029 1.346757
3.95E-07 3.71 E-07 3.58E-07 4.7E-07 7.55E-07 1.19E-06 4.6E-06 7.56E-05 7.92E-05 8.9E-05 6.34E-05 5.993E-05 7.53E-05
SS
Df
MS
F
P-value
F crit
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 0 O Unknown (10%): Angle of rotation, = +3.5 Temperature = 29 C (272)
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 2 4 6 8 10 15 20
Count 6 6 6 6 6 6 6
Sum 8.2 25.4 41.9 53.5 64.9 80.9 96.7
Average 1.366667 4.233333 6.9833 8.9166 10.816 13.483 16.116
Variance 0.7506 14.310 24.261 30.817 41.193 40.481 57.941
Fructose Glucose Sucrose Maltose Lactose Standard Mixture
7 7 7 7 7 7
128.8 52.9 69.9 48.5 40 31.4
18.4 7.5571 9.9857 6.9285 5.7142 4.4857
80.406 23.799 41.014 17.852 13.451 9.3580
ANOVA Source of Variation Rows Columns Errors Total
SS
Df
MS
F
P-value
F crit
953.655 887.146 161.641 2002.444
6 5 30 41
158.942 177.429 5.38804
29.499 32.930
2.66E 2.56E
2.4205 2.5335
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 (273)
Okonkwo et al. Int. J. Res. Chem. Environ. Vol.2 Issue 4 Oct. 2012(270-274) 2
scale. The reading for the standard mixture was also obtained in similar manner.
sensitivity (R = 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.
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.
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Polarimetry was the less sensitive method (R = 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.
Results and Discussion Table 1 presents the refractive index values of the various sugar standards and standard mixtures at different concentrations.
Fructose showed remarkable variation in 2 sensitivity (R = 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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The regression results of the refractometry is R = 2 0.99, while polarimetry regression for fructose is R = 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.
References
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.
2. Ray S. Biodegradable Polymer for Industrial Application. Ray Smith,. ISBN 0849334667 (2005).
1. The American Heritage Dictionary of the English Language, fourth edition. Copyright © 2007, 2000 by Houghton Mifflin Company, updated in 2009.Published by Houghton Mufflim Company.
3. McMillian J. D., preteatment of lignocellulocllsic biomass .In: Himmel,M.E, Baker,J.Ooverend, R.P(E.d.S)E Enzymaticscovernation of biomass fuel production American chemical soceity PP 292 to 234 (1994)
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.
4. www.dionex.comBiocolumns Retrieve 21/10/2011 5. Hassani A., Yegari R., Salehpour P. and Jassouri S. Comparisim of various sugar analysis methods in Penicillin – Gfermentation broth (2009) 6. Chavatte P., Clement F., Cash R., Grongnet J. E. Proceeding of the Annual Convention of AAEP, 44, 06 – 208 (1998)
Conclusion
7. Wyman C., Yang B., Dale B. E. and Elander R. T. Comparative sugar recovery Yield for pretreatment of corn stover and popr by leading technologies, and Enzymatic Hydrolysis of remaining solids. Annual meeting of American Institute of Chemical Engincerssalt Lake city(2007).
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
8. Association of Analytical Chemists: Official method of th dition, 550-594 (AOAC, 1980). analysis, 13 E
This shows that different sugars at different mixtures can be determined without discrimination using refractometry.
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