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Spectrophotometry
By: Prince Dario De Guzman5, Margarita Jai B. Cobangco1 Ernesto Rafael C. Dayrit2, Denise Victoria DG. De
Guzman3, and Mellanie Claire C. De Guzman4
Group 3, 2F Medical Technology, Faculty of Pharmacy, University of Santo Tomas, España, Manila
Abstract
This experiment seeks to analyze, evaluate, and validate the effects of temperature on invertase activity.
Enzymes are proteins that act as catalysts by lowering the activation energy in specific chemical reactions. A specific type
of enzyme, Invertase, hydrolyses sucrose into glucose and fructose. To determine the effects of temperature, two different
kinds of enzymes – denatured and stock solution were both immersed in varied temperatures of hot water bath to facilitate
hydrolysis of sucrose. Dinitrosalicylic acid works on reduction-oxidation method and due to its change in coloration;
absorbance can be measured using spectrophotometric analysis. The absorbance was measured by setting the
wavelength of the standard samples at 540 nm and zero for its corresponding blank solutions. The data gathered showed
the relationship of absorbance to the temeperature and acid-hydrolyzed sucrose concentration. Consequently, it was
inferred that enzymes have an optimal temperature at which they function most effectively.
x=y
1 1.67 -0.013
2 3.33 -0.007
3 5 0.001
4 6.67 0.009
5 8.33 0.347
6 10 0.816
"which is
the
Figure 3-2. Graph of HSC in DNS Colorimetric Method
note: The calibration curve is (y=0.043x)
e"0.043
With the use of the calibration curve of that
graph, the group was able to derived the
specific concentration of the hydrolyzed
sucrose under different temperatures.
2. Boyer, Rodney. (2006). Concepts in Biochemistry (3rd ed.)
John Wiley and Sons Asia Pte Ltd. 131- 155
Figure 3-3. Graph of Derived Conc. vs. Temperature 3. Campbell, M.K. & Farell, S.O. (2009). Biochemistry (6th ed.)
Philippines: Cengage Learning Asia Pte Ltd. 143- 150
This figure above proved that the 4. Mckee, J.R. & Mckee, T. (2009). Biochemistry: The
derived concentration from the calibration Molecular Basis of Life (4th ed.). New York: Oxford University
Press. 184- 195, 202- 219
curve is correct due to the bell-like orientation
5. Reiner, J.M. (1969). Behavior of enzyme Systems. New
formed by the linear sequence of the graph. York: Van Nostrand Reinhold. 45- 54, 113- 116, 261- 284.