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Inversion of Sucrose
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Introduction & Theory
A chemical enzyme is a chemical that promotes a chemical reaction, but does not itself react to form products. Enzymes are not consumed in a chemical reaction, but are simply used in a step of a larger sequence of reactions. The enzyme reacts with a molecule briefly, then the catalyst allows another molecule in the reaction mixture to react with it, cleaving the enzyme and allowing it to react again with a molecule of reactant.
In the simplest case, the reaction is as follows if the enzyme is denoted E, the reactant S (substrate), the enzyme-reactant complex ES, and the product P with reaction rates kx.
destruction of ES, ES may be determined by
Where Km is a convenient way of expressing the constant
The increased rate of reaction in the presence of an enzyme is a result of an enzyme's ability to lower activation energy barriers within the reaction. An enzyme cannot cause a reaction that would otherwise be thermodynamically impossible, but it can speed reactions that are normally so slow that they do not appear to be occurring to reasonable rates. This is a result the fact that enzymes are not permanently changed over the course of a reaction, only used; hence they cannot donate energy to propel a reaction.
The goal of this experiment is to determine the rate of conversion of sucrose to glucose and fructose. This rate will be measured indirectly by measurement of changes in the optical rotation of the reaction mixture.
In this experiment, a thermostated temperature bath (set at 25±0.1 C) and a polarimeter are used along with a standard assortment of reaction flasks and paraffin. Specifically, 4 volumetric flasks (2 x 100 mL, 2 x 50 mL), and 2 flasks (500 mL, 250 mL) will be used. Cane sugar, distilled water, 4 M HCl, and 4 M chloroacetic acid will be used in preparing solutions.
A polarimeter consists of a light source which passes through a polarizing filter to produce plane polarized light at a constant angle. This light is passed through an accurately known length of sample and is passed through another, moveable, polarizing filter and to an eyepiece. When liquid that is not optically active is in the sample chamber, the light is completely blocked when the two filters' polarizing planes are perpendicular. Using this as a calibration, the rotation relative to this point required to block the light of a given optically active sample completely is the angle of rotation.
The only dependent observable in this experiment is the rotation angle given by the polarimeter. Coupled with time, the angle of rotation indicates the extent to which the reaction has proceeded.
Optical rotation is a phenomenon that occurs when plane polarized light is rotated as it passes through a substance. This effect is not uncommon in organic molecules, and is easily measured with a pair of polarizing filters and a light source. By determining the rotation of the products and of the final reactants, the composition of a combination of reactants and products can be determined. This allows the extent of the reaction to be measured in real time.
In this experiment, acid is used to catalyze the conversion of sucrose to glucose and fructose. Under the conditions used, this takes place fairly slowly and the rotation angle can be seen to shift gradually from that of the products to that of the reactants.
Using volumetric flasks, 100 mL each of 20% cane sugar in distilled water and 4 M HCl should be placed in a thermostated 25C bath to equilibrate. Calibrate the polarimeter with distilled water while waiting for the solutions to reach a constant temperature.
Ensure that the equipment is ready to receive the samples, as quick measurements are essential to the accuracy of the results. Start a stop watch as the two are mixed in a 500 mL flask, cover with paraffin, and mix rapidly. Quickly rinse the polarimeter cell twice with the solution and then fill completely, avoiding air bubbles. Record the angle of rotation and the time every minute for the first then minutes, every two minutes for the next twenty minutes, and every four minutes for the next twenty minutes. After taking the first reading, place the unused sucrose solution in the bath for later use.
While waiting for the last ten minutes, place two 50 mL volumetric flasks in the bath, one containing 20% cane sugar solution and the other with 4 M choloracetic acid. After an hour has passed, record the final angle of rotation and time. Finally, recheck the calibration of the polarimeter with distilled water to find any drift.
Repeat the previous procedure with the sucrose and chloroacetic acid in the water bath. This time however, record the angle and time every two minutes for the first ten minutes, and every ten minutes for fifty additional minutes.
Check the rotation of the leftover solution from the first trial and use this value as for both solutions. Finally, thoroughly clean everything that came in contact with the solutions as they are corrosive.
Contamination and changes in concentration can affect the rate of the reaction, so every receiving flash must be clean, dry, and rinsed with small amounts of solutions a few times before the solution is transferred to it. Also, for the reaction rate to be constant, the samples must be kept at constant temperature and not agitated except as necessary for mixing.
The procedure given in the handout calls for use of a 20% sucrose solution. In the event that this solution will need to be prepared, specific amounts of sucrose and water will need to be determined. Percent compositions of this type are generally based on weight percentages. Starting with 200 mL distilled water, with a weight of 20 g, the weight of sucrose needed would be 20% of 20 g, or 4 g.
Hydrochloric acid is corrosive, avoid contact with skin and mucous membranes. Inhalation of vapors may cause severe irritation of the respiratory tract, as well as cause tissue damage if inhaled in high concentrations. In the event of skin contact, flush thoroughly with water. If ingested, seek medical attention immediately and do not induce vomiting. Contact with eyes should be treated by flushing of affected area with copious amounts of water for at least 15 minutes.
Chloroacetic acid is corrosive, avoid contact with skin and mucous membranes. In the event of skin contact, flush thoroughly with water. If ingested, seek medical attention immediately and do not induce vomiting. Contact with eyes should be treated by flushing of affected area with copious amounts of water for at least 15 minutes.
Sucrose is generally considered safe, although in crystalline form it can irritate eyes and mucous membranes due to its abrasive nature. In the presence of oxidants, sucrose in the solid form may burn violently. In the event of fire, any type of fire extinguisher may be used, and the inhalation of smoke must be avoided.
1. Safety data taken from the internet at "http://hazard.com/msds",
2. D.P. Shoemaker, C.W. Garland, J.W. Nibler, Experiments in Physical Chemistry, 6th ed., The McGraw-Hill Companies (1996).