Experiment: Carbohydrates and Optical Activity

Transcription

1 Experiment: Carbohydrates and Optical Activity This lab will explore properties of sugars, one type of carbohydrate. In this lab you will build molecular models of three types of sugars and explore the stereoisomers associated with these sugars. Recall that stereoisomers have the same connectivity of atoms but the atoms are arranged differently in space. These different spatial arrangements can lead to two different types of stereoisomers: enantiomers and diastereomers. You will also encounter meso compounds as you build models. Stereoisomers exhibit optical activity. Ordinary light consists of light vibrating in all different directions. Plane polarized light vibrates only in one plane. If a compound can rotate the plane of rotation, it is said to be optically active. In this lab you will test three different concentrations of sugar solution to determine the optical rotation. Then you will determine the identity of an unknown sugar by measuring its optical rotation. The rotation of an optically active compound is determined by use of an instrument called a polarimeter. This instrument shines a beam of plane-polarized light on a solution. An optically active sample rotates the plane of this plane polarized light, either to the left (levorotatory) or to the right (dextrorotatory). The amount of observed rotation for a given solution is directly proportional to: the concentration of the optically active compound the length of the tube holding the solution. If either of these two factors is changed, the observed rotation will also change. The specific rotation is defined as: [α] T D α = C measured (for a solution) Where, αmeasured = the degree of rotation measured with the polarimeter (+) for dextrorotatory and (-) for levorotatory l = the path length of the sample holder in decimeters (usually 1.00 or 2.00 dm) C = the concentration of the sample in terms of grams solute/ml of solvent The symbol [α] T is used for specific rotation, where the D stands for the sodium D-line (589 D nm) and the T refers to the temperature at which the determination was made. The specific rotation is a constant for an optically active substance at a given temperature. Finally, there are several qualitative tests that can be used to determine the characteristics of a carbohydrate in solution. In this lab you will perform one of these classic tests: Benedict's test. This test is used to determine if a carbohydrate is a reducing carbohydrate. 17

2 A reducing sugar can reduce certain chemicals as the sugar is oxidized. (Recall that oxidation and reduction must occur together.) A sugar with an aldehyde group or one that can isomerize into an aldehyde are considered reducing sugars. All monosaccharides and most disaccharides, except sucrose, can be oxidized. When the ring structure of a carbohydrate opens, the aldehyde group can be oxidized. Ketoses can also be oxidized, because the keto group isomerizes to give an aldehyde. isomerization ( s pecifically, tautomerization ) C 2 O C O C O C O ketose aldose ( which can now reduce Benedict ' s reagent ) Benedict's reagent contains Cu 2+ ions that are reduced to Cu 1+ when the carbonyl group is oxidized. Cu 1+ ions form a brick red precipitate, Cu2O, in aqueous solution. The remaining solution varies from green through yellow to red, depending on the initial concentration of the carbohydrate. A positive test indicates that the carbohydrate is, indeed, a reducing carbohydrate. Pre-lab Preparation 1. Write Fischer projections for the following sugars and identify any chiral carbons with a *: D-erythrose and L-erythrose 2. Define enantiomer, diastereomer and meso compound. 3. Give the value for the specific rotation for sucrose and provide a reference. 4. Carefully review how to take a polarimeter reading as described below. Experimental Procedure! Safety Considerations! Benedicts reagent is harmful if ingested and can be an irritant. 18

3 Part A: Modeling of Carbohydrates Write all answers to the questions below in your lab manual. 1. Build a models of D-erythrose and L-erythrose (see pre-lab) using the following scheme: C = black, = yellow, O = red, CO = green, C2O = purple or blue Are these models superimposable (identical)? Are they mirror images? Based on this, are they enantiomers or diastereomers? Consider your definitions above. 2. Now, for one of your structures, switch the place of the and O on one carbon only. Compare this to the other molecular model. Are these models superimposable (identical)? Are they mirror images? Based on this, are they enantiomers or diastereomers? 3. Now create models for the following: C 2 O O O C 2 O O O C 2 O C 2 O Does these structures contain chiral carbons (mark with an *). Are these models mirror images? Are they superimposable (identical)? What type of molecule is this? (See prelab defintitions.) Part B: Optical Rotation Measurements 1. Measure out an assigned amount of sucrose. This value will range from approximately 5-8 grams. Record the exact mass and then carefully transfer the sucrose from the weighing paper to a 50.0 ml volumetric flask, which will be set out for you. (If you spill any sucrose, you must begin again.) 2. Add a few milliliters of de-ionized water to the volumetric flask and swirl to begin dissolving the sugar. Continue adding water until you are a few milliliters from the etched line denoting the 50 ml level. From that point, add water dropwise until the bottom of the meniscus is just touching the line. (If you go past the line, you must begin again after rinsing the flask with water.) Stopper, invert, and shake the volumetric flask at least 20 times to insure complete uniformity of the sample. 3. Obtain one of the 2 decimeter polarimeter tubes and pour the solution carefully into the tube. Tip up the end of the tube in each direction to release any air bubbles. 4. Place the cylinder into the polarimeter and take a reading. This is done by turning the pointer while looking in the eye piece through the sample. You will see that there are semicircles visible in your sample. When you have the correct value, both half-circles will look equally dim. A slight adjustment in either direction will make one half go dark and the other light. 19

4 This intermediate value, where both halves look equal in intensity, is the correct setting. (You should try adjusting it to the extremes of rotation to get a feel for what you are seeing. When you are far away from the correct value, the sample may look completely dark or completely light. This is not correct, even though both halves look equally dark. The correct setting is the point where adjusting in either direction causes one half-circle to becomes light and the other dark. Experiment with this to be sure you are comfortable.) 3. Read the angle of rotation through the upper eyepiece of the polarimeter. This eyepiece magnifies the polarimeter scale. If the zero line of the movable scale lies to the left of the zero of the fixed scale, the angle of rotation is negative and the sample is levorotatory. If it lies to the right, the angle of rotation is positive and the sample is dextrorotatory. Each line to the left or right of zero on the fixed scale represents one degree of rotation. Using the Vernier scale on the polarimeter allows you to determine the angle to the nearest tenth of a degree. The example below will show how to read the angle of rotation including use of the Vernier scale. The marker that gives you the number before the decimal is the zero line on the bottom scale, which slides past the fixed, upper scale. For example, in the example below, the zero line of the sliding scale is between on the main scale (first arrow). This means our reading must be 12.something. The number after the decimal is obtained by looking along the sliding scale to see which of the 10 lines matches up perfectly with any of the lines in the upper scale. This line on the sliding scale represents the digit after the decimal. ere, the 6 th line on the sliding scale (second arrow) matches with a line on the upper scale, so our reading is Notice that we don t care which line on the upper scale matches; that is not relevant to our reading A levorotatory sample would be read using the left-side of the scales. Be careful to realize that the numbers are increasing to the left. Test yourself by taking a reading for the scale shown in the pre-lab. 5. Calculate the specific rotation for sucrose with the correct number of significant figures. On the board, share your data with the rest of the class. You will need to provide your sample mass, polarimeter reading and calculated specific rotation. Be sure to collect every else s data in your notebook as well. Part C: Identification of an Unknown Sugar 1. Repeat the above procedure using 2 grams of your assigned unknown sugar. Remember to record the exact mass used. 20

5 2. Calculate the specific rotation for your unknown and compare to the reference values below to identify the sample. Table of Possible Unknown Sugars Sugar Specific Rotation D-Arabinose -105 D-Fructose D-Galactose D-Glucose D-Lactose D-Maltose +136 D-Mannose D-Xylose Part D: Benedict s Test 1. Place 0.5 ml (10 drops) of 1% glucose solution in to a clean test tube. 2. Add about 2 ml of Benedict s reagent and heat the mixture in a boiling water bath for up to six minutes. Record your observations. 3. Repeat the test for the following 1% solutions of sugars: sucrose, lactose and starch. Post-Lab and Report Requirements 1. ow did the measured values for optical rotation change as the concentration increased? And was there a trend in the optical rotation values for the specific rotation (calculated)? ow did your specific rotation compare to the reference value in your pre-lab? 2. Give the Unknown number, the specific rotation value and your identification. Comment on the accuracy of your measurement compared to the reference value. 3. Which of your samples contained reducing sugars based on your Benedict s test? Would lactose be expected to give a positive or negative result with Benedict s reagent? Summarize your results and conclusions in your conclusion section. 21