Biomolecules: Identification of an Unknown Sugar
In this lab period, you will determine the identity of an unknown sugar. You will conduct up to three experiments to collect the data needed to prove the identity of the unknown. The data that might be obtained includes the time for an osazone to form, the decomposition point and the specific rotation of the sugar.
Osazone Formation
When one equivalent of an aldehyde or ketone (i.e., compounds that contain a carbonyl group) reacts with phenylhydrazine, the product is called a phenylhydrazone. For example, the reaction between benzaldehyde and phenylhydrazine yields the phenylhydrazone of benzaldehyde as shown in Figure 1.
Figure 1.Preparation of a phenylhydrazone.
When glucose, a pentahydroxyaldehyde or aldohexose, reacts with an excess of phenylhydrazine, the product is not a phenylhydrazone but a phenylosazone or simply osazone. Anosazone contains two phenylhydrazone groupings. Figure 2 shows the reaction of glucose with an excess of phenylhydrazine.
Figure 2. Preparation of an osazone from D-glucose.
Mannose, a pentahydroxyaldehyde that differs in configuration from glucose only at C-2, gives an osazone with exactly the same structure as that of the osazone of glucose as shown in Figure 3.
Figure 3. Preparation of an osazone from D-mannose.
How can these reactions help us determine whether we have glucose or mannose as an unknown? The answer is by timing the reactions. The
osazone from glucose forms in approximately five minutes; whereas, the osazone from mannose forms in less than one minute. Thus, if we allow our unknown glucose or mannose to start forming an osazone at exactly the same time we start osazone formation with authentic samples of glucose and mannose, our unknown will form its osazone at the same time as one of the known compounds but not the other. Thus, our sugar forms its osazone simultaneously with one of the known sugars. Osazone formation is a chemical property that is useful for the identification of an unknown sugar.
Decomposition Point
Most sugars do not melt; they decompose. When table sugar or sucrose is heated, a black color often appears because the sucrose has decomposed. Thus, when a sugar is heated in a melting point capillary tube, the result in not a melting range of temperatures, but a temperature at which the sugar appears to decompose. The temperature at which the sugar decomposes is the decomposition point. Like melting points, decomposition points are a function of the compound, and different sugars have different decomposition points. Thus, decomposition points are useful physical constants for identifying unknown sugars.
Specific Rotation
During the first semester, you learned the meaning of specific rotation. The specific rotation of a compound is the rotation in a 1-decimeter tube of a solution made by dissolving one gram of the compound in 100-mL solvent. Thus, specific rotation is a defined term. We usually do not measure exactly one gram in 100-mL of solvent. We usually calculate the specific rotation, which is a constant. The symbol for specific rotation is a Greek alpha in square brackets, []. The observed rotation is simply the Greek alpha, . Thus, the specific rotation is calculated by equation 1, where c is the concentration in grams per 100 mL and l is the path length in decimeters. Because [] is a defined term, it has units of degrees (o) instead of units implied by equation 1.
[]D = /(cl) (1)
he specific rotation of sugars cannot be determined immediately after dissolving them in water. Sugars must be allowed to spend several days in the aqueous solution before the specific rotation is obtained. To understand why this is so, we need to understand the term mutarotation. Mutarotation is the equilibration of the two anomers of the sugar in solution. Glucose exists in cyclic forms (two anomers) known as -glucopyranose and-glucopyranose, depending on the configuration at the anomeric carbon atom. These forms equilibrate (come to a position of equilibrium) in water so that the ratio of alpha to beta forms remains constant. Thus, the specific rotation of the sugar after it undergoes mutarotation is a constant, which is the literature value. Figure 4 shows the equilibria involved in the mutarotation of D-glucose.
Figure 4. Mutarotation of glucose.
At equilibrium, the ratio of glucose is about 37/63. The specific rotation of the mixture is +53o, which is the value calculated from an observed rotation by equation 1. You will prepare an aqueous solution of your unknown one week in advance of determining its specific rotation to allow for mutarotation. The specific rotation is another physical constant that is useful in determining the identity of a sugar.
Table 1 lists sugars and properties that can be used to identify them. You will determine the identity of one of the sugars, which will be issued as an unknown.
Table 1. Sugars.
Name of Sugar / Decomp.Temperature
(oC) / []D20 / Approximate
time for osazone to form (min.)
D-glucose (hydrated) / 90 / +48 / 4-5
maltose (hydrated) / 100 / +129 / soluble
D-fructose (hydrated) / 104 / -92 / 2
D-mannose / 132 / +14 / 0.5
D-xylose / 145 / +19 / 7
D-Glucose (anhydrous) / 146 / +53 / 4-5
L-arabinose / 160 / +113 / 10
maltose (anhydrous) / 165 / +129 / soluble
sucrose / 185 / +66 / 45
lactose / 203 / +113 / soluble
Procedure
Preparation of Solution for Specific Rotation
(To be conducted one week before observing the rotation)
1. Obtain an unknown sugar, show the instructor the number of the unknown and record it in your lab notebook.
2. Keep about 0.3 g of the unknown for next week’s experiments.
3. Tare a 25-mL volumetric flask on an analytical balance that measures to four decimal places (instrument room).
4. Transfer the remainder of the unknown sugar (about 3 g) to the volumetric flask and record the mass of the unknown.
5. Add distilled water to the volumetric flask until the water level reaches about 2 cm below the etched mark on the flask.
Water will be added to the mark at the next lab meeting. The sugar will undergo mutarotation during this time.
6. Stopper the volumetric flask, mark it with your initials, and save it in the designated location until next week.
Specific Rotation of the Unknown Sugar
1. Retrieve the 25-mL volumetric flask that contains your unknown sugar.
2. Add distilled water to the mark on the flask.
The bottom of the meniscus should appear to touch the etched mark when the neck of the flask is viewed from the side.
3. Shake the flask to ensure the contents are thoroughly mixed.
4. Carefully fill a clean polarimeter tube with the unknown sugar solution.
The tube diameter is flared at one end. Fill at the narrow end.
After sealing the tube,gently rotate it so that any air bubbles present collect in the flared end.
5. Measure the observed rotation and calculate the specific rotation [] by equation 1.
Take care to use the correct units of path length and concentration in the calculations.
Osazone Formation
The key to a good result with this experiment is to prepare the six test tubes as fast as possible, to ensure the contents of each test tube are thoroughly mixed, to place all of the test tubes in the water at the same time, and to remove a test tube only after the osazone has formed in it.
1. Prepare a boiling-water bath by filling a large beaker (i.e., one that can hold six or seven 6-in test tubes) half full of water and heating the beaker on a hot plate.
2. Place a 0.1-g sample of your unknown and five known sugars in separate 6-in test tubes marked for identification.
The five known sugars that form osazones are glucose, fructose, mannose, sucrose, and xylose.
3. Add the following reagents to each test tube in the following order.
a. 4 mL distilled water
b. 0.5-mL saturated sodium bisulfite, NaHSO3, solution
c. 0.3-g solid sodium acetate
4. Mix the contents of each tube thoroughly with a stirring rod. Clean the stirring rod after each usage by dipping it in a beaker of water and drying it with a paper towel.
Note: Speed is important in the next three steps. Read them before you do them and then do them as fast as possible.
6. Ensure the water in your bath is boiling, and the water level in the beaker rises above the liquid level of the solutions in the test tubes.
5. Add0.2-g solid phenylhydrazine hydrochloride as quickly as possible to the six tubes in the following order: (1) mannose, (2) fructose, (3) glucose, (4) xylose, (5) sucrose, and (6) your unknown.(The reaction will start as soon as you add this reagent, so be quick.)
6. Place all six test tubes in the boiling water at the same time and record the start time to the nearest second.Do not remove any test tube from the water unless you observe a precipitate—any color change can be regarded as a precipitate)
The five known sugars will form osazones over a 45-min period, starting with mannose (turns white almost immediately and precipitates in about 30 sec) and ending with sucrose (yellow precipitate in about 45 min).
7. Constantly monitor the test tubes. As soon as the osazone of mannose forms, remove its test tube. If your unknown is the next to precipitate, you have mannose as an unknown. Next, fructose’s osazone will precipitate. Remove its test tube. If you have fructose as an unknown, your unknown will precipitate following fructose’s. Remove each known as its osazone forms, leaving your unknown and the remaining candidates in the hot water. The unknown’s osazone should precipitate immediately after the authentic sample precipitates.
It is not necessary to wait for the osazones of sugars that precipitate after your unknown has precipitated. You are finished as soon as you identify your unknown.
8. Some osazones are water soluble. See Table 1. If you have one of these sugars as an unknown, you must conduct at least one of the other two experiments to identify it.
Decomposition Point
1. Using a melting point apparatus, determine the decomposition point of your unknown sugar.
2. Compare the decomposition temperaturewith those in Table 1.
Carbohydrate Questions
Stu No___ Sec____Last name______, First ______
Number of Unknown Sugar______My unknown is: ______
(In the multiple-choice questions, put an X for all answers that are correct.)
1. The - and - forms of D-glucopyranose may be described as:
__A. anomers
__B. epimers
__C. enantiomers
__D. diastereomers
2. What is the generic name of the sugar derivative you made in lab?
(i.e., what do you get when you react a sugar with phenylhydrazine?)
Ans.______
3. A furanose is a cyclic form of a sugar that contains a furan-like structure. Furan is:
__A. a five-member ring.
__B. a cyclic ether.
__C. a five-member lactone.
__D. an ester.
4. Draw a Fischer structure of glucose.
5. Draw the structure of -D-glucopyranose in a chair conformation.
6. Puta star (*) on the anomeric carbon atom on your structure of problem 5.
7. A ketotetrose contains:
__A. an aldehyde
__B. a ketone
__C. four carbon atoms.
__D. two primary alcohols.
8. An aldopentose contains:
__A. an aldehyde
__B. a ketone
__C. a primary alcohol.
__D. five carbon atoms.
9. Draw structure ofL-erythrose in a sawhorse projection.
10. Name the seven D-sugars that are diastereomers of D-glucose.
1.______
2.______
3.______
4.______
5.______
6.______
7.______
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Lab 14