Experiment 15EXPERIMENT 15 Carbohydrates endnonreducing endPlease note that not all disaccharides or polysaccharides contain a reducing end.An example is sucrose (shown below), which does not have a hemiacetal (or hemiketal) group on either of its ends: CHOPolysaccharides, such as amylose or amylopectin, do have a hemiacetal group on one of their terminal ends, but they are mainly nonreducing substances because there is only one reducing group present for every 2,00010,000 monosaccharidic units.In such a low concentration, the reducing group does NOT give a positive test with Benedicts or Fehlings reagent.
On the other hand, when a nonreducing disaccharide (sucrose) or a polysaccharide such as amylose is hydrolyzed, the glycosidic linkages (acetal) are broken and reducing ends are created.
Hydrolyzed sucrose (a mixture of Dglucose and Dfructose) will give a positive test with Benedicts or Fehlings reagent as well as hydrolyzed amylose (a mixture of glucose and glucosecontaining oligosaccharides).The hydrolysis of sucrose or amylose can be achieved by using a strong acid such as HCl or with the aid of biological catalysts (i.e., enzymes).
Starch can form an intense, brilliant, dark blue or violet colored complex with iodine.The straight chain component of starch (or amylose) gives a blue color, while the branched component (or amylopectin) yields a purple color.In the presence of iodine, amylose forms helixes, where the iodine molecules assemble as long polyiodide chains.The helixforming branches of amylopectin are much shorter than those of amylose.
Therefore, the polyiodide chains are also much shorter in the amylopectiniodine complex than in the amyloseiodine complex.The result is a different color (purple).
When starch is hydrolyzed and broken down to small carbohydrate units, the iodine will not give a dark blue (or purple) color.The iodine test is used in this experiment to indicate the completion of the hydrolysis.In this experiment you will investigate some chemical properties of carbohydrates in terms of their functional groups.
Reducing and Nonreducing Properties of Carbohydrates Aldoses (polyhydroxy aldehydes).
All aldoses are reducing sugars because they contain free aldehyde functional groups.The aldehydes are oxidized by mild oxidizing agents (e.g., Benedicts or Fehlings reagent) to the corresponding carboxylates.For example: R-CHO + 2CuNaOH + Cu
(from Fehlings reagent)
(red precipitate) Ketoses (polyhydroxy ketones).All ketoses are reducing sugars because they have a ketone functional group next to an alcohol functional group.
Hemiacetal functional group (potential aldehydes).Carbohydrates with hemiacetal functional groups can reduce mild oxidizing agents such as Fehlings reagent because hemiacetals can easily form aldehydes through mutarotation.Hydrolysis of Acetal Groups Disaccharides and polysaccharides can be converted into monosaccharides by hydrolysis.For example: Lactose + H D-galactose + D-glucose
ProcedureReducing or Nonreducing Carbohydrates
Place approximately 2 mL (approximately 40 drops) of Fehlings solution (20 drops each of solution part A and solution part B) into each of five labeled test tubes.Add 10 drops of each of the following carbohydrates to the corresponding test tubes as shown in the following table.Test Tube No Name of Carbohydrate 1 Glucose 2 Fructose 3 Sucrose 4 Lactose 5 Starch Place the test tubes in a boiling water bath for 5 min.A 600 mL (or any available large) beaker containing about 200 mL of tap water and a few boiling stones is used as the bath.Record your results on your data sheet.Which of those carbohydrates are reducing carbohydrates? Remember to use boiling stones; they prevent bumping.Handle the hot test tubes with a test tube holder and the hot beaker with Hydrolysis of Carbohydrates Hydrolysis of sucrose (acid versus base) Place 3 mL of 2% sucrose solution in each of two labeled test tubes.
To the first test tube (#1), add 3 mL of water and 3 drops of dilute sulfuric acid solution (3 M HTo the second test tube (#2), add 3 mL of water and 3 drops of dilute sodium hydroxide solution (3 M NaOH).To avoid burns from the acid or the base, use gloves when dispensing these reagents.Heat the test tubes in a boiling water bath for about 5 min.Then allow both solutions to cool to room temperature by carefully placing in a test tube rack.To the contents of test tube #1, add dilute sodium hydroxide solution (3 M NaOH) (about ten drops) until red litmus paper turns blue.
When using litmus paper, do NOT place the litmus into your solution; instead, use your glass stirring rod, dipped into the test tube solution, to spot the litmus.Test a few drops of each of the two solutions (test tubes #1 and #2) with Fehlings reagent following the procedure that is described for carbohydrates above.
Record your results on your data sheet.Acidcatalyzed hydrolysis of starch Place 5.0 mL of starch solution in a 150 x 15
mm test tube and add 1.0 mL of dilute sulfuric acid (3 M H).Mix it by gently shaking the test tube.Heat the solution in a boiling water bath for about 5 min.
Using a clean medicine dropper, transfer about 3 drops of the starch solution into a white spot plate and then add 2 drops of iodine solution.Observe the color of the solution.If the solution gives a positive test with iodine solution (the solution should turn blue), the hydrolysis is not complete and you should continue heating.Transfer about 3 drops of the boiling solution at 5 min.
intervals for an iodine test Note: Rinse the medicine dropper very thoroughly before each test).When the solution no longer gives the characteristic blue color with iodine solution, stop heating and record the time needed for the completion of hydrolysis on the data sheet
. Data and Calculations for Experiment 15Reducing or nonreducing carbohydrates Test Tube Substance Color Observati
Chem 415 experiment 1EXPERIMENT 1- QUALITATIVE ANALYSIS OF CARBOHYDRATES A carbohydrate is an organic compound with the general formula Cm(H2O)n, that is, consists only of carbon, hydrogen and oxygen, with the last two in the 2:1 atom ratio.Carbohydrates make up the bulk of organic substances on earth and perform numerous roles in living things.
The carbohydrates (saccharides) are divided into four chemical groups: monosaccharides, disaccharides, oligosaccharides and polysaccharides.Polysaccharides serve for the storage of energy (e.g., starch in plants and glycogen in animals) and as structural components (e.g., cellulose in plants and chitin in arthropods).
Structural polysaccharides are frequently found in combination wit proteins (glycoproteins or mucoproteins) or lipids (lipopolysaccharides).
The 5-carbon monosaccharide ribose is an important component of coenzymes (e.g., ATP, FAD and NAD) and the backbone of the genetic molecule known as RNA.
The related deoxyribose is a component of DNA.
Saccharides and their derivatives include many other important biomolecules that play key roles in the immune system, fertilization, preventing pathogenesis, blood clotting and development .This experiment aims to introduce you with the identification of unknown carbohydrates.To gain maximum benefit, observations should be related, as far as possible, to the structure of the substances examined.
Some important points: 1.
Most of the tests and reactions described are not quantitative and volumes are approximate, despite these facts some tests do not work if quantities greatly in excess of those stated are used.
DO NOT place your pipettes in reagent bottles as this leads to contamination.3.
In most tests, it is important to apply a control test using water instead of the solution under examination.If you are in doubt about the result of a test, perform the reaction with a suitable known compound.4.
In this experiment, sugar samples are given in their solid state.To perform each procedure, you should prepare your own sugar solution by taking very small amounts of solid sugars.5.
When you need to boil your sample in a test tube, prepare a hot water in a large beaker and put your test tube inside the beaker.DO NOT forget to put boiling chips in the beaker.
TESTS ON CARBOHYDRATES: 1) containing carbohydrates in a combined form, based on the dehydration of the carbohydrate by sulfuric acid to produce an aldehyde (either furfural or a derivative), which then condenses with the phenolic structure resulting in a red or purple-colored compound.Procedure: - Apply this test two different carbohydrate solutions of your own choice, preferably to one monosaccharide and one polysaccharide.- Place 2 mL of a known carbohydrate solution in a test tube, add 1 drop of -naphthol in ethanol).- Pour 1-2 mL of conc.H2SO4 down the side of the test tube, so that it forms a layer at the bottom of the tube.
- Observe the color at the interface between two layers and compare your result with a control test. A brown color due to charring must be ignored and the test should be repeated with a more dilute sugar solution.Questions: Write the reaction step(s) involved in this test? Give an example of a protein structure that would give positive test with
2) Solubility Tests:
- Apply this test to all carbohydrates provided.- Observe the solubility of the carbohydrates both in water and ethanol. DO NOT depend on your solubility observations during identification of your unknown compound.
3) Carbohydrates as Reducing Sugars: A reducing sugar is any sugar that, in a solution, has an aldehyde or a ketone group.The enolization of sugars under alkaline conditions is an important consideration in reduction tests.The ability of a sugar to reduce alkaline test reagents depends on the availability of an aldehyde or keto group for reduction reactions.A number of sugars especially disaccharides or polysaccharides have glycosidic linkages which involve bonding a carbohydrate (sugar) molecule to another one, and hence there is no reducing group on the sugar; like in the case of sucrose, glycogen, starch and dextrin.In the case of reducing sugars, the presence of alkali causes extensive enolization especially at high pH and temperature.This leads to a higher susceptibility to oxidation reactions than at neutral or acidic pH.These sugars, therefore, become potential agents capable of reducing Cu+2 to Cu+, Ag+ to Ag and so fort.Most commonly used tests for detection of reducing sugars are
a) Test: s Solution (deep blue colored) is used to determine the presence of reducing sugars and aldehydes.
Perform this test with fructose, glucose, maltose and sucrose.Procedure: - of Fehling solution B (solution of potassium tartrate).- Add 2 mL of the sugar solution, mix well and boil. Try to see the red precipitate of cuprous oxide that forms at the end of the reaction.
Questions: What is the function of tartrate? Some disaccharides such as maltose are reducing agents, whereas others, such as sucrose are not.Explain briefly by incluiding the structures of the sugars.
b) , is slightly acidic and is balanced so that is can only be reduced by monosaccharides but not less powerful reducing sugars.Disaccharides may also react with this reagent, but the reaction is much slower when compared to monosaccharides.Perform this test with glucose, maltose and sucrose.Procedure: - To 1- - Boil for 5 min.
in a water bath and allow to stand. You will observe a brick-red cuprous oxide precipitate if reduction has taken place.Questions: or negative? Explain your answer by giving reasons and structures.
c) distinguished from aldoses via their ketone/aldehyde functionality.
If the sugar contains a ketone group, it is a ketose and if it contains an aldehyde group, it is an aldose.This test