Experiment #10

Test for Carbohydrates

Group 5

INTRODUCTION Carbohydrates are called carbohydrates because the carbon, oxygen and hydrogen they contain are generally in proportion to form water with the general formula Cn(H2O)n. Molecules consist of carbon, hydrogen and oxygen atoms. A major food source and a key form of energy for most organisms. When combined together to form polymers, carbohydrates can function as long term food storage molecules, as protective membranes for organisms and cells, and as the main structural support for plants and constituents of many cells and their contents. (Nordqvist, 2009) Carbohydrates make up the bulk of organic substances on earth and perform numerous roles in living things (Calgan, 2012). A carbohydrate is called an organic compound, because it is made up of a long chain of carbon atoms. Sugars provide living things with energy and act as substances used for structure. When sugars are broken down in the mitochondria, they can power cell machinery to create the energy-rich compound called ATP (adenosine triphosphate). (Rader, 2012) 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 with 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. (Kayitmazer, 2012) Carbohydrates are dehydrated when treated with strong mineral acids under nonoxidizing conditions. The free or potentially free aldehyde or ketonic groups of monosaccharides and certain disaccharides are readily oxidized by mildly alkaline solutions of cupric ions; such as Benedict's reagent (copper sulfate, sodium carbonate, and sodium citrate). These carbohydrates are called reducing sugars because they reduce cupric (Cu+2)ions to form cuprous (Cu+1) oxide, Cu2O, a yellow to brick-red precipitate. The aldoses are oxidized to aldonic acids (glucose forms gluconic acid), as well as other products. The ketoses yield a mixture of lower molecular weight oxidation products. (Brooks, 2009)

Methodology
This section shows the procedures done by the students to test for the presence of Carbohydrates. A. Molisch Test To 3 separate test tubes containing 1 ml. each of glucose, sucrose and starch, 3 drops of naphthol solution is added. While the test tube is inclined, slowly add 1 ml. of conc. Sulfuric acid. B. Anthrone test To 3 separate test tubes containing 1 ml. each of glucose, sucrose and starch, 1 ml. of Anthrone reagent is added. The solution is mixed thoroughly and placed in a boiling water bath for 3 minutes. C. Benedicts test To 5 separate test tubes containing 1 ml. each of glucose, fructose, sucrose, lactose and starch, 1 ml. of Benedicts solution is added. After shaking the solutions, it is placed in a boiling water bath for 5 minutes. D. Nylanders test To 5 separate test tubes containing 1 ml. each of glucose, fructose, sucrose, lactose and starch, 1 ml. of Nylanders solution is added. The test tubes are placed in a boiling water bath for 5 minutes. E. Barfoeds test To 5 separate test tubes containing 1 ml. each of glucose, fructose, sucrose, lactose and starch, 1 ml. of Barfoeds reagent is added. After shaking the solutions, the test tubes are placed in a boiling water bath until it produces the positive results. F. Mucic Acid test To 10 ml. of galactose and lactose solutions in separate evaporating dish, 5 ml. of conc. Nitric acid is added. Then the solution is evaporated under a fume hood to a volume of 2 ml. while cooling, the evaporating dish are covered with a watch glass. After setting it aside, 5 ml. of distilled water is added and then the solution was transferred into clean test tubes. G. Seliwanoffs test In separate test tubes containing 1 ml. of fructose and sucrose, 1 ml. of Seliwanoffs reagent is added. The test tubes are placed in a boiling water bath afterwards.

H. Aniline Acetate test In this test, 3 ml of arabinose was added with 2 ml. of conc. HCl then the solution was gently boiled for a minute. A filter paper moistened with aniline acetate is now introduced upon the mouth of the test tube. I. Bials test In a test tube with 1 ml. of arabinose, 1 ml. of Bials reagent is added. The mixture was gently boiled afterwards. When the solution was cooled, 1 ml. of amyl alcohol was added.

Results and Discussion

A. General Color tests

1. Molisch test The illustration above show the equation involved in furfural formation from the Molisch test. The furfurals further react with -naphthol present in the test reagent to produce a purple product. Molisch test show positive reults for all carbohydrates, monosaccharides give a rapid positive test while disaccharides and polysaccharides react slower. Molischs Test is a sensitive chemical test for all carbohydrates, and some compounds 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. (Kayitmazer, 2012) 2. Anthrone test Anthrone react with dextrins, monosaccharides, disaccharides, polysaccharides, starch, gums and glycosides. A blue-green coloration of the solution is obtained during the Anthrone test. Carbohydrates are dehydrated with concentrated H2SO4 to form Furfural, which condenses

with anthrone to form a green color complex which can be measured by using colorimetrically at 620nm (or) by using a red filter. (Calgan, 2012) B. Tests for 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. Most commonly used tests for detection of reducing sugars are Fehlings Test, Benedicts Test and Barfoeds Test. (Calgan, 2012) Name of test Benedicts test Nylanders test Barfoeds test 1. Benedicts test Composition of reagent Cu++ Bismuth subnitrate Cupric Acetate Substance result obtained test Red ppt. Green coloration/ppt. Reddish ppt.

General Equation Reaction with Glucose C6H12O6 + Cu ++ + 5 OH C6H12O7 + Cu2O + 3 H2O

Red coloration of the solution showed positive result. Reducing sugars are oxidized by the copper ions in the Benedicts solution to form a carboxylic acid and a reddish precipitate of cuprous oxide within three minutes in alkaline medium. The Benedict's test allows us to detect the presence of reducing sugars (sugars with a free aldehyde or ketone group). All monosaccharides are reducing sugars; they all have a free reactive carbonyl group. Some disaccharides have exposed carbonyl groups and are also reducing sugars. Other disaccharides such as sucrose are non-reducing sugars and will not react with Benedict's solution. (Miles, 2010)

2. Nylanders test Nylanders test is used to test glucose levels. Its composition, bismuth subnitrate, will react to the reducing sugar to produce a color for identification. It produced black precipitate with Glucose, Fructose and Lactose. 3. Barfoeds test

General Equation Reaction with Lactose: C12H22O11 + 2Cu++ + 2H2O C12H22O12 + Cu2O + 4H+

Reducing sugars are oxidized by the copper ion in solution to form a carboxylic acid and a reddish precipitate of copper (I) oxide. Prolonged boiling may hydrolyse disaccharides (slow reacting) to give a false positive reaction. The precipitate of cuprous oxide is less dense than Benedicts test, therefore, its recommended to leave the tube to stand to allow the precipitate to settle. Barfoed's test is a copper reduction test but unlike Benedict's test, it is carried out in an acid (pH 4.6) rather than alkaline solution. (Koffuor, 2012) Barfoeds reagent, cupric acetate in acetic acid 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. (Calgan, 2012) C. Mucic acid test for Galactose and Lactose HC=O-(CHOH)4-COOH + 2 HNO3 HOC=O-(CHOH)4-COOH + H2O + 2 NO2

Galactose is a monosaccharide. When combined with glucose, through a dehydration reaction, the result is the disaccharide lactose. Galactose and glucose are the two monosaccharide sugar components that make up the disaccharide sugar, lactose. Oxidation of most monosaccharides by nitric acid yields soluble dicarboxylic acids. However, oxidation of galactose yields an insoluble mucic acid. Lactose will also yield a mucic acid, due to hydrolysis of the glycosidic linkage between its glucose and galactose subunits. (Kayitmazer, 2012) D. Seliwanoffs Test for Ketoses Seliwanoffs Test distinguishes between aldose and ketose sugars. Ketoses are 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 is based on the fact that, when heated, ketoses are more rapidly dehydrated than aldoses. (Calgan, 2012) The reagent is a solution of resorcinol in concentrated HCl. The acid when heated along with a sugar will produce furfural or hydroxymethylfurfural, which further reacts to give a red color. Ketoses react more quickly than aldoses and thus the reaction time is a means of separation or detection. Ketoses react within 1 minute of heating while aldoses will require several minutes. Disaccharides containing fructose should react intermediately between that of fructose alone and one of the aldoses. (Luckie, 2009) E. Tests for Aniline Pentoses 1. Aniline Acetate test Pink coloration on the moistened filter paper indicated positive results for the presence of Pentoses. The aniline acetate test is a chemical test to identify the presence of certain carbohydrates. These carbohydrates may be converted (by hydrochloric acid) to furfural, which reacts with aniline acetate to produce a bright pink color. (Muliken, 1904) 2. Bials test Bials Test is to determine the presence of pentoses (5C sugars). The components of this reagent are resorcinol, HCl, and ferric chloride. In this test, the pentose is dehydrated to form furfural and the solution turns bluish or green and a precipitate may form. (Baldwin, 1955) Conclusion A. General Color test Glucose, sucrose and starch gives off positive result with both Anthrone and Molischs test since glucose is a monosaccharide, sucrose contains dissacharide molecules composed of monosaccharide glucose, and starch is a polysaccharide. B. Tests for Reducing Agents Sucrose, a non-reducing sugar and Starch, a polysaccharide, are negative for Benedicts test. Glucose, Lactose, and Fructose had red precipitate on both Benedicts and Barfoeds test, indicating positive result. Sucrose is also positive for Barfoeds test. These carbohydrates contain monosaccharides/disaccharides which reacted with the composition of the Benedicts and Barfoeds reagent.

C. Mucic Acid Test Galactose and Lactose both produced pointed prisms that can be seen under a microscope. Oxidation of galactose yielded an insoluble mucic acid. Lactose also yielded a mucic acid, due to hydrolysis of the glycosidic linkage between its glucose and galactose subunits. D. Seliwanoffs test Fructose produced a red color faster than Sucrose. Which makes us conclude that fructose is a ketose while sucrose is an aldose. Since fructose reacted faster than the sucrose. E. Tests for pentoses Arabinose is positive for both Aniline Acetate and Bials test. Arabinose is a carbohydrate/monosaccharide with 5 carbon atoms including an aldehyde functional group.

Part 3: Questions
1. Why do many proteins give off positive results for Molisch test? Molischs Test is a sensitive chemical test for all carbohydrates, and some compounds containing carbohydrates in a combined form, based on the dehydration of the carbohydrate by sulfuric acid to produce an aldehyde (Calgan, 2012). It is possible that aldehydes are also released when mixing proteins, as they often have complex and hybrid structures that could include aldehydes. 2. From what other sources are pentoses obtained? Pentoses can be obtained through nucleic acid, gum arabic, plum and cherry gums, and wood gums. 3. How would you test for milk in a milk chocolate bar? You can test the presence of milk by Sodium Citrate test. Also, various methods are available for measuring milkfat, including the Gerber test, which is typically used for routine quality control, and the Roese-Gottlieb test, which is the standard in most countries. Oftentimes, test results vary because not all tests measure free fatty acids or phospholipids. The presence of free fat is measured as it often results from the loss of emulsion stability in whole milk and fat-filled milks.

4. How can Seliwanoffs test be used to distinguish fructose from sucrose? Seliwanoffs test is utilized to differentiate between aldose and ketose sugars.The reagents consist of resorcinol and conc. HCl, acid hydrolysis of polysaccharides and oligosaccharides thus yielding simpler sugars. A ketose is present if the mixture turns red. Ketoses are distinguished from aldoses through their ketone/aldehyde functionality. When heated, ketoses are more rapidly dehydrated than aldoses. The dehydrated ketose then reacts with the resorcinol to produce a deep cherry red color. Aldoses may react slightly to produce a faint pink color. Fructose and sucrose are two common sugars which give a positive test. Sucrose, a disaccharide consisting of fructose and glucose, also gives a positive test. This is due to the presence of concentrated HCl, which hydrolyses sucrose into its monosaccharide subunits (glucose and fructose). Fructose which is a ketose will react and precipitate faster than sucrose. 5. Of what use is Barfoeds test in identifying unknown sugar? Barfoeds test will be helpful in identifying reducing sugars since Barfoeds reagent, cupric acetate in acetic acid is balanced so that is can only be reduced by monosaccharides but not less powerful reducing sugars. 6. What is the chemical name and formula of the ppt. obtained as positive result in Benedicts and Barfoed test? In Benedicts and Barfoeds test, oxidation of the sugars by copper ions will yield a reddish precipitate which is copper (I) oxide or cuprous oxide.

Benedicts test forming red ppt. of Cu2O

Barfoeds test forming red ppt. of Cu2O

REFERENCES:
Sattler Louis. Limitations of the Anthrone Test for Carbohydrates. Date retrieved, July 17, 2013 from http://pubs.acs.org/doi/abs/10.1021/ja01164a521. Miles, Donald. Intro to Macromolecules: Benedicts test for reducing sugars. Date retrieved, July 19, 2013 from http://www.biosci.ohiou.edu/introbioslab/Bios170/170_2/benedict.htm. Amiad, Hussein. (2012). Carbohydrates date retrieved, July 19, 2013 from http://elearning.najah.edu/OldData/docs/29763_Carbohydrates.pdf.. Koffuor, George. Barfoed's Test. Qualitative Tests in Carbohydrates date retrieved July 19,2013 from http://open.umich.edu/education/med/oernetwork/pharmacology/biopharm/barfoedstest/2012 Luckie, Douglas. LB-145: CELL AND MOLECULAR BIOLOGY. TESTS BASED ON DEHYDRATION OF CARBOHYDRATES. Date retrieved, July 19,2013 from https://www.msu.edu/course/lbs/145/luckie/Lab1.html Mulliken, Samuel. (1911). A Method for the identification of pure organic compounds by a systematic analytical procedure based on physical properties and chemical reactions. Published by J. Wiley & Sons, Inc. date retrieved, July 19,2013 from Google Books link page 33. Calgan, Dilek. General Biochemistry. EXPERIMENT 1- Test for Carbohydrates. Date retrieved: July 19, 2013, from http://www.chem.boun.edu.tr/webpages/courses/Chem415/Chem%20415%20Experiment%201. pdf Kayitmazer, Basak. Biochemistry 415. Analysis of the presence of Carbohydrates. Date retrieved: July 16, 2013, from http://www.chem.boun.edu.tr/webpages/faculty/bkayitmazer/bkayitmazer.html Baldwin, E (1995). Cole's Practical Physiological Chemistry. published by Heffer, Cambridge.