Experiment 6: Qualitative test of carbohydrates
Introduction
Carbohydrates are the most abundant bio molecules on Earth. Each year, photosynthesis converts more than 100 billion metric tons of CO2 and H2O into cellulose and other plant products. Certain carbohydrates (sugar and starch) are a dietary staple in most parts of the world, and the oxidation of carbohydrates is the central energy-yielding pathway in most non-photosynthetic cells. Insoluble carbohydrate polymers serve as structural and protective elements in the cell walls of bacteria and plants and in the connective tissues of animals.
Carbohydrates are an essential component of our diet and an important source of energy for us. Most of the things generally included in our diet have a large ratio of carbohydrates present. Also the patients suffering from Diabetes excrete huge amounts of sugar in their urine which needs to be estimated. The purity of carbohydrates can also be checked by methods which can qualitatively estimate particular sugars. Hence the need for carbohydrate estimation arose.
In this experiment, students will be given solutions containing fructose, glucose, lactose, galactose, ribose, sucrose, starch and two unknown compounds. Students are required to test the solutions by using a series of reagent that will yield a color change after reacting with specific functional groups of the compounds being tested. The unknown solutions will be one of the above solutions
MATERIALS
Fructose, Glucose, Lactose, Galactose, Ribose, Sucrose, Starch, 2 unknown solutions, Molisch's reagent, Benedict's reagent, Barfoed's reagent, Seliwanoff's reagent, Bial's reagent, Lugol's iodine reagent (IKI), 10% ferric chloride hexahydrate (FeCl3), 3g of orcinol and concentrated HCl.
METHODS
The following qualitative tests are performed on 0.2 M solutions (unless otherwise tested) of starch, sucrose, glucose, lactose, galactose, ribose, and ribulose. The devised scheme is used in the prelab section to identify an unknown solution. The unknown will be one of the above solutions or a mixture of two of the above solutions.
Molisch test for carbohydrates
The molisch test is a general test for the presence of carbohydrates. Molisch reagent is a solution of α-naphtol in 95% ethanol. This test is useful for identifying any compound which can be dehydrated to furfural or hydromethylfurfural in the presence of H2SO4. Furfural is derived from the dehydration of pentose and pentosans, while hydromethylfurfural is produced from hexoses and hexosans. Oligosaccharides and polysaccharides are hydrolysed to yield their repeating monomers by the acid. The α-naphtol reacts with the cyclic aldehydes to form purple colored condensation products. Although this test will detect compounds other than carbohydrates (i.e. glycoproteins), a negative result indicated the absence of carbohydrates.
2 ml of the solution is put into a clean test tube.
Two drops of Molisch reagent are added into the test tube and been mixed thoroughly.
The test tube is inclined and 5 ml of concentrated H2SO4 been gently poured down the side of the test tube.
The purple color is observed at the interface of the sugar which positive result is indicated
Benedicts's test for reducing sugars
Alkaline solutions of copper are reduced by sugars having a free aldehyde or ketone group, with the formation of colored cuprous oxide. Benedicts's solution is composed of copper sulfate, sodium carbonate, and sodium citrate (pH 10.5). The citrate will form soluble complex ions with Cu++, preventing the precipitation of CuCO3 in alkaline solutions.
Benedicts's reagent : Generally use a commercial reagents, but to make it from scratch, first 100 g sodium carbonate and 173 g sodium citrate dehydrate are dissolved in a final volume of 850 ml water. Slowly, with stirring, 17.3 g copper sulfate pentahydrate is added in 100 ml of water. The final volume is brought to one liter. The commercial reagent, at least, seems to be stable for years.
1 ml of the solution is put into a clean test tube
5 ml of Benedicts's reagent is added into the test tube and been mixed thoroughly.
The test tube is placed in a boiling water bath and been heated for 3 minutes.
The test tube is removed and allowed to be cooled.
The color ranges is observed from green to yellow to orange to brick-red depending on the amount of reducing sugar in the sample.
Barfoed's test for monosaccharides
This reaction will detect reducing monosaccharides in the presence of disaccharides. This reagent uses copper ions to detect reducing sugars inan acidic solution. The same color changes is looked as in Benedicts's test.
Barfoed's reagent: This looks like Benedicts's but differs somewhat. The reagent is prepared by dissolving 70 g copper acetate monohydrate and 9 ml glacial acetic acid (pH 4.6) in water to a final volume of one liter. The reagent is stable for years.
1 ml of the solution is put into a clean test tube.
3 ml of Barfoed's reagent is added into the test tube and been mixed thoroughly.
The test tube is placed in a boiling water and been heated for 3 minutes.
The test tube is removed and allowed it to be cooled.
The formation of green, red, or yellow precipitates are observed which positive result are indicated.
Lasker and Enkelwitz test for ketoses
The Lasker and Enkelwitz test utilizes Benedicts's solution, although the reaction is carried out at a much lower temperature. The color changes that are seen during this test are the same as with Benedicts's solutions. Dilute sugar solutions are used with this test (0.02 M)
1 ml of the solution is put into a clean test tube.
5 ml of Benedicts's reagent is added into the test tube and been mixed thoroughly.
The test tube is placed in a water bath and been heated at 55°C for 10-20 minutes.
The test tube is removed and allowed to be cooled.
The formation of green, red, or yellow precipitate are observed which the positive results indicated. Ketopentoses demonstrated a positive reaction within 10 minutes, while ketohexoses take about 20 minutes to react.
Seliwanoff's test for ketoses
Seliwanoff's Reagent: 1 g resorcinol is dissolved in 330 ml concentrated HCl, dilute to one liter (approximately 4M HCl final). This reagent seems to be stable for more than a year, though we usually make less than the recipe specifies.
1 ml of the solution is put into a clean test tube.
5 ml of Seliwanoff's reagent is added into the test tube and been mixed thoroughly.
The test tube is placed in boiling water bath and been heated for 4 minutes
The test tube is removed and allowed to be cooled.
The formation of orange or red color (not precipitate) is observed which the positive results are indicated.
Bial's test for pentoses
Orcinol formed colored condensation products with furfural generated by the dehydration of pentoses and pentosans. The dilute sugar solutions is used with this test (0.02 M) if necessary.
Bial's reagent: 3 g of orcinol is dissolved in 500 ml concentrated HCl, 2.5 ml of a 10% solution of ferric chloride hexahydrate (FeCl3) are added and been diluted to one liter with water, this is approximately 6 M HCl . The reagent is probably stable for a few weeks. The "classical" Bial's reagent is made with a liter of concentrated HCl, undiluted with water. It gives a slightly stronger reaction, and considerably faster (30-60 seconds), but is much less stable, and the fumes are much more a problem with concentrated than with 6 M HCl. The reaction even seems to work, more slowly and with less intense color, if the final HCl concentration is only 4 M.
2 ml of the solution is put into a clean test tube.
5 ml of Bial's reagent is added into the test tube and been mixed thoroughly.
The test tube is placed in a boiling water bath and been heated for less than 5 minutes.
The test tube is removed and allowed to be cooled.
The formation of green to blue colored solution or precipitate is observed which the positives results are denoted.
Iodine test for starch and glycogen
The use of Lugol's iodine reagent (IKI) is useful to distinguish starch and glycogen from other polysaccharides. Lugol's iodine yields blue-black color in the presence of starch. Glycogen reacts with Lugol's reagent to give a brown-blue color. Other polysaccharides and monosaccharides yield no change; the test solutions remains the characteristics brown-yellow of the reagent. It is thought that starch and glycogen from helical coils. Iodine atoms can then fit into the helices to form a starch–iodine complex. Starch in the form of amylose and amylopectin has less branches than glycogen. This means the helices of starch are longer than glycogen, therefore binding more iodine atoms. The result is that the color produced by a starch-iodine complex is more intense than that obtained with a glycogen-iodine complex.
5 ml of the solution is put into a clean test tube.
2-3 drops of Lugol's iodine reagent are added into the test tube and been mixed thoroughly.
A brown-blue color is observed which the positive result is indicated for glycogen while a blue-black color is for starch. The brown color of the test reagent is the negative test.
RESULTS
Type of CHO
Result (positive or negative)
Molisch Test
Benedicts's
Test
Barfoed's Test
Lasker and Enkelwitz Test
Seliwanoff's
Test
Bial's Test
Iodine
Test
Fructose
+
+
+
+
+
-
-
Glucose
+
+
+
-
-
-
+
Lactose
+
+
-
-
-
-
-
Galactose
+
-
+
-
+
-
-
Ribose
+
-
+
-
-
-
-
Sucrose
+
-
-
-
+
-
-
Starch
+
-
-
-
-
-
-
Unknown 1
+
-
-
-
-
-
-
Unknown 2
+
-
+
-
-
-
-
Discussion
Based on this experiment, we conducted different type of carbohydrate test which are Molisch, Benedicts, Barfoed, Lasker and Enkewitz, Bial and Iodine tests on different carbohydrates. For Molisch test, the result shown are positive on all types of carbohydrates. Molisch's 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.
For Benedicts's test, based on the table, the positive results only shown on fructose, glucose and lactose. Theoritically, in the presence of even small quantities of reducing sugars the entire body of the solution will be filled with a precipitate which is red. But in the case of non-reducing sugar like sucrose, the solution will remain perfectly clean.
For Barfoed's test, experimentally mostly the results shown are positive, but the negative results shown only on lactose, sucrose and starch also on solution unknown 1. Barfoed's 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.
For Lasker and Enkelwitz test, only the fructose shows the positive result. Theoretically, the Lasker and Enkelwitz test tested for ketoses.This, of course,ruled out Glucose, as it is an aldose.Glucose and only fructose react positively but no positive result shown for glucose. The Lasker and Enkelwitz test also utilizes Benedict's solution, although the reaction is carried out at a much lower temperature. The color changes that are seen during this test are the same as with Benedict's solution.
For Seliwanoff's test only fructose, galactose and sucrose shows the positive results based on the table. Theoretically the Seliwanoff's 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. Example of ketose is monosaccharide such as glucose, fructose and galactose which will gives rapid positive result but unfortunately no positive test result shown on glucose in this experiment maybe due to some error.
For Bial's test, all carbohydrates show the negative result. Theoretically, the formation of a bluish product indicates the positives result but no bluish product formed in this test. All other colors indicate a negative result for pentoses. Hexoses generally react to form green, red, or brown products.
For Iodine test, based on the table only glucose gave a positive result. Iodine test is an indicator for the presence of starch. Iodine solution (iodine dissolved in an aqueous solution of potassium iodide) reacts with starch producing a blue-black color. This is due to there might be a slightly starch contained in glucose which gave a positive result on this test.
There may have been some errors occured down to contamination but with what remains unknown. As can be seen from the table of results shown above, the unknown gave two positive results. It gave a positive result under the Molisch test indicating that it is a carbohydrate and it also gave a positive result under the Benedict's test indicating that it is a reducing sugar. In addition to this it tested positive under the iodine test indicating the presence of starch. Unkown solution gave negative results under the Seliwanoff's test which indicates it is not Fructose or glucose. It also gave a negative result under the Bial's test which indicates that it is a hexose sugar and not a pentose sugar. As previously mentioned the starch gave a blue result under Benedict's test which is not what was expected from a reducing sugar, as it would have been expected to produce an orange precipitate. This may have been down to contamination but with what remains unknown.
Conclusion
References
Dreywood, R. (1946). Qualitative Test for Carbohydrate Material. Industrial & Engineering Chemistry Analytical Edition, 18(8), 499-499. http://dx.doi.org/10.1021/i560156a015
http://www.biologydiscussion.com/carbohydrates/test/qualitative-and-quantitative-tests-for-carbohydrates/13042
https://fulltimes.wordpress.com/carbohydrates-test/
