UNBiol6

School Science Lessons
Biology experiments
Food, food tests, plant physiology
Updated: 2009-09-28
Please send comments to: J.Elfick@uq.edu.au

Table of contents
9.3.11.0 Food, food tests
9.109.0 Plant physiology

9.3.11.0 Food, food tests
Food chemistry, Topic 19
2.34 Three kinds of food (Primary)
3.98 Elements in foods
4.2.0 Fermentation processes in food production
4.2.11 Glycemic index (GI) GI value and GI load
9.228 Body Mass Index (BMI)

6.5.12 Tests for sugars
6.5.13 Tests for carbon dioxide with bromothymol blue
9.3.11 Tests for oxidase and peroxidase in plant tissues
9.3.12 Tests for zymase and catalase in yeast
9.132 Tests for starch, iodine tests for starch
9.132a Amylose and amylopectin
9.134 Tests for carbohydrates, Molisch's test (alpha-naphthol test), Solubility in water
9.135 Tests for cellulose, iodine tests for cellulose
9.136 Tests for cellulose, solubility tests for cellulose
9.137 Tests for fats and oils
9.138 Tests for nitrogen compounds in food, soda lime test
9.139 Tests for proteins, biuret reaction, Millon's reagent, xanthoproteic reaction
9.140 Tests for simple sugars, reducing sugars, Fehling's test
9.141 Tests for reducing sugars, Benedict's tests for reducing sugars, urine test
9.142 Tests for starch, Fehling's tests for starch
9.143 Tests for vitamin C (L-ascorbic acid)
9.144 Tests for wood
9.154 Tests for carbon dioxide using limewater
9.182 Tests for glucose and starch with "Testape"
12.18.5.1a Dehydration of sugar by sulfuric acid
16.3.1.6 Tests for wood, hemicellulose, lignin
16.4.1.0 Tests for organic acids and alcohols
16.4.5 Tests for fats. proportion of fats in foods
16.4.6 Tests for gases from burning hydrocarbons
16.4.7 Tests for saturated hydrocarbons, bromine water test
16.4.8 Tests for saturated hydrocarbons, alkaline potassium manganate (VII) solution test
16.4.9 Tests for saturated hydrocarbons, acidified potassium manganate (VII) solution test
16.6.1 Tests for proteins, heat test for proteins
16.6.2 Tests for proteins, burning test for proteins
16.6.4 Tests for albumin and gelatine
16.6.5 Tests for proteins, biuret test
16.6.6 Tests for proteins, xanthoproteic test
16.6.7 Tests for proteins, Millon's test
16.6.8 Tests for proteins, Albustix test strips
16.6.10 Tests for proteins, Sakaguchi's arginine test
16.6.11 Tests for sulfur in proteins
16.10.1 Tests for starch breakdown to sugars, hydrolysis of starch, iodine test, Fehling's test

9.109.0 Plant physiology
9.3.10 Activity of diastase
9.3.11 Tests for oxidase and peroxidase in plant tissues
9.3.12 Tests for zymase and catalase in yeast
9.3.14 Action of lipase in castor oil seeds
9.3.15 Moisture content of plant organs and ash content of plant dry matter
9.128 Heat different foods
9.129 Hydrolysis of starch by dilute hydrochloric acid
9.130 Hydrolysis of starch by salivary amylase (ptyalin)
9.131 Hydrolysis of sucrose by dilute acids
16.4.1.1 Carboxylic acids, fatty acids and their salts, Carboxylic acids group: (-COOH), suffix: -oic acid
16.4.1.1.1 Dicarboxylic acids, two carboxyl groups (-dioic acid)
16.4.1.1.2 Tricarboxylic acids, citric acid
16.4.1.1a Vitamin A
16.4.1.2 Vitamin B1
16.4.1.3 Vitamin C (ascorbic acid)
16.4.1.4 Vitamin D
16.4.1.5 Vitamin E
16.4.2 Prepare ethanoic acid (acetic acid) ionization reaction
16.4.3 Prepare ethanedioic acid-2-water (oxalic acid) ionization reaction
16.4.4 EDTA, ethylene diamine tetra acetic acid, (HOOC.CH₂)₂N(CH₂)₂N(CH₂.COOH)₂16.4.4.1 Ion exchange resins
16.7.15 Commercially available test reagents
16.7.16 Artificial sweeteners
16.8.1 Reactions of benzene
16.8.2 Prepare ferric tannate with tea leaves
16.8.3 Extraction of caffeine and benzoic acid from soft drinks, e.g. cola and lemonade
16.9.1 Burn carbohydrates, fats and proteins
16.9.2 Heat food with copper (II) oxide

6.5.12 Tests for sugars
See 9.140: Tests for simple sugars, reducing sugars, Fehling's test
Maize, sugar beet and sprouting onion bulbs are suitable for sugar tests because they contain stored simple sugar rather than the large starch molecule. Cut pieces 2 cm long and put in 2 mL sugar test solution in a Pyrex test-tube and boil the mixture. Make the sugar test solution from 173 g of sodium citrate, 200 g of crystalline sodium carbonate, and 17.3 g of crystalline copper (II) sulfate. Dissolve the carbonate and citrate in 100 mLwater. These substances will dissolve faster if the water is warmed. Dissolve the copper (II) sulfate in 100 mL water and slowly pour this solution into the carbonate citrate solution. Cool and add water to make 1 litre of test solution. Show the colour change by dissolving a little cane sugar in 10 mLwater in a test-tube. Add saliva that will change the cane sugar (sucrose) into a simple sugar (glucose). Add 3 mL of the test solution and boil over a heat source. A yellowish or reddish precipitate forms when simple sugar is present.

6.5.13 Tests for carbon dioxide with bromothymol blue
Bromothymol blue solution is used to show the presence of carbon dioxide. Fill four test-tubes three quarters full of water. Add 25 drops of bromothymol blue to each tube. Put a sprig of Elodea or other small water plant in two of the tubes. With a drinking straw, blow bubbles into one tube not containing a plant, and then into one with a plant. Note the colour change that shows the presence of carbon dioxide. Put stoppers in the four test-tubes and note the changes within 15 minutes to an hour. Repeat the experiment, but put the tubes in a dark place, a closed desk.

9.3.10 Activity of diastase
1. Prepare active diastase
1.1. Buy taka-diastase from the chemist and prepare a 0.1% solution, or,
1.2. Germinate barley grains on damp filter paper until the shoots begin to emerge, crush with a mortar and pestle, add 50 mL water then filter. The filtrate will contain active diastase.
2. Boil half the active diastase solution. Prepare a 1% solution of starch and place 5 mL in one each of two test-tubes. Add an equal quantity of the unboiled or boiled diastase extract to the two test-tubes. Periodically take a drop of the mixture from each tube and test its reaction with dilute iodine on a white tile. At first the blue-black starch colour occurs in both test-tubes. However, this colour in the test-tube containing unboiled enzyme is soon replaced by reddish colours, and finally no colour, showing that starch has been converted to simpler substances, mostly to sugars. The temporary production of red colours occurs because of the formation of intermediate substances, e.g. dextrin. The mixture containing boiled enzyme will continue to give the starch reaction. Apply the Fehling's test to each test-tube. The above experiment will proceed faster if the tubes are placed in a water bath at a temperature of 30^oC to 40^oC. Compare the time required for the reaction to reach completion at room temperature and at the higher temperature.

9.3.11 Tests for oxidase and peroxidase in plant tissues
These enzymes may be detected by means of a 1% solution of gum guaiacum in 60% alcohol. Pound different plant tissues with water using a mortar and pestle. Decant the extract into a white dish and test with the gum guaiacum. If oxidase is present, a blue oxidation product of a constituent of the gum will be formed. If no colour change occurs, add hydrogen peroxide (5 vols) to the same mixture. A blue colour indicates that peroxidase is present. Potato and carrot give the oxidase reaction, cabbage and turnip give the peroxidase reaction. Tissues containing oxidase often turn brown when cut surfaces are exposed to the air.

9.3.12 Tests for zymase and catalase in yeast
1. Place a mixture of fresh yeast, glucose and water in the proportion of 1:1:10 in a saccharimeter or double test-tube set at 30^oC to 40^oC. A gas forms that turns limewater milky. Ethyl alcohol is present in the fermenting mixture. The fermentation of the sugar occurs under the influence of the zymase complex of enzymes. Repeat the experiment with other sugars, e.g. sucrose.
2. To show that yeast contain a very active catalase enzyme, set up a double test-tube with the inner tube filled with 2 vols hydrogen peroxide. Add 1 g yeast. Observe the rapid evolution of a gas. Test the gas with a glowing splint to show it is oxygen.

9.3.14 Action of lipase in castor oil seeds
Shell about 10 g seeds and divide into two portions. Use a mortar and pestle to crush one portion with 4 g of castor oil and 5 mL water. Treat the second portion in the same way, but use 5 mL N/10 sulfuric acid instead of the 5 mL water. Allow both to stand for about one hour, then to each add 25 mL of alcohol. Then titrate the free acid in each with a normal solution of caustic soda, using phenolphthalein as an indicator. Note that the second portion, which was pounded with acid, contains a much greater amount of free acid. This shows that the activity of lipase present in the seeds has been accelerated in an acid medium.

9.3.15 Moisture content of plant organs and ash content of plant dry matter
1. Measure the moisture content of plant organs, e.g. leaves, tubers by heating shredded or ground weighed samples in an oven at 95^oC to 100^oC. Leave the samples in the oven overnight, then cool them in a desiccator and weigh them again. Replace the samples in the oven for two hours and then weigh them again Calculate the percentage moisture content of the original material.
2. Test cabbage or other leaves, storage organs such as carrot or potato, or seeds. Heat 10 g samples in dishes in an oven at 100^oC. Before taking samples shred materials such as cabbage or carrot and grind seeds in a mill. Leave the samples in the oven overnight, then cool in a desiccator and weigh again. Replace the material in the oven for a further few hours and then weigh again, to make sure that all moisture has been driven off. Calculate the percentage moisture content of the original material.

9.128 Heat different foods
See 19.3.4.3: Non-enzymatic browning, caramelization
See 19.3.4.4: Non-enzymatic browning, the Maillard reaction
1. Prepare in separate test-tubes small samples of the following:
1.1 Carbohydrate, e.g. starch or sugar
1.2 Fat, e.g. butter
1.3 Protein, e.g. meat.
Heat the samples gently, then more strongly until they begin to burn. Burning carbohydrates have a smell of caramel. Burning fats produce acrolein that makes the eyes water. Heated proteins produce ammonia-like compounds with different odours. Continue heating each sample until only a residue of carbon remains. The three food samples decompose on heating to leave a black solid, carbon. Clean the test-tubes without delay.
2. Put a small sample of starch in a test-tube. Hold the test-tube almost horizontally and heat the starch over a flame. Be careful! Hot glass can cause severe skin burns so wear thick protective gloves and handle with care. Note the liquid that appears nearest the flame at the bottom of the test-tube. Test the liquid for water with cobalt (II) chloride. The cobalt chloride turns blue so the liquid is water.
3. Repeat the experiment with the same quantity of the following:
3.1 Granulated sugar
3.2 Olive oil
3.3 Boiled egg white.
When testing the egg white, note the appearance of a thick, white mist and an ammonia-like smell. Hold a moistened strip of red litmus paper in the mist. It turns blue. Only the egg white produces this reaction because only it contains nitrogen.
4. Heat food with copper (II) oxide in a small test-tube. Copper oxide releases oxygen to the food. Test the gas in the test-tube with limewater by withdrawing gas with a medicine dropper, teat pipette, then expel the gas as a bubble through limewater. The gas is carbon dioxide. Copper (II) oxide releases oxygen to the food. Also, note the water condensed in the cooler parts of the test-tube.
5. Measure the ash content of plant dry matter. Weigh a 2 g powdered sample of plant dry matter into a crucible. Heat over a Bunsen burner in a fume cupboard, gently at first, and then strongly. Continue heating until only the ash remains as an almost white residue of salts. After cooling, weigh the crucible again and calculate the percentage ash content in the dry matter.
6. Measure the moisture content of plant organs, e.g. cabbage leaf, carrot, potato. Shred materials such as cabbage or carrot and grind seeds in a mill. Heat 10 g samples in dishes in an oven at 100^oC. Leave the samples in the oven overnight, then cool in a desiccator and weigh again. Use safety glasses and insulated heat-proof gloves when handling the hot dishes. To make sure that all moisture has been driven off, replace the material in the oven for two hours, and then weigh again. Calculate the percentage moisture content of the original material.
7. Put glucose into a test-tube then heat gently strongly until only a black residue of carbon remains. Clean the test-tube immediately.
8. Put glucose in a test-tube. Note its crystalline state at room temperature. Add water and sand, then heat until it decomposes to carbon dioxide and water. Taste is sweet. Cane sugar is crystalline starch and cellulose. Cane sugar is readily soluble in water, cellulose is not. Starch is insoluble in cold water but in hot water it forms a solution that may set like a jelly when cooled. All three carbohydrates decompose on heating to form carbon as a black solid.

9.129 Hydrolysis of starch by dilute hydrochloric acid
See also: 16.3.1.5 Starches, amylum, glycogen | See 16.3.1.2 Fehling's tests for reducing sugars and aldehydes in solution, glucose and fructose
Do not allow students to handle concentrated hydrochloric acid.
1. Do Fehling's tests for simple sugars on a 1% starch solution. No reaction occurs if the starch is pure. Add 10 drops of concentrated hydrochloric acid to 10 mL of 1% starch solution in a test-tube. Stand the test-tube in boiling water for 10 minutes then leave to cool. Take 5 mL of this solution and neutralize it by adding 1 mL of sodium hydroxide solution. Do Fehling's tests for simple sugars. If the test is not positive, heat the solution for a longer period and test again. The starch is converted to simple reducing sugars by acid hydrolysis. Compare the results at room temperature and 10^oC above room temperature.
2. Test the results with "Testape". Tear off a small piece of "Testape". Lay it on the bench and add drops of the solutions. After 30 seconds, compare the colours of the test paper with the colour chart on the Testape dispenser.
3. Do the Fehling's test on a colloidal solution of starch. Note the reaction is negative. Then hydrolyse a portion of the starch solution by boiling with equal volume of dilute sulfuric acid for 10 minutes, stirring all the time. Test the solution periodically by applying the iodine test on a drop on a tile. Note the stages of colour changes. Finally neutralize, apply the Fehling's test.

9.130 Hydrolysis of starch by salivary amylase (ptyalin)
Plants have alpha amylase and beta amylase (diastase in brewing malt). Animals have only alpha amylase, pancreatic amylase, salivary amylase (ptyalin). The amylase enzyme hydrolyses the 1,4-glycosidic bonds in starch to produce reducing sugars.
You may have to seek approval to work with human saliva because it can spread disease. Instead of using human saliva, use salivary amylase from a laboratory supply company.
1. Prepare a dilute saliva solution by rinsing 20 mL of warm water in the mouth for one minute then spit it into a beaker. Use a teat pipette to add 2 mL of dilute saliva to 10 mL of fresh 1% starch solution. Stir the solution thoroughly. Record the time of adding the saliva. At five minute intervals put two drops of the saliva starch solution in a test-tube or on a white tile and add a drop of iodine solution. Note the colour of the solution. Wash the dropper between each test. For each successive test the blue colour decreases because starch is being converted to glucose sugar. Saliva contains salivary amylase (ptyalin) a catalyst that converts starch to the simple sugar maltose and water.
2. Put three drops of the starch and saliva solution into a test-tube. Add 3 mL of Fehling's reagent and heat the solution until almost boiling. Note the colour of the precipitate. For each successive drop of starch and saliva solution tested at five minute intervals. The brick-red precipitate increases, showing that the amount of glucose sugar is increasing. The enzyme salivary amylase in the saliva is breaking down starch into glucose sugar.
3. Remove a drop of the starch and saliva solution and put it on a white tile. Put a drop of iodine solution on the drop of starch and saliva solution. Note the colour of the solution. For each successive drop taken out at each five minute interval, the blue colour decreases, showing that starch is being converted to glucose sugar. Wash the dropper between each test.

9.131 Hydrolysis of sucrose by dilute acids
See also 6.3.1.4: Disaccharides
Do Fehling's tests for simple sugars on a 1% cent sucrose solution. No reaction occurs with sucrose solution if it is pure. Add 10 drops of 10% hydrochloric acid to the 1% cent sucrose solution. Boil the solution for two minutes and leave to cool. Add 10 drops of sodium hydroxide solution to neutralize the acid. Do Fehling's tests for simple sugars. Stand the test-tube in boiling water. The reaction produces a red precipitate in the sucrose solution. The intensity of the colour depends on the extent of the hydrolysis.

9.132 Tests for starch, iodine tests for starch
See diagram 2.26: Iodine tests for starch | See also 16.3.1.5: Starches, amylum, glycogen | See diagram 2.0: Starch grain in potato cell | 16.7.8 Iodine tests for starch
Be Careful! Heat alcohol with an electric heater or use a water bath. Do NOT use a Bunsen burner!
1. Sugar, the products of photosynthesis, and the large starch molecules formed from many sugar molecules are present in leaves. A simple starch test consists of applying a dilute iodine solution and watching for the typical blue-black colour that shows that starch is present. The iodine solution is prepared by dissolving 10 g of potassium iodine in 100 mL deionized water and adding 5 g of iodine. Tubers, potatoes or a starch paste may be used to show the colour change.
2. When testing leaves softening the leaf cells by boiling in water for a few minutes is necessary. Then the leaf is put in boiling alcohol until the pigments that will mask the reactions are removed from the leaf. Chlorophyll is usually removed in 5-8 minutes but fleshy leaves may take longer or require a change of alcohol for adequate removal of pigments. The iodine solution should react with the starch within 15 minutes.
3. Iodine is scarcely soluble in water, so iodine solution is iodine dissolved in potassium iodide solution. To make iodine solution, I₂/ KI, dissolve 15 g of potassium iodide in 20 mL of water. Then dissolve 3 g of iodine crystals in this solution and make up to 1 litre with demineralized water. Use safety glasses and nitrile chemical-resistant gloves when weighing solid iodine because it is harmful and corrosive. Once in solution the amounts of iodine used are minute.

9.132a Amylose and amylopectin
Different starches contain different proportions of amylose and amylopectin, both . Amylose, a long-chain polymer of glucose, gives the deep blue colour with iodine, but amylopectin, a long-chain many branched polymer of glucose, gives a red-brown colour with iodine. Tincture of iodine antiseptic is a solution of iodine in ethanol.

1.1 Boil a half full test-tube of water. Add 1 g of powdered laundry starch and continue boiling. Cool the solution then add drops of iodine solution. The liquid appears black but if hold it up to the light it appears dark blue. Pour out half the solution then reheat the test-tube. The blue colour disappears. Cool the test-tube under a water tap. The blue colour reappears.
1.2 Do the iodine tests for starch on a solution of glucose. Pure glucose sugar does not react with iodine solution.
1.3 Do the iodine tests for starch in a waterweed. Put a well developed shoot of waterweed in a 600 mL beaker filled with water. Put the beaker in the sunlight or expose it to electric light (e.g. from a microscope lamp) after 2 hours detach a leaf from the upper end of the shoot using the tweezers and place it in a drop of chloral hydrate solution on a microscope slide. Immediately add one drop of iodine potassium iodide solution. Mount a coverslip. Examine the slide under high power. The assimilation starch can be seen in the chloroplasts of the waterweed in the form of small blue black dots. It has been stained that colour by the iodine potassium iodide solution which is used as a stain for identifying starch.
1.4 Do the iodine tests for starch on thin leaves. Gather the leaves immediately after they have been exposed to several hours of daylight. Put the leaves in boiling water to kill the cells. Heat a beaker of water to boiling. Turn off the Bunsen burner or electric heater then put a test-tube of methylated spirit in the hot water to boil the alcohol. The boiling point of alcohol is lower than the boiling point of water, so if the water is hot enough the alcohol will boil. Put the leaves in the hot methylated spirit until the chlorophyll pigment that can mask the reaction is removed to the alcohol. When the leaf is almost white, put it in the iodine solution. Note the deep blue colour. Be careful! Use safety glasses and insulated heat-proof gloves.
1.5. Repeat the test on several types of leaves and plant storage organs, e.g. potato tubers, sweet potato, carrot, onion, apple, banana. The test works better with cooked starch because heat breaks the walls of the starch grains in the plant cells. Test leaves at different times of the day after different exposure to sunlight. Test variegated leaves and different coloured leaves. Test different parts of plants by adding the iodine solution to a cut surface.
1.6 Add drops of iodine solution to some solid glucose, sucrose, cotton wool, laundry starch, bread, potato and rice. The test is positive for the last four substances only. The blue colour produced when iodine is added to starch is characteristic of starch. Margarine may contain a small quantity of starch to differentiate it from butter. If liver sausage contains starch, it is adulterated.
1.7. Do the iodine tests for starch on the cut surface of a tuber, e.g. potato, and note the blue-black colour formed. Scrape the cut surface of a potato tuber. Put a scraping as big as a pin head in a drop of water on a microscope slide and apply a coverslip. Examine the tissue under the low power. Put a drop of iodine solution on the microscope slide to touch one side of the coverslip. Touch a piece of absorbent paper to the other side of the coverslip to draw the iodine solution across. Keep looking down the microscope and see the starch grains turning blue. Examine the starch grains in the cells under high power. Focus up and down on a starch grain to see the layers. Examine other examples of starch grains, e.g. bean seed, rice grain.

9.134 Tests for carbohydrates, Molisch's test (alpha-naphthol test), Solubility in water
1. Molisch's test (alpha-naphthol test)
Add 0.1 mL 5% ethanolic alpha-naphthol to 5 ml of medium and swirl the test-tube to mix the solutions. Carefully pour concentrated sulfuric acid down the side of the test-tube. A violet ring or purple colour at the junction of the the liquids indicates carbohydrates. ( H. Molisch 1856-1937)
2. Solubility in water
2.1 Dissolve carbohydrates in water. Heat the water if necessary. Glucose and sucrose (cane sugar) are soluble in water. Cellulose is not soluble in water. Starch is insoluble in cold water, but in hot water it forms a solution that may set like a jelly when cooled.
2.2 Tie a teaspoonful of plain wheat flour in a fine cloth, e.g. a handkerchief, and pummel it up and down in a dish of water. Allow the white suspension in the dish to settle then decant the water. Do the iodine tests for starch on the precipitate. Examine the sticky mass left inside the cloth. It is mainly gluten and cellulose

9.135 Tests for cellulose, iodine tests for cellulose
See also 6.3.1.6: Cellulose, hemicellulose, lignin, tests for wood
Do not allow students to handle concentrated sulfuric acid. Use safety goggles and nitrile chemical-resistant gloves.
Add iodine solution to cotton wool in a beaker. The cotton wool turns yellow. Drain off the iodine solution. Add drops of concentrated sulfuric acid. The cotton wool turns a deep blue. Cotton wool is almost pure cellulose. Test a slice of onion bulb under the microscope.

9.136 Tests for cellulose, solubility tests for cellulose
Do not allow students to handle concentrated hydrochloric acid. Use safety goggles and nitrile chemical-resistant gloves.
Cellulose is soluble in the following:
1. Solution of zinc oxide in concentrated hydrochloric acid
2. Ammoniacal copper carbonate dissolved in dilute ammonia solution
3. Schweizer's reagent (not "Schweitzer") is made by dissolving 0.3% solution of precipitated copper (II) hydroxide solution in a 20% dilute ammonia solution to form tetraammine copper dihydroxide, cuprammonium hydroxide [Cu(NH₃)₄](OH)₂) complex ion Cu(NH₃)₄²⁺. The reagent forms a deep azure solution, Eduard Mathias Schweizer (1818 -1860) discovered this method of dissolving cellulose in copper tetra-ammine.

9.137 Tests for fats and oils
Examples of fats: butter, margarine, beef dripping, mutton dripping, suet and tallow. Examples of oils: olive oil, castor oil, linseed oil, coconut oil. Oils are oily liquids at room temperature that float on water.
1. Paper tests for fats
1.1 Mark two pencil crosses on writing paper, 10 cm apart. Use a pipette to put a drop of olive oil (fat) on one cross and use another pipette put a drop of water on the other cross. Compare both crosses after 12 hours and 24 hours. Note how the cross on which the oil was dropped can be distinguished. Fat makes a translucent mark on paper.
1.2 Cut different foods and press the cut surface on white paper, e.g. walnut, hazelnut, coconut, sausage, butter, boiled egg white, sugar lump. Note which foodstuffs make a translucent mark and so contain fat.
2. Sudan III tests for fats
The fat soluble dye Sudan (III) stains triglycerides, C₂₂H₁₆N₄O, 1-((4-(phenyldiazenyl)phenyl)diazenyl)naphthalen-2-ol. It is carcinogenic
2.1 Half fill a test-tube with water, add drops of Sudan III solution, shake the test-tube and note the faintly pink colour. Be careful! Use safety goggles and nitrile chemical-resistant gloves when handling stains. Add the same number of drops of olive oil, shake the solution and leave to stand in a test-tube rack. Wait for the oil to settle to the top of the test-tube. Note the colour of the oil and the water.
2.2 Add a drop of Sudan III solution to cut seeds and nuts, e.g. castor bean seed, sunflower seed. Cut a very thin slice of the seed and examine under high power.
3. Fat is not soluble in water, but is soluble in ether, chloroform and acetone. Students should not be allowed to work with ether or chloroform because these chemicals are very volatile, so students should not do this test.

4. Apply osmic acid to the cut surface of nuts and seeds. A black colour forms to show the presence of oil. Osmic acid is an acute poison if ingested, inhaled or in contact with the skin. So this test should NOT be done in a school laboratory.
5. Heat drops of oil in a Pyrex test-tube. Oil decomposes on heating to leave carbon.

9.138 Tests for nitrogen compounds in food, soda lime test
See diagram 16.9.3: Test with moist litmus paper
Put crushed cheese or meat in a test-tube with soda lime. Mix the food and soda lime then heat the mixture. Note the pungent odour of ammonia at the mouth of the tube. Test with moist litmus paper. Red litmus paper turns blue. The food produces ammonia gas, so the nitrogen in the ammonia must have come from the food. Soda lime is a mixture of sodium hydroxide and calcium hydroxide and is used as a laboratory drying agent.

9.139 Tests for proteins, biuret reaction, Millon's reagent, xanthoproteic reaction
Prepare a protein solution by shaking the white of an egg in its own volume of water. Also, shake ground pea seeds with water for several minutes, filter the mixture and keep the filtrate.
1. Biuret reaction
Add drops of 10% sodium hydroxide solution to protein solution then add drops of copper (II) sulfate solution. Note the violet colour
2. Millon's reagent (Millon's solution)
In some school systems this test is not allowed because this reagent contains mercury (I) nitrate. Do not prepare Millon's reagent.
Add drops of Millon's reagent to protein solution then heat the solution. The protein precipitates and turns pink when heated.
3. Xanthoproteic reaction
Do not allow students to handle concentrated nitric acid.
Add concentrated nitric acid to the protein solution then heat the solution with care.Note the yellow colour. Cool the mixture under the tap then add drops of concentrated ammonia solution. The colour intensifies to orange. Repeat the experiment with expressed plant juice, pieces of plant tissue and slices of plant tissue on a microscopic slide. Be careful! Concentrated ammonia it can cause skin burns and has a very strong odour when a large amount of the gas (50 parts per million) is in the air. Low levels of ammonia may harm some asthmatics and other sensitive individuals. Students should not do this test. 4. Heat proteins
Heat a protein solution and note any changes. The change is similar to the change in the white of an egg when it is boiled.

9.140 Tests for simple sugars, reducing sugars, Fehling's test
See also 16.3.7: Fehling's tests for aldehydes in solution
1. Tests for glucose and fructose
1.1 Mix equal parts of Fehling's A solution and Fehling's B solution. Take 3 mL of this deep blue solution and add 3 mL of 1% glucose solution. Stand the test-tube in boiling water for some minutes or warm the solution gently over a Bunsen burner, with constant shaking. The blue colour gradually disappears and a bright red precipitate of copper oxide forms to indicate the presence of glucose. A red precipitate shows that glucose is a reducing agent. Fructose gives the same reaction.
1.2 Add glucose crystals to a test-tube a quarter filled with water. Close the test-tube with your thumb and shake until the glucose dissolves. Pour into a second test-tube the same quantity of Fehling's A solution and Fehling's B solution. Heat the contents of the test-tube not at the bottom. but a just below the surface of the liquid. Hold the test-tube so the mouth points away from people. As soon as the solution boils, add the contents to the glucose solution. Heat the contents of the test-tube. A green then a brick-red (orange-red) precipitate forms of copper (I) oxide forms that shows the presence of glucose sugar. Fructose and methanal give the same reaction. Repeat the experiment by testing cane sugar or beet sugar (sucrose) starch and cellulose. They do not change the colour of Fehling's solution.
1.3 Test juice squeezed from crushed leaves, stems, and fruit. The test is positive, showing the presence of simple sweet-tasting sugars. However, roots and seeds test negative because they contain mainly starch. Repeat the experiment with crumbled cake, biscuit, rice, starch and other common foods.
2. Test sucrose
2.1 Do Fehling's test on a 1% cent solution of sucrose, cane sugar or beet sugar. No reaction occurs with sucrose solution if it is pure. Hydrolyse the solution of sucrose in a test-tube by adding 10 drops of dilute hydrochloric acid to the 1% cent sucrose solution, Boil for a few minutes, cool and add 10 drops of sodium hydroxide solution to neutralize the acid. Add freshly made Fehling's A and B solutions. Stand the test-tube in boiling water. The reaction produces red precipitate in the sucrose solution. The intensity of the colour depends on the extent of the hydrolysis. If no red colour appears, again add acid and boil the solution until a red colour appears.
2.2 To a 3 mL sample of 1% sucrose solution, add 10 drops of dilute hydrochloric acid. Boil for a few minutes, cool and add 10 drops of sodium hydroxide solution to neutralize the acid and then 3 mL of deep blue Fehling's solution. Stand the test-tube in boiling water. If no red colour appears, again add acid and boil the solution until a red colour appears.
3. Test starch
3.1 Test a dilute starch solution. Starch does not react with Fehling's reagent.
3.2 Hydrolyse starch solution by boiling. with equal volume of dilute sulfuric acid for about 10 minutes, with constant stirring. Neutralize and apply the Fehling's test.
4. Test cellulose
Cellulose does not change the colour of Fehling's reagent.
5. Test plant parts
5.1 Test juice squeezed from crushed leaves, stems, and fruit. The test is positive indicating the presence of simple sweet-tasting sugars. However, roots and seeds test negative because they contain mainly starch. Repeat the experiment with crumbled cake, biscuit, rice, starch and other common foods.
5.2 Test plant organs for glucose and fructose, e.g. seeds, leaves, roots, stems, tubers. Extract some of the juice from the plant organ to be tested. Cut the tissues into fine pieces, and then crush the material with a pestle and mortar. If little juice is expressed, add a few mL water and continue to crush. Then perform Fehling's test on the plant extracts obtained.

The following solutions must be prepared by school staff before the experiment. Do not ask students to prepare these solutions or weigh out sodium hydroxide. Use safety glasses and nitrile chemical-resistant gloves. The test was invented by H.C. von Fehling (1812-1885).
6. Prepare Fehling's reagent
6.1 Simple sugars, e.g. glucose and fructose, reduce blue copper (II) oxide in Fehling's reagent to brick-red copper (I) oxide. To make Fehling's A solution, dissolve 17 g of copper (II) sulfate crystals in water and make up to 250 mL. To make Fehling's B solution, dissolve 87 g of sodium potassium tartrate-4-water (Rochelle salt) and 35 g of sodium hydroxide in water and make up to 250 mL. Just before doing the test, prepare Fehling's reagent by mixing equal volumes of Fehling's A solution and Fehling's B solutions to form a clear deep blue solution.
6.2 The reagent is made up in separate parts, Fehling's solution A and Fehling's solution B. Fehling's A is prepared by dissolving 34.6 g of copper sulfate in 500 mL deionized water. Fehling's B is prepared by, dissolving 175 g of Rochelle salt and 50 g of sodium hydroxide in 500 mL distilled water. The complete reagent is prepared when required for use by mixing equal quantities of the A and B solutions.

9.141 Tests for reducing sugars, Benedict's tests for reducing sugars, urine test
See 16.3.1.4.1: Reducing sugars and nonreducing sugars
Benedict's tests for reducing sugars uses a citrate ion-Cu²⁺ complex. Benedict's reagent is a mixture of copper (II) sulfate, hydrated sodium citrate and hydrated sodium carbonate. Add Benedict's reagent to a test solution and heat to boiling. A high concentration of reducing sugars gives a red precipitate and a low concentration gives a yellow precipitate. Benedict's test is more sensitive than Fehling's test and is easier to do because only one solution is needed. However, it may be more expensive. Nowadays, Benedict's test is used instead of Fehling's tests for detecting reducing sugars. This test was discovered by S. R. Benedict (1884-1936).
The oxidation methods for blood glucose are based on the reducing properties of glucose. Copper reduction tests are among the oldest methods for glucose determination. In a hot alkaline solution, glucose will reduce cupric salts to cuprous salts. The quantity of cuprous salts produced is directly proportional to the glucose concentration. Other procedures make use of the reduction of alkaline ferricyanide, which is yellow, to a ferrocyanide, which is colorless. The decrease of yellow color is dependent upon the glucose concentration.
Urine test
Add 8 drops of urine to 5 mL of Benedict's reagent, heat to boiling for two minutes and leave to cool. Reducing sugars produce precipitates: light green turbidity 0.1-0.5% sugar, green precipitate 0.5-1.0 % sugar, yellow precipitate 1.0 to 2.0% sugar, red precipitate > 2.9% sugar.

9.142 Tests for starch, Fehling's tests for starch
1. No reaction occurs with starch solution if it is pure. Test 1% pure starch solution with Fehling's test. The test is negative. Add 10 drops of concentrated hydrochloric acid to 10 mL of 1% starch solution. Stand the test-tube in boiling water for 10 minutes then leave to cool. Take 5 mL of this solution and neutralize by adding 1 mL of sodium hydroxide solution. tests for Fehling's solution. If the test is positive, the starch is converted to reducing sugars by acid hydrolysis. If the tests for glucose is not positive, heat for a longer period and test again.
2. Do the Fehling's test on a colloidal solution of starch. Note the reaction is negative. Then hydrolyse a portion of the starch solution by boiling with equal volume of dilute sulfuric acid for 10 minutes, stirring all the time. Test the solution periodically by applying the iodine test on a drop on a tile. Note the stages of colour changes. Finally neutralize, apply the Fehling's test.
3. Prepare a 1% suspension of starch in a little water, notice how the starch breaks up but does not dissolve. Boil the suspension and again examine. Add one drop of iodine solution to 5 mL of water and then add several drops of starch paste. The blue colour produced is the best tests for starch. Apply the Fehling's test and note the result.
4. Tie a teaspoonful of plain wheat flour in a fine cloth, like a handkerchief, and pummel it up and down in a saucer of water. Allow the white suspension in the dish to settle and decant the water. Test the solid for starch. Examine the sticky mass left in the cloth. It is mainly gluten and cellulose.

9.143 Tests for vitamin C (L-ascorbic acid)
Vitamin C is a water-soluble vitamin, essential for the formation of collagen in connective tissue. Vegetables should be cooked quickly in as little water as possible to retain nutrients. Sailors deprived of vitamin C during long voyages developed scurvy and suffered bleeding gums, lack of wound healing and anaemia, leading to death.
1. DCPIP (2,6-dichlorophenolindophenol) is a blue dye decolorized by ascorbic acid, but a pink colour may remain. To prepare the solution use 0.0162g of DCPIP per litre of water (0.1% DCPIP solution). Add 1 mL of DCPIP solution to an ascorbic acid solution or a solution of crushed vitamin C tablets. Add more ascorbic acid, vitamin C, until the blue solution turns colourless. DCPIP is reduced by ascorbic acid.
2. Test fresh fruits or vegetables, e.g. lemon juice or spinach, by crushing them with a mortar and pestle, shaking with 20 mL of water and testing the extracts with DCPIP solution.
3. Boil the same fruits or vegetables or fruits in water for ten minutes. Crush the boiled fruit or vegetable, add 20 mL of water, then test with DCPIP solution. Note which crushed fruit or vegetable contains the most ascorbic acid. Also, test the cooking water they were boiled in.
4. Test lemon or orange drinks, lemon cordial, blackcurrant juice, pickles, cucumber, and sauerkraut for vitamin C.
5. Test whether ascorbic acid is destroyed in an acidic or a basic solution.
6. Crush boiled fruit or vegetable, add 20 mL of water, then test with DCPIP solution. Note which fruit or vegetable extract contains the most ascorbic acid.
7. Test lemon or orange drinks, lemon cordial, blackcurrant juice, pickles, cucumber, and sauerkraut for vitamin C.
8. Test whether ascorbic acid is destroyed in an acidic or a basic solution. Some people add bicarbonate of soda (sodium hydrogen carbonate) to the water when boiling vegetables to make them look more green. However, this chemical destroys vitamin C.

9.144 Tests for wood
See also 16.3.1.6: Cellulose, hemicellulose, lignin, tests for wood
Put drops of a colourless solution of aniline sulfate or aniline chloride on the cut surface of a piece of wood. Note the bright yellow colour that shows the position of wood tissue, xylem. Cut across the stems of herbaceous plants, e.g. sunflower, pumpkin, celery, and apply aniline chloride solution to the cut surfaces. Look for any evidence of wood and record its position in the stem.

9.154 Tests for carbon dioxide using limewater
See diagram 3.34.1: Limewater tests for carbon dioxide
Prepare the weak alkali calcium hydroxide solution, limewater, by adding solid calcium hydroxide, slaked lime, to demineralized water. Shake the solution vigorously and leave to stand. Calcium hydroxide solid is only slightly soluble in water. When a white solid has settled as a fine white sediment, decant the clear limewater above the sediment. To replenish the limewater, add more demineralized water to the sediment in the stock bottle, shake and leave to settle. The settling process may take several days.
Pass carbon dioxide through the clear limewater. The solution becomes milky because of a fine precipitate of calcium carbonate.
Ca(OH)₂ (aq) + CO₂ (g) --> Ca(CO₃)₂ (s) + 2HCl (l)
Pass more carbon dioxide through the limewater. The solution becomes clear again because of the formation of soluble calcium hydrogen carbonate.
CaCO_{3 (s)} + CO₂ (g) + H₂O (l) --> Ca(HCO₃)₂ (aq)
Pass air through freshly-made limewater. After a long time may see a faint cloudy precipitate. The air contains about 0.4% carbon dioxide. Use a drinking straw to exhale into the limewater. A cloudy precipitate soon forms because exhaled breath contains about 4% carbon dioxide.

9.182 Tests for glucose and starch with "Testape"
1. Prepare two same-size pieces of dialysis tubing. Hold the end under water until it is soft. Tie a knot in the end and pull so that the knot is tight. Hold the other end under water until it is soft. To open the tubing, rub the fingers back and forth until it opens. Half fill beaker 1 with glucose solution. Half fill beaker 2 with starch solution. Half fill each piece of dialysis tubing with demineralized water and put one piece in beaker 1 and the other piece in beaker 2. Cover each beaker with a watch glass, and leave overnight. Pour one finger breadth of the starch solution into a test-tube. Add two drops of iodine solution. The solution becomes blue-black. Pour one finger breadth of the glucose into a test-tube. Tear off a small piece of "Testape", and dip it in the glucose solution. A green colour shows glucose. The next day, use "Testape" to test the glucose solution in beaker 1 and the liquid in the dialysis tubing. Both test positive. Add drops of iodine to the starch solution in beaker 2 and to the liquid in the tubing. Only the liquid in beaker 2 tested positive. The liquid in the dialysis tubing in beaker 1 tested negative. Glucose can pass through the wall of dialysis tubing but starch cannot.

16.4.1.1a Vitamin A
Vitamin A is a group of substances including retinol and retinal. When light strikes the retinal / opsin complex in the retina, a double bond in retinal is converted from (cis-) to (trans-) to send a signal to the optic nerve. Vitamin A is used to maintain epithelial tissue. Normal blood contains 15 to 60 mg retinol per 100 mL of serum. The vitamin A precursor is beta carotene.

16.4.1.2 Vitamin B1
See diagram 16.4.1.2: Thiamine
Vitamin B1, thiamine, is a water-soluble factor that is a cofactor for many enzymes, e.g. enzymes that release carbon dioxide from beta-keto acids. Thiamine occurs in the brown coating of unpolished rice and other cereal grains.

16.4.1.3 Vitamin C (ascorbic acid)
See diagram 16.4.1.3: L-Ascorbic acid (vitamin C)
Vitamin C, ascorbic acid, is the water-soluble antioxidant and used for formation of collagen, bone, teeth, and tendons and for amino acid metabolism. Lack of ascorbic acid results in scurvy that can be prevented by a Recommended Daily Allowance, RDA of 60 m for young adult males.

16.4.1.4 Vitamin D
Vitamin D is a group of compounds including pyridoxine, pyridoxal and pyridoxamine. The latter two are cofactors for some metabolic enzymes for catalysis, biosynthesis and degradation of amino acids.

16.4.1.5 Vitamin E
Vitamin E, alpha-tocopherol, an oil soluble anti-oxidant found in polyunsaturated oils in amounts necessary to protect the them against oxidation.
See 19.2.1.6: Antioxidant phenols, antioxidants, vitamin E, beta-carotene
See diagram 19.2.1.6: BHT, BHA, TBHQ, propyl gallate, Vitamin E

16.4.1.0 Tests for organic acids and alcohols
Organic acids contain the carboxyl group COOH. They have different properties, e.g. compound solubility tested by adding acid to water, litmus and other indicator reactions, conductivity tests, and reaction when heated. For example (+) tartaric acid decomposes when heated, but other organic acids sublime. Increase in molecular weight of organic acids results in decrease in solubility and solidification.
Test solutions of the alcohols and acids with litmus to show that alcohols do not ionize whereas organic acids do ionize to some extent in water solution.

16.4.1.1 Carboxylic acids, fatty acids and their salts
See: Formic acid, See: Acetic acid
Fatty acids are aliphatic monocarboxylic acids derived from or contained in esterified form in an animal or vegetable fat, oil or wax. Natural fatty acids usually have an unbranched chain of 4 to 28 carbons that may be saturated or unsaturated. All acyclic aliphatic carboxylic acids. may be called fatty acids. Carboxylic acids have the group -CO.OH, i.e. a carbonyl group attached to an hydroxyl group. Oxo acids have a carboxy group and an aldehyde or ketone group in the same molecule, e.g. HC(=O)CH₂CH₂CH₂C(=O)OH, 5-oxopentanoic acid. Carboxylic acids, one carboxyl group (R-COOH, RC(=O)OH) (-oic acid) fatty acids, e.g. methanoic acid (formic acid) (HCOOH) ethanoic acid (acetic acid) (CH₃COOH, CH₃C=OOH)

16.4.1.1.1 Dicarboxylic acids, two carboxyl groups (suffix: -dioic acid), e.g. ethanedioic acid (oxalic acid) [(COOH)₂], propanedioic acid (malonic acid) HOOCCH₂COOH, butanedioic acid (succinic acid) [(CH₂)₂(COOH)₂], butanoic acid (n-butyric acid) C₃H₇COOH, hexanedioic acid (adipic acid) [(CH₂)₄(COOH)₂]

16.4.1.1.2 Tricarboxylic acids, citric acid
Citric acid, C₆H₈O₇, 2-hydroxypropane-1,2,3-tricarboxylic acid (HOOCCH₂C(OH)(COOH)CH₂COOH) occurs in fruits, e.g. lemons, and prepared by fermentation using Aspergillus, used for drink flavouring.

16.4.2 Prepare ethanoic acid (acetic acid) ionization reaction
See 16.4.1.1: Carboxylic acids, fatty acids and their salts
Ethanoic acid (acetic acid, CH₃COOH) is a weak acid. Only a small proportion of it breaks into ions in aqueous solution, Ka = 1.76 X 10^-5.
Put sodium acetate-3-water in a Pyrex test-tube. Add 1 mL of concentrated sulfuric acid and heat gently. Test any vapour with moist litmus paper. Blue litmus turns red. Cautiously smell the vapours and note the characteristic odour of acetic acid.
Ionization reaction, Ka = 1.76 X 10^-5
CH₃COOH + H₂O <--> H₃O⁺ + CH₃COO^-However, although acetic acid is only partly dissociated in water, in a more basic solvent, e.g. liquid ammonia, it is completely dissociated.
CH₃COOH + NH₃ --> NH4⁺ + CH3COO^-

16.4.3 Prepare ethanedioic acid-2-water (oxalic acid) ionization reaction
See diagram 16.4.3 | See 16.4.1.1.1: Dicarboxylic acids
Ionization reaction
H₂C₂O₄ + H₂O <--> H₃O⁺ + HC₂O₄^-, Ka = 3.8 X 10^-2
HC₂O₄^- + H₂O <--> H₃O⁺ + C₂O₄^2-, Ka = 5.0 X 10^-5
Ethanedioic acid-2-water (oxalic acid) a colourless crystal (HOOC-COOH.2H₂O) exists in many plants, e.g. rhubarb that can be used as a laxative.
Be careful! This experiment must be done in a fume cupboard. The reaction will be violent, so use very small quantities of chemical.
Add concentrated nitric acid to sucrose in a beaker. The sucrose starts dissolving. Heat the mixture in a fume cupboard. All the sucrose gradually dissolves. Meanwhile, the nitric acid decomposes to turn the solution yellow, and produces much white smoke. Along with the temperature rise, the solution colour becomes deeper and deeper and a large amount of reddish brown gas is released. By controlling heating, evaporate the solution nearly to dryness, and volatilize the reddish brown gas as thoroughly as possible. Cool the beaker in water and snowflake like crystals of ethanedioic acid-2-water (oxalic acid) appear. Do not use an excessive quantity of nitric acid. Otherwise, the time for heating would be overlong and the nitric acid would not decompose completely, leading to a yellow product.
sucrose + concentrated nitric acid ---> dehydrated ethanedioic acid-2-water (oxalic acid).

16.4.4 EDTA, ethylene diamine tetra acetic acid, (HOOC.CH₂)₂N(CH₂)₂N(CH₂.COOH)₂See diagram 16.4.4: EDTA molecule
A chelate is a metal ion bound to two or more atoms of a chelating agent (sequestering agent), e.g. the simple chelating agent 1,2-diaminoethane (ethylenediamine), NH₂.CH_2.CH₂.NH₂forms bonds to a metal ion through its nitrogen atoms. Porphyrin chelates include haeme. in haemoglobin, bonded to iron (II) ion, and chlorophyll bonded to Mg (II) ion. Similarly vitamin B-12 has cobalt (II) ion bonded to a chelating agent.
The synthetic chelating agent EDTA can form complexes with calcium and magnesium ions. So it can form the calcium complex [Ca(EDTA)]^2-. The sodium salt used as an antidote for metal poisoning, an anticoagulant, enzyme deactivation, bactericide, industrial processes. The EDTA disodium salt is: (HOOC.CH₂)₂N(CH₂)₂N(CH_2.COO.Na)₂.2H₂O.
EDTA is used in the food industry to deactivate the enzymes containing metal ions that cause food spoilage, loss of colour and loss of flavour. Similarly EDTA can be used to dissolve the calcium carbonate scale caused by hard water and prevent stored blood from clotting by sequestering calcium ions. As a treatment for lead poisoning calcium disodium EDTA exchanges its chelated calcium for lead and the resulting lead chelate is excreted. If a ligand is defined as a small molecule that binds to a larger molecule, then chelates can be said to bring about the complexation of a ligand. The terms ligand, chelate, chelating agent and sequestering agent are used in slightly different ways in chemistry, medicine, and general industry.
If EDTA = H₄Y, then the disodium dihydrate form = Na₂H₂Y.2H₂O
H₂Y^2- + Ca²⁺ <--> CaY^2-+ 2H⁺Industrial synthesis of EDTANH_2.CH₂.CH_2.NH₂ + 4 H.CHO + 4 Na.CN + 4 H₂O → (Na.OOC.CH₂)₂N(CH₂₎₂N(CH₂.COO.Na)₂ + 4 NH₃1,2-diaminoethane (ethylenediamine) + methanal (formaldehyde) + sodium cyanide + water --> sodium salt + ammonia (Na.OOC.CH₂)₂N(CH₂₎₂N(CH_2.COO.Na)₂ + 4 HCl → (HOOC.CH₂)₂N(CH₂₎₂N(CH_2.COOH)₂ + 4 NaCl
sodium salt + hydrochloric acid --> EDTA + sodium chloride

16.4.4.1 Ion exchange resins
Let RZ = the resin, an organic polymer matrix. Charged groups are bound to the resin.
Cation exchange resin, H⁺ form, to remove cations, e.g. Ca²⁺, from solution
2RZ-SO₃^- H⁺ + Ca²⁺ <--> (RZSO₃^-)₂Ca²⁺ + 2H⁺
Anion exchange resin, OH- from, to remove anions, e.g. Cl^-, from solution
RZ-N(CH₃)₃⁺ OH^- + Cl^- -->RZ-N(CH₃)₃⁺ Cl^- + OH^-To "soften" water, usually only a cation exchange resin is used. If both a cation exchange resin and an anion exchange resin are used with tap water to remove ionic salts by ion-exchange, the resulting solution is deionized water, a cheaper alternative to distilled water.

16.4.5 Tests for fats, proportion of fats in foods
Heat 5 mL of the fat with 5 mL alcohol and two flakes of sodium hydroxide.
Continue heating until the layers merge. Pour into 200 mL water and stir. Repeat the experiment with chocolate and estimate what fraction of chocolate is fat.

16.4.6 Tests for gases from burning hydrocarbons
Hydrocarbons burn in excess air to form carbon dioxide and water. The reaction is exothermic. Methane burns with a clear flame. Ethene (ethylene) and ethyne (acetylene) burn with a smoky luminous flame. This flame can be seen above a chimney at many petrol refineries where excess ethene is burned off.
Light a natural gas burner or pour drops of cigarette lighter fuel in an evaporating basin and ignite it. Hold a dry test-tube containing ice over the burning gas. Water from the combustion condenses on the test-tube. Add limewater and shake. The milky precipitate shows the presence of carbon dioxide.
CH₄ (g) + 2O₂ (g) ---> CO₂ (g) + 2H₂O (g)
In insufficient oxygen, the poisonous gas carbon monoxide forms.
2CH₄ (g) + 3O₂ (g) ---> 2CO (g) + 4H₂O (g)
In excess air acetylene (ethyne) burns with a hot white flame. If the acetylene is mixed with oxygen in an oxy-acetylene torch, a temperature of 3 000^oC can be produced.
2C₂H₂ (g) + 5O₂ (g) ---> 4CO₂ (g) + 2H₂O (g)

16.4.7 Tests for saturated hydrocarbons, bromine water test
Shake the following gases with bromine water in a test-tube closed with a stopper:
1. Methane
No reaction
2. Ethene (ethylene)
The reddish brown colour of the bromine water disappears and 1,2-dibromethane (ethylene dibromide) forms. Remove the stopper and notice the characteristic odour of 1,2-dibromethane (ethylene dibromide).
H₂C=CH₂ + Br₂ ---> Br.CH₂.CH₂.Br (ethylene dibromide)
3. Ethyne (acetylene)
Decolorizes bromine water more slowly than ethene (ethylene).

16.4.8 Tests for saturated hydrocarbons, alkaline potassium manganate (VII) solution test
Dissolve 0.1 g anhydrous sodium carbonate in 1 mL of 1% potassium manganate (VII) solution. Add five drops to a test-tube containing the paraffin. Attach a stopper and shake.
1. Methane
No reaction
2. Ethene (ethylene)
Green then brown precipitate of manganese (IV) oxide (manganese dioxide) forms. The solution contains ethylene glycol (ethane-1,2-diol). Ethylene glycol is used in antifreeze mixtures in car radiators.
H₂C=CH₂ + H₂O + (O) ---> HO.CH₂.CH₂.OH
ethene (ethylene) + water ---> ethylene glycol
3.Ethyne (acetylene)
A green then brown precipitate of manganese (IV) oxide (manganese dioxide) forms. The solution contains ethylene glycol.
H₂C=CH₂ + H₂O + (O) ---> HO.CH₂.CH₂.OH

16.4.9 Tests for saturated hydrocarbons, acidified potassium manganate (VII) solution test
Add 1 mL of dilute sulfuric acid to 0.5 mL of 1% potassium manganate (VII) solution. Add five drops to a test-tube containing the paraffin. Attach a stopper and shake.
1. Methane
No reaction
2. Ethene (ethylene) and ethyne (acetylene)
A colourless solution of ethylene glycol forms.
H₂C=CH₂ + H₂O + (O) ---> HO.CH₂.CH₂.OH
ethene (ethylene) + water ---> ethylene glycol

16.6.1 Tests for proteins, heat test for proteins
See 16.3.6.0 Proteins, peptides, amino acids
Proteins decompose when heated to form carbon and a mixture of gases. One gas is usually ammonia. Proteins are slightly soluble in cold water, but are more soluble in hot water. When the hot solution is cooled, it may set like a jelly. Heat proteins until they char. Smell the gases that form

16.6.2 Tests for proteins, burning test for proteins
Burn feathers or hairs and note the gases that form. These proteins contain sulfur. Try the same test on samples of fats and carbohydrates to observe if the results are different enough to detect proteins.

16.6.4 Tests for albumin and gelatine
1. Heat albumin and gelatine in separate test-tubes. They decompose when heated, producing carbon and a mixture of gases one of which usually, but not always, is ammonia.
2. Add small quantities of albumin and gelatine to water. Shake. Warm the mixture and leave to cool. They are sparingly soluble in cold water but are more soluble in hot water. When the hot solution is cooled, it may set like a jelly.
3. Add to small quantities of albumin and gelatine to Biuret solution, potassium hydroxide solution and then a few drops of copper (II) sulfate solution. They produce a deep blue violet colour when treated with solution of copper (II) sulfate and an alkali. This is called the Biuret test for proteins.
4. Add drops of Millon's solution to albumin and gelatine and warm. They produce a white precipitate, a brick-red colour develops on warming the mixture.

16.6.5 Tests for proteins, biuret test
Biuret, NH₂CONHCONH_2,is formed from heated urea, crystallizes as NH₂CONHCONH₂.H₂O. Alkaline solution of biuret gives red-violet colour with copper (II) sulfate solution because of reaction with peptide bonds but no reaction if solution contains amino acids. The concentration of the colour is proportional to the amount of protein so the biuret test is approximately quantitative. So if the sample contains soluble protein, the reagent turns from light blue to purple but if the reagent remains light blue, the sample does not contain protein.
1. Add an equal volume of 40% sodium hydroxide solution to any protein solution, e.g. egg albumin, dried milk, gelatine. Add drops of dilute copper (II) sulfate solution with a light blue colour. The reaction produces a violet colour.
2. Add an equal volume of 1% potassium hydroxide solution + a few drops of 1% copper sulfate solution to the sample solution. If the solution turns purple, protein is present in the sample.

16.6.6 Tests for proteins, xanthoproteic test
Do protein tests on plant material by using expressed juice, aqueous extracts, pieces of tissue, or on a microscopic slide on which thin slices or sections of the tissue are placed. To a protein solution add one third of that volume of concentrated nitric acid. Heat gently to boiling with care. The precipitate changes from white to yellow. Cool the mixture under the tap and add drops of concentrated ammonia solution. The reaction produces an orange colour. Positive results come from proteins containing an aromatic group, e.g. phenylalanine, tyrosine, tryptophane.

16.6.7 Tests for proteins, Millon's test
Add drops of Millon's reagent and heat.
Be careful! Millon's Reagent contains mercury (I) nitrate in nitrous acid. This document does not recommend the use of mercury salts in school experiments. However, the amount of mercury (I) nitrate in drops of Millon's Reagent is very small.
Add drops of Millon's reagent to equal number of drops of a protein solution. Proteins form a white precipitate that turns pink when heated. A brick-red precipitate indicates the presence of the amino acid tyrosine.

16.6.8 Tests for proteins, Albustix test strips
Albustix strips are test papers dipped into buffered tetrabromophenol blue solution as an indicator solution. The indicator on the Albustix strip can combine with proteins. This change will in turn change the colour of the strip from yellow to shades of green. They are used by doctors to test the proteins present in a sample of human urine both quickly and semi-quantitatively. Protein in the urine may suggest kidney disease in which the glomerular membrane allows passage of serum albumin and some serum globulin from the plasma resulting in oedema. Thestest is most sensitive to albumin. Normal urtine contains 20 mg / 100 L . Dip an Albustix strip in a protein solution and observe the change in its colour.

16.6.10 Tests for proteins, Sakaguchi's arginine test
Make a 5 mL test solution alkaline with drops of sodium hydroxide solution. Add 5 drops of 2% alpha-naphthol in alcohol solution the one drop of sodium hypochlorite or bleaching powder solution. A carmine colour indicates the presence of arginine.

16.6.11 Tests for sulfur in proteins
Add drops of lead acetate solution to 5 mL of egg albumen test solution. Then add sodium hydroxide solution until the lead hydroxide precipitate forms then dissolves. Heat to boiling. A brown-black precipitate of lead sulfide indicates the presence of the amino acid cystine.

16.7.15 Commercially available test reagents
1. Tests for carbohydrates: dextrin, fructose, galactose (+)glucose, inositol, inulin, lactose, maltose, mannitol, ribose, soluble starch, sorbitol, sucrose. 2. Tests for proteins: casein, egg albumen, fibrin, gelatin, haemoglobin, peptone. 3. Tests for: L-arginine, L-asinine, L-aspargine, L-cystine, 2-aminopentanedioic acid (L-glutamic acid, glutamic acid, aspartic acid, L-aspartic acid, aminoethanoic acid (glycine, amino-acetic acid) L-histidine monohydrochloride, L-leucine, L-lysine hydrochloride (L-lysine monohydrochloride) DL-methionine, DL-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophane, L-tyrosine, L-valine. 4. Clinistix strips for (+) glucose in urine. 4. Albustix strips for protein (albumen) in urine. 5. Ketostix strips for ketones in urine

16.7.16 Artificial sweeteners
Non-sucrose sweeteners, e.g. "EQUAL", contain lactose and aspartic acid, so that 1 sachet 16 kJ = 2 level teaspoonfuls of "sugar", sucrose (140 kJ).

16.8.1 Reactions of benzene
See 16.3.4.0: Aromatics, aromatic compounds
1. Add 1 mL bromine water to 5 drops of benzene in a test-tube. The bromine water is not decolorized, unlike ethylene and acetylene with bromine water.
2. Put 5 drops of benzene in 2 test-tubes. In one of the test-tubes add iron filings. Add 3 drops of bromine water to each test-tube., Hydrogen bromide forms in both test-tubes but more in the test-tube containing the iron filings ha acts as a catalyst.
C₆H₆ + Br₂ --> C₆H₅Br + HBr
3. Add 5 drops of benzene to 1 mL of acidified potassium permanganate solution. The permanganate solution decolorizes only when the mixture is heated. The benzene is oxidized to lower molecular weigh molecules.

16.8.2 Prepare ferric tannate with tea leaves
Tannin is a mixture of organic chemicals related to polyhydroxy-benzoic acids. Tannin has a bitter taste and is astringent, i.e. it contracts the mouth. It is found in the bark and other tissues of many plants probably to control grazing. It is used to prepare black ink and leather from animal hides.
1. Add 200 g (2 tea bags) of dried tea to 250 mL of boiling water.
2. Add an unused pad of steel wool to 100 mL of vinegar, boil for 10 minutes, then strain through cotton wool in a filter funnel. Leave to cool then add 1 mL of hydrogen peroxide solution to produce a brown red, indicating iron (III).
3. Add equal volumes of solution 1. to solution 2. to produce a black solution of ferric tannate.
2H⁺ + Fe --> Fe²⁺ + H₂
2H⁺ + 2Fe²⁺ + H₂O₂ --> 2Fe³⁺ + 2H₂O
Fe³⁺ + tannic acid --> ferric tannate

16.8.3 Extraction of caffeine and benzoic acid from soft drinks, e.g. cola and lemonade
See 1.10.0A: Purine group of alkaloids, caffeine
1. Isolation of caffeine
Add 2 g of sodium carbonate to 50 mL of a cola (kola) drink in a 1 litre conical flask. Add 50 mL of dichloromethane (methylene chloride) and swirl gently for five minutes. Do not shake. Transfer into a separating funnel and leave to settle for 10 minutes). Drain the lower methylene chloride layer into a 250 mL conical flask. Add 50 mL more dichloromethane to the separating funnel and enclose with a stopper. Carefully invert the separating funnel 3 times to allow any remaining caffeine to be extracted into the dichloromethane layer. Again drain the lower methylene chloride layer into the 250 mL conical flask. Add 5 g of anhydrous magnesium sulfate to remove the water when it forms insoluble hydrated magnesium sulfate. Filter the now clear dichloromethane through cotton wool pad into a 250 mL beaker. Evaporate the dichloromethane on a water bath in a fume cupboard or distil it off to recover the solvent. Weigh the remaining precipitate. Test the precipitate by putting a small amount on a watch glass and mix with 3 drops of concentrated hydrochloric acid. Be careful! Add small crystals of potassium chlorate. Mix with a glass rod and evaporate to dryness on a water bath in a closed fume cupboard. Leave the watch glass to cool then moisten the residue with 2 drops 2 M ammonia solution. The residue turns purple
2. Isolation of benzoic acid
Pour half a drink-can of lemonade is poured into a 1 L conical flask and add 2 drops of dilute hydrochloric acid. Add 50 mL dichloromethane then swirled gently for five minutes. Pour into a separating funnel and leave to allowed to settle for 5 minutes. Drain the solvent layer into a 100 mL beaker and leave to evaporate in a fume cupboard. A residue of benzoic acid remains.

16.9.1 Burn carbohydrates, fats and proteins
See 19.3.4.3: Non-enzymatic browning, caramelization
See 19.3.4.4: Non-enzymatic browning, the Maillard reaction
1. Heated proteins produce ammonia like compounds with different odours. Burning carbohydrates have a smell of caramel. Burning fats produce acrolein that prepares the eyes water.
2. Heat separately, until beginning to burn, small samples of: 1. carbohydrate, e.g. starch or sugar 2. fat, e.g. butter 3. protein, e.g. meat. Note the difference in smells produced. Continue heating all samples until a residue of carbon remains.

16.9.2 Heat food with copper (II) oxide
Heat food with copper (II) oxide. Water condenses on the cooler parts of the tube. Test the gas in the test-tube with limewater by withdrawing some gas in a teat pipette and passing it through lime water. The gas is carbon dioxide. Copper (II) oxide releases oxygen to the food.

16.10.1 Breakdown starch to sugars, hydrolysis of starch, iodine test, Fehling's test
(C₁₂H₂₀O₁₀)_n + nH₂O + H⁺ --> nC₁₂H₂₂O₁₁ + nH₂O + H⁺ --> 2nC₆H₁₂O₆
starch.--> maltose.--> glucose
1. Put 10 mL of dilute starch solution into a test-tube. Add to this 1 mL of saliva and stir this into the starch solution. Record the time of adding the saliva. At 5 minute intervals remove three drops by means of a dropper and put them on a clean white tile taking care to keep them from running into other. The dropper must be washed between each test. Put some iodine solution on each drop. The decreasing intensity of the blue colour shows the decreasing amount of starch. To tests for increasing amounts of sugar, put three drops of the reaction mixture into a small test-tube. Add 3 mL of Fehling's solution and heat this mixture almost to boiling point. The test should show that there is more sugar after boiling.
2. Boil cut potato in water then let cool. Filter the solution to separate the soluble amylase from the insoluble amylopectin of the starch grains. Add tincture of iodine to the filtered starch solution An intense blue colour occurs. The solution contains beta-amylase, C₆H₁₀O₅that forms a complex with iodine: (beta-amylase)_p (I^-) (I₂)_r(H2O)_s [where r < p < s].