Biochemistry, Tests for food.

School Science Lessons
(UNBiol6)
2024-10-27

Biochemistry, Caffeine, Food tests
Please send comments to: j.elfick@uq.edu.au
Contents
9.1.0 Biochemistry
9.2.0 Caffeine
9.3.0 Tests for food, food tests

9.1.0 Biochemistry
9.1.1 Burn carbohydrates, fats and proteins
9.1.2 Carbonyls
9.1.3 Carboxylic acids, fatty acids and their salts
9.1.4 Dicarboxylic acids
9.1.5 Effect of temperature on the rate of reaction of trypsin
9.1.6 Ethanoic acid, acetic acid, CH3COOH
9.1.7 Vinegar, CH3COOH
9.1.8 Heat food with copper (II) oxide
9.1.9 Isolation of benzoic acid in lemonade
9.1.10 Prepare ethanoic acid
9.1.12 Prepare oxalic acid

9.2.0 Caffeine, experiments
9.2.1 Approximate caffeine content of beverages, mg /cup
9.2.2 Caffeine, extraction with supercritical carbon dioxide, critical point
9.2.3 Extraction of caffeine and benzoic acid from soft drinks
9.2.4 Isolation of caffeine in cola (kola), soft drink

9.3.0 Tests for food, food tests
9.3.1 Tests for acetic acid in vinegar
9.3.2 Tests for albumin and gelatine
9.3.3 Tests for aldehydes, Tollens' test
9.3.4 Tests for amylose and amylopectin
9.3.5 Tests for ash content of plant dry matter.
9.3.6 Tests for breakdown of starch to sugars
9.3.7 Tests for carbohydrates, Molisch's test
9.3.8 Tests for cellulose
9.3.9 Tests for diastase activity
9.3.10 Tests for glucose, Tollens' test
9.3.10a Tests for oxidation of glucose, blue bottle experiment
9.3.11 Tests for fats and oils
9.3.12 Tests for lignin
9.3.13 Tests for lipase
9.3.14 Tests for plant tissues water content
9.3.14a Tests for plant tissues oxidase and peroxidase
9.3.15 Tests for nitrogen content in food, soda lime test
9.3.16 Tests for organic acids and alcohols
9.4.0 Tests for proteins
9.5.0 Tests for starch
9.3.17 Tests for sugars, sugar test solution
9.3.18 Tests for sulfur in proteins
9.3.19 Tests for tannic acid in tea
9.3.20 Tests for unsaturation
9.3.21 Tests for vitamin C, DCPIP
9.3.22 Tests for wood
9.3.23 Tests for zymase and catalase in yeast.

9.4.0 Tests for proteins
9.4.1 Tests for proteins, Albustix test strips
9.4.2 Tests for proteins, biuret test
9.4.3 Tests for proteins, burning test
9.4.4 Tests for proteins, heat test
9.4.5 Tests for proteins, Millon's test
9.4.6 Tests for proteins, Sakaguchi's arginine test
9.4.7 Tests for proteins, xanthoproteic test

9.5.0 Tests for starch
9.3.6 Tests for breakdown of starch to sugars
9.5.2 Tests for glucose and starch, "Testape"
9.5.3 Tests for hydrolysis of starch, dilute hydrochloric acid
9.5.4 Tests for hydrolysis of starch, salivary amylase
9.5.5 Tests for starch in potato tuber cells
9.5.6 Tests for starch, iodine tests

9.3.20 Tests for unsaturation
9.3.33.1 Tests for unsaturated fats, acidified potassium manganate (VII) solution
9.3.33.2 Tests for unsaturated fats, bromine water test
9.3.33.3 Tests for unsaturated hydrocarbons, acidified potassium manganate (VII) solution
9.3.33.4 Tests for unsaturated hydrocarbons, alkaline potassium manganate (VII) solution
9.3.33.5 Tests for unsaturated hydrocarbons, Baeyer's test
9.3.33.6 Tests for unsaturated hydrocarbons, bromine water test
9.3.33.7 Tests for unsaturated hydrocarbons, ignition test.

9.1.1 Burn carbohydrates, fats and proteins
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.
Note the difference in smells produced.
Continue heating all samples until a residue of carbon remains.

9.1.2 Carbonyls
Carboxyl group: COOH
Carbonyl ( >C=O), organic compound functional group
Carbonyls, >C=O, are either aldehydes (RCHO, suffix: -al), or ketones (RR'C=O, suffix: -one)
Carbonyl groups are strongly polar so the boiling temperatures are higher than similar sizes of alkanes.
But they are not as high as similar sizes of alcohols, because of the hydrogen bonding between alcohol molecules.
As the length of the carbon chains increase in carbonyls the solubility in water decreases.
Shorter chain carbonyl compounds mix with water, because of the hydrogen bonding between the oxygen of the carbonyl compound and water.
Hydrogen bonds can form between propanone and water molecules.
Carbonyl compounds are unsaturated (C=O) so addition reactions may occur.
For example, the reaction between an aldehyde and hydrogen cyanide in the presence of potassium cyanide (not permitted in schools).
RCHO + HCN --> CN-RCH(OH)
The -CN group is called nitrile in organic chemistry. but is called cyanide in organic chemistry.
Aldehydes, but not ketones, can be oxidized easily to carboxylic acid with acidified potassium dichromate (K2Cr2O7 / H⁺) as oxidizing agent.
RCHO + [O] --> RC(OH)O
Reducing agents can reduce an aldehyde to a primary alcohol and a ketone to a secondary alcohol.
Test for carbonyls by mixing with an acid solution of Brady's reagent (2,4-dinitrophenylhydrozone) in methanol to form derivatives.
Then purify the derivatives by recrystallization, then consult tables of the melting points of the different derivatives.
Weak oxidizing agents can oxidize aldehydes, but not ketones.

9.1.3 Carboxylic acids, fatty acids and their salts
Fatty acids are open chain aliphatic monocarboxylic acids derived from, or 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 (fatty acids), R-(COOH)n, contain the carboxyl group -CO.OH, i.e -COOH, carbonyl group attached to an hydroxyl group.
They are weak acids, e.g ethanoic acid (acetic acid) CH3COOH, so the general formula is RCOOH.
An anion formed from carboxylic acid is called a carboxylate.
The carbonyl group (carboxy) is -COOH.
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.
For example, methanoic acid (formic acid) (HCOOH) and ethanoic acid (acetic acid) (CH3COOH, CH3C=OOH).

9.1.5 Effect of temperature on the rate of reaction of trypsin
Enzyme: 0.5% solution of trypsin
Substrate: powdered fat free lite milk skim milk suspension
(Change substrate concentration by diluting the suspension.)
The action of trypsin on the milk suspension results in a transparent solution.
1. Use a plastic pipette to add 10 drops of milk from a 20℃ water bath to a test tube.
2. Place the bottom of the test tube on the black cross printed on a white laminated card.
3. Using a clean plastic pipette, to add 2 drops of trypsin from a container in the 20℃ water bath to the test tube and start a stop watch.
4. Look directly down the test tube and note the time taken for the solution to become clear, i.e. when the black cross appears through the test tube.
5. Repeat the experiment using water baths set at 30℃, 40℃, 50℃, 60℃ steps and enter the results in a table of results.
Note at which temperature was the reaction the fastest / slowest.
6. Draw a graph of temperature against time.
Note: For most experiments, students are advise to not look directly down a test-tube, but look from the side!
See diagram 1.13: Smelling a gas

9.1.6 Ethanoic acid, acetic acid,CH3COOH
Ethanoic acid, CH3COOH, CH3CO2OH, acetic acid, main content of vinegar, food code E260, derived from anaerobic fermentation of carbohydrates
Pure acetic acid is called "glacial acetic acid", because it contains >1% water and forms ice-like crystals on cooling.
Common ion effect, sodium ethanoate and ethanoic acid: 17.5.9.1
Ethanoic acid, (acetic acid), ionization reaction
Ethanoic acid, Rubber chicken bone, Bouncy egg: 12.3.12
Heat of neutralization with a calorimeter: 14.1.5, (See: 5.)
Prepare ethyl acetate (ethyl ethanoate): 16.4.2
Prepare ethanoic acid: 9.1.7
Storing acids, acetic acid: 3.3.1
Tests for acetates: 12.11.1
Acetic acid, CH3CO2H, CH3COOH, ethanoic acid, weak acid, Ka small, colourless, mobile liquid (RD. glacial acetic acid, 17 M), 1.05 gm cm^-3, BP. 118^oC, solidifies at 16.7^oC, miscible with water, pungent odour, "glacial" ethanoic acid, (vinegar, eisel, is about 5% acetic acid, sour wine), E260 (anti-bacterial medicine, used to clean toilets, stain remover, dissolves grease, mild disinfectant).
Common name: white vinegar (5%)
Vinegar is a weak 4-5 % solution of acetic acid, C2H4O2.
Pure acetic acid is a colourless liquid that solidifies to ice-like crystals in cold weather, so it is called "glacial" acetic acid.
Acetic acid is a weak acid, but, like citric acid and tartaric acid, it forms carbon dioxide with chalk, baking soda, and forms hydrogen with iron filings and zinc.
Acetic acid is used in the manufacture of artificial silk, non-flam celluloid.
By treating copper with acetic acid in the presence of air a green-blue pigment forms called verdigris, i.e. impure copper acetate.
Acetic acid, CH3COOH, glacial acetic acid, analytical reagent, white vinegar, photographic stop bath, volatile organic compound, toxic if ingested, irritant vapour, corrosive colourless liquid, strong vinegar-like odour, flammable, > at 39^oC forms explosive vapour / air mixture.
Topical medicines, treat warts, make chemical compounds, antimicrobial agent, latex coagulant, food preservative, solvent in solid fuels for barbecues.
Glacial acetic acid, 17 M, 99%, 120 mL of concentrated solution for 1 litre of 2 M solution
Glacial acetic acid, 99.5%, packing 25/225 kg in plastic drum
Acetic acid, > 13 M
Acetic acid, glacial > 80%
Acetic acid 2-13 M, 10-89%
Acetic acid < 2 M, < 10% solution, vinegar, Not hazardous
Ethanoic acid-2-water, glacial acetic acid, fixative, pure acid is called "glacial"
Experiment
A weak acid can dissolve the calcium in a bone or egg shell
1. The rubber chicken bone experiment.
Select a chicken bone, e.g. a "drumstick (tibia and fibula) or a "wishbone", furcula, the forked bone.
Clean any tissue off the bone, wash it with warm salt water and dry it.
Try to bend the bone between your fingers.
Cover a chicken bone with vinegar or dilute acid hydrochloric acid.
Change the solutions each day for 2 - 5 days.
The vinegar reacts with the calcium in the bone to form soluble calcium acetate.
Dry the bone with absorbent paper.
Try to bend the bone between your fingers.
The bone can be bent and even tied into a knot, because all the calcium has been removed.
CaCO3 + 2CH3COOH --> Ca(CH3COO)2 + H2O + CO2
2. The bouncy egg experiment.
Cover a fresh egg with vinegar or dilute acid hydrochloric acid.
Change the solutions each day for 7 days.
Pick up the decalcified egg and drop it to show that it will bounce and not break.
3. Decolorize vinegar
Boil vinegar with decolorizing charcoal.
Ordinary charcoal is not very successful.
Neutralize vinegar with ammonia then half fill an evaporating basin with it and add a piece of litmus paper.
Add dilute ammonia to the evaporating basin then stir the solution until the litmus paper turns blue.
Transfer the liquid, now a solution of ammonium acetate, to a test-tube, add drops of hydrogen peroxide then heat the mixture until it boils.
The brown colour of the solution disappears, destroyed by the oxygen from the hydrogen peroxide.
Use the remaining liquid for the next experiment.

9.1.7 Vinegar, Acetic acid, CH3COOH
Acidity of vinegar and wine: 12.8.4.3
Experiments
Rubber chicken bone, Bouncy egg: 12.3.0.6
Prepare mayonnaise and salad dressing emulsions: 16.7.12
Prepare casein plastic from milk: 3.100
Prepare verdigris with copper and vinegar: 12.11.1
Prepare vinegar from wine: 19.1.5
Prepare vinegar with Acetobacter aceti: 4.2.6
Tests for acetic acid in vinegar: 9.3.2
Vinegar
"Kitchen Science", vinegar, baking soda, cornstarch, lemon (toy product)
Low cost, Distilled white vinegar (FCC grade), 5% acetic acid, approximately 0.83 M
Vinegar (acetic acid), Acetic acid, Low cost: distilled white vinegar
Acetic acid, Low cost: distilled white vinegar
Use vinegar + Condy's crystals to neutralize bad smells.
Use vinegar + kerosene for floor polish.
Use vinegar, added to shoe cleaner for quick drying tennis shoes.
Use vinegar, brown vinegar, solution to remove shine from seats of skirts or blue serge suits.
Use vinegar, brushed on inside of tight shoes, to make them fit.
Use vinegar to clean bathroom tiles, glass, spectacles, washing machine.
Use vinegar to remove "fur" from kettles, gravy stains on clothes, stains on marble, cooking smells.
Use vinegar to marinate meat, make soil more acid, treat upset stomach.
Vinegar bacteria medium: 9.2.22

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

9.1.9 Isolation of benzoic acid in lemonade
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 5 minutes.
Pour into a separating funnel and leave to allowed to settle for 5 minutes.
Drain the solvent layer into a 100 mL beaker, leave to evaporate in a fume cupboard, and a residue of benzoic acid remains.

9.1.10 Prepare ethanoic acid
Ethanoic acid (acetic acid, CH3COOH), is a weak acid.
Only a small proportion of it breaks into ions in aqueous solution, Ka = 1.76 × 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 × 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^-.

9.1.12 Prepare oxalic acid
See diagram 16.4.3: Melting point of fat or oil.
Ethanedioic acid-2-water, C2H2O4, oxalic acid, colourless crystals (HOOC-COOH.2H2O).
It occurs in many plants, e.g. rhubarb that can be used as a laxative.
Experiment
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.
With the temperature rise, solution colour becomes 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).

9.2.1 Approximate caffeine content of beverages
Brewed coffee 40-180 mg/cup, depends on length of time brewed
Percolated coffee cup 150 mL, Caffeine content 100 mg
Drip coffee cup 150 mL, Caffeine content 80 to 200 mg
Instant coffee cup 150 mL, Caffeine content 60 to 70 mg (30 to 120 mg)
Energy drink with caffeine, e.g. Red Bull, Caffeine content 80 mg / can
Cocoa cup 150 mL, Caffeine content 4-5 mg
Dark chocolate bar 100 g Caffeine content 20-25 mg
Milk chocolate bar, Caffeine content 3-6 mg
Kola, Cola drink-can 375 mL, Caffeine content 35 to 55 mg
Tea cup 150 mL, Caffeine content 50 mg (20-90 mg depends on length of time brewed)
Decaffeinated tea cup 150 mL, Caffeine content 2 mg
Coffee beans (green), Caffeine content 0.8 to 1.8% mg
Tea leaves (undried), Caffeine content 0.8 to 2.1% mg
In some countries, the legal limit for caffeine content is 55 mg / 375 mL (cola drink-can)
The fatal dose of caffeine is about 10 mg.
Caffeine is in analgesic preparations containing Ergotamine for the relief of migraine headaches.
Caffeine is found in coffee Coffea arabica, tea Camellia sinensis, yerba mate Ilex paraguariensis, guarana Paullinia cupana Cola Cola nitida.
Theobromine is found in chocolate Theobroba cacao.

9.2.2 Caffeine, extraction with supercritical carbon dioxide
Materials can exist in three states: solid, liquid and gas.
You can change from one to another by altering temperature and / or pressure.
If you increase temperature and pressure enough, the distinction between liquid and gas will disappear at the critical point.
No phase boundary between liquid and gas exists, because at extremely high temperatures and pressures, the liquid and gaseous phases become indistinguishable.
The critical point of water is 374^oC, 218 atmospheres of pressure.
At above 31.1^oC and 72.9 atmospheres of pressure carbon dioxide behaves like liquid and like a gas.
It spreads out like a gas to fill the available space and can dissolve substances as if it was a liquid.
The low temperature is convenient, because it can be used with substances that would be damaged by the high temperature.
Supercritical carbon dioxide is used to remove caffeine from tea and coffee, an extract substances from hops, essential oils, and environmental pollutants.
It is also used for dry cleaning.
Supercritical carbon dioxide is used to extract caffeine from coffee beans, extract nicotine from tobacco, extract oil from oilseeds, e.g. soy bean and sunflower, extract the natural insecticide pyrethrins from the perennial plant pyrethrum, (Chrysanthemum cinerariaefolium).

9.2.3 Extraction of caffeine and benzoic acid from soft drinks
Extraction of caffeine and benzoic acid from soft drinks, e.g. cola and lemonade
16.3.22 Purine derivatives, True alkaloids, (See: Caffeine)
See diagram 16.21.10: Purines (See: Caffeine).

9.2.4 Isolation of caffeine in cola
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 5 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.

9.3.1 Tests for acetic acid in vinegar
Cool the neutralized vinegar remaining from the previous experiment under the tap.
Add to it drops of ammonium iron (III) sulfate or iron (III) chloride (ferric chloride) in solution.
The liquid turns a bright red colour.

9.3.2 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 and shake the mixture..
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.

9.3.3 Tests for aldehydes, Tollens' test
The test for aldehydes uses Tollens' reagent, a solution of silver nitrate in ammonia solution.
Its is used for silver mirror tests.
Aldehydes with Tollens' reagent forms a metallic silver mirror as the aldehyde is oxidized and the silver is reduced
RCHO + [O] --> RCOOH
Ag⁺ + e- --> Ag.
1. Be careful because silver salts are expensive! Do not keep the Tollens' reagent after the test, because it can explode on standing.
Prepare the Tollens' reagent, just before doing the test.
After doing the test wash the unused Tollens' reagent down the sink with lots of water.
Do this test in a fume cupboard.
Prepare Tollens' reagent
Be careful! Tollens' reagent evaporated to dryness is explosive.
Add 1 drop of dilute sodium hydroxide solution to 1 mL silver nitrate solution.
When a brown precipitate of silver oxide form, add drops of dilute ammonia solution, NH3 (aq) ("ammonium hydroxide"), solution until the precipitate dissolves.
2. Clean a test-tube with water and acetone.
Add Tollens' reagent then 3 drops of acetaldehyde.
Warm the test-tube in a beaker of water and a silver mirror of silver deposits.
This reaction can be used to "silver plate" small objects or coins.
CH3CHO (aq) + Ag2O (s) --> CH3COOH (aq) + 2 Ag (s).
3. Put 5 mL of 5% (w/w) silver nitrate solution in a test-tube.
Add 5 drops of 0.4 M sodium hydroxide solution and shake gently.
Add 1 M ammonium hydroxide drop by drop, with gentle shaking, until the precipitate just dissolves.
4. Use Tollens' reagent with formaldehyde to reduce silver ions to the metal to form a silver mirror on the inside of a clean test-tube.

9.3.4 Tests for amylose and amylopectin
Different starches contain different proportions of amylose and amylopectin.
Amylose, a long chain polymer of glucose, gives a deep blue colour with iodine.
Amylopectin, a long chain many branched polymer of glucose, gives a red-brown colour with iodine.
Tincture of iodine antiseptic, formerly sold in pharmacies, is a solution of iodine in ethanol.
Experiments
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.
2. Iodine tests for starch on a solution of glucose.
Pure glucose sugar does not react with iodine solution.
3. 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, use tweezers to detach a leaf from the upper end of the shoot 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 and eExamine 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 that is used as a stain for identifying starch.
4. 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.
5. Iodine tests for starch on several types of leaves and plant storage organs
Use 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.
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 4 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.

9.3.5 Tests for ash content of plant dry matter
The term "ash content" on food packets refers to the percentage weight of the residues on heating, but no "ash" is added to the food.
Experiments
1. After tests for moisture, heat dry remainder to 575^oC to decompose remaining organic compounds, leaving ash residue.
It consists of oxides and salts containing anions, e.g. chlorides and other halides, phosphates, sulfates.
It also contains cations, e.g. calcium, iron, magnesium, and manganese, potassium, sodium.
2. 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.

9.3.6 Tests for breakdown of starch to sugars
Salivary amylase enzyme breaks down starch into the reducing sugars (+) glucose and maltose.
Reducing sugars do not react with iodine solution and starch does not react with Fehling's solution.
The sugars reduce copper (II) in Fehling's solution to brick-red copper (I) oxide.
Experiments
1. Prepare a clear solution of laundry starch by adding a mixture of 1g starch in 10 ml of water to 500 mL of boiling water.
Leave the solution to cool to room temperature.
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.
After 2 minutes, use a dropper to put 2 drops of the solution on a white tile.
At 5 minute intervals, remove 3 drops with a dropper and put them on a clean white tile, taking care to keep them from running into each other.
The dropper must be washed between each test.
2. To test for starch, add iodine solution and note the intensity of the blue black colour.
The decreasing intensity of the blue colour shows the decreasing amount of starch.
3. To test for increasing amounts of sugar, put 3 drops of the reaction mixture into a small test-tube.
Add Fehling's No. 1 and No. 2 solutions and heat this mixture almost to boiling point.
Note the intensity of the brick-red colour increasing with time.
Repeat the experiment every 2 minutes with clean droppers.
Note the decreasing intensity of the blue colour that shows that starch is being used up.
Keep doing the test until it shows that there is more sugar after boiling.

9.3.7 Tests for carbohydrates, Molisch's test
Molisch's test (α-naphtha test), Solubility in water
Molisch's test: α-naphthol in ethanol + unknown solution + concentrated acid --> violet ring
Naphthol may contain carcinogenic impurities.
It should be used only by senior students and not where food is being prepared.
Experiments
1. Molisch's test (α-naphthol test) (Hans Molisch. 1856-1937, Austria).
Add 0.1 mL 5% ethanolic α-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 liquids indicates carbohydrates.
2. Solubility in water
* 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.
* 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.
3. Boil the same fruits or vegetables or fruits in water for ten minutes.
Test the boiled fruits or vegetables with DCPIP solution, the test for vitamin C, ascorbic acid.
Also, test the cooking water they were boiled in.
4. Crush the 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.
5. Test lemon or orange drinks, lemon cordial, blackcurrant juice, pickles, cucumber, and sauerkraut for vitamin C.
6. Test whether ascorbic acid is destroyed in an acidic or a basic solution.
Cooks may 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.
Vegetables should be cooked quickly in as little water as possible to retain nutrients.
Vitamin, C is a water-soluble vitamin essential for the formation of collagen in connective tissue.
Sailors deprived of vitamin, C during long voyages developed scurvy, bleeding gums, lack of wound healing, anaemia, leading to death.

9.3.8 Tests for cellulose
1. Iodine test for cellulose
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.
Cellulose does not change the colour of Fehling's solution.
2. Fehling's test for cellulose- no colour change
3. 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:
Experiments
1. Solution of zinc oxide in concentrated hydrochloric acid
2. Ammoniacal copper carbonate dissolved in dilute ammonia solution
3. Schweizer's reagent, (not "Schweitzer"): Dissolve 0.3% solution of precipitated copper (II) hydroxide solution in a 20%, dilute ammonia solution.
It forms tetraammine copper dihydroxide, cuprammonium hydroxide [Cu(NH3)4](OH)2) complex ion, Cu(NH3)4²⁺.
The reagent forms a deep azure solution.
Mathias Eduard Schweizer, 1818-1860, Switzerland, discovered this method of dissolving cellulose in copper tetra-ammine.
Cellulose does not change the colour of Fehling's solution.

9.3.9 Tests for diastase activity
Diastase is a group of enzymes, α-amylase or β-amylase, or γ-amylase, that hydrolyses the breakdown of starch to maltose.
Diastase was originally extracted from the barley mash used to brew beer.
Diastase occurs in seeds and in the pancreas.
Taka-diastase from Aspergillus oryzae powder is sold to laboratories for research.
Experiments
1. Prepare active diastase
Buy taka-diastase from the chemist and prepare a 0.1% solution, or,
Germinate barley grains on damp filter paper until the shoots begin to emerge.
Crush the shoots with a mortar and pestle, add 50 mL of 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.
Then finally, it is replaced by 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.
3. Diastase is an amylase that acts on starch and breaks it into simple sugars.
Working concentrations suggested by the supplier: 5 mL starch (1%), 5 mL diastase (1%).
To monitor the progress of the reaction, place 2 drops iodine solution in each of 6 tile wells.
Immediately after you mix the starch and diastase, transfer 2 drops of the mixture to the first well.
Repeat this at 30 s intervals in succeeding wells.
To confirm the presence of reducing sugars, e.g. glucose, use Benedict's test.

9.3.10 Tests for glucose, Tollens' test
Dissolve 2.8 g of silver nitrate in 170 mL of deionized water to prepare an approximate 0.1 mol per litre solution.
Dissolve 3.7 h potassium hydroxide in 85 mL of deionized water to prepare an approximate 0.8 mol per litre solution.
Dissolve 0.75 g of glucose in 17 mL deionized water.
Add drops of 880 ammonia to the silver nitrate solution in a test-tube, until a brown precipitate forms.
Continue adding 5 mL of the 880 ammonia until the precipitate dissolves leaving a colourless solution of Tollens' reagent.
It contains the ion Ag(NH3)2⁺ (aq).
Add the glucose solution to the Tollens' reagent and shake the test-tube until the solution turns brown then forms a silver mirror inside the test-tube.
The aldehyde glucose reduces the Ag⁺ (aq) ions to silver metal.
Pour the contents of the test-tube down the sink and flush it down the sink with much water.
C6H12O6, i.e. as aldehyde:
CH2OH(CHOH)4CHO (aq) + 2Ag(NH3)2⁺ (aq) + 3OH^- (aq) --> 2Ag (s) + CH2OH(CHOH)4CO2^- (aq) + 4NH3 (aq) + 2H2O (l)
Show that this test reaction does not occur with propanone, a ketone.
Use Tollens' reagent to show the reduction of silver nitrate solution by formic acid to form a mirror inside the test-tube.

9.3.10a Tests for oxidation of glucose, blue bottle experiment
In a sodium hydroxide solution, the aldehyde glucose is oxidized by oxygen gas, to gluconic acid, then forms sodium gluconate.
Methylene blue acts as an oxygen transfer catalyst and is reduced to colourless leucomethylene blue.
Leucomethylene blue is then oxidized by oxygen in the air to methylene blue again.
Methylene blue, C16H18ClN3S, a thiazine dyestuff, is blue when oxidized and colourless when reduced.
Experiments
1. Solution 1: Add 2.5 g glucose (dextrose) + 2.5 g sodium hydroxide + 1 mL 0.1% solution methylene blue to 500 mL water.
Solution 2: Add 5 g glucose + 5 g NaOH + 1 mL 0.1% solution methylene blue to 500 mL water.
Note that the blue colour of Solution 2 disappears faster than in Solution 1.
The blue colour appears at the surface of the solutions, because of oxygen in the air.
Shake the flasks and the blue colour returns.
Solution 2: Dissolve 0.05 g of methylene blue in 50 mL 0.1% ethanol.
Solution 2: Dissolve 6 g of NaOH (or 8 g KOH), in 300 mL water in a conical flask at above 25^oC.
Stir to dissolve then the add 5 mL of Solution 1.
The blue solution turns colourless.
Close the flask and shake to dissolve air in the solution, or pour the solution from a height.
The colour changes to blue then fades back to colourless.
Repeat the shaking many times and note the colour changes.
Leave the solution for some hours and shake again.
The solution turns yellow and no colour change occurs after shaking.
Repeat the experiment. but instead of shaking the flask, pass nitrogen gas or natural gas through the solution.
No colour change occurs, because oxygen was not dissolved as in the shaking.
2. Repeat with other dyes:
2.1 Phenosafranine solution is red when oxidized and colourless when reduced.
Use 6 drops of 0.2% solution in water that becomes pink on shaking and colourless when standing, after some time.
2.2 Phenosafranine, 6 drops of 0.2% solution in water + 20 drops of 0.1% methylene blue in ethanol.
The solution becomes pink on shaking, then purple with more shaking, then blue.
On standing, the sequence of colours reverses.
2.3 Indigo carmine solution becomes brown-red on gentle shaking and pale green on more shaking.
On standing the sequence of colours reverses.
2.4 Resazurin (red to colourless) is dark blue when first added to the solution, then becomes pale blue, then becomes pink purple on shaking.
Resazurin has dichromatism (polychromatism), the hue of the colour depends both on the concentration of the absorbing substance and the thickness of the medium the light passes through.

9.3.11 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.
Experiments
1. Paper tests for fats
* 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.
* 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.
* Rub foods with absorbent brown paper to indicate the spread of 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.
Make a saturated solution of Sudan III in a 1:1 solution of 70% ethanol and acetone.
Dilute this solution by preparing a 6:4 solution with ethanol.
* 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.
* 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.
* Solvent for Sudan III
Put drops of Sudan III dye in a test-tube.
Add water and shake the test-tube.
The dye does not dissolve.
Add a little oil or melted butter and shake the test-tube.
The dye dissolves in fats and in oils.
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 they 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.
6. Tests for fats and oils, Wijs' solution
Prepare Wijs' solution, Toxic by all routes, avoid vapours from brown-red crystals
Dissolve 26 g of sublimated iodine in 200 mL of glacial acetic acid.
Heat the solution gently and then leave to cool.
Put 20 mL of potassium iodide solution and 100 mL of distilled water in a 300 mL Erlenmeyer flask.
Add 20 mL of the iodine solution.
To test fats and oils for the iodine value or iodine number, then to determine the degree of unsaturation in fatty acids.
Iodine values: coconut oil about 9, olive oil about 85, linseed oil about 200.6.
7. Tests for fats proportion in foods
Heat 5 mL of the fat with 5 mL alcohol and 2 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.

9.3.12 Tests for lignin
1. Use 1,3,5-trihydroxybenzene, 1% solution in ethanol, as wood stain, for a bright red colour.
2. Put sections of plant stem in phloroglucinol solution for 30 seconds.
Use a mounted needle to transfer the section to a drop of concentrated hydrochloric acid for two seconds.
Mount the section in glycerine and apply a coverslip.
Observe the lignin in xylem or woody tissue stained bright red.

9.3.13 Tests for lipase
Lipase is an esterase enzyme that hydrolyses fats (glycerides) to form glycerol and fatty acids.
It occurs in milk and milk products and is often included in detergent solutions.
Ingestion of large amounts is harmful.
Lipases are produced by the bacteria Bacillus megabacterium and Bacillussubtilis, and the fungus Rhizopus stolonifera.
Experiments
1. Test for activity of lipase in castor oil seeds
Shell 10 g of castor oil seeds and divide into 2 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 portions 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 that 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.
2. Test for activity of lipase in milk
Lipase can break down the fat in milk into fatty acids.
Make the milk alkaline by the adding a weak solution of sodium bicarbonate.
If the indicator phenolphthalein is present, the progress of the reaction can be observed.
Working concentrations suggested by the supplier:
2 mL milk (fresh or UHT, full cream, homogenized), 7 drops 0.5 M sodium carbonate, 5 drops phenolphthalein (1%), 1 mL lipase (5%).

9.3.14 Tests for plant tissues water content
1. Measure moisture content of plant tissues, by heating shredded weighed samples 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 2 hours and then weigh them again.
Calculate the percentage moisture content of the original material.
2. Test cabbage or other leaves, storage tissues, e.g. 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.
3. Measure the moisture content of plant tissues, 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 2 hours, and then weigh again.
Calculate the percentage moisture content of the original material.

9.3.14a Tests for plant tissues oxidase and peroxidase
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, but cabbage and turnip give the peroxidase reaction.
Tissues containing oxidase often turn brown when cut surfaces are exposed to the air.

9.3.15 Tests for nitrogen content 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.

9.3.16 Tests for organic acids and alcohols
Organic acids contain the carboxyl group COOH.
Different properties include: compound solubility tested by adding acid to water, litmus reactions, conductivity tests, and reactions 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.

9.3.17 Tests for sugars, sugar test solution
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 sugar test solution from 173 g sodium citrate, 200 g of crystalline sodium carbonate, and 17.3 g crystalline copper (II) sulfate.
Dissolve the carbonate and citrate in 100 mL of water.
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 mL of water 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.

9.3.18 Tests for sulfur in proteins
Add drops of lead acetate solution to 5 mL of egg albumen test solution.
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, C6H12N2O4S2

9.3.19 Test for tannic acid in tea.
When tea has been brewed for a long time it develops a bitter taste, because of tannic acid dissolved out of the tea leaves.
Test tea left standing for a time by pouring one or two drops into a test-tube.
Add 4 cm of water.
Add drops of ammonium iron (III) sulfate solution.
A black precipitate of iron tannate forms.

9.3.21 Tests for vitamin C, DCPIP
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.
Vitamin C test material: dichlorophenol / indophenol.
Experiments
1. DCPIP, solution, tablets (2,6-dichlorophenolindophenol), is a blue dye decolorized by ascorbic acid, but 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.
It is a toxic chemical and should not be used where food is being prepared.
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.
Cooks may 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.

9.3.22 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.

9.3.23 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 an active catalase enzyme, use a double test-tube with the inner tube filled with 2 vols hydrogen peroxide.
Add 1 g yeast and observe the rapid evolution of a gas.
Test the gas with a glowing splint to show it is oxygen.

9.3.33.1 Tests for unsaturated fats, acidified potassium manganate (VII) solution
Acidified potassium manganate (VII) solution test
The fat sample, e.g. coconut oil, palm oil, sunflower oil, should first be dissolved in a warm solvent, e.g. ethanol or Volasil (octamethylcyclotetrasiloxane), 5 drops added to 5 drops.
Add drops of 0.0005 M potassium manganate (VII) solution acidified with 1 M sulfuric acid.
Swirl the test-tube when adding the drops and note the number of drops used before the purple solution changes to colourless, then settles to a faint purple colour.
The number of drops of acidified potassium manganate (VII) solution needed to produce the faint purple colour tint is a crude estimation of the relative number of double bonds in the fat, i.e. the degree of unsaturation of the fat.

9.3.33.2 Tests for unsaturated fats, bromine water test
The fat sample, e.g. coconut oil, palm oil, sunflower oil, should first be dissolved in a solvent, e.g. ethanol or Volasil (octamethylcyclotetrasiloxane), 5 drops added to 5 drops.
Add drops of bromine water in a fume cupboard or under a fume hood or near an open window.
Swirl test-tube when adding drops and note number of drops used before yellow-orange bromine water changes to yellow tint.
The number of drops of bromine water needed to produce the yellow tint is a crude estimation of the relative number of double bonds in the fat, i.e. the degree of unsaturation of the fat.
However, some students cannot recognize this permanent yellow tint.

9.3.33.3 Tests for unsaturated hydrocarbons, acidified potassium manganate (VII) solution
Add 1 mL of dilute sulfuric acid to 0.5 mL
of 1% potassium manganate (VII) solution.
Add 5 drops to a test-tube containing the paraffin.
Attach a stopper and shake.
1. Methane
No reaction
2. Ethene (ethylene)
A colourless solution of ethylene glycol forms.
H2C=CH2 + H2O + [O] -->HO.CH2.CH2.OH
ethene (ethylene) + water -->ethane-1, 2-diol (ethylene glycol).

9.3.33.4 Tests for unsaturated hydrocarbons, alkaline potassium manganate (VII) solution test
Dissolve 0.1 g anhydrous sodium carbonate in 1 mL of 1% potassium manganate (VII) solution.
Add 5 drops to a test-tube containing the paraffin.
Attach a stopper and shake.
1. Methane
No reaction
2. Ethene (ethylene)
Dark purple manganate (VII) ion (MnO4^-), oxidizes carbon-carbon double bonds and is reduced to dark green manganate (VI) ion (MnO4^2-), then further reduced to a black-brown precipitate of manganese (IV) oxide (manganese dioxide).
The solution contains ethane-1, 2-diol (ethylene glycol).
Ethylene glycol is used in antifreeze mixtures in car radiators.
H2C=CH2 + H2O + [O] -->HO.CH2.CH2.OH
ethene (ethylene) + water -->ethane-1, 2-diol (ethylene glycol) [O] = oxygen from an oxidizing agent, in organic chemistry equations
3. Alkaline potassium manganate (VII) solution oxidizes any hydrocarbon side chain attached to a benzene ring, with long heating, to a single -COOH group, to the main product benzoic acid.

9.3.33.5 Tests for unsaturated hydrocarbons, Baeyer's test
Change in colour of the reagent (purple permanganate to brown manganese dioxide) redox reaction.
Add 0.1 g or 0.2 mL of the paraffin to 2 mL of water or ethanol, + 2% aqueous potassium permanganate solution, drop by drop while shaking until the purple colour of the permanganate persists.
The positive test is the gradual disappearance of the purple colour and the appearance of a brown suspension of MnO2, or the solution turns red-brown.
The test works for most aldehydes, formic acid, formic acid esters, phenols, mercaptans and thioethers.

9.3.33.6 Tests for unsaturated hydrocarbons, bromine water test
Alkene or alkyne (double bonds or triple bonds) + bromine water, yellow colour disappears.
However, aromatic compounds do not decolorize bromine water, because they are very stable compounds.
Experiments
Shake the following gases with bromine water in a test-tube closed with a stopper:
1. Methane, CH4
No reaction
2. Hexane, C6H14, does not decolorize bromine water
3. Ethene (ethylene)
The yellow orange colour of the bromine water disappears and colourless 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)
Another test unsaturated hydrocarbons uses for bromine in carbon tetrachloride + potassium permanganate to cause decolorization.
However, carbon tetrachloride is not allowed in a school science laboratory.

9.3.33.7 Tests for unsaturated hydrocarbons, ignition test
Ignite a substance in an evaporating basin and observe the smoke over the flame.
The darker or more sooty the smoke, the more unsaturated, e.g. aromatic compound.
If the air is clear over a luminous flame, the compound is saturated, e.g. n-hexane.

9.4.1 Tests for proteins, Albustix test strips
Albustix strips are test papers dipped into buffered tetrabromophenol blue solution (C19H10Br4O5S), 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 when the glomerular membrane allows passage of serum albumin and serum globulin from the blood plasma.
The test is most sensitive to albumin.
Normally it contains 20 mg / 100 L.
Dip an Albustix strip in a protein solution and observe the change in its colour.
One end of an albustix strip is impregnated with an inert base containing tetrabromophenol blue, buffered to pH3 with a citrate buffer.
Saturation of the strip with protein causes a colour change yellow to green to blue to show concentration of protein in the solution.

9.4.2 Tests for proteins, biuret test
A "biuret test" is a general term for any reaction for a test for proteins where sodium hydroxide solution is added then copper sulfate solution.
So a "biuret test" may not need to use biuret, NH2CONHCONH2, at all!
Prepare a protein solution by shaking the egg white in its own volume of water.
Also, shake ground pea seeds with water for several minutes, filter the mixture and keep the filtrate
Experiments
1. Add 1 mL of 2 M sodium hydroxide solution and 1 mL of copper sulfate solution to the sample solution.
If the solution turns purple, protein is present in the sample.
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.
3. 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.
4. Biuret test using biuret
Biuret, NH2CONHCONH2, is formed from heated urea, and crystallizes as NH2CONHCONH2.H2O.
An alkaline solution of biuret gives red-violet colour with copper (II) sulfate solution, because of reaction with peptide bonds.
However, but no reaction if the solution contains amino acids.
The concentration of the colour is proportional to the amount of protein (Beer-Lambert law), so the biuret test is almost quantitative.
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.
The biuret test detects peptide bonds between amino acids.
5. Prepare biuret solution with potassium hydroxide, copper (II) sulfate and potassium sodium tartrate (KNaC4H4O6.4H2O).
If the blue reagent turns violet it has detected proteins.
If it turns pink it has detected short chain polypeptides.
6. Dissolve 3 g of copper sulfate and 12 g of potassium tartrate in 1 litre of deionized water.
Slowly add 600 mL of 10% sodium hydroxide with constant stirring.
A purple colour indicates protein.
7. 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.
8. Biuret 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.
Add drops of 10% sodium hydroxide solution to protein solution then add drops of copper (II) sulfate solution.
Note the violet colour.

9.4.3 Tests for proteins, burning test
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.

9.4.4 Tests for proteins, heat test
Experiments
1. 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.
2. Heat a protein solution and note any changes.
The change is similar to the change in the egg white when it is boiled.

9.4.5 Tests for proteins, Millon's test
Millon's solution is made by dissolving mercury in concentrated nitric acid and diluting with water.
When heated with phenolic compounds it gives a red coloration.
The test is especially for tyrosine and proteins containing tyrosine.
Experiments
1. Prepare a protein solution by shaking the egg white in its own volume of water.
Also, shake ground pea seeds with water for several minutes, filter the mixture and keep the filtrate.
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.
2. 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.
3. 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.
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.
5. 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.
Add drops of Millon's reagent to protein solution then heat the solution.
The protein precipitates and turns pink when heated.

9.4.6 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% α-naphthol in alcohol solution the one drop of sodium hypochlorite or bleaching powder solution.
A carmine colour indicates the presence of arginine.

9.4.7 Tests for proteins, xanthoproteic test
Do not allow students to handle concentrated nitric acid.
In some school systems, this test is not recommended for use in schools.
Yellow xanthoprotein is formed by hot nitric acid with albumin or protein.
Add ammonia to change to a deep orange-yellow colour to identify proteins.
Prepare a protein solution by shaking the egg white in its own volume of water.
Also, shake ground pea seeds with water for several minutes, filter the mixture and keep the filtrate.
Experiments
1. Do protein tests on plant material by using expressed juice, aqueous extracts, pieces of tissue, or on thin slices on a microscope slide
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.
2. 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!
3. 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.
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.
Low levels of ammonia may harm some asthmatics and other sensitive individuals.
Students should not do this test.

9.5.2 Tests for glucose and starch, "Testape"
Prepare 2 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.
Cover each beaker with a watch glass, and leave overnight.
Pour one finger breadth of the starch solution into a test-tube.
Add 2 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 beaker1 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.

9.5.3 Tests for hydrolysis of starch, dilute hydrochloric acid
1. Tests for hydrolysis of starch, dilute hydrochloric acid
Do not allow students to handle concentrated hydrochloric acid.
Experiments
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.
Hydrolyse a portion of the starch solution by boiling with equal volume of dilute sulfuric acid for 10 minutes, and keep stirring.
Test the solution periodically by applying the iodine test on a drop on a tile.
Note the stages of colour changes.
Neutralize the solution and apply the Fehling's test.
4. 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 2 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.

9.5.4 Tests for hydrolysis of starch, salivary amylase
Tests for hydrolysis of starch, salivary amylase
Plants have α-amylase and β-amylase (diastase in brewing malt).
Animals have only α-amylase, pancreatic amylase, salivary amylase (ptyalin, alpha-amylase).
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.
Instead of using human saliva, use salivary amylase from a laboratory supply company.
Experiments
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 5 minute intervals, put 2 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 3 drops of the starch and saliva solution into a test-tube.
Add 3 mL of Fehling's solution and heat the solution until almost boiling.
Note the colour of the precipitate.
For each successive drop of starch and saliva solution test at 5 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 5 minute interval, the blue colour decreases, showing that starch is being converted to glucose sugar.
Wash the dropper between each test.
Tests for hydrolysis of starch
(C12H20O10)n + nH2O + H⁺ --> nC12H22O11 + nH2O + H⁺ --> 2nC6H12O6
starch --> maltose --> glucose
Experiments
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 3 drops by means of a dropper and put them on a clean white tile taking care to keep them from running into each 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.
2. To test for increasing amounts of sugar, put 3 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.
3. 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, C6H10O5 that forms a complex with iodine: (beta-amylase)p (I^-) (I2)r(H2O)s [where r < p < s].
Salivary amylase enzyme breaks down starch into the reducing sugars (+)glucose and maltose.
Reducing sugars do not react with iodine solution and starch does not react with Fehling's solution.
The sugars reduce copper (II) in Fehling's solution to brick-red copper (I) oxide.
4. Prepare a clear solution of laundry starch by adding a mixture of 1g starch in 10 ml of water to 500 mL of boiling water.
Leave the solution to cool to room temperature.
5. 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.
After 2 minutes use a dropper to put 2 drops of the solution on a white tile.
At 5 minute intervals, remove 3 drops with a dropper and put them on a clean white tile taking care to keep them from running into each other.
The dropper must be washed between each test.
6. To test for starch, add iodine solution and note the intensity of the blue black colour.
The decreasing intensity of the blue colour shows the decreasing amount of starch.
7. To test for increasing amounts of sugar, put 3 drops of the reaction mixture into a small test-tube.
Add Fehling's No. 1 and No. 2 solutions and heat this mixture almost to boiling point.
Note the intensity of the brick-red colour increasing with time.
Repeat the experiment every 2 minutes with clean droppers.
Note the decreasing intensity of the blue colour that shows that starch is being used up.
Keep doing the test until it shows that there is more sugar after boiling.

9.5.5 Tests for starch in potato tuber cells
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.

9.5.6 Tests for starch, iodine tests
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.
Different starches contain different proportions of amylose and amylopectin, both polymers of glucose.
Amylose gives the deep blue colour. but amylopectin gives a red-brown colour.
Tincture of iodine antiseptic is a solution of iodine in ethanol.
Store an iodine container inside a second container, because the lid of the iodine container may deteriorate.
When testing for the enzyme digestion of starch, when starch is present, iodine is dark blue.
If the blue colour lightens or disappears, this indicates starch is breaking down.
Factors such as temperature, pH and concentration can affect the rate of breakdown of starch.
However, how to quantify the degree of breakdown, remains a problem.
See diagram 2.26: Drawing stain across.
Be Careful! Heat alcohol with an electric heater or use a water bath.
Do NOT use a Bunsen burner!
Experiments
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.
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 β-amylase, C6H10O5, forms a complex with iodine: (β-amylase)p (I^-), (I2)r(H2O)s [where r < p< s].
3. Do the iodine test for starch on a solution of glucose.
Pure glucose sugar does not react with iodine solution.
Iodine is poisonous and should not be used where food is being prepared.
4. Sugar, the products of photosynthesis, and the large starch molecules formed from many sugar molecules are present in leaves.
For a simple starch test add a dilute iodine solution and note the typical blue-black colour that shows that starch is present.
Use tubers, potatoes or a starch paste to show the colour change.
5. 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 need a change of alcohol for removal of pigments.
The iodine solution should react with the starch within 15 minutes.
6. Purchase: Iodine, trace metals basis, I2.
Purchase: Potassium iodide, ACS reagent, 99.0%, KI.
Store iodine container inside a second container, because the lid of the iodine container may deteriorate.
Iodine is scarcely soluble in water, so iodine solution is iodine dissolved in potassium iodide solution.
Use safety glasses and nitrile gloves when weighing solid iodine, because it is harmful and corrosive.
Once in solution the amounts of iodine used are minute.
Specific tests for starch giving blue-black colour and general stain.
Iodine solution kills and fixes living material and makes cytoplasm and nucleus more visible.
Lignin walls in xylem stained brown, leaving cellulose walls of parenchyma relatively unstained.
Stains proteins brown, nuclei dark brown, chloroplasts brown, but black if starch present, cellulose cell walls yellow, lignified cell walls deep yellow to brown.