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2010

Emergency Phone Numbers/ Acil Telefonlar

110 Fire/tfaiye112 Ambulance/Salk Acil Servis118 Unknown Dials/Bilinmeyen numaralar153 Municipal Office/Zabta154 Traffic police/Alo Trafik155 Police/Polis156 Gendarme/Jandarma187 Natural gas / Doalgaz arzaAIBU District Security and Ambulance-AB ii Gvenlik ve Ambulans: 03742541933

Poison Information Center/Zehir Danma Merkezi

Cemal Grsel Street/ Caddesi No: 18 Shhiye/ANKARAPhone/Tel: 08003147900-4 lines -4hat(Toll Free-cretsiz)Phone/Tel: 03124337001(cretli-with toll)

Hospitals in Bolu

I.Baysal Faculty of Medicine/Tp Fakltesi 03742534656Bolu I.Baysal State Hospital/Devlet Hastanesi 03742154450Krolu State Hospital/Devlet Hastanesi 03742704575

Hospitals in the Environ Provinces/evre Hastaneler

Dzce Tp Fakltesi(Dzce Medical Faculty) 0380 542 13 90Ankara Atatrk State Hospital/Devlet Hastanesi Bilkent/Ankara 0312 291 25 25Ministry of Health/Ankara Hospital-Salk Bakanl Ankara Hastanesi ,Cebeci-Ankara, 0312363 33 30Hacettepe University Hospital-Hacettepe niversitesi 0312 310 35 45Bayndr Hospital(private)- zel Bayndr Hastanesi-Stz 0312 287 90 00Ankara Gven Hospital(Gven Hastanesi):Kavakldere, 0312 457 25 25

stanbul Marmara University Hospital(Marmara niversitesi Hastanesi), 0216 327 10 10Anadolu Salk Merkezi(Anatolian Health center-Associated with Johns Hopkins center inthe US)Gebze 0262 44 44 276Toyotasa Hastanesi-Toyotasa Hospital Adapazar 0264-2293160 / 61

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SELECTED GLASSWARES AND BASIC INSTRUMENTS IN THE LABORATORY

Erlenmayer flask Beaker Bunsen burner

Loop Incubator Autoclave

Vortex for test tube mixing UV-VISIBLE spectrophotometer

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Glass rod Pasteur pipettes

GENERAL RULES FOR SAFETY

The biochemistry laboratory is a place of adventure and discovery. Some ofthe most important events in scientific history have happened in laboratories. Theantibiotic powers of penicillin were discovered in a laboratory. The plastics usedtoday for clothing and other products were first made in a laboratory. The list isendless.

One of the first things any scientist learns is that working in the laboratorycan be an exciting experience. However, the laboratory can also be quite dangerousif proper safety rules are not followed at all times. In order to prepare yourself for asafe year in the laboratory, read over the following safety rules. Then read them asecond time. Make sure you understand each rule. If you do not, ask your teachingstaff to explain any rules you are unsure of. You may even want to suggest furtherrules in the section labeled "Other Safety Rules" mentioned in Section I.

A. Dress Code

1. Many materials in the laboratory can cause eye injury. To protect yourselffrom possible injury, always wear safety goggles or glasses whenever youare working with chemicals, burners, or any substance that might get intoyour eyes.

2. Laboratory aprons or coats should be worn everytime in the laboratoryand keep them buttoned.

3. Tie back long hair in order to keep it away from any chemicals, bunsenburners, and candles, or other laboratory equipment.

4. Any article of clothing or jewelery that can hang down and touch chemicalsand flames should be removed or tied back before working in the laboratory.Sleeves should be rolled up.

5. Sandals and open toe shoes will not protect the feet from any spill.

B. General Safety Rules

1. Read all directions for an experiment several times. Follow the directions

exactly as they are written. If you are in doubt about any part of theexperiment, ask your teaching staff for assistance.

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2. Never perform activities that are not authorized by your teaching staff. Alwaysobtain permission before "experimenting" on your own.

3. Never handle any equipment unless you have specific permission.4. Take extreme care not to spill any material in the laboratory. If spills occur,

ask your teaching staff immediately about the proper clean-up procedure. Never simply pour chemicals or other substances into the sink or trashcontainer.

5. Never eat or drink in the laboratory. Wash your hands before and after eachexperiment.

6. There should be no loud talking or horseplay in the laboratory.7. When performing a lab, make sure the work area has been cleared of purses,

books , jackets, etc.8. DO NOT SIT ON THE LABORATORY BENCH ZONE.9. Know the location and use of all safety equipment (goggles, aprons, eyewash,

fire blanket, fire extinguishers, etc.)10. Read your assignment before coming to class and be aware of all safety

precautions. Follow the directions.

C. Heating and Fire Safety

1. Never heat any chemical that you are not instructed to heat. A chemical thatis harmless when cool can be dangerous when heated.

2. Always maintain a clean work area and keep all materials away fromflames. Never leave a flame unattended.

3. Never reach across a flame.4. Make sure you know how to light a Bunsen burner. (Your teaching staff will

demonstrate the proper procedure for lighting a burner.) If the flame leapsout of a burner towards you, turn the gas off immediately. Do not touch the

burner. It may be hot. And never leave a lighted burner unattended!5. Always point a test tube that is being heated away from you and others.

Chemicals can splash or boil out of a heated test tube.6. Never heat a liquid in a closed container. The expanding gases produced

may blow the container apart, injuring you or others.

7. Never pick up any container that has been heated without first holding theback of your hand near it. If you can feel the heat on the back of your hand,the container may be too hot to handle. Always use a clamp or tongs whenhandling hot containers. Hot glassware looks the same as cool glassware.

D. Using Chemicals Safely

1. Never mix chemicals for the "fun of it." You might produce a dangerous, possibly explosive substance. No unauthorized experiments should beperformed.

2. Never touch, taste, or smell any chemical that you do not know for a fact isharmless. Many chemicals are poisonous. If you are instructed to note the

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fumes in an experiment, always gently wave your hand over the opening of acontainer and direct the fumes toward your nose. Do not inhale the fumesdirectly from the container.

3. Use only those chemicals needed in the activity. Keep all lids closed when achemical is not being used. Notify your teaching staff when chemicals arespilled.

4. Dispose of all chemicals as instructed by your teaching staff.5. Be extra careful when working with acids or bases. Pour such chemicals

over the sink, not over your work bench.6. When diluting an acid, always pour the acid into water. Never pour water

into the acid.7. Rinse any acids off your skin or clothing with water. Immediately notify

your teaching staff of any acid spill.8. Never use mouth-pipetting.

9. Be sure you use the correct chemical. Read the label twice.10. Do not return any excess back to the reagent bottle.11. Do not contaminate the chemical supply.12. Keep combustible materials away from open flames (alcohol, carbon

disulfide, and acetone are combustible).13. Do NOT use the same spatula to remove chemicals from two different

containers. Each container should have a different spatula.14. When you remove the stopper from a bottle, do NOT lay it down on the

desk, but place the stopper between your two fingers and hold the bottle sothe label is in the palm of your hand so drips won't ruin the label, etc. Boththe bottle and the stopper will be held in one hand. Be sure and rinse anydrips that might have gotten on the outside of the bottle.

15. Be careful not to interchange stoppers from two different containers.16. Replace all stoppers and caps on the bottle as soon as you finish using it.17. Mercury spills must be informed to the teaching staff in the laboratory and

cleaned up immediately. Use the new mercury sponge clean up kits put outby various companies.

E. Using Glassware Safely

1. Glass tubing should never be forced into a rubber stopper. A turning motionand lubricant will be helpful when inserting glass tubing into rubber stoppersor rubber tubing. Your teaching staff will demonstrate the proper way toinsert glass tubing.

2. When heating glassware, use a wire or ceramic screen to protect glasswarefrom the flame of a Bunsen burner.

3. If you are instructed to cut glass tubing, always fire polish the ends

immediately to remove sharp edges.

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4. Never use broken or chipped glassware. If glassware breaks, notify yourteacher and dispose of the glassware in the proper trash container.

5. Never eat or drink from laboratory glassware. Always thoroughly cleanglassware before putting it away.

F. Using Sharp Instruments

1. Handle scalpels or razor blades with extreme care. Never cut any materialtowards you: always cut away from you.

2. Notify your teaching staff immediately if you are cut in the laboratory.

3. Properly mount, dissecting specimens to the dissecting pan before making acut.

G. Electrical Equipment Rules

1. Batteries should never be intentionally shorted. Severe burns can be causedby the heat generated in a bare copper wire placed directly across the batteryterminals. If a mercury type dry cell is shorted, an explosion can result.

2. Never deliberately shock yourself or another person. Susceptibility toshock and possible resulting injury is unpredictable because of the many

physical and physiological variables.3. Turn off all power when setting up circuits or repairing electrical equipment.4. Never use such metal articles as metal rulers, metal pencils or pens, nor wear

rings, metal watchbands, bracelets, etc. when doing electrical work.5. When disconnecting a piece of electrical equipment, pull the plug and not

the wire.6. Use caution in handling electrical equipment which has been in use and has

been disconnected. The equipment may still be hot enough to produce aserious burn.

7. Never connect, disconnect, or operate a piece of electrical equipment with

wet hands or while standing on a wet floor.

H. End-of-Experiment Rules

1. When an experiment is completed, always clean up your work area andreturn all equipment to its proper place.

2. Wash your hands after every experiment.3. Make sure all candles and burners are turned off before leaving the

laboratory. Check that the gas line leading to the burner is off as well.

I. Other Safety Rules

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1. Do not use hair spray or hair mousse during or even before coming tolaboratory class. These are highly flammable and might cause automaticignition when in close proximity to a heat source.

2. Synthetic fingernails are also highly flammable and should not be worn inthe lab.

Experiment I:

How to prepare solutions in the laboratory: The Concentration, Molarity and

Normality Concept

The concentration refers to the relative amount of a substance in a given volume of

solution; but in some cases alternative expressions are used. Different ways of expressingconcentration are listed below, which are mostly used in biochemical calculations.

1. Percent solution: Per cent concentration refers to a number of parts in 100 parts ofsolution

Weight percent (% w/w) = weight of soluteweight of solution x 100

%5 (w/w) NaCl solution is the one containing 5 g NaCl in 100 g of solution.

Volume percent (% v/v) = volume of solutevolume of solution x 100

%5 (v/v) NaCl solution of ethanol in water is the one containing 5 mL NaCl in 100ml of solution.

Weight-volume percent (% w/v) = weight of solutevolume of solution x 100

%5 (w/v) NaCl solution is the one containing 5 gr NaCl in 100 mL of solution.

Example: How many grams of NaCl are necessary to prepare 500 mL of a 10 % (w/v)solution?

2. Concentration expression for very dilute solutions

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Miligram percent (%mg) is the number of miligram present in 100 mL of solution.

mg % = mg of solute100 mL solution

Parts per million (ppm) means the number of miligrams of solute per liter of solution.

ppm (liquids) = mg of solute = mg of solute = mg of solute103g 103mL 1 L solution

for example: 1 L of solution that contains 5 ppm CdCl2 contains 5 mg of CdCl2.

Example : How can you prepare 250 mL 100 ppm solution ?

Molarity

The term molarity is defined as the number of moles of solute per liter of solution.

M= n (number of moles)V

number of moles = mass in gramsmolecular weight

Dilute solutions are often expressed in termsof milimolarity and micromolarity.

1 mmole = 10-3 moles1 mole = 10-6 moles1 nmole = 10-9 moles1 pmole = 10-12 moles

Example : How many moles of NaCl are present in 150 mL of a 1.5 M solution? (M.W.NaCl : 58.5 )

How many grams of solid NaOH are required to prepare 500 mL of 0.04 M solution ?Express the concentrationof this solution in terms of % (w/v) (M.W. of NaOH : 40 )

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V1 = volume of initial solutionC1 = concentration of initial solutionV2 = volume of desired (final diluted solution)C2 = concentration of desired (final diluted solution)

Example :You have 5M NaOH. You need 100 mL of 1M solution. How much 5M would berequired ?

Can you prepare 50 mL of a 2 N solution from a 5 N stock solution?

Can you dilute 7 mL of a 5 % solution to a 3 % solution ?

LITERATURE CITED

1. Segel, I.H. Biochemical Calculations, Wiley Publishing Co. New York, 1976.

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EXPERIMENT II

SAPONIFICATION(SOAP MAKING)

Fatty acids are straight-chain monocarboxylic acids. The most common fatty acids

range in size from 10-20 carbons and most often have an even number of carbon atoms

including the carboxyl group carbon. The carbon-carbon bonds in saturated fatty acids are all

single bonds, while unsaturated fatty acids have one or more carbon-carbon double bonds in

their chains. One example of a saturated fatty acid is palmitic acid, CH3-(CH2)14-CO2H. Fatty

acids are seldom found as free molecules in nature but are most often a part of a larger

molecule called a triglyceride. Triglycerides consist of a three-membered carbon chain

(glycerol backbone) with a fatty acid bonded to each of the three carbon atoms in the glycerol

backbone.

Soap is produced by the saponification (hydrolysis) of a triglyceride (fat or oil)

(Figure 1). The triglyceride is reacted with a strong base such as sodium or potassium

hydroxide to produce glycerol and fatty acid salts. The salt of the fatty acid is called a soap.

The bond between the fatty acid and the glycerol backbone is referred to as an ester linkage.

In the saponification process the ester linkage is broken to form glycerol and soap.

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Figure 1. Saponification of a triglyceride.

Saponification value (or "saponification number", also referred to as "sap" in short)

represents the number of milligrams ofpotassium hydroxide orsodium hydroxide required to

saponify 1g of fat under the conditions specified. It is a measure of the average molecular

weight (or chain length) of all the fatty acids present. As most of the mass of a fat/triester is

in the 3 fatty acids, it allows for comparison of the average fatty acid chain length. If there

are more moles of potassium hydroxide (KOH) used for 1 gram of fat then there are more

moles of the fat. Therefore the chain lengths are smaller, due to the equation: The chemical

compound potassium hydroxide (KOH), sometimes known as caustic potash, potassa, potash

lye, and potassium hydrate, is a metallic base. Saponification of a lipid with potassium

hydroxide. Fats consist of a wide group of compounds that are generally soluble in organic

solvents and largely insoluble in water. The molecular mass of a substance (less accurately

called molecular weight and abbreviated as MW) is the mass of one molecule of that

substance, relative to the unified atomic mass unit u (equal to 1/12 the mass of one atom of

carbon-12). In chemistry, especially biochemistry, a fatty acid is a carboxylic acid often with

a long unbranched aliphatic tail (chain), which is either saturated or unsaturated.

Number of moles = mass of oil/relative atomic mass.

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http://www.nationmaster.com/encyclopedia/Potassium-hydroxidehttp://www.nationmaster.com/encyclopedia/Sodium-hydroxidehttp://www.nationmaster.com/encyclopedia/Saponificationhttp://www.nationmaster.com/encyclopedia/Fathttp://www.nationmaster.com/encyclopedia/Molecular-weighthttp://www.nationmaster.com/encyclopedia/Molecular-weighthttp://www.nationmaster.com/encyclopedia/Fatty-acidhttp://www.nationmaster.com/encyclopedia/Potassium-hydroxidehttp://www.nationmaster.com/encyclopedia/Sodium-hydroxidehttp://www.nationmaster.com/encyclopedia/Saponificationhttp://www.nationmaster.com/encyclopedia/Fathttp://www.nationmaster.com/encyclopedia/Molecular-weighthttp://www.nationmaster.com/encyclopedia/Molecular-weighthttp://www.nationmaster.com/encyclopedia/Fatty-acid

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The relative atomic mass would be smaller with smaller fatty acid chains, meaning more

moles. The higher the saponification value, the smaller the chain lengths.

MATERIALS AND THE METHOD

Caution: Oil will be hot, and may splatter or catch fire. Wear goggles at all times.

Preparation of the hot water bath

1. Assemble a hot water bath by filling an 800 mL beaker approximately 3/4 full with water

and begin heating the water with a bunsen burner.

2. Add three or four boiling chips to the water in the hot water bath to prevent the water from

boiling over.

Preparing the reaction mixture

3. In a 150 mL beaker add the following ingredients.

A. 15 mL of oil (or 10 g of solid shortening)

B. 20 mL of 20% NaOH

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C. 10 mL of ethanol (Methanol can be used but since it is extremely toxic, ethanol is

recommended.

D. 3 boiling chips (These will help prevent the mixture from boiling over while it is being

heating.)

4. Note the total volume (level) in the 150 mL beaker and how many layers the ingredients

initially form.

5. Begin heating the reaction mixture by clamping the beaker and contents in the hot water

bath. Heat the mixture for about 25 minutes after the water comes to a slow boil. The 150

mL beaker should be clamped so that the reaction mixture is below the level of the water in

the water bath. Maintain the water level in the water bath by adding water as needed.

6. Using a stirring rod, stir the reaction mixture frequently so that it does not boil over.

7. Maintain the total volume of the reaction mixture by adding small quantities of 1:1

(volume/volume) ethanol-deionized water.

8. After the initial 25 minute heating there should be no separation of layers in the beaker.

Testing the reaction mixture

9. Test the reaction mixture to determine if the saponification process is complete by carefully

placing a few drops of the reaction mixture in a 6-inch test tube. Add 10 mL of cold water. If

fat droplets form, add 5 mL of the 20% NaOH and 5 mL of ethanol to the beaker and continue

to heat for an additional 10 minutes, or until no fat droplets form upon testing.

Isolating the soap-CAUTION!!!!: Remember that the beaker and clamp are hot!

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10. When the saponification process is complete, turn the bunsen burner off, add 25 mL of

deionized water to the beaker and place the beaker on the bench top to cool for about 5-6

minutes. Then place the soap reaction beaker into an ice bath and cool for about 10 minutes.

11. At this point, measure out about 50 mL of saturated NaCl solution and cool it also for

about 5-6 minutes in the ice bath.

12. After the cooling time is complete for the soap reaction mixture, decant any liquid from

the beaker. (Be careful not to pour off the soap.)

13. Next add, add the 50 mL cold, saturated NaCl solution to the soap beaker and stir

thoroughly with a glass rod. This process separates the soap from the glycerol and excess

base and is called "salting out."

14. Collect the solid soap, using a Buchner funnel. (Note: Decant as much liquid off before

adding the solid soap to the Buchner funnel.)

15. While the air is being drawn over the soap in the funnel, wash the soap with two 20 mL

volumes of ice cold deionized water. Continue to draw air over the soap for another 3

minutes.

Soap disposal

16. Place the soap in the designated container.

17. Thoroughly rinse all of the glassware with water before storing.

LITERATURE CITED

1. Segel, I.H. Biochemical Calculations, Wiley Publishing Co. New York, 1976.

2. Perry and Bassow, Chemistry, Laboratory Manual, 1985.

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Experiment III

The Gelatinization and Retrogradation of Starch:

Objectives:

In this experiment, youll be able:

-To realize the process of sol and gel formation in starch-water dispersions.

-To predict how sugars, acids, lipids, and other ingredients affect the quality

characteristics of starch-containing products.

-To predict how processing, production, preparation, and storage of starch sols and gels

affect the quality characteristics of the final product.

To realize processing and modification of starches and relate this to dispersion

characteristics.

To be aware of the molecular and granule structure of various native starches to starch

paste and gel characteristics.

INTRODUCTION

Starch is found in almost every typical meal. For a truly useful nutrient it is critical

that there be some understanding of what starch is and how preparation processes will

influence it. Starch is a polysaccharide (meaning "many sugars") made up of glucose units

linked together to form long chains. The number of glucose molecules joined in a single

starch molecule varies from five hundred to several hundred thousand, depending on the type

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of starch. Starch is the storage form of energy for plants, just as glycogen is the storage form

of energy for animals. The plant directs the starch molecules to the amyloplasts, where they

are deposited to form granules. Thus, both in plants and in the extracted concentrate, starch

exists as granules varying in diameter from 2 to 130 microns. The size and shape of the

granule is characteristic of the plant from which it came and serves as a way of identifying the

source of a particular starch.

The structure of the granule of grain is crystalline with the starch molecules orienting

in such a way as to form radially oriented crystals. This crystalline arrangement is what gives

rise to the phenomenon of birefringence. When a beam of polarized light is directed through

a starch granule, the granule is divided by dark lines into four wedge-shaped sections. This

cross-hatching or cross is characteristic of spherocrystalline structures.

There are two types of starch molecules: amylose and amylopectin. Amylose averages

20 to 30 percent of the total amount of starch in most native starches. There are some

starches, such as waxy cornstarch, which contain only amylopectin. Others may only contain

amylose. Glucose residues united by a 1,4 linkage form the linear chain molecule of amylose.

Amylose is the linear fraction and amylopectin is the branched fraction.

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Amylose molecules contribute to gel formation. This is because the linear chains can

orient parallel to each other, moving close enough together to bond. Probably due to the ease

with which they can slip past each other in the cooked paste, they do not contribute

significantly to viscosity. The branched amylopectin molecules give viscosity to the cooked

paste. This is partially due to the role it serves in maintaining the swollen granule. Their side

chains and bulky shape keep them from orienting closely enough to bond together, so they do

not usually contribute to gel formation. Different plants have different relative amounts of

amylose and amylopectin. These different proportions of the two types of starch within the

starch grains of the plant give each starch its characteristic properties in cooking and gel

formation.

Starch in its processed, commercial form is composed of starch grains or granules with

most of the moisture removed. It is insoluble in water. When put in cold water, the grains

may absorb a small amount of the liquid. Up to 60 to 70C, the swelling is reversible, called

as the retrogradation of starch. The degree of reversibility is dependent upon the particular

starch. With higher temperatures in irreversible swelling called gelatinization begins.

Starch begins to gelatinize between 60 and 70 C, the exact temperature dependent is

the specific starch. For example, different starches exhibit different granular densities, which

affect the ease with which these granules can absorb water. Since loss of birefringence occurs

at the time of initial rapid gelatinization (swelling of the granule), loss of birefringence is a

good indicator of the initial gelatinization temperature of a given starch. The largest granules,

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which are usually less compact, begin to swell first. Once optimum gelatinization of the

grains has occurred, unnecessary agitation may fragment the swollen starch grains and cause

thinning of the paste.

The gelatinization range refers to the temperature range over which all the granules are

fully swollen. This range is different for different starches. However, one can often observe

this gelatinization because it is usually evidence by increased translucency and increased

viscosity. This is due to water being absorbed away from the liquid phase into the starch

granule.

Raw starch that has not had moisture added does not undergo gelatinization. By

definition, gelatinization is a phenomena which takes place in the presence of heat and

moisture. The dry raw starch, if heated, would undergo dextrinization. This certainly would

affect the starch paste viscosity and starch gel strength. The paste viscosity would be

decreased and gel strength decreased.

If a "limited amount" of moisture is added to the raw starch you may get partial

gelatinization. This condition exists in baked products. If a typical starch paste is allowed to

stand undisturbed, inter-molecular bonds begin to form, causing the formation of a semirigid

structure or gel. This gel is a structure of amylose, molecules bonded to one another and,

slightly, to the branches of amylopectin molecules within the swollen granule. This

phenomenon is sometimes called retrogradation. The conditions of gelation is critical to the

ultimate rigidity of the final product. Gelation of starch occurs due to its 3-D structure. With

heat removal, retrogradation of mixture to a paste-like mass of gel is formed.

PROCEDURE (Materials and method)

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Materials:

Starch, Beaker, Glassware petri plates, Glass baget, Spoon

a) Weigh 10 grams of starch, mix it with 100 mL distilled water and heat it until swelling at

100 C

b) Weigh 10 grams of starch, mix it with 100 mL distilled water and heat it until swelling at

50-60 C.

c) Take a piece of starch solution and put it onto the glassware petri plates

d) Do not forget to look at the starch crystals under microscope.

LITERATURE CITED

1.Muhrbeck, P. and Eliasson, AC. 1987. Influence of pH and ionic strength on the viscoelastic

properties of starch gels- a comparison of potato and cassava starches. Carbohydrate Polymers

7: 291-300

2. URL 1: http://food.oregonstate.edu/learn/starch.html(Cited 04.12.2008)

3. Johnson, J.M., E.A. Davis, and J. Gordon. 1990. Interactions of starch and sugar water

measured by electron spin resonance and differential scanning calorimetry. Cereal Chemistry

67(3): 286-291.

4. Bean, M.M. and W.T. Yamazaki. 1978. Wheat starch gelatinization in sugar solutions. I.

Sucrose: Microscopy and viscosity effects. Cereal Chemistry 55(6): 936-944.

Experiment IV

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Determination of Vitamin C by Iodine Titration

Vitamin C - Ascorbic Acid

Vitamin C (ascorbic acid) is an antioxidant that is essential for human nutrition.

Vitamin C deficiency can lead to a disease called scurvy, which is characterized by

abnormalities in the bones and teeth. Many fruits and vegetables contain vitamin C, but

cooking destroys the vitamin, so raw citrus fruits and their juices are the main source of

ascorbic acid for most people.

One way to determine the amount of vitamin C in food is to use a redox titration. The

redox reaction is better than an acid-base titration since there are additional acids in a juice,

but few of them interfere with the oxidation of ascorbic acid by iodine.

The chemistry works like this: Iodine reacts with vitamin C. When this happens, both

the iodine and vitamin C turn into different chemicals. If a sample contains vitamin C, iodine

added to the sample will react with it and no longer be iodine. At the same time, the vitamin C

that reacts with the iodine also is no longer vitamin C. When the amount of iodine added to

the sample is greater than the amount of vitamin C that was there, all the vitamin C will be

destroyed and there will be some iodine left over. So to find out how much vitamin C is in a

sample, you can add small amounts of iodine, until the iodine you add no longer disappears as

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soon as it is added. The starch plays another role. Starch and iodine can pair up to make a

dark blue material. In this combination, the iodine can still react with vitamin C. When that

happens, the starch-iodine pair does not exist any more (because the iodine is gone), so the

dark blue color disappears as well. So if you add the starch-iodine solution to the sample with

vitamin C, the blue color of the sample will disappear until you have added enough iodine to

react with all of the vitamin C. When that happens, the blue color will persist. It is much

easier to see the blue color of the starch-iodine solution than to see the faint yellow color of a

weak iodine solution. So the starch acts as an "indicator" for iodine. It makes it easy to see if

any iodine is in the sample.

Iodine is relatively insoluble, but this can be improved by complexing the iodine with

iodide to form triiodide:

I2 + I- I3-

Triiodide oxidizes vitamin C to form dehydroascorbic acid:

C6H8O6 + I3- + H2O --> C6H6O6 + 3I- + 2H+

As long as vitamin C is present in the solution, the triiodide is converted to the iodide

ion very quickly. However, when all the vitamin C is oxidized, iodine and triiodide will be

present, which react with starch to form a blue-black complex. The blue-black color is the

endpoint of the titration.

This titration procedure is appropriate for testing the amount of vitamin C in vitamin C

tablets, juices, and fresh, frozen, or packaged fruits and vegetables. The titration can be

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performed using just iodine solution and not iodate, but the iodate solution is more stable and

gives a more accurate result.

Purpose

The goal of this laboratory exercise is to determine the amount of vitamin C in

samples, such as fruit juice.

Procedure

Preparing Solutions

1% Starch Indicator Solution

1. Add 0.50 g soluble starch to one-liter near-boiling distilled water.

2. Mix well and allow to cool before use.

Iodine Solution (Lugol solution can also work for this)

1. Dissolve 5.00 g potassium iodide (KI) and 0.268 g potassium iodate (KIO3) in

200 mL of distilled water.

2. Add 30 mL of 3 M sulfuric acid.

3. Pour this solution into a 500 mL graduated cylinder and dilute it to a final

volume of 500 mL with distilled water.

4. Mix the solution.

5. Transfer the solution to a 600 mL beaker. Label the beaker as your iodine

solution.

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Vitamin C Standard Solution

1. Dissolve 0.250 g vitamin C (ascorbic acid) in 100 mL distilled water.

2. Dilute to 250 ml with distilled water in a volumetric flask. Label the flask as

your vitamin C standard solution.

Standardizing Solutions

1. Add 25.00 ml of vitamin C standard solution to a 125 mL Erlenmayer flask.

2. Add 10 drops of 1% starch solution.

3. Rinse your buret with a small volume of the iodine solution and then fill it.

Record the initial volume.

4. Titrate the solution until the endpoint is reached. This will be when you see the

first sign of blue color that persists after 20 seconds of swirling the solution.

5. Record the final volume of iodine solution. The volume that was required is

the starting volume minus the final volume.

6. Repeat the titration at least twice more for the sample. The results should

agree within 0.1 mL.

Experiment V

Benedict's Test for Reducing Sugars

The Benedict's test allows us to detect the presence of reducing sugars (sugars with a

free aldehyde or ketone group). All monosaccharides are reducing sugars; they all have a

free reactive carbonyl group. Some disaccharides like maltose have exposed carbonyl groups

and are also reducing sugars (less reactive than monosaccharides). Other disaccharides such

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as sucrose are non-reducing sugars and will not react with Benedict's solution. Starches are

also non-reducing sugars.

Note that the -1,4 linkage will prevent

the left-hand ring from opening up. Inorder to open up, the red C-O bond must

break, which requires that the hydrogenthat was on the green oxygen goes back tothe red oxygen, but it's not there anymore.

O C5

\ /C1 C4/ \ / \

C2 O C3

Let's use maltose as a specific example.The left-hand part of the molecule is stuckin the ring position. The right-hand ring,on the other hand, still can open because

the hydrogen on the #1 OH can and willmove back to the oxygen in the ring, thus,opening the ring and forming the double

bond of the aldehyde group.

So, one ring can continue to open and this disaccharide can continue to act as a reducing

sugar. However, one ring is stuck in the closed position because the hydrogen that originally

came from the #5 OH and moved to the #1 OH when the ring closed was lost in the

dehydration reaction and is not available to move back and open the ring. Note that only half

of the glucose rings in maltose can open and close and form the double bond that allows for

the reducing reaction. Consequently, maltose and other similar disaccharides will only reduce

half as quickly and half as much as an equal weight of or other similar monosaccharides.

That's something to keep in mind when you do your experiment for this laboratory.

SUCROSE:

When these two OH's react by an enzyme-catalyzed dehydration reaction, the resulting

product is sucrose.

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CuSO4 Cu+++ SO4--

2 Cu++ + Reducing Sugar Cu+

(electron donor)

Cu+ Cu2O (precipitate)

The final color of the solution depends on how much of this precipitate was formed,

and therefore the color gives an indication of how much reducing sugar was present.

Rating Scale: Increasing amounts of reducing sugar

blue green orange red reddish brown

(-) (+) (++) (+++) (++++)

To Test For Sugar:

1) Add 2 ml of unknown/control solution.

2) Add 1.5 ml of Benedicts solution (WEAR GLOVES + GOGGLES!!).

3) Mix gently. Make sure tube is labelled on the top as the label will soak in water!

4) Place test tube in a boiling water bath.

5) Record the color development in 3-5 min using the rating scale.

LITERATURE CITED

1.http://apbio.savithasastry.com/Units/Unit%201/labs/chp5_macromolecule_lab.doc(Cited:

06.01.2009)

Experiment VI

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Starch Test For Polysaccharides

Amylose in polysaccharides is responsible for the formation of a deep blue

color in the presence of iodine. The iodine molecule slips inside of the amylose coil.

To Test For Starch:

1) Add 1 ml of unknown/control in a test tube.

2) Add 1 drop of Iodine solution

3) Record color development against a white paper placed behind the test tube.

Brown (-) Blue-Black (++++)

LITERATURE CITED

1.http://apbio.savithasastry.com/Units/Unit%201/labs/chp5_macromolecule_lab.doc(Cited:

06.01.2009)

Experiment VII

Biurets Test for protein determination:

The Biuret Reagent is made of sodium hydroxide and copper sulfate. The blue reagent

turns violet in the presence of proteins, and changes to pink when combined with short-chain

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polypeptides. In this test for proteins there is a reaction between the copper ions and the

amino groups in the peptide bonds.

To test for proteins:

1) Add 2 ml of unknown/control solution to a test tube.

2) Add 4 drops of Biurets Reagent (WEAR GLOVES AND GOGGLES!!).

3) Mix gently.

4) Record color development against a white background.

Blue (-) Slight Purple/Pink (+) Violet (++++)

REFERENCES

1.http://apbio.savithasastry.com/Units/Unit%201/labs/chp5_macromolecule_lab.doc(Cited:

06.01.2009)

Experiment VIII

Brown paper test for fats:

Lipids make a translucent spot on a brown paper bag.

1) Rub the unknown/control on a brown paper bag mark the spot and label.

2) Dry thoroughly a) at room temperature for 30 min. b) at 120 C for 30 min.

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3) Hold against light.

4) Record appearance of translucent spot.

No spot (-) Translucent Spot (++++)

REFERENCES

1.http://apbio.savithasastry.com/Units/Unit%201/labs/chp5_macromolecule_lab.doc(Cited:

06.01.2009)

Experiment IX

DNA Spooling only performed with the fruit strawberry or kiwi!

The liquid detergent causes the cell membrane to break down and dissolves the lipids

and proteins of the cell by disrupting the bonds that hold the cell membrane together. The

detergent causes lipids and proteins to precipitate out of the solution. This is filtered out in

the cheese cloth. Next, NaCl enables nucleic acids to precipitate out of an alcohol solution

because it shields the negative phosphate end of DNA, causing the DNA strands to come

closer together and coalesce.

Procedure:

1. Add 2 grams of table salt (NaCl) and 10 mL of cleaning detergent (any brand) into a

100-mL measuring cylinder containing 90 mL distilled water. Swell to mix the

contents completely. This is the cell-lysis buffer.

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2. Cut your fruit into small slices (about 10 mm thick) and smush it in a zipped bag for 2

min. Add 2 mL of the cell lysis buffer and mush it again for 1 min. Some fruits may

need blending. [Warning: too much blending would shear the DNA molecules.]

3. Filter the slurry from the liquid through a cheese cloth (sitting in a filter funnel) into a

100-mL beaker. Each group should collect about 3 mL. [This filtering separates the

cell wall material and protein (remains in the cheese cloth) from DNA, which is now

in solution.]

4. Add equal volume (3 mL) of ice-cold ethanol slowly onto the surface of the fruit

extract carefully. [The ethanol must be ice cold - kept in the freezer overnight

beforehand.]

5. Immerse a bamboo skewer with a bent tip at the interface of the two layers (alcohol

and fruit layer), stir slowly and continuously in a small circle (clockwise direction) to

collect the DNA thread at the tip of the Pasteur pipette. [DNA doesn't dissolve in

ethanol - it comes out of the lower layer into the upper layer. DNA has the appearance

of white mucus.]

LITERATURE CITED

1.http://apbio.savithasastry.com/Units/Unit%201/labs/chp5_macromolecule_lab.doc

(Cited: 06.01.2009)

Experiment X

Thin-layer chromatography

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Thin layer chromatography, which is typically abbreviated as TLC, is a type of liquid

chromatography that can separate chemical compounds of differing structure based on the rate

at which they move through a support under defined conditions.

TLC is useful in detecting chemicals of security concern, including chemical weapons,

explosives, stabilizing chemicals for rocket propellants, and illicit drugs. For example, the

Forensic Service Center of Lawrence Livermore National Laboratory has designed a

computerized and portable TLC machine that can be taken to the field, and which has the

ability to analyze 20 samples at a time. Analysis can be completed within 30 minutes.

TLC as it is still practiced today was introduced by Justus Kirchner in 1951. From its

beginning, the technique was an inexpensive, reliable, fast, and easy to perform means of

distinguishing different compounds from each other. The method was qualitativeit showed

the presence of a compound but not how much of the compound was present. In the late

1960s, TLC was refined so that it could reliably measure the amounts of compounds. In other

words, the technique became quantitative. Further refinement reduced the thickness of the

support material and increased the amount of the separating material that could be packed into

the support. In High Performance TLC (HPTLC) the resolution of chemically similar

compounds is better than with conventional TLC, and less sample is required. HPTLC

requires specialized analysis equipment, and so is still not as popular or widespread as

conventional TLC.

In TLC a solution of the sample is added to a layer of support material (i.e., grains of

silica or alumina) that has been spread out and dried on a sheet of material such as glass. The

support is known as the plate. The sample is added as a spot at one end of the plate. The

plate is then put into a sealed chamber that contains a shallow pool of chemicals (the solvent),

which is just enough to wet the bottom of the plate. As the solvent moves up through the

plate support layer by capillary action, the sample is dragged along. The different chemical

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constituents of the sample do not move at the same speed, however, and will become

physically separated from one another. The positions of the various sample constituents and

their chemical identities are determined by physical methods (i.e., ultraviolet light) or by the

addition of other chemical sprays that react with the sample constituents.

i. Adsorbent:

The most common adsorbent used is Silica gel (silicic acid combined with a small

amount of gypsum as a binding agent). Many other material have also proven useful for

specific purposes; for example, alumina for the separation of steroids and water soluble

vitamins and Kieselgur for separation of sugars.

ii. Solvents:

Although it is frequently possible to select a good solvent system by the empirical

methods, some time and effort may be saved by consideration of some principles of the

chromatographic separation. Of particular importance in TLC are the elutropic series ofsolvents. This is a series of solvents arranged in the order of their eluting power; that is

having increasing ability to remove compounds from an adsorbent. A knowledge of the

relative adsorbent characteristics of the class of compounds to be separated is also important.

For example, saturated hydrocarbons are adsorbed poorly while unsaturated hydrocarbons are

adsorbed according to the increase in number of their double bonds.

iii. Development:

The apparatus used for development must have certain features:

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a)be capable of retaining any of a multitude of mixtures of original solvents.

b)be air tight, to insure maintenance of a solvent saturated atmosphere.

c) be relatively easy to handle.

iv. Visualization:

Compounds separated can be visualized by:

a)natural colour.

b)spray reagents; yields coloured derivatives on reaction with the separated spots.

c) iodine vapour.

d) fluorescence and UV absorption.

e) autoradiography.

EXPERIMENTAL PROTOCOL

Handle the plates carefully. NO FINGER PRINTS. These will be shown after

exposing to I2 vapour.

1. Using the template provided, mark the plates with a sharp pencil, as shown below(in the

figure).

2. Line the chamber with chromatography paper. Prepare 202 mL of solvent system (Hexane:

Ether: Acetic acid 60:40:1) in a 500 mL Erlenmayer flask. Mix and pour ~150 mL into the

chamber. Cover and let the chamber saturate while loading the plates.

3. With a 10 L capillary pipette, spot 1-2 L of chlorophyll standard onto the TLC plate, as

shown. Make sure the spot remains smaller than 4 mm in diameter. Move on to the other

standards. After the spots have dried, repeat loading each standards until you have loaded

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approx. 10 L each. Also, load 10 L of your lipid extract on one spot, and then the

remainder of the extract as a line (i.e., a series of spots).

4. Let dry the spots. Make sure that the loading area is above the solvent. Place the plates in

the chamber to develop.

5. Immediately close the cover and let run for approximately 30 min, until the solvent front

has reached the upper line.

6. Remove the plate and leave to dry in the rack in the fume hood. Discard the solvent in the

waste container provided, remove the chromatography paper and leave in the chamber. Leave

the chamber in the fume hood to dry.

7. Now place the plate in the iodine tank in the fume hood. You will see the lipids as yellow

spots after about 5 min or so.

8. Mark the edges of the spots with a pencil. Make a tracing on onion skin paper for a record.

9. Scrap off lipid fractions as shown, place in weighing paper, fold and roll to grind the

clumps.

10. Meanwhile prepare columns to elute the lipids from silica gel. To do this insert a small

amount of glass wool into a pasteur pipette, label the pipette and leave on the stand provided.

11. Carefully transfer the silica gel having different lipids into appropriate columns. Keep 7

mL glass vials beneath the columns.

12. Drip 1 mL of chloroform into each column except for the chlorophyll columns. To the

latter, add >1 mL of 100 % methanol.

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13. Shake the vials well. Place them in the fume hood evaporating set-up and evaporate off

the solvent under a stream of N2(Ask the instructor before using the N2 tank).

14. Store the dry lipids under N2.

15. Label and leave the separated lipid in the -20 C freezer until transmethylation (day 2).

Results

You should have obtained a good separation of the lipid classes, similar to the plate

shown below. However, differences in the solvent composition will lead to altered mobility

of the different components. A common problem occurs when the TLC tank is not

completely sealed, and the solvents can slowly evaporate. Since evaporation rate depends on

the boiling point, solvents like hexane will evaporate faster. As a result, the mobile phase

becomes more polar. Nevertheless, a positive identification of your spots should always be

possible since you ran standards on the same plate as well.

LITERATURE CITED:

URL 1: http://www.espionageinfo.com/Te-Uk/Thin-

Layer-Chromatography.html(Cited 06.01.2009)

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URL 2: http://www.sfu.ca/bisc/bisc-429/TLC.html(Cited 06.01.2009)

EXPERIMENT XI

THE ELECTROPHORESIS

Electrophoresis on polyacrylamide gels in the presence of detergent sodium dodecyl

sulfate[CH3 (CH2)10 CH2 OSO3 Na], abbreviated SDS is a rapid and often employed

technique for the determination of the molecular weights of polypeptide chains.

In addition, the electrophoresis is a common laboratory technique used for separating

DNA fragments. DNA samples are placed in a special gel and subjected to an electric field.

Because DNA is negatively-charged, it moves toward the positive electrode. The DNA

fragments that are shortest will travel farthest, while the longest fragments will remain closest

to the origin. Using the same basic principles, electrophoresis can also be used to separate

RNA and proteins.

First, follow our step-by-step instructions to build a gel electrophoresis chamber using

inexpensive materials from local hardware and electronics stores. Then follow the below

procedure to simulate DNA electrophoresis using food colors from your kitchen pantry.

Materials you will need:

Electrophoresis chamber, gel form and comb, Power supply that produces 50-150 volts.Agar

Agar granules, Salt solution, Commercial food colors, Filter paper circles (cut out with hole

punch), Tweezers, Masking tape, Water.

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MELTING THE AGAR

Add 1 gram agar to 100 milliliters of water in a glass container(a small canning jar).

Cover the container with plastic wrap. Pierce a small hole in the plastic for ventilation.

Heat the solution in the microwave oven on high power until it comes to a boil. Watch the

solution closely; agar foams up and boils over easily. Remove the container (protect your

hand with a pot holder or folded paper towel) and gently swirl it to re-suspend any settled

agar. Continue this process until the agar dissolves completely. Cool the agar until you can

comfortably touch the flask.

POURING THE GEL

Place tape across the ends of the gel form and place the comb in the form.

Pour cooled agar into the form. The agar should come at least half way up the comb teeth.

Immediately rinse and fill the agar flask with hot water to dissolve any remaining agar.

When the agar has solidified, carefully remove the comb. Remove the tape from the ends of

the gel form.

LOADING THE SAMPLES

Make a written record of which sample you will load in each well of the gel. You may

find it helpful to load samples in every other well. Place the gel form on a black or dark

surface to help you see the wells in the agar. Fold filter paper circle in half and hold sideways

using tweezers. Dip filter paper into full-strength food color to saturate. Gently ease the filter

paper into the well. Be careful not to puncture the bottoms of the wells as you load each

sample. Repeat for remaining colors.

Setting up the Gel Chamber

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Place the gel in the electrophoresis chamber. Make sure that the wells are closest to

the negative (black) electrode. To prepare the buffer, add a pinch of salt to one liter of tap

water, deionized water, or distilled water (the water source that works for you may depend on

your local water quality) and swirl to dissolve. Fill each half of the chamber, adding solution

until it is close to the top of the gel. Gently flood the gel from the end opposite the wells to

minimize sample diffusion. Place the lid on the chamber and connect the electrode leads to

the power supply. Connect the black lead to the negative terminal and the red lead to the

positive terminal.

Running and Analyzing the Gel

Turn on the power supply and adjust the voltage to 50-100 volts.

Run the gel for 5-10 minutes. Once the dyes have moved through the gel, turn off the power

supply, disconnect the electrode leads, and remove the chamber lid. Remove the gel from the

electrophoresis chamber and analyze your results. Did some colors move further than others?

Did some colors separate into two?

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REFERENCES:

1. Arn, E. (1985) Experimental Biochemistry Laboratory Manual, Middle East

Technical University, Department of Biological Sciences, Ankara, TURKEY.

2. URL 1: http://learn.genetics.utah.edu/content/labs/gel/electrophoresis/ (Cited:

06.01.2009)

Experiment XII:

The effect of glucose concentration on the growth of bakers yeast under aerobic

and anaerobic conditions

In this experiment you will be able to observe the effect of glucose concentration on

yeast growth. Different glucose concentrations (1% and 10%) were added to yeast extract

broth and the growth will be monitored under:

a) aerobic conditions

b) anaerobic conditions(in anaerobic jar)

At the end you are expected to compare the ethanol production and the pH.

Experiment XIII:

The effect of starch and aspartame concentration on the growth of bakers yeast under

aerobic and anaerobic conditions

In this experiment you will be able to observe the effect of starch and aspartame

concentration on yeast growth. Different glucose and fructose concentrations (1% and 10%)

were added to yeast extract broth and the growth will be monitored under:

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a)aerobic conditions b)anaerobic conditions(in anaerobic jar)

At the end you are expected to compare the ethanol production and the pH.

Experiment XIV

Spectrophotometry

Spectrophotometry is an instrument used for measuring the transmission or reflection

of light by comparing various wavelengths of the light. Also it is regarded as an analytic

technique by which chemical substances are identified by sorting gaseous ions by mass using

electric and magnetic fields. Different types of spectrophotometry is available like visible

(360-700 nm wavelength), UV-visible spectrophotometry, atomic absorption

spectrophotometry, mass spectrophotometry, Fourier transform infrared spectrophotometry

etc. For example, a mass spectrometer uses electrical means to detect the sorted ions, while a

mass spectrograph uses photographic or other nonelectrical means; either device is a mass

spectroscope. The process is widely used to measure masses and relative abundances of

different isotopes, to analyze products of a separation by liquid or gas chromatography, to test

vacuum integrity in high-vacuum equipment, and to measure the geological age of minerals.

In analytical chemistry, atomic absorption spectroscopy is a technique for

determining the concentration of a particular metal element in a sample. Atomic absorption

spectroscopy can be used to analyze the concentration of over 62 different metals in a

solution.

Mass spectrometry begins by ionizing the molecules in the target sampleremoving

one or more electrons to give them a positive charge. Molecules must be charged so they can

be accelerated. The principle is the same as that used in a television or fluorescent light bulb:

Charged particles are accelerated by being pulled toward something of the opposite charge.

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In the mass spectrometer, the speed the molecules attain during acceleration is proportional to

their mass (actually, their mass-charge ratio). By determining the speed of the molecules,

researchers can calculate their mass.

Turbid samples have large backgrounds which make obtaining accurate absorbance

readings very difficult. The more turbid the sample, the more incorrect the answer obtained

by an ordinary spectrophotometer will be. And, one cannot know that the results are incorrect

with the ordinary spectrophotometer without further quantitative measurements.

For many current applications, a spectrometer is increasingly becoming the

measurement device of choice. Unlike other methods which give "single point" measurements

for each calibration and unknown sample (i.e., pH, or single element Atomic Absorption), the

spectrum of a sample contains many data points. Every response value in a spectrum has

some relation to the properties or constituent(s) that make up the measured sample. Using a

spectrum of a sample that has many data points has some distinct advantages over single point

measurement techniques. One of the most important factors is that there are many more

measurements per sample (spectral data points) to use in generating the calibration equations.

As anyone who has performed quantitative analysis knows, the more measurements per

sample, the more accurate the results. The problem for the analyst is to discover what those

relationships are, and use a calibration model that reflects them accurately. One advantage of

using spectroscopy as a measurement technique is that the Beer-Lambert Law (also know as

Beer's Law) defines a simple linear relationship between the spectrum and the composition of

a sample. This law, which should be familiar to all spectroscopists, forms the basis of nearly

all other chemometric methods for spectroscopic data. Simply stated, the law claims that

when a sample is placed in the beam of a spectrometer, there is a direct and linear relationship

between the amount (concentration) of its constituent(s) and the amount of energy it absorbs.

In mathematical terms:

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Where is the samples Absorbance value at specific wavelength (or frequency) ,

, is the absorptivity coefficient of the material (constituent) at that wavelength, b or ( l ) is

the pathlength through the sample(in cuvette) and C is the concentration. The absorptivity

coefficient for every material is different, but for a given compound at a selected wavelength,

this value is a constant. The only problem that remains is to discover the "real" value of that

constant.

In this experiment you will be able to learn about the basics of an ordinary

spectrophotometer. The sample is the sour cherry juice; and you are responsible to meet with

the spectrophotometry by means of the original juice and its diluted form(50 mL sour cherry

juice will be diluted to 1:1; 1:5; 1:20 and 1:100) and it will be monitored through the

spectrophotometer by recording its absorbance.

LITERATURE CITED

1. http://www.olisweb.com/products/upgrades/dw2.php(Cited 05.08.2009)

2. http://www.yourdictionary.com/spectrophotometer (Cited 05.08.2009)

Experiment XV

Protein determination by Lowry method

The Lowry protein assay method for protein concentration determination is one of the

most venerable and widely-used protein assays. The Lowry method was first described in1951 by Lowry et al. (Lowry et al.,1951).

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Hydrolysis is probably the most accurate method of determining protein concentration

followed by amino acid analysis. Most other methods are sensitive to the amino acid

composition of the protein, and absolute concentrations cannot be obtained. The Lowry

procedure is sensitive, and is moderately constant from protein to protein. The Lowry protein

estimation has been so widely used that it is a completely acceptable alternative to a rigorous

absolute determination in almost all circumstances in which protein mixtures or crude extracts

are involved.

The method is based on both the Biuret reaction, in which the peptide bonds of

proteins react with copper under alkaline conditions to produce Cu+, which reacts with

the Folin reagent, and the FolinCiocalteau reaction, which is poorly understood but in

essence phosphomolybdotungstate is reduced to heteropolymolybdenum blue by the copper-

catalyzed oxidation of aromatic amino acids. The reactions result in a strong blue color,

which depends partly on the tyrosine and tryptophan content. The method is sensitive down

to about 0.01 mg of protein/mL, and is best used on solutions with concentrations in the range

0.011.0 mg/mL of protein.

Equipment and Reagents:

Spectrophotometer

Solution A

Solution B

Solution C

Bovine Serum Albumin(BSA)

Procedure

Modified Lowry Method

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Solution A:

Weigh out 2 g Na-K-tartrate and 100 g Na2CO3 and dissolve in 500 mL 1N NaOH and

complete with distilled water to 1 liter.

Solution B:

Weigh out 2 g Na-K-tartrate and 1 g CuSO4.5H2O, dissolve in 90 mL distilled water and add

10 mL 1N NaOH.

Solution C:

Mix Folin Ciocalteu reagent with distilled water(1:15)

Dilute the protein sample to 1 mL with distilled water and add 0.9 mL solution A.

Allow the test tubes in a water bath at 50 C for 10 minutes.

Cool to room temperature and add 0.1 mL solution B and leave at room temperature at least

10 min.

Add 3 mL solution C rapidly to ensure mixing within 1 second, heat the tubes again at 50 C

for 10 minutes and cool to room temperature.

Read the absorbance at 650 nm.

Prepare a standard curve using bovine serum albumin(BSA) as a stock solution.

LITERATURE CITED

URL 1: http://www.molecularstation.com/protein/lowry-protein-assay/ (Cited 07.01.2009)

Experiment XVI

Heat Transfer

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In this experiment you will be able to observe the heat conduction and heat

convection, which are the milestones of the thermodynamic principles.

Procedure

Heat up 250 mL of water at the cooking pan for a)1 min b) 3 min c) 5 min d) 10 min and

record the temperature 1) at the inner portion of the lid b) at the inner portion of the bottom.

Experiment XVII

The detection of proteins in the urine

Proteins are normally not present in the urine. The normal concentration of proteins

in urine is around 0-10 mg/dL. Sometimes proteins may be present in the urine due to

defects of the metabolism. The kidney does two important jobs in the body. It filters out the

waste products in the blood so it can be released in the form of urine. It also reabsorbs those

materials the body still needs which got past the original filtering system. However, there

are certain things in the blood which do not get past the filtering system of the kidney simply

because they are too big. The red and white blood cells are a good example. These cells are

entirely too large to pass through the tiny "holes" of the filter. This is good thing because we

would otherwise need a blood transfusion every time we went to the bathroom.

Proteins are large molecules which help make up our muscles, important parts of our

immune system, and many other portions of our bodies. Most proteins are also too large to

pass through the filtering system of the kidney. And since they are not supposed to pass into

the kidney, there is no mechanism for proteins to be reabsorbed if they make it in there.

Therefore, if protein is detected in the urine, it means there is something going on with the

filter (called the glomerulus) that is allowing the proteins to pass. Proteins give pink color

with the strong acids like nitric acid. The aim of this experiment is to detect the proteins in

the urine.

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Materials : fresh urine, test tube, pipette tips, strong acid, gloves.

Method:

1. Put 5 mL of fresh urine into a test tube.

2. Take 2 mL from the nitric acid by a pipette.

3. Dip the pipette containing nitric acid into the test tube containing the nitric acid.

Discard the pipette containing nitric acid into the tube.

4. Do not mix the tube.

5. Remove the pipette tip from the tube.

6. After a minute, a pink ring was formed just in the middle of the test tube.

7. This means that the urine contains protein.

Experiment 18

The dialysis of the proteins:

Introduction:

The protein sample, in solution, is sealed in the semi-permeable membrane bag,

manufactured with varying pore sizes which is then placed in a large volume of buffer,

normally 200 500 times the volume of the sample. Due to the gradient between the sample

and the buffer, the molecules in the sample attempt to flow from the dialysis bag where only

those smaller than the pore size actually leave the sample. The power of dialysis lies in its

ability to cleanse a sample of a number of contaminants at once, including small proteins, and

small contaminating molecules such as salt.

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The key to a successful separation based on dialysis is the selection of an optimal pore

size. Membrane pore sizes are typically measured in Daltons such that pores measuring 1,000

Daltons will retain molecules a molecular mass of 1,000 Daltons.

Various dialysis methods are commonly employed for the crystallization ofproteins.

Typical procedures include the use of dialysisbags and the dialysis buttons. The general

principle involved is that the protein solution is graduallybrought to a point ofsupersaturation

by imposing a gradient ofionic strength or organic solvent concentration across thewall ofthe

dialysis membrane. However, in some cases, the imposition of this gradient across the

dialysis membrane can result in the formation ofa large numberofcrystal nucleation sites,

thereby giving rise to a reduction in the maximum size ofthecrystals which can be obtained.

Materials:

1. Intestine.2. A piece of comfort string.3. The protein source, mentioned by your laboratory research assistant.

Method:

1. Put adequate amount of distilled water into a beaker according to the size of the

intestine that you have used.

2. Pour a definite(known) amount of milk into the intestine and tighten it closely by the

comfort string.

3. Watch the motion of the molecules outside the membrane of the intestine by turbidity.

4. Compare the untreated milk with the dialyzed milk according to its protein

concentration by Biuret method.

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LITERATURE CITED:

1.http://en.wikibooks.org/wiki/Proteomics/Protein_Sample_Preparation/Contaminant_Elimin

ation

Experiment 19:

The ammonium sulfate precipitation of the proteins:

Introduction:

Ammonium sulfate precipitation is a method ofprotein

purification by alteringsolubility

of protein. It is a specific case of a more general technique known as salting out.

Ammonium sulfate

is commonly used as its solubility is so high that salt solutions with high ionic strength are

allowed.

The solubility of proteins varies according to the ionic strength of the solution, and

hence according to the salt concentration. Two distinct effects are observed: at low salt

concentrations, the solubility of the protein increases with increasing salt concentration (i.e.

increasing ionic strength), an effect termed salting in. As the salt concentration (ionic

strength) is increased further, the solubility of the protein begins to decrease. At sufficiently

high ionic strength, the protein will be almost completely precipitated from the solution

(salting out).

Since proteins differ markedly in their solubilities at high ionic strength, salting-out is

a very useful procedure to assist in the purification of a given protein. The commonly used

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salt is ammonium sulfate, as it is very water soluble, has no adverse effects upon enzyme

activity. It is generally used as a saturated aqueous solution which is diluted to the required

concentration, expressed as a percentage concentration of the saturated solution (a 100%

solution).

In the preliminary test, the ammonium sulfate concentration is increased stepwise,

and the precipitated protein is recovered at each stage. This is usually done by adding solid

ammonium sulfate, but calculating how much ammonium sulfate to add to a solution at one

concentration to achieve a desired higher concentration is tricky, since addition of ammonium

sulfate significantly increases the volume of the solution. The amount to add can be

determined either from published nomograms or by using an online calculator. Each protein

precipitate is dissolved individually in fresh buffer and assayed for total protein content and

amount of desired protein. The aim is to find the ammonium sulfate concentration which will

precipitate the maximum proportion of undesired protein, whilst leaving most of the desired

protein still in solution.

The precipitated protein is then removed by centrifugation and then the ammonium

sulfate concentration is increased to a value that will precipitate most of the protein of interest

whilst leaving the maximum amount of protein contaminants still in solution. The

precipitated protein of interest is recovered by centrifugation and dissolved in fresh buffer for

the next stage of purification.

This technique is useful to quickly remove large amounts of contaminant proteins, as

a first step in many purification schemes. It is also often employed during the later stages of

purification to concentrate protein from dilute solution following the procedures such as gel

filtration.

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Material:

1. The protein source, mentioned by your laboratory research assistant.

Method:

1.Take aliquots of the extract, ideally 20-50 mL place in beakers and pre-chill to 4 C.

2.Calculate the amount of ammonium sulphate required to give 20, 30, 40, 50, 60, 70 and

80% saturation from the equation given in the ammonium sulfate precipitation table. Weigh

out the required amounts of ammonium sulphate and ensure all lumps are removed(use a

pestle and mortar).

3.Slowly add the ammonium sulphate to each aliquot whilst stirring(use a magnetic follower

and stirrer). Leave each aliquot stirring for one hour at 4 C.

4.Centrifuge each aliquot at 3000 g for 40 minutes.

5.Remove the supernatants and drain the pellets. Dissolve the pellets in buffer(e.g.

phosphate-buffered saline(PBS) or 50 mM Tris-HCl, pH=8.). Use the same volume for each

pellet, approximately twice the volume of the largest pellet.

6.If undissolved material remains, centrifuge at 3000 g for 15 minutes.

7. Assay the supernatants for total protein using by Biuret(Dialyse the supernatants if

ammonium sulfate interferes with the assay).

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8.Plot the concentration of total protein and the protein of interest against % saturation of

ammonium sulphate. For an ammonium sulphate cut choose the maximum % saturation

which does not precipitate the protein of interest, and the minimum % saturation that

precipitates all the protein of interest.

The process will be repeated in the coming week with the denatured proteins by pH or

temperature.

LITERATURE CITED:

1.http://www.absoluteastronomy.com/topics/Ammonium_sulfate_precipitation(cited:05.05.2009)

2. Harris, E.L.V. and Angal, S. Protein purification methods:A practical approach. IRL PressInc. Oxford, 1993.

EXPERIMENT 20

PROTEIN DENATURATION IN EGGS

Protein molecules carry out many important tasks in living systems. Most important, the

majority of proteins are quite specific about which task they perform. Protein structure is what

dictates this specificity, and the three-dimensional (tertiary) structure is particularly important.

When this specific three-dimensional structure is disrupted, the protein loses its functionality

and is said to have undergone denaturation.

The interactions, such as hydrogen bonding, that dictate the tertiary structure of proteins are

not as strong as covalent chemical bonds. Because these interactions are rather weak, they can

be disrupted with relatively modest stresses.

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If a solution containing a protein is heated, it will reach a temperature at which properties

such as viscosity or the absorption of ultraviolet (UV) light will change abruptly. This

temperature is called the melting temperature of the protein (because the measurement is

analogous to that made for the melting of a solid). The melting temperature varies fordifferent proteins, but temperatures above 41C (105.8F) will break the interactions in many

proteins and denature them. This temperature is not that much higher than normal body

temperature (37C or 98.6F), so this fact demonstrates how dangerous a high fever can be.

A familiar example of heat-caused denaturation are the changes observed in the albumin

protein of egg whites when they are cooked. When an egg is first cracked open, the "whites"

are translucent and runny (they flow like a liquid), but upon heating they harden and turn

white. The change in viscosity and color is an indication that the proteins have beendenatured.

Factors other than heat can also denature proteins. Changes in pH affect the chemistry of

amino acid residues and can lead to denaturation. Hydrogen bonding often involves these side

changes. Protonation of the amino acid residues (when an acidic proton H + attaches to a lone

pair of electrons on a nitrogen) changes whether or not they participate in hydrogen bonding,

so a change in the pH can denature a protein.

An egg white before the denaturation of the albumin protein causes the transucent substance

to change in color and viscosity.

The heat-caused denaturation in albumin protein in egg whites causes the once translucent,

runny substance into one that is white and firm.

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Changes in salt concentration may also denature proteins, but these effects depend on several

factors including the identity of the salt. Some salts, such as ammonium sulfate, tend to

stabilize protein structures and increase the melting temperature. Others, such as calcium

chloride, destabilize proteins and lower the melting temperature and are called chaotropic.Salts in this category can also be used in the laboratory to help purify proteins that are being

studied, by lowering their solubility and causing them to "salt out."

Experimental:

Materials

Eggs

Cooking pan

Tap waterLowry solution

Procedure:

Boil some tap water and put some eggs into the water very after boiling. Observe the eggs

with respect to an egg which is not boiled, served as control.

Calculate the protein content by using Lowry solution with respect to control.

LITERATURE CITED

http://www.chemistryexplained.com/Co-Di/Denaturation.html(Cited: 17.02.2011)

EXPERIMENT 21

Determination of water holding capacity

Water holding capacity was determined according to a method byGould and others (1989).An amount of 3 g of the flour sample (dried) was weighed into a centrifugal tube, added with30 mL distilled water and mixed using the vortex mixer (Vortex V1 Plus, Boeco, Germany)for 30 s. The sample was allowed to hydrate for 2 h at room temperature.

This was followed by centrifugation using a bench top centrifuge (Kubota 5100 Bench TopCentrifuge, Fujioka, Japan) at 2800 rpm for 10 min. The supernatant was discarded and the

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hydrated sample was weighed. Results were expressed as

LITERATURE CITED

Gould JM, Jasberg BK, Cote GL. 1989. Structure-function relationships of alkaline-peroxidetreated lignocellulose from wheat straw. Cereal Chem66:2137.

EXPERIMENT 22

Determination of oil holding capacity

Oil holding capacity was determined according to the method ofCaprez and others (1986). Asample of 3 g flour (dried) was weighed into a centrifugal tube, added with 30 mL of corn oil,and mixed using a vortex mixer (Vortex V1 Plus, Boeco, Hamburg, Germany) for 30 s. Thesample was allowed to stand for 1 h at room temperature. This was followed by centrifugationin a bench top centrifuge (Kubota 5100 Bench Top Centrifuge) at 2800 rpm for 10 min. The

supernatant was discarded and the pellet was weighed. Results were expressed as:

LITERATURE CITED

Caprez A, Arrigoni E, Amado R, Neukom H. 1986. Influence of different types of thermal

treatment on chemical composition and physical properties of wheat bran.J Cereal Sci 4:2339.

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