GUIDES IN BIOCHEMISTRY EXPERIMENTS SBK3013 / TBK4013

2011

DEPARTMENT OF BIOLOGY

DR SHAKINAZ DESA

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Laboratory safety will be explained during the first practical session.

THE REPORT CONTENT

Your report MAY consist of

1. Practical no. :

2. Experiment title - write a title that present your experiment, for example: The

determination of two protein sample using Biuret and Lowry assay

3. Abstract - summary of the experiment which should include objectives, brief

introduction, brief methods, analyzed results, conclusion.

4. Introduction – highlight on the main subject of interest

5. Materials and methods – do not copy from the manual. Write in your own

sentences. You may use diagrams, flow chart etc.

6. Results – analyzed the data and you may present the results in form of charts,

table, figure, and photo.

7. Discussion – answer the questions of what, why, when, where, how.

8. References – state author, book/journal, year, page number(s), and publisher.

All reports must be submitted in two weeks after the practical. Your instructor will

inform you the suitable due date.

Guide Title

1 Acid Base Experiment

2 Protein assay

3 Enzyme experiment

4 Experiment on vitamin C

5 Experiment using lipid

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GUIDE 1: ACID BASE EXPERIMENT

1. Acid Base Titration

Weak acid is different from strong acid because it cannot dissociate

completely in water. Weak acid such as acetic acid and organic acid can be

partially dissociated. Therefore there is only small amount of weak acid

molecules dissociated, while in strong acid, all acid molecules exist as H+

and A- in a solution. Due to this property, H

+ concentration in weak acid

depends on the coefficient of equilibrium.

We can measure the pH by emerging the tip of pH meter into the solution

and read the value in the recorder. The higher concentration of H+ , the

lower the pH value.

Objective

1. to observe the property of weak acid with pH changes

2. to learn how to use pH meter correctly

3. to learn how to prepare buffer system

4. to experience how to titrate acid-base

Materials

0.1 M acetic acid =4.76

0.1M phosphoric acid 2.15, 7.20, 12.35

0.1M amino glicine acid=2.53 (carboxylic) 9.78 (amino)

0.1M NaOH

Calibrated pH meter

Test Acid

25

Methods

1. Fill in 0.1M NaOH in a burette

2. Titrate 25 ml of the three acids separately with NaOH.

3. Measure the pH every time you add 1 ml of NaOH.

4. Record your findings.

Data analysis

1. Plot a graph of pH versus volume of base. Discuss the graph.

2. Determine the pKa for each acid from the graph

3. How do you determine the pKa?

4. Why must you choose the point of inflection at the graph?

5. Why is the graph different for each acid?

2. Application: Making pH indicator

This experiment explores the extraction of natural indicators from common

flowers, fruits, and vegetables and the pH at which these natural indicators

change color. Some indicator solutions and papers will indicate both an acid

and a base, while others are specific to just one. In this lab you will be

making a test paper that will indicate the presence of acid or base. It works

only when it is wet with dH2O. You can make your own scale by dipping the

paper into known pH solution. You can use the color scale to compare.

Materials

i. 0.1 M HCl solution (add 8.3 mL of concentrated HCl solution to

enough deionized or distilled water to make 1.0 L of solution)*

ii. 0.1 M NaOH solution (dissolve 4.0 g NaOH in enough deionized or

distilled water to make 1.0 L of solution)*

iii. 2-propanol (isopropyl alcohol) /acetone /deionized or distilled water

iv. Fruits/vegetables/flowers (dark colors are preferred).

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Caution should be used when working with solutions of hydrochloric acid

and sodium hydroxide. Both can be irritating to the skin. Goggles must be

worn throughout the experiment. If it is necessary to heat the extract in

order to concentrate the indicator, do not heat those containing the

flammable solvents, 2-propanol or acetone, over an open flame. Use a

water bath or hot plate.

Extracting the indicator

1. Select plant parts that are most pigmented.

For flowers, only a few petals are normally needed. For fruits and

vegetables, finely chopped pieces should be used. Use only that portion

of the fruit or vegetable that is most pigmented.

2. Add about 10 mL of solvent and macerate.

3. You may leave it overnight to enhance the extraction, if necessary.

Suggested solvents: water, 2-propanol, 50-50 water/2-propanol mixture,

or acetone. One of these should easily extract the color. If the extracts

are very dilute, the color can be concentrated by heating the opened

bag in a warm water bath or by pouring the extract in a beaker and

evaporating on a hot plate.

4. Filter and collect the filtrate from the macerated plant samples.

Testing the pH range of the indicator

1. Label 13 test tubes from 1 to 13.

2. Place 9.0 mL of distilled or deionized water in all test tubes except #1

and #13.

3. Prepare solutions in the acid range in the following manner:

a. Place 10.0 mL of 0.1 M HCl in test tube #1. (pH = 1)

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b. Transfer 1.0 mL of 0.1 M acid from test tube #1 to test tube #2

and mix thoroughly. (pH = 2)

c. Transfer 1.0 mL of acid solution from test tube # 2 to test tube

#3 and mix thoroughly. (pH = 3)

d. Continue making the serial dilutions by transferring 1.0 mL of

the most recently diluted acid solution to the next test tube

until six acid solutions of pH 1 to 6 have been prepared. Be

sure to mix each thoroughly before the transfer.

4. Add 10.0 mL distilled or deionized water to test tube #7. (pH = 7)

5. Prepare solutions of base in the following manner:

a. Place 10.0 mL of 0.1 M NaOH in test tube #13. (pH = 13)

b. Transfer 1.0 mL of 0.1 M NaOH from test tube #13 to test tube

#12 and mix thoroughly. (pH = 12)

c. Continue making serial dilutions of the base going from pH 12

down to pH 8 by transferring 1.0 mL of the most recently

diluted basic solution to the next test tube and mixing

thoroughly each time.

6. Label the wells of a spot plate from 1 to 13. Transfer a few drops of

each of the solutions prepared in steps 3, 4, and 5 to the

corresponding well in the spot plate.

7. Add a drop or two of the flower/fruit/vegetable extract indicator to

each well. Observe the pH at which the indicator changes color.

Testing the pH of other liquids

Once the pH ranges of the indicators have been determined, they can be

used in acid-base titrations or to test the pH of household chemicals.

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GUIDE 2: PROTEIN EXPERIMENT

1. Protein Assays

This exercise introduces students to methods of determining protein

concentrations: absorbance at 540 nm for the Biuret and 750 nm for the

Lowry assays.

Prepared solutions of gelatin at 1, 2, 3, 4, 5, and 6 mg/mL in water for

Biuret assay. Prepare 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/L for Lowry method.

Procedure:

a) Biuret assay:

Mix 0.50 mL of protein with 2.50 mL of Biuret reagent. Measure the

absorbance at 540 nm after 10 minutes.

Biuret reagent:

i. Add, with stirring, 300 mL of 10% (w/v) NaOH to 500 mL of a solution

containing 0.3% copper sulfate pentahydrate and 1.2% sodium

potassium tartarate, then dilute to one liter.

ii. The reagent is stable for a few months but not a year.

iii. Adding one gram of potassium iodide per liter and storing in the dark

makes it last indefinitely.

A540: Measure the absorbance of 1 mL at 540 nm. Plot the standard curve.

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b) Lowry assay:

Mix 0.25 mL of protein with 2.5 mL of Lowry reagent 1. After 10 minutes,

add 0.25 mL of Lowry reagent 2 and mix well immediately. After 30

minutes, measure the absorbance at 750 nm. Plot the standard curve.

Lowry reagent 1: Mix one volume of reagent B (0.5% copper sulfate

pentahydrate, 1% sodium or potassium tartrate) with 50 volumes of

reagent A (2% sodium carbonate, 0.4% NaOH). Both reagents A and B are

supposed to be stable for a long time but I have had a problem with

precipitation in reagent B that seems to remedied by adding a little NaOH.

Lowry reagent 2: Dilute commercial Folin-Ciocalteu phenol reagent with an

equal volume of water. Stable for a few days or weeks.

All series should include a zero protein (water) tube (reagent blank).

Test Sample:

Test sample can be sample with protein. Be creative in setting an

experiment.

Substitute the protein in both tests with a test sample.

Measure the absorbance and compare the concentration using both

methods.

Questions:

1. Under what conditions would this methodology not be appropriate?

2. Describe (in brief) three alternative methods of determining protein

concentration.

3. What is an "appropriate blank" and why?

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GUIDE 3: EXPERIMENT ON ENZYME

Enzyme Kinetics

In this laboratory we will examine the kinetics of α-amylase as found in

saliva. Enzyme reacts differently in different environment. The properties of

the environment may influence the rate of an enzyme to react. For

example, pH, temperature or substrate concentration. In this lab, we will

determine the effect of such to amylase.

1. Preparation of standard reference

Procedure:

1. Prepare starch solutions from the stock solution (1.0 mg/ml) into

dilutions of 0.01, 0.025, 0.05, 0.1, 0.3, 0.5, 0.7, and 1.0 mg/ml from

the starch stock solution.

2. Iodine solution is prepared by adding 5 g potassium iodide to 100 ml

water. The dissolved potassium iodide is added with 1 g of iodine and

is allowed to dissolve.

3. Prepare a standard curve of Absorbance (@ 590 nm) vs.

Concentration of a starch/iodine mixture. Use the following table as

guide.

Table 1 Preparation of starch/iodine mixture for standard curve determination

Test

tube

8 ml starch

of x mg/ml

Water

(ml)

Iodine

(ml)

Measure the

absorbance

at 590nm

1 0 9 1

2 0.01 1 1

3 0.025 1 1

4 0.05 1 1

5 0.1 1 1

6 0.3 1 1

7 0.5 1 1

8 0.7 1 1

9 1.0 1 1

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2. The effect of substrate concentration

Experiment of starch hydrolysis in different substrate concentration

experiment must be prepared as the following table.

Question: What will you use to prepare the blank/reference?

Table 2

Data analysis

1. Calculate starch concentration for each sample after hydrolysis (SF)

through use of the standard curve. The initial starch concentration (S0)

is already known. [S] = (So) – (SF)

2. The velocity (rate of digestion) of the reaction for each sample can be

calculated as:

V = ∆ S/ ∆ t = (S0 - SF) / 10 minutes

3. Prepare a table showing rate of hydrolysis (V) at different the starch

concentrations.

4. Plot a Michaelis-Menten graph.

5. Prepare a graph of 1/starch concentration (x-axis) versus 1/rate of

digestion (y-axis). This type of reciprocal graph displaying enzyme

kinetics is a Lineweaver-Burke plot.

6. State the value of Vmax and Michaelis constant Km from your graph.

Test

tube

8 ml starch

of x mg/ml

Water

(ml)

Amylase

(ml)

Iodine

(ml)

1 0 8 1 1

2 0.01 0 1 1 Place all test

3 0.025 0 1 Incubate 1 tubes in an

4 0.05 0 1 each 1 ice bath.

5 0.1 0 1 sample 1 Measure the

6 0.3 0 1 at 35°C for 1 absorbance at

7 0.5 0 1 10 minutes 1 590nm

8 0.7 0 1 1

9 1.0 0 1 1

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The y-intercept of the Line-weaver Burke plot is the reciprocal of the

maximum velocity of the reaction (Vmax). The x-intercept is the negative

reciprocal of the Michaelis constant. (Km).

3. The effect of temperature

Prepare as the following for the experiment of different temperature.

Table 3

Data analysis

Plot the Lineweaver-Burke line for the result of 20, 28, 35 and 40°C.

Compare all three plots.

What are the values of Vmax and Km for all plots?

Test

tube

8 ml starch

of x mg/ml

Water

(ml)

Amylase

(ml)

Iodine

(ml)

1 0 8 1 1

2 0.01 0 1 Incubate 1 Place all test

3 0.025 0 1 each 1 tubes in an ice

4 0.05 0 1 sample 1 bath.

5 0.1 0 1 at 20, 28, 1 Measure the

6 0.3 0 1 35,40°C for 1 absorbance at

7 0.5 0 1 10 minutes 1 590nm

8 0.7 0 1 1

9 1.0 0 1 1

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4. The effect of pH

Prepare the following for the experiment using different pH.

(CAUTION: DO NOT ADD THE ENZYME BEFORE ADDING BUFFER pH)

Table 4

Data analysis

What are the values of V for all pH?

Compare the velocity for each of the pH test

Test

tube

Starch

0.5 mg/ml

2 ml

buffer of

pH x

Amylase

(ml)

Iodine

(ml)

1 5ml 4 1ml Incubate 1 Place all test tubes

2 5 5 1 each sample 1 in an ice bath.

3 5 6 1 at 35°C 1 Measure the

4 5 7 1 for 10 1 Absorbance at

5 5 8 1 minutes 1 590nm

6 5 9 1 1

7 5 10 1 1

blank 5 3 ml of dH2O 1

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GUIDE 4: EXPERIMENT ON VITAMIN C

1. Measuring Vitamin C using starch-iodine test

We will measure the amount of vitamin C in many different types of foods.

The chemical reaction we will use to measure the amount of vitamin C uses

one of its functions in the body. Vitamin C involves in our cells oxidation-

reduction reactions. Vitamin C can react with iodine. Therefore we will

measure the amount of vitamin C by adding iodine to our food extracts

until the vitamin C can bind no more iodine.

Iodine in excess of the vitamin C will react with a starch solution you

will add to the extract to produce a bluish-black color. The addition of a

chemical to measure another chemical is called a titration.

Materials:

1. Food sources of vitamin C: for example juices, extraction of plants,

flowers, fruits, grains, and vegetables, vitamin C tablet or

cooked/treated food sample (boiled/refrigerated/grilled)

2. Starch solution (1%): Mix 1 g starch in 100 ml boiling H2O. Boil for one

minute while stirring. Stir until completely dissolved (this solution will

be cloudy).

3. Iodine solution: Mix 0.6 g potassium iodide in 500 ml H2O. Mix 0.6 g

iodine in 50 ml of ethyl alcohol. These two iodine solutions should be

mixed well before combining. Combine the two iodine solutions and

add an additional 450 ml of H2O.

4. Hydrochloric Acid (HCl) 1 M, (5 ml)

5. Blender

6. Filter/ cheesecloth

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Procedure:

1. Preparing the vitamin C extracts:

i. Chop food material into small pieces and place into

blender.

ii. Add 100 ml of distilled water to the blender.

iii. Blend using the highest speed until the material is

thoroughly ground.

iv. Strain the ground extract

v. Measure 30 ml of the strained extract into a 250 ml

Erlenmeyer flask or beaker.

2. Measuring vitamin C in the food sample:

i. Place 30 mL of the food extracts solution in a 250 ml flask

or beaker.

ii. Add 2 drops of the 0.1 M HCl to the flask.

iii. Add 5 ml of the starch solution to the flask.

iv. Fill a burette with the iodine solution.

v. Record the initial volume reading.

vi. Add the iodine solution in 1 ml increments to the flask

while swirling the flask.

vii. Add iodine until the solution stays blue-black for 15

seconds.

viii. Record the volume reading on the burette.

3. Comparing cooked food and raw food’s vitamin C

Does the way you prepare your food affect the vitamin C available to

be ingested? Vitamin C is a water-soluble vitamin. Would cooking

food by boiling in water affect the vitamin C content? If vitamin C is

lost during the cooking process, where does it go? What types of

experiments could you design to test your hypothesis? You will be

testing your hypothesis to determine if vitamin C content is changed

during cooking or if different ways of food preparation yield different

amounts of vitamin C.

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i. Food can be prepared according to your creativity. For

example, you can boil or steam or place in a freezer. You

can also prepare the food by different exposing time to

heat etc.

ii. Chop food material into small pieces and place into

blender.

iii. Obtain your data using the same method in previous

section.

iv. Record the volume reading on the burette.

v. Compare the relative amounts of ascorbic acid present in

the samples you are testing.

vi. Compare your results with those of other members of the

class. What do the results show?

Questions:

i. What juices or drinks had the most vitamin C?

ii. Did the drinks have the vitamin C that they advertised on

the labels?

iii. What food sources had the most vitamin C?

iv. What families or groups had the most vitamin C?

v. Did plants that you do not normally eat have vitamin C?

vi. Did heat affect the vitamin C content of food?

vii. Did heat increase or decrease the vitamin C levels?

viii. What way of food preparation would be the most

nutritious?

ix. Do you have any ideas now to get more vitamins from your

meals?

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2. Application: Magic Writing

Materials

Beaker

Iodine

Lemon/Lime juice

Notebook paper

Cup

Art brush

Procedure:

STEP A: IODINE SOLUTION

1. Pour 100 ml water into a 500ml-beaker.

2. Add 10 ml of Iodine to the water and stir.

STEP B:

1. Cut a section from the notebook paper.

2. The paper must fit inside a 500ml-beaker

STEP C: VITAMIN C SOLUTION

1. Squeeze the juice of the lemon/lime into another beaker

STEP D:

1. Dip the art brush into the lemon/lime juice

2. Write a message on the piece of paper.

3. Allow the juice to dry on the paper.

4. Submerse the paper in the iodine solution in the bowl.

RESULTS

Observe your findings. Why do you get the result?

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GUIDE 5: EXPERIMENT USING LIPID

1. Saponification of triglycerides

Triglycerides are composed of three fatty acids linked to glycerol by fatty

acyl esters (-O-CO-R). The fatty acids may be saturated (no C=C double

bonds) or unsaturated. Liquid triglycerides are oils, while solid triglycerides

are fats.

Saponification: By heating a triglyceride in aqueous potassium hydroxide

(KOH) the fatty acyl esters can be cleaved off (hydrolysis) leaving behind

glycerol and the potassium salt of the fatty acid. The process is called

"saponification" (or soap formation) since the potassium salts of fatty acids

are in fact "soaps".

The "saponification number" is used as an indicator of fatty acid chain

length in triglycerides. The value is simply a measurement of the mg of KOH

required to complete the hydrolysis of one gram of fat or oil.

Triglycerides containing high fatty acids number will have a lower

saponification number than triglycerides with low fatty acids number. In

this laboratory you will evaluate one of several triglycerides to determine

the saponification number. In this experiment, we will make soap by the

same process, called saponification, but will use modern ingredients.

In the process of making soap, animal fat, which is a triglyceride, is

hydrolyzed by the action of a strong base, such as sodium hydroxide, and

heat. The resulting products are soap and glycerol

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Materials:

Triglyceride sample: e.g. Coconut oil, corn oil, palm oil, margarine, butter

Solvent (1:1 ethanol/ether)

0.5M KOH

Phenolphthalein

0.5M HCL

WARNING: KOH is a very strong corrosive agent and can cause serious

burns on contact. Use appropriate eye / hand protection during this

procedure.

Methods: TO BE CARRIED OUT IN A FUME HOOD

1. Place 1.0 g of the sample triglyceride in to a small beaker and

dissolve in 4 ml of solvent (solvent is 1:1 ethanol / ether).

2. Transfer dissolved triglycerides to a small distillation flask and wash

the beaker twice with 1 ml of solvent (1:1 ethanol/ether) to collect all

residual material. Add the "wash" to the distillation flask.

3. Add 25 ml of 0.5M KOH /ethanol solution

4. Measure the exact volume of your mixture. Set up a second system

as a "Control" (or reference) with 25 ml of the 0.5M KOH/ethanol

solution plus additional 1:1 ethanol/ether solvent for a final volume

identical to your experimental solution.

5. Set up a reflux condenser on each flask and place in boiling water

bath for 30 minutes. The hydrolysis will occur during this period.

6. Allow flasks to cool. Add three drops of indicator solution

(phenolphthalein, 10 g/L) to both flasks and titrate with 0.5M HCl

solution.

7. The molar difference between the amount of 0.5M HCl required to

neutralize the "Control" and the amount of HCl required to neutralize

the test sample equals the amount of 0.5M KOH used in the

saponification process.

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8. Calculate the weight (mg) of KOH used to saponify the 1 g sample.

Determine the saponification number for tested samples

9. Obtain the final results from the other groups for each sample and

prepare a table to summarize the results.

Use the following table to assist you to calculate the saponification number

Sample Initial

volume

of HCl

Blank –

Final

Volume

of HCl

Blank

Initial

volume

of HCl

Sample –

Final

Volume

of HCl

Sample

Mol for

blank

(Molarity

x

����Volume

HCl

[blank])

Mol for

sample

(Molarity x

����Volume

HCl

[sample])

Mol of

reacted

KOH or

Mol KOH

Saponification

number =

Mol KOH x 56.1 x

103

gram of

fat

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2. Application: Making Soap

CAUTION: Safety goggles must be warned throughout this experiment.

This lab is intended to be done in a school lab, with adult supervision.

Sodium hydroxide is very caustic, and can cause severe burns to the

skin, especially when hot.

Materials:

*80 ml of 6 M NaOH solution

15 grams of fat

75 ml of distilled water

**300 ml hot sodium chloride solution

100-ml graduated cylinder

Stirring rod

400-ml beaker

250-ml beaker

* To make 6 molar sodium hydroxide, dissolve 19.2 grams of NaOH in

enough water to make a total volume of 80 ml.

** This is just a saturated solution of NaCl.

Methods:

1. Obtain 40 ml of 6 molar NaOH and 17.5 grams of fat

2. Place 40 ml of the NaOH solution and the fat in a 250-ml beaker.

3. Heat to boil over the lowest flame that will sustain the boiling process.

Stir the mixture constantly to avoid spattering. If spattering occurs,

remove the flame and continue stirring the mixture. Replace the flame

and continue heating after the spattering stops.

4. Continue boiling and stirring for about 20 minutes, or until it appears

that most of the water has been evaporated.

5. Then carefully add the remaining 20 ml of NaOH solution and

continue boiling for an additional 20 minutes or until most of the

water has boiled off. DO NOT LET IT BOIL DRY.

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6. As the crude soap cools, a waxy solid should form. Add to it about

12.5 ml of distilled water and about 50 ml of hot, saturated sodium

chloride solution.

7. Stir the mixture, breaking up lumps with your stirring rod.

8. Decant the wash solution by pouring it through a wire screen, which

will trap small soap particles.

9. Repeat the wash process twice. After the final washing, press the soap

between two sheets of paper towelling to expel as much water as

possible

Questions:

1. What is the relationship between saponification and phase (liquid /

solid) of a triglyceride?

2. Why do triglycerides with longer fatty acids have a lower

saponification number than those with shorter fatty acids?

3. Why is the difference in the molar amount of HCl used to neutralize

the control and the amount of HCl used to neutralize the sample

equivalent to the molar amount of KOH used to saponify the test

sample?

4. Why do soaps disperse grease?