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Biochemistry
Laboratory Manual

A compilation of experiments
and exercises

Compiled by:
Honeylynn E. Edles
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TABLE OF CONTENTS
Laboratory Safety Rules and

Smith, GOB 2nd Ed, McGraw Hill

Regulations............................................................................. 3

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Globally Harmonized System............................................................................................... 4

Exercise # 1
The Cell………………………………………………………………………………………....................................... 5

Exercise # 2
Concentration…………........................................................................................................ 9

Experiment # 1
Preparation of Buffers....................................................................................................... 13

Experiment # 2
Water and Its Properties................................................................................................... 15

Experiment # 3
Carbohydrates…………………………………………………………………..................................................17

Experiment # 4
Lipids……………………......................................................................................................... 23

Experiment # 5
Analysis and Denaturation of Proteins.............................................................................. 26

Experiment # 6
Chromatographic Analysis of Proteins…………...................................................................... 31

Experiment # 7
Analysis of Urine…………………………………………...................................................................... 34

REFERENCES………………........................................................................................................ 37

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Laboratory Safety Rules and Regulations

1. The chemistry laboratory is a place for serious work. Do not perform activities without your teacher’s
permission. Never work alone in the laboratory. Work only when your teacher is present.

2. Study your lab activity before you come to the lab. If you are in doubt about any procedures, ask your
teacher for help.

3. Safety goggles and a laboratory apron must be worn whenever you work in the lab. Gloves should be
worn whenever you use chemicals that cause irritations or can be absorbed through the skin.

4. Contact lenses should not be worn in the lab, even if goggles are worn. Lenses can absorb vapors and
are difficult to remove in an emergency.

5. Long hair should be tied back to reduce the possibility of it catching fire.

6. Avoid wearing dangling jewelry or loose, draping clothing. The loose clothing may catch fire and either
the clothing or jewelry could catch on chemical apparatus.

7. Wear shoes that cover the feet at all times. Bare feet or sandals are not permitted in the lab.

8. Know the location of the fire extinguisher, safety shower, eyewash, fire blanket, and first aid kit. Know
how to use the safety equipment provided for you.

9. Report any accident, injury, incorrect procedure, or damaged equipment immediately to your
teacher.

10. Handle chemicals carefully. Check the labels of all bottles before removing the contents. Read the
labels three times: before you pick up the container, when the container is in your hand, and when you
put the bottle back.

11. Do not return unused chemicals to reagent bottles.

12. Do not take reagent bottles to your work area unless specifically instructed to do so. Use test tubes,
paper, or beakers to obtain your chemicals. Take only small amounts. It is easier to get more than to
dispose of excess.

13. Do not insert droppers into reagent bottles. Pour a small amount of the chemical into a beaker.

14. Never taste any chemical substance. Never draw any chemicals into a pipette with your mouth.
Eating, drinking, chewing gum, and smoking are prohibited in the laboratory.

15. If chemicals come into contact with your eyes or skin, flush the area immediately with large
quantities of water. Immediately inform your teacher of the nature of the spill.

16. Keep combustible materials away from open flames. (Alcohol and acetone are combustible.

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Globally Harmonized System

As of June 1, 2015, the Hazard Communication Standard requires pictograms on labels to alert users of
the chemical hazards to which they may be exposed. Each pictogram consist of a symbol o a white
background framed within a red border and represents a distinct hazard(s). The pictogram on the label
is determined by the chemical hazard classification.

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Answer Sheet
Exercise # 1
The Cell

Name:_________________________________Date Submitted:_______________
Yr and Sec: ____________________________ Date Performed:_______________
Group No. ____________________________ Score:________________________

Theory:

The basic unit of structure and function in the human body is the cell. Each of a cell’s part, or organelles,
as well as the entire cell, is organized to perform a specific function. Cells have the ability to metabolize,
grow and reproduce, move and response to stimuli. The cells of the body differ in shape, size and in
specific role in the body. Cells that are similar in structure and function form tissues, which in turn,
construct the various body organs.

I. Using the following list of terms, correctly label all the cell parts indicated by leader lines. Then select
different colors for each structure and use them to color the coding circles and the corresponding
structure in the illustration.

A. . Animal Cell

o Nucleus o Golgi Bodies

o Nucleolus o Smooth Endoplasmic reticulum
o Nuclear membrane o Rough Endoplasmic reticulum
o Plasma Membrane o Lysosome
o Cytoplasm o Filamentous Cytoskeleton (microtubules)
o Mitochondrion

II. Anatomy of an Animal Cell

Complete the following table to fully describe the

Cell Structure Location Function

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1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

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B. Plant Cell

o Nucleus o Golgi Bodies

o Nucleolus o Smooth Endoplasmic reticulum
o Nuclear membrane o Rough Endoplasmic reticulum
o Cell Wall o Lysosome
o Chloroplast o Vacuole
o Mitochondrion

III. Anatomy of a Plant Cell

Complete the following table to fully describe the various cell parts.
Cell Structure Location Function

1.

2.

3.

4.

5.

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6.

7.

8.

9.

10.

11.

Answer Sheet
Exercise # 2
Concentration

Name:_________________________________Date Submitted:_______________

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Yr and Sec: ____________________________ Date Performed:_______________

Group No. ____________________________ Score:________________________

Theory

The concentration of a solution is the amount of solute present in a given amount

of solvent, or a given amount of solution.

1. A sample of 0.892 g of potassium chloride (KCl) is dissolved in 54.6 g of water. What is

the percent by mass of KCl in the solution?

2. Calculate the molality of a sulfuric acid solution containing 24.4 g of sulfuric acid in 198 g
of water. The molar mass of sulfuric acid is 98.09 g

3. How would you prepare a 1.5% NaBr (w:v) aqueous solution?

4. In a biochemical assay, a chemist needs to add 3.81 g of glucose to a reaction mixture.

Calculate the volume in milliliters of a 2.53 Mglucose solution she should use for the
addition.

5. The concentrated sulfuric acid we use in the laboratory is 98.0 percent H 2SO4 by mass.
Calculate the molality and molarity of the acid solution. The density of the solution is
1.83 g/mL

Experiment No. 1
Preparation of Buffers

Theory:

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A buffer solution is one that is resistant to change in pH when small amounts of strong acid or
base are added. For example, when 0.01 mole of strong acid or base are added to distilled water, the
pH drops to 2 with the acid and rises to 12 with the base. If the same amount of acid or base is added to
an acetic acid – sodium acetate buffer, the pH may only change a fraction of a unit.
Many buffers are prepared by combining a weak acid and its conjugate (acetic acid and
sodium acetate) or a weak base and its conjugate (ammonia and ammonium chloride). In general, the
pH range in which a buffer solution is effective is +/- one pH unit on either side of the pKa. The
Henderson–Hasselbalch provides the information needed to prepare a buffer.
[ conjugatebase ]
pH= pKa+log
[ weakacid ]
There is a limit to the amount of acid or base that can be added to a buffer solution before one
of the components is used up. This limit is called the buffer capacity and is defined as the moles of acid
or base necessary to change the pH of one liter of solution by one unit.

Buffer Capacity = (number of moles of OH- or H3O+ added)

(pH change)(volume of buffer in L)

Materials:

Graduated cylinder, 100 mL 0.10 M K2HPO4

Beaker, 400 mL 0.20 M KH2PO4
Volumetric Flask, 100 mL 0.10 M CH3COOH
Pipette 0.02 M NaOH
pH Meter Sodium Acetate
Bottle

Safety:

 Always wear an apron and goggles in the lab.

 Report any spills so they may be cleaned up.

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Sample Computation

How would you prepare 10mL of a 0.01M phosphate buffer, pH 7.40, fromstoc solutions of 0.10M
KH2PO4and 0.25M K2HPO4? pKa of KH2PO4 = 7.20. Prepare 10 mL of a 0.01 M phosphate buffer, pH 7.70,
from stock solutions of 0.1 M K2HPO4and 0.2 M KH2PO4. (pKa for the weak acid = 7.20).

1. Use the Henderson Hasselbalch equation to find the ratio of A to HA.

pH = pKa + log [A-] / [HA]
7.40 = 7.20 + log [A-] / [HA]
0.20 = log [A-] / [HA]
1.584893192 = [A-] / [HA]*
*Since [A-] / [HA] = 1.584893192, we can say that [A-] / [HA] = 1.584893192/1. In this case [A-] =
1.584893192; [HA] = 1.

2. Calculate the decimal fraction (part/whole) of each buffer component.

A-= 1.584893192 / (1.000 + 1.584893192)
= 1.584893192/ 2.584893192 = 0.61313682
HA = 1.000 / 2.584893192= 0.38686318

3. Find the molarity (M)of each component in the buffer by simply multiplying the
molarity of the buffer by the decimal fraction of each component.
MA- = 0.01M x 0.61313682 = 0.006131368M
MHA= 0.01M x 0.38686318 = 0.003868632M

4. Calculate the moles of each component in the buffer.

Moles = Molarity x Liters of buffer
molesA- = 0.006131368M x 0.01L = 6.131 x 10 -5 moles
molesHA= 0.003868632M x 0.01L = 3.869 x 10 -5 moles

5. Calculate the volume of each stock solution required to make the buffer
Liters of stock = moles of the buffer component / Molarity of the stock
LA- = 6.131 x 10-5 moles / 0.25 M = 2.452 x 10-4 L = 245µL
LHA= 3.869 x 10-5 moles / 0.10 M = 3.86 9 x 10-4 L = 387µL

6. To prepare this buffer, one would use appropriately-sized pipets to measure and
transfer each component to a 10mL volumetric flask and bring the solution to
volume with dH2O.

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Procedure:

A. Phosphate Buffer

1. Prepare 100 mLof a 0.01 M phosphate buffer, pH 7.70, from stock solutions of 0.1 M K 2HPO4and
0.2 M KH2PO4. (pKa for the weak acid = 7.20).

a. Use the Henderson-Hasselbalch equation to calculate the volume of each stock solution
needed.
pH = pKa + log [conjugate base] / [weak acid]
b. Check your calculations with other students. See the instructor if there is uncertainty.
c. Make the solution and check the pH of a portion of your buffer solution using the pH meter.

B. Acetate Buffer

2. Prepare 100 mLof 0.01 M acetate buffer,pH 3.80, from stock solutions of 0.1 M acetic acid and
0.02 M sodium hydroxide. pKa acetic acid= 4.76.

1. Use the Henderson-Hasselbalch equation to calculate the volume of each stock solution
needed.
pH = pKa + log [conjugate base] / [weak acid]
2. Check your calculations with other students. See the instructor if there is uncertainty.
3. Make the solution and check the pH of a portion of your buffer solution using the pH meter.
4. Calculate the exact volume of the 0.01 M acetate buffer required to make 100 mLof a
0.0005 M acetate buffer.
1. Prepare this new buffer using the following equation to aid you in your calculations. C 1V1=C2V2
2. Check the pH of this new buffer.

C. Bicarbonate Buffer

3. Prepare 100 mL of 0.200 bicarbonate buffer, pH 10 by calculating the mass of sodium

bicarbonate and sodium carbonate

a. Use the Henderson-Hasselbalch equation to calculate the volume of each stock solution
needed.
pH = pKa + log [conjugate base] / [weak acid]
b. Check your calculations with other students. See the instructor if there is uncertainty.
c. Make the solution and check the pH of a portion of your buffer solution using the pH meter.

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Report Sheet
Experiment # 1
Preparation of Buffers

Name:_________________________________Date Submitted:_______________
Yr and Sec: ____________________________ Date Performed:_______________
Group No. ____________________________ Score:________________________

DATA:
Show your calculation:
A. Preparation of Phosphate Buffer

Calculated Volume
K2HPO4
KH2PO4

Actual pH of the buffer: _____________________

B. Preparation of Acetate Buffer

Calculated Volume
CH3COOH
KH2PO4

Actual pH of the buffer: _____________________

C. Preparation of Bicarbonate Buffer

Calculated Volume
CH3COOH
KH2PO4

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Actual pH of the buffer: _____________________

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GUIDE QUESTIONS:

1. How would you relate the importance of pH to the body fluids?

2. Calculate the pH if 0.30M acetic acid, with Ka = 1.8 x 10 -5 is added to 0.20 M sodium
acetate.

3. On the laboratory shelf are 250mM solutions of both acetic acid and sodium
hydroxide. How would you make a 100 mL solution of 25mM acetate buffer of pH
5.50 using these stock solutions?

4. In the phosphate buffer system containing K2HPO4and KH2PO4, what is the weak
acid? What is its conjugate base?

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Experiment No. 2
Water and its Properties
THEORY
Water is important to all living systems. It serves as a natural solvent for mineral ions
and other substances. It is also the dispersion medium for colloidal cells like protoplasm. It
serves as a medium for most biochemical reactions, and is the most abundant component of
cells. Except for bone tissues and enamel, water constitutes about 70% of the human body.

MATERIALS
NaCl Margarine citric acid powder String
Sugar Ethanol NaHCO3 powder Semi-permeable
Gelatin CHCl3(any 5% AgNO3 membrane
CuSO4 nonpolar solvent Water cellophane
10% NaOH 5% CuSO4

PROCEDURE
1. Water as a universal solvent
a. Put about 0.5 grams of the following substances into six separate test tubes:
NaCl, sugar, gelatin, CuSO4, margarine, ethanol
b. Add 1 mL water to each test tube and shake vigorously to dissolve the substance. To
substances that did not dissolve, add another 1 mL of water and shake again. Add
another 1 mL to the solids that still not dissolve and shake again.
c. Repeat the solubility test using CHCl3 instead of water.
2. Water as a good medium for biochemical reactions.
a. Mix about 0.1 gram of dry, powdered citric acid and sodium bicarbonate (NaHCO 3) in
a dry test tube. Observe if a chemical reaction occurs.
b. Add about 10 mL of water to the mixture and note what happens.
3. Properties of Water Solutions
Dialysis
a. Obtain a dialysis bag and soak in clean water for about 10 minutes.
b. Fill the bag with 30mL of 1% starch – NaCl mixture, tie the bag and rinse thoroughly
with water.
c. Put the bag in a beaker containing deionized water.
d. Adjust the set-up such that levels of fluids inside and outside the bag are the same.
e. After 1 hour, test 1mL of the dialyzate (water in the beaker) with a few drops of
0.1M AgNO3.

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Figure 1. Dialysis set-up

Report Sheet
Experiment # 2
Water and its Properties

Name:_________________________________Date Submitted:_______________
Yr and Sec: ____________________________ Date Performed:_______________
Group No. ____________________________ Score:________________________

DATA

1. Solubility of substances in water and CHCl 3

Substances Solubility in water Solubility in CHCl3

1. NaCl

2. Sugar

3. Gelatin

4. CuSO4

5. Margarine

6. Ethanol

2. Water as a good medium for biochemical reactions.

Observation:

3. Properties of Water Solutions

Test Observation Inference

5% AgNO3

GUIDE QUESTION
Enumerate the different functions of water in living systems

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Experiment No. 3
Carbohydrates
THEORY

Carbohydrates are a major source of energy from our diet. It is composed of the elements C, H,
and O. It is also called saccharides, which means “sugars. Carbohydrates are produced by photosynthesis
in plants. Glucose are synthesized in plants from CO 2, H2O, and energy from the sun. Carbohydrates are
oxidized in living cells (respiration) to produce CO 2, H2O, and energy.
Monosaccharides are the simplest carbohydrates. Disaccharides consist of two
monosaccharides. Polysaccharides contain many monosaccharides

MATERIALS
Test tubes Barfoed’s solution Fehling’s A and B
Beaker Seliwanoff’s Test Conc HCl
Pneumatic trough Iodine solution Dil NaOH
Hot plate Molisch reagent Red litmus paper
Benedct’s solution Conc sulfuric acid Droppers

PROCEDURE
A. Benedicts Test
1. Prepare a boiling water bath and label eight clean small test tubes.
2. In separate test tubes add 1 mL of the Benedict’s reagent. To each test tube add 5 drops of the test
carbohydrate solution. Mix the samples.
3. Place all of the test tubes at the same time into the boiling water bath.
4. Note and record how long it takes for the red Cu2O precipitate to form; also note if the blue
Benedict’s reagent color disappears.
5. After 10 minutes remove all the tubes. Keep the boiling water bath going for the remaining three
experiments.

B. Barfoed’s Test
1. Use the boiling water bath from before and label a new set of 8 clean small test tubes.
2. In separate test tubes add 1 mL of the Barfoeds’s reagent. To each test tube add 10
drops of the test carbohydrate solution. Mix the samples.
3. Place all of the test tubes at the same time into the boiling water bath.
4. Note and record how long it takes for the red Cu2O precipitate to form.
5. After 10 minutes remove all the tubes.

C. Seliwanoff Test
1. Use the boiling water bath from before and label a new set of 8 clean small test tubes.
2. In separate test tubes add 1 mL of the Seliwanoff’s reagent. To each test tube add 3 drops
of the test carbohydrate solution. Mix the samples.
3. Place all of the test tubes at the same time into the boiling water bath.
4. Note and record how long it takes for the first clear red colored solution to form.
5. Remove all the tubes as soon as the first positive test is seen as prolonged heating (in
excess of 5 minutes) may cause spurious results.

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D. Iodine test
1. Place 3 drops of each test carbohydrate solution in separate wells of a clean spot
plate.
2. Add 1 drop of the iodine solution to each test carbohydrate solution.
3. Note and record the color of each sample.

E. Molisch Test
1. To 2ml of a known solution add 1 drop of Molisch’s reagent (10% α-naphthol in ethanol).
2. Down the side of a sloping tube slowly pour 1-2ml of conc. H 2SO4, so that it forms a layer at the
bottom of the tube.
3. Observe the color at the interface between two layers.

F. Fehling’s Test:
1. To 1ml of Fehling’s solution A (aqueous solution of CuSO 4) add 1ml of Fehling’s solution B (solution
of sodium potassium tartrate)
2. Add 2ml of the sugar/carbihydrate solution, mix well and boil.

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Report Sheet
Experiment # 3
Carbohydrates

Name:_________________________________Date Submitted:_______________
Yr and Sec: ____________________________ Date Performed:_______________
Group No. ____________________________ Score:________________________

DATA

A. Benedict’s Test
Test Sample Observation Result (+/-)
Sucrose
Fructose
Maltose
Ribose
Brown Sugar
Starch
Rice w/o saliva
Rice w/ saliva
Rice w/ saliva (boiled)
Bread w/o saliva
Bread w/ saliva
Bread w/ saliva (boiled)

B. Barfoed’s Test
Test Sample Observation Result (+/-)
Sucrose
Fructose
Maltose
Ribose
Brown Sugar
Starch
Rice w/o saliva

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Rice w/ saliva
Rice w/ saliva (boiled)
Bread w/o saliva
Bread w/ saliva
Bread w/ saliva (boiled)

C. Seliwanoff’s Test
Test Sample Observation Result (+/-)
Sucrose
Fructose
Maltose
Ribose
Brown Sugar
Starch
Rice w/o saliva
Rice w/ saliva
Rice w/ saliva (boiled)
Bread w/o saliva
Bread w/ saliva
Bread w/ saliva (boiled)

D. Iodine Test
Test Sample Observation Result (+/-)
Sucrose
Fructose
Maltose
Ribose
Brown Sugar
Starch
Rice w/o saliva
Rice w/ saliva
Rice w/ saliva (boiled)
Bread w/o saliva

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Bread w/ saliva
Bread w/ saliva (boiled)

E. Molisch Test
Test Sample Observation Result (+/-)
Sucrose
Fructose
Maltose
Ribose
Brown Sugar
Starch
Rice w/o saliva
Rice w/ saliva
Rice w/ saliva (boiled)
Bread w/o saliva
Bread w/ saliva
Bread w/ saliva (boiled)

F. Fehling’s Test
Test Sample Observation Result (+/-)
Sucrose
Fructose
Maltose
Ribose
Brown Sugar
Starch
Rice w/o saliva
Rice w/ saliva
Rice w/ saliva (boiled)
Bread w/o saliva
Bread w/ saliva
Bread w/ saliva (boiled)

GUIDE QUESTIONS

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1. Suppose you saw no sign of a color change in the Benedict’s test, no sign of a red precipitate
after 10 minutes with the Barfoed’s test, and a dark red colored solution with the Seliwanoff
test,.

Indicate which sugar(s) you could have: ________________________________________

2. Suppose you saw a red precipitate with the Benedict’s test, a red precipitate after 2 minutes
with the Barfoed’s test, and a straw-colored solution after more than 5 minutes with the
Seliwanoff’s test.

Indicate which sugar(s) you could have: ______________________________________

3. Suppose you saw a red precipitate with the Benedict’s test, no sign of a red precipitate after
10 minutes with the Barfoed’s test, and a straw-colored solution after more than 5 minutes
with the Seliwanoff’s test.

Indicate which sugar(s) you could have: __________________________________________

4. What has amylase in the saliva done to the carbohydrate in bread and rice? Be specific.

5. Is there a different result between the boiled and unboiled saliva? Explain

Experiment No. 4
Lipids

THEORY
Lipids are poorly soluble in water but they dissolve in organic solvents such as benzene and
chloroform. Their functions are to act as metabolic fuel, as stored forms of energy, and as components
of cellular membranes. Lipids are classified into fatty acids, glycerides, spingolipids, and steroids.
Fatty acids are the smallest unit of lipids. They are further divided into saturated and unsaturated.
Saturated or single bonded fatty acids are usually solid at room temperature. Unsaturated fatty acids
contain double bonds and are liquid at room temperature. Fatty acids are long chained carboxylic acids
which, when ionized, cause the formation of H+ and RCOO- anion. Fatty acids react with NaOH producing
soaps.

MATERIALS

Palm Oil Vegetable Oil Coconut Oil Olive Oil

Sesame Oil Canola Oil Pork Oil Cottonseed Oil
Linseed Oil Cod Liver Oil Peanut Oil Lard
Margarine Butter Stearic Acid Acetone
Iodine solution Petroleum ether Detergent Test tubes
Erlenmeyer flask Beaker Balance Hot plate

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Dropper pipette

PROCEDURE
1. The Solubility of Fats.
a. The following procedure is to be repeated for each of the samples of lipids at your table.
b. Place 5 drops of and oil or a small sample of your lipid into each of three separate test
tubes.
c. To the first tube add 5 ml. of water, to the second 5 ml. of acetone and to the third add
5 ml. of water to which a pinch of soap has been added.
d. Shake each tube well and allow to stand for a few minutes.
e. Observe whether solution or emulsification has occurred. Instead of using soap add a
small quantity of protein to the lipid and water mixture
2. A Test for the Degree of Saturation of Fatty Acids.
a. Add 5 ml. of your liquid lipids into separate test tubes and a control tube with 5 ml. of
water.
b. To each tube slowly and carefully add the halogen water dropwise, shaking the tube
after each addition. The halogen solution should be added just until it fails to be
decolorized.
c. Record the number of drops needed to bring about full decolorization.
d. Compare your results with those of your neighbors at other tables and order your lipids
in terms of increasing degrees of unsaturation.

3. Extraction of Lipids from Food

1. Accurately weigh 20 g of solid food. Note the nutrition facts of the food.
2. Record the weight of your sample
3. Crush and crumble the food into small pieces and place it in an Erlenmeyer Flask
4. Add 25 mL of petroleum ether to the flask containing the food sample and swirl the flask
for several minutes to get the lipids to dissolve in the petroleum ether.
5. While waiting for the lipids to dissolve, weigh a clean 100 mL beaker.
6. Record the mass of the beaker to the nearest 0.01 g
7. Carefully decant the petroleum ether from the flask containing your food sample to the
weighed beaker. (Pour only the liquid, not the solid portion of the food.
8. Place the beaker on the hot plate and evaporate all petroleum ether.
9. When all pet ether is evaporated,allow the beaker to cool. Put the beaker in an ice
bath.
10. After the beaker is cooled, dry it outside and get the mass of the sample.
11. Calculate the mass extracted from the food sample and determine the weight percent
fat in that food.

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Report Sheet
Experiment # 4
Lipids

Name:_________________________________Date Submitted:_______________
Yr and Sec: ____________________________ Date Performed:_______________
Group No. ____________________________ Score:________________________

DATA

Solubility of Lipids
Sample Lipid Water Acetone Soap

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Test for the Degree of Saturation

Sample Lipid Number of drops Color after Saturated or
adding I2 Unsaturated

Extraction of Lipids from Food

Food Sample: _______________________________________-
Mass of Mass of Lipid Mass of Mass of Lipid Extracted
Food Sample and beaker Beaker

Weight percent of lipid in food: ____________________________

Guide Questions
1. Determine the number of Calories (kcal) of energy derived from fat in 100g your food sample by
multiplying 9 Cal/g fat times the answer for % lipid obtained. How does your result compared to
your data.

2. Note the difference fats tested for unsaturation. List them from the most unsaturated to the
least unsaturated.

3. From your observations, which is the better emulsifying agent, soap or detergent?

Experiment No. 5
Analysis and Denaturation of Proteins

THEORY

The cell is composed mainly of proteins, and their repeating unit, the amino acid. Proteins
function as biological catalyst or enzymes, transporters of oxygen and hormones.
There are four levels of proteins structure: the primary, secondary, tertiary and quaternary level. The
primary structure is composed of single-covalently bonded amino acids. There are two types of
secondary structure, the α-helix and the β-sheets. The alpha helix structure contains one strand of

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amino acid that is bonded by intermolecular bonds. Two chains that are linked by hydrogen bonds
describe the beta sheet structure. The tertiary structure of the protein refers to the combination of
both.
Denaturation of Proteins
Denaturation is the unfolding of the complex secondary, tertiary and quaternary structure of
proteins. Heat, strong acids and organic solvents can denature proteins. Heat causes the atoms within
the protein molecule to vibrate more rapidly, causing the hydrogen bonds and hydrophobic interactions
to break. Strong acids break salt linkages by ionizing the carboxylic group, and the alcohol denatures the
protein by disrupting the hydrogen bonds.
Heavy metals like silver, lead and mercury also denature the proteins by combining the free carboxylate
anions of the acidic amino acid with the metal, causing precipitation. This is the rationale in the use of
proteins (egg white) as antidote for heavy metal poisoning.

MATERIALS

Test tubes Watch glass Millon’s reagent 95% ethanol

Test tube holder Water bath 0.1% NaNO2 2% mercuric
Test tube brush Graudated cylinder Hopkin Cole’s chloride
Test tube rack Cork reagent 2% lead actetate
Dropper 5% albumin Conc. H2SO4 2% silver nitrate
Pipette 5% gelatine 10% NAOH Picric acid
Aspirator Conc HNO3 5% lead acetate Tannic acid
Spatula Conc NH4OH Peptone Conc HCl
Stirring rod 5% phenol Ammonia water Egg albumin
Electrical stove 5% Amino acid sol. 0.2% urea

PROCEDURE
I. Color Test for Amino Acids and Proteins
A. Ninhydrin Test
Place 15 drops of amino acid and protein solutions in separate test tube. Add 16 drops
of ninhydrin solution. Heat the test tubes using a water bath.
B. Xanthoproteic Test
Place 20 drops of amino acid and protein solutions in separate test tube. Add 5 drops of
conc HNO3 into each test tube. Notice the formation of white precipitate. Mix it
thoroughly in a water bath and note the changes in color. Allow it to cool and add few
drops of conc NH4OH. Note the intensity of color.
C. Millon’s Test
Place 20 drops of amino acid and protein solution in separately-labeled test tubes. Add 4
drops of Millon’s reagent into each test tube. Heat in boiling water bath for 10 mins
then allow to cool by placing the tube in running tap water. Then add 4 drops of freshly
prepared 0.% NaNO2 and warm gently. Note any change in the color of the precipitate.
D. Hopkin’s Cole Test
Place 1 mL each amino acid and protein solution in separately-labelled test tubes. Add 4
drops of Hopkin’s Cole reagent. Incline the test tube in the test tube rack. Carefully
allow 2 ml of conc sulphuric acid to slide down the side of the inclined test tube so that
it will form a layer below the protein solution. Take note of the color on the junction of
the two liquids

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E. Lead Acetate Test

Place 1 ml each of amino acid and protein solution in separately labelled test tubes. Add
5 drops of 10% NaOH and 3 drops of 5% lead acetate solution into each test tube. Shake
and heat in boiling water bath. Describe the color of the precipitate
F. Biuret Test
Place 2 mL each of amino acid and protein solution separately-labeled test tubes. Add
1mL of 10% NaOH solution and about 5 drops of CuSO 4 solution. Observe the color that
develops.
II. Protein Denaturation
A. Coagulation
1. By Heating
Place 1 mL of albumin and gelatine in a test tube. Heat the test tube in a boiling water bath for
10 mins with constant stirring. Allow it to cool. Add 3mL distilled water to the test tubes. Filter
both tubes separately, then test both filtrates using Biuret reagent. Compare the results
2. Inorganic Acids
Label two test tubes as 1 and 2, and add 3mL of 5% albumin into each test tube. To test tube
1, add conc HCl drop by drop, shaking after each addition. Record the number of drops of
the acid until a precipitate is formed. Then add an excess amount of HCl and take note
whether it will increase or dissolve the precipitate formed. Repeat the same procedure in
test tube 2, but this time use conc, H2SO4.
3. Alcohol
Place 1 mL each of 5% solutions of albumin and gelatin in separately-labeled test tubes. Add
5 mL of 95% ethanol in each test tube, mix and note the formation of precipitation.
III. Precipitation
1. By Heavy Metal Salts
In three test tubes, place 3 mL of egg albumin solution. To test tube 1, add 5 drops of
lead acetate solution. Add excess drop of lead acetate solution and note whether the
precipitate is increased or dissolved. Repeat the procedure by adding 5 drops of silver
nitrate to test tube 2 and add 5 drops of mercuric chloride to the third test tube.
Describe the results.
2. By Alkaloidal Reagents
Place 3 mL of egg albumin solution in two test tubes. In test tube 1, add 2 mL of tannic
acid solution and test tube 2 add 2mL of picric acid solution. Describe the protein
solution in each test tube after the addition of alkaloidal reagents.

Report Sheet
Experiment # 5
Analysis and Denaturation of Proteins

Name:_________________________________Date Submitted:_______________
Yr and Sec: ____________________________ Date Performed:_______________
Group No. ____________________________ Score:________________________

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DATA

I. Colorimetric Test for Amino Acids

A. Ninhydrin Test
Test Sample Observation +/-
Tyrosine
Tryptophan
Lysine
Glycine
Glutamic Acid
Serine
Arginine
Egg Albumin
Gelatin

B. Xanthoproteic Test
Test Sample Observation +/-
Tyrosine
Tryptophan
Lysine
Glycine
Glutamic Acid
Serine
Arginine
Egg Albumin
Gelatin

C. Millon’s Test
Test Sample Observation +/-
Tyrosine
Tryptophan
Lysine
Glycine
Glutamic Acid
Serine
Arginine
Egg Albumin
Gelatin
D. Hopkin’s Cole Test
Test Sample Observation +/-
Tyrosine
Tryptophan
Lysine
Glycine
Glutamic Acid
Serine

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Arginine
Egg Albumin
Gelatin

E. Lead Acetate Test

Test Sample Observation +/-
Tyrosine
Tryptophan
Lysine
Glycine
Glutamic Acid
Serine
Arginine
Egg Albumin
Gelatin

F. Biuret Test
Test Sample Observation +/-
Tyrosine
Tryptophan
Lysine
Glycine
Glutamic Acid
Serine
Arginine
Egg Albumin
Gelatin

II. Protein Denaturation

A. Coagulation
Test Sample Observation Biuret Reagent (+/-)
Gelatin
Albumin

B. Inorganic Acid
Test Sample # of drops of HCl # of drops of H2SO4
1 mL Albumin
2 mL Albumin
3 mL Albumin

C. Alcohol
Test Sample Observation

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Gelatin
Albumin

III. Precipitation
A. Heavy Metals
Heavy Metal Observation
Lead Acetate
Silver Nitrate
Mercuric Chloride

B. Alkaloidal Reagent
Heavy Metal Observation
Tannic Acid
Picric Acid

Guide Questions
1. Surgical instruments are sterilized by heating them, and alcohol is used as disinfectant in
cleansing the skin prior to an injection. Why are those methods successful in killing harmful
organisms?

2. Explain why egg whites and milk are used as antidotes for heavy metal poisoning.

3. Explain why picric acid and tannic acids are used in treatment of burns.

4. What is the colored precipitate obtained in the sulphur test (lead acetate test)?

Experiment No. 6
Chromatographic Analysis of Amino Acids

THEORY

Proteins are complex compounds that contain amino acids. They are hydrolyzed by acids,
alkalies or enzyme activity. They are broken down to simpler molecules and finally to their individual
amino acid components. Another technique in hydrolyzing of proteins is by chromatography.

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The separation process is based on the fact that porous solids adsorbs different substances to
different extremes depending upon their polarity. The term “Adsorption” refers to the adhesion or
stickiness of a substance to the surface of another substance, as opposed to the term “absorption”
which refers to a substance penetrating into the inner structure of another substance. A mixture to be
separated is first applied to an immovable porous solid (like paper, or alumina, or fine silica sand) called
the stationary phase. The components of the mixture then get “washed” along the porous solid by the
flow of a solvent called the mobile phase. The mobile phase can be liquid (as in column, paper, or thin-
layer chromatography) or it can be a gas (as in gas chromatography).
Substances can be identified by the heights they reach on the completed chromatogram by
calculating Rf (rate of flow or retention factor) values. The Rf value is a constant for a given substance
under the same experimental conditions. The Rf value may be calculated from the following equation.
The Rf value itself is unitless.

MATERIAL
Diet soda Aspartame (artificial sweetener)
0.12% Aspartic Acid 0.12% Phenylalanine
3M HCl activated charcoal
Mobile phase (6:2:2 1-butanol: glacial acetic acid:distilled water
2% Ninhydrin test tube
Chromatography paper 400 mL beaker
Glass funnel aluminum foil
Capillary tube graduated cylinder
Spot plate filter paper

PROCEDURE

A. Hydrolysis of Aspartame and Expired Diet Soda

a. Weigh 0.1 g of Aspartame and add 1 mL of HCl in a test tube
b. Heat the test tube gently in a warm water bath (95C-100C)
c. Add activated charcoal and filter using a filter paper and glass funnel
d. Transfer the filtrate to a clean test tube.
e. Do the same procedure using 1 mL of diet soda to have a simulated “expired diet soda”.

2.Chromatography
1. Obtain a 9 x 14 cm piece of chromatography paper.
2. Use a pencil to draw the origin line about 1.5 cm from the bottom of the long edge of the paper.
3. Use a pencil to make 8 small evenly spaced vertical marks every 1.5 cm along the length of the
origin line, starting about 1.5 cm from the edge of the paper. Number these marks 1 through 6
just below each with a pencil.
4. On each well of the spot plate, place3 drops of aspartic acid, phenylalanine, hydrolyzed
aspartame, unhydrolyzed aspartame, diet soda and “expired diet soda”
5. Using a toothpick or capillary tube, apply a tiny spot on each vertical mark. Make sure the spot is
dry before respotting. Spots should not exceed one mm in diameter. Note the number of spots
for each sample.

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Spot No. Sample No of Spotting Times

1 Aspartic Acid 6
2 Phenylalanine 6
3 Hydrolyzed aspartame 12
4 Unhydrolyzed aspartame 12
5 Diet soda 12
6 Expired diet soda 12

6. Roll the chromatography paper into a cylinder with the origin line at the
bottom and the dye spots on the outside of the cylinder. Use two
fingers of one hand to hold the ends of the paper close together, about
2 mm apart. Staple the top ends and then staple the bottom ends. The
ends of the paper should not touch.
7. In a 400 mL beaker, put 25 mL of the mobile phase solvent and place
the rolled paper inside the beaker.
8. Cover the chamber with aluminum foil.
9. Watch the eluent as it moves up the paper and see what happens as it comes into contact with
the ink and food colors. Leave the paper in the beaker until the eluent front is about 1.5 cm
from the top of the paper.
10. When the eluent front nears the top, remove the chromatogram and open the cylinder by
tearing the paper at the staples. Set the chromatogram on an empty beaker or a paper towel to
dry.
11. Spray 2 % ninhydrin solution on the paper.
12. Dry the paper in a oven with a temperature of 80C for about 5 minutes.
13. Mark the spots that have developed on the chromatography paperby tracing them with a pencil`

Report Sheet
Experiment # 6
Chromatographic Analysis of Amino Acids

Name:_________________________________Date Submitted:_______________
Yr and Sec: ____________________________ Date Performed:_______________

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Group No. ____________________________ Score:________________________

DATA

A. Hydrolysis of Aspartame
Observation during heating:

Observation after filtration

B. Preparation of Expired Diet Soda

Observation during heating:

Observation after filtration

C. Spotting the Chromatography paper

Spot No. Color of the Spot Distance Distance Rf Value

travelled by the Travelled by the
Spot Solvent
1
2
3
4
5
6

Guide Questions

1. Why should you avoid getting your fingertips on the chromatography paper?
How will it interfere?

2. Comment on the amino acid content of the diet soda and “expired” amino acid based on your
chromatography results.

Experiment No. 7

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Analysis of Urine

THEORY
Urine is formed by the kidneys as they function to remove waste products and foreign materials.
It keeps the level of ions in the blood serum at constant value. These substances include ammonia, urea,
proteins from pathogens, uric acid and ions of hydrogen, sodium potassium, calcium, chloride and
sulfate.

MATERIALS
Urine sample 5% CuSO4
3M NaOH NH4OH
Benedict’s Reagent 6M Nitric acid
3M sulfuric acid 6M NaOH
1:1 glacial acetic acid and 10% sodium nitroprusside litmus paper
Test tubes graduated cylinder
pH paper ammonia
Water bath stirring rod
Evaporating dish watch glass
Stirring rod dropper
0.1M silver nitrate

PROCEDURE
A. Physical Properties
1. Place 20 drops of urine in a test tube.
2. Note the odor/ smell, color and clarity of the urine.
3. Dip a piece of pH paper to determine the pH of the urine
B. Biuret Test – Proteins
1. Place 20 drops of urine in a test tube.
2. Add 3 drops of 6M NaOH and 2 drops of 5% CuSO 4.
3. Swirl the solution gently.
C. Benedict’s Test – Carbohydrates
1. Place 20 drops of urine sample in a test tube.
2. Place 20 drops of Benedict’s solution and mix thoroughly
3. Heat the test tubes in a boiling water bath for 5 minutes.
4. Remove the test tubes from the bath and allow them to cool.
D. Test for Ketone Bodies
1. Place 20 drops of urine in a test tube.
2. Add 10 drops of 1:1 glacial acetic acid and 10% sodium nitroprusside.
3. Allow 10 drops of concentrated ammonia on the side of the walls of the test tubes.
4. Allow the solution to stand for 2 minutes.
E. Test for Bicarbonate
1. Place 20 drops of urine in a test tube/
2. Tilt the test tube and 2 drops of H2SO4.
F. Test for Chloride
1. Place 20 drops of urine in a test tube.
2. Acidify the sample by adding drops of 6M nitric acid
3. Add 5 drops of 0.1 M silver nitrate

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*Your instructor may assign different types of urine.

Report Sheet
Experiment # 6
Chromatographic Analysis of Amino Acids

Name:_________________________________Date Submitted:_______________
Yr and Sec: ____________________________ Date Performed:_______________
Group No. ____________________________ Score:________________________

DATA

A. Physical properties

Smell: _______________________________ Color:________________________________

Clarity: ______________________________ pH:__________________________________

B. Biuret Test
Urine Sample Observations Inference

C. Benedict’s Test
Urine Sample Observations Inference

D. Test for Ketone Bodies

Urine Sample Observations Inference

E. Test for Bicarbonates

Urine Sample Observations Inference

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F. Test for Chlorides

Urine Sample Observations Inference

GUIDE QUESTIONS

1. What is your overall assessment on the health of the subjects’ health?

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REFERENCES

 Lontoc, B. M. et.al. 2000. Biochemistry Laboratory Workbook

 CH104 Paper and TLC.pdf
 PREP of Buffers at desired pH_Carroll lab Chap 3(ex
www.xula.edu_Chemistry_documents_biolab_Caroll...(14-12-03pdf)
 Chang, R. 2010. General Chemistry, McGraw Hill, New York, USA

Compilation of Biochemistry Experiments and Exercises