1
PREFACE
iii
LABORATORY SAFETY GUIDELINES
iv
EXPERIMENT 1A THE CELL
4
EXPERIMENT 1B QUALITATIVE DETERMINATION OF SUBCELLULAR COMPONENTS 9
EXPERIMENT 2A PROTEIN
14
EXPERIMENT 2B PROTEIN DENATURATION
19
EXPERIMENT 3A ENZYME
25
EXPERIMENT 3B FACTORS AFFECTING ENZYMATIC REACTIONS
30
EXPERIMENT 4A NUCLEIC ACID
35
EXPERIMENT 4B ANALYSIS OF NUCLEIC ACID
40
EXPERIMENT 5A LIPIDS
45
EXPERIMENT 5B ANALYSIS OF BRAIN LIPIDS
52
EXPERIMENT 6A GENERAL TESTS FOR CARBOHYDRATES
57
EXPERIMENT 6B QUALITATIVE ANALYSIS OF CARBOHYDRATES
62
EXPERIMENT 7A URINALYSIS: NORMAL CONSTITUENTS
67
EXPERIMENT 7B URINALYSIS: PATHOLOGICAL CONSTITUENTS
72
2
EXPERIMENT 1A
THE CELL
Biochemistry is a study of the molecules found in living organisms and the
chemical processes these molecules undergo. To fully understand its concept, it is
necessary to be acquainted on the different cellular organelles where these processes
are occurring. This can be done by disrupting the cell through homogenization then
separation through differential centrifugation. By centrifugation, the organelles are
separated according to its density. This experiment lets you perform the
homogenization and centrifugation techniques and will prepare the samples to be used
in the next experiment.
OBJECTIVES
 To be able to isolate the components of cell
 To identify the biomolecule components of cell organelles
 To appreciate the biochemical systems that maintain life
PRE-LAB ASSIGNMENT
1. What is differential centrifugation?
2. Look for the densities of the different organelles.
WHAT TO BRING
7 grams chicken liver (keep frozen before use)
MATERIALS
Beakers
Test tubes
Blender
Test tube rack
Scalpel
Stirring rod
Graduated cylinder
Test tube brush
Centrifuge
Filter paper
REAGENTS
0.025M sucrose solution
Distilled water
3
PROCEDURE
A. HOMOGENIZATION OF LIVER
1. Thaw the liver first.
2. Wash the thawed liver with distilled water then with sucrose solution.
3. Place the liver in a filter paper to thoroughly dry it.
4. Minced the liver in a watch glass using scalpel and transfer in a beaker.
5. Add 35ml of sucrose solution and homogenize using a blender for 5-10
minutes.
6. Transfer the homogenate in test tubes for centrifugation.
B. DIFFERENTIAL CENTRIFUGATION
1. Centrifuge the homogenate at 2rpm in 5 minutes.
2. Decant and label the residue as sediment I.
3. Centrifuge the supernatant at 5rpm in 15 minutes and decant. Label residue
as sediment II.
4. Centrifuge the supernatant at 8rpm for 20 minutes and decant. Label residue
as sediment III and supernatant as supernatant III.
5. Seal the samples and keep in refrigerator until next lab period.
See diagram for the flow:
HOMOGENIZED
LIVER SAMPLE
CENTRIFUGE (5mins)
SEDIMENT I
SUPERNATANT
CENTRIFUGE (15mins)
SUPERNATANT
SEDIMENT II
CENTRIFUGE (20mins)
SUPERNATANT III
SEDIMENT III
4
POST-LAB QUESTIONS
1. Explain the process of homogenization and the purposes of each solvent added.
2. What is the significance of the different speed being used in each level of
centrifugation?
3. What organelles are separated in each centrifugation?
CONCLUSIONS
5
EXPERIMENT 1B
QUALITATIVE DETERMINATION OF SUBCELLULAR COMPONENTS
Any complex living organism originates from simple elements like carbon,
hydrogen, oxygen and may be nitrogen or phosphorus. These elements combine to form
the biomolecules like carbohydrates, proteins, nucleic acids, and lipids. These
biomolecules in turn, make up each organelle found in the living cell. In order to analyze
the composition of these organelles, one should have an idea of the molecular
composition of the organelle itself. The different tests that will be encountered in this
experiment will give you an idea of the composition of the cell itself.
OBJECTIVES
 To perform the different qualitative tests in the differentiated samples in 1A
 To identify the biomolecules present in the samples
 To be acquainted on the different chemical tests that may be encountered in
biochemistry laboratory
PRE-LAB ASSIGNMENT
Give the purpose and the positive indicator of the different tests found in this
experiment.
WHAT TO BRING
Samples prepared from Experiment 1A
MATERIALS
Beakers
Test tube brush
Stirring rod
Test tube holder
Graduated cylinder
Filter paper
Centrifuge
Water bath
Test tubes
Hot plate
Test tube rack
6
REAGENTS
Diphenylamine solution
6M NaOH
Molisch Reagent
0.5% CuSO4
Conc. H2SO4
Sudan III reagent
PROCEDURE
SAMPLES:Sediment I, Sediment II, Sediment III and Supernatant III.
For sediment samples, dissolve in 10ml of homogenizing solution
A. DISCHE DIPHENYLAMINE TEST
1. Place 2 ml each of samples in a test tube.
2. Add 3 ml of diphenylamine solution to each test tube and mix.
3. Heat in water bath for 10 minutes and cool in an ice bath. A clear tube
means absent, a blue color indicates DNA while greenish color indicates
RNA.
B. MOLISCH TEST
1. In two test tubes place 1ml each of the samples.
2. Add to each tube, 3-5 drops of Molisch reagent and mix well.
3. To each test tube, add 1 ml concentrated sulfuric acid along the side of the
walls of the inclined tube and do not mix to observe the change in color at
the junction of the two liquids. Record your observation.
C. BIURET TEST
1. Mix 1.0 ml of sample and 10 drops of 6M sodium hydroxide solution in a
test tube.
2. Add 2 drops of 0.5% copper sulfate solution. Mix well and record your
observation.
D. SUDAN III TEST
1. Place the last portion of residue II in a test tube.
2. Add dichloromethane drop by drop until all sample dissolves.
3. Add one drop of Sudan III reagent.
4. Red color will appear if fat is present.
7
DATA SHEET
Name: ______________________________
Date: ________________
Yr. & Sec.: ______________________ Group No. ____
Rating: _______________
QUALITATIVE DETERMINATION OF SUBCELLULAR COMPONENTS
EXPERIMENT 1B
TESTS
OBSERVATIONS
RESULTS
A. DISCHE TEST
B. MOLISCH TEST
C. BIURET TEST
D. SUDAN TEST
8
POST-LAB QUESTIONS
1. What are the substances tested in each test? What are its theoretical results?
2. What biomolecules are present in the samples analyzed based on the results of
your experiment?
3. Explain the results gathered from each test in comparison to the theoretical or
expected results.
CONCLUSIONS
9
EXPERIMENT 2A
PROTEINS
Protein is the most abundant substance in the cell next to water, comprising 15%
of its over-all mass. Protein is composed of amino acids as its building block. It is linked
together with peptide bonds with a positive charged nitrogen-containing group at one
end and a negatively charged carboxyl-group. Along the chain is a series of different side
chains from different amino acids. Some side chains are neutral, some are acidic, some
are basic, and some are classified as polar or nonpolar. The different tests in this
experiment will help you identify the different types of amino acids present in a protein
sample.
OBJECTIVES
 To perform qualitative test for different types of proteins
 To identify proteins based on the different tests performed
 Relate the test results to the chemical structure of each protein or amino acid
PRE-LAB ASSIGNMENT
For the following tests, research on what is the purpose of the test, the reagents
involved in the test, and its positive indicator:
A. Biuret Test
B. Ninhydrin Test
C. Hopkins-Cole Test
D. Sakaguchi Test
E. Xanthoproteic Test
WHAT TO BRING
Aspartame or Equal (5 tabs or 1 sachet)
1 fresh chicken egg
1 small tetra pack evaporated milk
10
MATERIALS
Graduated cylinder
Test tube holder
Beaker
Test tube brush
Stirring rod
Hot plate
Test tubes
Water bath
Test tube rack
REAGENTS
Distilled water
Hopkins-Cole reagent
6M NaOH
Concentrated H2SO4
50% NaOH
0.2% α-naphthol solution
0.5% CuSO4 solution
Sodium hypochlorite
Ninhydrin solution
Concentrated HNO3
PROCEDURE
A. SAMPLE PREPARATION
1. Egg Albumin Solution – Mix 5 ml of egg white in 45ml water
2. Aspartame or Equal – Dissolve 5 tabs in 15.0ml water or 1 sachet in 15.0
ml water
3. Evaporated Milk – Mix 10ml of evaporated milk in 40ml water
B. HOPKINS-COLE TEST
1. Add 2.0 ml of Hopkins-Cole reagent to 2.0 ml of protein sample in a test
tube and mix thoroughly.
2. Tilt the test tube and carefully pour along one side the tube 1.0 ml of
concentrated sulfuric acid.
3. Hold the test tube in an upright position and observe the color of the ring
formed at the interface of the two liquids. (if no ring is visible, gently
agitate the tube to cause a very slight mixing at the surface)
4. Record your observations.
11
C. BIURET TEST
1. Mix 1.0 ml of protein sample and 10 drops of 6M sodium hydroxide
solution in a test tube.
2. Add 2 drops of 0.5% copper sulfate solution. Mix well and record your
observation.
D. NINHYDRIN TEST
1. Add 10 drops of ninhydrin solution to 2.0ml of protein sample. Mix
thoroughly.
2. Heat the test tubes in boiling water bath until a color change is observed.
3. Compare the results.
E. SAKAGUCHI TEST (Strictly observe the order of addition of reagents)
1. To 5.0-ml of protein sample, add 1.0 ml of 6M sodium hydroxide solution.
Mix thoroughly.
2. Add 1.0-ml of 0.2% α-naphthol solution. Mix thoroughly.
3. After 3 munities, add 5 drops of sodium hypochlorite.
4. Immediately note the color of the resulting solution because it fades
quickly. Record observations.
F. XANTHOPROTEIC TEST
1. Add 0.5-ml of concentrated nitric acid to 1.0-ml protein sample.
2. Mix with a stirring rod and warm in water bath for 5 minutes. Note the
color of precipitate
3. Cool the contents then make it basic by adding 50% sodium hydroxide.
4. Note the changes.
12
DATA SHEET
Name: ______________________________
Date: ________________
Yr. & Sec.: ______________________ Group No. ____
Rating: _______________
PROTEINS
EXPERIMENT 2A
TESTS
OBSERVATIONS
RESULT
A. HOPKINS-COLE
TEST
B. BIURET TEST
C. NINHYDRIN
TEST
D. SAKAGUCHI
TEST
G. XANTHOPROTEIC TEST
13
POST-LAB QUESTIONS
1. What is the principle behind each test? Give general chemical equation.
2. What group of amino acids is identified in each test?
3. Account for the difference in color intensity between each samples (if
there are any)
4. Are all the proteins positive for all tests? Why or why not?
CONCLUSIONS
14
EXPERIMENT 2B
PROTEIN DENATURATION
Protein denaturation is the modification in conformation of protein accompanied
by disruption and possible destruction of secondary, tertiary and quaternary structure
of protein. This is brought upon by different types of agents like heat, mechanical
disturbance, inorganic and organic substances and etc. Loss of solubility in water is the
frequent consequence of protein denaturation. When denatured protein precipitates
out, it is called coagulation. This experiment help you understand the effects brought
about by some of these agents that cause denaturation of proteins.
OBJECTIVES
 To observe the effects of several denaturing reagents on a protein sample
 To differentiate the effect of these denaturing reagents to the protein sample
PRE-LAB ASSIGNMENT
1. Define denaturation.
2. What physical and chemical agents are capable of denaturing proteins? Give the
type of bonds or attractive forces disrupted by these agents.
WHAT TO BRING
1 fresh chicken egg
MATERIALS
Graduated cylinder
Test tube holder
Beaker
Test tube brush
Stirring rod
Hot plate
Test tubes
Water bath
Test tube rack
15
REAGENTS
Distilled water
10% BaCl2
Albumin
Concentrated H2SO4
95% ethanol
Concentrated HNO3
70% ethanol
Picric acid
10% AgNO3
Tannic acid
10% CuSO4
Trichloroacetic acid
PROCEDURE
A. Sample Preparation for Proteins
Egg Albumin Solution – Mix 5 ml of egg white in 45ml water
Standard – 2 ml sample of the prepared albumin solution (untreated)
B. EFFECT OF HEAT
1. Place to 2.0 ml of albumin solution in a test tube and heat in boiling water
for 5 minutes.
2. Compare the results with the standard.
C. EFFECT OF ALCOHOL
1. Label two test tubes as 1 and 2.
2. Add 3.0 ml of albumin solution in each test tube.
3. To tube no.1, add 5 ml of 95% ethanol and to tube no. 2, 5 ml of 70%
ethanol.
4. Compare the results with the standard.
5. Record your observations as no precipitation, slight precipitation, and
heavy precipitation.
16
D. EFFECT OF HEAVY METALS
1. Place 2.0 ml of albumin solution in 3 separate test tubes and add the
following reagents:
a.
1 ml of 10% AgNO3
b. 1 ml of 10% CuSO4
c.
1 ml of 10% BaCl2
2. Shake all the test tubes.
3. Note the color of the precipitates formed. Set aside for 5 minutes.
4. Tabulate and compare the results with the standard.
5. Decant the supernatant liquid and test the solubility of a small portion of
the precipitate in 5.0 ml water.
E. EFFECT OF STRONG MINERAL ACIDS
1. Place 2.0 ml of albumin solution in 2 separate test tubes and add the
following reagents:
a. 1 ml of concentrated H2SO4
b. 1 ml of concentrated HNO3
2. Mix thoroughly.
3. Compare the results with the standard.
F. EFFECT OF ALKALOIDAL REAGENTS
1. Place 2.0 ml of albumin solution in 2 separate test tubes and add the
following reagents:
a. 1 ml of picric acid
b. 1 ml of tannic acid
c. 1 ml of trichloroacetic acid
2. Shake all test tubes gently. Note the color of precipitate formed.
3. Compare with the standard.
17
DATA SHEET
Name: ______________________________
Date: ________________
Yr. & Sec.: ______________________ Group No. ____
Rating: _______________
PROTEIN DENATURATION
EXPERIMENT 2B
DENATURING
OBSERVATIONS
AGENTS
COMPARISON TO
STANDARD
HEAT
95% Ethanol
ALCOHOL
70% Ethanol
AgNO3
HEAVY
CuSO4
METALS
BaCl2
18
H2SO4
STRONG
MINERAL
ACIDS
HNO3
Picric acid
ALKALOIDAL
Tannic acid
REAGENTS
Trichloroacetic acid
19
POST-LAB QUESTIONS
1. Explain how each of the agents causes denaturation of the protein.
2. Explain the differences of the reagents in each test.
3. Give ONE practical application of each agent presented.
CONCLUSIONS
20
EXPERIMENT 3A
ENZYMES
Enzymes are substances that act as a catalyst for biochemical reactions by finding
a pathway which has lower activation energy. Each enzyme has an active site where the
chemical reactions occur. The reactant of the said chemical reaction is called substrate.
One amazing property of enzymes is its high specificity; it only acts to a specific
substrate or group of substrates. For example, alcohol dehydrogenase is an enzyme that
oxidizes group of alcohols to aldehyde to carboxylic acid. On the other hand, enzyme
like lactase is only specific for the substrate lactose that breaks it to glucose and
galactose. In this experiment, you will experience the action of some enzymes to its
specific substrate.
OBJECTIVES
 Demonstrate the catalytic action of enzymes through different organic specimen
 Recognize the specificity of enzymes to its substrate
 Identify the products of each reaction through different color tests
 Describe the role of enzyme in digestive processes
PRE-LAB ASSIGNMENT
For the following enzymes, research on the role or function of each enzyme in
biological system and give one example:
A. Amylase
B. Oxidase
C. Protease
D. Rennin
WHAT TO BRING
Potato
Saliva
Evaporated milk
Cheesecloth
21
MATERIALS
Graduated cylinder
Test tube brush
Beaker
Hot plate
Stirring rod
Water bath
Test tubes
Thermometer
Test tube rack
Blender
Test tube holder
REAGENTS
Distilled water
0.2% HCl solution
1% starch solution
Casein
Benedict’s reagent
Calcium chloride solution
Phosphate buffer with pH 6.0
Saturated ammonium oxalate
Freshly prepared 0.2% Catechol solution
1% rennin solution
1% pepsin
PROCEDURES
A. AMYLASE
1. Put 1.0 ml of 1% starch solution on each two test tubes.
2. To one test tube, add 1.0ml saliva.
3. Mix both thoroughly and place in a water bath set for 37oC for 10 minutes.
(Make sure temperature does not raise more than 40oC)
4. Set aside and cool.
5. Add to each test tube 3.0 ml Benedict’s reagent and warm in a boiling
water bath for 5 minutes. Note the color and precipitate of both test
tubes.
B. OXIDASE
1. Wash and peel a potato.
2. Grate the potato into 100ml of water. Blender may be used.
22
3. Transfer the potato solution onto cheesecloth and filter the extract
through a filter paper.
4. Add 5 ml each of filtrate into two test tubes.
5. Place on test tube in water bath for 10 minutes and set aside the other
test tube for comparison.
6. Place 0.5ml of phosphate buffer with pH 6.0 and 2 ml freshly prepared
0.2% Catechol solution into both test tubes.
7. Allow the test tubes to stand for 30 minutes and observe the result after.
C. PROTEASE
1. Place 5 ml each of 1% pepsin dissolved in 0.2% HCl solution into two
separate test tubes.
2. Heat the test tubes to boiling for 1 minute.
3. Let it cool.
4. To one test tube, add 0.5 g casein.
5. Place both test tubes in a beaker of water at 40oC for at least 30 munities.
6. Add 0.5ml of CaCl2 solution to each test tube and compare the results.
Take note of the precipitation.
D. RENIN
1. Place 5 ml each of evaporated milk in two test tubes.
2. To one test tube, add 12 drops of saturated ammonium oxalate and mix.
3. Add 1ml of 1% rennin solution to both test tubes and heat in water bath at
40oC for 20 minutes.
4. Observe and compare the results.
23
DATA SHEET
Name: ______________________________
Date: ________________
Yr. & Sec.: ______________________ Group No. ____
Rating: _______________
ENZYMES
EXPERIMENT 3A
ENZYME
OBSERVATIONS
RESULT
A. AMYLASE
B. OXIDASE
C. PROTEASE
D. RENIN
24
POST-LAB QUESTIONS
1. Explain the mechanism of enzyme action on its substrate presented in the
experiment. Identify which one is the substrate and the product of each digestion
performed.
2. Explain the specificity of enzymes on its substrate.
3. Give the theoretical results of each test performed and explain the results based
on the theoretical results.
CONCLUSIONS
25
EXPERIMENT 3B
FACTORS AFFECTING ENZYME ACTIVITY
Enzymes are known to catalyze a lot of biochemical reactions. Some enzymes can
increase the reaction rate many million times faster than without a catalyst. An example
is OMP, orotidylate decarboxylase, catalyzing 1017 times faster than uncatalyzed
reaction. This means that reaction that would take 78 million years will only take 18
milliseconds with the enzyme OMP. Enzymes are mostly globular proteins. Some are
simple proteins, some are conjugated proteins. Their action on the substrate can be
controlled by adjusting the temperature, pH, or substrate or enzyme’s concentration.
Further, since they are proteins, they also affected by agents that causes them to
undergo denaturation. This experiment shows the effect of these factors on enzymatic
reactions.
OBJECTIVES
 Describe the effect of the factors presented in the experiment on enzyme’s
activity.
 Relate the factors to human enzymatic metabolic activities.
PRE-LAB ASSIGNMENT
Give the factors affecting enzyme activity and give the normal conditions for each
factor to make enzyme perform in its optimum activity.
WHAT TO BRING
10 – 15 ml of Saliva
REAGENTS
Distilled water
Buffer solution at pH 4
1% starch solution
Buffer solution at pH 7
Iodine
Buffer solution at pH 10
26
MATERIALS
Graduated cylinder
Test tube brush
Beaker
Hot plate
Stirring rod
Water bath
Test tubes
Thermometer
Test tube rack
Blender
Test tube holder
PROCEDURES
A. EFFECT OF TEMPERATURE
1. Label three clean and dry test tubes as A1, A2 and A3. Into each test tube
place 1.0 ml of saliva.
2. Place A1 in an ice bath, place A2 in water bath with temperature of
maintaining 37oC, and place A3 in boiling water, all for 5 minutes.
3. To each test tube, add 2.0 ml of 1% starch and leave for 10 minutes.
4. After 10 minutes, add 2 drops of iodine.
5. Note and record the color. Rate the intensity of color from 1- 3, 1 being
colorless.
B. EFFECT OF pH
1. Label three clean and dry test tubes as B1, B2 and B3. Into each test tube
place 2.0 ml of 1% starch solution.
2. Place in B1 2.0ml of buffer solution at pH 4, place in B2 2.0ml of buffer
solution at pH 7, place in B3 2.0ml of buffer solution at pH 10.
3. Make sure in this step, the saliva is added simultaneously to all test tubes.
To each test tube, add 1.0 ml saliva.
4. Warm all test tubes in 37oC water bath for 10 minutes.
5. After heating, add 2 drops of iodine solution.
6. Note and record the color. Rate the intensity of color from 1- 3, 1 being
colorless.
27
C. EFFECT OF ENZYME CONCENTRATION
1. Label four clean and dry test tubes as C1, C2, C3 and C4. Into each test
tube place 2.0 ml of 1% starch solution.
2. Place one drop of iodine solution in each test tube and stir.
3. In another test tube, place 2.0 ml saliva and warm at 37 oC for 5 minutes.
4. To test tube C2, add 3 drops of saliva and record the time of digestion
(become colorless). Use C1 as the control (no digestion).
5. Repeat step 4 for test tube C3 using 6 drops of saliva and C4 using 10
drops of saliva.
6. Record the time of digestion and rate the degree of digestion from 1-4, 1
being no digestion.
28
DATA SHEET
Name: ______________________________
Date: ________________
Yr. & Sec.: ______________________ Group No. ____
Rating: _______________
FACTORS AFFECTING ENZYME ACTIVITY
EXPERIMENT 3B
FACTORS
VARIATIONS
OBSERVATIONS
RESULT
A1
A. EFFECT OF
TEMPERATURE
A2
A3
B1
B. EFFECT OF pH
B2
B3
C1
C. EFFECT OF
ENZYME
C2
CONCENTRATION
C3
29
POST-LAB QUESTIONS
1. Explain how the factors affect the enzyme activity and relate to human metabolic
activities.
2. Give the theoretical results of each factor and explain of it coincides with the
results of your experiment.
3. Give possible causes of errors.
CONCLUSIONS
30
EXPERIMENT 4A
NUCLEIC ACID
Nucleic acid is one of the biomolecules which is compose of monomer units
called nucleotides. A nucleotide is a three-subunit molecule composing of a pentose
sugar bonded to the phosphate group and a nitrogen-heterocyclic base. If the
nucleotide is made up of a ribose sugar, it is named as ribonucleotide. If it is a
deoxyribose sugar then it is named as deoxyribonucleotide. The polymers are called
ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) respectively. In this experiment
you will learn how to extract DNA from plant tissue.
OBJECTIVES
 Extract DNA from plant tissue
 Describe each procedure done during the extraction
 Identify the sugar in the extracted DNA through a qualitative test
PRE-LAB ASSIGNMENT
1. Differentiate DNA from RNA
2. What are the factors that stabilize and destabilize the DNA structure?
WHAT TO BRING
2 white onion bulbs or 2 pieces banana or 5 pieces strawberries
Coffee filter paper or gauge
MATERIALS
Graduated cylinder
Test tube rack
Beaker
Test tube holder
Stirring rod
Test tube brush
Blender
Hot plate
Test tubes
Water bath
31
REAGENTS
Distilled water
Diphenylamine solution
95% ethanol
Standard Saline Citrate Solution
1% glucose solution, 1% ribose solution,
1% deoxyribose solution
PROCEDURE
***Make two sets of experiment, one for today’s analysis and one for the next session
A. Preparation of materials and reagents
1. Place 20ml 95% ethanol in the refrigerator to chill it. You will use this in
procedure no. 12.
2. Homogenizing solution. In a 250-ml beaker add 120ml hot distilled water,
1.5 gram NaCl, and 5 ml dishwashing liquid. Mix them together using a
clean stirring rod slowly to avoid foaming of the soap.
B. Extraction of DNA
1. Coarsely chop the onions or banana in small cubes (do not chop too finely)
and blend for 30 seconds.
2. Add 50ml homogenizing solution and blend again.
3. Filter the mixture in a beaker through a coffee filter or gauze. When you
filter, try to keep the foam from getting into the filtrate.
4. Take the 95% ethanol out of the freezer and place slowly about 15ml to
the filtrate using a graduated cylinder.
5. Let the mixture stand for 5-10 minutes undisturbed. Observe for a bubble
formation and DNA will precipitate out of the solution. DNA will appear as
white filamentous material in the ethanol layer.
***For the other set-up, leave it overnight.
6. Spoon the DNA with stirring rod and add SSC solution.
32
C. Analysis of the extracted DNA: Diphenylamine Test
1. Prepare four test tubes with the label: glucose, ribose, deoxyribose, and
extracted DNA.
2. Place 2 ml each of the following solutions that corresponds their labels: 1%
glucose solution, 1% ribose solution, 1% deoxyribose solution, and 1%
extracted DNA solution.
3. Add 3 ml of diphenylamine solution to each test tube and mix.
4. Heat in water bath for 10 minutes and cool in an ice bath.
33
DATA SHEET
Name: ______________________________
Date: ________________
Yr. & Sec.: ______________________ Group No. ____
Rating: _______________
NUCLEIC ACID
EXPERIMENT 4A
PROCEDURE
OBSERVATIONS
RESULT
A. EXTRACTION
B. ANALYSIS
34
POST-LAB QUESTIONS
1. What is the general structure of ribonucleotide and deoxyribonucleotide. Explain
the difference.
2. What are the levels of the structure of either RNA or DNA?
3. Explain the purposes of each step done in the procedure of extracting DNA.
4. What is the purpose of diphenylamine test? What is the chemical reaction
involved in this test?
CONCLUSIONS
35
ANALYSIS OF NUCLEIC ACIDS
EXPERIMENT 4B
Most of the cellular DNA is found in the nucleus of the cell. These DNA molecules
are compose of a series of nucleotides, arranged accordingly. Nucleotides, the building
blocks of nucleic acid are joined by phosphodiester bonds. The monomeric nucleotides
of DNA are composed of adenine, guanine, cytosine and thymine. While in RNA another
nucleotide is present, uracil. Adenine and guanine are called purine bases while
cytosine, thymine and uracil are called pyrimidine bases.
The qualitative analysis of DNA sample will depend on the hydrolysis of its chain
to nucleotides and hydrolysis of nucleotides to phospohorus, deoxyribose, and purine or
pyrimidine bases. To analyze the individual components of nucleotide, different tests
are performed in this experiment.
OBJECTIVES
 To qualitative test the DNA sample gathered from experiment 4A
 Differentiate unhydrolyzed DNA to acid hydrolyzed DNA
PRE-LAB ASSIGNMENT
1. Give the chemical reaction for the hydrolysis of DNA using sulfuric acid.
2. Look for the positive/expected results of the following tests:
a. Benedict’s Test
b. Test for Purine
c. Bial’s Test
d. Test for Phosphate
WHAT TO BRING
Marble for test tube cover
36
MATERIALS
Graduated cylinder
Test tube rack
Beaker
Test tube holder
Stirring rod
Test tube brush
Blender
Hot plate
Test tubes
Water bath
REAGENTS
Distilled water
1% silver nitrate
10% sulfuric acid
Bial’s reagent
Benedict’s reagent
6N nitric acid
1% ammonium hydroxide
Ammonium molybdate
PROCEDURES
A. Acid hydrolysis
1. Divide the gathered DNA into two test tubes. Labeling test tube A for
hydrolyzed and B for unhydrolyzed.
2. In test tube A, place 10ml of 10% sulfuric acid. To test tube B, add 10ml
distilled water and divide into four test tubes. Set aside.
3. Cover the test tube A with marble and heat at 60oC in a water bath for one
hour. Maintain the temperature.
4. After heating, cool test tube A at room temperature and centrifuge at
2rpm for 10 minutes.
5. Discard precipitate and divide it into four test tubes for the following tests.
37
B. Analysis of DNA
***Perform these tests for the two test solutions: Test tube A and Test tube B
1. BENEDICT’S TEST
a.
Neutralize the test solution by adding small amount of solid
Na2CO3. Test with litmus paper. Let it stand for 2 minutes then
decant.
b.
To 1ml of the test solution add 0.5mL of Benedict’s reagent. Boil
in a water bath for 5 minutes or longer. Observe the results
2. TEST FOR PURINE BASES
a. Treat 1 ml of the test solution with 1% ammonium hydroxide until
basic. Test with litmus paper. Add few drops of 1% silver nitrate
solution. Let the mixture stand undisturbed and look for
precipitate.
3. BIAL’S TEST
a.
Mix 1 ml of test solution with 0.5ml of Bial’s reagent.
b.
Heat in a boiling water bath for 5-10 minutes until you see visible
result.
4. TEST FOR PHOSPHATE
a.
To 1 ml of test solution add 1% ammonium hydroxide until basic.
Test with litmus paper.
b.
Acidify with 6N nitric acid. Test with another litmus paper.
c.
Add 1 ml of ammonium molybdate and heat in a water bath.
Observe result.
38
DATA SHEET
Name: ______________________________
Date: ________________
Yr. & Sec.: ______________________ Group No. ____
Rating: _______________
ANALYSIS OF NUCLEIC ACIDS
EXPERIMENT 4B
TESTS
OBSERVATIONS
RESULT
A
A. BENEDICTS
B
A
B. TEST FOR PURINE
B
A
C. BIAL’S TEST
B
A
D. TEST FOR
PHOSPHATE
B
39
POST-LAB QUESTIONS
1. What substances are tested in each analysis done? Explain why results showed
positive or negative.
2. Compare the results of test tube A and B and explain.
3. Cite some factors that may cause errors in the experiment.
CONCLUSIONS
40
LIPIDS
EXPERIMENT 5A
Lipids, one of the major biomolecules in living cells have no common structure
unlike proteins, carbohydrates, and nucleic acids. Lipids are defined as organic
substances that are insoluble (or sparingly soluble) in water but soluble in organic
solvents. This physical characteristic as well as the chemical properties of lipids depends
on the presence of carboxyl groups, number of double bonds, number of hydroxyl
groups, and length of carbon chains.
Referring to its structure, lipids can be divided into simple, compound or derived
lipids. Simple lipids are esters of fatty acids and alcohol. Compound lipids are esters of
fatty acids and alcohol that contain other functional group. While derived lipids are
lipids that contain hydrocarbon rings and a long hydrocarbon side chain. The building
blocks of lipids differ from each type but the most common is the fatty acid. This
experiment helps you observe and understand the different properties of lipids.
OBJECTIVES
 Learn how to characterize lipids through different tests
 Identify the type of lipids based on chemical properties
PRE-LAB ASSIGNMENT
1. Identify the lipids that are part of simple, compound and derived lipids.
2. Draw the structures of the lipid samples in the experiment.
3. How will the iodine tests the unsaturation of a substance?
4. What is Acrolein test? What it is for? What is its positive indicator?
WHAT TO BRING
Olive oil
Butter
Coconut oil
Lotion
Lecithin
41
MATERIALS
Filter paper
Test tube rack
Graduated cylinder
Test tube holder
Beaker
Test tube brush
Stirring rod
Hot plate
Test tubes
REAGENTS
Distilled water
1M NaOH
Dichloromethane
I2 in KI
Cyclohexane
Potassium bisulfate
1M HCl
PROCEDURES
A. TRANSLUSCENT EFFECT
1. In a small filter paper, drop a lipid sample. Do this to the five samples
separately. If sample is gel-like, dissolve in 1ml dichloromethane.
2. Allow the spots to dry.
3. After drying, hold the filter paper against a light and see any translucent
spot. Record your observation.
B. SOLUBILITY
1. In four different test tubes add 1ml each of water, dichloromethane,
cyclohexane, 1M HCl and 1M NaOH.
2. Add to each test tube three drops of lipid sample and mix thoroughly.
Make sure you place strictly three drops of sample on each solvent as
solubility is affected by amount.
3. Observe if sample is thoroughly dissolved. See separation of layers to
detect miscibility. Record your observations.
4. Repeat the procedure with the four different samples.
42
C. TEST FOR UNSATURATION
1. In six different test tubes place 3ml of ether. Label the test tubes with your
different lipid sample and the sixth as negative control.
2. Add the lipid sample in each test tube while add none to the sixth. Mix
thoroughly.
3. To the six test tubes add 3 drops of I2 in KI.
4. Shake again and note the changes in color.
D. ACROLEIN TEST
1. In a clean crucible, place 0,5gram of potassium bisulfate and one to two
drops of lipid sample. Then cover.
2. Heat the sample slowly and note the odor produced.
43
DATA SHEET
Name: ______________________________
Date: ________________
Yr. & Sec.: ______________________ Group No. ____
Rating: _______________
LIPIDS
EXPERIMENT 5A
A. TRANSLUSCENT EFFECT
SAMPLES
OBSERVATIONS
RESULTS
Olive oil
Coconut oil
Lecithin
Butter
Lotion
B. SOLUBILITY
SAMPLES
WATER
DICHLORO
METHANE
CYCLOHEXANE
DILUTE
DILUTE
HCl
NaOH
Olive oil
Coconut oil
Lecithin
Butter
Lotion
44
C. TEST FOR UNSATURATION
SAMPLES
OBSERVATIONS
RESULTS
Olive oil
Coconut oil
Lecithin
Butter
Lotion
D. ACROLEIN TEST
SAMPLES
OBSERVATIONS
RESULTS
Olive oil
Coconut oil
Lecithin
Butter
Lotion
45
POST-LAB QUESTIONS
1. What is the purpose of translucent spot test? What is detected by this test? Is it
conclusive? Why?
2. Explain the solubility test by comparing the structures of the lipid samples and
the solvents.
3. Explain why results in the Test for Unsaturation are positive or negative.
4. What is the principle behind Acrolein test? Explain why results showed positive or
negative.
CONCLUSIONS
46
EXPERIMENT 5B
ANALYSIS OF BRAIN LIPIDS
The brain has the second highest component of lipids and is 50% of the brain’s
dry weight. It virtually does not contain trigelycerides but has plenty of membrane
lipids. One of the functions of lipids in the brain is to determine the protein function and
synaptic throughput in neurons. According to study, membrane lipids have a crucial role
in major depression and anxiety. Other than membrane lipids, there are also other lipids
that function as biomessenger like steriod hormones and eicosanoids. This experiment
will determine the lipid composition of a brain sample.
OBJECTIVES
 Distinguish the different types of lipids found in the brain
 Characterize brain lipids through different tests
PRE-LAB ASSIGNMENT
1. Draw the general structure of phospholipids, sphingoglycolipids, and cholesterol.
2. Identify the positive indicators of the following tests: Ninhydrin test, Sudan III
test, Ammnonium molybdate test and Salkowski test.
WHAT TO BRING
Calf or Pig brain
1ml Egg albumin
1 gel capsule Lecithin
MATERIALS
Filter paper
Test tube rack
Graduated cylinder
Test tube holder
Beaker
Test tube brush
Stirring rod
Hot plate
Test tubes
47
REAGENTS
Distilled water
Molisch reagent
Dichloromethane
Concentrated H2SO4
Methanol
Ninhydrin solution
Ether
6N HNO3
95% ethanol
Ammonium molybdate
Acetone
Sudan III
5% glucose
PROCEDURES
A. EXTRACTION OF BRAIN LIPIDS. SHOULD BE DONE ONE DAY BEFORE THE THIS
LABORATORY SESSION
1. Chop 100 g of fresh brain tissues and measure its volume.
2. Homogenize the brain tissue with dichloromethane-methanol (2:1) solvent
3. Place in Erlenmeyer flask and add ether enough to cover it.
4. Cover tightly (ether is highly volatile) and set aside for the next day
laboratory session.
5. After a day, decant the incubated sample.
RESIDUE I
1.
Get a small portion of the incubated brain and add 10ml hot 95%
ethanol (about 60 degrees)
2.
Mix thoroughly and decant
3.
Discard the residue and label supernatant as Supernatant II. Use
this in Molisch Test.
SUPERNATANT I
a. Add acetone slowly until precipitation is complete. Complete
precipitation happens when no cloudy formation will appear after
adding a drop of acetone.
b. Filter the mixture. And label residue and Residue II.
48
c. Divide the residue into four parts. Use this in Ninhydrin test,
Ammonium molydbate Test, Sudan III Test and Salkowski Test.
**If samples are too large, make sure to use only a small portion.
B. QUALITATIVE TESTS
1. MOLISCH TEST
1.
In two test tubes place 1ml each of the samples: 5% glucose and
Supernatant II
2.
Add to each tube, 3-5 drops of Molisch reagent and mix well.
3.
To each test tube, add 1 ml concentrated sulfuric acid along the
side of the walls of the inclined tube and do not mix to observe
the change in color at the junction of the two liquids. Record your
observation.
2. NINHYDRIN TEST
1.
In two test tubes place 2.0 ml of samples: Residue II and egg
albumin solution (1:10 ratio)
2.
Add 10 drops of ninhydrin solution to 2.0ml of sample. Mix
thoroughly.
3.
Heat the test tubes in boiling water bath until a color change is
observed.
4.
Compare the results.
3. AMMONIUM MOLYBDATE TEST
1.
In two test tubes place 2.0 ml of samples: Residue II and one drop
of lecithin.
2.
Add 1.0 ml of 6N nitric acid to the samples.
3.
Heat the mixture in boiling water bath for 5 minutes.
4.
Add 1.0 ml ammonium molybdate solution and heat again for 5
minutes.
5.
Note the color changes and compare.
49
4. SUDAN III TEST
1.
Place the last portion of residue II in a test tube.
2.
Add dichloromethane drop by drop until all sample dissolves.
3.
Add one drop of Sudan III reagent.
4.
Red color will appear if fat is present.
5. SALKOWSKI TEST (Make sure all apparatus used here is free of water)
1.
Dissolve the fourth portion of residue II in dichloromethane drop
by drop.
2.
Add equal amount of drops of concentrated sulfuric acid and
shake gently.
3.
Let liquid layers separate and observe color change at the
interface.
50
DATA SHEET
Name: ______________________________
Date: ________________
Yr. & Sec.: ______________________ Group No. ____
Rating: _______________
ANALYSIS OF BRAIN LIPIDS
EXPERIMENT 5B
TESTS
OBSERVATIONS
RESULTS
A. MOLISCH TEST
B. NINHYDRIN TEST
C. AMMONIUM
MOLYBDATE
TEST
D. SUDAN III TEST
E. SALKOWSKI TEST
51
POST-LAB QUESTIONS
1. What types of lipids are found in your brain sample? How will you justify your
answer?
2. Are the results in this experiment coincides with the expected outcome? Why?
3. State some possible errors encountered in the experiment.
CONCLUSIONS
52
EXPERIMENT 6A
GENERAL TEST FOR CARBOHYDRATES
Of all the organic carbon on Earth, more than half of those are the carbohydrates
starch and cellulose. Both are polymers of glucose. Animals and humans possess
enzyme in the body that breaks down starch into glucose molecules. On the other hand,
animals (except termites) and humans don’t have enzyme cellulase to break down
cellulose. Carbohydrates’ building blocks are called monosaccharide or sugar. Each
monosaccharide is joined by glycosodic bond. Monosaccharide is a polyhydroxy
aldehyde (aldose) or a polyhydroxy ketone (ketose) with three or more carbons. They
can be in their linear or cyclic form. The chemical properties of the carbohydrates
depend on what types of monosaccharides are present in their polymeric chain. This
experiment identifies the carbohydrates in the extracted liver sample.
OBJECTIVES
 Extract carbohydrates from liver sample
 Perform and familiarize the tests for identification of carbohydrates
 Understand the principle behind each step of extraction
PRE-LAB ASSIGNMENT
1. What is the classification of monosaccharides? Give one example of each.
2. Draw the Fischer and Haworth projections of glucose.
3. Determine the positive indicator of each Molisih test and Iodine Test.
WHAT TO BRING
Chicken liver
MATERIALS
Graduated cylinder
Test tube holder
Beaker
Test tube brush
Stirring rod
Water bath
Test tubes
Hot plate
Test tube rack
Mortar and pestle
53
REAGENTS
Distilled water
Molisch reagent
0.1% acetic acid
Conc. H2SO4
Ethanol
Iodine
5% glucose
PROCEDURES
A. SAMPLE PREPARATION
1. Weigh 3 grams of chicken liver and place in a beaker.
2. Place 2 ml of boiling water while mixing it for 2 minutes to precipitate the
proteins.
3. Transfer the mixture in a mortar and grind until no lump is visible.
4. Add to the mixture another 3ml of distilled water and transfer in a beaker.
5. To the beaker, add 1ml of 0.1% acetic acid.
6. Heat the mixture in a water bath for 30 minutes.
7. Filter immediately while still warm.
8. To the filtrate, add 5 to 10 drops of ethanol to further precipitation. Filter
solution if cloudy.
B. QUALITATIVE TESTS
1. MOLISCH TEST
a. In three test tubes place 1ml each of the samples: 5% glucose,
water, and prepared liver sample.
b. Add to each tube, 3-5 drops of Molisch reagent and mix well.
c. To each test tube, add 1 ml concentrated sulfuric acid along the
side of the walls of the inclined tube and do not mix to observe the
change in color at the junction of the two liquids. Record your
observation.
54
2. IODINE TEST
a. In three test tubes place 1ml each of the samples: 5% starch, water,
and prepared liver sample.
b. Add to each tube, 3-5 drops of Iodine and mix well. Record the
color.
c. Warm the test tubes in water bath until there is change in color.
d. Immediately remove the test tubes and cool at room temperature.
e. Record the color of your solutions.
55
DATA SHEET
Name: ______________________________
Date: ________________
Yr. & Sec.: ______________________ Group No. ____
Rating: _______________
GENERAL TEST FOR CARBOHYDRATES
EXPERIMENT 6A
PROCEDURE
RESULTS
OBSERVATIONS
A. PREPARATION
B. MOLISCH TEST
C. IODINE TEST
56
POST-LAB QUESTIONS
1. Explain how the carbohydrates are extracted from the liver.
2. Explain Molisch test. Give the chemical reaction of this test and explain the
result. Is it positive or negative, why?
3. Explain Iodine test. Give the chemical reaction of this test and explain the
result. Is it positive or negative, why?
CONCLUSIONS
57
EXPERIMENT 6B
QUALITATIVE TESTS FOR CARBOHYDRATES
Carbohydrates can be classified into monosaccharide, the monomer unit of
carbohydrates, disaccharide if two monosaccharides are joined, oligosaccharide if few
monosaccharides are joined or polysaccharide if it is already compose of large number
of monosaccharides. Several tests can be used to determine whether a carbohydrate is
(a) a ketone monosaccharide (ketose) or an aldehyde monosaccharide (aldose), (b) a
monosaccharide or a disaccharide, (c) a reducing disaccharide or a non-reducing
disaccharide, or (d) if it is a polysaccharide or not.
Glucose in the urine is normally present in a very small amount (about 0.010.03g/100ml urine). When the amount exceeds this level, glucosuria happens and can
be an indication of diabetes. Diabetes is a disease wherein the level of blood glucose or
blood sugar in humans is very high. This happens when human body does not make
insulin (hormone that regulates the metabolism of carbohydrates), type I or does not
make or use insulin well, type II. You will identify in this experiment reducing and nonreducing sugars in the samples through the tests presented.
OBJECTIVES
 Perform the different tests for different types of carbohydrates
 Understand the principle behind each test and its purpose
 Qualitatively detect sugar content from a urine sample of a diabetic person
PRE-LAB ASSIGNMENT
1. Look for the specific purpose of the different tests presented in the experiment
and its positive indicator.
2. Search on the normal values and abnormal values of glucose in urine
WHAT TO BRING
Urine samples:
Random urine (RU) – taken from any time of the day who did not fast
Diabetic urine (DU) – taken any time of the day from diabetic person who did not fast
58
MATERIALS
Graduated cylinder
Test tube holder
Beaker
Test tube brush
Stirring rod
Water bath
Test tubes
Hot plate
Test tube rack
REAGENTS
5% glucose
Benedict’s reagent
5% sucrose
Barfoed’s reagent
5% fructose
Bial’s orcinol reagent
5% lactose
Seliwanoff’s reagent
5% xylose
PROCEDURES
A. BENEDICT’S TEST
1. Label 6 test tubes with the following samples: glucose, sucrose, fructose,
lactose, RU, and DU.
2. Place in each test tubes 1 ml of Benedict’s reagent.
3. Add 1 ml each of the samples and mix thoroughly.
4. Warm the test tubes and remove immediately if color changes occur.
5. Record observations according to the pattern below.
DATA
COLOR OF SOLUTION
INTERPRETATION
(-)
Blue
Absent
(+)
Green, slight yellow ppt*
Present, trace
(++)
Green, thick yellow ppt
Present, about 1g/100mL
(+++)
Yellow, orange ppt
Present, about 2g/100mL
Orange, orange to red ppt
Present, more than 2g
(++++)
*ppt = precipitate
59
B. BARFOED’S TEST
1. Label 4 test tubes with the following samples: glucose, sucrose, fructose,
and lactose.
2. Place in each test tubes 1 ml of Barfoed’s reagent.
3. Add 1 ml each of the samples to the four labeled test tubes respective
their names and mix thoroughly.
4. Heat for 30 seconds. Note the changes and record.
C. BIAL’S ORCINOL TEST
1. Label 3 test tubes with the following samples: glucose, fructose, and
xylose.
2. Place in each test tubes 1 ml of Bial’s orcinol reagent.
3. Add 1 ml each of glucose, sucrose, fructose, and lactose to the three
labeled test tubes respective their names and mix thoroughly.
4. Boil gently for 5 minutes.
5. Look for green colored solution and red colored solution and record.
D. SELIWANOFF’S TEST
1. Label 4 test tubes with the following samples: glucose, sucrose, fructose,
and lactose.
2. Place in each test tubes 1 ml of Seliwanoff’s reagent.
3. Add 1 ml each of glucose, sucrose, fructose, and lactose to the four labeled
test tubes respective their names and mix thoroughly.
4. Boil for 30 seconds.
5. Observe color changes and record.
60
DATA SHEET
Name: ______________________________
Date: ________________
Yr. & Sec.: ______________________ Group No. ____
Rating: _______________
QUALITATIVE TESTS FOR CARBOHYDRATES
EXPERIMENT 6B
WRITE (+) for positive results and (-) for negative results. For Benedict’s test, follow
instruction in the procedure. Write N/A if not applicable.
SAMPLES’ RESULTS
TESTS
GLUCOSE
FRUCTOSE SUCROSE
LACTOSE
XYLOSE
RU
DU
A. BENEDICT’S
B. BARFOED’S
C. BIAL’S
D. SELIWANOFF’S
61
POST-LAB QUESTIONS
1. Explain the results gathered from each test by presenting the principle behind it.
What makes the test positive? Explain why some samples exhibit positive results
and why some have negative results.
2. Compare the urine from a normal person and a diabetic person. If they are the
same or not, lay down some explanations of the results.
CONCLUSIONS
62
EXPERIMENT 7A
URINALYSIS – NORMAL CONSTITUENTS
Urine is one of the waste products of metabolism in the body. The presence or
absence of a substance in the urine could be used as a diagnostic test for a disorder or
disease. Complete urine analysis or urinalysis consists of three parts: physical
properties, chemical properties and urine sediment findings. The average urine
production in adult humans is around 1.4L per person, where 91-95% is water. The rest
of the remaining percentages of urine content are the different chemical substances
which are by-products of metabolism. In this experiment you are going to examine the
inorganic constituents of urine.
OBJECTIVES
 Analyze a urine sample
 Identify the inorganic constituents of urine sample through the tests
 Correlate the findings to clinical significance
PRE-LAB ASSIGNMENT
1. What are the normal physical properties of urine?
2. What are the normal inorganic constituents of urine? What are its normal values?
3. Determine the positive indicator on each test involve.
WHAT TO BRING
URINE SAMPLES should be taken at most an hour before the analysis
Random urine sample from a group mate
Urine sample from a sick person
MATERIALS
pH paper
Graduated cylinder
Filter paper
Beaker
Evaporating dish
Stirring rod
63
Test tubes
Test tube brush
Test tube rack
Water bath
Test tube holder
Hot plate
REAGENTS
2% K2C2O4
Dilute HCl
10% NH4OH
20% NaOH
Dilute HNO3
10% NaOH
10% AgNO3
0.05% CuSO4
10% BaCl2
Sodium nitroprusside
PROCEDURES
A. COLOR AND pH
1. Place urine sample in a test tube and identify its color.
2. Place a pH paper strip to determine its pH.
3. Record observations.
B. CATION ANALYSIS
1. Calcium. Place 3 ml sample in a test tube. To the sample, add 3 drops of
2% potassium oxalate solution. If precipitate will form, calcium is present.
Set aside for the next procedure
2. Magnesium. Filter the mixture from the previous step. Test the filtrate for
complete precipitation by adding dropwise potassium oxalate solution. If
no more precipitate will form, filter the mixture. To the filtrate, add drop
by drop 10% ammonium hydroxide until basic. Set aside and observe for
precipitation.
C. ANION ANALYSIS
1. Chlorides. Place 2 ml sample in a test tube. Add dropwise dilute nitric acid
until acidic. Add 2-3 drops of silver nitrate and observe for the formation
of white precipitate.
64
2. Sulfates. Place 2 ml sample in a test tube. Add dropwise dilute
hydrochloric acid until acidic. Add 2-3 drops of 10% Barium Chloride and
observe for the formation of white precipitate.
3. Phosphates. Place 5 ml sample in a test tube. Add dropwise dilute
ammonium hydroxide until basic. Precipitate formation signifies presence
of phosphate ions.
D. ORGANIC COMPONENTS’ ANALYSIS
1. Urea. Evaporate in a water bath 10ml of urine until volume is reduced to
one-third. Cool and add 2 ml of 20% sodium hydroxide. Mix well. To the
mixture add 2-3 drops of 0.05% copper sulfate.
2. Creatinine. Nitroprusside Test. Place 2 ml of urine sample in a test tube.
Add 2-3 drops of sodium nitroprusside. Add 3-5 drops of 10% sodium
hydroxide to make it basic. Observe the results.
65
DATA SHEET
Name: ______________________________
Date: ________________
Yr. & Sec.: ______________________ Group No. ____
Rating: _______________
URINALYSIS – NORMAL CONSTITUENTS
EXPERIMENT 7A
WRITE (+) for positive results and (-) for negative results.
SAMPLES’ RESULTS
TESTS
NORMAL URINE
SICK URINE
COLOR
pH
Calcium
Magnesium
Chloride
Sulfate
Phosphate
Urea
Creatinine
66
POST-LAB QUESTIONS
1. What does color of urine implies?
2. How does temperature affect the volume of urine excretion?
3. How does diet affect the urine composition?
4. How does urine differ from a normal person to a sick person?
CONCLUSIONS
67
EXPERIMENT 7B
URINALYSIS – PATHOLOGICAL ANALYSIS
There are several conditions where urine can have abnormal components. These
are called abnormal characteristics since these substances shouldn’t be found in the
urine sample of a healthy person. Conditions like proteinuria, oliguria, dysuria or
glucosoria are only some of the examples. In order to understand these conditions, one
should know the substances involve in these diseases. This experiment will let you
analyze a urine sample and determine the substances present in that sample. After
such, if there are abnormal substances present in that sample, you can roughly diagnose
the condition of the body.
OBJECTIVES
 Differentiate normal urine versus abnormal urine
 Identify the abnormal substances present in a given urine sample
 Correlate the findings to clinical significance
PRE-LAB ASSIGNMENT
1. Identify all abnormal substances that may be present in urine. Name the
conditions of such presence.
2. Identify the positive indicators on each test involve in this experiment.
WHAT TO BRING
URINE SAMPLES should be taken at most an hour before the analysis
Random urine sample from a group mate
Urine sample from a sick person
MATERIALS
Graduated cylinder
Test tubes
Beaker
Test tube rack
Stirring rod
Test tube holder
68
Test tube brush
Hot plate
Water bath
REAGENTS
Dilute NaOH
10% NH4OH
6M NaOH
5% Sodium nitroprusside
0.05% CuSO4
Saturated benzidine solution
Concentrated HNO3
Glacial acetic acid
Benedict’s Reagent
3% H2O2
PROCEDURES
TEST SAMPLES: In each analysis, prepare two test tubes with labels:
Normal Urine (NU) and Abnormal Urine (AU)
A. BIURET’S TEST
1. Place 1 ml each of the test samples in previously labeled test tubes.
2. Add to each test tube 10 drops of 6M sodium hydroxide and mix
thoroughly.
3. Add 2 drops of 0.5% copper sulfate solution. Mix well and record your
observation.
B. HELLER’S RING TEST
1. Place 1.5ml of concentrated nitric acid each in previously labeled test
tubes.
2. Slowly without disturbing, add the 2ml test sample on the inner wall of the
test tube.
3. Observe for a formation of fluffy white ring at the junction of two liquids.
C. BENEDICT’S TEST
1. Place 2 ml of Benedict’s solution each in previously labeled test tubes.
2. Add 1 ml each of the test samples in the tubes and mix thoroughly.
69
3. Boil in water bath for 2-3 minutes and set aside to cool.
4. Record observations according to the pattern below:
DATA
COLOR OF SOLUTION
GLUCOSE AMOUNT
(-)
Blue
< 0.1% or <100 mg/dL
(+)
Green, slight yellow ppt*
0.5% or 150 mg/dL
(++)
Green, thick yellow ppt
1.0% or 1,000 mg/dL
(+++)
Yellow, orange ppt
2.0% or 2,000 mg/dL
Orange, orange to red ppt
>2.0% or >2,000 mg/Dl
(++++)
D. LEGAL’S TEST
1. Place 1 ml each of the test samples in previously labeled test tubes.
2. Add dropwise dilute sodium hydroxide to make it slightly basic.
3. Add few drops of 5% sodium nitroprusside and equal drops of glacial
acetic acid.
4. Observe color change. A ruby-red or violet-red color indicates acetone. If
yellow color is observed, acetone is absent.
E. TEST FOR BILE ACID AND SALTS
1. Place 1 ml each of concentrated nitric acid in previously labeled test tubes.
2. By means of a dropper, slowly add 3-ml of test sample in each tube
without disturbing it.
3. Note the color of the rings (green nearest the urine, then blue, then violet,
red, and reddish-yellow nearest the acid) if positive.
F. TEST FOR BLOOD
1. Heat 2 ml of test sample to boiling. Cool then add equal amount of
saturated benzidine solution in glacial acetic acid.
2. Add 1 ml of 3% hydrogen peroxide.
3. Presence of blue/green color produced in less than 10 minutes signifies
blood in the sample.
70
DATA SHEET
Name: ______________________________
Date: ________________
Yr. & Sec.: ______________________ Group No. ____
Rating: _______________
URINALYSIS – PATHOLOGICAL ANALYSIS
EXPERIMENT 7B
WRITE (+) for positive results and (-) for negative results.
SAMPLES’ RESULTS
TESTS
NORMAL URINE
SICK URINE
BIURET
HELLER
BENEDICT
LEGAL
BILE SALTS AND
ACIDS
BLOOD
71
POST-LAB QUESTIONS
1. What substances are present in the two urine sample?
2. What are the differences of the two urine sample?
3. From the gathered results, what might be the condition of the two sample urine?
CONCLUSIONS
72