For instructional purposes only • 1st Semester SY 2020-2021 61

Exercise No. 9:
Chemistry of Urine
Introduction
Examining a sample of urine can give such Information about the processes
occurring within the body. The amounts of electrolyte, uric acid, and glucose
can all lead to conclusions about the functioning of the kidneys, liver and the
general state of health of the individual.
IONS IN URINE
Urine normally consists of about 96% water. The other 4% consists of waste
products being eliminated from the cells of the body to maintain proper
osmotic pressure, electrolyte levels and pH. Urine normally contains the
inorganic ions Cl -, SO42-, PO43-, K+, Na+, NH4+, and Ca2+. Organic components
normally found include urea and uric acid. Urea is an end product of protein
metabolism and uric acid is an end product of purine metabolism.

Urea

The presence of Na+ and K+ ion can be determined by flame tests. The
present of other electrolytes will also be determined. Uric acid will be
detected through the formation of uric add crystals.
GLUCOSE
Glucose, if present, can be detected by Benedict’s test. Glucose may show up
in urine (glucosoria) when high amounts of glucose accumulate in the blood
and the renal threshold is exceeded. Conditions such as diabetes mellitus and
liver damage may be indicated.
pH and SPECIFIC GRAVITY OF URINE
Urine has usually has a pH around 6.0, although this varies considerably with
diet and activity and can have a range from 4.6 to 8.0 at different times. Urine
normally has a light yellow color derived from pigments formed by the
breakdown of bilirubin obtained from the destruction of red blood cells. The
normal range for specific gravity is 1.005 to 1.030. In this experiment, you will
test the pH of a urine sample, measure its specific gravity and note its color.
KETONE BODIES
Ketone bodies such as acetone and acetoacetic acid may appear in the urine
when large amounts of fat are metabolized for energy purposes due to an

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 62 CHEM 132.2: Biochemistry Laboratory

insufficiency of glucose in the diet or an inability to utilize glucose as in

diabetes mellitus. Ketone bodies are associated with certain diets (low
carbohydrate), starvation, diabetes mellitus, and liver damage. High levels of
protein (proteinuria) may indicate disease or damage to the kidneys or urinary
tract.

Learning Outcomes
1. To test urine for pH, specific gravity, and the presence of electrolytes
and organic compounds.
2. To test urine for the presence of abnormally occurring compounds of
proteins, glucose, and ketone bodies.

Materials
 Urine samples (normal ammonium molybdate 0.1 M (NH4)2C2O4
and pathological) sol’n
 Urinometer 0.1 M AgNO3 (NH4)2SO4 (s)

 pH meter 0.1M BaCl 2 5% nitroprusside

reagent
 1% urease Beakers Conc. NH 4OH

 Conc. HCl Blue cobalt glass Benedict’s reagent

 litmus paper 6M HNO3 6M HCl

 Test tubes Flame-test wire

 Plastic wrap 1M HOAc

Procedure

Collection of Urine. Use a dean, dry bottle or a beaker to collect a sample (50
mL) or urine. A morning sample is preferable. If you will not be testing the
urine sample immediately, store it in a refrigerator until the laboratory hour.
You will carry out the following urinalysis on your own urine sample and on a
pathological sample prepared by your instructor.

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For instructional purposes only • 1st Semester SY 2020-2021 63

A. COLOR, pH and specific gravity

A-1 Obtain 50 mL of your own urine and 50 mL of a pathological urine
sample. Describe the color of the urine.
A-2 Determine the pH of the urine.
A-3 Determine the specific gravity of the urine sample with a urinometer.

B. UREA
Place 5 mL of each urine sample in separate test tubes. Add 2 mL 1%
urease solution to each. Let the mixtures stand for 1 hour. Heat the test
tubes gently while you hold a piece of moistened litmus paper across the
top of the test tubes. The evolution of ammonia will turn the paper blue,
indicating the presence of urea. Record the results.
=O

Urease

H2O + NH2CNH2 2NH3 + CO2

Urea Ammonia

C. URIC ACID
Place 25 mL of each urine samples in small, separate beakers. Add 20
drops of conc. HCl to each. CAUTION: HCl MUST BE HANDLED
CAREFULLY. USE GOOGLES. Cover the beaker with plastic wrap. Let the
solutions stand until the next laboratory period. Look for the appearance
of any uric acid crystals. Record results.

D. ELECTROLYTES
D-1 Chloride Place 3 mL of each urine sample in separate test tubes. Add
1 mL 6M HNO3 and 10 drops 0.1 M AgNO3. A white precipitate (AgCl)
confirms the presence of chloride.

D-2 Sulfate Place 3 mL of each urine sample in separate test tube. Add 1
mL 6M HNO3 and 10 drops of 0.1M BaCl 2. A white precipitate (BaSO4)
confirms the presence of sulfate.

D-3 Phosphate Place 5 mL of each urine sample in separate test tubes.

Add 2 mL 6M HNO3 and 3 mL ammonia molybdate solution. Heat gently.
A yellow precipitate confirms the presence of phosphate.

D-4 Sodium Clean a flame-test wire in 6M HCl and then dip the wire in
one of the urine specimens. Heat the loop of the wire in a flame. A bright-
yellow flame indicates the presence of sodium ion.
Potassium Dip a flame-test wire into one of the specimens again.
Cover your eye with a blue cobalt glass square, and observe the color of
the flame through the glass as you heat the wire. The appearance of a

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 64 CHEM 132.2: Biochemistry Laboratory

deep red flame through the glass indicates the presence of potassium.
Repeat the flame tests with each urine sample.

D-5 Calcium To 3 mL of each urine sample, add 5 drops 1M HOAc and 2

mL 0.1M (NH 4)2C2O4. A cloudy, white precipitate of calcium oxalate
confirms the presence of calcium.

E. GLUCOSE
Place 8 drops of each urine sample in separate test tubes. Add 5 mL
Benedict’s reagent to each. Place the test tubes in boiling water bath for 5
minutes. Record any changes in color. If glucose is present, estimate the
amount.

Color with Benedict’s mg% mg/dL

Blue 0.10 100
Blue-Green 0.25 250
Green 0.50 500
Yellow 1.00 1000
Orange 2.00 2000
Commercially available test strips such as Clinistix or tablets such as
Clinitest may also be used to test for the presence of glucose. Follow
directions on the Package.
F. KETONE BODIES
Place 5 mL of each urine sample in separate test tube. Saturate each
with ammonium sulfate, (NH 4)2SO4. Add 5 drops nitroprusside reagent.
CAUTION: TOXIC. Tip the test tube and CAREFULLY add 20 drops of
concentrated NH 4OH down the side. The presence of a purple ring where
the layers meet indicates the presence of ketone bodies.
Commercially available test strips such as Ketosix may also be used to
test for the presence of ketone bodies. Follow directions on the package.
G. PROTEINS
PIace 5 mL of each urine, sample in separate test tubes. Filter urine first if
it is not clear. CAREFULLY heat the upper one-half of the liquid to boiling
using the Bunsen burner. Add 5 drops 1M HOAc. Heat the upper portion
for another 1-2 minutes. The formation of a white cloudy precipitate
indicates the presence of protein.
Commercially available test strips such as Albustix may also be used to
determine the presence of protein (albumin} in the urine samples.
H. BAR GRAPH OF ELECTROLYTES
Use graph paper to draw a bar graph of the cations and anions typically
found in body fluids.

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For instructional purposes only • 1st Semester SY 2020-2021 65

Guide questions and instructions on how to prepare

and submit the laboratory

Additional Resources

Use References style here

References

Robyt, J.F. and B.J. White. 1987. Biochemical Techniques: Theory and Practice. Brooks/Cole
Pub. Co. : Monterey, California; pp.407
Timberlake, K. 1988. Laboratory Manual for Chemistry. Harper Collins Publishers Inc. : New
York, pp 277-372.

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Mission: Development of a highly competitive human resource, cutting-edge scientific knowledge TP-IMD-04
and innovative technologies for sustainable communities and environment. V0 07-15-2020
No.