BIOCHEMISTRY

CHEMISTRY OF URINE
PURPOSE
1. Test urine for pH, specific gravity, and the presence of electrolytes and organic
compounds.
2. Test urine for the presence of abnormally occurring compounds of proteins, glucose, and
ketone bodies.
MATERIALS
urine samples (normal and pathological)
urinometer
blue cobalt glass
pH paper
6M HNO3
1% urease
flame-test wire
conc. HCl
1M HOAc
litmus paper
0.1M (NH4)2C2O4
test tubes
(NH4)2SO4(s)
plastic wrap
5% nitroprusside reagent
beakers
conc. NH4OH
0.1M AgNO3
Benedicts reagent
0.1M BaCl2
6M HCl
ammonium molybdate solution
OPTIONAL: Reagent strips or tablets such as Clinistix, Clinitest, Ketostix, Albustix
DISCUSSION OF EXPERIMENT
Examining a sample of urine can give such information about the processes occurring
within the body. The amounts of electrolytes, 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.

O
NH2 C NH2

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 acid crystals.

GLUCOSE
Glucose, if present, can be detected by Benedicts 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 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 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.
LABORATORY ACTIVITIES
Collection of Urine. Use a clean, 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.
1. 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 results.
O

H2O

NH2CNH2
urea

urease

2NH3 +
ammonia

CO2

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 GOGGLES.
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 0.1M AgNO3. A white precipitate (AgCl) confirms the presence of
chloride.

D-2

Sulfate
Place 3 mL of each urine sample in separate test tubes. Add 1 mL 6M
HNO3 and 10 drops of 0.1M BaCl2. 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 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
(NH4)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 Benedicts
reagent to each. Place the test tubes in a boiling water bath for 5 minutes. Record any
changes in color. If glucose is present, estimate the amount.
Color with Benedicts
Blue

mg%

mg/dL

0.10

100

Blue-green
Green
Yellow
Orange

0.25
0.50
1.00
2.00

250
500
1000
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 tubes. Saturate each with
ammonium sulfate, (NH4)2SO4. Add 5 drops nitroprusside reagent. CAUTION: TOXIC. Tip
the test tube and CAREFULLY add 20 drops of concentrated NH4OH 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
Place 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.

LABORATORY RECORD
A. COLOR, pH AND SPECIFIC GRAVITY
Own Urine

Pathological Urine

A-1

Color

______________________

________________________

A-2

pH

______________________

________________________

A-3

Specific
Gravity

______________________

________________________

What is your interpretation of the above tests?

B. UREA
Own Urine
Effect on litmus

Pathological Urine

____________________

______________________

What is your interpretation of the urea test?

C. URIC ACID
Crystals

Own Urine

Pathological Urine

___________________

______________________

What is your interpretation of the uric acid test?

Is uric acid normally found in urine? Why?

D. ELECTROLYTES
Indicate the presence of electrolytes as follows:
Not present ( - )
Present ( + )
Strongly Present (++)
Own Urine

Pathological Urine

D-1

Cl-

__________________

______________________

D-2

SO42-

__________________

______________________

D-3

PO43-

__________________

______________________

D-4

Na+

__________________

______________________

K+

__________________

______________________

Ca2+

__________________

______________________

D-5

What is your interpretation of the above tests?

E. GLUCOSE

Own Urine

Pathological Urine

Benedicts

__________________

______________________

Estimate of mg%

__________________

______________________

Estimate of mg/dL

__________________

______________________

Test paper (_________)

Name

__________________

______________________

What is your interpretation of results?

When would glucose be found in a urine sample? Why?

E. KETONE BODIES
Own Urine

Pathological Urine

Color of ring

__________________

______________________

Test paper(_________)
name

__________________

______________________

What is your interpretation of the test for ketone bodies?

When would ketone bodies be found in a urine sample? Why?

F. PROTEIN
Own Urine

Pathological Urine

Appearance

__________________

______________________

Test paper (___________)

Name

__________________

______________________

What is your interpretation of the protein test results?

When would protein appear in a urine sample?

G. BAR GRAPH OF ELECTROLYTES

The following lists the component in a typical urine sample at a pH of 5.5. Draw a
bar graph to represent this data.
Concentration

(mol/L) of Electrolytes in Urine pH 5.5

Cations

Anions

Nonelectrolytes

Na+

170

Cl-

175

Urea

300

K+

90

HPO43-

45

Creatinine

10

Ca2+

SO42-

60

Mg2+

Organic
acids

45

NH4+

60