INTRODUCTION
Many  kidney  and other medical problems can be detected by routine urinalysis, which  includes chemical pH measurement,  tests to detect protein, sugar, and ketones and microscopic examination to detect red and white blood cells (not done in this experiment). In a doctor office,  dipsticks or strips  (strips of plastic  impregnated with chemicals) are routinely used in  urine testing. The chemicals in the stick react with substances in the urine and change color.
Protein  presence   in urine (proteinuria) is usually a sign  of kidney disease, but it may occur normally after strenuous exercise.
Glucose  in the urine (glucosuria)  is usually caused by diabetes.
Ketones in the urine (ketonuria)  may appear as  a result of  starvation,  uncontrolled diabetes, and  alcohol intoxication. Ketones are formed when the body  breaks down fat.
In some instances, doctors prescribe specialized tests for the levels of  inorganic ions, such as chloride, sulfate, calcium, ammonium, and phosphate. Although  normal urine contains these ions, some medical disorders (e.g. resulting from biochemical dysfunction) may cause either lower or increased concentration of some of these  ions.
                                                                                                                                        Useful link on urinary system
About this experiment
SAFETY

You will perform the  chemical tests on artificial urine samples, which include normal and pathological urine.
Normal urine samples will only  be   tested for the presence of  inorganic ions (qualitative tests).
Pathological urine samples and  the unknown sample  will only be tested  for  protein, glucose and ketones. Pathological urine sample may contain  one, two, or all three of the  above chemical species. Therefore all three:  proteinuria, ketonuria and glucosuria tests must be done on pathological and unknown sample.
You may also be able to perform a strip (dipstick)  test, based upon availability of strips.

PRELAB
1. What does it mean when  we say that a urine sample tested positive for ketones?
 

2.  List three most dangerous chemicals used in this experiment and  the health hazards associated with  them.
 
 

3 A. What equipment is required to test a normal urine sample for chloride, sulfate and calcium ion?
 
 

B. How is this different from what is used in the tests for ammonium  and phosphate ions?
 
 

4.  Explain briefly why normal urine contains inorganic ions (hint:  where do they come from?)

PROCEDURE
A. Qualitative Ion  Tests:  Normal  Urine Tested
Use  a glass or porcelain well plate for the tests 1- 3. Use 3 drops of urine sample for tests 1-3
Use a test tube for tests 4 and  5.
1.  Chloride  ion
Place 3 drops of urine sample in  a well.  Add 1 drop  of 3 M nitric acid to  the sample.  Does the color change  with the addition of the acid?  Add 1  drop of 0.1 M silver nitrate solution.
A white ppt indicates the presence of chlorides = positive  for Cl^- ion.
2.  Sulfate ion
Place 3 drops of urine sample in  a well.  Add 1 drop  of 3 M  hydrochloric acid to the sample. Carefully heat to boiling and add 2-3 drops of barium chloride solution.
A white precipitate indicates the presence of sulfates = positive for SO₄^2-  ion.
3.  Calcium ion
Place 3 drops of urine sample in  a well. Add 2 drops of  saturated  potassium (or ammonium) oxalate  to the sample.
A cloudy white precipitate ( may take up to 5 minutes to appear) indicates the presence of calcium ions = positive  for Ca²⁺ ion.
4.  Ammonium ion
Using a calibrated beral pipette  provided with a bottle, place  about 1 mL of  urine in a test tube. Add 0.7 mL of 6 M sodium hydroxide ( NaOH)  to the sample.  Carefully heat the liquid in a test  tube (do not boil!) while holding a moist piece of red litmus paper  inside the test tube but not touching the walls.
If the  paper turns blue and  ammonia smell can be detected, ammonium ion is present in the sample= positive test for NH₄⁺  ion.
5.  Phosphate ion
Add 2 drops of 3 M nitric acid to 1 mL of  urine in a test tube.  Add 10 drops of ammonium molybdate solution and place in water bath heated to  65^oC (use test tube holder!).
It may take a while, but a cloudy yellow precipitate which  clumps and settles constitutes a positive result for PO₄^3- ion.
B.  Tests for Protein, Glucose and  Ketones: Pathological Urine  and Unknown Sample  Tested ( test both simultaneously)
6.   Proteinuria :
Heat 2 mL of  proper pathological urine in a test tube by holding with a  test tube holder over a Bunsen burner flame.  Add 1 or 2 drops of glacial acetic acid to dissolve any precipitated phosphates. Reheat.
The  presence of any white precipitate indicates  protein presence = proteinuria= positive for protein
7. Ketonuria:
place 1 ml of urine in test tube and add sufficient nitroprusside reagent to saturate.  Shake
tube vigorously.  Cautiously add  of concentrated  ammonium hydroxide to form an separate layer on top.  Note  the color at the interface of the two layers.
Purple color indicates the presence of ketones = positive test result for ketones.
8.  Glucosuria:
Add 4 drops of  urine to 2 mL of Benedict's reagent in a test tube.  Heat in a boiling water bath  for 5 min.  Cool to room temperature.
A clear blue solution indicates no glucose or other reducing  sugar = negative test result  for glucose
Green  color  = 0.25% glucose;  yellow  color =  1% ,   orange color =   between 1% and  2%,   brick red color = more than 2%

DATA SHEET
A. NORMAL URINE SAMPLE
For the following normal urine constituents place (+) if the  test result is positive or (-) if it is negative (constituent not found).

ION	RESULT  (+) OR (-)	OBSERVATIONS
1.Chloride
2. Sulfate
3. Ammonium
4. Calcium
5. Phosphate

B. PATHOLOGICAL URINE SAMPLE  AND  UNKNOWN

TEST	PATHOLOGICAL URINE SAMPLE TEST RESULT	UNKNOWN #____________
PROTEINURIA	positive (+) or  negative (-) Observations:	positive (+) or  negative (-) Observations:
kETONURIA	positive (+) or  negative (-) Observations:	positive (+) or  negative (-) Observations:
GLUCOSURIA	0.25%                    1-  2%              >2% Observations:	0.25%                      1-  2%              >2% Observations:

 

OPTIONAL : STRIP- TEST FOR PROTEIN, GLUCOSE AND KETONES ( if strips available)
To perform a strip test  immerse a multi purpose urine test strip in a sample of urine for 1-2 seconds making sure that the entire test area of the strip comes in contact with the urine. After 1 minute compare the  colors with the color code on the box  for  ketone and   protein  and record results/values  corresponding to those colors. After 3 minutes, compare the color  on the glucose line  and record the corresponding result/value on your data sheet.

Unknown # ___________________

                               Strip Color         Result/ value

Protein:
 

Ketones:
 
 

Glucose:
 

POSTLAB
1. Briefly explain the  biochemical mechanism by which ketones are produced in the body.
 
 
 
 
 
 
 
 
 
 
 
 

2. Normal urine does not  contain detectable levels of  glucose. Why?