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DEPARTMENT OF PURE AND APPLIED CHEMISTRY

Visayas State University, Baybay, Leyte


CHEM 31 Biochemistry
Laboratory Report

Name: Loise Mariel C. Bibera Date Performed: 05/25/15

Lab. Schedule: M-F 7:00-10:00 Date Submitted: 06/01/15

Group No: 4 Score:

Experiment No. 9

Chemistry of Urine

I. Objectives:
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.

II. Results
A. Color, pH and Specific Gravity

Test Urine Sample


A-1 Color Light Yellow
A-2 pH 5
A-3 Specific Gravity 1.004

B. Urea
Effect on Litmus Blue

C. Uric Acid

Crystals No Crystals Formed


D. Electrolytes

D-1 Cl-
D-2 SO42- Not Present (-)
D-3 PO43-
D-4 Na+ Strongly Present (++)
K+ Present (+)
D-5 Ca2+ Present

E. Glucose

Benedicts Test Blue


Estimate of mg % 0.10 mg %
Estimate of mg/dL 100 mg/dL

III. Discussion

The excretion of urine, is very important for different bodily functions. It regulates the
balance of water in the body. On the other, substances are released along with the urine that
are produced during metabolism and are no longer needed by the body. These also include
toxic substances, which may have been absorbed from food, or medicines. The composition of
urine can vary greatly and constantly fluctuates with dietary intake (food and water) and
metabolic activity. Urine consists mostly of water with various organic and inorganic substances
such as urea, uric acid, creatine, sodium chloride, ammonia, sulfates, and phosphates as its
principal ingredients. By examining urine, indications for diseases of the urinary system can be
detected and it can also provide evidence of metabolic diseases like diabetes or some type of
kidney or liver disease. In this experimental procedure basic qualitative tests have been
conducted on a freshly collected urine sample. This is to observe some of its physical properties
and detect the presence of certain compounds or chemicals which may be indicative of an
underlying disease.

A. Color, pH and Specific Gravity

The physical characteristics of urine include observations and measurements of color,


specific gravity and pH. Visual observation of a urine sample can give important clues as to
evidence of pathology.

COLOR

The color of normal urine is usually light yellow to amber. Generally the greater the
solute volume the deeper the color. The yellow color in urine is due to chemicals called
urobilins. These are the breakdown products of the bile pigment bilirubin. Bilirubin is itself is a
breakdown product of the heme part of hemoglobin from worn-out red blood cells. Deviations
from normal color can be caused by certain drugs and various vegetables such as carrots,
beets, and rhubarb. An unusual urine color can be a sign of disease. For instance, deep red to
brown urine is an identifying characteristic of porphyria, a rare, inherited disorder of red blood
cells. The urine specimen is colored light yellow thus, it is considered normal.

pH

On testing its pH, the collected urine sample gave a pH of 5 which is slightly acidic. Normal,
freshly-voided urine may have a pH range of 5.5 - 8.0. The pH of urine may change with diet,
medications, kidney disease, and metabolic diseases such as diabetes mellitus.

Specific Gravity

Aside from pH the specific gravity of urine was also determined by using urinometer. The
specific gravity of a solution is the ration of the weight of a given volume of the solution (urine)
to the weight of an equal volume of water. It indicates the concentration of dissolved solids
such as urea, phosphates, chlorides, proteins present in the urine. Normal specific gravity is
1.005 - 1.030 with most normal falling between 1.010 and 1.025. The higher the number the
more concentrated the urine.

B. Urea

Urea is a waste product of many living organisms, and is the major organic component
of human urine. This is because it is at the end of chain of reactions which break down the
amino acids that make up proteins. These amino acids are metabolized and converted in the
liver to ammonia, CO2, water and energy. But the ammonia is toxic to cells, and so must be
excreted from the body. So the liver converts the ammonia to a non-toxic compound, urea,
which can then be safely transported in the blood to the kidneys, where it is eliminated in urine.

The presence of urea in urine can be tested through the addition of NaOH to the urine
sample this reaction will produce ammonia. The evolution of ammonia on the heated urine
sample will turn the red litmus to blue, indicating the presence of urea. The equation for the
conversion of urea to ammonia is shown below.

2NaOH + (NH2)2CO 2NH3 + Na2CO3


Sodium hydroxide + urea ammonia (g) + sodium carbonate

C. Uric Acid

Uric acid is a heterocyclic compound of carbon, nitrogen, oxygen, and hydrogen with
the formula C5H4N4O3. It forms ions and salts known as urates and acid urates, such as
ammonium acid urate. Uric acid is a product of the metabolic breakdown of certain foods that
contain purine nucleotides, such as liver, anchovies, mackerel, dried beans, beer and wine. High
blood concentrations of uric acid can lead to gout, a type of arthritis. The chemical is associated
with other medical conditions including diabetes and the formation of ammonium acid
urate kidney stones. For this part concentrated HCl was added to urine this is to acidify the
solution even further because uric acid will tend to be insoluble in extremely acidic pH levels.
Crystals of uric acid will be observed if uric acid is present. The tightly covered mixture was
allowed to sit for 24 hours for uric acid crystals to form. After sitting the mixture for a day
presence of crystals was not observed, indicating a low uric acid or no amount at all. This might
be an indication of kidney problems, meaning that the kidneys arent able to get rid of the uric
acid well enough.

D. Electrolytes

Electrolytes are chemicals that form electrically charged particles (ions) in body fluids.
These ions carry the electrical energy necessary for many functions, including muscle
contractions and transmission of nerve impulses. Many bodily functions depend on electrolytes;
optimal performance requires a consistent and adequate supply of these important nutrients.

Urine normally contains these electrolytes which include Cl-, SO4-, PO4-, K+, Na+, and
Ca+. Any of the excess electrolytes are then filtered out by the kidneys and particularly out
through the urine. The types of test conducted to indicate the presence of such ions is
discussed below.

Chloride

Chloride is the relative anion (negatively charged ion) that accompanies sodium. This
electrolyte is absolutely necessary in maintaining the osmotic tension in both blood and
extracellular fluids. Chloride can be obtained from the diet with foods containing certain
amounts of chloride (e.g table salt). The test involved the addition of a solution of silver nitrate
to the sample of urine that has been acidified with nitric acid. This is to prevent silver from
reacting with carbonates which also produce a white precipitate giving an inaccurate result.
Having the solution acidified formation of a white precipitate indicates the presence of chlorides.
The equation for this reaction is shown below:

Cl- + AgNO3 AgCl (s) + NO3-


Sulfate

Sulfates are considered to be among the most important macronutrients in cells, being
the major source of sulfur in many living organisms. They are metabolically inert which means
they do not possess catalytic function in human energy metabolism. To know their presence the
procedure made use of the solubility rules. By adding in a solution of barium chloride to
acidified urine barium chloride was precipitated from the solution. The equation for this reaction
is shown below:

SO42- + BaCl2 BaSO4 (s) + 2Cl-


Phosphate

Building and repairing bones and teeth, aid in muscle contraction and helps nerve
function are some of the functions of phosphates. This was tested in the experimental
procedure through reaction of the acidified urine sample with ammonium molybdate, which
produced a yellow precipitate indicating the presence of phosphates. The ammonium molybdate
((NH4)2MoO4) forms a precipitate of ammonium phosphomolybdate ((NH4)3PO4.12MoO4) which
is a bright yellow compound that is extremely insoluble even in dilute nitric acid.

Sodium and Potassium

Sodium and potassium are the chief cations (positively charged ion) outside and within
muscle cells. The existences of each were identified using flame tests. . A flame test wire was
dipped into some hydrochloric acid and heated to red hot in the flame of a Bunsen burner. This
was done to remove any forms of contaminants and other residues that may affect the result.
The wire was then dipped into the urine solution and then heated. The formation of a yellow
flame indicated the presence of sodium and the formation of a red flame as seen through a
cobalt glass square indicated the presence of potassium in the urine sample.

Calcium

Calcium is the most abundant mineral in the human body. Normal heart rhythm, healthy
nerve transmission, and strong muscle contractions require a constant blood calcium
level. Same with sulfate this test implies the solubility rules. After acidifying urine it was then
reacted with ammonium oxalate. The formation of a white precipitate of calcium oxalate
indicated a positive result for the urine sample. Shown below is a simple equation for the
reaction:

Ca2+ + (NH4)2C2O4 CaC2O4 + 2NH4+


E. Glucose

The presence of glucose in urine is called glycosuria. This condition indicates that the
blood glucose level has exceed the renal threshold. This condition may occur in diabetes
mellitus. Benedicts reagent was added to the sample and was boiled for about 5 minutes. If
glucose is present, the estimate amount can be assed with the values given from the manual.
From the observations on the tested sample there is no relative difference from its initial blue
color. From the table the blue color is approximately equivalent to 0.10 mg or 100 mg/dL
glucose present in the urine sample which is of a low concentration.

F. Ketone Bodies
When the body metabolizes fats incompletely, ketones are excreted in the urine
resulting in ketonuria. The ketone test is based on the development of colors ranging from light
pink to maroon when ketones react with nitroprusside. Ketonuria may be present in diabetes
and starvation or fasting. Since ketones will evaporate at room temperature, urine should be
tightly covered and refrigerated if not tested promptly. In normal urine, ketone bodies will not
be present. The urine sample tested negative for ketone bodies. This test is also known as
Rotheras Test for testing the presence of urine. This was done by saturating with ammonium
sulfate in order to concentrate the ketone bodies to the center of the solution and to prevent
any side rections. Nitroprusside was then added to this mixture, followed by the gradual
addition of ammonium hydroxide or ammonia solution. The presence of a purple ring where the
layers meet indicates the presence of ketone bodies. The purple ring is a product of the
complex between the ketone and the nitroprusside in the presence of ammonia.

G. Proteins

Protein in the urine is called proteinuria. This is an important indicator of renal disease,
but can be caused by other conditions as well. Usually healthy individuals dont contain proteins
in their urine because their excess proteins are well regulated by the kidneys. To test whether
there are proteins present in the sample drops of HOAc was added. The addition of HOAc
provided a development of minor turbidity. After the test solution was heated very small
amounts of white precipitate was observed. Having such small amounts of precipitate indicates
that the sample only contained trace amounts of proteins.

IV. Conclusion

A clinical examination of urine can provide a convenient, cost effective and non-invasive means
of assessing kidney function and providing an overall assessment of our body's health.

V. References

Exercise 13: Chemical Examination of Urine

http://www.pkdiet.com/pdf/Urinary%20pH%20testing.pdf
Complete Urinalysis

http://www.highlands.edu/academics/divisions/scipe/biology/faculty/henderson/APII/Lab%20m
anual/urinalysis_lab.htm

Why is Urine Yellow?

http://sxxz.blogspot.com/2006/07/why-is-urine-yellow.html#sthash.RYkqLoNi.dpuf

Urea

http://www.ch.ic.ac.uk/rzepa/mim/environmental/html/urea_text.htm

Uric Acid

http://en.wikipedia.org/wiki/Uric_acid

Electrolyte Replenishment - Why Its So Important and How to Do It Right


http://www.hammernutrition.com/knowledge/electrolyte-replenishment-why-it-146-s-so-
important-and-how-to-do-it-right.1274.html

"Protein Urine Test"


http://www.healthline.com/health/protein-electrophoresis-serum#Overview1