EXPERIMENT II

BLOOD
Date of Works: October 1st, 2015
A. OBJECTIVES

1. Understanding of main component that contained in blood.

2. Skill of making blood plasma and blood serum.
3. Skill in doing test to blood plasma, test of Fe in hemoglobin, test of albumin
and globulin in blood serum and test of non protein substance in blood serum.
B. INTRODUCTION
Blood is a fluid present in the blood vessels of the color red. Blood serves as a
conveyance which takes oxygen from the lungs to be distributed to all body
tissues, to transport carbon dioxide from the tissues to be removed through the
lungs, taking nutrients from the small intestine to be circulated and distributed to
all tissues of the body, secrete substances not useful for the body to be removed
through the skin and kidneys, as the body's defense against disease, spread the
heat throughout the body. (Syaifuddin, 2006).

Pict 1. Blood

Blood is a liquid tissue that consists of two parts, namely blood plasma and
blood cells. Blood cell consists of three types of erythrocytes, leukocytes, and
platelets. Overall blood volume is one-twelfth of body weight or about five liters.
Approximately 55% is blood plasma, while the remaining 45% consists of blood
cells. The nature of which blood has an osmotic pressure of 28mmHg, a viscosity
of 1.7 at 37 C and a pH of 7.0 to 7.8 (Pearce, 2006).
Blood plasma contains about 90% water. Among the various types of
substances dissolved in water are inorganic salts. Sometimes - sometimes called
1

blood electrolytes and blood plasma contained in the form of dissolved ions. The
combined concentration of these ions is important in the maintenance of osmotic
balance 'These ions assist in supporting the blood PH. Be some ions also have a
pH of 7.4 in humans. The ability of the brain and nerves to function normally also
depends on the concentration of ions key in interstitial fluid (Campbell, 2002).
Blood components that have assignment to transport oxygen is pigment with
chromoproteida structure, contain of globin protein and hem color substance. Hem
is derivate of protophorpin with Fe2+ ion included prosthetic group. Haemoglobin
(Hb) is blood color substance composed of group prosthetic protohem
(ferroprotophorfirin) and globin protein. Protohem in every animal is same, only
its protein. Blood is coagulaned easily, to prevent it add anti-coagulaned substance
such as oxalate compound, sitrate or EDTA.
The main function of blood in circulation is as a transport medium, regulating
body temperature, maintenance of fluid balance, as well as erythrocytes alkaline
balance in his life remains in the body. Red blood cells capable of transporting
effectively without leaving their functions within the network as well as its
presence in the blood just passed away. Blood also has a so-called blood platelets
or platelets that play a role in blood clotting process (Hilman et al., 2005).
Blood clotting events by Dr. Karl Landsteiner in 1901 serve as the basis for
classification of blood by the ABO system. Landsteiner discovered that the
erythrocytes of some individuals will clot when mixed with blood serum from
another individual, but this happened to everyone. Blood clotting mechanism is
going to undergo platelet clumping (agglutination) due to tissue damage or injury.
Platelet clots are called thromboplastin. Ca ions contained blood preformance
causing Factor VII into konvertin. Then reacted with konvertin thromboplastin
and Ca ions transform prothrombin into thrombin, but only slightly. Thrombin
binds to Ca ions and in the presence of thrombin then accelarator plasma globulin
serum accelarator from inactive to active. Prothrombin turns into thrombin and
fibrinogen activate. Fibrinogen will turn into fine threads, called fibrin (Campbell
et al., 2006).
Some functions of blood in the body:
1. Breathing: transport oxygen from the lungs to the tissues of carbon dioxide
from the tissues to the lungs.
2. Nutrition: tranport adsorbed substances through the intestinal wall.
3. Excretion: tranport remnants of metabolism to the kidneys, lungs, skin and
intestines for disposal.
4. Set the temperature to flatten body heat.
5. Set the acid-base balance in the body.
6. Tranport hormones and metabolites.
i, 2009)
.

C. Apparatus and Reagent

Apparatus:
Burner
Tripod
Gauze
Cuvet centrifuge
Centrifuge
Reaction tube
Filter paper
Porcelain cup
Stirrer
Reagent:
Blood
Acetic acid glacial
Dilute NaCl
Zn(OH)2
CaCl2 20%
Dilute HCl
Nitric acid concentrate
Dilute potassium rodanide
Pottasium ferricyanide
Saturated of ammonium sulfate
Solid of ammonium sulfate
Glycerol
Solid of sodium carbonate
CH3COOH 2%
Dilute ammonium oxalate 10%
Red chlor phenol indicator
Dilute HNO3
Dilute AgNO3
Dilute CuSO4
Dilute BaCl2

D. Procedure
1. Manufacture of Blood Plasma and Blood Serum

Prepare fresh

Add 1 ml Zn(OH)2

chicken blood

in kuvet centrifuge

Filter the

Centrifuge 10 minutes

precipitate, take 2

until form precipitate

ml of fitrate.
Add 30 ml NaCl and
2 drops CaCl so form
precipitate

Filter precipitate.
The filtrate called
blood serum.

2. Fe in Hemoglobin Test

Take 10 drops blood

Heated until all

in porcelain cup

burnt out

Pour mixture in

Prepare test tube

porcelain cup, stir until

of HCl and HNO3

ash dissloved
In different test tube,
add K4[Fe(CN)6] in tube

Observe and
write the result

I, KCNS in Tube II

3. Non Protein Substance Test

a. Ca2+ Test (Calcium Ion)
Take 1 ml filtrate

Add 1 ml

put into test tube

ammonium oxalate
Observe and write

b. Cl Test (Chloride Ion)

the result

Take 1 ml filtrate

Add 1 ml AgNO3

put into test tube

and HCl

c. Glucose Test

Observe and write

the result

Take 1 ml filtrate

Add 2 drops glycerol

put into test tube

and Na2CO3

Observe and write

Add 2 drops CuSO4

2result
d. SO4the
Test
(Sulfat Ion)

+ Fehling A & B

Take 1 ml filtrate

Add several drops

put into test tube

BaCl2

E. Observation Data
1. Fe in Hemoglobin Test

Observe and write

the result
+/+

Treatment
Before blood serum was
heated
Ash + HCldilute + HNO3p.a

Observation
Blood become ash
Light yellow color solution

Tube 1 + K4[Fe(CN)6]

Greenish blue color

solution and precipitate
Brownish orange color
solution and red precipitate

Observation
Muddy white color solution

+/+

Observation
Muddy white color solution

+/+

Tube II + KCNS

2. Ca2+ Test (calcium ion)

Treatment
Blood serum + (NH4)2C2O4

3. Cl- Test (chloride ion)

Treatment
Blood serum + AgNO3 +
HNO3

4. Glucose Test
Treatment
Blood serum + glycerol +
Na2CO3 + Fehling A&B

Observation
Blue color solution. After
heated, solution produce
red precipitate.

+/+

Observation
Colorless solution

+/+

5. SO42- Test (sulfate ion)

Treatment
Blood serum + BaCl2

F. Discussion
1. Manufacture of blood plasma and blood serum
Blood plasma is made of 10 ml of fresh chicken blood were added 1 ml
solution of Zn (OH) 2 to 5% in the cuvette centrifuges. Then do the centrifuge
for 10 minutes until the resulting precipitate. Addition of Zn (OH) 2 function
so that blood does not clot quickly. The precipitate is filtered with filter paper.
Sediment left on the filter paper is called packed cell and the filtrate is called
called blood plasma.
Blood serum made from 2 ml of blood plasma were added 30 ml of 0.9%
NaCl solution and then added another 2 drops of 20% CaCl2 solution so that
the resulting sludge then the precipitate is filtered with filter paper. Sediment
called fibrinogen, and the filtrate is called blood serum.
Plasma is generated crimson blood serum viscous and clear yellow.
2. Fe in Hemoglobin Test
In this test done warming up of blood plasma burning all. It is intended
that the molecule / organic compounds in the blood disappear leaving only
inorganic elements such as Fe2+ which undergoes oxidation to Fe3+ due to
warming. There are two circumstances oxidizing Fe atom is +2 and +3 (ions
Fe2+ and Fe3+). Hemoglobin normally carries Fe2+, but sometimes oxidized to
Fe3+ ions.
After warming continued leaching process by using dilute HCl and
HNO3p.a, with a ratio of 3: 1 and, stirring constantly warming. Forming fluid

that is clean, then divided into two test tubes clean and dry to test the presence
of Fe in the blood. Reactions that occur are:
Fe2+ Fe3+ + ePict 2. Reaction of Fe oxidation

The addition of aqua regia (HCl and HNO 3) serves to bind Fe. The
reaction is:
Fe3 + + 3Cl- FeCl3
Pict 3. Reaction of addition aqua regia in Fe

In the first tube, the filtrate is added a solution of K4 [Fe (CN) 4] produces
a blue-green solution. The reaction as follow:
Fe3 + + K4[Fe(CN)6] FeK[Fe(CN)6] + 3K+
Pict 4. Reaction of Fe and potassium perrocyanide

On the tube II, the filtrate plus KCNS solution produces brownish orange
solution. The reaction is as follows:
Fe3+ + 3KCNS Fe(CNS)3 + 3K+
Pict 5. Reaction of Fe and potassium rodanide

But after a few minutes, on the tube I changed color to bluish green and
the second tube changes color to orange. This is because there are reactions
that take place.
3. Ca2+ Test (Calcium Ion)
Testing was done using 1 ml of filtrate / blood serum is added to the
(NH4)2C2O4 10%, where the compound is specific to the function more
calcium so that calcium can be bound by oxalic resulting in no free calcium to
help the process of blood coagulation. In the Ca2+ positive test contained in the
blood is evident with white turbid solution. His reaction was as follows:
Ca2+ + (NH4)2C2O4 CaC2O4(s) + 2NH4+
Pict 6. Reaction of calcium ions and ammonium oxalate

4. Cl- Test (Chloride Ion)

In this test is used as much as 1 ml of blood serum were added AgNO 3 and
HNO3 which AgNO3 will form the acid atmosphere convert into the
hydrochlorid acid. The hydrochloric acid will react AgNO3 to form white
precipitate AgCl. In this case the blood that tested positive for Cl -. Reactions
that occur are:

HCl + AgNO3 AgCl(s) + HNO3

Pict 7. Reaction of HCl and ammonium oxalate

5. Glucose Test
In this test, a blood serum was added to the mixed solution of Fehling A
and B then to accelerate the reaction, the solution is heated to form a brick red
solution and brick red precipitate is also formed. This indicates that the blood
positive containing glucose. Reactions that occur are:

C H

C OH

H C
HO C

OH
H

H C
+ Cu2+ + 4OH-

HO

OH
H

H C

OH

H C

OH

H C

OH

H C

OH

CH2OH

+ Cu2O + 2H2O

CH2OH

Pict 8. Reaction of blood + fehling A & B

6. SO42- Test (Sulfate Ion)

In this test, a blood serum was added dilute BaCl2. These additions serve
to white precipitate of sulfate ions. Based on observations, showed positive
results that the blood contains sulfate ions with the formation of a white
precipitate. Reactions that occur are:
BaCl2 +

SO42-

BaSO4

+ 2Cl-

white precipitate
Pict 9. Reaction of BaCl2 and sulfate ions

G. Conclussion and Suggestion

Conclusion :
1. The main component of blood is the blood cells and blood plasma.
2. Blood plasma contains Fe3+ ions in hemoglobin.

3. Blood serum showed positive for Ca2+, Cl-, glucose, and SO42- ions.
Suggestion :
1. Read MSDS before doing experiments.
2. To efficiency of time in experiment, member of group do the experiment of
every test, then show the result in other member of group.
3. When heated blood in Fe test, make sure that all blood was burnt out
become ash because it will give effect in the result of experiment.
H. References
Campbell, N.A., J.B. Reece., M.R. Taylor., and E.J. Simon. 2006. Biology.
Concepts and Connection, Fifth Edition. Pearson Education, Inc. Benjamin
Cummins, San Fransisco.
Hilman, R.S., Ault K.A., and Rinder H.M. 2005. Hematology in Clinical Practice
4th ed. The Washington Manual Hematology. New York.
Pearce C.E. 2006. Anatomi dan Fisiologi Untuk Paramedic. PT Gramedia Pustaka
Utama. Jakarta.

Semarang, October 8, 2015

Know,
Lab Work Lecturer

Student

Samuel Budi Wardhana, S.Si, M.Si

Candra Tri Kurnianingsih

NIP.

NIM. 4301413032

APPENDIX
Answering Question
1. The purpose of heating with glacial acetate in experimental blood test is to
remove crystal water content, outlines of its binding globulin and protein
together to form hematin chloride NaCl.
2. The function of dilute NaCl solution on an experimental blood test crystal is to
form hematin chloride and accelerate chloride reacts.
3. Reaction that may happen from Fe test in hemoglobin:
a. Fe3+ + K4[Fe(CN)6] FeK[Fe(CN)6] + 3K+
b. Fe3+ + KCNS Fe(CNS)3 + 3K+
4. The pupose of heating blood until burned perfectly on Fe test in hemoglobin is
to oxidize Fe2+ to Fe3+ and eliminate organic compounds.
5. The function of mixed dilute HCl with concentrated HNO 3 in experimental Fe
in hemoglobin test is as a solvent metals other than Fe so as to facilitate the
oxidation of Fe2+ into Fe3+. The name mixed is aqua regia.
6. Protein components that exist in blood serum are albumin and globulin.
7. The uses of a concentrated solution of ammonium sulphate and ammonium
sulphate solid on test of albumin and globulin in blood serum:
- (NH4)2SO4 solid for salting out process.
- (NH4)2SO4 concentrated to obtain the content of albumin and globulin
better (apart from the other impurities).
8. The meaning of salting out is a method of separation and blood serum protein
fractions with different salt concentrations.
9. Reaction that may happen in the experimental test of non-protein substance in
blood serum:
Ca2+ + (NH4)2C2O4 CaC2O4(s) + 2NH4+

a.

Reaction of calcium ions and ammonium oxalateCl-

HCl + AgNO3 AgCl(s) + HNO3

b.

Reaction of HCl and ammonium oxalate

c.

C H
H C
HO C

C OH

OH
H

H C
+ Cu2+ + 4OH-

HO

OH
H

H C

OH

H C

OH

H C

OH

H C

OH

+ Cu2O + 2H2O

CH2OH

CH2OH

Reaction of blood + fehling A & B

d.

BaCl2 +

SO42-

BaSO4

+ 2Cl-

white precipitate
Reaction of BaCl2 and sulfate ions

10. Part of blood serum crude suspension that forms is fibrinogen.

11. Blood serum contains glucose and amino acid transport because blood as food,
through the metabolism of glucose and amino acids, carbohydrates raised in
the form of glucose and protein raised in the form of amino acids.