Laboratory Work Report Date : 22nd February 2018

General Biochemistry Time : 1500 - 1800

Lecturer : Puspa Julistia P
Assistant : Janson Calvin

BIOPHYSICS

Group 16

Darryl Yee Zhi Heng (B04178009)

Roshini Muniandy (B04178014)
Lai Yoke Ping (B04178022)
Natasha Anne Binti Mohammed Noor Azam (B40178034)

DEPARTMENT OF BIOCHEMISTRY
FACULTY OF MATHEMATICS AND NATURAL SCIENCES
BOGOR AGRICULTURAL UNIVERSITY
2018
 INTRODUCTION

Density is defined as the ratio between mass and volume or mass per unit
volume or mass per unit volume. It is a measure of how much stuff an object has
in a unit volume (cubic meter or cubic centimetre). Mass is a measure of how
much stuff an object contains, and volume is the measure of how much space an
object occupies in three- dimensional space. The density of an object or quantity
of matter is its mass divided by its volume. This is usually measured under
standard conditions for temperature and pressure: one factor affecting the density
of a material is how concentrated the atoms are in a given volume.

The surface tension of a liquid results from an imbalance of intermolecular

attractive forces, the cohesive forces between molecules: a molecule in the bulk
liquid experience cohesive forces with other molecules in all directions. A
molecule at the surface of a liquid experiences only net inward cohesive forces.
As temperature decreases, surface tension increases. Surface tension decreases
strong; as molecules become more active with an increase in temperature
becoming zero at its boiling point and vanishing at critical temperature. Adding
chemical to a liquid will change its surface tension characteristic (Burdon 2014).

Cohesion, water is attracted to water. Adhesion, water is attracted to other

substances. Adhesion and cohesion are water properties that affect every water
molecule on earth an also the interaction of water molecules with molecules of
other substances. Cohesion and adhesion are the stickiness that water molecules
have for each other and for other substances.

An emulsion is a colloid of two or more immiscible liquids where one

liquid contains a dispersion of the other liquids. Is a special type of mixture made
by combining two liquids that normally don’t mix? Types of emulsions, oil in
water emulsion (o/w): oil is present as the dispersed phase and water as the
dispersion medium (Hadadi 2016). Water in oil emulsion (w/o): water forms the
dispersed phase, and the oil acts as the dispersion medium. Fresh milk is a natural
emulsion. Milk is an emulsion of fat in water (Lauffer 2012).

A colloid is a type of homogeneous mixture where the dispersed particles

do not settle out. When colloids are present in as solution, it is known as a
colloidal solution. These colloid solutions can be categorized into two groups as
lyophilic and lyophobic depending on the interaction between colloids and the
liquid (solvent). Lyophilic colloids they have a high affinity for the liquid the are
dispersed in. when the colloids are mixed with a suitable solvent, a high attraction
force arises between the solvent molecules and the colloid particles. Lyophobic
colloids there is no attraction between the colloids and the liquid and
thermodynamically unstable. These colloid tend to form aggregates when added
to a liquid. But the stability can be increased by adding a surface active agent.
Example of lyophobic berlin blue colloid.
 A buffer is a solution containing either a weak acid and its salt or a weak
base and its salt, which is resistant to a change in PH. In other words, a buffer is
an aqueous solution of either a weak acid and its conjugate base or a weak base
and its conjugate acid. Buffer are used to maintain a stable pH in a solution, as
they can neutralize small quantities of additional acid base. For example human
blood is an excellent example natural buffer solution. Buffer solutions are of two
types’ acidic buffer and basic buffer.

Buffer capacity is a measure of the efficiency of a buffer in resisting

changes in PH. The buffer capacity is expressed as the amount of strong acid or
base, in gram- equivalents, that must be added to 1 litter of the solution to change
its PH by one unit. It is magnitude of the resistance of a buffer to change in the
PH

β=∆β / ∆ ᴩH

β is a buffer capacity

∆β is a strong acid or base added

∆ ᴩH is the change in ᴩH

Buffer capacity is directly proportional to concentration of buffer components.

Solute is defined as the substances that is dissolved in a solution. For

solution of fluids, the solvent is present in greater amount than the solute. There
are three type of solutions that can occur in body based on solute concentration:
isotonic, hypotonic, and hypertonic. Water moves readily across cell membranes
through special protein-lined channels, and if the total concentration of all
dissolved solute is not equal on both sides, there will be net movement of water
molecules into or out of the cell. Isotonic, cells placed in water that neither gain
nor lose water through osmosis because the concentration of solutes in the cell are
equal to the solutes outsides. Example of hypertonic, would be the oceans because
the solutes (salts) outside of the cells are greater than inside of the cells.
Freshwater habitats are examples of hypotonic because the freshwater has less
solutes than inside of the cell.

The red blood cell, lyses and crenation, these erythrocytes looks as if they
have projections extending from a smaller central area, like a spiked ball. The
crenation may be either large,, irregular spicules of acanthocytes, or smaller, more
echinocytes. When cell surface antigens by flow cytometry, the cells in whole
blood are usually first la- baled, and then the red blood cells are lysed either with
ammonium chloride or commercial whole blood lysing solution (Scallan 2012).
Objective:

Determine density of a solution. Observe surface tension on various

solutions. Observe characteristic differences on various emulsions. Observe
characteristics differences on various colloids. Create various buffer solutions.
Observe osmotic pressure effects towards a solution.

METHODOLOGY

Time and Place for Practicum

The practicum was conducted in the biochemistry laboratory of FMIPA of

Bogor Agricultural University. The practicum was held on Thursday, February
15th,2018 and February 22nd 2018. The time given for the practicum was from 3.00
pm – 6.00 pm.

Apparatus and Materials

There were a few apparatus used in this experiment. To determine the

density of a solution, the apparatus used were a hydrometer, measuring cylinders,
Mohr pipette, volumetric pipette, bulb, thermometer, densitometer, beakers and
weighing scale. For the surface tension experiment, the apparatus used were watch
glass and needles. The apparatus that were used for the emulsion test were test
tubes, microscope, mortar, slides, cover slips and dropper.
There were also a few materials used in this experiment. Solutions used in
this experiment were 0.3% NaCl solution, 0.9% NaCl solution, 5% NaCl solution,
5% glucose solution, coconut oil, faucet water, 1% albumen solution, urine, bile,
river water, detergent, alcohol, distilled water, and soapy water. Coconut oil, Arab
gum, fresh milk, and margarine were used to study the emulsion system. Sudan
red acted as an indicator in this experiment.

Procedure

Density of various natural solutions

To measure the density of various solutions, the density for various types of
liquids had been measured using a hydrometer. The density of distilled water,
0.3% NaCl solution, 0.9% NaCl solution, 5% NaCl solution, 5% glucose solution,
coconut water, faucet water, 1% albumen solution and urine were measured. The
temperature of solution samples were recorded with a thermometer. All the data
were recorded in a table.

The surface tension of a liquid

To determine the surface tension of a liquid, a needle was placed on a watch glass
and then filled up with distilled water slowly until the needle floats. The step is
then repeated using distilled water with bile, coconut water, river water and
detergent. The results obtained were observed and recorded.

The number of droplets and surface tension

The pipette was rinsed and cleaned thoroughly using distilled water before the
experiment began. The pipette was then held in a straight and upright position for
2 minutes. Then, the number of droplets of 1 mL of distilled water dropped from a
pipette was counted and recorded. The experiment was repeated by using 20%
NaCl solution, alcohol and detergent.

Emulsion of coconut oil and water/soap

The same volume of oil and water was added in a test tube and shaken until the
solution became homogeneous. To identify the components of coconut oil in the
emulsion, a few drops of Sudan red was added in the solution. The stability of the
emulsion was compared when the solution was shaken longer and excessively.
The solution was then observed under the microscope. The experiment was
repeated using coconut oil and soap.

Emulsion of coconut oil and Arab gum

1g of Arab gum was weighed and mixed with 5mL of coconut oil in a mortar. The
Arab gum was crushed until it became a homogeneous mixture and then 3 ml of
distilled water was added. It was stirred until it became concentrated and 5 ml of
distilled water was added slowly while stirring it. The stability of emulsion was
observed in a test tube. A drop of emulsion was observed under the microscope.

Natural emulsion

Milk is a natural emulsion. The stability of the emulsion was observed in a test
tube. A drop of the emulsion was observed under the microscope.
Industrious emulsion

Margarine was added with a drop of Sudan red and observed under the
microscope. The type of emulsion of each sample was determined after observing
each of them under the microscope.

Apparatus and Materials

Many types of apparatus were used in this experiment. As for the colloid
experiment, a 250 ml beaker glass was used to contain the solutions. A 10 ml
pipette, test tubes and other beaker glasses were used as well. As for the osmotic
pressure experiment, test tubes and a dropper were used. A microscope was also
used to observe the specimen. As for the buffer experiment, a pH meter was used
to observe the pH values. A 10 ml pipette and few test tubes were used to measure
and hold liquids. A stirrer was also used to mix the liquids.

Many materials were used to conduct the experiment. As for the colloid
experiment, the materials used were distilled water, ferrocyanide kalium 0.2 N,
ferrichloride 0.02 N, ferrihydroxide 33%, NaCl 10%, Mg SO4 salt to prepare the
gelatin colloid 2%, starch colloid 2%, berlin blue colloid 0.2 N. Some of the
colloids were also used in the precipitation experiment. As for the osmotic
pressure experiment, NaCl 0.3%, 0.9%, 5% and blood were used. Acetic acid 0.1
1 1
N, sodium acetate 0.1 N, Na2 HPO 4 M and KH 2 PO 44 M were used in the
15 15
buffer experiment.

Procedure

Lyophilic Colloid Solution- Gelatin Colloid 2%

25 ml H2O with 2g of gelatin is added into a 250 ml beaker. The gelatin was left
for a few minutes until all gelatin had absorbed the water and expanded. Then 75
ml of boiling water was added and stirred. The results were recorded.

Lyophilic Colloid Solution - Starch Colloid 2%

A mixture involving 10 ml of cold water with 2g of starch was created and it was
stirred until it became homogeneous. After that, 70 ml of boiling water was added
and stirred. The results were recorded.

Lyophobic Colloid – Berlin Blue Colloid

10 ml of ferrocyanide kalium 0.2 N mixture and ferrichloride 0.02 N were

channeled to a 100 ml beaker glass using a pipette. It was stirred until it became
homogeneous. Then, an estimated amount of 5 ml was extracted and poured into a
test tube. Water was added when necessary to observe if there are any sediments
formed. The results were recorded.

Lyophobic Colloid – Ferrihydroxide Colloid

200 ml of boiling water was poured and then 1 ml of ferrihydroxide 33% was
channeled to it using a pipette. The colour that appears after the solution becomes
homogeneous was observed. The results were recorded.

Precipitation of Lyophilic Colloids with NaCl 10%

A few millilitre of 10% NaCl was added into one of the lyophilic colloids to form
a precipitate. Distilled water was added if a saturated sediment was obtained. Mg
SO4 salt was also added until a saturated solution was obtained if there was no
production of sediment. The results were recorded.

Precipitation of Colloids Lyophobic with NaCl 10%

A small amount of NaCl 10% was added to a lyophobic colloid. This step was
continued until a precipitate was formed. The result was recorded.

Properties of Colloid Solution

A solution of 15% gelatin was set up and then distributed to 4 test tubes. The test
tubes were froze until a formation of gel could be seen. Berlin blue solution, Eosin
solution, giemsa solution and a CuSO 4 5% colloid solution were prepared. Those
solutions had been stored in a refrigerator for one night. The results were
recorded. To prevent melting, the precaution taken was that the gel was kept in a
proper way.

Buffer experiment- Standard acetate buffer

A mixture of acetic acid 0.1 N with sodium acetate 0.1 N was prepared using the
following amounts respectively ; 9.25 ml with 0.75 ml, 8.20 ml with 1.80 ml, 6.30
ml with 3.70 ml, 4.00 ml with 6.00 ml and 2.10 ml with 7.90 ml. The pH of the
solutions was measured after they were stirred until they became homogeneous.
The results were recorded.

Buffer experiment- Standard phosphate buffer

1 1
A mixture of M Na2 HPO 4with M KH 2 PO 44 was prepared using the
15 15
following amounts respectively; 2.50ml with 9.50ml, 1.20ml with 8.80ml, 2.65ml
with 7.35ml, 5.00ml with 5.00ml, 7.15ml with 2.85ml. The pH of each of the
solutions were measured. The results were recorded.
Osmotic pressure of liquid red blood cells

Three test tubes, one containing NaCl 0.3%, another NaCl 0.9%, and the other
NaCl 5% were prepared. Two to three drops of blood were placed in each test
tube. The precipitation formed between the three test tubes were observed. The
solution was observed under a microscope. The observations and results were
recorded.

RESULT AND DISCUSSION

Density

In this experiment, we measured the density of multiple different solutions

using a hydrometer. Based on our observation and calculation, tap water and NaCl
0.3% solution has a density of 1.004 g/cm3, distilled aqua has a density of 1.002
g/cm3, NaCl 0.9% solution has a density of 1.006 g/cm 3, NaCl 5% solution has a
density of 1.028 g/cm3, coconut water has a density of 1.021 g/cm 3, albumin 1%
solution has a density of 1.008 g/cm 3, and glucose 5% solution has a density of
1.012 g/cm3. The density increases from distilled aqua, tap water and NaCl 0.3%
solution, NaCl 0.9% solution, albumin 1% solution, glucose 5% solution, coconut
water, and NaCl 5% solution. The results were recorded in table 1.

According to Frelund, distilled aqua has a density of 0.997 g/cm 3 at 25℃

(Frelund et al. 2012). On the other hand, according to Kumar and Sheetal,
Glucose 5% solution has a density of 1.016 g/cm 3 at 25℃ (Kumar and Sheetal
2016). There is a slight inaccuracy when we compare the density of the solutions
from our results to the literature. This may be caused by contamination of the
solutions, the hydrometer sticking to the sides of the glass bottle due to adhesion
and human error such as marking the water level to the hydrometer inaccurately
while it was in the water. Moreover, the solutions have varying densities due to
different concentrations. Kumar and Sheetal also stated that the higher the
concentration of a dissolved substance, the higher the density of the solution
(Kumar and Sheetal 2016).

Based on the results, distilled aqua, tap water, albumin 1% solution, NaCl
0.3% and 0.9% solutions have low densities due to low concentrations of
dissolved substances in them. Distilled aqua has the lowest density due to the fact
that it have little to no dissolved substance in it. In contrast, density of tap water is
slightly above water because it contains contaminants and minerals. NaCl 5%
solution has the highest density in comparison to NaCl 0.3% and 0.9% solutions
because it contains the highest concentration of solute. Besides, glucose 5%
solution has high density as well due to its high concentration. According to
Montalbán, the higher the molecular weight of an ionic solution, the higher the
density (Montalbán et al. 2017). Thus, for the case of coconut water, its density is
high as due to the presence of ions thus it has a higher molecular weight.
Table 1. Density of Solute
Type of Tsolute Tdensitometer Measured Correction Correction
Solute Density factor Density
Tap water 25℃ 20℃ 1.002 g/cm3 0.002 g/cm3 1.004 g/cm3
Distilled 25℃ 20℃ 1.000 g/cm3 0.002 g/cm3 1.002 g/cm3
aqua
NaCl 0.3% 25℃ 20℃ 1.002 g/cm3 0.002 g/cm3 1.004 g/cm3
NaCl 0.9% 25℃ 20℃ 1.004 g/cm3 0.002 g/cm3 1.006 g/cm3
NaCl 5% 25℃ 20℃ 1.026 g/cm3 0.002 g/cm3 1.028 g/cm3
Coconut 22℃ 20℃ 1.020 g/cm3 0.001 g/cm3 1.021 g/cm3
water
Albumin 25℃ 20℃ 1.006 g/cm3 0.002 g/cm3 1.008 g/cm3
1%
Glucose 25℃ 20℃ 1.010 g/cm3 0.002 g/cm3 1.012 g/cm3
5%

Formulas

Tsolute−Tdensitometer
CF = ×10−3
3
25 ℃−20 ℃
CF of Tap water= ×10−3
3
CF of Tap water=0.00166 ρ
CF of Tap water ≈ 0.002 ρ

Correction Density=Measured Density+CF

Correction Density of Tap water=1.002 ρ+0.002 ρ
Correction Density of Tap water=1.004 ρ

In this experiment, we measured the density of urine by using a

hydrometer. Based on our observation and calculation, urine have a density of
1.019 g/cm3. When compared to the solutions from table 1, urine have the third
highest density after NaCl 5% and coconut water. The results were recorded in
table 2.

According to Bettelheim, urine densities varies among individuals due to

factors such as amount of food consumed, type of food consumed, volume of
water intake, dissolved substances in the urine and others (Bettelheim et. al 2015).
Thus, the higher the concentration of solute, the higher the density and vice versa.
When a person’s urine density is between 1.002-1.030 g/cm3, it means that their
kidneys are functioning normally. Furthermore, if the urine density is more than
1.010 g/cm3, it indicates mild dehydration and as the density increase, the
dehydration level increases (Bettelheim et al. 2015). When we compare our results
to the literature, this result shows that our subject has normal functioning kidneys
and that he is slightly dehydrated.
Table 2. Density of Urine
Type of Tsolute Tdensitometer Measured Correction Correction
Solute Density factor Density
Urine 28℃ 20℃ 1.016ρ 0.003ρ 1.019ρ
Surface tension

In this experiment, we measured the surface tension of NaCl 20%, alcohol

soap, and distilled aqua by comparing the number of drops by using a pipette.
Based on our observation, the number of drops taken for NaCl 20% solution was
23 drops, 65 drops for alcohol, 19 drops for kerosene, 47 drops for soap solution,
and 31 drops for distilled aqua. The results were recorded in table 3.

According to Burdon, the surface tension of a liquid increases when the

concentration of dissolved substances on its surface layer is lesser than the
concentration of dissolved substances in its solution (Burdon 2014). With this in
mind, NaCl 20% solution have the lower amount of drops because it have the
lesser concentration of dissolved substances on its surface layer than in its
solution when compared to soap solution. Moreover, the higher the molecular
bonding in the solution, the higher the cohesion forces thus having higher surface
tension (Burdon 2014). Alcohol have higher number of drops because the
hydrogen bonding of alcohol is not as strong when compared to the hydrogen
bonding in distilled aqua. This shows that the surface tension in alcohol is lower
than in distilled aqua. Thus, the lower the number of droplets from the pipette, the
higher the surface tension of the solution. Unfortunately, from our data, kerosene
have low number of drop which indicates a higher surface tension and this does
not correlate to our literature. Oil is nonpolar, so hydrogen bonds do not form
between the molecules, so the surface tension should be lesser than distilled aqua.
This error might have happened due degradation of the kerosene which was seen
by the brownish colour.

Table 3. Liquid Surface Tension

Type of Solute ∑ drops
NaCl 20% 23
Alcohol 65
Kerosene 19
Soap 47
Distilled aqua 31
Emulsion

In this experiment we have observed five types of emulsion, which are

emulsion of palm oil and water, emulsion of palm oil and detergent, gum Arabic-
palm oil emulsion, fresh milk and margarine. In the emulsion of palm oil and
water, the solution is not stable. In this emulsion, palm oil is forming drops and
water is the media of dissolving the palm oil droplets This is one of the examples
of oil-in-water (O/W) emulsion. In the emulsion of palm oil and detergent, the
solution is considered stable. Here palm oil is acting as the dissolving compound
and detergent as the dissolved compound. Emulsion of palm oil and soap is
water-in-oil (W/O) emulsion. To differentiate O/W and O/W emulsion, we can
look at the continuous phase and the dispersed phase. If the continuous phase is
water, it is the O/W emulsion, and vice-versa for the W/O emulsion. In gum
Arabic-palm oil emulsion, the emulsion is stable because gum Arabic acts as a
stabiliser. Gum Arabic, also known as acacia gum, is widely used in the food
industry because it has low viscosity (Saha D. and Bhattacharya S. 2010).
Milk is an example of natural emulsion. The dissolved compound in milk
is the cream and the dissolved compound is skim milk. Milk is a stable emulsion
because it has fats in large droplet form. The density of milk fat and its aqueous
phase has a difference, and this causes the fat droplets to rise through the milk.
Milk fat globules are special in such a way that they will cluster together when
they rise. This will in turn form larger fat particles (Vaclavik VA. et al. 2014).
Margarine is also observed in this experiment. Margarine is an industrial emulsion
and it has high stability. Margarine consists of a water-in-oil emulsion, and has
tiny fat droplets spread throughout a fat phase in a stable crystalline form.

Table 4. Emulsion
Emulsion Water + Detergent + Gum Arabic + Milk Margarine
Palm oil Palm oil Palm oil

Stability -- -- + + +

Type W/O O/W W/O O/W W/O

Dissolved Palm oil Detergent Gum Arabic Cream Water,skim

compound milk

Dissolving Water Palm oil Water Skim milk Vegetable oils,

compound fat
 Picture

Colloid

Colloid sedimentation by salt solution

A colloid is a solution that has particles with size ranging between 1 and
1000 nm in diameter, but still can remain evenly distributed throughout the
solution. In colloids, one substance is evenly dispersed in another substance. The
substance being dispersed is referred to as being in the dispersed phase, while the
substance in which it is dispersed is in the continuous phase or also called as
dispersion medium. Lyophilic substances usually consist of sols of organic
substances like gelatin, starch and gum. Lyophobic substances include sols of
inorganic substances such as ferrihydroxide, Berlin blue, and arsenic.
In this experiment, we have done several colloid sedimentations by salt
solution. The first two was done by adding several ml of NaCL 10% into starch
and gelatin colloid respectively. Since both starch 2% and gelatin 2% colloids are
lyophilic, there is no sedimentation formed when NaCL was added. Both lyophilic
colloids added with NaCL 10% are relatively stable as there are strong forces of
interaction between colloidal particles and the liquid. No precipitation occurs
when NaCL and MgSO4 were added into both gelatin and starch colloids.
On the other hand, lyophobic colloids added with NaCL 10% are more
unstable compared with lyophilic colloid. This is because the forces of attraction
between colloidal particles and liquid are weaker. Precipitate was produced in
both ferrihydroxide and Berlin blue colloids when NaCL and MgSO4 were added.
Precipitation occurs because the particles of dispersed phase carry some charge.
When electrolyte like NaCL is added, the colloidal particles will take up the ions
carrying opposite charge from the electrolyte. This causes the colloidal particles to
change their charge to neutral and the neutral particles will come closer together
and form precipitate.
Table 5. Colloid sedimentation
Colloid Gelatin colloid Starch colloid 2% Berlin Blue colloid Ferrihydroxide
2% colloid

Type Lyophilic Lyophilic Lyophobic Lyophobic

Dissolved Gelatin Starch Berlin blue Ferrihydroxide

compound

Dissolving NaCl + NaCl + MgSO4 NaCl NaCl

compound MgSO4

Stability +++ +++ + +

Precipitate - - Blue precipitate Orange precipitate

formed formed

Picture

Colloid solution characteristics

Gelatin 15% solution is prepared and 5mL of the solution was inserted into
4 test tubes. In each test tubes, Berlin blue, CuSO4, eosin, and giemsa solution
was added respectively. Colloid solution characteristics were then observed. We
noticed that only Berlin blue could not diffuse into the gelatin 15% solution when
mixed. This is because Berlin blue is lyophobic colloid while gelatin solution is
lyophilic solution. The forces of interaction between lyophobic and lyophilic
colloids are weak or even no interaction. Lyophobic solutions have weak affinity
towards their medium of dispersion.
On the other side, when CuSO4, eosin and giemsa were added into gelatin
15% solution, we can see that the 3 solutions have started to diffuse to the gelatin
15% solution. The reason behind this is because all the 3 solutions are lyophilic
and gelatin solution is also lyophilic. Gelatin 15% solution can form strong forces
of interaction with all CuSO4, eosin and giemsa solution. Lyophilic solutions
have higher affinity towards their medium dispersion, hence they can diffuse into
the gelatin solution.

Table 6. Characteristics of Colloid

Colloid Gelatin 15% +

Berlin Blue CuSO 4 5% Eosin Giemsa

Type Lyophobic Lyophilic Lyophilic Lyophilic

Picture
Buffer

Table 7. Data of pH of standard Acetate Buffer

Volume of acetic Volume of Measured pH Theoretical pH Buffer capacity
acid / mL sodium acetate /
mL

9.25 0.75 4 3.7 0.78

8.20 1.80 4 4.1 0.86

6.30 3.70 5 4.5 0.95

4.00 6.00 5 4.9 1.03

2.10 7.90 5 5.3 1,12

A buffer solution (more precisely, pH buffer or hydrogen ion buffer) is an

aqueous solution consisting of a mixture of a weak acid and its conjugate base, or
vice versa. Its pH changes very little when a small amount of strong acid or base
is added to it. Buffer solutions are used as a means of keeping pH at a nearly
constant value in a wide variety of chemical applications. In nature, there are
many systems that use buffering for pH regulation. For example, the bicarbonate
buffering system is used to regulate the pH of blood. Acetic buffer and phosphate
buffer solutions were made in this experiment (Machado et al. 2008). The
standard acetic buffer solution in this experiment was made using acetic acid and
sodium acetate. The results of making standard acetic buffer is as the table above.
Example of calculation of 0.75ml sodium acetate and 9.25ml acetic acid :

Mole of acetic acid :

n
M= V
n = M ×V

1l
n = 0.1 M × 9.25 ml × 1000ml
n = 0.925 M
Mole of sodium acetate :

n
M= V
n = M×V

1l
n = 0.1 M × 0.75ml × 1000 ml
=0.075

5
Ka of Acetic acid = 1.76  10

mol of acetic acid


[ H ] = Ka × mol of sodium acetate

0.925 M
5
1.76  10 × 0.075 M
=
-4
= 2.171 10


pH = - log [ H ]
-4
= - log 2.171 10
= 3.663

pKa = -log [Ka]

5
= -log 1.76  10
= 4.754

pH
Buffer capacity = pKa
 3.663
= 4.754
= 0.78

Buffer capacity is a measure of the efficiency of a buffer in resisting changes

in pH. Conventionally, the buffer capacity (β) is expressed as the amount of
strong acid or base, in gram-equivalents, that must be added to 1 liter of the
solution to change its pH by one unit. In practice, smaller pH changes are
measured and the buffer capacity is quantitatively expressed as the ratio of acid or
base added to the change in pH produced (e.g., mEq./pH for x volume). The data
on table 8 shows that the theoretical pH and buffer capacities are different. This
result is due to the difference in volume of the solution used. The buffer capacity
for standard acetate buffer is 0.78. If the volume of acetic acid acid is more than
sodium acetate, the pH value will be lower and the pH will be higher when
volume of acetic acid is smaller than sodium acetate. The higher the pH value, the
bigger the buffer capacity.

The phosphate buffer is a neutral buffer with a pH range of 7. the standard

phosphate buffer is produced with the mixture of potassium dihydrogen phosphate
(KH2PO4) and disodium phosphate (Na2HPO4). The result of making standard
phosphate buffer is as follows.

Table 8. Data of pH of Standard Phosphate Buffer

Volume of Volume of Measured pH Theoretical pH Buffer capacity
Na2HPO4 KH2PO4

0.50 9.50 5 5.92 0.823

1.20 8.80 5 6.34 0.880

 2.65 7.35 6 6.42 0.891

5.00 5.00 6 7.21 1.001

7.15 2.85 6 7.60 1.005

Example of calculation of 0.50ml Na2HPO4 and 9.50ml KH2PO4 :

Mole of KH 2 PO 4 :

n
M= V
n = M ×V

1 1l
n = 15 M × 9.50 ml × 1000 ml
n = 0.633 M

Mole of Na2HPO4 :

n
M= V
n = M ×V

1 1l
n = 15 M × 0.50 ml × 1000 ml
n = 0.033M
8
Ka of Na2HPO4 = 6.23  10

mol of KH 2 PO 4
H ] = Ka × mol of Na 2 HPO 4


0.633M
8
6.23  10 × 0.033M
=
6
= 1.1950  10

pH = -log [ H ]
6
= -log 1.1950  10
 = 5.922
pKa = -log [Ka]
8
= -log 6.23  10
= 7.206

pH
Buffer Capacity = pKa

5.922
= 7.206
= 0.823

The data in table 9 illustrates that the theoretical pH and buffer capacity are
different. The standard phosphate buffer solution has a buffer capacity of 0.823.
The results are obtained according to different volume of the solution used. If the
volume of Na2HPO4 is higher than the volume of KH2PO4 , the pH will be higher.
The higher the pH value, the bigger the buffer capacity.
Besides, the pH has a slight difference when comparing the pH measured and
the theoretical pH. This is probably cause by parallax error or inaccurate
measurement during experiment.

The three major buffer systems of our body are carbonic acid bicarbonate
buffer system, phosphate buffer system and protein buffer system. The body's
chemical buffer system consists of three individual buffers out of which the
carbonic acid bicarbonate buffer is the most important. Cellular respiration
produces carbon dioxide as a waste product. This is immediately converted to
bicarbonate ion in the blood. On reaching the lungs it is again converted to and
released as carbon dioxide. While in the blood, it neutralises acids released due to
other metabolic processes. In the stomach and deudenum it also neutralises gastric
acids and stabilises the intra cellular pH of epithelial cells by the secretions of
bicarbonate ions into the gastric mucosa (Kreig et al. 2014). Phosphate buffer
system operates in the internal fluids of all cells. It consists of dihydrogen
phosphate ions as the hydrogen ion donor (acid) and hydrogen phosphate ion as
the ion acceptor (base). If additional hydroxide ions enter the cellular fluid, they
are neutralised by the dihydrogen phosphate ion. If extra hydrogen ions enter the
cellular fluid then they are neutralised by the hydrogen phosphate ion. Protein
buffer system helps to maintain acidity in and around the cells. Haemoglobin
makes an excellent buffer by binding to small amounts of acids in the blood,
before they can alter the pH of the blood. Other proteins containing amino acid
histidine are also good at buffering. The main purpose of all these buffers is to
maintain proper pH within the body system so that all biochemical process can
take place.

Osmotic Pressure

Osmotic pressure is the minimum pressure which needs to be applied to a

solution to prevent the inward flow of its pure solvent across a semipermeable
membrane (Atkins et al. 2010). It is also defined as the measure of the tendency of
a solution to take in pure solvent (which belongs to the solution under discussion)
by osmosis. Potential osmotic pressure is the maximum osmotic pressure that
could develop in a solution if it were separated from its pure solvent by a
selectively permeable membrane. The phenomenon of osmosis arises from the
propensity of a pure solvent to move through a semipermeable membrane and into
its solution containing a solute to which the membrane is impermeable. This
process is of vital importance in biology as the cell's membrane is semipermeable.
The osmotic pressure was conducted using red blood cells of cow mixed with
NaCl of three different concentrations which are NaCl 0.3%, NaCl 0.9%, and
NaCl 5%. The results of observation of the effect of osmotic pressure is shown in
the table below.

Table 9. Osmotic Pressure

NaCl solution (%) 0.3 0.9 5
Observation -Hypotonic -Isotonic -Hypertonic
-Hemolysis -No changes -Crenation
-Precipitate -Less precipitate -No precipitate
formed formed formed
Photos
Photos from
literature

Theoretically, the osmotic pressure in the cell will be higher compared to

the outside of the cell after the cell is immersed in 0.3% NaCl solution, causing
water to enter the erythrocyte by osmosis process. This will make the cell to
become turgid and it will burst. At this situation, it is called as hemolysis and
there is precipitate formed. The cell is said to be hypotonic. Red blood cells will
become isotonic when immersed to 0.9% NaCl solution. This is because the
concentration of the cell is almost the same as the surroundings. Hence, the
erythrocyte does not have any changes in shape because there is no net flow of
water in or out of the red blood cell as the osmotic pressure remains the same and
will have less precipitate. Lastly, the osmotic pressure outside the cell will is
higher than the inside of the cell when immersed in 5% NaCl solution, this cause
water to move out of the erythrocyte by osmosis. The red blood cell will shrink
and undergo plasmolysis. It is said to be hypertonic.

CONCLUSION

In conclusion, throughout this experiment, we have learnt about density,

surface tension, emulsion, colloids, buffer and also osmotic pressure. Density is
the mass over volume of a substance and it is proportional to the concentration of
a substance. Surface tension of a fluid is the resistance of fluid surface’s thin layer
upon the force to change the surface range of the fluid. Surface tension is
dependent on the dissolved substances in the fluid. There are two types of
emulsion we have learnt, which is the water-in-oil and oil-in-water emulsions.
Colloids are also categorized into two types, lyophilic and lyophobic. Lyophilic
colloids will not form precipitation when electrolytes like NaCl is added, while
lyophobic colloids will form precipitation. When lyophobic colloid is mixed with
lyophilic colloid, the lyophobic colloid will not diffuse into the lyophilic colloid
as they have different characteristics. For buffer, the buffer capacity is higher if
the pH of the buffer is high. We have also studied about the red blood cell fluid
osmotic pressure. In NaCl 0.3%, the osmotic pressure of the solution is higher
than that in the blood cells, hence the solution is hypotonic and the blood cells
will undergo hemolysis. In NaCl 0.9%, the osmotic pressure of the solution is
same as that in the blood cells, hence the solution is said to be isotonic and the red
blood cells will remain unchanged. In NaCl 5%, the osmotic pressure of the
solution is lower than that in the blood cells, thus the solution is hypertonic and
the red blood cells will undergo crenation.

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