Kingdom of Saud Arabia
King Abdulaziz University
Girls Collage of Science
Biochemistry Department
Metabolism (1)
BIOC 211
Practical Manual
Name:
Name:
Computer
Computer No.:
No.:
Section:
Section:
1
Contents
Lab
#
1
Experiment
Page
Expressing concentration of solutions
2
13
3
Calorimetric determination of glucose by the 3,5dinitrosalicylic acid method
Estimation of carbohydrate by Anthrone method
4
Quantitative estimation of pentoses
19
5
Determination of Vitamin C in foods by iodometric assay
22
6
Assay of tissue glycogen
25
7
Estimation of total lipid by colorimetric method
28
8
Estimation of cholesterol by liberman-burchard method
31
9
Determination of saponification number
34
2
2
17
Experiment 1
Expressing Concentration Of Solutions
In chemistry, concentration is the measure of how much of a given substance there is
mixed with another substance, but most frequently the concept is limited to homogeneous
solutions, where it refers to the amount of solute in the solvent.
To concentrate a solution, one must add more solute (e.g. alcohol), or reduce the amount
of solvent (e.g. water). By contrast, to dilute a solution, one must add more solvent, or reduce
the amount of solute.
Unless two substances are fully miscible there exists a concentration at which no further
solute will dissolve in a solution. At this point, the solution is said to be saturated.
Qualitative description
These glasses containing red dye demonstrate qualitative changes in concentration. The
solutions on the left are more dilute, compared to the more concentrated solutions on the
right.
concentration is described in a qualitative way, through the use of adjectives such as
"dilute" for solutions of relatively low concentration and of others like "concentrated" for
solutions of relatively high concentration
Quantitative notation:
3
There are a number of different ways to quantitatively express concentration; the most
common are listed below. They are based on mass, volume, or both.
Molarity
Molarity is probably the most commonly used unit of concentration. It is the number of moles of solute
per liter of solution.
1. Solid material
molarity =
moles
of
solute
volume of solution (liter)
moles of solute= weigh
gm
molecular weight
M = weigh
gm . 1000
.
molecular weight
volume of solution (ml)
2. Liquid material:
Molarity of liquid = Percentage x
Specific gravity
x 1000 x volume (ml)
Molecular weight x volume of solution (ml)
Example:
What is the molarity of a solution made when water is added to 11 g CaCl2 to make 100 mL of
solution?
Normality
Normality is equal to the gram equivalent weight of a solute per liter of solution. A
gram equivalent weight or equivalent is a measure of the reactive capacity of a given
molecule. Normality is the only concentration unit that is reaction dependent.
Example:
1 M sulfuric acid (H2SO4) is 2 N for acid-base reactions because each mole of sulfuric
acid provides 2 moles of H+ ions. On the other hand, 1 M sulfuric acid is 1 N for sulfate
precipitation, since 1 mole of sulfuric acid provides 1 mole of sulfate ions.
4
For example, hydrochloric acid (HCl) is a monoprotic acid and thus has 1 mol = 1 gram
equivalent. One liter of 1 M aqueous solution of HCl acid contains 36.5 grams HCl. It is
called 1 N (one normal) solution of HCl. It is given by the following formula:
1. Solid material:
Gram equivalent = weight
Equivalent weight
.
Equivalent weight = molecular weight
valence
N = weight
Equivalent weight
. . 1000
..
volume of solution (ml)
2. Liquid material:
Normality of liquid = Percentage x
Specific gravity
x 1000 x volume (ml)
Equivalent weight x volume of solution (ml)
Table of measures units
Units
Symbol
Uses
Convert to other unit
Liter
L
volume
1L = 1000ml=106µl. I.E
(1Lx 1000 = 1000ml, 1Lx1000000= 106µl) and
(1dLx100=100 ml).
Gram
Gm
mass
1gmx 1000= 1000 mg= 106µg
1mg/1000= mg, 1mgx1000= 1000µg
5
1- Acids and Bases
Acids are compounds that contain hydrogen and can dissolve in water to release hydrogen
ions into solution. For example, hydrochloric acid (HCl) dissolves in water as follows:
H2O
H+(aq)
HCl
Cl-(aq)
+
Base is substances that dissolve in water to release hydroxide ions (OH-) into solution. For
example, a typical base according to the Arrhenius definition is sodium hydroxide (NaOH):
H2O
Na+(aq)
NaOH
+
OH-(aq)
Arrhenius's theory explains why all acids have similar properties to each other (and,
conversely, why all bases are similar): because all acids release H+ into solution (and all
bases release OH-).
Neutralization
As you can see from the equations, acids release H+ into solution and bases release OH-. If
we were to mix an acid and base together, the H+ ion would combine with the OH- ion to
make the molecule H2O, or plain water:
H+(aq)
+
OH-(aq)
H2O
The neutralization reaction of an acid with a base will always produce water and a salt, as
shown below:
Acid
Base
Water
6
Salt
HCl
+
NaOH
H2O
+
NaCl
2- pH
pH is the concentration of hydrogen ions present. Acids increase the concentration of
hydrogen ions, while bases decrease the concentration of hydrogen ions (by accepting them).
The acidity or basicity of something, therefore, can be measured by its hydrogen ion
concentration.
The pH scale is described by the formula:
pH = -log [H+]
Note: concentration is commonly abbreviated by using square
brackets, thus [H+] = hydrogen ion concentration. When
measuring pH, [H+] is in units of moles of H+ per liter of
solution.
The pH scale ranges from 0 to 14. Substances with a pH between 0 and less than 7 are
acids (pH and [H+] are inversely related - lower pH means higher [H+]). Substances with a pH
greater than 7 and up to 14 are bases (higher pH means lower [H+]). Right in the middle, at
pH = 7, are neutral substances, for example, pure water. The relationship between [H+] and
pH is shown in the table below alongside some common examples of acids and bases in
everyday life.
7
Acids
Neutral
Bases
[H+]
pH
Example
1 X 100
0
HCl
1 x 10-1
1
Stomach acid
1 x 10-2
2
Lemon juice
1 x 10-3
3
Vinegar
1 x 10-4
4
Soda
1 x 10-5
5
Rainwater
1 x 10-6
6
Milk
1 x 10-7
7
Pure water
1 x 10-8
8
Egg whites
1 x 10-9
9
Baking soda
1 x 10-10
10
Tums® antacid
1 x 10-11
11
Ammonia
1 x 10-12
12
Mineral lime - Ca(OH)2
1 x 10-13
13
Drano®
1 x 10-14
14
NaOH
8
- pH indicator
A pH indicator is a halochromic chemical compound that is added in small amounts
to a solution so that the pH (acidity or basicity) of the solution can be determined visually.
Normally, the indicator causes the color of the solution to change depending on the pH.
- pH meter
PH can measured by: 1. pH meter
2. PH strips
3. titration
pH meter:
A pH meter is an electronic instrument used to measure the pH (acidity or alkalinity)
of a liquid (though special probes are sometimes used to measure the pH of semi-solid
substances). A typical pH meter consists of a special measuring probe (a glass
electrode) connected to an electronic meter that measures and displays the pH reading.
3- Buffer
A buffer solution is an aqueous solution consisting of a mixture of a weak acid and its
conjugate base or a weak base and its conjugate acid. Buffer solutions are used as a means of
9
keeping pH at a nearly constant value in a wide variety of chemical applications. Many life
forms thrive only in a relatively small pH range; an example of a buffer solution is blood.
Method :
1. Prepare 20 ml of HCL (0.5 M)?
2. Prepare 20 ml of NaOH (2 N)?
Homework:
What is the molarity and normality of a solution made when water is added to 10 gm of H 2SO4 to
make 100 mL of solution?
Results
10
Experiment 2
Spectrophotometer
A spectrophotometer is one of the scientific instruments commonly found in many
research and industrial laboratories. Spectrophotometers are used for research in physics,
molecular biology, chemistry, and biochemistry labs.
The spectrophotometer is measure the amount of light of a specificed wavelength which
passes through a medium. The amount of light absorbed by a medium is proportional to the
concentration of the absorbing material or solute present. Thus the concentration of a colored
solute in a solution may be determined in the lab by measuring the absorbency of light at a
given wavelength. Wavelength (often abbreviated as lambda) is measured in nm. The
spectrophotometer allows selection of a wavelength pass through the solution.
At the spectrophotometer, you should have two cuvettes in a plastic rack. Solutions
which are to be read are poured into cuvettes which are inserted into the machine. One should
be marked "B"for the blank and one "S" for your sample
Here is a summary of the steps of operation of a spectrophotometer:
1- Power >>>>>>>>Turn on power.
2-Warmup>>>>>>Allow about 5 minutes when first turned on.
11
3-Wavelength>>>>>Select appropriate wavelength.
4- Zero>>>>>
With sample holder empty and closed, adjust meter needle to 0%T
(or infinite A) using zero control knob.
5- Blank >>>>Fill tube half full with water. Place in sample holder and close cover.
Adjust meter needle to 100%T (or 0 A) using light control knob.
6- Standard>>>>Measure absorbance (or %T) of known solution. Fill tube half full with
sample of known concentration. Place in sample holder and close cover. Read
absorbance value (or %T) from meter. Repeat this step if making a calibration curve
or verifying proportionality (Beer's Law).
7- Sample>>>>>Measure absorbance (or %T) of solution with unknown concentration
as in previous step.
Light source
slit
condenser
cuvette
filter
Lens
12
photocell
galvanometer
1- Serial dilution
A serial dilution is the stepwise dilution of a substance in solution. Usually the
dilution factor at each step is constant, resulting in a geometric progression of the
concentration in a logarithmic fashion. A ten-fold serial dilution could be 1 M, 0.1 M,
0.01 M, 0.001 M... Serial dilutions are used to accurately create highly diluted solutions
as well as solutions for experiments resulting in concentration curves with a
logarithmic scale
Serial dilutions are widely used in experimental sciences, including biochemistry,
pharmacology, microbiology, and physics, as well as in homeopathy.
Dilution factor= Final volume
Initial volume
2- Standard curve
A standard curve is a quantitative research tool, a method of plotting assay data that is used
to determine the concentration of a substance, particularly proteins and DNA. It can be used
in many biological experiments.
13
For example a standard curve for protein concentration is often created using known
concentrations of bovine serum albumin. The assay procedure may measure absorbance,
optical density, luminescence, fluorescence, radioactivity, or something else.
EXPERIMENT 3___
_______
Calorimetric Determination of Glucose by the 3,5dinitrosalicylic acid Method.
Principle:
Several reagents have been employed which assay sugars by using their reducing properties.
This method tests for the presence of free carbonyl group (C=O), the so-called reducing
sugars. This involves the oxidation of the aldehyde functional group present in, for example,
glucose and the ketone functional group in fructose. Simultaneously, 3,5-dinitrosalicylic acid
(DNS) is reduced to 3-amino-5-nitrosalicylic acid under alkaline conditions, as illustrated in
the equation below:
The chemistry of the reaction is complicated since standard curves do not always go through
the origin and different sugars give different color yields. The method is therefore not
suitable for the determination of a complex mixture of reducing sugar.
Materials:
1. Standard Glucose Solution:
14
0.1g anhydrous glucose is dissolved in distilled water and then raised the volume to 100 ml
with distilled water.
2. Dinitro salicylic acid reagent:
a. Solution "a" is prepared by dissolving 300g of sodium potassium tartarate in about 500 ml
distilled water.
b. Solution "b" is prepared by dissolving 10 g of 3,5-dinitrosalicylic acid in 200 ml of 2N
NaOH solution.
c. The dinitrosalycilate reagent is prepared by mixing solutions a & b and raising the final
volume to 1 litre with distilled water.
Procedure:
1. Pipette in duplicate the following reagents into a series of dry-clean and labelled test tubes
and as indicated in the following table, take Section A.
SECTION A
SECTION B
ml. H2O
bbbbbB
BB
Tube
No.
ml. Stand.
Glucose.
ml. H2O
ml.
Dinitrosalicylic
reagent
1
0.0
1.0
2.0
7.0
2
0.2
0.8
2.0
7.0
3
0.4
0.6
2.0
7.0
4
0.6
0.4
2.0
7.0
5
0.8
0.2
2.0
7.0
6
1.0
0.0
2.0
7.0
2. After replacing the above mentioned solutions as in section A in the labelled tubes, shake
well and then place them in a boiling water bath for 5 minutes.
3. Cool the tubes thoroughly and then add 7.0 ml of distilled water to each tube as indicated
in section B of the previous table, Read the extinction (Optical density) of the colored
solutions at 540 nm using the solution in tube 1 as a blank (control).
Note: All the tubes must be cooled to room temperature before reading since the extinction is
sensitive to temperature change.
15
4. Record the readings in section B, and plot the relationship between the optical density and
the concentration of glucose solution. See whether there is a linear relationship between the
concentrations of glucose solutions and their corresponding optical densities.
5. Use the already prepared standard curve for the determination of the unknown
concentration of the glucose solution provided and tissue extract form exp.6 or any other
unknown reducing sugar sample.
Name:
No.
Experiment 3:
Results Sheet
The concentration of standard glucose solution :
mg/ml
- After conducting your test, fill the following table :
Tube Concentration
(Mg/ml)
No.
Absorbance
(At 540 nm)
1
2
3
4
5
6
7
- Plot the standard curve of the absorbance (y- axis) against the concentration ( x-axis )
- Use this plot to estimate the concentration of your unknown glucose sample.
- Express your results in mg/dl , mg% , g/ml and g/l
16
Name:
No.
Experiment 3:
Results Sheet
17
EXPERIMENT 4 ___
_______
Est imation of carbohydrate by Anthrone
Method.
The anthrone reaction is the basis of a rapid and convenient method for
the determination of carbohydrates, either free or present
polysaccharides.
Principle :
Carbohydrates are dehydrated by concentrated H2SO4 to form
furfural.Furfural condenses with anthrone to form a blue-green colored
complex solution shows an absorption maximum at 620nm, which is
measured colorimetrically. note that some carbohydrates may give other
colors.
The extinction depends on the compound investigated, but is constant for
a particular molecule.
Materials :
1. Anthrone reagent (0.2% in conc. H2SO4).
2. Glucose (10mg/100ml).
Procedure :
1. Pipette out into a series of test tubes different volumes of glucose
solution and make up the volume to 1ml with water.
2. Add 4ml of anthrone reagent to each tube.
3. mix well.
4. Cover the tubes with marbles on top to prevent loss of water by
evaporation.
5. Keep the tubes in a boiling water bath for 10 minutes.
6. cool to room temperature.
7. Measure the optical density at 620 nm using a blank tube containing
1ml water and 4ml reagent.
8. Draw the standard cure and determine the concentration of unknown
glucose solution
18
in
Name:
No.
Experiment 4:
Results Sheet
19
EXPERIMENT 5__ _
_______
Quantitative Estimation of Pentoses
Principle:
When pentoses are heated with conc. HCl , furfural is formed which
condenses with orcinol in the presence of ferric ions to give a blue-green
color.
CH3
OH
HO
orcinol (3,5-dihidroxytoluene)
Materials :
1- Orcinol reagent. (Dissolve 1.5 g of orcinol in 500 ml of conc. HCl and
add 20 drops of a 100g/l solution of FeCl3.)
or 1.5 g Orcinol + (0.5 g FeCl3 + 500 ml conc. HCl)
Procedure:
Carry the experiment in two test tubes one for the standard and the other
for the unknown.In each tube place the following:
1- 7.5 ml Orcinol reagent.
2- 2.5 ml sample. Shake well.
3- Heat for 25 minutes in a boiling water bath with a marple on top of
each tube (use a glass stopper).
4- Cool to room temperature in cold water.
5- Read at 665 nm.
20
Calculation:
Standard : Ribose 2mg/ml (0.2%) in water.
Concentration of unknown = Absorbance of unk x Conc of std
Absorbance of std
21
Name:
No.
Experiment 5:
Results Sheet
Concentration of standard pentose solution :
Calculations:
Ast.=
Aun.=
22
mg/ml
Experiment 6
Determination of Vitamin C in Foods
By Iodometric Assay
Definition:
Vit. C is a water soluble vitamin that is necessary for normal growth and development.
Alternative Names:
Ascorbic acid
Food Sources:
Green peppers, Citrus fruits, Strawberries, Tommatos and white potato.
Function:
1- Promote healthy teeth and gums
2- Helps for absorption of iron
3- Promote wound healing
Recommended daily allowance (RDAs) of vit. C is = 70mg/day.
Side Effects:
Deficiency → Scurvy
Increased intake → Diarrhea
Principle:
Vit. C can be assayed by direct titration with iodine.
Procedure:
1- measure 10ml of juice
2- add 10 drops of starch
3- titer with 0.1 N iodine → blue color
23
Calculation:
1 L of 1 N iodine ≡ 88.06 ascorbic acid
1 ml of 0.1 N iodine = 88.06/ 10 x 1000= 0.008806
g% ascorbic acid ≡ Z x 0.008806 x 100/ 10ml (volume of juice)
Z = end point of titration
24
Results
25
EXPERIMENT 7___
_______
T he assay of t issue glycogen
Principle:
Glycogen is released from the tissue by heating with strong alkali and
precipitated on the addition of ethanol. Sodium sulphate is added as a co
precipitant to give a quantitative yield of glycogen.
The polysaccharide is then hydrolyzed in acid and the glucose released is
estimated.
Materials:
1. Heart, liver, and muscle from a freshly killed rat.
2.potassium hydroxide (300 g/l)
3. Calibrated centrifuge tubes (10 ml).
30
4. Boiling water bath.
24
5. Saturated Na2 S04.
20 ml
6. Ethanol (95% v/v).
250 ml
7. Volumetric flasks (100 ml).
24
8. Test tubes calibrated at 10 ml.
100 ml
9. HCl (1.2 mol/l.).
100 ml
10. Marbles.
11. Phenol red indicator solution.
12 ml
12. NaOH (0.5 mol/l).
250 ml
13. Reagents for the estimation of glucose (Experiment 1).
Procedure:
Isolation of glycogen:Accurately weigh the complete heart and muscle and about 1.5 g of
liver. Place the tissues into a calibrated centrifuge tube containing 2
ml of KOH (300 g/l) and heat in a boiling water bath for 20 min with
occasional shaking. Cool the tubes in ice, add 0.2 ml of saturated
Na2 SO4, and mix thoroughly. Precipitate the glycogen by adding 5
26
ml of ethanol (95% v/v), stand on ice for 5 min, and remove the
precipitate by centrifugation. Discard the supernatant and dissolve
the precipitated glycogen in about 5 ml of water with gentle
warming, then dilute with distilled water to the 10 ml calibration
mark and mix thoroughly. In the case of the fed animals, transfer the
liver sample quantitatively to a 100 ml volumetric flask and make up
to the mark with water.
Hydrolysis and estimation of glycogen:Pipette duplicate 1 ml samples of the glycogen solutions into test
tubes calibrated at 10 ml, add 1 ml of HCl (1.2 mol/l), place a marble
on top of each tube, and heat in a boiling water bath for 2 h. At the
end of this period, add 1 drop of phenol red indicator and neutralize
carefully with NaOH (0.5 mol/l) until the indicator changes from
yellow through orange to a pink color. Dilute to 5 ml with distilled
water and determine the glucose content by the 3.5 dinitrosalisylic
acid method (Experiment
1 ).Then use the standard curve you
obtained to estimate the concentration of glucose per100 g sample.
27
Name:
No.
Experiment 7:
Results Sheet
Calculate the amount of glycogen in the liver sample, using the standard
curve you plotted in experiment 1.
28
EXPERIMENT 8____
_______
Estimation of total lipids by colorimetric
method.
Principle:
Lipids react with sulfuric acid to form carbonium ions which
subsequently react with the vanillin phosphate ester to yield a purple
complex that is measured photometrically at 540 nm. The intensity of the
colour is proportional to the Total lipids concentration.
Materials :
1. Vanillin reagent, 0.04M. Dissolve 6.1 g of vanillin in water and
dilute to 1 liter. This solution is stable for about 2 months in a
brown bottle at room temperature.
2. . Phosphovanillin reagent. Add 350 ml of the vanillin reagent and
50 ml of water to a flask. Add with constant stirring, 600 ml of
concentrated (85%) phosphoric acid. This solution is also stable for
about 2 months in a brown bottle at room temperature.
3. Sulfuric acid, concentrated, reagent grade.
4. Standard solution. A good U.S.P. grade of olive oil may be used
as a standard. In two tarred 100 ml volumetric flasks add
approximately 0.5 and 1 ml of the olive oil and weigh again to
obtain the exact weight of oil added. (It is time consuming to try to
weigh out exactly 500 mg , or any other definite weight, of the oil;
the approximate amounts are added. and the exact weight
determined.) The above standards should be about 500 and 1,000
mg/dl. Dissolve the oil in absolute ethanol and dilute to the mark
with the ethanol. This solution is stable for about month in the
refrigerator.
5. Standard solution of cholesterol (1g/100 ml acetone)
29
Procedure:
In separate tubes add 20 l of water (blank), 20 l of samples, and 20 l
of standards. To each tube add 0.2 ml of concentrated sulfuric acid. Mix
well, preferably on a vortex mixer. Place all tubes in boiling water bath
for 10 min, remove, and cool in water to room temperature. To each tube
add 10 ml of the phosphovanillin reagent and mix well. Incubate at 370C
in a water bath for 15 min. Cool and read standards and samples against
blank at 540 nm.
Calculations :
Concentration of unknown = Absorbance of unk x Conc of std
Absorbance of std
30
Name:
Experiment 8:
No.
Results Sheet
Concentration of standard cholesterol solution :
mg/ml
Concentration of standard olive oil solution :
mg/ml
1)
Ast. Cholesterol =
Aun cholesterol =
2)
Ast. Olive Oil =
Aun Olive Oil =
31
EXPERIMENT 9
___
Estim ation of Cholesterol Liberman – Bu rchard
Reaction
Principle :
Cholesterol is readily soluble in acetone, while most complex lipids are
insoluble in this solvent.
Blood or serum is extracted with an alcohol-acetone mixture which
removes cholesterol and other lipids and precipitates protein. The
organic solvent is removed by evapotation on a boiling water bath and
dry residue dissolved in chloroform. The cholesteror is then determined
colorimetrically using the Liebermann-Burchard reaction.
Acetic anhydride reacts with cholesterol in a chloroform solution to
produce a characteristic blue-green color.
The exact nature of the
chromophore is not known but the reaction probably includes
esterification of the hydroxyl group in the 3 position as well as other
rearrangement in the molecule.The cholesterol is determined
colorimetrically using the Libermann – Burchard reaction.
Materials :
1. Serum or blood.
2. Alcohol-acetone mixture (1:1).
3. Chloroform.
4. Acetic anhydride-sulphuric acid mixture
(30:1 mix just before use, Care!).
5. Stock cholesterol solution (2 mg/ml in
chloroform).
6. Working cholesterol solution. (Dilute the
above solution one in five with chloroform to
give a solution of 0.4 mg/ml.)
Procedure :
32
25 ml
21
500 ml
930 ml
250 ml
11
1- Place 10 ml of the alcohol-acetone solvent in a centrifuge tube and
0.2 ml of serum or blood.
2- Immerse the tube in a boiling water bath with shaking until the
solvent begins to boil.
3- Remove the tube and continue shaking the mixture for a further 5
min.
4- Cool to room temperature and centrifuge.
5- Decant the supernatant fluid into a test tube and evaporate to
dryness on a boiling water bath.
6- Cool and dissolve the residue in 2 ml of chloroform.
7- Add 2 ml of acetic anhydride-sulphuric acid mixture to all tube and
thoroughly mix.
8- Leave the tubes in the dark at room temperature and read the
extinction at 680 nm.
9- Carry the experiment in a test tube one for the standard:
a-0.2 ml of standard then 2 ml of chloroform.
b-Add 2 ml of acetic anhydride-sulfuric acid mixture and
thoroughly mix.
c- Leave the tube in the dark at room temperature and read the
extinction at 680 nm.
Calculation:
Determine the concentration of the unknown sample according to the
following equation:
Concentration of unknown = Absorbance of unk x Conc of std
Absorbance of std
33
Name:
No.
Experiment 9:
Results Sheet
Calculate the concentration of cholesterol in your sample.
34
EXPERIMENT 10____
_______
Determination of Saponification Number.
Principle:
On refluxing with alkali, triacylglycerols (fatty acid esters) are
hydrolyzed to give glycerol and potassium salts of fatty acids (soap).Such
process is known as, Saponification . The saponification
equation is
shown below:
O
CH2
CH
O
O
C
O
R
C
R
Saponification
O
CH2
O
C
R
+
3 KOH
CH2 OH
CH
H
e+
at
Water
OH
CH2 OH
+
3 RCOONa
Soap
Fat
The saponification value is the number of milligrams of KOH required to
neutralize the fatty acids resulting from the complete hydrolysis of 1g of
fat.
The saponification value gives an indication of the nature of the fatty
acids constituent of fat and thus, depends on the the average molecular
weight of the fatty acids constituent of fat. The greater the molecular
weight (the longer the carbon chain),
the smaller the number of fatty acids is liberated per gram of fat
hydrolyzed and therefore, the smaller the saponification number and vice
versa.
Materials:
1- Fats and oils (olive oil, coconut oil, sesame oil, and butter)
2- Fat solvent (equal volumes of 95% ethanol and ether)
3-Alcholic KOH (0.5 mol/liter)
35
4-Boiling water bath.
5-Phenolphethalein.
6-Hydrochloric acid (0.5 mol/liter)
7-Burettes (10 ml and 25 ml)
8-Conical flasks (250ml)
Procedure:
1- Accurately take 0.5 ml of oil in a 250 ml conical flask (flask 1).
2- Add 10 ml of alcoholic KOH in flask 1 and in another flask (flask 2)
add 10 ml of KOH only.
3- Heat flask 1 on a boiling water bath for 10 min.
4- Leave to cool to room temperature.
5- Add 10 drops of Ph. Ph. as indicator in each flask
6- Titrate with 0.5 mol/liter HCl until the pink color disappears.
7- Record your readings as T ml (flask 1) for test and B ml (flask 2) for blank.
Calculations:
The difference between the blank and the test reading gives the number of
milliliters of KOHrequired to saponify 1g fat.
You can use this formula to calculate the saponification value:
1ml (0.5 N HCl ) = 28.05 mg KOH
( B-T ) =
S
saponification value (S) = ( B-T ) x 28.05 =
Wt. of fat (1g
36
mg KOH/1g
Name:
No.
Experiment 10:
Results Sheet
1- Calculate the Saponification value -for your test oil.
2- Record the results your friends have obtained for other oils.
1- your results:
37
38
39