STANDARD SOLUTION AND STANDARDISATION

1. Experiment Objectives
1. To prepare a primary and secondary standard solutions
2. To do standardisation of secondary standard solution

2. Theory
Titration is a common laboratory method of quantitative chemical analysis that is
used to determine the unknown concentration of a known reactant. A reagent, called the
titrant, of a known concentration (a standard solution) and volume is used to react with a
solution of the analyte or titrant, whose concentration is unknown.
A standard solution is a solution containing a precisely known concentration of an
element or a substance.
There are two kind of standard solution:
1. Primary standard solution.
2. Secondary standard solution.
Primary standard solution:
1. Standard solution that is accurate enough and is not calibrated by or subordinate
to other standards.
2. A primary standard is typically a reagent which can be weighed easily, and which
is so pure that its weight is truly representative of the number of moles of
substance contained.
Examples: oxalic and boric acid.
Secondary standard solution:
1. Its concentration is able to change and must be calibrated by primary standard.
2. A reagent which is prepared from solid or liquid materials which do not have
high purity.
Examples: hydrochloride acid and sodium hydroxide solution.
By using a calibrated burette to add the titrant, it is possible to determine the exact
amount that has been consumed when the endpoint is reached. The endpoint is the point
at which the titration is complete, as determined by an indicator. This is ideally the same
volume as the equivalence point—the volume of added titrant at which the number of
moles of titrant is equal to the number of moles of analyte. Many methods can be used to

Analytical Chemistry Laboratory 1

 indicate the endpoint of a reaction; titrations often use visual indicators (the reactant
mixture changes color). In simple acid-base titrations a pH indicator may be used, such as
phenolphtalein, which becomes pink when a certain pH (about 8.2) is reached or
exceeded. Another example is methyl orange, which is red in acids and yellow in alkali
solutions. Not every titration requires an indicator. In some cases, either the reactants or
the products are strongly coloured and can serve as the "indicator". For example, a redox
titration using potassium permanganate (pink/purple) as the titrant does not require an
indicator. When the titrant is reduced, it turns colourless. After the equivalence point,
there is excess titrant present. The equivalence point is identified from the first faint
persisting pink colour (due to an excess of permanganate) in the solution being titrated.

3. Materials
1. NaOH p.a.
2. H2C2O4.H2O
3. Na2B4O7.10 H20
4. HCl p.a
5. Phenophtalein
6. Methyl orange
7. Distilled water

4. Experiment Apparatus
1. Measuring flask 100 ml
2. Beaker glass 250 ml
3. Erlenmeyer 125 ml
4. Burette 50 ml
5. Volumetric pipette 10 ml, 25 ml
6. Measuring pipette 10 ml
7. Pipette
8. Weighing bottle

5. Procedures
a. Preparation of oxalic acid solution:
1. Weight the oxalic acid ( H2C2O4 .2H2O) as much as 290 – 310 mg.
2. Dilute it with distilled water of 75 mL in the 250 mL beaker glass.

Analytical Chemistry Laboratory 2

 3. Put it in to the measuring flask and shake appropriately until homogeneous.

b. Preparation of sodium hydroxide:

1. Weight 500 mg of NaOH in the weighing bottle.
2. Pour 50 mL of distilled water to the beaker glass and mix with the NaOH.
3. Put 7.5 mL of NaOH solution to the beaker glass containing 100 mL of distilled
water.

c. Standardisation of sodium hydroxide solution:

1. Put 25 mL of oxalic acid solution to the Erlenmeyer flask and drop an indicator
of phenophtalein.
2. Fill the burette with sodium hydroxide solution.
3. Drop the titrant (sodium hydroxide) to the solution of oxalic acid until the
endpoint is reached.
4. Record the volume of sodium hydroxide when the colour of analite solution
changes.
5. Standardisation is done 3 times (no. 1-4).
6. Calculate the average volume of sodium hydroxide.

d. Preparation of boric acid solution:

1. Weight the borax (Na2B4O7 .10 H2O) as much as 500 – 700 mg.
2. Put the borax into a glass beaker and dissolve with 75 ml of distilled water.
3. Move over borax solution into the flask size 100 ml and shake until
homogeneous.

e. Preparation of HCl 0.1 N solution:

1. Note the density and concentration of concentrated HCl from concentrated HCl
bottle.
2. Pour distilled water into a flask size 100 ml, up to approximately half the volume
of the measuring flask.
3. Take as many x ml concentrated HCl (from calculation) with a measuring pipette
and put into the measuring flask which has been filled with distilled water.
4. Add distilled water into a measuring flask up to mark the line and shake until
homogeneous.

Analytical Chemistry Laboratory 3

 f. Standardisation of HCl 0.1 N solution:
1. Take 25 mL of boric acid solution and pour into the Erlenmeyer. Drop a few of
methyl orange.
2. Pour HCl solution into the burette.
3. Drop HCl solution into boric acid solution in the Erlenmeye until the endpoint is
reached. Record volume of HCl solution.
4. Repeat 3 times the procedure 1-3.
5. Calculate average volume of boric acid solution.

6. Data Analysis
1. Calculation of normality of oxalic acid solution

Normality =

Wg grek 1
 g
x2 x
M gmol gmol litre

2W grek 2W
 
M 75l 75 M

2. Calculation of normality of NaOH solution

g eq ( NaOH ) = g eq (Ox. Acid)
VNaOH NNaOH = Vox. acid Nox. acid

Vox. acid 2 W
N NaOH 
VNaOH 75M
3. Calculation of normality of boric acid solution:

Normality =

Wg grek 1
 g
x2 x
M gmol gmol 75 litre

Analytical Chemistry Laboratory 4

 2W grek 2W
  N
M 75 75 M

4. Calculation of volume of concentrated HCl

For preparing HCl 0,1 N solution as many V mL, therefore concentrated HCl which
must be taken = X ml.
mgek mg
0 ,1 x V ml x 36 ,5
ml mgmol
X 
mgek
1
mgmol
X  3 ,65 V mg

Concentration of concentrated HCl = k %

Density of concentrated HCl = L g/ml
3 ,65 V mg
X 
g 1000 mg k
L x x
ml g 100
3 ,65V
X  ml
10 x k x L

5. Calculation of normality of HCl solution

g eq ( HCl ) = g eq ( Boric acid )
VHCl NHCl = VBoric acid . NBoric acid
VBoric acid 2W
N HCl 
VHCl 75M

7. Report
a. Discussion
- Indicate which includes primary and standard solution in the experiment!
- Why does the secondary standard solution need to be standardised?
- Explain the working principles of the titration process?
- When will the endpoint occur?

Analytical Chemistry Laboratory 5

 b. Preliminary Report

PRELIMINARY REPORT
STANDARD SOLUTION AND STANDARDISATION

Group:
No. Name Student's number
1. ………………………. ……………………
2. ………………………. ……………………
3. ………………………. ……………………

Day/Date:

1. Materials
No. materials Density Purity Mass Volume
1. NaOH p.a. …………… …… …… ………
2. HCl p.a. …………… …… …… ………
3. Na2B4O7.10H2O …………… …… …… ………
4. H2C2O4.2H2O …………… …… …… ………

2. Data
No. Solution Volume, mL
I II III
1. NaOH ……… ……. …….
2. H2C2O4.2H2O ……… ……. …….
3. HCl ……… ……. …….
4. Na2B4O7.10H2O ……… ……. …….

Surakarta, ……………….
Students,
1…………..
2…………..
3…………..

Analytical Chemistry Laboratory 6

 POTENTIOMETRIC ANALYSIS

1. Experiment Objectives
1. To investigate the properties of polybasic acids or salts.
2. To determine level of acid or salt in a polybasic potentiometry.

2. Theory
Polybasic acid is the acid in solution will have more than one level of ionization,
with ionization constant respectively. For example, a weak acid swapped n, constant
level and ionization are as follows:

HnA + H2O  H3O+ + Hn-1A- K a1 

H O H
3

n 1 A 
H n A
Hn-1A- + H2O  H3O+ + Hn-2A2- K a2 
H O H A  
n2
2

H A 
3

n 1

and so on until finally:

HA(n-1) + H2O  H3O+ + A-n K a2 

H O A   n

HA 
3
( n 1 )

Therefore, when the acid titrated with strong base flavors of one (eg NaOH) then
it gained more than one equivalence point of the reaction and pH of equivalent point as
follows:
HnA + NaOH  NaHn-1A + H2O pH1 = ½ (pKa1 + pKa2)
NaHn-1A + NaOH  Na2Hn-2A + H2O pH2 = ½ (pKa2 + pKa3)
and so on until finally:
Nan-1A + NaOH  NanA + H2O pHn = ½ (pKw + pKan + log (G))
If the Ka value is very small (very weak acid) then there is no indicator that can
be used to determine when the final equivalence point. This can be addressed among
others by a potentiometric titration.
Potentiometric titration is a titration where the final titration not using an
indicator but determined by measuring the change in electrode potential or pH changes
(in particular the addition of each titrant solution).
Determination of titration end point / equivalence point can be made by:

Analytical Chemistry Laboratory 7

 1. Creating graphs:
a. E versus V
b. pH versus V
2. Creating a graph of E/V versus V or pH/V then looking at the maximum
or minimum point of the graph.
3. Crossing the graph 1 and 2.
And vice versa, if a solution of sodium salt of a polybasic acid is titrated with HCl then
the equivalence point also more than one.

3. Materials
1. Buffer solution
2. Standard solution of NaOH 0,1 N
3. Standard solution of oxalic acid (from 1st experiment)
4. Solution of H2SO4, Na2SO3, NaHCO3
5. Distilled water
6. Phenolphtalein indicator

4. Experiment Apparatus
1. pH meter
2. Burette
3. Erlenmeyer
4. Pipette
5. Magnetic stirrer
6. Beaker glass

5. Procedures
1. Make a standard solution of 0.1 N NaOH and then standardized with a standard
solution of oxalic acid.
2. Turn on pH meter and leave it for + 15 minutes. While waiting period, wash the
electrode with distilled water and dry with wipe paper carefully.
3. Insert the electrodes into a beaker glass filled with buffer solution of pH 4 or 7 or 9
and turn the key 4 on USE and turn the key 3 on the pH indicated the needle 5 on the

Analytical Chemistry Laboratory 8

 scale. If the needle does not point at pH 4 or 9 then turn the key 1 so that the needle
indicates the pH suitable buffer solution that is inserted into the beaker glass.
4. Repeat step no. 3, until without turning or changing the position of the needle button
1, it has already a scale showing the actual amount of solution pH.
5. Take 10 ml of H2SO4 solution samples or other samples and add to the volume of 50
ml distilled water and pour into a beaker glass and put the magnetic stirrer. Put the
electrode that had been washed and dried into dilute solution of H2SO4 and turn the
key 3 and measure the pH of dilute H2SO4.
6. Add standard solution of 0.1N NaOH from a burette. Make a note the change in pH
for each addition of titrant V ml until the equivalence point.
7. Do the same procedures for each sample.
8. After all the works are completed, turn off the pH meter, and soak the electrode in
distilled water.
9. Make a graph of pH versus volume of NaOH and pH/V versus volume of NaOH
and determine the equivalence point of the neutralization reaction.
10. Calculate the concentration of sample titrated.

6. Data Analysis
Normality of NaOH
grek ( NaOH ) = grek (Oxalic acid )
VNaOH NNaOH = Voxalic acid . Noxalic acid
Voxalicacid 2W
N NaOH 
V NaOH 75 M

7. Report
a. Discussion
- Why does a polybasic acid have the level of ionization? Write ionization reaction
sequence used for the experiment of polybasic acid!
- Write the reaction that occurs when the polybasic acid used in the experiment is
titrated with aqueous NaOH!
- Why does a polybasic acid perform pH changes during titration? How is the
trend? (See the curves)

Analytical Chemistry Laboratory 9

 b. Preliminary Report

PRELIMINARY REPORT
POTENTIOMETRY

Group :
No. NAME NIM
1. ………………………. ……………………
2. ………………………. ……………………
3. ………………………. ……………………

Day/Date :
Data of Standardization of NaOH solution
Volume of NaOH solution= …………….ml
No. Volume of oxalic acid
1. …………………….
2. …………………….
3. …………………….
Average = ………………..

Data of Experiment
pH
Volume of NaOH,
No. H2SO4 Na2SO3 NaHCO3
ml
1 2 3 1 2 3 1 2 3
1 0
2 2 (example)
3 4 (example)
etc. 6 (example) etc.

Analytical Chemistry Laboratory 10

Graphs (made on millimeter/graph paper)

pH
and
pH/V

Surakarta, ………………………
Students
1……………..
2……………..
3……………..

Analytical Chemistry Laboratory 11

 POLARIMETRIC ANALYSIS

1. Experiment Objectives
1. To create a polarimetric standars curve based on standard solution known its
concentration.
2. To determine an unknown concentration of an active optic solution using polarimetric
method.

2. Theory
White light is polychromatic light consisting of different wavelengths that can vibrate all
directions. It can be converted into monochromatic light consisting of only one wavelength by
using a filter or special light sources. Monochromatic light is called polarised light. Interaction
of a certain organic compounds with polarised light was analysed with a polarimeter. While the
polarimeter is an instrument used to measure the magnitude that occurred due to the interaction
of organic compounds with polarised light.
Optically active materials are compounds that can rotate the polarisation of plane
polarized light. Optical substance is characterised by an asymmetrical carbon atom or
chiral C atoms in organic compounds, for example: quartz (SiO2), fructose.
Essentially monochromatic light have a lot of vibration plane. The plane of
vibration will be perpendicular to the plane. The vibration plane can be mechanically
separated into two planes which are mutually perpendicular. The polarised light is a
compound that has a one-way vibration which are perpendicular to its direction.
The basic principle of polarimetry is the measurement of optical spin degree of a
substance that cause rotation of vibrating field polarised beam. There are two kinds of
rotation of vibrating field polarised beam by optically active compounds, namely:
1. Dexro rotary (+), if the direction of rotation to the right or according to a clockwise.
2. Levo rotary (-), if the direction of rotation to the left or counterclockwise.
The beam has a vibration direction and the direction of propagation in all
directions with a variety of colors and wavelengths, known as polychromatic.
To produce monochromatic light, then use a filter or source particular light. This
monochromatic light will pass through a prism, a crystal which has particular screen that
prevent the passage of light, so that the light has only one direction of vibration which is
known as polarised light.

Analytical Chemistry Laboratory 12

 If a light is passed on a solution, a solution will continue the light which
vibrational direction is unidirectional and absorb the perpendicular to this direction. Here
the solution is used as a polarisator. Finally the light that comes out of solution are
polarised light field.
Specific rotation is symbolized with []t therefore can be formulated:
[]t =  / dc
Where :
 = the angle polarized by a solution with a concentration of c grams of
solute per mL of solution observed
d = distance of the solution (dm)
c = concentartion (gram/mL)
t = temperature (oC)
 = wave length, if use Na light at room temperature 20oC is notated D, ie.
589,3 nm.
By using the same tube, so the concentration of substances can be determined by a
standard curve.

3. Materials
1. Sucrose
2. Distilled water

4. Experiment Apparatus
1. Polarimeter instrument
2. Flask size 100 mL
3. Pipette
4. Erlenmeyer
5. Weighing bottle
6. Glass stirrer
7. Glass funnel

5. Procedures
1. Prepare 100 mL of sucrose solution in 5 flasks using 5, 10, 15, 20, dan 25 gram of
sucrose.

Analytical Chemistry Laboratory 13

 2. Fill fully the cuvet with distilled water and make sure no bubble in the cuvet.
3. Rotate prism analyser until you get a light plane. Do this measurement 3 times.
Record this position as zero point.
4. Repeat procedure no 2-3 using other solutions. Each solution is required 3
observations. Do not forget to clean the cuvet using distilled water every time you
change solution.
5. Repeat procedure no 2-3 using sample unknow its concentration.
6. Create calibration curve of optical rotation value vs standard solution concentration.
7. Find the sample concentration from that curve.

6. Report
a. Discussion
- Explain how the principle polarimeter!
- What is the value of optical rotation for distilled water? Why?
- What is the effect of sucrose concentration on the value of optical rotation?
- How do you determine the sample concentration?

b. Preliminary Report

PRELIMINARY REPORT
POLARIMETRIC ANALYSIS
Group :
No. Name Student's number
1. ………………………. ……………………
2. ………………………. ……………………
3. ………………………. ……………………

Date:
Data
No. Concentration Value of optical rotation
I II III average
1. 0 …………… …………. ………….. ………
2. 5 mg/100 mL …………… …………. ………….. ………

Analytical Chemistry Laboratory 14

 3. 10 mg/100 mL …………… …………. ………….. ………
4. 15 mg/100 mL …………… …………. ………….. ………
5. 20 mg/100 mL …………… …………. …………. ………
6. 25 mg/100 mL …………… …………. ………….. ………
7. Sample …………… …………. ………….. ………
Surakarta, ………………………
Students
1……………..
2……………..
3……………..

Analytical Chemistry Laboratory 15

 COMPLEXOMETRIC ANALYSIS

1. Experiment Objectives
1. To understand the principles of complexometric quantitative analysis.
2. To investigate and determine the magnesium concentration in samples.

2. Theory
One of chemical reaction types that can be the basic of titrimetry method
implementation is reaction of complex ion formation, which is soluble and little
dissociation able. Some metallic ions such as cobalt, nickel, zinc, cadmium and mercury
(II) react with nitrogen compounds (example: ammonia and tri-en) form the stabilized
complexes. Meanwhile, other metallic ions such as aluminum, plumbom and bismuth are
suitable to react with compounds contain oxygen as electron donator.
Several compounds that contain both nitrogen and oxygen can be reacted with
some metallic ions to produce the stabilized complexes. Ethylene Diamine Tetra Acetic
Acid (EDTA) is a popular one.

3. Materials
1. EDTA
2. MgSO4.7H2O as sample
3. NH4Cl A.R
4. NH4OH
5. EBT indicator
6. Tri ethanolamine
7. Ethanol
8. Distilled water

4. Experiment Apparatus
1. Volumetric flask
2. Digital balance
3. Burette
4. Measuring pipette
5. Beaker glass 250 ml

Analytical Chemistry Laboratory 16

 6. Erlenmeyer
7. Volume pipette
8. Drop pipette
9. Stirring glass

5. Procedures
a. Preparation of EDTA Solution 0.1 M
EDTA (9.365 g) is dissolved in 100 ml of distilled water in beaker glass. The solution
is moved into volumetric flask (250 ml), and the amount of distilled water be added
until limited line of volumetric flask. The volumetric flask is shaken to homogenate
that solution well.

b. Preparation of Buffer Solution pH 10

NH4Cl A.R (17.5 gram) is dissolved in 142 ml of the high concentration solution of
NH4OH; and then the amount of distilled water is added until the limited line of
volumetric flask. The volumetric flask is shaken in order to get homogeneous
solution.

c. The Process Steps Used to Determine the Magnesium Concentration in Samples.

Mixture of both sample solution and distilled water are 10 ml each is prepared in
Erlenmeyer, added by 0.9 ml buffer solution ( pH = 10) and EBT indicator (1 – 2
drops). Titration process can be conducted, the EDTA solution 0.1 M titrate that
sample until its color is changed from red to blue. As a notice, be careful in treating
process particularly near an equivalent point in order to get valid data.

6. Data Analysis
In the preparation of EDTA solution 0.1 M, equation below is can be used.
m1
M1 
BM1 . V
The quantitative analysis of magnesium can be determined by using this correlation:
1 ml 0.1 M EDTA ~ 2.432 mg Magnesium
The weight of magnesium = (2.432 x V1) mg
Where:
M1 = the molarity of EDTA (mmole/ml)

Analytical Chemistry Laboratory 17

 m1 = the weight of EDTA (gram)
BM1 = the molecular weight of EDTA (mg/mmol)
V = Initial volume of EDTA (ml)
V1 = Volume of EDTA solution 0.1 M (ml)

7. Report
a. Discussion
- Describe and explain clearly the basic or fundamental principles of
complexometric analysis.
- Describe, analyze and explain all of results (include reaction and changes).
- What is a complex ion? Give the appropriate explanation!

b. Preliminary Report

PRELIMINARY REPORT
COMPLEXOMETRIC ANALYSIS

Group:
No. Name Student’s Number
1. ………………………. ……………………
2. ………………………. ……………………
3. ………………………. ……………………

Analytical Chemistry Laboratory 18

Day/Date:
Determining the amount of magnesium in samples uses complexometric analysis
method.
Volume of sample :
The molarity of EDTA standard solution :
No Volume of sample Volume of EDTA solution 0.1 M

1
2
3

Surakarta, ………………………….
Students
1……………………..
2……………………..
3……………………..

Analytical Chemistry Laboratory 19

 CARBONATE ANALYSIS

1. Experiment Objective
To determine the concentration of carbonate in samples by using both qualitative and
quantitative analysis methods.

2. Theory
The qualitative analysis method of the carbonate concentration can be realised by adding
certain reagent to produce precipitated carbonate. The precipitated carbonate is dissolve
in especially solvent. While, acidimetric method is used to analyse carbonate
quantitatively. In the titration process, chloride acid is used as titran. Besides, indicator
agents are very important in that process because they can indicate when the process has
to be finished. The determining of indicators is based on pH of equivalence.

3. Materials
1. Sodium hydroxide as sample
2. Chloride acid
3. Barium chloride
4. Calcium chloride
5. Methyl orange as indicator
6. Phenolphthalein
7. Sodium borax
8. Distilled water

4. Experiment Apparatus
1. Beaker glass
2. Erlenmeyer
3. Volumetric Flask
4. Burette
5. Measuring pipette
6. Volume Pipette
7. Weight Bottle
8. Funnel Conical

Analytical Chemistry Laboratory 20

 9. Stirring Glass
10. Drop pipette

5. Procedures
a. Standardisation of the Chloride Acid Solution.
Sodium borax are put, weigh 500 - 600 gram accurately. Then, that sodium borax is
poured into beaker glass, add 75 ml distilled water to dissolve that compound. The
solution is moved into volumetric flask (100 ml), add the distilled water until limited
line, and do not forget to shake. A 25 ml sodium borax solution is moved from that
flask into erlenmeyer by using a pipette, add 2 drops of methyl orange indicator. Pour
a chloride acid standard solution into burette, titrate sample until approach
equivalence and note volume of chloride acid solution are used. Do these steps at least
twice more.

b. The Carbonate Qualitative Analysis.

A 3 to 4 flakes of NaOH is put into a weight bottle, then weigh it accurately. The
NaOH solution can be made by dissolving those flakes into distilled water in flask
(250 ml). Solution or sample is moved into erlenmeyer, heat at certain temperature
(70oC). The barium chloride solution 10% is added into sample until the end of
forming precipitation or sedimentation. That mixture is cooled, analyze both physical
properties and the amount of precipitation and write on experimental paper. Do these
steps for another compound (ex: calcium chloride to replace barium chloride), in wich
the process is carried out without heating.

c. The Carbonate Qualitative Analysis in Sample.

A 25 ml of carbonate solution (as a sample) is put into Erlenmeyer, add methyl orange
indicator and titrate sample by chloride acid solution. Volume of chloride acid
solution (V1) is written on the experimental report. Do these steps at least twice more.
Meanwhile, a 25 ml of carbonate solution (as a sample) is put into Erlenmeyer, add
phenolphthalein indicator and titrate sample by chloride acid solution. Volume of
chloride acid solution (V2) is written on the experimental report. Do these steps at
least twice more

Analytical Chemistry Laboratory 21

6. Data Analysis
a. Standardisation of Chloride Acid Standard Solution.
The equation below can be used to determine normality of solution:
V1 N 1
N2 
V2

b. The Quantitative Analysis of Carbonate in Sample

There are two equation related to calculate the percentage of carbonate in sample:
mass of carbonate 
(V1  V2 )  N chloride acid  BE carbonate  (number of watering processes)

m CO3
the percentage of carbonate   100%
W
where:
m CO3 = mass of carbonate in sample

W = mass of sample

7. Report
a. Discussion

- Describe, analyze and explain all of results (include reaction and changes).
- What are the meaning of both equivalent point and the end of titration? Give
an appropriate explanation!

b. Preliminary Report

PRELIMINARY REPORT
CARBONATE ANALYSIS

Group:
No. Name Student’s Number
1. ………………………. ……………………
2. ………………………. ……………………
3. ………………………. ……………………

Analytical Chemistry Laboratory 22

Day/Date:
1. Properties of materials :
Name of Weight Purity  Volume
No reagent gram % g/cm3 ml
1
2
3
4
5
6
7
8

2. The Qualitative Analysis Data

No Step of Experiment Change

Conclusion:
__________________________________________________________

3. The Quantitative Analysis Data

a. The Chloride Acid Standardisation
Volume, ml
No Solution
1 2 3
1. Chloride acid

2. Sodium borax

Analytical Chemistry Laboratory 23

 b. Carbonate Analysis
Volume, ml
No Solution
1 2 3
Carbonate sample
1.
Chloride acid
2.

Surakarta, ………………………
Students
1……………..
2……………..
3……………..

Analytical Chemistry Laboratory 24

 COLORIMETRIC ANALYSIS

1. Experiment Objective
To determine levels of a substance with a colorimetric method.

2. Theory
Colorimetry is a method of chemical analysis based on comparison of color
intensity of a solution with the color standard solution. This method is more advantageous
than other methods, because it requires chemicals, time and content of the relatively small
sample that is less than 1% of substances or elements that exist in small amounts in the
samples of the pure solvent.
Colorimetric analysis using a colorimeter. Colorimeter equipped with a
photometer to replace eye cells called photo electric colorimeter, which can generate
currents with a strength that depends on how much light is absorbed by the solution. But
this type of equipment is relatively expensive. If as a heat source used white light that
made simple, it is called colorimeter.
There are several kinds of methods of colorimetric analysis, among others:
1. Dilution method
The sample solutions and standards respectively in Nessler tubes diluted to be seen
directly from the side having the same color.
2. Standard series method
Solution to be analyzed contained in Nessler tube color (directly by eye) compared
with a series of standard solution with the same volume.
3. Colorimetric titration method
Standard solution was added by titration to the reagent until the color generated is
similar to the sample solution and each solution of equal volume.
4. Balancing method
In methods 2 and 3 above thick layer (the surface liquid height) at t1 and t2, so that
when the same color can be defined concentration of 1 is equal to the concentration
of 2 (c1 = c2). Meanwhile in the method of balancing a thick layer of modified until the
color is the same:
t1. c1 = t2. c2 or c2 = t1. c1 / t2
t = height of liquid surface
c = concentration of solution

Analytical Chemistry Laboratory 25

 Determination of Fe(III) using Colorimetric Method
Ferric ion reacts with thiocyanate to produce the red color of the complex compound is
formed:
Fe 3+ + 6 CNS-  [ Fe(CNS)6]3-
For the reaction Fe3+ is complete use excessive thiocyanate, while strong acid is necessary
to avoid the occurrence of hydrolysis.
Fe3+ + 3 H2O  Fe(OH)3 + 3 H+
3. Materials
1. Ferric ammonium
2. Concentrated HCl
3. KCNS 10%
4. Distilled water
4. Experiment Apparatus
1. Reaction tube
2. Measuring pipette 10 ml
3. Pipette drops
4. Measuring flask 1 L, 100 ml
5. Burette
6. Glass watch

5. Procedures
1. Make a solution of Fe3+ standard (1 mL = 0.1 mg Fe3+). Weigh accurately 0.4520 g
ferric ammonium alum, dissolved in distilled water to taste, then move into a 500 mL
measuring flask. Add 5 mL of concentrated HCl, then dilute with water to 500 mL.
2. A total of 10 mL standard solution of the above (no. 1) is inserted into the measuring
flask 100 mL (1 mL = 0.01 mg Fe3+), add water, then shaken and put into a burette
3. Make a color standard solution.
Prepare a test tube (eg 5 pieces) and give the serial number, input the standard
solution of Fe3+ at the top (no. 2) into each test tube with a volume rise, for example:
1 mL, 2 mL, 3 mL, 4 mL, and 5 mL. Add 5 mL of solution KCNS 10%, dilute with
distilled water to 20 mL, shake until blended, observe the color of each solution in a
test tube.

Analytical Chemistry Laboratory 26

 4. A total of 5 mL sample solution (provided by the lecturer / assistant) is inserted into
another tube, add 5 mL KCNS 10% solution, dilute with distilled water to 20 mL,
shaken until a homogeneous solution.
5. a. Compare the color of solution no. 4 with the color of a row of standard solution of
Fe3+ (no. 3). If the color of solution no. 4 together with one standard solution of
Fe3+, the concentration of solution no. 4 are the same as the standard solution.
b. If the color standard solution with no. 4 has not been there the same, then dilute a
standard solution whose color a little more condensed with water (from the burette)
and shaken until the color the same color with no. 4, note the liquid surface height.
Calculate the concentration of solution no. 4.

6. Data Analysis
Determination of Fe using Colorimetry
1. For the same volume of solution, at the time of color the same (5a):
Concentration of sample = concentration of standard
Fe = Fe standard solution sample = concentration x volume of standard solution
standard solution
2. For a different solution volume, at the same solution color (5b):
Concentration of sample = concentration of the diluted standard
The concentration of the diluted standards = c1 x t1 / t2
c1 = standard concentration at first (before dilution)
t1, t2 = height of fluid surface at first, the end

7. Report
a. Discussion
- The principle of colorimetric analysis related to Fe content.
- Compare the Fe content of the results of colorimetric analysis with the actual
concentration that made by assistant. Explain the causes of deviations that may
occur.

Analytical Chemistry Laboratory 27

 b. Preliminary Report

PRELIMINARY REPORT
COLORIMETRIC ANALYSIS
Group :
No. Name Student's number
1. ………………………. ……………………
2. ………………………. ……………………
3. ………………………. ……………………
Day/Date :
Determination of Fe3+ using Colorimetry
Weight of ferri ammonium = …………………………
No Volume of Vol. of standard Fe3+ t1 t2
sample At initial/Color no.
1. 20 mL 20 mL/ …….. ………. ……….
2. 20 mL 20 mL/ …….. ………. ……….
3. 20 mL 20 mL/ …….. ………. ……….
4. 20 mL 20 mL/ …….. ………. ……….
5. 20 mL 20 mL/ …….. ………. ……….

Surakarta, ………………………
Students,
1……………..
2……………..
3……………..

Analytical Chemistry Laboratory 28

 PERMANGANOMETRIC ANALYSIS

1. Experiment Objectives
1. To make a standard solution of KMnO4 and standardization of KMnO4 solution
2. To use a standard solution of KMnO4 for the analysis of iron and calcium salts.

2. Theory
Potassium permanganate, KMnO4, is a strong oxidant, the permanganate ion is
violet. By the influence of sunlight, potassium permanganate breaks down into MnO4-. In
acid solution, permanganate ion to give the reaction (Vogel, 1985):
 
Mn O4  8H   5 e 
 Mn2  4 H 2O

From the equation it can be noted:

1 gmol g
1 Normal KMnO 4   31,606
5 L L

H2SO4 is used for acidification, not HCl, because HCl will oxidize chloride (Cl- )
to chlorine (Cl2) as the reaction below:
2 MnO4- + 10 Cl- + 16 H+ → 2 Mn++ + 5 Cl2 + 8 H2O

KMnO4 is not a primary standard solution, therefore the standard solutions must
be standardized before use. On the standardization of KMnO4 solution, it can be used
Na2C2O4, H2C2O4.2H2O, As2O3, K4Fe(CN)6.3H2O. The use of primary standard of
Na2C2O4 and H2C2O4.2H2O solutions are more common than others.

The reactions are:

5 Na2C2O4 + 2 KMnO4 + 8 H2SO4 → 2 MnSO4 + K2SO4 + 5 Na2SO4 + 8 H2O + 10 CO2
5 Na2C2O4 + 2 KMnO4 + 3 H2SO4 → 2 MnSO4 + K2SO4 + 8 H2O + 10 CO2

The reactions above show that C2O42- - 2 e → 2 CO2

Therefore,

Analytical Chemistry Laboratory 29

 134,02
1 grek Na2C2O4   67,01 gram
2
126,07
1 grek H 2C2O4 2 H 2O   63,04 gram
2

The Use of KMnO4 Standard Solution

Achmad (2001) said that standard solution of KMnO4 can be used for a variety of
analysis, i.e.:
1. Analysis the mixture of ferri and ferrous in solution.
Determining the mixture of Fe3+ and Fe2+ can be carried out in two stages, ferro can
be done directly while the ferri should be reduced to ferrous. The way is set the Fe 2+
in solution followed by a set Fe3+.
2. Analysis of iron content in iron ore.
3. Analysis of calcium salts.
Calcium salt (Ca) as Ca-oxalate is precipitated with added (NH4)2C2H4, and the
sediment occurred is filtered, washed and continued by titration using KMnO4. The
reactions that occur in the titration are:

CaC2O4 + H2SO4 → CaSO4 + H2C2O4

H2C2O4 + 2 MnO4- + 6 H+ → 2 Mn2+ + 10 CO2 + 8 H2O
4. Analysis of MnO2 in manganese.
5. Analysis of hydrogen peroxide.

3. Materials
1. Sulfuric acid
2. H3PO3
3. KMnO4
4. SnCl2
5. Distilled water
6. HCl
7. Na2C2O4.2H2O
8. (NH4)2C2O4
9. MnSO4.4H2O
10. H2SO4
11. Ammonia

Analytical Chemistry Laboratory 30

4. Experiment Apparatus
1. Glass watch
2. Volume pipette 10 ml
3. Beaker glass
4. Erlenmeyer 250 ml
5. Electrical heater
6. Measuring glass 10 ml
7. Sinter glass
8. Thermometer 100 °C
9. Sample bottle (brown)
10. Water bath heater
11. Desiccator
12. Burette
13. Measuring flask 100 ml
14. Funnel

5. Procedures
a. Make a solution of 0.1 N KMnO4
Weigh a 1.6 gram in a watch glass, move into the beakerglass, add 500 ml water, then
cover with a large watch glass and dissolve. Heat solution to boiling for 15-30
minutes, then cooled as room temperature, filtered cold solution with a given
sinterglass or funnel glass wool (not allowed with filter paper). The filtrate obtained is
stored in a clean brown bottle. When the solution will be used, it must be standardized
first.
b. Standardisation of KMnO4 0,1 N standard solution
Standardisation performed with sodium oxalate, by weighing exactly 0.6701 g
Na2C2O4 crystal and dried (at temperature of 105º-110º C for two hours, then cooled
in a desiccator). Then add distilled water into a 100 ml flask until the line mark, shake
it until homogeneous. Take 10 ml with a pipette volume the Na2C2O4 solution above,
pour into the erlenmeyer. Add 4-6 ml of 2N H2SO4, heat in a water bath at
temperature around 75-80 ºC. Solution was titrated with KMnO4 in a state of hot pink
to arise that are not lost until one minute. Titration performed three times (the
difference titration no more than 0.1 ml).

Analytical Chemistry Laboratory 31

 c. Analysis ferro ion.
Take 10 ml sample solution mixture of Fe3+ and Fe2+, add 10 ml of 1 N H2SO4
solution, then titrate with 0.1 N KMnO4 until there is a pink color.
Reduction Fe3+ → Fe2+ using SnCl2
Take 10 ml sample solution, add 4 ml of dilute HCl (1:1), heat to boiling, and bright
yellow solution. Then dropped into a solution of SnCl 2 drop by drop while stirring,
until the yellow colour disappeared or changed to green. This means that the reduction
of Fe3+ → Fe2+ has been perfect. Solution which has been reduced rapidly cooled, add
40 ml water and 8 ml of 5% HgCl2 solution, resulting in white precipitate. Add
another 80 ml hot distilled water 6-8 ml of preventive solution Zimmermann
Reinhardt (made by: 50 gram MnSO4 4H2O dissolved in 250 ml distilled water, a
mixture of 100 ml of concentrated H2SO4 with 300 ml distilled water, 100 ml H3PO3).
Do a titration with KMnO4 solution until the pink colour.

d. Analysis Fe content in iron ores

Weigh 2 grams of iron ore samples and dissolved in 100 ml dilute HCl (1:1) in erlenmeyer
equipped with a short funnel. Through the funnel add 100 ml of cold distilled water, then
strain the filtrate collected in 250 mL erlenmeyer, the residue was washed with dilute HCl.
The filtrate obtained and added distilled water to 250 ml. Take 10 ml, move in erlenmeyer
and heat until boiling, do like the way to reduce Fe3+ → Fe2+. Once reduced add 80 mL
distilled water and 6-8 ml of preventive solution.

e. Analysis of calcium salts

Take 10 ml of saline solution pour into beakerglass, the solution is made acidic, add
MO indicator drop by drop. Then bring to a boil while stirring, add 10 ml of 5%
ammonium oxalate, heated to a temperature 70-80 ºC, add a solution of ammonia
(1:1) drop by drop until the alkalis. To finalize crystal deposition (digestion), allow
the solution of approximately one hour in a warm place. Then filtered with ash-free
filter paper (Whatman filter paper), test whether there is Ca that has not been
precipitated with ammonium oxalate solution. Wash precipitate with cold distilled
water until the sediment is free from oxalate and chloride ions. Punch a hole edge of
filter paper located on the funnel containing the precipitate CaC2O4 with spatula, wash
precipitate with hot water, the filtrate put in erlenmeyer. Clean the remaining
sediment by flushing a solution of 2N H2SO4, put in erlenmeyer rinse with hot

Analytical Chemistry Laboratory 32

 distilled water. The precipitate dissolved and dilute to 50 ml. Further do a titration
with 0.1 N KMnO4 while is hot.

6. Data Analysis
Calculate the content of ferri, ferrous, iron in iron ore and appropriate levels of Ca by the

calculation as follows:

If needed V1 ml 0.1 N KMnO4

Fe2+ = 0,1 x V1 x 56 mgram/ 10 ml
0,1  V1  56
 mgram
10

If needed V2 mL 0.1 N KMnO4

Fe 3 
0,1  V2  0,1  V1  mLgrek / mL
10
 0,1  V2  0,1  V1   56 mgram / mL

If needed V1 mL 0.1 N KMnO4 0,1 N

Fe3  0,1  V mgrek / mL


0,1  V BA Fe10 gram / mL
1000

250 0,1V  AW Fe  10
In 250 mL, Fe 3 weight  gram / 250 mL
100 1000
250 0,1  V AW Fe  10
So, Fe in iron ore  100 1000  100 %
2

Analytical Chemistry Laboratory 33

 If needed Vrata-rata ml 0.1 N KMnO4

Ca 2   0,1  V mgrek
0,1 V  40
 mgram / mL
2
mgrek garam Ca  mgrek KMnO4
N Ca  VCa  0,1  V  mgrek
0,1  V
N Ca          grek / L
VCa

7. References
a. Achmad, S, 2001, Analisis Kuantitatif, Fakultas MIPA Universitas Gadjah Mada,
Yogyakarta

b. Vogel, 1985, Buku Teks Analisis Anorganik Kualitatif Makro dan Semimikro, Edisi
ke-5, PT Kalman Media Pusaka, Jakarta.

8. Report
a. Discussion
- The basic principles of analysis and the application of permanganometric analysis in
industry.
- Relationship to experiments already carried out.

b. Preliminary Report

PRELIMINARY REPORT
PERMANGANOMETRIC ANALYSIS

Group :
No. Name Student's number
1. ………………………. ……………………
2. ………………………. ……………………
3. ………………………. ……………………

Analytical Chemistry Laboratory 34

Day/Date :
Volume of KMnO4 for:
Analysis of ferri = ….mL
Analysis of ferro = ….mL
Analysis of Fe content in iron ore = .…mL
Analysis of Ca = ….mL

Surakarta, ………………………
Students
1……………..
2……………..
3……………..

Analytical Chemistry Laboratory 35

 PROTEIN ANALYSIS

1. Experiment Objective
To determine the nitrogen content of organic substances by using Kjeldahl method.

2. Theory
Protein is large biomolecules made up of α-amino acid residues linked together
by amide, or peptide, bonds. Twenty amino acids are commonly found in proteins; all are
α-amino acids and all except glycine have stereo-chemistry similar to that of L sugars
(McMurry, 1996).
Polypeptides are copolymers of amino acids, i.e., different amino acids are joined
to each other in a specific sequence. Proteins are naturally occurring large polypeptides,
often but not always in aggregation with one of more other polypeptides and/or with other
types of molecules or ions.

H O H O H O H O

H2N C C OH + H2N C C OH H2N C C N C C H + H2O

R1 R2 R1 H R2
amino acid 1 amino acid 2 peptide bond

Nitrogen is one of the five major elements found in organic materials such as protein.

Kjeldahl Method for Nitrogen Determination

The Kjeldahl method is the accepted reference method for the determination of
protein in food, and was first published On March 7, 1883 by Johan Kjeldahl, Head of
Chemistry Department, Danish Brewing Company, Carlsberg, Denmark. Since then, his
method has been extensively studied, modified, and improved upon. Today, the Kjeldahl
method for the determination of organic nitrogen is the worldwide standard for the
purpose of calculating the protein content in both human food and animal food.
According to Blamire (2003) the central basis used in this procedure is the
oxidation of the organic compound using strong sulfuric acid. As the organic material is
oxidized the carbon it contains is converted to carbon dioxide and the hydrogen is
converted into water.

Analytical Chemistry Laboratory 36

 The nitrogen, from the amine groups found in the peptide bonds of the
polypeptide chains, is converted to ammonium ion, which dissolves in the oxidizing
solution, and can later be converted to ammonia gas.
The Kjeldahl method for nitrogen analysis is composed of three distinct steps. These are
digestion, distillation, and titration.
The method involves the estimation of the total nitrogen in the food (it does not directly
measure protein), and the conversion of the percentage nitrogen to protein, assuming that
all the nitrogen in the food is present as protein, using a conversion factor based on the
percentage nitrogen in the food protein.

% Protein = %NxF
F = conversion factor
= 100/(%N in food protein)
For dairy products, F = 100/15.68
= 6.38
For soya products, F = 5.71

A. The Digestion Process

A general equation for the digestion of an organic sample is shown below as one
basic example:
Organic-N + H2SO4 → (NH4)2SO4 + H2O + CO2 + other sample matrix by- products
A number of interrelated digestion conditions determine the rate of reaction and
the completeness of the breakdown of nitrogen to ammonium sulfate. Among these are
heat input to the acid digestion mixture, amount of inorganic salt added to elevate the acid
boiling temperature, reflux rate of H2SO4 in the neck of the digestion flask, length of
digestion, and catalyst addition. Adjusting any one of these factors has an influence on the
others. Proper digestion conditions for a given sample matrix are achieved through
establishing a balance of these factors in a controlled and repeatable fashion. In addition,
if the sample contains nitrate or nitrite nitrogen, it is possible to chemically pretreat the
digest to include or exclude this nitrogen source from the analysis as desired in a
particular situation.

Analytical Chemistry Laboratory 37

 B. Distillation
The majority of the NH3 is distilled and trapped in the receiving acid solution
within the first 5 or 10 minutes of boiling. But depending on the volume of the digestion
mixture and the method being followed, 15 to 150 mL of condensate should be collected
in the receiving flask to ensure complete recovery of nitrogen. Further extension of the
distillation times and volumes collected simply results in more water being carried over to
the receiving solution. Excess water does not change the titration results. Distillation
times and distillate volumes collected should be standardized for all samples of a given
methodology. The rate of distillation is affected by condenser cooling capacity and
cooling water temperature, but primarily by heat input. Typically the heating elements
used for distillation have variable temperature controllers. A distillation rate of about 7.5
mL/minute is most commonly cited in accepted methods. Connecting bulbs or expansion
chambers between the digestion flask and the condenser is an important consideration to
prevent carryover of the alkaline digestion mixture into the receiving flask. The slightest
bit of contamination of the receiving solution can cause significant error in the titration
step. When very low levels of nitrogen are being determined, it is advisable to
“precondition” the distillation apparatus prior to distillation. This can be done by
distilling a 1:1 mixture of ammonia-free water and 50% NaOH for 5 minutes just before
sample distillation to reduce contamination from atmospheric ammonia.

C. Titration
To quantify the amount of ammonia in the receiving solution. The amount of
nitrogen in a sample can be calculated from the quantified amount of ammonia ions in the
receiving solution.

3. Materials
1. Sample
2. K2SO4(crystal)
3. CuSO4(crystal)
4. H2SO4
5. Distilled water
6. Zn
7. Phenolphthalein indicator
8. NaOH 45%

Analytical Chemistry Laboratory 38

 9. NaOH 0,1 N
10. Methyl orange/red indicator
11. HCl 0,1 N

4. Experiment Apparatus
1. Kjeldahl flasks, 500 to 800 mL Kjeldahl digestion unit with fume removal manifold
Kjeldahl distillation apparatus-Kjeldahl flask connected to distillation trap by rubber
stopper. Distillation trap is connected to condenser with low-sulfur tubing. Outlet of
condenser should be less than 4 mm diameter.
2. Erlenmeyer flask, 500 mL
3. Analytical balance, sensitive to 0.1 mg
4. Burette

5. Procedures

A. Digestion
1. Weigh approximately 1.5 g ground sample into digestion flask, recording weight (W)
to nearest 0.1 mg. Add 10 g potassium sulfate and 0.2 g anhydrous copper sulfate.
Then add 25 mL sulfuric acid. (Add additional 1.0 mL sulfuric acid for each 0.1 g fat
or 0.2 g other organic matter if sample weight is greater than 1 g)
2. Place flask on preheated burner (adjusted to bring 250 mL water at 25 oC to rolling
boil in 5 min).
3. Heat until white fumes clear bulb of flask, swirl gently, and continue heating for 90
min for copper catalyst or 40 min for CuSO4/TiO2 mixed catalyst.
4. Cool, cautiously add 250 mL distilled water and cool to room temperature (less than
25oC). Note: If bumping occurs during distillation, volume of water may be increased
to ca. 275 mL.

B. Distillation
1. Prepare titration flask by adding appropriate volume (V HCl) accurately measured
acid standard solution to amount of water so that condenser tip is immersed (try 15
mL acid and 70 mL water if undecided). For reagent blank, pipet 1 mL of acid and
add approximately 85 mL water. Add 3 to 4 drops methyl red indicator solution.
2. Add 2 to 3 drops of tributyl citrate or other antifoam agent to digestion flask to reduce
foaming.
Analytical Chemistry Laboratory 39
 3. Add another 0.5 to 1.0 g alundum granules.
4. Slowly down side of flask, add sufficient 45% sodium hydroxide solution
(approximately 80 mL) to make mixture strongly alkali. (Do not mix until after flask
is connected to distillation apparatus or ammonia will be lost.)
5. Immediately connect flask to distillation apparatus and distill at about 7.5 boil rate
(temperature set to bring 250 mL water at 25oC to boil in 7.5 min) until at least 150
mL distillate is collected in titrating flask.
6. Remove digestion flask and titrating flask from unit, rinsing the condenser tube with
distilled water as the flask is being removed.

C. Titration
Titrate excess acid with standard sodium hydroxide solution to orange endpoint (color
change from red to orange to yellow) and record volume to nearest 0.01 mL
(VNaOH). Titrate the reagent blank (B) similarly.

6. Calculations
One mole of NH3 coming from the digestion mixture (and hence from the original
protein) will neutralize exactly one mole of the acid in the trapping flask.
The first calculation, therefore, is to find the number of moles of NH 3 that have been
produced and then trapped from your sample(s).
The calculations for % N or % protein must take into account which type of receiving
solution was used and any dilution factors used during the distillation process. The
equations given here are in long form. They are often simplified in the published standard
methods. In the equations below, “N” represents normality. “mL blank” refers to the
milliliters of base needed to back titrate a reagent blank if standard acid is the receiving
solution, or refers to milliliters of standard acid needed to titrate a reagent blank if boric
acid is the receiving solution. When standard acid is used as the receiving solution, the
equation is:
If the sample weight is in milligrams, the molecular weight of nitrogen should be changed
to 1400.67. When HCl is used as the receiving solution the equation is
(www.ExpotechUSA.com):
% Nitrogen =
( mL s tan dard acid  mL blank ) x N of acid x 1.4007

Weight of sample in g

Analytical Chemistry Laboratory 40

 7. Reference

a. McMurry, J., 1996, Organic Chemistry, 4th. Ed., Brooks/Cole Publishing

Company, USA.

b. http://www.rosesci.com/Products/ChemicalAnalysis/Kjeldahl Chemistry-overview.htm,
Accessed on 26 February 2011 at 4.15 am

c. Blamire, J., 2003, e-learning for Quantiative Analysis Kjeldahl Method, http:
//www.brooklyn.cuny.edu/bc/ahp/SDKC/Chem/SD_KjeldahlMethod.html. Accessed on
26 February 2011 at 4.18 pm

d. http://www.ExpotechUSA.com, A Guide to Kjeldahl Nitrogen Determination

Methods and Apparatus, an Industry Service Publication, on 28 February 2011 at
1.15 am

8. Report
a. Discussion
- Record your results and calculate yields.
- Discuss your results and interpret your data.
- The yield should be calculated for every reaction that you perform. In this cases it
may be useful to distinguish between the yield and the yield of a reference
protein.

b. Preliminary Report

PRELIMINARY REPORT
PROTEIN ANALYSIS

Group:

No Name Student's number

1 ……………………………... …………………………………….
2 ……………………………... …………………………………….
3 ……………………………... …………………………………….

Analytical Chemistry Laboratory 41

Day/Date:
__________________________________________________________________
1. Materials

No Compound Specific Gravity Percent Weight Volume

Concentration
1 K2SO4 ........................... ...................... ................. .................
2 CuSO4 .......................... ...................... ................. .................
3 H2SO4 .......................... ...................... ................. .................
4 Zn .......................... ...................... ................. .................
5 NaOH .......................... ...................... ................. .................
6 HCl .......................... ...................... ................. .................

2. Experimental Data
No Component Unit Name
1 Weight of sample ……………………….. g
2 Normality of HCl ……………………….. N
3 Volume of NaOH ………………………. mL
4 Volume of HCl ………………………. mL

Students,
1……………..
2……………..
3……………..

Analytical Chemistry Laboratory 42

 SAPONIFICATION AND ACID VALUE OF FAT AND OIL

1. Experiment Objectives
1. To calculate the number of saponification value of fat and oil sample.
2. To calculate the number of acid value of fat and oil sample.

2. Theory
Chemical structure of oil, fat and lipid is an ester of fatty acid and alcohol. If
glycerol is alcohol the ester is known as triglyceride which almost constitutes in oils and
fats. If alcohol is a long chain of high molecular weight monohydroxy alcohol the ester is
known as wax e.g. beeswax.

O O

OH C R1 CH2 O C R1
CH2OH O O
- 3 H2O
CHOH + OH C R2 R2 C O C H
O
O
CH2OH
CH2 O C R3
OH C R3

Glycerol Fatty acid Triglyceride

Triglycerides are the main constituents of vegetable oils and animal fats.
Triglycerides have lower densities than water (they float on water), and at normal room
temperatures may be solid or liquid. When solid, they are called "fats" or "butters" and
when liquid they are called "oils". A triglyceride, also called triacylglycerol (TAG), is a
chemical compound formed from one molecule of glycerol and three fatty acids.
Animal fats such as butter, beef, pork, and poultry fats and vegetable oils such as
corn, peanut, sun flower, palm oils, olive oils are triacylglycerols or triglycerides. These
are triesters of glycerol (IUPAC name 1,2,3-propanetriol). Each of the three OH groups of
glycerol forms an ester group by reaction with the COOH group of a fatty acid to form
the triacylglycerol. A variety of triacylglycerol are possible. Simple triacylglycerols are
those in which R1, R2, and R3 are the same, i.e., three molecules of the same fatty acid
react with glycerol. Complex triacylglycerol are those in which R1, R2, and R3 are

Analytical Chemistry Laboratory 43

 different. Naturally occurring triacylglycerols are complex triacylglycerols (Odian and
Blei, 1994).

Composition of lipids (Odian and Blei, 1994; Gazy, 2009):

Lipid is a solution, it is solvent is the glycerides (saponifiable part) and it is solute is non-
glycerides (non-saponifiable part).
1. Non-saponifiables (or unsaponifiables)
It is that part of lipid which is not affected by alkalihydroxides during saponificaiton
of lipid and can be extracted by organic solvents after saponifcation. They are non-
volatile on drying at 80°C. It rarely exceeds 2%. Nonsaponifiable lipids do not
undergo hydrolytic cleveage into smaller molecules.
2. Saponifiable part
It is the major part of lipid as it constitutes about 99% of lipid. Therefore the chemical
and physical properties of lipids vary with variations of the glyceride part. Variations
of glyceride part are due to its fatty acid composition. Saponifiable lipids contain at
least one ester group, which undergoes hydrolysis in the presence of an acid, a base,
or an enzyme. Hydrolysis by base is referred to as saponification. Hydrolysis cleaves
a saponifiable lipid into two or more smaller molecules.

The chemical examinations of lipids involve reactions with ester linkage of the
glycerides or free carboxylic acid, hydroxyl group of hydroxyl acids as well as the double
bonds of the hydrocarbon chain of fatty acids, in order to differentiate between the
different types of lipids, through determination of some chemical constants.
Reaction with carboxylic acid (- COOH) group: acid value, saponification value, and
ester value.
A. Acid Value
Acid value (AV) is an important indicator of fat and oil quality. Acid value is
expressed as the amount of potassium hydroxide (in milligrams) necessary to
neutralize free fatty acids contained in 1 g of the substance (ISO 660 1983 E). Edible
oil contain > 1%. Pharmaceutical oil must not have any acidity.
Significance: acid value is the measure of hydrolytic rancidity. In general, it gives an
indication about edibility of the lipid

Analytical Chemistry Laboratory 44

 B. Saponification Value
This method is used to determine the total acid content, both free and combined, of
tall oil. (Acid number only measures the free acid). The combined acids are primarily
esters formed by reaction with the neutral components present in the original tall oil.
The saponification value is therefore a measure of tall oil quality. It is determined by
measuring the alkali required to saponify the combined acids and neutralize the free
acids.
Saponification value is expressed by potassium hydroxide in mg required to saponify
one (1) gram of fat. It depends on the kind of fatty acid contained in the fat.

Rancidity Testing
Rancidity in food and feedstuff may result from oxidation of the lipid component
of the sample, microbiological deterioration of the sample or both. Because the lack of a
universally accepted definition of rancidity, no single rancidity test will meet every
client's needs. Oils are said to become rancid when they undergo a degradation process
known as oxidation. A variety of chemical compounds such as peroxides, aldehydes and
free fatty acids are created as oil oxidizes. The tests that follow are those most frequently
requested to monitor or predict oxidative degradation.
Here we will be test a sample of fatty acid. The sample is first saponified by
adding 0.5mol/L potassium hydroxide ethanol, and then the excessive potassium
hydroxide is titrated with 0.5mol/L HCl until the endpoint is reached. End point is
determined by the maximum inflexion point on titration curve.
Acid value indicates the proportion of free fatty acid present in oil or fat and may
be defined as the number of milligrams of caustic potash required to neutralize the acid in
1 gm of the sample. The normal acid value for most samples lies within 0.5. If any titrable
acid other than a fatty acid is present in the sample, it will be an error. A high acid value
indicates a stale oil or fat stored under improper conditions.

3. Materials
1. Potassium hydroxide, 0.5 N solution in ethyl alcohol, standardized to ± 0.005
2. Hydrochloric acid, 0.5 N solution, standardized to ± 0.005.
3. Phenolphthalein indicator, 1% (visual titration only).
4. Sample (oil, margarine)

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4. Experiment Apparatus
1. Erlenmeyer flask, 250-mL with S/T 24/40 neck and reflux condenser.
2. Pipette, 25-mL.
3. Burette
4. Hot plate
5. Stop watch

5. Procedures
A. Saponification Value
1. Weigh 5 g of sample, to the nearest 0.01 g, into a 250-mL Erlenmeyer flask.
2. Using a pipette, add 50 mL of 0.5 N ethanolic potassium hydroxide, and fix a
cooling pipe to the flask.
3. Gently heat the flask occasionally shaking while adjusting the heat so that backflow
ethanol will not reach the top of cooling pipe. Reflux for saponification around 60
minutes.
4. After heated for 60 minutes, immediately cool it, and titrate between 60 and 70ºC
with 0.5 N HCl using phenolphthalein indicator before the test liquid is solidified
5. Run a blank test in the same manner (without sample following step 1 to 4 above)
for 3 times to obtain mean value of titration volume of 0.5 N HCl.

B. Acid Value
1. Weigh 5 g of sample and transfer it solution from the burette. The appearance of
pink color indicates the into 250 mL Erlenmeyer flask.
2. Using a pipette, add 50 mL of 95% of neutralized alcohol solution to the sample
solution. Heat this mixture for 10 minutes by using the heater.
3. Take the solution after 10 minutes and add 1 or 2 drops of phenolphthalein
indicator. Titrate this against the KOH end point.

6. Calculation

Saponification Value 
 A  B  x N x 56 ,1
W

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 Where:

A = HCl, for blank, mL

B = HCl, for sample, mL
W = weight of sample (dry basis), g
N = normality HCl solution
56.1 = equivalent weight of potassium hydroxide
Cautions in measurement:
When the sample is heated in flask for saponification, gently heat it so that backflow
ethanol will not reach the top of cooling pipe fitted to the flask.

mL of KOH x N KOH x MR KOH

Acid value 
W

7. References
a. ASTM D464 Saponification Number of Naval Stores Products Including Tall Oil and
Other Related Products.

b. ASTM D5558 Standard Test Method for Fat and Oil

c. Firestone, D (Ed.), Official Methods and Recommended Practices of the American Oil
Chemists Society, 4th ed., American Oil Chemists Society, Champaign, 1996, Method
Ca 5a–40. Free Fatty Acids.

d. Gazy, A. A. K., 2009, Lipids Analysis of Oils and Fats, Pharmaceutical Analytical
Chemistry, Faculty of Pharmacy- University of Alexandria

e. ISO 660 1983 E. Animal and Vegetable Fats and Oils. – Determination of Acid Value
and Acidity, ISO, Geneva, 1983.

f. Odian, G. and Blei, I., 1994, Schaum’s Outline of Theory and Problems of General,
Organic, and Biological Chemistry, McGraw Hill, Inc., USA.

8. Report
a. Result and Discussion
- Record your results and calculate yields.
- Discuss your results and interpret your data.
- The yield should be calculated for every reaction that you perform. In this cases it
may be useful to distinguish between the yield and the yield of a reference oil.

Analytical Chemistry Laboratory 47

 b. Preliminary Report

PRELIMINARY REPORT
SAPONIFICATION AND ACID VALUE OF FAT AND OIL

Group:

No Name Student's number

1 ……………………………... …………………………………….
2 ……………………………... …………………………………….
3 ……………………………... …………………………………….
Day/Date:
__________________________________________________________________
3. Materials
No Compound Density Percent Weight Volume
Concentration
1 Ethanol ....................... ...................... ................. .................
2 KOH ....................... ...................... ................. .................
3 HCl ....................... ...................... ................. .................

4. Experimental Data
Saponification Value
No Component Unit Name
1 Weight of sample ……………………….. g
2 Normality of HCl ……………………….. N
3 Volume of blank titration ………………………. mL
4 Volume of sample titration ………………………. mL

Acid Value
No Component Unit Name
1 Weight of sample ……………………….. g
2 Normality of KOH-ethanol ……………………….. N
3 Volume of sample titration ………………………. mL

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 Students,
1……………..
2……………..
3……………..

Analytical Chemistry Laboratory 49