DEPARTMENT OF CHEMICAL TECHNOLOGY AND

FOOD

DMK 4023
INSTRUMENTATION IN OIL AND FAT
ANALYSIS
LABORATORY MANUAL

NAME: ____________________________________________

REGISTRATION NO.: _______________________________

CLASS: ___________________________________________

1
2
 PRACTICAL TOPIC PAGE

Preface 4

General Laboratory Rules 5

Format & Marking Scheme of Laboratory Report 6

Anisidine Value in Oil Sample Using Uv-Spectrometer 7

Analysis Of Triacylglycerol Using Hplc-Pda 9

Enzymatic Glycerolysis-Production Of Monoacylglrcerol From 10

Cocoa Butter

Analysis Of The Thermal Properties Differential Scanning 11

Calorimetry (Dsc)
13
Determination Of Fat Content- Soxhlet Method

3
 PREFACE
This laboratory manual is prepared and distributed as a practical guide for Semester 4 students in the Diploma
of Chemical Technology Programmed, who are following the DMK 4023 module.

The contents of this practical guide are arranged so as to expose students to the practical aspects of topics
covered in their lectures. Practical are arranged in accordance to the prescribed module syllabus and the
equipment, and facilities that are available in the laboratory.

To successfully carry out any practical, students are expected to:

 read beforehand the relevant practical instructions provided in this manual before entering the
laboratory.
 receive further explanation and instructions that are given before using any apparatus and material in
the laboratory.
 identify and gather materials that are needed, before beginning the session.
 complete the practical session in accordance to the allocated time.

Due to space constraints, explanations provided in this practical manual are necessarily limited in scope and
depth. Students are therefore expected to read further, referring to recommended books and other materials,
before carrying out a practical or preparing its report.

Laboratory reports are considered to be a primary means of presenting experimental findings in a clear and
concise way. As such the preparation of laboratory reports are given due emphasis in the DMK4023 module.
Students are encouraged to make use of books and other reference materials in their preparation of every
laboratory report. In particular, students should take note of written comments from the lecturer, upon the
return of their laboratory reports. These are a form of feedback, which is a very important means by which
students improve on their practical and report writing skills.

It is hoped that students will derive much benefit from this practical manual, in their endeavor to master the
fundamental concepts and practical skills which are covered in the DMK4023 Inorganic and Physical Chemistry
module.

4
 GENERAL
LABORATORY RULES
1. Students are NOT ALLOWED to enter the laboratory without the supervision of the laboratory
attendant or lecturer.

2. On entering the laboratory, before beginning any laboratory activity, all students are required to wear
their laboratory coats (buttoned up completely).

3. Students are required to wear shoes in the laboratory. Sandals, slippers and other such footwear are
prohibited in the laboratory.

4. Do not run, joke or banter in thelaboratory. Do not carry out any activity that may endanger self or
friend.

5. Long sleeved clothing should be worn neatly buttoned or with sleeves rolled up to the elbows.

6. Female students with long hair, who do not wear head scarves must have their hair tied up.

7. Experiments can be started upon receiving instructions from the lecturer or laboratory assistant.
Students should not begin if still unclear or doubtful about the experimental procedure.

8. Concentrate on what you are doing, when handling chemicals or laboratory equipment. Follow ALL
prescribedsteps carefully, including safety proceduresto prevent accidents.

9. Do not light a Bunsen burner if there is inflammable material nearby.

10. Unfinished or leftover chemicalsand samples should be kept in a properly prepared container for
recycling or disposal.

11. After use, all equipment or glassware must be cleaned and returned to its proper place. Do not leave
dirty apparatus in the sink.

12. Report to the lectureror laboratory assistant there is any breakage of glass equipment or apparatus. Do
not throw any broken glass into the sink.

13. Wash hands and any other part of the bodythat have been exposed to poisonous or dangerous
chemicals, with soap and plenty of water, especially if any itchiness is felt.

14. Before leaving the laboratory make sure all equipment, lights, air conditioning, water taps and gas taps
are turned or switched off.Wash your hands thoroughly with soap and water before leaving the
laboratory.

15. Students are not to eat and drink in the laboratory. Laboratory apparatus are not to be used as food
containers.

16. In the event of fire or other emergency, act calmly and do not rush for the laboratory exits.

17. Do not enter the laboratory to conduct any experiment if you are not feeling well. Ask the lecturer’s
permission to be exempted from the practical session and take arest.

5
 FORMAT & MARKING SCHEME OF
LABORATORY REPORT

Each laboratory report must be handed in latest by one week after the day the practical session is held. The
report should contain the following:

Front Page
A. Student’s identity
Name, Registration No., Class and names of group members.
B. Details of Practical
Practical No.,date on which it is carried out and actual date of report submission.

Inside the Report

C. Practical No., Title andObjective(s)(5marks)
D. Introduction(10marks)
This section explains the theoretical andconceptual aspects of the practical. You are NOT allowed to
copy from the explanation given in the practical manual. If you do that, 2 markswill be deducted
from your mark for the whole report.
E. Apparatus, chemicals and samples (5 marks)
This section lists all the apparatus, reagents and samples used in the practical.
F. Method (5 marks)
This section explains the steps followed in the practical. All the steps should be shown in the form of
an organisational chart, in the active voice.
G. Results (30marks)
All results and observations are to be recorded in this part of the report. The results should be
presented in the form of tables which are appropriate to the need of the particular practical.
H. Discussion (30marks)
This is a very important section.You have to refer to other texts before you can write it. This is where
you discuss the observations and results that you have obtained. Were you successful in achieving
your stated objective(s) or not? Are your results in agreement with the theoretical basis of the
practical?If not you need to suggest plausible reasons why not. Were there mistakes in methodology
made and resulting experimental errors caused? If so, state the corrective measures that should have
been taken. If the results are in full agreement with their theoretical basis, you should explain the
mechanismsor how the results were derived. This means that you must prove your results
theoretically.
I. Conclusion (10marks)
This section should be concise. It shows the results obtained based on the stated objective(s) of
the practical.
J. References(5marks)
Record all the reference materials that you have used in writing the report here, in the recommended
format.
K. Questions(Marks given here are included in the marks for discussion.)
If questions are given, answer them here.

6
PRACTICAL 1

Title : ANISIDINE VALUE IN OIL SAMPLE USING UV SPECTROMETER

Aim : To calculate the anisidine value in oils

Introduction : The secondary stage of oxidation occurs when the hydro-peroxides decompose
to form carbonyls and other compounds, in particular aldehydes. These are
what gives the oil a rancid smell, and they are measured by the AV.
The lower the AV, the better the quality of the oil.
The AV test is a good way to measure secondary oxidation products and should
be used together with a primary test like PV.

The p-Anisidine Value analysis provides a rapid and reliable way to verify the
goodness and the real oxidation stage of fats and oils being sold, purchased or
processed.
The p-Anisidine Value analysis (AV) deals with fats and oils and with the
oxidation processes occurring in them, the undesirable series of chemical
reactions involving oxygen that degrades their quality. Oxidation generates a
sequence of breakdown products, starting with primary oxidation products
(peroxides, dienes, free fatty acids) then secondary products (carbonyls,
aldehydes, trienes) and then tertiary products.

Prosedure.

Preparation p-anisidine reagent

The crystallized p-anisidine was then dissolved in acetic acid in the relation 0.25 g p-anisidine
per 100ml glacial acetic acid.

Measuring of anisidine value by UV spectrophotometer

1. One gram of the oil sample was weighed into 25 ml volumetric flask.
2. Isooctane was added up to 25 ml and the content was mixed.
3. The absorbance of the solution was registered in at 350 nm wavelength in a
spectrophotometer in 1 cm cells. A reference cell with isooctane (solvent) was used as a
blank.
4. Pipette exactly 5 ml of the fat solution into one test tube and exactly 5 ml of the solvent into
second test tube. By using micropipette add 1 ml of p-anisidiine reagent to each test tube. Shake
te tube to homogenize the solution and reagent. After 10 minutes, measure the absorbance at
350nm

7
 5. A reference cell with isooctane with added p-anisidine reagent was used as a blank.(second
test tube)
The anisidine value
A.V =25 X (1.2 As –Ab)
W
As= the absorbance of the sample after reaction with p-anisidine
Ab= the absorbance of the sample solution
W= weight iin gram of sample

8
PRACTICAL 2

Title : ANALYSIS OF TRIACYLGLYCEROL USING HPLC-PDA

Aim : To analyses of triacylglycerol in oils using HPLC-PDA

Introduction : Triacylglycerols, also known as triglycerides, are the simplest lipids formed
by fatty acids. It is made up of three fatty acids ester linked to a single
glycerol. Most triacylglycerols contain two or three different fatty acids.
Triacylglycerols are nonpolar, hydrophobic, and insoluble in water. This is
due to the ester linked bond between the polar hydroxyls of glycerol and
the polar carboxylates of the fatty acids. Common triacylglycerols are
vegetable oils, dairy products, and animal fat

Triacylglycerol in vegetable oils can be separated according to the

equivalent carbon number (ECN) by using reversed phase high
performance liquid chromatography system with reflective index(RI)
detector. Generally, ECN is defining according to the following equation:

ECN = CN - 2n
CN = the number of carbon
n = number of double bond
*TAG with low ECN will detect first because have short chain and more
double bond.

Apparatus: HPLC , injector,PDA Detectors ,Column: C18

Reagents: Acetone HPLC grade,acetonnitrile HPLC grade,chloroform

Sample: Palm oil, soybean oil
Prosedure.

1. 500mg of sample weight in 10 ml volumetric flask with solvent(mobile phase)

2. Prepare the mobile phase: Acetone: acetonnitrile.(63.5:36.5)
3. C18 column used.
4. The flow rate is 1ml/min, temperature in detector set at 40°C
5. The injection volume of sample solution is 10 µL.
6. Compare the retention time with authentic TAG references.

9
PRACTICAL 3

Title : ENZYMATIC GLYCEROLYSIS-PRODUCTION OF

MONOACYLGLRCEROL FROM COCOA BUTTER

Aim : To produce of enzymatic glycerolysis-production of monoacylglrcerol

from cocoa butter
Introduction : Structured TAGs (sTAGs) are defined as TAGs which are modified
chemically or enzymat-ically to change the fatty acid composition and/or
positional distribution in the glycerolbackbone. In a stricter definition,
sTAGs are referred particular molecular species of TAGswith defined
molecular structure (i.e. specific fatty acid residues in specific
positions).Molecular structure of TAGs influences their metabolic fate in
organisms (i.e. digestionand absorption) as well as their physical
characteristics (e.g. melting points) and crys-tallinity. Consequently,
designing sTAGs with particular chemical structure, it is possibleto control
the behavior of TAGs, thereby improving the nutritional and
pharmaceuticalproperties of TAGs.

The catalytic actionof lipases is reversible. They catalyze hydrolysis in an

aqueous system, but also esterification (reverse reaction of hydrolysis) in a
microaqueous system, where water content is very low.
Transesterification is categorized into four subclasses according to the
chemical specieswhich react with the ester. Alcoholysis is the reaction with
an ester and an alcohol, while acidolysis is the one with an ester and an
acid.Interesterification is a reaction between two different esters, where
alcohol and acid moiety is swapped. In Aminolysis an ester isreacted with
an amine, generating an amide and an alcohol.

Reagents: Glyserol, lipase, chlorofom, sodium sulphate.

Sample: Palm oil, soybean oil, cocoa butter
Prosedure.

1. Weight 1.432 g glyserol, 5.00 g cocoa butter and pipette 0.057ml water into 25 ml boiling flask
2. Put the flask into oil bath at 50 C for 5 minute while stirring.
3. Add lipase and keep strirring 30 minutes.
4. Remove the flask and take out the magnetic stirr.
5. Seperating enzyme from the reaction mixture using sintered glass filter funnel
6. Collect the product into100 ml beaker and add 50 ml chlorofom and stir
7. Tranfer the solution into separating flask and wash it by extracting with 3 time 10 ml water.
8. Dried the sample by adding 1.0 g sodium sulphate.

10
PRACTICAL 4

Title : ANALYSIS OF THE THERMAL PROPERTIES DIFFERENTIAL

SCANNING CALORIMETRY (DSC)

Aim : To Analysis of the Thermal Properties Differential Scanning Calorimetry

(DSC)

Introduction : Differential Scanning Calorimetry (DSC) is a thermal analysis technique to

measure the temperature and heat flows associated with phase transitions
in materials, as a function of time and temperature. The measurements can
provide both quantitative and qualitative information concerning physical
and chemical changes that involve endothermic (energy consuming) and
exothermic (energy producing) processes, or changes in heat capacity
can be used for both QC and R&D purposes.

It is important that there be good thermal contact between the sample and
the pan. As the samplepan reaches a temperature where a phase transition
(melting, for instance) occurs, it requires moreheat than the reference pan
in order to maintain the specified temperature gradient; some heat
isrequired to bring about the phase transition itself. Similarly, upon
cooling, the sample gives off heatduring recrystallization, while the
reference does not. The DSC therefore measures heat flow in andout of a
sample as a function of temperature, giving precise measurement of phase
transitiontemperatures.

11
Sample: Palm oil and peanuts

Prosedure.

1. Using a small mortar and pestle, grind the solid samples into a fine powder.
2. Transfer ~10-mg portion to a sample pan and record its mass.
3. Tap the sample pan and/or spread the sample out evenly using a spatula, being careful
not spill any sample.
4. Place a pan cover evenly over the sample and place the sample in the press.
5. Pull the press lever down to crimp the samplepan/cover.
6. Clean up the mortar and pestle with water and a Kimwipe.
7. For the sample, the reference is an empty pan with a cover.

12
PRACTICAL 5

Title : DETERMINATION OF FAT CONTENT- SOXHLET METHOD

Aim : To determine of percentage fat content in nuts using soxhlet method

Introduction : The term “lipid” refers to a group of compounds that are soluble in water,
but show variable solubility in a number of organic solvents.
The lipid content of food determined by extraction with one solvent may
be quite different from the lipid content as determined with another solvent
of different polarity.

Fat content is determined by often by solvent extraction methods, but it is

also can be determined by non-solvent wet extraction methods and by
instrumental methods that rely on the physical and chemical properties of
lipids.
The method of choice depends on a variety of factors, including the nature
of the sample, the purpose of the analysis and instrumentation available.

Reagents: petroleum ether

Sample: peanut, food sample

13
Prosedure.

1. Weight a 250 ml round bottom boiling flask

2. Weight accurately 2.00g of sample on filtered paper and fold it before placed a timble
3. Cover the timble with cotton
4. Put the timble in soxhlex apparatus.
5. By using beaker, pour 200ml petroleum ether into soxhlet apparatus.
6. Put the boiling flask with the extraction apparatus on heating mantle and assemble yhe
condenser.
7. Ensure the water tap is open
8. Heat the apparatus around 65C for 2.5 hour.
9. Evapporate the solvent to dryness using rotary evaporator.
10. Cool down the flask and calculate the percentage of fat in the sample.

FAT CONTENT(%) = (WEIGHT OF FLASK + FAT)- (WEIGHT OF FLASK) X 100

(WEIGHT OF SAMPLE

14