Food Analysis

Efendi Oulan Gustav Hakim Nata Buana, S.TP. M.Eng.

Aprilia Fitriani, S.TP., M.Sc.
 Course Contract
• 80% minimum attendance. Fail to do so
Component Percentage (%) without prior notification, you’ll get D.

Quiz 10 • Assignment could be either paper work or

presentation.
Assignment 20
•Your participations during class are
Middle Term 30 highly encouraged

Final Term 30 Grade Score Grade Score

A 86 C 56
Activity 10
AB 78,5 CD 48,5
Total 100 B 71 D 41
BC 63,5 E 0
 Course Content
Schedule Chapter Lecturer

1 3 April 2018 Sampling Technique EOG

2 10 April 2018 Moisture & Total Solids APF

analysis
3 17 April 2018 Carbohydrate Analysis APF
4 24 April 2018 Protein Analysis EOG
5 1 Mei 2018 Lipid Analysis APF
6 8 Mei 2018 Mineral & Ash Analysis EOG
7 15 Mei 2018 Vitamin Analysis APF
UTS
 Course Content
Time Chapter Lecturer

8 29 Mei 2018 Mass Spectometry APF

9 5 Juni 2018 Chromatography APF
10 12 Juni 2018 Pigment Analysis EOG
11 19 Juni 2018 Antioxidant Analysis APF
12 26 Juni 2018 Physical Properties Analysis EOG
13 3 Juli 2018 Toxin and Food Analysis EOG
14 10 Juli 2018 Electrophoresis and Immunological
EOG
Based Analysis
UAS
Lipid Analysis

Aprilia Fitriani
Major Food Compounds

Lemak

Protein

Karbohidrat

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Lipid?

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 Lipid Definition
Kelompok lipid yang sangat bersifat hidrofobik.
Contoh: Trigliserida

Kelompok lipid yang memiliki gugus hidrofobik

dan hidrofilik
Contoh: Mono- dan di-gliserida

Asam lemak rantai pendek (C1-C4) larut dalam

air dan tidak larut dalam pelarut
Contoh: as. butirat

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Trigliserida

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 Fat
Content of
Selected
Food

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 Fat
Content of
Selected
Food

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 Lipid Analysis Methods
Solvent Extraction Methods

• Continuous
• Semi-continuous
• Discontinuous

Non Solvent Wet Extraction Method

• Babcock method
• Gerber method

Instrumental Method

• GC
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Solvent Extraction Methods
Sample preparation

• Predrying sample
• Particle size reduction
• Acid hydrolysis

Solvent selection

• Ideal solvent
• Ethyl ether, petroleum ether, pentane & hexane

Extraction method

• Continuous
• Semi-continuous
• Discontinuous
• Total lipid by GC

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What is the Purpose of
Sample Preparation in
Lipid Analysis?

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Sample Preparation
Depends on the type of food and the type and
nature of lipids in the food.

The extraction method for lipids in liquid milk is

generally different from that for lipids in
solid soybeans.

To analyze the lipids in foods effectively,

knowledge of the structure, the chemistry, and
the occurrence of the principal lipid classes and
their constituents is necessary.

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Sample Preparation Predrying sample

Lipid cannot be
Solvent effectively extracted

Solvent
Hydrophobicity and
hygroscophycity
solvent

Moist food

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 Sample Preparation Predrying sample

- Conventional drying
- Lipids become bound
to proteins and
carbohydrate
High temperature - Bound lipids are not
easily extracted with
organic solvents
Low temperature
- Oven drying vacuum
- Break the fat-water
emulsion
- Easy to grinding
Dried food
- Increase of surface
area

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 Sample Preparation Particle size reduction

It can be difficult to extract

lipids from whole soybeans
because of the limited
porosity of the soybean hull.

- Particle size
- Adequate grinding is very
important

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 Sample Preparation Particle size reduction

- The extraction of the ground seeds with selected solvent after repeated
grinding at low temperature to minimize lipid oxidation
- For better extraction, the sample and solvent are mixed in a high-speed
comminuting device such as a blender
- Such products may best be ground after freezing with liquid nitrogen

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 Sample Preparation Acid Hydrolysis

- Lipids in foods such as dairy, bread, flour, and animal products is

bound to proteins and carbohydrates.
- Direct extraction with nonpolar solvents is inefficient
- Such foods must be prepared for lipid extraction by acid hydrolysis

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Sample Preparation Acid Hydrolysis

Source : Nielsen, 2009

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Sample Preparation Acid Hydrolysis

• The sample can be predigested by refluxing for 1

1 h with 3 N hydrochloric acid

• Added Ethanol and solid hexametaphosphate

2

• The acid hydrolysis of two eggs requires 10 ml of

HCl and heating in a water bath at 65◦C for 15–25
examples min or until the solution is clear

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 Solvent Selection – ideal solvent
• Should have a high solvent power for
lipids and low or no solvent power for
proteins, amino acids, and carbohydrates
• Easy to evaporate
• No residue
• Relative low boiling point
• Nonflammable and nontoxic both for
liquid and vapor states
• Easy to penetrate sample particle
• Inexpensive
• Non hygroscopic

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It is difficult to find
an ideal fat
solvent to meet all of
these requirements

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 Solvent
• Ethyl Ether • Petroleum Ether
• Boiling point : 34.6oC • Boiling point : 35-38oC
• Better solvent for fat than • More hydrophobic than
PE ethyl ether
• Expensive than other • Less hygroscopic & less
solvent flammable than ethyl ether
• Hygroscopic • Cheap
• Form peroxide • Selective for more
• Greater danger explosion hydrophobic lipid
& fire hazard

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Extraction Method - Goldfish Continuous Solvent
Extraction Method
method
Principle

• Solvent from a boiling flask continuously flows over the sample

held in a ceramic thimble.
• Fat content is measured by weight loss of the sample or by weight
of the fat removed

Adventages

• Faster
• more efficient extraction than semicontinuous extraction methods

Disadventages

• Cause channeling which results in incomplete extraction

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 Continuous Solvent
Extraction Method Extraction Method

Goldfish method

Source : Nielsen, 2009

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 Procedure
Goldfish Method
Place 60 mL PE/EE in
Weigh thimble beaker extraction

Place 2-3 g sample in Place beaker on heater

thimble apparatus

Weigh thimble Extraction 4 hour

Lower temp, cooling

Weigh extraction beaker sample

Place thimble into glass Remove extraction beaker,

holding tube dry overnight

Up to condenser apparatus Dry at 100oC, 30 min

Cooled in desiccator,
weigh
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 Extraction Method - Continuous Solvent
Extraction Method
Calculation
Weight of fat in sample = (beaker + fat) − beaker

% fat on dry weight basis = (g of fat in sample) x 100

g of dried sample

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Extraction Method – Semi-Continuous Solvent
Extraction Method
Soxhlet Method
Principle
• The solvent builds up in the extraction chamber for 5–10
min and completely surrounds the sample and then siphons
back to the boiling flask.
• Fat content is measured by weight loss of the sample or by
weight of the fat removed

Adventages
• This method provides a soaking effect of the sample and
does not cause channeling

Disadventages
• This method requires more time than the continuous method

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 Semi-Continuous Solvent
Extraction Method Extraction Method

Soxhlet method

Source : Nielsen, 2009

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Extraction Method – Semi-Continuous Solvent
Extraction Method
sample preparation

• If sample has more than

10% of water, sample
should dry to get
constant weight on 95-
100oC, 5 hour, under
pressure ≤ 100 mmHg

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 Extraction Method – Semi-Continuous Solvent
Extraction Method
Procedure
Weigh sample 2-5 g in extraction thimble and
cover with glass wool

Weigh boiling flask

Lower heat temperature

Place EE/PE in boiling flask

Dry boiling flask, 100oC, 30 Min

Assemble boiling flask, soxhlet flask, condenser Cooled in desiccator

Extraction 4-16 hour Weigh

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Extraction Method - Semi- Continuous
Solvent Extraction
Calculation Method

% fat on dry weight basis = (g of fat in sample) x 100

g of dried sample

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Extraction Method – Discontinuous Solvent
Extraction Method
Mojonnier Method
Principle

• Fat is extracted with a mixture of ethyl ether and petroleum ether in a

Mojonnier flask, and the extracted fat is dried to a constant weight and
expressed as percent fat by weight.
• Primarily to dairy foods, and applicable to other foods (fat in flour and pet
food)

Adventages

• Does not require removal of moisture from the sample

• It can be applied to both liquid and solid samples

Disadventages

• Involve an acid hydrolysis with HCl, followed by extraction with a

combination of EE & PE

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 Extraction Method Discontinuous Solvent
Extraction Method

Mojonnier fat extraction flask

Source : Nielsen, 2009

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Extraction Method – Discontinuous Solvent
Extraction Method
sample preparation
• Weigh test portion,
homogeneous sample at 20oC
by pouring back and forth
between clean beaker glass.
If there is a bubble, warm the
sample into 38oC in
waterbath. Cool the sample
to 20oC before transferring in
test portion.

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Extraction Method – Discontinuous Solvent
Extraction Method
blanko preparation
• A pair of reagent blanks must
be prepared every day. For
reagent blank determination,
use 10 ml of distilled water
instead of milk sample. The
reagent blank should be
<0.002 g. Duplicate analyses
should be <0.03% fat.

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Extraction Method – Discontinuous Solvent
Extraction Method
Mojonnier Procedure
Weigh 10 g sample into mojonnier flask
Centrifuge 30 sec, 600 rpm

Add 1.5 mL or more NH4OH

Pour solution from mojonnier flask into weigh
mojonnier fat dish
Add 10 mL ethanol 95%, shake for
90 sec
Perform 2nd & 3rd extraction with same way

Add 25 mL EE, shake for 90 sec

Evaporate solvent on the electric hot plate
≤ 100oC
Add 25 mL PE, shake for 90 sec
Dry the dish in the oven to get constant
weight
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 Extraction Method - Discontinuous
Solvent Extraction
Calculation Method

% fat = 100 x {[(wt dish + fat) – (wt dish)] – (avg wt blank residue)}
wt sample

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Extraction Method – Non-Solvent Wet
Extraction Method
Babcock Method

Principle

• Adding sulfuric acid to determine fat in milk sample

Disadventages

• Does not determine phospholipids in milk products

• Use for milk without sugar or chocolate without modification

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 Non-Solvent Wet
Extraction Method Extraction Method

Babcock extraction method

Source : Nielsen, 2009

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Extraction Method – Non-Solvent Wet
Extraction Method
Babcock Procedure
Pipette 17.6 mL milk into babcock test bottle

Add 17.5 mL H2SO4

Centrifuge 5 min, 50-60oC

Add hot water

Read the nearest from the graduation mark of

the bottle

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Extraction Method – Non-Solvent Wet
Extraction Method
Garber Method

Principle

• Adding sulfuric acid and amyl alcohol to determine fat in

milk sample

Adventages

• Simple, faster and wide application than Babcock method

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 Non-Solvent Wet
Extraction Method Extraction Method

Garber extraction method

Source : Nielsen, 2009

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Extraction Method – Non-Solvent Wet
Extraction Method
Garber Procedure
Add 10 mL H2SO4 15-21oC into Gerber milk bottle

Add 11 mL milk

Add 1 mL amyl alcohol

Tighten the stopper & shaking

Centrifuge 4 min

Waterbath 60-63oC, 5 min, read the fat

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Instrumental Method – GC (FID)
Principle

• This method based on the saponification – esterification –

methylester analysis using GC.
• FID (Sensitive; Wide of response; Good response for
organic compound; Destruction sample)
Adventages
• Can be used for all food materials
• Rapid & Sensitive
• Simple

Disadventages
• Expensive

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Instrumental Method – GC (FID)

• Detector : Flame Ionization Detector

• Column : fused silica capillary (HP-88, HP-INNOWAX,
SP-2560, CP-Sil 88, etc) ID : 0.25 mm, thickness 0.2-0.25
μm.
• Injector temperature : 200oC
• Detector temperature : 250-300oC
• Gas : hydrogen / helium
• Hydrogen flow : 30 mL /minutes
• Oxygen flow : 400 mL/minutes
• Nitrogen flow : 30 mL/minutes
• Injection sample : 1 μL
• Internal standard : asam margarat (C17:0)

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 Sample preparation – GC (FID)

• External standard :
To identify fatty acid type, and to determine
retention time
• Internal standard :
To quantitative analysis
Saponification : Hydrolysis lipid/fat into fatty
acid
Esterification : Fatty acid  FAME
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GC – Saponification Procedure
Weight lipid (100-105 g) + internal standard 1 mL

Add 1.5mL 0.5 M NaOH in methanol

Homogenization with vortex

Flow N2

Homogenization with vortex

Heat on 80-100oC, 5 min, cooled

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 GC – Esterification Procedure
Liquid sample + 2 mL BF3 methanol

Take hexane layer into vial

Homogenization with vortex

Place some Na2SO4

Flow N2

Heat on 80-100oC, 30 min, cooled Transfer heptane layer into new vial

Sample ready to analysis

Place 1 mL hexane than vortex

Place 3mL saturated NaOH

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 Quiz
1. Pengujian analisis lemak dengan metode goldfish menghasilkan
kacang tanah kering memiliki kadar lemak sebesar 60 % bk.
Sampel yang digunakan sebanyak 2 g. Berapakah berat lemak
awal dalam sampel tersebut?
2. Jelaskan makna dari channeling pada proses ekstraksi lemak
dengan metode goldfish!
3. Jelaskan fungsi asam sulfat dan isoamil alkohol dalam analisis
lemak dengan metode ekstraksi garber!

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