SFA3091

FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

29

PROXIMATE ANALYSIS

EXPERIMENT 1: DETERMINATION OF MOISTURE CONTENT

OBJECTIVE
To determine moisture content in food.
_
INTRODUCTION
Water is the major component of most foods and serves a variety of functions. It influences
the structure, appearance and taste of food. The moisture content of a food also influences
its spoilage process. Each food has its own characteristics water content. The determination
of moisture content is one of the most fundamental and important analytical procedures in
food analysis. The approximate water content of a food can affect the choice method of
analysis.
The moisture (or total solids) content of foods is important for a variety of reasons. Moisture
is an important factor in food quality, preservation, and resistance to deterioration. Moisture
content of foods can be determined by a variety of methods. In this experiment, several
methods to determine the moisture content of foods will be used and the results compared.
PRINCIPLE
The sample is heated (according to the drying method) and the loss of weight is used to
calculate the moisture content of the sample.
METHOD A: FORCED DRAFT DRYING OVEN
Sample: Minced Meat
Apparatus/Instrument/Chemicals/Samples

Analytical balance
Dessicator
Crucible with lid
Oven, set at 105C
Minced meat
Tongs

Procedure
1) Perform this experiment in duplicates.
2) Dry the crucible and its lid in the oven at 105C overnight and transfer to the
dessicator to cool (approx 30 min)
Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

29

3)
4)
5)
6)
7)

Weigh the crucible.

Weigh about 5 g sample into crucible. Spread the meat.
Replace the lid and weigh the crucible and its contents (WI)
Place the crucible with its lid slipped to one side. Dry for 16h or overnight at 105C
After drying, use a pair of tongs to transfer the crucible and lid to the dessicator to
cool (approx 45 min). Reweigh the crucible, lid and its dried content.
8) Replace the crucible with its lid partially covered in the oven for 1h. Transfer to the
dessicator to cool, then weigh the dish and its content again. If the weight obtained at
this step is less than that obtained at Step 7, it means the sample was not sufficiently
dried. In this case, repeat this step until constant weight is obtained (W2).
Calculations
Moisture(%)= w1-w2 x 100
W1
Where, w1 = weight (g) of sample before drying
W2= weight (g) of sample after drying

Calculate percentage moisture (wt/wt):

% moisture = (wt of H20 in sample / wt of sample ) x 100

% moisture = (wt of wet sample + pan ) ( wt of dried sample + pan )
( wt of wet sample + pan ) ( wt of pan )

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

X 100

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

METHOD B : MOISTURE ANALYZER (AND METHOD)

Sample: flour
***Performed sample in triplicate.
Apparatus/Instrument/Chemicals/Sample

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

Page

of

29

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

Page

of

29

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

29

EXPERIMENT 2: DETERMINATION OF MINERAL CONTENT

OBJECTIVE
To determine mineral content in tea leaves and coffee powder.
Introduction
Ash is the inorganic residue remaining after the water and organic matter have been
removed by heating in the presence of oxidizing agents, which provides a measure of the
total amount of minerals within a food. Analytical techniques for providing information about
the total mineral content are based on the fact that the minerals (the analyte) can be
distinguished from all the other components (the matrix) within a food in some measurable
way. The most widely used methods are based on the fact that minerals are not destroyed
by heating, and that they have a low volatility compared to other food components. The three
main types of analytical procedure used to determine the ash content of foods are based on
this principle: dry ashing, wet ashing and low temperature plasma dry ashing. The method
chosen for a particular analysis depends on the reason for carrying out the analysis, the type
of food analyzed and the equipment available. Ashing may also be used as the first step in
preparing samples for analysis of specific minerals, by atomic spectroscopy or the various
traditional methods described below. Ash contents of fresh foods rarely exceed 5%, although
some processed foods can have ash contents as high as 12%, e.g., dried beef.
1.

Dry ashing for the majority of the samples,

Dry ashing is incineration at high temperature (525C or higher) accomplished in a

muffle furnace. Crucible selection becomes critical in ashing because type depends on the
specific use. Quartz crucibles are resistant to acids and halogens, but not alkali. Porcelain
crucibles resemble quartz crucibles in their properties but will crack with rapid
temperature changes. These crucibles usually used because they are relatively
inexpensive. Steel crucibles are resistant to both acids and alkalis and are inexpensive, but they
are composed of chromium and nickel, which are possible sources of contamination. Platinum
crucibles are inert are the best crucibles but they are currently far too expensive for routine use
for large number of samples.
The advantages of conventional dry ashing are that it is a safe method, it requires
no added reagents or blank subtraction, and little attention is needed once ignition begins. A
large number of crucibles can be handled at once, and the resultant ash can be used for
analyses like individual elements, acid-insoluble ash, and water-soluble and insoluble ash. The
disadvantages are the length of time required (12 - 18 h, or overnight) and expensive
equipment. There will be a loss of the volatile elements and interactions between mineral
components and crucibles. Volatile elements at risk of being lost include arsenic, boron,
cadmium, chromium, copper, iron, lead, mercury, nickel, phosphorus, vanadium and zinc.
2.
Wet ashing for samples with high fat content (meats and meat products) as a
preparation of elemental analysis,
Wet ashing is a procedure for oxidizing organic substances by using acids and oxidizing agents
or their combinations. Minerals are solubilised without volatilization. Wet ashing is preferable
to dry ashing for specific elemental analysis. The oxidation time is short and requires a hood,
hot plate, long tongs and safety equipment. Nitric and perchloric acid acids are preferable,
Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

29

but a special perchloric acid hood is necessary. The disadvantages of wet ashing are that it
takes almost constant operator attention, use of corrosive reagents and only small numbers
can be handled at any one time.
3.
Low-temperature plasma dry ashing for preparation of samples when volatile elemental
analyses are conducted.
Apparatus
Crucible (or similar porcelain or metal
dishes)

Sample
Hot plate

Muffled furnace
Procedure
1. Dry a representative sample (weigh accurately to nearest mg ~ 3 5 g of samples)
in a crucible in an oven at 130C overnight. Char the sample on an electric hot
plate or over a low flame in a fume cupboard until it has ceased smoking.
2. Place the above crucibles (containing charred sample) in a cold muffle oven and
bring the temperature to 550C.
3. Ignite the sample 12- 18 h (or overnight) at 550C.
4. Turn off the muffle furnace and wait to open until the temperature has dropped to at
least 250C, preferably lower. Open door carefully to avoid losing ash that may be
light and fluffy.
5. Use safety tongs to quickly transfer the crucibles to a dessicator with a porcelain
plate and desiccant. Cover crucibles, close the dessicator, and allow crucibles to
cool prior to weighing.
References
1.

Pearson, D (1976). General Methods. The Chemical Analysis of Foods.

Group Limited London.

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

Longman

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

29

EXPERIMENT 3 : DETERMINATION OF FAT CONTENT

OBJECTIVE
To determine fat content in meatball and peanut.
INTRODUCTION
The term lipid refers to a group of compounds that are sparingly soluble in water, but show
variable solubility in a number of organic solvents (e.g., ethyl ether, petroleum ether,
acetone, ethanol, methanol, benzene). The lipid content of a food determined by extraction
with one solvent may be quite different from the lipid content as determined with another
solvent of different polarity.
The solvent extraction methods used for fat analysis are:
a) Soxhlet method conventional
b) Mojonnier method
c) Soxhtherm - automated
PRINCIPLE
To determine the fat content of a food sample, the protein component is digested with boiling
hydrochloric acid to break the lipo-protein bonds. The digestion solution is filtered and the fat
remaining in the filter after the drying period is extracted with petroleum ether or n-hexane.
After the evaporation of the solvent, the residue is dried and weighed. The solvent is distilled
and the dried residue is weighed. The fat content is calculated based on the given formula.

Apparatus/Instrument/Chemicals/Samples

Mechanical blender
Analytical balance
Desiccator, with drying agent, e.g. Blaugel
Soxtherm micro/macro and multistat
Drying oven
Cotton Wool, chemically clean and fat-free

Procedure
1.

Preparation of the extraction beakers

3 -5 boiling stones are put into each extraction beaker. The beakers are dried in a drying
oven for about an hour at 103C 2C. After cooling off in the desiccator to room
temperature, weigh the beaker with a precision of +/-0.1mg.

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE
2.

Page

of

29

Sample Preparation

A representative average sample of at least 2 g should be used. The sample is mixed and
homogenized in the mixer at least twice. The sample can be stored in an airtight container to
prevent decay and any change in content. Prior to the analysis weigh 10g of the sample on a
filter paper and fold into a predried extraction thimble. Cover the thimble with cotton wool.
3.

Extraction

Put the thimble in the specified beaker. Any remaining fat traces on the watch glass have to
be taken up with some cotton wool, damp with extraction agent, and put into the extraction
thimble as well. After adding 140-ml extraction agent the sample is extracted in the
Soxtherm automatic using the following program:
Solvent:
Boiling Point:
Amount of Solvent:

Petrol ether 40/60

Boiling range 40 - 60 C
micro100ml/ macro 150 ml

Parameter for the Program:

Program Step
T-Classification
Extraction Temperature
Reduction Interval
Reduction Pulse
Hot Extraction
Evaporation A:
Rinsing Time

Parameter
200C
150C
4 min
4s
30 min
5 Intervals
70 min

Comment

Sample must be completely immersed

Level of solvent ca. 1-2 cm below the
thimble

After the program is finished, the extraction beakers are dried in the drying oven in an
upright position for 60 min at 103C 2C. Then, place them in a desiccator, leave them to
cool down to room temperature and weigh with a precision of +/- 1 mg. In order to check the
consistency of the weight, the sample is left to dry for another 30 min and weighed again
after cooling down. This procedure is repeated as long as two successive weighings show
no more than 1 mg difference. Should the weight increase then the previous lower value
should be taken. Extraction, drying, and weighing have to be done immediately after each
other.
4.

Calculation

The total fat content (w) in g/100 g (corresponds to %) of the sample is calculated using the
following formula:
(m2 m1) * 100
w =
m0
m1: Mass of the empty extraction beaker with boiling stones in g
Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

29

m2: Mass of the extraction beaker with fat after drying in g

m0: Weight at the start of the analysis in g
The result is expressed to two decimal places.
5.

References

1. AOAC. 2005. Official Methods of Analysis. 18th Edition

2. Gerhardt training manual
3. Suzanne Nielen, S. 2003. Food Analysis. Third Edition. Springer Science + Business
Media, Inc.
4. Suzanne Nielen, S. 2003. Food Analysis Laboratory Manual. Kluwer Academic/ Plenum
Publishers.

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

10

29

EXPERIMENT 4: DETERMINATION OF PROTEIN CONTENT

OBJECTIVE
To determine a protein content in fish and tempe.
INTRODUCTION
The protein content of foods can be determined by numerous methods. The Kjeldahl,
nitrogen combustion (Dumas) and infrared spectroscopy methods for protein analysis are
based on nitrogen determination. The methods are from the Official Methods of Analysis of
AOAC International (1), and are used commonly in research laboratories working on
proteins.

PRINCIPLE

The Kjeldahl procedure can be basically divided into three parts: (1) digestion, (2) distillation,
(3) titration. In the digestion step, organic nitrogen is converted to an ammonium in the
presence of a catalyst at approximately 370C. In this experiment, the sample is digested in
H2SO4, using Copper-based catalyst, converting N to NH3 which is distilled and titrated.
Sulfuric acid

Protein

(NH4)2SO4
Heat, catalyst

In the distillation step the digested sample is made alkaline with NaOH and the nitrogen is
distilled off as NH3. This NH3 is trapped in a boric acid solution.

(NH4)2SO4 + 2NaOH

2NH3 + Na2SO4 + 2H2O

NH3 + H3BO3 (boric acid)

NH4 + H2BO3
(borate ion)

The amount of ammonia nitrogen in this solution is quantified by titration with a standard HCl
solution. A reagent blank is carried through the analysis and the volume of HCl titrant
required for this blank is subtracted from each determination.

H2BO3- + H+

Date:
Name:

30.12.09
CWZ

H3BO3

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

11

29

This analysis determines total nitrogen and not usable nitrogen and this is the reason it is
called a crude protein analysis.
Method Reference
German official collection of methods 35 LMBG L 06.00 7, L 07.00 7, L 08.00 7
AOAC Official Method 928.08, Nitrogen in Meat, Kjeldahl Method, alternatively II
Apparatus/Instrument/Chemicals/Samples

Sulfuric acid 98% min.

Catalyst tablets to be used: Kjeltabs CX

Caustic soda 32%

Boric acid solution 2%

Indicator Solution M5 (Merck) or similar

Standard acid 0.1N or c = 0.1 mol/, alternatively sulfuric acid 0.1N or c = 0.05 mol/l

Mechanical comminuting instrument

Analytical balance (0.001 g)

Kjeldahl digestion block Kjeldatherm, Turbotherm, flask heater for Kjeldahl flask with
wide neck opening

Vapodest distillation System

Burette, 50 ml nominal capacity, with a scale on 0.05 ml or titration system (not with
the Vap 50) or pH meter with combined electrode

Procedure

1. Sample Preparation
1. Weigh accurately 2.00g comminuted sample as a start on a piece of a filter paper.
2. Store the sample air tight so that any changes or decay of the composition is
avoided. Prior to the analysis the sample should be at room temperature. The
examination of the thus prepared sample has to be done within the following 24 h.

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

12

29

2. Digestion chemicals

1. The chemicals are added. Sulfuric acid is used to wash down any sample residue,
which might remain at the glass walls.
Chemicals
Sulfuric acid

20 ml

Kjeltabs

Indicator solution
M5
Standard acid 0.1N or c=0.1mol/L;
alternatively sulfuric acid 0.1N or
c=0.05mol/L
3. Digestion with Kjeldatherm

When working with a Kjeldatherm-System with 250 ml Kjeldatherm-digestion tubes,

the following digestion parameters are recommended:

Time in

Temperature

min

in C

40

400

Comments

Digestion tubes are put into the preheated block

and time it takes for the sample to become
translucent

30

400

Dehydrate the sample

Foaming during the digestion has to be expected, however, the foaming should not
go higher then 2/3 of the glass.

If excessive reactions should occur, take out the insert rack.

During the digestion black particles remaining at the glass wall are washed back with
condensing sulfuric acid.

The sample glass has to be translucent after the digestion in order to obtain good
results.

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

13

29

4. Digestion with Turbotherm

When working with a Turbotherm-System with 250 ml Kjeldatherm-digestion tubes,

the following program parameter are recommended:

Time in

Power in %

Comment

100

Heating up of the system to bring digestion solution

min
10 to 15

to boiling
60

70 to 80

Digestion solution is turning translucent after ca 20

to 30 min

5. Digestion with Flask heater

For Serial Flask Heater with 500 or 750 ml Kjeldahl flask with wide neck opening the
following procedure is recommended:

Time in

Power

Comments

min
20

50

1,5

Heating up till the digestion solution is boiling

After

20-30

translucent.

min.
Wash

the

sample

down

should

remaining

turn

sample

particles with condensing sulfuric acid into the

flask.

6. Suction

During the entire digestion period the scrubber should be on. About 1200 ml of a
15% caustic soda is recommended for the washing bottle; this amount is sufficient to
neutralize digestion gases of about 60 digestions.

The cooling off period after the lifting of the insert rack or the cooling off period after
turning off the heating is about 30 minutes; during this time the scrubber should be
working as well.

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

14

29

7. Distillation

After the digested sample has cooled off the water steam distillation is done according to
the following program:

Program parameter

Vap 50

Water addition in s

NaOH addition in s

Reaction time in s

Distillation time in s

240

Steam output in %

100

Suction sample in s

25

Boric acid addition in s

6s

Suction receiver in s

25

Titration

Auto

Calculation

Auto

8. Titration (is done automatically when using the Vap 50)

3 - 4 drops of an indicator mixture M 5 are added to the receiving solution and it is

then titrated with 0.1 N titration acid till the color changes from green to grey/violet.

If the determination of the endpoint is done with a pH-meter or a titrator, the addition
of the indicator mixture is obsolete.

9. Blank Value

For the determination of the blank value the analysis (digestion and distillation) is run
just using the given chemicals.

The consumption of those chemicals has then to be taken into account when the
calculation is done.

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE
10.

Page

of

15

29

Calculation

1.4007 * c * (V - Vb)
%N=
Sample weight (g)

c : Concentration of the standard-acid solution: Hydrochloric acid 0.1N or c = 0.1

mol/l
Alternative: sulfuric acid 0.1N or c = 0.05 mol/l
V: Consumption of the standard acid in ml (Sample)
Vb: Consumption of the standard acid in ml (Blank Sample)
% raw protein= % N * 6.25
Nitrogen to Protein Conversion Factors for Various Foods
Factor
Egg or meat

6.25

Dairy products

6.38

Wheat

5.70

Other cereal grains or oilseeds

6.25

Almonds

5.18

Peanut and Brazil nuts

5.46

Other tree nuts and coconut

5.30

Soybean products

6.08

References

1. Gerhadt manual training

2. AOAC. 2005. Official Methods of Analysis. 18th Edition
3. Nielsen, S. S. 2003. Food Analysis. 3rd Edition. Springer Science

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

16

29

EXPERIMENT 5 : DETERMINATION FIBRE CONTENT

OBJECTIVE
To determine fibre content in whole bran bread and spinach.
DETERMINATION OF CRUDE FIBRE USING FIBREBAG (GERHARDT METHOD)
Apparatus/Instrument/Chemicals/Samples

Hotplate
1L Beaker glass without spout and glass condenser with riffle
FibreBag Carousel for 6 FibreBags with bayonet coupling
Bag with 100 FibreBags
Glass spacer
Accessory: crucible for incineration
Drying Chamber, Temp. 105C
Muffle furnace, Temp. 600C
Water heater
Desiccator
Timer or alarm clock
Analytical balance
Fume cabinet
Sulfuric acid c (H2SO4) = 0.13 mol/l
Potassium hydroxide solution c (KOH) = 0.23 mol/l
Petroleum ether, boiling range 40 to 60
Water distilled or demineralized
Sodium Hydroxide c (NaOH) = 0.313 mol/l
Hydrochloride acid c (HCl) = 0.1 mol/l

System Description:
1.

Preparation
a. FibreBag is dried at 105 1C for 1h in the drying chamber. The weight of the
FibreBag is the value A for the balance protocol. When storing the FibreBags in a
desiccator they only have to be dried once and then, can be weighted directly.
b. Put 1g sample into the FibreBag and weight with 1 mg preciseness; this gives value
B for the weighing protocol. A determination of the blank value should be done
parallel to the regular analysis. The value should be 1 mg/FibreBag. The dry matter
of the sample should be determined separately and is important for the calculation of
the content (result related to the dry matter).
c. Put the glass spacer into the FibreBag and insert the bag in carousel.
d. De-fatting of the sample, especially important for samples with a fat content of 5 %:
Immerse the carousel three times in a row into 100ml 40/60 petroleum ether. By
Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

17

29

turning it as well as moving it up and down the sample is defatted. This facilitates the
washing and filtration, which will follow. Furthermore, no crude fibre content is lost.
Throw away the first petroleum ether fraction but the following can be reused. After a
short drying process in the fume cupboard (about 2 minutes), immerse the carousel
in the first washing solution.
2.

Washing - Phase I ( Instrument method )

a. Measure 360 ml H2SO4 = 0.13mol/l into the first beaker.
b. Attach handling tool to the carousel and lower it gently into the beaker.
c. Mix it by rotating the carousel for about 1 minute so that the sample is entirely
soaked and make sure that the FibreBag is filled with washing solution.
d. Place the beaker on the hotplate, which has been preheated for about 5 minutes.
e. Bring it to a boil by setting it full (takes about 3-5 minutes); reduce the hotplate setting
when it starts to boil (at about 90C).
f.

Adjust the hotplate setting to obtain a very gentle simmering for about 30 minutes.
During this boiling stage the sample should float freely in the FibreBag.

g. This can be helped by gently rotating the carousel with the handling tool or by softly
swirling the beaker.
h. After exactly 30 minutes from the boiling point remove the beaker from the hotplate.
Also, take the carousel out of the beaker using the handling tool thus draining the
acid from the FibreBag.
i.

Washing out of the acid:

Discard the acid and solubles within the beaker.
Rinse the carousel several times with hot water.

3. Washing Phase II ( Instrument Method )

a. Measure 360 ml potassium hydroxide solution c (KOH) = 0.23 mol/l into the beaker.
b. Attach handling tool to the carousel and lower it gently into the beaker of solution.
Mix it by rotating the carousel for about 1 minute so that the FibreBag is filled
completely with the solution.
c. Place again the extraction beaker on a preheated hotplate.
d. Again, bring it to a boil by setting it full (takes about 3-5 minutes); reduce the hotplate
setting when it starts to boil.
e. Adjust the hotplate setting to obtain a very gentle simmering for about 30 minutes.

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE
f.

Page

of

18

29

During this boiling stage the sample should float freely in the FibreBag. This can be
helped by gently rotating the carousel with the handling tool or by softly swirling the
beaker.

g. After exactly 30 minutes from the boiling point remove the beaker from the hotplate.
h. Also, take out the carousel from the beaker using the handling tool thus draining the
solution from the FibreBags.
i.

Washing out the alkalis:

Discard the alkali and soluble within the beaker.
Rinse the carousel several times with hot water. (Check by using pH-indicator
paper)
Dry the FibreBags by wiping them with a paper towel or by rotating the carousel
in an empty beaker,

4. Drying of the FibreBags

a. Take out the drained FibreBags of the carousel and put into a crucible, which has to
be pre-ashed at 600C and weighted (value F for the balance protocol). Place it into
a drying chamber overnight at 105C. FibreBag after digestion and crucible is value
C.
5.

Incineration of Samples
a. Incinerate the FibreBags at 600C for at least 4 hours or overnight. The resulting
vapours are not hazardous!
b. After the incineration, weight the crucible, which was left, to cool off in the desiccator
and obtain value for the weighing protocol. value D.

6. Calculation:
The crude fibre is the non-solubles which remain after digestion with acids and alkalis
minus the content of ash and is calculated as follows:
% Crude Fibre

Blank Value E

((C - A) - (D - E)) x 100

B
D-F

Meaning:
A = Mass FibreBag in g
B = Mass Sample weight in g (has to be adjusted according to dry content)
C = Mass Crucible and dried FibreBag after digestion in g
D = Mass Crucible and Ash in g
E = Blank Value of the empty FibreBag in g
F= Mass Crucible in g

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

19

29

Results/ Weighing protocol: Blank value E in g: ................

FibreBag
Number

Samplenumber

A in g

F in g

B in g

C in g

D in g *

Crude
Fibre (%)

1
1
3
4
4
5
6
* minus blank value E in g
Comment: A small beaker can also be used instead of a crucible.

7.

Validation
The development of the FibreBags analysis has shown that it is of vital importance that
certain parameters have to be strictly observed. Thus, it is recommended to strictly
observing the times given for cooling, heating and boiling.
This also goes for:
Amount of Sample
Concentration of Acids and Alkalis
Times for drying and incineration and temperatures
No other solvents than Petroleum ether

References
1. Nielsen, S.S. 1994. Introduction to the Chemical Analysis of Foods. Boston: Jones
and Bartlett Publishers, Inc.
2. Gerhadt manual training.

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

20

29

EXPERIMENT 6 : GAS CHROMATOGRAPHY

Introduction

Gas chromatography (GC) has many applications in the analysis of food products.
GC has been used for the determination of fatty acids, triglycerides, cholesterol, gases,
water, alcohols, pesticides, flavor compounds, and many more. While GC has been used for
other food components such as sugars, oligosaccharides, amino acids, peptides, and
vitamins, these substances are more suited to analysis by high performance liquid
chromatography. GC is ideally suited to the analysis of volatile substances that are thermally
stable. Substances such as pesticides and flavor compounds that meet criteria can be
isolated
from a food and directly injected into the GC. For compounds that are
thermally unstable, too low in volatility, or yield poor chromatographic separation due to
polarity, a derivatization step must be done before GC analysis. The two parts of the
experiment described here include the analysis of alcohols that require no derivatization
step, and the analysis of fatty acids which requires derivatization. The experiments specify
the use of capillary columns, but the first experiment includes conditions for a packed
column.
Reading assignment
Reineccius, G.A. 2003. Gas chromatography. Ch. 29, in Food Analysis, 3rd ed. S.S. Nielsen
(Ed.), Kluwer Academic, New York.
METHOD A: DETERMINATION OF METHANOL AND HIGHER ALCOHOLS BY GAS
CHROMATOGRAPHY
Introduction
The quantification of higher alcohols, also known as fusel oils, in wine and distilled spirits is
important because of the potential flavor impact of these compounds. These higher alcohols
include n-propyl alcohol, isobutyl alcohol, and isoamyl alcohol. Some countries have
regulations that specify maximum and/or minimum amounts of total higher alcohols in certain
alcoholic beverages. Table wine typically contains only low levels of higher alcohols but
dessert wines contain higher levels, especially if the wine is fortified with brandy.
Methanol is produced enzymatically during the production of wine. Pectin-methylesterase hydrolyzes the methyl ester of -1, 4-D-galcaturonopyranose. The action of this
enzyme, which is naturally present in grapes and may also be added during vinification, is
necessary for proper clarification of the wine. White wines produced in the United States
contain less methanol (4-107 mg/L) compared to red and rose wines (48-227 mg/L).
Methanol has a lower boiling point than the higher alcohols, so it is more readily volatilized
and elutes earlier from a gas chromatography (GC) column.
Methanol and higher alcohols in distilled liquors are readily quantitated by gas
chromatography, using an internal standard such as benzyl alcohol, 3-pentanol, or n-butyl
alcohol. The method outlined below is similar to AOAC Methods 968.09 and 972.10
[Alcohols (Higher) and Ethyl Acetate in Distilled Liquors].

Objective
Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

21

29

Determine the content of methanol, n-propyl alcohol, and isobutyl alcohol in wine by gas
chromatography, using benzyl alcohol as the internal standard.
Principle of Method
Gas chromatography uses high temperatures to volatilize compounds that are separated as
they pass through the stationary phase of a column and are detected for quantitation.
Chemicals
________________________________________________________________________
CAS No.
Hazards
Benzyl alcohol
100-51-6
harmful
Ethanol
64-17-5
highly flammable
Isobutyl alcohol
78-83-1
irritant
Methanol
67-56-1
extremely flammable
n-Propyl alcohol
71-23-8
irritant, highly
flammable
________________________________________________________________________

Reagents
(** It is recommended that these solutions be prepared by the laboratory assistant before
class.)

Ethanol, 16% (vol/vol) with deionized distilled water (dd) water, 100 ml**
Ethanol, 50% (vol/vol) with dd water, 3100 ml**
Ethanol, 95% (vol/vol) with dd water, 100 ml**
Stock solutions**
Prepared with known amounts of ethanol and fusel alcohols or methanol;
1. 10.0 g of methanol and 50% (vol/vol) ethanol to 1000 ml.
2. 5.0 g of n-propyl alcohol and 50% (vol/vol) ethanol to 1000 ml.
3. 5.0 g of isobutyl alcohol and 50% (vol/vol) ethanol to 1000 ml.
4. 5.0 g of benzyl alcohol in 95% (vol/vol ethanol to 100 ml.

Working standard solutions**

Prepared from stock solutions, to contain different amounts of each of the fusel
alcohols; aliquots of these are used to get standard curves. Prepare four working
standards by combining:
1. 0.5 ml of stock solutions 1, 2, and 3 with 4.5 ml of 50% (vol/vol) ethanol plus 16%
(vol/vol) ethanol to 100 ml.
2. 1.0 ml of stock solutions 1, 2, and 3 with 3.0 ml of 50% (vol/vol) ethanol plus 16%
(vol/vol) ethanol to 100 ml.
3. 1.5 ml of stock solutions 1, 2, and 3 with 1.5 ml of 50% (vol/vol) ethanol plus 16%
(vol/vol) ethanol to 100 ml.
4. 2.0 ml of stock solutions 1, 2, and 3 with 16% (vol/vol) ethanol to 100 ml.
(Note: The final concentration of ethanol in each of these working standard solutions
is 18% (vol/vol) ethanol.)

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

22

29

Hazards, Precautions, and Waste Disposal

The alcohols are fire hazards; avoid open flames, breathing vapors, and contact with skin.
Otherwise, adhere to normal laboratory safety procedures. Wear safety glasses at all times.
Aqueous waste can go down the drain with a water flush.
Supplies
(Used by students)

Mechanical pipettor, 1000 l, with tips

Round bottom flask, 500 ml
Syringe (for GC)
6 Volumetric flasks, 100 ml
4 Volumetric flasks, 1000 ml

Equipment

Analytical balance
Distillation unit (heating element to fit 500 ml round-bottom flask; cold water
condenser)
Gas chromatography unit:

________________________________________________________________________
Column
DB-wax (30 m, 0.32 nm ID, 0.5 um film thickness) (Agilent Technologies,
PaloAlto, CA) or equivalent (capillary column), or 80/120 Carbopack BAW/5%
Carbowax 20M, 6 ft x in OD x 2mm ID glass column (packed column)
Injector
Temperature 200C
Column
Temperature 70C to 170C@5C/min
Carrier gas
He at 2 ml/min (N2 at 20 ml/min for packed column)
Detector
Flame ionization
Attenuation 8 (for all runs)
________________________________________________________________________
ID=inner diameter
OD=outer diameter
BAW=base and acid washed
Procedure
(Instructions are given for single standard and sample analysis, but injections can be
replicated.)

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

23

29

I. Sample Preparation
1. Fill a 100-ml volumetric flask to volume with the wine sample to be analyzed
2. Pour the wine into a 500-ml round bottom flask and rinse the volumetric flask several
times with dd water to complete the transfer. Add additional water if necessary to bring the
volume of sample plus dd water to ca. 150 ml.
3. Distill the sample and recover the distillate in a clean 100-ml volumetric flask. Continue
the distillation until the 100-ml volumetric is filled to the mark
4. Add 1.0 ml of each stock benzyl alcohol solution to 100 ml of each working standard
solution and wine sample to be analyzed.
II. Analysis of Sample and Working Standard Solutions
1. Inject 1 l of each sample and working standard solution in separate runs on the GC
column (split ratio 1: 20). (For packed column, inject 5.0 l.)
2. Obtain chromatograms and data from integration of peaks.
Data and Calculations
1. Calculate the concentration (mg/L) of methanol, n-propyl alcohol, and isobutyl alcohol in
each of the four working standard solutios (see example calculation below).
Alcohol concentration (mg/L)
________________________________________________________________________
Working standard
methanol
n-propyl alcohol
isobutyl alcohol
________________________________________________________________________
1
2
3
4
________________________________________________________________________

Example calculations:
Working standard solution #1- contains methanol + n-propyl alcohol + isobutyl alcohol, all in
ethanol
Methanol in stock solution #1:
10g methanol = 1 g
= 0.01 g
1000ml
100ml
ml
Working standard solution #1 contains 0.5 ml of stock solutions #1.
= 0.5 ml of 0.01 g methanol/ml
=0.005 g methanol= 5 mg methanol
That 5 mg methanol is contained in 100 ml volume.
= 5 mg/100 ml= 50 mg/1000 ml
= 50 mg methanol/L
Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

24

29

Repeat procedure for each alcohol in each working standard solution.

Peak height ratios for alcohol peaks at various concentrations of methanol, n-propyl alcohol,
and isobutyl alcohol, with benzyl alcohol as internal standard:
________________________________________________________________________
Peak Height Ratio1
____________________________________________________________
Alcohol Conc. Methanol
n-Propyl Alcohol
Isobutyl Alcohol
(mg/L)
Benzyl Alcohol
Benzyl Alcohol
Benzyl Alcohol
________________________________________________________________________
25
50
75
100
150
200
unknown
sample
________________________________________________________________________
1
Give individual values and the ratio.
2. Calculate the peak height or peak area ratios for methanol, n-propyl alcohol, and isobutyl
alcohol, compared to the internal standard, for each of the working standard solutions and
the wine sample. To identify which is the methanol, n-propyl alcohol, and isobutyl peak, see
the chromatogram that follows. Note that data from automatic integration of the peaks can
be used for these calculations. Report the ratios in a table as shown below. Show an
example calculation of concentration for each type of alcohol.
3. Construct standard curves for methanol, n-propyl alcohol, and isobutyl alcohol using the
peak height ratios. All lines can be shown on one graph. Determine the equations for the
lines.
4. Calculate the peak ratios for methanol, n-propyl alcohol, and isobutyl alcohol in the wine
sample, and their concentrations in mg/L.

Questions
1. Explain how this experiment would have differed in standard solutions used,
measurements taken, and standard curves used if you had used external standards rather
than an internal standard.
2. What are the advantages of using an internal standard rather than external standards for
this application, and what were the appropriate criteria to use in selecting the external
standard.

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

25

29

EXPERIMENT 7 : HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Figure: A Flow Scheme for HPLC

INTRODUCTION
This experiment is designed to introduce the use of the technique of Solid Phase Extraction
(SPE) to clean up samples and the technique of HPLC for performing a separation and
quantification of caffeine in beverages.
Solid-phase extraction (SPE) is a separation technique used to extract compounds
from a mixture of impurities. SPE is used to concentrate and purify samples for analytes of
interest from several matrices. The separation ability of solid phase extraction is based on
the preferential affinity of desired analyte, usually, to a solid phase through which the test
sample is passed. The solid phase is selected so that the impurities in the sample are unretained on the solid phase (adsorbent/stationary phase) while the analyte of interest is
retained on it. Analytes that are retained on the stationary phase can then be eluted from the
solid phase extraction cartridge with the appropriate solvent.
High-performance liquid chromatography (HPLC) is a separation technique used to
separate components of a mixture from each other by taking advantage of a variety of
physiochemical interactions of analytes in the mixture between two phases. One phase is
held
stationary in a column while the mixture to be analyzed is introduced into another phase that
is
moved over the stationary phase (mobile phase). Different components of the sample are
carried forward at different rates by the moving liquid phase, due to their differing
interactions with the stationary and mobile phases. There are a number of different kinds of
chromatography, which differ in the mobile and the stationary phase used.
A detector measures response changes between the solvent itself, and the solvent
together with sample when passing through it. The electrical response is digitized and sent
to a data system. Less volatile and larger samples can be used with HPLC. It was
discovered that better separation of the components of the mixture occurs if the particles in
the stationary phase are very small. However, it was also found that if very small particles
were used in the column, then the liquid passed very slowly through the column. Therefore,
a pump is used to force the liquid through the column.
Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

26

29

Caffeine is a stimulant that is commonly found in many foods and drinks that we
consume. Caffeine has a mildly addictive effect on the body; it is therefore interesting to
know exactly how much caffeine is in certain beverages. One way to analyze caffeine
content in beverages is by using high-performance liquid chromatography (HPLC).
OBJECTIVE
1. To determine caffeine content in tea and coffee using high performance liquid
chromatography (HPLC).
2. To familiarise the students with HPLC system.
DETERMINATION OF CAFFEINE IN FOOD SAMPLES USING HPLC
1. Apparatus/Instrument/Chemicals/Samples

Analytical balance
Round bottom flask, 250ml 2 pcs for duplicate analysis.
Heating mental 2 pcs for duplicate analysis
Reflux 2 pcs for duplicate analysis
Volumetric flask, 250 ml 2 pcs for duplicate analysis
Volumetric flask, 10 ml 3 pcs.
Disposable plastic syringe
Pasteur pipettes
Accubond C18-SPE, 6ml, 1000mg.
Manifold
Sample vials, 2 ml for auto sampler
Nylon Syringe filter, Diameter 13 mm with 0.45 um pore size
Filtration assembly for mobile phase with 0.45 um nylon membrane filters.
Hydrochloric acid, 10% v/v
Methanol, HPLC Grade as mobile phase
Acetic Acid solution, 1% as mobile phase
Stock solution: 50mg caffeine in 100ml (500 ppm) deionised water.
Standard solutions 0.05 mg/ml (50ppm), 0.10 mg/ml (100ppm), 0.15 mg/ml
(150ppm) and 0.5 mg/ml (500 ppm) caffeine.
0.5 mg/ml: 0.05 g caffeine standard in 100 ml deionised water (Stock
Solutions)
0.15 mg/ml: Take 3.0 ml from stock solutions and pipette into 10ml volumetric
flask. Then dilute with deionised water until reach to the mark.
0.10 mg/ml: Take 2.0 ml from stock solutions and pipette into 10ml
volumetric flask. Then dilute with deionised water until reach to the mark.
0.05 mg/ml: Take 1.0 ml from stock solutions and pipette into 10ml volumetric
flask. Then dilute with deionised water until reach to the mark.

HPLC System:
Agilent 1200 Series.
VWD Detector, Wavelength 280 nm, flow rate 0.9 ml/min.
Injection volume 20 ul.
Column: Zorbax Eclipse XDB-C18, 4.6 X 150 mm, 5
Mobile phase: 65% Methanol, 35% deionised water with acetic acid (1%).
2. Procedure
Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

I.

Page

of

27

29

Sample extraction
1. Weigh accurately 1g sample and transfer into 250 ml round bottom flask. Reflux
with 100 ml of 10% HCL for 1 hour.
2. Transfer contents of flask into a 250 ml volumetric flask and make up to mark with
deionised water.
3. Prepare spiking sample (Duplicate): Take 1.0 ml from stock solutions (standard)
and pipette into 10ml volumetric flask and top up with sample solution until mark.

II.

Solid Phase Extraction

1.

2.
3.
4.
5.
6.
7.
8.

Activate an SPE cartridge stationary phase sorbent - (Accubond C18) to by

passing 6mL 50% methanol through the sorbent. Make sure to keep the sorbent
wet. Turn off the vacuum as methanol level approaches the top of packing.
Pass 3mL of water through the packing; keep the sorbent wet and turn off the
vacuum as water level approaches the top of packing.
Pipette 1.00mL of sample solution to the SPE cartridge and apply a vacuum,
keep the sorbent wet. Save the filtrate.
Reintroduce the filtrate into the SPE tube and repeat the previous step, this time
draw all liquid through.
Draw air through the column for 3-5 minutes or until dry.
Elute the analyte from the column with 10 mL (5mL 2) of mobile phase into a
small flask.
Filter the sample through 0.45 um syringe filter into 2ml vials.
Run samples together with standard solution through HPLC.

Calculation
1. Draw the calibration curve of caffeine standard (Concentration Vs Area).

2. Get the concentration of samples (X) from the calibration curve.

3. Calculate according to the equation given as below:
Caffeine (mg/ml) =
Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

____

x 250 x 10

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

28

29

Weight of samples

Lab Report
The lab report should contain:
1. Your chromatograms from the computer
2. Your calibration graph for caffeine with R2.
3. The concentration of caffeine in each of the beverages (remember to include appropriate
dilution factors), as well as amount of caffeine in a typical serving of each beverage (you
decide what a typical serving is!). Show all calculations.
4. Percent recovery for spiked sample. Show all calculation.
5. Answers to the following questions
Describe three types of detectors for HPLC.
What is the difference between reverse phase and normal phase chromatography?
Why does the pH effect the retention time for caffeine?
What values did you find on the web for the amounts of caffeine in your sample? Be
sure to give the web address you used.
References
1. MS 1235: 1991
2. Nielsen, S.S. 1994. Introduction to the Chemical Analysis of Foods, Boston: Jones and
Barlett Publisher, Inc
3. Abdul Mumin, Kazi Farida Akhter,. Zainal Abedin, Zakir Hossain. 2006: Determination of
Caffeine by Solid Phase Extraction and High Performance Liquid Chromatography (SPE
HPLC) Malaysian Journal of Chemistry, Vol. 8, No. 1, 045 - 051

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual

SFA3091
FOOD ANALYSIS LABORATORY PROCEDURE

Page

of

29

29

METHOD ON REPORTING RESULT FOR CAFFEINE ANALYSIS

SAMPLE

CAFFEINE (PPM)
FROM
CHROMATOGRAM (X)

FORMULA
CALCULATION

X
x 250
Sample weight (g)
X
x 250
Sample weight (g)
X
x 250 x 10
Sample weight (g)
X
x 250 x 10
Sample weight (g)
X
x 250 x 10
Sample weight (g)
X
x 250
Sample weight (g)

1. Sample 1
(Direct)

2. Sample 2
(Direct)

3. Sample (SPE)
1

4. Sample (SPE)
2

5. Spiked (SPE)
with 50ppm
caffeine
6. Spiked
(without SPE)
with 50ppm
caffeine

ACTUAL
CONCENTRATION OF
CAFFEINE IN mg/100g
sample
.
Caffeine (mg/100g) =
average std dev
.

.
Caffeine (mg/100g) =
average std dev
.

a. The amount of caffeine in spiked sample (sample 5 and 6); TRUE VALUE = 50 ppm
+ (9 x [ ] caffeine per ml)

b. % Recovery for SPE sample = |Result sample 5 TRUE VALUE| x 100

TRUE VALUE

c. % Recovery for without SPE sample = |Result sample 6 TRUE VALUE| x 100
TRUE VALUE

Date:
Name:

30.12.09
CWZ

Revision:

1 (13.01.11)
NBH

File: Food analysis manual