proximate analysis

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FOOD ANALYSIS LABORATORY PROCEDURE 1 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 105ºC Minced meat Tongs

Procedure

1) Perform this experiment in duplicates.2) Dry the crucible and its lid in the oven at 105ºC overnight and transfer to the

dessicator to cool (approx 30 min)3) Weigh the crucible.

Date: 30.12.09 Revision: 1 (13.01.11) File: Food analysis manualName: CWZ NBH

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4) Weigh about 5 g sample into crucible. Spread the meat.5) Replace the lid and weigh the crucible and its contents (WI)6) Place the crucible with its lid slipped to one side. Dry for 16h or overnight at 105ºC7) 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 100W1

Where, w1 = weight (g) of sample before dryingW2= 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 ) X 100

( wt of wet sample + pan ) – ( wt of pan )

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METHOD B : MOISTURE ANALYZER (AND METHOD)

Sample: flour

***Performed sample in triplicate.

Apparatus/Instrument/Chemicals/Sample


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EXPERIMENT 2: DETERMINATION OF MINERAL CONTENT


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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 (525°C 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, but a special perchloric acid hood is necessary. The disadvantages of wet ashing are that it


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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)

Muffled furnace

Sample

Hot plate

Procedure1. Dry a representative sample (weigh accurately to nearest mg ~ 3 – 5 g of samples)

in a crucible in an oven at 130°C 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 550°C.

3. Ignite the sample 12- 18 h (or overnight) at 550°C.4. Turn off the muffle furnace and wait to open until the temperature has dropped to at

least 250°C, 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. Longman Group Limited London.


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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 – conventionalb) Mojonnier methodc) 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.


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 103°C ± 2°C. After cooling off in the desiccator to room temperature, weigh the beaker with a precision of +/-0.1mg.


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2. 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: Petrol ether 40/60Boiling Point: Boiling range 40 - 60 °CAmount of Solvent: micro100ml/ macro 150 ml

Parameter for the Program:

Program Step Parameter CommentT-Classification 200°CExtraction Temperature 150°CReduction Interval 4 minReduction Pulse 4 sHot Extraction 30 min Sample must be completely immersedEvaporation A: 5 IntervalsRinsing Time 70 min 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 103°C ± 2°C. 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) * 100w = m0

m1: Mass of the empty extraction beaker with boiling stones in g


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m2: Mass of the extraction beaker with fat after drying in gm0: 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 Edition2. Gerhardt training manual3. 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.


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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 370ºC. In this experiment, the sample is digested in

H2SO4, using Copper-based catalyst, converting N to NH3 which is distilled and titrated.

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.

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.


Protein (NH4)2SO4

(NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O

NH3 + H3BO3 (boric acid) NH4 + H2BO3-

(borate ion)

H2BO3- + H+ H3BO3

Sulfuric acid

Heat, catalyst

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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


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.

2. Digestion chemicals


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1. The chemicals are added. Sulfuric acid is used to wash down any sample residue,

which might remain at the glass walls.

3. Digestion with Kjeldatherm

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

the following digestion parameters are recommended:

Time in

min

Temperature

in ° C

Comments

40 400 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.


Chemicals

Sulfuric acid 20 ml

Kjeltabs 2

Indicator solution

M5

Standard acid 0.1N or c=0.1mol/L;

alternatively sulfuric acid 0.1N or

c=0.05mol/L

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4. Digestion with Turbotherm

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

the following program parameter are recommended:

Time in

min

Power in % Comment

10 to 15 100 Heating up of the system to bring digestion solution

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

min

Power Comments

20 3 Heating up till the digestion solution is boiling

50 1,5 After 20-30 min. the sample should turn

translucent. Wash down remaining 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.


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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 9

NaOH addition in s 8

Reaction time in s 0

Distillation time in s 240

Steam output in % 100

Suction sample in s 25

Boric acid addition in s 6 s

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.


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10. 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


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EXPERIMENT 5 : DETERMINATION FIBRE CONTENT

OBJECTIVE

To determine fibre content in whole bran bread and spinach.

DETERMINATION OF CRUDE FIBRE USING FIBREBAG (GERHARDT METHOD)


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. 105°C Muffle furnace, Temp. 600°C 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 ±1°C 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 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.


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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.

f. 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.


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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 600°C and weighted (value F for the balance protocol). Place it into a drying chamber overnight at 105°C. FibreBag after digestion and crucible is value C.

5. Incineration of Samplesa. Incinerate the FibreBags at 600°C 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 = ((C - A) - (D - E)) x 100 B

Blank Value E = D - F

Meaning:

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

F= Mass Crucible in g

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

FibreBag Sample-number

A in g F in g B in g C in g D in g * Crude Fibre (%)


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Number

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.


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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-methyl-esterase hydrolyzes the methyl ester of α-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


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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. HazardsBenzyl alcohol 100-51-6 harmfulEthanol 64-17-5 highly flammableIsobutyl alcohol 78-83-1 irritantMethanol 67-56-1 extremely flammablen-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.)


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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 µl, 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 ¼ in OD x 2mm ID glass column (packed column)

InjectorTemperature 200˚CColumnTemperature 70˚C to 170˚C@5˚C/minCarrier gas He at 2 ml/min (N2 at 20 ml/min for packed column)Detector Flame ionizationAttenuation 8 (for all runs)________________________________________________________________________

ID=inner diameterOD=outer diameterBAW=base and acid washed

Procedure

(Instructions are given for single standard and sample analysis, but injections can be replicated.)


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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 µl of each sample and working standard solution in separate runs on the GC column (split ratio 1: 20). (For packed column, inject 5.0 µl.)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________________________________________________________________________1234________________________________________________________________________

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


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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________________________________________________________________________255075100150200

unknownsample

________________________________________________________________________1Give 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.


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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 un-retained 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 heldstationary in a column while the mixture to be analyzed is introduced into another phase that ismoved 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.


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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


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I. 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. 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.

2. Pass 3mL of water through the packing; keep the sorbent wet and turn off the vacuum as water level approaches the top of packing.

3. Pipette 1.00mL of sample solution to the SPE cartridge and apply a vacuum, keep the sorbent wet. Save the filtrate.

4. Reintroduce the filtrate into the SPE tube and repeat the previous step, this time draw all liquid through.

5. Draw air through the column for 3-5 minutes or until dry.6. Elute the analyte from the column with 10 mL (5mL× 2) of mobile phase into a

small flask.7. Filter the sample through 0.45 um syringe filter into 2ml vials.8. 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) = X ____ x 250 x 10

Weight of samples


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Lab Report

The lab report should contain:

1. Your chromatograms from the computer2. 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: 19912. Nielsen, S.S. 1994. Introduction to the Chemical Analysis of Foods, Boston: Jones and

Barlett Publisher, Inc3. 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


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METHOD ON REPORTING RESULT FOR CAFFEINE ANALYSIS

SAMPLE CAFFEINE (PPM) FROM CHROMATOGRAM (X)

FORMULA CALCULATION

ACTUAL CONCENTRATION OF CAFFEINE IN mg/100g sample

1. Sample 1 (Direct)

X . x 250 Sample weight (g)

Caffeine (mg/100g) =average ± std dev

2. Sample 2 (Direct)


3. Sample (SPE) 1

X . x 250 x 10 Sample weight (g)

Caffeine (mg/100g) =average ± std dev

4. Sample (SPE) 2

X . x 250 x 10Sample weight (g)

5. Spiked (SPE) with 50ppm caffeine

X . x 250 x 10 Sample weight (g)

6. Spiked (without SPE) with 50ppm caffeine


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


proximate analysis

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