Chapter 2- Overview of basic/classical methods of food analysis

1.Classical Method of Food Analysis

 Titrimetric Analysis
 Gravimetric Procedure
 Solvent Extraction
 Refractometry
2. Instrumental methods
Titrimetric Analysis
 Titrimetric analysis involves determination of the volume of a solution of
accurately known concentration, which is required to react quantitatively with
the measured volume of the solution of a substance, concentration of which is to
be determined.
 The solution of accurately known concentration is called standard solution. The
mass of the substance dissolved in the solution of unknown concentration is
calculated from the volume of the standard solution used, the chemical equation
and the relative molecular masses of the reacting compounds.
 The reagent of known concentration is called titrant and the substance being
titrated is termed as titrand.
 To carry out titrimetric analysis, standard solution is usually added from the long-
graduated tube called burette.
 The process of adding the standard solution to the solution of unknown
concentration until the reaction is just complete is called titration.
 The point at which reaction is completed is called equivalence point or the
theoretical or stoichiometric end point.
 It is not possible all the time to take standard solution in the burette. You will
come to know about it later in this unit in the titration of sodium hydroxide with
oxalic acid.

Detection of End Point

 The end point is detected either by some physical change produced in the
reaction mixture itself or by the addition of an auxiliary reagent, known as
indicator; alternatively some other physical measurement may be used.
 At the completion of the reaction, the indicator shows a visible change e.g.
(colour change or turbidity) in the solution being titrated.
 In an ideal titration, the visible end point coincides with the stoichiometric or
theoretical end point; but in practice usually some small difference occurs. This
represents titration error.
 Indicator and the experimental conditions selected should be such that the
difference between the visible end point and the theoretical end point is
minimum.

Requirement For a Reaction in the Titrimetric Analysis

  The substance of which amount is to be determined by titrimetric analysis
must react completely and rapidly with the other reagent in stoichiometric
proportion.
 The reaction should be fast and there must be alteration in physical or
chemical property of the solution at the equivalence point, which can be
detected by an indicator, or by measuring the potential difference or current
etc.

Acidimetry and alkalimetry

 Titrimetric analysis can be carried out for various types of reactions. In this unit
you will learn only about neutralisation reactions.
 These involve titrations of acids and bases. Standard solutions of acids
(acidimetry) and bases (alkalimetry) are used in these titrations.
 In quantitative estimation through titrimetric analysis, concentration of solution
is expressed in terms of molarity.
 It is number of moles of solute dissolved in 1 litre of solution.

Standard Solution
 A solution of exactly known concentration is called standard solution. Any
substance, which is stable at room temperature and does not react with solvent
in which it is dissolved, can be directly weighed to prepare its standard solution.
 Description and preparation of these solutions is given below:

Primary and secondary standards

 A primary standard is a compound of sufficient purity in which total amount of
impurities does not exceed 0.01-0.02%.
 The standard solution can be prepared by direct weighing of a sample of
primary standard followed by its dissolution in water (or solvent) to obtain a
definite volume of solution. The substance to be used as a primary standard
should also satisfy the following requirements:
o It must be easily available in pure and dry form.
 o It should not undergo change in air i.e. it should not be hygroscopic, oxidised
by air or affected by gases such as carbon dioxide present in the atmosphere
or lose water of crystallization, so that it can be stored safely.
o It should be easy to detect the impurities present in it.
o It should have high relative molecular mass so that weighing errors are
neglible.
o Its reaction with another substance should be instantaneous and
stoichiometric.
o The substance should be readily soluble in water.

 It is difficult to obtain an ideal primary standard. Therefore, substances having

characteristics nearer to the primary standards are usually employed.
 Unstable hydrated salts, as a rule, should not to be used as primary standards.
 However, sodium carbonate, sodium tetraborate, potassium hydrogen
phthalate, oxalic acid, ferrous ammonium sulphate etc. can be used as primary
standards because of their sufficient stabilities.
 A solution of secondary standard is the one which may be used for
standardization after finding out its exact concentration by titration against a
standard solution of primary standard.
 A secondary standard cannot be used for preparing standard solution by direct
weighing.
 Sodium hydroxide and potassium permanganate are examples of secondary
standards.
 Before starting titrimetric analysis, you should be familiar with some techniques
such as, weighing by using chemical balance, preparing standard solution,
measuring volume by using burette and pipette.

Indicators in Acid Base Titration

 Acid base indicators are sensitive to pH change. For most acid base titrations, it is
possible to select indicators which exhibit colour change at pH close to the
equivalence point.
 We will discuss here about only two indicators – phenolphthalein and methyl
orange.
Phenolpthalein
 Phenolpthalein is a weak acid, therefore it does not dissociate in the acidic
medium and remains in the unionised form, which is colourless.
  In the acidic medium, equilibrium lies to the left. In the alkaline medium, the
ionisation of phenolphthalein increases considerably due to the constant
removal of H+ ions released from HPh by the OH– ions from the alkali.
o So the concentration of Ph– ion increases in the solution, which imparts pink
colour to the solution.

 For a weak acid vs strong alkali titration, phenolphthalein is the most suitable
indicator.
 This is so because the last drop of added alkali brings the pH of the solution in the
range in which phenolphthalein shows sharp colour change.
Methyl orange
 Methyl orange is a weak base and is yellow in colour in the unionised form.
Sodium salt of methyl orange is represented as follows:

 The anion formed from the indicator is an active species, which on accepting a
proton (i.e acting as Bronsted Lowry base) changes from the benzenoid form to
the quinonoid form.
  The quinonoid form is deeper in colour and thus is responsible for the colour
change at the end point. This is illustrated in the following manner:

Choice of Indicator
 In the titration of strong acid and a weak base, methyl orange is chosen as
indicator.
 When titration between strong base and weak acid is to be performed then
phenolphthalein is a good indicator.
 In this case alkali is dropped from the burette and acid is taken in the tiration
flask.
 Colour of the solution taken in the titration flask changes from colourless to
pink. This change of colour is easily perceptible to the human eye.
 If we take alkali in the titration flask, the colour change will be from pink to
colourless and accuracy in noting the colour change may be less.
 In the titration of strong acid versus strong base anyone of the above indicators
can be used.
 For the titration of weak acid vs weak base no indicator is available.

Titratable Acidity Apparatus

 a- potentiometric titration b- colorimetric titration

Redox Titration
 Redox Titration is a laboratory method in which we can find a concentration of a
given analyte. It is based on a redox reaction between the analyte and the titrant
and sometimes may involve a potentiometer or a redox indicator as a part of
their process.
 When we say that this titration is based on a redox reaction between the analyte
and the titrant, then it means that it is based on an oxidation-reduction reaction
between the analyte and the titrant.
 Now, as we know redox titrations are based on redox reactions, and redox
reaction is basically the oxidation-reduction reaction.

 In every redox reaction, both reduction and oxidation must occur.

 Substance that gives electrons is the reducing agent or reductant. Substance that
accepts electrons is the oxidizing agent or oxidant.
 Reaction of ferrous ion with chlorine gas Overall, the number of electrons lost in
the oxidation half reaction must equal the number gained in the reduction half
equation.

Precipitation Titration
 Precipitation titration is the method that involves the formation of precipitate
during titration.
 In the precipitation titration, the titrant reacts with the analyte and forms an
insoluble substance, this titration is continued till the consumption of the last
drop of the analyte.
 The process of titration is used to check the purity of synthesized compounds
mainly in pharmaceuticals. Through precipitation titration the precipitates are
formed during the process of titration.
 To determine the concentration of chloride ions in a certain solution, the
solution can be titrated with a silver nitrate solution of known concentration.
 The white-colored precipitate of silver chloride is formed.
 The quantity of silver ions used to determine the endpoint is equal to the
quantity of originally present chloride ions.
 Reactions involved in the process is given below:
AgNO3 +Cl- → AgCI+NO3-
 Principle: In the precipitation titration the quantity of participating reagent
added is equal to the precipitate.
 Quantity of reagent added = quantity of the precipitate obtained

Types of Precipitation Titration

 There are mainly three types of precipitation titrations:
o Volhard’s Method
o Fajan’s Method
o Mohr’s Method
Volhard’s Method:
 This method was given by the German Chemist Jacob Volhard. In this method
anions like halides, phosphate, and chromate are determined in an acidic
medium by using silver ions.
 To prevent the precipitation of iron ions as hydrated oxide it is important to
perform this titration in an acidic medium.
 In this method of reaction with the excess of silver nitrate, the chloride present
in the solution is converted into silver chloride.
 The leftover silver nitrate from the reaction is again treated with potassium
thiocyanate solution.
 The excess of thiocyanate is made to react with an indicator which gives a red
color on reacting with ferric ammonium sulfate indicator and a complex is
formed.
Fajan’s Method:
 This method was given by American chemist Kazimierz Fagan.
  In this method, the indicator absorption method is used. In this method,
dichlorofluorescein worked as an indicator and methyl chloride ions that are
present in excess are absorbed on the silver chloride surface.
 The endpoint is determined by the green suspension of AgCl which turns pink
which is a complex of AgCl and indicator.

Mohr’s Method:
 This method was given by German Chemist Karl Friedrich Mohr.
 This is a direct titration method in which silver nitrate is used as titrant and
solution of chloride ion is used as analyte.
 The indicator used in the titration is Potassium chromate.
 The reddish-brown precipitate is formed at the end of the reaction of silver ions
and chromate ions, after the consumption of all the chloride ions.

Gravimetric Procedures
 Gravimetric analysis, a method of quantitative chemical analysis in which the
constituent sought is converted into a substance (of known composition) that can
be separated from the sample and weighed.
 The steps commonly followed in gravimetric analysis are:
o preparation of a solution containing a known weight of the sample,
o separation of the desired constituent,
o weighing the isolated constituent, and
o computation of the amount of the particular constituent in the sample from
the observed weight of the food substance.

Solvent Extraction
 The fact that lipids are soluble in organic solvents, but insoluble in water,
provides the food analyst with a convenient method of separating the lipid
components in foods from water soluble components, such as proteins,
carbohydrates and minerals.
 In fact, solvent extraction techniques are one of the most commonly used
methods of isolating lipids from foods and of determining the total lipid content
of foods.
Sample Preparation
The preparation of a sample for solvent extraction usually involves a number of steps:
 Drying sample. It is often necessary to dry samples prior to solvent extraction,
because many organic solvents cannot easily penetrate into foods containing
water, and therefore extraction would be inefficient.
 Particle size reduction. Dried samples are usually finely ground prior to solvent
extraction to produce a more homogeneous sample and to increase the surface
area of lipid exposed to the solvent.
o Grinding is often carried out at low temperatures to reduce the tendency for
lipid oxidation to occur.
 Acid hydrolysis. Some foods contain lipids that are complexed with proteins
(lipoproteins) or polysaccharides (glycolipids).
o To determine the concentration of these components it is necessary to break
the bonds which hold the lipid and non-lipid components together prior to
solvent extraction.
o Acid hydrolysis is commonly used to release bound lipids into easily
extractable forms, e.g. a sample is digested by heating it for 1 hour in the
presence of 3N HCl acid.
 Solvent Selection. The ideal solvent for lipid extraction would completely extract
all the lipid components from a food, while leaving all the other components
behind.
o In practice, the efficiency of solvent extraction depends on the polarity of the
lipids present compared to the polarity of the solvent.
 o Polar lipids (such as glycolipids or phospholipids) are more soluble in polar
solvents (such as alcohols), than in non-polar solvents (such as hexane).
o On the other hand, non-polar lipids (such as triacylglycerols) are more
soluble in non-polar solvents than in polar ones.
o The fact that different lipids have different polarities means that it is
impossible to select a single organic solvent to extract them all.
o Thus the total lipid content determined by solvent extraction depends on the
nature of the organic solvent used to carry out the extraction: the total lipid
content determined using one solvent may be different from that
determined using another solvent.
o In addition to the above considerations, a solvent should also be inexpensive,
have a relatively low boiling point (so that it can easily be removed by
evaporation), be non-toxic and be nonflammable (for safety reasons).
o It is difficult to find a single solvent which meets all of these requirements.
o Ethyl ether and petroleum ether are the most commonly used solvents, but
pentane and hexane are also used for some foods.

Batch Solvent Extraction

 These methods are based on mixing the sample and the solvent in a suitable
container, e.g., a separatory funnel.
 The container is shaken vigorously and the organic solvent and aqueous phase
are allowed to separate (either by gravity or centrifugation).
 The aqueous phase is then decanted off, and the concentration of lipid in the
solvent is determined by evaporating the solvent and measuring the mass of
lipid remaining: %Lipid = 100 � (Mlipid/Msample).
 This procedure may have to be repeated a number of times to improve the
efficiency of the extraction process.
 In this case the aqueous phase would undergo further extractions using fresh
solvent, then all the solvent fractions would be collected together and the lipid
determined by weighing after evaporation of solvent.
 The efficiency of the extraction of a particular type of lipid by a particular type of
solvent can be quantified by an equilibrium partition coefficient, K =
csolvent/caqueous, where csolvent and caqueous are the concentration of lipid
in the solvent and aqueous phase, respectively.
 The higher the partition coefficient the more efficient the extraction process.

Semi-Continuous Solvent Extraction

 Semi-continuous solvent extraction methods are commonly used to increase the
efficiency of lipid extraction from foods.
  The Soxhlet method is the most commonly used example of a semi-continuous
method.
 In the Soxhlet method a sample is dried, ground into small particles and placed
in a porous thimble.
 The thimble is placed in an extraction chamber, which is suspended above a
flask containing the solvent and below a condenser.
 The flask is heated and the solvent evaporates and moves up into the
condenser where it is converted into a liquid that trickles into the extraction
chamber containing the sample.
 Eventually, the solvent builds up in the extraction chamber and completely
surrounds the sample.
 The extraction chamber is designed so that when the solvent surrounding the
sample exceeds a certain level it overflows and trickles back down into the
boiling flask.
 As the solvent passes through the sample it extracts the lipids and carries them
into the flask.
 The lipids then remain in the flask because of their low volatility. At the end of
the extraction process, which typically lasts a few hours, the flask containing the
solvent and lipid is removed, the solvent is evaporated and the mass of lipid
remaining is measured (Mlipid).
 The percentage of lipid in the initial sample (Msample) can then be calculated:
%Lipid = 100 � (Mlipid/Msample).
 A number of instrument manufacturers have designed modified versions of the
Soxhlet method that can be used to determine the total lipid content more
easily and rapidly (e.g. Soxtec).

Continuous Solvent Extraction

 The Goldfish method is similar to the Soxhlet method except that the extraction
chamber is designed so that the solvent just trickles through the sample rather
than building up around it.
 This reduces the amount of time required to carry out the extraction, but it has
the disadvantage that channeling of the solvent can occur, i.e., the solvent may
preferentially take certain routes through the sample and therefore the
extraction is inefficient.
 This is not a problem in the Soxhlet method because the sample is always
surrounded by solvent.

Accelerated Solvent Extraction

  The efficiency of solvent extraction can be increased by carrying it out at a
higher temperature and pressure than are normally used.
 The effectiveness of a solvent at extracting lipids from a food increases as its
temperature increases, but the pressure must also be increased to keep the
solvent in the liquid state.
 This reduces the amount of solvent required to carry out the analysis, which is
beneficial from a cost and environmental standpoint.
 Special instruments are available to carry out solvent extraction at elevated
temperatures and pressures.

Supercritical Fluid Extraction

 Solvent extraction can be carried out using special instruments that use
supercritical carbon dioxide (rather than organic liquids) as the solvent.
 These instruments are finding greater use because of the cost and environmental
problems associated with the usage and disposal of organic solvents.
 When pressurized CO2 is heated above a certain critical temperature it becomes
a supercritical fluid, which has some of the properties of a gas and some of a
liquid.
 The fact that it behaves like a gas means that it can easily penetrate into a
sample and extract the lipids, while the fact that it behaves like a fluid means
that it can dissolve a large quantity of lipids (especially at higher pressures).
 Instruments based on this principle heat the food sample to be analyzed in a
pressurized chamber and then mix supercritical CO2 fluid with it.
 The CO2 extracts the lipid, and forms a separate solvent layer, which is
separated from the aqueous components.
 The pressure and temperature of the solvent are then reduced which causes the
CO2 to turn to a gas, leaving the lipid fraction remaining.
 The lipid content of a food is determined by weighing the percentage of lipid
extracted from the original sample.

Nonsolvent Liquid Extraction Methods.

 A number of liquid extraction methods do not rely on organic solvents, but use
other chemicals to separate the lipids from the rest of the food.
 The Babcock, Gerber and Detergent methods are examples of nonsolvent liquid
extraction methods for determining the lipid content of milk and some other
dairy products.

Babcock Method
  A specified amount of milk is accurately pipetted into a specially designed flask
(the Babcock bottle).
 Sulfuric acid is mixed with the milk, which digests the protein, generates heat,
and breaks down the fat globule membrane that surrounds the droplets,
thereby releasing the fat.
 The sample is then centrifuged while it is hot (55-60oC) which causes the liquid
fat to rise into the neck of the Babcock bottle.
 The neck is graduated to give the amount of milk fat present in wt%.
 The Babcock method takes about 45 minutes to carry out, and is precise to
within 0.1%.
 It does not determine phospholipids in milk, because they are located in the
aqueous phase or at the boundary between the lipid and aqueous phases.

Gerber Method
 This method is similar to the Babcock method except that a mixture of sulfuric
acid and isoamyl alcohol, and a slightly different shaped bottle, are used.
 It is faster and simpler to carry out than the Babcock method.
 The isoamyl alcohol is used to prevent charring of the sugars by heat and
sulfuric acid which can be a problem in the Babcock method since it makes it
difficult to read the fat content from the graduated flask.
 This method is used mainly in Europe, whilst the Babcock method is used mainly
in the USA.
 As with the Babcock method, it does not determine phospholipids.

Detergent Method
 This method was developed to overcome the inconvenience and safety concerns
associated with the use of highly corrosive acids.
 A sample is mixed with a combination of surfactants in a Babcock bottle.
 The surfactants displace the fat globule membrane which surrounds the
emulsion droplets in milk and causes them to coalesce and separate.
 The sample is centrifuged which allows the fat to move into the graduated neck
of the bottle, where its concentration can then be determined.

Instrumental methods
 The are a wide variety of different instrumental methods available for
determining the total lipid content of food materials.
 These can be divided into three different categories according to their
physicochemical principles:
o measurement of bulk physical properties,
 o measurement of adsorption of radiation,
o measurement of scattering of radiation.
 Each instrumental methods has its own advantages and disadvantages, and range
of foods to which it can be applied.