UNIT THREE

3. Choosing an Analytical Technique

 different analytical techniques available to
determine a particular property of a food

 It is therefore necessary to select the most

appropriate technique for the specific application

 The analytical technique selected depends on:

 property to be measured,
 type of food to be analyzed, and
 reason for carrying out the analysis.
 Analytical procedure may already be routinely
used in the laboratory or company where you
are working
 Alternatively, it may be possible to contact an
expert who could recommend a certain
technique, e.g., a University Professor or a
Consultant
 Often it is necessary to consult scientific and
technical publications
 There are a number of different sources where
information about the techniques used to
analyze foods can be obtained:
 3.1 Books

 Food analysis books may provide a general

overview of the various analytical procedures
used to analyze food properties or they may
deal with specific food components or
physicochemical characteristics.
 Consulting a general textbook on food analysis
is usually the best place to begin to obtain an
overview of the types of analytical procedures
available for analyzing foods and to critically
determine their relative advantages and
disadvantages.
 Food Analysis, 2nd Edition. S.S. Nielsen, Aspen
Publishers
 Food Analysis: Theory and Practice. Y. Pomeranz
& C.E. Meloan, Chapman and Hall
 Food Analysis: Principles and Techniques. D.W.
Gruenwedel and J.R. Whitaker, Marcel Dekker
 Analytical Chemistry of Foods. C.S. James, Blackie
Academic and Professional
3.2. Tabulated Official Methods of Analysis
A number of scientific organizations have been
setup to establish certain techniques as official
methods, e.g. Association of the Official Analytical
Chemists (AOAC) and American Oil Chemists
Society (AOCS).
Normally, a particular laboratory develops a new
analytical procedure and proposes it as a new
official method to one of the organizations.
• The method is then tested by a number of independent
laboratories using the same analytical procedure and
type of equipment stipulated in the original proposal.
• The results of these tests are collated and compared
with expected values to ensure that the method gives
reproducible and accurate results.
• After rigorous testing the procedure may be accepted,
modified or rejected as an official method.
• Organizations publish volumes that contain the
officially recognized test methods for a variety
of different food components and foodstuffs.
• It is possible to consult one of these official
publications and ascertain whether a suitable
analytical procedure already exists or can be
modified for your particular application.
 3.3. Journals
Analytical methods developed by other scientists are
often reported in scientific journals, e.g., Journal of Food
Science, Journal of Agriculture and Food Chemistry,
Journal of the American Oil Chemists Society, Analytical
Chemistry.
Information about analytical methods in journals can
often be obtained by searching computer databases of
scientific publications available at libraries or on the
Internet (e.g., Web of Science, Medline).
 3.4. Equipment and Reagent Suppliers

• Many companies that manufacture equipment and

reagents used to analyze foods advertise their products
in scientific journals, trade journals, trade directories.
• These companies will send you literature that describes
the principles and specifications of the equipment or
test procedures that they are selling, which can be used
to determine the advantages and limitations of each
technique.
 3.5. Internet
• The Internet is an excellent source of information on the
various analytical procedures available for analyzing food
properties.

• University lecturers, book suppliers, scientific organizations,

scientific journals, computer databases, and equipment and
reagent suppliers post information on the web about food
analysis techniques.

• This information can be accessed using appropriately selected

keywords in an Internet search engine.
 3.6. Developing a New Technique

• In some cases there may be no suitable techniques

available and so it is necessary to develop a new one.
• This must be done with great care so as to ensure that
the technique gives accurate and reliable
measurements.
• Confidence in the accuracy of the technique can be
obtained by analyzing samples of known properties or
by comparing the results of the new technique with
those of well-established or official methods.
 1.4. Selecting an Appropriate Technique

Some of the criteria that are important in selecting a technique

are listed below:

1. Precision: A measure of the ability to reproduce an answer

between determinations performed by the same scientist (or
group of scientists) using the same equipment and experimental
approach.

2. Reproducibility: A measure of the ability to reproduce an

answer by scientists using the same experimental approach but
in different laboratories using different equipment.
3. Accuracy: A measure of how close one can actually measure
the true value of the parameter being measured, e.g., fat
content, or sodium concentration.

4. Simplicity of operation: A measure of the ease with which

relatively unskilled workers may carry out the analysis.

5. Cost: The total cost of the analysis, including the reagents,

instrumentation and salary of personnel required to carry it out.

6. Speed: The time needed to complete the analysis of a single

sample or the number of samples that can be analyzed in a given
time.
7. Sensitivity: A measure of the lowest concentration of a
component that can be detected by a given procedure.
8. Specificity: A measure of the ability to detect and quantify
specific components within a food material, even in the
presence of other similar components, e.g., fructose in the
presence of sucrose or glucose.
9. Safety: Many reagents and procedures used in food
analysis are potentially hazardous e.g. strong acids or
bases, toxic chemicals or flammable materials.
10. Destructive/Nondestructive: In some analytical methods the
sample is destroyed during the analysis, whereas in others it remains
intact (undamaged).
11. On-line/Off-line: Some analytical methods can be used to measure
the properties of a food during processing, whereas others can only
be used after the sample has been taken from the production line.

12. Official Approval: Various international bodies have given official

approval to methods that have been comprehensively studied by
independent analysts and shown to be acceptable to the various
organizations involved, e.g., ISO, Association of the Official Analytical
Chemists (AOAC) , American Oil Chemists Society (AOCS).
13. Nature of Food Matrix: The composition, structure and
physical properties of the matrix material surrounding
the analyte often influences the type of method that can
be used to carry out an analysis, e.g., whether the matrix
is solid or liquid, transparent or opaque, polar or non-
polar.
• If there are a number of alternative methods available for
measuring a certain property of a food, the choice of a
particular method will depend on which of the above
criteria is most important.
• For example, accuracy and use of an official
method may be the most important criteria in a
government laboratory which checks the validity of
compositional or nutritional claims on food
products, whereas speed and the ability to
make nondestructive measurements may be more
important for routine quality control in a factory
where a large number of samples have to be
analyzed rapidly.
 UNIT FOUR

INSTRUMENTATION IN FOOD
ANALYSIS
 Why do We Need Instrumentation in Food
Analysis?

• Due to complex nature of food matrix, it often becomes

impossible to accurately analyze one component in the presence
of others using the classical method of analysis.

• This may lead to inaccurate and unreliable results and

sometimes erroneous and false results because of lack of
specificity and sensitivity of classical method.

• Therefore, in order to achieve the reliability of results, today the

instrumental analytical techniques have become mandatory in
development, quality control and safety exports of food
products and meeting the regulatory norms of food products.
 Classical methods
I. Titrimetric analysis
II. Gravimetric procedure
III. Solvent extraction
IV. Refractometry

Advantage
• Low cost, simplicity of operation no need to calibrate and
requirement of calibration of modern analytical
instruments ,
• Disadvantage
• Lack of sensitivity
• Lack of specificity of certain constituents
 Titrimetric technique
• Volume of a solution of known concentration (standard)
required to completely react with a solution (food) of
unknown concentration
• Stoichiometric point
– estimated by change in colour of indicator chemical
• Acid-base titration’s
• Redox titration’s
• Precipitation titration’s
• Solution of unknown concentration prepared from food to be
analyzed
• Titrand = solution in titration flask
• Titrant = solution added from burette
• Acid-base titration’s = Vitamin C in orange juice, citric acid,
tartaric acid
 Acid-base titration's
• Measure of Titratable Acidity (TA) of liquid foods
by using standard sodium hydroxide in the
presence of indicators ; phenolphthalein (dye).
– endpoint (@ certain pH )
• The actual point of colour change known as the
end point may not represent the stoichiometric
point (titration error)
• The object of titration is to determine the
amount of acid which is exactly equivalent
chemically to the amount of base present.
• A solution of known concentration acid or
base is added from a burette to a solution
of unknown concentration of base or acid.
• Stoichiometric; Calculation of the quantities
of reactants and products in a chemical
reaction.
• The quantitative relationship between
reactants and products in a chemical
reaction.
Titratable acidity apparatus
 Redox titration
• Two half reactions one reduction, one oxidation
• Example: determination of sulphur dioxide in
foods
– sulphur dioxide is oxidised and iodine reduced;
– SO2 + H2O  SO3 + 2H+ + 2e-
SO3 + H2O  H2SO4
I2 + 2e-  2I-
Summary: SO2 + I2 + 2H2O  2I- + 2H+ + H2SO4
– end point starch indicator is purple colour
• Reduction = broadly addition of electrons or
more narrowly removal of oxygen atoms or
addition of hydrogen atoms.
• Oxidation = broad sense removal of electrons or
more narrowly addition of oxygen atoms or
removal of hydrogen atoms.
 Gravimetric procedures
• Weight of food constituent is measured after appropriate
treatment.
– moisture
– ash
– total dietary fiber
• Moisture by oven drying - dry sample weighed
• Ash by using muffle furnace to convert all organic material to
carbon dioxide and water leaving minerals such as sodium
chloride and potassium chloride which is weighed
• Total dietary fibre - series of enzymatic treatments to remove
all digestible components, indigestible residue weighed
• AOAC method for “total dietary fibre” determination
Crude fibre analysis
• Bread crushed through 1mm sieve, Dried and
delipidated with ether, Boiled in sulphuric acid.
• Filtered then boiled in alkali, Residue filtered and
washed.
• Dried and weighed, ashed and re-weighed to give
“crude fibre” corrected for in-organics.
 Solvent Extraction Methods
• Constituents of food extracted by non-polar
solvents
– used for fat content determination
• solvent separated
• solvent removed
• residue weighed

Soxhlet method boils with solvent such as

petroleum ether, hexane etc…
 Methods of Instrumental Techniques
Sample preparation requirement for instrumental analytical
techniques

Few instrumental techniques can measure samples directly

without pretreatment.
• The samples must be treated to make them compatible
with the instrumental technique selected.
• Transformation of the sample into a form that can be
measured using the selected technique is termed sample
preparation.
• Sample selection and preparation usually represent the
largest investment of time in the implementation of an
analytical method.
• It is important to realize that the majority of
instrumental techniques require the sample to be in
a liquid phase.
• For solid samples the techniques used to transfer
the analyte into the liquid phase includes :
 dissolution
 extraction
 decomposition (sometimes termed digestion) or some
combination of these.
For Liquid or gas phase sample preparation includes:
• Filtration: to remove particulate matter
• Extraction: to remove an analyte from a complex matrix
• Chromatographic separation: to remove interferences from the
analyte are all commonly used for liquid or gaseous samples
Some considerations during sample preparation:
 The integrity of the analyte must be kept by considering
the possibility of contamination, loss, or chemical and
physical changes to the analyte.
 The expected concentration of the analyte in the sample
with the smallest statistically detectable concentration of
the instrument used for the measurement is called the
limit of detection (LOD).
 Care must be exercised to prevent dilution of the analyte
to a concentration below that of the LOD of the method.
 It may be necessary to enrich the sample concentration
to make an accurate measurement, a step termed
preconcentration.
 Classification of Instrumental Techniques

• The classification of instrumental techniques will be made

by considering the type of interaction of the instrument
with a chemical or physical property of the analyte.

The instrumental techniques are generally classified into:

• Chromatographic Techniques ,
HPLC
GC
AAS
• Spectroscopic Techniques
• Thermal methods of analysis
 Introduction
• Chromatography is the term used to describe a
separation technique in which a mobile phase carrying a
mixture is caused to move in contact with a selectively
absorbent stationary phase.

The two main chromatographic are GC and HPLC

• In  GC we have both mobile and stationary phase

• The comparison of retention times gives GC its

analytical usefulness.
• RETENTION TIME (tR )

 The time between sample injection and an analyte reaching a

detector at the end of the column.

• Different analyte = different retention time

• Rt A = Rt B on one column or one mobile phase

Unlikely Rt A = Rt B on second column or second mobile phase

Should be compared to a known standard

 Gas Chromatography Basics
• Gas Liquid Chromatography (GLC)

• Mobile phase does not interact with analyte

• Separation occurs by interaction of analyte differentially with

stationary phase and temperature.

• Chromatography is used to separate mixtures of substances into

their components. ... They all have a stationary phase (a solid, or
a liquid supported on a solid) and a mobile phase (a liquid or a
gas).
• The mobile phase flows through the stationary phase and carries
the components of the mixture with it.
 Gas Chromatography
• Gas Chromatography (GC) is one of the most versatile
analytical techniques used in the food industry.
• GC is used to separate volatile organic components in a
mixture.
• It enables fast separation and identification of components
in a complex mixture using appropriate detectors.
• Substances to be analyzed by GC must be volatile and must
be thermally stable below 3500C.
• Samples are rapidly injected by means of a hypodermic
micro syringe through a silicone rubber septum into the
column.
 Basic Components of Gas Chromatography

 Carrier Gas
 Sample Inlet
 Column
 Ovens

 Detectors
 Data acquisition system
Basic Components
 Gas Carrier
• Carrier gas is the means to move constituents of a
sample through the column and yet the choice of
possible gases is restricted.

• The choice of a practical carrier gas is simple: nitrogen or

helium.

• The gases nitrogen or helium come from cylinder or

bottled gas supply.

• The carrier gas has to be cleaned over molecular sieve

beds (to reduce moisture).
 Sample Inlet

• Gas samples can be injected into the column using gas-

tight syringes or using rotary gas switching valves that
offer enormous flexibility for GC instruments.
• Sample is loaded into a loop and then, with a change in
the valve position, is swept into the column under flow
of the gas source.
Injection
 Column
• General types of column in GC

Packed column (easily emptied and refilled when efficiency

is reduced)
Capillary column (capillary longer, more expensive and
more sensitive)

• Capillary columns are much more effective that packed

columns
 oven
• Most gas chromatographs are equipped
with ovens to keep the column at
temperatures from 40 to 350oC.

• Resistive wire coil that radiates into the

inner volume of the oven.
 Detector
• Detectors that are used in GC are
• FID, ECD
• The Flame Ionization Detector(FID) relies upon
the formation of gaseous ions from organic
molecules combusted in a hydrogen–air flame.
• Electron Capture Detector (ECD)is especially
sensitive to and selective for compounds
containing Cl2, O2, and other electronegative
element.
Working Principles of Gas Chromatography
• Once GC has separated a mixture, the components
can be identified using known retention times
• Retention time variation depending on:

- The nature of and the interactions between the

solute/analyte and the stationary and mobile phases.
- The flow rate of the carrier gas
- The temperature of the column
- The length and diameter of the column
 Interpretation of data
• Generally chromatographic data is presented as a
graph of detector response (y-axis) against retention
time (x-axis), which is called a chromatogram.

• The area under a peak is proportional to the amount

of analyte present in the chromatogram.
• By calculating the area of the peak using the
mathematical function of integration, the
concentration of an analyte in the original sample
can be determined.
 Applications Of Gas Chromatography
• Gas Chromatography can be applied for analysis and
determination of different compounds in food products such
as :
1) Cholesterol, Fatty acid profiling and Trans fat analysis;
2) Antioxidants and Preservatives like TBHQ, Benzoic acid,
Sorbic acid Acetic acid, etc;
3) Analysis of residual pesticides and environmental
contaminants;
4) Characterization of flavours and fragrances; and
5) The Gas Chromatographic profiling of the essential volatile
oils gives a reasonable ‘fingerprint’ which can be used to
characterize the identity of the particular oil.
 Limitation Of Gas Chromatography
• Limited to volatile samples.
• Not suitable for samples that degrade at
elevated temperatures (thermally labile).

• Requires MS detector for analyte structural

elucidation (characterization).

• Most Non-MS Detectors are destructive.

 LIQUID CHROMATOGRAPHY
• Liquid chromatography covers a variety of separation
techniques such as liquid solid (adsorption
chromatography), liquid-liquid (partition
chromatography), ion exchange, size exclusion etc…
High Performance Liquid Chromatography (HPLC)
• HPLC is used to separate, identify, and quantify polar
and non-volatile compounds when in mixture.
• HPLC utilizes a column that holds liquid stationary
phase, a pump that moves the mobile phase (s) through
the column, and a detector that shows the retention
times of the molecules.
 Characteristic Features of HPLC
• Fast, accurate and high power of resolution.

• Results are repeatable and reproducible.

• Facilitates determination of multiple components in a single run.

• Provides method of choice for thermally unstable and high molecular

weight compounds.

• Separated components can be easily collected from the mobile phase

for further characterization, as it is a non-destructive technique.

• Suitable for both aqueous and non-aqueous sample.

HPLC is an excellent technique for separation of chemical
compounds with high degree of specificity and selectivity
and is basically a highly improved form of column
chromatography.
• Instead of a solvent being allowed to drip through a column
under gravity, as in the case classical column
chromatography it is forced through under high pressures
of up to 400 atm, which makes it much faster.
• The technique is not limited by the volatility or stability of
the sample compound.
 Basic Components of the HPLC

Figure.1.Schematic diagram of a HPLC Instrument

1) Mobile phase reservoir, filtering
• The most common type of solvent reservoir is
a glass bottle.
• Most of the manufacturers supply these
bottles with special caps, Teflon tubing and
filters to connect to the pump inlet and to the
purge (clear) gas (helium) used to remove
dissolved air.
• Helium purging and storage of the solvent
under helium is not sufficient for degassing
aqueous solvents. It is useful to apply a
vacuum for 5-10 min. and then keep the
solvent under a helium atmosphere.
 2) Pump
The role of the pump is to force a liquid (called the
mobile phase) through the liquid chromatograph at a
specific flow rate, expressed in milliliters per min
(mL/min).
High pressure pumps are needed to force solvents
through packed stationary phase beds. Smaller bed
particles require higher pressures.
There are many advantages to using smaller particles,
but they may not be essential for all separations.
The most important advantages are: higher resolution,
faster analyses, and increased sample load capacity.
However, only the most demanding separations require
these advances in significant amounts.
 3 )Injector

 Sample introduction can be accomplished in various

ways.
 The simplest method is to use an injection valve.
 In more sophisticated LC systems, automatic sampling
devices are incorporated where the sample is introduced
with the help of auto-samplers and microprocessors
 The injector serves to introduce the liquid sample into
the flow stream of the mobile phase.
 Typical sample volumes are 5-to 20-microliters (μL).
 An auto-sampler is the automatic version for when the
user has many samples to analyze or when manual
injection is not practical.
4) Column

Key Point –Proper choice of column is critical for success in HPLC

Typical HPLC columns are 5, 10, 15 and 25 cm in length and are filled
with small diameter (3, 5 or 10 μm) particles. usually 4.6 mm;

Types of columns in HPLC:

Analytical [internal diameter (i.d.) 1.0 -4.6-mm; lengths 15 –250 mm]
Preparative (i.d. > 4.6 mm; lengths 50 –250 mm)
Capillary (i.d. 0.1 -1.0 mm; various lengths)
Nano (i.d. < 0.1 mm, or sometimes stated as < 100 μm)

Materials of construction for the tubing

Stainless steel (the most popular; gives high pressure capabilities)
Glass (mostly for biomolecules)
PEEK polymer (biocompatible and chemically inert to most solvents)
Column
5 ) Detector
 HPLC There are many detection principles used to detect
the compounds eluting from an HPLC column.

The most commonly used detector in LC is the ultraviolet

absorption detector

Other detectors in common use include

 Photo Diode Array UV detector (PAD),
 Refractive index (RI),
 Fluorescence (FLU),
 Electro capture (EC).
6 )Data system
 Since the detector signal is electronic, using
modern data collection techniques can aid the
signal analysis.
 The main goal in using electronic data systems
is to increase analysis accuracy and precision,
while reducing operator attention.
 There are several types of data systems, each
differing in terms of available features. In
routine analysis, where no automation (in
terms of data management or process control)
is needed, a pre-programmed computing
integrator may be sufficient.
 Application of HPLC in food quality control
• HPLC finds wide applications in food, both for
profiling and analysis of various components such as:
1) Amino acids profiling, peptides and proteins.
2) Lipids and alcohols.
3) Carbohydrates and carbohydrate profiling, sweeteners.
4) Fat soluble and water soluble vitamins, carotenoids.
5) Organic acids and organic bases.
6) Residues of toxin( aflatoxins in peanuts), Antimicrobial and veterinary
drugs, pesticides in fruits and vegetables, etc.
7) Pigments, colorants and phenolic compounds.
8) Bittering substances.
9) Additives, preservatives, antioxidants and stabilizers in processed food
products.
 Working principle of HPLC
• The sample to be analyzed is introduced, in small
volumes, into the stream of mobile phase.
• The solution moved through the column is slowed by
specific chemical or physical interactions with the
stationary phase present within the column.
• The velocity of the solution depends on the nature of the
sample and on the compositions of the stationary
(column) phase.
• The time at which a specific sample elutes (comes out of
the end of the column) is called the retention time; the
retention time under particular conditions is considered
an identifying characteristic of a given sample.
 Interpretation of Data Obtained from the
HPLC
• One of the more essential purposes of chromatographic
methods is to determine the quantity of components in a
sample solution.
• Quantitative analysis is based on the determination of the
magnitude of the detector signal for the component of
interest.
• The signal should change linearly with the quantity of
compounds, and the signal can be measured as the height
or area of a chromatographic peak.
• Most HPLC instruments purchased in the market include
integrators or computers that can perform acquisition
and management.
• Precision of quantitative analysis in HPLC is
affected by several factors, such as the accuracy of
injection, and the processes of separation and
detection.
• Qualitative analysis (identification) and quantitative
analysis (determination)can be performed using the
information contained in the chromatogram.
• Qualitative analysis: What are components A, B
and C ?
• Quantitative analysis: What is the concentration
of components A, B and C?
 Comparison of HPLC and GC
HPLC in some respect is more versatile than GC
since:
1) It is not limited to volatile and thermally stable
substances.
It can accommodate thermally unstable, nonvolatile
compounds and inorganic ions.
2) The choice of mobile and stationary phase is wider.
3) GC is better from the point of view of speed and
simplicity of equipment.
The analysis cost is also significantly cheaper as HPLC
requires highly pure solvents that are costly.
 Spectroscopy Technique
• Spectroscopy pertains to the dispersion of alight into
its component colors (i.e. energies) via prism or the
study of the interaction between radiation and matter as
a function of wavelength (λ).
• This technique utilizes interactions between
electromagnetic radiation and matter to provide
information about food properties, e.g., molecular
composition, structure, dynamics and interactions.
Spectrophotometer
 Instrument used to measure the absorbance of light
by compounds within a liquid.
• Interaction between electromagnetic radiation and atoms or
molecules in food.
• Measure radiation emitted or absorbed
– absorption based on Beer-Lambert Law “amount of
light absorbed by a solution is proportional to the
concentration and length of the solution”.
• Used to detect and measure different food constituents;
• Absorption - used in estimation of proteins, carbohydrates,
mineral elements, vitamins and pectin.
• Emission - less widely used in food analysis, estimation of some
minerals.

spectrophotometer.jpg
Sample holders ( Cells)
 SPECTROSCOPIC INSTRUMENTS
The most commonly used spectroscopic
instruments for food analysis are:
• Atomic-Absorption Spectroscopy (AAS)
• UV- Visible Spectroscopy
• Inductively Coupled Plasma (ICP)
• Nuclear Magnetic Resonance Spectroscopy
(NMR)
• Fourier Transform Infrared Spectroscopy (FTIR
 UV-Visible Spectroscopy
• UV-visible spectroscopy is an important tool for the
chemical profiling of food products after extraction of
the components in the suitable solvent.
Atomic Absorption Spectroscopy (AAS)
• Atomic absorption spectrometry is a very popular
method for assessing the concentration of metals and
minerals that may be present in the food products.
• This technique allows measuring all the elements of
periodic table.
• It encompasses a wide variety of techniques and
provides rapid, sensitive and selective determination of
elemental composition.
• What is AAS?

Atomic absorption spectroscopy is a

quantitative method of analysis that is
applicable to many metals and a few
nonmetals.
 What is AAS ?

• The technique was introduced in 1955 by Walsh

in Australia (A.Walsh, Spectrochim. Acta, 1955,
7, 108)

Alan Walsh 1916-1998

• An atomic absorption spectrophotometer
consists of a light source, a sample
compartment and a detector.

Sample
Compartment

Light Source Detector

Atomic absorption spectrometer has five basic
components, which are:

1) A light source (cathode lamp)

2) A sample cell (absorption cell)

3) Monochromator

4) Detector

5) Output unit
• In this technique, the elements in the sample are
brought into their ionized form in solution by using
the wet digestion, dry ashing or suitable microwave
assisted digestion system and then aspirated through
a nebulizer into the high temperature flame where
the sample gets converted into gaseous atoms.
• The source of light is usually a hollow cathode lamp,
which is composed of the element being measured.
Each element requires a different lamp.
• The hollow cathode lamp produces emission lines
specific for the element used to construct the
cathode.