Sampling Techniques of

UNIT 11 SAMPLING TECHNIQUES OF Food Products

FOOD PRODUCTS
Structure
11.0 Objectives
11.1 Introduction
11.2 Sample Collection
11.2.1 Homogenous Versus Heterogeneous Populations
11.2.2 Manual Versus Continuous Sampling
11.2.3 Importance of Sample Collection
11.2.4 Errors in Analytical Results due to Improper Sampling
11.2.5 Risks Associated with Sampling
11.3 Sampling Standards
11.4 The Sampling Plan
11.4.1 Understanding a Sample Plan
11.4.2 Statistical Approaches
11.5 Sampling Techniques/Methods
11.5.1 Probability Sampling
11.5.2 Non-probability Sampling
11.5.3 Types of Sampling
11.5.4 Operating Characteristic (OC) Curves
11.5.5 Requirements of Good Sampling Methods
11.5.6 Cost of Sampling
11.5.7 Problems in Sampling
11.6 Three Class Sampling Plan
11.7 Preparation of Sampling Plans
11.7.1 Model Sampling Plan
11.8 Sub Sampling for Analysis and Taking the Test Portion
11.8.1 Composite Lab Sample Preparation
11.8.2 Opinions of Experts
11.9 Sample Preparation for Analysis
11.9.1 Precautions to be Followed while Preparing a Sample for Analysis
11.10 Difficulties in Sampling
11.10.1 Example for Effect of Sampling on Analytical Result
11.11 Sample Accountability
11.11.1 Documentation
11.11.2 Chain of Custody Form
11.11.3 Sample Receipt and Handling
11.11.4 Monitoring of Samples
11.12 Retention of Samples and Records
11.12.1 Identify the Properties of Retained Samples
11.12.2 Retention Period
11.13 Case Study
11.14 Let Us Sum Up
11.15 Key Words
11.16 Answers to Check Your Progress Exercises
11.17 Some Useful Books

11.0 OBJECTIVES
After reading this unit, we shall be able to:
• state the importance of sampling in analysis of food products;
• enlist standards and guides on sampling;
• prepare sampling plans;
• describe different sampling techniques; and
• devise ways to draw a representative sample from lots.
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Food Analysis
11.1 INTRODUCTION
To control food quality and acceptance within satisfactory limits, it is
important to monitor the vital characteristics of raw materials, ingredients, and
processed foods. This could be done by evaluating all foods or ingredients
from a particular lot, which is feasible if the analytical technique is rapid and
non-destructive. However, it is usually more practical to select a portion of the
total product volume and assume the quality of the selected portion is typical
of the whole lot.
Obtaining a portion, or sample, that is representative of the whole is referred to
as sampling, and the total quantity from which a sample is obtained is called
the population. Adequate sampling technique helps to ensure that sample
quality measurements are an accurate and precise estimate of the quantity of
the population. By sampling only a fraction of the population, a quality
estimate can be obtained more quickly and with less expense and personnel
time than if the total population were measured. The sample is only an estimate
of the value of the population, but with proper sampling technique, it can be a
very accurate estimate.

11.2 SAMPLE COLLECTION

It is important to clearly define the population that is to be sampled. The
population may vary in size from a production lot, a day's production, to the
contents of a warehouse. Extrapolating information obtained from a sample of
a production lot to the population of the lot can be done accurately, but
conclusions cannot be drawn from data describing larger populations, such as
the whole warehouse.
Populations may be finite, such as the size of a lot, or infinite, such as in the
number of temperature observations made of a lot over time. For finite pop-
ulations, sampling provides an estimate of lot quality. In contrast, sampling
from infinite populations provides information about a process. Fig.11.1
represents the sampling for physical and chemical characteristics of food.
Regardless of the population type, that is, finite or infinite, the data obtained
from sampling are compared to a range of acceptable values to ensure the
population sampled is within specifications.
11.2.1 Homogeneous Versus Heterogeneous Populations
The ideal population would be uniform throughout and identical at all
locations. Such a population would be homogeneous. Sampling from such a
population is simple, as a sample can be taken from any location and the
analytical data obtained will be representative of the whole. However, this
occurs rarely, as even in an apparently uniform product, such as sugar syrup,
suspended particles and sediments in a few places may render the population
heterogeneous. In fact, most populations that are sampled are heterogeneous.
Therefore, the location within a population where a sample is taken will affect
the subsequent data obtained. However, sampling plans and sample preparation
can make the sample representative of the population or take heterogeneity into
account in some other way.
11.2.2 Manual Versus Continuous Sampling
To obtain a manual sample the person taking the sample must attempt to take a
"random sample" to avoid human bias in the sampling method. Thus, the

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sample must be taken from a number of locations within the population to Sampling Techniques of
ensure it is representative of the whole population. For liquids in small Food Products
containers, this can be done by shaking prior to sampling. When sampling
from a large volume of liquid, such as that stored in silos, aeration ensures a
homogeneous unit. Liquids may be sampled by pipetting, pumping, or dipping.
However, when sampling grain from a rail car, mixing is impossible and
samples are obtained by probing from several points at random within the rail
car. Such manual sampling of granular or powdered material is usually
achieved with triers or probes that are inserted into the population at several
locations. Errors may occur in sampling, as rounded particles may flow into
the sampling compartments more easily than angular ones. Similarly,
hygroscopic materials flow more readily into the sampling devices than does
non hygroscopic material. Horizontal core samples have been found to contain
a larger proportion of smaller-sized particles than vertical ones. Continuous
sampling is performed mechanically.
Qualitative Characteristics
(e.g. commodity defects )

Inspection of isolated lots. Inspection of a continuous series of lots.

E.g., inspection of the aspects of a piece of fruit, E.g., inspection of the aspects of a piece of fruit,
or of a can in isolated lots. or of a can in continuous lots.

To be sampled by attribute sampling plan for To be sampled by attribute sampling plans for
isolated lots. continuous lots.

Quantitative characteristics
(e.g., compositional characteristics)

Inspection of isolated lots Inspection of continuous series of lots

Bulk Item Bulk Item

e.g. fat content of e.g. sodium e.g.: fat content e.g. sodium
milk in a tank content of a of milk in a tank. content of a
dietary cheese. dietary cheese.

To be sample by Sampling by To be sampled To be sampled

variable sampling attributes. by variable by attribute
plans for sampling plans sampling plans
isolated lots for continuous for continuous
series of lots. series of lots.

Fig. 11.1: Sampling Flowchart for Chemical and Physical Characteristics

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Food Analysis 11.2.3 Importance of Sample Collection
The reliability of analytical data thus obtained depends on several factors,
sampling being the major factor. Current analytical methods require only few
grams of food sample to analyze. Thus, it is necessary that a sample be as
representative of the population as possible.
There are three basic activities involved in analysis of food products:
• Collection of representative sample.
• Sample preparation.
• Analysis using appropriate methods and instruments.
These activities, although independent in nature, yet can have decisive
influence on each other. Furthermore, each of these activities have their own
potential sources of variations that contribute to the uncertainty level
associated with any analytical result. Thus, care must be taken to identify the
sources of variation and minimize or avoid them while accomplishing any
activity. On the part of the laboratories, it is therefore necessary to develop a
plan for the proper performance of each activity, and then establish quality
standards and written procedures in compliance with the standards. Many
times, the activity of sampling falls outside the purview of a laboratory’s
mandate or control. This is especially true in commercial testing laboratories
where the “first contact” is the arrival of samples. To improve the overall
quality of the analytical process, a laboratory must do all it can to receive
appropriate, applicable, defensible samples. The development of appropriate
plans will depend upon an understanding of the problems involved in each
activity, and then the application of reasonable judgements in seeking
solutions.
It should be noted that sampling terminology and procedures used may vary
between companies and between specific applications. However, the principles
described in this Unit are intended to provide a basis for understanding,
developing, and evaluating sampling plans and sample handling procedures for
specific applications encountered.
A sample should represent a population as adequately as possible. To ensure
proper sampling, the analysts need to be consulted time to time concerning
proper sample size, suitable containers for sampling or the use of appropriate
preservatives to prevent any spoilage or transformation in a sample before
analysis. One common cause of lack of precision or lab-to-lab variation in
analytical results for a particular population can be traced back to erroneous
sampling. For example, in case of grapes, a laboratory sample size of meager
3 kg berries represents the whole population of > 10000 kg in 1 hectare
vineyard area. Thus, if the sample collected is not representative, then there
will be sample-to-sample variation in results. When significant difference in
results occurs among laboratories which have supposedly analyzed the same
sample, a serious conflict may arise questioning the competence and credibility
of the laboratories. Many of these situations can be avoided if samples are
collected according to a rational plan that gives some assurance that the sample
delivered to the laboratory represents the composition of the parent lot.
There are at least two ways to measure a given lot of goods: one, that we often
assume to be the “proper” way, is to find its “true value”, by which we mean
its average value. The other way, often discovered accidentally as a result of
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“poor” sampling, is to measure its variability. So called proper sampling of Sampling Techniques of
drug dosage forms, for example, may involve compositing 20 tablets, by which Food Products
the majority of the tablets could be used to dilute and conceal the fact that
several of them are severely sub- or super- potent. Similarly, two lots of grain
may have been purposely, but ineffectively, mixed in an attempt to reduce the
average level of a contaminant. Sampling that led to the laboratory finding
inconsistent results would reveal the attempt to dilute an illegal product.

11.2.4 Errors in Analytical Results due to Improper Sampling

Few studies have been conducted on the distribution of error among the three
activities: sampling, sample preparation, and analysis. In one such study,
which involved analysis of 20 nanogram/gram concentration of aflatoxin in a
lot of peanuts, the error contributed by the sampling step was as high as 67%
of the total variance, in comparison to 20% and 13% errors contributed by the
analyst and the analytical procedure, respectively. In a field study conducted
at the National Research Centre for Grapes, Pune, the pesticide residues in
grape samples analyzed in 15 individual grape bunches collected out of 1 acre
area showed above 50% sampling-induced variations. The results of such
experimentations are not unusual and it illustrates the proportion of error that
can be attributed to sampling. For peanuts, the distribution of aflatoxin can
vary widely, with a few peanuts accounting for most of the contamination.
Similarly, in case of the field sampling of grapes, the pesticide residues might
have deposited in variable concentrations in different grape bunches and thus
when they were analyzed separately, showed variable results. The important
point in these examples is to show that sampling error can play a very
significant part in the overall error in the analytical system.

11.2.5 Risks Associated with Sampling

There are two types of risks associated with sampling. Both should be
considered when developing a sampling plan. The consumer risk describes the
probability of accepting a poor quality population. This should happen rarely
(<5% of the lots) but the actual acceptable probability of a consumer risk
depends on the consequences associated with accepting an unacceptable lot.
These may vary from major health hazards and subsequent fatalities to a lot
being of slightly lower quality than standard lots. Obviously, the former
demands a low or no probability of occurring whereas the latter would be
allowed to occur more frequently. The second risk i.e., vendor risk is the
probability of rejecting an acceptable product. As with consumer risk, the
consequences of an error determine the acceptable probability of the risk. An
acceptable probability of vendor risk is usually 5-10%.

11.3 SAMPLING STANDARDS

Data obtained from an analytical technique are the result of a stepwise
procedure from sampling, to sample preparation, laboratory analysis, data
processing, and data interpretation. There is a potential for error at each step
and the uncertainty, or reliability, of the final result depends on the cumulative
errors at each stage. Variance is an estimate of the uncertainty. The total
variance of the whole testing procedure is equal to the sum of the variances
associated with each step of the sampling procedure and represents the
precision of the process. Precision is a measure of the reproducibility of the
data. In contrast, accuracy is a measure of how close the data are to the true
value. The most efficient way to improve accuracy is to improve the reliability
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Food Analysis of the step with the greatest variance. Frequently, this is the initial sampling
step. The reliability of sampling is dependent more on the sample size than on
the population size. The larger the sample size, the more reliable the sampling.
However, sample size is limited by time, cost, sampling methods, and the
logistics of sample handling, analysis, and data processing.
It should be noted that sampling terminology and procedures used may vary
between companies and between specific applications. Several standards and
recommendations provide the ways and means to sample a particular lot.
ISO specifies various standards and guidelines for drawing samples and data
interpretation for samples. Few such standards related to food are mentioned in
Table 11.1.
Table 11.1: Standards and Guides of Sampling

ID of Standard/Guide Title of Standard/Guide

ISO 2854 : 1976 Statistical Interpretation of data- techniques of estimation and

tests relating to means and variances.

ISO 2859-0 : 1995 Sampling procedures for inspection by attributes-Part 0 ;

Introduction to the ISO 2859 Attribute sampling system

ISO 2859-1 : 1999 / Sampling procedures for inspection by attributes-Part 1;

Sampling plans indexed by Acceptable Quality Level (AQL) for
IS 2500 (Part I) : 1989 lot-by-lot inspection.

IS 2500 (Part II) : 1965 Sampling inspection procedures.

ISO 2859-2 : 1985 Sampling procedures for inspection by attributes- Part 2;

Sampling plans indexed by Limiting Quality (LQ) for isolated lot
inspection.

ISO 3494 : 1976 Statistical interpretation of data – Power of tests relating to mans
and variances.

ISO 3951 : 1989 Sampling procedures and charts for inspection by variables for
per cent non-conforming.

ISO 5725-1 : 1994 Application of statistics- Accuracy (trueness and precision) of

measurement methods and results- Part 1; General principles and
definitions.

ISO 7002 : 1986 Agricultural food products-Layout for a standard method of

sampling a lot.

ISO 8423 : 1991 Sequential sampling plans for inspection by variables for per cent
non-conforming (known standard deviation).

ISO 8422 : 1991 Sequential sampling plans for inspection by attributes.

ISO/ TR 8550 : 1994 Guide for the selection of an acceptance sampling system,
scheme or plan for inspection of discrete items in lots.

ISO 10725 : 2000 Acceptance sampling plans and procedures for the inspection of
bulk material.

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 Sampling Techniques of
ISO/ FDIS 11648/1 Statistical aspects of sampling from bulk materials – Part 1; Food Products
General principles.

ISO/ DIS 14560 Acceptance sampling procedures by attributes – specified quality

levels in non-conforming items per million.

Other IS specifications Specifications published by BIS pertaining to individual products

contain the details of lot size and corresponding number and
quantity of samples to be drawn and tested for conformance.

Since all standards are subject to revision, parties to agreements based up on

these guidelines should ensure that the most recent editions of the standards
are always applied.
An example for flour sampling as per AOAC method is presented here. The
AOAC Method 925.08 (6) describes the method for sampling flour from
sacks. The number of sacks to be sampled is determined by the square root of
the number of sacks in the lot. The sacks to be sampled are chosen according
to their exposure. The samples that are more frequently exposed are sampled
more often than samples that are exposed less. Sampling is done by drawing a
core from a corner at the top of the sack diagonally to the center. The sampling
instrument is a cylindrical, polished trier with a pointed end. It is 13 mm in
diameter with a slit at least one third of the circumference of the trier. A
second sample is taken from the opposite corner in a similar manner. The
cores are stored for analysis in a clean, dry, airtight container that has been
opened near the lot to be sampled. The container should be sealed immediately
after the sample is added. A separate container is used for each sack.
Additional details regarding the container and the procedure also are described
below.
Title 21 CFR specifies the sampling procedures required to ensure that specific
foods conform to the standard of identity. In the case of canned fruits, 21 CFR
145.3 defines a sample unit as "container, a portion of the contents of the
container, or a composite mixture of product from small containers that is
sufficient for the testing of a single unit". Furthermore, a sampling plan is
specified for containers of specific net weights. The container size is
determined by the size of the lot. A specific number of containers must be
filled for sampling of each lot size. The lot is rejected if the number of
defective units exceeds the acceptable limit. For example, out of a lot
containing 48,001 to 84,000 units, each weighing 1 kg or less, 48 samples
should be selected. If six or more of these units fail to conform to the attribute
of interest the lot will be rejected. Based on statistical confidence intervals,
this sampling plan will reject 95% of the defective lots examined, that is, 5%
consumer risk.
Table 11.2: Minimum Number of Primary Samples to be taken from a Lot
(Ref: CODEX doc. CAC/GL 33)

Name of Commodity Minimum Number of Primary Samples to be taken

from a Lot

1. Meat and Poultry

a) Non-suspect lot 1

b) Suspect lot Determined according to Table 11.3

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Food Analysis 2. Other products

a) Products packaged in bulk, 1

which can be assumed to be
(A lot may be mixed by grading or manufacturing
well mixed or homogeneous.
process)

b) Products packaged in bulk, For products comprised of large units, the minimum
which may not be well mixed number of primary samples should comply with the
or homogeneous. minimum number of units required for the laboratory
sample.

Either Weight of lot, kg

<50 3

50-500 5

>500 10

or, Number of cans, cartons,

containers in the lot

1-25 1

26-100 5

> 100 10

Table 11.3: Number of randomly selected primary samples required for a given
probability of finding at least one non-compliant sample in a lot of meat or
poultry for a given incidence of non-compliant residue in the lot
(Ref: CODEX doc. CAC/GL 33)

Incidence of Non- Minimum number of samples (no ) required to detect a

compliant non- compliant residue with a probability of
Residues in the Lot
% 90% 95% 99%
90 1 - 2
80 - 2 3
70 2 3 4
60 3 4 5
50 4 5 7
40 5 6 9
35 6 7 11
30 7 9 13
25 9 11 17
20 11 14 21
15 15 19 29
10 22 29 44
5 45 59 90
1 231 299 459
0.5 460 598 919
0.1 2302 2995 4603

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Notes: Sampling Techniques of
Food Products
a) The table assumes random sampling.
b) Where the number of primary samples indicated in Table 11.2 is more than about
10% of units in the total lot, the number of primary samples taken may be fewer
and should be calculated as follows:
no
n=
1 + (no-1 ) / N
Where, n = minimum number of primary samples to be taken
no = number of primary samples given in Table 11.2
N = number of units, capable of yielding a primary sample in the lot.
c) Where a single primary sample is taken, the probability of detecting a non-
compliance is similar to the incidence of non-compliant residues.
d) For exact or alternative probabilities, or for a different incidence of non-
compliance, the number of samples to be taken may be calculated from:
1 – p = ( 1 – i )n
Where, p is the probability and i is the incidence of non-compliant residue in the lot
(both expressed as functions, not percentages) and n is the number of samples.
Table 11.4: Sampling Procedure and Minimum Amount (composite/bulk) to be Sampled
from lots

Crop Type Sampling Procedure Example Minimum

Quantity
Root, tuber Take samples from all areas Beet (red, sugar, fodder), 5 kg
and bulb of the crop. Remove as onions, parsnips, potatoes, (and not less
vegetables much adhering soil as sweet potatoes, turnips than 5 items)
possible from samples but
do not wash.
(Note: In some cases, where Carrots, radish, spring 2 kg
leaf parts are used as stock onions.
feed, they may need to be
sampled separately).
Take the sample from all Brassica (cabbage, 5 kg
areas of the crop. Sample cauliflower, broccoli, (and not less
parts of the crop exposed to kohlrabi, curly kale). than 5 items)
the spray and also those
apparently protected by
foliage.
Leafy, stem, Remove as much soil as Asparagus, brussel 2 kg
fruiting and possible from crops such as sprouts, celery, chicory,
legume celery, but do not wash. lettuce, spinach, turnip
vegetables tops
Cucumber, melon, 5 kg
squashes, eggplant (and not less
than 5 items)
Peppers, tomatoes, 2 kg
gherkins
Beans, peas, etc. (with 2 kg
pods)
All tree and Select fruit from all parts of Apples, citrus, peaches, 5 kg
bush fruit, the tree/bush, high and low, pears
including and from both sides of the
vines, small row, and select fruits
fruits and according to abundance
berries whether in each segment or
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Food Analysis the whole tree/bush. More
fruit should therefore be
selected from the more
densely laden parts of the
crop.
Sample parts of the crop Cherries, nuts, olives, 2 kg
exposed to the spray and plums.
also those apparently
protected by foliage.
Take large and small fruits, Bush fruit (all types), 2 kg
perfect or slightly grapes, strawberries
blemished, but not so small
or blemished that they
would not normally be
saleable.
Cereal Cut not less than ten small Maize (grain and cobs) 2 kg
Grains areas (approximately 0.1 m2)
chosen randomly from all
areas of the crop. Cut stalks
about 10 cm above the
ground. Remove grain from
the straw.
Oil Seeds Collect the heads when they Sesame, canola, 1 kg of seeds
have reached the stage of soybeans, sunflower
maturity at which they are
normally harvested and if
convenient thresh to remove
the seeds.

Table 11.5: Description of Primary samples and min. size of laboratory sample
(Ref: CODEX doc. CAC/GL 33)
S. No Commodity Examples Nature of Primary Minimum Size of
Classification Samples to be taken Lab Sample
1. Primary food commodities of animal origin
1.1 Large mammals Cattle Whole or part of 0.5 kg
Whole or half carcass Sheep diaphragm,
supplemented by
Usually 10 kg or more Pigs cervical muscles, if
Necessary

1.2 Small mammals Rabbits Whole carcass or hind 0.5 kg, after
Whole carcass quarters removal of skin
and bone

1.3 Mammal meat parts, Quarters Whole unit(s),or a 0.5 kg, after
loose Chops portion of a large unit removal of bone
fresh/chilled/frozen
Steaks
Packaged or other wise
Shoulders
1.4 Mammal meat parts, Quarters Either a frozen cross- 0.5 kg, after
bulk frozen Chops section of a container removal of bone
or the whole (or
portions of individual)
meat parts
2. Poultry fats
2.1 Birds, at slaughter Chickens Units abdominal fat 0.5 kg
whole or part-carcass Turkeys from at least three birds

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2.2 Bird meat parts Legs Either visible fat, 0.5 kg Sampling Techniques of
Breast – trimmed from units Food Products
muscle
Whole units or portions 2 kg
where fat is not
trimmable
2.3 Bird fat tissue in bulk Units taken with a 0.5 kg
sampling device from
at least three portions
2.4 Poultry eggs Whole eggs 12 whole eggs

2.5 Liquid, frozen or dried Whole units 0.5 kg

egg products
3. Processed foods of animal origin
3.1 Mammal or bird Ham Packaged units or a 0.5 kg or 2 kg if
comminuted, cooked, sausage / representative cross fat content
canned, dried, rendered Minced section from a < 5%
or otherwise processed beef / container or units
products including multi (including juices, if
ingredient products Chicken any) taken with a
paste sampling device
3.2 Liquid milks, milk Packaged units 0.5 L or
powders, ice creams etc. 0.5 kg
3.3 Cheese Units > Whole unit 0.5 kg
0.3 kg
Units < Whole unit 0.3 kg
0.3 kg
4. Herbs Fresh Whole units 0.5 kg
Dried Whole units 0.1 kg
5. Feed commodities of plant origin
5.1 Legume animal Whole units 1 kg (at least 10
feeds and other units)
forages and fodders
5.2 Straw, hay and Units taken with a 0.5 kg (at least
other dry products sampling device 10 units)
6. Processed foods of plant origin
6.1 Products of high Packages or units 0.1 kg
unit value taken with a
sampling device
6.2 Solid products of Hops Packages or units 0.2 kg
low bulk density Tea taken with a
sampling device
6.3 Other solid Bread Packages or units 0.5 kg
products Flour taken with a
Dried fruits sampling device
Vegetable
6.4 Liquid products Oils Packages or units 0.5 L or
Juices taken with a 0.5 kg
sampling device

"
Check Your Progress Exercise 1
Note: a) Use the space below for your answers.
b) Check your answers with those given at the end of the unit.
15
Food Analysis 1) What is the importance of sample collection in analysis of food products?
………………………………………………………………………………
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2) What are homogenous and heterogeneous populations?
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11.4 THE SAMPLING PLAN

11.4.1 Understanding a Sample Plan
Sampling is generally done for a specific purpose and the purpose may indeed
suggest or dictate the nature of any sampling plan. The International Union of
Pure and Applied Chemistry (IUPAC) defines a sampling plan as "a
predetermined procedure for the selection, withdrawal, preservation,
transportation, and preparation of the portions to be removed from a lot as
samples". A sampling plan should be a well-organized document that
establishes the required procedures for accomplishing the program's objectives.
It should address the issues of who, what, where, why, and how. The primary
aim of sampling is to obtain a sample, subject to constraints on size, that will
satisfy the sampling plan specifications. A sampling plan should be selected on
the basis of the sampling objective, the study population, the statistical unit, the
sample selection criteria, and the analysis procedures. Factors determing the
choice of a sampling plan are enlisted in Table 11.6. The two primary
objectives of sampling are often to estimate the average value of a
characteristic and determine if the average value meets the specifications
defined in the sampling plan. The presence of a well designed plan is important
because it provides a consistent model to guide people performing the
sampling activity, and it serves as a reminder of the important elements in this
part of the overall sample analysis program.
Table 11.6: Factors Affecting Choice of Sampling Plans

Factors to be considered Questions

Purpose of inspection a Is it to accept or reject the lot?
b Is it to measure the average quality of the lot?
c Is it to determine the variability of the product?
Nature of Product a Is it homogeneous or heterogeneous?
b What is the unit size?
c How consistently have past populations met specifications?
d What is the cost of the material being sampled?
Nature of the test method a Is the test critical or minor?
b Will someone become sick or die if the population fails to
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 pass the test? Sampling Techniques of
c Is the test destructive or non-destructive? Food Products

d How much does the test cost to complete?

Nature of the population a Is the lot large but uniform?
being investigated b Does the lot consist of smaller, easily identifiable sublots?
c What is the distribution of the units within the population?

11.4.2 Statistical Approaches

In many sampling programs, statistical approaches are not given the requisite
attention. Percentage sampling systems that specify a fixed percentage of a lot,
say 5 or 10%, do not provide the quality protection that is often assumed.
Statistical sampling theory furnishes the means to analyze the relationship
between a lot of goods and the samples that are drawn from it. It can be used
to estimate population measure, or “parameters,” such as variance and
correlation, from knowledge of corresponding samples quantities. The
importance of sampling is recognized in ISO 17025, (this standard will be
discussed in detail in Course 5, Block 3) which requires that test reports make
reference to the sampling procedure used by the laboratory or the submitting
body.

Check Your Progress Exercise 2

"
Note: a) Use the space below for your answers.
b) Check your answers with those given at the end of the unit.
1) Sampling plan is very important in sampling. Why?
………………………………………………………………………………
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2) Mention the purposes that are served by a sampling plan.
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11.5 SAMPLING TECHNIQUES/METHODS

There are several sampling methods/techniques in common use. These are
probability sampling, non-probability sampling, bulk sampling, and acceptance
sampling. These are described in brief below:

11.5.1 Probability Sampling

Probability sampling is used when a representative sample is desired, and uses
principles of statistical sampling and probability i.e. elimination of human bias.

17
Food Analysis It is a random selection approach that tends to give each unit an equal chance
of being selected.
Simple random sampling requires that the number of units in the population
be known and each unit is assigned a number. A specific quantity of random
numbers between one and total number of population units is selected. Sample
size is determined by lot size and potential impact of a consumer or vendor
error. Units corresponding to the random numbers are then analyzed as an
estimate of the population.
Systematic sampling is used when a complete list of sample units is not
available, but when samples are distributed evenly over time or space, such as
on a production line. The first sample is selected at random and then every nth
unit after that.
Stratified sampling involves dividing the population into overlapping
subgroups so that each subgroup is as homogenous as possible. Group means,
therefore, differ from each other as much as possible. Random samples are
then taken from each subgroup. The procedure provides a representative
sample because no part of the population is excluded and it is less expensive
than simple random sampling.
Cluster sampling entails dividing the population into clusters or subgroups so
that cluster’s characteristics are as identical as possible, that is, the means are
very similar to each other. Any heterogeneity occurs within each cluster.
Clusters should be small and having a similar number of units in each cluster.
The clusters are sampled randomly and may be either totally inspected or sub-
sampled for analysis. This sampling method is more efficient and less
expensive than simple random sampling, if populations can be divided into
homogenous groups.
Composite sampling is used to obtain samples from bagged products such as
flour, seeds, and larger items in bulk. Two or more samples are combined to
obtain one sample for analysis that reduces differences between samples. For
example, FDA composite 12 and at least six subsamples, respectively, for the
sample to be analyzed for compliance with nutrition labeling regulations.

11.5.2 Non-probability Sampling

Non-probability sampling is used when it is not possible to collect a
representative sample, or a representative sample is not desired. For example,
in case of adulteration such as rodent contamination, the objective of the
sampling plan may be to highlight the adulteration rather than collect a
representative sample of the population. The sample collector uses judgement
rather than statistical considerations in the selection of the sample. The unusual
or unexpected characteristics in a population could be selected to be identified.
This type of sampling is done in many ways, but in each case the probability of
including any specific portion of the population is not equal because the
investigator selects the samples without estimating sampling error.
Judgement sampling is solely at the discretion of the sampler and therefore is
highly dependent on the person taking the sample. This method is used when it
is the only practical way of obtaining the sample. This method may present a
better estimate of the population than random sampling if sampling is done by
an experienced individual and limitations of extrapolations from the results are
understood.
18
Convenience sampling is performed when ease of sampling is the key factor. Sampling Techniques of
The first pallet in a lot or the sample that is most accessible is selected. This Food Products
type of sampling will not be representative of the population, and therefore is
not recommended.
Restricted sampling may be unavoidable when the entire population is not
accessible. For example, if sample is to be taken from a loaded truck, but the
sample is not a representative of the entire population.
Quota sampling is the division of a lot into groups representing various
categories, and samples are then taken from each group. This method is less
expensive than random sampling but also is less reliable.

11.5.3 Types of Sampling

A) Bulk sampling
Bulk sampling involves the selection of a sample from a lot of material that
does not consist of discrete, identifiable or constant units. Sampling may be
performed in static or dynamic situations. Bulk sampling poses special
problems requiring certain decisions to be made: the number of increments to
be taken, the size of the increments, from where in the pile or stream they
should be drawn, the sampling device to be used, and how to reduce the
increments taken to a reasonable size of sample for delivery to the laboratory.
B) Acceptance sampling
Acceptance sampling differs from the previous types and involves the
application of a predetermined plan to decide whether a lot of goods meet
defined criteria for acceptance. The risks of accepting “bad” or rejecting
“good” lots are stated in conjunction with one or more parameters, for
example, quality indices of the plan. Statistical plans can be designed to
regulate the probabilities of rejecting good lots or accepting bad lots.
Refer Tables 11.2 and 11.3 in the annexure.
There are two broad categories of acceptance sampling: sampling by attributes
and sampling by variables.
Sampling by attributes
In sampling by attributes, the unit of product is classified as defective or non-
defective, or the number of defects in a unit of product is counted with respect
to a given requirement. Or, the sampling is performed to decide on the
acceptability of a population based on whether the sample possesses a certain
characteristic, for example, Clostridium botulinum contamination in canned
goods. An example of net weight determination may serve to explain the
differences between the two categories. In attribute sampling, each unit that
weighs 1 pound or more is accepted, and each unit that weighs less than 1
pound is rejected. If the number of rejects exceeds a predetermined number,
the lot is rejected. If the number of rejects is less than the predetermined
number, the lot is accepted.
Sampling by variables
In variable sampling, sampling is performed to estimate quantitatively the
amount of a substance (e.g., salt) or a characteristic (e.g., color) on a contin-
uous scale. The estimate obtained from the sample is compared with an
19
Food Analysis acceptable value (i.e., previously determined) and the deviation measured. This
type of sampling usually produces data that have a normal distribution such as
in the per cent fill of a container and total solids of a food sample. In general,
variable sampling requires smaller sample size than attribute sampling and
each characteristic should be sampled for separately when possible.

11.5.4 Operating Characteristic (OC) Curves

Operating Characteristic (OC) curves are used extensively in acceptance
sampling. The OC curve shows the relationship between the quality and the per
cent of lots expected to be acceptable for the quality characteristic inspected.
In other words, the OC curve is a graph of lot defectives against the probability
that the sampling plan will accept the lot. Fig. 11.2 depicts OC curves for an
ideal sampling plan.

Fig. 11.2: Operating Characteristic Curve

The Operating Characteristic (OC) curve shows the probability of acceptance,

Pa, for any level of lot quality. On the horizontal axis is the quality
characteristic. This OC curve enables you to evaluate the probability of
acceptance for any true lot quality level-on a what-if basis. This way, you can
design sampling plans that perform the way you want.
We can interpret the curve according to this example:
1) If the lot quality is 0.093 fraction defective, then the probability of
acceptance, Pa, is 0.05.
2) If the lot quality is 0.018 fraction defective, then the probability of
acceptance, Pa, is 0.95.

11.5.5 Requirements of Good Sampling Methods

Samples are useful for their intended purpose when they are taken in a manner
consistent with generally recognized good sampling techniques and good
sampling practices. This requires the following:
• Inspection of the lot before sampling.
• Use of suitable sampling devices for the particular commodity and type of
sample desired.
• Use of suitable containers to hold the sample.
• Maintenance of the integrity of the sample and associated records.
• Use of adequate precautions in preserving, packing and delivery of the
sample to the lab in a timely manner.
20
• Provision of appropriate storage conditions for the sample both prior to and Sampling Techniques of
following analysis. Food Products

All of these factors, along with others such as cost versus benefits analysis, and
a review of program objectives and regularity requirements, are to be assessed
and brought together in a sampling plan that serves as a guide to management,
as well as to operating personnel as a firm plan to achieve quality in sampling.

11.5.6 Cost of Sampling

The attention of users is drawn upon relation between the efficiency and size of
sample. For a given Acceptable Quality Level (AQL), the smaller the sample
size, the smaller the cost of sampling, but the worse the efficiency, that is the
risk to wrongly accepting a lot increases and worsens the damage in trade.

11.5.7 Problems in Sampling

Analytical data never are more reliable than the sampling technique. Sampling
bias, due to non-statistically viable convenience, may compromise reliability.
Errors also may be introduced by not understanding the population distribution
and subsequent selection of an inappropriate sampling plan.
Unreliable data also can be obtained by non-statistical factors such as poor
sample storage resulting in sample degradation. Samples should be stored in a
container that protects the sample from moisture and other environmental
factors that may affect the sample (e.g., heat, light, air). To protect against
changes in moisture content, samples should be stored in an airtight container.
Light sensitive samples should be stored in containers made of opaque glass, or
the container wrapped in aluminum foil. Oxygen sensitive samples should be
stored under nitrogen or an inert gas. Refrigeration or freezing may be
necessary to protect chemically unstable samples. However, freezing should be
avoided when storing unstable emulsions. Preservatives (e.g., mercuric
chloride, potassium dichromate, and chloroform) can be used to stabilize
certain food substances during storage.
Mislabeling of samples causes mistaken sample identification. Samples should
be clearly identified by markings on the sample container in a manner such that
markings will not be removed or damaged during storage and transport. For
example, plastic bags that are to be stored in ice water should be marked with
water-insoluble ink.
If the sample is an official or legal sample the container must be sealed to
protect against tampering and the seal mark easily identified. Official samples
also must include the date of sampling with the name and signature of the
sampling agent. The chain of custody of such samples must be identified
clearly.

11.6 THREE CLASS SAMPLING PLAN

Another type of sampling used frequently by regulatory agencies to determine
acceptance or rejection of a lot (often defined as the quality of product
produced under essentially the same conditions but representing no more than
one day’s production) is the three-class sampling plan. This approach is often
used when assessing microbiological contamination of foods. In this case, “n”
is the number of samples, usually selected at random from the lot, the
21
Food Analysis numerical value “m“ represents acceptable concentrations, the numerical value
“M” represents un-acceptable concentrations, and “c” is the maximum
allowable number of marginally acceptable sample units such that if this
number is exceeded, the lot is considered as un-acceptable. While “m”
separates sample units of acceptable quality from those of marginally
acceptable quality, “M” separates sample units of marginally acceptable
quality from those of defective quality.
For enforcement purposes, the sampling technique used should be the same as
the sampling technique used to set the standard. For example, minimum
reportable limits for particles are based on composite samples and not on
individual lots.

11.7 PREPARATION OF SAMPLING PLANS

The development of quality sampling plans is a science in itself and has been
given consideration by a number of organizations. One plan format that
deserves serious consideration, developed by the International Organization for
Standardization, is shown with comments in ISO/TC 34, ISO/DIS 7002.2,
“Agricultural food products- Layout for a standard method of sampling from a
lot “(1988).
It can serve as a starting point or check list for developing a sampling plan for
most commodities. The title and headings from sections in the monograph are
as below:

11.7.1 Model Sampling Plan

Agricultural food products- Layout for a standard method of sampling from
a lot.
1) Title (short but appropriate for index identification)
2) Introduction (describing the purpose of the plan)
3) Scope (describing the breadth of coverage of the plan)
4) Field of application (products to be covered; where sampling will be done)
5) References (documents, the validity of the plan with reference to other
requirements)
6) Definitions (specific terms associate with a particular matrix)
7) Principle (statistical basis of the method of sampling)
8) Administrative arrangements
a) Sampling personnel
b) Representation of parties concerned
c) Health, safety and security precautions
d) Preparation of the sampling report
9) Identification and inspection of the lot prior to sampling (important in
survey sampling for identification, condition of the lot and selection of
method of sampling).
10) Sampling equipment and ambient conditions (proper tools such as use of
sterile equipment for aseptic sampling).
22
11) Sample containers and packing (essential to prevent contamination and Sampling Techniques of
damage during shipment or storage). Food Products

12) Sampling procedures (as dictated by the plan objectives).

a) Sampling size (adequate for all analytical testing to be done.
Refer Tables 11.2 to 11.4 in Annexure)
b) Taking the sample.
c) Preparation of bulk samples (Table 11.3) and reduced samples
(Table 11.4).
d) Selection of samples of pre-packaged products.
13) Packing, sealing and marking of samples and sample containers
(identification of units and to establish chain-of-custody).
a) Filling and sealing sample containers.
b) Labeling or marking (including signature of sampling personnel).
c) Packing samples for storage or transportation.
14) Precautions during storage and transportation of samples.
15) Sampling report
a) Administrative details.
b) Details of unit packs or enclosure containing the lot.
c) Material samples.
d) Marking and sealing of samples.
16) Annexure (supplemental information, if necessary).

Check Your Progress Exercise 3 "

Note: a) Use the space below for your answers.
b) Check your answers with those given at the end of the unit.
1) Mention some sampling methods/techniques related food products?
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…
……………………………………………………………………………….
………………………………………………………………………………
2) What is probability sampling?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…
………………………………………………………………………………

23
Food Analysis 3) Describe advantages and disadvantages of the plans- Sampling by
attributes and sampling by variables?
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…
……………………………………………………………………………….
4) Write down the application flow of three- class sampling plan?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…
5) Mention some basic requirements of good sampling methods?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…
6) How much quantity of sample is required for the following items?
Root and bulb vegetables, Cereal grains, Poultry eggs, Liquid milk, Liquid
products.
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…

11.8 SUB SAMPLING FOR ANALYSIS AND

TAKING THE TEST PORTION
If the test portion analyzed does not represent the sample or the lot from which
it was taken, in that case, even the best analysis could give misleading
information. Distortions introduced at this point will carry through the path of
analysis and adversely affect the final results and the conclusions drawn from
them. There are generally two choices in analytical sub sampling:
• Preparation of a composite laboratory sample (if multiple units are
submitted for analysis).
• Examination of individual units.

11.8.1 Composite Lab Sample Preparation

A composite lab sample is one in which the individual units or representative
portions of units are mixed to form a uniform mixture. Portions are then taken
24
from the composite for analysis. Compositing saves analytical time and in Sampling Techniques of
some types of contract testing it may be the procedure specified. If the results Food Products
indicate that there may be a problem, it will likely be necessary to go back and
analyze individual samples. Compositing is not the procedure of choice when
there is a chance that an individual unit that constitutes a public health or
safety threats will not be detected (there are some exceptions) or where a unit
at or outside of tolerance level will not be detected because of matrix dilutions.
Multiple unit lab sampling is indicated when the possible range of values
among individual units is considered significant or it is desirable to establish
the variability of the lot.
Refer to Tables 11.4 and 11.5 for the details commodities and quantity of
sample required.

11.8.2 Opinions of Experts

You den and Steiner (Statistical Manual of the AOAC, AOAC International,
Arlington, VA, p 41) observed that, “Many materials are notoriously difficult
to sample. Often the variability among samples is the controlling factor in the
confidence placed in the analytical result.” They note further that
A mistake sometimes made is to composite several samples and then to run
repeat determinations on this composite sample. The analyst may be happy
with several results that are in close agreement because only the analytical
error is involved in the results. And some may put their faith in the result
admittedly, if the individual samples were of the same weight and properly
mixed, the same average will result whether the samples are analyzed
individually or repeats are made on the composition. Using the composite
sample effectively conceals the between-sample variation. It should be
mandatory to run the samples individually, for only by doing so will anybody
be in a position to make any statistical statements about the results, no matter
how good the analytical procedure.
Somewhat similar view of sub sampling for analysis is expressed in an article
published in Chemical and Engineering news by an ad hoc sub committee of
The American Chemical society for “Dealing with the Scientific Aspects of
Regularity Measurements.”
This report observes that the number of samples to be analyzed in a given
situation usually is limited by the resources available for collection of samples
or for their analysis. However, the reliability of the result generally increases
with the square root of the number of samples analyzed. For this reason,
analysis of multiple samples are preferred over single samples since, single
samples give no information on the homogeneity of the lot that was sampled. In
addition, for single samples, the sampling error is also confounded with the
analytical error. As a result, if the total number of determinations must be
fixed, multiple independent single samples are preferred over replicate
aliquots per a single random sample. In any case, the sampling decisions
should be a priori decision and should be based on the question at issue.
In addition to the number of sub samples taken for analysis, it is essential that
each be prepared in a way that achieves homogeneity and is handled in a
manner that prevents alteration from the original composition. Obviously,
failure to prepare a homogeneous sub sample at this point will affect the results
of the analysis regardless of the method used.
25
Food Analysis
11.9 SAMPLE PREPARATION FOR ANALYSIS
Every type of material that is to be prepared for analysis presents its own
practical difficulties. The requirements for suitable sample preparation are
dictated by the consistency and the chemical characteristics of the analyte and
the matrix, and by the distribution of the analyte in the sample. Even seemingly
homogeneous materials such as liquids may be subject to sedimentation or
stratification. Thus, vigilance and care are the watch words to ensure
homogenity.

11.9.1 Precautions to be followed while Preparing a Sample

for Analysis
Mixing: Single phase liquids can generally be mixed, stirred, shaken or
blended. Dry particulate materials can be reduced in the volume by coning and
quartering, by rolling and quartering, or by the use of a splitter, such as a refill.
A variety of implements and machines are available for sample disintegration,
such as mills, grinders and cutters. Care in their use is necessary to prevent loss
of dust or change in composition through partial separation of components.
Screening can be used to improve the efficiency of particle size reduction,
followed by mixing to attain homogeneity. Sampling errors can occur even in
well mixed particulate mixtures especially in trace analysis if the particles
differ appreciably in size or physical properties.

Cleanliness of equipment used in process

Every piece of equipment used in the preparation of a sample must be
examined critically to ensure their cleanliness, so that they do not contaminate
or decompose or cause any physical loss of the sample while processing.
Grinders were mentioned above as contributing to the loss of finer particles as
dust. They have been known to segregate materials with in the mix by size as
well, with the finer material, collecting beneath the blade e.g., Metal screens
can pass fine particles, but retain powder that adheres to the screen materials.
Glass containers and laboratory apparatus can adsorb certain materials and
may require surface treatment. Plastic containers can retain contaminants, such
as animal hairs, while the rest of the sample is transferred with apparent ease.
In the other words, validation of a method of analysis, includes, most certainly
validation of the method of sample preparation and storage.

Changes in physical characteristics

Loss or gain of moisture during processing can be a problem. Loss can be
minimized by keeping samples covered with plastic or aluminum foil. A cold
product can be protected from gaining moisture by allowing the sample to
come to room temperature before preparation begins. High fat samples such as
nuts may be difficult to grind without clogging up the grinder; one technique
that is used is to freeze the samples prior to grinding.
Changes in chemical characteristics
When volatile organic constituents are present in any sample, processing may
be difficult and needs special care, e.g. maintaining chilled condition to
prevent any loss of volatile constituents. Similarly, in case of photo-sensitive
chemicals (e.g. natural product pesticides), it is required to process a sample
under darkness to prevent degradation on exposure to light.
26
Portions for sampling Sampling Techniques of
Food Products
As a general guide, food samples are analyzed in the form they are commonly
consumed. Inedible portions, such as stones (e.g. for mango), nutshells, or fish
bones are removed and discarded prior to analysis, and suitable note made of
how the sample was prepared. The technique used for setting the standard
should be used to ensure comparability.

Sampling for Trace metals

Trace metals analysis can present significant problems, For example, the trace
metals can be distributed unequally between liquid and solid phases in pickles,
canned vegetables and canned fruits. Obviously, such irregular distribution of
metals can pose problems for the analyst in establishing the level of metal
residues in the product, as well as for those concerned with setting tolerances.
Thus, it becomes necessary to analyze both the solid and liquid phases.

Check Your Progress Exercise 4 "

Note: a) Use the space below for your answers.
b) Check your answers with those given at the end of the unit.
1) Write down some precautions while preparing a sample for analysis?
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…
……………………………………………………………………………….

11.10 DIFFICULTIES IN SAMPLING

As mentioned earlier, one of the most difficult problem in sampling from a lot,
and in subsequent lab subsamples, is trying to obtain a representative sample
for the analysis of aflatoxins in raw agricultural commodities. Aflatoxin
contamination exhibits a highly erratic distribution, with a reduction in
heterogeneity as the food or feed is reduced in particle size. After it was
recognized that there was a high rate of variability and within same samples
from the same lot, there was a moment towards the collection of larger and
larger samples. Sample sizes started, for peanuts with 1 kg, and the size
increased as more reliable results were required by food procedures (increasing
sample size reduces the number of good lots that are likely to be rejected and
the number of bad lots accepted).

11.10.1 Example for Effect of Sampling on Analytical Result

At the present time in the United States, the sample taken from a lot of shelled
peanuts of 144 pounds; three 48 pounds samples with portions taken at random
from the lot. Examination in the lab is by sequential analysis with first 48
pounds sample ground in a subsampling mill and test portions examined in
duplicate. If the average of the test portions is below the established tolerance
(set by US Food and Drug Administration), the lot is passed. If the average is
above the acceptance level, the lot is rejected. If the findings fall between the
two figures the second 48 pound sample is comminuted and the analysis
27
Food Analysis repeated. If a decision cannot be made to accept or reject the lot, the third 48
pounds sample is prepared, assayed, and the cumulative results considered.
The foregoing example point out dramatically the need for attention to lot
sampling, lab subsampling, and sample preparation for analysis. While this is a
rather extreme case, it illustrates that sampling problems cannot be ignored or
treated indifferently. In Canada, while the specified sample sizes are smaller,
ranging from 12 to 20 kg. depending on the commodity and the lot size, and
minimum number of sampling sites are also stipulated to address the erratic
distribution of aflatoxin contamination.

11.11 SAMPLE ACCOUNTABILITY

11.11.1 Documentation
A laboratory sample is generally the starting point for analytical work. The
sample may be delivered by mail, courier, flight, or directly by the collector. It
may arrive in any of various containers and conditions: frozen, packed in ice,
or at room temperature. The package may be sealed or unsealed, and the
sample itself may be spoiled or broken. The sample may or may not be
accompanied by appropriate documentation to advise the laboratory regarding
purpose, test parameters and the conditions of storage, etc.
Once a sample is received, all the circumstances and conditions must be
documented as they could have bearing upon the quality or the significance of
the test results. It is important for appropriate quality analysis that sample
arrives in proper condition with meaningful documents. Procedures for these
must be established, continually reviewed, and enforced, to keep poor sample
handling and delivery to a minimum level. To avoid any future legal
complications, the laboratories are advised to protect themselves with the
cautionary statement in the test report indicating that the results relate only to
the sample that was tested.

11.11.2 Chain of Custody Form

In most organizations specific sampling procedures are written and the sample
collectors are trained regarding their responsibilities. The first important
activity is the documentation to ensure product traceability. The sample should
be easily identifiable and placed under seal and packing. Shipping and delivery
instructions are followed to effect delivery to the laboratory. The
documentation consists of a chain-of-custody that accompanies the sample as it
moves through the laboratory and subsequent administrative handling. This
form is usually prepared in multiple copies for distribution to various units in
the organization, may be supplemented with affidavits, dealer’s statements,
bills or other relevant information that concerns the sample, its origin, the
transfers from one custodian to the next and the sample’s significance or
importance. Information such as sample number, product name and
identification, reason for collection, description of the sample and of the
method of collection, size of the lot from which the sample was taken, codes,
shipment information, collection date, name of the collector, means of
transportation, and whether or not sealed are supplied with the sample. If the
sample is sealed, the seal includes the sample number, date the seal was
affixed, and the collector’s signature. The seal is attached to the package in
such a way that it must be broken before the sample can be obtained.

28
11.11.3 Sample Receipt and Handling Sampling Techniques of
Food Products
The next step in the sample accountability system is receipt of the sample in
the laboratory. A dependable record of sample handling is important so that the
sample is accepted by a sample custodian who documents the action by
completing a sample accountability record. This document should contain the
sample number, the name of the product and date received, indicate who
received it, describe the method of shipment or delivery, describe the packages
received and their condition, and provide space for recording various storage
locations before and after analyses. Deliveries of the sample or portions of the
sample to the analyst, and its return, will also be recorded on this form. There
will be a signed statement concerning the final disposition of the reserve
sample. A two-part form can be used for this purpose; one copy remains with
the sample custodian and the other moves with the sample through the
laboratory and is used by a supervisor for sample management purposes. Some
laboratories use a sample receiving log book for sample control. The
information entered in the log book is essentially the same as that described for
the two-part form.

11.11.4 Monitoring of Samples

The sample accountability in a laboratory can be monitored by a simple
computer program; a unique label should be generated and affixed to the
sample container, and all the pertinent sample information should be entered
into the computer database. The information entered at log–in becomes part of
the data base, which is then built up through the manual or automatic addition
of sample handling information and analytical data. Worksheet pages or
reports can be calculated and printed, and the data base itself latter queried and
manipulated for various information and reporting purposes. Regardless of the
recording system used, the analytical information generally reported includes a
description of the sample, subsampling procedure sample preparation methods
used, deviations from methods, validation and recovery experiments (if
performed), standards used, source of reference materials, raw data,
calculations and description of the reserve sample and how it was prepared for
storage after the completion of the analysis. In addition, pertinent supporting
documents such as chromatograms, spectra, and other charts are suitably
identified with instruments identification, operating conditions, analyst name,
sample number and date. If the reserve sample is sealed, the information
placed on the seal is shown in the report. The sample is then returned to the
sample custodian to be stored for whatever future action may be necessary, or
until the sample is destroyed.

11.12 RETENTION OF SAMPLES AND RECORDS

After an analysis is complete and the results reported, the laboratory needs a
written policy for guidance on the retention of the samples and the associated
records. For samples and records that may be involved in litigation, the storage
period can extend for years. For the majority of samples, fortunately, this is not
usually the case. The objective should be to destroy samples as soon as it can
be analyzed, with certainty that they will no longer be required for further
testing or as evidence. The records may be disposed after they are no longer
legally or administratively important.

29
Food Analysis 11.12.1 Identify the Properties of Retained Samples
It is very important for the laboratory management to determine whether or not
the materials being discarded are hazardous in nature. Although samples
themselves may not be hazardous, acid digestions and organic solvent
extractions certainly can be hazardous. Sample management includes the
proper disposal of samples and laboratory preparations. Standard operating
procedures for samples for sample disposal are essential.

11.12.2 Retention Period

Storage periods, obviously, must be determined by each facility depending on
its obligations, but clear policy must be in place to prevent both the destruction
of important items, and the accumulation of what is essentially junk. From a
quality assurance point of view, the improper destruction of active samples or
records is low quality performance in violation of policy, and the Quality
Assurance (QA) program must provide a means to detect such actions in an
effort to prevent their recurrence.

11.13 CASE STUDY

In a 500 T consignment of imported frozen animal carcasses, 300 T labeled as
produced by A and 200 T labeled as produced by B is to be checked for
residues.
Assumed facts:
i) The carcasses are from an exporter whose products have recently been
associated with excessive residues of Permethrin (fat soluble) and
Diflubezuron (non-fat soluble).
ii) Carcasses in lot A have trimmable fat, where as those in lot B do not.
iii) The sampling plan is to provide a 95% probability of detection if 10% of
the carcasses contain excessive residues.
iv) There is no legal requirement to prepare replicate lab samples.
v) Sampling records are in hard copy form.
vi) Rendering of fat tissue for extraction of lipid acceptable under national
law.
Consequent actions and discussions:
i) The consignment is sampled as two separate, suspect lots, A and B
ii) Table 11.3 shows that 29 lab samples should be taken and therefore, as
far as practicable, 29 carcasses are selected at random from each lot.
iii) From each selected carcass in lot A, a minimum of 0.5 kg of adhering
fat tissue is taken as a (primary) lab sample and a minimum of 0.5 kg of
meat (meat does not include bone) is taken as a separate (primary) lab
sample.
iv) The carcasses in lot B have no trimmable fat and 29 samples of 2 kg
meat are taken.
v) As each lab sample is taken, it is placed in a new polythene bag,
securely labeled and sealed, and the sample record completed. The
samples are sent to the lab, ensuring that they do not thaw.

30
 Copies of the sample records are given to the owner/custodian of the Sampling Techniques of
consignment. Copies are sent with the samples and also retained by the Food Products
sampling officer.
vi) Fat tissue lab samples from lot a are rendered, the lipid collected and
aliquots (analytical portions) analyzed for Permethrin residues. The
results are expressed on a whole fat tissue basis.
vii) Bones, if any, are removed from the meat lab samples, which are
minced before the determination of Diflubenzuron residues in
analytical portions. The results are expressed on the basis of whole
meat without bones.
viii)If meat samples from both lots contain Diflubenzuron ≤ 0.05 mg/kg
and all samples from lot A contain < 1 mg/ kg Permethrin, lot B is
acceptable and lot a is acceptable with respect to Diflubenzuron
residues.
ix) If 3 of the 29 fat samples of lot A contain Permethrin > 1 mg/kg,
replicate analytical portions of fat from these 3 lab samples are
analyzed. Taking into account the analytical uncertainty, if the results
conform that the MRL is exceeded, if the 3 carcasses do not comply
with the MRL, where as the other 26 do comply with the MRL.
x) If the entire lot is not to be rejected on this basis, lab samples of fat
tissue from the remaining carcasses in lot A may be taken for analysis,
in order to separate the acceptable carcasses for those that are
unacceptable.

11.14 LET US SUM UP

Actions for the management and control of sampling, sample preparation, and
sample analysis are summarized below:
1) Work with appropriate persons to develop sampling plans for the various
types of products delivered to the laboratory for analysis.
2) Establish sub sampling procedures for various products, giving
consideration to the use of composites or individual unit examinations,
based on the variability to be expected among sample units and the
resources available for their analysis.
3) Prepare guidelines for sample preparation for analysis that will minimize
composition change.
4) Choose the appropriate sampling selection method to achieve the intended
purpose of taking samples.
5) Describe subsampling within the lab in written procedures which analysts
have the responsibility to follow.
6) Manipulation and preparation must be done with care to avoid losses of
material.
7) Develop a system to ensure sample accountability.
8) Provide policy for the management of samples when they are no longer
needed.

31
Food Analysis 9) Maintain written procedures for sample disposal taking into consideration
hazardous waste regulations.

11.15 KEY WORDS

The definitions of sampling terms used in this document are mostly those
specified in ISO 7002. Some of the more commonly used terms in acceptance
sampling are described in this section.
Lot : A definite quantity of some commodity
manufactured or produced under conditions, which
are presumed uniform for the purpose of this
document.
Consignment : A consignment is a quantity of some commodity
delivered at one time. It may consist in either a
portion of a lot, either a set of several lots.
Sample : Set composed of one or several items (or a portion of
(Representative matter) selected by different means in a population
sample) (or in an important quantity of matter). It is intended
to provide information on a given characteristic of
the studied population (or matter), and to form a
basis for a decision concerning the population or the
matter or the process, which has produced it.
A representative sample is a sample in which the
characteristics of the lot from which it is drawn are
maintained. It is in particular the case of a simple
random sample where each of the items or
increments of the lot has been given the same
probability of entering the sample.
Sampling : Procedure used to draw or constitute a sample.
Empirical or punctual sampling procedures are
sampling procedures, which are not statistical-based
procedures that are used to make a decision on the
inspected lot.
Total Estimation : In the estimation of a parameter, the total estimation
Error error is the difference between the calculated value
and the true value of the parameter. The total
estimation error is due to:
i) Sampling error ii) Measurement error
iii) Rounding-of-values vi) Bias of the estimator
or sub-division
in to the classes
Sampling Error : Part of the total estimation error due to one or
several of the following parameters:
• The heterogeneity of the inspected characteristics.
• The random nature of a sampling.
• The known and acceptable characteristics of the
sampling plans.

32
 Sampling Techniques of
Acceptable Quality : The inspection of a lot using either an attributes or Food Products
Level (AQL) variables sampling plan will allow a decision to be
made on the quality of the lot.
AQL for a given sampling plan is the rate of the non-
conforming items at which a lot will be rejected with
a low probability, usually 5%.
Limiting Quality : For a given sampling plan is the rate of non-
(LQ) conforming items at which a lot will be accepted
with a low probability, usually 10%.
Sampling Plan : A pre-determined procedure for the selection,
withdrawal, preservation, transportation and
preparation of the portions to be removed from a lot
as samples.

11.16 ANSWERS TO CHECK YOUR PROGRESS "

EXERCISES
Check Your Progress Exercise 1
Your answer should include following points:
1) The quality of a small sample analyzed is attributed to the lot, which the
sample represents. If the sample does not represent the population
adequately and efficiently, then the results obtained may not truly represent
the quality of the lot.
2) Homogenous population would be uniform and identical at all locations.
These populations/samples in which the composition would vary at
different locations is heterogenous population.
Check Your Progress Exercise 2
Your answer should include following points:
1) It is very important to arrive at a well designed sampling plan because it is
a guide to the people who are going to perform sampling. It also serves as a
reminder of the important elements in the over all analytical program.
2) a) Sampling plan is a guide for the whole analytical program.
b) It is a means for operating on a planned basis which reduces variation.
c) It serves as a reference document for similar activities in the future.
d) A document for comparison of performances against objectives.
e) It also serves as a source for imparting training.
Check Your Progress Exercise 3
Your answer should include following points:
1) i) Probability sampling,
ii) It is a means for operating on a planned basis which reduces variation,
iii) Non-Probability sampling,

33
Food Analysis iv) Bulk sampling,
v) Acceptance sampling,
vi) Sampling by attributes, and
vii) Sampling by variables.
2) Probability sampling is used when a representative sample is desired, and
uses principles of statistical sampling and probability, a random selection
approach that tends to give each unit an equal chance of being selected.
3) Sampling by attributes:
Advantages: 1. No condition on the mathematical law of distribution of the
variable inspected. 2. Greater simplicity of the processing the results on the
sample.
Disadvantages: 1. Less effective than variables plans for a same sample
size of n increments (Least Quality, LQ is higher). 2. More costly than
variables plans because the collected sample requires more increments than
those required, for the same efficacy, by a variables plan.
Sampling by variables:
Advantages: 1. More effective than attributes plans for the same sample
size of n increments (LQ is lower) for the same AQL, they are less
expensive than attributes plans because the sample collected requires fewer
increments than those required for a same efficacy, by attributes plans.
Disadvantages: They cannot be used in all cases because to validate the
calculation formulas of the inspected variable must necessarily or
approximately follow a normal law.
4) Set the values of m, M, n and c → Collect the sample with n items →
Inspect each item in the sample → Accept the lot if: number of marginally
defective items (i.e. a concentration of micro-organisms between m and M)
≤ c. Immediately reject the lot if the concentration of the micro-organisms
in any item > M and / or the number of marginally defective items > c.
Where, m: accptable concentration; M: unacceptable concentration;
c: maximum allowable number of marginally acceptable sample units; and
n is the number of sample units selected randomly from the lot.
5) a) The lots shall be thoroughly inspected before sampling to design a good
sampling plan.
b) Suitable sampling devices shall be identified and used while sampling.
c) Compatible containers shall be identified and used. The material of the
container shall not cause any undue contamination to the quality of
sample collected. For example, Non-sterile containers shall not be used
while the sample has to undergo microbiological tests.
d) Suitable packing and delivery method.
e) Provision of appropriate environmental conditions. For example, When
there is a need to determine Volatile Organic Constituents in a sample
of water, it has to preserved/transported at a temperature 4 to 8oC.
6) Root and bulb vegetable- 5 kg, cereal grains- 2 kg, poultry eggs- 12 eggs,
liquid milk- 0.5 L, liquid products- 0.5 L

34
Check Your Progress Exercise 4 Sampling Techniques of
Food Products
Your answer should include following points:
1) a) Use a suitable method for homogenization. For example, Liquids can
be homogenized by stirring, shaking or by blending and take an aliquot.
Solids can be homogenized by grinding, pulverizing and volume
reduced by coning and quartering.
b) Use clean and suitable sampling devices and containers. For example,
A scoop used for sampling a food shall be sterile.
c) A glass container is not compatible for collecting water sample
intended for the determination of metals, since metals like sodium are
absorbed by glass.
d) Moisture content of sample changes with surrounding temperature.
Suitable precautions shall be taken to retain the originality.
e) Maintain suitable environmental conditions to minimize expected
chemical changes if any. For example, A food sample for
microbiological enumeration shall be collected in a sterile container
and be stored/transported in chilled condition.
f) If the sample is presented in both liquid and solid phases, homogenize
both phases before a test portion is taken.

11.17 SOME USEFUL BOOKS

Nielsen, S. Suzanne (2003). Food Analysis Laboratory Manual, 3rd Edition,
CHIPS Publishers, U.K.
International Organization for Standardization (ISO): www.iso.org
Codex Alimentarius (CODEX), www.codexalimentarius.net
Agriculture and Processed Food Products Export Development Authority
(APEDA), Ministry of Commerce and Industry: www.apeda.com
Bureau of Indian Standards (BIS), Ministry of Consumer Affairs, Food and
Public Distribution: www.bis.org.in / www.fcamin.nic.in
Export Inspection Council of India (EIC), Ministry of Commerce and Industry:
www.eicindia.org
Prevention of Food Adulteration Act (1954), 24th Edition, 2003, Ministry of
Health and Family welfare: www.mohfw.nic.in

35
 Food Analysis
UNIT 12 PHYSICAL AND CHEMICAL
ANALYSIS OF FOODS
Structure
12.0 Objectives
12.1 Introduction
12.2 Physical Properties
12.2.1 Specific Gravity/Density
12.2.2 Specific Heat Capacity
12.2.3 Surface Tension
12.2.4 Viscosity
12.2.5 Refractive Index
12.2.6 Filth
12.2.7 Particle Size
12.3 Chemical Properties
12.3.1 Moisture
12.3.2 Water Activity
12.3.3 Protein
12.3.4 Fat
12.3.5 Volatile Oil
12.3.6 Crude Fiber
12.3.7 Dietary Fiber
12.3.8 Total Ash
12.3.9 Acid Insoluble Ash
12.3.10 Sulphated Ash
12.3.11 Reducing and Non-Reducing Sugars
12.3.12 Starch
12.4 Physical and Chemical Properties of Oils and Fats
12.4.1 Acid Value and Free Fatty Acids
12.4.2 Unsaponifiable Matter
12.4.3 Melting Point
12.4.4 Solid-liquid Ratio
12.4.5 Specific Gravity
12.4.6 Titre Value
12.4.7 Colour
12.4.8 Iodine Value (Wij’s)
12.4.9 Saponification Value
12.4.10 Acetyl Value and Hydroxyl Value
12.4.11 Reichert-Meissl (RM) Value
12.4.12 Polenske Value
12.4.13 Rancidity
12.5 Let Us Sum Up
12.6 Key Words
12.7 Terminal Questions
12.8 Answers to Check Your Progress Exercises
12.9 Some Useful Books

12.0 OBJECTIVES
After reading this unit, we shall be able to:
• explain the physical properties of foods;
• analyze the food for its chemical composition;
• explain the importance of physico-chemical properties of foods;
• define the chemical constants of oils and fats; and
• discuss the advantages and disadvantages of a particular techniques used
36 for food analysis.
 Physical and Chemical
12.1 INTRODUCTION Analysis of Foods

Analysis plays an important role in assessment and maintenance of food

quality and safety, both in industry as well as for enforcement authorities at
national and international levels. Earlier, food analysis was concerned with
food adulteration only. Now-a-days there is an increasing tendency to examine
food from a more positive and broader view point. Processed foods are
produced within the limits of prescribed manufacturing formulations, set also
to comply with legal and/or other requirements. In many food laboratories,
most of the routine work is confined to proximate (e.g. moisture, protein,
carbohydrate, lipid, fiber, ash) analysis and the analysis of additives and
contaminants. This is done by analysis of the product at different stages of
processing starting at the farm level. The regulatory requirements for the
analysis of food additives and contaminants at very low level have necessitated
the development of instrumental techniques suitable for rapid assessment. In
case of proximate analysis, the methods may vary for different category of
food products. Hence, the results obtained for a particular food constituent
depends on the procedure adopted. However as long as the same standard
procedure is applied to the same food each time, the results are usually
repetitive and thus provide an adequate basis for interpretation.
In food industry, various food components and parameters are analyzed in both
raw and processed products. Knowledge of chemical composition of food is
important to health, well being and safety of the consumers. Knowledge of the
chemical and biochemical composition of food is useful to manufacturers in
understanding the importance of various nutritional constituents so that the
amount of essential nutrients may be maintained or improved during and after
processing. The knowledge of the principles of different food analysis
techniques is useful for selection of appropriate technique for analyzing a
particular food. The compilation of physical and chemical techniques in this
unit would be helpful in understanding the basic principles of food analysis.

12.2 PHYSICAL PROPERTIES

The physical properties of food products have not received adequate attention
from the food scientists, although a thorough understanding of the physical
changes and principles involved in delivering food products from farm to
consumers is essential. Important physical parameters are briefly described
below:

12.2.1 Specific Gravity/Density

The specific gravity of a substance is the ratio of density of the material to the
density of water at a specified temperature. Specific Gravity can be expressed
as
SG = ρ / ρH2O
where
SG = specific gravity
ρ = density of fluid or substance (kg/m3)
ρH2O = density of water (kg/m3)

37
 Food Analysis It is common to use the density of water at 4 oC (39oF) as reference - at this
point the density of water is at the highest - 1000 kg/m3 or 62.4 lb/ft3. Water is
the standard for solids and liquids, while hydrogen is the standard for gases.
The density of a liquid at a particular temperature is the mass (e.g. gram) of
unit volume (e.g. 1 mL) of the liquid.

The density of solid indicates the weight of a substance held in a unit volume.
Densities of liquids are generally measured either by weighing a definite
volume of the liquid in a density bottle or pyknometer or by determining the
buoyancy acting on a sinker immersed in a liquid. The same principal is used
in lactometer for determining density of milk. When sufficient liquid is
available, the density can be determined by means of hydrometers or more
accurately by means of the Westphal balance.

12.2.2 Specific Heat Capacity

Specific heat capacity of a substance is the amount of heat required to raise the
temperature of that substance by one degree centigrade. Specific heat of a
substance helps in calculating the amount of heat required to raise the
temperature of a substance by a certain amount. Unit of specific heat capacity
is J g-1K-1 or cal g-1K-1
For example, how much heat would be required to raise the temperature of 100
g of water from 30°C to 65°C?

Formula:
Units of heat (Calories) = mcΔT, where m is the mass in g, c is specific heat
capacity and ΔT corresponds to change of temperature
T

= 100 g × 0.94 × 35°C

= 3290 calories

12.2.3 Surface Tension

Surface tension is defined as the force acting upon a line of unit (1cm) length
on the surface of a liquid. Surface tension is a state of stress at the surface of a
liquid, which occurs due to inward force of attraction on the surface molecules
as result of which the upper surface of a liquid behaves like a stretched
membrane. Surface tension is a manifestation of the forces of attraction that
hold the molecules together in the liquid (or solid) state; thus liquid droplets
tend to become spheres – the form of least surface area – because of the
material cohesion of molecules. High surface tension is found with liquids that
have strong cohesive forces and consequently high boiling points whereas
volatile organic liquids have low surface tension. For a given liquid, the
surface tension decreases with rise of temperature and becomes zero at critical
point.

Surface tension may be measured by several ways:

A. Capillary rise method: If a capillary tube is placed in a liquid it is found
that the liquid rises in the tube. To determine the height, to which the liquid
38
 rises in the tube, it is measured by cathetometer or travelling microscope. Physical and Chemical
Surface tension is determined by following equation: Analysis of Foods

Surface tension (γ) = r × h × d × g

2
where,
r = radius of capillary tube
d = density of the liquid
h = height to which the liquid rises, and
g = acceleration due to gravity.
B. Drop-weight method: This is one of the simplest method. The size of drop
issuing from a capillary orifice is governed by the surface tension of the
liquid. The instrument employed is called a stalagmometer. The surface
tension of a liquid food material like milk can be determined by comparing
with water at same temperature. A tube of uniform bore is used and the
number of drops falling per unit of time are counted and compared to that
of water. Hence, a liquid showing 200 drops as compared to water with 100
drops would have a surface tension one-half as great as that of water.
C. Torsion balance method: A more accurate method is the platinum ring
procedure. The surface tension can be measured by the force required to
detach a horizontal ring of platinum wire from the surface of a liquid. The
ring is connected to a delicate balance and the pull required to draw it out
of a liquid is measured by a torsion balance.

12.2.4 Viscosity
Viscosity is a property of a liquid closely related to the resistance to flow. It is
the frictional effect due to the passage of one layer of fluid (liquid/gas) over
another. The coefficient of viscosity is defined as the force required per unit
area to maintain unit difference of velocity between two parallel planes in the
fluid at 1cm apart. The kinematic viscosity of a liquid is equal to the ratio of
the dynamic viscosity and density of the liquid at the same temperature. The
unit of kinematic viscosity is stokes (S) and the unit of dynamic viscosity is
dynes/cm2 (Poise). Viscosity of liquid may be determined by any method that
will measure the resistance to shear offered by the liquid.
When a liquid flows through a capillary tube of radius r for a time t, under a
constant pressure head p, the volume of liquid v issuing from the tube is given
by :

v = π × p × t × r4
8×l×η
Where,
η = Coefficient of viscosity, and
l = length of the tube.
It is difficult to determine the absolute coefficient of viscosity for a liquid,
however; the relative viscosity of a liquid with respect to water may be
determined. The simplest way to do this is to make use of Ostwald viscometer.
The viscosity may be determined by the method of the falling sphere, which
depends on the time taken for a sphere to fall through a given distance. This is
39
 Food Analysis used for the measurement of viscous liquids. The viscosity of liquid is
calculated by the following equation.

η (D-d) × t
=
ηs (D-d) × ts
Where,
η= Coefficient of viscosity of the liquid,
D= density of sphere,
d= density of liquid, and
t= time taken. The subscript s refers to a standard liquid, the viscosity of
which is known.
A wide range of viscosity can also be measured by Brookfield viscometer
using different spindle and speed. Brookfield viscometer is usually suitable for
the measurement of highly viscous liquids.

12.2.5 Refractive Index

The measurement of refractive index of certain substances is helpful in
identifying and establishing their purity, in determining the molecular structure
of organic compounds and quantitative analysis of certain types of solutions.
The Abbe refractometer is particularly used in food analysis and in the testing
of oils. It covers a wide range of refractive indices and uses a very small
amount of sample. In making a refractive index measurement, the beam of
light is usually passed from air through the solid or liquid medium being
measured and then through a glass prism and out into the air again. The
refractive index may be calculated from the angle through which a telescope
must be turned in order to pick up the emerging beam on a cross hair. The
angle measured includes the refraction at the liquid-glass interface and at the
glass-air interface.

12.2.6 Filth
Any foreign matter in product associated with objectionable conditions or
practices in production, storage, or distribution included are filth, decomposed
material and miscellaneous matter viz., sand, soil, glass or other foreign
substances excluding bacterial counts.
A. Filth: Any objectionable matter contributed by animal contamination of
product such as rodent, insect, or bird matter; or any other objectionable
matter contributed by unsanitary conditions.
B. Heavy filth: Heavier filth material is separated from food products by
sedimentation techniques based on different densities of filth, food
particles and immersion liquids such as chloroform. e.g., insect and rodent
excreta pellets and pellets fragments, sand and soil. When chloroform is
mixed vigorously with the plant material and allowed to separate, most of
the plant tissues rise and the heavy extraneous matter settles to the bottom,
which can be separated by draining on filter cloth.
C. Light filth: Lighter filth particles that are oleophillic and are separated from
product by floating them in an oil-aqueous liquid mixture e.g., insect
fragments, whole insects, rodent hairs and feather barbules. It is difficult to
wet all the insect parts without creating frothy emulsion of the plant
40
 material. Droplets of oil adhering to sides of trap flask may prevent insects Physical and Chemical
from rising. To overcome it, the oil is worked thoroughly into the water- Analysis of Foods
food mixture without incorporating air. If an emulsion is formed, capryl
alcohol or ethanol may be used to break the emulsion.
D. Sieved filth: Filth particles of specific size ranges are separated
quantitatively from the product by use of selected sieve mesh sizes.

12.2.7 Particle Size

Particle size analysis means the separation of a sample of material into
fractions of different average diameters. Hand sieving is the most common
method. The sieves are arranged in a nest, the outer casing of each sieve fitting
the casing of the sieve below it and the nest provided with a top cover and
bottom blank pan. The weighed sample is placed on the coarsest sieve and the
whole nest is given a preliminary shaking after which each sieve is shaken
separately to complete the separation. Both time and intensity of shaking must
be kept as uniform as possible. A mechanical shaker is preferable to hand
shaking since time and intensity of shaking can be exactly repeated from
sample to sample. For separation finer than 200 mesh elutriation is used.
Elutriation is the separation of material by the action of a rising current of
fluid. Microscopic sizing analysis is a form of direct counting and is
theoretically more accurate than either screening or elutriation. However, the
method is only a guide, since the quantity of material visible in the
microscopic field is very minute.

Check Your Progress Exercise 1 "

Note: a) Use the space below for your answers.
b) Check your answers with those given at the end of the unit.
1) What is filth? Briefly discuss the separation technique for the heavy filth?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…
……………………………………………………………………………….
2) Correlate heat and specific heat?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…
……………………………………………………………………………….
41
 Food Analysis 3) How can you measure the viscosity of liquid food product with low
consistency?
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…
……………………………………………………………………………….
……………………………………………………………………………….
4) Differentiate the specific gravity and density of foods?
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…
………………………………………………………………………………
……………………………………………………………………………….
5) Define surface tension? Suggest a simple method for its determination?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…

12.3 CHEMICAL PROPERTIES

12.3.1 Moisture
Water, the simplest of all constituents of foods, is one of great concern to
producer, consumer and chemist. The weight of food has little significance
unless taken into consideration with the water content. The accurate
determination of moisture poses many challenges. One of the problems is the
difficulty of separating all of the water from the food sample, resulting in
underestimation of moisture content. On the other hand, harsher conditions to
remove all moisture from a food may simultaneously cause decomposition of
the product, which may result in the production of water along with/or a loss in
sample mass. Thus the accuracy of the method would be severely in question.
Most of the methods for the estimation of water in foods depend on the loss in
weight on heating. An exposure to the air of the drying oven causes the
oxidation of certain oils and other constituents; a gain in weight of such
constituents offsets the loss in weight due to moisture. To obviate the error, the
drying should be performed in vacuum. The loss in weight on heating is not
entirely because of water as other volatile substances evident to the sense of
smell are present in most foods, although the amount is too small. Significant
loss of volatile compounds from the food is another potential difficulty in the
determination. Most of the spices however contain notable quantities of
volatile oil that pass off with the water.
42
Analytical methods of moisture determination can be classified in two ways. Physical and Chemical
Analysis of Foods
1) Direct methods: Moisture analysis normally involves removing water
from the food samples by drying, distillation, extraction and its quantity is
measured by weighing, titration and so forth, e.g., oven drying, vacuum
drying, freeze drying, distillation method, Karl Fischer method, chemical
desiccation, thermo-gravimetric analysis and gas chromatography.
2) Indirect methods: The indirect methods must be calibrated against
standard moisture values that have been precisely determined using direct
methods, e.g., refractometry, infrared absorption, near infrared reflectance
spectroscopy, microwave absorption, dielectric capacitance, mass
spectrometry, NMR spectroscopy, neutron scattering method, etc.

I) Air-Oven Drying Method

It is one of the most common and widely used methods for routine moisture
determination. The ovens should be thermally regulated to ±0.5°C and have
minimal temperature variations (<±3°C) within the oven. The main criterion of
food for moisture determination by air-oven drying is that sample should be
thermally stable and should not contain significant amount of volatile
compounds.

II) Vacuum Oven Drying Method

It is the standard and most accurate drying method for moisture analysis of
foods. The AOAC methods generally recommend that moisture content of food
can be determined by heating foods at 98 to 102°C at a pressure of 25-100 mm
Hg for 2-6 h. Lower temperatures (60-70°C) can be used for heat sensitive/
sugary food products in sugar to prevent decomposition used for products like
jam, confectionery etc.

III) Distillation Method

Two types of distillation procedures exist for moisture determination;

(a) Direct distillation and (b) Reflux distillation.
a) Direct Distillation
In this method, a food is heated in a liquid immiscible in water and has a high
boiling point (e.g., mineral oil). The water in the food like spices and herbs
distils directly from this liquid, condenses and collects in a graduated tubes; the
volume of the water removed is then measured e.g., Spices, herbs.
b) Reflux Distillation
It makes use of the azeotropic properties of solvent mixtures. During heating,
water and an immiscible solvent (toluene or xylene) distil off together at a
constant ratio and frequently at a temperature lower than the boiling point of
either component. For example, the boiling points of water and toluene are
100°C and 110.6°C, respectively, but the boiling point of the binary mixture is
85°C; the distillation ratio of the mixture is 20% water and 80% toluene. As
water is denser than toluene, the water is again collected in a suitable
measuring apparatus where it separates and has its volume measured. A
representation of the reflux distillation apparatus is shown in Fig. 12.1.

43
 Food Analysis
Dimension in millimeters
and capacity in millilitres

Guard tube containing

Silica gel (5.2.5)

Detail of graduated
measuring tube (5.2.3)

Liebig-West
condenser (5.2.4)

Graduated measuring
tube (5.2.3)

Flask (5.2.1)

Fig. 12.1: Dean and Stark Apparatus for Moisture Determination

IV) Karl Fischer Titration Method

The Karl Fishcer (KF) titration has become a standard method for the moisture
analysis in liquids and solids due to its selectivity, high precision and speed.
The method is particularly suitable for food where heating methods give erratic
results. This method has been approved for dried vegetables, oils and fats,
cocoa products and liquid molasses. The KF titrimetric method is based upon
the quantitative reaction of water with an anhydrous solution of sulphur
dioxide and iodine dissolved in pyridine and an alcohol. The Karl Fischer
reagent consists of iodine, pyridine, SO2 and methanol. The titration is
conducted either by volumetric method (where the end point is brown colour,
determined visually) or by coulometric titration, where the end point is
determined by a potentiometer. Food samples may be directly introduced into
the reaction vessel if the water is easily accessible to the reagent. In solid foods
where water is not accessible, the water is frequently extracted into anhydrous
methanol and then estimated.
V) Refractometry
The refractive index is a ratio of the sine of the angle of incidence made by a
ray in air to the sine of the angle of refraction made by the ray in the material
being tested. The refractive index increases as the moisture content decreases.
By measuring the refractive index of a solution, the moisture content can be
rapidly determined using an appropriate calibration curve. Refractometry is
best suited for high sugar products viz., fruit products, syrups and honey. For
solid or semi-solid foods, the sample can be homogenized with an anhydrous
solvent (e.g., isopropanol) and then the refractive index of the solution can be
44
measured using a refractometer. The moisture content of the sample may be Physical and Chemical
calculated using the calibration curve (produced by measuring refractive index Analysis of Foods
of solutions containing the same solvents with known amounts of added water)
and the mass of food homogenized in the solvent.

12.3.2 Water Activity

The water activity (aw) of a food describes the energy status of the water in the
food. It is the ratio of vapour pressure of water in a sample to saturation vapour
pressure at sample temperature. The water activity is not influenced by the
total quantity of water in a sample but only by that fraction which is least
tightly bound. Temperature affects aw due to changes in water binding,
solubility of solution in water and the state of sample matrix. Most high
moisture foods exhibit negligible change with temperature. The water activity
controls all aspects of microbial growth by lengthening the lag phase of
microbial growth. The water activity influences non-enzymatic browning, lipid
oxidation, degradation of vitamins, enzymatic reactions, protein denaturation,
starch gelatinization and starch retrogradation. The water activity is usually
measured by using different hygrometers viz., hair hygrometer, resistance
sensor, capacitance sensor, dew point hygrometer, etc.

12.3.3 Protein
All natural foods contain protein, although trace amounts are found in honey
and maple sugar. The quantification of total protein in food and food products
can be performed directly or by determining total nitrogen from conversion of
crude protein using a suitable conversion factor. The protein content is
calculated from the total nitrogen determined by either Kjeldahl method or
Dumas/Pregl-Dimas method. Amides (abundant in young shoots), ammonium
salts, nitrates, lecithin, nucleic acid, purines of tea, coffee, cocoa and meat
extracts in addition to protein contain nitrogen in varying proportions.
Although small, these compounds thus add error to the calculated protein
estimate. However, the protein calculated by factor is a valuable figure, not
only because it represents approximately the true protein present but also
because it is an index of the content of other groups. The protein content can
also be determined directly by formal titration, UV spectrophotometry, Lowry
method, Dye binding method, IR spectrophotometry, NMR spectroscopy,
turbidimetry, refractometry, etc.
I) Direct Method
Since foods contain mixtures of proteins, the methods for the direct
determination of proteins need to be calibrated against a reference standard for
nitrogen, e.g. Kjeldahl method.
i) Formal Titration Method
When formaldehyde is added to neutralized aqueous solution containing
protein, the –NH2 group of protein converts to methylene-amino group
(-N=CH2-) with the release of proton. This may be titrated.
ii) Spectrophotometric Method
The Lowry method is based on the amplification of the biuret reaction
(complex of cupric ions with protein) by subsequent reduction of the Folin
phenol reagent (mixed acids of phosphomolybdic and phosphotungstic) by
45
 Food Analysis tyrosine and tryptophan. This redox reaction is accompanied by the formation
of a blue colour (λabs 745 – 750 nm), which is highly pH dependant (10-10.5).
II) Indirect Method

i) Kjeldahl Method
It has wide acceptance for the determination of protein in food products. The
method follows three steps:
Digestion – Decomposition of organic matter by heating in the presence of
concentrated sulphuric acid, the end product is ammonium sulphate solution.
Distillation – Ammonium sulphate is converted into gaseous ammonia by
addition of an excess base, followed by boiling and condensation of the
ammonia in a receiving solution (acid).
Titration – Quantification of the unreacted acid in the collecting vessel.
The rate of digestion and the completeness of the breakdown of nitrogenous
compounds to ammonium sulphate mainly depends upon the heat input,
amount of boiling point elevator of acid (alkali sulphate), addition of catalyst
(mercury, cupper sulphate, titanium dioxide), oxidant (hydrogen peroxide),
reflux rate of sulphuric acid and length of digestion.
Ammonia is liberated from the acid digestion mixture by distillation in the
presence of alkali (50% NaOH). A total recovery of ammonia from the digest
can be obtained within 5 to 20 min by direct distillation and about 10 min by
steam distillation.

Fig. 12.2: Kjeldahl Nitrogen distillation assembly

ii) Dumas Combustion Method

The protein content of foods can be estimated by the determination of
elemental nitrogen using instruments based on the Dumas principle. In these
instruments, the nitrogen containing constituents of the sample are combusted
at high temperature about 1000°C in the presence of oxygen to oxides of
nitrogen (NOx) and then reduced over copper or tungsten to gaseous nitrogen
which is measured by gas solid chromatograph using thermal conductivity
detector. This method offers significant advantage over Kjeldahl method i.e.,
shorter analysis time (3-4 min), but these instruments appear to have limited
usefulness for some food products because they can only deal with very less
46 amount of sample.
12.3.4 Fat Physical and Chemical
Analysis of Foods
The oils and fats from oilseeds and fruits as well as from animal fatty tissues
correspond quite closely with those extracted by diethyl ether. Practically, all
the sterols and phosphorus containing organic compounds notably the lecithins
are extracted with the glycerides. Essential oil and resins are the chief
constituents of the ether extract of certain spices. Similarly, pepper contains
nitrogenous ether soluble substance, piperine (alkaloids). Other solvents viz.,
chloroform, carbon tetrachloride, carbon disulphide and petroleum distillates
of lower or higher boiling points dissolve fats and oils and can be used but the
yield and composition of the extract differ somewhat with the solvent. Free fat
can be extracted by the less polar solvents such as petroleum ether and diethyl
ether, whereas the bound fat requires more polar solvents viz., alcohols for
their extraction. The bound fat may be broken down by hydrolysis or other
chemical treatment to yield free fat. Hence, the amount of extracted fat found
in food products will depend on the method of analysis used.
I) Direct Solvent Extraction Method
The free fat content can be conveniently determined in foods by extracting the
dried and ground material with petroleum ether or diethyl ether in Soxhlet
extraction apparatus (Fig. 12.3). Extraction in the presence of alcohols causes
the release of lipoidal substances bound to proteins and carbohydrates viz.,
phospholipids and glycolipids. Hence, maximum extraction is obtained by a
mixture of polar and non-polar solvents. This procedure co-extracts water and
water soluble substances. Hence, the residue after solvent removal and the
addition of anhydrous sodium sulphate needs to be extracted with petroleum
ether.

BULB TYPE
CONDENSER

SYPHON

EXTRACTION
THIMBLE

ROUND BOTTOM FLASK

BOILING AID

HEATING HOOD

Fig. 12.3: Soxhlet extraction apparatus

II) Solubilization Extraction Method

Bound fat can be made free if the food sample is dissolved completely prior to
extraction with polar solvents. Dissolution of the food can be achieved by acid
or alkaline hydrolysis.
In acid hydrolysis method, the sample is heated on a steam bath with dilute
HCl and boiled for 30 min. The sample solution is filtered through a wet filter
paper and washed with hot water. The filter paper is then oven dried and
placed directly into a Soxhlet apparatus and extracted with ethyl or petroleum
ether or dichloromethane.
47
 Food Analysis In alkali hydrolysis method (Rose Gottlieb method), the material is treated
with ammonia and alcohol in cold and the fat is extracted with diethyl ether-
petroleum ether mixture. The alcohol precipitates the protein, which dissolves
in the ammonia; the fat can then be extracted with ether. Petroleum ether is
then added as it reduces the proportion of water and hence all non-fatty
substances.
(All dimensions in millimeters)

Fig. 12.4: Fat extraction Rose Gottlieb tubes with fitting

III) Volumetric Method

These involve dissolving the sample in sulphuric acid and centrifuging out the
fat in specially calibrated glass vessels (butyrometers). The Gerber method is
commonly employed for the routine determination of fat in milk and dairy
products.

12.3.5 Volatile Oil

The method involves distilling the volatile oil over with boiling water,
condensing and collecting the oil in a measured volume of xylene in a
graduated tube. (Fig. 12.5).

48
 Physical and Chemical
Analysis of Foods

All Dimension in millimeters.

Fig. 12.5: Apparatus for determination of volatile oil

12.3.6 Crude Fiber

The crude fibre representing the cell wall material left after boiling with dilute
acid and alkali in the process, is a mixture of cellulose, lignin and pentosans,
together with sand, silica and other mineral matter locked in the tissues and
little nitrogenous matter after grinding and defatting, boiling with sulphuric
acid solution, and separation and washing of the insoluble residue. This residue
is boiled with sodium hydroxide solution, separated, washed, and dried and the
insoluble residue is then weighed. The loss in mass on incineration is also
noted.

12.3.7 Dietary Fiber

The current surge of interest in dietary fibre is attributed to its prophylactic and
curative properties against colon cancer, coronary heart disease, obesity,
gallstones, constipation, bowel irregularities and even hemorrhoids. The food
industry is responding to the desires of today’s consumers for fibre-rich
products that can be used to foster their health, vitality and well-being. Dietary
fiber refers to a macro-constituent of food, which includes the remnants of
edible plant cells including polysaccharides (cellulose, hemicellulose, gums,
mucilage, pectin), lignin, and associated substances viz., oligosaccharides,
waxes, cutin and suberin that are resistant to digestion in the alimentary tract of
human being. Dietary fiber is separated from other constituents of food by
means of enzymatic hydrolysis while crude fiber is separated by means of acid
and alkali hydrolysis of other food constituents.
49
 Food Analysis I) Total Dietary Fibre (TDF)
Duplicate test portions of dried foods (fat extracted if containing >10% at) are
gelatinized with heat stable α-amylase and then enzymatically digested with
protease and amyloglucosidase to remove protein and starch. Four volumes of
ethyl alcohol are added to precipitate soluble dietary fibre. Total residue is
filtered, washed with 78% ethyl alcohol, 95% ethyl alcohol, and acetone. After
drying, residue is weighed. One duplicate is analyzed for protein and other is
incinerated at 525°C and ash is determined.

Total dietary fibre (%) = Weight of residue–weight (protein + ash) × 100

Weight of sample
II) Soluble Dietary Fibre (SDF)
Duplicate test portions of dried foods, (fat extracted if containing >10%fat), are
gelatinized with heat stable α-amylase and then enzymatically digested with
protease and amyloglucosidase to remove protein and starch. Insoluble dietary
fibre is removed by filtering and washing residue with water. Soluble dietary
fibre in filtrate is precipitated by adding 95% ethyl alcohol to filtrate.
Precipitate is filtered and washed with 78% ethyl alcohol, 95% ethyl alcohol,
and acetone, dried and weighed. One duplicate is analyzed for protein and
second is incinerated at 525°C to determine ash. Soluble dietary fibre = Weight
residue – weight (protein + ash).
III) Insoluble Dietary Fibre (IDF)
Duplicate test portions of dried foods, (fat extracted if containing >10%fat), are
gelatinized with heat stable α-amylase and then enzymatically digested with
protease and amyloglucosidase to remove protein and starch. Enzyme digest is
filtered and residue is washed with warm water, dried and weighed. Insoluble
dietary fibre residue value is corrected for protein, ash and blank.

12.3.8 Total Ash

Ash refers to the inorganic residue remaining after total incineration of organic
matter. The ash content is an indicator of product quality and the nutritional
value of food products. When a high ash figure suggests the presence of an
inorganic adulterant, it is advisable to determine the acid insoluble ash.
I) Dry Ashing
Dry ashing is the most standard method for determining the ash content of a
food sample. The sample is commonly ignited at 550-600°C to oxidize all
organic materials without flaming. The inorganic residue that does not
volatilize at that temperature is called ash. The ash content is determined from
the loss of weight, which occurs from complete oxidation of sample.
II) Wet Ashing
Wet ashing is usually used for the elemental analysis. Wet ashing commonly
employs concentrated nitric acid and perchloric acid or nitric acid and
sulphuric acid to oxidize the organic matter of the food sample. These acids
are partially removed by volatilization and the soluble minerals remain
dissolved in nitric acid. Any silica present is dehydrated and made insoluble.
However, great care must be taken when using perchloric acid, because it can
be explosive on contact with water.
50
12.3.9 Acid Insoluble Ash Physical and Chemical
Analysis of Foods
The acid insoluble ash is a measure of the sandy matter and maxima are
prescribed for herbs and spices. Acid insoluble ash is determined by dissolving
ash in dilute hydrochloric acid (10% w/w), the liquid filtered through an
ashless filter paper and thoroughly washed with hot water. The filter paper is
then ignited in the original dish, cooled and weighed.

12.3.10 Sulphated Ash

This involves moistening the ash with concentrated sulphuric acid and igniting
gently to constant weight. The sulphated ash gives a more reliable ash figure
for sample containing varying amount of volatile inorganic substances that
may be lost at the ignition temperature used.

12.3.11 Reducing and Non-Reducing Sugars

I) Lane and Eynon Volumetric Method
Reducing sugar and non-reducing sugar (after inversion) reduces the copper in
Fehling’s solution to the red precipitate cuprous oxide. The sugar content in a
food sample is estimated by determining the volume of unknown sugar
solution required to completely reduce a measured volume of Fehling’s
solution. The Fehling’s solution is an alkaline solution of copper sulphate
(CuSO4. 5H2O, 6.9%) and Rochelle salt Sodium Potassium Tartrate
(KNaC4H4O6.4H2O, 34.6%). In this method, methylene blue is used as an
oxidation-reduction indicator of the end point. The methylene blue is added to
the reaction mixture of sugar and Fehling’s solution. Its use is based on the fact
that it is reduced and completely decolourized by minute amounts of reducing
sugar or invert sugar but not so long as any cupric salt is present. The reduction
is carried out in a flask in which the liquid is kept boiling constantly to prevent
reoxidation. In acid-base indicators, the change is often in the nature of colour
i.e. in its position in the spectrum; but with oxidation-reduction indicators it is
a change in intensity of colour.
II) Colorimetric Method
Food sample is clarified with the help of suitable clarifying agent and the
filtrate has to be free from protein and fat. Phenol solution and concentrated
sulphuric acid are added to an aliquot portion of the filtrate, thus producing a
colour which is proportional to the amount of carbohydrate present, which is
measured photometrically at a wavelength of 490 nm.

12.3.12 Starch
After the sugars present in the sample are leached out, starch is hydrolyzed
using acid or enzyme and then the sugar is estimated.
I) Acid Hydrolysis Method
The sugar is leached out by precipitating starch with alcohol and the starch is
hydrolyzed with conc. hydrochloric acid and the resulting reducing sugar is
determined by titrimetric or by colorimetric method.
Starch (%) = % Reducing sugar × 0.90
II) Enzymatic Hydrolysis Method
Sugars are removed by leaching with alcohol. If much fat and proteinaceous
materials are present, the sample is treated with hot ethanolic KOH and washed 51
 Food Analysis with 80% ethanol. The starch in the residue is gelatinized and incubated with
amyloglucosidase enzyme at pH 4.5 which converts the starch to glucose
which is measured enzymatically using glucoxidase.
Let us answer a few questions before we move to the next section of physical
and chemical testing of Fats and Carbohydrates. Microscopic field is very
minute.

# Check Your Progress Exercise 2

Note: a) Use the space below for your answers.
b) Check your answers with those given at the end of the unit.
1) What are the different techniques used for moisture determination in food
products?
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…
……………………………………………………………………………….
2) Describe the principle of Kjeldahl method used for protein determination?
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…
……………………………………………………………………………….
3) Differentiate water content and water activity of food?
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…
……………………………………………………………………………….
4) Differentiate crude fibre and dietary fibre?
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…
……………………………………………………………………………….
5) What is the significance of ash content in foods?
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…

52
…………………………………………………………………………….…
6) Explain the role of methylene blue indicator in the determination of sugar? Physical and Chemical
Analysis of Foods
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…

12.4 PHYSICAL AND CHEMICAL PROPERTIES

OF OILS AND FATS
Modern advances in oils and fats technology and the nutrition science have led
to the need for greater awareness of the composition and structure of dietary
lipids and many new advanced test methods and analytical procedures have
been introduced. This section describes the general methods used to examine
oils and fats for their physical and chemical properties and methods for
assessing their quality criteria.

12.4.1 Acid Value and Free Fatty Acids

Acid value is the amount of KOH in milligram, required to neutralize the free
fatty acids present in 1 g of the oil or fat. It is determined by directly titrating
the material in an alcoholic medium with aqueous sodium or potassium
hydroxide solution. Free fatty acid is calculated as oleic, lauric, ricinoleic or
palmitic acids. Acid value when expressed, as mg of KOH/g of fat should be
not more than 4.0 in virgin oils and 0.6 in non-virgin oils.
Free fatty acid (%) = Titre volume × normality of NaOH × 28.2
(as oleic acid) Weight of sample
Acid Value = % FFA × 1.99

12.4.2 Unsaponifiable Matter

Unsaponifiable matter is that fraction of oils and fats which is not saponified
by caustic alkali, but is soluble in ordinary fat solvents. The material is
completely saponified with alcoholic potassium hydroxide solution and
extracted with petroleum ether. The petroleum ether extract is washed with
aqueous alcohol and then again with water. The washed ether extract is
evaporated and the residue weighed. Unsaponifiable matter is this residue
minus the fatty acid present in it, which is determined by titration with sodium
hydroxide solution in alcoholic medium.

12.4.3 Melting Point

Oils and fats are chiefly mixtures of glycerides. They do not exhibit either a
definite or a sharp melting point. Therefore, the term melting point does not
imply the same characteristics that it does with pure crystalline substances.
Fats pass through a stage of gradual softening before they become completely
liquid. The melting point is, therefore, defined by the specific conditions of the
method by which it is determined. The melting point is determined by taking
the solid fat inside a small capillary tube and sample may be compared by
measuring the temperature at which under specified conditions a column of fat
fixed length rises in an open capillary tube under a definite pressure (slip
point).
53
 Food Analysis 12.4.4 Solid-liquid Ratio
This provides information on the extent of saturation of triglycerides in fat, e.g.
the extent of hydrogenation of oil or the suitability of fat for a particular use.
The ratio can be measured by dilatometry. This is based on the measurement of
isothermal expansion of the fat. The sample is melted, put in an enclosed
calibrated glass tube known as a dilatometer and solidified under standardized
conditions. The temperature of the solidified fat is then raised in 5°C stages
and the volume of the fat measured each time until it is almost completely
molten. By plotting the change in volume against temperature a melting
dilation graph is obtained. However, determination of solid-liquid ratio in fat
by Nuclear Magnetic Resonance (NMR) method is more suitable and accurate.

12.4.5 Specific Gravity

The specific gravity may be determined with a specific gravity bottle or
pyknometer. The temperatures at which the specific gravity is determined shall
be reported, namely, sp gr 30°C/30°C or sp gr 95°C/30°C.

12.4.6 Titre Value

When the molten fatty acids are cooled and begin to solidify, the latent heat of
fusion is liberated and consequently a sudden rise in temperature can be
observed. The sample is prepared from the fat by saponification and
subsequent liberation of the fatty acids by dilute mineral acid. The washed and
dried fatty acids are transferred to a test tube of specified dimensions in which
they are cooled and agitated with a stirrer until solidification becomes
noticeable. The highest/simplify temperature recorded, after mixing has been
discontinued, is known as the titre value.

Fig. 12.6: Assembly of Apparatus for Titre Test

54
Determination of Titre — Fill the low-form beaker with water up to two-thirds Physical and Chemical
of its capacity. Adjust the temperature of water between 15°C and 20°C below Analysis of Foods
the expected titre point when it is not above 35°C, and at 20 ± 1°C when it is
35°C or higher. Fill the test-tube up to the mark with the fatty acid preparation
at a temperature 10 to 12°C higher than the expected titre point. Insert the titre
thermometer in the centre of the sample and adjust its height so that its
immersion mark coincides with the top surface of the fatty acid layer. When
the temperature of the fatty acid comes down to about 10°C higher than the
titre point, set the stirrer moving in a vertical direction at a rate of about 60
complete up and down motions per minute. The temperature of the fatty acid
gradually comes down and stirring is continued until the temperature remains
constant for 30 seconds. The stirring is stopped when the temperature begins to
rise and the stirrer is raised out of the sample. The highest temperature
recorded by the thermometer during this rise is the titre point. Duplicate
determinations should agree within 0.2°C.

12.4.7 Colour
This method determines the colour of oils by comparison with Lovibond
glasses of known colour characteristics. The colour is expressed as the sum
total of the yellow and red slides used to match the colour of the oil in a cell of
the specified size in the Lovibond tintometer. The colour may also be
measured in a spectrophotometer using carbon tetrachloride as blank at the
wavelength of maximum absorption.

12.4.8 Iodine Value (Wij’s)

Iodine value is a measure of level of unsaturation in fat. Iodine value is the
amount of iodine in (g) absorbed per 100 g of the oil or fat. The material is
treated, in carbon tetrachloride medium, with a known excess of iodine
monochloride solution in glacial acetic acid (Wijs solution). The excess of
iodine monochloride is treated with potassium iodide and the liberated iodine
estimated by titration with standard sodium thiosulphate solution.
Iodine value (mg/100g)= Titre volume ( blank – sample) × normality of Na2S2O3 ×12.69
Weight of sample

I Cl
‫׀‬ ‫׀‬
- CH=CH- + ICl → - H-C -C-H-
H2O + ICl → IOH + HCl
KI + HOH → KOH + HI
2Na2 S2O3 + I 2→ Na2S4O6 + 2NaI

12.4.9 Saponification Value

When fat is saponified by refluxing with a known excess of alcoholic
potassium hydroxide solution, the triglycerides hydrolyze, while glycerol and
soap are formed. The alkali consumed for this hydrolysis is a measure of the
saponification value, which is determined by titrating the excess alkali with
standard hydrochloric acid. Saponification value is defined as the amount of
KOH in mg required in saponifying completely 1 g of oil or fat. It is also a
measure of the mean molecular weight of the fatty acids originally bound as
triglycerides. 55
 Food Analysis Saponification value = titre value (blank – sample) × normality of HCL × 56.1
Weight of sample

12.4.10 Acetyl Value and Hydroxyl Value

The acetyl value of fats or oils defined as the amount of KOH in mg required
for the neutralization of the acetic acid obtained by the saponification of 1 g of
the acetylated product.
The process consists in acetylating the oil or fat with a measured quantity of
acetic anhydride in pyridine decomposing the excess anhydride by boiling with
water and then, after the addition of sufficient butyl alcohol to give a
homogeneous solution, titrating with alcoholic alkali. A control test with the
acetic anhydride and pyridine without the oil or fat provides a measure of the
acetic anhydride available for acetylation; a similar test with the oil or fat and
the pyridine without the acetic anhydride provides a measure of the free fatty
acid present. From the figures obtained, the acetyl value or the hydroxyl value
of the fat is calculated.

12.4.11 Reichert-Meissl (RM) Value

Reichert Meissl value is a measure of water soluble steam volatile fatty acids
chiefly butyric and caproic acids. RM value is the number of ml of 0.1N NaOH
solution required to neutralize the steam volatile water soluble fatty acids
distilled from 5 g of oil or fat under the precise conditions. The material is
saponified by heating with glycerol sodium hydroxide solution and then split
by treatment with dilute sulphuric acid. The volatile acids are immediately
steam distilled. The soluble volatile acids in the distillate are filtered out and
estimated by titration with standard sodium hydroxide solution. RM value is a
unique test used for evaluation of adulteration of milk fat with other fat.

12.4.12 Polenske Value

Polenske value differs from the RM value in that it is a measure of ‘steam
volatile’ but of water insoluble fatty acid like caprylic, capric and lauric acids
present in oils and fats. The condenser, the 25-ml cylinder and the receiver
used in the Reichert-Meissl value determination are washed into the filter
paper through which the distillate was filtered for that determination. After
rinsing, the residue on the filter paper is taken up with ethyl alcohol and
titrated with standard sodium hydroxide solution.

12.4.13 Rancidity
The reactions that take place when a fat becomes rancid are only partly
understood. It appears that the more common type of rancidity that results in
the formation of a rancid odour and taste is due to oxidation at the double
bonds. Apparently, peroxides are formed in the early stages of the process and
these are later decomposed into aldehydes, ketones and acid fragments. The
evaluation of rancidity can be done by determining different oxidation
products.
I) Kreis Test: The method employs phloroglucinol as a reagent in ether in
the presence of concentrated hydrochloric acid. If epihydrinaldehyde is
present, a reddish addition product is formed, indicating rancidity. Addition
reaction of aldehydes with phloroglucinol can be determined using
56 spectrophotometric detection at 540 nm.
II) Peroxide Value: The peroxide value is an indicator of oxidative rancidity Physical and Chemical
in fats. However, the incipient stages of rancidity can be detected by this Analysis of Foods
test before the spoilage can be detected organoleptically. The peroxide
value is a measure of the peroxides contained in a sample of fat, expressed
as milli-equivalents of peroxide oxygen per kg of the material. The
material in an acetic acid-chloroform medium is treated with an aqueous
solution of potassium iodide. The liberated iodine is titrated with standard
sodium thiosulphate solution. The peroxide value of fresh oil is less than
10.

Peroxide value (meq/1000g) = Normality of Na2S2O3 × titre volume × 1000

Weight of sample
H+/∆
ROO + KI → ROH + KOH + I2
2Na2S2O3 + I2 + starch → starch – I2 + Na2S4O6 + 2NaI

7.5±2.5
60±2
B
80:2

37·5 : 2·5 00

70:10
70:10

75±5
75±5

160:5
520:5 A
12: 0·5 00

300:5
45±5
E
30: 2 00

110 ml

D 100 ml

All dimensions in millimeters.

Fig. 12.7: Reichert-Meissl Distillation Apparatus

Check Your Progress Exercise 3 "

Note: a) Use the space below for your answers.
b) Check your answers with those given at the end of the unit. 57
 Food Analysis 1) What are the physical parameters used for the quality evaluation of oils and
fats?
………………………………………………………………………………
………………………………………………………………………………
…………………………………………………………………………….…
……………………………………………………………………………….
2) Define the iodine value? What is the significance of iodine value?
………………………………………………………………………………
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3) Define RM value? Briefly discuss its method of determination?
………………………………………………………………………………
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4) Which test will you perform to evaluate the rancidity in oil?
………………………………………………………………………………
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…………………………………………………………………………….…
……………………………………………………………………………….

12.5 LET US SUM UP

The physical and chemical analysis techniques used for food analysis are
enumerated in this unit. The principles and methods of determining different
physical parameters of food products are discussed in this unit. The chemical
analysis of foods includes proximate analysis and ultimate analysis. The
proximate analysis consists of determining the moisture, fat, protein, sugar,
starch, ash and crude fibre or dietary fibre. The suitability of different chemical
analysis techniques for determining different food constituents is briefly
discussed. However, the ultimate analysis that refers to the determination of a
particular element or a compound present in the material is out of the scope.
The physico-chemical properties of oils and fats are also included in this unit.

12.6 KEY WORDS

D : A
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58
n p Physical and Chemical
g e Analysis of Foods
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59
 Food Analysis t

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12.7 TERMINAL QUESTIONS

1) Describe the importance of filth in food products? Briefly discuss the
method of determination of filth?
2) Discuss the method of gradation of cereals according to the size of grains?
3) Describe at least three different techniques for the measurement of
moisture in food products?
4) Explain the different factors responsible for the error in protein
determination by Kjeldahl method?
5) Discuss the different steps involved in the determination of total dietary
fibre in foods?

78
6) Describe the effect of water activity on different physico-chemical Physical and Chemical
properties of foods? Analysis of Foods

7) How can you determine starch in foods containing higher content of sugar?
8) Name the different physico-chemical parameters oil by which we can
evaluate the purity of oil?

12.8 ANSWERS TO CHECK YOUR PROGRESS "

EXERCISES
Check Your Progress Exercise 1
Your answer should include following points:
1) Filth is any objectionable matter contributed by animal contamination of
product such as rodent, insect or bird matter or any other objectionable
matter contributed by insanitary conditions. Heavy filth can be separated
by sedimentation based on different densities of filth, after immersing food
particles in solvents like chloroform.
2) Specific heat is the number of calories (heat) required to raise the
temperature of one gram of substance by 1°C. Heat = Weight × specific
heat × change of temperature.
3) Ostwald viscometer is suitable for measuring the viscosity of liquid food
product with low consistency. Ostwald viscometer is a capillary viscometer
which gives more accurate result than any other viscometer.
4) The specific gravity of a substance is the ratio of its weight to the weight of
an equal volume of another substance taken as standard while density is the
ratio of mass to a fixed volume of a material at a particular temperature.
5) The surface tension is defined as the force acting upon a line of unit (1cm)
length in the surface of the liquid. The falling drop method is a simple
method used to measure the surface tension of a liquid.
Check Your Progress Exercise 2
Your answer should include following points:
1) More frequently moisture in food products is determined by methods like
oven drying, vacuum drying, distillation, Karl Fischer titration, etc.
2) The Kjeldahl method for protein determination in foods follows three basic
steps: (a) Digestion – decomposition of organic matter by heating in the
presence of concentrated sulphuric acid, the end result is ammonium
sulphate solution. (b) Distillation – Ammonium sulphate is converted into
gaseous ammonia by addition of an excess base, followed by boiling and
condensation of the ammonia in a receiving solution. (c) Titration –
quantification of the ammonia released from the digest.
3) The water activity is not determined by the total quantity of water in a
sample but only by that fraction which is least tightly bound.
4) Dietary fiber is separated from other constituents of food by means of
enzymatic hydrolysis while crude fiber is separated by means of acid and
alkali hydrolysis of other food constituents.
79
 Food Analysis 5) The ash content is an indicator of product quality and the nutritional value
of food products; e.g. milk powder with high ash content indicates
adulteration with alkali neutralizer.
6) In Lane and Eynon titration method for sugar estimation, methylene blue is
used as an oxidation-reduction indicator of the end point. Its use is based
on the fact that it is reduced and completely decolourized by minute
amounts of reducing sugar or invert sugar but not so long as any cupric salt
is present.
Check Your Progress Exercise 3
Your answer should include following points:
1) The important physical parameters used for the quality evaluation of
oils/fats are specific gravity, refractive index, melting point, colour, etc.
2) Iodine value is defined as the number of g of iodine absorbed per 100 g of
the oil or fat under specified conditions. Iodine value indicates the level of
unsaturation in fat.
3) RM value is defined as the number of ml of 0.1N NaOH solution required
to neutralize the steam volatile water soluble fatty acids distilled from 5 g
of oil or fat under the precise conditions. This is determined by saponifying
fat by heating with glycerol sodium hydroxide solution and then split by
treatment with dilute sulphuric acid. The volatile acids are immediately
steam distilled. The soluble volatile acids in the distillate are filtered out
and estimated by titration with standard sodium hydroxide solution.
4) Definitely rancid fat proclaims itself by odour and taste but it is not so
simple to determine the presence of rancidity in the early stages of
development. The rancidity in fat or oil can be evaluated either by Kreis
test or by determining peroxide value.

12.9 SOME USEFUL BOOKS

AOAC (2005). Official Methods of Analysis of AOAC International, 18th
Edition, (W, Horwitz, G.W. Latimer, eds.), Suite 500, Gaithersburg, Maryland,
USA.
Bureau of Indian Standards (2008). Indian Standards Institution, Manak
Bhavan, New Delhi.
Findlay, A. and Kitchener, J.A. (1962). Practical Physical Chemistry. 8th Edn.,
Longmans Publishers, London.
Kirk, R.S. and Sawyer, R. (1999). Pearson’s Composition and Analysis of
Foods. 9th Edition. Addison Wesley Longman Ltd., England.
Kirschenbauer, H.G. (1944). Fats and Oils: An Outline of their Chemistry and
Technology. Reinhold Publishing Corporation, New York.
Nollet, L.M. (2004). Physical Characterization and Nutritional Analysis.
In: Handbook of Food Analysis, Vol. I, 2nd Edn., Mercel Dekker, Inc., New
York.

80
Ranganna, S. (2003). Handbook of Analysis and Quality Control for Fruit and Physical and Chemical
Vegetable Products. 2nd Edition. Tata McGraw-Hill Publishing Company Analysis of Foods
Limited, New Delhi.

81
Food Analysis
UNIT 13 INSTRUMENTATION IN FOOD
ANALYSIS
Structure
13.0 Objectives
13.1 Introduction
13.1.1 Need for Food Analysis
13.1.2 Why do We Need Instrumentation in Food Analysis?
13.2 Selecting an Appropriate Instrumental Technique
13.2.1 Criteria for Selecting a Technique
13.2.2 Instrumental Techniques in Food Analysis
13.3 Chromatographic Techniques
13.3.1 Gas Chromatography
13.3.2 Detector for Gas Chromatography
13.3.3 Sampling Techniques
13.3.4 Applications of Gas Chromatography
13.4 Liquid Chromatography
13.4.1 Characteristic Features of HPLC
13.4.2 Comparison of HPLC and GC
13.4.3 A Typical Modern Liquid Chromatograph
13.4.4 Detectors for HPLC
13.4.5 Applications of HPLC
13.5 Thin Layer Chromatography
13.6 High Performance Thin Layer Chromatography (HPTLC)
13.7 Hyphenated Techniques
13.7.1 Gas Chromatography-Mass Spectrometry (GC-MS)
13.7.2 Liquid Chromatography-Mass Spectrometry (LC-MS)
13.8 Spectroscopic Techniques
13.8.1 Distribution of Energy in Atoms and Molecules
13.8.2 Characteristics of Electromagnetic Waves
13.8.3 Interaction of Radiation with Matter
13.8.4 Measurement Modes
13.9 Spectroscopic Instruments
13.9.1 UV-Visible Spectroscopy
13.9.2 Atomic-Absorption Spectroscopy (AAS)
13.9.3 Inductively Coupled Plasma – Optical Emmission Spectrophotometry
(ICP- OES/MS)
13.9.4 Nuclear Magnetic Resonance Spectroscopy (NMR)
13.9.5 Fourier Transform Infrared Spectroscopy (FT-IR)
13.10 Thermal Methods of Analysis
13.10.1 Thermogravimetry
13.10.2 Differential Thermal Analysis (DTA)
13.10.3 Differential Scanning Calorimetry (DSC)
13.11 Key Words
13.12 Answers to Check Your Progress Exercises
13.13 Some Useful Books

13.0 OBJECTIVES
After reading this unit, we shall be able to:
• reason advanced instrumentation required in food analysis;
• outline principles of instrumental techniques used in food analysis; and
• describe applications of instrumental technique in analysis of macro and
micro food components.
62
13.1 INTRODUCTION Instrumentation in
Food Analysis

13.1.1 Need for Food Analysis

Food analysis is the discipline dealing with the development, application and
study of analytical procedures for characterizing the properties of foods and
their constituents. All food products whether raw or processed are analyzed to
provide information about a wide variety of different characteristics, including
their composition, structure, physicochemical properties and sensory attributes.
In fact the food is analyzed for a variety of reasons, e.g. compliance with legal
and labeling requirements, assessment of product quality, determination of
nutritive value, detection of adulteration, research and development.
Food safety is an issue of prime importance. With the growing concerns about
the food and health safety, the food regulatory authorities in different countries
have imposed stringent mandatory norms for the presence of various toxicants,
which if present beyond a prescribed residual level might prove hazardous to
human health. Moreover, with the implementation of WTO and globalization,
it has become important that all food products for export out of the country
should meet the regulatory norms of the prescribed limits of different toxicants
in various food products. As, government bodies regulate the permitted levels
of contaminant compounds; much of this advancement has been driven by
increased sensitivity and specificity of determination technique e.g. using
analytical instruments.
Food Analysis serves as a unique and invaluable tool for all food scientists,
technologists and regulatory authorities for quality assurance and control of
food products, to study the different aspects of food products.
Food is a complex matrix consisting of different components. These
components can be categorized into different categories which are listed as
given below:
1) Nutrients: e.g. Proteins, Amino acids, Total cholesterol, Trans Fats and
Lipid profile, Carbohydrates, Sugars, Dietary fiber, Vitamins, Minerals etc.
Depending upon the food product some of them may be present at high
concentration levels while others may be present at low concentration
levels of parts per million.
2) Additives: e.g. Colors, Dyes, Stabilizers, Antioxidants, Flavors and
Fragrance, Preservatives, etc.
The additives are added to the food products for the purpose of giving the
food products desired appearance, texture, flavor and extending the shelf
life. The additives are usually present at very low concentration levels.
3) Adulterants: They are added intentionally to the food products mostly for
the purpose of cost benefits and they may be present at higher as well as
lower amounts. They may be safe or sometimes highly toxic, such as,
argemone in mustard oil, sudan red in chillies, animal cholesterol in ghee,
low cost vegetable oil in high cost vegetable oil etc.
4) Contaminants and Toxicants: Toxicants can be classified into Physical
toxicants (e.g. glass, wood, metal, insect matter etc.); Biological toxicants
such as microbes and pathogens; and Chemical toxicants such as residual
pesticides, residual antibiotics, mycotoxins, and environmental pollutants
like PAH (polycyclic aromatic hydrocarbons), PCB (polychlorinated
63
Food Analysis biphenyls), Dioxins, toxic metals etc. Most of the times these contaminants
are not added intentionally but find there way into the food products from
environmental pollution or if proper practices are not being followed
during agriculture, animal breeding, storage or processing. The various
toxicants are present at low levels of concentration and if present beyond a
certain prescribed level of concentration in food products may prove to be
highly toxic or carcinogenic to humans.

13.1.2 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. More often than not, interferences are encountered during
the measurement of minor components in the presence of the components
present in bulk quantities. All 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.

13.2 SELECTING AN APPROPRIATE

INSTRUMENTAL TECHNIQUE
There are usually a number of different analytical techniques available to
determine a particular property of a food material. It is therefore necessary to
select the most appropriate technique for the specific application. The
analytical technique selected depends on the property to be measured, the type
of food to be analyzed and the reason for carrying out the analysis.

13.2.1 Criteria for Selecting a Technique

Some of the criteria that are important in selecting an instrumental analytical
technique are listed below:
• Precision: A measure of the ability to reproduce a result by a specific
analyst (or group of analysts) using the same equipment and experimental
approach kepping other conditions unchanged.
• Reproducibility: A measure of the ability to reproduce result using the
same experimental approach in same as well as different laboratories using
same/different equipment.
• Accuracy: A measure of how close one can actually measure the value to
the true value of the parameter being measured.
• Simplicity of operation: A measure of the ease with which relatively
unskilled workers may carry out the analysis.
• Speed: Analysis of single sample or the number of samples in a given time.
• Sensitivity: A measure of the lowest concentration of the component that
can be detected by a given procedure.
• 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.
64
• Nature of food matrix: The composition, structure and physical properties Instrumentation in
of the matrix material surrounding the analyte often influences the type of Food Analysis
method that can be used to carry out an analysis e.g. whether the matrix is
solid or liquid, transparent or opaque, polar or nonpolar.
If there are a number of alternatives 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.

13.2.2 Instrumental Techniques in Food Analysis

Analysis of food products for the majority of the parameters can be undertaken
using different instrumental techniques as described below.
• Chromatographic Techniques
• Hyphenated Techniques
• Spectroscopic Techniques
• Thermal methods of analysis

13.3 CHROMATOGRAPHIC TECHNIQUES

Chromatography is defined as a process by which solutes are separated by a
dynamic differential migration in a system consisting of two or more phases,
one of which moves continuously in a given direction called as mobile phase
and in which the individual substances exhibit different mobilities by reason of
differences in adsorption, partition, solubility, vapour pressure, molecular size
or ionic charge density. The individual substances thus separated can be
identified or determined using appropriate technique. Chromatographic
techniques can broadly be classified into:
1) Gas Chromatography (GC)
Gas chromatography is applied to volatile organic compounds. The mobile
phase is a gas and the stationary phase is usually a liquid on a solid support or
sometimes a solid adsorbent.
2) Liquid Chromatography (LC)
Liquid chromatography is used to separate analytes in solution including metal
ions and organic compounds. The mobile phase is a solvent and the stationary
phase is a liquid on a solid support, a solid, or an ion-exchange resin.

13.3.1 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 analysed by GC
must be volatile and must be thermally stable below 350°C.
A schematic diagram of a gas chromatograph showing various components is
presented in Fig. 13.1. Samples are rapidly injected by means of a hypodermic
micro syringe through a silicone rubber septum into the column. The sample
injection port, column, and detector are heated to temperatures at which the
65
Food Analysis sample has a vapour pressure of at least 10 torr, usually about 50ºC above the
boiling point of the highest boiling solute. The injection port and detector are
usually kept at higher temperature than the column to promote rapid
vaporization of the injected sample and prevent sample condensation in the
detector. For packed columns, liquid samples of 0.1 to 10 μL are injected. For
capillary columns, volumes of only about 1/100 these sizes must be injected
because of the lower capacity (albeit greater resolution) of the columns.
Sample splitters are included on chromatographs designed for use with
capillary columns that deliver a small fixed fraction of the sample to the
column, with the remaining part going to waste.
The response is detected in the form of peaks. Separation occurs as the vapor
constituents equilibrate and partition between carrier gas and the stationary
phase. The carrier gas is a chemically inert gas available in pure form such as
argon, helium, or nitrogen.
The sample is automatically detected as it emerges from the column (at/or a
constant flow/ gas pressure rate), using a variety of detectors whose response is
dependent upon the composition of the vapor. The chromatographic peaks are
recorded as a function of time. By measuring the retention time (elapsed time
in minutes between the time a sample is injected and the time the
chromatographic peak reaches maximum intensity) and comparing this time
with that of a standard of the pure substance, it may be possible to identify the
peak (agreement of retention times of two compounds does not guarantee the
compounds are identical). The area under the peak is proportional to the
concentration, and so the amount of the substance can be quantitatively
determined. The peaks are often very sharp and, if so, the peak heights can be
compared with a calibration curve prepared in the same manner.

Fig 13.1: Schematic Diagram of a Gas Chromatograph

13.3.2 Detector for Gas Chromatography

Various types of detectors are used for different applications:
• Flame Ionisation Detector (FID)
• Electron Capture Detector (ECD)
66
• Nitrogen Phosphorous Detector (NPD) Instrumentation in
Food Analysis
• Thermal Conductivity Detector (TCD)
A) Flame Ionisation Detector (FID): FID is the most widely used of all
detectors because, it is reliable, easy to maintain and operate, rugged and
responds to almost all classes of compounds but, insensitive to water. The
response is roughly dependent upon number of carbon atoms in the
molecule. Hydrogen and air are the required gases for the ignition of flame.
An organic compound is burnt in a flame produced by H2 and air which in
turn produces ions.
Organic sample + H2 + O2 -----> CO2 + H2O + (ions)+ + e-
The ions produced are collected by a pair of polarized electrodes inside the
detector and the current is produced which is directly proportional to the
concentration. The current is amplified and then recorded.
B) Electron Capture Detector (ECD): ECD is the most sensitive detector for
halogenated compounds, anhydrides, peroxide, conjugated carbonyls,
nitrites, nitrates and organometallics, but, insensitive to hydrocarbons,
alcohols, ketones and amines. Solvent containing analyte from the column
is passed over a β (electron)-emitter (radioactive compound that emits
electrons, e.g. 63Ni). Electron from the emitter causes ionization of carrier
gas producing a burst of electrons, which produce a constant current
between a pair of electrodes. But, when a solute with higher electron
effinity is eluted from the column, some of the electrons are captured and
the current flow is reduced in proportion to the concentration of the eluting
compound.
C) Nitrogen Phosphorous Detector (NPD): NPD is a selective detector for
nitrogen and phosphorous containing organic compounds, e.g.
organophosphorus and carbamate pesticides. NPD is similar in design to
FID. In this detector nitrogen and phosphorus containing molecules exiting
the column collide with electrically heated ceramic thermoionic bead
which is positioned near the jet orifice and undergo catalytic surface
chemical reaction. Ions created in this reaction are attracted towards a
collector electrode, amplified and output is given to data system.
D) Thermal Conductivity Detector (TCD): This detector is used for the
analysis of permanent gases. It responds to all gases and vapours with a
thermal conductivity different from that of carrier. In this detector, no
separate fuel is required. This detector consists of two pair of heated
elements, each as an arm of a Wheatstone bridge over which the column
effluent and a reference gas stream flow. When there is only reference gas,
balance of conductivity in the bridge is maintained. But when the analyte
reaches the filament, the thermal conductivity is changed, causing
unbalanced bridge which provide signal.

13.3.3 Sampling Techniques

There are different types of sampling techniques normally employed for GC
analysis of volatile samples:
1) Headspace analysis.

67
Food Analysis 2) Thermal Desorption.
3) Purge and Trap technique
1) Headspace Analysis
A sample in a sealed vial is equilibrated at a fixed temperature, for example 10
min. and the vapour in equilibrium above the sample is sampled and injected
into the gas chromatograph flavour analysis etc.
2) Thermal Desorption
Thermal Desorption (TD) is a technique in which solid or semisolid samples
are heated under a flow of inert gas. Volatile and semi volatile organic
compounds are extracted from the sample matrix into the gas stream and
introduced into a gas chromatograph. Samples are typically weighed into a
replaceable PTFE tube liner, which is inserted into a stainless steel tube for
heating.
Thermal desorption is well suited for dry or homogenous samples such as food
packaging films, spices, coffee flavour profile, voletile organics in wine,
mushrooms, fruits, honey etc. solid foods, cosmetics, ointments, and creams.
3) Purge and Trap Technique
The Purge and Trap technique is a variation of thermal desorption analysis in
which volatiles are purged from a liquid sample placed in a vessel by bubbling
a gas (e.g. air or nitrogen) through the sample and collecting the volatiles in a
sorbent tube containing a suitable sorbent. The trapped volatiles are then
analyzed by thermally desorbing them from the sorbent. This is a form of
‘Headspace’ analysis in which analytes are concentrated prior to introduction
into the GC.
Purge and Trap is suitable for non-homogenous samples, since fairly large and
high moisture samples can be taken. Examples include foods such as pizza or
fruits. The measurements of malodorous organic volatiles in the headspace
vapor above a sample of stored food, is used to determine whether it still meets
the “freshness” requirements.

13.3.4 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.

13.4 LIQUID CHROMATOGRAPHY

Liquid chromatography covers a variety of separation techniques such as liquid
solid (adsorption chromatography), liquid-liquid (partition chromatography),
68
ion exchange, size exclusion, thin layer, high performance thin layer and paper Instrumentation in
chromatography; all involving a liquid mobile phase. In this unit, we would Food Analysis
learn more about the liquid chromatographic techniques used specially for the
purpose of food analysis i.e.
• High Performance Liquid Chromatography (HPLC)
• Thin Layer Chromatography (TLC)
• High Performance Thin Layer Chromatography (HPTLC)
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.
● Normal phase chromatography
In this technique, the stationary phase is polar. A non-polar mobile phase, such
as n-Hexane, methylene chloride, or chloroform is used. The stationary phase
is bonded silioxane with a polar functional group, e.g. Cyano, diol, amino,
dimethylamino, etc. These phases retain polar compounds in preference to
non-polar compounds.
● Reversed phase chromatography
In Reversed Phase Chromatography (RPC) relatively non-polar stationary
phase is used, with a polar mobile phase constituting of methanol, acetonitrile,
tetrahydrofuran, etc., usually in combination with water. Methanol and
acetonitrile are most common. The water content is varied for adjusting the
polarity. Methanol is used for acidic compounds and acetonitrile for basic
compounds. Tetrahydrofuran is used for those with larger dipoles. These
solvents are UV transparent and have low viscosity. The most common bonded
phases are n-octaldecyl (C18 ) or n-octyl (C8) bonded polysiloxanes.

13.4.1 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.

13.4.2 Comparison of HPLC and GC

HPLC in some respect is more versatile then 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.
69
Food Analysis 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.
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.
13.4.3 A Typical Modern Liquid Chromatograph
A Typical Modern Liquid Chromatograph Consists of:
i) Solvent delivery system which includes a pump
ii) Sample injection system
iii) Column and Detector
iv) Chromatography software and Computer
Compounds are separated by injecting a sample mixture into the column. The
different components in the mixture pass through the column at different rates
due to differences in their partitioning behavior between the mobile and the
stationary phase. As the sample solution flows through the column with the
mobile phase, the components present in the injected sample solution migrate
according to the non-covalent interactions of the compounds with the column.
The mobile phase could be run in isocratic and gradient modes. A separation
by HPLC, which employs constant composition of mobile phase throughout
the chromatographic run, is called an isocratic elution and in case two or more
solvents of different polarities are used in different proportions during the
chromatographic run the method is known as gradient elution.
The stationary phase in HPLC refers to the solid support contained within the
column through which the mobile phase continuously flows. Columns
containing various types of stationary phases are available commercially. For
most of the separation in food analysis the columns normally used are Normal
phase (e.g. silica) or the Reverse phase (e.g. C8, C18). Samples are injected into
the HPLC via an injection port. In modern HPLC systems, the sample injection
is typically automated.
13.4.4 Detectors for HPLC
In order to detect the compounds as they elute from the column there are many
types of detectors that can be used with HPLC. Some of the common detectors
include: Ultra Violet (UV), Photo Diode Array (PDA), Refractive Index (RI),
Fluorescent, and Mass Spectrometric (MS) detector.
A) Refractive Index (RI): Detectors measure the ability of sample molecules
to bend or refract light. This property for each molecule or compound is
called its refractive index. For most RI detectors, light proceeds through a
bi-modular flow-cell to a photo detector. One channel of the flow-cell
directs the mobile phase passing through the column while the other directs
only the mobile phase. Detection occurs when the light is refracted due to
samples eluting from the column, and this is read as a disparity between the
two channels.

70
B) Ultra-Violet (UV): Detector measure the ability of a sample to absorb Instrumentation in
radiation in the UV region. This can be accomplished at one or several Food Analysis
wavelengths.
C) Photo Diode Array (PDA): Detector produces a three-dimensional graph
that assists in examining the purity of a peak. The chromatographic peak is
supported by authentication through UV-VIS spectrum.
D) Fluorescent: Detector measure the ability of a compound to absorb and
then re-emit light at given wavelengths. Each compound has a
characteristic fluorescence. The excitation source passes through the flow-
cell to a photodetector while a monochromator measures the emission
wavelengths.
E) Mass Spectroscopy (MS) Detectors: The sample compound or molecule
is ionized, passed through a mass analyzer, and the ion current is detected.
There are various methods for ionization.
solvent
reservoir

processing unit
pump to
and display
produce high
pressure

signal to processor

sample detector
injection
HPLC tube

waste

Fig. 13.2: Schematic Diagram of HPLC

13.4.5 Applications of HPLC

HPLC finds wide applications in food, both for profiling and analysis of
various components such as:
1) Amino acids profiling, peptides and hproteins.
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 Mycotoxin, Antimicrobial and veterinary drugs, pesticides, etc.
7) Pigments, colorants and phenolic compounds.
8) Bittering substances.
9) Additives, preservatives, antioxidants and stabilizers in processed food
products.

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Food Analysis
13.5 THIN LAYER CHROMATOGRAPHY
Thin Layer Chromatography (TLC) is an important chromatographic
technique, frequently used for qualitative identification and initial screening of
components. A sample is spotted onto the plate or a strip with a micropipette
and the plate or the strip is placed in a suitable solvent to develop the
chromatogram. The solvent is drawn up the plate by capillary action, which
moves the sample components up the plate at different rates, depending upon
their solubility and their degree of retention by the stationary phase. Following
development, the individual spots are noted or made visible by treatment with
a reagent that forms a coloured derivative. For example, amino acids and
amines are detected by spraying the plate with a solution of ninhydrin,
resulting in a blue or purple coloured spot. If the solute compound is
fluorescent, they can be detected by exposing to UV light. The spots generally
move at a certain fraction of the rate at which the solvent moves and they are
characterized by the Rf value.
Distance solute moves from the point of application
Rf =
Distance solvent front moves
Rf value is characteristic for a given stationary phase and solvent combination.
Since the separation and identification spots on TLC is based upon visual
observation, at certain times, if the product analyzed contains large number of
components, the method may suffer from poor resolution due to the closely
lying or the overlapping spots and poor specificity. Therefore, the results may
sometimes be uncertain, misleading or inaccurate.
watch glass

thin layer chromatography breaker

plate

pencil line

solvent
spot of mixture
Fig. 13.3: Schematic Diagram of a thin Layer Chromatographic Technique

13.6 HIGH PERFORMANCE THIN LAYER

CHROMATOGRAPHY (HPTLC)
HPTLC enhances the power of thin layer chromatography by improving the
speed and efficiency of separation by the development of instrumentation to
automate sample application, development of the chromatogram, and
detection, including accurate and precise in situ quantitation.
HPTLC is a selective, precise, and accurate chromatographic technique for
fingerprint analysis of food products. This involves densitometric evaluation
after resolving the components of the sample on silica gel plates with very fine
72
particle size. For densitometric evaluation, peak areas are recorded at the Instrumentation in
appropriate wavelength. HPTLC has the advantages of many fold possibilities Food Analysis
of qualitative and quantitative detection in analyzing food products with
accuracy and precision.
Applications
HPTLC can be used for a large number of applications in food industry for
screening purposes, e.g.:
• Determination adulterants in food products such as argemone in mustard
oil.
• Separation of food colours in food samples.
• Determination of residues of mycotoxins in food products.

Check Your Progress Exercise 1 "

Note: a) Use the space below for your answers.
b) Check your answers with those given at the end of the unit.
1) Define Chromatography technique?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
2) Explain the applications of Gas Chromatography?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
3) Name the detectors used for HPLC and GC.
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………

13.7 HYPHENATED TECHNIQUES

In the past two decades, combining chromatographic separation system on-line
with a spectroscopic detector in order to obtain structural information on the
analytes present in a sample has become the most important approach for the
identification and/for confirmation of the identity of target and unknown
chemical compounds. For most (trace-level) analytical problems in research
field of food products, the combination of column liquid chromatography or
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Food Analysis capillary gas chromatography with a mass spectrophotometer has become the
preferred approach for the analysis of food products. Two very important
examples of hyphenated procedures that are used in food analysis are GC-MS
and LC-MS. The information obtained from GC-MS or LC-MS is not only
useful for quantitation but also provide confirmation of the analyte, specially if
the residue is present at trace level. In GC and HPLC analysis, the results
sometimes might be misleading as compounds having similar chemical nature
often elute at approximately the same retention time.
Mass spectrometry is an analytical technique that identifies the chemical
composition of a sample on the basis of the mass to charge ratio of charged
particles. The technique has both qualitative and quantitative uses, including:
• Identifying unknown compounds by the mass and mass fragmentation
pattern, which are usually specific in nature. Identification is also possible
on comparison to standard mass spectrometry libraries, which usually
accompanies GC-MS instruments. In addition, it is also possible to develop
own library of standard reference compounds that can be utilized in
identifying compounds in unknown samples.
• Determining the isotopic composition of elements in a compound.
• Determining the structure of a compound by observing its fragmentation
pattern.
• Quantifying the amount of a compound.

Fig. 13.4: Schematic Diagram of GC-MS

The underlying principle of mass spectrometry is that as charged particles ions

pass through electric and magnetic fields, their paths vary according to their
mass-to-charge ratios. Devices which operate according to this principle are
called mass spectrometers. The design of a mass spectrometer has three
essential modules: an ion source, which transforms the molecules of a sample
into a variety of ionized fragments; an analyzer, which sorts the ions by their
masses, using internally generated electric and magnetic fields; and a detector,
which measures the value of some indicator quantity and thus providing data
for calculating the abundances of respective ion fragments.

13.7.1 Gas Chromatography-Mass Spectrometry (GC-MS)

The capabilities of integrated gas chromatography-mass spectrometry are
almost unique in meeting the requirements for analytical methods which are
74
not only highly sensitive but also specific and reliable in providing information Instrumentation in
on specific compounds as a function of their concentrations. Food Analysis

The GC portion of this system provides separation of volatile organic solutes

in a mixture in the gas phase. As each solute exits the GC column, it is diverted
into a mass spectrometer which is capable of monitoring both the amount and
identifying the chemical nature of the solute. In this way, both quantitative and
qualitative information about the mixture can be obtained.
The MS portion of the system takes each gaseous solute eluting out of gas
chromatograph and ionizes it in an electron beam. The ions formed by a
specific solute will depend on the nature of the bonds in the molecule, and it is
possible to get both ionized molecules and its fragments. The ions thus formed
are then directed down a separator which isolates and counts the ions
according to mass. The sequence and relative intensity of the mass peaks give
information about the chemical identity of the solute. The absolute intensity of
the peaks provide information about the amount of substance present.
Applications of Gas Chromatography Mass spectrometry:
Gas chromatography is useful for a number of applications in food analysis
specially from the point of view of safety of the food products. The technique
is useful for:
1) Pesticide residue analysis in all raw and processed food products.
2) Analysis of environmental contaminants, such as polyclorinated biphenyls,
polyaromatic hydrocarbons, dioxins, etc. in food products.
3) Fatty acid profiling in oils and fats.
4) Flavours and fragrance in food products.
5) Volatiles and other residual solvents in food packaging material.

13.7.2 Liquid Chromatography-Mass Spectrometry (LC-MS)

Liquid Chromatography-Mass Spectrometry (LC-MS) is an analytical
technique that combines the physical separation capabilities of HPLC coupled
with the confirmation by mass spectrometry. LC-MS is a powerful technique
used for many applications which has very high sensitivity and specificity.
Generally, its application is oriented towards the specific detection and
potential identification of chemicals in the presence of other chemicals in a
complex mixture.
LC-MS technique is used when very high sensitivity and specificity at trace
residue level is required.
Types of Mass analyzer used :
There are a different types of mass analyzers that could be used in LC/MS.
• Single Quadrupole,
• Triple Quadrupole,
• Ion Trap,
• TOF (Time of Flight), and
• Quadrupole-time of flight (Q-TOF).
75
Food Analysis The quadrupole and ion trap instruments are highly sensitive and hence used
for target-oriented residue analysis; whereas the TOF and Q-TOF instruments
are mainly used for accurate mass analysis and structure identifications of
target and non-target compounds, metabolite identifications, etc.
Applications of Liquid Chromatography-Mass Spectrometry (LC-MS)
LC-MS is routinely used for detection of :
1) Mycotoxins: LC-MS is routinels used for detection of: Toxins produced by
different fungi, e.g. Aspergillus sp., Fusarium sp., Penicillium sp., etc.
Some of the mycotoxins regularly analyzed in food samples include
Aflatoxins B1, B2, G1 and G2, ochratoxin A, etc.
2) Residual drugs and antibiotics in different food products.
3) Banned dyes and colourants e.g. Sudan dyes in different food products.
4) Residual pesticides in raw and processed food products.

13.8 SPECTROSCOPIC TECHNIQUES

Spectroscopy pertains to the dispersion of an object's light into its component
colors (i.e. energies) via prism or the study of the interactions 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. A variety of the instruments that are commonly
used to analyze food materials based on spectroscopy includes, UV-visible,
fluorescence, atomic, infrared and nuclear magnetic resonance spectrometer.

13.8.1 Distribution of Energy in Atoms and Molecules

Atoms and molecules can only exist in a limited number of discrete energy
levels: they cannot have energies between these levels, i.e., their energy levels
are quantized. Each molecular species has a unique set of energy levels that
depends on its unique atomic structure (electrons, protons, neutrons) and
molecular structure (type and arrangement of atoms and bonds). The lowest of
these energy levels is referred to as the ground state, while higher levels are
referred to as excited states. The potential energy of an atom or molecule is
usually defined relative to the ground state (which is arbitrarily taken to have
zero energy). The potential energy of a molecule is made-up of contributions
from a number of different sources: electronic, vibrational, rotational,
translation and nuclear.
• Electronic Energy Levels: Electrons in an atom are arranged into a
number of different shells and sub-shells. An electron can move from one
of these sub-shell levels to another by absorbing or emitting radiation of
appropriate energy. The system is then said to have undergone an
electronic transition. Electronic transitions may involve electrons that are
in inner shells (higher energy) or outer shells (lower energy) of atoms.
• Vibrational Energy Levels: Molecules (but not atoms) can vibrate in a
number of different modes, e.g., the atoms can compress or stretch along
the axis of a bond, or they can bend symmetrically or asymmetrically. Each
of these vibrations occurs at a characteristic frequency (energy) that
depends on the mass of the atoms and the strength of the bonds involved.
76
• Rotational Energy Levels: Molecules often contain chemical groups that Instrumentation in
are capable of rotating around certain bonds at fixed frequencies (and Food Analysis
therefore energies). Each group has a specific number of frequencies at
which it rotates and therefore has a specific number of quantized rotational
energy levels. The rotation frequency is determined by the mass of the
atoms involved and their distance from the axis of rotation.
• Nuclear Energy Levels: The nuclei of certain atoms have a property
known as spin. A (charged) spinning nucleus generates a small magnetic
field and can be thought of as a small magnet. Normally, this magnet can
be orientated in any direction, but in the presence of an external magnetic
field it can only align itself either with or against the field, i.e., it is
quantized. Transitions between the different energy levels within the nuclei
can be made to occur by applying radiation of a specific energy to the
sample.
• Translational Energy Levels: Atoms and molecules are in continual
translational motion because of the thermal energy of the system.
Translational energy levels are quantized, however, the differences
between the energy levels are so small that the molecules act as though the
energy is distributed continuously.

13.8.2 Characteristics of Electromagnetic Waves

Electromagnetic waves may be thought of as small packets of energy (photons)
that move through space with wave-like properties, i.e., they exhibit wave-
particle duality (e.g. photoelectric effect). They consist of oscillating electric
and magnetic fields that are perpendicular to one another, and to the direction
of propagation. The sinusoidal variation in the amplitude of the electric vector
of the wave can be plotted as a function of time (at a fixed position within a
material) or as a function of distance (at a fixed point in time). A
monochromatic (single wavelength) electromagnetic wave that propagates
through a vacuum can be described completely by its frequency, wavelength
and amplitude (or parameters derived from these):
• The frequency (v) of a wave is the number of cycles per second (Hz = s-1).
• The period (T) of a wave is the time taken to complete a cycle: T = 1/v.
• The wavelength λ is the distance between successive maxima/minima of a
wave.
• The wave number (cm-1) is the number of cycles per unit distance.
• The amplitude (A) of a wave is the maximum magnitude of the electric
vector.
• The intensity (I) of a wave is proportional to the square of the amplitude. It
is the amount of energy passing through a given area per second.
Increasing the intensity of an electromagnetic wave increases the number
of quanta passing a given area per second, not the energy of each
individual quantum.
• The velocity (c) of an electromagnetic wave is the distance travelled per
second: c = v λ The velocity of an electromagnetic wave travelling through
a vacuum is the speed of light c = 3 × 108 ms-1. The velocity of an
electromagnetic wave traveling through a material is always less than that
in a vacuum. The refractive index of a material is equal to cvacuum/cmaterial.
77
Food Analysis • The energy (E) of the photons in an electromagnetic wave is related to the
frequency of the wave:
E = hv = h/T = hc/ λ
where, h = Planks constant (6.6262 × 10-34 J s). These expressions can be
used to relate the energy of an electromagnetic wave to its frequency,
period, wavelength or wave number. This relationship indicates that
monochromatic radiation (i.e., radiation of a single frequency) contains
photons that all have the same energy.
The electromagnetic spectrum consists of radiation that ranges in
wavelength from 10-12 m (high energy) to 104 m (low energy). The physical
principles and mathematical description of radiation across the whole of
the electromagnetic spectrum is the same, however, it is convenient to
divide it into a number of different regions depending on the origin of the
waves, i.e., cosmic rays, gamma rays, x-rays, ultraviolet, visible, infrared,
microwaves, and radio waves.

13.8.3 Interaction of Radiation with Matter

Spectroscopic techniques utilize the fact that atoms and molecules have a
discrete set of energy levels and that transitions can only occur between them.
When an electromagnetic wave propagates through a material, the atoms or
molecules can absorb energy and move to an excited state if the photons in the
wave have energies that are exactly equal to the difference between two energy
levels (ΔE = hv). Alternatively, if an excited atom or molecule emits energy in
the form of radiation the waves emitted must have energies that are exactly
equal to the difference between two energy levels (ΔE = hv). The energy of the
photons in different regions of the electromagnetic spectrum corresponds to
different types of energetic transitions that can occur in atoms and molecules,
e.g., electronic, rotational, vibrational, translational, nuclear transitions.
Electromagnetic radiation can therefore be used to probe different molecular
characteristics of matter. The atomic or molecular origin of the transitions that
occur between different energy levels in matter, the region of the
electromagnetic spectrum that these transitions correspond to, and the
spectroscopic techniques that can be used to measure these transitions are
summarized below:

Transition Region of e/m Spectroscopy Technique

Spectrum

Electronic (kJ mol-1) UV-Visible UV, Visible, Atomic

Fluorescence

Vibrational (10 kJ mol-1) Near and Mid Infrared

Infrared

Rotational (0.1 kJ mol-1) Far Infrared, Infrared

Microwaves

Nuclear (10-6 kJ mol-1) Radio Waves Nuclear Magnetic

Resonance (NMR)

78
The difference between electronic energy levels is greater than between Instrumentation in
vibrational energy levels, which is greater than between rotational energy Food Analysis
levels. Thus higher energy radiation (shorter wavelength) is needed to cause
transitions between electronic levels than between vibrational or rotational
levels. In practice, a molecule can be thought of as having a number of
different electronic energy levels, with rotational and vibrational energy levels
superimposed on them.
I) Absorption
Absorption is the process by which energy is transferred from an
electromagnetic wave to an atom or molecule and causes it to move to an
excited state. Absorption can only occur when an atom or molecule absorbs a
photon of light that has energy, which exactly corresponds to the difference
between two energy levels, i.e., it must be quantized. At room temperature, the
ground state of atoms and molecules is usually the most probable and stable
state and hence transitions usually occur from the ground state to higher energy
levels. At higher temperatures, more of the higher energy levels are occupied
and so, transitions between higher energy levels may also become important.
If an atom or molecule is subjected to electromagnetic radiation of different
wavelengths (energies) it will only absorb photons at those wavelengths which
correspond to exact differences between two different energy levels within the
material. A plot of the fraction of photons absorbed at a particular wavelength
versus the energy of the photons at that wavelength is called an absorption
spectrum. Conventionally, the axis of absorption spectra are specified in terms
of easily measurable quantities: x-axis shows transmittance or absorbance
(rather than fraction of photons absorbed); y-axis shows wavelength, frequency
or wave number (rather than photon energy).
II) Emission
Emission of radiation is the reverse of absorption, occurring when energy from
an atom or molecule is released in the form of a photon of radiation. When a
molecule is raised to an excited state it will only exist in this state for a very
short time before relaxing back to the ground state. This is because it will
always try to move to its lowest energy state. There are two important
relaxation processes through which an excited molecule can dissipate its
energy:
• Non-radiative decay: This is the most common way that an excited
molecule loses its energy. Energy is dissipated in a number of small
(quantized) steps due to transfer of energy from the excited molecule to
surrounding molecules in the form of kinetic energy (heat). Nevertheless,
the heat generated is usually so small that it has little effect on the overall
temperature of the system.
• Radiative decay: In some cases, an atom or molecule loses its energy in
the form of a photon (emission). This is the case in atomic emission
spectroscopy.
Sometimes both of these processes occur together. In fluorescence
spectroscopy, a molecule absorbs electromagnetic radiation, which causes it to
move into an excited state. It then returns to the ground state by dissipating
some of its energy in the form of non-radiative decay and the rest in the form
of a photon of radiation. The photon emitted is therefore of lower energy
79
Food Analysis (longer wavelength) than the incident wave. Usually, an electron decays to the
lowest energy level in the excited electronic state, and then returns to the
ground state.

13.8.4 Measurement Modes

The design of an analytical instrument based on spectroscopy depends on the
nature of the energetic transitions involved (e.g., electronic, vibration, rotation,
translation, nuclear), the nature of the radiative process involved (e.g.,
absorption, emission, fluorescence) and the nature of the food matrix (e.g.,
absorbing or non-absorbing). These factors determine the wavelength
(frequency) of electromagnetic radiation used, the way that the electromagnetic
radiation is generated and the way that the electromagnetic radiation is
detected. Some commonly used designs are highlighted below:
• Emission: The sample being analyzed is energetically stimulated (e.g., by
heating or application of radiation) and the amount of electromagnetic
radiation produced by the sample is measured at different wavelengths,
e.g., atomic emission spectroscopy, NMR, fluorescence.
• Transmission: An electromagnetic wave generated by the analytical
instrument is propagated directly through the sample and the reduction in
its amplitude due to interaction with the sample is measured at different
wavelengths, e.g., atomic absorption spectroscopy, infrared transmission
measurements, UV-visible spectrophotometery.
• Reflection: An electromagnetic wave generated by the analytical
instrument is reflected from the surface of the sample and the reduction in
its amplitude due to interaction with the sample is measured at different
wavelengths, e.g., infrared reflection measurements, color measurements.

13.9 SPECTROSCOPIC INSTRUMENTS

The most commonly used spectroscopic instruments for food analysis are:
• UV- Visible Spectroscopy
• Atomic-Absorption Spectroscopy (AAS)
• Inductively Coupled Plasma (ICP)
• Nuclear Magnetic Resonance Spectroscopy (NMR)
• Fourier Transform Infrared Spectroscopy (FTIR)

13.9.1 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. Chemical
profiling using W-visible spectroscopy complemented with chemical profiling
with other chromatographic technique can be used as reference for quality
control of food products.

80
 Instrumentation in
Food Analysis

Fig. 13.5: Schematic Diagram of UV-visible Spectroscopy

A beam of light from a visible and/or UV light source is separated into its
component wavelengths by a prism or diffraction grating. Each
monochromatic (single wavelength) beam in turn is split into two equal
intensity beams by a half-mirrored device. One beam, the sample beam passes
through a small transparent container (cuvette) containing a solution of the
compound being studied in a transparent solvent. The other beam, the
reference passes through an identical cuvette containing only the solvent. The
intensities of these light beams are then measured by electronic detectors and
compared. The intensity of the reference beam, which should have suffered
little or no light absorption, is defined as I0. The intensity of the sample beam
is defined as I. Over a short period of time, the spectrometer automatically
scans all the component wavelengths in the manner described. The ultraviolet
(UV) region scanned is normally from 200 to 400 nm, and the visible portion
is from 400 to 800 nm. If the sample compound does not absorb light of a
given wavelength, I = I0. However, if the sample compound absorbs light then
I is less than I0, Absorption may be presented as transmittance (T = I/I0) or
absorbance (A= log I0/I). If no absorption has occurred, T = 1.0 and A= 0.
Most spectrometers display absorbance on the vertical axis, and the commonly
observed range is from 0 (100% transmittance) to 2 (1% transmittance). The
wavelength of maximum absorbance is a characteristic value, designated as
λmax. Different compounds may have very different absorption maxima and
absorbances.
The most commonly used solvents are water, ethanol, hexane and cyclohexane.
Solvents having double or triple bonds, or heavy atoms are generally avoided.

81
Food Analysis 13.9.2 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.
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. The lamp is filled with an inert gas like argon or neon.
When a potential is applied, the gas is ionized and is driven towards the
cathode and cause the metal atoms to sputter all the surface of the cathode and
produce specific atomic emission lines.
The characteristic emission lines produced by the source i.e. hollow cathode
lamp are absorbed by the atoms which get excited and are raised to higher
energy level. As the sample passes through the flame, the beam of light passes
through the monochromator. The monochromator isolates the specific
spectrum line emitted by the light source through spectral dispersion and
focuses it upon a photo multiplier detector where light signal is converted into
an electrical signal. The process of electrical signal is fulfilled by a signal
amplifier. The signal could be displayed for readout or further fed into a data
station.

lens lens detector

monochromator
hollow atomized
cathode lamp sample

readout amplifier

Fig. 13.6: Schematic of an Atomic-Absorption Experiment

The greater amount of sample present, the higher the absorbance energy.
Different flames can be achieved by using different mixtures of gases,
82
depending on the desired temperature and burning velocity. Some elements can Instrumentation in
only be converted to atoms at high temperatures. Even at high temperatures, if Food Analysis
excess oxygen is present, some metals form oxides that do not re-dissociate
into atoms. To inhibit their formation, conditions of the flame may be modified
to achieve a reducing, non-oxidizing flame. The most widely used flames are
air-acetylene and nitrous oxide-acetylene flame. The nitrous oxide-acetylene
flame has a higher temperature as compared to air-acetylene flame.
A) Electrothermal Atomization
In determination of certain elements (Ag, Be, Ca, Fe, Pb and Sn), flame atomic
absorption spectroscopy may not be very sensitive. Use of electro thermal
atomization provides sensitivity up to the levels of parts per billion (ppb) to
subparts per billion levels. This type of atomization requires a graphite furnace,
which is a graphite tub, where the sample is rapidly atomized after thermal pre-
treatment. To maintain a dense fraction of free ground state elements in the
optical path, an inert gas atmosphere is used. Since the dilution and expansion
effects of flame cells are avoided, and the atoms have a longer residence time
in the optical path, a higher peak concentration of atoms is obtained.
B) Vapour Generation- Atomic Absorption Spectroscopy (AAS-VGA)
Vapour Generation- Atomic Absorption Spectroscopy (AAS-VGA) is used for
the determination of hydride-forming elements, such as As, Hg, Sn, etc., using
quartz tube atomizer. VGA-AAS is highly sensitive and effective method for
quantification of toxic metals in food products and can be used for the
determination of these elements at ppm to ppb levels.
Applications of Atomic-absorption spectroscopy
1) Atomic absorption spectroscopy can be used to analyze the concentration
of esssential minerals and toxic metals in all raw and processed food
products.

13.9.3 Inductively Coupled Plasma – Optical Emmission

Spectrophotometry (ICP-OES/MS)
Inductively Coupled Plasma (ICP) is an analytical technique used for the
detection of trace metals in Food samples. The primary goal of ICP is to get
elements to emit radiations of characteristic wavelength that can then be
measured.
An ICP requires that the elements, which are to be analyzed, in the solution
form. An aqueous solution is preferred over an organic solution, as organic
solutions require a special accessory prior to introduction of a sample into the
ICP. Solid samples are also discouraged, as clogging of the instrument can
occur. The nebulizer transforms the aqueous solution into an aerosol. The light
emitted by the atoms of an element in the ICP must be converted to an
electrical signal that can be measured quantitatively
An ICP typically includes the following components:
• Sample introduction system (nebulizer)
• ICP torch
• High frequency generator

83
Food Analysis • Transfer optics and spectrometer
• Computer interface
ICP is an atomic emission technique using argon plasma as an excitation
source. The sample is introduced into a premix spray chamber, where it is
directed up the central tube of the ICP ‘‘torch’’. The torch consists of
concentric tubes with independent argon streams flowing through each tube.
The top of the torch is centered within a Radio Frequency (RF) induction coil,
which is the source of energy for the system. After ignition, the plasma is
propagated through inductive coupling with the RF field generated from the
coil. The ICP torch is designed specifically to promote penetration of the
plasma skin by the sample, allowing sample atoms to experience the full
energy of the plasma source. The high temperatures provided by the ICP
provide excellent sensitivities for refractory elements and also essentially
eliminate chemical interferences. Like all emission techniques, there are no
source lamps. By monitoring several wavelengths, either all at once or in a
programmed sequence, many elements can be determined in one automated
analysis. ICP emission, therefore, offers significant speed advantages over
atomic absorption for multi element analysis. Except for the refractory
elements, which may be substantially better than even graphite furnace AA,
ICP detection limits are comparable to flame atomic absorption. The high
temperatures of the ICP carry one disadvantage. The plasma is so effective in
generating excited state species that the rich emission spectra produced
increase the probability of spectral interferences. High resolution
monochromators and sophisticated software for background and inter element
correction are used to deal with this potential problem. Another limitation of
ICP emission is the initial cost of the instrument. The price for basic ICP
systems starts at about the same level as the prices for top-of-the-line
automated AA systems. More sophisticated instrument can cost two to four
times the price of basic systems.

Mass discriminator
and Detector
Sample Introduction
and Aerosol
Generation
Ionization by
Argon Plasma

Data Analysis

Fig. 13.7: Schematic Diagram of ICP – OES

84
13.9.4 Nuclear Magnetic Resonance Spectroscopy (NMR) Instrumentation in
Food Analysis
NMR spectroscopy exploits the magnetic properties of certain nuclei. The most
important applications for the organic chemist are proton NMR and carbon-13
NMR spectroscopy. In principle, NMR is applicable to any nucleus possessing
spin.
Much information can be obtained from an NMR spectrum. Analysis of a
NMR spectrum provides information on the number and type of chemical
entities in a molecule. A bit more detail + enclosures followed.
The impact of NMR spectroscopy on the natural sciences has been substantial.
It can, among other things, be used to study mixtures of analytes, to understand
dynamic effects such as change in temperature and reaction mechanisms, and
is an invaluable tool in understanding protein and nucleic acid structure and
function. It can be applied to a wide variety of samples, both in the liquid and
the solid state.

13.9.5 Fourier Transform-Infrared Spectroscopy (FT-IR)

The mid-IR region covers the range from 4000 to 400 cm-1. The basic principle
of the IR technique is that various organic functional groups absorb infrared
light at specific wavelengths. Thus, since every organic molecule has a unique
chemical composition, it also has a unique infrared spectrum. Biological
samples are composed of proteins, carbohydrates, lipids and nucleic acids.
Since these molecules contain functional organic groups, the IR spectrum
consists of bands from all these components.
The infrared spectrum is very complex, and it contains a large amount of
information. To evaluate the data, it is necessary to use multivariate statistical
analysis.
Some of the applications of FT-IR Spectroscopy for food analysis include:
1) Characterization of essential oils in various spices
2) Evaluation of trans fats in oils and fats

Check Your Progress Exercise 2 "

Note: a) Use the space below for your answers.
b) Check your answers with those given at the end of the unit.
1) What do you understand by Hyphenated techniques? List any two
examples of this technique?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
2) Which Spectroscopic technique is used for analyzing the concentration of
metals and minerals in food products?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
85
Food Analysis 3) What are the application of FT-IR Spectroscopy for food analysis?
………………………………………………………………………………
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………………………………………………………………………………

13.10 THERMAL METHODS OF ANALYSIS

Most foods are subjected to variations in their temperature during production,
transport, storage, preparation and consumption, e.g. pasteurization,
sterilization, evaporation, cooking, freezing, chilling etc. Temperature changes
cause alterations in the physical and chemical properties of food components
which influence the overall properties of final product e.g., taste, appearance,
texture and stability. Chemical reaction such as hydrolysis, oxidation or
reduction may be enhanced, or physical changes, such as evaporation, melting,
crystallization, aggregation or gelation may occur. A better understanding of
the influence of the temperature on the properties of food enables food
manufacturers to optimize processing condition and improve product quality. It
is therefore important for food scientist to have analytical technique to monitor
the changes that occur in food. These techniques are often grouped under the
general heading thermal analysis, at present the term thermal analysis is
usually reserved for a narrow range of technique that measures changes in the
physical properties of foods with temperature e.g. mass, density, rheology, and
heat capacity.
A variety of differential analytical technique have been deployed to monitor
changes in the physical properties of food components that occurs in response
to controlled changes in temperature. A number of most important of these
thermal analysis techniques are described below:

13.10.1 Thermogravimetry
Thermogravimetry technique continuously measure the mass of a sample as it
is heated or cooled at a controlled rate, or it is held at a particular temperature
for a period of time with reference to a control sample. Thermogravimetry is
useful for monitoring process that involves a change in the mass of food or
food component, e.g., drying, liberation of gasses, absorption of moisture. To
study the various types of processing and storage condition that a food might
normally experience, thermogravimetric instruments have been specially
designed to allow measurements to be carried out under specific environments
e.g., controlled pressures or atmosphere. Gravimetric instruments typically
consist of a sensitive balance situated within a container whose pressure,
temperature and gaseous environment can be carefully controlled. The ability
to carefully control the temperature, pressure and composition of the gases
surrounding a sample is extremely valuable for food scientist, because it allows
them to model processes such as drying, cooking, and uptake of moisture
during storage.
Differential Thermal Analysis and Differential Scanning Calorimetry
DTA and DSC are techniques rely on changes in the heat absorbed or released
by a material as its temperature is varied at a controlled rate. These changes
occur when components within a food undergo some type of phase transition
86
(e.g., crystallization, melting, evaporation, glass transitions, conformational Instrumentation in
change) or a chemical reaction (e.g. oxidation, hydrolysis) Food Analysis

13.10.2 Differential Thermal Analysis (DTA)

DTA is defined as a technique for recording the difference in temperature
between a substance and a reference material against time or temperature as
the two specimen are subjected to identical temperature regimes in a
environment heated or cooled at a controlled rate. A typical instrument consists
of two measurement cells that are located in a temperature controlled
environment, whose temperature can be varied in a programmed manner. The
sample to be tested is placed into the ‘sample cell’ while a reference material
of known thermal properties (often distilled water) is placed in the "reference
cell". The two cells are then heated or cooled at a controlled rate. The small
difference in temperature between a "sample cell" and "reference cell"
(ΔT =T sample- T reference) is measured using accurate thermocouples placed
below the cells as the temperature of the external environment (T external) is
varied in a controlled fashion. The output of the instrument is therefore a plot
of ΔT versus T external. The position of the peak provides information about the
temperature that the transition occurs. The under a peak depends on the amount
of material involved in the transition and the enthalpy change per unit amount
of material.

Fig. 13.8: Differential Thermal Analysis

13.10.3 Differential Scanning Calorimetry (DSC)

DSC is a technique for recording the energy required to keep a zero
temperature difference between a sample cell and a reference cell that are
heated or cooled at a controlled rate. The thermocouples constantly measure
the temperature of each cell and heaters supply heat to one or other of the cells
so that they both have exactly the same temperature. If a sample were to
undergo a phase transition it would either absorb or release heat. DSC data is
therefore reported as the rate of energy absorption (Q) by the sample relative to
the reference material as a function of external temperature. Information about
thermal transitions that occur within a sample is obtained by analyzing the Q
versus T external thermo gram. It should be noted that it is possible to measure
the change in the heat release by a material as a function of time under
isothermal (constant temperature) conditions.

87
Food Analysis
Area = ∆ H melt

Area = ∆ H cryst
dQ
dt

Tg Tcryst Tmelt T

Fig. 13.9: An illustrated Thermogram

Applications
• Determining Specific heat capacity: The Specific heat capacity is an
important physical quantity in food industry, since it is the amount of
energy that must be supplied or withdrawn from a material in order to
increase or decrease its temperature by a 1ºC. The knowledge of Specific
heat capacity of a material is therefore important in the design of processes
such as chilling, freezing, warming, sterilization and cooking.
• Phase transitions: DSC and DTA are routinely used in the food industry
to characterize phase transitions in foods, for e.g. crystallization, melting,
glass transitions and conformational changes. They can be used to provide
information about the temperature at which transitions occur. When a
material changes its physical state from solid to liquid (melting) or from
liquid to solid (crystallization) it absorbs or gives out heat respectively.
Pure substances usually have very sharp melting or crystallization points
and therefore all the heat is absorbed or evolved over a narrow range of
temperatures, leading to sharp DSC or DTA peak. Many food components
are chemically complex materials and therefore the phase transitions occur
over a wide range of temperature, e.g. edible oils contain a wide variety of
different triacylglycerols each with its own melting points. Peaks from food
oils may also be complicated by the fact that triacylglycerols can crystallize
in more than one different crystalline structure i.e. they are polymorphic.

# Check Your Progress Exercise 3

Note: a) Use the space below for your answers.
b) Check your answers with those given at the end of the unit.
1) Name different types of Thermal analysis methods used in food?
………………………………………………………………………………
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88
2) Describe briefly the various applications of Thermal method of analysis in Instrumentation in
food? Food Analysis

………………………………………………………………………………
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13.11 KEY WORDS

Chromatography : It is a technique where solutes are separated
by a dynamic differential migration in a
system consisting of two or more phases
where one is mobile phase and the other is a
stationary phase.
Spectroscopy : Spectroscopy pertains to the dispersion of a
light 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).
Mass Spectrometry : Mass spectrometry is an analytical technique
that identifies the identity of a chemical
substance on the basis of the mass to charge
ratio of fragmented charged particles.
HPLC : It is a separation technique with a solid
stationary phase (column) and a liquid mobile
phase.
GC : It is a separation technique with a solid
stationary phase (column) and gaseous mobile
phase. Generally used for volatile compounds
analysis.
AAS : Atomic spectroscopy is the study of
absorption and emission characterictics of
geseous atoms of elements.
ICP : Elements are excited by Argon plasma to
gaseous ions which are further identified and
quantified by emission spectroscopy or mass
spectrometry.
Rheological Technique : It is the study of deformation and flow in food
or material in response to an applied load or
deformation.
Hyphenated Technique : It is the combination of two or more analytical
techniques, e.g. chromatographic separation
system (HPLC/GC) on-line with a
spectroscopic detector (MS), in order to obtain
structural information of the analytes present
in a sample.
89
Food Analysis
NMR Spectroscopy : NMR spectroscopy is the technique of
studying structural properties of compounds
such as type and nature of bonds using the
magnetic properties of certain nuclei
possessing spin.
Wheatstone Bridge : It is an electrical circuit. In wheat-stone
bridge four resistance R1, R2, R3 and R4 are
connected end to end with each other to form
a closed loop. A sensitive glavanometer “G”
is connected between their junctions. The
circuit is provided with two keys “K1” and
“K2”. Generally wheat-stone bridge is used to
determine unknown resistances.

# 13.12 ANSWERS TO CHECK YOUR PROGRESS

EXERCISES
Check Your Progress Exercise 1
Your answer should include following points:
1) It is a technique where solutes are separated by a dynamic differential
migration in a system, consisting of two or more phases where one is
mobile phase and the other is a stationary phase.
2) Gas Chromatography can be applied for analysis and determination of
different compounds in food products such as:
a) Cholesterol, Fatty acid profiling and Trans fat analysis;
b) Antioxidants and Preservatives like TBHQ, Benzoic acid, Sorbic acid
Acetic acid, etc;
c) Analysis of residual pesticides and environmental contaminants;
d) Characterization of flavours and fragrances; and
e) 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.
3) Detector used for gas chromatography are:
Flame Ionisation Detector (FID), Electron Capture Detector (ECD),
Nitrogen Phosphorous Detector (NPD) Thermal Conductivity Detector
(TCD)
Detector used for HPLC are:
Ultra Violet (UV), Photo Diode Array (PDA), Refractive Index (RI),
Fluorescent, and Mass Spectrometric (MS) detector.

Check Your Progress Exercise 2

Your answer should include following points:

90
1) It is the combination of two or more analytical techniques, e.g. Instrumentation in
chromatographic separation system (HPLC/GC) on-line with a Food Analysis
spectroscopic detector (MS), in order to obtain structural information of the
analytes present in a sample.
Examples: GC-MS, LC-MS
2) Atomic Absorption Spectroscopy
3) Applications of FT-IR Spectroscopy for food analysis include:
a) Characterization of essential oils in various spices.
b) Evaluation of trans fats in oils and fats.
Check Your Progress Exercise 3
Your answer should include following points:
1) Thermogravimetry
Differential Thermal Analysis
Differential Scanning Calorimetry (DSC)
2) a) Thermal method of analysis is useful in measuring specific heat
capacity of a material.
b) DSC and DTA are used to characterise phase transition in foods e.g.
crystallization, melting, glass transition and conformational changes.

13.13 SOME USEFUL BOOKS

S.S. Nielsen, (1998). Introduction to Food Analysis. Aspen Publishers - The
best general overview of food analysis techniques currently available. .
Y. Pomeranz and C.E. Meloan. Food Analysis: Theory and Practice. Chapman
and Hall - General overview of food analysis techniques.
D.W. Gruenwedel and J.R. Whitaker. Food Analysis: Principles and
Techniques. Marcel Dekker - General overview of food analysis techniques.
C.S. James. Analytical Chemistry of Foods. Blackie Academic and
Professional - General overview of food analysis techniques.
Official Methods of Analysis, Association of Official Analytical Chemists -
Officially recognized methods of analysis for many food components.

91
Food Analysis
UNIT 14 SENSORY EVALUATION OF FOOD
PRODUCTS
Structure
14.0 Objectives
14.1 Introduction
14.2 Selection of Panel
14.2.1 Types of Panel
14.2.2 Methodology for Sensory Evaluation
14.3 Maintaining Suitable Environmental Conditions
14.3.1 Laboratory Set-up and Equipment
14.4 Sample Preparation
14.5 Types of Tests
14.5.1 Analytical Tests
14.5.2 Affective (Preference and Acceptance) Tests
14.6 Applications of Sensory Evaluation
14.7 Key Words
14.8 Answers to Check Your Progress Exercises
14.9 Some Useful Books

14.0 OBJECTIVES
After reading this unit, you would be able to:
• underline the importance and significance of sensory evaluation;
• enlist quality control aspects of sensory evaluation;
• Evaluate and assess the quality of food products based on the sensory
parameters; and
• know the relevance and significance of sensory evaluation of food products
and use sensory evaluation as a tool for improvements in quality of food
products besides creating the consumer acceptance.

14.1 INTRODUCTION
As we all know, the sensory behavior of food products is the ultimate criterion
for the acceptability of any product by the consumer. Unless the food products
meet the desired standards of taste, flavor, texture, etc., the consumer will not
accept the products. In other words, quality of food products to a consumer
means the sensory behavior of products. Overall quality of food products
depends on factors such as quantity, nutritional parameters, physico-chemical
and physico-mechanical parameters, several other hidden attributes and
sensory properties. At the time of buying any food product, we should look for
nutritional parameters, like calories, the vitamins, the minerals, the proteins,
and other ingredients, etc. At the same time, we also remain concerned with
the presence of undesirable substances in the food products, for example, all
the toxic and allergic ingredients. Since the undesirable constituents in food
products may cause serious health hazards, usually, these parameters are
regulated by stringent government guidelines and norms. Labelling of
nutritional parameters is required by law in most countries.

92
The most important parameter for both the processors and consumers is the Sensory Evaluation of
sensory quality. As the name suggests, the term ‘sensory’ is related to senses of Food Products
the human being. Sensory quality is important to processor, since it attracts
consumers but it is equally important to the consumers, since it satisfies their
aesthetic and gustatory senses.

Keeping in view, the importance of the sensory quality, the sensory attributes
needs to be evaluated. Evaluation of sensory quality has been defined as “a
scientific discipline used to evoke, measure, analyze and interpret reactions to
those characteristics of foods and materials as they are perceived by the senses
of sight, taste, touch and hearing”.

Sensory evaluation of products is assuming increasing significance, as it

provides information for several purposes i.e. quality control, assessment of
process variation, cost reduction, product improvement, shelf-life, new product
development and analysis of market.

To derive maximum benefits out of sensory evaluation, it is necessary to

follow the methodology in its full scientific perspective. The basic steps to
perform the sensory analysis are:

a) Selection of the proper panel;

b) Maintaining suitable environmental conditions and use of standard

equipment for the test;

c) Obtaining representative samples;

d) Preparation and presentation of samples for evaluation in a manner that

ensures the uniformity and representation of the samples; and

e) Selection of the proper methods and statistical techniques.

14.2 SELECTION OF PANEL

Sensory evaluation is normally carried out by designed experiments under
proper environmental conditions by both trained and untrained panels. The
experts who have acquired the product-specific skills are not appropriate for
the general evaluation because of the risk of being biased.
Panels with different degree of training are required for different types of
sensory analysis. The degree of training required depends on a number of
considerations, such as degree of differences to be detected, number of
panelists required for the tests and time and value of the analysis to the product
type. Training is also necessary for descriptive and profile panels using special
procedures for specific products and situations.
For any sensory evaluation test, at least ten members should be selected in a
panel. The panel members should be recruited based on various criteria such
as:
a) Interest and motivation
Candidates who are interested in sensory analysis and who have the liking for
the test product or products to be investigated are likely to be more motivated

93
Food Analysis and hence, such kind of people are likely to become better assessors than those
who are without such essential interest and motivation.

b) Attitude

Candidates who are quite flexible in their eating habits should be considered.
Those with rigid attitudes with strong dislikes and likes towards food products
may not be suitable to become a member of the panel.

c) Knowledge and aptitude

Candidates having capacity to concentrate and those who can remain

unaffected from all the external influences and those having adequate
knowledge of all aspects of the product should be considered as panelists.

d) Health

The candidates must have good general health and should not have any
disabilities, which may affect their senses.

e) Ability to communicate/Personality characteristics

The candidates must have ability to communicate and describe the sensations
they have perceived while assessing a product. The candidates should be
reliable and honest in their approach.

14.2.1 Types of Panel

A) Trained Panel (Laboratory Panel)

The candidates should be carefully selected and trained and it need not be an
expert panel. Trained panels provide answers to two general questions relating
to the sensory properties of foods:

• Is there a difference between or among stimuli?, and

• What are the direction and the intensity of differences?

The trained panels should ideally have 5 to 10 members and the same should
be used in all developmental and processing studies. The panel should be able
to establish the intensity of a sensory characters of overall quality of a food.
The panel for flavor profile studies should have a higher degree of training for
detailed analysis of the flavor spectrum of complex processed foods.

B) Discriminative and Communicative (also known as D and C) Panel

(Semi-Trained Panel)

This type of panel should be constituted of people familiar with quality of

different classes of foods and thus is capable of discriminating differences and
communicating their reactions. The panelists may not be trained formally but
they should be capable of following instructions given at the evaluation
session. The panel should consist of about 25 to 30 members and should be
used to find the acceptability of preference of final products as a preliminary
screening programme to select a few for large scale consumer trials.

94
C) Untrained Panel (Consumer Panel) Sensory Evaluation of
Food Products
The members of the untrained panel should be selected at random from the
potential consumers in a market area. The number of panelists should be large
enough to ensure due representation of different age, sex, race and income
level group in the population of potential consumers. The findings should be
based on at least 100 independent judgements.

D. Qualifications for Panelists

The panelists particularly for the trained and Discriminative and

Communicative (D&C) panels should have the following qualifications:

a) Sound health without any defects affecting sensory perception;

b) Average sensitivity;

c) Capability of independent judgement;

d) Ability to concentrate, train and learn;

e) Intellectual curiosity and interest in quality evaluation work;

f) Willingness to spend time in evaluation and submission to periodic tests;

g) Freedom from prejudices in respect of a particular food product; and

h) Food enthusiast having the liking for trying different types of foods.

14.2.2 Methodology for Sensory Evaluation

Prior to the start of the sensory evaluation process, the following steps needs to
be accomplished:

a) Screening

The panel members should be selected keeping in view the products to be

evaluated. The prospective members should appear for a test designed so as to
pick the members of desired level of sensitivity.

b) Training

The panel members should undergo a period of training in the type of work
they shall be doing later. The members should be educated in special
vocabulary and they should be taught to be able to appropriately perceive and
express the sensory reactions. Testing sessions should be preceded by a few
informal orientation sessions in which the type of sample is introduced and
discussed and tentative decisions made about testing conditions, temperature,
quantity, mode of presentation, etc. Further, the language used to describe the
character notes of aroma, taste and overall quality should be developed and
tested. Reference standards for expressing amplitudes should also be discussed
in these orientation sessions.

95
Food Analysis c) Briefing of Panel

The panel members should be given clear and precise instructions before they
start testing. When a quality attribute is evaluated, the instructions should be
given in the scorecard. In case of rating tests, the panelist should be given clear
and precise instructions in respect of scale used to help anchor judgements in
respect of degree and direction of quality attributes and grade specification.
The instructions should not lead the panel to the identity of particular samples
or induce errors of anticipation.

# Check Your Progress Exercise 1

Note: a) Use the space below for your answers.
b) Check your answers with those given at the end of the unit.
1) Describe Sensory Evaluation?
………………………………………………………………………………
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2) What is a sensory panel?
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3) List the types of sensory panel?
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 Sensory Evaluation of
14.3 MAINTAINING SUITABLE ENVIRONMENTAL Food Products
CONDITIONS

14.3.1 Laboratory Set-up and Equipment

Fig.14.1 shows a typical layout of the sensory laboratory.

a) General

Environmental factors, where the sensory evaluation is to be done and samples

which need to be evaluated, should be suitably controlled. Sensory evaluation
should be conducted in quiet and well-lit rooms, free from any odors. The
rooms should be constructed to have comfort for concentrated prolonged
testing and ease of cleaning. Pleasing neutral shades and maintenance of
comfortable temperature and humidity conditions of the whole area or at least
the room where panel members are going to sit and discuss are desirable and
appealing. All these are essential to help the panel members develop interest
for carrying out the test. The testing area where booths are located should be
separated from sample preparation and wash rooms.

b) Reception and Briefing Room

As the name suggests, the reception and briefing room is the place where the
panel members are entertained first. This room should be well maintained and
equipped with comfortable chairs. It should be designed to ensure maintenance
of pleasant attitudes and it should minimize the congestion to the booths. Panel
members should assemble here and to start with, the panel members should be
briefed about the test, etc. here.

c) Panel Booths

These areas are the actual places for the tests. The booths should be located
between or adjacent to the reception and preparation rooms and should consist
of test booths of identical design, a separate table having natural daylight or
illuminated with special daylight bulbs for evaluation of colors of samples and
a table. The entry and exit to the panel booth area through independent doors is
often useful to avoid any communication between panel members, which may
lead to any bias while assessing the sample.

d) Preparation Room

The preparation room should be suitably separated from the testing room and it
should be equipped for preparing and serving samples. The room should have
facilities for cooking of samples with additional facilities for prepared food
storage cabinets -hot and cold. The kitchen ventilation should be such that
cooking odors are expelled from the laboratory and should not penetrate the
panel-booth area.

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Food Analysis

Exit

HOT COLD
EXHAUST HOOD ELECTRIC COLD STORE WATER WATER DRAIN
AND DRAIN
CONNECTED COOKING SINK SINK BOARD
REFRIGERATION BOARD
WINDOW

TO EXHAUST RANGE
INDICATION COLOUR
LIGHTS
DAY LIGHT OR
COLOURED LIGHTS PREPARATION AND SERVICE ROOM SHELVES
AS REQUIRED

Entrance to
SERVICE TABLE preparation
room

Exit

AIR TESTING BOOTH

CONDITIONER
VISUAL EXAMINATION
TABLE
Figure 1: Layout of a typical sensory laboratory
TESTING ROOM

DAY LIGHT FITTINGS

SHELVES
PIN BLACK
BOARD BOARD

Entrance to
panel booth
WINDOW

RECEPTION ROOM

FILING
CABINETS

Entrance
Fig. 14.1: Layout of a typical sensory laboratory

14.4 SAMPLE PREPARATION

Samples should be prepared in a way to bring out the difference in a particular
quality attribute under evaluation. All variables like temperature, time of
boiling, quantity and composition of water, blending, etc., should be controlled
to ensure identical method of preparation for all samples. Care should be taken
that no loss of flavor occurs and no foreign tastes or odors are imparted by the
procedure during preparation, storage, serving, etc.
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 Sensory Evaluation of
14.5 TYPES OF TESTS Food Products

The sensory tests may be broadly classified into two major categories i.e.:
1) Analytical tests: Analytical tests are based on the evaluation of differences
in clarity, quality and/or quantity of sensory characteristics of a product.
The panelists for carrying out the analytical tests are screened for interest,
ability to discriminate differences and reproduce results. They are trained
to function as a human analytical instrument
2) Affective tests: Affective tests are based on the evaluation preferences
and/or acceptance and/or opinions of product.
Both these categories have been summarized in Table 14.1

14.5.1 Analytical Tests

Analytical tests are used for laboratory evaluation of products in terms of
differences or similarities and for identification of sensory characteristics.
There are two major types of analytical tests – discriminative and descriptive.
Both the tests employ experienced and/or trained panelists. Potential panelists
are screened for selected personal traits, interests and ability to discriminate
differences and generate reproducible results.

Table 14.1: Types of Tests for Sensory Evaluation

Classification of Appropriate methods Type and No. of panelists
methods by function

A. ANALYTICAL TESTS

(a) Discriminative

(i) Difference test: • Paired-comparison • Normal sensory acuity.

Measures simply the • Duo-trio • Panel size depends on
difference between • Triangle product variability and
the methods. • Ranking judgement reproducibility.
• Rating difference/scalar • A recommended minimum
difference from control number is generally 5,
since any fewer could
represent too much
dependence upon one
individual’s responses.
(ii) Sensitivity test: • Threshold
Measures the ability • Dilution
of individuals to • Rating difference/
detect sensory scalar difference from
characteristics Control
(b) Descriptive Attribute rating
Measures qualitative • Category scaling • Sensory acuity
and/ or quantitative • Ratio scaling (Magnitude • Motivated
characteristics Estimation) • Trained or highly trained
• Flavor profile analysis
• Texture profile analysis
• Quantitative descriptive
Analysis

99
Food Analysis B. AFFECTIVE TESTS
• Paired-performance • Randomly selected
• Ranking • Untrained
• Rating • Representative of target
- Hedonic (verbal or facial) population
scale • Consumers of test product
- Food action scale • No recommended “magic
number” – minimum is
generally 24 panelists,
which is sometimes
considered rough product
screening; 50-100 panelists
usually considered
adequate
a) Discriminative Tests
There are two types of discriminative tests; difference and sensitivity test.
Difference tests measure whether samples can be differentiated at some
predetermined level of statistical probability. Sensitivity tests measure the
ability of individuals to detect sensory characteristics.
(i) Difference test : There are several types of Difference Tests.
• Paired Comparison Test
Two coded samples are evaluated simultaneously or sequentially in a balanced
order of presentation. This test is used to find simple difference and directional
difference in a specific characteristic and difference preference in consumer
analysis of foods. This is also applicable in training and testing of panelists.
In simple difference test, panelists are asked to test whether the samples in
each pair are the same or different. Whereas, in case of directional difference,
the panelists are asked to indicate which sample in the pair has greater or lesser
degree of intensity of a specified sensory attribute.
The data is evaluated using the evaluation card having the judgement of the panelist. A
typical evaluation card for the paired comparison tests is given below:
Format 1A: Specimen Evaluation Card For Paired Comparison Test
(Simple Difference)
Name: Date:
Product: Time:
You are given one or several pairs of samples.
Evaluate the two samples in the pair for difference in* ________________.
Indicate your judgement by crossing out words not applicable.
Pair No. Code No. of Pairs Your Judgement
01. -------- --------- Different/Not different
02. -------- --------- Different/Not different

Signature
*The panel organizer should indicate qualify attributes to be evaluated.

100
 Format 1B: Specimen Evaluation Card For-Paired Comparison Sensory Evaluation of
Food Products
(Directional difference/preference)

Name: Date:
Product: Time:
You are given one or several pair of samples.
Evaluate the two samples in the pair for difference/preference in* __________.
Indicate your judgement by crossing out words not applicable.
If different, indicate the Code No. of the sample which is more*___ /preferred.
Pair No. Code No. of Pairs Your Judgement If samples in a pair
are different, code
no. of sample,
this is more ___
preferred.
1. ______ ______ Different/Not different _________________
2. ______ ______ Different/Not different _________________

Signature
*The panel organizer should indicate the quality attributes to be evaluated.

The data is analyzed using binomial and multinomial distribution (probability)

tables for panel selection, product difference or preference and when and when
number of observations exceeds the table value, χ2-test or t-test for percentage
for product difference or preference has to be used.

• Duo-Trio Test
This test employs three samples, two identical and one different. One sample is
identified as the standard and presented first, followed by two coded samples,
one of which is identical to the standard. The judge is required to identify the
sample, which matches the standard.
The data is evaluated using the evaluation card having the judgement of the
panelist. A typical evaluation card for the duo-trio test is as below:

Format 2: Specimen Evaluation Card For-Duo-Trio Test

Name: Date:
Product: Time:
⇒ The first sample ‘R’ is the reference sample. Test it carefully.
From the pair of coded samples next given, judge which sample is the same as
‘R’.

101
Food Analysis Pair No. Code No. of Pairs Code No. of sample
Matching with ‘R’
1. _________________ _________________
2. _________________ _________________
3. _________________ __________________

Signature

The data is analyzed using binomial and multinomial distribution (probability)

tables for panel selection, product difference or preference. When number of
observations exceeds the table value, χ2-test or t-test for percentage for product
difference or preference has to be used.
• Triangle Test
This test employs three coded samples, two identical and one different,
presented simultaneously. None of the sample is identified as the standard. The
judge must determine which of the three samples presented differs from the
other two.
The data is analyzed using binomial and multinomial distribution (probability)
tables for panel selection, product difference or preference and when number
of observations exceeds the table value, χ2-test or t-test for percentage for
product difference or preference has to be used.
The data is evaluated using the evaluation card having the judgement of the
panelist. A typical evaluation card for the triangle test is as below:

Format 3: Specimen Evaluation Card For-Triangle Test

Name: Date:
Product: Time:
⇒ Two of the three samples are identical.
Determine the odd sample.
Pair No. Code No. of Samples Code No. of Odd Sample
1. ______ ______ ______ ___________________
2. ______ ______ ______ ___________________
3. ______ ______ ______ ___________________

Signature

• Ranking Test
This test is used to make simultaneous comparisons of several samples on the
basis of a single characteristic. A control needs to be identified; all test samples
to be coded. Samples (which may include control or standard) are presented
simultaneously and ranked accordingly.
102
The data is evaluated using the evaluation card having the judgement of the Sensory Evaluation of
panelist. A typical evaluation card for the ranking test is as given below: Food Products

Format 4: Specimen Evaluation Card for-Ranking Test

Name: ___________________ Date:______________________

Product:__________________ Time: _____________________

Please rank the samples in numerical order according to intensity of quality

attribute under test of the product or your preference.

Intensity/Ranking Code No. or

Preference Sample
First ____________________
Second ____________________
Third ____________________
Fourth ____________________

Comments: (Type of off-flavour, etc.)

Signature
The data obtained from ranking test is evaluated by adopting the following
statistical recommendations:
a) If the number of samples exceeds 7, adopt χ2-test;
b) Rank sum analysis has to be adopted for product difference/preference
when the number of observations is within 20;
c) χ2-test has to be used for product difference/preference;
d) Analysis of variance (ANOVA) is adopted for the ranks converted to
normal scores for multiple comparison.
(ii) Sensitivity Test: There are several ways of carrying out sensitivity test.
• Threshold test
These tests are usually expressed as absolute, and indicate the minimum
detectable level of concentration of a substance. Criteria of response in
determining threshold include detection threshold (awareness of change from
some neutral background) and recognition threshold (point at which the
stimulus becomes identifiable).

103
Food Analysis The identification threshold concentrations (sensitivity of individual panelists)
and just noticeable difference values are found from the panel data. The data
from the homogeneous panel is used for product evaluation by finding
arithmetic or geometric mean according to concentration series given.
The data is evaluated using the evaluation card having the judgement of the
panelist. A typical evaluation card for the threshold test is as given below:

Format 5: Specimen Evaluation Card for-Threshold Test

Name: ___________________ Date:______________________

Product:__________________ Time: _____________________

You receive a series of samples with increasing concentrations of one of the 4

taste qualities (sweet, salty, sour, bitter)*.
Start with Samples No. 1 and continue with Samples No. 2, No. 3, etc.
Retasting of already tested solutions is not allowed.
Describes the taste* and the feeling factors and give intensity scores.
Use the following intensity scale:
0 = None or pure water taste
? = Different from water, but taste quality not identifiable
X = Threshold very weak (identify the taste)
1 = Weak
2 = Medium
3 = Strong
4 = Very strong
5 = Extremely strong

Sample No. Description of Taste

and Feeling Factors
1 ____________________
2 ____________________
3 ____________________
4 ____________________
5 ____________________
6 ____________________
7 ____________________
8 ____________________
9 ____________________
10 ____________________
11 ____________________
12 ____________________

Signature
*To be modified for odour analysis.

• Dilution test
The dilution technique determines the smallest amount of test material that can
be detected when it is mixed with a standard material. The technique may
104
provide information on relative intensities of treatment at comparable dilution Sensory Evaluation of
levels. Dilution testing is limited to food products that can be made Food Products
homogeneous without affecting the factor being tested.
The data is evaluated using the evaluation card having the judgement of the
panelist. A typical evaluation card for the dilution test is as given below:

Format 6: Specimen Evaluation Card for-Dilution Test

Name: ___________________ Date:______________________

Product*:_________________ Time: _____________________

Assign scores for each sample for various characteristics.

Quality Maximum Code No. or Samples

Attributes Score

Colour 20 _______ _______ _______ _______

Consistency 20 _______ _______ _______ _______

Flavour 40 _______ _______ _______ _______

Absence of defects 20 _______ _______ _______ _______

Total score 100 _______ _______ _______ _______

Comments

Signature
*The weighted rating is a typical score applicable to orange marmelades. For other products
similar scales have to be worked out..

Data from the dilution test is analyzed by finding the arithmetic or geometric
mean for the group and expressing as dilution number or dilution index, which
is defined as the percentage or ratio of the test substance in one mixture when
the substance is just identifiable.
b) Descriptive Tests
Descriptive tests attempt to identify sensory characteristics and quantify them.
Panelists are selected on their ability to perceive differences between test
procedures.
Descriptive tests are based on two types of methodologies:
i) Attribute rating: It involves:
• Category Scaling
Coded samples are presented simultaneously or sequentially in a balanced
order, which differs among the individual panel members. Category scales
105
Food Analysis consists of a series of word phrases structured in ascending or descending
order of intensity and are used to measure the specific attributes (e.g.
sweetness, off-flavor etc.). For the purpose of analysis, successive digits are
later assigned to each point represented on the scale, usually beginning at the
end representing zero intensity. A statistical analysis (e.g. analysis of variance)
of the mean intensity scores for each sample is used to determine significant
differences among the mean scores for the sample represented.
• Ratio Scaling (Magnitude Estimation)
This test is used to estimate the relationship between physical intensity and
sensory magnitude. It can also be used for comparable ratings on specific
attributes among two or more products. The method permits the participants to
use a wide range of numbers of his/her own choice with the property that ratios
or proportions among the numerical assignments reflect ratios of sensory
intensities. The numerical ratings given to the first sample presented may be
any one of the subject’s choice, except zero or a negative value. Ratings given
to the succeeding samples should be in proportion to the rating assigned to the
first. The numbers assigned are subjected to statistical analysis after
normalization.
• Flavor Profile Analysis
The technique provides a written record of a product’s perceptible aroma and
flavor components, feeling factors and aftertastes. The panelist characterizes
individual aroma and flavor notes in the order perceived and assigns an
intensity value using a constant rating scale. A panel of four or five members is
normally used. Panelists independently examine the product under study,
record their impressions of aroma, flavor and aftertastes, then reports to a panel
leader in an open discussion. The final flavor profile, upon comparison with an
original profile, can show the effect of an ingredient substitution, a processing
change packaging, age etc.
• Texture Profile Analysis
This is a descriptive technique based on the principle of the flavor profile
method. It provides a systematic approach to measure the textural dimensions
of food in terms of mechanical, geometrical, fat and moisture characteristics.
The panel is composed of six to nine members. The findings of the panels are
recorded and a profile for similarities and differences is used for interpretation.
• Quantitative Descriptive Analysis
This technique utilizes an unstructured category scale and a panel of not less
than six trained panelists, and obtains repeated judgements from each panelist
for each test products.
Let us take an example of sensory evaluation for a sample of cookies. In order
to evaluate sensory attributes of cookies, following Descriptive Tests can be
undertaken:

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 Sensory Evaluation of
Texture Food Products

Surface 1 5 10
Smooth Rough

1 5 10
Dryness Oily Dry

1 5 10
Moisture absorption: None Much
Scale

Flavor

Sweet
1 5 10

Salty
1 5 10

Bitter
1 5 10

Scale

14.5.2 Affective (Preference and Acceptance) Tests

Affective tests refer to preference testing based on the measurement of
preference, or a measure from which relative preference may be determined,
e.g. pleasure-displeasure, like-dislike.
Preference may be defined as:
a) Expression of higher degree of liking, and
b) Choice of one object over others.
Preference is only one of the many factors involved in acceptance.
Acceptance may be defined as:
a) An experience or feature of experience, characterized by a positive attitude;
and/or
b) Actual utilization of the product.
Acceptability is inferred from scale ratings.

107
Food Analysis There are three types of affective tests; Paired – Performance Test, Ranking
Test and Rating Scale.

i) Paired – Performance Test

In the simplest application of a paired-performance test for preference, two
samples are presented, simultaneously or sequentially. The Panelist is
requested to express a preference based on the specific attributes, the reason
for preference may be included if desired. The method may also be applied to
make multiple paired – comparisons within a sample series, i.e. a standard
product vs each of several experimental products.

ii) Ranking Test

This is an extension of the paired-preference test approach. Three or more
coded samples are presented simultaneously, sufficient in amount so that the
panelist can check back on his or her first impression. The subject is asked to
assign an order to the sample according to his or her preference. The amount of
liking (or disliking) for individual samples cannot be adequately determined by
this method.

iii) Rating Scale

Scale ratings reflect panelist’s perceived intensities of a specified attribute
under a given set of conditions. Various rating scales have been developed and
used:
a) Hedonic Rating Scale
This test is used to measure the level of liking for food products by a
population. It may be applied in testing for presence or acceptance. The
method relies on panelist’s capacities to report directly and reliably, their
feelings of like and dislike. Several variations of the traditional nine-point
hedonic scale have been used effectively. These include:
• Reduced number of rating categories, although not fewer than five is
recommended.
• A greater number of “like” rating categories than “dislike”,
• Omission of the neutral rating categories by caricatures representing
degrees of pleasure and displeasure (facial hedonic scale), and
• Use of non-structured, non-numerical line scale anchored with “like” and
“dislike” on opposite ends.
The panelist is asked to evaluate each sample and mark the scales accordingly.
Instructions must not influence the panelist’s response. Hedonic scale ratings
are converted to numerical scores, and statistical analysis is applied to
determine difference in degree of liking between or among samples. A hedonic
rating test can yield both absolute and relative information about the test
samples. Absolute information is derived from the degree of liking or disliking
indicated for each sample and relative information is derived from the direction
and degree of difference between or among the sample scores.
The data from the hedonic scale ratings are evaluated by rank sum analysis,
t-test or chi square test.

108
 Sensory Evaluation of
Food Products
The data is evaluated using the evaluation card having the judgement of the
panelist. A typical evaluation card for the hedonic scale test is as below:

Format 7: Specimen Evaluation Card for-Hedonic Scale

Name: ___________________ Date:______________________

Product:__________________ Time: _____________________

Test this sample and check appropriate box how much you like or dislike.
Use the appropriate scale to show your attitude by checking at the point that
best describes your feeling about the sample.
Please give your reason for this attitude.
Remember you are the only one who can tell what you like.
An honest expression of your personal feeling will help us.

Code No.

Like extremely

Like very much

Like moderately

Like slightly

Neither like nor dislike

Dislike slightly

Dislike moderately

Dislike very much

Dislike extremely

Comments.

Signature

109
Food Analysis Format 8: Specimen Evaluation Card for-Hedonic Scale (facial)

Name: ___________________ Date:______________________

Product:__________________ Time: _____________________

Please check the box under the figure which best describes how you feel
about this product.

Signature

b) Food Action Scale Rating

This test may be used to measure the level of acceptance of food products by a
population. The scale is not applicable for rating specific characteristics; rather
it is a measure of general attitude towards food product. This rating scale
includes action as well as affective type statements. Nine successive rating
categories ranging from “I would eat this every time it, I have an opportunity”
to “I would eat this only if I were forced to” are represented. Samples are
presented sequentially in a balanced order, and the panelist is told to decide
which of the statements on the scale best represents his or her attitude. Subjects
are allowed to make their own inferences about the meaning of the scale
categories. The scale ratings are converted to numerical scores to facilitate
analysis of data.
The data is evaluated using the evaluation card having the judgement of the
panelist. A typical evaluation card for the dilution test is as below:
Let us take another example for sensory evaluation of a product for customer
acceptance and preference. For evaluating sensory attributes of a soft drink,
following Acceptance Tests can be undertaken:
Taste : Water-like
Strongly sweet
Sweet
Salty
Strongly salty
Medicinal
Viscosity : Less Viscous (e.g. water-like)
110
 Highly viscous (e.g. honey-like) Sensory Evaluation of
Food Products
Consistency : Homogeneous
Heterogeneous

Format 9: Specimen Evaluation Card for-Food Action Scale Rating

Name: ___________________ Date:______________________

Product:__________________ Time: _____________________

Indicate in appropriate box which of nine statements on the following scale

best represent your attitude towards the product.

Code No._______________

I would eat this every opportunity I had

I would eat this very often

I would frequently eat this

I like this and would eat it now and then

I would eat this if available but would not go out of my way

I don’t like it would eat it on an occasion

I would hardly ever eat this

I would eat this only if there were no other food choices

I would eat this only if I were forced to

Comments.
Note –– The word ‘eat’ may be replaced by ‘drink’, ‘buy’ or ‘use’.

Signature

14.6 APPLICATIONS OF SENSORY EVALUATION

The sensory evaluation tests are commonly being use in food industry as well
as certain other industrial applications as follows:
1) New Product Development
Some new products are unique, but most of them are imitations or variations of
some established standards. In either case, the product developer needs
information on acceptability to consumers as an input for marketing. The
acceptability of products can be evaluated following the given sequence:

111
Food Analysis a) Characterization of product prototype sample to determine uniqueness.
b) Evaluation of the experimental prototype samples to establish whether
differences exist among them.
c) Determination of whether the prototype samples meet the acceptability
requirements established for the product.
2) Product Improvement/Process change/cost reduction
Improvement in the products can be judged based on sensory evaluation in
following ways:
a) Difference tests to determine whether the experimental product is the same
or different from the control.
b) Affective test: If product differs, to establish whether the experimental
product is liked more than the control.
3) Quality Control
Representative samples are usually evaluated by difference tests and
descriptive tests to ensure that the end product is having all the required
qualities during production, distribution and marketing.
4) Storage Stability studies
These are conducted to establish information on product shelf life during
transportation, warehousing, retailing and during storage. Representative
samples are obtained, evaluated initially and then at specific time intervals of
storage. Sensory tests are also used to determine product storage stability such
as:
a) Difference tests to determine whether the storage samples are different
from the control (if no significant difference is found, product stability is
assumed).
b) Descriptive tests used alone or in conjunction with difference tests, to
characterize and/or quantify the changes that may have occurred during
storage.
c) Acceptance tests to determine the relative acceptance of stored product.
5) Product grading or Rating
This requires an accurate classification of samples according to the grade
standards defined for the product; as well as an evaluation of samples in
relation to each other. Category scoring or ratio scaling based on the presence
and intensity of selected characteristics may be used to measure samples
against standard specifications set for the product.

# Check Your Progress Exercise 2

Note: a) Use the space below for your answers.
b) Check your answers with those given at the end of the unit.
1) What is required for sensory evaluation test?
………………………………………………………………………………
………………………………………………………………………………
112
 ……………………………………………………………………………… Sensory Evaluation of
Food Products
………………………………………………………………………………
2) Which test measures quantitative characteristics of a product and how?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
3) What is Affective tests and what are the types of affective tests?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
4) What are the different types of rating scales?
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………
………………………………………………………………………………

14.7 KEY WORDS

Sensory Analysis : Examination of organoleptic attributes of a
product by the sense organs.
Sensory (adj.) : Relating to the use of the sense organs.
Organoleptlc (adj.) : Relating to an attribute of a product
perceptible by the sense organs.
Sensation (noun) : Subjective reaction resulting from sensory
stimulation.
Assessor (sensory) : Any person taking part in a sensory test.
(noun)
Attribute (noun) : Perceptible characteristic.
Acceptability (noun) : State of product favourably received by a
given individual or population, in terms of its
organoleptic attributes.
Acceptance (noun) : The act of a given individual or population of
finding that a product answers satisfactorily
to his/her/its expectations.

113
Food Analysis
Preference (noun) : Expression of the emotional state or reaction
of an assessor which leads him/her to find
one product better than one or several others.
Discrimination (noun) : Act or qualitative and/or quantitative
differentiation between two or more stimuli.
Hedonic (adj.) : Relating to like or dislike.
Quality (noun) : Collection of features and characteristics of a
product or service that confer its ability to
satisfy stated or implied needs.
Product (noun) : Edible or inedible matter which can be
evaluated by sensory analysis. Examples:
food products, cosmetics, textile and fabrics.
Bias (noun) : Systematic errors which may be positive or
negative.

# 14.8 ANSWERS TO CHECK YOUR PROGRESS

EXERCISE
Check Your Progress Exercise 1
Your answer should include following points:
1) Sensory Evaluation is defined as “a scientific discipline used to evoke,
measure, analyze and interpret reactions to those characteristics of foods
and materials as they are perceived by the senses of sight, taste, touch and
hearing”.
2) Sensory evaluation is normally carried out by designed experiments under
proper environmental conditions by both trained and untrained person and
they are called sensory panel.
3) There are three types of panels:
i) Trained Panel (Laboratory Panel)
ii) Discriminatative and communicative Panel (semitrained Panel)
iii) Untrained Panel (Cosumer Panel)

Check Your Progress Exercise 2

Your answer should include following points:
1) Selection of the proper panel:
i) Maintaining suitable environmental conditions and use of standard
equipment for the test;
ii) Obtaining representative samples;
iii) Preparation and presentation of samples for evaluation in a manner that
ensures the uniformity and representation of the samples; and
iv) Selection of the proper methods and statistical techniques.
114
2) Descriptive tests using scaling and profiling methods. Sensory Evaluation of
Food Products
3) Affective tests refer to preference testing based on the measurement of
preference, or a measure from which relative preference may be
determined, e.g. pleasure-displeasure, like-dislike.
There are three types of affective tests:
i) Paired – Performance Test
ii) Ranking Test
iii) Rating Scale (Hedonic Rating Scale, Food Action Scale Rating)
4) i) Hedonic Rating Scale
ii) Food Action Scale Rating

14.9 SOME USEFUL BOOKS

Sensory Evaluation Guide for Testing of Food and Beverage Products. By
Sensory Evaluation Division, Institute of Food Technologists; Food
Technology, November 1981; 50-59.
Handbook of Analysis and Quality Control for Fruits and Vegetable Products.
S. Ranganna, II Edn. 1994. Tata Mc Graw-Hill Publishing Co. N. Delhi.
Chapter 19: Sensory Evaluation.
IS 6273 – 1974 (Reaffirmed 2002): Guide for Sensory Evaluation of Foods;
Part I: Optimum Requirements.
IS 6273 – 1974 (Reaffirmed 2002): Guide for Sensory Evaluation of Foods;
Part II: Methods and Evaluation Cards.
IS 5126 – 1996/ISO 5492 - 1992 (Reaffirmed 2001): Sensory Analysis –
Vocabulary.
IS 8140 – 1976 (Reaffirmed 2002): Guide for Selection of Panel for Sensory
Evaluation of Foods and Beverages.

115