CHAPTER ONE

INTRODUCTION TO 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.
 The analytical procedures are used to provide information about foods: composition,
structure, physicochemical properties and sensory attributes.
Reasons for food analysis

1. Meeting government and industry regulations

Food analysis helps everyone from ingredients suppliers to product manufacturers meet and
maintain these standards. For example, in the USA peanut butter must contain at least 90%
peanuts, a maximum of 10% added ingredients such as salt and sugar and have a total fat content
of no more than 55%.
2. Protecting consumers
Consumer protection is front of mind in the food and beverage sector. Analytical techniques are
used to screen both ingredients and finished products before they hit consumer shelves. For
example, polymerase chain reaction (PCR) tests can be used to detect foodborne pathogens and
viruses in products such as lettuce. Laboratory tests are also carried out to detect toxins deposited
by pesticides and herbicides, as well as traces of metal, wood, glass and other contaminants.
3. Preventing food fraud
Food fraud is a global issue that affects consumers, product manufacturers and ingredients
suppliers. The 2013 horse meat scandal is one of the most high-profile cases of food fraud, with
the Food Safety Authority of Ireland initially detecting horse DNA in samples of frozen ready
meals and burgers supposedly containing beef.

4. Nutritional labelling
Nutritional labelling offers consumers in-depth information on the quality and content of food.
Most countries require food manufacturers to publish standardised nutritional labels covering fat,
cholesterol, carbohydrates, sodium, sugars and protein. This information helps consumers make
informed choices.
5. Consistency and quality control
While consistency can vary for small-batch products, maintaining the same overall properties is a
top priority for large-scale manufacturers. Frequent testing allows manufactures to meet the
expectations of consumers and ultimately, drive profits and increase their market share. Tests are
used to monitor everything from flavour and texture to natural sugar and starch concentrations,
which can vary in products such as potatoes.
6. Research and development
Food analysis is fundamental to research and development in the industry. For example, US-
based company Beyond Meat relies on food analysis techniques to develop new plant-based
beef products. The brand has established itself as a market leader and has forged partnerships
with global brands such as KFC, Pizza Hut and Panda Express.
The cannabis industry is subject to especially strict regulations, with analytical techniques used
to characterize both recreational and medicinal products.
ASSIGMENT
 Discuss the application of food analysis. (15marks).

CHAPTER TWO
METHODS OF FOOD ANALYSIS.
CONCENTRATION:
 concentration is the amount of solute present in the given quantity of solvent or solution
 METHODS TO EXPRESS CONCENTRATION
i. Mass percentage: Mass of solute per100g of solution
Mass % = (mass of solute / total mass of solution) X 100
ii. Volume percentage: volume of solute per100mlof solution
Volume % = (volume of solute/ total volume of
Solution) X 100
iii. Mass by volume percentage (w/V): Another unit which is commonly used in medicine and
pharmacy is mass by volume percentage. It is the mass of solute dissolved in 100 mL of the
solution.
Mass by Volume % = (mass of solute/ total volume of solution) X 100
iv. Parts per million: parts of a component per million parts of the solution.
Parts per million = (Number of parts of the component /Total number of parts of all components
of the solution) ×106
v. Mole fraction(x): It is the ratio of no. of moles of one component to the total no. of all the
components present in the solution. For binary solution:‐the no. of moles of A and B are nA and
nB respectively
vi. Molarity (M): No. of moles of solute dissolved in one litre of solution.
Molarity (M) = moles of solute/ vol. of solution in litre
SHORT FORM: MOLAR
vii. Molality (m):No. of moles of solute per kg of the solvent.
molality(m) = moles of solute/mass of solvent in kg
SHORT FORM: MOLAL
Molality is independent of temp. whereas molarity is a function of temp.
because vol. depends on temp. and mass does not.

SELECTING AN APPROPRIATE TECHNIQUE

Some of the criteria that are important in selecting a technique are listed below:
Precision: A measure of the ability to reproduce an answer between determinations performed
by the same scientist (or group of scientists) using the same equipment and experimental
approach.
Reproducibility: A measure of the ability to reproduce an answer by scientists using the same
experimental approach but in different laboratories using different equipment.
Accuracy: A measure of how close one can actually measure the true value of the parameter
being measured, e.g., fat content, or sodium concentration.
Simplicity of operation: A measure of the ease with which relatively unskilled workers may
carry out the analysis.
Cost: The total cost of the analysis, including the reagents, instrumentation and salary of
personnel required to carry it out.
Speed: The time needed to complete the analysis of a single sample or the number of samples
that can be analyzed in a given time.
Sensitivity: A measure of the lowest concentration of a 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.
Safety: Many reagents and procedures used in food analysis are potentially hazardous e.g. strong
acids or bases, toxic chemicals or flammable materials.
Destructive/Nondestructive: In some analytical methods the sample is destroyed during the
analysis, whereas in others it remains intact.
On-line/Off-line: Some analytical methods can be used to measure the properties of a food
during processing, whereas others can only be used after the sample has been taken from the
production line.
Official Approval: Various international bodies have given official approval to methods that
have been comprehensively studied by independent analysts and shown to be acceptable to the
various organizations involved, e.g., ISO, AOAC, AOCS.
Nature of Food Matrix: The composition, structure and physical properties of the matrix material
surrounding the analyte often influences the type of method that can be used to carry out an
analysis, e.g., whether the matrix is solid or liquid, transparent or opaque, polar or non-polar.

SAMPLING
  Sampling is the process of selecting a group of individuals from a population to study
them and characterize the population as a whole.
 The population includes all members from a specified group, all possible outcomes or
measurements that are of interest. The exact population will depend on the scope of the
study.
 Population. The whole of the material whose properties we are trying to obtain an
estimate of is usually referred to as the population.
 The sample consists of some observations drawn from the population, so a part of a
subset of the population. The sample is the group of elements who participated in the
study.
 Sample. Only a fraction of the population is usually selected for analysis, which is
referred to as the sample the sample may be comprised of one or more sub-
samples selected from different regions within the population.
 The sampling frame is the information that locates and defines the dimensions of the
universe.
 Laboratory Sample. The sample may be too large to conveniently analyze using a
laboratory procedure and so only a fraction of it is actually used in the final laboratory
analysis. This fraction is usually referred to as the laboratory sample

A GOOD SAMPLE SHOULD SATISFY THE BELOW CONDITIONS-

1. Representativeness: The sample should be the best representative of the population
under study.
2. Accuracy: Accuracy is defined as the degree to which bias is absent from the sample. An
accurate (unbiased) sample is one that exactly represents the population.
3. Size: A good sample must be adequate in size and reliability.

SAMPLING PLAN

 sampling plan should be a clearly written document that contains precise details that an
analyst uses to decide the sample size, the locations from which the sample should be
selected, the method used to collect the sample, and the method used to preserve them
prior to analysis.
 It should also stipulate the required documentation of procedures carried out during the
sampling process.
 The choice of a particular sampling plan depends on;
1. the purpose of the analysis
2. the property to be measured
3. the nature of the total population and of the individual samples
 4. The type of analytical technique used to characterize the samples.
 For certain products and types of populations sampling plans have already been
developed and documented by various organizations which authorize official
methods, e.g., the Association of Official Analytical Chemists (AOAC)
1 Purpose of Analysis
 The first thing to decide when choosing a suitable sampling plan is the purpose of the analysis.
Samples are analyzed for a number of different reasons in the food industry and this affects the
type of sampling plan used:
 � Official samples. Samples may be selected for official or legal requirements by
government laboratories. These samples are analyzed to ensure that manufacturers are
supplying safe foods that meet legal and labeling requirements. An officially sanctioned
sampling plan and analytical protocol is often required for this type of analysis.
 � Raw materials. Raw materials are often analyzed before acceptance by a factory, or
before use in a particular manufacturing process, to ensure that they are of an appropriate
quality.
 � Process control samples. A food is often analyzed during processing to ensure that the
process is operating in an efficient manner. Thus if a problem develops during processing it
can be quickly detected and the process adjusted so that the properties of the sample are not
adversely effected. Techniques used to monitor process control must be capable of producing
precise results in a short time. Manufacturers can either use analytical techniques that measure
the properties of foods on-line, or they can select and remove samples and test them in a
quality assurance laboratory.
 � Finished products. Samples of the final product are usually selected and tested to
ensure that the food is safe, meets legal and labeling requirements, and is of a high and
consistent quality. Officially sanctioned methods are often used for determining nutritional
labeling.
 � Research and Development. Samples are analyzed by food scientists involved in
fundamental research or in product development.� In many situations it is not necessary to
use a sampling plan in R&D because only small amounts of materials with well-defined
properties are analyzed.
2. Nature of Measured Property
 Once the reason for carrying out the analysis has been established it is necessary to clearly
specify the particular property that is going to be measured, e.g., color, weight, presence of
extraneous matter, fat content or microbial count. The properties of foods can usually be
classified as either attributes or variables. An attribute is something that a product either does
or does not have, e.g., it does or does not contain a piece of glass, or it is or is not spoilt. On
the other hand, a variable is some property that can be measured on a continuous scale, such
as the weight, fat content or moisture content of a material. Variable sampling usually
requires less samples than attribute sampling.
 The type of property measured also determines the seriousness of the outcome if the properties
of the laboratory sample do not represent those of the population. For example, if the property
measured is the presence of a harmful substance (such as bacteria, glass or toxic chemicals),
then the seriousness of the outcome if a mistake is made in the sampling is much greater than
if the property measured is a quality parameter (such as color or texture). Consequently, the
 sampling plan has to be much more rigorous for detection of potentially harmful substances
than for quantification of quality parameters.
3. Nature of Population
 It is extremely important to clearly define the nature of the population from which samples are
to be selected when deciding which type of sampling plan to use. Some of the important points
to consider are listed below:
 A population may be either finite or infinite.
 A finite population is one that has a definite size, e.g., a truckload of apples, a tanker full of
milk, or a vat full of oil.
 An infinite population is one that has no definite size, e.g., a conveyor belt that operates
continuously, from which foods are selected periodically.
 Analysis of a finite population usually provides information about the properties of the
population, whereas analysis of an infinite population usually provides information about the
properties of the process. To facilitate the development of a sampling plan it is usually
convenient to divide an "infinite" population into a number of finite populations, e.g., all the
products produced by one shift of workers, or all the samples produced in one day.
 A population may be either continuous or compartmentalized.
 A continuous population is one in which there is no physical separation between the different
parts of the sample, e.g., liquid milk or oil stored in a tanker.
 A compartmentalized population is one that is split into a number of separate sub-
units, e.g., boxes of potato chips in a truck, or bottles of tomato ketchup moving along a
conveyor belt. The number and size of the individual sub-units determines the choice of a
particular sampling plan.
 A population may be either homogenous or heterogeneous. ere
 A homogeneous population is one in which the properties of the individual samples are the
same at every location within the material (e.g. a tanker of well stirred liquid oil), whereas a
heterogeneous population is one in which the properties of the individual samples vary with
location (e.g. a truck full of potatoes, some of which are bad). If the properties of a population
were homogeneous then there would be no problem in selecting a sampling plan because
every individual sample would be representative of the whole population. In practice, most
populations are heterogeneous and so we must carefully select a number of individual samples
from diffnt locations within the population to obtain an indication of the properties of the total
population.
4. Nature of Test Procedure
 The nature of the procedure used to analyze the food may also determine the choice of a
particular sampling plan, e.g., the speed, precision, accuracy and cost per analysis, or whether
the technique is destructive or non-destructive. Obviously, it is more convenient to analyze the
properties of many samples if the analytical technique used is capable of rapid, low cost,
nondestructive and accurate measurements.

DEVELOPING A SAMPLING PLAN

After considering the above factors one should be able to select or develop a sampling plan
which is most suitable for a particular application. Different sampling plans have been designed
to take into account differences in the types of samples and populations encountered, the
information required and the analytical techniques used. Some of the features that are commonly
specified in official sampling plans are listed below.
Sample size. The size of the sample selected for analysis largely depends on the expected
variations in properties within a population, the seriousness of the outcome if a bad sample is not
detected, the cost of analysis, and the type of analytical technique used. Given this information it
is often possible to use statistical techniques to design a sampling plan that specifies the
minimum number of sub-samples that need to be analyzed to obtain an accurate representation of
the population. Often the size of the sample is impractically large, and so a process known
as sequential sampling is used. Here sub-samples selected from the population are examined
sequentially until the results are sufficiently definite from a statistical viewpoint. For example,
sub-samples are analyzed until the ratio of good ones to bad ones falls within some statistically
predefined value that enables one to confidently reject or accept the population.
Sample location. In homogeneous populations it does not matter where the sample is taken from
because all the sub-samples have the same properties. In heterogeneous populations the location
from which the sub-samples are selected is extremely important.
In random sampling the sub-samples are chosen randomly from any location within the material
being tested. Random sampling is often preferred because it avoids human bias in selecting
samples and because it facilitates the application of statistics. In systematic sampling the samples
are drawn systematically with location or time, e.g., every 10th box in a truck may be analyzed,
or a sample may be chosen from a conveyor belt every 1 minute. This type of sampling is often
easy to implement, but it is important to be sure that there is not a correlation between the
sampling rate and the sub-sample properties. In judgment sampling the sub-samples are drawn
from the whole population using the judgment and experience of the analyst. This could be the
easiest sub-sample to get to, such as the boxes of product nearest the door of a truck.
Alternatively, the person who selects the sub-samples may have some experience about where
the worst sub-samples are usually found, e.g., near the doors of a warehouse where the
temperature control is not so good. It is not usually possible to apply proper statistical analysis to
this type of sampling, since the sub-samples selected are not usually a good representation of the
population.
Sample collection. Sample selection may either be carried out manually by a human being or by
specialized mechanical sampling devices. Manual sampling may involve simply picking a
sample from a conveyor belt or a truck, or using special cups or containers to collect samples
from a tank or sack. The manner in which samples are selected is usually specified in sampling
plans.

PREPARATION OF LABORATORY SAMPLES

Once we have selected a sample that represents the properties of the whole population, we must
prepare it for analysis in the laboratory. The preparation of a sample for analysis must be done
very carefully in order to make accurate and precise measurements.

1. Making Samples Homogeneous

The food material within the sample selected from the population is usually
heterogeneous, i.e., its properties vary from one location to another.� Sample heterogeneity
may either be caused by variations in the properties of different units within the sample (inter-
unit variation) and/or it may be caused by variations within the individual units in the sample
(intra-unit variation). The units in the sample could be apples, potatoes, bottles of ketchup,
containers of milk etc.� An example of inter-unit variation would be a box of oranges, some of
good quality and some of bad quality.� An example of intra-unit variation would be an
individual orange, whose skin has different properties than its flesh. For this reason it is usually
necessary to make samples homogeneous before they are analyzed, otherwise it would be
difficult to select a representative laboratory sample from the sample. A number of mechanical
devices have been developed for homogenizing foods, and the type used depends on the
properties of the food being analyzed (e.g., solid, semi-solid, liquid).� Homogenization can be
achieved using mechanical devices (e.g., grinders, mixers, slicers, blenders), enzymatic methods
(e.g., proteases, cellulases, lipases) or chemical methods (e.g., strong acids, strong bases,
detergents).

2. Reducing Sample Size

Once the sample has been made homogeneous, a small more manageable portion is selected for
analysis. This is usually referred to as a laboratory sample, and ideally it will have properties
which are representative of the population from which it was originally selected. Sampling plans
often define the method for reducing the size of a sample in order to obtain reliable and
repeatable results.

3. Preventing Changes in Sample

Once we have selected our sample we have to ensure that it does not undergo any significant
changes in its properties from the moment of sampling to the time when the actual analysis is
carried out, e.g., enzymatic, chemical, microbial or physical changes. There are a number of
ways these changes can be prevented.
� Enzymatic Inactivation. Many foods contain active enzymes they can cause
changes in the properties of the food prior to analysis, e.g., proteases, cellulases, lipases, etc. If
the action of one of these enzymes alters the characteristics of the compound being analyzed then
it will lead to erroneous data and it should therefore be inactivated or eliminated. Freezing,
drying, heat treatment and chemical preservatives (or a combination) are often used to control
enzyme activity, with the method used depending on the type of food being analyzed and the
purpose of the analysis.
Lipid Protection. Unsaturated lipids may be altered by various oxidation reactions.
Exposure to light, elevated temperatures, oxygen or pro-oxidants can increase the rate at which
these reactions proceed. Consequently, it is usually necessary to store samples that have high
unsaturated lipid contents under nitrogen or some other inert gas, in dark rooms or covered
bottles and in refrigerated temperatures. Providing that they do not interfere with the analysis
antioxidants may be added to retard oxidation.
Microbial Growth and Contamination. Microorganisms are present naturally in many foods
and if they are not controlled they can alter the composition of the sample to be analyzed.
Freezing, drying, heat treatment and chemical preservatives (or a combination) are often used to
control the growth of microbes in foods.
Physical Changes. A number of physical changes may occur in a sample, e.g., water may be
lost due to evaporation or gained due to condensation; fat or ice may melt or crystallize;
structural properties may be disturbed. Physical changes can be minimized by controlling the
temperature of the sample, and the forces that it experiences.
4. Sample Identification
Laboratory samples should always be labeled carefully so that if any problem develops its origin
can easily be identified. The information used to identify a sample includes:
a) Sample description,
b) Time sample was taken,
c) Location sample was taken from,
d) Person who took the sample,
e) Method used to select the sample.
The analyst should always keep a detailed notebook clearly documenting the sample selection
and preparation procedures performed and recording the results of any analytical procedures
carried out on each sample. Each sample should be marked with a code on its label that can be
correlated to the notebook. Thus if any problem arises, it can easily be identified.

2.4. Data Analysis and Reporting

Food analysis usually involves making a number of repeated measurements on the same sample
to provide confidence that the analysis was carried out correctly and to obtain a best estimate of
the value being measured and a statistical indication of the reliability of the value. A variety of
statistical techniques are available that enable us to obtain this information about the laboratory
sample from multiple measurements.

1. Measure of Central Tendency of Data

The most commonly used parameter for representing the overall properties of a number of

measurements is the mean:

Here n is the total number of measurements, xi is the individually measured values and is the
mean value.
The mean is the best experimental estimate of the value that can be obtained from the
measurements. It does not necessarily have to correspond to the true value of the parameter one
is trying to measure. There may be some form of systematic error in our analytical method that
means that the measured value is not the same as the true value (see below). Accuracy refers to
how closely the measured value agrees with the true value. The problem with determining the
accuracy is that the true value of the parameter being measured is often not known. Nevertheless,
it is sometimes possible to purchase or prepare standards that have known properties and analyze
these standards using the same analytical technique as used for the unknown food samples. The
absolute error Eabs, which is the difference between the true value (xtrue) and the measured value
(xi), can then be determined: Eabs = (xi - xtrue).� For these reasons, analytical instruments should
be carefully maintained and frequently calibrated to ensure that they are operating correctly.

Sources of Error
There are three common sources of error in any analytical technique:
Personal Errors (Blunders). These occur when the analytical test is not carried out correctly: the
wrong chemical reagent or equipment might have been used; some of the sample may have been
spilt; a volume or mass may have been recorded incorrectly; etc. It is partly for this reason that
analytical measurements should be repeated a number of times using freshly prepared laboratory
samples.� Blunders are usually easy to identify and can be eliminated by carrying out the
analytical method again more carefully.
� Random Errors. These produce data that vary in a non-reproducible fashion from one
measurement to the next e.g., instrumental noise. This type of error determines the standard
deviation of a measurement. There may be a number of different sources of random error and
these are accumulative
� Systematic Errors. A systematic error produces results that consistently deviate from the
true answer in some systematic way, e.g., measurements may always be 10% too high. This type
of error would occur if the volume of a pipette was different from the stipulated value. For
example, a nominally 100 cm3 pipette may always deliver 101 cm3 instead of the correct value.
To make accurate and precise measurements it is important when designing and setting up an
analytical procedure to identify the various sources of error and to minimize their effects. Often,
one particular step will be the largest source of error, and the best improvement in accuracy or
precision can be achieved by minimizing the error in this step.

SAMPLING TECHNIQUES:

There are several different sampling techniques available, and they can be subdivided into two
groups-

1. Probability sampling involves random selection, allowing you to make statistical inferences
about the whole group.

There are four types of probability sampling techniques

 Simple random sampling

 Cluster sampling
 Systematic sampling
 Stratified random sampling
2. Non-probability sampling involves non-random selection based on convenience or other
criteria, allowing you to easily collect initial data. There are four types of Non-probability
sampling techniques.

 Convenience sampling
 Judgmental or purposive sampling
 Snowball sampling
 Quota sampling
Choosing Between Probability and Non-Probability Samples
The choice between using a probability or a non-probability approach to sampling depends on a
variety of factors:

1. Objectives and scope of the study

2. Method of data collection
3. Precision of the results
4. Availability of a sampling frame and resources required to maintain the frame
5. Availability of extra information about the members of the population
Probability Sampling

Probability sampling is normally preferred when conducting major studies, especially when a
population frame is available, ensuring that we can select and contact each unit in the population.
Probability sampling allows us to quantify the standard error of estimates, confidence intervals to
be formed and hypotheses to be formally tested.

The main disadvantage is Bias in selecting the sample and the costs involved in the survey.

Simple random sampling

In Simple Random Sampling, each observation in the population is given an equal probability of
selection, and every possible sample of a given size has the same probability of being selected.
One possible method of selecting a simple random sample is to number each unit on the
sampling frame sequentially and make the selections by generating numbers from a random
number generator.

Simple random sampling can involve the units being selected either with or without replacement.
Replacement sampling allows the units to be selected multiple times whilst without replacement
only allows a unit to be selected once. Without replacement, sampling is the most commonly
used method.

Ex: If a sample of 20 needs to be collected from a population of 100. Assign unique numbers to
population members and randomly select 20 members with a random generator. Train and test
split in ML

Applications

1. Train and test split in machine learning problems

2. Lottery methods
Advantages

1. Minimum sampling bias as the samples are collected randomly

2. Selection of samples is simple as random generators are used
3. The results can be generalized due to representativeness
Disadvantages
 1. The potential availability of all respondents can be costly and time consuming
2. Larger sample sizes
Systematic sampling

In systematic random sampling, the researcher first randomly picks the first item from the
population. Then, the researcher will select each nth item from the list. The procedure involved
in systematic random sampling is very easy and can be done manually. The results are
representative of the population unless certain characteristics of the population are repeated for
every nth individual.

Steps in selecting a systematic random sample:

1. Calculate the sampling interval (the number of observations in the population divided by
the number of observations needed for the sample)
2. Select a random start between 1 and sampling interval
3. Repeatedly add sampling interval to select subsequent households
Ex: If a sample of 20 needs to be collected from a population of 100. Divide the population into
20 groups with a members of (100/20) = 5. Select a random number from the first group and get
every 5th member from the random number.

Applications

1. Quality Control: The systematic sampling is extensively used in manufacturing industries

for statistical quality control of their products. Here a sample is obtained by taking an
item from the current production stream at regular intervals.
2. In Auditing: In auditing the savings accounts, the most natural way to sample a list of
accounts to check compliance with accounting procedures.
Advantages

1. Cost and time efficient

2. Spreads the sample more evenly over the population
Disadvantages

1. Complete population should be known

2. Sample bias If there are periodic patterns within the dataset
Stratified random sampling

In Stratified random sampling, the entire population is divided into multiple non-overlapping,
homogeneous groups (strata) and randomly choose final members from the various strata for
research. Members in each of these groups should be distinct so that every member of all groups
get equal opportunity to be selected using simple probability.

There are three types of stratified random sampling-

1. Proportionate Stratified Random Sampling

The sample size of each stratum in this technique is proportionate to the population size of the
stratum when viewed against the entire population. For example, you have 3 strata with 10, 20
and 30 population sizes respectively and the sampling fraction is 0.5 then the random samples
are 5, 10 and 15 from each stratum respectively.

2. Disproportionate Stratified Random Sampling

The only difference between proportionate and disproportionate stratified random sampling is
their sampling fractions. With disproportionate sampling, the different strata have different
sampling fractions.

3. Optimal stratified sampling

The size of the strata is proportional to the standard deviation of the variables being studied.

Ex: A company wants to do an employee satisfaction survey and the company has 300k
employees and planned to collect a sample of 1000 employees for the survey. So the sample
should contain all the levels of employees and from all the locations. So create different strata or
groups and select the sample from each strata.

Advantages

1. Greater level of representation from all the groups

2. If there is homogeneity within strata and heterogeneity between strata, the estimates can
be as accurate
Disadvantages

1. Requires the knowledge of strata membership

2. Might take longer and more expensive
3. Complex methodology
Cluster sampling

Cluster sampling divides the population into multiple clusters for research. Researchers then
select random groups with a simple random or systematic random sampling technique for data
collection and data analysis.

Steps involved in cluster sampling:

1. Create the clusters from the population data

2. Select each cluster as a sampling frame
3. Number each cluster
 4. Select the random clusters
After selecting the clusters, either complete clusters will be used for the study or apply the other
sampling methods to pick the sample elements from the clusters.

Ex: A researcher wants to conduct an academic performance of engineering students under a

particular university. He can divide the entire population into multiple engineering colleges
(Which are clusters) and randomly pick up some clusters for the study.

Types of cluster sampling:

1. One-stage cluster : From the above example, selecting the entire students from the
random engineering colleges is one stage cluster
2. Two-Stage Cluster: From the same example, picking up the random students from the
each cluster by random or systematic sampling is Two-Stage Cluster
Advantages

1. Saves time and money

2. It is very easy to use from the practical standpoint
3. Larger sample sizes can be used
Disadvantages

1. High sampling error

2. May fail to reflect the diversity in the sampling frame
Non-probability sampling

Non-Probability samples are preferred when accuracy in the results is not important. These are
inexpensive, easy to run and no frame is required. If a non-probability sample is carried out
carefully, then the bias in the results can be reduced.

The main disadvantage of Non-Probability sampling is “dangerous to make inferences about the
whole population.”

Convenience sampling

Convenience sampling is the easiest method of sampling and the participants are selected based
on availability and willingness to participate in the survey. The results are prone to significant
bias as the sample may not be a representative of population.

Applications

1. Surveys conducted in social networking sites and offices

Examples: The polls conducted in Facebook or Youtube. The people who are interested in taking
the survey or polls will attend the survey and the results may not be accurate as the results are
prone to significant bias.
Advantages

1. It is easy to get the sample

2. Low cost and participants are readily available
Disadvantages

1. Can’t generalize the results

2. Possibility of under or over representation of the population
3. Significant bias
Quota sampling

This method is mainly used by market researchers. The researchers divide the survey population
into mutually exclusive subgroups. These subgroups are selected with respect to certain known
features, traits, or interests. Samples from each subgroup are selected by the researcher.

Quota sampling can be divided into two groups-

1. Controlled quota sampling involves introduction of certain restrictions in order to limit

researcher’s choice of samples.
2. Uncontrolled quota sampling resembles convenience sampling method in a way that
researcher is free to choose sample group members
Steps involved in Quota Sampling

1. Divide the population into exclusive sub groups

2. Identify the proportion of sub groups in the population
3. Select the subjects for each subgroup
4. Ensure the sample is the representative of population
Ex: A painting company wants to do research on one of their products. So the researcher uses the
quota sampling methods to pick up painters, builders, agents and retail painting shop owners.

Advantages

1. Cost effective
2. Doesn’t depend on sampling frames
3. Allows the researchers to sample a subgroup that is of great interest to the study
Disadvantages

1. sample may be overrepresented

2. Unable to calculate the sampling error
3. Great potential for researcher bias and the quality of work may suffer due to researcher
incompetency and/or lack of experience
Judgement (or Purposive) Sampling
In Judgement (or Purposive) Sampling, a researcher relies on his or her judgment when choosing
members of the population to participate in the study. Researchers often believe that they can
obtain a representative sample by using sound judgment, which will result in saving time and
money.

As the researcher’s knowledge is instrumental in creating a sample in this sampling technique,

there are chances that the results obtained will be highly accurate with a minimum margin of
error.

Ex: A broadcasting company wants to research one of the TV shows. The researcher has an idea
of the target audience and he can choose the members of the population to participate in the
study.

Advantages

1. Cost and time effective sampling method

2. Allows researchers to approach their target market directly
3. Almost real-time results
Disadvantages

1. Vulnerability to errors in judgment by researcher

2. Low level of reliability and high levels of bias
3. Inability to generalize research findings
Snowball sampling

This method is commonly used in social sciences when investigating hard-to-reach groups.
Existing subjects are asked to nominate further subjects known to them, so the sample increases
in size like a rolling snowball. For example, when surveying risk behaviors amongst intravenous
drug users, participants may be asked to nominate other users to be interviewed.

This sampling method involves primary data sources nominating other potential primary data
sources to be used in the research. So the snowball sampling method is based on referrals from
initial subjects to generate additional subjects. Therefore, when applying this sampling method
members of the sample group are recruited via chain referral.

There are three patterns of Snowball Sampling-

1. Linear snowball sampling. Recruit only one subject and the subject provides only one
referral
2. Exponential non-discriminative snowball sampling. Recruit only one subject and the
subject provides multiple referrals
3. Exponential discriminative snowball sampling. Recruit only one subject and the subject
provides multiple referrals. But only one subject is picked up from the referrals
Ex: Individuals with rare diseases. If a drug company is interested in doing research on the
individuals with rare diseases, it may be difficult to find these individuals. So the drug company
can find few individuals to participate in the study and request them to refer the individuals from
their contacts.

Advantages

1. Researchers can reach rare subjects in a particular population

2. Low-cost and easy to implement
3. It doesn’t require a recruitment team to recruit the additional subjects
Disadvantages

1. The sample may not be a representative

2. Sampling bias may occur
3. Because the sample is likely to be biased, it can be hard to draw conclusions about the
larger population with any confidence
Finally,

1. Reducing sampling error is the major goal of any selection technique.

2. A sample should be big enough to answer the research question, but not so big that the
process of sampling becomes uneconomical.
3. In general, the larger the sample, the smaller the sampling error, and the better job you
can do.
4. Decide the appropriate sampling method based on the study or use case.

Physico-chemical methods of food analysis

 The pH, acidity, total soluble solids, pectin etc can be measured using these
methods. Hydrogen ion concentration can be estimated using pH meter. It consists of
a glass indicating electrode and a reference electrode to complete the electrical
circuit. Digital handheld pH meters also available for this purpose.
 The concentration of sugars in foods can be determined by refracto meter.
Refractometers are instruments used to measure substances dissolved in water and
certain oils. The refractometer works using the principle of light refraction through
liquids.
 Refractometers use this principle to determine the amount of dissolved solids in
liquids by passing light through a sample and showing the refracted angle on a scale.
The scale most commonly used is referred to as the Brix scale. The Brix scale is
defined as: the number of grams of pure cane sugar dissolved in 100 grams of pure
water (grams sugar/100 grams of sugar solution).
 Brix or Balling hydrometer gives directly the percentage of sugar by weight in the
syrup. It is always necessary to make a temperature correction since the hydrometers
are usually calibrated at 20°C. Each instrument used by canners usually covers a
 range of only 10° Brix, e.g. 10-20, 20-30, 30-40, 40-50, 50-60 Brix respectively and
are graduated in 1/10th divisions. Brix is defined as percent sucrose measured by a
Brix hydrometer.

ASSIGNMENTS
1. Explain the procedure applied in the cleaning of laboratory apparatus. (20marks).
2. Discuss the microbial methods of food analysis. (20marks).
3. Explain the texture evaluation in food analysis. (10marks)
CHAPTER THREE
DETERMINATION OF SPECIFIC FOOD
COMPONENTS (proximate analysis).
 Proximate analysis. Refers to the quantitative analysis of macromolecules in food. A
combination of different techniques, such as extraction, Kjeldahl, NIR are used to
determine protein, fat, moisture, ash and carbohydrates levels
DETERMINATION OF MOISTURE CONTENT

MCwb = Moisture content wet basis [%]

MCdb = Moisture content dry basis [%]
Wi = Initial weight
Wf = Final weight
Moisture content is one of the most commonly measured properties of food materials. It is
important to food scientists for a number of different reasons:
• Legal and Labeling Requirements. There are legal limits to the maximum or minimum amount
of water that must be present in certain types of food.
• Economic. The cost of many foods depends on the amount of water they contain - water is an
inexpensive ingredient, and manufacturers often try to incorporate as much as possible in a food,
without exceeding some maximum legal requirement
. • Microbial Stability. The propensity of microorganisms to grow in foods depends on their
water content. For this reason many foods are dried below some critical moisture content.
• Food Quality. The texture, taste, appearance and stability of foods depends on the amount of
water they contain.
 • Food Processing Operations. A knowledge of the moisture content is often necessary to predict
the behavior of foods during processing, e.g. mixing, drying flow through pipe or packaging.
Methods for Determination of Moisture in Foods
Methods for determination of moisture in food can be divided into four different classes.
1. Drying methods
2. Distillation procedure
3. Chemical assays
4. Physical procedure
1 Drying methods
 The procedures for determination of the moisture content specified in food standards
generally involve thermal drying methods.
 The material is heated under carefully specified temperature and the loss of weight is
taken as a measure of the moisture content of the sample.
 The value obtained for moisture depends on type of oven, temperature and length of
drying. Therefore, the methods provide same time approximate rather than accurate
moisture values. The rate at which moisture can be removed from the surface of a solid
phase is a function water vapour pressure and of the drying temperature. Practical
consideration dictates, however, selecting temperatures at which the decomposition of
organic compounds is minimised, and yet the time required for quantitative drying at the
selected temperature not unduly prolonged.
Advantages
Drying methods, however, are simple, relatively rapid, and permit the simultaneously analyzes of
large number of samples.
Disadvantages
1. Heating of a moist organic substances causes, in addition, volatilization of material and
formation of gaseous product by irreversible thermal decomposition of organic component.
2. Further weight changes resulting from oxidation phenomenon (i.e. oxidation of oils) occur.
3. Improperly maintained dessicator and dessicants can cause erratic results from pick up of
moisture during cooling.
4. Formation of a crust that is impervious to evaporation of moisture from the centre of a dried
sample.

Factors affecting the precision of moisture measurements by drying methods

The accuracy of moisture determinations is affected by
1. Drying temperature
2. Relative humidity of the drying chamber
3. Air movement in the drying chamber
4. Vacuum in the chamber
5. Depth and particle size of the samples
6. Drying oven construction
7. Number and position of samples in oven
8. Diameter and type of container (material of container)
2 Distillation methods
There are two types of distillation procedures.
 In one type, water is distilled from an immiscible liquid of high boiling point. The sample
suspended in a mineral oil having a flash point much above the boiling point of water is
heated to a predetermined temperature in a suitable apparatus. The water that distills off
is condensed and is collected in a suitable measuring cylinder.
 In the second type, the mixture of water and an immiscible solvent (i.e. xylene, toluene,
tetrachloroethane) distills off and is collected in a suitable measuring apparatus in which
water separates and its volume can be measured.
Liquids with a high specific gravity (tetrachloroethylene, carbon tetrachloride) eliminate fire
hazard and reduce the danger of overheating or charring, as sample floats on top of the liquid.
Advantages

Distillation methods cause less decomposition in some foods than drying at elevated
temperatures. However, chemical reactions produced by heat are reduced but not eliminated.
Disadvantages
Many difficulties may be encountered in the determination of moisture by the distillation
method. These include
1. Relatively low precision of the receiving measuring device.
2. Difficulties in reading the meniscus.
3. Adherence of moisture droplets to the glass.
4. Overboiling (especially with xylol).
5. Solubility of water in the distillation liquid.
6. Incomplete evaporation of water and underestimation of moisture contents.
7. Distillation of water soluble components.
8. Foods in powder form (cereals, flours, starches) tend to bump during the distillation through
overheating on the bottom of the flask.
The main objection to distillation procedures is that they are not adaptable to routine testing.
Some of the disadvantages can be overcome by the following interventions:
1. Adherence of water to the walls of the condenser tubes or sides of the receiving tubes can be
generally remedied by using thoroughly cleaned glassware.
2. Use of small amount of wetting agent will also improve meniscus reading.
3. Incomplete recovery of water due to the formation of an emulsion can sometimes be remedied
by adding small amounts of amyl alcohol or isobutyl alcohol.
4. To improve the moisture distillation, wide mouthed boiling flasks can be used.
5. Dispersing the tested material on diatomaceous earth or on filter – cell is useful with many
viscous foods rich in sugar or protein.
6. Bumping of powder foods can be overcome through the introduction of a small amount of dry
short fiber asbestos.
7. Adverse effects of heat can be reduced still further by selecting organic solvents with a boiling
point below that of water, such as benzene. Such a choice, however, lengthens the distillation
time.
8. For accurate results, standard apparatus and careful attention to specified procedures are
essential.

3 Chemical methods
Karl Fischer Reagent Titration
The time needed for dehydrated foods stimulated the search for more rapid and accurate methods
for determining moisture. The Karl Fischer reagent has proved to be quite adaptable for this
purpose.
Advantages
 It is a method of choice for determination of water in many low moisture foods such as
dried fruits and vegetables, candies, chocolate, roasted coffee, oils and fats.
 Superiority of the method was demonstrated in determining moisture in sugar rich foods
(honey) or foods rich both in reducing sugars and proteins.
 The procedure has been applied also to foods with intermediate moisture foods (bakery
doughs, baked products, fat rich cake mixes) and to foods with high levels of volatile oils.
 The Karl Fischer reagent has proved to be quite adaptable for moisture determination by
chemical method.
 The Karl Fischer method for moisture determination is based on the reaction which
involves the reduction of iodine by sulphur dioxide in the presence of water.
2 H2O + SO2 + I2 ------------- H2SO4 + 2 HI
As shown by the above reaction for each mole of water one mole of iodine is required.
 Methanol is used to dissolve iodine and pyridine is used to dissolve sulphur dioxide.
  Numerous variations have been proposed for the preparation of the Fischer reagent.
 The sample in which water is to be determined is dispersed in an appropriate solvent (i.e.
methanol, mixture of methanol-sulphur dioxide-pyridine etc.).
 The solution is then titrated with a solution of iodine in methanol.
 The excess of iodine that cannot react with water is in free form.
 Adding to the system a few drops of methylene blue gives a green end point.
 Interfering substances are ascorbic acid (oxidation), aldehydes, ketones (release water),
mercaptans, diacylperoxide, thioacids and hydrazines – fading end point.
 The determination of moisture is carried out in a non aqueous system.
 Fluids are delivered but with an automatic pipet or syringe.
 Viscous fluids or pastes are generally homogenized with a solvent.
 Solids are either homogenized with solvent or titrated as suspensions.
 Granular products must be pulverized.
Disadvantages
 The main difficulty in using the Karl Fischer method arises from the lack of complete
water extraction.
 Formaldehyde is found to be a more rapid and versatile extractant of water from foods
than methanol.
 Modification of the extraction procedure is exemplified by a method for the water
determination in dairy products, where in xylene or carbon tetrachloride is employed in
mixed solvent systems with alcohol.
4. Physical methods
 Infrared determination – based on measuring the absorption at wavelengths characteristic
of the molecular vibration in water.
 The most useful wavelengths are 3.0 & 6.1 μm.
 Gas chromatographic method: based on extracting the moisture with an organic solvent
and determining water in the extract by gas chromatography
 Nuclear magnetic resonance
 Electric method: Densitometric method
 Refractometric method
 Polarimetric method
 EXAMPLE.

1. As an analyst, you are given a sample of condensed soup to analyze to determine if it is

reduced to the correct concentration. By gravimetric means, you find that the concentration is
26.54% solids. The company standard reads 28.63%. If the starting volume were 1000 gallons at
8.67% solids and the weight is 8.5 pounds per gallon, how much more water must be removed?
2. Your laboratory just received several sample containers of peas to analyze for moisture
content. There is a visible condensate on the inside of the container. What is your procedure to
obtain a result?
3. You have the following gravimetric results:
 Weight of dried pan and glass disc is 1.0376 g,
 weight of pan and liquid sample is 4.6274 g,
 And weight of the pan and dried sample is 1.7321 g.
What was the moisture content of the sample and what is the percent solids?
Answers
1. The weight of the soup initially is superfluous information. By condensing the soup to 26.54%
solids from 8.67% solids, the volume is reduced to 326.7 gal
[(8.67%/26.54%) × 1000 gal]. You need to reduce the volume further to obtain 28.63% solids
[(8.67%/28.63%) × 1000 gal] or 302.8 gal. The difference in the gallons obtained is 23.9 gal
(326.7 gal − 302.8 gal), or the volume of water that must be removed from the partially
condensed soup to comply with company standards.
2. This problem focuses on a real issue in the food processing industry – when do you analyze a
sample and when don’t you? It would appear that the peas have lost moisture that should be
within the vegetable for correct results. You will need to grind the peas in a food mill or blender.
If the peas are in a Mason jar or one that fits a blender head, no transfer is needed. Blend the peas
to a creamy texture. If a container transfer was made, then put the blended peas back into the
original container. Mix with the residual moisture to a uniform blend. Collect a sample for
moisture analysis. You should note on the report form containing the results of the analysis that
the pea samples had free moisture on container walls when they arrived.
3. Note Equations [2]–[4]. To use any of the equations, you must subtract the weight of the dried
pan and glass disc.
Then you obtain 3.5898 g of original sample and 0.6945 g when dried. By subtracting these
results, you have removed water (2.8953 g).
Then (0.6945 g/3.5898 g) × 100 = 19.35% solids and (2.8953 g/3.5898 g) × 100 = 80.65% water.
 DETERMINATION OF ASH CONTENT.

 ash content is a measure of the total amount of minerals present within a food,
 Mineral content is a measure of the amount of specific inorganic components
present within a food, such as Ca, Na, K and Cl.

Determination of the ash and mineral content of foods is important for a number of
reasons:

 Nutritional labeling. The concentration and type of minerals present must often be
stipulated on the label of a food.
 Quality. The quality of many foods depends on the concentration and type of minerals
they contain, including their taste, appearance, texture and stability.
 Microbiological stability. High mineral contents are sometimes used to retard the growth
of certain microorganisms.
 Nutrition. Some minerals are essential to a healthy diet (e.g., calcium, phosphorous,
potassium and sodium) whereas others can be toxic (e.g., lead, mercury, cadmium and
aluminum).
 Processing. It is often important to know the mineral content of foods during processing
because this affects the physicochemical properties of foods.

Determination of Ash Content

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

Sample Preparation
  As with all food analysis procedures it is crucial to carefully select a sample whose
composition represents that of the food being analyzed and to ensure that its
composition does not change significantly prior to analysis.
 Typically, samples of 1-10g are used in the analysis of ash content.
 Solid foods are finely ground and then carefully mixed to facilitate the choice of a
representative sample.
 Before carrying out an ash analysis, samples that are high in moisture are often dried
to prevent spattering during ashing.
 High fat samples are usually defatted by solvent extraction, as this facilitates the
release of the moisture and prevents spattering.
 Other possible problems include contamination of samples by minerals in grinders,
glassware or crucibles which come into contact with the sample during the analysis.
For the same reason, it is recommended to use deionized water when preparing
samples.

. Dry Ashing

 Dry ashing procedures use a high temperature muffle furnace capable of maintaining
temperatures of between 500 and 600 oC.
 Water and other volatile materials are vaporized and organic substances are burned
in the presence of the oxygen in air to CO2, H2O and N2.
 Most minerals are converted to oxides, sulfates, phosphates, chlorides or silicates.
 Although most minerals have fairly low volatility at these high temperatures, some
are volatile and may be partially lost, e.g., iron, lead and mercury.
 If an analysis is being carried out to determine the concentration of one of these
substances then it is advisable to use an alternative ashing method that uses lower
temperatures.

The food sample is weighed before and after ashing to determine the concentration of ash
present. The ash content can be expressed on either a dry or wet basis:

Where MASH refers to the mass of the ashed sample, and MDRY and MASH refer to the original
masses of the dried and wet samples.

 There are a number of different types of crucible available for ashing food samples,
including quartz, Pyrex, porcelain, steel and platinum.
 Selection of an appropriate crucible depends on the sample being analyzed and the
furnace temperature used.
  The most widely used crucibles are made from porcelain because it is relatively
inexpensive to purchase, can be used up to high temperatures (< 1200oC) and are easy to
clean.
 Porcelain crucibles are resistant to acids but can be corroded by alkaline samples, and
therefore different types of crucible should be used to analyze this type of sample.
 In addition, porcelain crucibles are prone to cracking if they experience rapid
temperature changes.
 A number of dry ashing methods have been officially recognized for the determination
of the ash content of various foods (AOAC Official Methods of Analysis). Typically, a
sample is held at 500-600 oC for 24 hours.

 Advantages: Safe, few reagents are required, many samples can be analyzed
simultaneously, not labor intensive, and ash can be analyzed for specific mineral content.
 Disadvantages: Long time required (12-24 hours), muffle furnaces are quite costly to run
due to electrical costs, loss of volatile minerals at high temperatures, e.g., Cu, Fe, Pb, Hg,
Ni, Zn.

Recently, analytical instruments have been developed to dry ash samples based on
microwave heating. These devices can be programmed to initially remove most of the moisture
(using a relatively low heat) and then convert the sample to ash (using a relatively high heat).
Microwave instruments greatly reduce the time required to carry out an ash analysis, with the
analysis time often being less than an hour. The major disadvantage is that it is not possible to
simultaneously analyze as many samples as in a muffle furnace.

Wet Ashing

 Wet ashing is primarily used in the preparation of samples for subsequent analysis of
specific minerals
 It breaks down and removes the organic matrix surrounding the minerals so that they
are left in an aqueous solution.
 A dried ground food sample is usually weighed into a flask containing strong acids
and oxidizing agents (e.g., nitric, perchloric and/or sulfuric acids) and then heated.
 Heating is continued until the organic matter is completely digested, leaving only the
mineral oxides in solution.
 The temperature and time used depends on the type of acids and oxidizing agents
used.
 Typically, a digestion takes from 10 minutes to a few hours at temperatures of about
350oC. The resulting solution can then be analyzed for specific minerals.

 Advantages: Little loss of volatile minerals occurs because of the lower temperatures
used, more rapid than dry ashing.
 Disadvantages Labor intensive, requires a special fume-cupboard if perchloric acid is
used because of its hazardous nature, low sample throughput.

Low Temperature Plasma Ashing

  A sample is placed into a glass chamber which is evacuated using a vacuum pump.
 A small amount of oxygen is pumped into the chamber and broken down to nascent
oxygen (O2 � 2O.) by application of an electromagnetic radio frequency field.
 The organic matter in the sample is rapidly oxidized by the nascent oxygen and the
moisture is evaporated because of the elevated temperatures.
 The relatively cool temperatures (< 150oC) used in low-temperature plasma ashing
cause less loss of volatile minerals than other methods.

 Advantages: Less chance of losing trace elements by volatilization

 Disadvantages: Relatively expensive equipment and small sample throughput.

Determination of Water Soluble and Insoluble Ash

o As well as the total ash content, it is sometimes useful to determine the ratio of water
soluble to water-insoluble ash as this gives a useful indication of the quality of certain
foods, e.g., the fruit content of preserves and jellies.
o Ash is diluted with distilled water then heated to nearly boiling, and the resulting
solution is filtered.
o The amount of soluble ash is determined by drying the filtrate, and the insoluble ash
is determined by rinsing, drying and ashing the filter paper.

Comparison of Ashing Methods

 The conventional dry ashing procedure is simple to carry out, is not labor intensive,
requires no expensive chemicals and can be used to analyze many samples
simultaneously.
 Nevertheless, the procedure is time-consuming and volatile minerals may be lost at
the high temperatures used.
 Microwave instruments are capable of speeding up the process of dry ashing.
 Wet ashing and low temperature plasma ashing are more rapid and cause less loss of
volatile minerals because samples are heated to lower temperatures.
 Nevertheless, the wet ashing procedure requires the use of hazardous chemicals and
is labor intensive, while the plasma method requires expensive equipment and has a
low sample throughput.

Determination of Specific Mineral Content

 Knowledge of the concentration and type of specific minerals present in food

products is often important in the food industry.
 The major physicochemical characteristics of minerals that are used to distinguish
them from the surrounding matrix are: their low volatility; their ability to react with
specific chemical reagents to give measurable changes; and their unique
electromagnetic spectra.
 The most effective means of determining the type and concentration of specific
minerals in foods is to use atomic absorption or emission spectroscopy.
  Instruments based on this principle can be used to quantify the entire range of
minerals in foods, often to concentrations as low as a few ppm.
 For these reasons they have largely replaced traditional methods of mineral analysis
in institutions that can afford to purchase and maintain one, or that routinely analyze
large numbers of samples.
 Institutions that do not have the resources or sample throughput to warrant purchasing
an atomic spectroscopy instrument rely on more traditional methods that require
chemicals and equipment commonly found in food laboratories.
 Many of the minerals of importance to food scientists can be measured using one of
these traditional methods.

Sample preparation

 Many of the analytical methods used to determine the specific mineral content of foods
require that the minerals be dissolved in an aqueous solution.
 For this reason, it is often necessary to isolate the minerals from the organic matrix
surrounding them prior to the analysis.
 This is usually carried out by ashing a sample using one of the methods described in the
previous section.
 It is important that the ashing procedure does not alter the mineral concentration in the
food due to volatilization.
 Another potential source of error in mineral analysis is the presence of contaminants in
the water, reagents or glassware. For this reason, ultrapure water or reagents should be
used, and/or a blank should be run at the same time as the sample being analyzed.
 A blank uses the same glassware and reagents as the sample being analyzed and therefore
should contain the same concentration of any contaminants.
 The concentration of minerals in the blank is then subtracted from the value determined
for the sample.
 Some substances can interfere with analysis of certain minerals, and should therefore be
eliminated prior to the analysis or accounted for in the data interpretation.
 The principles of a number of the most important traditional methods for analyzing
minerals are described below.
 Many more traditional methods can be found in the AOAC Official Methods of
Analysis.

Gravimetric Analysis

 The element to be analyzed is precipitated from solution by adding a reagent that reacts
with it to form an insoluble complex with a known chemical formula.
 The precipitate is separated from the solution by filtration, rinsed, dried and weighed.
 The amount of mineral present in the original sample is determined from a knowledge of
the chemical formula of the precipitate.
 For example, the amount of chloride in a solution can be determined by adding excess
silver ions to form an insoluble silver chloride precipitate, because it is known that Cl is
24.74% of AgCl.
 Gravimetric procedures are only suitable for large food samples, which have relatively
high concentrations of the mineral being analyzed.
 They are not suitable for analysis of trace elements because balances are not sensitive
enough to accurately weigh the small amount of precipitate formed.

Colorimetric methods

 These methods rely on a change in color of a reagent when it reacts with a specific
mineral in solution which can be quantified by measuring the absorbance of the solution
at a specific wavelength using a spectrophotometer.
 Colorimetric methods are used to determine the concentration of a wide variety of
different minerals.
 Vandate is often used as a colorimetric reagent because it changes color when it reacts
with minerals.
 For example, the phosphorous content of a sample can be determined by adding a
vandate-molybdate reagent to the sample. This forms a colored complex (yellow-orange)
with the phosphorous which can be quantified by measuring the absorbance of the
solution at 420nm, and comparing with a calibration curve. Different reagents are also
available to colorimetrically determine the concentration of other minerals.

Titrations

EDTA compleximetric titration

 EDTA is a chemical reagent that forms strong complexes with multivalent metallic ions.
 The disodium salt of EDTA is usually used because it is available in high purity: Na 2H2Y.
 The complexes formed by metal ions and EDTA can be represented by the following
equations:

m2+ + H2Y2- � mY2- + 2H+

m3+ + H2Y2- � mY- + 2H+

m4+ + H2Y2- � mY + 2H+

 The calcium content of foods is often determined by this method.

 An ashed food sample is diluted in water and then made alkaline (pH 12.5 to 13).
 An indicator that can form a colored complex with EDTA is then added to the solution,
and the solution is titrated with EDTA.
 The EDTA-indicator complex is chosen to be much weaker than the EDTA-mineral
complex.
 Consequently, as long as multivalent ions remain in the solution the EDTA forms a
strong complex with them and does not react with the indicator.
 However, once all the mineral ions have been complexed, any additional EDTA reacts
with the indicator and forms a colored complex that is used to determine the end-point of
the reaction.
  The calcium content of a food sample is determined by comparing the volume of EDTA
required to titrate it to the end-point with a calibration curve prepared for a series of
solutions of known calcium concentration.
 If there is a mixture of different multivalent metallic ions present in a food there could be
some problems in determining the concentration of a specific type of ion.
 It is often possible to remove interfering ions by passing the solution containing the
sample through an ion-exchange column prior to analysis.

Redox reactions

 Many analytical procedures are based on coupled reduction-oxidation (redox) reactions.

 Reduction is the gain of electrons by atoms or molecules, whereas oxidation is the
removal of electrons from atoms or molecules.
 Any molecular species that gains electrons during the course of a reaction is said to
be reduced, whereas any molecular species that loses electrons is said to be oxidized,
whether or not oxygen is involved.
 Electrons cannot be created or destroyed in ordinary chemical reactions and so any
oxidation reaction is accompanied by a reduction reaction.
 These coupled reactions are called redox reactions:

Xn � Xn+1 + e-

(Oxidation reaction � loss of electrons)

Ym + e- � Ym-1

(Reduction reaction � gain of electrons)

Xn + Ym � Xn+1 + Ym-1

(Coupled reaction� transfer of electrons)

 Analysts often design a coupled reaction system so that one of the half-reactions leads to
a measurable change in the system that can be conveniently used as an end-point, e.g., a
color change.
 Thus one of the coupled reactions usually involves the mineral being analyzed (e.g., X =
analyte), whereas the other involves an indicator (e.g., Y = indicator).

For example, permanganate ion (MnO4-) is a deep purple color (oxidized form), while the
mangenous ion (Mn2+) is a pale pink color (reduced form). Thus permanganate titrations can be
used as an indicator of many redox reactions:

MnO4- + 8H+ + 5e- � Mn2+ + 4H20

(Reduction reaction)
(Deep Purple)

(Pale Pink)

The calcium or iron content of foods can be determined by titration with a solution of
potassium permanganate, the end point corresponding to the first change of the solution from
pale pink to purple. The calcium or iron content is determined from the volume of permanganate
solution of known molarity that is required to reach the end-point. For iron the reaction is:

5Fe2+ � 5Fe3+ + 5e- (Oxidation reaction)

MnO4- + 8H+ + 5e- � Mn2+ + 4H20 (Reduction reaction)

5Fe2+ + MnO4- + 8H+ � 5Fe3+ + Mn2+ + 4H20 � (Coupled reaction)

Potassium permanganate is titrated into the aqueous solution of ashed food. While there is
Fe2+ remaining in the food the MnO4- is converted to Mn2+ that leads to a pale pink solution. Once
all of the Fe2+ has been converted to Fe3+ then the MnO4- remains in solution and leads to the
formation of a purple color, which is the end-point.

Precipitation titrations

When at least one product of a titration reaction is an insoluble precipitate, it is referred to as

a precipitation titration. A titrimetric method commonly used in the food industry is
the Mohr method for chloride analysis. Silver nitrate is titrated into an aqueous solution
containing the sample to be analyzed and a chromate indicator.

AgNO3 + NaCl � AgCl(s) + NaNO3

The interaction between silver and chloride is much stronger than that between silver and
chromate. The silver ion therefore reacts with the chloride ion to form AgCl, until all of
the chloride ion is exhausted. Any further addition of silver nitrate leads to the formation of
silver chromate, which is an insoluble orange colored solid.

Ag+ + Cl- � AgCl (colorless)� - until all Cl- is complexed

2Ag+ + CrO42- � Ag2CrO4 (orange) �� - after all Cl- is complexed

The end point of the reaction is the first hint of an orange color. The volume of silver nitrate
solution (of known molarity) required to reach the endpoint is determined, and thus the
concentration of chloride in solution can be calculated.

Ion-Selective Electrodes

The mineral content of many foods can be determined using ion-selective electrodes (ISE).
These devices work on the same principle as pH meters, but the composition of the glass
electrode is different so that it is sensitive to specific types of ion (rather than H +). Special glass
electrodes are commercially available to determine the concentration of K +, Na+, NH4+, Li+,
Ca2+ and Rb+ in aqueous solution. Two electrodes are dipped into an aqueous solution containing
the dissolved mineral: a reference electrode and a ion-selective electrode. The voltage across the
electrodes depends on the concentration of the mineral in solution and is measured at extremely
low current to prevent alterations in ion concentration. The concentration of a specific mineral is
determined from a calibration curve of voltage versus the logarithm of concentration. The major
advantages of this method are its simplicity, speed and ease of use. The technique has been used
to determine the salt concentration of butter, cheese and meat, the calcium concentration of milk
and the CO2 concentration of soft drinks. In principle, an ion selective electrode is only sensitive
to one type of ion, however, there is often interference from other types of ions. This problem
can often be reduced by adjusting pH, complexing or precipitating the interfering ions.�

Finally, it should be noted that the ISE technique is only sensitive to the concentration
of free ions present in a solution.� If the ions are complexed with other components, such as
chelating agents or biopolymers, then they will not be detected.� The ISE technique is therefore
particularly useful for quantifying the binding of minerals to food components.� If one wants to
determine the total concentration of a specific ion in a food (rather than the free concentration),
then one needs to ensure that ion binding does not occur, e.g., by ashing the food.

4.3.6 Atomic Spectroscopy

The determination of mineral type and concentration by atomic spectroscopy is more

sensitive, specific, and quicker than traditional wet chemistry methods. For this reason it has
largely replaced traditional methods in laboratories that can afford it or that routinely analyze for
minerals.

Principles of Atomic Spectroscopy

The primary cause of absorption and emission of radiation in atomic spectroscopy

is electronic transitions of outer shell electrons. Photons with the energy associated with this
type of transition are found in the UV-visible part of the electromagnetic spectrum. In this
respect atomic spectroscopy is similar to UV-visible spectroscopy, however, the samples used in
atomic spectroscopy are individual atoms in a gaseous state, whereas those used in UV-visible
spectroscopy are molecules dissolved in liquids. This has important consequences for the nature
of the spectra produced. In atomic spectroscopy the peaks are narrow and well defined, but in
UV-visible spectroscopy they are broad and overlap with one another. The are two major reasons
for this. Firstly, because absorption or emission is from atoms, rather than molecules, there are
no vibrational or rotational transitions superimposed on the electronic transitions. Secondly,
because the atoms are in a gaseous state they are well separated from each other and do not
interact with neighboring molecules.
 The energy change associated with a transition between two energy levels is related to the
wavelength of the absorbed radiation: DE = hc/l, where, h = Planks constant, c = the speed of
light and l = the wavelength. Thus for a given transition between two energy states radiation of a
discrete wavelength is either absorbed or emitted. Each element has a unique electronic structure
and therefore it has a unique set of energy levels. Consequently, it absorbs or emits radiation at
specific wavelengths. Each spectrum is therefore like a "fingerprint" that can be used to identify
a particular element. In addition, because the absorption and emission of radiation occurs at
different wavelengths for different types of atom, one element can be distinguished from others
by making measurements at a wavelength where it absorbs or emits radiation, but the other
elements do not.

Absorption occurs primarily when electrons in the ground state are promoted to various
excited states. Emission occurs when electrons in an excited state fall back to a lower energy
level. Atoms can exist in a number of different excited states, and can fall back to one of many
different lower energy states (not necessarily the ground state). Thus there are many more
lines in an emission spectra than there are in an absorption spectra.

Atomic spectroscopy is used to provide information about the type and concentration of
minerals in foods. The type of minerals is determined by measuring the position of the peaks in
the emission or absorption spectra. The concentration of mineral components is determined by
measuring the intensity of a spectral line known to correspond to the particular element of
interest. The reduction in intensity of an electromagnetic wave that travels through a sample is
used to determine the absorbance: A = -log(I/Io). The Beer-Lambert law can then be used to
relate the absorbance to the concentration of atoms in the sample: A = a.b.c, where A is
absorbance, a is extinction cofficient, b is sample pathlength and c is concentration of absorbing
species. In practice, there are often deviations from the above equation and so it is often
necessary to prepare a calibration curve using a series of standards of known concentration
prepared using the same reagents as used to prepare the sample. It is also important to run a
blank to take into account any impurities in the reagents that might interfere with the analysis.

Atomic Absorption Spectroscopy

Atomic absorption spectroscopy (AAS) is an analytical method that is based on the

absorption of UV-visible radiation by free atoms in the gaseous state. The food sample to be
analyzed is normally ashed and then dissolved in an aqueous solution. This solution is placed in
the instrument where it is heated to vaporize and atomize the minerals. A beam of radiation is
passed through the atomized sample, and the absorption of radiation is measured at specific
wavelengths corresponding to the mineral of interest. Information about the type and
concentration of minerals present is obtained by measuring the location and intensity of the
peaks in the absorption spectra.

Instrumentation

The radiation source. The most commonly used source of radiation in AAS is the hollow
cathode lamp. This is a hollow tube filled with argon or neon, and a cathode filament made of the
metallic form of the element to be analyzed. When a voltage is applied across the electrodes, the
lamp emits radiation characteristic of the metal in the cathode i.e., if the cathode is made of
sodium, a sodium emission spectrum is produced. When this radiation passes through a sample
containing sodium atoms it will be absorbed because it contains radiation of exactly the right
wavelength to promote transition from one energy level to another. Thus a different lamp is
needed for each type of element analyzed.

Chopper. The radiation arriving at the detector comes from two different sources: (i)
radiation emitted by the filament of the lamp (which is partially absorbed by the sample); (ii)
radiation that is emitted by the atoms in the sample that have been excited to higher energy levels
by absorption of energy from the atomizer. To quantify the concentration of minerals in a sample
using AAS it is necessary to measure the reduction in amplitude of the beam of radiation that has
passed through the sample, rather than the radiation emitted by the excited sample. This can be
done using a mechanical device, called a chopper, in conjunction with an electronic device that
distinguishes between direct and alternating currents. The chopper is a spinning disk with a
series of slits which is placed between the radiation source and the sample. The radiation from
the light source is therefore continuously being switched on and off at a specific
frequency, i.e., it is an alternating current. On the other hand, the radiation emitted from the
excited atoms in the sample is constant i.e., it is direct current. The overall detected radiation is
therefore the sum of a varying component and a constant component. Electronic devices are
available which can separate alternating and constant current. These devices are used in AAS
instruments to isolate the signal generated by the light from that emitted by the atoms in the
sample.

Atomizer. Atomizers are used to convert the sample to be analyzed into individual atoms.
The atomization process is achieved by exposing the sample to high temperatures, and involves
three stages: (i) removal of water associated with molecules, (ii) conversion of molecules into a
gas, and (iii) atomization of molecules. At higher temperatures the atoms may become ionized,
which is undesirable because the atomic spectra of ionized atoms is different from that of non-
ionized ones. Consequently, it is important to use a high enough temperature to atomize the
molecules, but not so high that the atoms are ionized. Two types of atomizer are commonly used
in atomic absorption instruments: flame and electrothermal atomization.

 Flame-atomizers consist of a nebulizer and a burner. The nebulizer converts the solution
into a fine mist or aerosol. The sample is forced through a tiny hole into a chamber
through which the oxidant and fuel are flowing. The oxidant and fuel carry the sample
into the flame. The burner is usually 5 -10 centimeters long so as to give a long
pathlength for the radiation to travel along. The characteristics of the flame can be altered
by varying the relative proportions and types of oxidant and fuel used in the flame. Air-
acetelyne and Nitrogen oxide-acetylene are the most commonly used mixtures of oxidant
and fuel. Thus flames with different temperatures can be produced. This is important
because the energy required to cause atomization, but not ionization, varies from
substance to substance. Instrument manufactures provide guidelines with their
instruments about the type of flame to use for specific elements.
 In electrothermal AAS the sample is placed in a small graphite cup which is electrically
heated to a temperature (typically 2,000 - 3,000 oC) high enough to produce volatilization
and atomization. The cup is positioned so that the radiation beam passes through the
 atomized sample. The advantage of electrothermal atomizers is that smaller samples are
required and detection limits are lower. Major disadvantages are that they are more
expensive to purchase, have a lower sample throughput, are more difficult to operate and
have a lower precision than flame-atomizers.

Wavelength selector. A wavelength selector is positioned in the optical path between the
flame (or furnace) and the detector. It's purpose is to isolate the spectral line of interest from the
rest of the radiation coming from the sample, so that only the radiation of the desired wavelength
reaches the detector. Wavelength selectors are typically, monochromatic gratings or filters.

Detector/Readout. The detector is a photomultiplier tube that converts electromagnetic

energy reaching it into an electrical signal. Most modern instruments have a computer to display
the signal output and store the spectra.

Atomic Emission Spectroscopy

Atomic emission spectroscopy (AES) is different from AAS, because it utilizes the emission
of radiation by a sample, rather than the absorption. For this reason samples usually have to be
heated to a higher temperature so that a greater proportion of the atoms are in an excited state
(although care must be taken to ensure that ionization does not occur because the spectra from
ionized atoms is different from that of non-ionized atoms). There are a number of ways that the
energy can be supplied to a sample, including heat, light, electricity and radio waves.

Instrumentation

In AES the sample itself acts as the source of the detected radiation, and therefore there is no
need to have a separate radiation source or a chopper. The sample is heated to a temperature
where it is atomized and a significant proportion of the atoms is in an excited state. Atomic
emissions are produced when the electrons in an excited state fall back to lower energy levels.
Since the allowed energy levels for each atom are different, they each have characteristic
emission spectrum from which they can be identified. Since a food usually contains a wide
variety of different minerals, each with a characteristics emission spectrum, the overall spectrum
produced contains many absorption peaks. The emitted radiation is therefore passed through a
wavelength selector to isolate specific peaks in the spectra corresponding to the atom of interest,
and the intensity of the peak is measured using a detector and displayed on a read-out device.

Atomization-Excitation Source. The purpose of the atomization-excitation source is to

atomize the sample, and to excite the atoms so that they emit a significant amount of detectable
radiation. The two most commonly used forms of atomization-excitation sources in food analysis
are Flame and Inductively Coupled Plasma (ICP) devices.

 In flame-AES a nebulizer-burner system is used to atomize the minerals in the sample

and excite a large proportion of them to higher energy levels.
 In ICP-AES a special device is used that heats the sample to very high temperatures
(6,000 to 10,000 K) in the presence of argon ions. The minerals in the sample are not
ionized at these temperatures because of the high concentration of argon ions
 (Ar � Ar+ + e-) leads to the release of electrons that push the equilibrium towards the
non-ionized form of the mineral (M+ + e- � M).

Wavelength selectors. Wavelength selectors are used to isolate particular spectral lines,
which are characteristic of the material being studied, from all the other spectral lines. A number
of different types of wavelength selector are available including filters and gratings. A filter can
only be used to measure the intensity at a particular fixed wavelength, whereas a grating can be
used to measure the intensity at many different wavelengths. A filter can therefore only be used
to analyze for one type of mineral, whereas a grating can be used to measure many different
types of minerals.

Practical considerations

Prior to making atomic spectroscopy measurements a food sample is usually ashed. The
resulting ash is dissolved in a suitable solvent, such as water or dilute HCl, before injecting it
into the instrument. Sometimes it is possible to analyze a sample without ashing it first. For
example, vegetables oils can be analyzed by dissolving them in acetone or ethanol and injecting
them directly into the instrument.

Concentrations of mineral elements in foods are often at the trace level and so it is important
to use very pure reagents when preparing samples for analysis. Similarly, one should ensure that
glassware in very clean and dry, so that it contains no contaminating elements. It is also
important to ensure there are no interfering substances in the sample whose presence would lead
to erroneous results. An interfering substance could be something that absorbs at the same
wavelength as the mineral being analyzed, or something that binds to the mineral and prevents it
from being efficiently atomized. There are various techniques available for removing the effects
of these interfering substances.

Determination of protein content in food.

Protein analysis is important for:

1. Nutrition labeling
2. Pricing: The cost of certain commodities is based on the protein content as measured
by nitrogen content (e.g., cereal grains; milk for making certain dairy products, e.g.,
cheese).
3. Functional property investigation: Proteins in various types of food have unique food
functional properties: for example, gliadin and glutelins in wheat flour for bread making,
casein in milk for coagulation into cheese products, and egg albumen for foaming (see
4. Biological activity determination: Some proteins, including enzymes or enzyme
inhibitors, are relevant to food science and nutrition: for instance, the proteolytic enzymes
in the tenderization of meats, pectinases in the ripening of fruits, and trypsin inhibitors in
legume seeds are proteins. To compare between samples, enzymes activity often is
expressed in terms of specific activity, meaning units of enzyme activity per mg of
protein.

Protein analysis is required when you want to know:

 1. Total protein content
2. Content of a particular protein in a mixture
3. Protein content during isolation and purification of a protein
4. No protein nitrogen
5. Amino acid composition
6. Nutritive value of a protein
9.1.3 Content in Food

. Kjeldahl method

 The Kjeldahl method was developed in 1883 by a brewer called Johann Kjeldahl.
 A food is digested with a strong acid so that it releases nitrogen which can be determined
by a suitable titration technique.
 The amount of protein present is then calculated from the nitrogen concentration of the
food.
 The same basic approach is still used today, although a number of improvements have
been made to speed up the process and to obtain more accurate measurements
 . It is usually considered to be the standard method of determining protein concentration.

 Because the Kjeldahl method does not measure the protein content directly a conversion
factor (F) is needed to convert the measured nitrogen concentration to a protein
concentration. A conversion factor of 6.25 (equivalent to 0.16 g nitrogen per gram of
protein) is used for many applications, however, this is only an average value, and each
protein has a different conversion factor depending on its amino-acid composition.
 The Kjeldahl method can conveniently be divided into three steps: digestion,
neutralization and titration.

Principles

Digestion

 The food sample to be analyzed is weighed into a digestion flask and then digested by
heating it in the presence of sulfuric acid (an oxidizing agent which digests the food),
anhydrous sodium sulfate (to speed up the reaction by raising the boiling point) and a
catalyst, such as copper, selenium, titanium, or mercury (to speed up the reaction).
 Digestion converts any nitrogen in the food (other than that which is in the form of
nitrates or nitrites) into ammonia, and other organic matter to C02 and H20.
 Ammonia gas is not liberated in an acid solution because the ammonia is in the form of
the ammonium ion (NH4+) which binds to the sulfate ion (SO42-) and thus remains in
solution:

N(food) ® (NH4)2SO4 (1)

Neutralization
After the digestion has been completed the digestion flask is connected to a recieving flask by a
tube. The solution in the digestion flask is then made alkaline by addition of sodium hydroxide,
which converts the ammonium sulfate into ammonia gas:

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

The ammonia gas that is formed is liberated from the solution and moves out of the digestion
flask and into the receiving flask - which contains an excess of boric acid. The low pH of the
solution in the receiving flask converts the ammonia gas into the ammonium ion, and
simultaneously converts the boric acid to the borate ion:

NH3 + H3BO3 (boric acid) ® NH4+ + H2BO3- (borate ion) (3)

Titration

The nitrogen content is then estimated by titration of the ammonium borate formed with standard
sulfuric or hydrochloric acid, using a suitable indicator to determine the end-point of the
reaction.

H2BO3- + H+ ® H3BO3 (4)

The concentration of hydrogen ions (in moles) required to reach the end-point is equivalent to
the concentration of nitrogen that was in the original food (Equation 3). The following equation
can be used to determine the nitrogen concentration of a sample that weighs m grams using a xM
HCl acid solution for the titration:

(5)

Where vs and vb are the titration volumes of the sample and blank, and 14g is the molecular
weight of nitrogen N. A blank sample is usually ran at the same time as the material being
analyzed to take into account any residual nitrogen which may be in the reagents used to carry
out the analysis. Once the nitrogen content has been determined it is converted to a protein
content using the appropriate conversion factor: %Protein = F� %N.

Advantages and Disadvantages

Advantages. The Kjeldahl method is widely used internationally and is still the standard method
for comparison against all other methods. Its universality, high precision and good
reproducibility have made it the major method for the estimation of protein in foods.

Disadvantages. It does not give a measure of the true protein, since all nitrogen in foods is not in
the form of protein. Different proteins need different correction factors because they have
different amino acid sequences. The use of concentrated sulfuric acid at high temperatures poses
a considerable hazard, as does the use of some of the possible catalysts The technique is time
consuming to carry-out.

6.2.2. Enhanced Dumas method

Recently, an automated instrumental technique has been developed which is capable of rapidly
measuring the protein concentration of food samples. This technique is based on a method first
described by a scientist called Dumas over a century and a half ago. It is beginning to compete
with the Kjeldahl method as the standard method of analysis for proteins for some foodstuffs due
to its rapidness.

6.2.2.1. General Principles

A sample of known mass is combusted in a high temperature (about 900 oC) chamber in the
presence of oxygen. This leads to the release of CO2, H2O and N2. The CO2 and H2O are removed
by passing the gasses over special columns that absorb them. The nitrogen content is then
measured by passing the remaining gasses through a column that has a thermal conductivity
detector at the end. The column helps separate the nitrogen from any residual CO2 and H2O that
may have remained in the gas stream. The instrument is calibrated by analyzing a material that is
pure and has a known nitrogen concentration, such as EDTA (= 9.59%N). Thus the signal from
the thermal conductivity detector can be converted into a nitrogen content. As with the Kjeldahl
method it is necessary to convert the concentration of nitrogen in a sample to the protein content,
using suitable conversion factors which depend on the precise amino acid sequence of the
protein.

6.2.2.2. Advantages and Disadvantages

Advantages: It is much faster than the Kjeldahl method (under 4 minutes per measurement,
compared to 1-2 hours for Kjeldahl). It doesn't need toxic chemicals or catalysts. Many samples
can be measured automatically. It is easy to use.

Disadvantages: High initial cost. It does not give a measure of the true protein, since all nitrogen
in foods is not in the form of protein. Different proteins need different correction factors because
they have different amino acid sequences. The small sample size makes it difficult to obtain a
representative sample.

6.2.3. Methods using UV-visible spectroscopy

A number of methods have been devised to measure protein concentration, which are based on
UV-visible spectroscopy. These methods use either the natural ability of proteins to absorb (or
scatter) light in the UV-visible region of the electromagnetic spectrum, or they chemically or
physically modify proteins to make them absorb (or scatter) light in this region. The basic
principle behind each of these tests is similar. First of all a calibration curve of absorbance (or
turbidity) versus protein concentration is prepared using a series of protein solutions of known
concentration. The absorbance (or turbidity) of the solution being analyzed is then measured at
the same wavelength, and its protein concentration determined from the calibration curve. The
main difference between the tests are the chemical groups which are responsible for the
absorption or scattering of radiation, e.g., peptide bonds, aromatic side-groups, basic groups and
aggregated proteins.

A number of the most commonly used UV-visible methods for determining the protein content
of foods are highlighted below:

6.2.3.1. Principles

Direct measurement at 280nm

Tryptophan and tyrosine absorb ultraviolet light strongly at 280 nm. The tryptophan and tyrosine
content of many proteins remains fairly constant, and so the absorbance of protein solutions at
280nm can be used to determine their concentration. The advantages of this method are that the
procedure is simple to carry out, it is nondestructive, and no special reagents are required. The
major disadvantage is that nucleic acids also absorb strongly at 280 nm and could therefore
interfere with the measurement of the protein if they are present in sufficient concentrations.
Even so, methods have been developed to overcome this problem, e.g., by measuring the
absorbance at two different wavelengths.

Biuret Method

A violet-purplish color is produced when cupric ions (Cu2+) interact with peptide bonds under
alkaline conditions. The biuret reagent, which contains all the chemicals required to carry out the
analysis, can be purchased commercially. It is mixed with a protein solution and then allowed to
stand for 15-30 minutes before the absorbance is read at 540 nm. The major advantage of this
technique is that there is no interference from materials that adsorb at lower wavelengths, and the
technique is less sensitive to protein type because it utilizes absorption involving peptide bonds
that are common to all proteins, rather than specific side groups. However, it has a relatively low
sensitivity compared to other UV-visible methods.

Lowry Method

The Lowry method combines the biuret reagent with another reagent (the Folin-Ciocalteau
phenol reagent) which reacts with tyrosine and tryptophan residues in proteins. This gives a
bluish color which can be read somewhere between 500 - 750 nm depending on the sensitivity
required. There is a small peak around 500 nm that can be used to determine high protein
concentrations and a large peak around 750 nm that can be used to determine low protein
concentrations. This method is more sensitive to low concentrations of proteins than the biuret
method.

Dye binding methods

A known excess of a negatively charged (anionic) dye is added to a protein solution whose pH is
adjusted so that the proteins are positively charged (i.e. < the isoelectric point). The proteins
form an insoluble complex with the dye because of the electrostatic attraction between the
molecules, but the unbound dye remains soluble. The anionic dye binds to cationic groups of the
basic amino acid residues (histidine, arganine and lysine) and to free amino terminal groups. The
amount of unbound dye remaining in solution after the insoluble protein-dye complex has been
removed (e.g., by centrifugation) is determined by measuring its absorbance. The amount of
protein present in the original solution is proportional to the amount of dye that bound to it:
dyebound = dyeinitial - dyefree.

Turbimetric method

Protein molecules which are normally soluble in solution can be made to precipitate by the
addition of certain chemicals, e.g., trichloroacetic acid. Protein precipitation causes the solution
to become turbid. Thus the concentration of protein can be determined by measuring the degree
of turbidity.

6.2.3.2. Advantages and Disadvantages

Advantages: UV-visible techniques are fairly rapid and simple to carry out, and are sensitive to
low concentrations of proteins.

Disadvantages: For most UV-visible techniques it is necessary to use dilute and transparent
solutions, which contain no contaminating substances which absorb or scatter light at the same
wavelength as the protein being analyzed. The need for transparent solutions means that most
foods must undergo significant amounts of sample preparation before they can be
analyzed, e.g., homogenization, solvent extraction, centrifugation, filtration, which can be time
consuming and laborious. In addition, it is sometimes difficult to quantitatively extract proteins
from certain types of foods, especially after they have been processed so that the proteins
become aggregated or covalently bound with other substances. In addition the absorbance
depends on the type of protein analyzed (different proteins have different amino acid sequences).

6.2.4. Other Instrumental Techniques

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

6.2.4.1. Principles

Measurement of Bulk Physical Properties

  Density: The density of a protein is greater than that of most other food components, and
so there is an increase in density of a food as its protein content increases. Thus the
protein content of foods can be determined by measuring their density.
 Refractive index: The refractive index of an aqueous solution increases as the protein
concentration increases and therefore RI measurements can be used to determine the
protein content.

Measurement of Adsorption of Radiation

 UV-visible: The concentration of proteins can be determined by measuring the

absorbance of ultraviolet-visible radiation (see above).
 Infrared: Infrared techniques can be used to determine the concentration of proteins in
food samples. Proteins absorb IR naturally due to characteristic vibrations (stretching and
bending) of certain chemical groups along the polypeptide backbone. Measurements of
the absorbance of radiation at certain wavelengths can thus be used to quantify the
concentration of protein in the sample. IR is particularly useful for rapid on-line analysis
of protein content. It also requires little sample preparation and is nondestructive. Its
major disadvantages are its high initial cost and the need for extensive calibration.
 Nuclear Magnetic Resonance: NMR spectroscopy can be used to determine the total
protein concentration of foods. The protein content is determined by measuring the area
under a peak in an NMR chemical shift spectra that corresponds to the protein fraction.

Measurement of Scattering of Radiation

 Light scattering: The concentration of protein aggregates in aqueous solution can be

determined using light scattering techniques because the turbidity of a solution is directly
proportional to the concentration of aggregates present.
 Ultrasonic scattering: The concentration of protein aggregates can also be determined
using ultrasonic scattering techniques because the ultrasonic velocity and absorption of
ultrasound are related to the concentration of protein aggregates present.

6.2.4.2. Advantages and Disadvantages

A number of these instrumental methods have major advantages over the other techniques
mentioned above because they are nondestructive, require little or no sample preparation, and
measurements are rapid and precise. A major disadvantage of the techniques which rely on
measurements of the bulk physical properties of foods are that a calibration curve must be
prepared between the physical property of interest and the total protein content, and this may
depend on the type of protein present and the food matrix it is contained within. In addition, the
techniques based on measurements of bulk physicochemical properties can only be used to
analyze foods with relatively simple compositions. In a food that contains many different
components whose concentration may vary, it is difficult to disentangle the contribution that the
protein makes to the overall measurement from that of the other components.

6.2.5. Comparison of methods

As food scientists we may often be in a position where we have to choose a particular technique
for measuring the protein concentration of a food. How do we decide which technique is the
most appropriate for our particular application ? The first thing to determine is what is the
information going to be used for. If the analysis is to be carried out for official
purposes, e.g., legal or labeling requirements, then it is important to use an officially recognized
method. The Kjeldahl method, and increasingly the Dumas method, have been officially
approved for a wide range of food applications. In contrast, only a small number of applications
of UV-visible spectroscopy have been officially recognized.

For quality control purposes, it is often more useful to have rapid and simple measurements of
protein content and therefore IR techniques are most suitable. For fundamental studies in the
laboratory, where pure proteins are often analyzed, UV-visible spectroscopic techniques are
often preferred because they give rapid and reliable measurements, and are sensitive to low
concentrations of protein.

Other factors which may have to be considered are the amount of sample preparation required,
their sensitivity and their speed. The Kjeldahl, Dumas and IR methods require very little sample
preparation. After a representative sample of the food has been selected it can usually be tested
directly. On the other hand, the various UV-visible methods require extensive sample preparation
prior to analysis. The protein must be extracted from the food into a dilute transparent solution,
which usually involves time consuming homogenization, solvent extraction, filtration and
centrifugation procedures. In addition, it may be difficult to completely isolate some proteins
from foods because they are strongly bound to other components. The various techniques also
have different sensitivities, i.e., the lowest concentration of protein which they can detect. The
UV-visible methods are the most sensitive, being able to detect protein concentrations as low as
0.001 wt%. The sensitivity of the Dumas, Kjeldahl and IR methods is somewhere around 0.1 wt
%. The time required per analysis, and the number of samples which can be run simultaneously,
are also important factors to consider when deciding which analytical technique to use. IR
techniques are capable of rapid analysis (< 1 minute) of protein concentration once they have
been calibrated. The modern instrumental Dumas method is fully automated and can measure the
protein concentration of a sample in less than 5 minutes, compared to the Kjeldahl method which
takes between 30 minutes and 2 hours to carry out. The various UV-visible methods range
between a couple of minutes to an hour (depending on the type of dye that is used and how long
it takes to react), although it does have the advantage that many samples can be run
simultaneously. Nevertheless, it is usually necessary to carry out extensive sample preparation
prior to analysis in order to get a transparent solution. Other factors which may be important
when selecting an appropriate technique are: the equipment available, ease of operation, the
desired accuracy, and whether or not the technique is nondestructive.

6.3. Protein Separation and Characterization

In the previous lecture, techniques used to determine the total concentration of protein in a food
were discussed. Food analysts are also often interested in the type of proteins present in a food
because each protein has unique nutritional and physicochemical properties. Protein type is
usually determined by separating and isolating the individual proteins from a complex mixture of
proteins, so that they can be subsequently identified and characterized. Proteins are separated on
the basis of differences in their physicochemical properties, such as size, charge, adsorption
characteristics, solubility and heat-stability. The choice of an appropriate separation technique
depends on a number of factors, including the reasons for carrying out the analysis, the amount
of sample available, the desired purity, the equipment available, the type of proteins present and
the cost. Large-scale methods are available for crude isolations of large quantities of proteins,
whereas small-scale methods are available for proteins that are expensive or only available in
small quantities. One of the factors that must be considered during the separation procedure is
the possibility that the native three dimensional structure of the protein molecules may be
altered.

A prior knowledge of the effects of environmental conditions on protein structure and

interactions is extremely useful when selecting the most appropriate separation technique.
Firstly, because it helps determine the most suitable conditions to use to isolate a particular
protein from a mixture of proteins (e.g., pH, ionic strength, solvent, temperature etc.), and
secondly, because it may be important to choose conditions which will not adversely affect the
molecular structure of the proteins.

6.3.1. Methods Based on Different Solubility Characteristics

Proteins can be separated by exploiting differences in their solubility in aqueous solutions. The
solubility of a protein molecule is determined by its amino acid sequence because this determines
its size, shape, hydrophobicity and electrical charge. Proteins can be selectively precipitated or
solubilized by altering the pH, ionic strength, dielectric constant or temperature of a solution.
These separation techniques are the most simple to use when large quantities of sample are
involved, because they are relatively quick, inexpensive and are not particularly influenced by
other food components. They are often used as the first step in any separation procedure because
the majority of the contaminating materials can be easily removed.

Salting out

Proteins are precipitated from aqueous solutions when the salt concentration exceeds a critical
level, which is known as salting-out, because all the water is "bound" to the salts, and is
therefore not available to hydrate the proteins. Ammonium sulfate [(NH4)2SO4] is commonly
used because it has a high water-solubility, although other neutral salts may also be
used, e.g., NaCl or KCl. Generally a two-step procedure is used to maximize the separation
efficiency. In the first step, the salt is added at a concentration just below that necessary to
precipitate out the protein of interest. The solution is then centrifuged to remove any proteins that
are less soluble than the protein of interest. The salt concentration is then increased to a point just
above that required to cause precipitation of the protein. This precipitates out the protein of
interest (which can be separated by centrifugation), but leaves more soluble proteins in solution.
The main problem with this method is that large concentrations of salt contaminate the solution,
which must be removed before the protein can be resolubilzed, e.g., by dialysis or ultrafiltration.

Isoelectric Precipitation
The isoelectric point (pI) of a protein is the pH where the net charge on the protein is zero.
Proteins tend to aggregate and precipitate at their pI because there is no electrostatic repulsion
keeping them apart. Proteins have different isoelectric points because of their different amino
acid sequences (i.e., relative numbers of anionic and cationic groups), and thus they can be
separated by adjusting the pH of a solution. When the pH is adjusted to the pI of a particular
protein it precipitates leaving the other proteins in solution.

Solvent Fractionation

The solubility of a protein depends on the dielectric constant of the solution that surrounds it
because this alters the magnitude of the electrostatic interactions between charged groups. As the
dielectric constant of a solution decreases the magnitude of the electrostatic interactions between
charged species increases. This tends to decrease the solubility of proteins in solution because
they are less ionized, and therefore the electrostatic repulsion between them is not sufficient to
prevent them from aggregating. The dielectric constant of aqueous solutions can be lowered by
adding water-soluble organic solvents, such as ethanol or acetone. The amount of organic solvent
required to cause precipitation depends on the protein and therefore proteins can be separated on
this basis. The optimum quantity of organic solvent required to precipitate a protein varies from
about 5 to 60%. Solvent fractionation is usually performed at 0oC or below to prevent protein
denaturation caused by temperature increases that occur when organic solvents are mixed with
water.

Denaturation of Contaminating Proteins

Many proteins are denatured and precipitate from solution when heated above a certain
temperature or by adjusting a solution to highly acid or basic pHs. Proteins that are stable at high
temperature or at extremes of pH are most easily separated by this technique because
contaminating proteins can be precipitated while the protein of interest remains in solution.

6.3.2. Separation due to Different Adsorption Characteristics

Adsorption chromatography involves the separation of compounds by selective adsorption-

desorption at a solid matrix that is contained within a column through which the mixture passes.
Separation is based on the different affinities of different proteins for the solid
matrix. Affinity and ion-exchange chromatography are the two major types of adsorption
chromatography commonly used for the separation of proteins. Separation can be carried out
using either an open column or high-pressure liquid chromatography.

Ion Exchange Chromatography

Ion exchange chromatography relies on the reversible adsorption-desorption of ions in solution

to a charged solid matrix or polymer network. This technique is the most commonly used
chromatographic technique for protein separation. A positively charged matrix is called
an anion-exchanger because it binds negatively charged ions (anions). A negatively charged
matrix is called a cation-exchanger because it binds positively charged ions (cations). The buffer
conditions (pH and ionic strength) are adjusted to favor maximum binding of the protein of
interest to the ion-exchange column. Contaminating proteins bind less strongly and therefore
pass more rapidly through the column. The protein of interest is then eluted using another buffer
solution which favors its desorption from the column (e.g., different pH or ionic strength).

Affinity Chromatography

Affinity chromatography uses a stationary phase that consists of a ligand covalently bound to a
solid support. The ligand is a molecule that has a highly specific and unique reversible affinity
for a particular protein. The sample to be analyzed is passed through the column and the protein
of interest binds to the ligand, whereas the contaminating proteins pass directly through. The
protein of interest is then eluted using a buffer solution which favors its desorption from the
column. This technique is the most efficient means of separating an individual protein from a
mixture of proteins, but it is the most expensive, because of the need to have columns with
specific ligands bound to them.

Both ion-exchange and affinity chromatography are commonly used to separate proteins and
amino-acids in the laboratory. They are used less commonly for commercial separations because
they are not suitable for rapidly separating large volumes and are relatively expensive.

6.3.3. Separation Due to Size Differences

Proteins can also be separated according to their size. Typically, the molecular weights of
proteins vary from about 10,000 to 1,000,000 daltons. In practice, separation depends on
the Stokes radius of a protein, rather than directly on its molecular weight. The Stokes radius is
the average radius that a protein has in solution, and depends on its three dimensional molecular
structure. For proteins with the same molecular weight the Stokes radius increases in the
following order: compact globular protein < flexible random-coil < rod-like protein.

Dialysis

Dialysis is used to separate molecules in solution by use of semipermeable membranes that

permit the passage of molecules smaller than a certain size through, but prevent the passing of
larger molecules. A protein solution is placed in dialysis tubing which is sealed and placed into a
large volume of water or buffer which is slowly stirred. Low molecular weight solutes flow
through the bag, but the large molecular weight protein molecules remain in the bag. Dialysis is a
relatively slow method, taking up to 12 hours to be completed. It is therefore most frequently
used in the laboratory. Dialysis is often used to remove salt from protein solutions after they
have been separated by salting-out, and to change buffers.

Ultrafiltration

A solution of protein is placed in a cell containing a semipermeable membrane, and pressure is

applied. Smaller molecules pass through the membrane, whereas the larger molecules remain in
the solution. The separation principle of this technique is therefore similar to dialysis, but
because pressure is applied separation is much quicker. Semipermeable membranes with cutoff
points between about 500 to 300,000 are available. That portion of the solution which is retained
by the cell (large molecules) is called the retentate, whilst that part which passes through the
membrane (small molecules) forms part of the ultrafiltrate. Ultrafiltration can be used to
concentrate a protein solution, remove salts, exchange buffers or fractionate proteins on the basis
of their size. Ultrafiltration units are used in the laboratory and on a commercial scale.

Size Exclusion Chromatography

This technique, sometimes known as gel filtration, also separates proteins according to their size.
A protein solution is poured into a column which is packed with porous beads made of a cross-
linked polymeric material (such as dextran or agarose). Molecules larger than the pores in the
beads are excluded, and move quickly through the column, whereas the movement of molecules
which enter the pores is retarded. Thus molecules are eluted off the column in order of
decreasing size. Beads of different average pore size are available for separating proteins of
different molecular weights. Manufacturers of these beads provide information about the
molecular weight range that they are most suitable for separating. Molecular weights of unknown
proteins can be determined by comparing their elution volumes Vo, with those determined using
proteins of known molecular weight: a plot of elution volume versus log(molecular weight)
should give a straight line. One problem with this method is that the molecular weight is not
directly related to the Stokes radius for different shaped proteins.

6.3.4. Separation by Electrophoresis

Electrophoresis relies on differences in the migration of charged molecules in a solution when an

electrical field is applied across it. It can be used to separate proteins on the basis of their size,
shape or charge.

Non-denaturing Electrophoresis

In non-denaturing electrophoresis, a buffered solution of native proteins is poured onto a porous

gel (usually polyacrylamide, starch or agarose) and a voltage is applied across the gel. The
proteins move through the gel in a direction that depends on the sign of their charge, and at a rate
that depends on the magnitude of the charge, and the friction to their movement:

Proteins may be positively or negatively charged in solution depending on their isoelectic points
(pI) and the pH of the solution. A protein is negatively charged if the pH is above the pI, and
positively charged if the pH is below the pI. The magnitude of the charge and applied voltage
will determine how far proteins migrate in a certain time. The higher the voltage or the greater
the charge on the protein the further it will move. The friction of a molecule is a measure of its
resistance to movement through the gel and is largely determined by the relationship between the
effective size of the molecule, and the size of the pores in the gel. The smaller the size of the
molecule, or the larger the size of the pores in the gel, the lower the resistance and therefore the
faster a molecule moves through the gel. Gels with different porosity's can be purchased from
chemical suppliers, or made up in the laboratory. Smaller pores sizes are obtained by using a
higher concentration of cross-linking reagent to form the gel. Gels may be contained between
two parallel plates, or in cylindrical tubes. In non-denaturing electrophoresis the native proteins
are separated based on a combination of their charge, size and shape.

Denaturing Electrophoresis

In denaturing electrophoresis proteins are separated primarily on their molecular weight.

Proteins are denatured prior to analysis by mixing them with mercaptoethanol, which breaks
down disulfide bonds, and sodium dodecyl sulfate (SDS), which is an anionic surfactant that
hydrophobically binds to protein molecules and causes them to unfold because of the repulsion
between negatively charged surfactant head-groups. Each protein molecule binds approximately
the same amount of SDS per unit length. Hence, the charge per unit length and the molecular
conformation is approximately similar for all proteins. As proteins travel through a gel network
they are primarily separated on the basis of their molecular weight because their movement
depends on the size of the protein molecule relative to the size of the pores in the gel: smaller
proteins moving more rapidly through the matrix than larger molecules. This type of
electrophoresis is commonly called sodium dodecyl sulfate -polyacrylamide gel
electrophoresis, or SDS-PAGE.

To determine how far proteins have moved a tracking dye is added to the protein
solution, e.g., bromophenol blue. This dye is a small charged molecule that migrates ahead of the
proteins. After the electrophoresis is completed the proteins are made visible by treating the gel
with a protein dye such as Coomassie Brilliant Blue or silver stain. The relative mobility of each
protein band is calculated:

Electrophoresis is often used to determine the protein composition of food products. The protein
is extracted from the food into solution, which is then separated using electrophoresis. SDS-
PAGE is used to determine the molecular weight of a protein by measuring Rm, and then
comparing it with a calibration curve produced using proteins of known molecular weight: a plot
of log (molecular weight) against relative mobility is usually linear. Denaturing electrophoresis
is more useful for determining molecular weights than non-denaturing electrophoresis, because
the friction to movement does not depend on the shape or original charge of the protein
molecules.

Isoelectric Focusing Electrophoresis

This technique is a modification of electrophoresis, in which proteins are separated by charge on

a gel matrix which has a pH gradient across it. Proteins migrate to the location where the pH
equals their isoelectric point and then stop moving because they are no longer charged. This
methods has one of the highest resolutions of all techniques used to separate proteins. Gels are
available that cover a narrow pH range (2-3 units) or a broad pH range (3-10 units) and one
should therefore select a gel which is most suitable for the proteins being separated.

Two Dimensional Electrophoresis

Isoelectric focusing and SDS-PAGE can be used together to improve resolution of complex
protein mixtures. Proteins are separated in one direction on the basis of charge using isoelectric
focusing, and then in a perpendicular direction on the basis of size using SDS-PAGE.

6.3.5. Amino Acid Analysis

Amino acid analysis is used to determine the amino acid composition of proteins. A protein
sample is first hydrolyzed (e.g. using a strong acid) to release the amino acids, which are then
separated using chromatography, e.g., ion exchange, affinity or absorption chromatography.

Analysis of Carbohydrates

Importance of carbohydrate analysis

 Standards of Identity - foods must have compositions which conform to government

regulations
 Nutritional Labeling - to inform consumers of the nutritional content of foods
 Detection of Adulteration - each food type has a carbohydrate "fingerprint"
 Food Quality - physicochemical properties of foods such as sweetness, appearance,
stability and texture depend on the type and concentration of carbohydrates present.
 Economic - industry doesn't want to give away expensive ingredients
 Food Processing - the efficiency of many food processing operations depends on the type
and concentration of carbohydrates that are present

Classification of Carbohydrates

Monosaccharides

Monosaccharides are water-soluble crystalline compounds. They are

aliphatic aldehydes or ketones which contain one carbonyl group and one or more hydroxyl
groups. Most natural monosachharides have either five (pentoses) or six (hexoses) carbon atoms.
Commonly occurring hexoses in foods are glucose, fructose and galactose, whilst commonly
occurring pentoses are arabinose and xylose. The reactive centers of monosaccharides are the
carbonyl and hydroxyl groups.

Oligosaccharides

These are relatively low molecular weight polymers of monosaccharides (< 20) that are
covalently bonded through glycosidic linkages. Disaccharides consist of two monomers,
whereas trisaccharides consist of three. Oligosaccharides containing glucose, fructose
and galactose monomers are the most commonly occurring in foods.

Polysaccharides

The majority of carbohydrates found in nature are present as polysaccharides. Polysaccharides

are high molecular weight polymers of monosaccharides (> 20). Polysaccharides containing all
the same monosaccharides are called homopolysaccharides (e.g., starch, cellulose and glycogen
are formed from only glucose), whereas those which contain more than one type of monomer are
known as heteropolysaccharides (e.g., pectin, hemicellulose and gums).

7.3. Methods of Analysis

 A large number of analytical techniques have been developed to measure the total
concentration and type of carbohydrates present in foods (see Food
Analysis by Nielssen or Food Analysis by Pomeranz and Meloan for more details).
 The carbohydrate content of a food can be determined by calculating the percent
remaining after all the other components have been measured: %carbohydrates = 100 -
%moisture - %protein - %lipid - %mineral.
 Nevertheless, this method can lead to erroneous results due to experimental errors in any
of the other methods, and so it is usually better to directly measure the carbohydrate
content for accurate measurements.

7.4. Monosaccharides and Oligosaccharides

7.4.1. Sample Preparation

The amount of preparation needed to prepare a sample for carbohydrate analysis depends on the
nature of the food being analyzed. Aqueous solutions, such as fruit juices, syrups and honey,
usually require very little preparation prior to analysis. On the other hand, many foods contain
carbohydrates that are physically associated or chemically bound to other components, e.g., nuts,
cereals, fruit, breads and vegetables. In these foods it is usually necessary to isolate the
carbohydrate from the rest of the food before it can be analyzed. The precise method of
carbohydrate isolation depends on the carbohydrate type, the food matrix type and the purpose
of analysis, however, there are some procedures that are common to many isolation techniques.
For example, foods are usually dried under vacuum (to prevent thermal degradation), ground to a
fine powder (to enhance solvent extraction) and then defatted by solvent extraction.

One of the most commonly used methods of extracting low molecular weight carbohydrates
from foods is to boil a defatted sample with an 80% alcohol solution. Monosaccharides and
oligosaccharides are soluble in alcoholic solutions, whereas proteins, polysaccharides and dietary
fiber are insoluble. The soluble components can be separated from the insoluble components by
filtering the boiled solution and collecting the filtrate (the part which passes through the filter)
and the retentante (the part retained by the filter). These two fractions can then be dried and
weighed to determine their concentrations. In addition, to monosaccharides and oligosaccharides
various other small molecules may also be present in the alcoholic extract that could interfere
with the subsequent analysis e.g., amino acids, organic acids, pigments, vitamins, minerals etc. It
is usually necessary to remove these components prior to carrying out a carbohydrate analysis.
This is commonly achieved by treating the solution with clarifying agents or by passing it
through one or more ion-exchange resins.

 Clarifying agents. Water extracts of many foods contain substances that are colored or
produce turbidity, and thus interfere with spectroscopic analysis or endpoint
determinations. For this reason solutions are usually clarified prior to analysis. The most
commonly used clarifying agents are heavy metal salts (such as lead acetate) which form
insoluble complexes with interfering substances that can be removed by filtration or
centrifugation. However, it is important that the clarifying agent does not precipitate any
of the carbohydrates from solution as this would cause an underestimation of the
carbohydrate content.
 Ion-exchange. Many monosaccharides and oligosaccharides are polar non-charged
molecules and can therefore be separated from charged molecules by passing samples
through ion-exchange columns. By using a combination of a positively and a negatively
charged column it is possible to remove most charged contaminants. Non-polar
molecules can be removed by passing a solution through a column with a non-polar
stationary phase. Thus proteins, amino acids, organic acids, minerals and hydrophobic
compounds can be separated from the carbohydrates prior to analysis.

Prior to analysis, the alcohol can be removed from the solutions by evaporation under vacuum so
that an aqueous solution of sugars remains.

7.4.2. Chromatographic and Electrophoretic methods

Chromatographic methods are the most powerful analytical techniques for the analysis of
the type and concentration of monosaccharides and oligosaccharides in foods. Thin layer
chromatography (TLC), Gas chromatography (GC) and High Performance Liquid
chromatography (HPLC) are commonly used to separate and identify carbohydrates.
Carbohydrates are separated on the basis of their differential adsorption characteristics by
passing the solution to be analyzed through a column. Carbohydrates can be separated on the
basis of their partition coefficients, polarities or sizes, depending on the type of column used.
HPLC is currently the most important chromatographic method for analyzing carbohydrates
because it is capable of rapid, specific, sensitive and precise measurements. In addition, GC
requires that the samples be volatile, which usually requires that they be derivitized, whereas in
HPLC samples can often be analyzed directly. HPLC and GC are commonly used in conjunction
with NMR or mass spectrometry so that the chemical structure of the molecules that make up the
peaks can also be identified.

Carbohydrates can also be separated by electrophoresis after they have been derivitized to make
them electrically charged, e.g., by reaction with borates. A solution of
the derivitized carbohydrates is applied to a gel and then a voltage is applied across it. The
carbohydrates are then separated on the basis of their size: the smaller the size of a carbohydrate
molecule, the faster it moves in an electrical field.

7.4.3. Chemical methods

A number of chemical methods used to determine monosaccharides and oligosaccharides are

based on the fact that many of these substances are reducing agents that can react with other
components to yield precipitates or colored complexes which can be quantified. The
concentration of carbohydrate can be determined gravimetrically, spectrophotometrically or by
titration. Non-reducing carbohydrates can be determined using the same methods if they are first
hydrolyzed to make them reducing. It is possible to determine the concentration of both non-
reducing and reducing sugars by carrying out an analysis for reducing sugars before and
after hydrolyzation. Many different chemical methods are available for quantifying
carbohydrates. Most of these can be divided into three catagories: titration, gravimetric and
colorimetric. An example of each of these different types is given below.

Titration Methods

The Lane-Eynon method is an example of a tritration method of determining the concentration of

reducing sugars in a sample. A burette is used to add the carbohydrate solution being analyzed to
a flask containing a known amount of boiling copper sulfate solution and a methylene blue
indicator. The reducing sugars in the carbohydrate solution react with the copper sulfate present
in the flask. Once all the copper sulfate in solution has reacted, any further addition of reducing
sugars causes the indicator to change from blue to white. The volume of sugar solution required
to reach the end point is recorded. The reaction is not stoichemetric, which means that it is
necessary to prepare a calibration curve by carrying out the experiment with a series of standard
solutions of known carbohydrate concentration.

The disadvantages of this method are (i) the results depend on the precise reaction times,
temperatures and reagent concentrations used and so these parameters must be carefully
controlled; (ii) it cannot distinguish between different types of reducing sugar, and (iii) it cannot
directly determine the concentration of non-reducing sugars, (iv) it is sucseptible to interference
from other types of molecules that act as reducing agents..

Gravimetric Methods

The Munson and Walker method is an example of a gravimetric method of determining the
concentration of reducing sugars in a sample. Carbohydrates are oxidized in the presence of heat
and an excess of copper sulfate and alkaline tartrate under carefully controlled conditions which
leads to the formation of a copper oxide precipitate:

����������� reducing sugar + Cu2+ + base � oxidized sugar + CuO2 (precipitate)

The amount of precipitate formed is directly related to the concentration of reducing sugars in
the initial sample. The concentration of precipitate present can be determined gravimetrically (by
filtration, drying and weighing), or titrimetrically (by redissolving the precipitate and titrating
with a suitable indicator). This method suffers from the same disadvantages as the Lane-
Eynon method, neverthless, it is more reproducible and accurate.

Colorimetric Methods

The Anthrone method is an example of a colorimetric method of determining the concentration

of the total sugars in a sample. Sugars react with the anthrone reagent under acidic conditions to
yield a blue-green color. The sample is mixed with sulfuric acid and the anthrone reagent and
then boiled until the reaction is completed. The solution is then allowed to cool and its
absorbance is measured at 620 nm. There is a linear relationship between the absorbance and the
amount of sugar that was present in the original sample. This method determines both reducing
and non-reducing sugars because of the presence of the strongly oxidizing sulfuric acid. Like the
other methods it is non-stoichemetric and therefore it is necessary to prepare a calibration curve
using a series of standards of known carbohydrate concentration.

The Phenol - Sulfuric Acid method is an example of a colorimetric method that is widely used to
determine the total concentration of carbohydrates present in foods. A clear aqueous solution of
the carbohydrates to be analyzed is placed in a test-tube, then phenol and sulfuric acid are added.
The solution turns a yellow-orange color as a result of the interaction between the carbohydrates
and the phenol. The absorbance at 420 nm is proportional to the carbohydrate concentration
initially in the sample. The sulfuric acid causes all non-reducing sugars to be converted to
reducing sugars, so that this method determines the total sugars present. This method is non-
stoichemetric and so it is necessary to prepare a calibration curve using a series of standards of
known carbohydrate concentration.

7.4.4. Enzymatic Methods

Analytical methods based on enzymes rely on their ability to catalyze specific reactions. These
methods are rapid, highly specific and sensitive to low concentrations and are therefore ideal for
determination of carbohydrates in foods. In addition, little sample preparation is usually required.
Liquid foods can be tested directly, whereas solid foods have to be dissolved in water first. There
are many enzyme assay kits which can be purchased commercially to carry out analysis for
specific carbohydrates. Manufacturers of these kits provide detailed instructions on how to carry
out the analysis. The two methods most commonly used to determine carbohydrate concentration
are: (i) allowing the reaction to go to completion and measuring the concentration of the product,
which is proportional to the concentration of the initial substrate; (ii). measuring the initial rate of
the enzyme catalyzed reaction because the rate is proportional to the substrate concentration.
Some examples of the use of enzyme methods to determine sugar concentrations in foods are
given below:

D-Glucose/D-Fructose

This method uses a series of steps to determine the concentration of both glucose and fructose in
a sample. First, glucose is converted to glucose-6-phosphate (G6P) by the
enzyme hexakinase and ATP. Then, G6P is oxidized by NADP+ in the presence of G6P-
dehydrogenase (G6P-DH)

����������� G6P + NADP+ � gluconate-6-phosphate + NADPH + H+

The amount of NADPH formed is proportional to the concentration of G6P in the sample and
can be measured spectrophotometrically at 340nm. The fructose concentration is then determined
by converting the fructose into glucose, using another specific enzyme, and repeating the above
procedure.

Maltose/Sucrose

The concentration of maltose and sucrose (disaccharides) in a sample can be determined after the
concentration of glucose and fructose have been determined by the previous method. The
maltose and sucrose are broken down into their constituent monosaccharides by the enzyme a-
glucosidase:

����������� maltose + H2O � 2 glucose

����������� sucrose +H2O � glucose + fructose

The concentrations of glucose and fructose can then be determined by the previous method. The
major problem with this method is that many other oligosaccharides are also converted
to monosaccharides by a-glucosidase, and it is difficult to determine precisely which
oligosaccharides are present. This method is therefore useful only when one knows the type of
carbohydrates present, but not their relative concentrations. Various other enzymatic methods are
available for determining the concentration of other monosaccharides and
oligosaccharides, e.g., lactose, galactose and raffinose (see Food Analysis Nielssen).

7.4.5. Physical Methods

Many different physical methods have been used to determine the carbohydrate concentration of
foods. These methods rely on their being a change in some physicochemical characteristic of a
food as its carbohydrate concentration varies. Commonly used methods include polarimetry,
refractive index, IR, and density.

Polarimetry

Molecules that contain an asymmetric carbon atom have the ability to rotate plane polarized
light. A polarimeter is a device that measures the angle that plane polarized light is rotated on
passing through a solution. A polarimeter consists of a source of monochromatic light, a
polarizer, a sample cell of known length, and an analyzer to measure the angle of rotation. The
extent of polarization is related to the concentration of the optically active molecules in solution
by the equation a = [a]lc, where a is the measured angle of rotation, [a] is the optical activity
(which is a constant for each type of molecule), l is the pathlength and c is the concentration. The
overall angle of rotation depends on the temperature and wavelength of light used and so these
parameters are usually standardized to 20oC and 589.3 nm (the D-line for sodium). A calibration
curve of a versus concentration is prepared using a series of solutions with known concentration,
or the value of [a] is taken from the literature if the type of carbohydrates present is known. The
concentration of carbohydrate in an unknown sample is then determined by measuring its angle
of rotation and comparing it with the calibration curve.

Refractive Index

The refractive index (n) of a material is the velocity of light in a vacuum divided by the velocity
of light in the material (n = c/cm). The refractive index of a material can be determined by
measuring the angle of refraction (r) and angle of incidence (i) at a boundary between it and
another material of known refractive index (Snell�s Law: sin(i)/sin(r) = n2/n1). In practice, the
refractive index of carbohydrate solutions is usually measured at a boundary with quartz.� The
refractive index of a carbohydrate solution increases with increasing concentration and so can be
used to measure the amount of carbohydrate present. The RI is also temperature and wavelength
dependent and so measurements are usually made at a specific temperature (20 oC) and
wavelength (589.3nm). This method is quick and simple to carry out and can be performed with
simple hand-held instruments. It is used routinely in industry to determine sugar concentrations
of syrups, honey, molasses, tomato products and jams.

Density

The density of a material is its mass divided by its volume. The density of aqueous solutions
increases as the carbohydrate concentration increases. Thus the carbohydrate concentration can
be determined by measuring density, e.g., using density bottles or hydrometers. This technique is
routinely used in industry for determination of carbohydrate concentrations of juices and
beverages.

Infrared

A material absorbs infrared due to vibration or rotation of molecular groups. Carbohydrates

contain molecular groups that absorb infrared radiation at wavelengths where none of the other
major food constituents absorb consequently their concentration can be determined by measuring
the infrared absorbance at these wavelengths. By carrying out measurements at a number of
different specific wavelengths it is possible to simultaneously determine the concentration of
carbohydrates, proteins, moisture and lipids. Measurements are normally carried out by
measuring the intensity of an infrared wave reflected from the surface of a sample: the greater
the absorbance, the lower the reflectance. Analytical instruments based on infrared absorbance
are non-destructive and capable of rapid measurements and are therefore particularly suitable for
on-line analysis or for use in a quality control laboratory where many samples are analyzed
routinely.

More sophisticated instrumental methods are capable of providing information about the
molecular structure of carbohydrates as well as their concentration, e.g., NMR or mass
spectrometry.
7.4.6. Immunoassays

Immuoassays are finding increasing use in the food industry for the qualitative and quantitative
analysis of food products. Immunoassays specific for low molecular weight carbohydrates are
developed by attaching the carbohydrate of interest to a protein, and then injecting it into an
animal. With time the animal develops antibodies specific for the carbohydrate molecule. These
antibodies can then be extracted from the animal and used as part of a test kit for determining the
concentration of the specific carbohydrate in foods. Immuoassays are extremely sensitive,
specific, easy to use and rapid.

Analysis of Polysaccharides and Fiber

These indigestible polysaccharides form part of a group of substances known as dietary

fiber, which also includes lignin (which is a polymer of aromatic molecules). Consumption of
many types of dietary fiber has been shown to have beneficial physiologically functional
properties for humans, e.g., prevention of cancer, heart disease and diabetes.

7.5.1. Analysis of Starch

Starch is the most common digestible polysaccharide found in foods, and is therefore a major
source of energy in our diets. In its natural form starch exists as water-insoluble granules (3 -
60 mm), but in many processed foods the starch is no longer in this form because of the
processing treatments involved (e.g., heating).� It consists of a mixture of two
glucose homopolysaccharides: amylose (500-2000 glucose units) which is linear,
and amylopectin (>1,000,000 glucose units) which is extensively branched. These two kinds of
starch have different physiochemical properties and so it is often important to determine the
concentration of each individual component of the starch, as well as the overall starch
concentration.

Sample preparation. The starch content of most foods cannot be determined directly because
the starch is contained within a structurally and chemically complex food matrix. In particular,
starch is often present in a semi-crystalline form (granular or retrograded starch) that is
inaccessible to the chemical reagents used to determine its concentration. It is therefore
necessary to isolate starch from the other components present in the food matrix prior to carrying
out a starch analysis.

In natural foods, such as legumes, cereals or tubers, the starch granules are usually separated
from the other major components by drying, grinding, steeping in water, filtration and
centrifugation.� The starch granules are water-insoluble and have a relatively high density
(1500 kg/m3) so that they will tend to move to the bottom of a container during centrifugation,
where they can be separated from the other water-soluble and less dense materials. Processed
food samples are normally dried, ground and then dispersed in hot 80% ethanol solutions.
The monosaccharides and oligosaccharides are soluble in the ethanol solution, while the starch is
insoluble. Hence, the starch can be separated from the sugars by filtering or centrifuging the
solution. If any semi-crystalline starch is present, the sample can be dispersed in water and
heated to a temperature where the starch gelatinizes (> 65 oC). Addition of perchloric acid or
calcium chloride to the water prior to heating facilitates the solubilization of starches that are
difficult to extract.

Analysis methods. Once the starch has been extracted there are a number of ways to determine its
concentration:

 Specific enzymes are added to the starch solution to breakdown the starch to glucose. The
glucose concentration is then analyzed using methods described previously
(e.g., chromatography or enzymatic methods). The starch concentration is calculated
from the glucose concentration.
 Iodine can be added to the starch solution to form an insoluble starch-iodine complex that
can be determined gravimetrically by collecting, drying and weighing the precipitate
formed or titrimetrically by determining the amount of iodine required to precipitate the
starch.
 If there are no other components present in the solution that would interfere with the
analysis, then the starch concentration could be determined using physical
methods, e.g., density, refractive index or polarimetry.

The amylose and amylopectin concentrations in a sample can be determined using the same
methods as described for starch once the amylose has been separated from the amylopectin. This
can be achieved by adding chemicals that form an insoluble complex with one of the
components, but not with the other, e.g. some alcohols precipitate amylose but not amylopectin.
Some of the methods mentioned will not determine the concentration of resistant starch present
in the sample.� If the concentration of resistant starch is required then an additional step can be
added to the procedure where dimethylsulfoxide (DMSO) is added to dissolve the resistant starch
prior to carrying out the analysis.

7.5.2. Analysis of Fibers

Over the past twenty years or so nutritionists have become aware of the importance of fiber
in the diet. Liberal consumption of fiber helps protect against colon cancer, cardiovascular
disease and constipation. Adequate intake of dietary fiber is therefore beneficial to good health.
Dietary fiber is defined as plant polysaccharides that are indigestible by humans, plus lignin. The
major components of dietary fiber are cellulose, hemicellulose, pectin, hydrocolloids and lignin.
Some types of starch, known as resistant starch, are also indigestible by human beings and may
be analyzed as dietary fiber. The basis of many fiber analysis techniques is therefore to develop a
procedure that mimics the processes that occur in the human digestive system.

7.5.2.1. Major Components of Dietary Fiber

Cell Wall Polysaccharides

Cellulose occurs in all plants as the principal structural component of the cell walls, and is
usually associated with various hemicelluloses and lignin. The type and extent of these
associations determines the characteristic textural properties of many edible plant materials.
Cellulose is a long linear homopolysaccahride of glucose, typically having up to 10,000 glucose
subunits. Cellulose molecules aggregate to form microfibrils that provide strength and rigidity in
plant cell walls. Hemicelluloses are a heterogeneous group of
branched heteropolysaccharides that contain a number of different sugars in their backbone and
side-chains. By definition hemicelluloses are soluble in dilute alkali solutions, but insoluble in
water. Pectins are another form of heteropolysaccharides found in cell walls that are rich
in uronic acids, soluble in hot water and that are capable of forming gels.

Non Cell Wall Polysaccharides

This group of substances are also indigestible carbohydrates, but they are not derived from
the cell walls of plants. Non-cell wall polysaccharides include hydrocolloids such as guar and
locust bean gum, gum arabic, agar, alginates and caragenans which are commonly used in foods
as gelling agents, stabilizers and thickeners.

Lignin

Lignin is a non-carbohydrate polymer that consists of about 40 aromatic subunits which are
covalently linked. It is usually associated with cellulose and hemicelluloses in plant cell-walls.

7.5.2.2. Common Procedures in Sample Preparation and Analysis

There are a number of procedures that are commonly used in many of the methods for dietary
fiber analysis:

 Lipid removal. The food sample to be analyzed is therefore dried, ground to a fine
powder and then the lipids are removed by solvent extraction.
 Protein removal. Proteins are usually broken down and solubilized using enzymes,
strong acid or strong alkali solutions. The resulting amino acids are then separated from
insoluble fiber by filtration or from total fiber by selective precipitation of the fiber with
ethanol solutions.
 Starch removal. Semi-crystalline starch is gelatinized by heating in the presence of water,
and then the starch is broken down and solubilized by specific enzymes, strong acid or
strong alkali. The glucose is then separated from insoluble fiber by filtration or separated
from total fiber by selective precipitation of the fiber with ethanol solutions.
 Selective precipitation of fibers. Dietary fibers can be separated from other components
in aqueous solutions by adding different concentrations of ethanol to cause selective
precipitation. The solubility of monosaccharides, oligosaccharides and polysaccharides
depends on the ethanol concentration. Water: monosaccharides, oligosaccharides, some
polysaccharides and amino acids are soluble; other polysaccharides and fiber are
insoluble. 80% ethanol solutions: monosaccharides, oligosaccharides and amino acids
are soluble; polysaccharides and fibers are insoluble. For this reason, concentrated
ethanol solutions are often used to selectively precipitate fibers from other
components.�
  Fiber analysis. The fiber content of a food can be determined either gravimetrically by
weighing the mass of an insoluble fiber fraction isolated from a sample or chemically by
breaking down the fiber into its constituent monosaccharides and measuring their
concentration using the methods described previously.

7.5.2.3. Gravimetric Methods

Crude Fiber Method

The crude fiber method gives an estimate of indigestible fiber in foods. It is determined by
sequential extraction of a defatted sample with 1.25% H2SO4 and 1.25% NaOH. The insoluble
residue is collected by filtration, dried, weighed and ashed to correct for mineral contamination
of the fiber residue. Crude fiber measures cellulose and lignin in the sample, but does not
determine hemicelluloses, pectins and hydrocolloids, because they are digested by the alkali and
acid and are therefore not collected. For this reason many food scientists believe that its use
should be discontinued. Nevertheless, it is a fairly simple method to carry out and is the official
AOAC method for a number of different foodstuffs.

Total, insoluble and soluble fiber method

The basic principle of this method is to isolate the fraction of interest by selective
precipitation and then to determine its mass by weighing. A gelatinized sample of dry, defatted
food is enzymatically digested with a-amylase, amyloglucosidase and protease to break down the
starch and protein components. The total fiber content of the sample is determined by adding
95% ethanol to the solution to precipitate all the fiber. The solution is then filtered and the fiber
is collected, dried and weighed. Alternatively, the water-soluble and water-insoluble fiber
components can be determined by filtering the enzymatically digested sample. This leaves
the soluble fiber in the filtrate solution, and the insoluble fiber trapped in the filter. The insoluble
component is collected from the filter, dried and weighed. The soluble component is precipitated
from solution by adding 95% alcohol to the filtrate, and is then collected by filtration, dried and
weighed. The protein and ash content of the various fractions are determined so as to correct for
any of these substances which might remain in the fiber: Fiber = residue weight - weight of
(protein + ash).

This method has been officially sanctioned by the AOAC and is widely used in the food
industry to determine the fiber content of a variety of foods. Its main disadvantage is that it tends
to overestimate the fiber content of foods containing high concentrations of simple
sugars, e.g., dried fruits, possibly because they get trapped in the precipitates formed when the
ethanol is added.

7.5.2.4. Chemical Methods

In chemical methods, the fiber content is equal to the sum of all nonstarch monosaccharides plus
lignin remaining once all the digestible carbohydrates have been removed. Monosaccharides are
measured using the various methods described previously.

Englyst-Cummings Procedure

A defatted food sample is heated in water to gelatinize the starch. Enzymes are then added to
digest the starch and proteins. Pure ethanol is added to the solution to precipitate the fiber, which
is separated from the digest by centrifugation, and is then washed and dried. The fiber is then
hydrolyzed using a concentrated sulfuric acid solution to break it down into its
constituent monosaccharides, whose concentration is determined using the methods described
previously, e.g., colorimetrically or chromatographically. The mass of fiber in the original
sample is assumed to be equal to the total mass of monosaccharides present. The concentration
of insoluble and soluble dietary fiber can also be determined by this method, using similar
separation steps as for the total, insoluble and soluble gravimetric method mentioned above.

����������� This method can be used to determine the total, soluble and insoluble
fiber contents of foods, but does not provide information about the lignin content. This is
because lignin is not a polysaccharide, and so it is not broken down to monosaccharides during
the acid digestion. For most foods this is not a problem because they have low lignin
concentrations anyway. If a food does contain significant amounts of lignin then another method
should be used, e.g., the gravimetric method or more sophisticated chemical methods
(e.g., the Theander-Marlett method).