DEPARTMENT OF FOOD TECHNOLOGY, KYAMBOGO UIVERSITY

FOOD ANALYSIS
Introduction
Food analysis is the assessment of various food components using chemical, instrumental and
physical methods. The chemical composition of food is often determined to establish the
acceptability or nutritive value of a food product. The nature of the sample and the specific
reasons for analysis commonly dictate the choice of analysis method. Furthermore, the speed,
precision and accuracy are often key factors that determine the choice of the method.

The success of any analysis relies on the proper selection and preparation of the sample, and on
the appropriate calculations and interpretation of the data.

Food analysis can be divided into two broad classes;

▪ Qualitative analysis comprises of tests, which enables the scientists to determine the kind
of species/constituent present and their state of combination.
▪ Quantitative analysis. This provides means of determining components in a unit quantity
of a sample. i.e. how much of a component.
The main food components analysed include;
▪ Macronutrients- Proteins, Sugars, Lipids/fats and water content. These are determined by
use of proximate methods.
▪ Micronutrients- Vitamins and mineral salts. These are mainly determined using
instrumental methods E.g. Spectrophotometer (Atomic absorption spectrophotometer and
Atomic emission spectrophotometer) and Chromatography (Gas chromatography and
High-performance liquid Chromatography, HPLC)

Importance of Food Analysis

Food analysis is important in food industry because;
i. Determination of nutritive value of foods in terms of quality and quantity of food
nutrients.
ii. To detect adulteration in foods due to careless handling or dubious traders/food
processors

1
 iii. To determine or draw food composition tables
iv. To detect chemical residues in foods (Insecticides, pesticides used during food
production).
v. To detect chemical and microbial spoilage of food
vi. To determine food quality with the aim of meeting national and international standards
vii. For research and development purposes
viii. Determine naturally occurring toxic compounds in foods. E.g. gossypol (toxic phenolic
pigment, C30H30O8) in cotton seed cake seeds. Gossypol causes goitre in humans,
Glycogenic compounds in cassava.

Common Terms Used in Food Analysis

Qualitative analysis: An analysis in which we determine the identity of the constituent species in
a sample.
Quantitative analysis: An analysis in which we determine how much of a constituent species is
present in a sample.
Analytes: The constituents of interest in a sample.
Matrix: All other constituents in a sample except for the analyte.
A selective reaction or test is one that can occur with other substances but exhibits a degree of
preference for the substance of interest.
A specific reaction or test is one that occurs only with the substance of interest. Note: few
reactions are specific but many exhibit selectivity.

A technique is any chemical or physical principle that can be used to study an analyte/sample.
Many techniques have been used to determine lead levels. For example, in graphite furnace
atomic absorption spectroscopy lead is atomized, and the ability of the free atoms to absorb light
is measured; thus, both a chemical principle (atomization) and a physical principle (absorption of
light) are used in this technique.

A method is the application of a technique for the determination of a specific analyte in a

specific matrix. E.g. the graphite furnace atomic absorption spectroscopic method for

2
determining lead levels in water is different from that for the determination of lead in soil or
blood. Choosing a method for determining lead in water depends on how the information is to be
used and the established design criteria. For some analytical problems the best method might use
graphite furnace atomic absorption spectroscopy, whereas other problems might be more easily
solved by using another technique, such as anodic stripping voltammetry or potentiometry with a
lead ion-selective electrode.

A procedure is a set of written directions detailing how to apply a method to a particular sample,
including information on proper sampling, handling of interferents, and validating results. A
method does not necessarily lead to a single procedure, as different analysts or agencies will
adapt the method to their specific needs.

A protocol is a set of stringent written guidelines detailing the procedure that must be followed if
the agency specifying the protocol is to accept the results of the analysis. Protocols are
commonly encountered when analytical chemistry is used to support or define public policy. For
purposes of determining lead levels in water under the Safe Drinking Water Act, labs follow a
protocol specified by the Environmental Protection Agency.

Accuracy is a measure of how closely the result of an experiment agrees with the expected result.
Accuracy describes how close a measured value is to the “true” value. If a known standard is
available, accuracy is how close your value is to the known value.

The difference between the obtained result and the expected result is usually divided by the
expected result and reported as a percent relative error.

The Analytical methods may be divided into three groups based on the magnitude of their
relative errors. When an experimental result is within 1% of the correct result, the analytical
method is highly accurate. Methods resulting in relative errors between 1% and 5% are
moderately accurate, but methods of low accuracy produce relative errors greater than 5%.

3
Precision. When a sample is analyzed several times, the individual results are rarely the same.
Instead, the results are randomly scattered. Precision is a measure of this variability. The closer
the agreement between individual analyses, the more precise the results. It is important to realize
that precision does not imply accuracy. Precision describes the reproducibility of a result. If you
measure a quantity several times and the values agree closely with one another, your
measurement is precise. If the values vary widely, your measurement is not very precise. As with
accuracy, precision depends on those factors affecting the relationship between the signal and the
analyte.

Sensitivity. The ability to demonstrate that two samples have different amounts of analyte is an
essential part of many analyses. A method’s sensitivity is a measure of its ability to establish that
such differences are significant. Sensitivity is often confused with a method’s detection limit.
The detection limit is the smallest amount of analyte that can be determined with confidence.
The detection limit, therefore, is a statistical parameter.

Types of Experimental Errors

Three general types of errors are encountered in a typical laboratory experiment measurement:
random or statistical errors, systematic errors, and gross errors.
i. Random (or indeterminate) errors arises due to some uncontrollable fluctuations in
variables that affect experimental measurements and therefore has no specific cause.
These errors cannot be positively identified and do not have a definite measurable value;
instead, they fluctuate in a random manner. These errors affect the precision of a
measurement and are sometimes referred to as two-sided errors because in the absence of
other types of errors, repeated measurements yield results that fluctuate above and below
the true value. With sufficiently large number of experimental measurements, an evenly
distributed data scattered around an average value or mean is achieved. Thus, precision of

4
 measurements subject to random errors can be improved by repeated measurements.
Random errors can be easily detected and can be reduced by repeating the measurement
or by refining the measurement method or technique.

ii. Systematic (or determinate) errors are instrumental, methodology-based, or individual

mistakes that lead to “skewed” data, that is consistently deviated in one direction from
the true value. These types of errors arise due to some specific cause and does not lead to
scattering of results around the actual value. Systematic errors can be identified and
eliminated with careful inspection of the experimental methods, or cross-calibration of
instruments. A determinate error can be further categorized into two: constant
determinate error and proportional determinate error. Constant determinate error (ecd)
gives the same amount of error independent of the concentration of the substance being
analyzed, whereas proportional determinate error (epd) depends directly on the
concentration of the substance being analyzed (i.e., epd = K C), where K is a constant and
C is the concentration of the analyte. Therefore, the total determinate error (Etd) will be
the sum of the proportional and constant determinate errors, i.e., Etd= ecd+ epd

iii. Gross errors are caused by an experimenter’s carelessness or equipment failure. As a

result, one gets measurements, outliers, that are quite different from the other sets of
similar measurements (i.e., the outliers are so far above or below the true value that they
are usually discarded when assessing data. The “Q-test” is a systematic way to determine
if a data point should be discarded or not.

Safety Precautions for the Laboratory

Before you begin your work, it is necessary for you to know safe practices for the laboratory that
must be observed at all times, for your protection and the protection of others. You are expected
to read these rules thoroughly and keep in mind that these rules were written with someone else’s
blood
i. Never work alone in the laboratory.
ii. Smoking is not permitted in the building.
iii. Unauthorized experiments are prohibited.

5
 iv. Know the location and use of the fire extinguisher, safety showers and first aid kit.
v. It is required that you wear prescription glasses or safety glasses at all times in the
laboratory for your own protection. Contact lenses are particularly dangerous and they
must not be worn in the laboratory.
vi. Report all injuries to your instructor at once.
vii. Never taste chemicals or solutions.
viii. Use the fume hoods for all poisonous reactions or any reactions which produce noxious
gases.
ix. When diluting concentrated acid or base always add the concentrated acid or base to
water (never the reverse), while stirring the solution. Be very careful with sulfuric acid.
x. Keep an orderly, clean laboratory desk. Return glassware to the lab drawer when finished
using it to keep the work area from becoming cluttered.
xi. Place unneeded books, etc. on the shelves at the side of the laboratory.
xii. Waste containers are provided for the disposal of all solid chemicals and paper, etc.
xiii. Stock reagent bottles are placed on the side bench or beside the balances; leave them at
that position.
xiv. Always read the label twice before taking any chemical from a bottle. If you are not sure
if you have the right chemical, ask!
xv. When pouring reagents, hold the bottle so the label points upwards facing the palm of the
hand. The accumulation of reagent on bottle lip may be removed by touching the bottle
lip to the rim of the receiving vessel.
xvi. Avoid using an excess of reagent. If you happen to have measured out too much, see if
someone else can use the excess.
xvii. Due to possible contamination of the contents of a whole stock bottle, never return
unused chemical to the stock bottle.
xviii. Always check your glassware before you use it. If it is broken or cracked, exchange it for
a new one.
xix. There is one Container reserved for broken glass. All broken glassware should be placed
in this crock and no other.
xx. If corrosive chemicals or liquids come in contact with the skin or clothing, flood with
copious amounts of water for an extended period of time.

6
 xxi. Spilled chemicals should be wiped up immediately; spilled acid or base should be rinsed
with plenty of water and wiped up with a sponge and the sponge rinsed after.
xxii. Inserting glass tubing or thermometers through a rubber stopper - first lubricate the tube
and stopper with glycerol or water, then holding the tube near the end to be inserted insert
slowly while rotating the tube. BE VERY CAREFUL!
xxiii. When you are ready to leave the laboratory, your bench area should be rinsed off with a
wet sponge and the water, gas, and air valves shut off.
xxiv. The chemistry store room is out of bounds to students. If you require apparatus, ask your
instructor for it.

GENERAL STEPS IN ANALYTICAL PROCEDURES

Choice of method. Accuracy and economic factors are considered as the principal factors that
determine the kind of techniques to be employed. The method is governed by the kind of sample
being analysed and the number of components in the sample of which quantitative information is
needed.

Sampling. The sample analysed must reflect the composition of the material from which it is
taken. The sampling should consider the whole population in order to provide an accurate picture
of the study. The value of the analysis obtained will depend on how representative the sample is
of lot, batch or consignment.
It is important to take a representative sample because; It is difficult to work with large
population or size. Therefore, the quality of sample should correctly represent the quality of the
entire population. Food is naturally heterogeneous i.e. non-uniform hence taking a representative
sample is important

Preparation of sample for lab analysis.

The sample may be solid, liquid or gas. The process involved in analysis varies depending on the
state.
Solid Samples. Crush the solid sample to increase the surface area. The equal portions are then
decomposed using mineral acid mixture. The resulting solution is then transferred to a suitable
volumetric flask and made up to the mark.

7
Liquid Samples For liquid samples filter and evaporate the known volume of water. Digest the
residue using a three-acid system (HNO3, HF & HClO). The solution obtained is transferred to a
volumetric flask and made up to the mark.
Air Samples. For the air sample, a known volume containing air precipitation is evaporated and
the resulting residue is digested as stated above (liquid samples) to get the final solution into the
volumetric flask.
Selection technique. Usually the method employed is not specific for a single species. In other
words, the properties measured may a times be characteristics of other elements or compounds of
food components. So, the species must be separated by digesting.
Completion of analysis. After selecting the species of interest, analysis is completed by carrying
out specific tests for the isolated component.
Calculation and interpretation of the results; statistical methods are encouraged and therefore
indispensable in any analytical procedure. This can easily be done nowadays by employing
computer software. MSTAT, CPA GENSTAT, SAS, SPSS and other computer programs can be
used efficiently and effectively.

SAMPLING AND SAMPLE PREPARATION

1. Introduction to Sampling
Sample is a finite part of a population whose properties are studied to gain information about the
whole population.
Sampling is the act, process or technique of obtaining suitable representative parts or group of a
population or consignment for the purpose of determining parameters or characteristics of the
whole population.
A population is the totality of all actual or conceivable objects of the type under investigation.
A consignment is the total quantity of material in bags, cartons or other form of packaging,
which is received or dispatched at a given time. From a consignment, lots, increments, bulk
samples and laboratory samples can be obtained.
A lot is a stated quantity of uniform characteristics taken from the consignment.
An Increment is a small quantity of material taken from parts of a lot by a single operation of a
sampling device.

8
Test portion, also called specimen, test unit, aliquot, is that quantity of a material of proper size
for measurement of the property of interest.
A bulk sample is a sample obtained by combining and mixing the increments.
A laboratory sample is a sample intended for testing or analysis that is obtained from bulked
samples and is the one intended for analysis or inspection.

Samples of adequate amounts for all the intended determinations should be taken. They should
be taken from different positions over the whole lot. The lots should be mixed thoroughly
immediately before sampling. The individual samples taken should be mixed in closed
Appropriate sampling tools should be used, such as probes, triers, stainless steel borer. The
sampling tools and sample containers should be free from contamination. In some analyses, the
time of sampling should be considered.
Samples must be chosen so as to be representative of the population. This ensures that the
conclusions made after analysing the samples are valid.
A representative sample is obtained through the process of random sampling (also called
probability sampling) and is referred to as a random sample. The sample is obtained following
probability methods in order to ensure that each and every item of the population has an equal
chance of being represented.
It is worthless to undertake analysis on samples that are not representative.

Important factors to consider when selecting sampling technique/method

i) Purpose of the analysis to be undertaken: Either evaluation of average quality, acceptance
or rejection of the material, regulatory control.
ii) Type of the food i.e. liquid, solid, perishable or non-perishable
iii) Nature of the population, consignment or lot: Its size should be large enough usually size
n≥30, homogeneity, history, cost, etc.
iv) Nature of the test procedure: Destructive, no-destructive, time required for analysis, cost
of analysis etc.
v) Accuracy, whether it’s acceptable or un acceptable criteria

9
Importance of sampling in food analysis
Sampling is done in order to undertake desired analysis of a lot or population by careful study of
a small number of units (called samples) drawn from the lot or population. When inspection or
analysis is based on samples instead of the whole lot or population, this is economical and there
is reduced wastage of material, especially when the tests are destructive. There is also less
handling damage and fewer analysts involved.
Sources of error during Sampling and Analysis
i) Sampling error. This arise from taking non-representative sample as a result of
differences in variety, maturity levels, sample size etc.
ii) Changes in composition of the material during sample preparation like loss or gain of
moisture content, loss of volatile compounds during drying, component loss due to
enzymatic reaction
iii) Sample contamination during sample preparation such as addition of minerals during
grinding
iv) Data collection analysis error and interpretation of results (inaccurate measurement due
to faulty or malfunctioning instrument/procedure

2. Sampling Methods
There are various methods of obtaining samples for analysis. They include:

a) Probability sampling methods (random sampling methods)

The samples are obtained by random selection methods in accordance with the principles of
statistical sampling probability. Sampling bias associated with the sampling personnel is
eliminated. The procedures for obtaining the sample are laid down beforehand as part of the
sampling design. The four major methods of probability sampling are:
i) Simple random sampling
This is the sampling method whereby all items of the population have an equal and independent
chance of being included in the sample. Tables of random numbers are available and can be used
to obtain the samples in a predetermined manner.
ii) Stratified random sampling

10
This method separates the population elements into overlapping groups called strata, and simple
random samples are then obtained from each stratum. There is reduced variability of sampling
units within each stratum than in the population as a whole. This helps to provide increased
information for a given cost. In addition, separate estimates of population parameters can be
obtained for each stratum without additional sampling. The cost of sampling is less for stratified
random sampling than in simple random sampling because of administrative convenience.
E.g determination of vitamin C in sweet oranges of Rwanda. Stratify the oranges according to
horticultural varieties, where each variety becomes a stratum.

iii) Systematic sampling

This is the sampling method whereby a 1in k elements from a list of units is selected. This is
called a one in k-systematic sample. Consider N sampling units numbered serially from 1 to N,
from which a sample size of n is to be obtained. We find an integer k, called the sampling
interval. K=N/n. Then selected randomly a number of c between 1 and k. Then the required
systematic sample is given by c, c+k, c+2k, ..., c+(n-1)k. Systematic sampling provides a useful
alternative to simple random sampling and is easier to perform, is less subject to error and it
provides greater information per unit cost.
E.g. with a 100 cartons of bread, number them from 1 to 100. Sampling interval, k = 7. c = 3.
Then systematic sampling will take the boxes numbered c = 3, 3 + 7 = 10, 3 + (2 x 7) =17, 3 + (3
x 7) = 24, 3 + (4 x 7) = 31.

iv) Cluster sampling

This method involves dividing the population into a specified number of clusters. The elements
of each cluster will be as similar as possible because they are physical close together. The
number of elements in each cluster should be small compared to the population size, and the
number of clusters should be reasonably large. Any heterogeneity of the population is reflected
in the clusters. From the clusters, a sample of few clusters, called cluster sample, is obtained by
simple random sampling method.
Example:
A soft drink company wished to estimate the buying of their products by 5,000 households in a
town. The travel costs from one household to another were substantial and cluster sampling was

11
undertaken. The 5,000 households were listed in 500 geographical clusters of 10 households
each. Then from the 500 clusters, a simple random sample of 5 clusters was selected.
Cluster sampling is less costly than simple random sampling or stratified random sampling.

b) Non-probability (non-random) sampling methods

In these sampling methods, the investigator uses own discretion or judgement in deciding which
items are to be taken for the sample. Probabilities are not used. As a result, some items of the
population have a better chance than others of being selected. Thus, there may arise biases and
accuracy of the results cannot be determined. The commonly applied non-random sampling
methods are:
i) Judgement sampling
The investigator uses own judgement and experience regarding the population, lot or sampling
frame to decide which units will be taken. This causes non-uniformity and as such, no statistical
techniques can be applied to study the precision of the estimates.
Judgement sampling can be useful where practical difficulties of a particular situation occur and
are best comprehended by the sampler. If carefully applied by experienced and skilled
investigators, this method can yield valuable results.
ii) Quota sampling
It involves specifying in advance, quotas of the samples to be selected from different subgroups
of lots. Samples are then taken from the quotas using judgement or random sampling. Reliability
of the results depends on how well the quotas have been decided.
iii) Restricted sampling
Samples are taken from only the accessible lot or population. The restriction can be natural or
purposely created for convenience. The sample may not be representative. However, in certain
situations (such as a heavily packed warehouse) the physical placement of elements of a
population may create accessibility problems and can only be carried out by taking convenient
samples.
iv) Convenience sampling
Where samples are taken by any convenient way. It is also called grab sampling or haphazard
sampling. Any convenient portion (such as the top layer in a box, or units near the entrance) is

12
taken in a way the investigator likes. Statistical techniques are difficult to apply in such a case,
and the results may not be quite representative.
c) Bulk Sampling
Bulk sampling refers to sampling of material available in bulk form (gaseous, liquid or solid).
Example: Static materials in large tanks, rail cars, ships, stockpiles, silos, bulk heaps, packages,
bags. It is impossible to obtain a representative sample from a static heap unless the material can
be coned and quartered completely.
In dynamic situations, e.g. filling a bulk storage area, loading bulk transport, sampling can be
applied more conveniently than in static situations. Usually bulk material is sampled by taking
increments, blending the increments into composite samples and reducing the composite samples
to a size suitable for laboratory testing.
d) Acceptance Sampling
Acceptance sampling is the method of sampling whereby a lot is accepted or rejected upon
inspection of sample selected following a predetermined sampling plan. It provides the basis for
deciding whether to accept or reject a batch. A sampling plan must specify the following
parameters:
Sample size (n), which is the number of items selected for inspection.
Acceptance number (c), which is the largest number of defectives (or defects) in the sample that
will permit the lot to be accepted.
Probability of acceptance (Pa), which is the percentage of samples out of a long series of
samples that will cause the product to be accepted.
Operating characteristics (OC) curve: This is a graphical plot used in acceptance sampling that
shows the probability of acceptance for a product at all possible levels of percent defectives. It
shows the relationship between the quality of lots submitted for sampling and the probability of
their being accepted. As the acceptance number increases, the quality of the lots passed by the
sampling plan becomes poorer. As the sample size is increased, the discriminating power of the
sampling plan between good and bad lots increases. Every sampling plan has errors associated
with it. The errors are:
Producer’s risk is the risk of rejecting a good lot by the sampling plan.
Consumer’s risk is the risk of accepting a bad lot by the sampling plan.

13
e) Preparation of Samples
The purpose of sample preparation is to have a homogeneous and representative sample of
reduced size and amount for subsequent analysis. It is important to prevent changes in the sample
during sample preparation e.g. When determining moisture content, it’s important to prevent loss
or gain of water as this may give false result.

The various methods of sample preparation include:

A. Cleaning: Is necessary in order to removes dirt and contamination. Unwanted parts are
also sorted and discarded.
B. Drying: Reduces moisture content. Minimizes bacterial contamination and spoilage, and
prepares material for grinding.
C. Size reduction:
i) Grinding: For size reduction of hard or frozen material into fine powders. Mills or
pestle and mortars can be used. The powder can be sieved using screens of
varying pore diameters.
ii) Slicing, mincing, homogenization and blending can be done for size reduction of
moist materials.
Losses and contamination of the samples during size reduction should be controlled. Utensils
used should not contaminate the sample. Temperature increase during grinding and
homogenisation should be controlled.
Sample preparation also may also allow for concentrating the component of interest. By making
the component available for analysis or preserving the component

D. Enzyme inactivation: This is necessary in order to avoid breakdown of some compounds

such as lipids, sugars, proteins and vitamins in the food tissues following sampling.
Enzymes can be inactivated by:
i) Heating. Blanching treatments
ii) Drying at low temperatures (60 – 80 oC) under vacuum.
iii) Chilling or freezing

14
 iv) Use of chemicals such as 80% methanol or ethanol, 5 – 10% perchloric or
chloroacetic acid.
E. Storage: Good storage to avoid contamination, dessication, moisture sorption or
volatilisation.
F. Use of chemical preservatives.

PROXIMATE ANALYSIS OF FOODS

Introduction
A proximate analysis comprises the methods used to determine the major (proximate)
components of foods. These components amount to almost 100% of most food substances. The
other (non-proximate) food constituents, accounting for a minor quantity in most foods are
vitamins, color substances, volatile compounds etc. The proximate components and their
recognized proximate methods of determination are:

Most routine work in many food laboratories involves proximate analyses.

Importance of proximate analysis in foodstuffs

i) Quantitative determination of the major chemical components of foods
ii) Data from proximate analysis is useful in elaboration of food standards and food
legislation
iii) Proximate analysis data is useful in quality control and assurance of foods

15
 iv) The data is useful in nutritional evaluation and labeling of foods
v) Data is useful in monitoring food adulteration practices

DETERMINATION OF MOISTURE CONTENT OF FOODS

Moisture content is the measure of the amount of water in a food. Determination of moisture
content is one of the most common and important analyses in food laboratories. Simple yet one
of the most difficult from which to obtain accurate and precise data. The dry matter that remains
after moisture removal is known as total solids. Computation of nutritional value of foods
requires that you know the moisture content. Moisture data are used to express results of other
analytical determinations on a uniform basis i.e. dry weight basis and wet weight basis

Importance of moisture determination in foods

There are several reasons that contribute to the increasing importance of moisture determination
in foods:
i) Moisture is a major chemical component of foods. It accounts for relatively percentage of
certain foods, such as fluid whole milk (87 to 91%), fresh fruits (85 to 93%), ice-cream
(65%), sweet potatoes (70%), dry cereal grains (10 to 12%), and fresh eggs (75%).
ii) In some foods, moisture content is a constant value, which can be used as a criterion of
identity and quality control.
iii) Moisture content strongly influences quality of the food product especially organoleptic
attributes like taste, texture and appearance.
iv) Knowledge of moisture content is useful during food processing and helps to predict the
behavior of foods during processing, e.g. during mixing, drying, flow in pipes, packaging
and milling
v) Moisture content is useful for economic reasons, the cost of many food products depends
on the amount of water they contain, i.e. freight charges\transport
vi) Knowledge of moisture content helps to express analytical results on a uniform basis, and
in meeting standard requirements of composition
vii) Moisture content influences chemical reactions in foods, which may lead to spoilage as
well as the storage condition of a particular food product (caking)

16
 viii) Moisture content influences microbial growth in foods and subsequent microbial
Spoilage and poisoning of food i.e. microbial stability of foods.

Various methods of moisture determination in foods:

Drying methods of moisture determination in foods

Involves thermal drying methods. The material is heated under carefully specified conditions.
The loss in weight represents the moisture content of the sample. The loss in weight is influenced
by:
i) Type of drying oven
ii) Temperature
iii) Length of drying period
Drying methods are simple, relatively rapid and allow determination of large number of samples.
However, the results obtained provide approximate rather than accurate moisture values.
Loss of volatiles affects results. Irreversible thermal decomposition of organic compounds may
occur. Case hardening may occur, thus affecting efficiency of the method. Low drying
temperatures are required, in order to avoid decomposition of organic compounds,
casehardening, and at which quantitative drying is not unduly prolonged. The depth of food
substance in the drying fish affects efficiency of the drying process. Construction of the drying
oven, vacuum in the drying chamber, position of samples in the oven also affects results. Errors
may arise from loss or uptake of moisture by the sample during storage. For damp samples, a
two-stage drying procedure is recommended. A sub-sample is first dried gradually, and its
moisture loss is determined. Then it is ground and the remaining moisture is determined. This
method gives more accurate results but prolongs the determination than the one-stage procedure.

17
 ▪ Moisture dishes should be handled carefully.
▪ They should have tight covers.
▪ They should not be handled directly with fingers, in order to avoid greases.
▪ They should be transferred from oven to desiccator for cooling and weighed immediately
after attaining room temperature.
▪ The desiccator should have active desiccant (bluish silica gel).
The values of moisture content obtained may not be a true measure of the total water content of
the sample.
i) Volatile compounds may evaporate and be lost at the drying temperature
ii) Only a proportion of the water in the food may evaporate at the drying temperature. The
reminder may be bound or adsorbed water which is difficult to remove
iii) For foods rich in sugars, it is advisable to use low drying temperatures (700C) or use of
vacuum.
iv) Loss of weight of sample due to loss of water may be influenced by particle size and
weight of sample used, type of drying dish, and variation of temperature within the oven.
v) Drying ovens with fans give more accurate results.

The drying temperatures used in moisture determination ranges from 70-1550C, depending on
test material. Average drying time is from 1-6 hours or more. The drying can be done for a
specified time period or until a constant weight is attained.

Air-oven methods of moisture determination

1. Air-oven methods of various types are used for moisture determination. Convection type or
forced-draft air ovens are used, where the later are preferred.
Uniformity of temperature within the oven is important as well as precision of temperature
control ( 0.50C or less). Mechanical circulation of air within the oven ensures proper and
uniform heat distribution.

18
2. Vacuum-oven methods: Drying under vacuum (reduced pressure). The rate of drying is thus
increased.

Procedure of moisture determination by oven method

o Grind the test sample

o Dry and weigh the drying dishes accurately (Wo)
o Weigh the sample in the drying dish accurately (W1)
o Heat the sample and dish at 105-1100C for 1-2 hours
o Cool to room temperature in a desiccator.
o Weigh accurately (W2)

Weight of moisture evaporated

MC%= ---------------------------------------
Weight of sample used

Weight before drying (W1)-weight after drying (W2)

= 100  ----------------------------------------------------------------
Weight before drying (W1)- weight of container (W0)

Distillation methods of moisture determination

The distillation procedures for moisture determination.
▪ The sample is refluxed with an immiscible solvent (xylene, or toluene), with a higher
boiling point and a lower specific gravity than water.
▪ The distilled water is collected in a suitable graduated measuring apparatus in which the
water separates from the solvent and its volume is measures directly.
▪ The distilled water falls below the condensed solvent in a graduated receiver, in which,
the aqueous phase is measured by volume.

ADVANTAGES
Distillation methods causes less thermal decomposition of the sample than drying methods

19
Problems associated with distillation methods of moisture determination
▪ Accuracy of the receiving device may not be good enough
▪ Also difficult in reading the meniscus
▪ Adherence of moisture droplets to the glass, over boiling
▪ Solubility of water in the distillation liquid
▪ Incomplete evaporation of water and underestimation of moisture contents
▪ Distillation of water soluble contents
▪ Formation of emulsions

Procedure of distillation method of moisture determination:

1.Put a known weight into the boiling flask (5g)

2.Add about 75ml of xylene into the boiling flask
3. Connect the distillation apparatus
4. Heat the sample + xylene in the boiling flask for about 30-60 minutes
5. After the distillation is complete, wash down any adhering water on the glass by xylene
6. Read off the volume of the water from the graduated scale of the receiver

Volume of water distilled

%MC= -------------------------------  100
Weight of the sample

Dean and Stark apparatu

20
Determination of moisture content of foods by chemical methods (Karl Fischer titration
method)
Karl Fischer originally determined this method. Chemical methods of water determination are
particularly suitable for low-moisture foods such as dried powders, oils, and fats.
The method is also superior to other moisture determination in sugar rich foods. (e.g.: sugar,
honey) and in foods rich in reducing sugars and proteins.
The method is also useful in foods of intermediate moisture levels. E.g.: bread, and foods rich in
essential oils
The method is based on reduction of Iodine by sulfur dioxide in the presence of water.
2 H2O + SO2 + I2→ H2SO4 + 2 HI
Reaction of water with iodine and sulfur dioxide in pyridine- methanol solution.

Iodine solution in methanol and sulfur dioxide in pyridine are added to the water-containing
food. Titration is then carried out.

KARL FISCHER REAGENT

A solution of iodine in methanol
A solution of sulfur dioxide in pyridine

These solutions are mixed before use to get the Karl Fischer’s reagent.
The sample of foods is dissolved in a mixture of sulfur dioxide- pyridine-methanol and titrated
with a solution of iodine in methanol.
The Karl Fischer method must be standardized against another reference method.
Disadvantages of Karl Fischer method
▪ Ascorbic acid is oxidized to dehydroascorbic acid by the Fisher reagent
▪ Aldehydes and ketones tend to react with methanol forming acetals and releasing water
▪ Mercaptans, thioacids and hydralazines may cause interferences
▪ Inorganic compounds such as metal oxides, hydroxides, bicarbonates, chromates, borates,
and sulphides affect the water titration.
▪ The method requires specialized apparatus

21
Excess reagent is required to react with all the water in the sample before titration
Sample should be well ground to pass a 40-mesh sieve in order that the reaction between the
water in the sample and the reagent occurs completely.

Other chemical methods of moisture determination

Food is mixed with powdered calcium carbide. The water reacts to produce acetylene. The
quantity of acetylene produced can be measured by the loss in weight of the mixture.

DETERMINATION OF FIBRE CONTENT IN FOODS

Crude fibre: Is the insoluble and combustible organic residue that remains after a sample has
been treated with:
▪ Light petroleum ether
▪ Boiling dilute H2SO4 (1.25% W/V)
▪ Boiling dilute NaOH (1.25 % W/V)
▪ Dilute HCl (1%)
▪ Alcohol (ethyl)

Crude fibre consists of lignin, hemicellulose, and cellulose

Dietary fibre is the fibre components that are not broken down by the human alimentary tract
enzymes to form lower molecular weight compounds, which can be absorbed into the blood
stream. Dietary fibre includes hemicelluloses, pectic substances, gums, mucilages, lignin, and
cellulose.

Determination of crude fibre content by Hennenberg-Stohmann method.

▪ Grind sample
▪ Weigh about 2 g accurately (W0)
▪ Transfer sample to conical flask
▪ Add 200 ml of boiled 1.25% W/V H2SO4
▪ Boil for exactly 30 min under reflux condenser
▪ Filter the digest under vacuum suction

22
 ▪ Boil 200ml of 1.25% NaOH and use it to transfer the residue from the filter into the
conical flask
▪ Heat for exactly 30 min under reflux condenser
▪ Filter residue
▪ Wash the residue with 1% HCl and then with boiling water till free from acid
▪ Wash residue twice with alcohol and three times with petroleum ether
▪ Dry the residue at 1000C for 1 hour
▪ Cool to room temperature
▪ Weigh accurately (W1)
▪ Incinerate at 450-5000C for one hour to remove organic residues
▪ Cool desiccator to room temperature
▪ Weigh accurately (W2)

Determination of crude lipids in foods

Lipids have 3 main functions in foods:

A) Culinary
B) Physiological
C) Nutritional
Lipids have ability to carry odours in foods and hence contribute to palatability of foods.
Dietary lipids are the most compact chemical energy available to humans (1 g lipid= 9 kcal energy).

Methods of quantitative determination of lipids

Solvent extraction method

The most method is the SOXHLET method. It uses specialized apparatus called Soxhlet extractor.
The Soxhlet extractor is a liquid-solid extractor, which eliminates use of large solvent volume.
▪ The extracting solvent is placed in flask A
23
 ▪ The solvent is heated
▪ The solvent goes up the side arm B. C is closed off
▪ The solvent vapour is condensed in condenser and it drips onto the samples
▪ When the extraction thimble fills up to G, the solvent siphons back into flask A
▪ The process starts again
▪ The solvent is used over and over again. Hence, only a small amount of solvent is used
▪ Soxhlet extractor works well with solids but not liquids
N.B:
o Successful extraction requires that bonds between lipids and other compounds are broken so that
the lipids are set free and are solubilized.
o For this to occur successfully, the polarities of the solvent and the lipid should be similar.
o E.g.: Polar solvents for polar lipids, non-polar solvents for non-polar lipids
o Chloroform is a good solvent, but fails to quantitatively extract compound lipids (e.g.
glycolipids, proteolipids, lipoproteins.

Sample preparation for crude lipid determination

o Fragmentation by grinding, homogenization: Avoiding chemical modification of sample

constituents by heat or enzymes. This ensures complete extraction of the lipids. This reduction of
particle size is important.
o Solvent used should be pure, peroxide-free and in proper solute/solvent ratio.
o Sample should be dry, as solvent cannot penetrate tissues saturated with water.
Solvents: Diethyl ether, petroleum ether, butanol, chloroform (methanol water mixed solvents),
acetone); or combinations thereof.

Extraction of lipids
Direct extraction of lipids is often carried out in a Soxhlet apparatus. The prepared and weighed sample
is put in an extraction thimble (made from filler paper) and covered with defatted (by soaking in ether),
cotton wool to prevent small particles escaping from the thimble. The extraction thimble and the
extraction apparatus is filled with solvent
The extraction apparatus are assembled, the solvent flask, extraction flask and condenser.
The solvent flask is heated. Solvent vapors evaporate. They are condensed in the condenser and fall back
into sample in the extraction apparatus. The solvent extracts the lipids from the sample and carries them
into the solvent flask by a siphoning action each time the height of the siphon is attained. At the end of
extraction, the apparatus is disconnected. The solvent from the weighed solvent flask is evaporated,
flask is dried.
The increase of weight of the flask is calculated and expressed as % total. The content of the crude lipids
is calculated

W2-W1
% Total lipids= ---------------- X 100
W

Where: - W2= weight of extraction flask with dried fat (g)

-W1= Weight of empty extraction flask (g)
- W= Weight of sample (g)

24
Procedure of Soxhlet method

1. Weigh the extraction flask (after washing and drying)

2. Weigh the food sample (about 5 g)
3. Transfer the weighed sample into an extraction thimble
4. Dry at 105-1100C for one hour
5. Place the thimble into the extraction apparatus
6. Add ethyl ether into the extraction flask (2/3 of flask)
7. Set up the Soxhlet apparatus and start extraction
8. control temperature so that about 80 condensed drops fall down on the thimble per minute
9. Extract for 8-16 hours. Add fresh ether when necessary, so that the thimble is always full
10. Remove the apparatus from the heat
11. Evaporate the apparatus from the heat
12. Dry the flask with the residue lipids at 105-1100C for 1 hour
13. Cool to room temperature. Weigh accurately. Calculate lipids

Fractionation of extracted fats

Extracted lipids comprise of a heterogenous mixture e.g. of non-polar hydrocarbons, polar compounds,
etc. This mixture is difficult to analyze. The methods used in fractionation of fats are:
A)- Column Chromatography: Separation of various components of lipid extracts. Separation done on
columns filled with a packing material e.g. silica and the lipids are eluted with solvents.
B)- Thin layer chromatography (TLC): Simple rapid sensitive. The separated spots can be recovered for
further analysis.

Analysis of chemical characteristics of fats and oils

1. Determination of unsaturation (IODINE VALUE)

▪ Important for classification of fats and oils for trade and use
▪ Useful in control of manufacturing processes
▪ The generally accepted parameter for expressing the degree of carbon- to-carbon
unsaturation of a fat, oil, or derivative is iodine value (or Iodine number)
▪ IV is defined as grams of iodine that adds to 100g of sample
▪ The most important method for determining IV is called the WIJS method (developed in
1898). Other is Hamus method (1901)
▪ The WIJS method is the most widely used and gives accurate results. The Hamus method
gives results about 2-5% below WIJS

WIJS method:

1. 0.1-3.0 g of a sample are dissolved in 15 ml carbon tetrachloride

2. 25 mls of WIJS solution is added (WIJS solution = 9 g iodine trichloride, in a mixture of 700mls
glacial acetic acid and 300 mls carbon tetrachloride)

25
3. The flask is closed, mixture shaken thoroughly, and allowed to stand at 200C for about one hour
in darkness
4. About 20 ml of 10% KI solution and about 150 ml H2O are added. Unreacted halogen is titrated
with standard thiosulfate solution
5. The liberated iodine is titrated with an accurately standardized thiosulfate solution, using
STARCH indicator

(B-S) X 12.692
IV=-------------------------------
Weight of sample
B= titre for blank
S= titre for sample
N= Normality of Na2S2O3(sodium thiosulfate) solution

For a pure fat or oil, the theoretical IV can be calculated as:

2 X 126.92 X number of double bonds

IV= ------------------------------------------------------ X 100
Molecular weight

2Na2S2O3 + I2 → 2 NaI + Na2S4O6

Atomic weight of iodine= 126.9
I= 126.9
I2= 126.9 X 2
1 mole of Na2S2O3 reacts with ½ mole of I2=126.9 g
1 ml of 0.1 N Na2S2O3 reacts with 126.9 X 10-3g I2

(B-A) X f X 126.9 X 10-3

IV= ----------------------------------- X 100
Weight of fat

B= Titre for blank

A= Titre for sample
F= Factor of 0.1 N Na2S2O3

Gerber method (uses milk butyrometer)

Used for fat determination in milk. It involves solution of all milk components other than fat in
sulfuric acid using amyl alcohol to break the milk emulsion and prevent charring of fat layer.
The fat is estimated by centrifugation, whereby the fat is read in a graduated butyrometer. Fat is
separated from milk in a standard butyrometer by mixing it with sulfuric acid and iso-amyl alcohol
and then centrifugation to facilitate measurement of the fat separated.
The H2SO4 shall have a density of 1.815 g/ml at 200C. Iso-amyl alcohol will have a specific gravity
of 0-8 at 200C.

26
Procedure:

▪ Warm milk at 200C. Shake well

▪ 10 ml H2SO4 is pipetted into the butyrometer
▪ 11 ml of milk is pipetted into the butyrometer
▪ 1 ml of isomyl-alcohol is pipetted into the butyrometer
▪ Butyrometer is stoppered, shaken thoroughly, investing it a few times
▪ Butyrometer is immersed into a water bath at 650C + 20C for 15 min
▪ Butyrometer is then placed in a centrifugal separator and centrifuged at 700rpm for 3-5 min
▪ It is immersed again into the water bath at 6 + 20C for 5 min
▪ The meniscus is adjusted so that the line dividing the fat from the remains of the liquid is in a
graduation mark in the fat column
▪ Read the % fat directly from the scale

ASH DETERMINATION IN FOODS

Ash is the inorganic residue from incineration (total thermal decomposition) of organic
matter.

Importance of ash determination

▪ Indicator of food purity
▪ Criterion of identifying certain foods, since for most natural foods, total ash is
characteristic
▪ Can be used as a control against food adulteration
▪ Total ash is related to mineral content, which is of nutritional value.
▪ Important in dietary planning and food formulation

Type of food Ash contents (%)

Fluid dairy products 0.5-1.0%
Fresh fruits 0.2-0.8%
Fresh vegetables  1%
Beans Up to 4%
Wheat endosperm 0.5
Most nuts 1.5-2.5
Meat and poultry 1.0
Fresh fish (edible portion) 1-2%
Egg yolk 1.7%
Egg white 0.6
Sugar, honey 0.5

Minerals
Ash is composed of different types of minerals in different proportions in different foods.
These are: calcium, phosphorus, iron, sodium, magnesium, sulfur, cobalt, zinc,
manganese, …

27
 o Phosphorus rich foods→ most dairy products, grains, nuts, meat, fish, chicken,
eggs, legumes
o Iron-rich foods→most grains, flours, cereals, nuts, meats, seafoods, poultry, eggs,
legumes, fruits, vegetables, dairy products
o Sodium-rich foods→Salt, most dairy products, fruits, cereals, eggs, poultry
o Potassium-rich foods→vegetables
o Mg-rich foods→nuts, cereals
o Mn-rich foods→cereals, vegetables, some fruits, meat
o Cu-rich foods→Seafood, liver, cereals
o S-rich foods→Most protein-rich foods, some vegetables
o Co-rich foods→ fruits and vegetables
o Zn-rich foods→Some seafoods
Alkaline ash: describes presence of salts of organic acids that are converted an ashing to the
corresponding carbonates.
Total ash: useful as a nutritional parameter
Water-soluble ash: used as an indicator of fruit content of fruit preserves
Acid-insoluble ash: useful indicator of mineral matter after digestion of total ash with 10% HCl.

Dry ashing method for total ash determination

In a muffle furnace at high temperature of  5500C

It is used for total ash determination. Sometimes it is used before an elemental analysis for
individual minerals. Wet or liquid samples are first dried in ovens. Solid foods must be ground
into fine powder. Ashing crucibles (dishes) should be cleaned. The sample is heated for a fixed
period of time or until a constant weight. The dish containing the residue is cooled in a desiccator
and the amount of ash is determined by weighing.
o Crucible: The ashing dishes are made of quartz, Porcelain, steel, nickel, platinum, and
gold-platinum alloy. They should be resistant to heat, acid, and high temperatures of up
to 11000C. Platinum crucibles are the most commonly used, but platinum is expensive for
routine use. It has a high melting point (17730C), good heat conductivity, and high
chemical inertness. The ashing dishes should only be touched with tongs. They should be
cleaned thoroughly with hot dilute HCl after use. Ashing in porcelain dishes is common
at 400-7000C(average 5500C). The final ash should be carbon-free. It should be white-
gray in color.
Dry ashing may cause loss of volatile compounds. Dry ashing is however, the most satisfactory
method of ash determination. The ashing is done in a muffle furnace-controlled temperature that
starts from 2500C and is raised within about 1 hour gradually to 450-5500C where it is held until
decomposition of organic matter is complete.
N.B.: Losses of some mineral elements may occur during dry ashing thus cause errors of
analysis. E.g.: Arsenic, cadmium, chromium, copper, iron, mercury, lead, nickel, zinc,
phosphorus, and vanadium. They can volatilize at < 5500C. Liquid and moist foods should be
dried prior to ashing.

Procedure for dry ashing

a) Preparation of containers

28
 1. Clean the ashing dishes, dry them, weigh accurately

b) Preparation of the sample

2. For liquid foods, weigh in the dish and dry it up using a Bunsen burner and dry in
an oven at 1050C
3. For high MC foods, such as fruits, vegetables, meat; weigh in the crucible and dry
in an oven at 1050C
4. For samples that swell on heating, such as cereal flours, char the sample in a small
flame to release smoke.
5. For fats and oils, weigh in a crucible, and put fire on

c) Incineration
6. Weigh the clean dry ashing dish when empty = W0
7. Take 2-5 g sample into ashing dishes
8. Weigh the dish + sample accurately= W1
9. Incinerate at 5500C-6000C for 2-5 hours until gray/white ash is formed
10. Weigh accurately (W2)
11. Calculate the crude ash (%)

Weight of ash remaining

Crude ash%= -------------------------------------  100
Weight of sample

W2-W0
=----------------------  100
W1-W0

Wet ashing method (also called wet digestion) or wet oxidation

This method is mainly used for digestion of samples for determining trace elements and metallic
poisons. Use of a single acid is desirable, but may not be practical for complete decomposition of
organic matter. Sulfuric acid can be used, together with a salt, e.g.: potassium sulfate. Nitric acid
is a good oxidant, but small amounts of H2SO4 are added. Mixed acids are more commonly used.
E.g.: H2SO4 + HNO3, perchloric acid (HClO4) + nitric acid (HNO3), H2SO4 + HClO4

Procedure for Wet Ashing

1. Weigh 0.5-2.0 g of homogenized sample and put into acid-cleaned, quartz or borosilicate
test tubes
2. Add 1 mL of concentrated sub-boiling distilled nitric acid with 1-2 mL of deionized
distilled water.

29
 3. Place the test tubes on a Multi-block heater and heat at 80 °C overnight.
4. Treat digests with 1 mL of 50% hydrogen peroxide dropwise, and heat at 100°C for
several hours
5. Repeat the peroxide treatment until sample digests become clear.
6. Again, heat the digests for 12hrs at 80 °C
7. Add 1 mL of HCl into the digests and continue heating for 3-4 h.
8. Filter digests using ash-less filter paper (7 cm No. 41), and store in acid-cleaned
polyethylene test tubes until analysis
The sample obtained can be analyzed using instrumental analysis techniques such as atomic
absorption spectrometry to determine the amounts or presence of specific metals.

Determination of nitrogen and crude protein content of foods

Protein content in foods is usually determined using Kjeldahl method of nitrogen determination.
The Kjeldahl method is used to determine the nitrogen content in organic and inorganic samples.
For more than 100 years, the Kjeldahl method has been used for the determination of nitrogen in
a wide range of samples. The determination of Kjeldahl nitrogen is made in foods and drinks,
meat, feeds, cereals and forages for the calculation of the protein content. Also, the Kjeldahl
method is used for the nitrogen determination in wastewaters, soils and other samples.
It is an official method and it is described in different normatives such as AOAC, USEPA, ISO,
Pharmacopeias and different European Directives.

Digestion
The aim of the digestion procedure is to break all nitrogen bonds in the sample and convert all of
the organically bonded nitrogen into ammonium ions (NH4+). Organic carbon and hydrogen form
carbon dioxide and water.

30
In this process the organic material carbonizes which can be visualized by the transformation of
the sample into black foam. During the digestion, the foam decomposes and finally a clear liquid
indicates the completion of the chemical reaction. For this purpose, the sample is mixed with
sulfuric acid at temperatures between 350 and 380 ºC. The higher the temperature used, the faster
the digestion process. The speed of the digestion can be greatly improved by the addition of salt
and catalysts. Potassium sulfate is added in order to increase the boiling point of sulfuric acid
and catalysts are added in order to increase the speed and efficiency of the digestion procedure.
Oxidizing agents can also be added to improve the speed even further.

After digestion is completed the sample is allowed to cool to room temperature, then diluted with
water and transferred to the distillation unit.
Distillation
During the distillation step the ammonium ions (NH4+) are converted into ammonia (NH3) by
adding alkali (NaOH). The ammonia (NH3) is transferred into the receiver vessel by means of
steam distillation.

The receiving vessel for the distillate is filled with an absorbing solution in order to capture the
dissolved ammonia gas.
▪ Common absorbing solutions involve aqueous boric acid [B(OH)3] of 2-4%
concentration. The ammonia is quantitatively captured by the boric acid solution forming
solvated ammonium ions.

▪ Also, other acids can be used as precisely dosed volume of sulfuric acid or hydrochloric
acid that captures the ammonia forming solvated ammonium ions.

Titration
The concentration of the captured ammonium ions can be determined using two types of
titrations:

31
 ▪ When using the boric acid solution as absorbing solution, an acid-base titration is
performed using standard solutions of sulfuric acid or hydrochloric acid and a mixture of
indicators. Depending on the amount of ammonium ions present, concentrations in the
range of 0.01N to 0.5N are used. Alternatively, the end point can be determined
potentiometrically with a pH-electrode. This titration is called direct titration.

▪ When using sulfuric acid standard solution as absorbing solution, the residual sulfuric
acid (the excess not reacted with NH3) is titrated with sodium hydroxide standard
solution and by difference the amount of ammonia is calculated. This titration is called
back titration.

Procedure
Digestion
1. Shake the milk sample carefully so that it does not foam.
2. Weigh approx. 5 g of the homogeneous sample
3. Place the sample into a digestion flask.
4. Add 2 Kjeldahl tablets of 5 g of the Missouri catalyst.
5. Add 20 ml Sulfuric Acid 98%.
6. Carefully suspend the sample by gently swirling the tube
7. Bring the digestion tube/flask and mixture into the digestion unit and into a heating
block.
8. Heat the mixture (350 – 380 ºC) until white fumes can be seen.
9. Continue the heating for about 180 minutes.
10. The vapors of water and sulfuric acid are bubbled through a solution of sodium hydroxide
(scrubber) to neutralize them.
11. The digestion is finished when the sample will be totally transparent with a slightly blue
color due to the Cu from the catalyst.

32
 12. The sample is allowed to cool to room temperature and cautiously approx. 100 ml of
water is added, and then the content of the glass tube is transferred to the distillation unit.
Distillation
13. 50 ml of sodium hydroxide 50% solution is added to the sample to neutralize the pH and
to convert NH4+ into NH3.
14. NH3 is captured in a 50 ml of boric acid solution 4% that contains 6 – 7 drops of
Tashiro’s indicator.
15. When NH3 reacts with boric acid the solution turns from red violet to green (pH 4.4-5.8)
due to the color change of the indicator from acid to basic medium.
16. Around 150 ml of condensate is captured in the boric acid solution. (It can take approx. 5
minutes).
Titration
17. Titrate with HCl 0.25 mol/l until the solution has a slightly violet color.
Note: With the volume and concentration of HCl needed we can calculate the number of mol of
nitrogen atoms in the sample and then the % of protein in the milk sample.
Calculation
The calculations for % nitrogen or % protein must consider which type of receiving solution was
used and any dilution factors used during the distillation process. In the equations below, “N”
represents normality. “ml blank” refers to the milliliters of base needed to back titrate a reagent
blank if standard acid is the receiving solution, or refers to milliliters of standard acid needed to
titrate a reagent blank if boric acid is the receiving solution.
▪ When boric acid is used as the receiving solution the equation is:

▪ When standard acid is used as the receiving solution, the equation is:

The general conversion factor 6.25 is used for most foods. Other factors for specific foods are:
-Wheat: 5.70 -Rice: 5.95
-Milk: 6.38 -barley, oaths, and rye: 5.83

33
-Gelatin: 5.55 -maize: 6.25
- Soya: 5.71 -egg, meat, fish, and cereal products: 5.61-5.93
- Legume, root tuber, vegetable, fruit, and microbial foods: 5.14-6.26

Determination of carbohydrates in foods

Carbohydrates are the most abundant food components. They include:
o Monosaccharides. e.g.: D-glucose
o Oligosaccharides. E.g.: sucrose
o Polysaccharides
-Structural polysaccharides. E.g. cellulose
-Nutrient polysaccharides. E.g. starch
Some carbohydrates are natural sweeteners. Carbohydrates are food nutrients supplying energy.
Qualitative detection of carbohydrates in foods

1.Fehlings reaction: for reducing sugars.

Reducing sugars reduce cupric ions (Cu2+) to cuprous oxide (CuO), upon boiling of the sugar
with CuSO4 to form brown precipitate.
Add 2 ml of Fehlings solution to 5 ml sugar.
CuSO4 + reducing sugar → reddish brown precipitate
Boil 2 min

2. Tollen’s reaction: for reducing sugars

the aldehydic group of reducing sugar reduces Tollen’s reagent Ag(NH3)2OH to produce a silver
mirror coloration.
R-CHO
Ag (NH3) 2OH→ Ag mirror
Heat
Tollen’s reagent is reduced to produce silver by aldehyde group of reducing sugar.
R-CHO (aldehyde) + 2 Ag (NH3) 2OH → 2 Ag (precipitate) + R-COONH4 + H2O + 3 NH3
Tollen’s reagent + sugar solution →silver mirror (formed if reducing sugar is present)
Heat

34
 Slowly
In water bath
1. Moolish reaction: Sugars and carbohydrates give colour reactions with many phenols.
E.g.: Thymol, -naphtol, resorcinol.
i. Principle: sugars are dehydrated by concentrate H2SO4 and changed to
furfural derivatives. The furfurals react with sulphonated -naphtol to
form violet colour.
-Naphtol is the most commonly used phenol. Thymol can also be used.
-Requirements: -5%. -Naphtol alcohol solution
-Concentrate H2SO4
-0.1 % sugar solution
ii. Add 1 ml of 5% -naphtol to 5 ml of sugar solution
iii. Pour 1 ml of concentrate of H2SO4 to the sample gently
iv. Check whether violet ring is formed between the two layers, of (5% -
naphtol + sugar= H2SO4)
v. Mix well under cold water tap
vi. Check if the color of solution changes to blue-violet or not. If yes,
reducing sugars are present.

2. Inversion reaction:
-Principle: -sucrose is hydrolyzed into invert sugars, glucose and fructose in the ratio 1:1, by
acid.
-The product is called invert sugar
- Sucrose → invert sugar (Glucose + fructose): 1:1.
Acid inversion
Heat
-Reagents: 6NH2SO4
Sodium carbonate (Na2CO3)
Fehling’s solution (CuSO4)
0.1% sucrose solution
-Procedure: -Add 3 ml H2SO4 to 3 ml sucrose solution

35
 - Heat for 10 min a water bath
- Make a solution weak by adding Na2CO3
- Add 1 ml of Fehling’s solution
- Boil for 2 min → reddish brown precipitation

3. Iodo-starch reaction: test for starch:

Iodine is reacted with starch. Blue color appears.
Starch solution in a test tube + cool and add one drop of Iodine solution→color changes to blue.
Heat in a Bunsen until all blue colors disappears. Cool and if blue color re-appears. This
confirms that starch is present.
Other methods
Determination of sugars:
o Polarimetry methods: use of polarimeter. Measure direction and angular rotation of
light.
-Prepare sugar solution
-Clarify with lead acetate
-Filter
-Measure angle of rotation using polarimeter.

36