Physical and Chemical Methods

of Food Analysis
Assoc. Prof. Dr. PHAN TẠI HUÂN
Faculty of Chemical Engineering and Food Technology
Nong Lam University - HCMC

Lecture 2:
Sampling and Sample Preparation

• Reading : Chapter 3 in Book “Introduction to the Chemical

Analysis of Foods”, Suzanne Nielsen, Jones and Bartlett
Publishers, London, 1994.

1
 Introduction
• Evaluating all foods or ingredients from a particular
lot is feasible if the analytical technique is rapid and
nondestructive.
• It is usually more practical to select a portion of the
total product volume (population) and assume the
quality of the selected portion is typical of the whole
lot.
– Sampling is obtaining a portion, or sample, that is
representative of the whole lot.
– Adequate sampling technique helps to ensure that sample
quality measurements are an accurate and precise estimate
of the quality of the population.
3

Introduction
• A laboratory sample for analysis can be of any size or
quantity.
• Sampling terminology and procedures used may vary
between companies and between specific
applications.
• Need to know a basis for understanding, developing,
and evaluating sampling plans and sample handling
procedures.

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 Sampling Plans
• Sampling plan is defined as "a predetermined
procedure for the selection, withdrawal, preservation,
transportation, and preparation of the portions to be
removed from a lot as samples.
• It should address the issues of who, what, where, why
and how.
• The primary aim of sampling is to obtain a sample,
subject to constraints on size, that will satisfy the
sampling plan specifications.

Sampling Plans
• A sampling plan should be selected on the basis of
the sampling objective, the study population, the
statistical unit, the sample selection criteria, and the
analysis procedures.
• The two primary objectives of sampling are often to
estimate the average value of a characteristic and
determine if the average value meets the
specifications defined in the sampling plan.

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 Sampling Purposes
1. Nutritional labeling
2. Detection of contaminants and foreign matter
3. Statistical process control (Quality Assurance)
4. Acceptance of raw materials, ingredients, or products
(Acceptance Sampling)
5. Release of lots of finished product
6. Detection of adulterations
7. Microbiological safety
8. Authenticity of food ingredients, etc.

Factors that Affect the Choice of Sampling Plans

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Factors that Affect the Choice of Sampling Plans

Sampling Plans
• Sampling plans are designed for examination of
either attributes or variables.
– In general, variable sampling requires smaller sample size
than attribute sampling and each characteristic should be
sampled for separately when possible.
• Acceptance sampling plans determine if a shipment
of products or ingredients has enough quality to be
accepted. They are used for evaluation of attributes or
variables, or a combination of both.
– Selection of the plan depends on the expected overall lot
quality and sampling costs.

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5
 Attributes Sampling
• In attribute sampling, sampling is performed to
decide on the acceptability of a population based on
whether the sample possesses a certain characteristic,
for example, aflatoxin contamination in a peanut
butter.
• The statistical distribution of such a attribute
sampling plan is Hypergeometric, Binomial, or
Poisson.

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Variables Sampling
• In variable sampling, sampling is performed to
estimate quantitatively the amount of a substance or a
characteristic on a continuous scale.
• The estimate obtained from the sample is compared
with an acceptable value and the deviation measured.
• This type of sampling usually produces data that have
a normal distribution, such as in the percent fill of a
container and total solids of a food sample.

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 Acceptance Sampling
• Acceptance sampling can be performed by the food
processor before receiving a lot of materials from a
supplier, or by a buyer who is evaluating the
processor’s output.
• Lot acceptance sampling plans fall into the following
categories: single, double, multiple, sequential, and
skip plans

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Acceptance Sampling Plans

• Single sampling plans allow accept/reject decisions to
be made by inspection of one sample of a specified
size.
• In single sampling plans, the decision of accepting or
rejecting a lot is based just on one sample of items
taken at random.
• These plans are usually denoted as (n, c) plans for a
sample size n, where the lot is rejected if there are
more than c defective samples.

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7
 Acceptance Sampling
• Double sampling plans require the selection of two
sample sets.
– If the lot is of extremely high or low quality, acceptance or
rejection may be determined after evaluation of the first set
of samples.
– If the first sample indicates the lot is of intermediate
quality, a second sample set is taken. A decision on the
acceptability is then based on analysis of data from both
sample sets.

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Acceptance Sampling Plans

Example of a double sampling plan with two points where the decision
16
of acceptance or rejection can be made.

8
 Acceptance Sampling Plans
• The ultimate extension of multiple sampling is
sequential sampling.
• A multiple sampling chart must be developed to
relate the cumulative number of defects to the
number of samples taken from the lot.
• The chart consists of two parallel lines: a rejection
line and an acceptance line.
– When the cumulative number of defects lies above the
rejection line, the lot should be rejected.
– When the number of defects falls below the acceptance
line, the lot can be accepted.
• Sampling should continue until the number of defects
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crosses the acceptance or the rejection line.

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 Acceptance Sampling Plans
• In skip lot sampling only a fraction of the submitted
lots is inspected.
• Skip lot sampling is a money-saving sampling
procedure
• It can be implemented only when there is enough
proof that the quality of the lots is consistent.

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Risks Associated with Sampling

• The consumer risk describes the probability of
accepting it poor quality population.
• Consumer risk should happen rarely (<5% of the lots)
but the actual acceptable probability of a consumer
risk depends on the consequences associated with
accepting an unacceptable lot.
– Problem with major health hazards and subsequent
fatalities demands a low or no probability of occurring.
– A lot being of slightly lower quality than standard lots
would be allowed to occur more frequently.

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10
 Risks Associated with Sampling
• The vendor risk (producer risk) is the probability of
rejecting an acceptable product .
• As with consumer risk, the consequences of an error
determine the acceptable probability of the risk.
• An acceptable probability of vendor risk is usually 5-
10%.
• A sampling plan should be simple and flexible,
protect both the consumer and the vendor, and
provide for utilization of rejected lots.

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Sampling Procedures
• The reliability of analytical data is compromised if
sampling is not done properly.
• The use of the data to be obtained will determine the
sampling procedure.
• For example, details for the sampling of specific food
products are described in the Official Methods of
Analysis of AOAC International and in the Code of
Federal Regulations (CFR).

Example
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 Homogeneous versus Heterogeneous Populations
• The population which is uniform throughout and
identical at all locations would be homogeneous.
• Sampling from such a population is simple, as a
sample can be taken from any location and the
analytical data obtained will be representative of the
whole.
• However, most populations that are sampled are
heterogeneous.
– apparently uniform sugar syrup still have suspended
particles and sediments in a few places.

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Manual versus Continuous Sampling

• To obtain a manual sample the person taking the
sample must attempt to take a random sample to avoid
human bias in the sampling method.
• The sample must be taken from a number of locations
within the population to ensure it is representative of
the whole population.
• Liquids may be sampled by pipetting, pumping or
dipping
– For liquids in small containers, this can be done by shaking
prior to sampling.
– When sampling from a large volume of liquid, such as that
stored in silos, aeration ensures a homogeneous unit. 24

12
 Manual versus Continuous Sampling
• When sampling grain from a large container, mixing
is impossible and samples are obtained by probing
from several points at random.
• Manual sampling of granular or powdered material is
usually achieved with triers or probes that are
inserted into the population at several locations.
– Rounded particles may flow into the sampling
compartments more easily than angular ones.
– Hygroscopic materials flow more readily into the sampling
devices than do nonhygroscopic material
– Horizontal core samples have been found to contain a
larger proportion of smaller sized particles than vertical
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ones

www.starqualitysamplers.com/seed.php

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 Manual versus Continuous Sampling
• Continuous sampling is performed mechanically.
• Continuous sampling should be less prone to human
bias than manual sampling.

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 Statistical Considerations
Probability Sampling
• Probability sampling plans provide a statistically
sound basis for obtaining representative samples with
elimination of human bias and therefore is the most
desirable. The probability of including any item in
the sample is known and sampling error can be
calculated .
– For example: Simple random sampling, Systematic
sampling, Stratified random sampling, Cluster
sampling, Composite sampling.

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Probability Sampling
• Simple random sampling requires that the number
of units in the population be known and each unit is
assigned a number. A specific quantity of random
numbers between one and the total number of
population units is selected.
• Systematic sampling is used when a complete list of
sample units is not available, but when samples are
distributed evenly over time or space, such as on a
production line. The first sample is selected at
random and then every nth unit after that.

30

15
 Probability Sampling
• Stratified sampling involves dividing the population
into non-overlapping subgroups so that each
subgroup is as homogeneous as possible.
• Group means, therefore, differ from each other as
much as possible.
• Random samples then are taken from each subgroup.

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Probability Sampling
• Cluster sampling entails dividing the population into
subgroups, or clusters, so that the clusters'
characteristics are as identical as possible, i.e., the
means are as similar as possible.
• Any heterogeneity occurs within each cluster.
• Clusters should be small with a similar number of
units in each cluster.
• The clusters are sampled randomly and may be either
totally inspected or sub-sampled for analysis.

32

16
 Probability Sampling
• Composite sampling is used to obtain samples from
bagged products such as flour, seeds, and larger items
in bulk.
• Two or more samples are combined to obtain one
sample for analysis that reduces differences between
samples.
• For example, FDA requires a composite of 12
samples with at least six subsamples taken and
analyzed for compliance with nutrition labeling
regulations.

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Statistical Considerations
Nonprobability Sampling
• Nonprobability sampling is performed when a
representative sample of a population cannot be
collected,
• This type of sampling can be done in many ways, but
in each case the probability of including any specific
portion of the population is not equal because the
investigator selects the samples, without estimating
sampling error.
– For example: Judgment sampling, Convenience
sampling, Restricted sampling, Haphazard sampling,
Quota sampling. 34

17
 Nonprobability Sampling
• Judgment sampling is solely at the discretion of the
sampler and therefore is highly dependent on the
person taking the sample.
• Convenience sampling is performed when ease of
sampling is the key factor. The first pallet in a lot or
the sample that is most accessible is selected
• Haphazard sampling is selection of any portion.

35

Nonprobability Sampling
• Restricted sampling may be unavoidable when the
entire population is not accessible . This is the case if
sampling from a loaded boxcar but the sample will
not be representative of the population .
• Quota sampling is the division of a lot into groups
representing various categories, and samples are then
taken from each group. This sampling method is less
expensive than random sampling but also is less
reliable.

36

18
 Statistical Considerations
Mixed Sampling
• Mixed sampling combines random and nonstatistical
sampling.
• The population is subdivided by the investigator and
items from the groups are selected randomly.

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Estimating the Sample Size

• Confidence intervals (CI)

 SD
CI  x  Z  or CI  x  t 
n n
• Maximum error (E) is acceptable for a desired level
of confidence

σ
E  Z
n
38

19
 Estimating the Sample Size
• Sample size:
2
 σ
n   Z 
 E
• Maximum error (E) can be expressed
_ in terms of the
accuracy (γ) as: E  γ. x
2
 
 σ 
n  Z _ 
 γ. x 

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Estimating the Sample Size

• We want to test the concentration of sodium in a lot of a ready-
to-eat food product with a level of confidence of 95%. Some
preliminary testing showed an average content of 1000mg of
sodium per tray with an estimated standard deviation of 500.
Determine the sample size with an accuracy of 10%.

Data: Confidence level = 95% (α = 0.05) ⇒ z = 1.96;

γ = 0.1; x = 1000; σ = 500.
2
  2
 σ   500 
n   Z  _    1.96    96
 
γ. x   0.1 x 1000 

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 Problems In Sampling
• Analytical data never are more reliable than the
sampling technique.
• Unreliable data also can be obtained by nonstatistical
factors such as poor sample storage resulting in
sample degradation.
• Mislabeling of samples causes mistaken sample
identification. Markings will not be removed or
damaged during storage and transport.
• Official samples also must include the date of
sampling with the name and signature of the
sampling agent.
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(Holme and Peck, 1998)

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 Preparation of Samples
General Particle Size Reduction Considerations
• If the particle size or mass of the sample is too large
for analysis, it must be reduced in bulk or particle
size.
• The sample can be spread on a clean surface and
divided into quarters. The two opposite quarters are
combined. If the mass is still too large for analysis,
the process is repeated.
• This method can be modified for homogeneous
liquids by pouring into four containers, and can be
automated.
43

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 Boerner divider

• a

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http://www.cd3wd.com/CD3WD_40/VITA/GRAINLOS/GIF/PGLX157.GIF http://www.wheatflourbook.org/DesktopModules/IM.aspx?I=167&M=0

Preparation of Samples
Grinding
• Various mills are available for reducing particle size
for sample homogenization.
– To homogenize moist samples, bowl cutters, meal mincers,
tissue grinders, mortars and pestles, or blenders are used,
– While mortars and pestles, and mills are best for dry
samples.
• The final particles of dried foods should be 20 mesh
for moisture, total protein, or mineral determinations.
• Particles of 40 mesh size are used for extraction
assays such as lipid and carbohydrate estimation.
46

23
 Preparation of Samples
• Some foods are more easily ground after drying in a
desiccator or vacuum oven.
• Grinding wet samples may cause significant losses of
moisture and chemical changes.
• In contrast, grinding frozen samples reduces
undesirable changes.
• The grinding process should not heat the sample, and
therefore the grinder should not be overloaded to
control the heat produced through friction.
• Contact of food with bare metal surfaces should be
avoided if trace metal analysis is to be performed.
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Preparation of Samples in Laboratory

48

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 Types of Samples
• Analyte: The compound of interest in an analysis
• Matrix: The material in which the analyte is being
analyzed (the sample)

Phase Considerations:
Gas Phase: Volatile compounds
Aqueous Phase: Polar compounds (hydrophilic)
Organic Phase: Non-polar compounds (hydrophobic)

49
(A. Mitchell, 2009)

Analysis of Analytes
Typical Steps:
1. Extract: Extract the analyte from the matrix.
2. Clean-Up: Removing interfering substances, based
upon adsorption, partitioning
3. Modify: Improve analysis, based upon the chemical
reactivity of an analyte.
4. Resolve: Separate the analyte from other compounds
in the analysis.
5. Quantitate: Measure the amount of material against a
standard.
50
(A. Mitchell, 2009)

25
 Extraction Considerations
Polarity:
• Intermolecular interactions are largely a result of the
polarity of molecules and their ability to form
hydrogen bonds
• Ions:
– Cations and anions are permanently charged and generally
soluble as NaCl, KCl, NH4Cl...
• Molecules:
– Dipole Moment (μ) –

51
(A. Mitchell, 2009)

The Hydrogen Bond:

Abnormally strong dipole-dipole attraction (μ= 1.94 D)

52
(A. Mitchell, 2009)

26
 Extraction Considerations
Solubility
• Polar Solvents generally dissolve hydrophilic
analytes and charged species.
• Non-Polar Solvents generally dissolve hydrophobic
analytes and are often immiscible with water.
• Dielectric Constant [ε] describes the polarity of
solvents. ε measures the effect of the substance on the
transmissin of an electric field. Solvents of increasing
polarity exhibit greater ε.

53
(A. Mitchell, 2009)

Extraction Considerations
Dielectric Constant [ε]

54
(A. Mitchell, 2009)

27
 Extraction Considerations
• Ratio between two forces (F/F´)so there are no units
• Polar solvents, with a high tendency to get oriented in
an electric field, have large dielectric constants
– Water ε = 80.1
• Non-polar solvents with little orientation in an
electric field have very low dielectric constants
– Hydrocarbons ε = 1.89

55
(A. Mitchell, 2009)

Extraction Considerations
Solvents
• Solvents provide an environment for chemical
reactions to occur.
– Solvents are used to selectively extract chemicals
from complex mixtures.
– Solvents are used to separate and purify all kinds of
molecules.
• Solubility is a measure of how much solute will
dissolve into a solvent at a given temperature.

56
(A. Mitchell, 2009)

28
 Extraction Considerations
In general solvents should meet the following criteria:
• It should be inert to the reaction conditions
• It should dissolve the reactants and reagents
• It should be easily removed at the end of the reaction
(appropriate boiling point)
• It should be cost effective to use
• Relatively non-toxic

57
(A. Mitchell, 2009)

Extraction Considerations
Primary factors affecting the solubility of solutes in
solvents:
• Temperature
• Molecular size
• Functional Group Chemistry
• pH for organic acids and bases

58
(A. Mitchell, 2009)

29
 Extraction Considerations
• Natural materials contain chemicals with a wide range
of polarities and thus their selective extraction can be
achieved using solvents of the right polarity or
through the use of distillation.
Examples:
– Hexane for fats, waxes, steroids
– MeOH for pigments, alkaloids, tannins, flavonoids
– Water for salts, small sugars, proteins

59
(A. Mitchell, 2009)

Extraction Considerations
Liquid-Liquid Extraction
• Separation of compounds based upon their
distribution between two immiscible liquids.
– Oil/water, hexane/water
• Standard procedure in the lab performed using a
separatory funnel.
• Can be use to isolate a single component or mixture
from a complex matrix.
• The distribution of a solute between two phases is an
equilibrium condition described by partition theory.
60
(A. Mitchell, 2009)

30
 Extraction Considerations

Partition Coefficient:

A
KD  organic

A aqueous

61
(A. Mitchell, 2009)

Extraction Considerations
Partition Coefficient (KD)
• The KD describes the equilibrium concentrations of a
solute between two immiscible liquid phases
• KD measures the relative affinity of a compound for 2
solvents
– KD > 1 The organic phase is a better solvent for
compound A (hydrophobic).
– KD < 1 The aqueous phase is a better solvent for
compound A (hydrophilic).

62
(A. Mitchell, 2009)

31
 Extraction Considerations
The KD will depend upon:
– The solvent system
– The temperature (mild effect at room temp)
– Ratio of concentration therefore there are no units

63
(A. Mitchell, 2009)

Quiz
Edmodo,
Assignment 1 (10 + 5 minutes)

Determine how much of a 1.1 g sample of caffeine is

in each layer of a water:chloroform (100 mL v/v)
system. Calculate KD based upon solubility:
– Caffeine water solubility = 1.8 g / 100 ml
– Chloroform solubility = 18 g / 100 ml

64

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 Extraction Considerations
Solid-Liquid Extraction
• Physical process by which a compound (or mixture of
compounds) is transferred from a solid phase to a
liquid phase
– Tea/water
• Standard technique with wide application

65
(A. Mitchell, 2009)

Extraction Considerations

66
(A. Mitchell, 2009)

33
 67

68
(A. Mitchell, 2009)

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 Distillation Considerations
• Boiling point (Bp)

69
(A. Mitchell, 2009)

Distillation Considerations
Intermolecular forces (IMFs)
• For a molecule to enter the gas phase it must
overcome the IMFs that keep it in the condensed state
• The primary factors effecting the volatility of
molecules are:
– H-bonding
– Dipole-dipole interactions
– van der Waals - London Forces
– Size and Shape

70
(A. Mitchell, 2009)

35
 Distillation Considerations

71
(A. Mitchell, 2009)

Distillation Considerations

72
(A. Mitchell, 2009)

36
 Distillation Considerations

73
(A. Mitchell, 2009)

Distillation Considerations

74
(A. Mitchell, 2009)

37