Chapter II

Sampling,
sample handling
and
sample preparation
AAC811S
2022
1
 “A chemical analysis is
meaningless unless you begin
with a meaningful sample.”
D.C. Harris.

2
 Chapter Overview
1. The Importance of Sampling

2. Designing a Sampling Plan

3. Implementing the Sampling Plan

4. Cleaning/drying of equipment & labware

5. Reagents and Solvents

6. Sample Preparation
3
 1. The Importance of Sampling

 Why might a carefully designed analytical method give poor results?

• Because we may have failed to account for sample errors.

• If we collect the wrong sample (or lose/contaminate analyte during

sampling/sample preparation), then we introduce a determinate source
of error (inaccurate results).

• If we do not collect enough samples (or collect wrong sample size), then
our precision may suffer (high standard deviation).

 In this chapter we consider how collecting samples and preparing them

for analysis affects the accuracy and precision of our results.

4
SOURCES OF SAMPLES CONTAMINATION AND LOSS

CONTAMINATION WITH THE SAME SUBSTANCE BEING ANALYZED

(ENHANCEMENT)

ADSORPTION OF THE ANALYTE

CONTAMINATION WITH OTHER SUBSTANCE WHICH INTERFERE WITH

THE ANALYTE

CONTAMINATION WITH OTHER SUBSTANCE WHICH INDUCES CHEMICAL

CHANGE OF ANALYTE

CHEMICAL INSTABILITY DUE TO CHANGES OF PHYSICAL CONDITIONS

(E.G. T, P, pH)
5
 Analytical steps

Field sampling Customer or analyst

Homogenization
Steps in which the Analyst is
fully responsible
Lab sub-sampling
Avoid loss or
contamination
Sample Prep
Avoid error for each
analytical step
Analysis

Data interpretation Analyst or customer

6
 Required information prior to analysis
• In order to choose the correct combination of methods to comprise the
appropriate analytical procedure, some basic information is required:

 Physical state(s) of sample (aqueous, sludge, solid, etc.),

 Analytes (organic, inorganic),
 Detection limit (sensitivity),
 Analytical objective (e.g. total analysis or speciation),
 Sample containers,
 Preservations,
 Holding Times

• If the individual samples do not accurately represent the population from

which they are drawn (i.e. the target population) then even a careful
analysis must yield an inaccurate result (determinate sampling error).

• But, even with the right sample, indeterminate sampling errors may still
limit the usefulness of analysis.

7
• Each step of an analysis contributes to random error that affects the
overall standard deviation (S).

• If an analysis is divided into 2 steps (i.e. collecting and analyzing the

samples), each characterized by a standard deviation, then the
relationship between the overall variance, s2, and the variances due to
sampling (ssamp2) and the analytical method (smeth2) is

• This equation shows that the overall variance for an analysis may be
limited by either the analytical method or the collecting of samples.

• Unfortunately, analysts often try to minimize the overall variance by

improving only the method’s precision.

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 SEVEN STEPS OF THE
SAMPLING PROCESS

• The sample collection process may

be broken down into 7 consecutive
steps shown in the Figure.

• Each step, if not completely

understood and properly carried out,
can potentially invalidate the
collected samples and even nullify
the whole sampling effort.

9
Risks of sample degradation during sampling and
storage

Risks
Decomposition
Loss
Reactions
T, light, microbial
Volatilization activity

Chemical Chemical reaction with

reactions ambient reagents
O2 ,CO2 ,H2O

Between compounds Between sample compounds and

within the sample container
 2. Designing a Sampling Plan
Sampling:
 The process of collecting a representative sample for analysis.
 Act of selection of a material fraction which is representing or provide
information on the largest quantity of this material.
 Protocol in which a fraction of a substance is collected to provide a
representative sample of the entire material.

• For a quantitative analysis, the sample’s composition MUST accurately

represent the target population.

A sampling plan must support the goals of an analysis.

Five questions to consider:

1. From where within the target population should we collect samples?

2. What type of samples to collect?
3. What is the minimum amount of sample for each analysis?
4. How many samples should we analyse?
5. How to minimize the overall variance for the analysis? 11
 2.1 Where to Sample the Target Population

 A sampling error occurs whenever a sample’s composition is not

identical to its target population.

 If the target population is homogeneous, then we can collect

individual samples without giving consideration to where to sample.
 Unfortunately, in most situations the target population is
heterogeneous.

 Other target populations show both a spatial and a temporal

heterogeneity.

 If the analyte’s distribution within the target population is a concern,

then our sampling plan must take this into account.
 For example, homogenizing the target population which is, however,
impracticable in most cases and also destroys information about the
analyte’s spatial or temporal distribution within the target population.

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 Basic sampling approaches
A. Random Sampling
• The ideal sampling plan provides
an unbiased estimate of the target
population’s properties.
• A random sampling is the easiest
way to satisfy this requirement.
• Though simple, a truly random
sample is however difficult to
collect.
• Haphazard sampling (i.e. sampling
without a plan) is not random and
may reflect an analyst’s
unintentional biases.
• In collecting a random sample we
make no assumptions about the
target population, making this the
least biased approach to sampling.
• However, a random sample is often
time consuming and expensive
because we need a greater number
of samples to ensure that we meet
the sample representativity criteria.
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B. Judgmental (selective) Sampling
• We use prior information about
the target population to help
guide our selection of samples.

• Judgmental sampling is more

biased than random sampling,
but requires fewer samples.

• Judgmental sampling is useful if

we wish to limit the number of
independent variables influencing
our results.

• For example, if we are studying

the bioaccumulation of PCB’s in
fish, we may choose to exclude
fish that are too small or that
appear diseased.

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C. Systematic Sampling
• Random & judgmental sampling
represent extremes in bias and in
the number of samples needed to
characterize the target population.

• Systematic sampling falls in

between these extremes.

• Here, we sample the target

population at regular intervals in
space or time.

• When a population’s heterogeneity

is time-dependent, samples are
drawn at regular intervals in time.

• If a target population’s properties

have a periodic trend, a systematic
sampling will lead to a significant
bias if our sampling frequency is too
small. 15
Combinations of the three primary approaches to sampling are also
possible.

Systematic–Judgmental Sampling
 We use prior knowledge about a system to guide a systematic sampling
plan.
 Focused sampling; money and time saving.

Stratified (judgmental–random) Sampling

 Many target populations consist of distinct units, or strata.
 Here, we divide the target population into strata and collect random
samples from within each stratum.
 Advantage: Improved overall sampling variance.

Convenience Sampling
 We select sample sites using criteria other than minimizing sampling
error and sampling variance.
 In this case cost, expedience, and accessibility are more important than
ensuring a random sample.
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 2.2 What Type of Sample to Collect?

• There are three common methods for obtaining samples: grab

sampling, composite sampling, and in situ sampling.

• Grab sampling: collect a portion of the target population at a specific

time and/or location, ( i.e. a “snapshot” ).

• Composite sampling: collect a set of grab samples that are combined

into a single sample before analysis (for average for ex.).

 Grab and composite samples cannot be used to continuously monitor

a time-dependent change in the target population.

• In situ sampling: insert an analytical sensor into the target population.

 Allows continuous monitoring of the target population without
removing individual grab samples.

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2.3 How Much Sample to Collect?

• To minimize sampling errors, samples must be of an appropriate size.

• Too small samples may introduce a sampling error.
• Too large samples require more time and money to collect and analyse.

2.4. How Many Samples to Collect?

For normally distributed samples,

nsamp: number of samples.

Rearranging and substituting e (= X−μ), gives

This equation provides an estimate for the smallest number of samples that
will produce the desired sampling error.
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 3. Implementing the Sampling Plan

• Implementing a sampling plan involves 3 steps: (1) physically

removing the sample from its target population, (2) preserving the
sample, and (3) preparing the sample for analysis.

• After removing a sample from its target population, there is a risk for
chemical or physical changes in the sample before analysis.

• To prevent this problem, we often preserve samples before

transporting them to the laboratory for analysis.

• The initial sample is called the primary or gross sample.

• In many cases we cannot analyse the gross sample without first

reducing the sample’s particle size, converting the sample into a more
readily analysable form, or improving its homogeneity (Preparation).

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 3.1 Solution (liquid) samples

3.1.1 Sample Collection

 To collect a grab sample (surface

waters): submerge a capped bottle
below the surface, remove the cap
and allow the bottle to fill
completely, and replace the cap.

 Collecting a sample this way

avoids the air–water interface,
which may be enriched with heavy
metals or contaminated with oil.

 Samples at greater depths are

collected using a sample bottle
lowered to the desired depth (See
Figure).
Figure. A Niskin sampling bottle
for collecting water samples
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from lakes and oceans
 Sample containers are made from glass (e.g. borosilicate glass) or
plastic.

• Glass containers are costly, heavy, and easy to break.

 They are used for the analysis of pesticides, oil and grease, and
organics (often interact with plastic surfaces).

• Plastic containers are made from a variety of polymers : polyethylene

(PE), polypropylene (PP), polycarbonate (PC), polyvinyl chloride (PVC),
and Teflon® (PTFE).

 They are lightweight, durable, and inexpensive (except Teflon® ).

 They also are preferred for the analysis of trace metals (glass surfaces
easily adsorb metal ions).

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3.1.2 Sample Preservation and Preparation

• After removing a sample from its target population, its chemical

composition may change.

• To prevent that, samples are preserved by:

 controlling the solution’s pH and temperature,
 by limiting its exposure to light or to the atmosphere,
 by adding a chemical preservative.

• After preserving a sample, it may be safely stored for later analysis.

• The maximum holding time between preservation and analysis depends

on the analyte’s stability and the effectiveness of preservation.

• Most liquids do not need additional preparation before analysis.

• Solutions with complex matrices (e.g. blood) may need additional
processing to separate the analytes from interferents.

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23
 3.2 Gases

• E.g. Automobile exhaust, Emissions from industrial smokestacks,

Atmospheric gases, etc.
• Also included in this category are aerosol particulates.

3.2.1 Sample Collection

• Solid sorbents are used for volatile gases and semi-volatile gases.

• Filtration is used to collect aerosol particulates.

• Trapping and filtering allows for sampling larger volumes of gas and
stabilizes the sample between its collection and its analysis.

• In solid sorbent sampling, a pump pulls the air through a canister

packed with sorbent particles.

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25
3.2.2 Sample Preservation and Preparation

• There is generally little need for sample preservation or preparation.

• The chemical composition of a gas sample is usually stable when it is

collected using a solid sorbent, a filter, or by cryogenic cooling.

• When using a solid sorbent, gaseous compounds are released for

analysis by thermal desorption or by extracting with a suitable solvent.

• If the sorbent is selective for a single analyte, the increase in the

sorbent’s mass can be used to determine the amount of analyte in the
sample.

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 3.3 Solids

3.3.1 Sample Collection

• Bottom sediments are collected with a bottom grab sampler.

• An alternative method is a cylindrical coring device or a corer which is

used to prevent sample mixing and, therefore their vertical profile,
preserving information about how the sediment’s composition changes
with depth.

• Collecting soil samples at depths of up to 30 cm is easily done with a

scoop or shovel (though high sampling variance).

• Soil samples from depths greater than 30 cm are collected using an

auger that drills a hole to the desired depth.

• Larger particulate solids, such as ores, are sampled using a riffle.

• A sample thief is used for sampling smaller particulate materials, such

as powders.
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 A gravity corer in Sample thief
Bottom grab sampler operation

A four-unit riffle Auger

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3.3.2 Sample Preservation (solids)

• Without preservation, a solid sample may undergo a change in

composition due to the loss of volatile material, biodegradation, and
chemical reactivity (particularly redox reactions).

• Storing samples at lower temperatures makes them less prone to

biodegradation and to the loss of volatile material.

• To minimize the loss of volatiles, the sample container is filled

completely, eliminating a headspace where gases collect.

• Samples that have not been exposed to O2 are particularly susceptible to

oxidation reactions.

• For example, the contact of air with anaerobic sediments must be

prevented.

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Methods of preserving samples

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 Sample drying

Goal

- keep low humidity during storage

- limit biological degradation

- often required before grinding

DRYING TEMPERATURE

- oven at 40°C : to limit losses by volatilisation

- lyophilisation at -50°C : biotissues or volatile species (Hg, As, Se…)

Moisture: Determination of H2O content by weighing before and after drying

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Lyophilization (dry-freezing):
remove the water at low temperature and under vacuum

Applicable unstable/fragile samples

Good preservation for biotissues
Potential degradation during the process
Unsuitable for highly volatile compounds

Gamma irradiation

Remove all microorganisms

Good preservation of biotissues
Potential degradation during the process
Cryogenic freezing: instantaneous freezing at low
temperature (-196°C) after sampling

Avoid the microbial activity to develop

Good preservation of organometallic compounds
Need to maintain all the analytical steps at -196°C
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 Temperature for sample storage

Storage
Suitable sample type Unsuitable sample type
conditions

High enzymatic activity

Freezer
Most solid samples Matrix alteration
(-18°C)
during defreezing
Unstable compounds
Soils, minerals
Fridge
Fruits and fresh vegetables Enzymatic activity
(4°C)
Aqueous samples
Dry powders or granules
Room temp
(darkness) Minerals Fresh food
Stable compounds
More hygroscopic
Dessicator Hygroscopic matrix
than dessicant

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Reducing Particle Size

• Done by a combination of crushing and grinding the gross sample.

• A variety of tools are used depending on the particle’s size and

hardness: jaw crushers (for large particles); Ball mills, disk mills, and
mortars and pestles are (used to further reduce particle size).

• The gross sample is reduced to a uniform particle size by intermittently

passing it through a sieve.

• Those particles not passing through the sieve receive additional

processing until the entire sample is of uniform size.

• The resulting material is mixed thoroughly to ensure homogeneity.

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 Sample homogenization
Goal

increase the representativity of the sub-sample (aliquot) from the collected

samples

GRINDING

sampling a large quantity and crushing into a homogenesous fine powder

- reduce errors related to sub-

sampling

- improve the extraction

efficiency by increasing the
specific surface of the sample

35
 4. Cleaning/drying of equipment & labware

• Particularly relevant where a piece of equipment is used repeatedly.

• Also applicable to decontamination of equipment.

• The purpose is to ensure that the risk of contamination from previous

samples, chemicals, standards or the laboratory environment will be
minimized.

• In many cases, the process of cleaning introduces new chemicals to

whatever is being cleaned.

• Thus, the equipment must be well rinsed after cleaning and then dried.

• Care should be taken during cleaning to ensure that the cleaning process
does not cause more problems than the original contamination.

• When cleaning volumetric glassware avoid machine washing, extremes

of temperature and the use of strong acids/alkalis or surfactants.

36
 Drying

 There are 4 ways of drying

equipment: Drying Rack
 Physically drying the item with an
absorbent material,
 Rinsing with a volatile solvent &
allow the solvent to evaporate at RT,
 Driving off solvents at RT by using
an air stream,
 Driving off solvents at HT (more
convenient & usually safe).

 Most labs have commercially

glassware drying ovens.

 However, a hot oven is not Washers/

recommended for driving off volatile Dryers
organic solvents and in drying
volumetric glassware (risk of
irreversible expansion). 37
 5. Reagents and Solvents
• Reagents – reducing and oxidizing agents, indicators, drying agents, buffer
solutions, complexing agents, acidic and alkaline materials.

• Solvents – water, organic liquids and supercritical fluids.

• For each of these you need to consider a number of aspects, such as

grade, labelling, preparation, containment, storage, safety, stability and
disposal.

Grade

• Most lab chemicals are available in different grades, usually according to

the concentration of impurities that are present.

• The nature of the impurities may or may not be important, depending on

how the chemical is to be used.

38
 Applications for each purity level

Grade Specifics and Application

Analytical Simple distillation , glass bottles or PET; rough
characterization of element conc  1 ppm ); cleaning bath

Suprapure Double distillation, glass bottles eventually covered by

PET film, rough characterization  100 ppb; Analysis at ppm
down to ppb levels; Analysis of soils, sediments, sludges,
contaminated waters/ organisms…

Ultrapure Sub-boiling distillation, Teflon® bottles, complete

characterization < 500 ppt; Analysis at ppt levels (dust,
Cost and waters, uncontaminated samples, …
Purity

39
5.1 Labelling

• It ensures that the identity and status of reagents, chemical standards,

apparatus and equipment are always clear to users.

• Various requirements for a label:

 It should be securely attached to the body of the container, NOT the
closure;
 It should have sufficient space to record all relevant information;
 It should be sufficiently indelible or protected to prevent the information
becoming illegible due to spillage or soiling.

• Exceptions to the above:

 When preparing solutions by weight with identical ‘lidded’ containers, it is

important to identify the container and its cap.

 In some cases, writing directly on the container may be the best option.

40
5.2 Safety

• It is part of good lab practice (GLP) and needs to be optimized.

• Most countries have a list of substances to be controlled carefully and the

maximum level to which workers can be exposed.

• If the analyst cannot get potential hazards information from label, Hazard
data sheets must be available for each chemical from the manufacturer.

• Also, the information is likely to be listed in manufacturers’ catalogues.

• Failing this, there are various books which list chemical properties,
including The Merck Index.

• If all else fails, the chemist should check the lab Manual Safety Data Sheet
(MSDS) or assume the worst and treat the chemical with extreme caution.

41
5.3 Disposal

• Responsible disposal of chemicals, samples and consumables also is

an important aspect of GLP.

• It may be permissible to dispose of some chemicals directly down the

drain, flushed down with copious volumes of water.

• For other chemicals, specific disposal instructions, where available,

must be followed (i.e. collection of specific types of chemical waste in
containers for disposal by incineration, landfill, etc.)

• Storage areas, such as refrigerators, freezers and cupboards, should be

regularly checked to avoid build-up of unnecessary items.

• Reagents and standards, which have passed their expiry date, and
samples which need no longer be retained, should be appropriately
disposed of.

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 6. Sample Preparation

• Great reduction in analytical variability can be achieved through the use of

appropriate sample preparation procedures.

• Generally, a reduction in subsampling variance can be accomplished by

the sample particle size reduction and homogenization.

• In certain circumstances there could be loss of volatile analytes or

irreversible chemical changes during sample preparation.

• Unlike gases and liquids, which generally require little sample preparation,
a solid sample usually needs some processing before analysis.

Two reasons for this:

 Reducing the sample’s average particle size allows us to collect the same
number of particles with a smaller, more manageable mass (obtain a
reasonable ssamp).
 Many analytical techniques require that the analyte be in solution.
43
 6.1 Precautions Against Errors
 Keep Procedures as Simple as Possible
Minimise the number of operations between sample preparation and final determination.

Keep the Apparatus Used to a Minimum

Minimise the number of vessels used per determination. Try to work in one vessel if possible.

Work in a Clean Environment

Always perform the work in as clean an environment as practicable. Work in fume cupboards,
and preferably laminar flow cabinets, or even a 'clean room' if available. Exclude rubber and
metal equipment, and use dedicated apparatus and closed systems whenever possible.

Minimise the Quantities of Reagents Used

This might necessitate changing the technique employed. For ex., in the wet digestion of OM,
the use of a closed system will permit operation at HT, this compensates for smaller volumes of
the digesting acids.

Use Certified Reference Materials

Wherever possible, check the analytical procedure using CRMs and, if necessary, use them to
evaluate the steps of the analysis.

Check the Robustness of the Procedure .

The dependence of the results on temperature, duration of the process, and quantities of
sample or reagents should be studied.
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 SENSITIVITY REQUIREMENTS

Water samples Biotissues Solid samples

As Cu
Ni
ppm
Cr
Hg
Hg
As
Se Sn
ppb

Hg

ppt Sn
45
 6.2 FLOW CHART FOR TRACE ANALYSIS
INORGANIC INORGANIC ORGANIC ORGANIC
ANALYTE ANALYTE ANALYTE ANALYTE
INORGANIC ORGANIC INORGANIC ORGANIC MATRIX
MATRIX MATRIX MATRIX
SAMPLE
DISSOLUTION DESTRUCTION OF EXTRACTION
ORGANIC MATTER

SOLUTION

SEPARATION
OF ANALYTE
REMOVAL OF INTERFERENCES (CLEAN-UP)
PRE-CONCENTRATION
DETECTION
SIGNAL

QUANTIFICATION

RESULTS

CONFIRMATION

REPORT
46
 6.3 METHODS FOR SAMPLE PREPARATION
INORGANIC ANALYTES ORGANIC

DRY ASHING
WET DIGESTION
DECOMPOSITION
DISSOLUTION
CHELATING
ELECTRODEPOSITION

EVAPORATION, DISTILLATION

VOLATILIZATION
PRECIPITATION
EXTRACTION

47
Bringing Solid Samples Into Solution

• Distilled water is usually the solvent of choice for inorganic salts, but
organic solvents, such as methanol, chloroform, and toluene are useful
for organic materials.

• When a sample is difficult to dissolve, the next step is to try digesting it

with an acid or a base.

• Digestions are carried out in an open container, usually a beaker, using a

hot-plate as a source of heat.

• The main advantage of an open-vessel digestion is cost.

• Volatile reaction products, however, are lost (determinate error).

48
 6.3.1 SAMPLES PREPARATION - INORGANIC ANALYTE
DESTRUCTION OF ORGANIC MATRICES - COMPLETE OXIDATION OF MATRIX

DRY OXIDATION WET OXIDATION

COMBUSTION WET DIGESTION (using acids,
oxidizing agents)
DRY ASHING
OPEN ( Kjeldahl flask, reflux
OPEN (crucible)
condenser, ultrasonic digestion)
CLOSED (oxygen bomb)
ACID PRESSURE DECOMPOSITION
LOW TEMP. (flow of oxygen)
MICROWAVE (closed, open)
UV (open)

•No need for const. monitoring •Need for const. monitoring

•Slow •Rapid

•High temp. (>400C) •Lower temp.

•More volatilization •Less volatilization

49
•Small blank •Larger blank
• Organic materials can be decomposed by dry ashing.

• Sample is placed in a crucible and heated over a flame or in a furnace.

• C in the sample oxidizes to CO2 (and H2O, SO2, and N2 leave as gases).

• These gases can be trapped and weighed to determine their

concentration in the organic material.

• Often the goal of dry ashing is to remove the organic material, leaving
behind an inorganic residue, or ash, that can be further analyzed.

DRY ASHING – ELEMENTS LOST DURING DRY ASHING

Ag Ag Au Be Cd Cs Ge Hg Ir Li
Ni Pb Pd Pt Rb Sb Se Sn Zn
Underlined: volatile under 5000C

50
 Preparation of solid samples
Solid matrix

geological metalurgical biological botanical

mineralisation extraction

Heating blocks Stirring

Ultrasonic bath
Fusion
Heating blocks

Microwaves Microwaves
51
 Solid/liquid extraction: heating systems

Hot plate Water bath SOXLHET

 several samples simultaneously
 simple
 low reproductibility
 low temperature control
 simple
 continuous extraction
 matrix independent
 no filtration needed
 time consuming
 working at boiling temp
 large volume of solvent
52
 Solid/liquid extraction: heating plate

Heating by conduction
Advantages
simple
low cost
convection
Drawbacks
. . . slow
. .. low temp control
lab contamination
low reproducibility

External temp higher than acid boiling point loss of volatile species

53
 Wet mineralisation in closed Teflon bomb
Advantages
• Avoid loss of volatile species
• Low lab-contamination
• Rapid due to high temp and pressure
• Low consumption of reagents
Drawbacks
• Limited sample amount for organic matrices
CO2
• risk of explosion

54
 Microwave sample preparation systems

• Many digestions are now carried out in a closed container using

microwave (MW) radiation as the source of energy.

• Vessels for MW digestion are made in Teflon® (or fluoropolymer) and

fused silica.

• Both materials are thermally stable, chemically resistant, transparent to

microwave radiation, and capable of withstanding elevated pressures.

• The vessels are placed in a microwave (MW) oven and MW energy is

controlled by monitoring T and/or P within one of the vessels.

• A MW digestion has several important advantages over an open-vessel

digestion (HT; HP and prevents the loss of volatile gases).

• Disadvantages: inability to add reagents during the digestion, limited

sample’s size, and safety concerns due to the HP and corrosive
reagents.
55
 refluxing
Closed Open
Circulator Magnetron Wavelength
attenuator
cutoff

Wave guide
sample

Magnetron

sample

Multimode System Monomode System + focalised waves

Perfect control of energy

56
 Microwave systems for digestion/extraction

Open system Closed

system

57
 Rapidity of microwave-assisted hydrolysis
of biomaterials

enzymic hydrolysis
acetic acid leaching
TMAH hydrolysis
HCl-MeOH leaching
NaOH-MeOH hydrolysis
microwave assisted
TMAH hydrolysis
1 5 10 15 20 25
Time of solubilization, h
 Rapidity of microwave systems for Hg speciation
in sediments

Conventional
methods
(e.g.hot plates)
Distillation

Ultrasonic
Extraction
Supercritical
Fluid
Extraction (2-4 min)
Microwave-
Assisted
Extraction
0.1 1 10 100
Extraction time* (hour)
59
*Time required for quantitative extraction of MeHg+
 WET DIGESTION
Choice of Acids
• The acid must be compatible with the end determination. For example:
 oxidising acids cannot be used with a voltammetric determination.
 Sulphuric acid cannot be used with ICP-MS employing nickel cones.

• The main acids used for elemental dissolution are HCl, HNO3 and their
mixture known as 'aqua regia’.
• Other acids are less frequently used and only for specific purposes.
 They include H2SO4, HClO4, HF, and H3PO4.

• Acids can be used either in their concentrated form or diluted.

 'Concentrated' acids can range from about 30% solutions (HCl) to almost
100% purity (H2SO4).
 For dissolution, dilute acids usually refer to molarities from 0.1 to 2 M.
60
Nitric Acid (HNO3)

• Normally obtained as the 70% azeotrope with water.

 Azeotrope (or a constant boiling point mixture): is a mixture of two
or more liquids whose proportions cannot be altered or changed by
simple distillation.

• Commonly referred to as 'concentrated' but in fact HNO3 is

available as 55% and higher concentrations (must be used with
great care!!!).

• Employed for its oxidising as well as its acidic properties, and

because its salts are almost invariably soluble, It is used for almost
all matrices.
• Can form insoluble oxides of AI, Nb, Ta, Ti, Sn, Sb, and W.

61
Hydrochloric Acid (HCl)
• Usually supplied as a 36% solution in water, known as the concentrated
acid, but other concentrations (32% and. 39%) are available.

• It is non-oxidizing; in fact is reducing towards some higher oxidation

states of metals, eg. Ce(IV), Te(VI), Mn(IV), Mn(VII).

• It cannot be used for the destruction of organic matter but is widely

employed for the attack of inorganic matrices.
• Chlorides of the metals are soluble except for Ag, Hg, TI, and Pb.

• The main problem with HCl is that it can form volatile chlorides such as
those of Hg, Ge, As, Sb, and Se.

62
Aqua Regia

• A 3:1 mixture by volume of concentrated HCl and HNO3.

• It has much more powerful oxidising and complexing properties than

either of its acid constituents, due to the formation of chlorine and
nitrosyl chloride (NOCI).

• It is generally used for the decomposition of difficult matrices, other

than silicates, and for noble and other electronegative metals.

63
Sulphuric Acid (H2SO4)

• Frequently used for the destruction of organic matrices, because it

combines acidity, oxidative, and dehydrating properties.

• Seldom used for inorganic matrices (though useful for some tasks,
such as the dissolution of TiO2) but used for disolving certain
individual elements.

• The sulphates of of Ca, Sr, Ba, and Pb are insoluble in water but
soluble in H2SO4.

• Note: ’concentrated’ H2SO4 refers to not less than 95 - 98% of the acid.

64
Perchloric Acid (HClO4)

• Usually supplied either as the 60% soln or as 72%, azeotrope in water.

• A powerful acid: metal perchlorates (except that of K) are very soluble.
• Has little, if any, ability to form complexes.
• Used mainly for the dissolution of steel samples and for the destruction
of organic matrices.
• It has no oxidising properties in the cold but when hot, becomes one of
the most powerful lab oxidants.
 For this reason, if it is mixed with a significant amount of OM, especially
easily oxidisable matter, in the cold it can appear safe, but on heating
can cause explosive oxidation.
 It must therefore be used only for well-establish digestions or to
remove the last traces of OM.
 It is good practice to remove most of the OM with HNO3 prior to cooling
down the soln ready for treatment with HClO4.
• Hydrogen peroxide (H2O2) can often be used as a substitute for HClO4.

65
Hydrofluoric Acid (HF)
• Supplied for analytical use as 40 or 48% aqueous solutions.
• Must be handled with care, and gloves, entirely free from any pinholes,
must always be worn (causes very painful and slow to heal burns, and
passes through skin to attack bone).
• A weak, non-oxidising acid that forms volatile compounds with Si, Ge, Sn,
Ti, Zr, and As.
 Fluorides of Mg, Ca, Sr, Ba, AI, Pb, and Cr(III) are insoluble.
• Most generally used to destroy silicate matrices from which SiF4 and
excess HF can be expelled by fuming with H2SO4 or HClO4.
• Fuming with HClO4 has the advantage of converting any insoluble fluorides
into soluble perchlorates.
• After using HF, it is good practice to add boric acid (H3BO3) to combine
with any residual traces of fluoride.
• In general, DILUTION IS NOT RECOMMENDED AS A METHOD OF
NEUTRALISATION – EVEN DILUTE HF CAN BE DANGEROUS.

66
Typical materials used for neutralization of HF

67
Orthophosphoric Acid (H3PO4)

• A weak, non-oxidising acid supplied in a solution containing 85 to 88%

of H3PO4.

• Forms many insoluble phosphates, thus not generally used for

decomposition.

• However, due to its complexing properties, it is useful for dissolving

chromites, ferrites, uranium oxides, and the phosphate ores of the
lanthanide elements (e.g. La, Ce, Pm, Eu, Gd, etc.)

• Note that H3PO4 damages nickel cones.

68
Hazards of common chemicals used in the digestion of rocks/sediments
alongside HF

69
70
Properties of some common acids used
in digestions

71
Use of Acid Mixtures
• It is rare for only one acid to be used in the digestion of a matrix.

• Small amounts of other acids may be employed to fulfil a particular

function.

• The use of HF in the dissolulion of silicates has been mentioned, as

well as the associated addition of H3BO3 to complex residual fluoride.

• Niobium (Nb) and Tantalum (Ta) ores may also be decomposed with
HF, which forms complex fluoro ions of the metals.

• Certain Nb and Ta ores can be decomposed with HF and HCI mixtures

under slightly elevated pressure.

• A mixture of HF, HCl, and H3PO4 also decomposes Nb ores.

72
Common mixtures

 As established above, digestions are rarely carried out with one acid alone.
 They are usually done with mixtures.
 Below are a few common mixtures used in digests:

 Aqua regia (3 HCl to 1 HNO3 (v/v)) – oxidising agent, used to dissolve

metals, especially Pt & Au.
 Reverse aqua regia (3 HNO3 to 1 HCl (v/v)) – used for most organic
materials.
 HNO3:HCl:HF Used, in various ratios, for the dissolution of alloys, ores,
silicates, ash.
 Caro’s acid (addition of H2O2 to H2SO4) – forms persulfuric acid (H2SO5)
used to oxidize organic samples.
 H2SO4 / H3PO4 : commonly used for dissolution of alumina (Al2O3) and
materials containing alumina such as ceramics, catalysts, slags, and ores.
Provides HT at LP.
 HNO3 / H2SO4 : Combination used to enhance decomposition of organic
samples

73
DISSOLUTION CONDITIONS OF SOME COMMON ANALYTES

74
 METHODS OF DISSOLVING INORGANIC MATERIAL

LEACHING AND PARTIAL DISSOLUTION:

dissolution in water or diluted acids and complexation by org. ligands
TOTAL DISSOLUTION
75
FUSION
Fusion

• Inorganic samples that resist decomposition with acids or bases often

can be brought into solution by fusing with a large excess of an alkali
metal salt, called a flux.

• Fusion involves the complete dissolution of the sample in molten flux.

• Fusions are generally more aggressive than acid dissolution methods

and are suitable for refractory mineral dissolution.

• After mixing the sample and the flux in a crucible, they are heated to a
molten state and allowed to cool slowly to room temperature.

• The melt usually dissolves readily in distilled water or dilute acid.

• Fusion disadvantages: contamination from flux and crucible, loss of

volatile materials.

76
MATRICES FOR WHICH FUSION IS USED

77
 Lithium borate

Lithium metaborate

Sodium peroxide

Potassium pyrosulfate

Boron oxide

78
 WET DIGESTION - GENERAL PRECAUTIONS

1.Avoiding losses
Occlusion means fitting together or obstructing

2.Occlusion in metal vessels

(I Hg Au Pt Pd Fe – occlude with Pt)

3.Adsorption of elements by silica-based materials

(elements forming oxides during decomposition)

4.Volatilization
Volatile species: oxides of Os, Re, Ru
chlorides of Sb, As, Cr, Co, Cu, Ge,
Fe, Pb, Zn
hydrides of Sb, As, Se, Te
others – Cd, Hg
5.Contamination
79
 PRECONCENTRATION METHODS

EVAPORATION AND DISTILATION

VOLATILIZATION / HYDRIDES GENERATION

SOLVENT EXTRACTION

PRECIPITATION

ION-EXCHANGE AND ION CHROMATOGRAPHY

ELECTROPHORESIS

ELECTRODEPOSITION

STRIPPING ANALYSIS
80
 6.3.2 SAMPLE PREPARATION - ORGANIC ANALYTES

 COMPOSITION OF ORGANICS: C,H,O CO2 + H2O

and Cl, Br, F, P, S, Metals ……

 GROUPS WITH SIMILAR PROPERTIES

INTERFERENCES

 NUMBER AND VARIETY OF ORGANIC COMPOUNDS

 COMPOSITION CHANGES
INTERACTIONS (PHYSICAL AND CHEMICAL)
CHEMICAL REACTIONS

81
Range of Compounds for Which Methods are Required
•There are ~100 elements and 10-15 inorganic anions that are of interest to the inorganic
analyst, but there are, in theory, 106 or more organic compounds of potential interest!
•Legislation already exists or is proposed for the permitted levels of a wide range of
chemical types. For example:
 pesticides include >30 classes of chemical compounds, residues that are subject to
statutory control.
 Veterinary drug residues include at least 7 classes of antimicrobial compounds, as well
as a range of anabolic compounds.
 There are many other compounds such as PAHs, PCBs, phthalates, dioxins, and
mycotoxins, which are subject to legislative control.
•The substrates that have to be analyzed for trace organic compounds include:
(i) gases (air, stack gases, gaseous reaction mixtures, factory atmospheres…)
(ii) liquids (water, body fluids, oils, effluents, industrial process mixtures,…)
(iii) solids (food, soil, sludge, human and animal tissues, plant materials, plastics,
polymers, packaging materials,...)
• The complexity of analytes to be determined ranges from simple molecules such as CS2
to compounds having a relative molecular mass of thousands (e.g., macrolide
antibiotics).
82
 The Need for Multi-analyte Methods Covering Many Compounds
Within a Group

• There are areas of legislation (e.g. pesticide residues} that require the
measurement of increasing numbers of analytes in a given substrate.

• Purely on a cost basis there is a need to determine as many compounds

as possible in a single analysis.

• Thus , an ideal method for pesticide residues would be capable of

determining more than 200 individual compounds.

• This is obviously going to include analytes with a wide range of physical

and chemical properties, which means that it is unlikely that such a
method will be the optimum approach for every single analyte that has to
be determined.
83
The Need to Determine Both the Parent Compound and Metabolites
• Metabolites may have considerably different properties from their parent
compound. By their very nature, they are usually more water-soluble.
• It is often therefore extremely difficult to determine both types using the
same method.
• Analysis of metabolites is of particular importance when they have
different toxicological properties from the parent and they are included
in lists for which there are legal limits.

Problems with Co-extracted Interferences

• In inorganic analyses it is often the case that the sample matrix can be
completely destroyed (ashing).
• In organic analyses such fierce conditions would, in most cases, also
destroy the analyte.
• Other separation techniques must therefore be employed.

84
 Similarities in Properties of Many of the Compounds
to be Determined in a Single Sample

• When similar compounds exist in a sample

but need to be individually quantified, the
specificity of the method becomes crucial.
Examples of such analyses are:
1) Tetrachlorodibenzodioxin (TCDD), where
some 25 isomers exist but only one (the
2,3,7,8-isomer) is of toxicological interest.
2) the increasing importance of separating D-
and L-isomers of a particular compound.
 Many pharmaceuticals, agrochemicals, and
industrial intermediates exist as racemic
mixtures, although it may only be one
enantiomer that is biologically active.
85
Instability of Many Organic Compounds to Heat and/or Light and
their Susceptibility to Hydrolysis and Oxidation
Examples are mycotoxins, which are unstable to UV , and many
Sulphur-containing fungicides, which are readily oxidized.

Toxicity of the Analytes, the Solvents and Reagents

• Some examples of toxic analytes are the N-nitrosamines
(carcinogenic), some mycotoxins (mutagenic), and chloropropanols
(believed to be mutagenic).
• Commonly used solvents such as dimethyl formamide (DMF),
dimethyl sulfoxide (DMSO), acetonitrile are acutely toxic and there are
very few other organic solvents that do not have some form of acute
or chronic hazard associated with their use.

86
STAGES IN ORGANIC TRACE ANALYSIS

87
 6.3.2.1 Separation Versus Preconcentration

• There are two common analytical problems :

1) Matrix components that interfere with an analyte’s analysis;

2) Analyte with a concentration too small to analyse accurately.

• We can use a separation to solve (1).

• We also often use a separation to solve (2).

• For a separation in which we recover the analyte in a new phase, it may

be possible to increase the analyte’s concentration.

• This step in an analytical procedure is known as a preconcentration.

88
 6.3.2.2 Classifying Separation Techniques
We can separate an analyte and an interferent if there is a significant
difference in at least one of their chemical or physical properties.

89
Choice of Extraction Technique

• The majority of methods are based on the permeation of an

extracting solvent through the sample matrix.

• This solvent can either be an organic solvent or a supercritical

fluid.
A super critical fluid is the phase of a material at critical T and P of the material
State of matter in which the thermodynamic parameters of a substance are higher than its critical P and T

• The exception to this rule is in the analysis of volatile analytes.

• These can be released by methods such as distillation, static

headspace, dynamic headspace, and sublimation.

90
The questions that must be asked before selecting an extraction
method are the following:
(i) Is the extraction exhaustive?
(ii) Are there undesirable co-extractives?
(iii) Is the unit time taken for each extraction economical?
(iv) If the method is time consuming, is there a stage at which the
extract can safely be left overnight?
(v) Can the extraction be automated and/or coupled with the end-
determination?
(vi) What is the maximum amount of sample with which the technique
can cope?
(vii) Will the extract need to be concentrated? How difficult will this be?
(viii) Will the extract be compatible with the end-determination
technique?
91
 Solids

Extraction of environmental pollutants from solid or semi-solid matrices can

be divided into several categories based on the method of extraction, mode
of heating and presence or not of some type of agitation.

92
Soxhlet Extraction

The basic Soxhlet extraction apparatus consists of a solvent reservoir, an

extraction body, a heat source (e.g. an isomantle) and a water-cooled
reflux condenser.

93
Shake-Flask Extraction (SFE)

• Carried out by placing a soil sample into a glass container, adding

organic solvent, & then agitating/shaking for a pre-specified time-period.
• After extraction, the solvent containing the analyte is separated from the
matrix by means of centrifugation and/or filtration.
• In some instances, it may be advisable to repeat the process several
times with fresh solvent and then combine all of the extracts.

94
Ultrasonic Extraction (Sonication)
• Use of sound waves to agitate a sample immersed in an organic solvent
(uses a sonic probe or a sonic bath).
• The sample is placed in a glass container and organic solvent is added.
The system is sonicated for ~3 min, using the sonic bath or probe. The
solvent is separated by centrifugation/filtration and fresh solvent added.
The process is repeated 3X and all of the extracts are then combined.

95
Supercritical Fluid Extraction
(SFE)
• Relies on the diversity of
properties exhibited by a
supercritical fluid (SF) to
(selectively) extract analytes
from solid, semi-solid or liquid
matrices.
• Important properties offered
by a SF for extraction are:
(i) good solvating power, (ii)
high diffusivity and low
viscosity, and (iii) minimal
surface tension.

 The term SF is used to

describe any substance above
its critical T,P.
 At the critical point of the
phase diagram, no
liquefaction will take place on
raising P and no gas will be
formed on increasing T. 96
97
Microwave-Assisted Extraction
(MAE)

98
Pressurized Fluid Extraction (PFE)
Uses high T,P to extract analytes rapidly and efficiently from solid matrices.

99
Solubility and Mass transfer Effect

The following three factors are considered important:

 HTs increase the capacity of solvents to solubilize analytes.
 Faster diffusion rates occur as a result of increased temperatures.
 Improved mass transfer, and hence increased extraction rates, occur when
fresh solvent is introduced, i.e. the concentration gradient is greater
between the fresh solvent and the surface of the sample matrix.

Disruption of Surface Equilibria

Temperature Effects
 HTs can disrupt the strong solute–matrix interactions caused by vdW forces,
H-bonding, and dipole attractions of the solute molecules and active sites on
the matrix.
 Decreases in the viscosities and surface tensions of solvents occur at HTs,
allowing an improved penetration of the matrix, and improved extraction.

Pressure Effects
 HPs allows solvents to remain liquified above their BPs.
 Extraction from within the matrix is possible, as P allows the solvent to
penetrate the sample matrix. 100
Matrix Solid-Phase Dispersion (MSPD)

 Analogous to solid-phase extraction (SPE) described later.

 The sample is mixed with an SPE sorbent (e.g. C18) from the cartridge.
 The sorbent-sample mixture is returned to the cartridge and eluted as in
SPE.
 The main purpose of the C18 sorbent is to act as an abrasive, thus
disrupting the sample’s structure, and creating a large surface area for
solvent interaction.

101
EXTRACTION FROM SOLIDS

102
PERMEATION OF SOLVENTS DURING
BLENDING

103
 Liquids

Solvent Extraction

•Two common approaches:

 Extraction is carried out discontinuously (equilibrium is established
between 2 immiscible phases) or Continuously (equilibrium may not be
reached).

•The extraction efficiency is governed by the choice of the solvents.

 Using aqueous and organic (e.g. DCM, chloroform, toluene, etc.) pairs of
solvents, the more hydrophobic analytes prefer the organic solvent
while the more hydrophilic compounds prefer the aqueous phase.

•The more desirable approach is often reflected in the nature of the analyte.
 Example, if the method of separation to be used is RP-HPLC, then the
target analyte is best isolated in the aqueous phase (the analyte can
then be injected directly into the HPLC).
 For GC, the analyte is best isolated in organic solvent (organic phase
allows solvent evaporation to be used, thus analyte pre-concentration).

HPLC- High Performance Liquid Chromatography

104
The equilibrium process can be influenced by
several factors including:
 adjustment of pH (prevents ionization of acids
or bases),
 formation of ion-pairs with ionizable analytes,
 formation of hydrophobic complexes with
metal ions,
 adding neutral salts to the aqueous phase to
reduce the solubility of the analyte (‘salting
out’).

105
Liquid–Liquid Extraction (LLE)

• In discontinuous extraction, the most common approach uses a

separating funnel.
• The sample is distributed or partitioned between 2 immiscible solvents in
which the analyte and matrix have different solubilities.

• Advantage: wide availability of pure solvents and use of low-cost

apparatus.

106
107
 Example: LLE before GC analysis of RHg compounds

Organic phase
+ organic solvent - non polar compounds
+ alkylating agent (*) (NaBEt4…) - Alkylated metals

agitation

 purification
Aqueous phase Aqueous phase  preconcentration
- Non polar compounds - Polar compounds
- Polar compounds - Ionic compounds  slow
- Ionic compounds
- Ionic (alkylated) metals (*)
 large volume

* Derivatization
Ethylation
Hg2+ + 2NaB(C2H5)4  Hg(C2H5)2 + 2Na+ + 2‘‘B(C2H5)3’’

CH3Hg+ + NaB(C2H5)4  CH3HgC2H5 + Na+ + ‘‘B(C2H5)3’’

•Formation of Carbon — Mercury bonds
•Slow process 108
Solid-Phase Extraction (SPE)

•Also referred to as liquid–solid extraction, involves bringing a liquid or

gaseous sample into contact with a solid phase (sorbent) whereby the analyte
is selectively adsorbed onto the surface of the solid phase.
 The latter is then separated from the solution and other solvents are added.
 The 1st such solvent is usually a wash (removes adsorbed matrix
components).
 An eluting solvent is brought into contact with the sorbent to selectively
desorb the analyte.

•The SP sorbent is usually packed into small tubes or cartridges.

•It is also available in discs (like filter paper) which can be mounted in a
filtration apparatus.
 The sample-containing solvent is forced by pressure or vacuum through the
sorbent.
 The analyte is retained by the sorbent and the extraneous material is washed
from the sorbent by the passing of an appropriate solvent.
 The analyte can then be eluted from the sorbent by using a suitable solvent.
109
Types of SPE Media
SPE sorbents can be divided into 3 classes:
 Normal phase,
 Reversed phase and
 Ion-exchange.
SPE Cartridges
•Usually made of PP (glass and PTFE are also available) with a wide entrance,
through which the sample is introduced, and a narrow exit.
 The sorbent material (0.05 - 10 g) is positioned between 2 frits (20 μm pore
size PE), at the exit of the cartridge, which act to both retain the sorbent and
to filter out particulate matter.

•Solvent flow is carried out by using a side-arm flask apparatus (single

cartridge), whereas multiple cartridges (8 to 30) can be simultaneously
processed by using a commercial vacuum manifold.

•A variation on this type of cartridge system (syringe filter) is when a plunger

is inserted into the cartridge barrel.
 Here, the solvent is added to the syringe barrel and forced through the SPE
unit by using the plunger (effective for early method development, or if no
vacuum system is available).
110
111
Method of SPE Operation

The method of operation can be divided into 5 steps, each characterized by

the nature and type of solvent used, which in turn is dependent upon the
characteristics of the sorbent and the sample.

112
Factors Affecting SPE

 The number of active sites available on the sorbent cannot be exceeded

by the number of molecules of analyte or otherwise ‘breakthrough’ will
occur.
 Hence, it is important to assess the capacity of the SPE cartridge or disc
for its intended application.
 The flow rate of sample through the sorbent is important; too fast flow =
minimal time for analyte–sorbent interaction.
 It is normal, therefore, for an SPE cartridge to operate with a flow rate of
3–10 ml/min, whereas rates of 10–100 ml/min are typical for the disc
format.
 Once the analyte of interest has been adsorbed by the sorbent, it may be
necessary to wash the sorbent of extraneous matrix components prior to
elution of the analyte. Obviously, the choice of solvent is critical in this
step.
 For the elution step, it is important to consider the volume of solvent to
be used (as well as its nature).

113
114
Solid-Phase Microextraction (SPME)

• The process whereby an analyte is adsorbed onto the surface of a coated-

silica fibre (for pre-concentration) followed by desorption of the analytes
into a suitable instrument for separation (GC/HPLC) and quantitation.

• The fused-silica fibre (~1 cm) is connected to a stainless-steel tube for

mechanical strength.
 This assembly is mounted within the syringe barrel for protection when
not in use.
 For SPME, the fibre is withdrawn into the syringe barrel, and then inserted
into the sample-containing vial for either solution or air analysis.
 The fibre is then exposed to the analyte(s) by pressing down the plunger
for a pre-specified time.
 The fibre is later withdrawn back into its protective syringe barrel and
withdrawn from the sample vial.
 The SPME device is then inserted into the hot injector of the GC and the
fibre exposed for a pre-specified time.
 The heat of the injector desorbs the analyte(s) from the fibre prior to GC
separation and detection.
115
116
117
Purge-and-trap
• Bubble an inert gas through the sample, releasing (purging) the
volatile compounds.
• These compounds are carried by the purge gas through a trap
containing an absorbent material where they are retained.
• Heating the trap and back-flushing with carrier gas transfers the
volatile compounds to the GC.

118
Headspace sampling
• Place the sample in a
closed vial with an
overlying air space.
• After allowing time for
the volatile analytes to
equilibrate between the
sample & the overlying
air, extract a portion of
the vapor phase
(Syringe /SPME) and
inject it into the GC.
Static Headspace

Dynamic Headspace

119
Thermal desorption
• Place a solid in a tube.
• After purging to remove any O2, heat the sample.
• Volatile analytes are swept by an inert gas and carried to the GC.
• Because volatilization is not a rapid process, the volatile analytes
are often concentrated at the top of the column by cooling the
column inlet below RT (cryogenic focusing).
• Once the volatilization is complete, the column inlet is rapidly
heated, releasing the analytes to travel through the column.

120
EXTRACTION FROM LIQUIDS

121
 6.3.2.4 Purification after extraction

Filtration
Remove particles from the extract

Lipid removal
Remove lipids by solvent extraction

liquid/liquid extraction
Remove extract components by solvent affinity

HPLC
Separate extract components by chromatographic elution (molecular size,
polarity, charge ….)

Ultrafiltration
Separate extract components at specific cutoff (molecular size)

Etc …

122
ORGANIC GROUP SEPARATION TECHNIQUES – CLEAN-UP

123
124
Recall: STAGES IN ORGANIC TRACE ANALYSIS

125
 Chapter Summary

• An analysis requires a sample.

• Collected samples must accurately represent their target population,

and the sampling plan must provide a sufficient number of samples of
appropriate size to minimize errors.

• There are several considerations for sampling: the type of sample;

whether to collect grab samples, composite samples, or in situ
samples; whether the population is homogeneous or heterogeneous;
the appropriate size for each sample; and, the number of samples to
collect.

• Removing a sample from its population may induce a change in its

composition (use apropriate containers and preservation).

• We may need to separate the analyte from interferents.

• Separations take advantage of physical or chemical properties.
126