UNIT 2: SAMPLING AND SAMPLE PREPARATION

Topic Outline:

⚫ Obtaining the sample

⚫ Sample handling
⚫ Sample preparation - solids
⚫ Total sample dissolution
⚫ Fusion
⚫ Sample preparation - liquid samples, extracts and solutions of solids

Learning Objectives:

After careful study of this chapter, you should be able to do the following:

⚫ Describe sampling in chemical analysis.

⚫ Describe the methods of sample preparation in solid and liquid samples.
⚫ Understand the methods of fusion and dissolution.
⚫ Define relevant terms.

ANALYTICAL SAMPLES AND METHODS

Types of Samples and Methods

1. Sample Size

⚫ Macro analysis is used for samples whose masses are greater than 0.1
g.
⚫ A semimicro analysis is performed on samples in the range of 0.01 to
0.1 g, and samples for a micro analysis are in the range 10-4 to 10-2 g.
⚫ Ultramicro analysis is for samples whose mass is less than 10-4 g.

2. Constituent Types

⚫ Major constituent - analytes are present in 1 - 100% by mass. Many

gravimetric and volumetric methods are major constituent
determinations.
⚫ Minor constituent - species present in the range of 0.01 to 1% in the
sample.
⚫ Trace constituent - constituents are between 100 ppm (0.01%) and 1
ppb.
⚫ Ultratrace constituent - components present in amounts lower than 1
ppb

REAL SAMPLES

⚫ Complicated analysis involving real samples are due to the presence of

sample matrix.
⚫ Matrix components can react with the same reagents as the analyte, or
they can cause an instrument response that is not easily distinguished
from the analyte. These effects interfere with the determination of the
analyte.
⚫ Matrix effects - interferences that are caused by extraneous species in
the matrix.
⚫ Take note! Samples are analyzed, but species or concentrations are
determined.

OBTAINING THE SAMPLE

⚫ A chemical analysis is most often performed on only a small fraction of

the material of interest, for example a few milliliters of water from a
polluted lake.
⚫ The composition of this fraction must reflect as closely as possible the
average composition of the bulk of the material if the results are to be
meaningful. In other words, it must be a representative sample - it
must truly represent the bulk system.

⚫ The process by which a representative fraction is acquired is termed

sampling. Often, sampling is the most difficult step in the entire
analytical process and the step that limits the accuracy of the procedure.
⚫ The items chosen for analysis are often called sampling units or
sampling increments.

⚫ To avoid confusion, chemists usually call the collection of sampling units

or increments, the gross sample.
⚫ The gross sample is usually reduced in size and homogenized to create
the laboratory sample.
⚫ Composite samples - small samples that are mixed and made
homogeneous to give the final sample to be tested.
⚫ Selective samples - individual samples analyzed in a method.
⚫ Random sample - one sample taken from one location at random in the
bulk system.
Other designations for samples are:
⚫ Bulk sample
⚫ Primary sample
⚫ Secondary sample
⚫ Subsample,
⚫ Laboratory sample
⚫ Test sample.

For example, a water sample from a well may be collected in a large

bottle (bulk sample or primary sample) from which a smaller sample is
acquired by pouring it into a vial to be taken into the laboratory (secondary
sample, subsample, or laboratory sample), then pouring it into a beaker
(another secondary sample or subsample), before a portion is finally
carefully measured into a flask (test sample) and diluted to make the
sample solution.

Figure 2.1. Steps in obtaining a laboratory sample. Adapted from

Fundamentals of analytical chemistry (9th ed.) (page 156), by D. A. Skoog et al.,
2014, Brooks/Cole.
Statistically, the goals of the sampling process are:

1. To obtain a mean analyte concentration that is an unbiased estimate of the

population mean. This goal can be realized only if all members of the
population have an equal probability of being included in the sample.

2. To obtain a variance in the measured analyte concentration that is an

unbiased estimate of the population variance so that valid confidence limits
can be found for the mean, and various hypothesis tests can be applied. This
goal can be reached only if every possible sample is equally likely to be
drawn.

Both goals require obtaining a random sample → does not imply that
the samples are chosen in a haphazard manner. Instead a randomization
procedure is applied to obtain such a sample.

SAMPLE HANDLING

⚫ The key concept is that the sample’s integrity must be strictly maintained
and preserved.
⚫ Examples are adding of precipitate, refrigeration, specified holding time,
and specified storage container.

Chain of Custody

⚫ It is very important to document who has handled the sample, what

responsibility each handler has at various junctures between the
sampling site and the laboratory and what actions the sample handler
has taken relating to sample integrity while the sample was in his/her
custody.

Figure 2.2. Representation of the chain of custody of a sample. Adapted

from Analytical chemistry for technicians (4th ed.) (page 21), by J. Kenkel,
2014, Taylor & Francis Group, LLC
Maintaining Sample Integrity

⚫ It is important for all sample custodians to maintain the sample in its

original physical and chemical condition so that it remains
representative of the bulk system in terms of the analyte identity and
concentration.
The following must be avoided:
⚫ Loss of sample matrix or solvent through evaporation or other means
⚫ Loss of analyte through evaporation, chemical reaction, temperature
effects, bacterial effects, etc.
⚫ Contamination with additional analyte (or another chemical that would
⚫ Interfere with the analysis) through contact with other matrices or
chemical systems
⚫ Moisture absorption or adsorption by exposure to humid air.

SAMPLE PREPARATION - SOLIDS

1. Particle size reduction

⚫ It would be ideal if a solid’s particle size can be reduced to that of a fine
powder, if possible, so that the solvent contact can be as complete as
possible.
⚫ Techniques involving crushing, milling, grinding, or pulverizing.
⚫ If the solid sample is of a state that reducing to a powder is not possible
(such as with a polymer film or piece of organic tissue), then particle size
reduction may take the form of cutting, chopping, or blending, etc.

2. Sample Homogenization and Division

⚫ Obtaining a portion that is representative of the larger sample (and
therefore representative of the entire bulk system) requires that the
larger sample be homogeneous.
⚫ A mixing procedure and a procedure by which a sample is divided to
create the required portions may follow particle size reduction.

3. Solid-Liquid Extraction
⚫ An analyte may be present in one material phase (either a solid or liquid
sample) and, as part of the sample preparation scheme, be required to
be separated from the sample matrix and placed in another phase (a
liquid).
⚫ Such a separation is known as an extraction—the analyte is “extracted”
from the initial phase by the liquid and is deposited (dissolved) in the
liquid while other sample components are insoluble and remain in the
initial phase.
⚫ Solid-liquid extraction - if the sample is solid
⚫ Two forms of solid-liquid extraction: (1) the sample and liquid are
shaken (or otherwise agitated) together in the same container, the
resultant mixture filtered, and the filtrate, which then contains the
analyte, collected or; (2) fresh liquid is continuously cycled through the
solid sample via a continuous evaporation/condensation process over a
period of hours (that usually does not require an extra filtration step)
and the liquid collected (Soxhlet extraction).

TOTAL SAMPLE DISSOLUTION AND OTHER CONSIDERATIONS

⚫ If extraction is not feasible, total dissolution of a sample may be required.

Total dissolution involves the proper choice of an acid—one that will
indeed dissolve the total solid sample.
⚫ This will usually involve the use of acid–water mixtures, but sometimes
fairly concentrated acid solutions, any of which may accompanied by
heating:
1. Hydrochloric acid
➢ A saturated solution of hydrogen chloride gas, fumes of which are very
pungent.
➢ Concentrated HCl is 38.0% HCl and about 12 M.
➢ Used especially for dissolving metals (Fe and Zn metals), metal oxides
(Fe oxide ores), and metal carbonates not ordinarily dissolved by water.

2. Sulfuric acid
➢ An acid that is considered a stronger acid than HCl in many respects.
➢ Concentrated sulfuric acid is about 96% H2SO4 (the remainder being
water), or about 18 M, and is a clear, colorless, syrupy dense liquid.
➢ Sample dissolution application is limited mostly to organic material,
such as vegetable plants.
➢ It is not as useful for metals because many metals form insoluble sulfates.
It is the solvent of choice for the Kjeldahl analysis for such materials as
grains, and products of grain processing. It is also used to dissolve
aluminum and titanium oxides on airplane parts.

3. Nitric acid
➢ Very dangerous and corrosive and is aptly referred to as an oxidizing
acid.
➢ Frequently, oxides of nitrogen form in such a reaction, and noxious
brown, white, and colorless gases are evolved.
➢ Concentrated HNO3 is 70% HNO3 (16 M)
➢ Used for applications where a strong acid with additional oxidizing
power is needed.
➢ Used in metals such as silver and copper, as well as organic materials
such as in a wastewater sample

4. Hydrofluoric acidd
➢ Has some very useful and specific applications, but is also very
dangerous.
➢ Concentrated HF is about 50% HF (26 M)
➢ Excellent solvent for silica (SiO2)-based materials such as sand, rocks and
glass, and stainlees steel methods.
➢ It dissolves glass so it must be stored in plastic containers

5. Perchloric acid
➢ Oxidizing acid like HNO3, but is considered to be even more powerful in
that regard when hot and concentrated.
➢ Used for metals, since metal perchlorates are highly soluble, but it is
most useful for more difficult organic samples, such as leathers and
rubbers, often in combination with nitric acid.
➢ Can also be used for stainless steels and other more stable alloys.
➢ Commercial HClO4 is 72% (12 M)
➢ Very dangerous acid, especially when hot and in contact with organic
matter, and should only be used in a fume hood designed for the
collection of its vapors. Contact with alcohols including polymeric
alcohols such as alcohols should be avoided to prevent potential
explosion.

6. Aqua Regia
➢ Acid mixture that is prepared by mixing one part concentrated HNO 3
with three parts concentrated HCl.
➢ Most powerful dissolving agents known that it can dissolve gold,
platinum, and other stable alloys.

7. Acetic acid
➢ A weak acid used in the preparation of buffer solutions.
➢ Although weak, it is still a rather dangerous acid with serious strong
vinegar fumes.
➢ Concentrated acetic acid has a 99.5% concentration (17 M).
➢ Referred to as “glacial” acetic acid because it has a relatively high
freezing point and in a cooler laboratory environment will turn to a
solid.

8. Ammonium hydroxide
➢ A weak base with limited sample dissolution application.
➢ Like hydrochloric acid and acetic acid, it is very dangerous because o
fumes, in this case, stifling ammonia fumes.
➢ Concentrated ammonium hydroxide has a concentration of 58% (15 M).

FUSION

Fusion is the dissolving of a sample using a molten inorganic salt,

generally called a “flux,” as the solvent. This flux dissolves the sample and,
upon cooling, results in a solid mass that is then soluble in a liquid reagent.
The dissolving power of the flux is mostly due to the extremely high
temperatures (usually 300°C to 1000°C) required to render most inorganic
salts molten.

However, some problems may be encountered in doing fusion:

⚫ The flux must be present in a fairly large quantity in order to be
successful.
⚫ The measurement of the analyte must not be affected by this large
quantity.
⚫ While a flux may be an excellent solvent for difficult samples, it will also
dissolve the container to some extent, creating contamination problems.
⚫ Platinum crucibles are commonly used, but nickel, gold, and porcelain
have been successfully used for some applications.

Common fluxes are:

⚫ Sodium carbonate, Na2CO3
⚫ Lithium tetraborate, Li2B4O7
⚫ Lithium metaborate, LiBO2
⚫ Either of the three chemicals above and combination with other
compounds, such as oxidizing agents (nitrates, chlorates, and peroxides.
⚫ Acidic flux potassium pyrosulfate (K2S2O7) for dissolving particularly
difficult metal oxides.

SAMPLE PREPARATION: LIQUID SAMPLES, EXTRACTS, AND SOLUTIONS

OF SOLIDS

1. Liquid-liquid extraction
⚫ The liquid solution containing the analyte (usually a water solution) is
brought into contact with a liquid solvent (usually a nonpolar organic
solvent) that is immiscible with the first solvent.
⚫ Separatory funnel is used to contain the solution of two immiscible
liquids. After shaking the contents, two separate liquid phases (layers)
are formed. the two layers can then be separated from each other by
allowing the two layers to drain one at a time through the valve
(“stopcock”) at the bottom of the funnel. The desired solution is then
carried forward to the next step.

Figure 2.3. Separatory funnel containing two

immiscible liquid layers. Adapted from Analytical
chemistry for technicians (4th ed.) (page 31), by J.
Kenkel, 2014, Taylor & Francis Group, LLC

2. Solid-phase extraction
⚫ The liquid solution containing the analyte is passed through a cartridge
containing a solid sorbent.
⚫ The sorbent either retains the analyte or its matrix. If it is the analyte
that is retained by the sorbent, the cartridge is first washed with a
solvent that will sweep away interferences. The analyte is then eluted
with a stronger solvent and collected. If it is the matrix that is retained
by the sorbent, the analyte is eluted immediately and collected.

REAGENTS USED IN SAMPLE PREPARATION

⚫ A reagent is a substance used in a chemical reaction in an analytical

laboratory because of its specific applicability to a given system or
procedure.
⚫ Examples of purity of reagents:
➢ Primary standard: A specially manufactured analytical reagent of
exceptional purity for standardizing solutions and preparing reference
standards.
➢ ACS certified: A reagent that meets or exceeds the specifications of
purity put forth by the American Chemical Society. The certificate of
analysis is on the label.
➢ Certified reagent: A reagent that meets the standards of purity
established by the manufacturer. The certificate of analysis is on the
label.
➢ USP/NF: Reagents that meet the purity requirements of the U.S.
Pharmacopeia (USP) and the National Formulary (NF). Generally of
interest to the pharmaceutical profession, these specifications may not
be adequate for reagent use.
➢ Spectro grade or spectranalyzed: Solvents of suitable purity for use in
spectrophotometric procedures. A certificate of analysis is on the label.
➢ High-performance liquid chromatography (HPLC) grade: Solvents of
suitable purity for use in liquid chromatography procedures.
➢ Practical: Chemicals of sufficiently high quality to be suitable for use in
some syntheses. Organic chemicals of practical grade may contain small
amounts of intermediates, isomers, or homologues.
➢ Technical: Chemicals of reasonable purity for applications that have no
official standard for purity

⚫ Shelf-life - the date beyond which the composition of a reagent can no

longer be trusted.