Ch27 Sample Preparation

Chapter 27
Harris 8
th
Edition
Instrumental Analysis

Sample Preparation and
Statistical Concerns
Introduction
A chemical analysis is meaningless unless you begin with a meaningful sample.
To measure cholesterol in a dinosaur skeleton or herbicide in a truckload of
oranges, you must have a strategy for selecting a representative sample from a
heterogeneous material.
Figure 27-1 shows that the concentration of nitrate
in sediment beneath a lake drops by two orders of
magnitude in the first 3 mm below the surface. If you
want to measure nitrate in sediment, it makes an
enormous difference whether you select a core
sample that is 1 m deep or skim the top 2 mm of
sediment for analysis.

Sampling is the process of collecting a
representative sample for analysis.
1
Real samples
generally require some degree of sample
preparation to remove substances that interfere in
the analysis of the desired analyte and, perhaps, to
convert analyte into a form suitable for analysis.
2

Figure 27-1
Introduction
The terminology of sampling and sample preparation is shown in Figure 27-2. A
lot is the total material (dinosaur skeleton or truckload of oranges) from which
samples are taken. A bulk sample (also called a gross sample) is taken from the
lot for analysis or archiving (storing for future reference).

The bulk sample must be representative of the lot, and the choice of bulk
sample is critical to producing a valid analysis. Box 0-1 gave a strategy for
sampling heterogeneous material.
Sampling is the process of selecting a
representative bulk sample from the lot. Sample
preparation is the process that converts a bulk
sample into a homogeneous laboratory sample.

Sample preparation also refers to steps that
eliminate interfering species or that concentrate the
analyte.
Figure 27-2
Introduction
From the representative bulk sample, a smaller, homogeneous laboratory
sample is formed that must have the same composition as the bulk sample.

For example, we might obtain a laboratory sample by grinding the entire solid
bulk sample to fine powder, mixing thoroughly, and keeping one bottle of
powder for testing.
Besides choosing a sample judiciously, we must be
careful about storing the sample. The composition may
change with time after collection because of chemical
changes, reaction with air, or interaction of the sample
with its container.
Figure 27-2
Small portions (called aliquots) of the laboratory
sample are used for individual analyses. Sample
preparation is the series of steps that convert a
representative bulk sample into a form suitable for
analysis.
Introduction
Glass is a notorious ion exchanger that alters the concentrations of trace ions in
solution.

Therefore, plastic (especially Teflon) collection bottles are frequently employed.

Even these materials can absorb trace levels of analytes.
Figure 27-2
For example, a 0.2-M HgCl2 solution lost 40-95% of
its concentration in 4 h in polyethylene bottles.

A 2-M Ag+ solution in a Teflon bottle lost 2% of its
concentration in a day and 27% in a month.
Introduction
Plastic containers must be washed before use. Table 27-1 shows that
manganese in blood serum samples increased by a factor of seven when
stored in unwashed polyethylene containers prior to analysis.
In the most demanding trace analysis of
lead at 1 pg/g in polar ice cores,
polyethylene containers contributed a
measurable flux of 1 fg of lead per cm2
per day even after they had been
soaked in acid for seven months.

Steel needles are an avoidable source
of metal contamination in biochemical
analysis.
TABLE 27-1 Manganese concentration of
serum stored in washed and unwashed
polyethylene containers.
Introduction
A study of mercury in Lake Michigan found levels near 1.6 pM (1.6 x 10
12
M),
which is two orders of magnitude below concentrations observed in earlier
studies.

Previous investigators apparently unknowingly contaminated their samples. A
study of handling techniques for the analysis of lead in rivers investigated
variations in sample collection, sample containers, protection during
transportation, filtration, preservatives, and preconcentration procedures.

Each step that deviated from best practice doubled the apparent concentration
of lead in stream water. Clean rooms with filtered air supplies are essential in
trace analysis. Even with the best precautions, the precision of trace analysis
becomes poorer as the concentration of analyte decreases (Box 5-2).

Unless the complete history of any sample is known with certainty, the
analyst is well advised not to spend his [or her] time in analyzing it.

Your laboratory notebook should describe how a sample was collected and
stored and exactly how it was handled, as well as stating how it was analyzed.
27-1 Statistics of Sampling
For random errors, the overall variance, s
2
0
, is the sum of the variance of
the analytical procedure, s
2
a
, and the variance of the sampling operation,
s
2
s
:
To understand the nature of the uncertainty in selecting a sample for analysis, consider a
random mixture of two kinds of solid particles. The theory of probability allows us to
state the possibility that a randomly drawn sample has the same composition as the bulk
sample. It may surprise you to learn how large a sample is required for accurate
sampling.

Suppose that the mixture contains nA particles of type A and nB particles of type B. The
probabilities of drawing A or B from the mixture are
Origin of Sampling Variance
If n particles are drawn at
random, the expected number of
particles of type A is np and the
standard deviation of many
drawings is known from the
binomial distribution to be
How much sample corresponds to 10
4
particles? Suppose that the particles are 1-mm-
diameter spheres.

The volume of a 1-mm-diameter sphere is (4/3)p(0.5 mm)
3
= 0.524 L. The density of
KCl is 1.984 g/mL and that of KNO
3
is 2.109 g/mL, so the average density of the
mixture is (0.01)(1.984) + (0.99)(2.109) = 2.108 g/mL.

The mass of mixture containing 10
4
particles is (10
4
)(0.524 10
3
mL)(2.108 g/mL) =
11.0 g.

If you take 11.0-g test portions from a larger laboratory sample, the expected sampling
standard deviation for chloride is 9.9%.

The sampling standard deviation for nitrate will be only 0.1%.
How can you prepare a mixture of 1-mm-diameter particles? You might make such a
mixture by grinding larger particles and passing them through a 16 mesh sieve, whose
screen openings are squares with sides of length 1.18 mm (Table 27-2).

Particles that pass through the screen are then passed through a 20 mesh sieve, whose
openings are 0.85 mm, and material that does not pass is retained for your sample. This
procedure gives particles whose diameters are in the range 0.851.18 mm. We refer to
the size range as 16/20 mesh.

Suppose that much finer particles of 80/120 mesh size (average diameter = 152 m,
average volume = 1.84 nL) were used instead. Now the mass containing 10
4
particles is
reduced from 11.0 to 0.038 8 g. We could analyze a larger sample to reduce the
sampling uncertainty for chloride.
27-2
Sampling uncertainty arises from the random nature of drawing particles from a
mixture. If the mixture is a liquid and the particles are molecules, there are about 10
22

particles/mL.

It will not require much volume of homogeneous liquid solution to reduce the sampling
error to a negligible value. Solids, however, must be ground to very fine dimensions,
and large quantities must be used to ensure a small sampling variance.

Grinding invariably contaminates the sample with material from the grinding apparatus.

Table 27-3 illustrates another problem with heterogeneous materials. Nickel ore was
crushed into small particles that were sieved and analyzed. Parts of the ore that are
deficient in nickel are relatively resistant to fracture, so the larger particles do not have
the same chemical composition as the smaller particles. It is necessary to grind the
entire ore to a fine power to have any hope of obtaining a representative sample.
Table 27-3 illustrates another
problem with heterogeneous
materials. Nickel ore was crushed
into small particles that were
sieved and analyzed. Parts of the
ore that are deficient in nickel are
relatively resistant to fracture, so
the larger particles do not have the
same chemical composition as the
smaller particles. It is necessary to
grind the entire ore to a fine power
to have any hope of obtaining a
representative sample.
A well-mixed powder containing KCl and KNO
3
is an example of a heterogeneous
material in which the variation from place to place is random.

How much of a random mixture should be analyzed to reduce the sampling variance for
one analysis to a desired level?
Choosing a Sample Size
To answer this question, consider
Figure 27-3, which shows results for
sampling the radioisotope
24
Na in
human liver. The tissue was
homogenized in a blender but was
not truly homogeneous, because it
was a suspension of small particles in
water.
The average number of
radioactive counts per second
per gram of sample was about
237. When the mass of
sample for each analysis was
about 0.09 g, the standard
deviation (shown by the error
bar at the left in the diagram)
was 31 counts per second
per gram of homogenate,
which is 13.1% of the
mean value (237).
When the sample size was increased to about 1.3 g, the standard deviation decreased to
13 counts/s/g, or 5.5% of the mean. For a sample size near 5.8 g, the standard
deviation was reduced to 5.7 counts/s/g, or. 2.4% of the mean.
Equation 27-4 told us that when n particles are drawn from a mixture of two kinds of
particles (such as liver tissue particles and droplets of water), the sampling standard
deviation will be
n
= , where p and q are the fraction of each kind of particle
present. The relative standard deviation is
n
/n = = .

The relative variance, (
n
/n)
2
, is therefore
Noting that the mass of sample drawn, m, is proportional to the number of particles
drawn, we can rewrite Equation 27-5 in the form
in which R is the relative standard deviation (expressed as a percentage) due to
sampling and K
s
is called the sampling constant. K
s
is the mass of sample producing a
relative sampling standard deviation of 1%.
(27-5)
(27-4)
Let's see if Equation 27-6 describes Figure 27-3. Table 27-4 shows that mR
2
is approximately
constant for large samples, but agreement is poor for the smallest sample. Attributing the poor
agreement at low mass to random sampling variation, we assign Ks 36 g in Equation 27-6.
This is the average from the 1.3- and 5.8-g samples in Table 27-4.
(27-5)
(27-6)
(27-6)
We just saw that a single 0.7-g sample is expected to give a sampling standard deviation
of 7%. How many 0.7-g samples must be analyzed to give 95% confidence that the
mean is known to within 4%?

The meaning of 95% confidence is that there is only a 5% chance that the true mean lies
more than 4% away from the measured mean.

The question we just asked refers only to sampling uncertainty and assumes that
analytical uncertainty is much smaller than sampling uncertainty.

Rearranging Student's t Equation 4-6 allows us to answer the question:
Choosing the Number of Replicate Analyses
Rearranging Student's t Equation 4-6 allows us to answer the question:
Choosing the Number of Replicate Analyses
in which is the true population mean, is the measured mean, n is the number of
samples needed, is the variance of the sampling operation, and e is the sought-for
uncertainty.

Both the quantities s
s
and e must be expressed as absolute uncertainties or both must be
expressed as relative uncertainties.

Student's t is taken from Table 4-2 for 95% confidence at n-1 degrees of freedom.
Because n is not yet known, the value of t for n = can be used to estimate n. After a
value of n is calculated, the process is repeated a few times until a constant value of n is
found.
27-2 Dissolving Samples for Analysis
Once a bulk sample is selected, a laboratory sample must be
prepared for analysis (Figure 27-2). A coarse solid sample should
be ground and mixed so that the laboratory sample has the same
composition as the bulk sample. Solids are typically dried at
110C at atmospheric pressure to remove adsorbed water prior
to analysis. Temperature-sensitive samples may simply be stored
in an environment that brings them to a constant, reproducible
moisture level.
The laboratory sample is usually dissolved for analysis. It is important to dissolve the
entire sample, or else we cannot be sure that all of the analyte was dissolved.

If the sample does not dissolve under mild conditions, acid digestion or fusion may be
used. Organic material may be destroyed by combustion (also called dry ashing) or wet
ashing (oxidation with liquid reagents) to place inorganic elements in suitable form for
analysis.
Solids can be ground in a mortar and pestle like those shown in Figure 27-5. The steel
mortar (also called a percussion mortar or diamond mortar) is a hardened steel tool
into which the sleeve and pestle fit snugly. Materials such as ores and minerals can be
crushed by striking the pestle lightly with a hammer.

The agate mortar (or similar ones made of porcelain, mullite, or alumina) is designed
for grinding small particles into a fine powder. Less expensive mortars tend to be more
porous and more easily scratched, which leads to contamination of the sample with
mortar material or portions of previously ground samples.

A ceramic mortar can be cleaned by wiping with a wet tissue and washing with distilled
water. Difficult residues can be removed by grinding with 4 M HCl in the mortar or by
grinding an abrasive cleaner (such as Ajax), followed by washing with HCl and water.

A boron carbide mortar and pestle is five times harder than agate and less prone to
contaminate the sample.
Grinding
Grinding
Figure 27-5 Steel, agate, and boron carbide mortars and pestles. The mortar is the base
and the pestle is the grinding tool. In regard to boron carbide, the mortar is a
hemispheric shell enclosed in a plastic or aluminum body. The pestle has a boron
carbide button at the tip of a plastic handle.
A ball mill is a grinding device in which steel or ceramic balls are rotated inside a drum
to crush the sample to a fine powder. Figure 27-6 shows a Wig-L-Bug, which
pulverizes a sample by shaking it in a vial with a ball that moves back and forth.

For soft materials, plastic vials and balls are appropriate. For harder materials, steel,
agate, and tungsten carbide are used.
Grinding
Figure 27-6 Wig-L-Bug sample shaker and polystyrene vial and ball for pulverizing
soft materials. The arrow shows the direction of shaking.
A Shatterbox laboratory mill spins a puck and ring inside a grinding container at 825
revolutions per minute to pulverize up to 100 g of material (Figure 27-7). Tungsten
carbide and zirconia containers are used for very hard samples.
Grinding
Figure 27-7 Shatterbox laboratory mill spins a puck and ring inside a container at high
speed to grind up to 100 mL of sample to a fine powder.
Spex Shatterbox w/
tungsten carbide
Table 27-5 lists acids commonly used for dissolving inorganic materials. The
nonoxidizing acids HCl, HBr, HF, H
3
PO4, dilute H
2
SO
4
, and dilute HClO
4
dissolve
metals by the redox reaction:
Dissolving Inorganic Materials with Acids
Metals with negative reduction potentials should dissolve, although some, such as Al,
form a protective oxide coat that inhibits dissolution.

Volatile species formed by protonation of anions such as carbonate ( CO
3
2-
H
2
CO
3

CO
2
), sulfide (S
2
H
2
S), phosphide (P
3
PH
3
), fluoride (F
HF), and borate

(BO
3
3-
H
3
BO
3
) will be lost from hot acids in open vessels.

Volatile metal halides such as SnCl
4
and HgCl
2
and some molecular oxides such as
OsO
4
and RuO
4
also can be lost.

Hot hydrofluoric acid is especially useful for dissolving silicates. Glass or platinum
vessels can be used for HCl, HBr, H
2
SO
4
, H
3
PO
4
, and HClO
4
.

HF should be used in Teflon, polyethylene, silver, or platinum vessels. The highest-
quality acids must be used to minimize contamination by the concentrated reagent.
Substances that do not dissolve in nonoxidizing acids may dissolve in the oxidizing
acids HNO
3
, hot, concentrated H
2
SO
4
, or hot, concentrated HClO
4
.

Nitric acid attacks most metals, but not Au and Pt, which dissolve in the 3:1 (vol/vol)
mixture of HCl:HNO
3
called aqua regia.

Strong oxidants such as Cl
2
or HClO
4
in HCl dissolve difficult materials such as Ir at
elevated temperature. A mixture of HNO
3
and HF attacks the refractory carbides,
nitrides, and borides of Ti, Zr, Ta, and W.

A powerful oxidizing solution known as piranha solution is a 1:1 (vol/vol) mixture of
30 wt% H
2
O
2
plus 98 wt% H
2
SO
4
.

Hot, concentrated HClO
4
(described later for organic substances) is a dangerous,
powerful oxidant whose oxidizing power is increased by adding concentrated H
2
SO
4

and catalysts such as V
2
O
5
or CrO
3
.
HF causes excruciating burns.
Exposure of just 2% of your body to concentrated (48 wt%)
HF can kill you.
Flood the affected area with water for 5 min and then coat the
skin with 2.5% calcium gluconate gel kept in the lab for this
purpose, and seek medical help.
If the gel is not available, use whatever calcium salt is handy.
HF damage can continue to develop days after exposure.
Digestion is conveniently carried out in a Teflon-lined bomb (a sealed vessel) heated in
a microwave oven. The vessel in Figure 27-8 has a volume of 23 mL and digests up to
1 g of inorganic material in up to 15 mL of concentrated acid or digests 0.1 g of organic
material, which releases a great deal of CO
2
(g).

Figure 27-8 Microwave
digestion bomb lined with
Teflon. The outer container
retains strength to 150C
but rarely reaches 50C.
Microwave energy heats the contents to 200C in a minute. To prevent explosions, the
lid releases gas from the vessel if the internal pressure exceeds 8 MPa (80 bar).

The bomb cannot be made of metal, which absorbs microwaves. An advantage of a
bomb is that it is cooled prior to opening, thus preventing loss of volatile products.
Figure 27-8 Microwave
digestion bomb lined with
Teflon. The outer container
retains strength to 150C
but rarely reaches 50C.
Substances that will not dissolve in acid can usually be dissolved by a hot, molten
inorganic flux (Table 27-6). Finely powdered unknown is mixed with 2 to 20 times its
mass of solid flux, and fusion (melting) is carried out in a platinumgold alloy crucible
at 300 to 1 200C in a furnace or over a burner.
Dissolving Inorganic Materials by Fusion
The apparatus in Figure 27-9 fuses three samples at once over propane burners with
mechanical agitation of the crucibles.

When the samples are homogeneous, the molten flux is poured into beakers containing
10 wt% aqueous HNO
3
to dissolve the product.
Figure 27-9 Automated apparatus
that fuses three samples at once over
propane burners. It also provides
mechanical agitation of the Pt/Au
crucibles.
Crucibles are shown in the tipped
position used to pour contents out
after fusion is complete.
Most fusions use lithium tetraborate (Li
2
B4O
7
, m.p. 930
o
C), lithium metaborate
(LiBO
2
, m.p. 845C), or a mixture of the two.

A nonwetting agent such as KI can be added to prevent the flux from sticking to the
crucible. For example, 0.2 g of cement might be fused with 2 g of Li
2
B
4
O
7
and 30 mg
of KI.

A disadvantage of a flux is that impurities are introduced by the large mass of solid
reagent. If part of the unknown can be dissolved with acid prior to fusion, it should be
dissolved. Then the insoluble component is dissolved with flux and the two portions are
combined for analysis.

Basic fluxes in Table 27-6 (LiBO
2
, Na
2
CO
3
, NaOH, KOH, and Na
2
O
2
) are best used to
dissolve acidic oxides of Si and P. Acidic fluxes (Li
2
B
4
O
7
, Na
2
B
4
O
7
, K
2
S
2
O
7
, and B
2
O
3
)
are most suitable for basic oxides (including cements and ores) of the alkali metals,
alkaline earths, lanthanides, and Al.
TABLE 27-6
Fluxes for sample dissolution
TABLE 27-6
TABLE 27-6
Digestion of organic material is classified as either dry ashing, when the procedure
does not include liquid, or wet ashing, when liquid is used.

Occasionally, fusion with Na
2
O
2
(called Parr oxidation) or alkali metals may be carried
out in a sealed bomb. Section 27-4 discussed combustion analysis, in which C, H, N, S,
and halogens are measured.

Convenient wet-ashing procedures include microwave digestion with acid in a Teflon
bomb (Figure 27-8). For example, 0.25 g of animal tissue can be digested for metal
analysis by placing the sample in a 60-mL Teflon vessel containing 1.5 mL of high-
purity 70% HNO
3
plus 1.5 mL of high-purity 96% H
2
SO
4
and heating it in a 700-W
kitchen microwave oven for 1 min.

Teflon bombs fitted with temperature and pressure sensors allow safe, programmable
control of digestion conditions. An important wet-ashing process is Kjeldahl digestion
with H
2
SO
4
for nitrogen analysis.
Decomposition of Organic Substances
In the Carius method, digestion is performed with fuming HNO
3
(which contains
excess dissolved NO
2
) in a sealed, heavy-walled glass tube at 200300C.

For safety, the glass Carius tube should be contained in a steel vessel pressurized to
approximately the same pressure expected inside the glass tube.

For trace analysis, sample should be placed inside a fused silica tube inside the glass
tube. Silica provides as little as 110% as much extractable metal as glass.
Figure 27-10 shows microwave wet-ashing apparatus. Sulfuric acid or a mixture of
H
2
SO
4
and HNO
3
(15 mL of acid per gram of unknown) is added to an organic
substance in a glass digestion tube fitted with the reflux cap.
Figure 27- 10
Microwave
apparatus for
digesting
organic
materials by wet
ashing.
In the first step, the sample is carbonized for
10 to 20 min at gentle reflux until all particles
have dissolved and the solution has a uniform
black appearance.

Power is turned off and the sample is allowed
to cool for l2 min. Next, oxidation is carried
out by adding H
2
O
2
or HNO
3
through the
reflux cap until the color disappears or the
solution is just barely tinted.
If the solution is not homogeneous, the power is turned up and the sample is heated to
bring all solids into solution. Repeated cycles of oxidation and solubilization may be
required.

Once conditions for a particular type of material are worked out, the procedure is
automated, with power levels and reagent delivery (by the peristaltic pump)
programmed into the controller.
The high-pressure asher in Figure 27-11
uses a resistive heating element inside a
sealed chamber for digestion at
temperatures up to 270C under a
pressure up to 140 bar.

High pressure allows acids to be heated
to high temperature without boiling.
At high temperature, HNO
3
oxidizes organic matter without assistance from H
2
SO
4
,
which is not as pure as HNO
3
and is therefore less suitable for trace analysis.
Figure 27-11 High-pressure autoclave
allows digestion up to 270C without
H
2
SO
4
in open vessels inside autoclave.
Silica or fluoropolymer vessels inside the
sealed chamber are loosely sealed by
Teflon caps that permit evolved gases to
escape.

The bottom of the vessel is filled with 5
vol% H
2
O
2
in H
2
O. Hydrogen peroxide
reduces nitrogen oxides generated by
digestion of organic matter.
As an example, a 1-g sample of animal tissue could be digested in a 50-mL fused-silica
vessel containing 5 mL of high-purity 70 vol% HNO
3
plus 0.2 mL of high-purity 37
vol% HCl.

Metallic elements in the digestion solution could be measured at part per billion to part
per million levels by inductively coupled plasmaatomic emission.
Add fresh HNO
3
and repeat the evaporation several times. After the sample cools to
room temperature, add HClO
4
and heat again.

If possible, HNO
3
should be present during the HClO
4
treatment. A large excess of
HNO
3
should be present when oxidizing organic materials.
Figure 27-
12 Reflux cap for
wet ashing in an
Erlenmeyer flask.
The hole allows
vapor to escape, and
the spout is curved
to contact the inside
of the flask.
Wet ashing with refluxing HNO
3
-HClO
4

(Figure 27-12) is a widely applicable, but
hazardous, procedure.

Perchloric acid has caused numerous
explosions.

Use a good blast shield in a metal-lined fume
hood designed for HClO
4
. First, heat the sample
slowly to boiling with HNO
3
but without HClO
4
.
Bottles of HClO
4
should not be stored on
wooden shelves, because acid spilled on
wood can form explosive cellulose
perchlorate esters. Perchloric acid also
should not be stored near organic reagents
or reducing agents.
Figure 27-
12 Reflux cap for
wet ashing in an
Erlenmeyer flask.
The hole allows
vapor to escape, and
the spout is curved
to contact the inside
of the flask.
The combination of Fe
2+
and H
2
O
2
, called Fenton's reagent, oxidizes organic material
in dilute aqueous solutions. For example, organic components of urine could be
destroyed in 30 min at 50C to release traces of mercury for analysis.

To do so, a 50-mL sample was adjusted to pH 34 with 0.5 M H
2
SO
4
. Then 50 L of
saturated aqueous ferrous ammonium sulfate, Fe(NH
4
)
2
(SO
4
)
2
, were added, followed by
100 L of 30% H
2
O
2
.
Fenton's reagent generates OH radical and,
possibly, Fe
II
OOH as the active species.
Sample preparation is the series of steps required to transform a sample so that it is
suitable for analysis. Sample preparation may include
dissolving the sample,
extracting analyte from a complex matrix,
concentrating a dilute analyte to a level that can be measured,
chemically converting analyte into a detectable form, and
removing or masking interfering species.
27-3 Sample Preparation Techniques
Liquid Extraction Techniques
Solid-Phase Extraction
Preconcentration
Derivatization
In extraction, analyte is dissolved in a
solvent that does not necessarily dissolve
the entire sample and does not
decompose the analyte.
In a typical microwave-assisted
extraction of pesticides from soil, a
mixture of soil plus acetone and hexane
is placed in a Teflon-lined bomb (Figure
27-8 and Figure 27-13) and heated by
microwaves to 150C. This temperature
is 50 to 100 higher than the boiling
points of solvents at atmospheric
pressure. Pesticides dissolve, but the soil
remains behind. The liquid is then
analyzed by chromatography.
Figure 27-13 Extraction vessels in a microwave oven that processes up to 12 samples in under
30 min. Each 100-mL vessel has a vent tube that releases vapor if the pressure exceeds 14 bar.
Vapors from the chamber are ultimately vented to a fume hood. The temperature inside each
vessel can be monitored and used to control the microwave power.
Acetone absorbs microwaves, so it
can be heated in a microwave oven.
Hexane does not absorb
microwaves.

To perform an extraction with pure
hexane, the liquid is placed in a
fluoropolymer insert inside the
Teflon vessel in Figure 27-8.

The walls of the insert contain
carbon black, which absorbs
microwaves and heats the solvent.
Figure 27-13 Extraction vessels in a microwave oven that processes up to 12 samples in under
30 min. Each 100-mL vessel has a vent tube that releases vapor if the pressure exceeds 14 bar.
Vapors from the chamber are ultimately vented to a fume hood. The temperature inside each
vessel can be monitored and used to control the microwave power.
Supercritical fluid extraction uses a supercritical fluid (Box 25-2) as the extraction
solvent.

CO
2
is the most common supercritical fluid because it is inexpensive and it eliminates
the need for costly disposal of waste organic solvents. Addition of a second solvent
such as methanol increases the solubility of polar analytes.

Nonpolar substances, such as petroleum hydrocarbons, can be extracted with
supercritical argon. The extraction process can be monitored by infrared spectroscopy
because Ar has no infrared absorption.
Supercritical fluid extraction
Figure 27-14a shows how a supercritical
fluid extraction can be carried out.
Pressurized fluid is pumped through a heated
extraction vessel.

Fluid can be left in contact with the sample
for some time or it can be pumped through
continuously.

At the outlet of the extraction vessel, the
fluid flows through a capillary tube to release
pressure.

Exiting CO
2
evaporates, leaving extracted
analyte in the collection vessel.

Alternatively, the CO
2
can be bubbled
through a solvent in the collection vessel to
leave a solution of analyte.
Figure 27-14 (a) Apparatus
for supercritical fluid
extraction.
Figure 27-14b shows the extraction of
organic compounds from dust collected
with a vacuum cleaner from door mats
at the chemistry building of Ohio State
University.
Figure 27-14 b) Vessel for extracting
house dust at 50C with 20 mol%
methanol/80 mol% CO
2
at 24.0 MPa
(240 bar).
The chromatogram of the
extract in Figure 27-14c
exhibits myriad organic
compounds that you and I
inhale in every breath.
Figure 27-14 (c) Gas chromatogram of CH
2
Cl
2
solution of extract using
a 30 m 0.25 mm diphenyl0.05dimethyl0.95siloxane column (1 m
film thickness) with a temperature ranging from 40 to 270C and
flame ionization detection.
Figure 27-15 shows glassware for
continuous liquid-liquid extraction of
a nonvolatile analyte.

In Figure 27-15a, the extracting
solvent is denser than the liquid being
extracted. Solvent boils from the
flask and condenses into the
extraction vessel.

Dense droplets of solvent falling
through the liquid column extract the
analyte.
Figure 27-15 Continuous liquid-liquid
extraction apparatus used when extraction
solvent is (a) denser than the liquid being
extracted or (b) lighter than the liquid
being extracted.
When the liquid level is high enough,
extraction solvent is pushed through
the return tube to the solvent
reservoir.

By this means, analyte is slowly
transferred from the light liquid at the
left into the dense liquid in the
reservoir.

Figure 27-15b shows the procedure
when the extraction solvent is less
dense than the liquid being extracted.
Figure 27-15 Continuous liquid-liquid
extraction apparatus used when extraction
solvent is (a) denser than the liquid being
extracted or (b) lighter than the liquid
being extracted.
Solid-phase extraction uses a small
volume of a chromatographic
stationary phase or molecularly
imprinted polymer (Box 26-2) to
isolate desired analytes from a
sample.

The extraction removes much of the
sample matrix to simplify the
analysis.
Figure 27-16 Steps in solid-phase extraction.
Figure 27-16 shows steps in the solid-
phase extraction of 10 ng/mL of
steroids from urine.
First, a syringe containing 1 mL of
C18-silica is conditioned with 2 mL of
methanol to remove adsorbed organic
material (Figure 27-16a).

Then the column is washed with 2 mL
of water. When the 10-mL urine
sample is applied, nonpolar
components adhere to the C18-silica,
and polar components pass through
(Figure 27-16b).
Figure 27-16 Steps in solid-phase extraction.
The column is then rinsed with 4 mL of 25 mM borate buffer at pH 8 to remove polar
substances (Figure 27-16c). Then rinses with 4 mL of 40 vol% methanol/60% water
and 4 mL of 20% acetone/80% water remove less polar substances (Figure 27-16d).
Finally, elution with two 0.5-mL aliquots of 73% methanol/27% water washes the
steroids from the column (Figure 27-16e).
Figure 27-17 compares chromatograms of the
drug naproxen in blood serum with or without
sample cleanup by solid-phase extraction.

Without cleanup, serum proteins overlap and
obscure the signal from naproxen. Solid-phase
extraction removes most of the protein.

Solid-phase extractions can reduce solvent
consumption in analytical chemistry.

For example, a standard procedure approved by
the U.S. Environmental Protection Agency for
the analysis of pesticides in wastewater requires
200 mL of dichloromethane for the liquid-liquid
extraction of 1 L of water.
Figure 27-17 HPLC of naproxen in
blood serum with no cleanup (upper
trace) or with prior sample cleanup
(lower trace) by solid-phase extraction on
C8-silica.
The same analytes can be isolated by solid-
phase extraction on C18-silica disks.

The pesticides are recovered from the disks by
supercritical fluid extraction with CO
2
that is
finally vented into a small volume of hexane.
This one kind of analysis can save 105 kg of
CH
2
Cl
2
per year.
Figure 27-17 HPLC of naproxen in
blood serum with no cleanup (upper
trace) or with prior sample cleanup
(lower trace) by solid-phase extraction
on C8-silica.
The solid-phase extraction
cartridge used to preconcentrate
cocaine from river water is the
mixed-mode cation-exchange
reversed-phase sorbent at the
upper left in Figure 27-18.

This is one of a family of resins
whose backbone contains
lipophilic benzene rings and
hydrophilic pyrrolidone rings.
The resins are wettable by water
and have affinity for both polar
and nonpolar substances.
Cocaine purification and extraction
FIGURE 27-18 Water-wettable,
hydrophobic ion-exchange Oasis
polymer sorbents for solid-phase
extraction. [From Waters Corporation,
Milford, MA.]
FIGURE 27-18 Water-wettable,
hydrophobic ion-exchange Oasis
polymer sorbents for solid-phase
extraction. [From Waters Corporation,
Milford, MA.]
The four ion-exchange
derivatives are useful
for retaining and then
releasing different
types of analytes when
conditions such as pH,
solvent, and ionic
strength are changed.
The conventional plate at the upper
right has eight rows of 12 syringe-
like wells, each of which can
contain 5 to 60 mg of resin.

The Elution plate at the upper
left has 96 Pasteur-pipet-like wells
with a small volume, which can be
eluted by 25-50 L of solvent.
FIGURE 27-19 96-Well Plate and 96-well mElution
Plate for solid-phase extraction. [Courtesy Waters
Corporation, Milford, MA.]
Preconcentration of cocaine from 500 mL of river water was done with just 60 mg of
resin in a single syringe. For multiple samples or exploratory research, a 96-well plate
such as those shown in Figure 27-19 can be used.
Solid-phase extraction was used to preconcentrate and partially purify traces of cocaine
and benzoylecgonine at the opening of this chapter.

A 500-mL volume of river water was filtered, spiked with 10 ng of internal standard,
and acidified to pH 2.0 with HCl.

A solid-phase cation-exchange extraction cartridge containing 60 mg of resin was
conditioned before use by washing with 6 mL of CH3OH, 3 mL of deionized H2O, and
3 mL of H2O acidified to pH 2.0 with HCl.

River water was sucked through the cartridge at 20 mL/min. Liquid was removed from
the cartridge by suction for 5 min.

Analytes were then eluted from the cartridge with 2mL of CH3OH followed by 2 mL of
2% ammonia solution in CH3OH.

This process preconcentrates the sample by a factor of 500 mL/4 mL = 125.
Solid-phase extractions reduce solvent consumption in analytical chemistry.

One standard procedure for the analysis of pesticides in wastewater requires 200 mL of
dichloromethane for the liquid-liquid extraction of 1 L of water.

The same analytes can be isolated by solid-phase extraction on C18-silica disks.

The pesticides are recovered from the disks by supercritical fluid extraction with CO2
that is finally vented into a small volume of hexane.

This one kind of analysis can save 10
5
kg of CH2Cl2 per year.
Ion-exchange resins can capture basic or acidic gases.
Carbonate liberated as CO
2
from (ZrO)
2
CO
3
(OH)
2
xH
2
O
used in nuclear fuel reprocessing can be measured by
placing a known amount of powdered solid in the test tube
in Figure 27-19 and adding 3 M HNO
3
.

When the solution is purged with N
2
, CO
2
is captured
quantitatively by moist anion-exchange resin in the side
arm:
Preconcentration
Figure 27-19 Apparatus for trapping basic or acidic gases
by ion exchange.
Carbonate is eluted from the resin with 1 M NaNO
3
and
measured by titration with acid. Table 27-7 gives other
applications of this technique.
Preconcentration
TABLE 27-7 Use of ion-exchange resin for trapping gases .
Carbonate is eluted from the resin with 1 M NaNO
3
and measured by titration with
acid. Table 27-7 gives other applications of this technique.
Trace analysis often requires preconcentration of analyte to bring it to a higher
concentration prior to analysis. Metal ions in natural waters can be preconcentrated with
cation-exchange resin.

For example, a 500-mL volume of seawater adjusted to pH 6.5 with ammonium acetate
and ammonia was passed through 2 g of Chelex-100 in the Mg
2+
form to trap all the
trace-metal ions.

Washing with 2 M HNO
3
eluted the metals in a total volume of 10 mL, thereby giving a
concentration increase of 500/10 = 50. Metals in the HNO
3
solution were then analyzed
by graphite furnace atomic absorption, with a typical detection limit for Pb being 15
pg/mL.

The detection limit for Pb in the seawater is therefore 50 times lower, or 0.3 pg/mL.
Preconcentration
Figure 27-18 shows the effect of pH on the recovery of metals from seawater. At low
pH, H
+
competes with the metal ions for ion-exchange sites and prevents complete
recovery.
Preconcentration
Figure 27-18 The pH dependence of the recovery of trace metals from seawater by Chelex-100.
The graph shows the pH of the seawater when it was passed through the column.
Derivatization is a procedure in which analyte is chemically modified to make it easier
to detect or separate. For example, formaldehyde and other aldehydes and ketones in
air, breath, or cigarette smoke can be trapped and derivatized by passing air through a
tiny cartridge containing 0.35 g of silica coated with 0.3 wt% 2,4-
dinitrophenylhydrazine.
Derivatization
Carbonyls are converted into the 2,4-
dinitrophenylhydrazone derivative, which is
eluted with 5 mL of acetonitrile and analyzed by
HPLC.

The products are readily detected by their strong
ultraviolet absorbance near 360 nm.

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Chapter 27

HF), and borate

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