The Analysis of Real Samples
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
1)
2)
3)
4)
5)
6)
7)
8)
Steps of quantitative analysis
Selecting a method
Sampling
Preparing a laboratory sample
Defining replicate samples by mass or volume
measurements
Preparing solutions of the samples
Eliminating interferences
Performing measurements for analyte
concentration
Computing the results and estimating their
reliability
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 Choice of analytical method
1) Definition of the Problem
•
What is the concentration range of the analyte to be
determined?
What degree of accuracy is desired?
What other components are present in the sample?
What are the physical and chemical properties of the
gross sample?
How many samples will be analyzed?
•
•
Chemical abstract/SciFinder
Web of Science
•
•
•
The Analysis of Standard Samples
Using Other Methods
Standard Addition to the Sample
•
•
•
•
2) Investigating the Literature
3) Testing the Procedure
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 Contents in sampling plan
– Physically removing the sample from its target
population
– Preserving and handling the sample
– Storing the sample
– Preparing the sample for analysis
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 For Liquid Samples
•
•
•
Examples: beverages, urine, natural waters
Sampling tools: pipet, syringe, water sampler etc.
Preservation:
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 For Gas Samples
• Sampling tools: stainless steel canister,
Tedlar/Teflon bag, solid sorbent trap, filtering,
cryogenic trap etc.
• For analyte removing:
– Thermal desorption
– Extracting with solvent
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 For Solid Samples
• Sampling tools: Grab sampler(抓取式採樣器)、
Corer(鑽取式採樣器)、Scoops(杓 )/shovel(鏟)、
Thief (套管式採樣刀)
• Sample preservation:
– Low temperatures
– Zero headspace
– Adding Inert gas
– etc.
• Sample preparation:
– Reducing the particle size (to reduce sampling
variance)
– Reducing the sample size (quantity)
– Bringing solid samples into solution
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 Bringing Solid Samples into Solution
– Swirling and heating/Conventional
digestion/Wet digestion
– Flux/Dry ashing
– Microwave digestion
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 Classifying Separation Techniques
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Separations Based on Size
1. Filtration
• Gravity/Suction/Pressure
• Porous filter
• Dissolved phase/particulate phase
• Gravimetric analysis
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2. Dialysis
A method of separation that uses a semipermeable membrane.
* Frequently used to purify proteins, hormones, and enzymes.
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3. Size-exclusion chromatography:
• Also called gel permeation or molecular exclusion
chromatography
• A separation method in which a mixture passes through a bed
of porous particles, with smaller particles taking longer to pass
through the bed due to their ability to move into the porous
structure.
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Separations Based on Mass or Density
• If there is a difference in the mass or density of the analyte and
interferent, then a separation can use centrifugation.
• The sample, as a suspension, is placed in a centrifuge tube and
spun at a high angular velocity (high numbers of revolutions
per minute, rpm).
• Particles of equal density, heavier particles having greater
sedimentation rates.
• Particles are of equal mass, the highest density have the
greatest sedimentation rate.
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Example 1: Separated lysosomes from other components
1. Destroying the cell membranes.
2. Centrifuge at 15,000xg (15,000 times the Earth’s gravity) 20
min.
3. Isolate supernatant from the by decanting
4. Centrifuge the supernatant at 30,000xg for 30 min.
5. Leaving a residue of lysosomes.
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Example 2: Equilibrium–density–gradient centrifugation
1. Establish the density gradients, e.g., solutions
of CsCl. (density gradient 1.65 g/cm3 ~ 1.80
g/cm3)
2. Place the the sample, e.g., mixture of proteins,
RNA, and DNA, into the centrifuge tube.
3. After centrifugation.
3
Proteins,
with
a
density
of
less
than
1.3
g/cm
Protein
experience no sedimentation.
3 separates
DNA,
a
density
of
approximately
1.7
g/cm
DNA
as a band near the middle of the centrifuge tube.
3
RNA RNA, with a density of greater than 1.8 g/cm
collects as a residue at the bottom of the centrifuge
tube.
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Separations Based on Complexation
Reactions (Masking)
Masking:
A pseudo-separation method in which a species is prevented from
participating in a chemical reaction by binding it with a
masking agent to an unreactive complex.
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Cyanide is an appropriate
masking agent for Ni2+
because the formation
constant for Ni(CN)42– is
greater than that for the Ni–
EDTA complex.
Ni(CN)42– is relatively inert in the presence of EDTA.
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Separations Based on a Change of State
Changes in Physical State:
1. Fractional distillation
Boiling points versus
composition diagram
for a near-ideal solution.
When the analyte and interferent are miscible liquids, for example,
a low-boiling point analyte and a high-boiling point interferent.
*The lower boiling point, the higher equilibrium vapor pressure.
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Equipment for a
fractional distillation.
Temp.
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Changes in Physical State:
2. Recrystallization (fractional crystallization)
The solid sample is dissolved solvent, then cool down to promote
the growth of large, pure crystal.
The purified sample is isolated by filtration.
Sample is dried to remove any remaining traces of the solvent.
Additional recrystallizations if necessary.
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Changes in Chemical State:
1. Evaporation
SiO2
SiO2
4HF
SiF4
+ 2H2O
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Other types of
Changes in Chemical State:
2. Selective precipitation
3. Electrodeposition
4. Ion exchange
5. pH dependent precipitation
6. Complexation/extraction
7. pH dependent complexation/extraction
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Separations Based on a Partitioning
Between Phases
For a selective partitioning of the analyte or interferent between
two immiscible phases. a phase containing a solute is brought
into contact with a second phase, the solute partitions itself
between the two phases:
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1. Liquid–Liquid Extractions:
The density of the two immiscible liquids
determines which phase is the upper phase.
For aqueous-organic extractions:
Density Lower
than H2O
Density Higher
than H2O
Diethyl ether
Hexane
Toluene
Chloroform
Dichloromethane
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2. Solid-Phase Extractions:
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3. Solid-phase microextration (SPME):
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4. Continuous Extractions:
(Soxhlet extractor for example)
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5. Microwave extractions:
The sample is placed in a sealed
digestion vessel along with the
liquid extraction phase, and a
microwave oven is used to heat
the extraction mixture. The
extraction to take place at a higher
temperature and pressure, thereby
reducing the amount of time
needed for a quantitative
extraction.
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6. Purge and trap:
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7. Supercritical fluids extractions (SFE):
Supercritical fluid is a state of matter where a substance is
held at a temperature and pressure that exceeds its critical
temperature and pressure
For example, CO2 at 340 atm and 80oC has the properties
between those of a gas and a liquid.
Density of supercritical fluid is high than that of gas, allowing
good extraction ability from sample.
Viscosity of a supercritical fluid is significantly less than that
of a liquid solvent, allowing it to pass more readily through
particulate samples.
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End of Chapter 35-38
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