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INTRODUCTION TO

ANALYTICAL CHEMISTRY
(PART 2)
THE
ANALYTICAL PROCESS
THE STEPS IN
A CHEMICAL ANALYSIS
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Steps of the Analytical Process
6 basic main steps in chemical analysis
1. Problem definition - Identify the problem and
formulate questions
2. Method selection - Select method of analysis
3. Sampling - Obtain bulk sample and extract
smaller laboratory sample from bulk
4. Sample Preparation - Prepare sample for
analysis
5. Analysis Analytical measurement of the
analyte in question
6. Calculation and reporting - Calculate results,
interpret/present data and draw conclusion
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(1) Problem Definition
Need to translate general questions into
specific questions - to be answered by
chemical measurement
What information is needed?
What type of sample to be analysed?
How sensitive must the method be?
What degree of accuracy and
precision required?
How are interferences eliminated?
The answers will help determine the specific
techniques to be adopted
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Selection of method depends on a
number of factors:
Sample type, sample size & preparation
required
Skill and training of analyst
Tools/instruments available
Selectivity, precision, sensitivity required
Cost (budget) and speed
Time required/target deadlines
Availability of methods or standard
methods (chemical literature: Books,
journals, manuals, etc)
(2) Method Selection
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Standard methods of analysis are available in
published materials such as the following:
Journals
Analytical Chimica Acta Analytical Abstracts
Analytical Chemistry Analyst
Analytical Communications Talanta
J. of Association of Official Analytical Chemists (AOAC)
Journal of Chromatography
Journal of Chromatographic Science
Trends in Analytical Chemistry
Books
ASTM Book of Standards
Official Methods of Analysis of the AOAC
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Sampling is the process to obtain a small
representative and homogeneous sample
- Representative - content of analytical sample
reflects content of bulk sample
- Homogeneous content is the same throughout
the whole sample
Sampling is the most critical step because it can
limit the accuracy of measurements
Requires storage and preservation steps
Sampling methods depends on the samples
- type, size & homogeneity
- physical state (solid, liquid, gas)
- chemical state
(3) Sampling
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Sampling Steps
(1) Identify the population to be studied
(2) Collect a gross representative sample from
the population
(3) Reduce the gross sample to a laboratory
sample suitable for analysis
Common sampling methods:
Grab Sample A portion of sample removed
from the population
Composite Sample - Several grab samples
combined to form a single sample
In-situ Sampling - Sampling done within the
population (on-site/without removal)
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Standard methods for sampling are available in
reference materials such as:
ASTM (American Society for Testing and
Material)
APHA (American Public Health Association)
AOAC (Association of Official Analytical Chemists
International)
General guideline:
- Homogeneous parent samples Simple grab
sample approach taken at random and assumed
representative
- Heterogeneous parent samples - Several
samples have to be taken
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SAMPLING SOLIDS
Problem: Solid materials are
heterogenous making sampling difficult
The larger the particle size, the larger
the gross sample should be
Best to take 1/50 to 1/100 of the total
bulk or total population
Special sampling techniques are
required to obtain a representative
gross sample
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Homogenization of Solid Samples
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The smaller the particle size of the sample, the lower
the error in analysis
Crushing
Pulverizing
Grinding
Rendering the sample into a thoroughly
mixed powder
Sample mixtures
Homogeneous samples may become
inhomogeneous upon standing
It should be thoroughly mixed before an
aliquot is taken for analysis
Example sampling techniques to obtain
representative gross sample
Stockpile of cereals: take increment from
surface and work into the interior
Compact solids (metals and alloys): random
drilling or sawing across the metal at random
intervals and collect the `sawdust
Bulky material (ore, grain, coal): obtain a
random sample while the material is in motion
(eg conveyor belt), and periodically transfer
portion into a sample container
Shipment/cargo of bagged material (bags of
grain, cement, etc): obtain a small sample from
each bag or every 10
th
bag, and combine
samples
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This is a common method
used to reduce the gross
sample to a smaller
laboratory sample
The sample is crushed and
mixed to form a conical pile
The pile is flattened and cut
into equal quarters, and two
opposite quarters are
collected at random
The quartering process is
repeated until the desired
sample size is obtained
Cone and Quarter Method
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SAMPLING LIQUIDS
Liquid samples are homogeneous, thus
easier to sample
The gross sample can be relatively small
Sampling techniques will depend on the
types of liquid
Examples of liquid sampling techniques:
Small quantities of non homogeneous liquid
sample is shaken and sampled immediately
Large volume of liquids are sampled (i) after
a transfer or (ii) during discharge or (iii) if in
a pipe, after passing through a pump when
they have undergone thorough mixing
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Large stationary liquids (eg lakes, rivers) are
sampled at different depths using a thief
sampler (a special device for obtaining
aliquots at different level)
The separate aliquots of liquids can be
analyzed individually or can be combined
into one gross sample (composite sample)
For biological fluids, the timing of sampling
is very important (eg, Blood sample is
collected after the patient has fasted for a
number of hours to analyze for sugar)
Continued.
Examples of liquid sampling techniques
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Gases tend to be homogeneous
and a large volume of sample is
required (bcause of low density)
Examples
- Air analysis: Use `Hi-Vol
sampler that contain filters to
collect particulates
- Liquid displacement method:
Sample must be slightly
soluble in the liquid & does not
react with it
- Breath sample: Subject blows
into an evacuated bag
SAMPLING GASES
Devices for gas
sampling
Air Sampling Filters
Air/Hi Vol
Samplers
Dust Sampler
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SAMPLE STORAGE AND PRESERVATION
Gross sample must be transported from the
sampling point to the analytical laboratory
without a physical or chemical change in its
characteristics
Preservation is can be carried out right at the
sampling point or in the laboratory
Sample preservation help minimize:
- physical changes such as adsorption, diffusion,
volatilization
- chemical changes such as oxidation and
microbiological degradation
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Method Description
Keep in sealed
containers or
under vacuum or
nitrogen
Refrigerate or
freeze or protect
from light
- Prevent decomposition of
biological samples from
bacterial action or
sensitive samples from
oxidation
- Protect from light
- Avoid thermal degredation
of thermal labile samples
- Prevent loss of water from
hygroscopic sample
- Prevent loss of volatile
analytes from sample
Methods of Preservation & Storage
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Method Description
Add chemical
stabilizers:
antibacterials or
antioxidants
- Prevent sample
decomposition by
bacteria
- Prevent sample oxidation
Adsorption on a
solid phase
- To immobilize or
stabilize the analyte
Store in
appropriate
containers
- Teflon (PTFE) for ionic
analyte
- Glass for organic analyte
Acidify (Add 10%
HNO
3
upon
collection, pH<2)
- Prevent precipitation of
metals from water
samples
Sample preparation (sample pretreatment) is a step
in chemical analysis where the sample is brought
into the correct size or form for analysis
General principles:
Determine amount (Weight or Volume)
Prepare replicate samples for statistics
- Replicates samples analyzed in the same way, same
size, at the same time
Bring the analyte into the best chemical form for
assay method used Dissolve in solution
Bring analyte into the best concentration range
for the chosen method
Pretreat, separate, pre-concentrate, eliminate
interferences
(4) Sample Preparation
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Maximize RECOVERY During Sample Preparation
Recovery:
A measure of the amount of analyte in the assay
relative to the amount of analyte in the sample
Percentage Recovery or % recovery
= (analyte concentration in assay) x 100
(analyte concentration in sample)
How analytes can be lost from the sample during
sample preparation?
- Absorption (eg. metal ions can absorb on the glass
surface, organic compounds can absorbed on plastic
containers)
- Decomposition (eg. organic compounds can
decompose by oxidation)
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Sample Preparation for Solids
(1) Grind and sieve to a suitable size to obtain
required homogenous size
Homogenize
- Solid sample is placed in a blender (solvent
may be added) and sample is homogenized to
finely divided particles
- Useful for plant and animal tissue, food, and
environmental samples
- Aqueous or organic solvent can be used
- Finely divided particles promote more
efficient extraction
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Sample
Inorganic sample
Common organic
sample
Biological sample
Hygroscopic sample
or oxidizable sample
Heat sensitive
sample
Drying Conditions
- Heat at 110
o
C
- Depends on sample
(removes vapors)
- Heat at <100
o
C
- Dry in vacuum desiccator
or under nitrogen
- Freeze dry
(2) Dry samples to remove moisture @ 105-
110
o
C for 1-2 hr
- Side reactions or decomposition of samples
must be considered
- Follow by storage in a desiccator
(3) Solubilize the sample in solution (aka dissolution)
(i) Simple dissolution - solid is dissolved in solvent
without chemical change
(ii) Wet Digestion (aka acid treatment) - Heat
sample with mineral acids in open/closed
container or in a microwave digester
(iii) Fusion Technique - Heat with flux or acid until
molten state
(iv) Dry Ashing - Oxidize by slow heating in oxygen
at very high temperature (400-700
o
C in furnace)
Destructive - harsh methods that destroys the
sample matrix (for inorganic analyte or analyte
that can be converted to inorganic derivative)
Nondestructive/partially destructive - mild or
non evasive dissolution (for organic analytes)
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Wet digestion/Acid Treatment
Low cost but may lose analyte by volatilization
HCl : Carbonates, phosphates, oxides
H
2
SO
4
: Organic material at 300C
HNO
3
: Any metals not dissolve by HCl
HClO
4
: Steel
HF : Silica
Aqua Regia (3:1, HCl:HNO
3
) for most inorganic
HNO
3
+HCl+HF (5:15:3) for alloys
Grades of acids
Very High Purity Chemicals eg Ultra-Pure (NBS)
Analytical Reagents eg Certified AR
TM
(Fisher)
Chemically Pure (CP) eg CP
TM
(Sigma)
Practical Grade eg Purified
TM
(Sigma)
Commercial or Technical Grade
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Microwave Digestion
Rapid and efficient acid decomposition using
microwave energy in a specially designed oven
Advantages
- Sample contained within the digestion vessel
- Highly efficient and rapid (5-10 min)
- Volatile elements are retained in reaction
vessel
- Easy to automate: a computer controls the
pressure and the temperature
Disadvantages
- Inability to add reagents during the digestion
- Limited amount of sample (typically 1 g or less)
- Safety concerns due to the use of high
pressures and corrosive reagents
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Fusion Techniques
1. Inorganic samples are mixed with large
excess of alkali metal salts known as flux
(at a ratio of 1:10-20, sample:salts) in a
crucible and heated at a high temperature
until the substance fuse together in a
molten state
2. The melt is allowed to cool at room
temperature and dissolved in dilute acid
or water
(Used when acids fail to dissolve sample,
eg. silica, mineral oxides, steel)
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Types of flux
Base flux: Na carbonates, hydroxides,
borate for alkaline : pyrosulfates, boric
oxide, fluoride acids
Oxidizing Flux: Sodium peroxide or
nitrate/alkaline metal + Sodium Carbonate
Disadvantages: Contamination by flux
material, high salt content may complicate
analysis, high temperature result in loss of
analyte (evaporation), sample container
may react with flux material
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Several sources of error are encountered
in the sample dissolution step
Incomplete dissolution of the analyte
Losses of analyte by the volatilization
(evaporation)
Introduction of external analyte from a
solvent (solvent contamination)
Contamination from the reaction of the
solvent with vessel walls
Errors In Sample Dissolution
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a) Use a selective analytical technique (eg
ion selective eletrode)
b) Use selective derivatization that converts
the analyte into another chemical species
that can be measured more easily
c) Use Standard Solution of high-purity
standard materials
d) Remove the analyte from the sample
matrix by a separation or extraction
process
To Measure Analyte containing
Interfering species
Separation of analyte from matrix -
Eliminate interferences from samples*
Provide suitable selectivity
Required if many analytes are present
Cause preconcentration of analyte - good for
more sensitive or accurate measurement
Preconcentration -
Required if amounts of analyte(s) too small in
bulk material (eg trace element analysis)
Extraction + preconcentration (10-100 times)
- Improves sensitivity
- Eliminate interferences from the sample matrix*
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Separation and Preconcentration
Soxhlet extraction
Liquid-liquid extraction
Chromatography : Gas chromatography,
liquid chromatography, gel permeation
chromatography, TLC, Ion exchange
chromatography, Paper
chromatography
Electrophoresis : liquid/solid
Dialysis
Precipitation : liquid/solid
Addition of Masking agent
Summary of Separation Techniques
Soxhlet Extraction
Classical extraction method
(Apparatus named after developer)
Place sample in porous
thimble
Exhaustive reflux for
up to 1 - 2 days
Solution of analyte(s)
in volatile solvent
(eg. CH
2
Cl
2
, CHCl
3
etc)
Evaporate to dryness
or suitable
concentration for
separation/analysis
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Dilution and Matrix Matching
After dissolution and separation, the sample
may not be suitable for direct measurement
(1) The instrument or method requires a particular
type of sample form, concentration, solvent,
oxidation state, etc
(1) Some samples require modifications:
- pH adjustment with acid/base/buffer
- Adjustment of oxidation state using an
appropriate oxidising agent
- Dissolving in acid (trace metal analysis by
spectroscopic techniques often requres
nitric acid to avoid mass-spectral
interferences with chloride solution)
Measurement is often the simplest
stage of the analytical process
Use reagents of high purity (eg. analytical
grade, reagent grade, etc)
A blank measurement must be performed
for trace analysis (The results of the blank will
be subsequently subtracted from the raw
analytical measurement)
Choice of Analytical Measurements:
- Classical methods
- Instrumental methods
(5) Analytical Measurement
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Classical methods (gravimetric, volumetric)
- Accurate and precise to approx. 0.1% but
require large (mmol, mg) amount of analyte
Instrumental method (Spectroscopic, HPLC,
GC, etc)
- More selective and sensitive than classical
methods but less precise, accurate to
approx. 1%
- Based on measurement of the physical or
chemical property of an analyte directly
related to the concentration
- Rapid, may be automated and may be used
for the determination of multiple analytes
at a time
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(1) Determine the concentration of the
analyte in the analytical sample solution
(2) Use results to calculate the amount of
analyte in the original (bulk) sample
Evaluate the results
- Requires appropriate use of statistics
- Must be reasonable, reliable and related to the
problem as originally stated
(6) Calculating Results and Reporting
Data presentation must be understood,
conclusions clearly shown
Report results with accuracy & precision
(include standard deviation, mean value, etc)
Verify reports (Professional/charted chemist)
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ILLUSTRATIONS AND
PHOTOGRAPHS
OF SAMPLING
TECHNIQUES
AND APPARATUS
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Consider the different
flowrates, mixing and
turbulance (eg rainy and
dry season)
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GOOGLE EARTH
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In-situ testing
(Parameters such as pH, DO, turbidity)
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Composite Water
Sampler (with
rechargable battery, able
to adjust to desired
sample: 25 mL to 600
mL, time 5 min to 4 hrs)
Grab Sampler
(Van Dorn Sampler)
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Water Sampling
(How much water do you need?)
Depends on parameters
to be analyzed
Chlorophyll and TSS often
require the greatest
volume (> 1L)
Better be safe to have
too much water rather
than too little
Depends on the system
and how practical it is
to carry out large
volumes of water
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Water sampling
(Microbes)
Grab samples taken
with sterile containers
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Examples of Gas Samplers
Underground gas
sampler
(sampling gas leaks,
methane decomposition
in landfills, etc)
Hi-vol sampler
[Particulate (PM10) and
microbes on filters]
Gas
canister
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