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Modern Instrumentation Methods and Technique
1. Separation Methods (GC, HPLC)
2. Spectroscopy
3.
4.
5.
6.
7.
8.
Ultraviolet/ Visible Spectroscopy (UV-Vis)
Infrared (FTIR) Spectroscopy
Introduction to Nuclear Magnetic Resonance (NMR)
Atomic Absorption (AAS) and Flame Emission Spectroscopy (FES)
Elemental Analysis
Statistical methods
Chromatography
Chromatography is a physical method of separation in which the components to be separated are
distributed between two phases, one of which is stationary (stationary phase), while the other
(the mobile phase) moves in a definite direction.
Liquid
Gas
Column
Flat sheet
Major types:
--- gas chromatography (GC)
--- liquid chromatography (LC)
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Equilibration based on
stationary
phase
1. Adsorption
2. Partition
3. Ion exchange
4. Size dependent pore penetration
mobile
phase
stationary phase
mobile
phase
inert
solid
stationary
phase
Basic chromatographic process
Sample: mixture of components a, b, and c
SP= stationary phase
MP= mobile phase
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Basic chromatographic process
Sample: mixture of components a, b, and c
MP= mobile phase
SP= stationary phase
Detector
Elution is a process in which solutes are washed through a stationary phase by the movement of a mobile phase.
Eluate is the mobile phase that exits the column.
An eluent is a solvent used to carry the components of a mixture through a stationary phase.
a
b
c
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Chromatogram is a plot of some function of solute concentration versus elution time.
tR= retention time
time required for the analyte to reach the detector
tM= dead time (void time)
time it takes for an unretained species to pass through
a chromatographic column.
tS (or t’R): adjusted retention time; time the analyte
spends in the stationary phase
Retention factor, kA: related to the rate at which A migrates through a column
Selectivity factor, a, for solutes A and B is defined as the ratio of the
distribution constant of the more strongly retained
solute (B) to the distribution constant for the less
strongly held solute (A).
Skoog and West: Fundamentals of Analytical Chemistry 9E
Column efficiency
Number of theoretical plates: N
Height equivalent to
a theoretical plate: HETP or H
L= length of the column
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed.
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Column efficiency
Column resolution (Rs)
Quantitative measure of the ability of the column
to separate two analytes
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
Column resolution (Rs)
If WA = WB
Number of
theoretical
plates: N
Retention factor, k
Selectivity factor, a,
Christian, Dasgupta, Schug - Analytical Chemistry 7th ed.
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Effect of column length on the resolution.
Chemical Analysis - Modern Instrumentation Methods and Techniques 2nd ed - F. Rouessac, A. Rouessac (Wiley, 2007)
Column efficiency
Optimal flow rate
Indicator ----- band broadening
Variables That Influence Column Efficiency
--- Velocity of mobile phase
--- Diffusion coefficient in mobile phase
--- Diffusion coefficient in stationary phase
--- Retention factor
--- Diameter and uniformity of packing particles
--- Thickness of liquid coating on stationary phase
velocity of the mobile phase
components mix faster
than they migrate
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
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Optimization of chromatographic analysis
--- choice of column
--- length of column
--- diameter of the column
--- stationary phase composition
--- mobile phase composition
--- flow rate
--- temperature
-------------------
always required !!!!!!!
The resolution and the elution time
are the two most important dependent
variables to consider.
the resolution varies with the square
root of the column length
Effect of solvent on resolution and elution time
water/acetonitrile
50/50%
55/45
water/acetonitrile
60/40%
65/35
How could you separate these two components?
Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed.
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Retention factor, kA: related to the rate at which A migrates through a column
k is optimal for 1 and 2
k is optimal for 5 and 6
k is optimal first for 3 and 4,
Gradient elution
then for 5 and 6
Gradient elution: steady changes of the mobile phase composition (HPLC) or temperature (GC) during the chromatographic run.
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
Solve the following problem
Chromatographic response of an artificial mixture containing
6 essential oil components and methyl benzoate as an internal
standard.
Using the cinnamaldehyde peak
1. Determine the number of theoretical plates for the column.
2. Determine the height equivalent to a theoretical plate, if
a fused-silica column of 0.25 mm x 30 cm with 0.25 μm film
was used.
Cinnamaldehyde
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
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1. Determine the number of theoretical plates for the column.
2. Determine the height equivalent to a theoretical plate, if
a fused-silica column of 0.25 mm x 30 cm with 0.25 μm film
was used.
19.0
Cinnamaldehyde
= 28523
H = 10.5 μm
0.45
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
In what order wolud the following compounds be eluted from a column having an apolar stationary
phase using a protic polar solvent?
Benzene, acetone, and benzoic acid?
Apolar
molecule
Organic acid
Polar
molecule
Fastest: benzoic acid
Second: acetone
Last: benzene
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Consider two compounds for which tM= 1 min, t1= 11.30 min and t2=12 min. The peak
widths at half-height are 10 s and 12 s, respectively. Calculate the time the analytes
spend in the stationary phase. Calculate the column resolution.
tS= tR – tM=
Compound 1: 11.30 – 1 = 10.30 min
Compound 2: 12 – 1 = 11 min
=
(12 – 11.30)∙60 = 18 s
= 0.82
10 + 12 = 22 s
Gas chromatography (GC)
The stationary phase
--- gas
– solid (adsorption) chromatography
--- gas – liquid (partition) chromatography
porous solid
immobilized liquid
Analyte in the vapor state distributes between the stationary phase and the carrier gas.
Gas-phase equilibria are rapid, so resolution (and the number of plates) can be high.
Application:
--- all gases
--- volatile and thermostable liquids and solids
(e.g. nonionized organic molecules upto 25 carbon atoms,
organometallic compounds)
--- non-volatile compounds can be derivatized
https://www.agilent.com/en/products/gas-chromatography/gc-systems/7820a-gc-system
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Analyte in the vapor state distributes between the stationary phase and the carrier gas.
The most important factor in gas chromatography is
the selection of the proper column (stationary phase) for the particular separation to be attempted.
The nature of the liquid or solid phase will determine the exchange equilibrium with the sample
components; and this will depend on
--- the solubility or adsorbability of the analytes,
--- the polarity of the stationary phase and sample molecules,
--- the degree of hydrogen bonding, and specific chemical interactions.
Components of GC instruments:
1.
2.
3.
4.
Sample injector
Column
Detector
Data system
Christian, Dasgupta, Schug - Analytical Chemistry 7th ed.
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Components of GC instruments:
1.
2.
3.
4.
Sample injector
Column
Detector
Data system
The sample injection port, column, and detector are heated
to temperatures at which the sample has a vapor pressure of at
least 10 torr, usually about 50 °C above the boiling point of the
highest boiling solute.
The injection port and detector are usually kept
somewhat warmer than the column
to promote rapid vaporization of the injected sample and
prevent sample condensation in the detector.
Christian, Dasgupta, Schug - Analytical Chemistry 7th ed.
Carrier gas
free of
all traces of hydrocarbons,
water vapour and oxygen
The carrier gas is a chemically inert gas available in pure form such as argon, helium, or
nitrogen, or hydrogen. It has no significant influence on partition coefficient.
A highly dense gas gives best efficiency since diffusivity is lower, but
a low-density gas gives faster speed. The viscosity of the carrier gas and its flow rate
have an effect on the analytes’ dispersion in the stationary phase and on their diffusion
in the mobile phase.
The choice of gas is often dictated by the type of detector.
Flow rate is regulated by controlling the gas inlet.
--- enables reproducibility of the retention times
Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed.
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Carrier gas
free of
all traces of hydrocarbons,
water vapour and oxygen
The carrier gas is a chemically inert gas available in pure form such as argon, helium, or
nitrogen, or hydrogen. It has no significant influence on partition coefficient.
A highly dense gas gives best efficiency since diffusivity is lower, but
a low-density gas gives faster speed. The viscosity of the carrier gas and its flow rate
have an effect on the analytes’ dispersion in the stationary phase and on their diffusion
in the mobile phase.
The choice of gas is often dictated by the type of detector.
Flow rate is regulated by controlling the gas inlet.
--- enables reproducibility of the retention times
The nature of the carrier gas has no significant influence upon the values of the partition
coefficients K of the compounds between the stationary and mobile phases,
owing to an absence of interaction between the gas and solutes.
Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed.
Injector
Suitably sized sample inserted as a „plug” of vapour.
--- packed columns: 0.1 – 20 μl liquid --- direct vaporization
--- capillary columns: 2 order of magnitude smaller ----- split/splitless
Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed.
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Colums
Packed columns
Capillary columns
Colums
Packed columns
Capillary columns
steel, glass
1-3 m (3-6 mm), spherical support (0.2 mm)
high purity fused silica
12-100 m (0.1-0.5 mm/0.5 mm)
--- routine applications
--- increased capacity
--- easy manifacturing,
--- wide variety of stationary phases
--- higher resolution
--- higher speed
--- trace analysis
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Stationary phases
„like dissolve like”
Very polar
Non polar
Highly polar
Effect of temperature
45 °C
optimum resolution vs. elution time
low temp.
higher temp.
145 °C
Programmed
30 – 180 °C
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
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Detectors
universal vs. discriminating (selective)
destructive vs. non-destructive
single information vs. multiple information
tR
tR + structural info.
Detectors
Gas chromatography always
uses flow-through
detectors that automatically
detect the analytes as they
elute from the column;
the
majority of GC detectors are
destructive.
Flame ionization detector (FID)
General
Destructive
Mass-sensitive
Insensitive toward non-combustable gases
(H2O, CO2, SO2 NOx)
Not-sensitive to functional groups containing
electronegative elements.
(carbonyl, halogen, alcohol, amine)
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
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Detectors
Termal conductivity detectors
Simple, general use
Non-destructive
Relatively low sensitivity
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
Detectors
Electron Capture detectors
High sensitivity for halogen cont. comp.
Not-destructive
Radioactive source: β-emitter Ni-63
Sensitive to molecules such as halogens, nitro compounds, peroxides
Insensitive toward hydrocarbons, alcohols, amines
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
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Detectors
Mass spectrometry detectors (GC/MS)
Typical gas chromatogram of unleaded gasoline, a complex mixture, using a capillary column.
Christian, Dasgupta, Schug - Analytical Chemistry 7th ed.
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Application of GC
Applicable to relatively volatile and thermally stable molecules.
--- qualitative analysis
(purity)
--- quantitative analysis
(calibration, internal standards)
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
Christian, Dasgupta, Schug - Analytical Chemistry 7th ed.
https://www.legalhelplawyers.com/blood-alcohol-testing-drunk-driving-cases
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449
377
Accidently only 8 μL injected
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High Performance Liquid Chromatography (HPLC)
--- adsorption chromatography
--- partition chromatography
--- normal phase
chromatography
polar stationary phase and nonpolar or inremediate polarity solvents
silica
hexan, THF, etc....
--- reverse phase chromatography
nonpolar stationary phase and polar solvents
octadecyl silica
water – acetonitrile, methanol – water mixture
--- ion exchange chromatorgraphy
--- size exclusion chromatography
Application:
--- nonvolatile compounds
--- small molecules
--- macromolecules
--- ionic species
Chromatography In everyday’s life
- Forensic Testing
CSI and Law and Order? Investigating criminal caseases
Crime scene testing: blood or cloth sample
Arson verification: chemical residues after fire or explosions
(GC)
- Quality testing in food and pharmaceutical Industry
- Is it really beefmeet in the burger?
In 2013, In Europe more than 300 tonnes of horsemeat was sold as beefmeat.
- Performance Enhancing Drug Testing
Doping test from blood (HPLC)
http://lasvegas.csiexhibit.com/
https://en.wikipedia.org/wiki/Hamburger
http://post.jagran.com/iitroorkee-engineers-develop-sensor-to-give-dope-test-results-in-5-minutes-1312087343
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Basic components of a HPLC system
Christian, Dasgupta, Schug - Analytical Chemistry 7th ed.
Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed.
Guard columns --- usually contain the same packing as the analytical
Analytical columns
Stainless steel tubing
4.6 mm inner diameter
5 – 250 mm lengths
Christian, Dasgupta, Schug - Analytical Chemistry 7th ed.
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Stationary phases in HPLC
Spherical microporous
silica particles, 10 μm
CH3
Christian, Dasgupta, Schug –
Analytical Chemistry 7th ed.
Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed.
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The effect of column temperature upon compound separation.
(a) 25°C, (b) 35°C, (c) 45°C
Chemical Analysis - Modern Instrumentation Methods and Techniques 2nd ed - F. Rouessac, A. Rouessac (Wiley, 2007)
Detectors
UV detectors
Fluorescence flow cell
Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed.
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Chromatograms of a sample containing two compounds A and B, for which the UV spectra are different.
Schematics of a differential refractive index detector.
Chemical Analysis - Modern Instrumentation Methods and Techniques 2nd ed - F. Rouessac, A. Rouessac (Wiley, 2007)
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Comparison of HPLC and GC
Both methods:
--- efficient, highly selective, widely applicable
--- only small amounts of sample required
--- readily adapted to quantitative analysis
HPLC
--- can accommodate nonvolatile and thermally
unstable compounds
--- generally applicable to inorganic ions
GC:
--- simple and inexpensive equipment
--- rapid
--- easy to interface with mass spectrometry
--- not for macromolecules or salts
Thin-Layer Chromatography (TLC)
Planar form of chromy.
Stationary phase:
--- finely divided sorbent on glass,
metal (Al),
plastic sheet
--- multiple samples can be simultaneously
analysed
Three stages:
1. Sample spotting (0.5 – 5 μl)
2. Plate development
3. Detection
Christian, Dasgupta, Schug – Analytical Chemistry 7th ed.
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Stationary phase:
Mobile phase:
--- finely divided sorbent on glass,
metal (Al),
plastic sheet
--- if silica or alumina used as stationary phase
60 Å pore size silica
10 -12 μm mean particle size
200 – 250 µm sorbent layer thickness
10 – 15 cm migration distance
Developing
eluting power of solvents increases in the order of
their polarities
(e.g. from hexane to acetone to alcohol to water)
Detection of spots / Visualization
--- UV lamp (265 nm or 365 nm) --- fluorescent analyte
--- plate contains fluorescent zinc-salt
--- iodine vapours
--- colored species can be visually observed
Analyte is characterized by the Rf (retardation factor) value:
Resolution:
Rs=2(x1–x2)/(w1+w2)
Efficiency:
N=16(x1/w)2
w= spot diameter
Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed.
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Two-dimensional TLC
--- two successive elution with
two mobile phases
Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods
and Techniques 2nd ed.
Questions 1:
GC response of an artificial mixture containing 6 essential
Oil components and methyl benzoate as an internal standard.
1. Determine the number of theoretical plates for the column.
2. Determine the height equivalent to a theoretical plate, if
a fused-silica column of 0.25 mm x 30 cm with 0.25 μm film
was used.
Cinnamaldehyde
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
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1. Determine the number of theoretical plates for the column.
2. Determine the height equivalent to a theoretical plate, if
a fused-silica column of 0.25 mm x 30 cm with 0.25 μm film
was used.
19.0
= 28523
Cinnamaldehyde
H = 10.5 μm
0.45
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
Question 2:
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
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The largest loss of ibuprofen as a percentage
occurs between 1.0 and 1.5 hours.
Question 3:
How could you calculate the efficiency (N) of a TLC plate for a compound whose
migration distance is x and spot diameter is w?
How could you define the H value?
Resolution:
Rs=2(x1–x2)/(w1+w2)
Efficiency:
N=16(x1/w)2
w= spot diameter
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Question 4:
In what order would the following compounds be eluted from an alumina column
using n-hexane as the eluting solvent?
CH3CH2OH
CH3CHO
CH3CO2H
They would elute in increasing order of polarity:
fatest: acetaldehyde
second: ethanol
last: acetic acid
Christian, Dasgupta, Schug – Analytical Chemistry 6th ed.
Question 5:
Fastest: benzoic acid
Second: acetone
Last: benzene
Christian, Dasgupta, Schug – Analytical Chemistry 7th ed.
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Question 6:
What solvent would you choose to separate a group of hydrocarbons,
CH3(CH2)xCH3, on an alumina column?
A nonpolar solvent like hexane or heptane.
Christian, Dasgupta, Schug – Analytical Chemistry 6th ed.
Question 7:
What is normal-phase chromatography? Reversed-phase chromatography?
Stationary phase
Mobile phase
Normal phase chromatography:
polar
non-polar
non-polar molecule move faster
Reversed-phase chromatography:
non-polar
polar
polar molecule move faster
Christian, Dasgupta, Schug – Analytical Chemistry 6th ed.
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Question 8:
Christian, Dasgupta, Schug – Analytical Chemistry 7th ed.
Question 9:
Christian, Dasgupta, Schug – Analytical Chemistry 7th ed.
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Question 10:
Fastest: 1
Second: 3
Third: 2
Rouessac, Rouessac - Chemical Analysis Modern Instrumentation Methods and Techniques 2nd ed.
Question 11:
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
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tR= retention time
time required for the analyte to reach the detector
tM= dead time (void time)
time it takes for an unretained species to pass through
a chromatographic column.
tS (or t’R): adjusted retention time; time the analyte
spends in the stationary phase
Retention factor, kA
tR= 30 min
Selectivity factor, a
tR= 50 min
tM= 4 min
Skoog and West: Fundamentals of Analytical Chemistry 9E
original column
longer column
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Question 12:
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
Question 13:
Substances A and B have retention times of 16.40 and 17.63 min, respectively, on
a 30 cm column. An unretained species passes through the column in 1.30 min.
The peak widths (at base) for A and B are 1.11 and 1.21 min, respectively.
Calculate
(a) The column resolution,
(b) The average number of plates in the column,
(c) The plate height,
(d) The length of column required to achieve a resolution of 1.5, and
(e) The time required to elute substance B on the column that gives an Rs value of 1.5.
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
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a)
b)
c)
Retention factor, kB
d)
Selectivity factor, a
original column
longer column
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e)
Question 14:
Retention factor, kA
Void time is 1.05 min
Selectivity factor, a
0.03735
0.90337
N= [Rs∙4/(0.03735∙0.90337)]2
= Rs2∙14053.52
kB= [(10.5+22/60)‒1.05]/1.05= 9.3492
kA= [10.5‒1.05]/1.05= 9.0
α= 9.3492/9= 1.0388
Skoog, West, Holler, Crouch - Fundamentals of Analytical Chemistry 9th ed.
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