Department of Chemistry

Instrumental Analysis I

Introduction

By Dawit Alemu
 1. Analytical Separation Techniques and
Classical Method of Analysis
Analytical Methods
This can be divided in to two broad classes as classical and
instrumental method of analysis.
 Classical Methods
 Separation of analytes by precipitation, extraction, or
distillation.
 Qualitative analysis by reaction of analytes with reagents
that yielded products that could be recognized by their
colors, boiling or melting points, solubilities, optical
activities, or refractive indexes.
 Quantitative analysis by gravimetric or by titrimetric
techniques
 Cont…
Instrumental Methods
 Measurement of physical properties of analytes - such
as conductivity, electrode potential, light absorption or
emission, mass-to-charge ratio, and fluorescence-
began to be employed for quantitative analysis of
inorganic, organic, and biochemical analytes.
 Efficient chromatographic separation techniques are
used for the separation of components of complex
mixtures.
 Instrumental Methods of analysis (collective name for
newer methods for separation and determination of
chemical species.)
 Cont …
Table 1.1. Comparisons between the two methods
Classical Methods Instrumental methods

Simple to use Somewhat complex

Cheap equipment requirement Coasty and require continuous calibration

Based on absolute (direct) measurement Are not absolute (indirect way) in measurement

Special training not required to operate the Special training will require to operate the
instruments instruments

Consume large sample volume Small sample can be used

Time consuming Save time since it is automatic

 Separation techniques
Brainstorming:

 How do you define separation?

 What is analytical separation?

 Why separation is extremely important in chemical analysis?

 Separation techniques cont…

 Separation is the act of differentiating pure compound from

interferent or separating mixture to its components.
 Separations are extremely important in synthesis, industrial
chemistry, biomedical sciences, and chemical analysis.
 The goals of an analytical separation are usually :
 to eliminate or reduce interferences and
 to obtain pure components of a mixture so that
quantitative analytical information can be obtained about
complex mixtures.
 Cont …
Table 1.2 Separation Methods

Method Basis of Methods

Mechanical phase separation
Precipitation and filtration Difference in solubility of compounds formed
Distillation Difference in volatility of compounds
Extraction Difference in solubility in two immiscible liquids
Ion exchange Difference in interaction of reactants with ion-exchange
resin
Chromatography Difference in rate of movement of a solute through a
stationary phase
Electrophoresis Difference in migration rate of charged species in an
electric field
Field-flow fractionation Difference in interaction with a field or gradient applied
perpendicular to transport direction
 2. Introduction to Chromatographic
Separation

2.1. Historical Background

Mikhail Tswett, Russian Botanst,
1872-1919
In 1906 Tswett used to chromatography to
separate plant pigments
He called the new technique chromatography
because the result of the analysis was 'written
in color' along the length of the adsorbent
column
Chroma means “color” and graphein means to “write” Mikhail Tswett
 Original Chromatography Experiment of Tswett
Start: A glass End: A series of
column is filled colored bands is
with powdered seen to form,
limestone corresponding to
(CaCO3). the pigments in
Later the original plant
An EtOH extract
of leaf pigments extract. These
is applied to the bands were later
top of the column. determined to be
chlorophylls,
EtOH is used to
xanthophylls and
flush the pigments
carotenoids.
down the column.
 Definition of 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 through it in a definite direction.

 Chromatography is the term used to describe a separation

technique in which a mobile phase carrying a mixture is caused to
move in contact with a selectively absorbent stationary phase.

• The chromatographic process occurs due to differences in the

distribution constant of the individual sample components.
 Cont …

Components or terms of chromatography

 mobile phase: a solvent that flows through the supporting
medium.
• Q? Which kind of phases can be used as mobile phase?
 stationary phase: a layer or coating on the supporting medium
that interacts with the analytes.
• Q? Which kinds of phases can be used for stationary phase?
 supporting medium: a solid surface on which the stationary
phase is bound or coated.
 Cont …
Note:
 In a chromatographic separation the mobile and stationary
phase must have low interaction.
 Always when we select a mobile phase we should consider the
following:
 The mobile phase must be the least eluted species.
 The mobile phase could not wash the stationary phase.
 a chromatogram: is the visual output of the chromatograph.
 In the case of an optimal separation, different peaks or
patterns on the chromatogram correspond to different
components of the separated mixture.
2.2. Types (Classification) of Chromatography

There are a number of different kinds of chromatography, which

differ in the mobile and the stationary phase used.
Chromatography can be classified based on three criteria.
1. the physical means by which the stationary phase and mobile
phase are brought together.
2. the type of mobile phase and stationary phases
3. the type of interaction occur between the sample and
stationary phase
 Cont…

1. Based on the physical means by which the stationary phase

and mobile phase are brought together.

 Column chromatography
 Packed
 Open tubular

 Planar chromatography
Paper
TLC
 Cont …
 Column chromatography

Column chromatography is a separation technique in which the

stationary phase is within a tube.
 Planar chromatography

Paper Chromatography
 Paper chromatography is a technique that involves placing a
small dot or line of sample solution onto a strip of
chromatography paper.
 Cont…
Thin Layer Chromatography (TLC)
 TLC is a widely employed laboratory technique and is similar
to paper chromatography.
 However, instead of using a stationary phase of paper, it
involves a stationary phase of a thin layer of adsorbent like
silica gel, alumina, or cellulose on a flat, inert substrate.
 Compared to paper, it has the advantage of faster runs, better
separations, and the choice between different adsorbents.
 Cont …
Distance the solute move
Rf =
Distance the solvent front move

• Q? How you can identify if invisible spots could occur when

you do TLC?
• Q? Which types of mobile phases are used for column and
planar chromatography?
• Q? Which types of stationary phases can be used for column
and planar chromatography?
2. Based on the type of mobile phase and stationary phases
 Gas chromatography (GC) includes all chromatographic
methods in which gas is used as mobile phase. It uses only
stationary phases either coated or packed on a column.
 Liquid chromatography (LC) includes all chromatographic
techniques in which liquid is used as mobile phase. For LC a
stationary phase is either packed on a column or coated on a
column or a planar plate can be used.
 Supercritical fluid chromatography (SFC) includes
chromatographic techniques which uses supercritical fluid as
mobile phase. For this technique the stationary phase could be
organic species bonded on a solid surface.
 Cont …
Table 2.1. Classification of chromatography based on the type of
mobile phase and stationary phases

Chromatography technique Mobile phase Stationary phase

Liquid chromatography (LC)
a, Liquid-liquid chromatography Liquid Liquid immobilized on a solid
surface

b, Liquid-solid chromatography Liquid solid

Gas chromatography (GC)
a, Gas-liquid chromatography Gas Liquid immobilized on a solid
surface

b, Gas-solid chromatography Gas solid

Supercritical fluid chromatography Supercritical fluid Organic species bonded on a solid
(SFC) surface
 3. based on the type of interaction occur between the
sample and stationary phase
Table 2.2. Types of chromatography based on interaction between
sample and stationary phase
Type of chromatography Type of interaction occur

a. Adsorption chromatography Adsorption of a sample on a solid surface

b. Partition chromatography Partition of a sample between two

immiscible liquids

c. Ion-exchange chromatography Ion exchange

d. Size exclusion chromatography Partitioning/ Sieving

 Elution
• Note:
 Separation in chromatography is based on the elution of the
sample across the stationary phase by the mobile phase.
 Across the movement the components of the sample interacts
with the stationary phase differently.
 The solute which interacts strongly with the stationary
phase will be strongly retained. So the speed of this solute
in the column or plane is very slow and takes more time to
be eluted.
 The solute, which interacts with the stationary phase
weakly, will be weakly retained. So the solute will be
eluted easily.
 Cont ….
 A -is a solute which has a weak interaction with the stationary
phase and weakly retained.
 B –is a solute which has a strong interaction with the
stationary phase and strongly retained.
 Resolution

Z
Rs 
WA / 2  WB / 2
2Z
Rs 
WA  WB
2[(t R ) B  (t R ) A ]
Rs 
WA  WB
 2.4. Migration Rates of Solutes
• The effectiveness of a chromatographic column in separating
two solutes depends in part on the relative rates at which the
two species are eluted.
• These rates in turn are determined by the ratios of the solute
concentrations in each of the two phases.
• The separation is enhanced by altering the relative flow rate of
solutes. i.e.
o by increasing the flow rate of weakly retained species and
o decreasing the flow rate of strongly retained species.
 Factors affecting Migration Rates of Solutes
• Distribution Constant, K or D
Ain mobile phase Ain stationary phase
• The equilibrium constant K; for this reaction is called a
distribution constant, which is defined as
𝐶𝑠
𝐾=
𝐶𝑚
Where: 𝐶𝑠 - Concentration in molar of solute A in the
stationary phase
𝐶𝑚 - Concentration in molar of solute A in the mobile
phase
Note:
• Large K value is obtained when the solute concentration in the
stationary phase is large. This indicates the solute interacts
with stationary phase strongly.
 Cont…
• Retention time
o The retention time (tR) is the time between injection of a
sample and the appearance of a solute peak at the detector
of a chromatographic column.
o The dead time (void time) (tM) is the time it takes for an
unretained species to pass through a chromatographic
column.
o All components spend this (tM) amount of time in the
mobile phase.
o Separations are based on the different times tS that
components spend in the stationary phase.
 Velocities: Linear rate of solute migration!

Velocity = distance/time  length of column/ retention times

L
Velocity of solute:
v 
tR
L
Velocity of mobile phase:
 
tM
 The Relationship between Migration Rate and
Distribution Constant

To relate the migration rate of a solute to its distribution

constant, we express the rate as a fraction of the velocity of the
mobile phase.
v    fraction of solute spends in mobile phase
moles of solute in mobile phase
v   
total moles of solute
cM VM
v   
cM VM  cSVS
 Cont…

cM VM
v   
cM VM  cSVS
1
v   
1  cSVS / cM VM
cS
K  Distribution Constant
cM
1
v   
1  K VS / VM
 Cont…
Retention Factor :
It is the amount of time a solute spends in the stationary phase relative to the time it
spends in the mobile phase.
1
v   
1  K VS / VM
k ' A  K AVS / VM (Retention Factor)
1
v   
1 k 'A
L L 1
 
tR tM 1 k ' A
tR  tM Adjusted retention time
k 'A 
tM
 Cont…
Selectivity Factor:
The selectivity factor 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).
B retained more than A   >1
KB
  Distribution
Constant
KA
k 'B
  Retention factor
k 'A
(t R ) A  t M (t R ) B  t M
k 'A  and k ' B 
tM tM
(t R ) B  t M
  Retention time
(t R ) A  t M
 Band Broadening and Column Efficiency -
Theoretical Plates and Plate Theories

H  plate height
N  number of plates
L
N 
H
2
H 
L
L = length of column packing

  standard deviation
2
L
 variance per unit length.
 Cont…
Relation between column distance and retention
times

L  column length (distance)

  standard deviation in distance
t R  retention time
  standard deviation in time
 

L tR

 
L / tR
 Cont…
Relation between column distance and retention
times
 

L tR
L
 
tR
W  4
WL
 
4 tR
~96%
Tangent at  2 2
W2 L
Inflection point H  
L 16 t R2
 Cont…
Determining the Number
of Theoretical Plates

N  number of pates
2
t 
N  16  R 
W 
W1/2  t 
2

N  5.54  R 
 W1/ 2 
 2.5. Optimization of Resolution
Effect of Retention Factor and Selectivity Factor on Resolution
A useful equation that relates the resolution of a column to the
number of plates also contains the retention and selectivity factors
of pair of solutes on the column.
𝑁 𝛼−1 𝐾′ 𝐵
𝑅𝑠 = ( )( )
4 𝛼 1+ 𝐾′ 𝐵
Where: 𝐾 ′ 𝐵 is the retention factor of the strongly retained solute.
𝛼 is selectivity factor
Rearrange to solve for the number of theoretical plates, N:
𝛼 2 1+ 𝐾′ 2
𝑁= 16𝑅𝑠2 𝐵
𝛼−1 𝐾′ 𝐵
Effect of Resolution on Retention Time
The time required to obtain the resolution Rs is given by;
3
16𝑅𝑠2 𝐻 𝛼 2 1+ 𝐾′ 𝐵
(𝑡𝑅 )𝐵 =
µ 𝛼−1 𝐾′ 𝐵 2
 Factors affecting solutes separation in CC
( Factors affecting column efficiency)
Factor Effect
Particle size of solid stationary Decrease of size improves separation (but very small
phase (or of support) particles need high pressure).
Column dimensions Efficiency increases as ratio length / width increases.
Non uniform packing results in irregular movement
Uniformity of packing of solutes through column & less uniform zone
formation, (i.e. band broadning or tailing).
Increase in column temperature results in speed of
Column temperature
elution but does not improve separation (tailing).
Solvents should be of low viscosity (to give efficient
Eluting solvent resolution) & h igh volatility (to get rapid recovery of
the substances).
Solvent flow rate Uniform & low flow rate gives better resolution.
Continuity of flow Discontinuous flow disturbs resolution
Condition of adsorbent Deactivation of adsorbent decreases separation.
Concentration of solutes Substances of high concentration move slowly.
Column Efficiency
Kinetic variables
 Zone Broadening
Flow Rate of Mobile Phase

Liquid chromatography Gas chromatography

Note the differences in flowrate and plates height scales

 Zone Broadening
Kinetic Processes
Van - Deemter
Equation
λ and γ are constants
that depend on quality
of the packing.

B is coefficient of
longitudinal diffusion.

Cs and Cm are
coefficients of mass
transfer in stationary
and mobile phase,
respectively.

H  A  B /   (CS  CM )
Zone Broadening
Multiple Pathways

Eddy Diffusion: band

broadening process results
from different path lengths
passed by solutes.

1. Directly proportional to
the diameters of packing
2. Lower mobile-phase
velocity, smaller eddy
diffusion
Zone Broadening
Longitudinal Diffusion
• The higher the
Column , the smaller
the H
• Much smaller in
Diffusion LC than in GC
 1. the physical means by which the stationary phase and mobile
phase are brought together.
 Paper Chromatography
 In paper chromatography, the mobile phase is a solvent, and
the stationary phase is water held in the fibers of
chromatography paper.
 A solution of the mixture to be separated is spotted onto a strip
of chromatography paper (or filter paper) with a dropper.
 The chromatogram is developed by placing the bottom of the
paper (but not the sample spot) in a tank containing a suitable
solvent.
 The solvent is drawn up the paper by capillary action.
 The components of the mixture move up the paper with the
solvent at different rates due to their differing interactions with
the stationary and mobile phases.
 Cont …

Rf = Distance the solute moves/Distance the solvent front moves

Column Chromatography
In column chromatography, the mobile phase is again a
solvent, and the stationary phase is a finely divided solid,
such as silica gel or alumina.
Chromatography columns vary in size and polarity.
There is an element of trial and error involved in selecting a
suitable solvent and column for the separation of the
constituents of a particular mixture. A small volume of the
sample whose constituents are to be separated is placed on
top of the column.
The choice of the eluting solvent should ensure that the
sample is soluble. However, if the sample was too soluble
the mobile phase (solvent) would move the solutes too
quickly, resulting in the nonseparation of the different
constituents.
Cont …
• Thin Layer Chromatography
• In thin layer chromatography, the mobile phase is also a
solvent, and the stationary phaseis a thin layer of finely
divided solid, such as silica gel or alumina, supported on glass
or aluminium.Thin layer chromatography is similar to paper
chromatography in that it involves spotting the mixture on the
plate and the solvent (mobile phase) rises up the plate in the
chromatography tank.
• It has an advantage over paper chromatography in that its
separations are very efficient because of the much smaller size
of the particles in the stationary phase.
• Thin layer chromatography is particularly useful in forensic
work, for example in the separation of dyes fromfibres. Gas
chromatography and high performance liquid chromatography
are more sophisticated chromatographic techniques.