Analytical Separations

 What is separations science?

– A collection of techniques for separating complex mixtures of
analytes
– Most separations are not an analytical technique in their own right,
until combined with an analytical detector (often a type of
spectrometer)

 Key analytical branches discussed in this class: chromatography,

electrophoresis, extraction
 What is a Separation?
 Separations are key aspects of many modern analytical
methods. Real world samples contain many analytes,
many analytical methods do not offer sufficient selectivity
to be able to speciate all the analytes that might be
present.
 Most separation methods involve separation of the
analytes into distinct chemical species, followed by
detection:
( a + b + c + d + ……) (a) + (b) + ( c ) + (d) +……

( a + b + c + d + ……)
( a + b + c + d + ……)
DETECTION
COMPLETE SEPARATION
(a) + ( b + c + d+ …..)
PARTIAL SEPARATION
( a + b ) + ( b + a) + ……..
ENRICHMENT
Analytical Separations
Introduction
Analyte, component, or chemical species is a substance or chemical constituent that is
of interest in an analytical procedure.
Interferent, any substance whose presence interferes with an analytical procedure and
generates incorrect results
We can separate an analyte and an interferent if there is a significant difference in at
least one of their chemical or physical properties.

basis of separation separation technique(s)

size filtration; dialaysis; size-exclusion chromatography

mass or density centrifugation

complex formation masking

change in physical state distillation; sublimation; recrystalization

change in chemical state precipitation; electrodeposition; volatilization

partitioning between phases extraction; chromatography

Analytical Separations
 Basic Types of Separations
Liquid Column Chromatogrphy
Liquid-Liquid (partition) chromatography (LLC)
stationary and mobile phases (immiscible)
Liquid-Solid (adsorption) chromatography (LSC)
Ion exchange chromatography (IEC)
Exclusion chromatography (EC)
Gas-Liquid chromatography (GLC)
Gas-Solid chromatography (GSC)

Separation Methods Based on Phase Equilibria

Gas-Liquid Gas-Solid Liquid-Liquid Liquid-Solid
Distillation Adsorption Extraction Precipitation chrom
Sublimation Gas- Liquid Liq-Liq chrom Zone melting
Foam Fractionation Molecular sieves
Fractional crystallization
Ion Exchange
Adsorption
Exclusion
 Analytical Separations
Basic Types of Separations
Separation methods based on rate processes

Barrier Separation Field Separation Other

membrane filtration electrophoresis molecular distillation
dialysis ultracentrifugation enzyme degradation
electro-dialysis thermal diffusion destructive distillation
electro-osmosis electrodeposition
reverse osmosis mass spectrometry
gaseous diffusion

Particle Separation methods

Filtration
Sedimentation
Centrifugation
Particle electrophoresis
Electrostatic precipitation
Analytical Separations
Separation of species by distillation

Distillation is widely used to separate volatile analytes from nonvolatile interferents.

It is based on differences in the boiling points of the materials in a mixture.

Vacuum distillation is used for compounds that have very high boiling points.

Lowering the pressure to the vapor pressure of the compound of interest causes
boiling.

Molecular distillation occurs at very low pressure (<0.01 torr) such that the lowest
possible temperature is used with the least damage to the distillate.

Pervaporation is a method for separating mixtures by partial volatilization

through a nonporous membrane.

Flash evaporation is a process in which a liquid is heated and then sent through a
reduced pressure chamber where there is partial vaporization of the liquid.
Analytical Separations
Analytical Separations

masking agent elements whose ions are masked

Ag, Au, Cd, Co, Cu, Fe, Hg, Mn, Ni, Pd,
CN–
Pt, Zn

SCN– Ag, Cd, Co, Cu, Fe, Ni, Pd, Pt, Zn

NH3 Ag, Co, Ni, Cu, Zn

F– Al, Co, Cr, Mg, Mn, Sn, Zn

Au, Ce, Co, Cu, Fe, Hg, Mn, Pb, Pd, Pt,
S2O2−3S2O32−
Sb, Sn, Zn

Al, Ba, Bi, Ca, Ce, Co, Cr, Cu, Fe, Hg,
tartrate
Mn, Pb, Pd, Pt, Sb, Sn, Zn

oxalate Al, Fe, Mg, Mn

thioglycolic acid Cu, Fe, Sn

Analytical Separations
Solid phase extraction
In a solid phase extraction of a liquid sample, we pass the sample through a
cartridge that contains a solid adsorbent, several examples of which are shown
below
Analytical Separations
Solid phase extraction
The choice of adsorbent is determined by the species we wish to separate. Table
below provides several representative examples of solid adsorbents and their
applications.
absorbent properties and uses

 retains low to moderate polarity species from organic matrices

silica
 fat soluble vitamins, steroids

 retains polar compounds

aminopropyl
 carbohydrates, organic acids

 retains wide variety of species from aqueous and organic matrices

cyanopropyl
 pesticides, hydrophobic peptides

 retains wide variety of species from aqueous and organic matrices

diol
 proteins, peptides, fungicides

 retains hydrophobic species from aqueous matrices

octadecyl (C–18)
 caffeine, sedatives, polyaromatic hydrocarbons, carbohydrates, pesticides

octyl (C–8)  similar to C-18

 Analytical Separations
Extractions

1.) Definition
 The transfer of a compound from one chemical phase to another
- The two phases used can be liquid-liquid, liquid-solid, gas-solid, etc
- Liquid-liquid is the most common type of extraction

Immiscible
[ S ]2
K
liquids

[ S ]1

- The partitioning of solute s between two chemical phases (1 and 2) is

described by the equilibrium constant K

K is called the partition coefficient

 Analytical Separations
Chromatography

1.) Definition
 A separation technique
based on the different rates
of travel of solutes through a
system composed of two
phases
- A stationary phase
- A mobile phase

 Detect compounds
emerging in column by
changes in absorbance,
voltage, current, etc

Chromatogram (not spectrum)

 Analytical Separations
Chromatography
2.) System Components and Process
 Stationary Phase: the chemical phase which remains in the column (chromatographic
system)
 Mobile Phase (eluent): the chemical phase which travels through the column
 Support: a solid onto which the stationary phase is chemically attached or coated

Solute are separated in chromatography

by their different interactions with the
stationary phase and mobile phase
 Analytical Separations
Chromatography

2.) System Components and Process

Solutes which interact more strongly with

the stationary phase take longer to pass
through the column
Strongly Retained

Weakly Retained
Solutes which only weakly interact with
the stationary phase or have no
interactions with it elute very quickly
 Analytical Separations
Chromatography

3.) Chromatogram
 Chromatogram: graph showing the detector response as a function of elution
time.

Retention time
Non-retained solute
(void volume)

 Retention time (tr): the time it takes a compound to pass through a column
 Retention volume (Vr): volume of mobile phase needed to push solute through
the column

The strength or degree with which a molecule is retained on the column can
be measured using retention time or retention volume.
 Analytical Separations
Chromatography

4.) Fundamental Measures of Solute Retention

 Adjusted retention time (tr’): the additional time required for a solute to
travel through a column beyond the time required for non-retained
solute
t'r  t r  t m
where: tm = minimum possible time for a non-retained
solute to pass through the column

 Relative Retention (a): ratio of adjusted retention time between two

solutes
t'r 2
a
t'r1
where: tr2’ > tr1’ , so a > 1
- Greater the relative retention the greater the separation between two
components
 Analytical Separations
Chromatography

4.) Fundamental Measures of Solute Retention

 Capacity factor (k’):

t r  t m Vr  Vm
k' 
tm Vm

 The longer a component is retained by the column, the greater the capacity
factor
- Capacity factor of a standard can be used to monitor performance of a column

 Capacity factor is equivalent to:

time solute spends in stationary phase

k' 
time solute spends in mobile phase
 Analytical Separations
Chromatography

4.) Fundamental Measures of Solute Retention

 Capacity factor is equivalent to:

time solute spends in stationary phase moles of solute in stationary phase

k'  
time solute spends in mobile phase moles of solute in mobile phase

C sVs
k' 
C mVm

where: Cs = concentration of solute in the stationary phase

Cm = concentration of solute in the mobile phase
Vs = volume of the stationary phase
Vm = volume of the mobile phase
 Analytical Separations
Chromatography

4.) Fundamental Measures of Solute Retention

 Capacity factor is equivalent to:
C sVs
k' 
C mVm
Cs
Under equilibrium
K
C m (partition coefficient)
conditions
Vs
k'  K
Vm
Capacity factor is directly proportional to partition coefficient

Example: The retention volume of a solute is 76.2 mL for a column with Vm = 16.6 mL
and Vs = 12.7 mL. Calculate the capacity factor and the partition coefficient for this
solute.
t r  t m Vr  Vm
k' 
tm Vm
 Analytical Separations
Chromatography

5.) Efficiency of Separation

 The separation of two solutes in chromatography depends both on the width of
the peaks and their degree of retention

 The separation between the two solutes is given by their Resolution (Rs)
 Analytical Separations
Chromatography

6.) Why Bands Spread?

 Multiple Flow Paths – As solute molecules travel through the column, some
arrive at the end sooner then others simply due to the different path traveled
around the support particles in the column that result in different travel
distances.

Molecules enter the Molecules exit the column at

column at the same time different times due to different
path lengths
 Analytical Separations
Chromatography

7.) Types of Liquid Chromatography

 Adsorption Chromatography
- Solutes are separated based on their different abilities to adsorb to the
support’s surface

- Uses an underivatized solid support (stationary phase = solid support)

- Oldest type of chromatography, but not commonly used
 Analytical Separations
Chromatography

7.) Types of Liquid Chromatography

 Partition Chromatography
- Solutes are separated based on their different abilities to partition between the
stationary phase and mobile phase.

- Uses a solid support coated or chemically derivatized with a polar or non-

polar layer
- Most common type of liquid chromatography at present. Good for most
organic compounds
- Reversed Phase: stationary phase is non-polar
- Normal Phase: stationary phase is polar
 Analytical Separations
Chromatography

7.) Types of Liquid Chromatography

 Ion-Exchange Chromatography (Already discussed)
- Used to separate ions based on their different abilities to interact with the
fixed exchange sites.

- Uses a solid support containing fixed charges (exchange sites) on its surface
- Cation-Exchange: support with negative groups
- Anion-Exchange: support with positive groups
 Analytical Separations
Chromatography

7.) Types of Liquid Chromatography

 Size Exclusion Chromatography
- Separates large and small solute based on their different abilities to enter the
pores of the support

- Uses a porous support that does not adsorb solutes

- Commonly used to separate biological molecules or polymers which differ by
size (MW)
 Analytical Separations
Chromatography

7.) Types of Liquid Chromatography

 Affinity Chromatography
- Separates molecules based on their different abilities to bind to the affinity
ligand

- Uses a support that contains an immobilized biological molecule (affinity

ligand)
- Commonly used to purify and analyze biological molecules
- Most Selective type of Chromatography