CHE 311 SEPARATION

TECHNIQUES
Lecturer : Mr M.Chinyama
Office: 246/S114
Topics to be covered;
•Introduction to Liquid Chromatiography.
•Introduction to Gas Chromatography.
•Quantitative Analysis
NB: Assessment dates to be confirmed.
INTRODUCTION TO HIGH PERORMANCE
LIQUID CHROMATOGRAPHY
EARLY CHROMATOGRAPHY ==GLASS COLUMNS WITH DIAMETERS
1-5 CM AND LENGTHS 50 – 500 CM, WITH PARTICLES OF PACKING
150 -200 MICRO METERE, GRAVITY FLOW OR PERISTALTIC
PUMP TO APPLY MOBILE PHASE TO COLUMN
 RESULTS OF EARLY GLASS
COLUMN LIQUID CHROMATOGPHY

• broad peaks

• low flow rates, leading to

longer analysis time

• columns can only

tolerate low operating
pressures
VAN DEEMTER EQUATION
APPLIED TO LIQUID
CHROMATOGRAPHY
 ?? INCREASE FLOW RATE

• increase in plate height (H) ==decrease in

column efficiency
?? decrease particle size of packing
• In 1960s particle sizes reduced to 3 – 10 nm

• This requires sophisticated instrumentation

?
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
SCHEMATIC DIAGRAM:HPLC
• Components

• Solvent chamber(mobile phase)

• High pressure pump

 Injector port== sample introduction

 Analytical column==separation of analytes

 Detector=== analyte detection

EXAMPLE OF SEPARATION

characteristics:

• narrow, resolved peaks

•short analysis times

•Separation of complex
mixtures
 MOBILE PHASE/SOLVENT
SYSTEM

Isocratic elution: use of a constant mobile phase

composition to elute solutes

Gradient elution: changing composition of mobile phase

with time solvent programming‚ going from a weak
mobile phase to a strong one. Solvent change can be
stepwise, linear or non-linear
THE ELUOTROPIC SERIES
 Stationary phases

• What’s in the name???

• Liquid Chromatography=== separation technique-
mobile phase is a liquid
• STATIONARY PHASE== Several different kinds of
stationary phases are available.
• NB: Stationary phases components include solid
support,

LAAQ-B-LC001B
LAAQ-B-LC001B
LAAQ-B-LC001B
LAAQ-B-LC001B
 • NB: different types of LC based on the type of interaction
between the stationary phase and analytes/solutes.

LAAQ-B-LC001B
 HPLC Separation Modes

• Normal phase chromatography

• Reversed phase chromatography

NB: Classification is based on the POLARITY OF THE

STATIONARY PHASE AND MOBILE PHASE. It is mainly
applicable to Adsorption chromatography and Partition
chromatography.

LAAQ-B-LC001B
 Normal Phase / Reversed Phase

Stationary
Mobile phase
phase
Normal High polarity Low polarity
phase (hydrophilic) (hydrophobic)

Reversed Low polarity High polarity

phase (hydrophobic) (hydrophilic)

23

LAAQ-B-LC001B
 Normal Phase Chromatography

• the stationary phase has a high polarity

(hydrophilic) and the mobile phase has a
low polarity (hydrophobic)

24

LAAQ-B-LC001B
 Stationary phases used in
Normal phase

1. Liquid-solid (adsorption Chromatography)

Examples: Silica, alumina

LAAQ-B-LC001B
 2. liquid-liquid /partition
chromatography
• NB: Stationary phase is a thin film of polar liquid
covalently bonded to the solid-support ( Mostly silica).
Examples:,

• Cyano type: -Si-CH2CH2CH2CN

• Amino type: -Si-CH2CH2CH2NH2

LAAQ-B-LC001B
 • NB: Most commonly used bonded phase stationary
phases have been chemically bonded to silica solid
support

LAAQ-B-LC001B
 Mobile phase for Normal phase
Chromatography
• Non-polar (low polarity)
Examples; Hydrocarbons, such as hexane (C6H14)
and toluene (C7H8 ) Chloroform and ethers
NB: 1. The least polar analytes are eluted first.
2. The elution time decreases with increasing
solvent polarity.

LAAQ-B-LC001B
 • Application:

• Separation of polar compounds (amino acids and

peptides, alcohols, carboxylic acids). This are large
molecules and are mostly non-volatile.
• NB: polar stationary phase

LAAQ-B-LC001B
 • Polar Functional Groups

• -COOH
-Carboxyl groups
• -NH2
-Amino groups
• -OH
-Hydroxyl groups

LAAQ-B-LC001B
LAAQ-B-LC001B
 • 1. NB: Historically: Charcoal and other inert non-polar
materials used as stationary phase (Adsorption
chromatography).

• 2. Partition chromatography (bonded phase) NB:

Stationary phase is a thin film of non- polar liquid
covalently bonded to the solid-support ( Mostly silica).
• Examples:,

LAAQ-B-LC001B
 Mobile phase/Solvents

• NB: Polar mobile phase

• A mixture of water and organic solvents used
(water, acetonitrile, tetrahydrofuran, methanol,
acetone etc).
• NB; 1.The most polar analytes are eluted first.
• 2. Elution time increases with increasing solvent
polarity.

LAAQ-B-LC001B
 • R=C18 (otadecyl ), R=C8 (Octyl) & R=phenyl

LAAQ-B-LC001B
 • Application:
• Separation of non-polar compounds. This are large
molecules and are mostly non-volatile.

• NB: non-polar stationary phase

LAAQ-B-LC001B
 • Non-polar Functional Groups

• -(CH2)nCH3
-Alkyl groups
• -C6H5
-Phenyl groups

LAAQ-B-LC001B
 Adsorption Chromatography
Separates solutes based on their adsorption to
solid adsorbent/stationary phase

LAAQ-B-LC001B
LAAQ-B-LC001B
 HPLC detectors
• role of a detector =
• detect the analytes eluted from the
column and measures (quantifies) the
amount of the analyte

• Detection is based on ionization or other

changes (physical, optical or
electrochemical properties) of the
analyte
39
 Examples of HPLC detectors

o UV/Vis Absorbance Detector (solute property)

o Fluorescence Detector (specific to compounds which fluoresce=
solute property)
o Refractive Index Detector (universal to all compounds= bulk
property)
o Conductivity Detector (specific to any ionic compounds)
o Electrochemical Detector( electrochemically active compounds)
• Mass spectrometer (universal in full scan mode)

The selection of a detector is based on the chemical

nature of the analytes, the limit of detection required,
and often the availability and cost of the detector
41
Advantages of HPLC
 High sensitivity
 Quantitative analysis possible
• Separation of non-volatile high molecular weight species

• Thermally labile compounds analysis

Disadvantage of LC
LC is subject to greater peak or band-broadening.

42
 GAS CHROMATOGRAPHY
• Separation of a gaseous analyte or volatile
liquid by a gaseous mobile phase and a
solid or liquid stationary phase
 Characteristics/properties of analytes
analysed by GC
• Mainly organic, with MW <500
• Samples must be volatile, Low boiling point
• Usually non-polar
• examples
Volatile organic compounds : boiling points range
(50 – 260 deg)

Examples of gaseous samples

Airborne pollutants

Volatiles from liquid samples

Volatiles from solid samples

 Components of GC
• A gas chromatograph consists of 3
critical components:
• 1) an inlet for introduction of the
sample

• 2) a column to separate the analytes

• 3) detector to detect analytes as they

emerge from the end of the column
 Carrier Gas (Mobile phase)

 Carrier gas == a stream of gas to carry analytes over stationary

phase

The carrier gas must be chemically inert to GC hardware, column and

the sample.

Helium, nitrogen and hydrogen gases can be used.

• Helium
– Most common but expensive
– Compatible with most detectors
– (u opt= 35 cm/s)

• Hydrogen gas
– Fastest separation ((u opt= 50 cm/s)

– BUT can catalytically react with unsaturated

compounds on metal surfaces
– Forms explosive mixtures in air (flammable)

• Nitrogen gas- provides the highest max resolution (u

opt= 15 cm/s), but slow
 GC Columns
• GC use 2 main types of columns:
1.Capillary columns/Open tubular columns.
These columns can further be divided into
a)WCOT-Wall coated Open Tubular
( Stationary phase coated on the inner
wall of the column)
b)PLOT-Porous layer Open Tubular –
( Stationary phase coated on support
material). Also Called Support Coated
Open Tubular (SCOT)
• 2. Packed columns
• Packe
d

•
Capillary
• NB: Capillary columns are more efficient
gives and give good resolution compared
to packed columns. This is due to the
absence of the multiple path term (A) in
the van Deemter equation.
Example: Capillary Column
Chromatogram
 Variables That Affect Column
Performance (Reminder!!!!)
• Resolution in terms of capacity factors,
efficiency, and selectivity factor
First term related to the kinetics that lead to band broadening

Second term is a selectivity term Third term depends on properties of both the
that is only related to the solute and the column
properties of the two solutes
Column length
 Column internal diameter
• Efficiency is indirectly proportional to
internal diameter
• Smaller diameter gives better resolution
• Larger diameter allows for larger sample
capacity
• Optimal column to start analysis is 0.25-
0.32 mm ID
• Generally resolution is improved with
– Narrow columns
– Longer columns
 Stationary phases
• Similar to HPLC, GC uses both polar
and non-polar stationary phases.
Examples;
 Choice of stationary phase
• * like dissolves like*
– Non-polar columns, non-polar solutes
– Polar cpds, polar columns
• Use stationary phase with polarity similar to that of
analytes

• Stat phases can degrade with age and exposure to O2,

stat phase can also break off and increase tailing
 Stationary phase film thickness
• Most frequently used is 0.25-1.0 um
• Retention is directly proportional to film
thickness
• Thick films used to obtain high retention for
very volatile compounds
• Thick films bad for late eluting peaks
• Column bleed increases with thickness
• Capacity increases with increase in film
thickness
 Factors affecting GC separation
Volatility of components
Polarity of compounds
Flow rate of carrier gas

Column temperature

Stationary phase polarity

Column length
Column internal diameter
Stationary phase film thickness
b) Oven temperature (Column temp)

• Isothermal-constant temp throughout

• Sufficient for simple separations of analytes with
similar retention. Elution based on Boiling
point/MWE.g hydrocarbons.
Consider a complex mixture e.g petrol , made up of 10
different hydrocarbons, with different volatilities, can
separation be achieved with isothermal temp program?
• In GC, temp of column is raised during separation (temp
programming)

– to volatilise the sample

– decreases retention time of late-eluting components

e.g if column maintained at 150°C, the less volatile cpds

may not elute but if increased from 150 to 250°C all
cpds can be eluted and separation of peaks is uniform
• But not too high temp to
– Decompose analytes
– decompose stationary phase
• Column should be hot enough to provide
sufficient vapor pressure for analytes to be
eluted in a reasonable time
GC DETECTORS
Common detectors available for GC.
1. TCD (thermal conductivity detector).= operates
on the changes in the thermal conductivity of the gas
stream brought about by the presence of analyte
molecules.

2. FID (flame ionization detector) =based on the

production of ions when compounds are burned.

3. Mass spectrometer (universal in full scan mode)

 4. ECD (electron capture detector)= uses Ni-63 as

a radiation source to cause ionization of the
substance. Detector is sensitive to functional groups
containing electronegative species such as halogens
and nitro groups.
Flame Ionization Detector
Electron Capture
Detector(ECD)
Gas Chromatography-Mass
Spectrometry
 Why use GC?
• GC is fast

• Has high efficiency due to the fact that analytes

will have higher diffusitivities in gases

• Can be used to separate complex mixtures

• Good for quantitative analysis

• Utilizes sensitive detectors
 Limitations
• Limited to analysis of volatile compounds

• Not suitable for thermally labile

compounds

• May require derivatization for polar

compounds-increasing sample handling
steps and hence increases sample loss