PTT 202
ORGANIC CHEMISTRY FOR
BIOTECHNOLOGY
Lecture 3:
Separation Methods
Zulkarnain Mohamed Idris
zulkarnainidris@unimap.edu.my
Semester 1 2013/2014
Classification of Separation Methods
1. Chromatography
is adsorption based separation process
Used to
separate
biomolecules,
fine &
specialty
chemicals
Analytical
tools:
To determine
chemical
compositions
of sample
Preparative
tools:
To purify &
collect
one/more
components of
a sample
Chromatography
SEPARATION TECHNIQUES
AC
HIC
SF: Super Critical; GS: Gas-Solid; GL: Gas-Liquid ; A: Affinity ; HI:
Hydrophobic/Hydrophilic; NP: Normal-Phase; RP: Reverse-Phase; IE: Ion Exchange; SE: Size
Exclusion; GP: Gel Permeation; GF: Gel Filtration; TL: Thin-Layer. C: Chromatography
SEPARATION TECHNIQUES:
Choice of MethodsMolecular Size /Polarity
Chromatography
BASIC SEPARATION PRINCIPLES
1
Solutes in solution or
volatiles in gas are
placed in mobile
phase & passed
over a selected
‘adsorbent’ material
(stationary phase)
Mobile phase:
Continuous
flow of a
carrier liquid
or gas
Stationary
phase:
A bed of
solids or
immobilized
liquid
2
The solutes or
volatiles have
differential ‘affinity’
for the adsorbent
material & thus
separate
Chromatography
BASIC SEPARATION PRINCIPLES
Example 1:
Hydrophobic interaction
Example 2:
Ionic interaction
SEPARATION TECHNIQUES IN
CHROMATOGRAPHY
LIQUID CHROMATOGRAPHY
1
i.
ii.
iii.
iv.
v.
Affinity Chromatography
The most selective type of chromatography
employed.
Utilizes the specific interaction between one
kind of solute molecule and a second
molecule that is immobilized (ligand) on a
stationary phase.
For example, the immobilized molecule may
be an antibody to some specific protein.
When solute containing a mixture of proteins
are passed by this molecule, only the specific
protein is reacted to this antibody, binding it
to the stationary phase.
This protein is later eluted by changing the
ionic strength or pH.
“Lock-and-key concept”
Ligand
Specific protein
(e.g. antibody) binds to ligand
Stationary
phase
Non-specific
proteins
SEPARATION TECHNIQUES IN
CHROMATOGRAPHY
LIQUID CHROMATOGRAPHY
2
i.
ii.
iii.
iv.
v.
Hydrophobic Chromatography
Typically used for protein separations
Employs derivatized polymer resins, with
phenyl, butyl, or octyl ligand groups
Stationary
phase
Protein adhere to the hydrophobic surface
under high salt conditions and redissolve
Ligand
into the mobile phase as the salt
(e.g. phenyl group)
concentration is reduced
By increasing the salt concentration of
solvent, these hydrophobic patches of
protein become more exposed and
interact with hydrophobic ligands on the
packing.
the
the
can
HIC
HIC is sensitive to pH, salt used, buffer type
and temperature.
Stationary
phase
SEPARATION TECHNIQUES IN
CHROMATOGRAPHY
LIQUID CHROMATOGRAPHY
3
i.
ii.
iii.
iv.
v.
Reverse Phase Chromatography
Employs a hydrophobic phase bonded to the
surface of the resin – typically silica based
Hydrophobic solutes bind in higher proportion
in reversed phased
Hydrophobic phases that are bonded to silica
are typically actyil (C8), actyldecyl (C18),
phenyl, and methyl (C1)
Different chain lengths and densities of the
different bonded phases lead to more or less
hydrophobicity
Bare silica participate in separation by
interacting with hydrophilic molecules, or
hydrophilic domains of large molecules
Stationary
phase
Ligand
(e.g. actyldecyl)
Stationary
phase
SEPARATION TECHNIQUES IN
CHROMATOGRAPHY
LIQUID CHROMATOGRAPHY
4
i.
ii.
iii.
iv.
v.
Ion Exchange Chromatography
Biomolecules generally have charged groups
on their surfaces, which change with the pH
of the solution
Molecule reversibly binds to an oppositely
charged group of the packing material
Molecules with a higher charge density bind
more strongly to the packing
The bound sample may be selectively
removed from the stationary phase by
changing the pH or salt concentration of the
mobile phase
It is particularly effective for proteins
because they are amphoteric
>Charge
density
Stationary
phase
Ligand
<Charge
(e.g.
actyldecyl)
density
Stationary
Stationary
phase
phase
Competing
ions
SEPARATION TECHNIQUES IN
CHROMATOGRAPHY
LIQUID CHROMATOGRAPHY
5
i.
Size Exclusion Chromatography
Also referred to as gel permeation
chromatography (GPC) for non-aqueous elution
systems or gel filtration chromatography (GFC)
for aqueous systems.
ii.
Separates solutes on the basis of their size
iii.
No binding between the solutes and the resin
iv.
v.
vi.
Smaller molecules can partially or completely
enter the stationary phase.
Molecules with
different sizes
Stationary
phase
Smaller
molecules
Because these smaller molecules have to flow
through both, the interparticle space, as well as
through the pore volume, they will elute from the
column after the excluded sample components
Used for removing small molecules from protein
solution
Large
molecules
Pores
Types of Resins (Stationary Phase)
SILICA-BASED RESINS
Uncoated silica
i.
ii.
iii.
iv.
v.
vi.
vii.
Compatible with water or organic
solvent
Serves as a good reversible adsorbent
for hydrophilic compounds
Organic solvent used as mobile phase,
and water is added as the
chromatography progresses
Not typically stable at extremes of pH
Available with high surface area and
small particle size; being very rigid;
does not collapse under high pressures
Denature some proteins and irreversibly
bind others
Used for purification of many
commercial biotechnology products
Coated silica
i.
ii.
iii.
Particles
alkanes
coated
with
long-chain
Has a high affinity for hydrophobic
molecules, which increases as the
chain length of the bonded alkane
increases.
Many varieties of the same chain
length phase – polymerized, simple
monolayer and end-capped
Types of Resins (Stationary Phase)
POLYMER-BASED RESINS
Synthetic polymers
Styrene divinylbenzene :
i.
ii.
ii.
o
Very stable at pH extremes
Support
for
ion
exchange
chromatography because of its
stability and rigidity
Polyacrylamide:
i.
Natural Polymers
used less often, not used as a
polymer solid but as hydrogel and
used as a size exclusion gel
The crosslinking in polyacrylamide
can be controlled by the amount of
bisacrylamide added in suspension
mixture
o
o
Used in hydrogel for
chromatography resins.
a
low
pressure
Naturally hydrophillic
Compatible
biomaterials
with
proteins
and
other
Agarose :
i.
can be crosslinked to form a reasonably
rigid bead that is capable of tolerating
pressures up to 4 bar.
Dextran:
i.
Less rigid and used in size exclusion
ii.
Can be formed with very large pores
iii.
Capable of including antibody molecules
and virus particles
Types of Resins (Stationary Phase)
ION EXCHANGE RESINS
Resins that have been derivatized with an ionic group
Most commonly used ionic groups:
i. sulfoxyl (SO3-) - most acidic
ii. carboxyl (COO-)
iii. diethylaminoethyl (DEAE) (2C2H5N+HC2H5)
iv. quaternary ethylamine (QAE) (4C2H5N+) - most basic
i.
Cation exchangers
Acidic ion exchanger
i.
Anion exchangers
Basic ion exchangers
ii.
Carry a negative charge
ii.
Carry a positive charge
iii.
Attract positive counterions
iii.
Attract negative counterions
Selection of Mobile Phase
1. Compatibility with stationary phase:
-must no react chemically with the stationary phase or break the
bond linking it to the supporting materials.
-Extreme pH or strong oxidizing agents should normally be
avoided.
2. Compatibility with detection system:
-the mobile phase must not interfere with the detection system.
-the solvent used must not absorb significantly at the wavelength
used.
-e.g: absorption at 280 nm is frequently used to detect protein but
some solvents such as acetone absorb at this wavelength, so this
solvent must be avoided as a mobile phase.
Selection of Mobile Phase
3. Polarity:
-the major factor in selecting a mobile phase is the polarity of the
solute or analyte (or molecules that are going to be separated).
-the polarity of the mobile phase should be such that there is an
effective partition of the solute or analyte between the two phases
(stationary and the mobile phases).
-gradient elution is required in which the solvent strength (or the
ability the solvent to break adsorptive bonds and elute the solute
from the adsorbent) of the mobile phase is gradually changed
during the separation process by altering the solvents in the
mixture.
4. Pressure consideration:
-solvents chosen should achieved the desired separation without
requiring pressures too high for the system.
SEPARATION TECHNIQUES IN
CHROMATOGRAPHY
GAS CHROMATOGRAPHY (GC)
Gas chromatographic equipment
Chromatographic
separation system
Packed column
Capillary column
SEPARATION TECHNIQUES IN
CHROMATOGRAPHY
CROSS-SECTIONAL VIEWS OF
PACKED & CAPILLARY COLUMNS
SEPARATION TECHNIQUES IN
CHROMATOGRAPHY
SEPARATION PROCESS IN GAS CHROMATOGRAPHY
i.
ii.
iii.
iv.
v.
Separation
method
based
on
conversion of sample to vapor phase
Liquid film of solvent
+ samples
The sample vapor is introduced onto a
column (packed/capillary) containing
stationary phase material
Carrier gas
(He)
Column
The mobile phase is typically an inert
gas such helium (He), nitrogen (N2) and
hydrogen (H2)
Separation occurs on the basis of
interaction between the sample
components and the stationary phase
Because the sample must be
maintained in the vapor phase, the
column is contained within an oven.
Sample vapor
Solvent vapor
TYPES OF ADSORBENTS
(EXAMPLES: STATIONARY PHASE FOR GAS CHROMATOGRAPHY)
Composition
Polarity
Applications
Temp limits
100% dimethyl polysiloxane
(Gum)
Nonpolar
Phenols, Hydrocarbons, Amines,
Sulfur compounds, Pesticides, PCBs
-60oC to
325oC
100% dimethyl polysiloxane
(Fluid)
Nonpolar
Amino acid derivatives, Essential oils
0oC to 280oC
5% diphenyl 95% dimethyl
polysiloxane
Nonpolar
Fatty acids, Methyl esters, Alkaloids,
Drugs, Halogenated compounds
-60oC to
325oC
14% cyanopropyl phenyl
polysiloxane
Immediate
Drugs, Steroids, Pesticides
-20oC to
280oC
50% phenyl, 50% methyl
polysiloxane
Immediate
Drugs, Steroids, Pesticides, Glycols
60oC to
240oC
50% cyanopropylmethyl, 50%
phenylmethyl polysiloxane
Immediate
Fatty acids, Methyl esters, Alditol
acetates
60oC to
240oC
50% trifluoropropyl polysiloxane
Immediate
Halogenated compounds,
+Aromatics
45oC to
240oC
Polyethylene glycol – TPA
modified
Polar
Acids, Alcohols, Aldehydes acrylates, 60oC to
Nitriles, Ketones
240oC
Polyethylene glycol
Polar
Free acids, Alcohols, Ethers, Essential
oils, Glycols, Solvents
60oC to
220oC
SEPARATION TECHNIQUES IN
CHROMATOGRAPHY
Resolution in Chromatography: To measure the
ability of column to separate two peaks
Rs= (tR2-tR1)/0.5 (wb1+wb2)
Wb1
Wb2
SEPARATION TECHNIQUES IN
CHROMATOGRAPHY
The separation efficiency of a column can be
expressed in term of the number of theoretical
plates in the column, (N):
N= 16 (tR/wb)2
SEPARATION TECHNIQUES IN
CHROMATOGRAPHY
Resolution in Chromatography (Rs)
Example:
Ethanol and methanol are separated in a
capillary GC column with retention times of
370 and 385 s, respectively, and base width
(wb) of 16.0 and 17.0 s. Calculate the
resolution (Rs) and the number of plates (N).
SEPARATION TECHNIQUES IN
CHROMATOGRAPHY
Resolution in Chromatography (Rs)
Answer:
Rs= (tR2-tR1)/0.5 (wb1+wb2)
= (385-370)/0.5(17.0+16.0)
= 0.91
SEPARATION TECHNIQUES IN
CHROMATOGRAPHY
The number of plates (N)
Answer:
Use the longest eluting peak to calculate N:
N= 16 (385/17.0)2
= 8.21 x 103 plates
2. Dialysis
- A technique for separating macromolecules from small
-
solute molecules.
Refers to diffusion of the solute molecules through a
membrane which restricts the movement of large molecule
(depends on the pore size).
The passage of the small molecules is due to a
concentration gradient across the membrane.
Cellophane is frequently used for dialysis and it has a
pore size of approximately 4-8 µm, makes it
impermeable to molecules with relative molecular mass in
excess about 10 000 Da.
Dialysis
(Low concentrated)
3. Ultrafiltration
- The solvent and solute are
forced
through
the
membrane under pressure
and the movement of large
molecules is restricted by
the pore size.
- Various cellulose and
polycarbonate membranes
are available with pore
size down to 5 nm which
are capable of excluding
molecules with a relative
molecular mass of 50 Da.