This document provides an overview of liquid chromatography (LC). It discusses various aspects of LC including:
- Common stationary phases used in column and thin layer chromatography methods
- How separation is based on differences in solute partitioning between mobile and stationary phases
- Developments in instrumental LC, including the use of solvent gradients to improve separation
- Components of HPLC systems such as pumps, injection valves, and detectors
- Factors that influence chromatographic separation including stationary phase chemistry, solvent selection, temperature, and pressure
- Trends in smaller particle sizes and bonded stationary phases to improve column efficiency
ION EXCHANGE CHROMATOGRAPHY
ByM.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
ION EXCHANGE CHROMATOGRAPHY
ByM.Vharshini
B.Sc. Bio Medical Science
Sri Ramachandra University
ION EXCHANGE CHROMATOGRAPHY
Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger.
It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids.
Cations or Anions can be separated using this method.
PRINCIPLE
It is based on the reversible electrostatic interaction of ions with the separation matrix (i.e.)
The separation occurs by reversible exchange of ions between the ions present in the solution and those present in the ion exchange resin.
CLASSIFICATION OF RESINS
According to the chemical nature they classified as-
1. Strong cation exchange resin
2. Weak cation exchange resin
3. Strong anion exchange resin
4. Weak anion exchange resin
According to the Source they can -
Natural resins : Cation - Zeolytes, Clay
Anion - Dolomite
Synthetic resins: Inorganic & Organic resins
◘Organic resins are polymeric resin matrix.
The resin composed of –
Polystyrene (sites for exchangeable functional groups)
Divinyl benzene(Cross linking agent)-offers stability.
Ion exchange resin should have following requirements
»It must be chemically stable.
»It should be insoluble in common solvents.
» It should have a sufficient degree of cross linking.
»The swollen resin must be denser than water.
»It must contain sufficient no. of ion exchange groups.
Physical properties of ion exchange resins
Cross linking:
It affects swelling & strength & solubility
Swelling:
When resin swells, polymer chain spreads apart
Polar solvents → swelling
Non-polar solvents → contraction
Swelling also affected electrolyte concentration.
Particle size and porosity
Increase in surface area & decrease in particle size will increase the rate of ion exchange.
Regeneration
Cation exchange resin are regenerated by treatment with acid, then washing with water.
Anion exchange resin are regenerated by treatment with NaOH, then washing with water until neutral.
EXPERIMENTAL SETUP OF ION EXCHANGE CHROMATOGRAPHY
Metrohm 850 Ion chromatography system
Instrumentation of ion exchange chromatography
PRACTICAL REQUIREMENTS
1.Column
» glass, stainless steel or polymers
2.Packing the column
» Wet packing method:
A slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column. Care must be taken to avoid air bubbles.
3.Application of the sample
After packing, sample is added to the top of the stationary phase, use syringe or pipette.
This layer is usually topped with a small layer of sand or with cotton or glass wool to protect the shape of the organic layer from the velocity of newly added eluent.
4.Mobile phase
Acids, alkalis, buffers…
6.Stationary phase
The ionic
Ion exchange chromatography -SlideShareRIZWAN RIZWI
This ppt provide a good knowledge about ion exchange chromatography. I think this is very helpful for you .Here i have tried to explain a best way and simple method so guys you all enjoy this and gain your knowledge. And wish for me to provide more pptx for you all .at the end i want your experience give me suggestion if i made any mistake thank you .
Solubilities of Crystalline Drugs in Polymers: An Improved Analytical Method ...Simon Curtis
Solubilities of Crystalline Drugs in Polymers: An Improved Analytical Method and Comparison of Solubilities of Indomethacin and Nifedipine in PVP, PVP/VA, and PVAc - YE SUN,1 JING TAO,1 GEOFF G. Z. ZHANG,2 LIAN YU1
This is a type of chromatography in which similar charged ions are separated by using ion exchange resin, that exchanges ions according to their relative affinities.
Ion exchange chromatography -SlideShareRIZWAN RIZWI
This ppt provide a good knowledge about ion exchange chromatography. I think this is very helpful for you .Here i have tried to explain a best way and simple method so guys you all enjoy this and gain your knowledge. And wish for me to provide more pptx for you all .at the end i want your experience give me suggestion if i made any mistake thank you .
Solubilities of Crystalline Drugs in Polymers: An Improved Analytical Method ...Simon Curtis
Solubilities of Crystalline Drugs in Polymers: An Improved Analytical Method and Comparison of Solubilities of Indomethacin and Nifedipine in PVP, PVP/VA, and PVAc - YE SUN,1 JING TAO,1 GEOFF G. Z. ZHANG,2 LIAN YU1
This is a type of chromatography in which similar charged ions are separated by using ion exchange resin, that exchanges ions according to their relative affinities.
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The lecture is devoted to newbies in chemistry eager to learn the basics of nomenclature. The tips given re easy and can be practiced to get perfection.
Principles of chromatography
Chromatography’ is an analytical technique commonly used for separating a mixture of chemical substances into its individual components, so that the individual components can be thoroughly analyzed. There are many types of chromatography e.g., liquid chromatography, gas chromatography, ion-exchange chromatography, affinity chromatography, but all of these employ the same basic principles.
Chromatography is a separation technique that every organic chemist and biochemist is familiar with. I, myself, being an organic chemist, have routinely carried out chromatographic separations of a variety of mixture of compounds in the lab. In fact, I was leafing through my research slides and came across a pictorial representation of an actual chromatographic separation that I had carried out in the lab
1. First chromatographic method described (as a
non-instrumental method).
Since samples don’t need to be initially
vaporized, potentially any compound can be
assayed by this method.
Instrumental development lagged behind that of
GC because of difficulties in creating a stable
solvent flow.
The stationary phase is a solid. Separation is
due to a series of adsorption/desorption steps.
We’ll be spending
most of our time with
instrumental LC methods.
However, column and
planer (TLC) methods
can also be used.
Silica and alumina are common stationary Separation is based on solute partitioning
phases. between two liquid phases - (relative
solubility).
Both solute and solvent are attracted to the
polar sites on the stationary phase.
If solutes have differing degrees of
attraction to the phase, a separation is
possible.
2. This approach comes closest to our
countercurrent extraction model.
More highly retained species have a greater
affinity (solubility) for the stationary phase -
compared to the mobile phase (solvent)
Separation of solutes is based on differences in
this relative solubility.
The elution order will be somewhat The stationary phase has an ionically charged
2
reversed but not exactly - other 3 surface, opposite that of the eluents.
factors must also be considered.
4
5
-
2 1
+ + -
4 5
1
3
-
For instrumental LC, weak exchange
resins are typically used.
These are typically exchange groups
bound to a support.
The traditional exchange resin beads
would be crushed under normal HPLC
conditions.
3. Separation is based on molecular size.
Stationary phase is a material of controlled
pore size. Also called gel permeation.
Columns can be obtained that will This example
separate specific size ranges. shows
three general
25,000
50,000
Larger species will elute first - they can’t classes of
components.
pass through as many pores so their
5,000
path is shorter. The second
has a much
Useful for determining size and size range larger size
for polymers, proteins, ... distribution.
4. Optimum solvent strength or polarity can be
obtained by mixing solvents.
0.3 0.4 0.5 0.6 0.7
MeCl2
0 50 100
10
methanol
10 50
ACN
0 100
Snyder extended the method of solvent Polarity is just one factor. The other is solvent
blending for reverse phase. selectivity.
He recognized that polarity is just one
factor that you can play with Rs =
N
4
(! - 1)
( k’
1 + k’ )
It relies on the use of 4 solvents in selectivity polarity
developing the optimum separation. term term
5. proton acceptors
0.2 0.7
Not all solvents are truly usable.
II
Can’t be mixed at any proportion
I
May interact chemically
III
UV absorption or viscosity is too high
IV Toxic, too flammable
VI
VIII High vapor pressure
VII V
0.7 0.2 Too expensive
proton large
0.2 0.7
donors dipole
methanol - acid
acetonitrile - base
tetrahydrofuran - large dipole
water - polarity adjustment
All are low viscosity
available in high purity
UV transparent
miscible in each other
6. 2. Create blends using each of the other 41% ACN, 59% water 30% ACN, 70% water
k’ = 5 k’ = 10
solvents and water that have the same
solvent polarity.
3. Evaluate each solvent for improvements
in peak shape or movement of selective 11% ACN, 12% THF
21% MeOH, 79% water
peaks. k’ = 10
77% water, k’ = 10
4. A mix of any of the blended solvents is
then evaluated for optimum resolution.
• Unlike GC, variations in temperature
have minimal effect on an LC
separation.
• However, variations in solvent
polarity can greatly affect retention.
• This can be accomplished by altering
the solvent mix during an analysis.
Not all LC methods can make use of
gradient elution
ion exchange - yes
liquid-liquid - difficult
bonded phase - yes
size exclusion - no
adsorption - yes
7. Starting solvent should have a polarity that
adequately resolves the first few
components.
Final polarity should adequately resolve
the last few species in a timely manner.
Now play connect the polarity - attempt
various blending rates to separate the
remaining components.
c f Unlike GC equipment, many HPLC
a b d systems have a modular design - can
simply add a new ‘box’ to change/extent
capabilities.
e
There is also a wider range of how to do
a - gradient controller d - column/pre-column
things like produce a flow or gradient.
b - pump/dampening system e - detector
c - sample introduction f - data output
We’ll cover some of the basic approaches.
All solvents should be ‘HPLC’ grade. All solvents should be degassed prior to use.
This is a type of reagent grade material. This reduces the chances of bubbles being
It has been filtered using a 0.2 µm filter. formed in the column or detector. Oxygen
present at high pressure can also cause a
You can purchase it or produce it yourself. problem.
Filtered solvent helps extend pump life by Methods that can be used
preventing scoring. It also reduces the Displacement with a less soluble gas
chances of a column plugging. Applying a vacuum
Heating the solvent.
8. Each type of system has its own advantages and
disadvantages.
Is the solvent reservoir limited?
Does it produce pressure pulses?
Can a gradient be produced?
solvent b a
outlet gas inlet to column
c
a - syringe d - motor
b - seal e - fill system
d e
c - gearing
solvent
convection Another non-pulsating system with a limited reservoir.
current Stepper motor/gear system allows for very fine flow
baffle control.
g One of the most common type of systems.
Unlimited reservoir system but expensive.
c a - motor
d b - gear Another problem is that it produces variable
c - seal pressure - must reverse stroke to refill.
b f d - piston
e - solvent in
f - check valves
g - solvent out
a
e
pump fill
9. Since the pump must spend at least a portion of
its time filling, the is a pressure drop during this
phase.
This effect must be
start of fill minimized or your
peaks will all have
pulses in them.
That would greatly
affect your sensitivity
and detection limit
start of pump
One approach is to have a more rapid fill cycle One could also use two or more pumps working
compared to the pump cycle. in tandem.
This does not eliminate the problem but does
reduce it.
This is a more expensive option.
In-line metal coil system
Reduces pulse to +/- 3% at 240 psig.
Low cost system
tube is
flattened
Flow passes through tube
- possible contamination
Limited range - about +/- 50-100 psi.
10. T type metal coil.
adjustment
screw
pump column
With this design, flow does not pass through the Allows the user to minimize pulsing under
dampner. actual operational conditions.
It still has the previous limitations Can reduce pulses to < 0.1 %
external • With LC, temperature programming is
pressure not typically an option in dealing with
source
homologous series.
Pressure source
can be a gas or • Instead, we rely on altering the nature or
a liquid
polarity of the solvent - gradient elution.
Reduces pulses to < 0.1% • The controller is the device that allow
External pressure can be monitored and you to create the gradient program.
controlled by the system.
• Gradients are produced based on the
Most expensive approach but the best usually is. type of pumping system you have.
mixing tee
pump
controller
a b
11. • These can be a bit more complex than A very common approach is the use of sampling
with GC systems. valves and loops.
sample sample
• If you attempted a manual syringe
injection, expect to find the plunger shot vent vent
into the ceiling - you might be working
with pressures as high as 5000 psi.
• A simple approach would be to stop the
flow and inject manually - not too good. column
solvent solvent
You must use ‘zero dead volume’ valves. Automated syringes
syringe
Manual and automated valve systems are
available. check valve
Major limitation is fixed sample size.
The loop must be changed in order to This method allow for adjustment of sample size.
alter sample size - does not require that The motor driven syringe can provide sufficient
the flow be stopped. pressure to inject sample past the check valve.
• A small column added between the injection HPLC has seen significant improvement over the
system and the analytical column. last 10 years primarily due to improved column
technology.
• It helps prevent entry of materials that might
want to stay on the column from your sample
or solvent. Packings are more uniform and smaller.
• Used to extend column life Phases are commonly chemically bound to the
packing.
• Should be the same packing as the analytical
column. Packing methods have improved.
12. As packing size is decreased, efficiency and
pressure requirements are increased.
Common diameters for analytical work
diameter plates
10 µm 5000
5 µm 9000
3 µm 15,000
All are for a 15 cm x 4.6 mm id column
Today, most packing fall into four categories.
Silica or alumina
Bound phases on either alumina or silica.
Gels
Controlled-pore glass or silica
13. Strong cation - sulfonic acid group Gels - organic or aqueous based
Strong anionic - quarternary amine Controlled-pore - silica or glass
Weak anion - primary amine Must be selected based on pressure
requirements and size range required
Weak cation - COOH for your application.
A solute property detector.
Sample must exhibit absorption in UV/Vis range.
Solvent must not absorb significantly at the
measured wavelength.
Types Filter photometer - single "
Variable wavelength
Multiwavelength.
Dual photocell
Light source detector
fixed "#
dual filter
flowcell photodiode array
If the filter is replaced by a monochrometer, you end up The photodiode array allow you to simultaneously
with a variable wavelength UV/Vis system monitor a range of " or obtain complete spectra.
14. Bulk property detector - general purpose.
Waters design
Based on refraction of light as it passes from one mirror
light source
media to another. Presence of a solute
changes the refractive index of the solvent.
split
flow cell adjustment
solvent only sample present detector
control
Varian design flow
cell
adjustment detector
control
light source
Bulk property detector.
Measures changes in polarity of the
liquid phase passing through the
cell.
15. • Most frequently applied of electrochemical
Measures conductivity of the solvent. detectors.
Useful for solutions of ions
• A known potential is applied across a set of
electrodes - typically a glassy carbon type
• Ability to oxidize or reduce a species can be
measured.
• Typically limited to working with a specific
class of materials per analysis.
electrodes
Several electrodes and combinations can be
used. Allows for some interesting data.