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Chromatography is a combination of two words;
* Chromo – Meaning color
* Graphy – representation of something on paper (writing)
Chromatography
Invention of Chromatography
Mikhail Tswett
Russian Botanist(1872-1919)
Mikhail Tswett inventedchromatography in 1901 duringhis research on plant pigments.
He used the technique to separatevarious plant pigments such asChlorophylls, Xanthophylls andCarotenoids.
Original Chromatography Experiment
Later
Start: A glass
column is filled
with powdered
limestone
(CaCO3).
End: A series of
colored bands is
seen to form,
corresponding to
the different
pigments in the
original plant
extract. These
bands were later
determined to be
chlorophylls,
xanthophylls and
carotenoids.
An EtOH extract
of leaf pigments
is applied to the
top of the column.
EtOH is used to
flush the pigments
down the column.
It is a physical separation method of separation in which the components of a
mixture are separated by differences in their distribution between two phases, one
of which is stationary (stationary phase) while the other (mobile phase) moves
through it in a definite direction. The substances must interact with the stationary
phase to be retained and separated by it.
Definition of chromatography
Definition of chromatography
IUPAC definition :
Chromatography is a physical method of separation in which the components to be
separated are distributed between two phases, one of which is stationary while the other
moves in a definite direction.
The stationary phase may be a solid, or a liquid supported on a solid or gel, the
mobile phase may be either a gas or a liquid.
Mixture
Separate
Analyze
• Identify
• Purify
• QuantifyComponents
Chromatography is used by scientists to:
•Analyze – examine a mixture, its components, and their relations to one another
•Identify – determine the identity of a mixture or components based on known
components
•Purify – separate components in order to isolate one of interest for further study
•Quantify – determine the amount of the a mixture and/or the components
present in the sample
Chromatograph: Instrument employed for a chromatography.
Eluent: Fluid entering a column.
Eluate: Fluid exiting the column.
Elution: The process of passing the mobile phase through the column.
Flow rate: How much mobile phase passed / minute (ml/min).
Linear velocity: Distance passed by mobile phase per 1 min in the column (cm/min).
Mobile Phase – gas or liquid that carries the mixture of components through the
stationary phase.
Stationary Phase – the part of the apparatus that holds the components as they move
through it, separating them.
Real-life examples of uses for chromatography:
Pharmaceutical Company
Hospital
Law Enforcement
Environmental Agency
Manufacturing Plant
Retention time:
It is the characteristic time it takes for a particular analyte to passthrough the system (from the column inlet to the detector) under setconditions.
Retardation factor (R):
Fraction of an analyte in the mobile phase of a chromatographic system.
•Liquid Chromatography – separates liquid samples with a liquid solvent (mobile
phase) and a column composed of solid beads (stationary phase)
• Gas Chromatography – separates vaporized samples with a carrier gas (mobile
phase) and a column composed of a liquid or of solid beads (stationary phase)
•Paper Chromatography – separates dried liquid samples with a liquid solvent
(mobile phase) and a paper strip (stationary phase)
•Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent
(mobile phase) and a glass plate covered with a thin layer of alumina or silica gel
(stationary phase)
Paper and Thin Layer Chromatography
Later
The solvent moves up paper by capillary action,
carrying mixture components at different rates.
solvent
solvent
front
Thin Layer Chromatography
Here the mobile phase is a liquid
Flowing past a thin layer of powder on a solid support.
Substances that are less attracted to the solid or are more
soluble in the liquid move faster.
And so move further up the plate by the time that the process has been
stopped by taking the plate out of the liqiud. - larger Rf
Rf = distance moved by substance
distance moved by solvent front
For substances that are very soluble in the liquid Rf will be close to ....
For substances that are rather insoluble in the liquid Rf will be close to ....
1
0
How Does Chromatography Work?In all chromatographic separations, the sample is transported in a mobile phase. The mobile phase can be a gas, a liquid, or a supercritical fluid.
The mobile phase is then forced through a stationary phaseheld in a column or on a solid surface. The stationary phase needs to be something that does not react with the mobile phase or the sample.
The sample then has the opportunity to interact with the stationary phase as it moves past it. Samples that interact greatly, then appear to move more slowly. Samples that interact weakly, then appear to move more quickly. Because of this difference in rates, the samples can then be separated into their components.
Chromatography is based on a physical equilibrium that results when a
solute is transferred between the mobile and a stationary phase.
A
A
A
A
AA
A
AA
A
A
A
K = distribution
coefficient or
partition ratio
K =CS
CM
Where CS is the molar concentration of the solute in the stationary phase and CM is the molar concentration in the mobile phase.
Cross Section of Equilibrium in a column.“A” are adsorbed to the stationary phase.“A” are traveling in the mobile phase.
Flow
As a material travels through the column, it assumes a Gaussian
concentration profile as it distributes between the stationary packing phase
and the flowing mobile gas or liquid carrier phase.
In a chromatography column, flowing gas or liquid continuously replaces
saturated mobile phase and results in movement of A through the column.
Column is packed
with particulate
stationary phase.
Flow
Flow
Flow
Flow
In a mixture, each component has a different distribution coefficient, and thus spends a
different amount of time absorbed on the solid packing phase vs being carried along with
the flowing gas
More volatile materials are carried through the column more rapidly than less volatile
materials, which results in a separation.
The Elution of a Solute through a Chromatographic System
Series of absorption-extraction processEquilibrium between the two phasesThe distribution system is continuously thermodynamically driven towards equilibriumEquilibrium process between two phases is complicated
Distribution of K.E. between both the phases
Solute molecules leave the stationary phase when their K.E. isequal or greater than the P.E. of their interaction with thestationary phase
No.of molecules at the boundary (N1) K.E. in excess of the P.E. Associated with molecular interaction with stationary phase (EA)
No.of molecules at the boundary (N2) K.E. is less of the P.E. Associated with molecular interaction with stationary phase (EA)
At equilibrium N1 = N2
Elution Development in TLC
Development of a Thin layer Plate
Note: The first two components were not completely separated.
Peaks in general tend to become shorter and wider with time.
If a detector is used to determine when the components elute
from the column, a series of Gaussian peaks are obtained,
one for each component in the mixture that was separated
by the column.
Theoretical plate is a term coined by Martin &
Synge. It is based on a study in which they imagined that
chromatographic columns were analogous to distillation
columns and made up or numerous discrete but connected
narrow layers or plates. Movement of the solute down the
column then could be treated as a stepwise transfer.
Theoretical plates (N) measure how efficiently a
column can separate a mixture into its components.
This efficiency is based on the retention time of the
components and the width of the peaks.
The Theoretical Plate
wb
tR
N = 16( t R
w b
) 2
N = Number of theoretical plates (a measure of efficiency)
tR is the retention time; it is measured from the injection peak (or zero) to the intersection of the tangents.wb is the width of the base of the triangle; it is measured at the intersection of the tangents with the baseline.
When the retention time, tR, is held constant, the column that produces peaks with narrower
bases, wb, will be more efficient – have a greater N value.
Likewise a column that produces wider peaks will be less efficient – have a smaller N value.
This is because a smaller denominator, wb, will yield a larger overall number and a larger denominator will yield a smaller number.
Larger N Smaller N
tR
tR
wb wb
N = 16( t R
w b
) 2
Good for volatile samples (up to about 250 oC)
0.1-1.0 microliter of liquid or 1-10 ml vapor
Can detect <1 ppm with certain detectors
Can be easily automated for injection and data analysis
Gas Chromatography
Components of a Gas Chromatograph
Gas Supply: (usually N2 or He)
Sample Injector: (syringe / septum)
Column: 1/8” or 1/4” x 6-50’ tubing packed with small uniform size, inert support coated with thin film of nonvolatile liquid
Detector: TC - thermal conductivityFID - flame ionization detector
Gas Liquid Chromatography
Here the mobile phase is an unreactive gas ( eg Nitrogen) flowing through a tube.
And the stationary phase is an involatile liquidheld on particles of a solid support.
In the animation below the red molecules are more soluble
in the liquid (or less volatile) than are the green molecules.
In practice the Column is contained in a thermostatic oven. (Why ?)
About 1μL of liquid is injected into one end of the column.
As each component reaches the other end it is detected and registered on a chart recorder.
The Retention Time is characteristic of a particular substance. (for the same column, temperature, gas flow etc.)
The area under each peak indicates the relative quantities.
Oven
Detector
Injection
port
Nitrogen
cylinder
Column
Recorder
Chromatogram of petrol
Suggest identities of some of the unlabelled peaks.
Schematic of a Commercial Gas Chromatograph
HP 5890 Capillary Gas Chromatograph with Robotic Sample Injector and Data Station
General Settings for GC Startup program
Y axis: 1-5 v (0-1 volt is in bright light, 4-5 volt is dark)X axis: 0-400 seconds
Good: Peaks are smooth, well separated and elute quickly
Plot of GC Elution Data forDichloromethane and Chloroform
On 25 cm Tide Column
Poor: peaks are noisy, due to flickering flame, and elute slowly.
To fix: Adjust sensor so that it is looking at the blue portion
of the flame. (Verify the flame is blue.)
Plot of GC Elution Data forDichloromethane and Chloroform
On 25 cm Tide Column
The peak height is proportional to the amount
of material eluting from the column at any given time,
The area under the peak is a measure of the total
amount of material that has eluted from the column.
Electronic integrators are used for area measurement
in commercial GCs. We will be using ALGEBRA.
Determination of the Amount
of Sample Components Present
wb
h
Area = 1/2 wb h
The Gaussian curve can be approximated as triangular
in shape, to simplify area measurement.
NOTE: the height is measured to the top of the tangents,
which is above the actual curve peak.
GEL FILTRATION
Gel filtration separates molecules according to the differences in size as theypass through the filtration medium packed in the column.
It is well suited for biomolecules that are sensitive to pH ,concentration andharsh environment.
Parameters that affects gel filtration are, particle size, flow rate, packagingdensity, porosity of the particle and viscosity of the mobile phase.
MATERIALS REQUIRED
Cross linked dextrans (sephadex)
Agarose (sepharose)
Polyacrylamide
Porous glass gel.
APPLICATIONS
Fractionation (purification of the desired protein using suitable gel)
Molecular weight determination
ION EXCHANGE
Ion exchange chromatography is used to remove ions of one type from a mixture and replace them by ions of another type.
The basic principle is reversible competitive binding
ION EXCHANGERS
• Cation exchangers (negative ions – stationary)
• Anion exchangers (positive ions - stationary)
Four types of polymers are commonly used. They are,
• Synthetic hydrophobic polymer resins crosslinked with divinylbenzene.
• Naturally occuring as well as synthetic polymers(cellulose)
• Synthetic hydrophilic polymers
• Silica gel
AFFINITY CHROMATOGRAPHY
• Affinity chromatography includesbioaffinity, dye-ligand affinity andimmobilized metal ion afffinity techniques.
• It is based on the formation of the specificand reversible complexes between a pair ofbiomolecules.
HPLC
HPLC is a physical separation technique in which a sample dissolved in aliquid is injected into a column packed with small particles and it is separatedinto its constituent components
HPLC is probably the most important and widely used analytical techniquefor quantitative analysis of organics and biomolecules
HPLC is applicable to many kind of samples:
Most useful for pharmaceuticals, biomolecules, and labile organics
HPLC Instrumentation Overview
53
Principle Pattern An Example
Detector
Thermostatted Column Compartment
Autosampler
Binary Pump
Vacuum DegasserSolvent Cabinet
Solvent Reservoirs
Controller
Typical HPLC Unit
HPTLC
HPTLC is a sophisticated form of TLC.
Fastest of all chromatographic techniques.
Any combinations of stationary and mobile phases can beused.
Analytical HPTLC is used for micro preparative analysis(ie., separation of milligram scale for analysis of fraction )
Gives more sharper and compact bands with minimumdistance of migration.
Used for both qualitative and quantitative analysis.
Applications of HPLC Techniques
• A mixture of sugars containing fructose, sucrose, lactose, maltose have been separated
• on column Zorbax NH2 with 70% acetonitrile in aq. Phase with RI detector
• In environmental pollution analysis for ions in trace concentrations
• For Bioseparation, DNA, carbohydrates, lipids, proteins and amino acids
Rest for assignment…………..