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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…………..