CHROMATOGRAPHY
CHROMATOGRAPHY
This method is used for separation of mixtures of
compounds between two phases:
 Stationary ( solid or liquid located on neutral medium)
 Mobile( liquid or gas)
This method is used for separation of mixture of
compounds with different speed of migration a given
compounds in porous medium with different ability of
adsorption or ion exchange or different solubility in
solvents.
Metods of
chromatography
 GC, Gas chromatography)
 LC, Liquid chromatography)
 HPLC,
High Pressure liquid chromatography
 TLC, Thin layer chromatography)
Classification of chromtographic methods
1.
Because of physico – chemical forces:
 Adsorption chromatography
 Ion exchange chromatography
 Partition chromatography
 Gel (size exclusion) chromatography
 Affinity chromatography
 Capillary chromatography
2.
Because of techniques of preparation:
 Column chromatography
 Planar chromatography ( thin layer, paper, gel)
3.
Because of density of mobile phase:
 GC, Gas Chromatography)
 LC, Liquid Chromatography)
4.
Because of density of mobile and stationary phases:
 Gas – liquid chromatography ( GLC)
 Liquid – liquid chromatography ( LLC)
 Gas – solid chromatography (GSC)
 Liquid - solid chromatography (LSC)
Investigated compound is located in so
called mobile phase and migrate with it
across stationary phase, it means porous
medium wich is adsorbent, ion exchanger or
molecular sieve.
Chromatography is used for:




Separation of compounds from mixtures
Purification
Identification
Quantitative and qualitative analysis
Choice of the method depends on:
 Amount of analysed mixtures
 Kind of compounds
 Complexity of separation
Explanation:
Compound is placed on stationary phase.
Mobile phase passes through the stationary phase.
Mobile phase solubilizes the components.
Mobile phase carries the individual components a certain distance
through the stationary phase, depending on their attraction to
both of the phases.
Illustration of
Chromatography
Stationary Phase
Separation
Mobile Phase
Mixture
Components
Component
s
Affinity to Stationary
Phase
Affinity to Mobile
Phase
Blue
----------------
Insoluble in Mobile Phase
Black


Red


Yellow

        
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)
Adsorption Chromatography
Adsorption of molecules which are in contact with solid adsorbent can be
caused by
 Physical forces ( physical adsorption)
 Chemical interaction (chemisorption).
Classification of adsorbents:
1. Because of adsorption activity:
 Weak ( starch, saccharose)
 Middle ( calcium carbonate, sodium carbonate)
 Strong ( aluminium oxide, activated silica acid)
2. Because of chemical properties:
 acidic ( SiO2)
 basic ( CaO)
 neutral ( charcoal)
 Amphoteric ( Al2O3)
3. Because of chemical nature:




Organic
Inorganic
Mixed
Specyfic
4. Because of polarity :
 Highly polar ( aluminium oxide, silica oxide)
 Weak polar (calcium carbonate)
 Non polar ( Charcoal)
Adsorption ability depends on used solvent, adsorbent preparation and its activity
Component of the mixture is adsorbed stronger if its polarity is less different than
polarity of adsorber.
Loading on the column with polar adsorber more polar solvent than components
of separated mixture cause elution of these components from the column
Solvents used in chromatography are ordered with increasing
ability of elution adsorbed polar compounds:
1. Heksane ( the least polar)
2. Carbon tetrachloride (CCl4)
3. Benzene
4. Diethyl ether
5. Acetone
6. Chloroform
7. Ethyl acetate
8. Ethanol
9. Methanol
10.Water
Thin Layer Chromatography – TLC (partition)
Separation of the mixture depends on the difference on partition
coefficient of the mixture of components between two non miscible
phases, from which one is a liquid coated on a medium (stationary
phase), and a second one – mobile phase (liquid,gas).
Nernst law of partition:
K = Cs / Cc
K – Coefficient at equilibrium state, depends only on temperature and
properties of solutes in solutions, and does not depend on amount of
dissolved compound.
Cs – concentration of compound dissolved in stationary phase
Cc - concentration of compound dissolved in mobile phase
Thin Layer Chromatography – TLC (partition)
Simple an d fast method.
Processes responsible for separation:
- adsorption
- partition
- ion exchange
- Combination of three above.
Advantageous of TLC:
- short time of separation
- simplicity
- cheap
Preparation of chromatographic plates
Glass plates (1-2 mm thick)
For microanalytical purpose - 0.2 mm thick
Loading investigated solution on a plate:
- Using micropipette – load one small drop of a solution about 1 cm
above the edge of the plate such way to reach the smallest dot.
Chromatography chamber must be :
- Leak proof
- Saturated by solwent
Developing of chromatogram
-Developing occurs during migration of solvent
from start to the end on about 10 cm.distance
Solvent migrates because of:
 capillary forces ( up technique)
 gravity forces ( down technique)
Development of chromatogram
After withdrawal from the chamber and drying – act on the
plate by special reagent (detector) which can react with
components of the mixture and give coloured products (iodine,
concentrated sulphuric acid, detector which can give
fluorescence inUV).
Since the sample is separated in the column, different peaks on the chromatogram
correspond to different components in the sample mixture. The chromatograms
above show the results of separations of protein mixtures by ion exchange
chromatography. The lettered peaks correspond to different proteins (A =
ovalbumin, B = conalbumin, C = cytochrome c, D = lysozyme). The separation
corresponding to the chromatogram on the left was performed at pH 5.85, while
the one on the right was performed at pH 6.5. It is evident that operation
conditions such as pH and temperature have a significant effect on the output.
Chromatography application for quantitative analysis –
Rf coefficient.
Substance location on chromatogram is characterized by
Rf values. (ratio of fronts) = retention factor
Distance from START line to the middle of substance spot - A
Rf =
Distance from START line to the End line of solvent - B
END
B
A
START
Rf - is characteristic value for each substance.
This values are provided in text books and are for
use in substance characterization.
GAS CHROMATOGRAPHY
Fast and effective method of separation for volatile compounds
mixtures.
Separated compound is carry out by gas (mobile phase) through
the column filled with stationary phase.
Depends on stationary phase chromatography can be divided as :
 Partition gas chromatography ( stationary phase – liquid on
stationary carrier)
 Adsorption gas chromatography ( stationary phase –
adsorbent)
 Capillary gas chromatography ( stationary phase – liquid are
directly on the column walls.)
Schematic of gas chromatograph
Sample’s injection
Chromatogram =result
Carrier gas
Oven
Column
Column –stationary phase on stationary carrier.
Capillary column with about 0.25 mm diameter does not need carrier
material.
GAS CHROMATOGRAPHY cont.
Carrier gas ( hydrogen, helium, nitrogen, argon, carbon dioxide) is
introduced to the column.
Investigated sample or mixture is injected to the column. Stream of
gas is carrying samples to be separated. Compounds are divided
according to their retention factor between gas and liquid phase.
Separated substances are measured and registered at the outlet of the
column using detector system. Operating temperature is 0oC – 400oC.
Stationary phase – organic liquids with small vapor pressure.
Carrier – materials with large specific area and small adsorption
properties ( diatomaceous earth, clays).
Detectors in gas chromatography
Quantitative measurement of mixture components can be done by
using different chemical and physical properties of the compounds.
Therefore there are the following type of detectors:
1. Thermal conductivity detector – katarometr.
2. Flame-ionization detector - (FID)
3. β- ionization detector –radioactive . Its energy induces elements of
carrier gas.
4. Recombination detector – is radioactive.
Detector signals are registered by sensitive writing system.
As a result we are obtaining CHROMATOGRAM.
Chromatogram – it is graph showing relationship detector signal vs time
or gas volume.
Absorbancja
Czas
Peak area is proportional to the amount of single analyzed sample.
Gas chromatograph
Chromatography column in gas chromatograph
GCMS
Retention parameters
1. Retention time tR – time from the moment of peak appearance
to maximum
of substance amount
2. Retention volume vR – volume of carrier gas necessary to elute
compound in specific retention time.
3. Relative retention–retention obtained by standard added to
investigated substance.
Distribution ratio K:
cL
K=
nL
=
cG
nG
:
vL
vG
cL cG – concentration of substances in liquid and gas phase
nL, nG – amount of moles
vL, vG – volume of liquid and gas phase
Distribution ability of column is increasing with length of the
column, type and amount stationary phase, column
temperature, speed and pressure of carrier gas.
Gas chromatography is appled in qualitative and quantitative
chemistry.
Column chromatography – another type of chromatography
Chromatographic
columns