Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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INTRODUCTION TO CHROMATOGRAPHY
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
What is Chromatography?
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
Chromatography:Chromatography operates on the same principle as extraction, but one phase is held in place while the other moves past it.
Phase 2
Phase 1
Solvent extraction:Extraction is the transfer of a solute from one phase to another
Extraction of Uranyl Nitrate from water into ether Partition
coefficient:
S(in phase 1) S(in phase 2)
K
[S]
2
[S]
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
What is Chromatography?
Chromatography:Chromatography operates on the same principle as extraction, but one phase is held in place while the other moves past it.
Air flow
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
Air flow
What is Chromatography?
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
Air flow
What is Chromatography?
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
Air flow
What is Chromatography?
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
What is Chromatography?
Initial bandA and B solutes
Fresh solvent(eluent)
Column packing(stationary phase)
Suspended in solvent(mobile phase)
Solvent flowing out(eluate)
A
B
B
emerges
A emerges
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
What is Chromatography?
It is the collective term for a family of laboratory techniques for the separation of mixtures by distribution of its components between a mobile and stationary phase over time
All chromatographic separations depend on the reversible sorption and desorption of the
components of the mixture in the stationary and mobile phase
Mobile phase:solvent moving through the plate or column(liquid or gas)
Stationary phase:substance which is fixed in place for the chromatographic procedure
The partitioning of solutes between the mobile and stationary phases gives rise to separation
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
Chromatographic principle
Stationary phase
Mobile phase
Samplemixture
Equilibrium established at each point (ideally)
The molecules of the mixture interact with the molecules of the
Mobile and Stationary Phase
Retardation of rate of
movement of molecules
Each molecule interacts differently with MP and SP
Different distribution coefficients and different
net rates of migration
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
Purpose of Chromatography
The two are not mutually exclusive
Analytical ChromatographyAnalytical Chromatography – separate and identify or measure the relative proportions of components in a mixture
Preparative ChromatographyPreparative Chromatography - purify and collect one or more components of a sample for further use
300 L
Purifies 1 Kg of material
Scaling Up:
Large column radius
Small column radius=
Large mass
Small mass
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
Types of Chromatography
Chromatography is divided into categories on the basis of the
mechanism of interaction of the solute with the stationary phase
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
Adsorption Chromatography
Competition between a solid adsorbent and the
mobile phase
Gas Column Gas-Solid Chromatography GC/GSC
Liquid
Column
Liquid column Chromatography
High Performance Liquid Chromatography
LCHPLC
Planar layer
Thin Layer ChromatographyPaper Chromatography
TLCPC
Partition Chromatography
Competition between a liquid stationary phase and the mobile phase
Gas ColumnGas-Liquid Chromatography
Supercritical Fluid Chromatography
GC/GLCSFC
Liquid
Column
Liquid-Liquid Chromatography
High Performance Liquid Chromatography
LC/HPLC
Ion Exchange Chromatography
Competition between an ionic exchange resin stationary phase and liquid mobile phase
Liquid
Column
Ion-Exchange Chromatography
High Performance Ion Chromatography
IECIC/HPIC
Permeation Chromatography
Competition between a polymer matrix
stationary phase and liquid mobile phase
Liquid
ColumnGel Permeation
ChromatographyGPC
Stationary PhaseMobilePhase
StationaryPhase
SupportType of
Chromatography
Classification based on the chromatographic principle
Acronym
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
Column: Stationary Phase held in a tube and Mobile Phase forced through
Planar: Stationary Phase supported on a flat plate or in pores of paper. Mobile Phase moves through Stationary Phase by capillary action, gravity or electrosatic forces
Classification of Chromatography
Based on Stationary Phase Support
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
Chromatography
Mobile Phase = LiquidLiquid Chromatography
Mobile Phase = GasGas Chromatography
Stationary Phase = Solid
LSC
StationaryPhase = Liquid
LLC
StationaryPhase = Solid
GSC
StationaryPhase = Liquid
GLC
Adsorption or ion exchange
Partition of analytes
between two phases
Adsorption
Partition of analytes
between two phases
Classification according to the phases
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
Molecular characteristic
Physical propertySeparation technique
Polarity
volatility GLC
solubility LLC
adsorptivity LSC
Ionic chargeIon Exchange
Chrom.Electrophoresis
Size (mass)diffusion
Gel Permeation Chrom.Dialysis
sedimentation Ultracentrifugation
Shape ligand binding Affinity Chrom.
Classification according to physical features of the analytes
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
Methods based on Ionic Nature of the sample
Ion nature of the
sample is affected
by:
•Number & nature of ionisable groups•pH environment
•Presence of other ions
And results in:
Association of ions with opposite
charge
Movement in an electric field
Ion-exchange chromatography
ElectrophoresisCapillary
electrophoresis
Competition for ionic binding sites
on a resin
Direction & velocity of movement
depends on the sign & intensity of the
ionic charge
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
AdsorptionChromatography
Methods based on polarity (i.e. affinity of like-molecules for each other)
Solid/Liquid Liquid/Liquid Liquid/Vapour
Adsorption Solubility Solubility
Solid adsorbents
Two immiscibleliquids
A solutionand its vapour
LiquidChromatography
Gas-LiquidChromatography
Partition between two phases:
A major factor in separation is:
And the methods involve:
Methods are generallyknown as:
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
Movement due to this may be:
Restricted bypore size
Methods based on size
Diffusion Sedimentation
Dialysis
Ultracentrifugation
Fixed pore size
Gel filtrationMolecular exclusion (largest molecules come outfirst as not held by pores)
Size affects the rate of:
Balanced byeach other
Reduced by high density solvents
Maximal in lowdensity solvents
Gel permeationchromatography
Variable pore size
Isodensity Velocity
Diffusion
Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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Pusat Pengajian Sains Kimia & Teknologi Makanan, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia.
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