What is Chromatography ?
(Chromatography = Color Writing)
Chromatography is a broad range of physical
methods used to separate complex mixtures by
their distribution b/w two immiscible phases–
one that is stationary and one that moves.
Separate • Analyze
• Identify
• Purify
• Quantify
Mixture Components
The Chromatogram
E A
The time that a peak
Chromatograph appears (it’s retention
D
time) is diagnostic for a
C
B
given compound
Sample Mixture
Chromatogram
B
C
E The relative size of a
A
peak (area or height)
Abundance
is proportional to the
relative abundance of
the compound in the
D
mixture
0 5 10 15 20
Time (minutes)
Historical Account
❖1903 - Mikhail Tswett Separated Plant Pigments
❖1942 - Martin and Synge Developed
Partition Chromatography.
❖1952 - Martin and James Developed Tswett
Gas Chromatography (GC) Father of Chromatography
❖1958 - Beginning of Modern LC
❖1967 -Horvoth & Lipsky HPLC
Principle
Chromatography utilizes the ability of solute to
distribute itself b/w two phases, according to
distribution or partition coefficients (Kc)
Xm Xs
Kc = [X]s / [X]m
Xs = Concentration of the component in the stationary phase
Xm = Concentration of the component in the mobile phase
Classification Based on Phases
Liquid
Mobile Phase
Format Liquid-Liquid Chromatography Liquid-Solid Chromatography
(Partition) (Adsorption)
Stationary Phase Liquid Solid
Normal Phase Reverse Phase Normal Phase Reverse Phase
Mobile Phase Non-Polar Mobile Phase Polar
Stationary Phase Polar Stationary Phase Non-Polar
Mobile Phase Gas
Format Gas-Liquid Chromatography Gas-Solid Chromatography
(Partition) (Adsorption)
Stationary Phase Liquid Solid
Classification Based on Separation Modes
• Adsorption
• Partition
•Size Exclusion
•Ion Exchange
•Affinity
Adsorption Chromatography
(LSC,GSC)
Probably one of the oldest
types of chromatography
The equilibration of the
solute components b/w the
mobile and solid stationary
phase accounts for the
separation
Normal phase LS
Reverse phase LS
d- d+
30 m Si - O - H
Silica Gel
• In LSC separation mechanism involves the competition
of the solute components for the active sites on an
adsorbent such as Silica Gel
• Components elute in order of increasing polarity
Partition Chromatography
Solute equilibriates
between the mobile
phase and the liquid
stationary phase
Gel Permeation / Gel Filtration / Size
Exclusion Chromatography
⚫ Lacks an attractive interaction between the stationary phase and
solute
⚫ Porous gel separates the molecules
according to their size
⚫ GPC/GFC/SEC mainly used for the
separation of Polymers & Proteins
⚫ Separation based on molecular size in solution
⚫ Order of elution - larger to smaller
⚫ Relatively low resolution method
Gel-Filtration Chromatography –
Separation based on size
Four Basic Types of GELS
1. Dextran (Sephadex Type) Fractionation range MW
0 -800,000 Daltons
2. Polyacrylamide (Bio-Gels)
100-400,000 Daltons
3. Agarose (Sepharose Type and Bio-Gels)
10,000-150,000,000
4. Polyacrylamide-Dextran (Sephacryl Type)
5,000-8,000,000
Affinity Chromatography
• Most selective type of
chromatography .
• It utilizes the specific
interaction between one
kind of solute molecule
and a second molecule
that is immobilized on a
stationary phase.
• The immobilized
molecule may be an
antibody to some
specific protein.
Affinity Chromatography
•Bound proteins can
be eluted with the
small molecule or
with denaturing
reagents (urea,
guanidine, etc.)
•Small molecules
are attached to beads
and complex protein
mixtures are applied
Ion-exchange Chromatography
⚫ Ion-exchange separations are carried out in columns packed
with an ion-exchanger.
⚫ Solute ions of the opposite charge in the mobile liquid phase
are attracted to the ion-exchanger by electrostatic forces.
Cation exchange columns have a negative charge to attract cations.
Anion exchange columns have a positive charge to attract anions
Ion-exchange
Cation Exchanger
RSO-3 …… Na+ + +NH R/
3 RSO-3 ……
+NH
3R
/ + Na+
exchanger counter ion charged molecule bound molecular exchanged
to be changed ion ion
Anion Exchanger
(R)4N+ …… Cl- + -OOCR/ (R)4N+ …… -OOCR/ + Cl-
exchanger counter ion charged molecule bound molecular exchanged
to be changed ion ion
IEC – Separation based on charge
Column Chromatography
DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY
Solvent(mobile or
moving phase)
OOOOOOOOOOO
A+ B+ C Sample OOOOOOOOOOO
(A+B+C) OOOOOOOOOOO
OOOOOOOOOOO
OOOOOOOOOOO OOOOOA OOOO
OOOOOOOOOO Column OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO Solid Particles OOOOOOOOOO
OOOOOOOOOOO (packing material- OOOOOB OOOO
OOOOOOOOOO stationary phase) OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOC OOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOO
OOOOOOOOOOO OOOOOOOOOOO
OOOOOOOOOO OOOOOOOOOOO
Eluant (eluate)
Principles of Separation on a column
Column chromatography – an example of the equipment used in low-performance liquid
chromatography
Solvent reservoir
Column head
Column
Column packing
Porous glass plate
Sample is usually applied directly to the top of the column.
Detection is by fraction collection with later analysis of each fraction
Thin layer chromatography
TLC is method of separating mixture by eluting them
through a planar chromatographic bed then visualizing
the separating bed by staining or charring.
❖ Easy to learn, fast and versatile technique
❖ Used both for qualitative and quantitative purposes
❖ An indispensable tool in both quality control and research labs.
Out line of TLC method
1. Preparation of TLC Plates
i. Selection of plate (standard 20 x 5 or 20 x 20 cm )
ii. Selection of adsorbent and making the slurry (1:2 w/v)
(Properties of adsorbent can be modified)
iii. Applying thin layer to the support
( Spreading, pouring, spraying and dipping)
iv. Activation of plate (120-150 °C; 30 min )
2. Application of Sample
Microanalytical TLC ( 20 x20 cm; thickness 0.25mm)
o Sample volume = 0.1- 0.5mm3 applied as spot
o Loading ~ 12 mg per spot (1-5 mg ml-1 solution concentration are practicable )
Semipreparative TLC ( 20 x 20 cm; thickness 0.25mm)
o Sample is often applied as streak
o Loading ~ 4 mg
Preparative TLC ( 20 x20 cm; thickness 0.5-2mm)
o Sample is often applied as streak
o Loading ~ 250 mg
Spotting a TLC plate with sample
3. Development
✓ One-dimensional
( Asecding TLC & Desending TLC)
✓ Two-dimensional TLC
Running the TLC plate in solvent
✓ Radial or circular TLC
TLC: A Two-Component Mixture
solvent front
component B Less polar!
solvent front
component B
component A More polar!
component A
origin mixture origin origin
solvent front
Increasing Development Time
TLC: Qualitative Analysis
A B unknown
3. Location of Separated Substances
1. Visual Inspection
2. Chemical Methods
(Involve the application of derivatising agent/ locating reagent/ chromogenic reagent
➢ Specific reagents (DNP–test for carbonyl compounds)
➢ non-specific reagents (Iodine, sulphuric acid )
3. Physical Methods
➢ Ultraviolet detection (absorption & transmission measurements)
➢ Fluorescence measurements
➢ TLC scanners
4. Radiochemical Methods
➢ Autoradiography
Visualized under a UV light
TLC: Determination of Rf Values
solvent front
Rf of component A =
dA component B
dS
dS
dB
Rf of component B =
dB
component A
dS
dA
The Rf value is a decimal
fraction, generally only origin
reported to two decimal
places
Difference b/w Normal-TLC and HP-TLC
N-TLC HP-TLC
Thickness of plate layer 0.25 Thickness of plate layer 0.1mm
mm
Silica gel with 5-40 mm particle Refined silica gel with a mean 5
size distribution (1000 – 2000 mm narrowed particle size
theoretical plates per 5 cm distribution
migration) (10000 theoretical plates per 5 cm
migration)
Normal analysis speed Fast analysis (5 – 10 fold
reduction in analysis time)
---------- Improved sensitivity and
resolution
Stationary Phases in TLC
• Particle size
• Homogeneity OH
OH
OH
OH
OH Si
Si O
Si O
Si O O
Si O O
O O
O O
O Si Si
O O
Si
Si O O O
Si O O
O
O O
O
Si
Si O
O Silica (SiO2)
O O
O
Alumina
O OH OH OH OH
Al Al Al Al Al
O O O O O O
Acidic: -Al-OH
Neutral: -Al-OH + -Al-O-
Basic: -Al-O-
Solvents in TLC
Generally a solvent or solvent mixture of
lowest polarity consistent with good
separation or employed
Paper Chromatography
PC may be described as a technique for the
separation of substances using paper (a mat of
cellulose fibers) as a chromatographic sorbent with
a liquid mobile phase
The use paper as chromatographic medium is
usually regarded as a typical partition
chromatography
Developing the PC Chromatogram
⚫ Place the strips in the beakers
⚫ Make sure the solution does not come
above your start line
⚫ Keep the beakers covered
⚫ Let strips develop until the ascending
solution front is about 2 cm from the
top of the strip
⚫ Remove the strips and let them
dry
⚫ Locate the spots and quote results
Factors which govern PC separation
Propelling Forces
⚫ Solvent Flow
⚫ Solubility
Retarding Forces
⚫ Partition
⚫ Adsorption
Some Representative Separation by TLC and PC
Substance Solvent system Locating reagent
Unsaturated fatty acids Pet.ether/Et2O I2 (TLC)
Lipids Pet.ether/Et2O/AcOH I2 or 50% H2SO4 (TLC)
(80/20/1)
Hydrocarbon oils CHCl3/C6H6 Flrorescence or conc. H2SO4 (TLC)
Sterols CHCl3/Me2Co (95/5) 50% H2SO4 (TLC)
Sugars Et.OAc/AcOH/MeOH/H2O -Naphthol - H2SO4 (TLC)
(60/15/15/10)
Amino acids BuOH/EtOH/H2O Ninhydrin (TLC )
Vitamins Hexane/Acetone SbCl3 in AcOH (TLC )
Carotenoids, Chlorophyll PrOH/pet.ether/Et2O Visual (PC)
Co, Mn, Ni, Cu, Fe chlorids Acetone/conc. HCl/water Rubenic acid (PC)
(87/8/5)
Aminoacids BuOH/AcOH/water (40/10/50) Ninhydrin (PC)
