Chromatography

Is a technique used to separate and identify the

components of a mixture.

Works by allowing the molecules present in the

mixture to distribute themselves between a
stationary and a mobile medium.

Molecules that spend most of their time in the mobile

phase are carried along faster.
 Types of Chromatography
● Adsorption Chromatography
● Paper Chromatography
● Thin Layer Chromatography
● Ion Exchange Chromatography
● Gel Filtration Chromatography or Gel permeation Chromatography or
Molecular sieve Chromatography
● Advanced chromatography Techniques
Gas Liquid Chromatography
Here the mobile phase is an unreactive gas ( eg
Nitrogen) flowing through a tube.

And the stationary phase is an involatile liquid

held 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.
Chromatography
Chromatogram - Detector signal vs.
retention time or volume

Detector Signal
1 2

time or volume
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.

 Injection
port Recorder

Oven

Detector

Column
Nitrogen
cylinder
 Chromatogram of petrol

Suggest identities of some of the unlabelled peaks.

 Classification based on Mobile
Gas Chromatography
Phase
Pyrolysis GC -
heat solid materials
to 500 - 10000C
Gas - solid Gas - liquid so they decompose
into gaseous products
Stationary Phase

Sample MUST be volatile at temperatures BELOW 3500C

 Classification based on Mobile
Phase
Liquid chromatography (LC)

Column High performance Thin layer

(gravity flow) (pressure flow) (adsorption)
 Types of Chromatography
• Classification by mobile phase:
– Gas - Gas chromatography (GC)
• 1951 Martin and James (fatty acids)
– Liquid - Liquid chromatography (LC)
• 1964 Horvath (Yale) instrument
• 1966 Horvath and Lipsky (nucleic acid components)
– Supercritical fluid - Supercritical fluid chromatography
(SFC)
• 1958 Lovelock (Yale)
 Forces
• Adsorption - for polar non-ionic compounds

• Ion Exchange - for ionic compounds

– Anion - analyte is anion; bonded phase has positive charge
– Cation – analyte is cation; bonded phase has negative charge

• Partition - based on the relative solubility of analyte in mobile and stationary

phases
– Normal – analyte is nonpolar organic; stationary phase MORE polar than the mobile
phase
– Reverse – analyte is polar organic; stationary phase LESS polar than the mobile
phase

• Size Exclusion - stationary phase is a porous matrix; sieving

 Detectors
• UV-vis
• Refractive Index (RI)
• Mass spectrometry (MS)
• Electrochemical (EC)
– amperometric
• NMR - novel
 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 ....
1

For substances that are rather insoluble in the liquid

Rf will be close to ....
0
Principles of Paper Chromatography
• Capillary Action – the movement of liquid within the spaces of a porous
material due to the forces of adhesion, cohesion, and surface tension. The
liquid is able to move up the filter paper because its attraction to itself is
stronger than the force of gravity.

• Solubility – the degree to which a material (solute) dissolves into a solvent.

Solutes dissolve into solvents that have similar properties. (Like dissolves
like) This allows different solutes to be separated by different combinations
of solvents.

Separation of components depends on both their solubility in the mobile

phase and their differential affinity to the mobile phase and the stationary
phase.
Paper Chromatography Experiment
What Color is that Sharpie?
Developing the Chromatograms
0% 20% 50% 70% 100%
1. Dyes separated – purple and black
2. Not soluble in low concentrations
of isopropanol
3. Partially soluble in concentrations
of isopropanol >20%
1. Dye separated – blue
2. Not very soluble in low
concentrations of isopropanol
3. Completely soluble in high
concentrations of isopropanol
1. Dye separated – blue and yellow
2. Blue – Soluble in concentrations
of isopropanol >20%
3. Yellow – Soluble in concentrations
of isopropanol >0%
1. Dyes separated – red and yellow
2. Yellow –soluble in low concentrations of isopropanol and
less soluble in high concentrations of isopropanol
3. Red – slightly
soluble in low
concentrations
of isopropanol,
and more
soluble in
concentrations
of isopropanol
>20%
Alternative Experiments
Alternative Experiments
Alternative Experiments
 ION EXCHANGE

• Protein interact with stationary phase by charge-charge interaction

•Positively charged proteins adhere to negatively charged functional

groups(carboxylates,sulfates:cation exchanger)

•Tertiary or quaternary amines: anion exchanger

•Sequential elution, change of pH

•Diethyl aminoethyl(DEAE)cellulose(anion exchanger)

•Carboxymethyl(CM)cellulose(cation exchanger)
 SIZE EXCLUTION/GEL FILTERATION/GEL
PERMEATION

POROUS BEADS AS STATIONARY PHASE

STROKE RADIUS;FUNCTION OF MOLECULAR MASS AND

SHAPE

GREATER THE STROKE RADIUS FASTER WLII BE THE

ELUTION

GEL IS MADE OF DEXTRAN AGAROSE OR

POLYACRYLAMIDE
H igh
Performance
L iquid
C hromatography
H igh
Pressure
L iquid
C hromatography
H igh
Priced
L iquid
C hromatography
 Partitioning
• Separation is based on the analyte’s relative
solubility between two liquid phases

Mobile Phase Stationary Phase

Solvent Bonded Phase

 Gradient
Instrumentation
Controller

•
Pump Column
Detector
Injector
Mobile Phases
 HPLC - Modes
• Normal Phase.
- Polar stationary phase and non-polar
solvent.

• Reverse Phase.
- Non-polar stationary phase and a polar
solvent.
Common Reverse Phase Solvents
• Methanol CH3OH
• Acetonitrile CH3CN

• Tetrahydrofuran

• Water H2O
• Solid Support - Backbone for bonded phases.
– Usually 10µ, 5µ or 3µ silica or polymeric particles.
• Bonded Phases - Functional groups firmly linked
(chemically bound) to the solid support.
– Extremely stable
– Reproducible
• Guard - Protects the analytical column:
– Particles
– Interferences
– Prolongs the life of the analytical column

• Analytical - Performs the separation.

 Bonded Phases
• C-2 Ethyl Silyl -Si-CH2-CH3

• C-8 Octyl Silyl -Si-(CH2)7-CH3

• C-18 Octadecyl Silyl -Si-(CH2)17-CH3

• CN Cyanopropyl Silyl -Si-(CH2)3-CN

Choosing the Particle Physical Characteristics
Pore Size
• Average size of pores or cavities in particles, ranging from 60-
10,000Å

Bonding Type
• Monomeric - single-point attachment of bonded phase molecule
• Polymeric - multi-point attachment of bonded phase molecule

Carbon Load
• Amount of bonded phase attached to base material, expressed as %C

Endcapping
• “Capping” of exposed silanols with short hydrocarbon chains after the primary
bonding step
COLUMN DIMENSIONS
Effect on chromatography

Column Dimension
• Short (30-50mm) - short run times, low backpressure
• Long (250-300mm) - higher resolution, long run times
• Narrow ( 2.1mm) - higher detector sensitivity
• Wide (10-22mm) - high sample loading
PARTICLE SHAPE
Effect on chromatography
Spherical particles offer reduced back pressures and longer column life when using viscous mobile
phases like 50:50 MeOH:H2O.
PARTICLE SIZE
Effect on chromatography
Smaller particles offer higher efficiency, but also cause higher backpressure. Choose 3µm particles for
resolving complex, multi-component samples. Otherwise, choose 5 or 10µm packings.
SURFACE AREA
Effect on chromatography
High surface area generally provides greater retention, capacity and resolution for separating complex,
multi-component samples. Low surface area packings generally equilibrate quickly, especially
important in gradient analyses.
PORE SIZE
Effect on chromatography
Larger pores allow larger solute molecules to be retained longer through maximum exposure to the
surface area of the particles. Choose a pore size of 150Å or less for sample MW  2000. Choose
a pore size of 300Å or greater for sample MW > 2000.
BONDING TYPE
Effect on chromatography
Monomeric bonding offers increased mass transfer rates, higher column efficiency, and faster column
equilibration.

CH3 R
monomeric
OH + X Si (CH2)17 CH3 Si (CH2)17 CH3
bonding
CH3 R

OH CH3 O CH3
polymeric
+ X Si (CH2)17 CH3 Si
bonding
OH X O (CH2)17 CH3

Polymeric bonding offers increased column stability, particularly

when highly aqueous mobile phases are used. Polymeric bonding
also enables the column to accept higher sample loading.
CARBON LOAD
Effect on chromatography
Higher carbon loads generally offer greater resolution and longer run times. Low carbon loads shorten
run times and many show a different selectivity, as in Alltech’s Platinum line of packings.
ENDCAPPING
Effect on chromatography
Endcapping reduces peak-tailing of polar solutes that interact excessively with the otherwise exposed,
mostly acidic silanols. Non-endcapped packings provide a different selectivity than do endcapped
packings, especially for such polar samples.
Alltech’s Platinum™ EPS packings are non-endcapped to offer enhanced polar selectivity.
 High Capac
Default Col

Loadability

High Stabil

Stability at
High Samp

Suitable fo
High Resol

Consumpti
Goals

High Sensi

Fast Analy

Fast Eqilib
High Effici
Metho

Backpress

Low Mobil
Applicatio
(Good for

Extremes
>2000
Low
Particle Size
small (3µm) • •
medium (5µm) •
large (10µm) •

Column Length
short (30mm) • • • • •
medium (150mm) •
long (300mm) •

Column ID
narrow (2.1mm) • • medium (4.6mm) •
wide (22.5mm) •

Surface Area
low (200m2/g) • • •
high (300m2/g) • • •

Pore Size
small (60Å) • •
medium (100Å) •
large (300Å) •

Carbon Load
low (3%) •
medium (10%) •
high (20%) • • •

Bonding Type
monomeric • •
polymeric • • • •

Particle Shape
spherical • • • •
irregular •
• UV
Detectors
– Single wavelength (filter)
– Variable wavelength (monochromator)
– Multiple wavelengths (PDA)
• Fluorescence
• Mass Spectrometric
 Monochromator

Prism Diffraction grating

PDA
Fluorescence
 High Vacuum System

Ion Mass Data

Inlet source Analyzer Detector System
 A Supelcosil LC-PAH Columns. B
Conditions: A: 150mm x 4.6mm, 5µ. Conditions: B: 50mm x 4.6mm,
Syringe filter
• How to check Base line?
• How to purge Autosampler?
• What is Seal washing kit? Q?
• How to change Local mode to Remote mode
• How to change concentrations of port?
• Is there any special grade solvent (mobile
phase) for HPLC?
• Is there any degassing available apart from
automated degasser in system?