CHROMATOGRAPHIC

TECHNIQUES
the detector type
 Most Common Stationary Phases

1. Separation of mixture of polar compounds

Carbowax 20M (polyethylene glycol)

2. Separation of mixtures of non-polar compounds

OV101 or SE-30 (polymer of methylsilicone)

3. Methylester of fatty acids

DEGS (diethylene glycol succinate)
 Chromatogram of petrol

•The area under each peak is proportional to the amount of that

component.
•The identification of a component is done by matching its retention
time with that of a known substance under the same conditions.
• This is then confirmed by comparing the mass spectra of the two
substances.
Advantages of Gas Chromatography

• Very good separation

• Time (analysis is short)
• Small sample is needed - µl
• Good detection system
• Quantitatively analyzed
NORMAL PHASE.
- POLAR STATIONARY PHASE AND NON-POLAR SOLVENT.

REVERSE PHASE.
- NON-POLAR STATIONARY PHASE AND A POLAR SOLVENT.
 Chromatographic Techniques
High-Performance Liquid
Chromatography(HPLC) :

HPLC, has found great utility in

separating different hydrocarbon group
types and identifying specific constituent
types.
The general advantages of high-
performance liquid chromatography
method are:
(1) each sample is analyzed as received;
(2) the boiling range of the sample is
generally immaterial;
(3) the total time per analysis is usually
of the order of minutes; and
(4) the method can be adapted for on-
stream analysis. A modern self contained HPLC.
Common Reverse Phase Solvents
CH3OH
Methanol
CH3CN
• Acetonitrile

• Tetrahydrofuran

• Water H2O

For injecting the solvent through the column

Minimize possible flow disturbances

Limiting factor in precision of liquid
chromatographic measurement

Volumes must be small

.1-500 µL

Sampling loops
› interchangeable loops (5-500 µL at pressures up
to 7000 psi)
 Columns
• 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-

Si-(CH2)17-CH3

• CN Cyanopropyl Silyl -Si-(CH2)3-CN

Smooth-bore stainless steel or heavy-walled
glass tubing

Hundreds of packed columns differing in size

and packing are available

Add columns together to increase length

HPLC COLOUMN :-

Mostly optical

Equipped with a flow cell

Focus light beam at the center
for maximum energy
transmission

Cell ensures that the separated
bands do not widen
 Detectors
• UV
– Single wavelength (filter)
– Variable wavelength (monochromator)
– Multiple wavelengths
• Fluorescence
• Electrochemical
• Mass Spectrometric