CHROMATOGRAPHY Quantification is achieved on the basis of peak area coupled
with an appropriate calibration graph.
The area of each peak in a chromatogram can be shown to be
Principle
proportional to the amount of the analyte producing the peak.
1. Distribution coefficients or partition coefficient
The area of the peak may be determined by measuring the
the way in which a compound (the analyte) distributes height of the peak (hP) and its width at half the height (wh)
between two immiscible phases.
GAS CHROMATOGRAPHY
Principle
All chromatographic systems consist of the stationary phase,
which may be a solid, gel, liquid or a solid/liquid mixture that It exploits differences in the partition coefficients between a
is immobilised and the mobile phase, which may be liquid or stationary liquid phase and a mobile gas phase of the
gaseous, and which is passed over or through the stationary volatilised analytes as they are carried through the column by
phase after the mixture of analytes to be separated has been the mobile gas phase.
applied to the stationary phase. Its use is therefore confined to analytes that are volatile but
• Chromatographic analysis can be carried out on either a thermally stable.
qualitative or quantitative basis. The partition coefficients are inversely proportional to the
1) Qualitative analysis volatility of the analytes so that the most volatile elute first.
The objective of this approach is to confirm the presence of a The temperature of the column is raised to 50300oC to
specific analyte in a test sample. facilitate analyte volatilisation.
The use of either a mass spectrometer or nuclear magnetic The stationary phase consists of a high-boiling-point liquid
resonance (NMR) spectrometer as a detector so that structural material such as silicone grease or wax that is either coated
evidence for the identity of the analyte responsible for the onto the internal wall of the column or supported on an inert
peak can be obtained. granular solid and packed into the column.
2) Quantitative analysis There is an optimum flow rate of the mobile gas phase for
maximum column efficiency.
The objective of this approach is to confirm the presence of a
specific analyte in a test sample and to quantify its amount. Apparatus
known as porous layer open tubular (PLOT) columns, for
adsorption work.
Commonly used stationary phases include polyethylene
glycol (CP wax and DB wax, very polar) and methyl and
phenyl-polysiloxanes (BP1, non-polar; BP10, medium polar).
A. Columns: These are of two types:
a) Packed conventional columns
coiled glass or stainless steel.
They are packed with stationary phase coated on an inert silica
support.
Commonly used stationary phases include the polyethylene
glycols (Carbowax 20M, very polar), methylphenyl- and
methylvinylsilicone gums.
Apiezon L (non-polar) and esters of adipic, succinic and
phthalic acids. b-Cyclodextrinbased phases are available
for chiral separations.
The most commonly used support is Celite (diatomaceous
silica), which because of the problem of support–sample
interaction is often treated so that the hydroxyl groups that
occur in the Celite are modified.
b) Capillary (open tubular) columns
made of high-quality fused quartz.
They are of two types known as wall-coated open tubular
(WCOT) and support-coated open tubular (SCOT), also
