CHROMATOGRAPHY
INTRODUCTION
Analytical chemists have few tools as powerful as chromatography to measure
distinct analytes in complex samples. The power of chromatography comes from
its ability to separate a mixture of compounds, or “analytes”, and determine their
respective identity (chemical structure) and concentration. Chromatography can
be divided into three basic types that include gas, liquid, and supercritical fluid
chromatography.
“It is the technique in which the components of a mixture are separated based upon
the rates at which they are carried or moved through a stationary phase (column) by
a gaseous or liquid mobile phase”.
Liquid chromatography can further be divided into ion exchange, separations
based on size, and even extended to gel-based electrophoretic techniques. This
lecture will provide a basic introduction to different types of liquid and gas
chromatography.
The relationship between each type of chromatography is illustrated in
In a flow chart below
Branches of Chromatography
In general, each type of chromatography is comprised of two distinct
steps: chromatography (or separation of individual compounds in
distinct elution bands) and identification (detection of each elution
band). Gas chromatography is the process of taking a sample and
injecting it into the instrument, turning the solvent and analytes into
gaseous form, and separating the mixture of compounds into individual
peaks (and preferably individual compounds). Liquid chromatography
completes the same process except the separations occur in a liquid
phase.
Chromatography peaks (bands)
Individual band or peaks exit the column and identification
occurs by a relatively universal detector. One particularly
common detector for both gas and liquid chromatography is mass
spectrometry (MS) which transforms each analyte from a
chemically neutral species into a positive cation, usually breaking
various bonds in the process. Detecting the mass of the individual
pieces (referred to as fragments) allows for conclusive
identification of the chemical structure of the analyte.
Based on the mobile phase this technique can be
simply classified into two categories as: Liquid
Chromatography and Gas Chromatography. The
column which holds the stationary phase (which in
the form of small particles of the diameter of the
order in microns), plays unique role in these
processes. Usually silica is the base material for
producing this phase.
LIQUID CHROMATOGRAPHY (LC/HPLC)
Principle:
Early liquid chromatography was carried out in long glass columns with wide diameter. The
diameters of the stacked particles inside the column were of the order of 150- 200 microns
range. Even then, the flow rates (eluent time) of the mobile phase with the analyte were very
slow and separation times were long - often several hours! With the advent of latest technology
the particle diameters were reduced as small as to 10 microns with replacement of glass
columns with steel ones. The flow rate of the mobile phase was improved by applying high
pressure to the column using pumps and hence the performance was improved. This
development led the instrument to be mostly called as “High-performance Liquid
Chromatography” or “High-Pressure Liquid Chromatography” (HPLC). Though HPLC retains
major of the credits to the analytical side, the earlier one of simple Liquid Chromatography
still finds applications in the preparative purposes.
INSTRUMENTATION
The basic HPLC system consists of a solvent (mobile phase) reservoir, pump, degasser, injection device,
column and detector. The pump draws the mobile phase from the reservoir and pumps it to the column
through the injector. At the end of the column (effluent end), a detector is positioned.
Mostly UV absorption detector is used. In the case of analytical studies, after the detection the eluents are
collected in waste bottles. In the case of preparative studies the eluents are fractionally collected for
further studies. Most of the HPLC design will be the same as described for all the four main groups
previously described. However, there can be differences in selecting the specific detectors for particular
type of analysis, say for example, with ion-exchange chromatography, detectors commonly used are
conductivity detectors for obvious reasons. Other important detectors for HPLC separations include
refractive index detector, fluorescence detector and mass selective detector. The following is the most
generalized outlay of the HPLC system:
INSTRUMENTATION
DISADVANTAGES
Column performance is very sensitive, which depends on
the method of packing. Further, no universal and
sensitive detection system is available.
GAS CHROMATOGRAPHY (GC)
Principle:
Here an inert carrier gas (Helium or Nitrogen) acts as the mobile phase. This will carry the
components of analyte mixture and elutes through the column. The column usually contains an
immobilized stationary phase. The technique can be categorised depending on the type of
stationary phase as follow:
Gas Solid Chromatography (GSC) - here the stationary phase is a solid which has a large
surface area at which adsorption of components of the analyte takes place. The separation is
possible based on the differences in the adsorption power and diffusion of gaseous analyte
molecules. The application of this method is limited and is mostly used in the separation of the
low-molecular-weight gaseous species like carbon monoxide, oxygen, nitrogen and lower
hydrocarbons.
Gas Liquid Chromatography (GLC) - this is the most important and widely
used method for separating and determining the chemical components of
volatile organic mixtures. Here the stationary phase is a liquid that is
immobilized on the surface of a solid support by adsorption or by chemical
bonding. The separation of the mixture into individual components is by
distribution ratio (partition) of these anayte components between the gaseous
phase and the immobilized liquid phase. Because of its wide applications most
of the GCs are configured for the GLC technique.
Instrumentation
The instrumentation for GC is different from that of HPLC in that the
injection port, column and detector are to be heated to a pre-specified
temperature. Since the mobile phase here is a gas (carrier gas) the
components present in the analyte mixture should be vaporised, so that it
can be effectively carried through the column. The basic instrumentation for
GC includes a carrier gas cylinder with regulator, a flow controller for the
gas, an injection port for introducing the sample, the column, the detector
and the recorder. An outlay is as follow:
In the above illustration the injection port, column oven and detector are hot zones. The success of
this technique requires the appropriate selection of the column and the temperature conditions at
which the column to be maintained throughout the analysis. Basically the columns for GC are
classified as analytical columns and preparative columns. The analytical columns are of two types:
packed column and open-tubular or capillary column. Both differ in the way the stationary phases
are stacked inside.
In the instrumentation of GC detectors play unique role. There are a number of detectors, which vary
in design, sensitivity and selectivity. Detectors in GC are designed to generate an electronic signal
when a gas other than the carrier gas elutes from the column.
DISADVANTAGES
Samples must be volatile and thermally stable below about
4000 C. No single universal detector is available and most
commonly used detectors are non-selective. One should take
much care in the analytical steps starting from the selection of
the column, the detector and must define the temperatures of all
the three ports viz., injection port, column oven and detector.
An improper programming on these will lead to erratic results.