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ADVANCED ANALYTICAL

TECHNIQUES

Chromatographic Separations
by
Arslan Munir
Topics
• History
• Chromatographic Separation
• General description of Chromatography
• Classifying Chromatographic Methods
• Chromatograms
• Relative migration rates of solutes
• Distribution Constant
• Retention Time
History
• Russian botanist Tswett is generally referred to as the
father of chromatography.
• His work was published in 1906.
• Tswett used a liquid-adsorption column containing
calcium carbonate to separate plant pigments.
• Tswett invented the terms chromatography and
chromatogram.
• Chromatography means “color writing”
Chromatographic Separation
• Chromatography is a widely used method for separation,
identification, and determination of the chemical
components in complex mixtures.
• No other separation method is as powerful and generally
applicable as chromatography.
General description of Chromatography
• Components of a mixture are carried through the
stationary phase by the flow of a mobile phase.
• Separations are based on differences in migration rates
among the mobile-phase components.
• The stationary phase in chromatography is a phase that is
fixed in place either in a column or on a planar surface.
• The mobile phase in chromatography is a phase that
moves over or through the stationary phase carrying with
it the analyte mixture.
• The mobile phase may be a gas, a liquid, or a
supercritical fluid.
Chromatography
• Chromatography is a technique in which the components of
a mixture are separated based on differences in the rates at
which they are carried through a fixed or stationary phase
by a gaseous or liquid mobile phase.
Classifying Chromatographic Methods
Chromatographic methods are of two basic types.
• In column chromatography, the stationary phase is held in
a narrow tube, and the mobile phase is forced through the
tube under pressure or by gravity.
• In planar chromatography, the stationary phase is
supported on a flat plate or in the pores of a paper, and
the mobile phase moves through the stationary phase by
capillary action or under the influence of gravity.
• Column chromatographic methods can be further
subdivided according to the nature of mobile phase,
specially liquid, gas and supercritical fluid.
Chromatographic terms
• The analyte is the substance that is to be separated
during chromatography.
• A chromatograph is equipment that enables a good
separation e-g gas chromatographic or liquid
chromatographic separation.
• The retention time is the characteristic time it takes for a
particular analyte to pass through the system (from the
column inlet to the detector) under set conditions.
• The solute refers to the sample components in partition
chromatography.
• The solvent refers to any substance capable of
solubilizing other substances, especially the liquid mobile
phase in LC.
Chromatographic terms
• Elution is a process in which solutes are washed through a
stationary phase by the movement of a mobile phase.
• The mobile phase that exits the column is termed the eluate.
• An eluent is a solvent used to carry the components of a
mixture through a stationary phase.
Chromatographic Process
• A basic column chromatographic process may be described as
follows:
• A vertical hollow glass tube (the column) is filled with a suitable
finely powdered solid, the stationary phase.
• At the top of this column is placed a small volume of the sample
mixture to be separated into individual components.
• The sample is then taken up by continuous addition of the
mobile phase, which goes through the column by gravity,
carrying the various constituents of the mixture along with it.
• The process is called elution.
• If the components migrate at different velocities, they will
become separated from each other and can be recovered,
mixed with the mobile phase.
Column Chromatography
Chromatograph
Basic components of a chromatograph are as follows:
• Mobile phase supply
• Sampling system
• Column
• Detector
• Recorder or computer
data acquisition
Chromatograms
• If a detector that responds to solute concentration is
placed at the end of the column during elution and its
signal is plotted as a function of time (or of a volume of
added mobile phase), a series of peaks is obtained, such
a plot, called a chromatogram, is useful for both
qualitative and quantitative analysis.
• The positions of the peaks on the time axis can be used
to identify the components of the sample.
• The area under the peaks provide a quantitative measure
of the amount of each species.
Chromatograms
Chromatography Nomenclature
• The baseline is any part of the chromatogram where only
mobile phase is emerging from the column.
• The peak maximum is the highest point of the peak.
• The injection point is that point in time/position time
when/where the sample is placed on the column.
• The retention time (tr) is the time elapsed between the injection
point and the peak maximum.
• Each solute has the characteristic retention time.
• The retention volume (Vr) is the volume of the mobile phase
passed through the column between the injection point and the
peak maximum.
• Vr = Q*tr
• Where Q is the flowrate in ml/min
continue
• The peak width at half height (w0.5) is the distance
between each side of a peak measured at half the peak
height.
• The peak width at the base (wb) is the distance between
the intersections of the tangents drawn to the sides of the
peaks and the peak base geometrically produced.
Relative migration rates of Solutes
• The effectiveness of a chromatographic column in
separating two solutes depends on the relative rates at
which the two species are eluted.
• The rates are in turn determined by the ratios of the solute
concentrations in each of the two phases.
• All chromatographic separations are based on differences
in the extent to which solutes are distributed between the
mobile and the stationary phase. For the solute species A,
the equilibrium is described by the equation
A(mobile) ↔ A(stationary)
The equilibrium constant Kc for this reaction is called a
distribution constant,
which is defined as
𝐾𝑐 = 𝐴 𝑠/[A]m
Applications of Chromatography
• Chromatography is a powerful and versatile tool for
separating closely related chemical species.
• In addition, it can be employed for the qualitative
identification and quantitative determination of separated
species.