CHROMATOGRAPHY
WHAT IS CHROMATOGRAPHY?
The general name given to methods by which two or more
compounds in a mixture are physically separated by distributing
themselves between two phases:
- a stationary phase (solid or liquid supported on a solid) and
- a mobile phase (gas or liquid)
The mobile phase continuously flows around the stationary phase.
Separation results from differences in their affinity for the
stationary phase.
LIQUID CHROMATOGRAPHY
Small volume of sample introduced to one side of the column.
The mobile solvent phase moves the sample through the column
packing.
The individual components undergo adsorption and desorption on
the packing
 slowing their motion by different amounts depending on their
affinity for the packing
Each component is distributed between the stationary phase (s) and
the mobile phase (m) as it passes down the column according to:
Xm  Xs
The distribution coefficient of component X:
KX 
[ X ]s
[ X ]m
Large Kx  X favours the stationary phase
and will move slower through the column
Separation of Mixtures by Paper Chromatography
Stationary
phase
Mobile
phase
Separation of Mixtures by Column Chromatography
Mobile
phase
Stationary
phase
Component with
greater affinity
for stationary
phase
Can collect
the different
fractions
eluted
Can constantly monitor the eluted solution as a function of time or
volume – produce a chromatogram.
t0 = time for solvent to traverse the column
tr = retention time
tw = peak base width
h = peak height
Synthetic chemists use columns to separate their products
They collect different fractions at different times
GAS CHROMATOGRAPHY
Mobile phase: generally an inert gas such as Helium
Stationary phase: generally an adsorbent or liquid distributed over
the surface of a porous, inert support.
Suitable for vapour
phase samples
To vaporise sample
solutes and keep them in
the vapour phase
COLUMNS
Two general types of column:
1) Packed columns
 contain a finely divided, inert, solid support material coated with
liquid stationary phase.
 length ~ 1.5 - 10m and internal diameter ~2 - 4mm.
2) Capillary columns (or Open tubular columns)
 internal diameter ~ few tenths of a mm and length ~ up to 100 m
 wall-coated columns: walls act as support for the stationary
liquid phase
 support-coated columns: wall is lined with a thin layer of
support material onto which the stationary phase is adsorbed.
As before:
Column temperature:
For precise work, column temperature must be controlled to within
tenths of a degree.
Optimum temperature  dependant on boiling point of the sample.
Temperature programming - if a sample has a wide boiling range,
column temperature is increased (either continuously or in steps)
as separation proceeds.
Capillary column
in GC oven
SAMPLE INJECTION
For optimum column efficiency:
- sample should not be too large
packed columns: L to 20 L
capillary columns: ~1 L
- sample should be introduced as a "plug" of vapour
slow injection of large samples causes band broadening
and loss of resolution.
Most common injection method - a microsyringe is used to inject
sample through a rubber septum into a flash vapouriser port at the
head of the column.
The temperature of the sample port is usually about 50°C higher
than the boiling point of the least volatile component of the sample.
Capillary GC split/splitless injector:
Sample vapourises to
form a mixture of carrier
gas, vapourised solvent
and vapourised solutes.
Inject sample
A proportion of the
gas mixture passes
into the column
To the column
DETECTORS
There are many detectors used in GC. Different detectors will give
different types of selectivity.
DETECTOR
SELECTIVITY
Thermal conductivity Universal
Electron capture
Halides, nitrates, nitriles, peroxides, anhydrides,
organometallics
Photo-ionization
Aliphatics, aromatics, ketones, esters, aldehydes,
amines, heterocyclics, organosulphurs, some
organometallics
Flame ionization
Most organic compounds
Flame photometric
Sulphur, phosphorus, tin, boron, arsenic,
germanium, selenium, chromium
Hall electrolytic
conductivity
Halide, nitrogen, nitrosamine, sulphur
Examples of some GC
chromatograms
Effect of different columns on “separation ability”:
THEORETICAL PLATE MODEL
Divide a column into a large number of separate layers, called
theoretical plates.
Equilibration of the sample between the stationary and mobile
phase occurs in each plate.
The analyte moves down the column by transfer of equilibrated
mobile phase from one plate to the next.
NB: this is a theory to help us understand and explain what is
happening  the plates do not really exist
The number of theoretical plates (N) in a column can be found by
examining a chromatographic peak after elution:
N
5.55 t r 2
w 1/ 22
tr = retention time
w1/2 = peak width at half-height
Different retention times for different solutes  different numbers of
plates for different solutes in a mixture
w1/2
COLUMN EFFICIENCY
The column is more efficient in separation if:
- there are more plates
- the plate height is smaller
Calculate the Height Equivalent to a Theoretical Plate (HETP):
HETP 
L
N
L = length of column
To obtain optimal separations  sharp, symmetrical peaks must
be obtained.
 band broadening must be limited
RATE THEORY OF CHROMATOGRAPHY
 takes account of the time taken for the solute to equilibrate
between the stationary and mobile phase (plate model assumes
infinitely fast equilibration).
 resulting peak shape of a peak affected by the rate of elution
Also the different paths available to solute molecules as they travel
through the stationary phase affects peak shape.
Van Deemter equation for plate height:
B
HETP  A   C u
u
u = average velocity of mobile phase
A, B, C = factors which contribute to band broadening
A, B, C = factors which contribute to band broadening
A - Eddy diffusion
Solute molecules will take different paths through the stationary
phase at random. Different paths have different lengths
 band broadening
B - Longitudinal diffusion
Concentration of analyte is more at the center than the edges of the
column,  analyte diffuses out from the center to the edges
 band broadening
High velocity of the mobile phase
 analyte spends less time on the column
 decreases the effects of longitudinal diffusion
C - Resistance to mass transfer
If velocity of mobile phase is high and analyte has a strong affinity
for stationary phase
 analyte in mobile phase will move ahead of that in stationary
phase
 band broadening
Van Deemter plot:
plot of plate height vs. average linear velocity of mobile phase
 useful in determining the optimum mobile phase flow rate
To obtain high resolution, the three terms must be maximised:
1 Increase the number of theoretical plates.
- Lengthening the column  an increase in retention time and
increased band broadening - not desirable.
- Instead reduce the height equivalent to a theoretical plate
(HETP) by reducing the size of the stationary phase particles.
2 Improve capacity
 In GC by changing the temperature
 In LC by changing the composition of the mobile phase
3 Increase the selectivity by:
- changing mobile phase composition
- changing column temperature
- changing composition of stationary phase
- using special chemical effects
(e.g. including complexing agents in stationary phase to
complex with one of the solutes)
But FIRST optimise capacity!