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Background Theory - Chromatography and TLC

General Principles of Chromatography
Chromatography is a technique which is widely used to separate the different component
compounds from a mixture of substances. The term chromatography encompasses a number of
different techniques which, although they will be discussed separately, are all based on common
principles. Chromatography may be divided broadly into three kinds: adsorption, partition and
ion-exchange, and the simplest type of these is adsorption chromatography which will be used
in Expt2.

1. Adsorption Chromatography
absorb = solutes and/or solution permeate into a solid (e.g. sponge and water)
adsorb = solutes and/or solution interact and adhere only to the surface of a solid, (i.e.
they do not permeate into the solid, like paint on metal)
In this technique, the sample substance to be separated into its pure components is adsorbed
onto a solid support (the stationary phase) and separation of the mixture into its pure
components is achieved by elution with solvents (the mobile phase) of different polarity.
Adsorption chromatography may be carried out in two ways, by column, or thin layer
techniques. Both techniques will be used in the organic laboratory and they will be discussed
separately.
There are two opposite types of adsorption chromatography:
i. Normal phase:
mobile phase: non-polar liquid solvent (e.g. hexanes, ether, dichloromethane)
stationary phase: polar solid column packing (e.g. silica, alumina)
ii. Reverse phase:
mobile phase: polar liquid solvent (e.g. water, methanol, acetonitrile)
stationary phase non-polar solid column packing (e.g. C18 functionalized silica)
Reverse phase packings are now sold for microscale or preparatory scale chromatography, however
they are much too expensive for a student lab (even the normal phase silica gel is a bit expensive and
often researchers recover and clean it for reuse). Reverse phase is however the most common form of
column chromatography used in analytical technique called High Pressure Liquid Chromatography
(HPLC) where one column costs ~$1000 (too expensive for 2 nd year lab experiments). Reverse phase
TLC plates are also available but cost significantly more than normal phase silica TLC plates (see for
example the following link:
http://www.sigmaaldrich.com/catalog/product/aldrich/z292923?lang=en&region=CA (active 2015)

Column Chromatography (Normal Phase)

In column chromatography a finely divided adsorbent such as silica or alumina is placed in a
glass column, supported at the bottom by a wad of glass wool, as shown below.

A layer of sand is placed over the top of the adsorbent, and the whole
column is wetted with the solvent to be used. The sample to be purified is dissolved in the
minimum of solvent then applied evenly to the top of the column and allowed to drain into the
adsorbant (e.g. silica gel); if the sample is loaded with too much solvent, the bands of
component compounds will be too large and stay overlapped instead of separating completely
from each other before they reach the bottom of the column. The column is then eluted by
passing down the mobile phase solvents (the solvent system is usually a mixture of solvents).
In this way, weakly adsorbed substances will pass rapidly through the column while the more
strongly adsorbed substances will pass through at a slower rate, allowing separate elution of
each pure component as they come of the column at different times. Also, it is often necessary
to elute with a series of solvents of increasing polarity to ensure complete separation of the
components.
NOTE: Pipette Column Chromatography is identical to the column chromatography above
except that it is a smaller scale. Pipette columns can be run much more quickly than a regular
column, however attentiveness and skill is needed because, with no stopcock, the solvent flow
can not be stopped.

Choice of Adsorbent (Stationary Phase)

Silica gel (60 mesh or 200 - 400 mesh ; oxides of silicon) is the by far the most often used
adsorbent for column chromatography of organics. Silica gel, which is a white sandy solid when
dry, swells considerably when wetted with solvent; this property makes it an ideal stationary
phase since a gel has a huge internal surface area allowing significantly more adsorption
interactions per given volume than a non-gel substance; the following link describes the
preparation, structure and properties of silica gel:
http://www.chromatography-online.org/Stationary-Phases/Silica-Gel/Irregular/rs_3_41.php
http://www.chromatography-online.org/Stationary-Phases/Silica-Gel/Irregular/rs_3_42.php
http://www.chromatography-online.org/Stationary-Phases/Silica-Gel/Spherical/rs_3_43.php

(active 2015)

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The order in which the compounds are eluted will depend on how strongly they are adsorbed on
the surface of the stationary phase. The following link illustrates how the surface of the silica
particles is polar due to the presence of polar Si-OH functional groups (whereas the internal SiO-Si groups are non-polar):
http://www.chromatography-online.org/Stationary-Phases/Silica-Gel/Structure/rs_3_44.php
(active 2015)
Thus polar functional groups on sample molecules interact with the polar surface Si-OH
functional groups. Different sample molecules have different types of polar functional groups
and therefore a different strength of interaction with the silica surface, which is the basis of the
chromatographic separation technique; more polar sample molecules such as carboxylic acids,
alcohols, phenols and esters will migrate more slowly than less polar sample molecules such as
alkanes, alkenes, alkynes and aromatics with no hydroxyl (OH) groups.
Organic Functional Groups in Order of Increasing Polarity and Slower Migration for normal
phase chromatography (see table C4 p. 123 Lehmans Student Lab Companion 2nd ed.)
saturated hydrocarbons
unsaturated hydrocarbons
ethers
esters
halides
ketones ~ aldehydes
amines
alcohols
carboxylic acids

lowest polarity / weakest adsorption / fastest migration

highest polarity / strongest adsorption / slowest migration

note: NH2 groups H-bond strongly to silica and usually do not move at all because silica is acidic
and protonates the amines giving an ammonium salt (RNH3+) which is no longer soluble in the
organic solvent.
note: water "deactivates" silica by forming relatively strong hydrogen bonds to the polar Si-OH
surface sites which blocks the binding of sample molecules; therefore care must be taken to
ensure that the silica is properly dried and not exposed to water before using it, and solvents
must not contain any water.
Other adsorbents used in column chromatography, with different binding abilities are cellulose,
calcium oxide and alumina (aluminum oxide). Alumina, unless specially pretreated, is slightly
basic and hence strongly adsorbs acidic substances or materials capable of forming hydrogen
bonds to the basic oxygen atoms of the alumina. Compounds without the ability to form
hydrogen bonds but with substantial dipole moments will be somewhat less strongly adsorbed
due to electronic interactions between their dipoles and those of the alumina. Compounds with
neither acidic hydrogens nor dipole moments are only very weakly adsorbed due to induced
dipole-dipole interactions. The complete order for the strength of all these bonding interactions
is generally the following: Salt Formation > Coordination Complexes > Hydrogen Bonding >

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Dipole-Dipole Interactions > Van der Waals. Some examples of these bonding interactions with
alumina are shown:

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Choice of Solvent (mobile phase)
The choice of solvents used to elute the various components of the mixture from the column will
depend upon the polarity of the components in the mixture and the type of chromatography and
stationary phase being used. For normal phase chromatography of very weakly adsorbed (nonpolar) components, a non-polar solvent such as petroleum ether, hexanes or toluene would be
used. For more strongly adsorbed (polar) components, more polar solvents such as alcohols,
ether, ethyl acetate or dichloromethane might be used. Because polar solvents can adsorb to
the polar silica displacing the sample molecules into the mobile phase such that there is not as
much interaction with the silical, it is usually best to start with a solvent of lower polarity and, if
necessary, gradually increase the polarity.
Occasionally, some components adsorb so strongly that they can not be chromatographed and
only a very polar solvent such as ethanol, water or even acetic acid would be able to displace
the material from the column. It should be noted that the water deactivates silica because of
its high polarity and strong H-bonding interactions, thus water is never used as a mobile phase
in normal phase chromatography.
Keep in mind that normal phase has a polar adsorbent stationary phase so only works well with
a non-polar mobile phase (and vice-versa for reverse phase chromatography). That being said,
the usual strategy is to use a mixture of ~ 5% - 40% polar solvent in 50% - 95% non-polar
solvent. Such mixtures give both polar and non-polar characteristics to the mobile phase,
allowing the separation of a wider range of compounds. If you find this explanation a bit nonrigorous, that is because determining what solvent system will separate a particular sample
mixture is very much a trial and error procedure and the preceding considerations are used only
as guide to decide what systems to try first. If one is lucky, a suitable solvent system can be
found relatively quickly, however, sometimes weeks (or months!) must be spent finding a
suitable solvent mixture since there are hundreds of solvent systems (and mixture ratios) that
can be tried.
Common Solvents in Order of Increasing Elutive power (see table C4 Lehman 2nd ed.)

petroleum ether / hexanes

cyclohexane
benzene / toluene
dichloromethane / chloroform
diethyl ether
ethyl acetate

lowest polarity / lowest elutive power

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acetone
ethanol / methanol
water
acetic acid

highest polarity / highest elutive power

Thin layer Chromatography (TLC)

TLC is a form of adsorption chromatography for small amounts of sample mixture. The TLC
technique is extremely useful for analysis of a sample mixture to show how many components
are in the mixture and also provides a quick way to test out different solvent systems, without
wasting too much solvent or valuable product, to see which solvent system separates the
components most effectively.
In fact, TLC can be considered simply as upside down column chromatography, with the
solvent ascending the absorbent, rather than descending. Here, the solid adsorbent, silica gel
(alumina plates are also available but much less used), is spread out in a thin layer over a flat
plastic, glass or metal sheet. The sample mixture is dissolved in a volatile organic solvent and a
microdrop(s) of the solution is dabbed on to the bottom of the plate to form of a spot; the
solvent quickly evaporates making it seem as if the spot has disappeared, but the sample
molecules (solute) do not evaporate and remain on the plate and usually give a small dark spot.
A suitable solvent is then allowed to run up the sheet by capillary action, as shown below.

sample spots

~ 5 - 8 mm

During the elution with the solvent, each sample component will have a particular partitioning
ratio (time spent in the stationary phase vs. time in the mobile phase) such that each given
sample components spot moves up the plate a distance that is characteristic (i.e. reproducible)
of the pure substance, and these distances differ from one substance to the next. Once the
solvent has travelled to ~ 1 cm below the top of the plate, the TLC plate is removed from the jar.

Under an established set of conditions (solvent system, adsorbent, etc.), a given compound
always travels a fixed distance relative to the distance travelled by the solvent front. This ratio is
known as the Rf value (Retardation factor) and is given as a decimal fraction:
Rf = y / x
where,
x = distance (in cm) from origin to solvent front
y = distance (in cm) from origin to the centre of the sample spot (if too much sample had
been applied and the spot has a faint tail, then its 'centre of density' is estimated).
A mixture of substances will thus give rise to a series of spots, one corresponding to each
component. Compounds that adsorb weakly to the stationary phase, e.g. a non-polar
compound in normal phase silica gel chromatography, will move rapidly and give a high Rf
value. Conversely, compounds that adsorb strongly, e.g. polar compounds in normal phase
silica gel chromatography.
For column chromatography, the ideal solvent system is the one that moves the desired
component of the mixture to a TLC Rf of ~ 0.25-0.35 and will separate this component from its
nearest neighbor with a difference in TLC Rf values of at least 0.20.
NOTE: Shown below is a typical example of a TLC analysis of sample mixture and a sample of
a pure component. Lanes 1 and 2 have identical sample just spotted with a small vs. large
amount of sample respectively (likewise for lanes 3 and 4)
Q. What is the Rf value for the pure product component on this TLC plate?

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In preparative TLC work (~ 0.1 to 0.4 g compound loaded on to large glass TLC plates),
the components are recovered from the plate after development by using a spatula to scrape off
the compound + silica in the particular region of plate that the spot finished up at, followed by
the removal of the substance from the adsorbent by extraction with a suitable solvent. The pure
compound can then be analyzed to identify it.

2. Partition Chromatography
The second general type of chromatography is partition chromatography, consisting of gasliquid chromatography, liquid-liquid chromatography and paper chromatography (which is an
application of liquid-liquid chromatography). In gas chromatography (see Exp. 2), the moving
phase is a stream of an inert gas, such as nitrogen or argon. The sample under investigation is
volatilised and the vapour swept through a column of the stationary phase by the carrier gas.
Partition of the sample between the stationary and moving phases will occur. However, for liquid
chromatography, the system is eluted with a moving liquid leading to separation of the
components of a mixture by the partitioning of these components between the stationary and
moving liquid phases so that the components will move at differing rates through the column.
Paper chromatography is analogous to thin layer chromatography except that in this case, the
support material consists of a sheet of specially prepared paper, and the stationary phase is
considered to be water adsorbed on the paper. Elution with a solvent, measurement of the Rf
value and preparative work is carried out in the same way as for thin layer chromatography.

3. Ion-Exchange Chromatography
The third type of chromatography, ion-exchange chromatography, is of somewhat more limited
application in the chemistry lab, but is widely used in biochemistry. Here, the solid support
consists of a resin which can have either basic or acidic properties, and mixtures of acidic or
basic substances can be separated using these resins by eluting with buffers of different pH's.