Marian Danielle Fidelis S. Borongan
BS PHARM III
Phar 43L W 1:30-4:30
January 14, 2014
Chromatography is a method by which a mixture is separated by distributing its components
between two phases. The stationary phase remains fixed in place while the mobile phase carries the
components of the mixture through the medium being used. The stationary phase acts as a constraint on
many of the components in a mixture, slowing them down to move slower than the mobile phase. The
movement of the components in the mobile phase is controlled by the significance of their interactions
with the mobile and/or stationary phases. Because of the differences in factors such as the solubility of
certain components in the mobile phase and the strength of their affinities for the stationary phase, some
components will move faster than others, thus facilitating the separation of the components within that
Thin Layer Chromatography
Thin-layer chromatography (TLC) is a very commonly used technique in synthetic chemistry for
identifying compounds, determining their purity and following the progress of a reaction. It also permits
the optimization of the solvent system for a given separation problem. In comparison with column
chromatography, it only requires small quantities of the compound and is much faster as well.
Plates, also known as chromatoplates, can be prepared in the lab but are most commonly
purchased. They constitute the stationary phase in a TLC set up and are usually made up of silica
gel and alumina. The backing of TLC plates is often composed of glass, aluminum, or plastic. The
face of the TLC plate must not be touched as contamination from skin oils or residues on gloves
can obscure results. Instead, always handle them by the edges, or with forceps.
Proper solvent selection is perhaps the most important aspect of TLC. This will become the mobile
phase of the set up. A common starting solvent is 1:1 hexane:ethyl acetate. Varying the ratio can
have a pronounce effect on the Rf values. Choosing the appropriate mixture of solvent helps in the
separation and identification of the components in a mixture.
Spots are applied to the plate using very thin glass pipettes. The capillary should be thin enough to
apply a neat spot, but not so thin as to prevent the uptake of an adequate quantity of analyte.
Principle of Thin Layer Chromatography
Different compounds in the sample mixture travel at different rates due to the differences in their
attraction to the stationary phase, and because of differences in solubility in the solvent. By changing the
solvent, or perhaps using a mixture, the separation of components (measured by the Rf value) can be
Separation of compounds is based on the competition of the solute and the mobile phase for
binding places on the stationary phase. For instance, if normal phase silica gel is used as the stationary
phase it can be considered polar. Given two compounds which differ in polarity, the more polar compound
has a stronger interaction with the silica and is therefore more capable to dispel the mobile phase from
the binding places. Consequently, the less polar compound moves higher up the plate (resulting in a
higher Rf value). If the mobile phase is changed to a more polar solvent or mixture of solvents, it is more
capable of dispelling solutes from the silica binding places and all compounds on the TLC plate will move
higher up the plate. It is commonly said that "strong" solvents (eluents) push the analyzed compounds up
the plate, while "weak" eluents barely move them. The order of strength/weakness depends on the
coating (stationary phase) of the TLC plate. For silica gel coated TLC plates, the eluent strength increases
order: Perfluoroalkane (weakest), Hexane, Pentane,Carbon
tetrachloride, Benzene/Toluene, Dichloromethane, Diethyl
ether, Ethylacetate, Acetonitrile, Acetone, 2-
Propanol/n-Butanol, Water, Methanol, Triethylamine, Acetic acid, Formic acid (strongest). For C18 coated
plates the order is reverse. Practically this means that if you use a mixture of ethyl acetate and hexane as
the mobile phase, adding more ethyl acetate results in higher Rf values for all compounds on the TLC
plate. Changing the polarity of the mobile phase will normally not result in reversed order of running of
the compounds on the TLC plate. An eluotropic series can be used as a guide in selecting a mobile phase.
If a reversed order of running of the compounds is desired, an apolar stationary phase should be used,
such as C18-functionalized silica.
TLC is very simple to use and inexpensive. Undergraduates can be taught this technique and apply
its similar principles to other chromatographic techniques. There are little materials needed for TLC
(chamber,watch glass, capillary, plate, solvent, pencil, and UV-light). Therefore, once the best solvent is
found, it can be applied to other techniques such as High performance liquid chromatography. More than 1
compound can be separated on a TLC plate as long as the mobile phase is preferred for each compound.
The solvents for the TLC plate can be changed easily and it is possible to use several different solvents
depending on your desired results. As stated earlier, TLC can be used to ensure purity of a compound. It
is very easy to check the purity using a UV-light. Identification of most compounds can be done simply by
checking Rf literature values. You can modify the chromatography conditions easily to increase the
optimization for resolution of a specific component.
TLC plates do not have long stationary phases. Therefore, the length of separation is limited
compared to other chromatographic techniques. Also, the detection limit is a lot higher. If you would need
a lower detection limit, one would have to use other chromatographic techniques. TLC operates as an
open system, so factors such as humidity and temperature can be consequences to the results of your
In column chromatography, the stationary phase, a solid adsorbent, is placed in a vertical glass (usually)
column. The mobile phase, a liquid, is added to the top and flows down through the column by either
gravity or external pressure. Column chromatography is generally used as a purification technique: it
isolates desired compounds from a mixture.
Only the Silica gel and alumina are two adsorbents commonly used by the organic chemist for
column chromatography. Smaller particle size increases the surface area of the adsorbent thus
they are sold in different mesh sizes. Adsorbent particle size affects how the solvent flows through
the column. Smaller particles (higher mesh values) are used for flash chromatography; larger
particles (lower mesh values) are used for gravity chromatography.
The polarity of the solvent which is passed through the column affects the relative rates at which
compounds move through the column. A non-polar solvent is first used to elute a less-polar
compound. Once the less-polar compound is off the column, a more-polar solvent is added to the
column to elute the more-polar compound.Polar solvents can more effectively compete with the
polar molecules of a mixture for the polar sites on the adsorbent surface and will also better
solvate the polar constituents. Consequently, a highly polar solvent will move even highly polar
molecules rapidly through the column. If a solvent is too polar, movement becomes too rapid, and
little or no separation of the components of a mixture will result. If a solvent is not polar enough,
no compounds will elute from the column. Proper choice of an eluting solvent is thus crucial to the
successful application of column chromatography as a separation technique.
The column is where separation occurs since it is in this tool where the absorbent and the solvent
will be placed allowing the separation of a mixture.
Principle of Column Chromatography
When a mixture of mobile phase and sample to be separated are introduced from top of the
column, the individual components of mixture move with different rates. Those with lower affinity and
adsorption to stationary phase move faster and eluted out first while those with greater adsorption affinity
move or travel slower and get eluted out last. The solute molecules adsorb to the column in a reversible
Column chromatography can be used in both analytical and preparative applications. It can be used
to determine the number of components in a mixture and can also be used to separate and purify
substantial quantities of these components for subsequent analysis.
Properly setting up the column requires some technical skill and manual dexterity. It is very time
consuming and tedious, especially for large samples. Collecting vessels must be frequently switched and
solvent levels need to be topped up.
Gas chromatography is a term used to describe the group of analytical separation techniques used
to analyze volatile substances in the gas phase. In gas chromatography, the components of a sample are
dissolved in a solvent and vaporized in order to separate the analytes by distributing the sample between
two phases: a stationary phase and a mobile phase. Gas chromatography is one of the sole forms of
chromatography that does not utilize the mobile phase for interacting with the analyte. The stationary
phase is either a solid adsorbent, termed gas-solid chromatography (GSC), or a liquid on an inert support,
termed gas-liquid chromatography (GLC).
The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium, argon,
and carbon dioxide. The choice of carrier gas is often dependant upon the type of detector which is
used. The carrier gas system also contains a molecular sieve to remove water and other impurities.
Sample Injection Port
For optimum column efficiency, the sample should not be too large, and should be introduced onto
the column as a "plug" of vapour. The most common injection method is where 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. For packed columns, sample size ranges from tenths of
a microliter up to 20 microliters. Capillary columns, on the other hand, need much less sample,
typically around 10-3 mL. For capillary GC, split/splitless injection is used.
They contain the volatile sample to be analyzed.
Principle of Gas Chromatography
Gas Chromatography is used to separate volatile compounds in a mixture. The separated
compounds can be identified and quantified. To achieve the identification of different compounds, three
steps can be distinguished in a GC-MS system:
Depending on the sample (gas, liquid or solid), compounds in a mixture need to be volatilized or
extracted from the matrix by one of the injection techniques.
After injection of a mixture, separation is achieved in the capillary column. This column is coated
with a fluid or a solid support, the stationary phase. An inert gas, also called the mobile phase, is
flowing through the column. Depending on the phase equilibrium between the stationary and
mobile phase, compounds travel with different velocities through the column. The mixture becomes
separated, and as a result, individual compounds reach the detector with a different retention time.
By choosing a column, which separates on boiling point, polarity, size or stereochemistry, a wide
range of compounds can be separated.
Many different detectors (table 2) can be used for detection of the separated compounds. The mass
spectrometer combines a high sensitivity with the unique property of being able to determine the
molecular composition. Below, only the mass spectrometer will be discussed in more detail. The
other detectors are dedicated tools to analyse specific compounds.
Gas chromatography requires only very small samples with little preparation. It is good at
separating complex mixtures into components. Results are rapidly obtained with very high precision. It will
allow instrument only with the sensitivity to detect organic mixtures of low concentrations. And the
equipment is not very complex.
It has a very strong retention of low volatility or polar solutes. Catalytic changes may occur on GSC