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IP6 GROUPS 4, 5 and 6

From Greek chroma (color) and graphein (to
A group of methods used for the separation,
identification, and determination of chemical
components in a complex mixture
Can be defined as a procedure by which solutes are
separated by a differential migration process in a
system consisting two or more phases, one of which
moves continuously in a given direction and in which
the individual substances exhibit different motilities
by reason of differences in adsorption, partition,
solubility, vapor pressure, molecular size, or ionic
charge density
Stationary Phase
one of the phases; a fixed bed of large surface area
it must be small and homogenous as possible to allow frequent and
efficient sorption and desorption
Mobile Phase
a fluid that moves through or over the surface of the stationary phase

Involves an equilibrium between a vapor enriched in the more-
volatile component and a liquid condensate of the same
Mikhail Tswett a Russian-Italian botanist who pioneered
adsorption chromatography, used it for separation of plant pigments

Classification of Chromatographic Techniques
A. Adsorption Chromatography
a. Gas-Solid Chromatography
b. Liquid Column Chromatography
c. High-Performance Liquid Chromatography
d. Thin-Layer Chromatography
B. Partition Chromatography
a. Gas-Liquid Chromatography
b. Supercritical Fluid Chromatography
c. Liquid-Liquid Chromatography
d. Paper Chromatography
e. High-Performance Liquid Chromatography

C. Ion-Exchange Chromatography
a. Ion-Exchange Chromatography
b. High-Performance Liquid Chromatography
D. Permeation Chromatography
a. Size Exclusion Chromatography
E. Affinity Chromatography
a. DNA Affinity Chromatography
F. Electrophoresis
a. Capillary Electro-Chromatography
Theory of Chromatography
Rate theory
By Giddings
Considers the dynamics of the solute
particle as it passes through the void spaces
between the stationary phase particles in the
system as well as its kinetics as it is
transferred to and from the stationary phase

Plate theory
By Martin and Singe
Considers the chromatographic system as a
series of discrete layers of theoretical plates.
At each of these, equilibration of the solute
between the mobile and stationary phases
occurs. The movement of the solute is
considered as a series of stepwise transfers
from plate to plate.

Retardation Factor (Rf)
Chromatographic systems achieve their
ability to separate mixtures of chemicals by
selectively retarding the passage of
compounds to through the stationary phase
while permitting others to move more freely
Used for qualitative evaluation
Different chromatographic techniques have
different ways of calculating Rf
The ratio of the distance from the origin
traveled by the solute band to the distance
traveled by the mobile phase in a particular
time (for TLC)

Thin-Layer Chromatography
Differs from other techniques because
separation occurs on a planar surface, not in
an enclosed column and the mobile face is
drawn by capillary action
Not as effective in separation but has
advantages of speed, versatility, and simplicity
Normal Phase relatively polar stationary
phase and relatively non-polar mobile phase
Reverse Phase relatively non-polar
stationary phase and relatively polar mobile

Stationary Phase
Finely divided solid spread as a thin layer
on a rigid supporting plate (plastic, steel,
or aluminum)
Silica gel most frequently used
stationary phase
Surface is acidic due to the presence of
many silanol hydroxyl groups therefore
best suited for polar and acidic compound
Silica gel + silicone oil or octadecylsilyl
(ODS) used for reverse phase

Alumina (aluminum oxide) preferred for
separation of basic and weakly polar
Polyamide (nylon) strong H-bonding
Other less frequently used sorbents:
calcium phosphate, calcium carbonate,
diatomaceous earth (kieselguhr)
Binders to the backing plate: calcium
sulfate (gypsum), starch
carboxymethylcellulose, or polyvinyl

Mobile Phase
A liquid allowed to migrate across the
surface of the plate
Chosen using eluotropic series and
depends largely on the nature of the
solutes and stationary phase
Preferable to use a single solvent
rather than a multicomponent mixture
because as it moves up the plate the
concentration changes since it is
adsorbed preferentially
Should be volatile so the set-up can
be equilibrated and it can be
evaporated after plate development

Can be altered (largely empirical)
Substances with functional groups
similar to those of the solutes may be
added to increase the Rf value by
promoting the solubility in the mobile
Acids or bases may be added to affect
the charges on the solutes to prevent
tailing (the substance trails a line
behind it like a tail)

Preparation of Plates
Larger dimensions = more effective separation
Scrupulously cleaned
Fluorescent indicators such as zinc silicate is added to
aid in detection
Two different sorbents may be applied to form a
gradient of both to yield separations that otherwise
would be impossible (ex. silica gel and alumina)
Thicker layer = quantitative (greater volume but may
cause band-broadening)
Thinner layer = qualitative (more effective separation)

Sample Application and Development
Plates should be dried
Spots should be evaporated after every application
and should be as small as possible
Saturate the atmosphere of the jar with mobile phase
Mobile phase should be evaporated after
Multiple development to increase resolution, the
plate is redeveloped in the same direction after it is
Two-dimensional development complex
development used for complex mixtures

Detection Methods
Obvious highly colored samples
Fluorescence Quenching zinc silicate

A. Preparation of TLC Plates

B. Preparation of Samples and Standards
Prepare 1% (w/v) ethanolic solution for each
sample and standard

C. Preparation of the Mobile Phase
200 mL of 95% ethyl acetate and 5% acetic

D. Spotting the Plate

Preparing the sample
Small amount of the material was used (1% solution)
Spotting the TLC plate
TLC plate heated before spotting (Activation) to evaporate
So that any water molecule adsorbed by the Silica is removed. When water is absorbed, the site
of attachment for the sample is blocked.
Pencil is used
Since graphite is inert.
TLC plate handled gently and by the edge
Since the 100-mm-thick coating of silica gel can be easily scratched off or contamination of the
surface can occur with the oil, dead skin cells, etc. that our hands contain.
Spotted a couple of times
So as to ensure that sufficient amount of material is present.
Spot made as small as possible
So as to avoid streaking, spotting and other common errors which may affect the result.
Picking a solvent
Ethyl acetate has a polarity of 4.4 while acetic acid
has a polarity of 6.2 in accordance with the polarity index.
(A higher value refers to a more polar substance). Thus,
the mobile solvent used was relatively non-polar.
Stays at the bottom = Add more polar solvent
Runs with the mobile phase = Add more non polar solvent
E. Developing the TLC Plate

F. Visualization of the Spots

Developing the plate
Covered with aluminum foil
So as to prevent evaporation of the solvent
Saturate the atmosphere within the container
So as to ensure better separation
Spots should be above the developing liquid and not submerged
So as to avoid washing off the sample
Solvent shouldnt reach the top end of the plate
So that uncertainty in measurements for Rf will be avoided
Immediately draw a line across where the solvent can be seen
For a more accurate Rf value
Visualizing the results
Use of UV light
o Short wave UV: 280 100 nm
o Long wave UV: 400 315 nm
o TLC plates normally contain a fluorescent
indicator that makes them glow green under
UV light of wavelength 254 nm
o Quench the green fluorescence
o Yield dark purple or blue spots

Use of Iodine Chamber
o Adsorb iodine vapors
o Yield brown spots
Process of Thin-Layer Chromatography
1. Preparing the Sample
2. Spotting the TLC Plate
3. Picking a Solvent
4. Developing the TLC Plate
5. Visualizing the TLC Plate
6. Calculating the Rf Values

Retardation Factor (Rf)
A more polar substance will have a lower Rf
value while a less polar substance will have
a higher Rf value. This can be inferred from
the fact that the mobile phase used was
relatively non-polar.
The non-polar substance experiences
stronger attraction to the non-polar solvent
than the polar stationary phase. Thus, they
spend most of their time in the mobile phase
and travel a larger distance.
distance travelled = Rf value = polarity
Retardation Factor (Rf) Values
Group 1 Group 2 Group 3 Group 4 Group 5 Group 6
Caffeine Excedrin 0.35 0.44 0.46 0.35 0.42 0.43
Caffeine Standard 0.37 0.43 0.38 0.35 0.42 0.43
Caffeine Sample 0.39 0.40 0.43 0.39 0.42 0.43
Aspirin Excedrin 0.84 0.86 0.91 0.80 0.82
Aspirin Standard 0.88 0.91 0.90 0.82
Aspirin Sample 0.84 0.90 0.87 0.81
0.70 0.75 0.83 0.66 0.73 0.68
0.70 0.77 0.78 0.68 0.75 0.68
0.70 0.77 0.78 0.68 0.75 0.68
Retardation Factor (Rf)
Experimental data show that aspirin is the least polar among the
three, followed by acetaminophen, then caffeine, the most polar. That
being said, aspirin travelled the greatest distance and has the highest
Rf value.
Similar Rf values will not necessarily mean that the two components
are the same molecule. For it to be a valid comparison, the TLC
plates must be run under the exact same conditions for temperature,
stationary phase, and mobile phase.

It can be seen from the results that
the samples contain other
components (impurities) which may
mean that the purification process
done during the previous experiment
wasnt efficient.

Rf values vary with the structure and polarity of a
substance. The most polar yield the smallest value while
the least polar yield the greatest value. Experimental data
showed that caffeine is the most polar among the three,
followed by acetaminophen. Caffeine travelled the least
distance due to its affinity to the very polar stationary phase
and therefore interacted with it more. Aspirin is said to be
the least polar, having travelled the greatest distance due
to its high affinity to the slightly polar solvent.
Through Thin-layer Chromatography with the use of
Silica as the stationary phase and a solvent system
composed of 95% ethyl acetate and 5% acetic acid as the
mobile phase, we were also able to determine the purity of
the extracts we yielded from the previous experiment. The
Rf values of the standard were used to identify the
components of the extracts. Through the collected data, it
was seen that most of the extracts werent exactly pure.
Aside from not having the exact Rf values that their
standards did, some spots were also present, marking

In the next future experiments, we recommend the use
of other solvent systems as mobile phases, other
adsorbents as stationary phases and chromatographic
techniques other than TLC.

Larcia, L. (ed.) (2014). Laboratory Manual in
Pharmaceutical Chemistry 125.1. Manila: UPMCP

Philadelphia College of Pharmacy (2013). Remington: The
science and practice of pharmacy (22
ed.). Michigan:
Pharmaceutical Press.

Questions asked during the report:
1. What will happen if the TLC plates are left too long in the chamber?
-If the TLC plates are left too long in the chamber, all the spots will move up the top edge of the
plate, therefore making the Rf values inaccurate.

2. Where were the contaminants found in?
-Most of the contaminants were found in the extracts from the previous experiment.

3. What is the effect of contaminants on the Rf value?
- Contaminants do not affect the Rf value. They do however create additional spots and imply
impurity of the sample.

4. Why is the plate heated before spotting?
-So that any water molecule adsorbed by the Silica is removed. When water is absorbed, the
site of attachment for the sample is blocked.