It is TPLC training report

1. Ethiopian Institute of Agricultural Research
Agricultural Nutritional Research Laboratories Directorate
Agricultural Quality Research laboratory
Training report on Thin Layer and column Chromatography
May, 2015,
By Ayetenew Abita (Assistant researcher)
Agricultural Quality Research Laboratory
1. Introduction about chromatography
Chromatography is an analytical technique which is used for separation, identification and
determination of chemical components from their chemical mixture using two non-miscible
phases such as; mobile and stationary phase. The one, the stationary phase, a liquid or solid, is
fixed in the system. The other, the mobile phase, a fluid, is streaming through the
chromatographic system. Where, the molecules of the analytes are distributed between the
mobile and the stationary phase. When present in the stationary phase, they are retained, and are
not moving through the system. In contrast, they migrate with the velocity, v, of the mobile
phase when being there. Due to the different distribution of the particular analytes the mean
residence time in the stationary phase differs, too, resulting in a different net migration velocity.
This is the principle of chromatographic separation.
Principle of chromatographic separation: Different distribution of the analytes between mobile
and stationary phase results in different migration velocities. And the position of the distribution
equilibrium determines the migration velocity. It reflects the intermolecular interactions of the
analyte with the stationary and the mobile phase.

2. 1.1 Classification of chromatography
The differentiation is based on techniques of chromatography or principle or physical changes
used
1. The physical states of stationary phase and mobile phases.
2. The on principle of separation used.
3. The chemical nature of stationary phase and mobile phases used (polarity).
4. Based on the shape of stationary phase employed.
5. Based on purpose of chromatography experiment.
6. Based on physical or chemical character of the stationary phase.
1.1.1 Based on the physical state of both phases
These are broadly classified as homogeneous or heterogeneous. The chromatography systems
differ based on the physical states of the phases used.
♠ Homogeneous techniques have both stationary phase and mobile phase as liquid. Ex: Liquid-
liquid chromatography.
♠ Heterogeneous techniques employ different stationary and mobile phases. Ex: Solid-
liquid chromatography, Solid-Gas chromatography, Liquid-Gas chromatography etc.
1.1.2 Based on the principle of separation
Principle used in separation is considered i.e adsorption method or partition method.
♠ Adsorption chromatography: Here the sample molecules get separated due to greater affinity to
absorb the solid stationary phase compared to that of mobile phase. This principle works when
stationary phase is a solid and mobile phase is liquid solvent.
♠ Partition chromatography: Here the samples molecules get separated due to relative
differences of dissolution and partition into different phases/layers. The molecules with greater
partition or dissolution into mobile phase, is separated faster while that with partition into solid
phase liquid moves slower or latter. Here both stationary phase and mobile phase are liquid in
nature or liquid as stationary phase and gas as mobile phase. The liquid on stationary phase
exists as a thin layer on a solid background.

3. 1.1.3. Based on the chemical nature of the mobile and stationary phase
This differentiation is based on chromatography column i.e the nature of stationary phase inside
the column;
♠ Normal phase chromatography: Here the stationary phase is polar in nature and mobile phase
is in non-polar nature. Hence on elution, non-polar compounds are eluted first and polar
compounds later as they have greater affinity to stationary phase. Mostly used in column
chromatography technique
Ex: Normal phase column chromatography.
♠ Reverse phase chromatography: This is reverse to the above method. The stationary phase is
non-polar and mobile phase is polar in nature. In practice this reversed phase chromatography
is highly used in routine analysis as most of the substances like drugs etc. used in daily life are
polar in nature.
Ex: Reverse phase HPLC systems.
1.1.4. Based on the shape of stationary phase
The shape of stationary phases depends on the support used to place the stationary phase. Hence
based on the shape of stationary phase, there are two types like Column chromatography and
planar chromatography.
♠ Columnar chromatography is one where stationary phase is column shape. It is widely used in
types like High pressure liquid chromatography (also medium pressure liquid chromatography),
Column chromatography, And Gas chromatography etc. Development of chromatogram occurs
in volume aspect.
♠ Planar chromatography is one wherein a stationary phase is flat. The development occurs on
the planar surface (only area). This type of chromatography is used in Thin Layer
Chromatography (TLC), High Pressure Thin Layer Chromatography (HPTLC) and Paper
Chromatography.

4. 1.1.5. Based on the purpose of chromatographic experiment
This is one of the types of chromatography Here the idea of experiment different. This can be
done on both planar type and columnar type of chromatography. The types are
♠ Preparative chromatography: The amount of sample injected or applied is very large and
the separated and pure component is collected for use. The desired component of sample is not
disposed off. This is also exclusively applied in column types as preparative column
chromatography.
♠ Analytic chromatography: Here the sample size applied or injected is very small and the
intention is aimed to identify the components in the sample and also their individual
concentrations in the sample. The eluted sample from the outlet is disposed off.
1.1.6. Based on the physical or chemical characteristics of the stationary phase
This is especially followed in columnar chromatography where in the stationary phase used has
specific character like being porous or charged.
♠ Size exclusion chromatography: Here the stationary phase has pores in its matrix. Consider
molecules are allowed to pass through, those with large size travel a short path under mobile
phase influence and pass out of column first and vice-verse.
♠ Ion exchange chromatography: Here the stationary phase has definite charged ions. When the
sample is passed through, it retains all the molecules with opposite charge and leaves off
molecules with same charge. So to elute the bound molecules, you need to pass another mobile
phase with similar charge to stationary phase to recover the bound molecules. (like molecule
displacement method).

5. So the above mentioned types of chromatography are theoretically classified. But practically we
have only 12 types of chromatography
So over all available chromatography techniques for regular analysis include.
1. Column chormatography: It is similar to the pic shown above where you pour mobile phase
from top of the column to flow through the sample present on stationary phase in the
column to get separated.
2. High performance liquid chromatography (HPLC): Here mobile phase is pumped into the
column at a defined pressure and further the column particles are very small so the surface
area is high and better separation takes place. See HPLC applications
3. Gas chromatography (GC): Here gas is used as mobile phase. For details see gas
chromatography principle.
4. Ion-exchange chromatography: Here the mobile phase is charged and sample molecules
with similar charge present on the charged stationary phase get eluted out as the mobile
phase molecules with charge displace them.
5. Size exclusion chromatography. Here the column is loaded with charged some gel having
pores. A sample particle when poured along with mobile phase has to pass through the sieve
like net work of the stationary phase. In doing so, the larger particles elute out first and
smaller ones last. The reason is the smaller ones take longer path in the column stationary
phase while larger particles take short path to elute out.
6. Thin layer chromatography (TLC): Here the stationary phase is a thin layer and plate like.
7. High performance thin layer chromatography (HTLC): Similar to TLC by more efficient.
8. Paper chromatography. Here, column is paper either rectangular or circular.
9. Affinity chromatography.
10. LC-MS
11. GC-MS
12. Ultra high performance chromatograph.
From the above listed chromatographic techniques hands-on training was held in two
chromatographic techniques such as; Thin layer chromatography (TLC) and Column
Chromatography (CC).

6. 2. THE OVERALL OF HANDS-ON TRAINING
Isolation of essential oil component by CC
5g of was taken to make slurry by 100ml hexane. The column tube was packed by adding the
prepared slurry. And filter paper and small purified sand was added on the top of silica gel. After
that the pure hexane was eluted until it becomes stabilized. Then a few gram of essential oil was
taken and applied at the top of the column. To fractionate the mixture the column was washed
with 200ml of hexane and fractionates was collected. And washing of the column was continued
with ethyl acetate while collecting fractionate. Finally the solvent was evaporated and the weight
of fractionates was measured.
Isolation of curcuminoids from turmeric by column chromatography
First 5g of powdered was subjected to CC after the column was packed as described above.
Chloroform and methanol was used as mobile phase with ration of (95:5) the polarity was
increased gradually as 100ml (95:5), 100ml (75:25), 100ml (50:50), 100ml (25:75). And the
observed individual fractions were collected separately.
Preparation of silica gel-coated plate (PTLC)
By mixing 30g of silica with distilled water and shaking it thoroughly slurry was prepared. Then
slurry was spread on the 20cm x 20cm glass plate. And the coated plate was allowed to dry
overnight. Then after, plates were putted in oven at 100o
C for 1hr to activate the plate. Finally, it
was allowed to cool and used for analysis.

7. Determination of total gingerol content with PTLC
The mobile phase hexane, ethyl acetate (4:1) and transfer it to the chromatographic chamber.
Secondly, the mobile phase was allowed to evaporate and saturate. Third, approx 20mg ginger
oleoresin was dissolved with small amount of acetone and applied on PTLC in the form of band
using pipette. Fourth, plates were placed into the chamber and the plates were removed after the
mobile phase moved 2/3 of the distance on the stationary phase. Fifth, the separates were
visualized at day light, UV lamp ant 254nm and 366nm. Then, the major two separate bands
were scratched. After that, the scratched sample was collected in the flask and dissolved in 25ml
of acetone then filtered the solution to recover the total ginger from silica gel. Lastly, the total
gingerol was weighed and calculated the percentage yield as follows.
Quantitative Isolation of Aloin from Aloe Latex
First, the mobile phase with the ratio of chloroform: methanol (4:1) was prepared and allowed to
saturate for 2hrs. Secondly, 20mg of latex was measured and dissolved approximately 2ml of
methanol. Thirdly, when the mobile phase moves 2/3of the distance of stationary phase having
0.5mm PTLC plate it was out from the chamber and dried at room temperature. Fourthly, it was
visualized different colors on the plate in different distance. Fifthly, the day light (yellow band)
and UV light at 254nm or 366nm (dark band) was visualized. Sixthly, the band was scratched
carefully and dissolved in 25ml of methanol then filtered to separate the isolate from silica gel.
Then, the filtrate was dried on Rota Vapour at 40o
C temperature. Finally, the round bottom
containing the dried sample in it and calculated the percentage yield of the isolated compound as
follows.