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Florentinus Dika Octa Riswanto, M.Sc.

Fakultas Farmasi
Universitas Sanata Dharma
2018

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Paper Chromatography Thin Layer

Chromatography

http://mrswolfgang.wikispaces.com/file/view/photoggggg.jpg/65003922/512x384/photoggggg.jpg
http://www.labsource.com/ProdImg/14/14320.jpg

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An analyte migrates up or across a layer of stationary

phase
(most commonly silica gel),

under the influence of a mobile phase

(usually a mixture of organic solvents)

which moves through the stationary phase

by capillary action.

Watson, 1999
Pharmaceutical Analysis,
Churcill Livingstone,
Page 77

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Silica gel 60 GF254

Average pore size of the layer: 60 Å
G = Gypsum (13%) is used as binder
F = Layer contains a fluorescence/fosforescence
indicator
254 = Excitation wavelengths of fluorescence/
fosforescence indicators
(Spangenberg et al., 2011 )

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Fluorescence - organic compounds

- the emission of radiation has decayed within 10-8 s
after the exciting radiation is cut off
- can be incorporated in the adsorbent layers or applied
afterwards by spraying or dipping

Phosphorescence - inorganic compounds

- the decay phase lasts longer (because the electrons
return to the ground state from a forbidden triplet state)
- incorporated as homogeneously as possible into the
stationary phase

(Jork et al., 1990)

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Luminescence Phenomena

(Jork et al., 1990)

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Organic fluorescence indicators for aluminium oxide, silica gel and cellulose layers
(code F366, UV366) include:
• the sodium salt of 3-hydroxypyrene-5,8,10-trisulfonic acid
• the sodium salt of 3,5-dihydroxypyrene-8,10-disulfonic acid
• sodium fluorescein and fluorescein or 2',7'-dichlorofluorescein
• rhodamine B and rhodamine 6G
• Morin
• cyanine dyestuffs
• stilbene derivatives (e.g. diaminostilbenetriazine) and
• optical brighteners (Ultraphor WT BASF, Calcofluor R-white, Leukophor).

(Jork et al., 1990)

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The substances employed as inorganic phosphorescence

indicators (code F254, UV254) include
• blue (tin-activated strontium compounds),
• yellow (uranyl acetate)
• yellow-green (manganese-activated zinc silicate or zinc
cadmium sulfide)

(Jork et al., 1990)

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(Kealey and Haines, 2002)

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Normal phase :
Polarity of stationary phase > mobile phase
Stationary phase : silica
Mobile phase : non polar organic solvents

Reversed phase :
Polarity of mobile phase > stationary phase
Stationary phase : RP-18
Mobile phase : water-based mobile phase

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Adsorption chromatography:
Adsorption in TLC occurs at the surface of the particles of
the stationary phase, which are in contact with the mobile
phase

Partition chromatography :
In partition chromatography, separation depends on the
relative solubility of the sample components in two immiscible
solvents brought into contact with each other

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The dominant features in adsorption chromatography

are the surface phenomena,
and in partition chromatography the decisive role is played by
the distribution between the two liquid phases.

Therefore, in adsorption chromatography, the type, position, and number

of the solute’s functional groups control the separation,

while the solute’s overall polarity plays the same role

in partition chromatography.

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(Komsta, 2007)

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Sampel Sampel
preparation application

Prechromatographic

Derivatization steps

Postchromatographic
Visual and
photometric
examination

Chromatogram
–detection-

Chromatographic
development

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“just depend on its

reproducibility”

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Sampel Sampel
preparation application

Prechromatographic

Derivatization steps

Postchromatographic
Visual and
photometric
examination

Chromatogram
–detection-

Chromatographic
development

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O
H2N
OH
GABA
O H2C
O H2N
H2N O
H3C OH
H2N
OH
CH3 OH
Gabapentin Vigabatrin
Pregabalin

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HO
O
H2N O O
OH
H
S
Gabapentin H
O
N HO
O
O OH O
H2N o-phthalaldehyde
H3C OH
S

CH3 O
O HO N
Pregabalin
O OH
H3C
HS OH
H2 C CH3
3-mercaptopropionic acid CH2
S
O
H2N O
N
OH
OH
Vigabatrin

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ABSORBANCE VS REFLECTANCE

The Lambert-Beer law is not applicable for TLC evaluations, because

TLC plates scatter light (Spangenberg et al., 2011)

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Calculation of Rf-value.
• Spot 2 has an identical Rf and
color with standard 1 (St 1), but
it does not mean that spot 1 was
always identical with St 1.

• Spot 3 has an identical Rf – value

with St 2, but their colors were
not identical, so Spot 3 and St 2
are different compounds.

• Although Spot 1–4 showed as

one single spot, it does not mean
the spots contained only one
compound

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Z6 – Z0
Rf Z6 =
Zf – Z0

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(Spangenberg, 2011)

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(Dünnschichtchromatographie) = TLC

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OH

OH

HO O

OH

OH O
Rutin
(major active components in Ginkgo biloba)

Mobile phase:
Ethyl acetate : acetic acid : formic acid :
water (100:11:11:26)

Stationary phase:
HPTLC plates silica 60 F254

High-performance Thin-layer Chromatography for

the Analysis of Medicinal Plants (2007)
Eike Reich, Anne Schibli

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(Revathy et al., 2011)

http://www.sciencedirect.com/science/article/pii/S0940299310000369

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TERIMA
KASIH
octadika@yahoo.com dika_octa

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