Introduction of HPTLC

1. TLC-HPTLC
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2. Advantages of TLC-HPTLC
Instrumental Thin-Layer Chromatography (or Planar Chromatography)
is a modern separation technique distinguished by flexibility, reliability and
cost efficiency.
Together with HPLC and GC it belongs to the microanalytical methods, which
play an important role in research and routine laboratories.
In many cases instrumental Thin-Layer Chromatography offers a more
suitable solution and often it is used as confirmatory or alternative
technique.
Due to the off-line principle, Thin-Layer Chromatography offers:
• Flexibility
• Parallel separation of many samples with minimal time requirement
• Simultaneous visual evaluation of all samples and sample components
• Simplified sample preparation due to single use of the stationary phase
• Possibility of multiple evaluations of the plate with different parameters
because all fractions of the sample are stored on the plate.
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3. Important Applications of TLC-HPTLC
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4. 1. Sample Application Capillary used to spot solution of each
sample.
2. Development This is when the separation actually occurs.
3. Visualization Results will be viewed under UV light.
4. Interpretation of Results Comparison of retention factors.
Four Stages in TLC
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5. TLC plate
B. Dissolve solid
sample in suitable solvent
C. Use TLC capillary
to transfer and spot
dissolved sample
A. Draw “guide lines”
lightly with pencil
 1 cm
 1 cm
Sample Application
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6. Development of TLC Plate
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TLC plate
TLC Developing Chamber
Place spotted TLC plate
in developing chamber
Developing solution
is drawn up the plate
by capillary action
Remove TLC plate when
solvent reaches top line
Developing
solution
(mobile phase)
}
{keep covered}

7. Visualization of TLC Results
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A. Allow solvent to evaporate
from surface of TLC plate.
C. Mark spots with a pencil
while viewing under UV.
UV
B. View results under UV light.
look for grayish spots on the
fluorescent green background

8. Silica gel impregnated with
Fluorescent dye
Glows yellowish-green when irradiated
With 254 nm UV light
Aromatic and conjugated compounds
Absorb 254 nm light
UV absorbing compounds appears dark
On yellow-green background
Visualisation
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9. Interpretation of TLC Results
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Determine retention factors
(Rf) for each spot detected.
distance spot has moved
distance solvent has moved
____________ ___________
Rf = =
X
Y
Y
X
distance spot has moved
distance solvent has moved
_______________________
Rf = =
Z
Y Z
T
distance spot has moved
distance solvent has moved
_______________________
Rf = =
T
Y
x

10. Sorbents Mechanism Applications
Silica gels Adsorption Amino acids, alkaloids,
hydrocarbons, vitamins
Hydrocarbon modified
silicas
Modified
partition
Nonpolar compounds
Cellulose Partition Amino acids, nucleotides,
carbohydrates
Alumina Adsortption Hydrocarbons, alkaloids, food
dyes, lipids
Kiselguhrs Partition Sugars, fatty acids
Ion-exchange
celluloses
Ion-exchange Nucleic acids, nucleotides
Polyamide Adsorption
(H-bonding)
Anthocyanins, aromatic acids,
flavonoids
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11. 2-D TLC
In this technique, the spotted sample is first developed in
one direction; then, after drying, the plate is turned 90 and
developed again, but now with different mobile phase
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