Thin Layer Chromatography

2021/03/02
Collect: Share:
• 30 mL beaker (2) • Mortar and pestle
• Aluminum foil (2) • Scissors and tape
• Filter paper (2) • 7 mL sample bottle
• Dropper (1) • UV light
• Paper towel (1) • Capillary tube
• Tweezers (1) • Standard solutions
• TLC plate (4)

Prepare:
• Pencil, rulers, camera
• Green leaves

1
 Objective and Skills
◆ Objective: Using thin-layer chromatography (TLC) to
separate and identify mixtures
◆ Principle
– Base on the distribution differences between the
stationary phase and the mobile phase of compounds
– Stationary phase: silica gel coated with fluorescent
indicator F254
– Mobile phase:
• A solvent which causes all the spotted material to move
with the solvent front is too polar.
• One which will not cause any of the material in the spot to
move is not polar enough.
• Make an approximate judgment to choose the suitable
solvent (may adjust by using EA and n-hexane)
◆ Skill: Extraction, spotting, developing, visualization. 2
 Flow Charts
◆ Part I: Thin layer chromatography of leaf extract
Extract of green leaves ➔ Spotting on TLC plate
➔ Developing with two kinds of solvent ➔ Visualization
➔ Mark with pencil ➔ Calculate Rf
◆ Part II: Thin layer chromatography of Panadol
Prepare samples ➔ Spotting on TLC plate
➔ Developing with two kinds of solvent
➔ Visualize under UV ➔ Mark with pencil ➔ Calculate Rf

Extraction I. TLC of leaf extract II. TLC of Panadol 3

 Step 1 Preparation of Leaf
Extract
◆ Cut 5-6 pieces of dark green leaves
◆ Add 10 mL n-hexane/EA (4 : 1) as
solvent and grind with mortar and pestle
◆ Extract to soln. appears dark green
◆ May need more solvent for
vaporization during grinding
◆ Conduct extraction in fume hood for
organic vapor
◆ Use dropper to transfer leaf extract to 7-
mL sample vial and label it
◆ Share the extract and capillary tube
◆ Compare the color and components
4
of different kinds of leaf extract
 Step 2 Preparation of Panadol

◆ Share Panadol samples

◆ Acetaminophen standard

◆ Caffeine standard

◆ Panadol Extra

◆ Record the brand name and

ingredients

5
 Step 3 Prepare Two
Developing Chambers

* Use two 30 mL beaker

** Developing solvent
1) n-Hexane/EA (4 : 1)
2) n-Hexane/EA (1 : 4)

Prepare two developing chambers

◆ Put the filter paper liner inside the 30 mL beaker

◆ Add suitable amount (about 2 mL) of solvent to moist the filter paper
and adjust the solvent to a depth of 0.2-0.3 cm.
◆ Cover the beaker with aluminum foil and set aside to keep chamber
saturated with vapor
6
 Step 4 Spotting

I. Leaf extract sample

• Three kinds of leaf extract
• Repeat spotting on the
same point for three times
0.5 cm
Draw the line and Three spots
spot lightly evenly spaced
◆ Place thin layer plate on a clean paper towel
◆ Lightly draw a line with pencil about 0.5 cm height from bottom
◆ Label three points on line separated from each other about 0.5 cm
◆ Dip capillary tube into test soln. and draw sample in
◆ Touch lightly and briefly; keep spots small (ca. 2 mm) to get better separation
Note:
• Sample with high concentration may cause tailing
• However, repeat spotting several times for dilute sample 7
 Step 5 Developing TLC Plate
◆ Blow gently on the plate to evaporate the
solvent and keep the applied spots small Place
◆ Hold it with tweezers by its edges and place it squarely
squarely in the Solvent 1 beaker.
◆ Do not touch the plate on the side of beaker
◆ Solvent level must not be above the spots, or Filter
the spotted material may dissolve in solvent paper liner
◆ Cover the beaker securely with aluminum foil Solvent

Note:
◆ Develop leaf extract TLC plate in Solvent 1:
n-hexane / EA (4 : 1)
◆ Prepare another TLC plate and develop in
Solvent 2: n-hexane / EA (1 : 4)
◆ Compare the developing results 8
 Step 6 Visualization and
Calculation

(1) Visualize color spots (2) Under UV light

◆ Take out TLC plate when solvent front runs near the edge
◆ Line the solvent front and circle the compounds with pencil
◆ Visualize the colorless compounds under UV light with short wavelength
(254 nm)
◆ Calculate the Rf values of compounds
◆ Compare the results among various leaf extracts and developing systems9
 Step 7 TLC of Panadol
◼ Prepare two TLC plates with three sample
1) Acetaminophen standard
2) Caffeine standard
3) Panadol Extra
◼ Develop each plate in Solvent 1 & Solvent 2
◼ Visualize under 254 nm UV light
C
◼ Circle the spots and calculate the values of Rf A

On/off

UV lamp

Observation Height of compound

Rf =
Height of solvent front
Place for sample
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 Notice Brief report

◆ Conduct leaf extraction in fume hood for vaporization of organic

solvent.
◆ Use dropper to transfer solvent with approximate amount.
◆ Share leaf extract, capillary tubes, and Panadol samples with each
other. Be cautious about not mixing up the capillary tubes.
◆ Keep your TLC plate clean. Hold it with tweezers, place it on a paper
towel, and avoid touching the silica gel by hands.
◆ Capillary tube can be used repeatedly. Rinse the tube by dipping it in
clean solvent and withdrawn solvent out on paper towel several times.
◆ Shall the developed thin layer plate show edge effect, you may prepare
another TLC plate and develop it again.
◆ Remain and tape your TLC plates on report to show your separation
results and support your discussion.
◆ After class, recycle capillary tubes, organic solvent, and residue of leaf
extract. 11