Thin Layer Chromatography Collect: (2020/05/02) Sample bottle (50 - - PowerPoint PPT Presentation

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Thin Layer Chromatography

Collect:

• Sample bottle (50 mL)
• TLC plate (4)
• Paper towel (1)
• Filter paper (55 mm, 1)
• Dropper

(2020/05/02)

Prepare by yourself:

• Pencil, ruler, camera
• Green leaves
• Panadol

Share:

• Mortar and pestle
• Scissors and tape
• UV light
• Sample bottle (7 mL)
• Beakers (30 mL)
• Capillary tube
• Standard solutions

Prepare:

• Glass rod
• Graduated cylinder (7 mL)
• Tweezers

Objective and Skills

 Objective: Using thin-layer chromatography (TLC) to separate and identify mixtures  Pr Prin incipl ciple – 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)

 Sk Skil ill: l: Extraction, spotting, developing, visualization.

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Flow Charts

 Part I: Thin layer chromatography of leaf extract Extract of green leaves  Spotting on TLC plate  Developing the plate  Visualization  Outline with pencil  Calculate Rf  Part II: Thin layer chromatography of Panadol Dissolving samples  Spotting on TLC plate Developing with two kinds of solvent  Visualize under UV  Outline with pencil  Calculate Rf

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Extraction

• I. TLC of leaf extract
• II. TLC of Panadol

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

• rganic 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

• f different kinds of leaf extract

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Step 2 Preparation of Panadol

 Take one tablet of Panadol; record

the bran name and ingredients

 Grind to powder  Weigh 0.05 g Panadol powder into

7 mL sample vial and add 5 mL EA to dissolve

 Standards prepared by stock room  Acetaminophen standard  Caffeine standard

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Step 3 Prepare Development Chamber

 Cut a 55 mm filter paper to two pieces of rectangle shape  Put the filter paper liner inside the 50 mL sample bottle  Add suitable amount of solvent to moist the filter paper  Adjust the solvent to a depth of 0.2-0.3 cm (about 2 mL)  Cap the bottle and set aside to keep chamber saturated with

vapor

Development chamber * Development solvent

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• II. Panadol

Solvent 1: n-hexane/acetone (7 : 3) Solvent 2: EA/acetone (1 : 1)

• I. Leaf extract

Solvent 1: n-hexane/acetone (7 : 3)

0.2 cm

Step 4 Spotting the Plate

 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 be filled in  Touch lightly and briefly to the plate  Keep spots small (ca. 2 mm) to get better

separation

Draw the line and spotting lightly

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* Sample plate

Three spots evenly spaced

0.5 cm

• I. Leaf extract plate
• Three kinds of leaf extract
• II. Panadol sample plate

1) Acetaminophen standard 2) Caffeine standard 3) Panadol test solution

• Sample with high concentration

may cause tailing

• Repeat spotting for dilute sample

* Sample and capillary tube

Step 5 Developing TLC Plate

 Blow gently on the plate to evaporate the

solvent and keep the applied spots small

 Hold it with tweezers by its edges and place it

squarely in the solvent surface.

 Do not touch the plate on the sides of bottle  Solvent level must not be above the spots, or

the spotted material may dissolve in solvent

 Cap the bottle securely

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Solvent Filter paper liner Place squarely

Note:

 Develop leaf extract plate with Solvent 1 n-hexane/acetone (7 : 3)  Develop Panadol plate with previous Solvent 1: n-hexane/acetone (7 : 3)  Discard Solvent 1 to waste bin and rinse with small portions of Solvent 2:

EA/acetone (1 : 1)

 Replace filter paper liner and add suitable amount of Solvent 2:

EA/acetone (1 : 1) to run another Panadol plate

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Step 6 Visualization and Recording

 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

extract and developing system Line with pencil * Panadol plate under UV light

Rf = Height of compound Height of solvent front

Notice (condensed 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

• ther. 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.

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